PALLADIN'S PLANT PHYSIOLOGY EDITED BY BURTON E. LIVINGSTON Prof. V. I. Palladin i 9 i i / PLANT PHYSIOLOGY BY VLADIMIR I. PALLADIN PROFESSOR IN THE UNIVERSITY OF PETROGRAD AUTHORIZED ENGLISH EDITION Based on the German Translation of the Sixth Russian Edition and on the Seventh Russian Edition (1914) EDITED BY BURTON EDWARD LIVINGSTON, Ph. D. PROFESSOR OF PLANT PHYSIOLOGY AND DIRECTOR OF THE LABORATORY OF PLANT PHYSIOLOGY OF THE JOHNS HOPKINS UNIVERSITY Second American Edition With a Biographic Note and Chapter Summaries by the Editor 173 ILLUSTRATIONS PHILADELPHIA P. BLAKISTON'S SON & CO 1012 WALNUT STREET Copyright, 1923, by P. Blakiston's Son & Co. PRINTED IN U. S. A. BY THE MAPLE PRESS YORK PA A NOTE OF APPRECIATION 1 Doctor V. I. Palladin, Academician of the Russian Academy of Sciences and author of this book, died in Petrograd on February 3, 1922, after a pro- longed illness that culminated in aortic aneurism. His life work was a fine contribution to physiological science in general, and especially to plant physio- logy. His text-book on plant physiology was published in Russian, German, French, and English, and the marked excellencies of the book have made his name well known wherever this science is studied. But his greatest contribu- tion lies in his research publications. Palladin was born July 11, 1859, in Moscow and received his education in the First Gymnasium of Moscow and in the University of Moscow. He studied botany under Timiriazev and Gorozhankin, and published his first research contribution, "On the structure and capacity for swelling of cell walls and starch grains," in 1883 (Zapiski Moskovskogo Univ.). His disser- tation for the master's degree, conferred at the University of Moscow in 1887, is on "The significance of oxygen for plants" (Bull. Soc. Nat. Moscow, 1886), and that for the doctor's degree, conferred at the same university in 1888, is on "The influence of oxygen on the decomposition of proteinaceous substances in plants" (Dissert. Moscow Univ., 1889). The first teaching position held by the great Russian physiologist was in the Institute of Rural Economics and Forestry at Novaya Alexandria, whither he went in 1886. Three years later, after receiving the doctor's degree, he became professor of plant anatomy and physiology in the University of Kharkov. In 1897 he was appointed to a professorship in the University of Warsaw and was made director of the Pomological Garden of Warsaw. He was called to the University of Petrograd in 1900, as professor of plant physiology, where he remained until 191 7. In the last-named year Palladin removed to the Crimea, giving lectures in the newly founded university at Simferopol. Later he became director of the Nikitskii Botanical Garden at Jalta. He was elected to the Russian Academy of Sciences in 1906, and took active part in the work of the Academy, publishing many papers in its proceedings. Election as academician is the highest honor conferred on Russian scientists, and only a few receive this mark of great distinction. 1 This note is mainly based on a biographical sketch of Palladin. by Professor X. I. Kuz- netzov, in the ninth Russian edition of the Physiology. I have been helped in its preparation by Dr. Selman A. Waksman, of the New Jersey Agricultural Experiment Station, and by Mr. L. J. Pessin, of the Mississippi Agricultural and Mechanical College, as well as by Mr. D. X. Borodin, of the Xew York Office of the Russian Bureau of Applied Botany and Prof. X. Ivanov, of the University of Petrograd. — B. E. L. vii v iii A NOTE OF APPRECIATION Palladin's lectures were always precise and unusually clear. His text-books —on plant anatomy, plant physiology, and systematic botany— show his excel- lent style of presentation. In his teaching positions Palladin always attracted a group of enthusiastic students. He was a calm and polished leader, always pleasant to work with, who would not quarrel over unessential matters but who understood how to lead the advance persistently toward the finer and greater things. The remarkable precision of his scientific thinking, together with his indefatigable application, placed him at the head of a school of plant physiology that extends far beyond the boundaries of Russia. Although he was interested in and contributed to many different lines of botanical study, Palladin's main research publications were, from the time of his master's-degree dissertation at Moscow, devoted to the fundamental phe- nomena of respiration. His many papers on this subject — and those that appeared under joint authorship, with one or more of his colleagues or students —were not confined to the Russian language, and Palladin's name became familiar to readers of the leading French and German journals devoted to botany and to physiological chemistry. To the scientific world at large, as well as to plant physiologists of all nations Palladin's thorough elucidation of some of the most fundamental and baffling aspects of the respiration process will stand as his greatest achievement. Step by step, he and his followers gradually built up a new and clear picture of the chemistry of respiration as it apparently occurs in all living cells. The main points of the Palladin theory of respiration are somewhat as follows: Under the influence of enzymes, carbohydrates and similar sub- stances are anaerobically decomposed into carbon dioxide and incompletely oxidized organic compounds, these partial oxidations occurring partly at the expense of oxygen derived from the decomposition of water. The hydrogen produced by aqueous decomposition may sometimes be set free, or it may dis- appear in the reduction of some of the incompletely oxidized compounds just mentioned, but it is regularly oxidized in aerobic respiration, with the formation of water. The aerobic oxidation of hydrogen occurs by two stages: (i) This element combines with respiration pigments (acceptors of hydrogen), thus forming respiration chromogens. (2) The chromogens, in turn, are oxidized by free oxygen, under the influence of oxidizing enzymes, forming water and respira- tion pigments. Thus, in normal, or aerobic, respiration, the carbon dioxide produced is a product of anaerobic respiration (fermentation), while the water produced is a product of the oxidation, by free oxygen, of anaerobically pro- duced hydrogen. Anaerobic respiration occurs in all living cells, of animals as well as plants, while aerobic respiration is confined to those forms that are sup- plied with free oxygen and possess adequate oxidizing enzymes. This theory, with all the details that it implies, must be regarded as one of the most bril- liant achievements of physiological science, and it may be said to represent the main contribution Palladin made to the advance of appreciative human A NOTE OF APPRECIATION IX knowledge. It is remarkable that the great scholar was able to bring this phase of his studies to such a logical completeness within his lifetime. Palladin's inspiration still works in the minds and lives of his students, and his contributions to science have become a permanent part of the mental equip- ment of mankind. The results of his studies and the bent and trend of his clear thought have left a lasting effect, even upon dwellers in far countries. The publication of this second printing of the English edition of the Physiology furnishes a significant illustration of the unity of science, in space as well as in time, and of the true immortality of the scientific spirit. Burton E. Livingston. Desert Laboratory, Tucson, Arizona. August, 15, 1922. AUTHOR'S PREFACE TO THE GERMAN EDITION This text-book constitutes an improved and enlarged translation of the sixth Russian edition of mv Plant Physiology. There are already several ex- cellent text-books on this subject in German, but I venture to hope that the present volume will not be without worth, especially on account of the atten- tion here given to the chemical aspect of physiological processes, and also be- cause of certain peculiarities in the presentation of the subject-matter itself. It is my pleasant duty to express my hearty thanks to Professor E. Abder- halden, through whose friendly offices the publication of this edition was undertaken. For the translation of the book, my thanks are due to Messrs. Nicolai von Adelung, S. Kostytschew, Georg Ritter and O. Walther, and I am also indebted to the last three gentlemen for valuable advice. W. Palladin. XI EDITOR'S NOTE TO THE SECOND AMERICAN EDITION In this, the second American edition of Palladin's book, a few typo- graphical and other errors that have come to the editor's attention have been corrected. In a very few cases the wording of the text has been somewhat improved, especially where the old wording was not quite clear. Several new notes by the editor have been added, notably in Part II, Chapters V and VI. While a small number of additional references to the literature have been inserted, no attempt has generally been made to enlarge the scope or increase the number of the citations. The text and notes are generally the same as in the first Edition. The editor is glad to acknowledge valuable assistance re- ceived from Dr. Sam F. Trelease, and also to express his thanks to those readers who have called his attention to errors occurring in the first Edition. The publishers have assumed the responsibility for the index. One new feature has been added, in the form of a summary for each chapter. In preparing the- summaries it has been attempted to present a succinct but rather complete statement of the main features dealt with in the respective chapters, with the idea that these resumes may be useful to the student, espe- cially in reviewing the subject. It is suggested, also, that the summary of a chapter may be read with profit before reading the chapter itself, the summary thus serving as a sort of general background for the more detailed information gained by perusing the fuller presentation. In some cases new material has been introduced into the summaries, mainly to clear up a few vague transitions from one topic to another that occur in the text, and generally to help the stu- dent gain a logical and consistent view-point for the subject as a whole. The editor is alone responsible for the summaries. The Desert Laboratory, Tucson, Arizona, July 15, 1922. xin EDITOR'S NOTE TO THE FIRST EDITION The German edition of this book has gained many friends in institutions where plant physiology is taught and has supplied a need for elementary students not otherwise met. Its small size, together with its generally excellent arrange- ment and manner of presentation render it very well suited to the use of begin- ning students who really desire to obtain a general grasp of the subject in a comparatively short time. Its brevity, its conciseness and the readableness of its story are its first attractions, but a further examination reveals the facts that Palladin has been exceptionally thorough in much of his treatment, and that a wealth of well-chosen citations from the literature of plant physiology places in the reader's hands a ready guide to original sources. In the latter regard the text-books originating in our own language are usually deficient, thereby depriv- ing the student of one of his most important rights at the very start— the right to appreciate that the key to the science he is entering really lies in its literature, contributed to by many hundreds of serious workers writing in many languages. Palladin approaches the subject from the point of view of a student of physio- logical chemistry, and it is the chemical aspects of plant physiology that here receive greatest emphasis. Most workers in the science will doubtless agree that this is an excellent method of approach. One who has read the book under- standingly should be able to plan his own further development, with the aid of the current journals and other contributions, and he will hardly miss the main general idea of present-day physiology, that the future of the subject must rest largely in the development and application of the technique and methods of thinking that characterize the more fundamental sciences of chemistry and physics. If the German translation has proved to be well suited to the use of serious elementary students, it follows that they should make use of it. Here, however, lies a difficulty. It appears to be the present fashion for graduates of American colleges to be able to really read only the English language, so that the drudgery of virtually digging their way through a German text militates strongly against their becoming familiar with the subject-matter involved ; they are apt to fail to grasp the ideas because of a sort of blind struggle to understand the language. This being all too commonly the case, those who take up plant physiology or its applications need, especially, just such a short and scientific treatise as Palladin's book offers, but they need it in their own language, so that they may revert to it now and again without distraction. In this way the student's physiological habits of thought may continue to advance steadily while he is learning to read the foreign tongues that will be requisite for his future work. It was to fill this sort of need among students aiming to make some branch of plant physiology their specialty that an English translation of the German edition was originally XV Xvi EDITOR S NOTE TO THE FIRST EDITION undertaken by Miss Aleita Hopping, working in this Laboratory. Out of her translation the present book has developed. Aside from its usefulness to university students, Palladin's treatise ought to be of great value to more advanced investigators, especially as it furnishes a summary of a large amount of the literature of the subject, and it is hoped that the present edition may prove helpful to the many English-speaking workers who are engaged in physiological research as applied to agriculture and forestry. To specialists in its own field the book may serve as a convenient means of approach to Palladin's general interpretations. Finally, the numerous Russian references may help to open the domain of Russian science to English-speaking students and to emphasize the rapidly growing importance of Russian research in this subject. As this translation was nearing completion, Prof. Palladin very kindly furnished the editor with a copy of the seventh Russian edition, with those passages marked in which the latter differs from the sixth Russian edition (from which the German was directly derived), and it seemed desirable to make the present book conform with the author's latest alterations as far as possible. Dr. E. E. Free, also of this Laboratory, has made the necessary translations from the Russian, following Prof. Palladin's notations, and these alterations are included in the English text as here brought forth. The body of the text aims to be primarily a true translation of the German edition, and the original forms of expression have been retained in practically all cases where this was at all possible in English. The general attitude of the author is so obviously opposed to teleological reasoning that the non-teleological point of view has been made unmistakable in those few places where the German text might leave the reader uncertain in this regard. Palladin's writing is more free from teleological misinterpretations of the relations between conditions and results than is that in most of the text-books hitherto available, and this fact was one of the reasons for the undertaking of the present translation. It will doubtless be a long time before teleology may be deleted from physiological writing and thinking, but readers with a teleological point of view, who may still be satisfied with the consideration of results or effects, in place of conditions or causes that may be as yet unknown, will perhaps not object seriously to an em- phasis upon the conviction that permanent progress does not lie in this direction. Few other alterations have been made, these consisting mainly in some modifica- tions in the order of presentation, some slight additions that render certain statements more easily understood, and a very few changes in terminology that seemed desirable. Slight additions are sometimes indicated by being enclosed in brackets. Editorial notes have been added here and there, in the form of footnotes, which are uniformly signed "Ed." Footnotes not thus designated are Palladin's own. The editorial notes give such additional matter as has seemed desirable, either for completeness of presentation or for a better understanding by English- speaking readers. They constitute, in the aggregate, only a small portion of the volume. editor's note to the first edition xvn Palladin's treatment of the topics Growth, Movement and Reproduction (which make up the subject matter of Part II) is much less complete than is his treat- ment of Nutrition (Part I), and no attempt has been made by the editor to alter this characteristic of the book. The reader will appreciate the fact that there is available an enormous wealth of knowledge not seriously touched upon in Part II, which he will be able to approach through such other treatises as are mentioned in the list of books that follows this note. The entire manuscript has been read and criticised by Dr. H. E. Pulling, of this Laboratory, who has contributed much valuable advice in regard to some of the editorial additions. Since literature references are of prime importance in a book of this kind, and since the citations are not always clearly, fully, nor uniformly given, either in the German or in the Russian, it became necessary to verify these and correct them when necessary. This arduous task has been carried out by Mrs. Grace J. Livingston. Nearly all of the references have thus been verified, and the form of citation has been rendered uniform, as far as possible, throughout the work. Dr. Free has cared for the Russian citations. No attempt has been made to indicate what portions of any of the citations are due to correction or completion. Citations that it has been impossible to verify are given just as they appear in the German (or Russian), and are followed by an asterisk(*) to signify this. Some additional literature references have been inserted by the editor, these being generally enclosed in brackets, unless they occur in editorial notes. The rapidly increasing frequency of references to Russian authors in scien- tific literature is accompanied by much discrepancy in the English spelling of Russian proper names. This matter will require more serious attention from scholarly scientific writers in the future than has been accorded it in the past, and an attempt is here made at least to avoid the exacerbation of a condition that is already bad enough. The difficulty has perhaps arisen mainly through the fact that our acquaintance with Russian science is almost wholly based on writings in other foreign languages, especially in French and German. We have too frequently taken the German or French transliteration, as the case maybe, without regard to the fact that this almost always leads to mispronuncia- tion by the English reader. Thus, Pavlov often appears as Pawlow, which is as incorrect in English as it is correct in German. The name of the author of the present volume furnishes another example; we have W. Palladin where we should have V. Palladin. (In this particular case, the silent final e of the Rus- sian and of the French form of this name should be dropped in English, to avoid the resulting lengthening of the last syllable and even the misplacing of the accent, which is penultimate. The name is pronounced Pai-lad'-in, 1 like Aladdin.) In those cases where it is quite clear that a proper name ought to be regarded as Russian, an English spelling is here adopted that will lead to no serious ambig- uity as to pronunciation and that can be readily retransformed into the Rus- 1 This is authoritative, from Professor Palladin himself. xviii editor's note to the first edition sian. In these transliterations of Russian words into English the rules of the U. S. Library of Congress have been followed, with a few slight modifications, as follows: ia, tu, ie are all given as ia, in, ie; i, 'i and i are all given as i; the sign of the silent letter between two others (') is omitted (Krasnoselskaia is used instead of KrasnoseV skaia) and Yegunov is employed instead of Egunov, to insure proper pronunciation. When the name is not certainly Russian and when sev- eral spellings occur, the commonest form occurring in the German book is adopted. In those cases where the paper cited is in Russian the author's name is transliterated into English in the citation, as well as in the text, the title of the paper being translated into English unless a title in French or German is avail- able. In citations from languages other than Russian, author's names are given just as they occur in the publications cited. The two or three spellings that thus occur for the same Russian name are all given in the index, with the requi- site cross-references. Thus, references to Ivanov are all given under this spelling, but Ivanojj and Iwanow are also given, with the notation, " see Ivanov." The index is somewhat more comprehensive than is the case with the orig- inal, and authors' names have been inserted in the same alphabet with the names of subjects. This feature of the index amounts practically to a bibliog- raphy; references are given to all pages where the name in question is men- tioned, and those pages that bear footnote citations of this name are indicated by full-face type. A note on the form of citation employed in this volume, and a selected list of books bearing on plant physiology, are added after the present note. It is hoped that these additions, as well as the citations of the book itself, may prove serviceable to those who wish to acquire familiarity with the far-flung literature of a subject that embraces the principles of many separately named sciences, that brings into a single narrative such topics as ionization, adsorption, photo- synthesis, fermentation, the forcing of azalias and the keeping-qualities of apples. Laboratory of Plant Physiology of the Johns Hopkins University. FORM OF CITATION The form of citation employed in the footnotes uses (i) an Italic Roman numeral (followed by a comma) for the series number, (2) a black-face Arabic numeral (followed by a colon) for the volume number, (3) a superscript numeral for a subdivision of the volume, (4) Arabic numerals, in ordinary type, for the first and last page of the article cited (separated by a dash, and the second number followed by a period), and (5) an ordinary Arabic numeral for the year of publication (followed by a period). When several pairs of page numbers are given, as when an article is continued through several issues of the serial, these pairs are separated by commas. Where there is no volume number the volume has to be designated by its year number, and this is given in the place that would be occupied by the volume number, and in black-face type. Some- times this year number, for which the volume stands, is not the same as the rear of publication. In cases where a volume extends into more than one year, the year of publication of the volume frequently gives place, to two year numbers (separated by a dash). When adequate information was available a single year number is given in the cases just mentioned, referring to the year of pub- lication of the article cited rather than to the two or more years of the volume as a whole. Author's names are given in black-face type, the surname preceding the initials or given name Idem (black-face type) denotes a repetition of the author's name, or of the authors' names, next preceding. Ibid. (Italics) de- notes repetition of the name of the serial next preceding. The rather customary promiscuous scattering of capital letters through citations has been avoided; words or their abbreviations begin with capital letters only (1) when they are considered as beginning a sentence, (2) when they are proper names, (3) when they begin the proper name of a serial (as, Bot. gaz., Plant world), (4) when they are important words in the proper name of a society, institution, etc. (as, Roy. Soc. London, Missouri Bot. Gard.), or (5) when they are German nouns (compare Ann. bot., Compt. rend., Bot. Zeitsch., Jahrb.wiss. Bot.). The abbreviations employed for the names of serials appearing in the citations are, it is hoped, self-explanatory. When a citation appears more than once, it is given in full only in the first instance, and later occurrences include simply the author's name, the year, and (in brackets) a reference to the page of this book where the full citation may be found. XIX A CLASSIFIED LIST OF BOOKS FOR REFERENCE IN PLANT PHYSIOLOGY Physics, General Chemistry and Mathematics Bernthsen, A., A Text-book of Organic Chemistry, English translation, edited by J. J. Sudborough. 674 p. New York, 1907. Comstock, Daniel F., and Troland, Leonard T., The Nature of Matter and Electricity, an Outline of Modern Views. 203 p. New York, 191 7. Davenport, C. B., Statistical Methods, with Special Reference to Biological Variation. 3d ed. 225 p. New York, 1914. Holleman, A. F., and Cooper, H. C, A Text-book of Inorganic Chemistry. 6th Eng. ed. 527 p. Philadelphia, 1921. Holleman, A. F., and Walker, A. J., A Text-book of Organic Chemistry. 5th Eng. ed. 642 p. New York, 1920. Mellor, J. W., Higher Mathematics for Students of Chemistry and Physics, with Special Reference to Practical Work. 641 p. London, 1909. Northrup, E. F., Laws of Physical Science, a Reference Book. 210 p. Philadelphia, 191 7. Nutting, P. G., Outlines of Applied Optics. 234 p. Philadelphia, 191 2. Ostwald, Wilhelm, The Principles of Inorganic Chemistry. Translated by Alexander Find- lay. 3d ed. 801 p. London, 1908. ■ — — — , The Fundamental Principles of Chemistry. Translated by Harry W. Morse. 349 p. New York, 1909. , Introduction to Chemistry. Translated by William T. Hall and Robert S. Wil- liams. 36S p. New York, 1911. Willows, R. S., and Hatschek, E., Surface Tension and Surface Energy and their Influence on Chemical Phenomena. 114 p. 2d ed. London, 1919. Physical Chemistry and Colloid Chemistry Clark, W. M., The Determination of Hydrogen Ions. 2d ed. 480 p. Baltimore, 1922. Cohen, Ernst, Physical Chemistry for Physicians and Biologists. Translated by Martin Fischer. 343 p. New York, 1903. Findlay, Alexander, Osmotic Pressure. 84 p. London, 1913. Freundlich, Herbert, Kapillarchemie, eine Darstellung der Chemie der Kolloide und ver- wandter Gebiete. 591 p. Leipzig, 1909. Hatschek, E., An Introduction to the Physics and Chemistry of Colloids. 172 p. 4th (re- vised) ed. London", 1922. Jellinek, Karl, Lehrbuch der physikalischen Chemie. Vol. I, 715 p. Stuttgart, 1914. Vol. II, 909 p. Stuttgart, 1915. [Two more volumes to follow.] Lewis, William C. McC, A System of Physical Chemistry. 2d ed. 3 vols. 494, 403 and 209 p. London and New York, 1918, 1919, 1921. Nernst, Walther, Theoretical Chemistry from the Standpoint of Avogadro's Rule and Thermodynamics. Translated by Chas. Skeele Palmer. 697 p. London and New York, 1895. Ostwald, Wolfgang, A Handbook of Colloid Chemistry. 2d Eng. ed., translated from the 3d Ger. ed. by Martin Fischer, with notes by Emil Hatschek. 266 p. Philadelphia, 1919. xxi X.xii A CLASSIFIED LIST OF BOOKS , An Introduction to Theoretical and Applied Colloid Chemistry, "The World of Neglected Dimensions." Translation by Martin H. Fischer. 232 p. New York, 1917. , Die Welt der Vernachlassigten Dimensionen. 219 p. Dresden andLeipzig, 1915. Philip, J. C, Physical Chemistry, Its Bearing on Biology and Medicine. 326 p. London, 1920. Taylor, W. W., The Chemistry of Colloids and Some Technical Applications. 3d impres- sion. 328 p. London, 1918. van't Hoff, J. H., Lectures on Theoretical and Physical Chemistry. Translated by R. A. Lehfeldt. Part I, Chemical Dynamics. 254 p. Part II, Chemical Statics. 156 p. Part III, Relations Between Properties and Composition. 143 p. London, 1898, 1899, and 1900. Washburn, Edward W., An Introduction to the Principles of Physical Chemistry from the Standpoint of Modern Atomistics and Thermodynamics. 2d ed. 516 p. New York, 1921. Zsigmondy, Richard, Kolloidchemie. 281 p. Leipzig, 1912. Zsigmondy, Richard, Spear, Ellwood B., and Norton, John Foote, The Chemistry of Colloids. [Part I is an English translation of Zsigmondy's Kolloidchemie, translated by Spear. Part II consists of Industrial Colloid Chemistry (by Spear) and a chapter on Col- loidal Chemistry and Sanitation (by Norton).] 288 p. New York, 191 7. Soil Science and Climatology Cameron, F. K., The Soil Solution, the Nutrient Medium for Plant Growth. 136 p. Easton, Pa., 191 1. Clements, F. E., Aeration and Air-content, the Role of Oxygen in Root Activity. Carnegie Inst. Wash. Publ. No. 315. 183 p. 1921. Ehrenberg, Paul, Die Bodenkolloide. 563 p. Dresden andLeipzig, 1915. Hall, A. D., The Soil, an Introduction to the Scientific Study of the Growth of Crops. 3d ed. 352 p. London, 1920. Hann, Julius, Handbuch der Klimatologie. 3 vols. 394, 426 and 713 p. Stuttgart, 1 908-1 1. , Handbook of Climatology. Part I, General Climatology. Translated from 2d Ger. ed., with additional references and notes, by Robert De Courcy Ward. 437 p. New York and London, 1903. Hilgard, E. W., Soils, Their Formation, Properties, Composition, and Relations to Climate and Plant Growth. 593 p. New York, 1906. Mitscherlich, Eilh. Alfred, Bodenkunde fur Land und Forstwirte. 2teAufL 317 p. Ber- lin, 1913. Russell, Edward J., Soil Conditions and Plant Growth. 4th ed. 406 p. London and New York, 192 1. Ward, Robert De Courcy, Climate, Considered Especially in Relation to Man. 372 p. New York, 1908. Warrington, Robert, Lectures on Some of the Physical Properties of Soil. 231 p. Oxford, 1900. General Physiology, Physiological Chemistry and Physiological Physics Abderhalden, Emil, Handbuch der biochemischen Arbeitsmethoden. 9 vols. Berlin, 1910-19. , Biochemisches Handlexikon. Vols. 1-7, Berlin, 191 1. Vol. 8, 1913; vol. 9, 1915. [Includes very extensive literature references.] Bayliss, William Maddock, Principles of General Physiology. 3d ed. 862 p. London and New York, 1920. Includes an extensive bibliography. Czapek, Friedrich, Biochemie der Pflanzen. ite Aufl. 2 vols. Jena, 1905. [Includes very extensive citations of the literature.] 2te Aufl. 3 vol., (828 p.). Jena, 1913. [Only first vol. has appeared.] Effront, Jean, Enzymes and Their Applications. Translated by Samuel C. Prescott. 322 p. New York, 1902. A CLASSIFIED LIST OF BOOKS XX1U Euler, H., General Chemistry of the Enzymes. Translated by T. H. Pope. 323 p. New York, 1912. , Grundlagen und Ergebnisse der Pflanzenchemie, nach der Schwedischen Aus- gabe bearbeitet. I Teil, Das chemische Material der Pflanzen. 239 p. Braunschweig, 1908. II Teil, Die allgemeinen Gesetze des Pflanzenlebens. Ill Teil, Die chemischen Vor- gange im Pflanzenkorper. The last 2 parts in one vol. 298 p. Braunschweig, 1909. Haas, P., and Hill, T. G., An Introduction to the Chemistry of Plant Products. 3d ed. 414 p. London, 1921. Henry, Thomas Anderson, The Plant Alkaloids. 466 p. Philadelphia, 1913. Hober, Rudolf, Physikalische chemie der Zelle und der Gewebe. 4th (revised) ed. S08 p. Leipzig and Berlin, 19 14. Loeb, J., The Dynamics of Living Matter. 233 p. New York, 1906. , The Mechanistic Conception of Life: Biological essays. 232 p. Chicago, 191 2. , The Organism as a Whole, from the Physicochemical Viewpoint. 379 p. New York and London, 1916. Mathews, Albert P., Physiological Chemistry. 3d ed. 1154P. New York, 1920. McClendon, J. F., Physical Chemistry of Vital Phenomena, for Students and Investi- gators in the Biological and Medical Sciences. 240 p. Princeton, 191 7. [Includes an ex- tensive bibliography.] Onslow, M. W., Practical Plant Biochemistry. 178 p. Cambridge, 1920. Putter, August, Vergleichende Physiologic 721 p. Jena, 191 1. Verworn, Max, Allgemeine Physiologie, ein Grundriss der Lehre vom Leben. 6 ed. 766 p. Jena, 191 5. , General Physiology, an Outline of the Science of Life. Translated from the 2d Ger- man edition by F. S. Lee. 599 p. London, 1899. Plant Morphology and General Botany Chamberlain, C. J., Methods in Plant Histology. 3d ed. 314 p. Chicago, 1915. De Bar}-, Heinrich Anton, Comparative Anatomy of the Vegetative Organs of the Phanero- gams and Ferns. Translated and annotated by F. O. Bower and D. H. Scott. 659 p. Oxford, 1884. Ganong, Wm. F., A Text-book of Botany for Colleges. 604 p. New York, 191 7. Haberlandt, G., Physiological Plant Anatomy. Translated by M. Drummond. 777 p. London, 1914. Jordan, Edwin O., A Text-book of General Bacteriology. 7th ed. 744 p. Philadelphia and London, 19 21. Martin, J. N., Botany with Agricultural Applications. 2d ed. 604 p. New York, 1920. Molisch, Hans, Mikrochemie der Pflanze. 2d ed. 434 p. Jena, 1921. Palladin, W. I. [V. I.], Pflanzenanatomie. Nach der sten Russischen Aufl., iibersetzt und bearbeitet von S. Tschulok. 195 p. Leipzig and Berlin, 1914. Zimmermann, A., Botanical microtechnique. Translated by J. E. Humphrey. 296 p. New York, 1893. Schimper, A. F. W., Plant Geography Upon a Physiological Basis. Translated by W. R. Fischer. 839 p. Oxford, 1903. Stevens, W. C, Plant Anatomy from the Standpoint of the Development and Functions of the Tissues, and Handbook of Microtechnic. 3d ed., 399 p. Philadelphia, 1916. Plant Physiology Atkins, W. R. G., Some Recent Researches in Plant Physiology. 328 p. London and New York, 1916. Barnes, C. R., "Physiology." Vol. I, Part II (p. 295-484) of: Coulter, J. M., Barnes, C. R., and Cowles, H. C, A Text-book of Botany for Colleges and Universities. New York, 1910. XXIV A CLASSIFIED LIST OF BOOKS Brenchley, Winifred E., Inorganic Plant Poisons and Stimulants, no p. Cambridge, 1014. Darwin, Francis, and Acton, E. Hamilton, Practical Physiology of Plants. 3d ed. 340 p. Cambridge, 1901. Detmer, W., Das Pflanzenphysiologische Praktikum, Anleitung zu pflanzenphysiolo- gischen Untersuchungen. 456 p. Jena, 1865. , Practical Plant Physiology. Translated by S. A. Moor. 555 p. London, 1909. Dixon, H. H., Transpiration and the Ascent of Sap in Plants. 216 p. London, 1914. Duggar, B. M., Plant Physiology with Special Reference to Plant Production. 516 p. New York, 191 1. Errera, Leo, Cours de Physiologie moleculaire Recueillies et redigees par H. Schouteden. (Extrait du Recueil de l'lnst. Bot. de Bruxelles, tome VII.) 153 p. Bruxelles, 1907. Ganong, William E., A Laboratory Course in Plant Physiology. 2d ed. 265 p. New York, 1908. , The Living Plant, a Description and Interpretation of Its Functions and Structure. 47S p. New York, 1913. Goodale, George L., Physiological Botany. 499 P- New York, 1885. Grafe, Viktor, Ernahrungsphysiologisches Praktikum der hoheren Pflanzen. 494 p. Berlin, 1914. Green, J. R., An Introduction to Vegetable Physiology. 3d ed. 47° P- London, 191 1. Jorgensen, Ingvar, and Stiles, Walter, Carbon Assimilation, a Review of Recent Work on the Pigments of the Green Leaf and the Processes Connected with Them. New Phytologist Reprint No. 10. 180 p. London, 191 7. Jost, Ludwig, Lectures on Plant Physiology. Translated by R. J. H. Gibson. 564 p. Oxford, 1907. [This is translated from the 1st German edition; the following is to be used with it: Jost, Ludwig, Plant Physiology. Translated by R. J. H. Gibson. Supplement, incor- porating the alterations of the second edition of the German original. 168 p. Oxford, 1913-] Keeble, Frederick, assisted by M. C. Rayner, Practical Plant Physiology. 250 p. London, 1911. Kolkwitz, R., Pflanzenphysiologie, Versuche und Beobachtungen an hoheren und niederen Pflanzen, einschliesslich Bakteriologie und Hydrobiologie mit Planktonkunde. 258 p. Jena, 1914. Linsbauer, Ludw., and Linsbauer, Karl, Vorschule der Pflanzenphysiologie. 2te Aufl. 255 p. Wien, 191 1. Livingston, Burton E., The Role of Diffusion and Osmotic Pressure in Plants. 149 p. Chicago, 1903. Livingston, B. E., and Shreve, F., The Distribution of Vegetation in the United States, as Related to Climatic Conditions. Carnegie Inst. Wash. Pub. No. 284. 590 p., 75 pl-» including 2 colored maps. 1921. MacDougal, D. T., Practical Text-book of Plant Physiology. 352 p. New York, 1908. Nathansohn, A., Der Stoffwechsel der Pflanzen. 472 P- Leipzig, 1910. Osterhout, W. J. V., Experiments with Plants. 492 p. New York, 1908. Peirce, G. J., A Text-book of Plant Physiology. 2d ed. 291 p. New York, 1909. Pfeffer, W., The Physiology of Plants, a Treatise upon the Metabolism and Sources of Energy in Plants. Translated by A. J. Ewart. Vol.1. 632 p. Oxford, 1900. Vol. II, 296 p. Oxford, 1906. Vol. III. 451 p. Oxford, 1906. [This is the standard reference for the whole subject.] Pringsheim, Ernst G., Die Reizbewegungen der Pflanzen. 326 p. Berlin, 191 2. Sablon, LeClerc du, Traite de physiologie vegetale et agricole. 610 p. Paris, 1911. Timiriazeff, C. A., [Timiriazev, K. A.], The Life of the Plant. Translated from the 7th Russian edition by Anna Cheremeteff. 355 p. London, 191 2. Vines, Sydney Howard, Lectures on the Physiology of Plants. 710 p. Cambridge, 1886. TABLE OF CONTENTS PART I— PHYSIOLOGY OF NUTRITION CHAPTER I Assimilation of Carbon and of the Radiant Energy of the Sun by Green Plants Page i. Importance of the assimilation of carbon by green plants i 2. Exchange of gases 2 3. Chlorophyll 5 4. Pigments accompanying chlorophyll 19 5. Influence of light upon the decomposition of carbonic acid by plants 21 6. Products of photosynthesis 28 7. Assimilation of solar radiant energy by green plants 32 S. Influence of external and internal conditions upon photosynthesis 34 9. Nutrition of green plants by organic compounds 36 Summary 39 CHAPTER II Assimilation of Carbon and of Energy by Plants without Chlorophyll 1. General discussion 42 2. Assimilation of energy from organic compounds by plants without chlorophyll. ... 42 3. Assimilation of energy from inorganic substances by plants without chlorophyll. ... 47 4. Distribution of microorganisms in nature 52 5. Sterilization and disinfection 56 6. Pure cultures 58 Summary 61 CHAPTER III Assimilation of Nitrogen 1. The nitrogen of the air 64 2. The nitrogen of the soil 65 3. Nitrification in soils 67 4. Circulation of nitrogen in nature 72 5. Fixation of atmospheric nitrogen by the Leguminosae 73 6. Assimilation of atmospheric nitrogen by bacteria 78 7. Assimilation of nitrogen compounds by lower plants 79 Summary 79 17*7 1. XXVI TABLE OF CONTENTS CHAPTER IV Absorption of Ash-constituents Page i. Cultures in artificial media 82 2. Importance of the essential ash-constituents 84 3. Importance of the non-essential ash-constituents 85 4. Ash-analysis of plants 88 5. Microchemical ash-analysis 9° 6. The plant and the soil 9 2 Summary io2 CHAPTER V Absorption of Materials in General 1. Materials absorbed by plants io 4 2. Diffusion of gases io 4 3. Absorption of gases io 5 4. Diffusion of dissolved substances io 9 5. Absorption of dissolved substances 119 Summary .126 CHAPTER VI Movement of Materials in the Plaxt 1. General occurrence of movement of materials 13° 2. Movement of gases I 3° j. Movement of water and dissolved substances 133 4. The transpiration stream r 34 (a) Transpiration T 34 (b) Exudation pressure 140 (c) Movement of water in the stem I 43 5. Movement of organic substances v . . . I 48 Summary r 5° CHAPTER VII Material Transformations in the Plant 1. The cell as the physiological unit x 54 2. Proteins r 55 3. Enzymes z ^3 4. Protein decomposition in plants x 7° 5. Nitrogenous products of protein decomposition i?5 6. Protein synthesis in plants I 78 7. Alkaloids, toxins and antitoxins I 8i 8. Lipoids and phosphatides x 83 9. Carbohydrates I 85 10. Glucosides J 87 11. Organic acids x 88 12. The importance of water in plants x 88 13. The germination of seeds l &9 Summary I 9 2 TABLE OF CONTENTS XXV11 CHAPTER VIII Fermentation and Respiration Page General discussion 10S 2. Alcoholic fermentation 201 3. Other kinds of fermentation 209 4. Plant respiration 210 Apparatus for measuring plant respiration 215 1 6. Formation of water during respiration 217 7. Liberation of heat during respiration 218 8. Anaerobic, or intramolecular, respiration 220 9. Respiration chromogens 10. Respiratory enzymes 223 11. Materials consumed in respiration 227 12. Special cases of respiration in lower plants 230 13. Circulation of energy in nature 232 Summary 232 PART II— PHYSIOLOGY OF GROWTH AND CONFIGURATION CHAPTER I General Discussion of Growth 1 Anatomical relations of cell growth 241 2. Conditions favorable to growth 242 3. Apparatus for the study of growth 245 Summary 246 CHAPTER II Growth Phenomena That are Controlled by Internal Conditions 1. The grand period of growth 247 2. Growth of root, stem and leaf . 247 3. Tissue strains 251 Summary 251 CHAPTER III Influence of External Conditions on Growth and Configuration 1. Dependence of growth and configuration upon temperature 253 2. Dependence of growth and configuration upon the oxygen content of the surroundings 258 3. Influence of other gases on growth and configuration 260 4. Influence of moisture on growth and configuration 263 5. Dependence of growth and configuration upon light 274 6. Influence of gravitation on growth and configuration 292 7. Influence of nutrition on growth and configuration 299 8. Influence of wounding, traction and pressure on growth and configuration. ' 300 Summary 3°5 XXV111 TABLE OF CONTENTS CHAPTER IV Twiners and Other Climbing Plants Page i. Twiners 311 2. Non-twining climbers 312 3. Circumnutation 314 Summary 315 CHAPTER V Movements of Variation 1. General survey of plant movements 316 2. Autonomic movements of variation 316 3. Paratonic movements of variation 316 Summary 320 CHAPTER VI Development and Reproduction 1. Influence of external and internal conditions on development 322 2. Influence of internal conditions on development 329 3. Reproduction 331 Summary 337 Index 341 INTRODUCTION La physiologie est une des sciences les plu dignes de l'attention des esprits eleves par l'importance des questions, qu'elle traite, et de toute la sympathie des hommes de progrds par l'influence, qu'elle est destines a exercer sur le bienetre de lhumanite. — Claude Bernard. The aim of plant physiology is to gain a complete and thorough knowledge of all the phenomena occurring in plants, to analyze the complex life processes so as to interpret them in terms of simpler ones and to reduce them finally to the principles of physics and chemistry. It is evident from this statement that physiology is dependent upon physics and chemistry, and that progress in physiology depends, in great measure, upon progress in these two other sciences. Only since the end of the eighteenth century, when the principle of the con- servation of mass was formulated by Lavoisier, and chemistry became an exact science, did it become possible for physiology also to begin to assume this character. Since that time it has been possible to employ the balance in pre- cise studies of the materials that enter and leave plants. The well-known experiment of van Helmont (1577-1644), performed long before those of Lavoi- sier, may be cited as an early though but partially successful attempt to use the balance for determining the source of the materials found in the plant body. A willow branch weighing 5 pounds was potted in 200 pounds of dry soil and watered with rain-water. After five years the weight of the rooted branch was estimated to be 164 pounds, while the dried earth showed a loss in weight of only 2 ounces. Van Helmont concluded from this that the material of the plant was formed from water, but this inference is incorrect, since the surrounding air was not considered. He would have been justified in concluding, however, that the greater part of the non-aqueous material of plants does not come from the soil. Besides the discoveries of Lavoisier, another important event in the history of chemistry must be alluded to here, the synthesis of urea, accomplished by Wohler in 1828. Up to that time organic compounds had been obtained only from living organisms, and the idea prevailed that the synthetic preparation of such compounds from inorganic materials was impossible and that their forma- tion presupposed the participation of a special vital activity. Wohler's dis- covery, together with subsequently successful syntheses of various other organic compounds, have shown that no vital force is essential to the formation of such substances. The organic and inorganic compounds of carbon are often combined in a single group, but there is an essential difference between them for the physi- ologist; all organic substances contain a store of energy, since they give off heat xx ix XXX INTRODUCTION when burned, while the inorganic carbon compounds cannot be burned. The heat of combustion, measured in calories, serves as an index of the energy content of organic compounds. By a large calory, or kilogram-calory (Cal., or kg.-cal.) is meant the amount of heat necessary to raise the temperature of iooo g. of water from o° to i°C; by a small calory, or gram-calory (cal. or g.-cal.) is meant the amount of heat necessary to raise the temperature of i g. of water the same amount." The following table shows the amounts of heat obtained from the combustion of i g. of various substances, expressed in kilogram-calories. Hydrogen 34-6 Carbon 8.0 Linseed oil • 9.3 Ethyl alcohol (C 2 H 6 0) 7.1 Gluten flour 5.9 Ammonia (NHs) 5 ' Starch (CeHioOs) 4 Glucose (C6G12O6) 3 Asparagin (C4H2N2O3) 3.3 It is evident from this table that hydrogen develops much more heat during combustion than does carbon. The more oxygen the molecule of a substance contains, the less is its heat of combustion, and it is for this reason that ethyl alcohol develops more heat than starch. The introduction of hy- drogen into the molecule, on the contrary, produces a great increase in the heat of combustion; thus, oil develops more heat than does pure carbon, while ammonia, without any carbon at all — but because of its high hydrogen content — produces a far greater amount of heat than does either starch or glucose. Wohler's discovery led to a great advance in the physico-chemical interpreta- tion of physiological processes. But there were still other difficulties to overcome^ Many chemical reactions go on in plants and animals at the tempera- ture of the organism (i.e., about ordinary room temperatures), while the same reactions outside the organism occur only at much higher temperatures or with the aid of strong acids. For instance, as will be seen later, plant respiration is a process of oxidation or combustion, but it proceeds at medium temperatures, while ordinary combustion requires a very high temperature. While plant and animal substances outside of the organism generally undergo oxidation slowly at ordinary temperatures, with the oxygen of the air, they are oxidized much more rapidly in the organism, at the same temperatures. This dis- crepancy was explained by the theory of catalysis, advanced by Berzelius in 1836. Catalytic action, according to this author, is a process wherein certain substances (called catalyzers) are capable of accelerating chemical reactions be- tween other substances, by the presence of the catalyzer alone, independently a The gram-calory is frequently defined as the heat required to raise the temperature of a gram of water one degree Centigrade, but this is not precise, since the specific heat and the heat of vaporization of water vary with its temperature. The definition given in the text is that of the o-degree gram-calory. Other calories are in use, as the 15-degree gram-calory, the heat needed to alter the temperature of a gram of water from 14. 5 to i5.5°C, etc.— Ed. INTRODUCTION XXXI of its chemical affinities and without its being used up in the reaction. A substance is regarded as a catalyzer if it alters the velocity of a chemical reac- tion without itself appearing in the end-products. For instance, if a weak solution of sulphuric acid is allowed to act upon metallic zinc, the evolution of hydrogen is very slow if both reagents are very pure, but the addition of a few drops of platinic chloride is sufficient to cause a stormy evolution of the gas. The reaction proceeds, either in the presence or in the absence of the platinum salt, according to the equation, Zn + H 2 S0 4 = ZnS0 4 + H 2 . The platinic salt does not enter into the reaction and so acts simply as a catalyzer. Various kinds of catalyzers have now been shown to exist in plants and ani- mals, and these are called ferments 6 or enzymes. Enzymes, according to Wilhelm Ostwald, are catalyzers formed in the organism during the life of the cell, and it is with their help that the living organism effects most of its chemical processes. Not only are digestion and assimilation regulated entirely by enzymes, but the production of chemical energy by oxidation, at the expense of the oxygen of the air — a process forming the basis for the life activity of most organisms — is also made possible and directed by these catalyzers. It is well known that oxygen is a very inactive substance at the temperature of organisms and that the maintenance of the life process would be impossible without an acceleration of chemical reaction velocities. In plants special enzymes (oxydases) are indeed found that act, either within or without the organism, to produce the oxida- tion of various substances at room temperature. The attention of scientists was especially attracted by the enzymes of lower plants, such as yeasts and bacteria, these plants having been themselves desig- nated as "organized ferments." The most important discoveries in the physi- ology of yeasts and bacteria are due to Pasteur, who proved the absence of spontaneous generation in the lower organisms, developed a clear conception of the various kinds of fermentation, and devised perfect methods for the con- trol of infectious diseases. The worker in the shop, as well as the farmer in the field, the physician at the bedside, the veterinarian treating domestic ani- mals, the brewer handling his yeast, are all now guided by the ideas of Pasteur. A physical discovery that was very important to physiology must here be mentioned, the formulation of the principle of the conservation of energy, by Julius Robert Mayer, in 1840. Mayer demonstrated that no energy is lost in 6 The noun ferment should be dropped, as unnecessary and apt to be misleading. What were once called unorganized ferments are enzymes, and organized ferments (such as yeasts, bacteria, etc.) may be called by name or referred to as fermentation organisms. The word enzyme is frequently mispronounced; it should be pronounced as if spelled enzim, with the first vowel accented and the second short. The spelling enzym is better, but has not yet come into general use in English. — Ed. c Students of chemical physiology should be well acquainted with Pasteur's life and work. See: Vallery-Radot, Rene, The Life of Pasteur. Translated by Mrs. R. L. Devonshire. ix -f- 484 p. New York, 1915. — Ed. XXX11 INTRODUCTION the various chemical reactions, but that it is transformed from the potential into the kinetic condition, or vice versa. In the combustion of coal, for example, heat is liberated, while by the reverse process, the decomposition of carbon dioxide, heat is stored. Since combustibility is a characteristic of all organic compounds, their formation from carbonic acid must therefore be accompanied by an intake of heat and a storing of potential energy, which may be subse- quently liberated during combustion. In all investigations concerning the transformations of materials in plants it must be clearly stated whether energy is stored or released, since only thus can it be clear what is the meaning and im- portance of such transformations in the general activity of the organism. At first glance, some phenomena seem to present exceptions to the principle of the conservation of energy and to exhibit no quantitative relation between cause and effect. For example, a small spark may cause the explosion of an enormous amount of gunpowder and thus produce tremendous destruction. It might seem here that a small cause has entailed a great effect; in reality, however, the same amount of energy was liberated in the explosion as was originally present — in a potential form — in the gunpowder. The spark served only to initiate the change of this energy from one condition to the other. A small concussion of the air is often sufficient to cause the fall of a huge boulder from a great height, but the work thereby performed is exactly equal to the amount necessary to replace the boulder in its original position. The pressure of the air serves here as the trigger that produces the discharge. In considering the great importance of enzymes in the chemical processes of plants it must be realized that their part in the various reactions does not con- sist in a simple release. Bredig was quite right when he said, "We still find much vagueness in the text-books as to whether, in this matter of the contact action of substances such as acids and enzymes in the hydrolysis of esters, carbohydrates, glucosides, etc., we have to do with the initiation of a reaction incapable of occurring by itself, or only with the acceleration of a reaction that takes place so slowly (in the absence of the catalyzer) as to be almost imper- ceptible, but that is nevertheless already in operation. The question is, there- fore, to use a mechanical figure, whether the enzyme sets into operation a machine previously held at rest by a trigger-pin, or whether the enzyme serves only as a lubricant to hasten the action of the machine (the chemical reaction), which would otherwise be very slow and almost imperceptible, because of great resistance." 1 Enzymes accelerate reactions that would otherwise progress but slowly (Wilh. Ostwald) and they are thus comparable only to the " lubricant. " d On the other hand, the touch that causes a reaction-movement of the leaves of Mimosa pndica (the sensitive plant) may be regarded as a typical example of a discharge or release. The causes that produce certain phenomena and the conditions that first render them possible must also be differentiated. For instance, if solid calcium 1 Bredig, G., Die Elemente der chemischen Kinetik, mit besonderer Beriicksichtigung der Katalyse und der Fermentwirkung, Ergeb. Physiol, i: 134-212. 1902. d Enzymes frequently appear to alter the end-point of a reaction, so that it proceeds farther in their presence than without them. — Ed. INTRODUCTION XNX111 sulphate is mixed with solid barium chloride there is no reaction; when water is added, however, barium sulphate and calcium chloride are formed. This reaction is caused by the chemical attraction of the elements, the water acting only as a necessary condition. Thus releases, which are conditioning factors, must be distinguished from real causes/ Plants have an internal structure, being composed of cells of various forms and sizes. The life of an organism is the sum-total of the life activities of the individual cells composing it, and the study of plant physiology presupposes an acquaintance with the internal structure or anatomy of the plant. Familiarity with the miscroscope is essential in physiological study, since many important physiological questions can be solved by its use. For the study of many physiological phenomena — those of growth and en- largement, for example — a knowledge of the structure of the given plant and an acquaintance with the external conditions affecting it, are not sufficient; it must also be remembered that the plant has developed from a long series of ancestors whose form and mode of living have not been without effect upon the offspring. In these cases, therefore, heredity must be taken into account/ e The definition of the term cause involves difficulties. It is probably best to consider that all changes are determined (in quantity, rate and direction) by a set of controlling conditions, the cause — in the ordinary sense — being simply the last one of these necessary conditions to be fulfilled. For a discussion of this matter see: Verworn, Max, Kausale und Konditionale Welt- anschauung, Jena, 191 2. — Ed. f This is somewhat vague; the phenomena in question are assuredly conditioned at any given time by the internal and external conditions then prevailing. The nature of the ances- tors of a plant and the surroundings under which these lived are but secondary conditions, which have been influential in determining what are the present internal conditions (what the plant is now), but which are, in themselves, without any present direct influence upon its processes. The phenomena connoted by the term heredity have played an important role in determining the present internal conditions, and these latter, together with the present sur- roundings, are now influential in the determination of physiological phenomena. — Ed. PART I PHYSIOLOGY OF NUTRITION CHAPTER I ASSIMILATION OF CARBON AND OF THE RADIANT ENERGY OF THE SUN BY GREEN PLANTS §i. Importance of the Assimilation of Carbon by Green Plants.— Plants may be classified according to their color into two groups, those that are green and those that are not. The green color forms such a conspicuous char- acteristic of many plants that certain ones are sometimes spoken of as "greens." The general distribution of the green coloring would itself suggest that some important property must be connected with it, and such is indeed the fact; upon this green coloring depends one of the main cosmic functions of plants, the building up of organic compounds from inorganic substances. A simple ex- periment will show this. A seed is placed in quartz sand and is watered from time to time with a solution of mineral salts. A plant grows from the seed, blooms and bears fruit. Comparison of the amount of organic material origi- nally present in the seed, with the corresponding amount found in the grown plant, shows that the latter amount is very much greater. If follows that green plants are able to form organic compounds from inorganic ones. Animals, and plants without green pigment, generally lack this power; they obtain organic compounds only after these have been already manufactured by green plants. The formation of organic substances by green plants is thus not only important from the standpoint of plant physiology, but it acquires a much broader interest, since the whole animal kingdom, including even mankind, is dependent upon green plants. In a physiological sense, green plants form the connecting link between the animal and mineral kingdoms. Since all organic compounds are characterized by their carbon content and by their combustibility — the latter property implying that energy was stored up in their formation— the study of plant physiology may begin with an in- quiry as to the sources of the carbon and the energy necessary for the formation of organic compounds in the organism. The answer is derived mainly from the study of the assimilation of carbon dioxide. This process consists, essentially, in the absorption of carbon dioxide by the green parts of plants and in the elimination of oxygen, in sunlight. Since the volumes of the two gases involved in this proc- ess are found to be about equal, it follows that for each molecule of carbon dioxide absorbed a molecule of oxygen is eliminated; C0 2 = 2 + C (principle of Avogadro). The carbon remains in the plant and thus produces an increase in its weight, this process being a part of what is called nutrition. 2 PHYSIOLOGY OF NUTRITION Since the formation of carbon dioxide in the combustion of carbon is ac- companied by the liberation of heat, energy must be stored in the reverse process, the decomposition of carbon dioxide. From this it is clear why sun- light is so important in this decomposition; the energy of the sunshine ab- sorbed by the plant is partly used in the decomposition of carbon dioxide and in the synthesis of other carbon compounds. The green coloring matter, chlo- rophyll, serves as a screen which absorbs the sun's rays and makes this energy fixation possible. §2. Exchange of Gases. — Our first knowledge of the elimination of oxygen by green plants was obtained by Priestley, 1 in 1772. Since animals utilize ''dephlogisticated air" (as Priestley, its discoverer, called oxygen) and thus render the atmosphere unfit for the maintenance of combustion and respiration, he sought a reverse process by which the air might be improved, and he found this process in plants. He placed plants under a bell-jar of air that had been vitiated by animal respiration and was thus unfit for the maintenance of com- bustion and respiration, and found that after some time the air became again capable of supporting these processes. Unfortunately, however, subsequent repetition of this experiment did not always give the same result. Sometimes the plants improved the air, often they did not, and Priestley did not know the reason for these variations. It remained for Ingen-Housz 2 to show that the purifying of the air was effected only by the green parts of plants, and only in sunlight. The importance of this process in the life of the plant was still un- explained; it was regarded as a purposeful arrangement for the improvement of the air for animals. Ingen-Housz had no clear idea as to what gas is taken in by the plant, and even thought that the gas given off by metals under the action of acids might be thus improved by plants. Senebier 3 was later able to show that carbon dioxide alone is absorbed, and that this absorption is a nutritive process. De Saussure 4 then found that the volume of oxygen given out was equal to that of carbon dioxide taken in, that the decomposition of the last- named gas was most rapid when one part of it was present in eleven parts of air, and, finally, that an increase in the weight of the plant occurred as a result of this absorption and decomposition. All these questions were finally taken up by Boussingault, 5 in a series of precise experiments. The equality of the vol- umes of the exchanged gases was established. By an experiment upon the de- composition of carbon dioxide by green plants in a mixture of this gas and hy- drogen or nitrogen, Boussingault was able to show that the decomposition in question began immediately after the illumination of the apparatus, and ceased as soon as it was darkened. Phosphorus was used to show the presence of 1 Priestley, Joseph, Experiments and observations on different kinds of airs. 324 p. London, 1774. 2 Ingen-Housz, Jan, Experiments upon vegetables, discovering their great power of purifying common air in the sunshine, and of injuring in the shade and at night. London, 1779. [Ref. in Ger. ed. is ap- parently to Scherer's translation, 3 v., Vienna, 1786, 178S, 1790. This was from author's French ed., 1780.] 3 Senebier, J., Memoires physico-chimiques sur l'influence de la lumiere solaire pour modifier les etres des trois regnes de la nature et sur-tout ceux du regne vegetal. Geneve, 1782. Idem, Physiologie v6g6tale. Geneve, 1800. < Saussure, Nicolas Theodore de, Recherches chimiques sur la vegetation. Paris, 1804. 5 Boussingault, JeanB. J. D., Agronomie, chimie agricole et physiologie. 2nd ed. Paris, 1860-1891. ASSIMILATION OF CARBON 3 oxygen, a piece of this substance being exposed in the experiment chamber. As soon as light was allowed to enter the apparatus the formation of a white vapor indicated the presence of oxygen, and when the apparatus was darkened the fumes already formed disappeared and no more appeared, showing that the elimination of oxygen had ceased. [The fumes are suspended phosphorus pentoxide (P 2 5 ), which dissolves in water, forming phosphoric acid (H 3 P0 4 ), and thus disappears soon after the apparatus is darkened.] Since this experiment was performed in a closed chamber with a high car- bon dioxide content, it was questionable whether the results obtained might justify the conclusion that plants can utilize the small amount of carbon di- oxide in the air under natural conditions (0.028-0.04 percent.). To clear up this point, Boussingault placed a plant in a jar through which a current of air was passed. Analysis of the entering air and of that passing out showed that the plant was able, under favorable conditions of light, to absorb almost all of the carbon dioxide that entered the jar. Regarding this experiment of Boussin- gault, Timiriazev says: To what degree the precision of this experiment aroused the wonder of his contemporaries (as did most of Boussingault's researches) can best be shown by an anecdote which I heard from Boussingault himself. "The investigation was undertaken jointly with Dumas, with weighings and records independently made by each worker, in order to secure more reliable results. At first all went well, and the plants decomposed carbon dioxide as they were ex- pected to do. Then things suddenly changed. On a bright, sunny day, the plants began to produce carbon dioxide instead of decomposing it. In the evening we examined the result with astonishment and stared at each other in blank amazement. Involuntarily we remembered the misfortune that had attended Priestley when he attempted to repeat his famous experiment. Several days passed by. Then, one fine morning, Regnault, the famous physicist, who had been watching our experiment with much interest, began to laugh at our long faces and admitted that he had been to blame for our misfortune. Every day, while we were at lunch- eon, he had sneaked over to our apparatus and breathed into it, 'in order,' as he explained, 'to be convinced that you were not taking a u for an x, and could really determine such small amounts of carbon dioxide.' " l C0 2 De Saussure and Boussingault showed that the ratio ^— is generally equal to unity. However, it must be remembered that green plant parts also respire while they are assimilating carbon dioxide; that is, they carry on the reverse process, wherein carbon dioxide is eliminated and oxygen is combined. Al- though the process of respiration is much weaker than that of photosynthesis (or "carbon assimilation""), still each must be kept distinct and it must be 1 Timiriazev, K. A., From the field of plant physiology. Public lectures and addresses. [Russian.] Moscow, 1888. P. 245. " The term photosynthesis has now come into very general use among English and French physiologists, in place of the more cumbersome expressions previously employed, and there seems to be little room for doubt that it will eventually become universal. The word is of American origin. Barnes (Barnes, C. R., On the food of green plants. Bot. gaz. 18: 403- 411. 1893) suggested photosyntax, and the other and better form is due to McMillan, and it> general introduction to MacDougal. Ewart is partly right in the footnote he appended to his translation of Pfeffer's Plant Physiology (1 : 302. Oxford, 1900), but his objections do not appear valid as against the use of photosynthesis. Of course, this should include all possible forms of chemical synthesis brought about through the action of light, but the formation of 4 PHYSIOLOGY OF NUTRITION CO found out how the ratio -^r- 2 varies, independently of respiration. Bonnier U2 and Mangin 1 investigated this and found the value of the ratio to be really somewhat less than unity. So the plant gives off not only the equivalent of all the oxygen originally contained in the absorbed carbon dioxide, but also a smaller portion of oxygen arising from the water that is decomposed in photosynthesis. 2 As to methods of investigation, the decomposition of carbon dioxide can be detected in the following manner. A cut leaf is placed in a calibrated glass tube (Fig. 1), the upper end closed and the lower, open end dipping into mercury. Then a part of the air is removed by a rubber tube and the level of the mercury rises. The volume of the remaining air is read, after which some carbon dioxide is admitted from a gasometer and the gas volume is again determined. The apparatus is not placed in light and after some time the gas volume is once more recorded. The remaining carbon dioxide is removed by injecting some concentrated potassium hydroxide solution, and the diminished gas volume is again read; pyrogallol is next introduced, and a final reading, after the removal of oxygen by the pyrogallol, gives the amount of nitrogen that remains. The numbers obtained permit the determination of the amounts of carbon dioxide absorbed and of oxygen liberated. 3 A less exact method consists in counting the number of gas bubbles carbohydrate out of carbon dioxide and water is by far the most important form of photosyn- thesis, and the term may readily be qualified whenever need arises. Thus, we may distinguish chlorophyll photosynthesis of carbohydrate from other photosyntheses. The word assimilation has been employed in so many different senses that to attempt its use as a precise term in this connection here seems unadvisable. — Jorgensen and Stiles prefer, however, to employ the "rather non-committal expression," carbon assimilation, and they do so in their recent very excellent monograph on this subject, which should be referred to in connection with this entire chapter, and which should become familiar to every student of plant physiology. See: Jorgensen, Ingvar, and Stiles, Walter, Carbon assimilation, a review of recent work on the pigments of the green leaf and the processes connected with them. New phytolo- gist reprint No. 10. London, 191 7. (This is reprinted from a series of articles having same title, in New phytol. 14-16. 1015-17.) — Ed. 1 Bonnier, Gaston, and Mangin, Louis, L'action chlorophyllienne separde de la respiration. Ann. sci. nat. Bot. VII, 3 : 5-44- 1886. 2 It will be seen later that hydrogen and oxygen are actually assimilated, as well as carbon, in the photosynthetic process, the source of the hydrogen and of some of the oxygen being water, taken up from the soil. s For precise methods of gas analysis see: Bunsen, Robert W. E., Gasometrische Methoden. 2te Aufl. Braunschweig, 1877. Winkler, C A., Lehrbuch der technischen Gasanalyse. 1885. [Idem, Handbook of technical gas-analysis containing concise instructions for carrying out gas-analytical methods of proved utility. Translated with a few additions by George Lunge. London, 1885. Idem, same title, 2d English ed. from 3d German ed. London, 1902.] For physiological experiments, see especially: Doyere, M. L., Etudes sur la respiration. Ann. chim. et phys. Ill, 28: 5-50. 1850. Blackman, F. Frost, Experi- mental researches on vegetable assimilation and respiration. I. On a new method for investigating the carbonic acid exchanges of plants. Phil, trans. Roy Soc. London B186: 485-502. 1896. [Idem, same title. II. On the paths of gaseous exchange between aerial leaves and the atmosphere. Ibid. B186: 503-562. 1896.] Palladin, W., and Kostytschew, S., in Abderhalden's Handbuch der biochemischen Arbeitsmethoden 3 : 479- Berlin, 1910. ASSIMILATION OF CARBOX 5 (comparatively pure oxygen 1 ) given off, in light, from the cut end of a piece of the water plant Elodea, submerged in water saturated with carbon dioxide, as shown in Fig. 2. If a number of such green water plants are placed under water in sunlight and are covered by an inverted funnel, over the neck of which is inverted a test-tube of water (Fig. 3), the test-tube soon becomes filled with a gas that is nearly pure oxygen. Schutzenberger's reagent (a solution of indigo carmine or nigrosine, de- colorized by sodium sulphite) can also be used to demonstrate that oxygen is r~\ W Fig. 2. — Elimina- tion of oxygen bubbles by Elodea in sunlight. Fig. 3. — Collection of oxygen from water plants in light. Fig. I. — Leaf in position in a measuring tube, for demonstration of absorption of carbon dioxide and elimination of oxygen during photo- synthesis. liberated by water plants; this solution is yellow when prepared, but turns blue in the presence of oxygen. If a shoot of Elodea, or other aquatic, is placed in a dilute solution of this reagent and exposed to sunlight, the solution surround- ing the leaves becomes blue in a few minutes. 2 §3. Chlorophyll.— Since the decomposition of carbon dioxide is effected exclu- sively by the green parts of plants, the properties of the green pigment — called 1 This method was perfected by Kohl. See: Kohl, F. G., Die assimilatorische Energie der blauen und violetten Strahlen des Spektrums. Ber. Deutsch. Bot. Ges. IS : 111-124. 1897. ' Kny, L., Die Abhangigkeit der Chlorophyllfunction von den Chromatophoren und vom Cytoplasma. Ber. Deutsch. Bot. Ges. 15: 388-403. 1897. [See also: Kolkwitz, R., Pflanzenphysiologie. Jena, 1914- P. 3-1 6 PHYSIOLOGY OF NUTRITION chlorophyll by Pelletier and Caventou 1 — must be studied. Chlorophyll can be extracted from leaves by alcohol, but the solution thus obtained also contains several other pigments, as well as colorless substances, for the removal of which various methods have been devised. 2 The method of Fremy involves the precipitation of the alcoholic extract with barium hydroxide; the green precipi- tate is collected upon a filter and treated with alcohol until the yellow pigments, xanthophyll and carotin, are completely removed. The remaining precipitate is then decomposed by potassium hydroxide, according to the method of Timiriazev. 3 The green solution thus obtained is treated with ether, and dilute acetic acid is gradually added, with shaking, to neutralize the potassium hydroxide. As long as the reaction is alkaline the ether remains without color, but as soon as the hydroxide is neutralized the lower layer becomes yellow, since all the green pigment passes into solution in the ether above. The color of the ether solution is emerald green, more intense than that of the alcoholic solution; it is cherry red, however, in reflected light, while the yellow solution shows no fluorescence. Timiriazev was the first to succeed in separating chlo- royhyll from yellow pigments, out of the alcoholic chlorophyll extract. This chlorophyll is not the normal pigment, however, for it has been changed by the treatment employed in separating it. The method of Kraus 4 is based upon the relative solubilities of the pig- ments in alcohol and benzine. If benzine is gradually added, with shaking, to the green alcoholic extract diluted with water so as to be about an 85 -per cent, solution of alcohol, two sharply distinct layers are finally formed, an upper, green layer (benzine) and a lower, golden-yellow one (alcohol and water). By renewed shaking of the former solution, with further additions of alcohol, the green pigment can be practically freed from the yellow coloring matter. The green pigments 6 form an amorphous mass, readily soluble in alcohol, ether and naphtha. The solution is intensely fluorescent, appearing cherry red by reflected light and green by transmitted light. The chemistry of chloro- phyll has been largely worked out by Willstatter and his co-workers. Two closely related pigments are always associated to form chlorophyll, these having been termed chlorophyll a and chlorophyll b. 1 [Pelletier, [Joseph], and Caventou, [J. B.], Sur la matiere verte des feuilles. Ann. chim. et phys. 11,9: 194-196. 1818.] f 2 Willstatter, Richard, Chlorophyll und seine wichtigsten Abbauprodukte. Abderhalden s Handb. biochem. Arbeitsmeth. 2: 671-716. Berlin, 1910. Willstatter, Richard, and Hug, Ernst, Isolierung des Chlorophylls. Liebig's Ann. Chem. u. Pharm. 380: 177-21 1. 1911. s Timiriazev, K. A., Spectral analysis of chlorophyll. [Russian.] St. Petersburg, 1871.* [Haas and Hill give methods for obtaining chlorophyll, and present a good discussion. See: Haas, Paul, and Hill, T. G., An introduction to the chemistry of plant products. London, 192 1.] ■> Kraus, Gregor, Zur Kenntnis der Chlorophyllfarbstoff e und ihrer Verwandten. Stuttgart, 1872. b Some modifications have been made in this discussion of chlorophyll, so that it does not agree entirely with Palladin's presentation. . An attempt has been made to bring it more into accord with Willstatter and Stall's monograph. (Willstatter, Richard, and Stall, Arthur, Untersuchungen liber Chlorophyll, Methoden und Ergebnisse. Berlin, 1913-) For English resumes of this work, see: West, Clarence J., A review of Willstatter's researches on chloro- phyll. Biochem. bull. 3: 229-258. 1914. Willstatter, R., Chlorophyll. Jour. Amer. Chem. Soc. 38: 323-345. 1915— Ed. ASSIMILATION OF CARBON 7 Alcoholic solution of chlorohyll a is blue-green by transmitted light and blood-red by reflected light; it is said to fluoresce blood-red. Alcoholic solu- tion of chlorophyll b is yellow-green by transmitted light and fluoresces brown- red. This phenomenon of fluorescence (seen also in a solution of the red dye eosin, which fluoresces green) appears to be due to an alteration in the wave- length of radiant energy, brought about by a peculiar action on the part of the molecules in the solution. By this action the chlorophyll solution gives off energy of long wave-lengths (red light) when it is illuminated by energy of much shorter wave-lengths (green and blue light)/ Of the total green pigment, as obtained from leaves, about 72 per cent, is chlorophyll a and the rest chlorophyll b. The proportions vary somewhat, but the variation is not over 10 perc ent. Both form crystals. The two chloro- phylls'' have the following formulas, as so far known: Chlorophyll a, C 55 H 72 6 N 4 Mg Chlorophyll b, C 5 5H 7 o0 6 N 4 Mg It is seen that both contain magnesium, the content of this element being about 5.6 per cent., by weight. Iron is apparently necessary for the formation of chlorophyll in plants, but it is not a part of the pigment. Iron does occur, however, in the molecule of hemoglobin, which is somewhat closely related to chlorophyll, chemically. An explanation 6 of this is to be found in the fact that the actions of these two substances in the cell are directly opposed; for the analytic action of hemoglobin, iron is essential, while magnesium seems to take part in the synthetic processes effected by chlorophyll. 1 1 Willstatter, Richard, Zur Kenntniss der Zusammensetzung des Chlorophylls. Liebig's Ann. Chem. u. Pharm. 350: 48-82. 1906. Willstatter, Richard, and Benz, Max, Ueber krystallisirtes Chlorophyll. Ibid. 358: 267-287. 1908. c This explanation is not given by Palladin. For a discussion of the various theories regard- ing the color and fluorescence of plant pigments, see : Horowitz, B., Plant pigments. Biochem. bull. 4: 161-172. 1915. — Ed. d Stokes had long ago suspected that chlorophyll is a mixture of two green pigments. In this connection see: Stokes, G. G., On the supposed identity of biliverdin with chlorophyll, with remarks on the constitution of chlorophyll. Proc. Roy. Soc. London 13 : 144-145. 1864. Sorby, H. C, On comparative vegetable chromatology. Ibid. 21 : 442-483. 1873. On an interesting method for separating the yellow and green pigments by absorption in paper or in a column of calcium carbonate, see: Tswett, M., Physikalisch-chemische Studien liber das Chlorophyll. Die Adsorptionen. Ber. Deutsch. Bot. Ges. 24: 316-323- ia° 6 - Idem, Adsorptionsanalyse und chromatographische Methode. Anwendung auf die Chemie des Chlorophylls. Ibid. 24 : 384-393. 1906. Idem, Ueber die nachsten Saurederivate der Chlorophylline. Ber. Deutsch. Chem. Ges. 41/: 1352-1354. 1908.— Ed. e It is difficult to understand this as an explanation. It must not be understood that hemoglobin and chlorophyll are really very much alike; they differ very markedly, but give some of the same decomposition products. It is true that both are related to the interchange, between the organism and its surroundings, of carbon dioxide and oxygen, but the actions of the two substances do not appear to be similar in detail. The author refers here to the fact that they have similar component atomic groups, which may suggest that, in the phylogeny of animals and plants, both groups of organisms may have developed from a common ancestral form having a substance with the characters that are common to hemoglobin and chlorophyll. This is as far as such a theory may go at present. But see below, page u, ct seq.—Ed. 8 PHYSIOLOGY OF NUTRITION Almost a third of the chlorophyll molecule is composed of phytyl, the radical of phytol, 1 an unsaturated mono-hydric primary alcohol of the aliphatic series, having the composition C20H40O and the probable structure shown by the following diagram: CH 3 — CH— CH— CH— CH— CH— CH— C=C CH— CHoOH I I I I I I I I I CH3 CH3 CH 3 CH 3 CH 3 CH 3 CH3CH3 CH3 Phytol is readily oxidized in the presence of air. Willstatter suggests that it may be obtained from isoprene in the following way: 4C5H8 (isoprene) -f- H 2 + 3H2 = C 2 oH 40 (phytol). Carotin appears also to be related to isoprene. The phytyl of chlorophyll may be replaced by the ethyl group if the leaves are treated with ethyl alcohol. This replacement is effected by an enzyme known as chlorophyllase. 2 Another alcohol radical is present in both the chlorophylls, namely, methyl (CH3). They thus appear to be esters of a complex, dicarboxylic acid, one of the two carboxyls (COOH) being joined to phytyl and the other to methyl. Regarding the complex acids that form the basis of the chlorophylls, there still remain some uncertainties, but it appears to be related to a tricarboxylic acid that may be represented by the formula (C3iH 2 9N 4 Mg) (COOH) 3 , but one of the carboxyls is inactive, so that a dicarboxylic acid results. A general idea of the manner in which the magnesium atom is probably related to the other compo- nents of the molecule may be obtained from the following structural formula for etiophyllin, to which this fundamental acid is apparently closely related. CH=CH I I CH 3 — C— CH C— C C 2 H 5 — C— C C— CH yC C x _ C 2 Ho — C — C /C — C — C 2 H.-j >N— Mg— N CH 3 — C=C C=rC— CH 3 ! I CH 3 CH 3 When the phytyl group of chlorophyll a is replaced by the ethyl group (C2H5), a substance is obtained (C37H380 6 N 4 Mg) which Willstatter called ethyl chlorophyllide. This forms beautiful crystals, which were earlier mistaken for pure chlorophyll. Chlorophyll b reacts in a similar way. According to the 1 Willstatter, Richard, and Hocheder, Ferdinand, Ueber die Einwirkung von Sauren und Alkalien auf Chlorophyll. Liebig's Ann. Chem. u. Pharm. 354: 205-258. 1007. Willstatter, Richard, Mayer, Erwin W., and Huni, Ernst, Ueber Phytol. I. 7&j'<2. 378: 73-152. 1011. 2 Willstatter, Richard, and Stoll, Arthur, Ueber Chlorophyllase. Liebig's Ann. Chem. u. Pharm. 378: 18-72. 1911. ASSIMILATION OF CARBON 9 method of Monteverde, 1 these crystals may be obtained by treatment of tritu- rated leaves with 95 per cent, ethyl alcohol; after an hour the extract is filtered and the alcohol is removed by evaporation, either in air or in hydrogen. The crystals are separated from impurities and from the yellow pigments by means of distilled water and benzine. In the pure condition they form a dark green, almost black powder, with a bluish metallic luster. Their alcoholic solution is green, with a beautiful red fluorescence. Although the solution is unstable in light, the crystals can endure intense light for a long time without change. The following plants serve especially well as sources of ethyl chlorophyllide in the crystalline condition: Dianthns barbatus, Lathyrus odoratus, Galeopsis versicolor, G. tetrahit, Acacia lophantha, and Dahlia variabilis. Amorphous chlorophyll may be obtained from many other plants. Willstatter and Benz 2 obtained over 2 g. of ethyl chlorophyllide from 1 kg. of dry leaves. afiC Fig. 4. — -Absorption spectrum of ethyl chlorophyllide, 0.1 g. in 5 1. of alcohol. (After Willstatter.) The thickness of the layer employed is shown (in millimeters) at the left, the conventional letters of the Fraunhofer lines are at the top, and the wave-lengths (in 10 m**) are indicated below. The absorption spectrum of chlorophyll deserves special attention. Light of certain ranges of wave-length is more or less completely absorbed by the solution, so that dark bands appear in the spectrum. The absorption spectrum of every colored solution changes with its concentration. On this account the spectrum of chlorophyll solution must be determined either throughout a range of concentrations or by using layers of various thicknesses. Six absorption bands are found in the spectrum (Fig. 4) of ethyl chlorophyllide; arranged ac- cording to their intensities, they form the series: I, VI, V, II, III, IV. The first band, lying between the Fraunhofer lines B and C, is the most distinct; it appears in solutions of weaker concentration than are necessary to make the others evident. The absorption bands become broader with increasing con- 1 Monteverde, N. A., Ueber das Protochlorophyll. Acta Horti Petropolitani 13: 190-217. 1894. Borodin had obtained crystals from chlorophyll before they were described by Monteverde. See: Borodin, J., Ueber Chlorophyllkrystalle. Bot. Zeitg. 40: 608-610, 622-626. 1882. 2 Willstatter and Benz, 1908. [See note 1, p. 7-1 IO PHYSIOLOGY OF NUTRITION centration and finally merge into one another, so that only the red rays, between . 1 and B, and a part of the green can pass through a concentrated solution or a thick layer; finally, with still further increase in concentration or thickness 1 ■i ■ i f ' 1 1 1 1 ' 1 ' I ' 1 i ' 1 1 1 ' « Q o o & 3 O o in C r o o m t-i *t -J- B I 1 ll 1 ll B ,1 F i || G Pig. 5. — Absorption spectra of five different concentrations of chlorophyll a. {After Willstatter and Sloll.) Fig. 6. — Absorption spectra of five different concentrations of chlorophyll b. {After Willstatter and Sloll.) of layer, the green rays are also completely absorbed and only the rays between A and B are transmitted. All objects appear red when seen through a very concentrated solution or a very thick layer. ASSIMILATION OF CARBON II The absorption spectra of chlorophyll a and chlorophyll b, in acetone, are shown in Figs. 5 and 6, reproduced photographically, these being taken from Willstatter and S toll's monograph (Tafel VIII). Five different concentrations are employed, the strongest being represented by the lowest spectrum in each case. The Fraunhofer lines and wave-lengths (in mm) are shown above/ The spectrum of living leaves shows the same absorption bands as does the spectrum of an alcoholic solution of chlorophyll (ethyl chlorophyllide) ; in the former case the bands are merely displaced a little toward the infra-red end of the spectrum. 9 The researches of Schunck and Marchlewski 1 have contributed much to an understanding of the chemical character of chlorophyll. The action of hydro- chloric acid upon an alcoholic chlorophyll solution produces first chlorophyllan, then phylloxanthin, and finally phyllocyanin. The interesting substance phyl- lo porphyrin 2 (C 16 H 1S N 2 0, or C32H36N4O0) 3 is obtained by treating phyllocyanin with strong alkalies. Phylloporphyrin crystallizes in beautiful, dark red-violet p IG » — Absorption spectra of phylloporphyrin (i, 3, 5) and of hematoporphyrin (2, 4, 6); 1 and 2 in ether; 3 and 4 in hydrochloric acid; 5 and 6 in zinc chloride solution. (After Schunck and Marchlewski.) crystals, is slightly soluble in alcohol and ether, and more readily soluble in chloroform. The absorption spectrum of its ethereal solution (Fig. 7) exhibits seven absorption bands, the first of which lies to the right of the red region of the spectrum, between C and D, and is very distinct. Phylloporphyrin is of great interest because of its close relationship to 1 Schunk, E., and Marchlewski, L., Zur Chemie des Chlorophylls. Liebig's Ann. Chem. u. Pharm. 278: 329-345- 1894. * Schunck, E. and Marchlewski ; L., Zur Chemie des Chlorophylls. (Zweite Abhandlung.) Liebig's Ann. Chem. u. Pharm. 284: 81-107. 1805. 'Willstatter, Richard, and Fritzsche, Hermann, Ueber den Abbau von Chlorophyll durch Alkalien. Liebig's Ann. Chem. u. Pharm. 371: 33-124- 1909. ' These two figures are added by the editor. — Ed. It seems highly probable that the chlorophyll of living leaves exists in colloidal solution. (Herlitzka, A., Neben den Zustand des Chlorophylls in der Pflanze und iiber kolloidales Chlorophyll. Biochem. Zeitsch. 38: 321-330. 1912. Iwanowski, [D.], Ueber das Verhalten des lebenden Chlorophylls zum Lichte. Ber. Deutsch. Bot. ges. 31: 600-612. 1913). — Ed. 12 PHYSIOLOGY OF NUTRITION hematoporphyrin, which was obtained by Nentskii and Sieber from hemoglobin of animal blood. Hematoporphyrin has the composition Ci 6 Hi 8 N 2 0i, the dif- ference between it and phylloporphyrin, as represented by these formulas, con- sisting in the lower oxygen content of the latter. 1 The method used in the isolation of hematoporphyrin is also analogous to that employed for phyllopor- phyrin. The spectra of these two substances, in various solvents, 2 are almost identical, except that the absorption bands of hematoporphyrin sometimes appear slightly displaced toward the red (Fig. 7). Both hematoporphyrin and phylloporphyrin, when heated in a test-tube, form a vapor which gives a red color to pine sawdust moistened with hydro- chloric acid, a characteristic indication of the presence of the pyrrol ring (C4H5N): the characteristic odor of pyrrol may also be plainly recognized in this vapor. 3 It thus appears that chlorophyll (acting synthetically) and hemo- globin (acting analytically) are closely related, in that the pyrrol ring is common to both. It is of great interest also to note that the bile pigment bilirubin has the same percentage formula as hematoporphyrin (Ci6Hi 8 N 2 0,j). Further- more, Nentskii and Zaliesskii 4 succeeded in obtaining mesoporphyrin ivom hemin, the latter substance being formed by the action of acids upon hemoglobin. Mesoporphyrin has the composition Ci 6 Hi 8 N 2 2 , and stands between hemato- porphyrin and phylloporphyrin in oxygen content. By a further decomposition of hemin these authors obtained hemopyrrol (Ci3H 8 N), a volatile oil that turns red in air and changes into urobilin, which is also obtained from bilirubin. When Nentskii and Marchlewski 5 succeeded in obtaining hemopyrrol and urobilin from phylloporphyrin, the relationship between chlorophyll and hemoglobin was conclusively established. The atomic group common to both, as in the case of the bile pigments, occurs in hemopyrrol. The following diagram represents the relationship existing between these three groups of substances. Chlorophyll Hemoglobin Hematoporphyrin Phylloporphyrin Hemopyrrol ! Urobilin Bilirubin 1 For the difference in structure between the two compounds see: Willstatter, Richard, and Asahina, Yasuhiko, Oxydation der Chlorophyllderivate. Liebig's Ann. Chem. u. Pharm. 373 : 227-238. 1910. 2 Schunck, E., and Marchlewski, L., Zur Chemie des Chlorophylls. (Vierte Abhandlung.) Liebig's Ann. Chem. u. Pharm. 290: 306-313. 1896. 3 Schunck, E., and Marchlewski, L., Zur Chemie des Chlorophylls. (Dritte Abhandlung.) Liebig's Ann. Chem. u. Pharm. 288: 200-218. 1895. 4 Nencki, M., and Zaleski, J. Ueber die Reduclionsproducte des Hamins durch Jodwasserstoff and Phosphoniumjodid und iiber die Constitution des Hamins and seiner Derivate. Ber. Deutsch. Chem. Ges. 34 1 ' 997-IOIO. 1901. 6 Nencki, M., and Marchlewski, L., Zur Chemie des Chlorophylls. Abbau des Pyhllocyanins zum Hamopyrrol. Ber. Deutsch. Chem. Ges. 34"; 1687-1693. 1901. ASSIMILATION OF CARBON' 1 3 Results of this kind are exceedingly important in biochemistry, since they seem to illuminate the most remote period in the evolutionary development of organisms, and point to a common origin of the plant and animal worlds. Dar- win's theory of the origin of species is based upon the conception of variability in structure, influenced by environmental conditions in the struggle for existence. But the differences between organisms lie, not only in the form and structure of the various organs, but also in the chemical properties of the substances con- stituting the living cells. The character of the metabolic processes is dependent upon the nature of the intracellular substances, and these processes, in their turn, determine the configuration of the cells and their differentiation into organs. In other words, the form of the cell-complexes composing the different organs is determined by metabolism as this has been developed by the various organs in the struggle for existence, relative to various environmental condi- tions. With a change of conditions, their chemical constitution and their metabolism are modified, which explains why they frequently change their form also. Thus, to obtain a fundamental conception of the evolution of the organic world, not only the structure but also the chemical composition of the cells and the products of their metabolism must be considered. From this viewpoint the work of Schunck and Marchlewski, whereby the leaf and blood pigments are shown to be related chemically, though widely different as to function, is of great scientific interest. 1 According to Nentskii, 2 chlorophyll and hemoglobin arise from chromogens that are protein decomposition products. A substance called tryptophan is formed in protein decomposition by pancreatic juice; tryptophan is colored red by bromine and is related, in its percentage composition, to hematoporphyrin and the melanins. The decomposition products of chlorophyll can be separated, according to Willstatter, 3 into two groups. Those obtained by the action of acids contain no magnesium; the action of alkalies, on the other hand, results in such deriva- tives as glaucophyllin, rhodophyllin, pyrrophyllin, and phyllophyllin, all of which contain magnesium. If acids are allowed to act upon these latter substances, new compounds without magnesium arise, which are related to hematoporphyrin ; in this way phylloporphyrin is obtained from phyllophyllin. The action of acids upon chlorophyll itself gives phaophytin, in which the phytyl can be re- placed by the ethyl group, giving ethyl phceophorbide; chlorophyllin modified by the action of acid is designated as phseophorbide, and phseophytin may thus be termed phytyl-phaeophorbide. i Nencki, M., Sur les rapports biologiques entre la matiere colorante des feuilles ct celle du sang. Arch, sci. biol. St.-Petersbourg 5: 254-260. 1897. 2 Nencki, M., Ueber die biologischen Beziehungen des Blatt- und des Blutfarbstoffes. Ber. Deutsch. Chem. Ges. 2o 7// : 2877-2883. 1896. » Willstatter, Richard, and Pfannenstiel, Adolf, Ueber Rhodophyllin. Liebig's Ann. Chem. u. Pharm. 358: 205-265. 1908. Willstatter and Fritzsche, 1909. [See note 3. P- "•] Willstatter and Hocheder, 1907. [See note 1, p. 8.] Willstatter, Richard, and Stoll, Arthur, Spaltung und Bildung von Chlorophyll. Liebig's Ann. Chem. u. Pharm. 380: 148-154- I9H. Willstatter, Richard, and Isler, Max., Vergleichende Untersuchung des Chlorophylls verschiedener Pflanzen. III. Ibid. 380: 154-176. I9H- [The whole series of studies is summarized by Willstatter and Stoll, 1913- (See note 6, p. 6-^ 14 PHYSIOLOGY OF NUTRITION Among the other transformation products of chlorophyll, protophyllin de- serves attention; Timiriazev 1 obtained this by the action of nascent hydrogen. It is yellow or red in solution, according to the concentration. It is very easily oxidized, going over into chlorophyll; for this reason it must be preserved under carbon dioxide or hydrogen in sealed tubes. It is stable in hydrogen, in light as well as in darkness, but in carbon dioxide it is stable only in darkness; in light, with carbon dioxide, it becomes green and is transformed into chlorophyll. It must be supposed that carbon dioxide is decomposed in this case and that oxygen is liberated, at the expense of which the transformation and greening of the protophyllin occurs. Absorption bands in the orange and green regions of the spectrum, corresponding to bands II and IV of chlorophyll, are character- istic of protophyllin. It appears from many investigations that the formation of chlorophyll in plants is a very complicated process. Until the publication of the work of Liro 2 most authors failed to distinguish between the beginning of chlorophyll formation and the visible accumulation of this pigment in plants as they become green. This distinction is quite necessary. We shall first turn our attention to the conditions requisite for the formation of chlorophyll . Light may be mentioned as the first of these. Leaves of angio- sperms grown in darkness are always yellow, but such etiolated plants soon turn green when exposed to light. Seedlings of some conifers, 3 young fern fronds and some one-celled algae 4 are exceptions, for they become green in darkness; still, according to Liubimenko, conifer seedlings form much less chlorophyll in dark- ness than in light. Very weak light is sufficient for chlorophyll formation, and light of medium intensity is most favorable. Famintsyn 5 exposed a part of an etiolated plant to direct sunlight, while the intensity of the light falling upon the remaining portion was reduced by interposing sheets of paper; greening always occurred first in the reduced light. According to Wiesner this phenomenon is to be explained by supposing that decomposition and formation of chlorophyll occur simultaneously. In light of low or medium intensity the decomposition process is nearly absent, while in strong light active formation is accom- panied by rapid breaking down of chlorophyll, which results in less pronounced greening than occurs in diffuse light. Various parts of the spectrum have different effects upon the formation of chlorophyll, a matter which was carefully investigated by Wiesner. 6 He 1 Timiriazeff, C, La chlorophylle et la reduction de l'acide carbonique par les vegetaux. Compt. rend. Paris 102: 686-689. 1886. Idem, La protophylline dans les plantes etiolees. Ibid. 109: 414-416. 1889. Idem, La protophylline naturelle et la protophylline artificielle. Ibid. 120: 467-470. 1895. 2 Liro, J. Ivar, Ueber die photochemische Chlorophyllbildung bei den Phanerogamen. Ann. Acad. Sci. Fennicae (Helsinki) Ai: 1-147. 1909. 8 Lubimenko, W., Influence de la lumiere sur le developpement des fruits et des graines chez les vegetaux superieurs. Rev. gen. hot. 22: 145-175. 1910. 4 Artari, A., Ueber die Entwicklung der griinen Algen unter Ausschluss der Bedingungen der Kohlen- saure-Assimilation. Bull. Soc. Imp. Nat. Moscou 13: 3Q—47. 1900. Idem, Zur Ernahrungs-physiologie der grunen Algen. Ber. Deutsch. Bot. Ges. 19: 7-9 1901. ' Famintzin, A., Die Wirkung des Lichts auf das Ergunen der Pflanzen ("aus dem Bulletin 10: 548- 552.") Melanges biol. Acad. Imp. Sci. St.-P6tersbourg 6: 94-100. 1866. « Wiesner, Julius, Untersuchungen uber die Beziehungen des Lichtes zum Chlorophyll. Sitzungsber. (math.-naturw. Kl.) K. Akad. Wiss. Wien 69': 327-385. 1874. Idem, Die Entstehung des Chloro- phylls in der Pflanze. Wien. 1877. ASSIMILATION OF CARBON 15 employed double-walled bell- jars with colored liquids, as light screens for isolat- ing certain regions of the spectrum (Fig. 8). Solutions of potassium dichromate and of ammoniacal copper oxide [copper sulphate solution to which an excess of ammonia water is added] were most frequently used; the first, in medium con- centration, permits the passage of the rays of the less refrangible half of the spectrum (red, orange, yellow and a part of the green), while. the second trans- mits the remainder of the visible rays (the rest of the green and all of the blue and violet). Thus, by the use of these liquids, the spectrum is separated into two parts. [Of course the intensity of the light transmitted is considerably decreased.] In weak light plants become green sooner under the yellow solution, but in strong light more quickly under the blue. This may be explained by supposing that in weak light the formation of chlorophyll occurs almost exclusively, under the influence of the less refrangible rays, which are most favorable, while in strong light, besides chlorophyll formation, an active de- composition also takes place. Experiments upon the de- composition of alcoholic solutions of chlorophyll under colored bell-jars have shown that this process is especially pronounced in the less refrangible half of the spectrum; greening in plants is thus seen to be weaker in strong yellow- red fight because a very rapid destruction here accompa- nies the formation of chlorophyll. But another explana- tion is also possible: strong light may not act directly upon chlorophyll that has already been formed but may, some- how, have a harmful effect upon some process antecedent to chlorophyll formation; this might explain why less chlorophyll accumulates in strong light. Plants do not become green under the non-luminous heat rays. In order to separate this portion of the spec- trum, Tyndall's solution is used, iodine in carbon bisul- phide; in low concentrations the rays between Fraunhofer lines A and B are transmitted, but these, produce no green color. In ultra-violet light green- ing is very slight. The production of chlorophyll is dependent upon temperature. Medium temperatures are most favorable, and no greening occurs at very low or at very high temperatures. Wiesner obtained the following results from experiments with etiolated barlev seedlings. Time Required Temperature F0R Greening Deg.C. Hours 2 _ 4 (No greening) 4-5-'^' 7 - 2S 10 3-50 18-19 l6? 30 -s 8 37-38 4.oo 4C (No greening) S3! S3! Pig. 8. — Double- walled bell-jar with colored solution filling the space between the walls. i6 PHYSIOLOGY OF NUTRITION The autumn coloration of leaves is dependent upon light and upon the tem- perature of the air; chlorophyll is decomposed by sunlight in autumn, while its re-formation is hindered by the low temperatures then prevailing. According to Batalin, 1 the conifer Chamacyparis obtusa is especially interesting in this connection. Branches in sunshine have a golden-yellow color in the cold sea- son, while shaded ones remain green;'' at the margin between the shaded and un- shaded regions the different colors may often be seen in neighboring cells. The products of chlorophyll decomposition do not remain in the leaf but dif- fuse away. 2 This is shown by the following experiment: if an incision is made in a leaf in the autumn, while it is still green, so that the chlorophyll decomposi- tion-products are prevented from diffusing away, the part of the leaf above the cut remains green while the other parts turn yellow (Fig. 9). The presence of iron is a third condition necessary for the formation of chlorophyll. 3 Without iron, plants remain bright yellow, thus suffering from chlorosis. Pig. 9. — Gingko leaf in which autumnal coloration has been prevented in the upper part, by an incision. {After Stahl.) The presence of oxygen is an additional condition necessary for greening. Etiolated leaves in an oxygen-free chamber remain yellow, even in light. This is also true when the amount of oxygen is small; greening demands an excess of this gas. Ville 1 was able to show that the absence of necessary mineral salts in the soil results in the diminution of the chlorophyll and carotin contents of leaves. 1 Batalin, A., Ueber die Zerstorung des Chlorophylls in lebenden Organen. Bot. Zeitg. 32 : 433-439. 1874- - Stahl, Ernst, Zur Biologie des Chlorophylls; Laubfarbe und Himmelslicht, Vergilbung und Etiole- ment. Jena, 1909. 3 Gris, Eusebe, Nouvelles experiences sur Taction des composes ferrugineux solubles, appliques h. la veg6tation, et specialement au traitement de la chlorose et de la debilite des plantes. Compt. rend. Paris 19:1118-1119. 1844. Molisch, Hans, Die pflanze in ihren Beziehungen zum Eisen. Eine physiologische Studie. Jena, 1892. * Ville, Georges, Recherches sur les relations qui existent entre la couleur des plantes et la richesse des terres en agents de £ertilit6. Compt. rend. Paris 109: 397-400. 1889. h This may also be seen in the arbor vitae (Thuja occidentalis) of the northeastern United States in very cold, bright winter weather. — Ed. ASSIMILATION OF CARBON 1 7 Lesage and Schimper 1 found that an excess of mineral substances reduces the chlorophyll content, an effect that may be observed not only in halophytes, growing normally upon soils rich in salts, but also in other plants when watered with strong salt solutions. Finally, Palladin 2 pointed out that carbohydrates are essential to the formation of chlorophyll. As will be seen farther on, plants fall into tw T o groups according to the carbohydrate content of their etiolated leaves; in one group (for example, wheat), such leaves contain much soluble carbohydrate material, while in etiolated leaves of the other group (such as bean and lupine) carbohydrates are almost entirely absent. If etiolated leaves of these plants are removed and floated upon water in light, those of barley become green, while almost all the bean leaves and all those of lupine remain yellow. In the latter are floated, not upon water but upon a saccharose or glucose solution, then they also all become green. The greening of entire, completely etiolated bean plants in light is explained in this way, that carbohydrates migrate into the leaves from the stems. Besides saccharose and glucose, such substances as raffinose, fructose, maltose, glycerine, and some others, also produce greening 3 under these conditions. The concentration of these substances is important in this connection. 4 Greening occurs quickly with a saccharose solution of low or medium concentration. If the concentration is previously increased to 35 per cent., in darkness, the leaves remain yellow for several days when subsequently brought into the light, but greening occurs quickly in these leaves if they are transferred from the strong solution to one having a concentration of from 5 to io per cent. Single-celled algae are particularly well adapted to the study of the importance of various substances in the formation of chlorophyll. Cultures in light exhibit a considerable range of color (from yellow-green to intense, dark green) according to the composition of the nutrient solution used. 5 Thus greening, or the accumulation of chlorophyll, is a physiological process that proceeds only in living cells and under conditions favorable to life. The substance from which chlorophyll arises has not yet been isolated, but the existence of such a substance may be inferred from various observations. According to Monteverde and Liubimenko, 6 a pigment called chlorophyllogen is formed, independently of light, in the chromatophores of all green plants. It is said to arise from a colorless chromogen, leucophyllj of which little more is 1 Schimper, A. F. W., Die Indo-Malayische Strandflora. Jena, 1891. P. 9. 2 Palladin, W., Ergrunen und Wachsthum der etiolirten Blatter. Ber. Deutsch. Bot. Ges. 9: 229-232. 1891. 3 Palladin, W., Recherches sur la formation de la chlorophylle dans les plantes. Rev. g6n. Bot. 9 : 385- 394- 1897. 4 Palladin, W., Einfluss der Concentration der Losungen auf die Chlorophyllbildung in etiolirten Blat- tern. Ber. Deutsch. Bot. Ges. 20: 224-228. 1902. 5 Artari, Alexander, Ueber die Bildung des Chlorophylls durch griine Algen. Ber. Deutsch. Bot. Ges. 20: 201-207. 1902. Matruchot, L., and Molliard, M., Variations de structure d'une algue verte sous l'influence du milieu nutritif. Rev. gen. bot. 40: 1 14-130, 254-268. 1902. 6 Monteverde, N. A., and Lubimenko, V. N., Recherches sur la formation de la chlorophylle chez les plantes. [Text in Russian.] Bull. Acad. Imp. Sci. St.-Petersbourg VI, 5: 73-100. 191 1. 7 Sachs, J., Ueber das Vorhandensein eines farblosen Chlorophyll-Chromogens in Pflanzentheilen, welche fahig sind griin zu werden. Lotos 9: 6-14. 1859. Idem, same title. Chem. Centralbl., n. F. 4: 145- IS3. 1859. o l8 PHYSIOLOGY OF NUTRITION known. Chlorophyllogen is a very unstable substance, and its absorption spec- trum shows a great similarity, in the red region, to that of chlorophyll. Attempts to isolate it result in an artificial transformation-product, the proto- chlorophyll of Monteverde. 1 Like chlorophyll, protochlorophyll is a deep green pigment, which is fluorescent, appearing red by reflected light. The spectrum shows four absorption bands. The absorption spectra of alcoholic solutions of protochlorophyll on the one hand, and of alcoholic chlorophyll on the other, are different in that the absorption band between B and C in the second is absent in the first, and the one between C and D in the first appears slightly displaced toward the left in the second; the other bands practically agree. Although protochlorophyll is a transformation-product, it is still of interest, in so far as its existence indicates the presence of a mother-substance for chlorophyll; protochlorophyll itself cannot change into chlorophyll. Protochlorophyll arises independently of light, from chlorophyllogen. As to its presence in living cells, it is normally found in large quantities in the inner seed-coats of the Cucurbitaceae, especially inLuffa. A rapid transformation of chlorophyllogen into chlorophyll occurs in living plant cells under the influence of light. This process can also be observed in plants that have been killed. According to Liro, if etiolated leaves are care- fully killed so that at least some of the chlorophyllogen remains, and if they are then exposed to light, some formation of chlorophyll can still be observed. For the transformation of chlorophyllogen into chlorophyll, Liro and Isachenko 2 have shown that neither oxygen, favorable temperature conditions, nor even the presence of carbohydrates are necessary, but since greening is possible only with these conditions they are evidently necessary for the formation of chloro- phyllogen, or of the chromogen that gives rise to it. Chlorophyll may be formed from chlorophyllogen in the absence of light, as is exemplified by plants that turn green in darkness; in such cases the influence of chemical agents must replace the action of light. 3 Such are the chief results of the researches thus far carried out upon chloro- phyll and its formation. As to the role it plays in the chemical decomposition of carbonic acid and the formation of the first products of photosynthesis almost nothing is known. Schryver 4 suggests that the formaldehyde arising in the decomposition of carbon dioxide and water enters into combination with the chlorophyll. » Monteverde, 1894. [See Note, 1, p. 9.] Monteverde, N. A., Der Einfluss des Lichts auf die Gesch- windingkeit deT Chlorophyllbildung in Blattern etiolirter Pflanzen. Trav. Soc. Imp. Nat. St.-P6tersbourg 27' ': 131-142 [Russian], 143-145 [German abstract]. 1896. Idem, Das Protochlorophyll und Chlorophyll. [Title and abstract in German, article in Russian.] Bull. Jard. Imp. Bot. St.-Petersbourg 2: 179-182. [Abstract, p. 181-182.] 1902. Idem, Ueber das Absorptionsspectrum des Protochlorophylls. I. [Title and abstract in German, article in Russian.] Ibid. 7: 37-42 [Abstract, p. 42], 47-58. [Abstract, p. 5S~58]. 1907. ' Issatchenko, B., Sur les conditions de la formation de la chlorophylle. [Title and abstract in French, article in Russian.] Bull. Jard. Imp. Bot. St.-Petersbourg 6: 20-28 [Abstract, p. 27-28]. 1906. Idem, same title. Ibid. 7: 59-64 [Abstract, p. 64]. 1907. Idem, same title. Ibid. 9: 106-120 [Ab- stract, p. 119-120]. 1909. 3 Monteverde and Liubimenko, 1911. [See note 6, p. 17.] 4 Schryver, S. B., Photochemical formation of formaldehyde in green plants. Chem. news 101 : 64. 19TO. ASSIMILATION OF CARBON 1 9 As to the physics of the action of chlorophyll, it behaves as a sensitizer 1 and renders the energy of the absorbed light effective in the decomposition of car- bon dioxide. In an analogous manner the red light rays between lines B and C of the spectrum rapidly decompose silver salts in the presence of chlorophyll, although these salts are otherwise decomposed only by blue and violet rays. §4. Pigments Accompanying Chlorophyll. — Among the other pigments accompanying chlorophyll, special attention should be given to carotin. 2 Boro- din 3 was able to show that carotin (called erythrophyll by him) regularly ap- peared in alcoholic leaf extract when he allowed this to form crystals under the microscope. The chemical nature of carotin, and also some of the conditions of its forma- tion in leaves, were first made clear by the investigations of Arnaud 4 and of Willstatter and Mieg. 5 This pigment forms flat, rhombic crystals, which, with one-sided illumination, appear blue-green on the illuminated side and orange- red on the other. It is readily soluble in ether, chloroform and carbon bisul- phide, less so in benzine, slightly soluble in hot alcohol, almost insoluble in cold alcohol and insoluble in water. A carbon bisulphide solution of carotin is blood-red; dissolved in concentrated sulphuric acid, carotin is bluish-violet. It is a hydrocarbon, with the formula C 4 oH 56 , which is easily oxidized. It may be transformed into cholesterin. The carotin content of leaves varies with the season of the year. A series of experiments continued throughout the summer upon the leaves of stinging nettle and horse-chestnut showed that the carotin content is greatest during the flowering season, for both plants. The formation of carotin is also dependent upon light; green leaves of vetch contained 178.8 mg. of carotin, as compared to 34.0 mg. in the same quantity of etiolated leaves. It was shown by the work of Kohl 6 that carotin is widely distributed. It is not limited to the green parts of plants but occurs also in flowers, fruits, seeds and subterranean organs, and also in fungi. It may be extracted in large quan- tities from carrots. The function of carotin is not yet clear, but its tendency to unite with oxygen appears, at any rate, to be significant in connection with the photosynthetic process, where reduction of compounds containing oxygen is known to occur. • Tappeiner, H. von, Die photodynamische Erscheinung (Sensibilisierung durch fluoreszierende Stoffe). Ergeb. Physiol. 8: 698-741. 1909- 2 Escher, Heinr. H., Zur Kenntnis des Carotins und des Lycopins. Zurich, 1909. 104 p. (Zurich Poly- techn. Dissert. 1909-10.) [For a general discussion of the yellow pigments, see Haas and Hill, 1921. (See note 3, p. 6.)] 'Borodin, J., Ueber krystallinische Nebenpigmente des Chlorophylls. Bull. Acad. Imp. Sci. St.- Petersbourg 28: 328-350. 1883. 4 Arnaud, A., Recherches sur les matieres, colorantes des feuilles; identite de la matiere rouge orange avec la carotine, Cj 8 H 2 40. Compt. rend. Paris 100: 75 1-753- 1885. Idem, Recherches sur la composi- tion de ia carotine, sa fonction chimique et sa formule. Ibid. 102 : 1119-1122. 1886. Idem, Sur la pres- ence de la cholesterine dans la carotte; recherches sur ce principe immediat. Ibid. 102 : 1310-1322. 1886. Idem, Recherches sur la carotine; son role physiologique probable dans la feuille. Ibid. 109: 911-914. 1889. s Willstatter, Richard, and Mieg, Walter, Ueber die gelben Begleiter des Chlorophylls. Liebig's Ann. Chem. u. Pharm. 355: 1-28. 1907. « Kohl, Friedrich Georg, Untersuchungen uber das Karotin und seine physiologische Bedeutung in der Pflanze. Leipzig, 1902. 20 PHYSIOLOGY OF NUTRITION The absorption spectrum of carotin has two dark bands in the green-blue half of the spectrum (Fig. 10). A second yellow pigment accompanying chlorophyll is xanthophyll, an oxida- tion product of carotin, with the formula C 4 oH 56 02. 1 Lycopin ■ Karoiin . 70 85 Fig. io.— Absorption spectra of carotin and lycopin. (After Escher.) The Fraunhofer lines are indicated by the letters above and the wave-lengths (in io nn) are shown below; the thickness of layer employed is given (in mm.) at the left. Lycopin 1 is closely related to carotin and has the same percentage formula (C 4 oH 5 6) ; it is found in the fruit of the tomato {Solatium ly coper sicum). Three dark bands occur in the right half of its absorption spectrum (Fig. 10). 700 B C J L GOOD E 500 F 400 700 B C J L eooD I 1 500 F G 400 p IG II# Absorption spectra of carotin (above) and xanthophyll (below). (After Will- statter and Stoll.) The Fraunhofer lines and the wave-lengths (in /i/i) are shown on the upper line of each diagram. Red alga? contain phycoerythrin, a protein-like substance, which is readily soluble in water but insoluble in alcohol, ether, and carbon bisulphide. The 1 Montanari, Carlo, Materia colorante rossa del pomodoro. Le Stazioni Sperimentali Agrarie Italiane 37: 909-919. 1904. [Willstatter, Richard, and Escher, Heinr. H., Ueber den Farbstoff der Tomate. Zeitsch. physiol. Chem. 64: 47-61. 1910.] i The absorption spectra of carotin and xanthophyll, as given by Willstatter and Stoll (1913) [see note b, p. 6] are here reproduced as Fig. 11. It is questionable whether xanthophyll is actually formed by the oxidation of carotin. — Ed. ASSIMILATION OF CARBON 21 dark, bluish-red solution shows an orange-yellow fluorescence. It crystallizes from salt solutions in hexagonal red crystals. 7 ' Phycocyanin, 1 the blue pigment of the blue-green algae, Cyanophyceae, is likewise of protein nature; it is soluble in water and glycerine but insoluble in ether and alcohol, its crystals are indigo blue in color. The brown algae contain a pigment, phycophcein, 2 which is easily soluble in water; in concentrated solutions it is dark reddish-brown. fc Engelmann 3 studied the absorption spectra of bright-colored leaves of vari- ous plants, and Stahl 4 investigated the biological importance of their coloring/ §5. Influence of Light upon the Decomposition of Carbonic Acid by Plants. An acquaintance with the properties of the different rays of the sun's spectrum (Fig. 12) is prerequisite to an understanding of the researches devoted to this subject. Only the central part of the spectrum, approximately that portion lying between lines A and H, is visible to the human eye; on either side are in- visible rays, infra-red to the left and ultra-violet to the right. Of the visible rays, the yellow are the brighest, the brightness reaching a maximum at line D and decreasing to zero beyond A and H. Brightness does not, however, represent the character of the rays, but only that of the human eye. The en- ergy maximum in the prismatic solar spectrum is usually shown as falling in the region of the infra-red, as in Fig. 12. Nevertheless, recent work upon the dis- tribution of heat in the ordinary diffraction spectrum of sunlight shows the 1 Molisch, Hans, Das Phycocyan, ein krystallisirbarer Eiweisskorper. Bot. Zeitg. 53: I3I-I35- 1895. 2Schiirt, Franz, Ueber das Phycophasin. Ber. Deutsch. Bot. Ges. 5: 259-274. 1887. aEnglemann, Th. W., Die Farben bunter Laubblatter und ihre Bedeutung fur die Zerlegung der Kohlensaure im Lichte. Bot. Zeitg. 45 : 393-398, 409-419. 425-436, 44i-4SO. 457-469- 1887. * Stahl, E., Ueber bunte Laubblatter. Ein Beitrag zur Pflanzenbiologie. II. Ann. Jard. Bot. Buitenzorg 13: 137-216. 1896. ■» On phycoerythrin, see Haas and Hill, 1921. [See note 3, p. 6.] The best study of this pigment is that of Hanson. (Hanson, E. K., Observations on phycoerythrin, the red pigment of deep-water alga?. New phytol. 8: 337-344. 1909.) — Ed. k But it seems to have been shown that there is no such pigment as phycophasin in the living cells, this being a post-mortem product of the decomposition of a colorless chromogen. The brown color of the brown algae is at least partly due to the presence of carotin. In this con- nection see the following: Molisch, Hans, Das Phycoerythyrin, seine Krystalisirbarkeit und chemische Natur. Bot. Zeitg. 52: 177-189. 1894. Idem, Das Phycocyan ein Krystal- lisirbarer Eisweisskorper. Ibid. 53: 131-135- 1895. Idem, Ueber den braunen Farbstoff der Pha;ophyceen und Diatomeen. Ibid. 63' ': 131-144- 1905- Tswett, M., Zur Kenntnis der Phaeophyceenfarbstoffe. Ber. Deutsch. Bot. Ges. 24: 235-244. 1906.— Ed. 1 The antliocyanins, or anthocyans, are other pigments that may be mentioned here. They occur very commonly in flowers, leaves, stems, fruits, and even in roots, giving them a red, blue or purple color and frequently masking the green of the chlorophyll in leaves. They are red when acid and blue when alkaline. The color of red apples and many other fruits, of many red, blue and purple flowers, of the beet-root, of red cabbage, of young leaves of many plants, and of the bronze-colored leaves of the copper beech, are due to the presence of these pigments. They are often present along with chlorophyll, as in the case of red cabbage and the copper beech, and still other pigments frequently accompany them. They are soluble in water, alcohol and ether, and the color of the solution alters from red to purple or blue as the reaction is altered from acid to neutral or alkaline. For further information see: Haas and Hill, 1921. [See note 3, p. 6.] West, Clarence J., Plant pigments: The chemistry of plant pigments other than chlorophyll. Biochem. bull. 4: 151-160. 1915. — Ed. 2 2 • PHYSIOLOGY OF NUTRITION energy maximum to lie between lines B and C; 1 and, according to the latest researches, the position of this maximum is not constant but varies from the region of the red to that of the yellow-green, according to the hour of the day. Finally, chemically active or "actinic" rays, with a maximum in the violet region, are frequently differentiated. The term actinic rays really refers to the power of light to decompose silver salts, which is most pronounced in the blue-violet region of the solar spectrum. Many other compounds are decom- posed by light, however, frequently in other regions than the blue-violet, and the wave-lengths producing such decomposition are those that are absorbed by the substances decomposed: thus, chlorophyll is most rapidly decomposed by rays between B and C, exactly the ones most completely absorbed by chlorophyll. Therefore, the curve of chemical intensity, as usually given, has no importance excepting with reference to silver salts: there are no specific "chemical" rays. Fig. 12. — Graphs of the prismatic solar spectrum. PA, infra-red; AH, visible; HS, ultra-violet rays; PTS, temperature curve; ALH, curve of light intensity; DKS, curve of effect of light upon the decomposition of silver salts. Researches upon the influence of light on the decomposition of carbon di- oxide and water by plants fall into two groups. One group includes studies dealing with the qualitative side of the question, as to which rays or wave- lengths are most effective in the process. The other includes quantitative in- vestigations, as to how much energy is needed for this decomposition. The first qualitative work was done by Daubeny™ and Draper" the former using i Langley, [S. P.], Observations du spectre solaire. Compt. rend. Paris 95: 482-487. 1882. Idem, Energy and vision. Phil. mag. V, 27 : 1-23. 1889. [Sunlight as it reaches plants is so variable in both quality and intensity that each quantitative experiment on photosynthesis, etc., in natural illumination, should be carried out with very careful measurements of solar radiation. Nutting states that the sun's total radiation varies over a range of 8 per cent, of the mean, while the earth's atmosphere, even with a clear sky, absorbs from 20 to 50 per cent., and this varies from minute to minute and from hour to hour of the day. Nutting gives a table (p. 202) of mean solar energy quantities reaching the surface of the earth at Washington at noon, for 26 different wave-lengths, from 38s to 428^. (See Nutting, P. G., Outlines of applied optics. Philadelphia, 191 2.) The wave-length showing the maximum energy value also varies markedly in natural sunlight. For further information see: Abbot, C. G., and Fowle, F. E., Jr., Primary standard pyrheliometer. Ann. Astrophys. Observ. Smithsonian Inst. 2: 39-47- 1908. Idem, The value of the solar constant of radiation. Astrophys. jour. 33: 191-196. 19". Also see Pulling, H. E., Sun- light and its measurement. Plant World 22: 151-171, 187-209. 1919— Ed ••' Daubeny, Charles, On the action of light upon plants, and of plants upon the atmosphere. Phil, trans. Roy. Soc. London 126: 149-175. 1836. — Ed. " Draper, John W., On the decomposition of carbonic acid gas and the alkaline carbonates by the light of the sun. Phil. mag. Ill, 23: 161-175- l8 43- Idem, Scientific memoires. 473 p. New York, 1878. P. 184-185.— Ed. ASSIMILATION OF CARBON 23 light screens and the latter the prismatic spectrum. Both came to the con- clusion that plants decompose carbon dioxide most readily under the influence of the yellow light rays. Sachs 1 divided the spectrum into two nearly equal portions, by using a solution of potassium dichromate and one of ammoniacal copper oxide, and found that decomposition of carbon dioxide proceeded almost as energetically in the yellow portion of the spectrum as in direct sunlight, while very little decomposition occurred in the blue-violet region. It is seen, therefore, that it is not the so-called "chemical" rays that are needed for this process, but chiefly the less refrangible rays of the first half of the spectrum. Sachs determined the amount of oxygen given off, using the method of counting ing gas bubbles (Fig. 2). The next problem was to discover in what rays of the first half of the spectrum the decomposition of carbonic acid was most rapid. The most exact studies upon this point were carried out by Timiriazev, 2 who arranged his experi- ments as follows: Sunlight was reflected from a helio- stat into a dark chamber and was then broken up by a carbon bisulphide prism. Pieces of bamboo leaves were enclosed in glass tubes, with air containing 5 per cent, of carbon dioxide, and these tubes were placed in various regions of the spectrum — in the red between A and B, in the chlorophyll absorption band between B and C, in the orange, in the yellow, and in the green. At the conclusion of the experiment analyses of the gas were made, by means of a very sensitive appa- ratus capable of measuring extremely small amounts of gas. Timiriazev's results are graphically repre- sented in Fig. 13. The ends of the five ordinates, for the five positions in the spectrum where the tubes were exposed, are joined to form a curve, which represents ^onof carbon dioxide in the relative rates of decomposition of carbon dioxide different parts of the spec , . ~, trum. {After Txmniazev.) in these different regions of the spectrum. 1 he maxi- mum decomposition occurs in the red, between B and C, in the region where light is most strongly absorbed by chlorophyll. No decomposition occurs between A and B (the line m represents the amount of carbon dioxide eliminated during the experiment). These results were confirmed by Engelmann 3 and Reinke. 4 1 Sachs, J.,Wirkungen farbigen Lichts auf Pflanzen. Bot. Zeitg. 22 : 353-358. 361-367. 369-372. 1864. 2 Timiriazev, K. A., (C.) On the assimilation of light by plants. [Russian.) St. Petersburg. 1875. Timiriazeff, C, Recherches sur la decomposition de l'acide carbonique dans le spectre solaire. par les parties vertes des vegetaux. (Extrait d'un Ouvrage "Sur l'assimilation, de la lumiere par les vegetaux," St.-Petersbourg, 1875; publie en languerusse.) Ann. chim. et phys. V, 12: 355-396. 1877. s Engelmann, Th. W., Ueber Sauerstoffausscheidung von Pflanzenzellen im Mikrospectrum. Bot. Zeitg. 40 : 419-426. 1882. * [Reinke, J., Untersuchungen uber die Einwirkung des Lichtes auf die Sauerstoffausscheidung per Pflan- zen. II. Die Wirkung der einzelnen Strahlengattungen des Sonnenlichtes. Bot. Zeitg. 42: 17-29. 33~46. 40-59. 1884. See column 27. Idem, Die Zerstorung von Chlorophyll osungen durch das Licht und eine neue Methode zur Erzeugung des Normalspectrums. Ibid. 43: 65-70, 81-89, 97-ioi, 113-117, 129-137 1885. See column 84. Idem, Die Abhangigkeit des Ergrunens von der Wellenlange des Lichts ungsber (Math.-Naturw. Mitth.). K. Preuss. Akad. Wiss. Berlin. 1893 : 301-314- 1893I Fig. 13. — Graphs show- ; relative rates of decom- Sitz- 24 PHYSIOLOGY OF NUTRITION Engelmann was the originator of the bacterial method for the study of photosynthesis. It is well known that many bacteria are active only in the presence of oxygen, and that their movement ceases as soon as there is no oxygen present. If a filament of a green alga is placed in a culture of such bacteria, upon a slide, and if the preparation is protected by a cover glass and darkened, the movement of the bacteria eventually ceases because of lack of oxygen. If a solar spectrum is now projected upon the alga filament, under the microscope, it is seen that the movement of the bacteria is renewed in the neigh- borhood of both of the main chlorophyll absorption bands (Fig. 14), being espe- cially pronounced in the red and appreciably weaker in the blue. It is only in the spectral regions thus undicated, therefore, that an evolution of oxygen occurs, to which the bacteria respond. The degree of difference between the efficiences of the blue and red spectral regions was established by Timiriazev. 1 For this purpose he divided the aB C D Eb F I_ Fig. 14. — Bacterial movement in the regions of the absorption bands of chlorophyll. (After Englemann.) The dots indicate moving bacteria and the letters denote the Fraunhofer lines. 700 600 500 WO Fig. 15. — AB, distribution of heat energy in the solar spectrum. (After Langley.) 100—14, relative rates of car- bon-dioxide decomposition by leaves in red and in blue light. spectrum into two equal parts by means of a cylindrical lens and a prism with a very small angle of refraction. Flat-sided glass tubes containing pieces of leaves of equal area were placed in the bright bands of blue and yellow light thus obtained, and a gas analysis of the tube contents was made after three- quarters of an hour or an hour. If the intensity of carbon dioxide decomposi- tion in the less refrangible (red-yellow) light be taken as 100, then the corre- sponding intensity in the more refrangible (blue) light is 54. Thus the light absorbed by the leaves in the blue half of the spectrum is only about half as effective as that absorbed in the other half. The absorption spectrum of the leaves used in Timiriazev's experiment is presented in Fig. 15. It must be noted, however, that the two absorption bands are not of equal width, the one in the blue-violet region of the normal spectrum being more than three times as wide as the band between B and C. If each of the ratios mentioned above is 1 Timiriazev, C, Photochemische Wirkung der am Rande des sichtbaren Spektrums liegenden Strahlen. 1893. (Russian.)* ASSIMILATION OF CARBON 25 divided by the breadth of the corresponding effective absorption band, there is obtained for an average wave-length of the red region, 100, and for a similar average in the blue-violet, 14, a relation which is graphically represented in Fig. 15. Thus red light is relatively much more effective than blue-violet light. How can this difference be explained? Obviously the explanation is to be found in a consideration of the energy of the different wave-lengths expressed in terms of their respective heat values, and (as will be seen from comparison of the curve of decomposition of carbon dioxide with the Langley curve, AB, representing the heating effect of the various parts of the solar spectrum) both of these increase in the same direction. So the blue and violet rays have only a comparatively slight effect in the decomposition of carbon dioxide, be- cause, even though they are absorbed by chlorophyll, they represent only a very small amount of energy. The dependence of the process of decomposition of carbon dioxide upon the energy of the light rays was demonstrated in a still more detailed manner by the experiments of Rikhter. 1 Only light that is absorbed can decompose carbon dioxide, and those wave-lengths of the absorbed light are most effective which furnish the greatest amount of heat energy. Rikhter used solutions of potassium dichormate, ammoniacal copper oxide and potassium permanganate as light filters. The plant received the following relative amounts of light when placed behind the various filters: Potassium Dichro- Ammoniacal Copper Potassium Perman- Water mate Solution Oxide Solution ganate Solution 1000 491 *77 2 33° 100 3 & 47-5 The corresponding relative rates of carbon dioxide decomposition behind the same light screens proved to be, on the average, as follows: Potassium Dichro- Ammoniacal Copper Potassium Perman- Water mate Solution Oxide Solution ganate Solution 1000 494 168.0 249 100 34-4 48 The numbers in the two series agree so closely as to suggest that the amount of photosynthetic work accomplished by a ray of light is proportional to the amount of energy absorbed by the leaf, and is independent of the wave length of the ray and of its position in the spectrum. 2 1 Richter, Andre, Etude sur la photosynthese, et sur 1'absorption par la feuille verte des rayons de differentes longueurs d'onde. Rev. gen. bot. 14: 151-169. 211-218. 1902. Kohl, 1897. [See p. 5. note 1.] 2 See also: Kniep, H., and Minder, F., Ueber den Einfluss verschiedenfarbigen Lichtes auf die Koblen- saureassimilation. Zeitsch. Bot. 1 : 619-650. 1909. [Puriewitsch, K., Untersuchungen iiber Photosyn- these. Jahrb. wiss. Bot. 53: 210-254. I9I3-1 These statements apply to leaves and should not be interpreted as necessarily applying to chlorophyll, for leaves contain carotin, etc., which surely affect their power to absorb radiation. Some referencess on sunlight have been given in note 1, p. 22. See also: Iwanowski, D., Ein Beitrag zur physiologischen Theorie des Chlorophylls. Ber. Deutsch. Bot. Ges. 32:433-447- 1914-— Ed. 26 PHYSIOLOGY OF NUTRITION Carbon dioxide is thus seen to be decomposed most rapidly in green plants by the light rays between lines B and C. But when other pigments besides chloro- phyll are present, the maximum of this decomposition may fall in another part of the spectrum. 1 In the Cyanophyceae the maxumim occurs at D; the brown algae show a maximum between D and E, although the decomposition between B and C is here almost as great; finally, the red algae have a maximum between D and E also, but the decomposition between B and C is here very weak. These facts are in agreement with the distribution of the various algae, accord- ing to depth, in the ocean; while the surface layer of water is mainly inhabited by green algae, the red forms are found at very great depths. Spectroscopic investigations have shown that red light, which is essential to green algae, is quickly absorbed by water and that this light is entirely absent at no great distance below the surface. On the other hand, the green and blue rays, which are absorbed by the red algae, attain great depths. According to Engelmann, 2 plants that contain no chlorophyll may also decompose carbon dioxide, provided they contain another pigment; as, for in- stance, the purple bacteria. p Engelmann's theory of complementary pigments found confirmation in the interesting researches of Gaidukov 3 upon the influence of colored light upon the color of Oscillaria. This alga tends to assume the color complementary to that of the light acting upon it, and the longer the organism remains in the colored light the more pronounced is the response. The following kinds of illumination produced the following colorations in the organism. Color of Light Color of Alga Red Green Brownish-yellow Blue-green Green Reddish Blue Brownish-yellow The principle illustrated by this phenomenon was designated by Gaidukov as the law of complementary chromatic adaptation. The amount of light 6 necessary for the decomposition of carbon dioxide is 1 Engelmann, Th. W., Farbe und Assimilation. Bot. Zeitg. 41 : 1-13. 17-29. 1883. -Engelmann, Th. W., Die Purpurbacterien und ihre Beziehungen zum Licht. Bot. Zeitg. 46: 661-669, 677-689, 693-710, 709-720. 1888. 3 Gaidukov, N., Ueber den Einfluss farbigen Lichts auf die Farbung lebender Oscillarien. Abh. K. Preuss. Akad. Wiss. Berlin, 1902. Anhang, Phys. Abh. V., p. 1-36. * Kreusler, U., Ueber eine Methode zur Beotachtung der Assimilation und Athmung der Pflanzen und iiber einige diese Vorgange beeinflussende Momente. Landw. Jahrb. 14: 913-065. 1885. Timriazeff, C, Sur le rapport entre l'intensit6 des radiations solaires et la decomposition de l'acide carbonique par les vegetaux. Compt. rend. Paris 109: 370-382. 1889. Pantanelli, Enrico, Abhangigkeit der Sauerstoff- ausscheidung belichteter Pflanzen von ausseren Bedingungen. Jahrb. wiss. Bot. 39: 167-228. 1904. Lubimenko, W., Sur la sensibility de 1' appareil chlorophyllien des plantes ombrophiles et ombrophobes. Rev. g6n. Bot. 17: 381-415. 1915. Idem, concentration du pigment vert et l'assimilation chlorophyl- lienne. Ibid. 20: 162-177, 217-238, 253-267; 285-297. 1908. Idem, Production de la substance seche et de la chlorophylle chez les vegfitaux superieurs aux differentes intensites lumineuses. Ann. sci. nat. Bot. IX, 7: 321-415. 1908. p But Molisch's studies indicate that the purple bacteria are not capable of the photo- synthesis of carbohydrates from carbon dioxide and water. See: Molisch, Hans, Die Purpur- bakterien nach neuen Untersuchungen, eine mikrobiologische Studie. 92 p. ]ena, 1907. (A misstatement occurred here in the first printing.) — Ed. ASSIMILATION OF CARBON 27 closely related to the individual properties of the plant, some forms needing more and other less light. Trees were long ago differentiated by students of forestry into two types, heliophobous (shade plants) and heliophilous (non-shade plants); among the first are included, for example, Abies (fir), Taxus (yew), Fagus (beech), Tilia (linden); among the latter, Pinus (pine), Larix (larch), Betula (birch), Robina (locust). Schistostega osmundacea, a moss that grows in dark caves, may be mentioned as an example of plants that can thrive in extremely weak light. Its protonema has a very peculiar structure (Fig. 16), and, although existing in semi-darkness, it appears emerald green. Single filaments of the protonema, as they grow upward, each form a plate of cells lying at right angles to the direction of the impinging light. Each cell of this plate has the form of a lens and the chloro- plasts lie in the prolonged basal region. Acting like biconvex lenses, these cells concentrate the light of the half-dark cave sufficiently to allow carbon .4 B Pig. 16. — Schistostega osmundacea: A, protonema; B, diagram representing the path taken by rays of light as they enter and leave the cells of the protonema. dioxide decomposition by the chloroplasts. A part of the light is reflected, thus rendering the protonema luminous. In general, plants are adapted to the minimum of available light (Wiesner, Liubimenko). In heliophilous plants (which thrive best in bright sunshine) the rate of carbon dioxide decomposition increases continuously with increase in light intensity; 5 on the other hand, for heliophobous plants (which thrive in shade or in regions of low light intensity) there exists an optimum light intensity, and any increase beyond this optimum results in a decrease in the amount of carbon dioxide decomposed. This difference is related to the different amounts of chlorophyll contained in the two kinds of plants. Liubi- menko was able to show that heliophobous plants are richer in chlorophyll than are heliophilous ones. Within limits, the greater the amount of light « It is not to be understood that there are no optimum light intensities for carbon-dioxide decomposition in plants that grow best in bright sunshine, only that such optima are markedly higher than those for plants that grow best in shade.— Ed. 28 PHYSIOLOGY OF NUTRITION and the higher the temperature, the smaller is the amount of chlorophyll formed by the plant. §6. Products of Photosynthesis. 1 — The simplest equation that may repre- sent the exchange of gases in photosynthesis is CO" = C + Oo. The carbon is retained by the plant, combined with other elements in the form of organic sub- stances. The question now arises as to what are to be considered as the first products of photosynthesis. The investigations of Sachs 2 showed that the first visible product is starch. If leaves are kept for several days in darkness the starch completely disappears from the chlorophyll bodies, and if the leaves are then returned to light starch soon appears again. Small traces of starch may be recog- nized by the method of Bohm, whereby leaves are first decolorized by alcohol and then treated with caustic potash and iodine solution; the starch grains, greatly swollen by potassium hydroxide, are stained by iodine and thus become visible. If a part of the leaf is covered with tinfoil before it is exposed to light, and if, after the exposure, the leaf is decolorized with alcohol and then treated with iodine, the portion that was shaded becomes yellowish brown, while the rest of the leaf is blue or black, accord- ing to the amount of starch present (Fig. 17). The experiment becomes particularly striking if the whole leaf is covered with a piece of tinfoil, or cardboard, from which the letters of the word starch, etc., have been cut out as in a stencil; after the treatment described above, the letters stand out blue against a brown background/ According to Famintsyn, 3 algae may be very satisfactorily employed in this connection; the presence of starch may be shown after only half an hour's illumination from a bright lamp. According to Kraus, 4 algae may form starch in sunlight within a period of five minutes. As Godlewski 5 has shown, starch 1 Brown, H. T., and Morris, G. H., A contribution to the chemistry and physiology of foliage leaves. Jour. Chem. Soc. London 63: 604-677. 1893. 2 Sachs, J., Ueber den Einfluss des Lichtes auf die Bildung des Amylums in den Chlorophyllkornern. Bot. Zeitg. 20: 365—373. 1862. Idem, Ueber die Auflosung und Wiederbildung des Amylums in den Chlorophyllkornern bei wechselnder Beleuchtung. Ibid. 22: 280-294. 1864. 3 [Famintzin, A., Die Wirkung des Lichtes auf Algen und einige andere ihnen nahe venvandte Organismen. Jahrb. wiss. Bot. 6: 1-44. 1867. See P. 34.] 4 [Kraus, Gregor, Einige Beobachtungen uber den Einfluss des Lichts und der Warme aud die Starkeer- zeugung im Chlorophyll. Jahrb. wiss Bot. 7: 511-531. 1868.] 6 Godlewski, Emil, Abhangigkeit der Starkebildung in den Chlorophyllkornern von dem Kohlensaurege- halt der Luft. Flora, n. R. 31: 378-383. 1873. T The experiment should be performed in such manner that access of the carbon dioxide of the air to the stomata is clearly not hindered; otherwise the conclusion given is not logically substantiated. (See Ganong, W. F., A laboratory course in plant physiology. 2 ed., New- York, 1908. P. 86-90.) It is usually best to transfer the decolorized leaves from alcohol to water, then to an aqueous solution of potassium hydroxide, after which an aqueous solution of potassium iodide and iodine is added to bring out the color reaction. The iodine solution may be prepared by dissolving 5 g. of the iodide in water, then dissolving 1 g. of iodine in this, and diluting the resulting double solution to a volume of 1000 cc. or less. — Ed. Fig. 17. — Accu- mulation of starch in the illuminated portion of a leaf. The light-colored portion was shaded by tinfoil and the starch has been stained by iodine. ASSIMILATION OF CARBOX 20 can be formed in light only in the presence of carbon dioxide. In a closed chamber, illuminated but free from this gas, no starch was formed; indeed, if starch had been originally present its amount decreased under these con- ditions. The chloroplasts of some plants do not form starch at all, as is the case with laves of Allium cepa (onion), A.fistulosum, Asphodelns luteus, Orchis militaris, and Lactuca sativa (lettuce), but in all these instances glucose is formed instead of starch. According to whether starch ((C 6 Hio0 5 )n) or glucose (C 6 Hi 2 6 ) is con- sidered as the first product of photosynthesis, the chemical equation represent- ing the process may take one or the other of the two forms given below: (1) 6 C0 2 + 5 H 2 = C 6 H 10 O 5 + 6 2 . (2) 6 COo + 6 H 2 = C 6 H 12 6 + 6 2 . Timiriazev 1 showed by direct experiment that the formation of starch in light is brought about by the same rays of the spectrum as are effective in the decom- position of carbon dioxide. By means of a heliostat, a spectrum was thrown upon a leaf of a plant that had been previously exposed to darkness so as to free the leaves of starch; two strips of paper were fastened across the leaf with the spectrum falling between them, and upon these strips were recorded the positions of the Fraunhofer lines in the spectrum. At the end of the experi- ment, after the leaf had been decolorized by alcohol and stained with iodine, it became evident that starch formation had occurred exactly in the regions cor- responding to the absorption bands of chlorophyll. In such an experiment the band between lines B and C is especially pronounced, and a fainter iodine- starch color is noticeable in the orange-yellow region, this coloration gradually decreasing in intensity and ceasing not far beyond the D line. Thus starch is produced by those wave-lengths of light that cause the decomposition of carbon dioxide, the rays between B and C being most effective in both cases. Briosi 2 was unable to find starch in the leaves of Musa (banana) and Strelitzia, but found oil instead, and expressed the opinion that the latter was the first product of photosynthesis in these plants. Holle 3 and Godlewski 4 were able to prove, however, that this supposition is untenable. Baeyer 5 advanced the hypothesis that formaldehyde is really the first prod- uct of photosynthesis, and that carbohydrates arise from this by progressive condensation or polymerization. The formation of formaldehyde thus supposed is represented by the equation, C0 2 + H 2 = CH 2 + 0>. Baeyer based his supposition upon a discovery by Butlerow 6 that oxymethylene (C 3 H 6 3 ) is con- 1 Timiriazeff, C, Enregistrement photographique de la fonction chlorophyllienne par la plante vivante. Compt. rend. Paris no: 1346-1347. 1890. - [Briosi, Giovani, Ueber normale Bildung von Fettartiger Substanz im Chlorophyll. Bot. Zeitg. 31 : 520-533, 545-550. 1873-] 3 Holle, H. G., Ueber die Assimilationsthatigkeit von Strelitzia regina. Flora, n. R. 35 : 113-120, 154- 160. 161-168, 184-192. 1877. ' Godlewski, Emil, 1st das Assimilationsprodukt der Musaceen Oel oder Starke? Flora, n. R. 35 : 215- 220. 1877. 5 Baeyer, Adolf, Ueber die Wasserentziehung und ihre Bedeutung fur das Pflanzenleben und die Gah- rung. Ber. Deutsch. Chem. Ges. 3: 63-75- 1870. « [Butlerow, A., Bildung einiger Zuckerarten durch Synthese. Liebig's Ann. Chem. u. Pharm. 120: 295- 298. 1861. Idem, Formation synthetique d'une substance sucree. Compt. rend. Paris 53: 145-147. 1861.] 30 PHYSIOLOGY OF NUTRITION verted into a sugar-like substance in the presence of calcium and barium hydroxides. Reinke is of the opinion that the hydrate of carbonic acid and not the anhydride, is decomposed in the light, as indicated by the equation, H 2 C0 3 = CH 2 + O2. The same author 1 was successful in showing that substances possessing aldehyde characters generally occur in green plants, and Curtius and Reinke 2 succeeded in isolating a material of this sort and in identifying it chemically. Curtius and Franzen 3 isolated a-/3-hexylene-aldehyde from the leaves of Carpinus (horn-beam). This aldehyde shows the same carbon skeleton as does glucose, as becomes evident from a comparison of their struc- tural formulae: CH 3 — CH 2 — CH 2 — CH— CH— Cf (a-0-Hexylene-aldehyde) \H CHo— CH— CH— CH— CH— Cf (d-glucose). I I I I I X H OH OH OH OH OH Pollacci 4 found, furthermore, that the green parts of plants gave a positive aldehyde reaction with Schiff 's reagent only if they had been previously exposed to light and carbon dioxide ; if the plants had previously been deprived of both light and this gas they gave, as did also fungi, no reaction for aldehyde. 8 Formaldehyde can be utilized by green plants in the formation of carbohy- drates, but none is absorbed in darkness. 5 Walther Lob's 6 interesting researches have furnished experimental evidence in favor of Baeyer's hypothesis. He used a silent electric discharge as source of energy, instead of sunlight, and established the following principal reactions between carbon dioxide and water, etc. 1. 2 C0 2 = 2 CO + 2 2. CO + H 2 = C0 2 + H 2 3. H 2 + CO = H 2 CO 4. CO + H 2 = HCOOH 5. 3 2 = 20 d 6. 2 H 2 + 2O3 = 2 H»0 2 + 2 1 Reinke, J., Studien uber das Protoplasma. I— III. Untersuch. Bot. Lab. Gottingen 2 : 1-202. i88r. Idem, Studien uber das Protoplasma. 2te Folge. Ibid. 3: 1-76. 1883. 2 Curtius, Theodor, and Reinke, J., Die fluchtige, reducirende Substanz der griinen Pflanzentheile. Ber. Deutsch. Bot. Ges. 15: 201—210. 1897. 3 Curtius, Theodor, and Franzen, Hartwig, Aldehyde aus grunen Pflanzenteilen. I. Mitteilung. Ueber a-0-Hexylenaldehyd. Sitzungsber. (math.-naturw. Kl.) Heidelberg. Akad. Wiss. Jahrgang 1910, Abhandl. 20. 13 p. IQIO. « Pollacci, Gino, Intorno all' assimilazione clorofilliana delle plante. Atti 1st. Bot. Univ. Pavia //, 7: 1-21. 1902. On the synthesis of carbohydrates in chloroplasts see: Fischer, Emil, Synthesen in der Zuckergruppe. II. Ber. Deutsch. Chem. Ges. 27 111 : 3189-3232. 1894. P« 3 2 3°- 5 Grafe, Viktor, Untersuchungen uber das Verhalten gruner Pflanzen zu gasformigen Formaldehyd. II. Ber. Deutsch. Bot. Ges. 29: 10-26. 191 1. Idem, Die biochemische Seite der Kohlensaure-Assimila- tion durch die grune Pflanze. Biochem. Zeitsch. 32: 114-129. 1911. [Baker, Sarah M., Quantitative experiments on the effect of formaldehyde upon living plants. Ann. bot. 27: 411-442. 1913.] 6 Lob, Walther, Zur Kenntnis der Assimilation der Kohlensaure. Landw. Jahrb. 35 : 541-378. 1906. * On reactions for identifying formaldehyde in plant parts, see Haas and Hill, 1921. [See note 3, p. 6.] — Ed. ASSIMILATION OF CARBON 3 1 The formation of formaldehyde was limited by the last three (secondary) reactions; hydrogen combined more easily with oxygen, to form hydrogen peroxide, than with carbon monoxide. To obtain formaldehyde in greater quantity Lob added a reducing agent (salicylic aldehyde, pyrogallol or chlorophyll. Glycolic aldehyde (which represents the simplest sugar), as well as formic acid and formaldehyde, arises from the action of the silent discharge upon carbon monoxide, water, and hydrogen; 2(H 2 + CO) = CH 2 OH — CHO (glycolic aldehyde). By the concentration of its solution in vacuo this substance is readily transformed into a tetrose or hexose. 1 Stoklasa and Zdobnicky 2 found that formaldehyde was formed by the action of ultra-violet light upon water vapor and carbon dioxide in the presence of potassium hydroxide, but no carbohydrates were thus produced. Sugar was formed, however, under these same conditions, when hydrogen was present in the nascent state.' Sorbose is formed by the action of light upon a mixture of formaldehyde and oxalic acid. 3 Bonnier and Mangin, as has already been mentioned (see page 4), have shown that if the interchange of gases accompanying the process of photosyn- C0 2 . , , thesis is determined independently of respiration, the ratio -q- is found to be somewhat less than unity. From this we must suppose that substances other than carbohydrates and less easily oxidized than these, are formed in the leaves under the influence of sunlight. The supposition that proteins also arise in the process of photosynthesis has been frequently advanced. This is supported by the quantitative researches of Sapozhnikov, 4 in which he established the fact that an increase in protein occurs parallel with the accumulation of carbo- hydrates in light. Posternak 5 is of the opinion that oxymethyl-phosphoric acid is also formed in leaves in the presence of light. 'Bach, A., Sur 1'evolution biochimique du carbone. Arch. sci. phys. et nat. 5: 401-415. 520-535 1898. This deals with the theory of photosynthesis. 2 Stoklasa, J., and Zdobnicky, W., Photochemische Synthese der Kohlenhydrate aus Kohlensaurean- hydrid und Wasserstoff in Abwesenheit von Chlorophyll. Biochem. Zeitsch. 30: 433-456. 1011. 'Inghilleri, Giuseppe, Photochemische Synthese der Kohlenhydrate. I. Mitteilung. Bildung von Sorbose. Zeitsch. physiol. Chem. 71 : 105-109. 1911. * Saposchnikoff, W., Bildung und Wanderung der Kohlenhydrate in den Laubblattem. Ber. Deutsch. Bot. Ges. 8: 233-242. 1890. Idem, Beitrag zur Kenntniss der Grenzen der Anhaufung von Kohlenhy- draten in den Blattern. Ibid. 11: 391-393. 1893- Idem, Eiweissstoffe und Kohlenhydrate der grunen Blatter als Assimilations-producte. 61 p. Tomsk, 1894- [Russian.] [Rev. by Rothert in: Bot. Centralbl. 63: 246-251. 1895. s Posternak, S., Contribution a l'6tude chimique de l'assimilation chlorophyllienne. Sur le premier produit d organization de l'acide phosphorique dans les plantes a chlorophylle avec quelques remarques sur le r&le physiologique de l'inosite. Rev. gin. bot. 12: 5-24. 65-73- 1900. ( Further, on the artificial formation of formaldehyde, etc., from carbon dioxide and water, see: Berthelot, D., and Gaudichon, H., Synthese photochimique des hydrates de carbone aux depens des elements de l'anhydride carbonique et de la vapeur de l'eau, en l'absence de chloro- phylle; synthese photochimique des composes quartenaires. Compt. rend. Paris 150: 1690- 1693. 1910. For a review of this general subject, see: Spoehr, H. A., Theories of photosyn- thesis. Plant world 19: 1-16. 1916. It should be remembered that the reactions that take place in leaves may not be the same as those studied in vitro. Very little experimental work has been done on the photochemical changes to which chlorophyll itself is subject. — Ed. $2 PHYSIOLOGY OF NUTRITION According to Krasheninnikov 1 a definite relation holds between the amount of carbon dioxide decomposed and the concomitant increase in dry weight, as is evident from the following average values: for a square meter of leaf surface the amount of carbon dioxide decomposed was 2286 cc. or 4.48 g., while the corre- sponding increase in dry weight was 2.94 g. The increase in dry weight for each weight unit of carbon dioxide decomposed was found to have the values given below, for the different plant forms considered. Bamboo o . 60 Cherry-laurel o . 60 Sugar cane 0.67 Linden 0.74 Tobacco 0.68 It is seen that this ratio appears to be fairly constant. The formation of a carbohydrate with the composition C12H22O11 (like cane sugar) would give this ratio a value of 0.64. Investigations upon the first products of photosynthesis agree with plant analyses in showing that an assimilation of water occurs simultaneously with that of carbon dioxide. In every green plant the formation of organic substance in sunlight is accompanied by assimilation of carbon, hydrogen and oxygen. The bulk of the dry weight of the plant is due to these three elements; this dry weight is made up of about 45 per cent, carbon, 42 per cent, oxygen, 6.5 per cent, hydrogen, 1.5 per cent, nitrogen, and 5 per cent, mineral constituents. Thus plants obtain more than 90 per cent, of their dry weight from the carbon dioxide of the air and the water of the soil. §7. Assimilation of Solar Radiant Energy by Green Plants. — We have already seen that green plants are able, with absorption of sunlight, to build up combustible organic compounds out of non-combustible inorganic substances. The chloroplasts of green plants furnish conditions for this process. Animal heat and movement, the heat of fuels, the work of steam engines, are all due to the freeing of the radiant energy of the sun which was previously fixed by the chloroplasts. Julius Robert Mayer stated very clearly the role of green plants when he said: Nature has set for herself the task of seizing the sunlight in its flight, as it streams upon the earth, and of accumulating the most swiftly moving of all forms of energy by transforming it into a potential state. To accomplish this purpose she has covered the surface of the earth with living organisms that absorb sunlight into themselves and thus generate a permanent store of potential chemical energy. These organisms are plants, and the plant world forms a reservoir in which the fleeting rays of light are caught and cleverly hoarded for future use. 2 The following interesting anecdote is taken from the biography of the engi- neer Stephenson, and shows that he also was well acquainted with this role played by plants. On Sunday as people were returning from church, with Stephenson and Buckland among 1 Krascheninnikoff, Th., Ansammlung der Sonnenengergie in den Pflanzen. Moskow, 1901. [Russian. ]• 2 Mayer, Julius Robert, Die Mechanik der Warme. P. 34. Leipzig, 1911. (Ostwald's Klassiker no. 180.) ASSIMILATION OF CARBOX 33 them, the whole company stopped upon the terrace beside Drayton Castle to watch a railway train as it vanished rapidly in the distance, with a trail of white smoke behind it. "Well, Buckland," said Stephenson as he turned to the famous geologist, "Answer me a question, not a very easy one, perhaps. Can you tell me what sort of force it is that drives yonder train along?'' "Well," answered the geologist, "I should think that the force was one of your great engines." "Yes but what moves the engine?" 'Why, one of your Newcastle engineers, of course." "No, sunlight." "How can that be?" asked the doctor. "I assure you it is nothing else," replied the engineer. "It is light that has lain stored in the earth for many thousands of years; the light absorbed by the plant during its growth is essential for the condensation of carbon, and this light, which has been buried in the coal measures for so many years, is now unearthed and, being freed again as in this locomotive, serves great human ends." 1 Along with the accumulation of starch there occurs also a storage of poten- tial energy in the plant. Krasheninnikov 2 was able to demonstrate this rela- tion by direct experiment. Half-leaves were removed from the plant and their areas were measured, after which they were dried and burned, to determine the heat of combustion of their dry substance. The remaining half-leaves, also removed from the plant but still alive, were exposed to light for a time, and the amount of carbon dioxide which they decomposed was measured. They were then dried and their heat of combustion was also determined. Below are given the average values of all the determinations, calculated for an area of 1 sq. m. of leaf surface exposed to the light. Increase in dry weight 3 . 51 g. Increase in carbohydrates 2 .46 g. Increase in carbon 1 . 58 g. Increase in heat of combustion i5>35° g.-cal. Amount of carbon dioxide decomposed 5 . 626 g. From the data of this experiment Krasheninnikov calculated that there was an increase of from 2.2 to. 3.6. g.-cal. for each gram of carbon dioxide decom- posed." It is also desirable to know what proportion of the radiant energy falling upon the leaf is assimilated. The first calculation bearing upon this question was made by Becquerel, 3 with the following results, which represent the yearly amounts of assimilation for three different types of vegetation, per hectare (2.5 acres). 1 Mayer, Adolf Eduard, Lehrbuch der Agrikulturchemie. 5 Aufl. Heidelberg, 1001-1902. P. 35. ; Krascheninnikoff, 1001. [See note 1, p. 32.] 3 Becquerel, Alexandre E., La lumiere, ses causes et ses effects. Paris, 1 867-1 868. u On alterations in the areas of leaves when the latter are transferred from shade to sun- light, which may possibly have some influence on the magnitudes of such values as these, see: Thoday, D., Experimental researches on vegetable assimilation and respiration. V. A critical examination of Sachs' method for using increase of dry weight as a measure of carbon dioxide assimilation in leaves. Proc. Roy. Soc. London B82: 1-55. 1909. — Ed. 3 34 PHYSIOLOGY OF NUTRITION Kilograms of Carbon Assimilated Kind of Vogetatbon per Hectare Forest in Central Europe 1800 Well fertilized meadow 3500 Helianthus tuberosus (Jerusalem artichoke) 6coo From a series of calculations, Becquerel came to the conclusion that, in France, plants assimilate less than 1 per cent, of the radiant energy that reaches them. Timiriazev arrived at the same result, and Brown's 1 more recent determinations give a still smaller value. In the latter case a Helianthus leaf received on a sunny day 600,000 g.-cal. per square meter of leaf surface per hour. In the same time an equal surface of leaf produced 0.8 g. of carbohydrates, for the formation of which 3200 g.-cal. were necessary. Thus the leaf accu- mulated, by the photosynthetic process, barely 0.5 per cent, of the solar energy reaching it; viewed as a machine designed to produce organic compounds, its efficiency is thus seen to be far from high/ An excess of light has a retarding effect upon increase in dry weight. It appears that different rays of the spectrum are effective in different stages of the photosynthetic process. 2 The importance of light to plants is not confined to the photosynthesis of carbohydrate from carbon dioxide and water; light is necessary for very many kinds of chemical reactions taking place in plants. Among the investigations that already testify to this are those upon the influence of light in protein formation. Numerous other reactions that are influenced by light and that are purely chemical in nature furnish additional evidence upon this point. Ciamician and Silber 3 were able to establish the fact that very many oxidations, reductions, hydrolyses, polymerizations and condensations are effected by light; such changes may progress very rapidly when an inorganic substance is involved. 4 §8. Influence of External and Internal Conditions upon Photosynthesis — One of the most important of the external conditions upon which various physiological processes depend is the temperature of the surroundings. The influence of temperature upon the velocity of the greening process has been shown above. Photosynthesis, on the other hand, is only very slightly affected 1 Brown, H. T., Recherches sur la fixation du carbone par les feuilles et sur la diffusion de l'acide carbonique. Traduit librement de l'Anglais par M. E. Demoussy. Ann. agron. 27: 428-438. 1901. [The original paper is: Brown, Horace T., Opening address by the President of Section B (Chemistry), Brit. Assoc. Adv. Sci., Nature 60: 474-483. 1899. (See also correction: ibid. 60: 544. 1899.) Also published in: Rept. Brit. Assoc. Adv. Sci. 1899: 664-683. 1900. See also: Brown, H. T., and Escombe F., Static diffusion of gases and liquids in relation to the assimilation of carbon and translocation in plants. Phil', trans. Roy. Soc. London B193: 223-292. 1900. 1 2 Liubimenko, V. N., La quantite de pigment vert dans le grain de chlorophylle et l'energie de la photo- synthese. [Abstract in French, p. 263-266; text in Russian.] Trav. Soc. Imp. Nat. St.-Petersbourg Ser. ///, Sect. Bot. 41: 1—266. 1910. s Ciamician, G., Sur les actions chimiques de la lumiere. Bull. Soc. chim. France 4 (fasc. is): i-xxvii. 1908. [A special appendix to this fasc, bound at end of vol., separately paged. 1 [See also note 1, p. 180.] * Neuberg, Carl, Chemische Umwandlungen durch Strahlenarten. I. Mitteilung. Katalytische Reak- tionen des Sonnenlichtes. Biochem. Zeitsch. 13: 305-320. 1908. Idem, Ueber die Reaktion der Gallen- sauren mit Rhamnose bzw. <5-Methyl-furfurol. Ibid. 14: 349-350. 1908. Idem, Bemerkung iiber die "Glucothionsauren." Ibid. 16: 25c— 253. 1909. Idem, Notiz iiber Phytin. Ibid. 16: 406-410. 1909. " In such calculations as this it is to be noted that the plant does not absorb nearly all the energy reaching it and that all the organic material formed does not appear in the final deter- minations. — Ed. ASSIMILATION OF CARBON 35 by temperature. According to the investigations of Kreusler, 1 the decomposi- tion of carbon dioxide begins at temperatures almost as low as the freezing point and continues up to 5o°C. His data are presented below. Tempera- Amount of Tempera- Amount of Tempera- Amount of tur e CO2 De- ture C0 2 De- ture CO2 De- Deg. C. composed Deg. C. composed Deg. C. composed 2.3 1.0 20.6 2.6 37.3 2.3 7.5 1.7 25.0 2.9 41.7 2.0 11. 3 2.4 29.3 2.4 46.6 1.3 15.8 2.8 33.0 2.4 If the amount of carbon dioxide decomposed in a unit of time at 2.3 be repre- sented by unity it is seen that this rate is not yet equal to 3 at 25 . Such a rise of temperature increases the rate of respiration to many times its original value."' Great fluctuations in atmospheric pressure exert a marked influence upon photosynthesis. 2 The process of photosynthesis is dependent upon the amount of chlorophyll present in the leaves. 3 The anatomical structure of these organs is also of importance, the stomata playing a particularly pronounced role. Mangin 4 1 Kreusler, U., Beobachtungen uber die Kohlensaure-Aufnahme und -Ausgabe (Assimilation und Athmung) der Pflanzen. II. Mittheilung: Abhangigkeit vom Entwicklungszustand — Einfluss der Tem- peratur. Landw. Jahrb. 16 : 711-755. 1887. [Idem, same title. III. Mittheilung: Einfluss der Tempera- tur; untere Grenze der Wirkung. Ibid. 17: 161-175. 1888. Idem, Beobachtungen uber Assimilation und Athmung der Pflanzen. IV. Mittheilung: Verhalten bei hoheren Temperaturen; Kohlensaure-ausschei- dung seitens getodterer Exemplare; Kohlensaure Verbrauch, wenn Ober- und Unterseite der Blatter dem Licht Zugewendet. Ibid. 19: 649-668. 1890.] 2 Friedel, Jean, L'assimilation chlorophyllienne aux pressions inferieures a la pression atmospherique. Rev. gen. bot. 14: 337-355, 369-390. 1902. s Liubimenko, 1910. [See note 2, p. 34.] 4 Mangin, L., Sur le rdle des stomates dans l'entree ou la sortie des gaz. Compt. rend. Paris 105 : 879-881. 1887. w But Gabrielle Matthaei's very careful studies (Matthaei, Gabrielle L. C, Experimental researches on vegetable assimilation and respiration. III. On the effect of temperature on carbon dioxid assimilation. Phil, trans. Roy. Soc. London B197: 47-105. 1905) show- that the influence of temperature upon photosynthesis in leaves of Primus laurocerasus (cherry-laurel) is much more pronounced than is indicated by Kreusler's numbers. Her re- sults are shown below, the amounts representing hourly rates per 50 sq. cm. of leaf. Temperature, deg. C, -6 8.8 11.4 15 23^7 30.5 37.5 4Q-5 43-Q CO-j assimilated, g. 0.0002 0.0038 0.0048 0.00700.0102 0.0157 0.0238 0.0149 0.0102 From these data it appears that the process in question about doubles for each increase in temperature of io°C, thus agreeing with a large number of chemical reactions. (Van't Hoff, J. H., Lectures on theoretical and physical chemistry, translated by R. A. Lehfeldt. London, no date — author's preface dated 1898. Part I, p. 227 et seq.) See also: Blackman, F. F., and Matthaei, G. L. C, Experimental researches on vegetable assimilation and respira- tion. IV. A quantitative study of carbon-dioxide assimilation and leaf temperature in natural illumination. Proc. Roy. Soc. London B76. 402-460. 1905. Blackman, F. F., Optima and limiting factors. Ann. bot. 19: 281-295. *9°5- Idem, The metabolism of the plant considered as a catalytic reaction. Presidential Address, Bot. Sect. British Assoc, Dublin meeting, 1908. Also published in: Science, n.s. 28: 628-636. 1908. Two criticial reviews of published data on photosynthesis may also be mentioned here; the first (Brown, W. H., and Heise, G. W., The application of photochemical temperature coefficients to the velocity of carbon dioxide assimilation. Philippine Jour. Sci. 12, C (botany): 1-25. 1917.) interprets the data as indicating that temperature has little effect on the rate of the process, while the second (Smith, A. M., The temperature coefficient of photosynthesis : a reply to criticism. Ann. bot. 33: 517-536. 1919.) corroborates the interpretation that temperature has a pronounced effect on the rate. — Ed. 36 PHYSIOLOGY OF NUTRITION was able to show that when the stomatal pores are artificially plugged exchange of gases is retarded. A privet leaf (Ligustrum vulgaris), the upper surface of which was coated with petrolatum, decomposed 6.26 g. of carbon dioxide, but only 1.92 g. was decomposed by a similar leaf coated on the under surface. [Privet leaves have stomata only below, so that coating the upper surface did not close the pores.] Stahl 1 arrived at the same result. Parts of the lower surfaces of leaves that had been rendered free from starch were covered with a mixture of one part of beeswax and three parts of cocoa butter, and the leaves were then exposed to light; after being bleached with alcohol and then treated with iodine, the part that had been covered was brown, while the remainder of the leaf was dark blue (Fig. 18). Blackman's 2 results point to the same conclusion. The size of the stomatal openings is also important. 3 An adequate supply of water in the leaves is essential to the normal progress of photosynthesis; according to Sachs and Nagamatsz 4 no starch is formed by wilting leaves, a fact which Stahl believed to be due to the stomatal closure that accompanies wilting. This interpre- tation is supported by the observation that leaves in which the stomata remain open even in the wilted condition (Rumex aquaticus, Caltha palustris, Hydrangea hortensis, Calla palustris) still continue to accumulate starch after wilting has occurred. Finally, an excess of salts in the soil has a retarding effect upon the rate of carbon dioxide decomposition. Schimper found that watering with sodium chloride solu- tion caused development to cease in most plants (non- halophytes), through a checking of photosynthesis. Ac- F 1 g . 18 .—Privet cording to Stahl this, also, is due to stomatal closure, leaf, the unshaded P or- caused Dy excess f sa i ts . if the leaves are slightly tion ofwhichwas J ..... covered with cocoa wounded so as to facilitate entrance of carbon dioxide into butter during exposure t k e t i ssue starch accumulates about the wound margins. to light. This portion ' . " . shows no starch reac- True halophytes grow, though slowly, upon soils rich in tion with iodine. sa j tSj since ^^ stomata do not close at all. §9. Nutrition of Green Plants by Organic Compounds. — Green plants can also use as food organic compounds that are supplied from without. 5 This form of nutrition may go on simultaneously with the assimilation of carbon 1 Stahl, Ernst., Einige Versuche uber Transpiration und Assimilation. Bot. Zeitg. 52 : 11 7-146. 1894. - Blackman, F. Frost, Experimental researches on vegetable assimilation and respiration. — No. I. On a new method for investigating the carbonic acid exchanges of plants. Phil, trans. Roy. Soc. London Bi86 7 : 485-502. 1895. Idem, same title, No. 11. On the paths of gaseous exchange between aerial leaves and the atmosphere. Ibid. B: 186 7 : 503-562. 1895. See Sect. IV. 3 Kolkunov, V., Ueber die Abhangigkeit der Assimilation von der Grosse der Spaltoffnungen bei den Gramineen. [Abstract in German, pp. 381-382; text in Russian.] Jour. exp. Landw. 8: 369-382. 1907. * Nagamatsz, Atsusuke, Beitrage zur Kenntnis der Chlorophyllfunktion. Arbeit. Bot. Inst. Wurzburg 3: 389-407. 1888. 5 Apparently carbon monoxide cannot be assimilated; see : Krascheninnikoff, Th., La plante verte assimile- t-elle l'oxyde de carbone? Rev. gen. bot. 21: 177-193. 1909- ASSIMILATION OF CARBON 37 dioxide from the air, which is especially true in the case of insectivorous plants. 1 These latter are green and can assimilate carbon dioxide, but, at the same time, they are provided with a characteristic mechanism for catching and digesting insects (Fig. 19). In this class, for instance, belongs the widely distributed sundew {Drosera rotundi folia), which grows in bogs. Its leaves are covered with pin-shaped tentacles or glands, which secrete a sticky fluid. As an insect alights upon the leaf, the tentacles bend toward it, a copious flow of an acid liquid Fig. 19. — Above, a leaf of Drosera rotundifolia, whose tentacles on the left side have responded to a stimulus, and one of Nepenthes gracilis. Below, a leaf of Dionaea muscipula; A, open; B, closed, with an imprisoned earwig. (After Pfeffer.) containing a pepsin-like enzyme takes place, and the insect is digested. Sundew- can also digest and absorb lean meat and white of egg. In Nepenthes' 2 a part of the petiole is modified into a tankard-shaped structure with the leaf-blade acting as a cover. The hollow portion contains a weakly acid solution, in which imprisoned insects are digested. Each leaf of Dionaea muscipula con- sists of a flattened petiole and a round leaf-blade divided by the midrib into halves, like the halves of an open mussel, separated by an angle of from 60 to 1 Darwin, Charles R., Insectivorous Plants. London, 1875. - Clautriau, G., La digestion dans les urnes de Nepenthes. Recueil Inst. Bot. Bruxelles 5: 89-133. 1902. Vines, S. H., The proteolytic enzyme of Nepenthes (III). Ann. bot. 15 : 563-573- 1901. 38 PHYSIOLOGY OF NUTRITION 90 degrees. The free margin of each lobe is extended into sharp, slender teeth, and each lobe bears on its upper surface near the center three very elastic bristles. When an insect alights upon the leaf and touches a bristle, the valves quickly close together and a digestive fluid is secreted into the space between them. If the ability to derive nutrition from complex organic compounds, inde- pendently of photosynthesis, is a special characteristic of the insectivores, nevertheless other plants that utilize the carbon dioxide of the air can also assimilate complex organic substances. Green water-plants thrive especially well in harbors where the water is very rich in organic compounds, in the neighborhood of canals and sewer outlets; for example, the algae, Ulva lactuca, some species of the genera Bangia and Ceramium, and Cystoseira barbata. Also, some single-celled green algae are known to grow excellently and retain their green color in pure culture in darkness, with organic substances supplied. Finally, it was proved by Bohm and other observers 1 that even green leaves that have been previously deprived of starch are able to assimilate various organic substances from solution and thus to form starch in darkness. In this manner starch can be formed from saccharose, glucose, fructose, lactose, glycerine, dextrine, mannite, melampyrite, and adonite. 2 Sapozhnikov' 5 investigated this matter quantitatively. Leaves of Astrapcea wallichii, previously rendered starch-free, formed in seven days from 4.6 to 5.3 g. of starch, per square meter of leaf surface, when floating upon a 20-per cent, solu- tion of cane sugar in darkness. Here assimilation is not limited for forma- tion of starch, however; the amount of proteins also increases when leaves are grown upon cane-sugar solution in darkness, and respiration is accelerated. The ability to absorb organic compounds is even more pronounced in roots than in leaves. Many green plants possess mycorhiza (see Chapter IV) and grow on humus soils, and these probably assimilate organic materials. Light influences the absorption of organic compounds by green plants. 4 According to the experiments of Reinhardt and Sushkov 5 the accumulation of starch in leaves floating upon cane-sugar solution depends upon a variety of conditions. This process occurs rapidly only at medium temperatures, while starch that was previously present disappears at higher or lower temperatures, in spite of the supply of sugar. Among poisons, some (quinin) hasten the first appearance of starch but prevent its continued accumulation; others (0.5 per cent, of caffein) favor the accumulation of starch. 1 [Boehm, Josef. Ueber Starkebildung aus Zucker. Bot. Zeitg. 41 : 33-38, 49~54- 1883. P. 35- Idem, Starkebildung in den Blattern von Sedum spectabile Boreau. Bot. Centralbl. 37 : 193-201, 225-232. 1889. P. 200.] Nadson, G., The formation of starch from organic substances by chlorophyll-bearing plant cells [Russian]. Trav. Soc. Imp. Nat. St-P6tersbourg 20: (Sect, bot.): 73-122. 1889. 2 Treboux, O., Starkebildung aus Adonit im Blatte von Adonis vernalis. Ber. Deutsch. Bot. Ges. 27: 428-430. 1909. 3 Saposchnikoff, W., Ueber die Grenzen der Anhaufung der Kohlenhydrate in den Blattern der Weinrebe und anderer Pflanzen. (Vorlaufige Mittheilung.) Ber. Deutsch. Bot. Ges. 9: 293-300. 1891. P. 298. Idem, 1890, 1893. [See note 4, p. 31.] 1 Lubimenko, W., Influence de la lumiere sur l'assimilation des matieres organiques par les. plantes vertes. Bull. Acad. Imp. Sci. St.-Petersbourg VI, 1: 395-426. 1907. 6 Reinhard, [L. V.| and Suschkoff, Beitrage zur Starkebildung in der Pflanze. Beih. Bot. Centralbl. 18: 133-146. 1904-1905- ASSIMILATION OF CARBON 39 Experiments in which green plants were supplied with organic nitrogenous compounds, in a chamber free from carbon dioxide, gave negative results. 1 Summary i. Importance of Carbon Assimilation by Green Plants.— Green plants form organic compounds from inorganic ones. Non-green plants and animals are unable to do this and are therefore all ultimately dependent on green plants for organic sub- stances. . The study of plant physiology may begin by inquiring about photosynthesis of carbohydrates by the green parts of plants. These organic compounds are formed from carbon dioxide and water, by means of solar energy that is absorbed and trans- formed in the green tissues. Carbon dioxide is of course a carbon compound, but it is not combustible and is usually classed as inorganic. Combustible carbon compounds derived from organisms are capable of being burned in air because they are incom- pletely oxidized; when completing their oxidation these compounds absorb oxygen and produce carbon dioxide and water, and this process of combustion liberates energy (heat or light or both). A certain amount of sunlight energy is absorbed, and a corresponding amount of oxygen is eliminated, when carbon dioxide and water are combined by green plants, with the formation of carbon compounds. 2. Exchange of Gases. — Photosynthesis is accompanied by taking in of carbon dioxide and giving out of oxygen, as well as by absorption of solar energy, and the ratio of the amount of absorbed carbon dioxide to the amount of oxygen eliminated in the same period has been found to have a value somewhat less than unity. The process results in decomposition of carbon dioxide and water, and in the union of the carbon, the hydrogen, and some of the oxygen, to form carbohydrates; the rest of the oxygen is given off. 3. Chlorophyll. — The two green pigments that make it possible for carbohydrate photosynthesis to occur in green plant tissues when light is properly supplied are called chlorophyll, or, more correctly, the chlorophylls. Photosynthesis of carbo- hydrates from carbon dioxide and water does not occur in tissues that do not contain these pigments. The green pigments are named chlorophyll a and chlorophyll b. They occur in green leaves in about the proportions 72 to 28, by weight. Dissolved in ethyl alcohol, the first appears blue-green, the second yellow-green, by transmitted light. Both are fluorescent, the first appearing blood-red, the second brown-red, by reflected light. The two are alike in that each molecule contains 55 atoms of C, 4 atoms of N, and a single atom of Mg. The molecule of chlorophyll a contains 72 atoms of H and 5 atoms of O, while that of chlorophyll b contains 70 atoms of H and 6 atoms of O. Iron is necessary for the formation of the chlorophylls in plants, but does not occur in the pigments themselves. The chlorophylls absorb light more or less completely according to the wave- lengths of the light that is supplied. Light of wave-lengths from about 640 to about 680 ft (red) is most completely absorbed. With wave-lengths shorter than about 475 fj. (blue to ultra-violet) absorption is almost as complete. The spectrum of chlorophyll solution shows, between these two, several other ranges of wave-lengths, with less complete absorption, and very strong solutions show complete absorption throughout the entire range of visible light.— The chlorophylls are chemically some- « Grafe, Victor, Untersuchungen iiber die Aufnahme yon Stickstoffhaltigen organischen Substanzen durch die Wurzel von Phanerogamen bei Ausschluss der Kohlensaure. Sitzungsber. (math.-naturw. Kl.) K. Akad. Wiss. Wien 118': II35-"S3. 1000. 40 PHYSIOLOGY OF NUTRITION what related to hemoglobin (which occurs in red blood-corpuscles of animals) ; they give several of the same decomposition products. For the formation of chlorophyll in leaves, etc., the following conditions are essen- tial: (i) light (within the limits of the visible spectrum and with different intensities for different kinds of plants); (2) temperature (from about o°C. to about 45°C. as general limits; the range is usually narrower, differing for different kinds of plants); (3) iron (but the supply must be very small or poisoning results); (4) oxygen; (5) salts derived from the soil (containing K, Ca, Mg, N, P, S) ; (6) water-soluble carbohydrates. 4. Pigments Accompanying Chlorophyll. — Several other pigments accompany the chlorophylls, especially carotin and xanthophyll, which are generally present in cells with the green pigments, but often occur in the absence of the latter. Carotin is a hydrocarbon, with the formula C 40 H 56 . It forms crystals that appear blue-green by reflected light and orange-red by transmitted light. It is insoluble in water, readily soluble in ether, carbon bisulphide, etc., and is readily oxidized. In leaves it varies in amount, according to the light intensity, temperature, etc. It occurs in all parts of plants. — Xanthophyll resembles carotin but contains some oxygen; it has the formula 4oCH 56 09. 5. Influence of Light in Carbohydrate Photosynthesis. — Light impinging on leaves is partly reflected, partly absorbed, and partly transmitted. Only that which is absorbed can influence chemical processes within the leaves. The absorbed portion may have various qualities (according to the proportions of the different ranges of wave-length that are present) and various total intensities. The range of wave- lengths approximately corresponding to our visual range of red and orange appears usually to be most effective in furnishing energy for photosynthesis, but the rest of the visible range of wave-lengths is not without effect. The proportional distribution of total light energy among the several ranges of wave length varies greatly in nature. When the relations between light quality and carbohydrate photosynthesis are to be dealt with, it is necessary to consider the energy-supplying power of any wave-length range of absorbed light. It has been suggested that the rate of the process may be proportional to the energy value of the absorbed light, other conditions being adequate and constant throughout the series of comparisons. The absorbing power of chloro- phyll-bearing tissue, for the different ranges of wave-lengths, is greatly influenced by the amount of chlorophyll present and by the presence of pigments other than chloro- phyll — also by the cell structures of the tissues. — Considering simply the total intensity of sunlight, carbohydrate photosynthesis proceeds with intensities between a minimum and a maximum, with an optimum intensity somewhere within the range. Shade- plants (as beech) have a low range of intensities for the process, while sun-plants (as pine) have a high range. Cave mosses thrive with very weak illumination. 6. Products of Carbohydrate Photosynthesis. — If a living green plant that forms starch be kept in darkness till all starch has disappeared from the chlorophyll-bearing cells, and if it be then exposed to suitable light, starch grains soon appear in the cells. But starch is not the first product of the photosynthetic process, for starch is formed from a water-soluble sugar (such as dextrose), not directly from carbon dioxide and water. There are plants that do not form starch, and these show an increased amount of sugar when they are brought into light after a prolonged period in darkness. A supply of carbon dioxide is of course necessary, in the surrounding air, and the form- ation of sugar or starch proceeds parallel to the absorption of carbon dioxide by the plant in this kind of a test. Of course the active cells are plentifully supplied with water, which is the other necessary material. Besides sugar, a prominent product of ASSIMILATION OF CARBON 4 1 this process is oxygen, most of which escapes from the green tissues into the. surroundings. 6a. Chemistry of Carbohydrate Photosynthesis. — Baeyer's hypothesis supposes that carbon dioxide and water are decomposed, that some free oxygen is produced, and that the remaining carbon, hydrogen, and oxygen are combined to form formalde- hyde (CH2O), the latter being polymerized, with the formation of dextrose (C 6 Hi 2 6 ). The hypothesis is represented by the equations: (1) CO2 + H 2 = CH 2 + 2 and (2) 6CH2O = C 6 Hi 2 6 . Traces of formladehyde have been found in green tissues, and green plants in light have been experimentally shown to be able to increase their carbohydrate content when supplied with this substance as the only source of carbon. But formaldehyde is a violent poison and can never accumulate considerably in living tissues. It is supposed that this substance generally polymerizes as rapidly as it is formed. If the hypothesis is true, light appears necessary for the polymeriza- tion of formaldehyde, as well as for its formation and for the antecedent decomposition of carbon dioxide and water. Many other hypotheses have been suggested, and the chemistry of this photosynthetic process is still to be worked out. — More than 90 per cent, of the dry weight of the plant is derived from the carbon dioxide and water used in the process here considered; the rest is derived from mineral salts absorbed from the soil solution. 7. Assimilation of Solar Radiant Energy by Green Plants. — The formation of carbohydrates in green plants necessarily results in the storage of potential energy, in an amount equivalent to the energy that would be freed by the complete oxidation or burning of the carbohydrates formed. The fuel values of wood and coal are pro- portional to the potential energy stored in these substances and set free when they are burned. This energy is a part of that which was absorbed from sunlight when the plants from which these fuels have been derived were growing. The stored solar energy of coal has lain dormant for ages, that of wood generally for years. Cal- culations indicate that 2.2-3.6 gram-calories of energy is stored for each gram of carbon dioxide decomposed in photosynthesis. Experiments have shown, however, that plants accumulate, as potential energy in their carbon compounds, less than 0.5 per cent, of the radiant energy that reaches them as sunlight. 8. Influence of Conditions on Carbohydrate Photosynthesis. — Internal conditions influencing the rate of carbohydrate photosynthesis are: (a) the amount of chlorophyll present; (b) anatomical and histological structure, especially arrangement and size of stomata; (c) condition of stomata — whether open, closed, partly closed, etc.; (d) turgor condition— whether the leaf is wilted, etc. (this is perhaps covered by c) ; (e) the rate at which products of the process leave the leaf; (/) the ability of the leaf to absorb light (may be included under b) ; (g) leaf temperature. External conditions influencing the rate of this process are: (a) the rate of supply of carbon dioxide; (b) the quality — wave-lengths — of the light received; (c) the rate of light-energy absorption — intensity of each group of wave-lengths and time during which leaf is exposed to them; (d) the temperature of the surroundings — which mainly controls leaf temperature; (c) other external conditions whose influence is not yet so well understood. 9. Nutrition of Green Plants by Organic Compounds. — Some green plants (as, for example, the insectivorous forms, Drosera, Nepenthes, Dionaea, etc.) are able to absorb considerable amounts of ready-made carbohydrates, etc., from the surround- ings. Many other green plants have this ability to a smaller degree. Of course the non-chlorophyll-bearing parts of green plants, regularly, derive their carbohydrates from the tissues that bear chlorophyll. CHAPTER II ASSIMILATION OF CARBON AND OF ENERGY BY PLANTS WITHOUT CHLOROPHYLL §i. General Discussion. — Most plants that are without chlorophyll and are, in consequence, unable to assimilate the energy of sunlight, do not have the power to transform non-combustible inorganic substances into organic compounds. As will appear later, in order to form their various organic substances, green plants require (besides carbon dioxide from the air and water from the soil) nitrogen, potassium, calcium, magnesium, iron, sulphur and phosphorus, all of which occur in the form of various salts in the soil. From the preceding dis- cussion of chlorophyll (see Chapter I) it appears that no plant without chloro- phyll can utilize the energy of sunlight to manufacture combustible organic matter out of such substances. Most non-green plants must use, as sources of both energy and material, organic compounds that have already been formed; they are thus more nearly related to animals than to green plants, as far as their nutrition is concerned. But organic compounds are not the only substances that can be oxidized. This property belongs also to various inorganic substances, such as ammonia, hydrogen sulphide and hydrogen, which thus contain stored energy. As we have previously seen (page xxviii), the heat of combustion of ammonia is greater than that of starch. The researches of recent years have shown that such substances can serve as sources of nutrition for certain plants without chlorophyll. On the basis of their mode of nutrition, plants without chlorophyll may be divided into two groups: (i) plants that derive their energy from organic compounds, and (2) plants that derive it from inorganic substances. §2. Assimilation of Energy from Organic Compounds by Plants without Chlorophyll. — Most bacteria, yeasts, fungi and the non-green seed-plants obtain their nutrition from previously formed organic compounds. To study the nutri- tional requirements of these forms, culture media containing various nutritive sub- stances are employed. It was formerly thought that the same nutrient medium should be suitable for all the simpler non-green forms, but this is not so. In higher plants, specialization — i.e., adaptation to surrounding conditions— is accompanied by peculiarities of external form as well as of anatomical structure. On the other hand, the lower plants, such as bacteria and yeasts, are marked by their structural similarity and simplicity. It was supposed, therefore, that such similarity of structure was accompanied by a similarity in the characteristic life processes, and this, in turn, led to the supposition that the nutritive processes must be more or less uniform in these lower forms. The most recent investigations have shown, however, that, in spite of the simple structure of microorganisms 42 ASSIMILATION OF CARBON 43 (more properly, just because of this very simplicity) they usually exhibit far- reaching physiological peculiarities. Each one of these organisms carries out its own little work, but it constitutes a very important link in the processes of nature. For example, the presence of two kinds of bacteria appears to be requisite for the oxidation into nitric acid of the ammonia present in the soil. One of these (Nitrosomonas) carries the oxidation as far as nitrous acid, the other (Nitro- bacter) oxidizes this to nitric acid. Ammonia is essential as nutrient material for the first form and nitrous acid is a waste. But this by-product constitutes an essential food substance for the other form. Is it possible, then, to conceive of some nutrient medium that would be equally well suited for the nutrition of both these bacteria? This question must receive a negative answer; a medium must be used that is favorable only to the microorganism under investigation, and that is especially adapted to its particular requirements. The use of such media is highly important if pure cultures are desired. This use has been desig- nated by Vinogradskii as the method of "selective culture." A culture is selective if it promotes only a certain func- tion, or if it promotes a function which is as restricted as possible. The more closely limited or exclusive are the conditions, the more favorable will these conditions be for one species possessing a particular property or function, in contrast to others not so endowed, and the growth of these latter in a medium thus alien to them will be quite impossible or at least very difficult. In thus assisting the desired microorgan- isms in their struggle for existence, we in- crease their numbers in our cultures and Fig. 20.— Various forms of bacteria, thereby render their discovery easier. When a specific bacterium has once been found, it is thus usually possible to discover also the method by which it may be isolated in pure culture. On this general principle is based the now frequent employment of many different kinds of nutrient substrata, both liquid and solid. The first attempt to prepare an artificial nutrient medium for microorganisms, was made by Pasteur, 1 whose solution for the culture of yeast had the following composition: water, 100 g.; ammonium tartrate, 1 g.; saccharose, 10 g.; and yeast ash, 0.075 g. Meat extract is used most commonly for the culture of bacteria (Fig. 20). The addition of gelatine to peptone bouillon (10 per cent, of gelatine in winter and 15 per cent, in summer) produces a solid substratum. Agar-agar may be used instead of gelatine. Besides the various kinds of meat extracts, milk, blood serum, yeast water, beer-wort and other similar materials may be used. Among other things, cylinders cut from potato tubers may be employed as solid media. 1 Pasteur, Louis, Memoire sur la fermentation alcoolique. Ann. chim. et phys. ///, 58: 323-426. i860. 44 PHYSIOLOGY OF NUTRITION Beer-wort is the best nutrient medium for the culture of yeast. 1 Other liquids are used, however, among which may be mentioned Pasteur's solution as given above, grape juice, the juice of various other fruits and berries, and other materials containing sugar. Hansen has carried out very exhaustive studies upon yeasts and has established, among others, the following important species. 2 Saccharomyces cerevisice I. Hansen. An English top-fermentation yeast, which produces, in beer-wort at room temperature, from 4 to 6 per cent, of alcohol. In the resting condition the plant consists of single cells, which begin to multiply by budding when placed in beer- wort. The young generation con- sists of large spherical or oval cells (Fig. 21). After the temination of the primary fermentation a scum appears on the surface of the fermenting liquid and on this a continuous membrane of yeast-cells is formed. The general appearance of these cells is different from that of the sedimentary forms; much elongated cells are found here (Fig. 22). In the surface membrane of old cul- tures occur very much elongated cells that are entirely unlike the young sedi- ment cells from which they have developed (Fig. 23). This film formation Pig. 21. — Saccharomyces cerevisice I. Fig. 22. — Saccharomyces cerevisice I. Sur- Young cells from the sediment of the beer- face film at i5-i6°C. {After E. Hansen.) vat. {After E. Hansen.) furnishes a striking example of the great variability in form, that is characteristic of yeast cells. In order to obtain ascospores young cultures must be used, and it is also essential that air be plentifully supplied. Little plaster of Paris disks prepared with special moulds are used for this purpose. These are placed in small, shallow glass pans (Petri dishes), covered with similar pans of slightly greater diameter, and then sterilized. A few drops from a day-old culture of yeast cells are placed upon one of these plaster disks. Sterilized water is poured into a dish around the disk, to keep the latter constantly moist. x\fter some time the ascospores are formed. Temperature exerts a pronounced in- fluence upon their formation. With the same temperature, ascospores of different species develop at different rates, and this fact is made use of in indenti- 1 Jorgensen, Alfred P. C, Die Mikroorganismen der Garungsindustrie. 4te Aufl. Berlin, 1898. Idem, Microorganisms and fermentation. Philadelphia, 191 1. Lindner, Paul, Mikroskopische Betriebskontrolle in den Garungsgewerben. 2te Aufl., Berlin, 1898. (ste Aufl., Berlin, 1909.) [Hansen, Emil Chr., Prac- tical studies in fermentation. Transl. by Alex. K. Miller. 227 p. London and New York, 1896. — See also the references on brewing, etc., given on p. 181.] The Carlsberg Laboratory in Copenhagen is especially interested in the study of fermentation organisms. It publishes a journal devoted to this study, entitled " Meddeledser fra Carlsberg Laboratories" 2 More information upon top and bottom fermentation will be found in Chapter VIII of this Part. ASSIMILATION OF CARBnX 45 Fig. 23. — Saccharomyces cerevisice I. Film of an old culture. (After E. Hansen.) EM Fig. 24. — Saccharomyces pastorianus I. Fig. 25. — Saccharomyces pastorianus Ascospores. (After E. Hansen.) III. Young cells of the sediment. (After E. Hansen.) 46 PHYSIOLOGY OF NUTRITION fying the different yeasts, particularly in technical analysis for distinguishing wild from cultivated forms. Saccharomyces pastorianus I. Hansen (Fig. 24). This is a bottom-fermenta- tion yeast and consists mainly of elongated cells, but round and oval cells also occur. This yeast is frequently present in the air in breweries. It imparts to the beer a disagreeable, bitter taste and an unpleasant odor. Saccharomyces pastorianus III. Hansen. This top-fermentation yeast produces a turbid condition in beer (Fig. 25). Saccharomyces anomalus Hansen. This species is distinguished by its char- acteristic ascospores, which have the form of hemispheres, with projecting rims at their bases (Fig. 26). Besides the species mentioned here, which are among those thoroughly investigated by Hansen, a great many other yeasts, both wild and cultivated, are known. Some of the cultivated varieties are employed in the brew- ing industry, some in distilleries, some in the manufacture of berry or fruit wines, and still others in the preparation of compressed yeast for bakers' use. The moulds (Fig. 27) are not very exacting as to their nutrition, Pig. 26.- -Saccharomyces oospores. anomalus. As- A B Fig. 27. — A, Penicillium glaucum; B, Asper- gillus glaucus. A conidiophore, in each case. for they can grow upon a very great variety of materials. Among artificial liquid media for mould culture, Raulin's 1 solution is the best known; its formula follows: Water 1 500 . o g. Saccharose 70 . o g. Tartaric acid 4 • o g. Ammonium nitrate 4 ■ o g. Ammonium phosphate o.6g. Ammonium sulphate o . 25 g. Potassium silicate o . 07 g. Potassium carbonate o.6g. Magnesium carbonate o . 4 g. Zinc sulphate o . 07 g. Ferric sulphate o . 07 g. Fermentation phenomena often accompany the nutrition of the moulds and bacteria. There is still very little known concerning the nutrition of the higher fungi. 1 Raulin, Jules, Etudes chimiques sur la vegetation. Ann. sci. nat. Bot. V, 1 1 : 93-299. 1 869. ASSIMILATION OF CARBON 47 Almost the only definitely known fact concerning the nutrition of seed- plants without chlorophyll is that some are saprophytes and others parasites. The former utilize decomposition products from plants and animals, while the latter attach themselves to living plants and derive nourishment therefrom. » The widely distributed dodder (species of Cuscuta) is an example of a para- site. It is parasitic upon nettles, hops and many other plants (Fig. 28). Parasitism exhibits such a high state of development in some flowering plants without chlorophyll that they possess neither root nor stem, nor have they any leaves. The entire plant body here resembles a fungus in its struc- ture, consisting of branching filaments each composed of a row of cells, very similar to fungus hyphae. The Balanophorese, Hydnoreae and Rafflesiaceae, are examples of such plants. The hypha-like body of these plants develops within various trees and derives nourishment therefrom after the manner of Fig. 28. — Section of stem of Cuscuta europaa, attached, by means of its haustorium, to the stem of a nettle. E represents the epidermis of the nettle. many fungi. The flower buds and flowers of these non-green parasites appear upon the branches of the host only during the flowering season of the latter. It then appears, at first glance, as though the plant infested by the parasite were bearing two kinds of flowers. In reality, however, some of these are the true flowers of the host plant, while the others belong to the parasite. Fig. 29 shows a portion of an underground stem of a host plant, bearing its own flower buds and a mature flower of a parasite, Hydnora africana. §3. Assimilation of Energy from Inorganic Substances by Plants without Chlorophyll. — Some bacteria are so constituted as to be able to obtain their energy from oxidizable inorganic substances that are common on the earth. Of these the nitrifying bacteria, which oxidize ammonia into nitric acid, are the most important. The absence of organic substances is necessary for their successful growth. Vinogradskii succeeded in obtaining a pure culture of 48 PHYSIOLOGY OF NUTRITION nitrifying bacteria only, by preparing a nutrient solution containing no organic substances. This nutrient medium 1 contained i g. of ammonium sulphate and i g. of potassium phosphate, dissolved in a liter of water. From 0.5 to 1.0 g. of basic magnesium carbonate was added to each 100 cc. of this solution. Nitrifying bacteria were able to develop excellently in this medium; they oxidized ammonia to nitric acid and formed an appreciable quantity of organic substance, thus assimilating the carbon dioxide of the air without the agency of sunlight. Bacteria that need organic substances for their nutrition could not develop in such a medium. Fig. 29. — Hydnora africana. t, part of the underground stem of the host plant; bl, one of the mature flowers; bl', bl", flower buds of the parasite. (H natural size.) {After Sachs.) Without the agency of sunlight as source of energy, green plants are unable to produce organic substance from the inorganic materials that serve as nutrients for these forms. As has been said, there are other inorganic substances, however (such as ammonia and hydrogen sulphide) that can serve as sources of energy for such plants as the bacteria just mentioned. These substances are common in nature, being frequently of organic origin as decomposition products of complex organic compounds, and, although they do not contain carbon (which is present in all organic compounds), yet they do possess the power 1 [Winogradsky, S., Recherches stir les organismes de la nitrification. I. Ann. Inst. Pasteur 4 : 213-231 1890. Idem, same title, II. 76^.4:257-275. 1890. Idem, same title. III. Ibid. 4: 760-771. 1890. Idem, same title, IV. Ibid. 5 : 52-100. 1891. [Idem, same title, V. Ibid. 5: 577-616. 1891. See No. IV, especially.] ASSIMILATION OF CARBON 49 to burn readily; i.e., to liberate heat. On this account these oxidizable in- organic substances can supply energy for these bacteria. Thus, nitrifying bacteria utilize ammonia, and sulphur bacteria make use of hydrogen sulphide. To obtain a solid substratum for cultures where organic substances must be avoided, silicic acid 1 may be used instead of gelatine or agar-agar. Vinogradskii 2 also proved that bacteria living in sulphur springs, as Beggia- toa and some other species, use hydrogen sulphide as a source of energy. This is first oxidized only to sulphur and water; H 2 S + O = H 2 + S. The sul- phur thus formed accumulates within the cells, to be further oxidized, in the presence of carbonates (e.g., calcium carbonate), to form calcium sulphate and carbonic acid. The sulphur bacteria play a very important role in the economy of nature; without them the circulation of sulphur might be impossible. In order to obtain sulphur bacteria, freshly cut pieces of roots of Butomus umbellalus . with the mud clinging to them, are placed in a deep vessel, in from 3 to 5 1. of water; some calcium sulphate is added and the vessel is left uncovered at room temperature. After several days the formation of hydrogen sulphide is evident, consequent upon the decomposition of calcium sulphate by various bacteria contained in the mud. Some time after the appearance of hydrogen sulphide the development of sulphur bacteria begins. They usually collect at some distance from the free surface of the liquid and, as they move upwards and downwards, they sometimes absorb hydrogen sulphide and sometimes oxygen. When grown upon a microscope slide, in a liquid containing hydrogen sul- phide, the sulphur bacteria assemble to form a ring, about a millimeter from the edge of the cover glass. If the drop of liquid is not covered they do not develop at all. There is therefore a definite optimum of oxygen supply for these bac- teria. According to the researches of Yegunov, 3 this point is well brought out by growing them in deep vessels. A bacterial membrane is formed at a cer- tain distance from the surface of the liquid and short, tassel-like outgrowths project downwards from this membrane. A part of such a membrane with its projections is shown, enlarged, in Fig. 30. If these outgrowths are examined with a horizontal microscope it becomes evident that they consist of bacterial cells that are moving up and down with a boiling motion, like water in a spring. The occurrence of hydrogen sulphide is not confined to bogs and sulphur springs, for this substance is also found in the sea. The water of the Black Sea below a depth of about 200 m. becomes richer in hydrogen sulphide as the depth increases. One hundred liters of water, collected at the depths given, contained the following amounts of hydrogen sulphide. 1 Omeliansky, V., Sur la culture des microtes mitrificateurs du sol. Arch. sci. biol. St.-Petersbourg 7: 291-302. iSoo- - Winogradsky, Sergius, Ueber Schwefelbacterien. Bot. Zeitg. 45: 480-507. 513-523. 520-539, 545-559. 569-576, 585-594, 606-610. 1887. Nathansohn, Alexander, Ueber eine neue Gruppe von Schwe- felbacterien und ihren Stoffwechsel. Mittheil. Zool. Sta. Neapel 15: 655-680. 1902. Beijerinck, M. W., Ueber die Baketerien welche sich im Dunkeln mit Kohlensaure als Kohlenstoffquelle ernahren konnen. Centralbl. Bakt. II. 11: 493-599. 1904. Omelianski, W., Ueber eine neue Art farbloser Thiospirillen. Ibid. II. 14: 769-772. 1905. ! Yegounow, M., Sur les sulfobacteries des limans d'Odessa. Arch. sci. biol. St.-Petersbourg 3: 381- 397. 1895. Idem, Die Mechanik und Typen der Teilung der Bakterienscharen. Centralbl. Bakt. //, 4: 97-109. 1898. 4 5° PHYSIOLOGY OF NUTRITION Depth in the Black Sea, meters 215 432 2040 2525 H 2 S Context per 100 l. cc. 33 222 555 655 In the mud of the sea-bottom are therefore going on various kinds of fermenta- tion, which are accompanied by the elimination of hydrogen sulphide." Only because of the presence of sulphur bacteria is the hydrogen sulphide prevented from reaching the upper layers of water. Nitrifying and sulphur bacteria use ammonia and hydrogen sulphide, which are injurious to other organisms, and aid in preventing the accumulation of these substances upon the surface of the earth; oxidizing them to nitric and sulphuric Fig. 30. — Part of a membrane of sulphur bacteria, magnified n times. (After Yegunow.) acids, they bring these substances again into the general circulation of materials in nature. Besides ammonia and hydrogen sulphide, hydrogen is also produced in large amounts by the decomposition of complex organic compounds, and yet it is present only in minimal quantities in the atmosphere. According to various determinations, the amount of hydrogen in the air varies between 0.0003 an d 0.0 1 per cent. It therefore appears that processes must occur on the earth, by which hydrogen is combined and so started anew in the general circulation of materials. The researches of Kaserer 1 have shown that there are special bacteria that utilize hydrogen. Viewed from the standpoint of thermo-chemistry, hydrogen represents the best nutrient substance. Its heat of combustion is eight times that of starch; a gram of starch gives out during combustion but 4.0 kg.-cal., of heat, while a gram of hydrogen gives out 34.6 kg.-cal. (see page xxviii). Certain soil bacteria, such as Bacillus pantotrophus and Bacillus oligocarbophilus, 1 Kaserer, Hermann, Die Oxydation des Wasserstoffes durch Mikroorganismen. Centralbl. Bakt. // 16: 681-696, 769-775. 1906. Lebedeff, A. F., Ueber die Assimilation des Kohlenstoffes bei wasserstoff- oxydierenden Bakterien. Ber. Deutsch. Bot. Ges. 27: 598-602. 1909. Nabokich, A. J., and Lebedeff, A. F., Ueber die Oxydation des Wasserstoffes durch Bakterien. Centralbl. Bakt. 11, 17 : 350-355- 1907. " This deduction is of course not strictly accurate; although perhaps most of the hydrogen sulphide, ammonia and hydrogen in nature is of organic origin, these substances are also pro- duced, to some extent at least, quite independently of organisms. — Ed. ASSIMILATION OF CARBON 5 1 utilize hydrogen. 1 The former can derive its nourishment from organic com- pounds but it can also grow in purely inorganic media, in which case it assimilates carbon dioxide and hydrogen from the atmosphere and forms for- maldehyde according to the equation, H 2 C0 3 + 2H2 = CH 2 + 2H0O. Niklevskii 2 has isolated two bacteria (Hydrogenomonas nitrea and H. flava) that can live upon an inorganic substratum with an atmosphere of hydrogen and oxygen containing some carbon dioxide. They form organic compounds from hydrogen and carbon dioxide, which are then oxidized to carbon dioxide and water during respiration. The assimilation of hydrogen ceases when they are grown upon organic substances. In all cases here described, of nutrition of bacteria by inorganic substances, the production of organic compounds occurs without the agency of sunlight. The formation of hydrogen, hydrogen sulphide and ammonia (by reduction of oxidized compounds existing in nature, such as water, sulphuric acid and nitric acid), goes on at the expense of radiant energy assimilated in green leaves, however. Therefore it is indirectly at the expense of this energy that nitrifying bacteria, sulphur bacteria and hydrogen bacteria are able to exist. 6 1 Methane (CH4), which is frequently given off during the putrefaction of organic substances, can also serve as a nutrient material for some bacteria. [See: Sohngen, N. L., Ueber Bakterien, welche Methan als Kohlenstoff nahrung und Energiequelle gebrauchen. Centralbl.Bakt.il, 15:513-517. 1906.] 2 Niklewski, Bronislaw, Ueber die Wasserstoffoxydation durch Mikroorganismen. Jahrb. wiss. Bot. 47: 113-142- 1910. 6 In the foregoing discussion the terms "combustible" or " oxidizable " and "non-combus- tible" or "non-oxidizable" substances should be considered as synonymous with the more ac- curate ones "substances of high energy content" and "substances of low energy content." Although plant physiology has never yet received adequate treatment from the standpoint of energy transformations, some of the more general principles of such a treatment are well recog- nized and are pertinent in the present connection. Energy can no more be destroyed or created than can matter, so that when compounds of high energy content (carbohydrates, proteins, etc.) are formed from compounds of lower energy content (carbon dioxide, water, inorganic salts, etc.) energy must be supplied from some source other than the reacting sub- stances themselves. Since the reverse process yields energy it is conceivable that some of the energy obtained by the oxidation of large organic molecules may enter into reaction by which other complex compounds may be formed. This appears to take place to some extent in green plants, in the formation of proteins, cellulose, etc., and in parasites and saprophytes. It is also conceivable that other substances that yield energy upon oxidation may enter into analogous reactions. That this possibility is realized in the cases of some bacteria seems to be true, and is one of the chief contributions that the investigation of these forms has made to general physiology. Beggiatoa, which the author mentions, appears to be able to form complex organic molecules from carbonates by means of the energy derived from the oxida- tion of hydrogen sulphide. (See: Keil, Friedrich, Beitrage zur Physiologie der farblosen Schwefelbakterien. Cohn's Beitrage zur Biol. d. Pflanzen 2: 335-372- 1912.) Bacteria that produce hydrogen sulphide must derive the necessary energy from other reac- tions that yield energy, as from the oxidation of carbohydrates. Many other colorless bacteria are similar in this respect. Besides the authors already cited in the text, see: Keil, 1912 (just cited). Hinze, G., Thiophysa volutans, ein neues Schwefelbakterium. Ber. Deutsch. Bot., Ges. 21: 309-316. 1903. Molisch, Hans, Neuc farblose Schwefelbakterien. Centralbl. Bakt. H- 33 : 55 -02 - 191 2. Lauterborn, Robert, Eine neue Gattung der Schwefelbakterien (Thyoploca schmidlei. nov. gen., nov. spec.). Ber. Deutsch. Bot. Ges. 25: 238-242. 1907. Other bacteria oxidize sulphites, the liberated energy apparently enabling them to form ;> 2 PHYSIOLOGY OF NUTRITION §4. Distribution of Microorganisms in Nature. — The study of microorganisms is possible only with the aid of the microscope, and their discovery was impos- sible until magnifying glasses became available. The Columbus who discovered the world of the lowest organisms, which are ordinarily invisible, was a Dutch lens-maker of Delft, Anton van Leeuwenhoek. He succeeded in making mag- nifying glasses that magnified 100 and even 150 diameters. When, in 1675, he examined a drop of rain water that had stood for several days in a barrel, using one of his glasses, he observed a vast number of extremely small organisms moving hither and thither in the water. The number of these organisms ap- proached 10,000 in a single drop. No such organisms were to be seen in freshly collected rain water, and Leeuwenhoek therefore concluded that the germs of these must have fallen into the water from the air. The question then arose as to the origin of these extremely small organisms, and this became the subject of a very lively polemic. It is well known that infusions of most organic materials, such as meat and vegetable matter, de- compose very easily. Microscopical examination of material undergoing de- composition always shows the presence of microorganisms. The promptness with which they appear led to the conclusion that we have here a spontaneous generation (generatio spontenea) of the lowest forms of life out of various organic substances. The theory of spontaneous generation has had many adherents, even until recent times. Thus, van Helmont (1 577-1644) was the author of a recipe for the production of mice from meal. It was maintained that maggots (fly larvae) arise by spontaneous generation in meat. Even after it had been provided by exact experimentation that neither mice nor maggots can be produced de novo, and that such forms must arise by propagation, still the conviction persisted for a long time that the tiny, microscopic organisms may develop by spon- taneous generation. As early as 1776 Spallanzani proved experimentally that this theory was incorrect. He showed that no animalcules appeared in an her- metically sealed vessel containing an infusion of organic material, no matter how long this was allowed to stand, provided the infusion had been first boiled for three-quarters of an hour. After such a vessel had been opened, however, the contents soon began to putrefy; because germs entered from the air, as Spallanzani maintained. Although the adherents of the theory of spontaneous complex carbon compounds from mineral carbonates and bicarbonates. (See Nathansohn, 1902, and Beijerinck, 1004. [Note 2, p. 49.]) In addition to these there are still others that oxidize ferrous compounds to the ferric form. 1 See: Winogradsky, S., Ueber Eisenbak- terien. Bot. Zeitg. 46: 261-270. 1888. Molisch, Hans. Die Eisenbakterien. Jena, 1910. Lieske, Rudolf, Beitrage zur Kenntnis der Physiologie von Spirophyllum ferrugineum Ellis, einen typischen Eisenbakterium. Jahrb. wiss. Bot. 49: 91-127. 1911. Idem, Untersuch- ungen uber die Physiologie eisenspeichernder Hyphomyceten. Ibid. 50: 328-354. 1911. Since the forms, or kinds, of energy are mutually transformable it is possible that energy for the syntheses that occur in organisms may be derived not only from chemical reactions and light but also from other immediate sources, such as the radiant energy of heat and electri- city. The heat of the medium in which the reactions occur is of course a very important source of energy, not generally discussed in this connection. — Ed. ASSIMILATION OF CARBON 53 generation were not convinced by the experiment of Spallanzani, nevertheless it received a practical application at the hands of a French cook, Francois Appert. who started a factory for making preserves. He found that it was possible to keep meats, vegetables and liquids unspoiled for unlimited periods of time, if these materials were placed in hermetically sealed jars and then heated in boiling water. Appert published his experiments in a book which passed through many editions; 1 the book brought him fame, the preserves brought him a fortune. We have here a conspicuous example of the dependence of technical arts upon theoretical knowledge; Spallanzani, in solving the purely philosophical question of the origin of living things on the earth, thereby gave Appert the opportunity to found a new industry. Since the objection was raised against Spallanzani's experiment, that the closed vessels contained an inadequate supply of air and that the quality of what air there was must be greatly impaired by the high temperature, Franz Schultze performed the following experiment in 1836. A glass flask (Fig. 31) half full Fig. 31. — Arrangement of bottle and potash bulbs in Schultze's experiment. of an organic infusion and tightly closed with a cork stopper, through which two bent glass tubes were passed, was subjected to active boiling for some time. While hot steam was still escaping from both tubes he attached a potash bulb to each, one filled with potassium hydroxide solution and the other with sulphuric acid, after which the apparatus was allowed to cool. Twice a day, for three months thereafter, air was drawn through the flask, entering through the sulphuric acid and passing out through the alkali. No organisms of any kind were found in the solution. All the germs present in the entering air were removed by the sulphuric acid. In this experiment the air retained its usual composition and was not heated. But this experiment did not seem to be entirely convincing, and it was only by the remarkable investigations of Pasteur that the question of spon- taneous generation was finally and conclusively settled in the negative. Pas- 1 [Appert, Charles, L'art de conserver pendant plusieurs annees toutes les substances animales et veg£- tales. 2nd ed. Paris, 1811. Idem, Le livre de tout les menages ou l'art de conserver pendant plusieurs anees les substances animales et vcgetales. 3rd ed. Paris, 1813. A 5th ed. was published in 1842, or earlier. Xone of these has been seen in preparing this note; the references are taken from: Catalogue gen6ral des livres imprimes de la Bibliotheque Xationale, Paris 3: 736. 1899. — Ed. 54 PHYSIOLOGY OF NUTRITION teur (1857) closed glass flasks of various solutions with cotton plugs and sub- jected them to prolonged boiling. If the boiling had been continued sufficiently long the solution in the flasks remained unchanged and free from microorganisms for an indefinite period of time. The air that entered the flasks during cooling was filtered through the cotton plugs, in which all the germs that it originally held were left behind. Since the spores of some bacteria withstand a single, though long-continued boiling, this operation must sometimes be repeated several times, and even under pressure, in order to kill all organisms originally present. Pasteur carried out a number of his experiments in glass flasks espe- cially arranged with two necks (Fig. 32). One of the necks bore a short piece of rubber tubing, which was closed by a bit of glass rod. The other neck was drawn out into a narrow tube, bent twice upon itself. Both were open during the boiling of the liquid. While boiling was still going on the wide tube was plugged, after which boiling was stopped and the apparatus was cooled, air entering through the narrow tube. The solution re- mained unchanged indefinitely, since all spores con- tained in the entering air were caught in the narrow bend of the tube. However, if the glass stopper was momentarily removed, thus allowing a very small number of microorganisms to enter the flask, then the solution immediately began to decompose. De- composition is brought about in such an experiment as a result of the rapid multiplication of the micro- organisms that have been introduced. To demonstrate conclusively that the theory of spontaneous generation is untenable, it remained still to prove that microorganisms and their spores really do occur in the air in great abundance. This question was also worked out by Pasteur in the most exact manner. He took a series of flasks, filled to a third of their volume with nutrient solution, brought the contents to boiling and then sealed them by fusing the glass of their narrow necks. The flasks were then placed in positions where he wished to investigate the air, and the sealed ends were then broken off, thus allowing air to enter. The flasks were then resealed. If the air entering a flask was free from germs, then the liquid remained unchanged, but if the entering air contained microorganisms or their spores, then decomposition began. In this way Pasteur proved that the air of deep cellars and high mountains is most nearly pure. It need not be con- cluded, however, that the air is absolutely free from organisms in those cases where the liquid remains unchanged in such experiments; it is quite possible that spores may be contained in the air but they may be able to develop in the particular nutrient medium chosen. Many exact investigations have now been made upon the distribution of microorganisms in the air. The table given below presents the average results from ten years of observation (1885-1894) upon the number of microorganisms Fig. 32. — Pasteur flask. ASSIMILATION OF CARBON ;>;> in a cubic centimeter of air in the Park of Montsourie. In the same table are shown the corresponding numbers, averages from ten years of observations, in one of the squares in Paris (Place Saint-Gervais). The numbers are much larger in cities than in the country. Season of Year Bacteria Winter. . Spring. Summer Autumn. 170 295 345 195 Place Saint Gervais (Paris) Moulds Bacteria i4S i95 246 230 43°S 8080 9845 S665 Moulds 1345 2275 2500 2185 Microorganisms occur not only in the air but also in water and soil. The water of rivers always contains bacteria, these being especially numerous in the vicinity of cities. The following numbers of bacteria were found in a cubic centimeter of water from the rivers and at the localities cited below. River Rhone River Spree above Lyons 75 below Lyons 800 above Berlin 4,3°° below Berlin 97>4oo Microorganisms also occur in rain water, in snow and in hail. The soil always contains microorganisms, their number naturally depending upon the amount of organic material present. Many more are found near the surface than in the deeper layers. The following table gives an idea of their distribution at various depths in. a soil covered with forest growth (at Pfingst- berg, in the vicinity of Potsdam). These are the numbers of microorganisms found in a cubic centimeter of soil from various depths at different times of the vear. Depth below Soil Surface, meters May 27 June 15 Nov. 3 0.0 150,000 140,000 55,ooo 0.5 200,000 145,000 75,000 1 .0 2,000 1,000 7,000 2 .0 2,000 100 3-o 3,000 700 1,500 4-5 100 100 PHYSIOLOGY OF NUTRITION Bacteria are present in all foods, milk furnishing especially favorable condi- tions for their development. When fresh this liquid generally contains no bac- teria, but they develop very quickly from spores that fall from the air. Thus a cubic centimeter of milk that had stood since milking at a temperature of i5.5°C, contained the following numbers of bacteria per cubic centimeter. Hours after Milking 4 9 24 Bacteria per cc. 34,000 1 00,000 4,000,000 The intestinal tract of man is densely populated with bacteria, which fre- quently cause decomposition of foods in the intestine. We are thus not only externally surrounded by bacteria, but are even internally infested with them. This seems to explain why these organisms appear so promptly in all kinds of organic material that they decompose. §5. Sterlization and Disinfection. 1 — In view of the fact that microorganisms are so universally present, all objects used in handling them must be absolutely free from spores or germs of any kind, especially if pure cultures of a certain species are desired. This is accomplished by sterilization. Such small objects Fig. 33. — Dry-air sterilizer heated by gas. as knives, scissors, glass rods, forceps, slides and cover glasses, platinum needles, etc., may be sterilized by heating in a gas or alcohol flame. Platinum instru- ments may be brought to a red heat but for other objects a few moments in the flame suffices, so that germs clinging to the surface may be destroyed. A dry- ing oven, or dry-air sterilizer, is used for the sterilization of larger objects (Fig. 33). This is usually equipped with double walls, the products of combustion 1 Abel, Rudolf V. L., Taschenbuch fur den bakteriologischen Praktikanten. [Abel's Laboratory hand- book of bacteriology. Tr. from 10th German ed. by M. H. Gordon. London, 1907.] Kiister, Ernst, Anleitung zur Kultur der Mikroorganismen fur den Gebrauch in zoologischen, botanischen, medizinischen und landwirtschaftlichen Laboratorien. Leipzig and Berlin, 1907- ASSIMILATION OF CARBON 57 from the gas flame below passing between the two walls and thus rendering the heating uniform. Objects that cannot endure dry heat are sterilized in a steam sterilizer, such as Koch's apparatus. This is a cylinder of tinned sheet iron or copper with a cover above. The lower part is filled with water and the objects to be sterilized are placed upon a perforated rack in the upper part. A burner below the cylinder heats the water to boiling and the contained objects are sterilized by water vapor at ioo°C. The apparatus is covered with felt or asbestos, to retard the escape of heat. d Fig. 34. — Arnold steam sterilizer. Instead of a steam sterilizer the autoclave is frequently used for steriliza- tion (Fig. 35). This is nothing more than a Papin's digester, operating with superheated steam, under pressure up to two atmospheres or more and at temperatures of from ioo° to i34°C. or higher. At a temperature of i2o°C. sterilization need last only fifteen minutes. At a temperature of 130 all germs are instantly killed, so that repeated treatment, necessary in the case of steam sterilization, is here superfluous. c For most satisfactory work the oven should have an automatic temperature-regulator, various forms of which are available for gas. Electrically heated, automatically regulated ovens are also obtainable, some of which are so well insulated that but little heat escapes to the exterior. — Ed. d One of the various forms of the Arnold type of steam sterilizer is most convenient and efficient in operation. (Fig. 34.) This keeps but a small amount of water boiling at any one time and a large portion of the water that is boiled away is condensed and returned to the reservoir. — Ed. 53 PHYSIOLOGY OF NUTRITION Liquids may also be sterilized by filtration. The most convenient arrange- ment for this purpose is the Chamberland filter, a hollow cylinder of porous porcelain, closed at one end. The liquid to be sterilized is passed, under pres- sure, through the porous walls of the previously sterilized filter. Various disinfecting materials are also used for the chemical destruction of microorganisms. The most effective of these is corrosive sublimate, or mercuric chloride (HgCl 2 ). A solution of i g. of mercuric chloride in a liter of distilled water is thus used in bacteriological laboratories. The hands of the worker and also his implements are disinfected with this solu- tion, which is also employed to destroy cultures that are not needed. A solution of one part of the salt in 300,000 parts of water prevents the development of the bacillus of splenic fever, Bacillus anthracis. Sulphurous acid, chlorinated lime [also known as bleaching powder; it con- tains calcium hypochlorite], hydrofluoric acid and its salts, boric acid, ozone, hydrogen per- oxide, milk of lime, and phenol, or carbolic acid, are also suitable for use as disinfectants. 6 §6. Pure Cultures. — To study microorgan- isms with respect to their developmental history and their physiological process it is necessary to obtain them in a pure culture. 1 A pure culture is one known to contain only a single, definite species of organism. Such a culture can be obtained only by fulfilling two conditions. The* first consists in the exercise of sufficient precaution to prevent the entrance of germs from the air into the sterilized culture medium; the second is the derivation of the culture from a single cell. A culture in which all the micro- organisms are quite similar is nevertheless not to be termed a pure culture unless it has been derived from a single cell, since very many microorganisms with entirely different physiological properties 1 Pure cultures may be purchased from several establishments, among which may be mentioned the following: Krals Bakteriologisches Laboratorium, Prag I, Kleiner Ring II; Institut fur Garungsgewerbe, Berlin N, Seest:asse 6s; Jorgensens Laboratorium, Kopenhagen, Frydendalsvej 30; Zentralstelle fur Pilz- kulturen, Amsterdam. [They may be obtained from the Laboratory of the American Museum of Natural History, New York, and from Parke, Davis and Co., Detroit. —Ed.] 'To the substances mentioned in the text may be added: iodine, sodium sulphite and Dakin's recent discovery, paratoluene-sodium-sulphochloramide (on the American market under the trade-name chlorazene, though it was called "chloramine" by Dakin [British med. jour., Aug. 25, 1915, also Jan. 29, 1916]). Chlorine, bromine, and potassium per- manganate are also used as disinfectants. It should be noted, however, that antiseptics or disinfectants that are useful in some cases may be useless or even harmful in others. Numerous references on this subject are given in the Index Medicus, Carnegie Inst., Wash. — Ed. Fig. 35. — Autoclave. The top is hinged and may be raised after releasing the locking clamps. ASSIMILATION OF CARBON 59 have exactly the same form. On the other hand, a culture obtained from a single cell is called a pure culture, even though the microorganisms therein contained exhibit diverse forms, since we now know that one and the same species of bacterium or yeast can assume different forms, according to its developmental stage and the influence of the medium in which it is grown. The method most frequently used for the production of pure cultures is that of dilution. This method was first used, in its original form, by Lister 1 in 1878, to obtain a pure culture of lactic acid bacteria. It was carefully elaborated for yeasts by the Danish bacteriologist, Hansen in 1881/ Let it be supposed that we have a fermenting beer-wort with many different species of yeasts, and that these are to be separated, so that each species may be had in pure culture. After shaking the liquid, several drops are taken up in a sterilized pipette and transferred to a Freudenreich flask (Fig. 36) partly filled with sterilized water. This flask is of glass, with a capacity of from 25 to 30 cc, and is closed by means of a glass cap shaped like a short, inverted thistle-tube, the small opening of which is plugged with cotton. To obtain a uniform distribution of the yeast cells throughout the liquid, the flask is thoroughly shaken, after which a drop of the contents is transferred, upon the bent end of a platinum wire, to the surface of a microscope cover glass which is marked off into small squares. Here the drop is spread out into a thin layer, and the number of cells present is determined by counting. A van Tieghem cell, or moist chamber, is used for this purpose (Fig. 37). This consists of a slide upon which a glass ring (c) is sealed with vaseline. A small quantity of water (d) is introduced into the chamber so that microorganisms clinging to the under side of the cover glass (a) may not become desiccated. The cross-ruled cover glass is sealed to the glass ring with vaseline, the culture drop hanging from its lower surface (b). The divisions marked upon the cover glass facilitate the counting of the cells under the microscope. Suppose that twenty cells are found upon the cover glass. The drop of liquid is again transferred, by means of the platinum hook, to a fresh Freunden- reich flask containing 40 cc. of sterilized water. After vigorous shaking about 1 cc. of this liquid is transferred (with a pipette) into each of forty Freudenreich flasks containing sterilized beer-wort. Since the original drop con- Fig. 36 — Freudenreich flask. a- c- mm 7 T tained only twenty cells, we should expect that Fig. 37. — Moist chamber, or J J ' ^ . van Tieghem cell, for microscopic the yeast would, in all probability, develop only in work, a, cover glass; b, position twenty f the flasks while the other twenty would 01 drop of medium; c, wall of . . chamber made of section of glass remain sterile. It is also highly probable that the bottom^of Ihambe / solution in ne w generation has arisen from only a single cell in those flasks where growth does occur. All 1 Lister, Joseph, On the lactic fermentation and its bearings on pathology. Trans. Pathol. Soc. London 20: 425-467. 1878. 'Hansen, 1896. [See note 1, p. 44.]. — Ed. 6o PHYSIOLOGY OF NUTRITION this is only highly probable, however, and not definitely established. Hansen employed this method in his work with yeasts. Flasks containing freshly inoculated beer-wort are vigorously shaken and then allowed to stand. The cells sink to the bottom and begin to multiply, so that, after a time, whitish colonies of cells become visible with the unaided eye. If a flask shows but one such colony it follows that only a single cell was introduced, since it is highly improbable that two cells might have settled together after the shaking. If, on the other hand, two or three cells have been introduced into the flask, then two or three colonies, respectively, develop. In order to secure pure yeast cultures, solid substrata may also be em- ployed, which make it possible to follow, under the microscope, the development of a colony from a single cell. For this purpose a drop from a young yeast cul- ture—previously shaken — is introduced into a small flask of sterilized water. From this is inoculated, by means of the tip of a platinum wire, another flask containing beer-wort and gelatine, warmed to 45°C. The latter is vigorously shaken and then a drop of the liquid is transferred to a circular cover glass (30 mm. in diameter), which has been marked off into numbered squares, and the cover is laid over a glass ring to form a moist chamber or van Tieghem cell. The yeast cells are held immovable in the hardened gelatine so that it may now be Fig. 38. — Pasteur flask; a slightly different form from that of Fig. 32, p. 54. Fig. 39. — Petri dish. Fig. 40. — Showing insertion of needle into solid medium in inverted tube, to make stab inoculation. noted in which squares single ones lie, and the development of colonies from these may be readily followed. When the colonies become clearly visible to the un- aided eye, one of them is removed from the cover glass and placed in a flask of nutrient solution. The colony is lifted on the end of a bit of flame-sterilized platinum wire, held by means of forceps, and the wire, with its colony, is dropped into the flask. During this operation the cover glass must be held with the drop on its under side, to prevent infection from the air. If a large quantity of pure culture is desired, a portion of a young culture a day old, obtained as just de- ASSIMILATION OF CARBON 6 1 scribed, is transferred with a pipette to a Pasteur flask (capacity about 200 cc.) of sterilized beer- wort (Fig. 38). After a day the contents of this flask are poured into second flask (capacity about 500 cc.) also filled with sterile beer- wort. Solid as well as liquid nutrient media are used for pure cultures of bacteria. In' the case of liquid media the dilution method described above is used to separate the cells. With solid media, which are very valuable for the pro- duction of pure cultures, Petri dishes are used for this purpose (Fig. 39). Each dish consists of two shallow glass pans (9 or 10 cm. in diameter), one being a little larger than the other and forming a cover for it. A trace of the mixed culture is introduced into a flask containing, for instance, a mixture of bouillon and gelatine, at 3o°C, after which the flask is shaken, and the contents are then poured into the dish and the latter is covered. After some time each bacterial cell builds a colony around itself, which can be seen by the unaided eye or with a simple magnifying glass. When a pure culture of a certain microorganism is finally obtained, then any number of pure cultures of that form may be readily prepared. Inoculations of liquid nutrient media are effected by means of a glass rod, a platinum wire or a pipette, with all the requisite precautions. Inoculations of solid media may take the form of either stab or streak cultures. To make a stab culture a platinum needle is dipped in the original culture and is then thrust upward into the solid medium held in an inverted test-tube (Fig. 40). F6r a streak cul- ture, a test-tube of solid medium with a slanting surface is prepared, and the point of the inoculating needle is drawn across this surface. Summary 1. General. — Plants without chlorophyll cannot form carbohydrates from carbon dioxide and water by means of the energy of sunlight. They derive energy, as well as material, from chemical compounds. Such plants may be divided into two groups: those of one group get energy from organic compounds alone (these compounds having been previously made by green plants), those of the other group derive energy from inorganic substances. Cells with chlorophyll utilize sunlight energy to form carbo- hydrates (and oxygen) out of carbon dioxide and water, while cells without chlorophyll either get carbohydrates (or related organic compounds) ready-made from their surroundings, being unable to utilize either sunlight energy or carbon dioxide, or else they derive energy from inorganic compounds and thereby form their carbohydrates and related compounds out of carbonates or carbon dioxide and water. 2. Non-green Plants That Derive Energy Only from Organic Compounds.— Yeasts, fungi, non-green seed plan s, the non-green portions of ordinary green plants, and most bacteria, derive their energy supply exclusively from ready-made organic com- pounds. These ompounds also supply carbon, which is of course as essential for non-green cells as for cells with chlorophyh 1 .- The microorganisms of this group are very important in nature, being largely responsible for decay and putrefaction. They live by decomposing the organic sub- stances produced by other organisms, including green plants. They may be dis- tinguished from one another by the nature of the substances required for their growth, and they may be grown in artificial nutrient media, such as Pasteur's culture solution 62 PHYSIOLOGY OF NUTRITION for yeast. These organisms are either saprophytic (living on dead material from other organisms) or parasitic (living on tissues that are still alive). There are also a few saprophytes and parasites among flowering plants. Dodder (Cuscuta) is an example of a parasite of this kind. Mushrooms are examples of large saprophytic forms. 3. Non-green Plants That Derive Energy from Inorganic Compounds. — This group is composed of certain kinds of bacteria that are able to oxidize inorganic com- pounds and thus secure a supply of energy. Of these, nitrifying bacteria are very important. They oxidize ammonia to nitric acid. They must be grown in surround- ings free from carbohydrates and other organic substances, but they require carbon dioxide (or carbonates) and oxygen. They form carbohydrates and other organic com- pounds out of water and carbonates or carbon dioxide, somewhat as do green plants, but their source of energy is very different. Another example of this group is furnished by the sulphur bacteria (as Beggiatoa), which oxidize hydrogen sulphide to sulphur and water, thus securing an energy supply. The sulphur produced is finally oxidized into sulphates, such as calcium sulphate. The sulphur bacteria grow in the presence of organic material. Some hydrogen bacteria (Hydrogenomonas) can form organic material from hydrogen, oxygen, and carbon dioxide, in the absence of organic compounds. Hydrogen is oxidized, thus supplying energy. In the presence of organic compounds hydrogen is not oxidized, and these bacteria are then to be considered as belonging to the preceding group. This whole matter of the carbon nutrition of plants may be stated as follows: Apparently all organic compounds in plants are formed, directly or indirectly, from carbohydrates (such as sugars). (1) The carbohydrates used may be formed in cells with chlorophyll, out of carbon dioxide and water, and by means of sunlight energy (2) The carbohydrates used may be formed in cells without chlorophyll, out of carbon dioxide (or carbonates) and water, by means of energy obtained through the oxidation of inorganic substances such as ammonia, sulphur dioxide, hydrogen, etc. (3) The carbohydrates used may be derived from the surroundings, either ready-made or else by the decomposition of other organic compounds that are themselves supplied ready- made in the surroundings. These other organic compounds may also be used directly, without the preliminary step of forming carbohydrates. There are just two general sources of energy for plant activities, (a) sunlight and (b) energy derived from the oxidation of substances; and the substances oxidized may be either organic or inorganic. 4. Microorganisms in Nature. — Since Spallanzani's time it has been known that all organisms are formed by the reproduction of other organisms, and that the micro- organisms found everywhere in nature arise in this way. On the basis of this principle Appert originated the art of preserving foods by sterilization. If all organisms in a preparation are killed at the start, and if no more are allowed to enter from without, there will be no living ones in the preparation. Fermentation and the decay of foods are caused by microorganisms, and these substances may therefore be preserved by sterilizing and then hermetically sealing them. This whole proposition was finally clearly worked out by Pasteur, who showed, among many other things, that the micro- organisms that cause fermentation in foods, etc., originate from individuals of the same forms, which fall in from the air, etc. The air generally contains large numbers and many kinds of microorganisms as do also soil, water, the human alimentary tract, etc. 5. Sterilization and Disinfection.— To obtain objects or material absolutely free from living microorganisms sterilization is necessary. In many cases this is done by dry heat. In other cases steam is used, especially in a closed chamber, such as the ASSIMILATION OF CARBON 63 autoclave. The heat must be applied for an adequate period, and the temperature must be sufficiently high. Liquids are frequently sterilized by passing them through a suitable filter (such as the Chamberland), which retains the bacteria, etc. Steril- ization may also be accomplished by the use of antiseptics or disinfectants, such as mercuric bichloride, phenol, etc. These simply poison the microorganisms. 6. Pure Cultures. — Pure cultures of any given kind of microorganism may be obtained by inoculating a suitable sterile medium with a single cell of the form desired, and allowing this to develop without the entrance of any other cells. Unless obtained in this way, a culture cannot be surely considered as pure. Single-spore inoculation is generally accomplished by repeated dilution of a liquid medium that contains the particular form desired. For this sort of work special technique has been devised. CHAPTER III ASSIMILATION OF NITROGEN 1 §i. The Nitrogen of the Air. — Atmospheric air is fourth-fifths free nitrogen and it contains very small amounts of ammonia. We owe the first experiments upon the assimilation of free nitrogen to Boussingault, a who grew various plants from the seed in nitrogen-free, ignited sand to which was added some ash from seeds of the kind of plants employed. He placed the porous culture pot in a shallow glass dish supported above the bottom of a larger glass pan, in which stood a large bell-jar, covering the cultures. (See Fig. 41.) Some sulphuric acid was placed in the large pan, to prevent the en- trance of ammonia from the outside air into the bell- jar. Two glass tubes were introduced under each jar, one to supply distilled water 6 to the dish in which the pot stood, the other to provide the necessary carbon dioxide to the air-space within the bell-jar. There was thus no source of nitrogen within the bell-jar, other than the free nitrogen of the air. The amount of nitrogen in the seed was determined, at the beginning of the experiment, by analy- sis of a control portion of the same kind of seed. The apparatus was exposed to light, and at the close of the experiment (after two or three months) the nitrogen content of the mature plant was deter- mined, and no increase in this element could be detected. It follows from this that free nitrogen is not assimilated by ordinary higher plants when these are cultivated in the soil without microorganisms. 1 A complete summary of the work upon nitrogen assimilation up to 1879 is given in: Grandeau, L.. Cours d'agriculture de l'ecole forestiere. Chimie et physiologie applies a l'agriculture et a la sylviculture I. La nutrition de la plante. Paris, 187c-* a Boussingault, 1860-91. [see note 5, p. 2.] Idem, De Taction du salpetre sur la vege- tation. Ann. sci. nat. Bot. IV, 4: 32-46. 1855. Idem, Recherches sur l'influence que l'azote assimilable des engrais exerce sur la production de la matiere vegetale. Ibid. IV, 7: 5-20. 1857.— Ed. 6 It should be mentioned, however, that, while distilled water should not add anything but water and the atmospheric gases to the organism, yet it may extract other materials. Thus seedlings grown in distilled water give off salts, etc., by diffusion into the surrounding medium. (See, further, note b, p. 83.) — Ed. 64 Fig. 41. — Arrangement of Boussin- gault, for growing a plant in nitrogen-free soil, without access of ammonia from the air. The large pan contains sulphuric acid (forming a seal) ; water is supplied through the tube at the right and carbon dioxide through the one at the left. ASSIMILATION OF NITROGEN 65 Experiments upon the assimilation of ammonia from the air by leaves were carried out by Sachs, c by Schlosing^ and by Adolf Mayer. e The upper parts of the plant were isolated from the soil and received the ammonia as the car- bonate, in solution. All the plant parts so treated exhibited a higher nitrogen content than the corresponding organs in the controls without ammonia thus supplied. This kind of nitrogen assimilation is of almost no importance under natural conditions, however, since the ammonia content of the air is exceedingly small. According to Schlosing a volume of 100 cu. m. of air contains, on the average, only 2.4 mg. of ammonia. §2. The Nitrogen of the Soil. — The nitrogen of the soil occurs as organic compounds, ammonium salts and nitrates/ The experiments of Boussingault and those of many agricultural chemists have shown that ordinary plants (with the exception of certain forms, especially the legumes, which will be dis- cussed later) obtain their nitrogen exclusively from the soil, and that all three kinds of nitrogen compounds of the soil are beneficial to plants. Soils poor in nitrogen, and thus unproductive, can often be made productive by addition of any of these three forms of nitrogen compounds, but this result can usually be best and most quickly attained by the addition of nitrates. Therefore, the various nitrates generally serve as the best source of nitrogen for higher plants. The question arises whether all nitrogen compounds of the soil are taken up directly by the plant or first undergo some alteration. In order to answer this question we must consider some of the properties of soils. According to Boussingault 1 kg. of soil contained the following amounts of nitrogen: Source of Soil Kind of N-compoxjnd LlEBFRAUENBERG Nancy Mettais Organic nitrogen gravis 2 . 101 0.019 0.029 grams 1-432 0.004 0.040 grams 1.223 0.004 0?^ Nitrogen of ammonium salts Nitrate nitrogen 5 • Most of the soil nitrogen thus has the form of organic compounds, which are decomposition products from the decay of animal and plant materials. The e Sachs, J., as cited by Robert Hoffman, Ueber die Aufnahme des Kohlensauren Ammoniaks der Luft durch die Pflanzenblatter. Jahresb. Agrikulturchem. 3: 78-80. 1862. — Ed. d Mayer, Adolf, Ueber die Aufnahme von Ammoniak durch oberirdische Pflanzentheile. Landw. Versuchsst. 17: 329-397. 1874. — Ed. ' Schloesing, Th., Sur l'absorption de l'ammoniaque de l'air par les vegetaux. Compt. rend. Paris 78: 1 700-1 703. 1874. Also see: Atwater, W. O. Ueber die Assimilation von Stickstoff aus der Atmosphare durch die Blatter der Pflanzen. Landw. Jahrb. 14: 621-632. 1885.— Ed. f Nitrites also occur, but in small amount. — Ed. 5 66 PHYSIOLOGY OF NUTRITION nitrogen of ammonium salts forms the smallest part. The ammonia of the soil is derived partly from the decomposition or organic nitrogenous compounds and partly from the air. According to Schlosing's investigations, ammonia gas is vigorously absorbed from the air by both dry and moist soils. Dry soils, it is true, soon become saturated with ammonia, but this is not so for moist soils, for the ammonia absorbed is gradually converted into nitric acid. A soil surface of i hectare (2.5 acres) can absorb yearly from 53 to 63 kg. of ammonia from the air. Besides organic compounds and ammonia, every soil also contains nitric acid or its salts. According to Boussingault's exact investigations nitric acid is formed in the soil at the expense of other nitrogenous compounds. A known quantity of damp soil, of known composition, was placed in a large carboy, which was sealed in 1859 and not reopened until 1871. At the conclusion of the experiment the soil in the carboy was again analyzed. The results are presented in the following table. Year 1859 1871 Difference Total nitrogen grams 0.4722 0.4520 -0.0202 Nitric acid nitrogen grams 0.0029 0.6178 +0.6149 grams 0.00075 0.16000 + 0.15925 The nitric acid was at least mainly formed from other nitrogenous compounds present in the soil. Moreover, during the progress of the experiment a part of the nitrogen of the soil diffused into the air of the enclosed space. Bous- singault showed in later experiments that very many kinds of organic materials (e.g., meat, blood, horn, bone, wool, etc.), if added to the soil, serve as sources for the formation of nitrates. Conditions thus exist in the soil which render possible the transformation of a great many kinds of nitrogen compounds into nitric acid or nitrates. Now the question arises, how is it that, in spite of the continuous formation of nitric acid, there is never more than a small quantity of this substance present in the soil? An answer is obtained from a consideration of the phenomena of absorption of various compounds by the soil. 17 The soil takes substances out of solution and retains them, so that a solution filtered through a soil layer becomes less concentrated. The first investigator to direct his attention to this phenomenon and to recognize its importance in agriculture was Bronner (1836), who describes the following experiment. A bottle with a small opening in the bottom is filled with fine sand or with half-dry, sifted garden-soil. Dark ill-smelling manure extract is gradually poured into the bottle until the entire soil-mass is saturated. The liquid issuing below is almost entirely odorless and * This is partly the phenomenon now generally termed adsorption.- — Ed. ASSIMILATION OF NITROGEN 67 colorless and has lost all the readily recognizable characteristics of manure extract. More exact studies show that not all compounds are thus retained by the soil; while ammonium salts are absorbed, nitrates easily pass through. This characteristic of nitrates, their ability to be washed out of soils, explains the small nitrate content of the soil. All of the nitrates not absorbed by plants are washed down by the rain into the deeper soil layers. Of all the nitrogenous substances occurring in the soil, the organic materials and ammonium salts form, so to speak, the nitrogen stock of the soil. These are firmly held and so act as a constant source of nitrates, which may be absorbed by plant roots. The investigations of Kostychev 1 have shown that organic nitrogenous compounds of humus do not consist solely of decomposition products of plant and animal substances but are mainly proteins, such as are the constituents of living organisms. In the leaf-mould formed by oak leaves that had been de- composing for twelve months the nitrogen content was 2.98 per cent., of which 2.73 per cent, was protein nitrogen and only 0.25 per cent, was made up of simpler nitrogenous compounds. These experiments constitute a new proof that the processes going on in the soil are not exclusively chemical, without the intervention of living cells, but are also physiological in their nature, being connected with the life-processes of organisms. The same author has shown that the phosphorus of the soil appears mainly in complex organic compounds such as are constituents of the lowest organisms. By virtue of its abundant bacterial life, the soil is practically a living mass.* §3. Nitrification in Soils.— The ability of the soil to produce nitric acid or nitrates from various more complex nitrogenous compounds depends upon various conditions. One of these, according to Schlosing, is free access of oxygen. Equal amounts of the same soil were confined in five vessels, and a current of gas was passed through each vessel. The gas passed through the first vessel was pure nitrogen, so that this soil was without oxygen. The other vessels, II, III, IV and V, received mixtures of nitrogen and oxygen containing 6, n, 16, and 21 per cent, of the latter gas, respectively. The amount of nitrate present in the soil was determined for each vessel at the beginning and end of the experiment. The results of these determinations, expressed as nitric 1 Kostytschew, P., Ueber die Mikroorganismen des Bodens. Kurlandische Land- und Forstwirtsch. Zeitg. (Riga) 5: 13-14. 1890. A On the nature of the organic matter of the soil see the following: Schreiner, Oswald, and Shorey, Edmund C, The isolation of harmful organic substances from soils. U. S. Dept. Agric, Bur. Soils, Bull. 53. 53 p. Washington, 1909. Idem, Chemical nature of soil organic matter. Ibid. Bull. 74, 48 p. Washington, 1910. Schreiner, Oswald, and Skinner, J. J., Nitrogenous soil constituents and their bearing on soil fertility. Ibid. Bull. 87. 84 p. Washington, 191 2. Trusov, A., The formation of humus by means of vegeta- ble substances. [Russian.] Selskoie khoziaistvo i liesovodstvo (Economie agricole et syl- viculture) Petrograd 246: 233-245. 1914. Rev. in: Month, bull, agric. intell. and pi. diseases 6: 540-541. 1915- Abo rev. in: Exp. sta. rec. 34: 619. 1916. Idem, same title. [Russian.] Selskoie khoziaistvo i liesovodstvo (Economic agricole et sylviculture) Petro- grad 248: 409-437. 1915. Rev. in: Month, bull, agric. intell. and pi. diseases 7: 46-47. 1916. Also rev. in: Exp. sta. rec. 34: 516. 1916. — Ed. 68 PHYSIOLOGY OF NUTRITION acid, are presented below. The soil without oxygen thus lost its whole content of nitrate, and those supplied with oxygen formed additional amounts, the quantity formed increasing with the amount of oxygen supplied. Vessel Number and Oxygen Content of Gas Nitric Acid Present in the Soil Loss Gain Nov. 18, 1872 July 3, 1873 mg. 64 64 64 64 64 mg. 00 263 286 267 289 mg. 64 mg. II. 6 per cent, oxygen III. ii per cent, oxygen IV. 16 per cent, oxygen V. 21 per cent, oxygen 199 222 203 225 Nitrification in soils is due to bacterial action, as Schlosing and Mlintz 1 have shown. These authors took a large-bore glass tube a meter long, filled it with a mixture of sand and lime and allowed sewage water containing ammonia to percolate slowly through it. After some days nitrate could be indentified in the filtrate. The ammonia of the water was oxidized in its passage through the tube. They also subjected the soil contained in the tube to the action of chloroform vapor during the percolation, to determine whether this oxidation was effected by the soil itself or by microorganisms contained therein. The re- sult was a cessation of nitrification, the filtrate containing ammonia instead of nitrates in this case. Since the chloroform probably only repressed the vitality of the soil bacteria, without influencing purely chemical processes, Schlosing and Muntz concluded that the process of nitrification in the soil is caused by bacteria. After many investigators had vainly endeavored to obtain the nitrifying bacteria of soil in the pure culture, Vinogradskii 2 was at length successful in this, as have been mentioned above (page 48). Further investigations by Vinogradskii showed that the nitrification of ammonia and ammonium salts to nitrates is effected in the soil not by one but by two species of bacteria. One form produces nitrites (N0 2 ) from ammonium salts, and the other produces nitrates from nitrites. Vinogradskii proposed to reserve the term Nitrobacteria for all those bacteria that have to do with converting ammonium into nitrate. Investigation of the morpho- logical characteristics of nitrite-formers from different sources shows that they belong to different species. The difference between the nitrite-formers of 1 [Schloesing, Th., and Muntz, A., Sur la nitrification par les ferments organis6s. Compt. rend. Paris 84:301-303. 1877. Idem, same title. Ibid. 85 : 1018-1020. 1877. Idem, same title. Jbtd. 86: 892- 9S s Winog'radsky, S., Recherches sur les organismes de la nitrification. I, II, III. IV and V. : 1890. [See note I, p. 48.] Idem. Contributions a la morphologie des organismes de la nitrification. [Russian and French.] Arch. sci. biol. St.-Petersbourg 1 : 87-137. 1892. ASSIMILATION OF NITROGEN 6 9 the Old World and of the New is so great that it has even been necessary to distinguish two different genera, each with several species. The nitrite bac- teria of the Old World constitute the genus Nitrosomonas, with two species (N. europaa, N.javanensis) and local varieties. Those of the New World form the genus Nitrosococcus. A third genus, Nitrobader, 1 includes those bacteria that oxidize nitrites to nitrates. The work of Vinogradskii led to the supposition that these organisms might get their carbon as magnesium carbonate, but Godlewski 2 showed that such is not the case. Even with magnesium carbonate (MgC0 3 ) present, no carbon assimilation occurs in an atmosphere devoid of carbon dioxide. The nitrify- ing bacteria are thus shown to obtain their carbon from the carbon dioxide of the air. Further investigations of Vinogradskii and Omelianskii 3 cleared up the re- lation of nitrifying organisms to various organic compounds that check their growth. In the following table are given, for each of the two kinds of bacteria and for several organic compounds, the concentrations of the latter that just begin to retard growth and those that check it completely. Nitrite Formers Concentration Just Retard- ing Growth Inhibiting Growth Nitrate Formers Concentration Just Retard- ing Growth Inhibiting Growth Glucose. . . Peptone . . . Asparagin . Ammonia . 0.025-0.050 0.025 0.05 0.2 0.2 o-3 0.05 0.8 0.05 0.0005 0.2-0.3 0.5-1.0 0.015 Vinogradskii and Omelianskii state: "The action of the above-named sub- stances, in preventing nitrification, is so pronounced and becomes evident at such low concentrations, that these substances are not to be considered even as neutral in this case, although they are usually regarded as nutrients in bacteri- ology; on the contrary, their action is quite analogous to that of the substances that are known as antiseptics." If the presence of organic substances checks the process of nitrification, then no nitrifying of organic nitrogenous compounds is to be expected in pure cul- tures of nitrobacteria. According to Omelianskii 4 these organisms are entirely lacking in ability either to break down organic nitrigenous compounds by split- ting off ammonia, or to oxidize the nitrogen of these compounds directly. Or- * On methods for pure cultures of nitrifying bacteria, see: Omeliansky, 1899. [See note I, p. 49.1 2 Godlewski, Emil, O nitryfikacyi ammoniaku. Krakow. 1896.* » Winogradsky, S., and Omeliansky, V., L'influence des substances organiques sur le travail des microbes nitrificateurs. Arch. sci. biol. St.-P6tersbourg 7: 233-271. 1899. » Omeliansky, V., Sur la nitrification de l'azote organique. Arch. sci. biol. St.-Petersbourg 7 : 272-290. 1899- 7° PHYSIOLOGY OF NUTRITION ganic nitrogen can be nitrified by nitrobacteria only after it has been changed into ammonia or ammonium salts. The cooperation is thus necessary, of at least one of the bacterial forms that give rise to ammonia from organic com- pounds. Omelianskii was able to obtain nitrification of bouillon if he inocu- lated the medium with three species of bacteria at the same time: Bacillus ramosus, Nitrosomonas and Nitrobacter. If only B. ramosus and Nitrosomonas are introduced the process is limited to the formation of nitrous acid (nitrites), while B. ramosus and Nitrobacter produce only ammonia. Inoculation with Nitrosomonas and Nitrobacter leaves the bouillon unchanged. All these rela- tions may be shown by a diagram, reproduced below, in which the bacteria that decompose organic compounds to form ammonia are represented by a, those that form nitrites are represented by b, and those that oxidize nitrous to nitric acid (nitrites to nitrates) are represented by c. Organic Nitrogen Ammonia Nitrogen Nitrite Nitrogen Nitrate Nitrogen a + b -f- c a + b a -f- c b + c No alteration of organic nitrogen. Fig. 42. — Comparison of the effect of nitrate and of ammonium salts on growth of plants in bog-soil, which is poor in lime. O, no fertilizer; NO 3, nitrate added; NH 3 . ammonium salts added. (After P. Wagner.) Now that we have become acquainted with the process of nitrification, we may consider the question whether higher plants are able to obtain their nitro- gen only as nitrates or whether they can assimilate ammonium salts directly, without previous nitrification of the latter. Recent discoveries favor the view that nitrates act chiefly, if not exclusively, as the source of nitrogen for such plants. The experiments of Wagner 1 have shown that nitrates and ammo- nium salts have different effects according to the nature of the soil employed. Turnips were grown in vessels of a bog-soil very poor in calcium. In one series of experiments some of the vessels contained no nitrogen fertilizer, others each 1 Wagner, Paul, Dimgungsfragen unter Beriicksichtigung neuer Forschungsergebnisse. Heft. IV. Ber- lin, 1898. 72 p. ASSIMILATION OF NITROGEN 71 contained 2 g. of nitrogen as nitrates, and still others each contained about 2 g. of nitrogen as ammonium salts. In a second series calcareous marl was added throughout, in addition to the fertilizers mentioned above. The results of this experiment are brought together in the following table (see also Fig. 42). Fertilizer Dry Yield Increase in Yield, Compared with Cul- ture without Nitrogen Without lime With lime grams Without addition of nitrogen .... 6.3 2 g. of nitrogen as nitrate 94-4 2 g. of nitrogen as ammonium salt. 29.4 Without addition of nitrogen 9.6 \ 2 g. of nitrogen as nitrate 92 .0 I 2 g. of nitrogen as ammonium salt . . 86 . 7 grams 88.1 23.1 82.4 77.1 Thus, ammonium salts have but little value as fertilizers for soils poor in lime. But soils rich in lime show almost as good yields with ammonium salts as with nitrates (Fig. 43). These experiments show that nitrate fertilizer is suitable Pig. 43. — Comparison of the effect of nitrate and ammonium salts on growth of plants in soil rich in lime. O, no fertilizer; NO3, nitrate added; NHi, ammonium salts added. (After P. Wagner.) for many different kinds of soils whereas ammonia fertilizer is suitable for only a limited number. There are two reasons for this: first, if we suppose that the ammonia is all oxidized to nitric acid before assimilation, then free nitric acid may be produced in the soil that lacks calcium (as in the first series of ex- periments just described), and this acid retards the growth of the plants as well as the nitrification process. Secondly, if we suppose that a part of the ammonia is assimilated unchanged, then free acid may again accumulate in the soil lack- ing calcium; for ammonium salts are physiologically acid, their basic radicals being absorbed by the plants to a greater extent than are their acid radicals. 1 1 This is more fully considered in Chapter IV. 72 PHYSIOLOGY OF NUTRITION The presence of calcium carbonate prevents the accumulation of free acid in both cases. Such experiments with natural soils cannot answer the question regarding the direct assimilation of ammonia. Sterilized soils must be used, in which the nitrifying process is eliminated. The experiments of Pitsch, 1 Breal, 2 and Kos- sovich, 3 who used sterilized soils, gave positive results. §4. Circulation of Nitrogen in Nature. — The investigations of Boussingault and of Schlosing and Mtintz [see note c, page 64, and notes c, d, e, page 65] established the view that higher plants can assimilate only combined nitrogen. Free nitrogen should thus have absolutely no value for green plants, in spite of the enormous amount of it present in the air. Schlosing pictures the circula- tion of nitrogen in nature in the following way. Nitric acid (HN0 3 ) formed in the soil is taken up by plants and transformed into proteins and other organic compounds, which, in their turn, serve for the nutrition of animals. These compounds of nitrogen finally return to the soil as decomposition products of plants and animals, and are there again oxidized to nitrates. The nitrate of the soil, that is not assimilated by plants, is washed into the deep soil layers by pre- cipitation water and finally reaches the sea, where it is changed back into am- monium salts by the life activities of marine organisms. Ammonia evaporates, with water vapor, from the surface of the sea, and is again taken up from the atmosphere by plant leaves or by the soil, and in this way re-enters the general circulation. All these transformations of combined nitrogen have no effect upon the total amount of it occurring in nature. Natural processes are known, however, which lead to the decomposition of nitrogenous compounds and to the liberation of molecular nitrogen. Thus, in the complete combustion of nitroge- nous organic compounds the total nitrogen is eliminated as nitrogen gas. The decomposition of organic compounds in the soil is also accompanied by the liberation of free nitrogen. The total amount of combined nitrogen in nature is diminished by these processes and, for this reason, many authors have sought some natural process that might lead to fixation of free nitrogen. Nitrogen is one of the elements that form only weak combinations with other elements. Until recently chemistry could name but three kinds of nitrogen fixation that might be of importance in nature: (1) An electric spark discharge effects the union of nitrogen and oxygen (Cavendish). (2) A silent electrical discharge causes the union of nitrogen with organic substances (Berthelot). (3) During the evaporation of water a small amount of nitrogen combines with hydrogen from the water and produces ammonium nitrate (Schonbein). Only the first of these three possibilities has real significance in nature, namely the fixation of atmospheric nitrogen during thunderstorms. 1 Pitsch, Otto, Versuche zur Entscheidung der Frage ob saltpetersaure Salze fur die Entwicklung der landw. Kulturgew a cb.se unentbehrlich sind. II. Landw. Versuchsst. 42: i~95- i893- Pitsch, O., and Haarst, J. Van, same title as above, III. Ibid. 46: 357-382. 1896. 2 Breal, E., Contribution a l'etude de l'alimentation azot6e des vegetaux. Ann. agron. 19: 274-293. 1893. . 3 Kossowitch, P., Ammoniaksalze als unmittelbare Stickstoff Quelle fur pflanzen. [Abstract in German, p. 637-638. Text in Russian.] Jour exp. Landw. 2 : 625-638. 190 1. ASSIMILATION OF NITROGEN 73 Recent technical advance has made it possible to obtain larger amounts of nitrogen compounds from atmospheric nitrogen. By oxidation of the latter, with the electric current, nitric acid is obtained on a large scale. By passing nitrogen through glowing calcium carbide, calcium cyanamide is formed, accord- ing to the equation: CaC 2 + 2N = CaCN 2 + C. The German commercial name of this product in the raw state is " Kalkstickstoff ,n and it is used as a nitrogen fertilizer. What has been attained by man only after much travail is commonly ac- complished by plants, however, for we now know a number of plants that can assimilate atmospheric nitrogen. §5. Fixation of Atmospheric Nitrogen by the Leguminosae.— All legumes are able to develop normally, producing a rich harvest with a high nitrogen content, without the addition of any nitrogenous compounds to the soil, as the exact studies of Lawes and Gilbert have shown. If we cultivate some sort of grain or legume for many years in succession on the same field without applying fer- tilizer, the nitrogen content of the crop finally reaches a certain minimum, beyond which it does not alter. Addition of mineral fertilizers without nitrogen is almost without effect upon the yield of grain, the nitrogen content remaining almost the same as before. This is entirely different in the case of the legumes; the same mineral fertilizer without nitrogen produces a marked increase in the nitrogen content of this crop. Two series of experiments by P. Wagner 2 are illustrated in Figs. 44 and 45, one with peas and the other with oats, the experimental conditions being the same in both cases. The containers marked O contained no fertilizer at all, those marked KP contained potassium and phosphoric acid (P0 4 ), and those marked KPN contained potassium, phosphoric acid and nitrogen as nitrate. Comparison of these figures reveals a distinct difference between the legumes and the grains in their relation to fertilizers. The growth of oat plants is seen to be very slight in the unfertilized culture, and the addition of potassium and phosphoric acid produces no improvement; while the addition of these together with potassium nitrate produces excellent growth (Fig. 44) . The behavior of the pea plants is entirely different. These do not need nitrate fertilizer, addition « Frank, A., Die Nutzbarmachung des freien Stickstoffs der Luft fur Landwirtschaft und Industrie. Zeitsch. angew. Chem. 16: 536-539- 1903. Gerlach, M., Die Nutzbarmachung des atmospharischen Stickstoffes. Illustr. landw. Zeitg. 1904. Nos. 5 and 7.* Review by Vogel in: Centralbl. Bakt. //. 12 : 495-497- 1904. [See also review in: Exp. sta. rec. 15: 25. 1003-C4.] 2 Wagner, P., Ergebnisse von Dunungungsversuchen in Lichtdruckbildern mit erlauterndem Vortrage viber die rationelle Dungung der land wirtschaftlichen Kulturpflanzen. 2te Aufl. Darmstadt, 1891. •Lawes J. B., and Gilbert, J. H., The sources of the nitrogen of our leguminous crops. Jour. Roy. Agric. Soc. England III, 2: 657-702. London, 1891. Idem, The Rothamsted memoirs on agricultural chemistry and physiology. 7 v. London, 1886-1899. Idem, same title. 3 v.London, 1 890-1 893. Hall, A. D., The book of the Rothamsted experiments. 294 p. New York, 1905. For a brief discussion of this whole matter see: Russell, E. J., Soil condi- tions and plant growth. 4 th ed. London and New York 406 p., 192 1. Russell's ex- cellent bibliography includes references to a number of the papers of Lawes and Gilbert. These papers have all been collected and published in the Rothamsted Memoirs, and Lawes and Gilbert's results are summarized by Hall. — Ed. 74 PHYSIOLOGY OF NUTRITION Fig. 44. — Growth of oats with various fertilizers. O, without addition to the soil; KP with addition of potassium and phosphoric acid; KPN, with addition of potassium, phosphoric acid and potassium nitrate. (After P. Wagner.) Compare with Fig. 45- Fig. 45. — Growth of peas with various fertilizers. 0, without addition to the soil; KP, with addition of potassium and phosphoric acid; KPN, with addition of potassium, phos- phoric acid and potassium nitrate. (After P. Wagner.) Compare with Fig. 44. ASSIMILATION OF NITROGEN 75 of potassium and phosphoric acid being sufficient to produce normal growth. In this case the total need of nitrogen is supplied from the air (Fig. 45). The results of Lawes and Gilbert and those of Wagner thus seem to dis- agree with the conclusions reached by Boussingault (see page 64). This is explained by the fact that Boussingault used sterilized soils, whereas the other authors, just referred to, worked with unsterilized soil under natural condi- tions. The reason for the entirely different behavior of legumes in sterilized soils and in unsterilized soils has been discovered in a series of remarkable in- vestigations conducted by Hellriegel and Wilfarth. 1 In their experiments, various legumes grew quite normally in soils that lacked nitrogen, provided these soils were not previously sterilized. Growth was checked, however, in sterilized, nitrogen-free soils, because of lack of nitrogen. Addition to the sterilized soil of a small quantity of an infusion from unsterilized soil produced normal growth of the plants and resulted in a crop rich in nitrogen. If the added infusion was previously boiled, however, then its addition produced no effect at all; the plants were retarded in their develop- ment and the harvest showed no increase in nitrogen. The soil used in preparing the infusion must be taken from a field upon which plants of the kind used in the experiment have been cultivated; for ex- ample, if peas are employed the soil used for the water extract must be obtained from a field where peas have previously been grown. Legumes growing under natural condi- tions have small tubercles upon their roots (Fig. 46). Hellriegel and Wilfarth ob- served that these tubercles developed only in unsterilized soil, or in sterilized soil only if it had been treated with infusion of un- sterilized soil. Tubercles never develop in uninoculated sterilized soils. From their studies Hellriegel and Wilfarth came to the conclusion that the formation of root tubercles is the result of a symbiosis between the legumes and lower organisms, and that these very tubercles are directly influential in the assimila- tion of atmospheric nitrogen by leguminous plants. A cross-section of a legume root, through one of these tubercles, shows that the greater part of the tubercle consists of parenchymatous tissue (Fig. 47). The inner cells are very different from the outer ones. The former constitute 1 Hellriegel, H., and Wilfarth, H., Untersuchungen uber die Stickstoffnahrung der Gramineen und Leguminosen. Beilage Zeitschr. Rubenzucker-Indust. d. deutsch. Reich. 234 p. November, 1888. Fig. 46. — Root system of pea plant, with tubercles (if.) 76 PHYSIOLOGY OF NUTRITION the so-called bacterioid tissue and are characterized by thin cell walls and high content of protein. The protein substances occur in the cells as small, bacteria- like rods, which are branched in the older tubercles. These are the so-called bacterioids. The cells of the outer parenchyma layers contain little reserve material, and only those adjacent to the bacterioid tissue are filled with starch grains. The tubercle is covered on the outside by a layer of cork, and branches of the vascular bundles of the root extend into the tubercle. Beijerinck 1 and Prazmovskii 2 have succeeded in securing tubercle bacteria in pure culture. When transferred to a nutrient solution, the young bacteria, or the modified cells called bacterioids, begin to divide and multiply rapidly. The newly formed organisms appear to be in no way different from ordinary bacteria, and they show the same kind of movement. Prazmovskii has given them the name Bacterium radicicola. This writer has studied the developmental history of the tubercles of the pea plant. If sterilized soil in which young pea seedlings are growing is inocu- lated with a pure culture of Bacterium radicicola, an accumulation of bacteria in the root-hairs becomes noticeable after several days. This mass of bacteria then becomes enclosed in a sheath, forming a sack-like body that enlarges and Fig. 47. — Cross-section of a root tubercle of lupine, showing bacterioid tissue (the elon- gated area below) surrounded by root parenchyma. The dark lines above the bacterioid area represent vessels that penetrate from the uninjured root to the hypertrophied tubercle. penetrates through the root-hair into the root parenchyma as a bacterial fila- ment. Having advanced into the root, this filament begins to branch rapidly. A lively division of the cells of the root parenchyma proceeds at the same time, in the neighborhood of the bacterial filament, which results in a swelling in this region of the root and in the formation of a tubercle. The branches of the fila- ment occupy the central portion of the tubercle. The filament sheath finally disintegrates and the bacteria thus liberated enter the cell sap. Here they beijerinck, M. W., Die Bacterien der Papilionaceen-Knollchen. Bot. Zeitg. 46: 725-735. 741-750, 757-77L 781-790, 797-802. 1888. 2 Prazmowski, Adam, Die Wurzelknollchen der Erbse. I. Teil. Die Aetiologie und Entwickelungs- geschichte der Knollchen. Landw. Versuchsst. 37 : 161-238. 1890. ASSIMILATION OF NITROGEN 77 enlarge and become branched, thus becoming mature bacterioids. At this time the vascular bundles develop in the tubercle. The bacterioid tissue becomes depleted after a time, its contents being used up by the plant. The bacterial cells collect in groups in the remaining portions of the infection-filaments and become enclosed in a hard sheath. The spore-like colonies thus formed fall away after the destruction of the tubercle and are capable of infecting other roots the following spring. Kossovich 1 sought to solve the question as to what organs of legumes absorb atmospheric nitrogen. He carried out two series of experiments, in one case depriving the leaves, and in the other case the roots, of nitrogen. He came to the conclusion that nitrogen is absorbed by the roots. Infection of legumes with cultures of Bacterium radicicola does not always have a favorable influence upon the growth of these plants. If the inoculation occurs late in the growing season (in July), the result is an abundant formation of root tubercles, but the plants, instead of growing better, grow more poorly than do uninfected individuals. The action of the bacteria is merely parasitic in this case. Microscopic investigation shows that the transformation of bacteria into bacterioids does not occur here, and it was for this reason that Nobbe and Hiltner 2 believed assimilation of atmospheric nitrogen to be corre- lated with the formation of the bacterioids. Long-continued cultivation upon nutrient gelatine (from spring until midsummer) is said to make Bacterium radicicola more vigorous and to deter its transformation into bacterioids after it enters the root. Plants inoculated late in the season, being already partially exhausted at this time, are too weak to produce this change in the infecting organism. Investigation of the tubercle bacteria of various legumes leads to the conclu- sion that there are many varieties of these organisms. In order to obtain a healthy development of Robinia pseudacacia in soil without available nitrogen, inoculations must be made with cultures from Robinia tubercles; infection with bacteria from pea and lupine tubercles has no effect at all. But inoculation with cultures from Cytisus tubercles has almost as good an effect as inoculation with cultures of the bacteria of Robinia itself. 3 Certain non-leguminous plants also assimilate atmospheric nitrogen by symbiosis with bacteria, and the tubercles may be formed in other regions of the plant besides the root system. For example, the leaves of some of the tropical Rubiaceae are characterized by numerous rounded, tubercle-like thicken- ings, which contain peculiar bacterial cells (Mycobacterium rubiacearum) . These bacteria fix nitrogen from the air in the same general manner as do the root- tubercle bacteria of legumes. 4 (See Fig. 48.) 1 Kossowitsch, P., Durch welche Organe nehmen die Leguminosen den freien Stickstoff auf ? Bot. Zeitg. 50: 697-702, 713-723, 720-738, 745-756, 771-774- 1892. 2 Nobbe, F., and Hiltner, L., Wodurch werden die knollchenbesitzenden Leguminosen befahigt, den freien atmospharischen Stickstoff fur sich zu verwerten? Landw. Versuchsst. 42 : 450-478. 1893. 8 Nobbe, F., Schmid, E., Hiltner, L., and Hotter, E., Versuche iiber die Stickstoff-Assimilation der Leguminosen. Landw. Versuchsst. 39: 327-359. 1891. * [Faber, F. C. von, Das erbliche Zusammenleben von Bakterien und tropischen Pnanzen. Jahrb. wiss. Bot. 51: 285-375. 19 1 2.] •8 PHYSIOLOGY OF NUTRITION §6. Assimilation of Atmospheric Nitrogen by Bacteria. — The work of Berthelot 1 rendered assimilation of free nitrogen by the bacteria of the soil very probable, but we owe the final solution of this problem to Vinogradskii 2 and Beijerinck. 3 Vinogradskii caused the development of nitrogen-fixing A Pig. 48. — A. Leaves of Pavetla indica, showing nodules, which contain nitrogen-fixing bacteria. B. Cells of Mycobacterium rubiacearum from leaf nodules of Pavetta zimmermanniana. Magnified to 3000 diameters. (After Faber.) 1 Berthelot, Marcellin, Fixation de l'azote atmospherique sur la terre vegetale. Ann. chim. et phys. 13: S-14. 15-73. 74-78, 78-92, 93-H9. 1888. 8 Winogradsky, S., Sur l'assimilation de l'azote gazeux de l'atmosphere par les microbes. Compt. rend. Paris 116: 1385-1388. 1893. Idem, same title. Ibid. 118: 353-355- 1894. 'Beijerinck, M. W., Ueber oligonitrophile Mikroben. Centralbl. Bakt. //, 7: 561-582. 1901. Freund- enreich, Ed. von, Ueber stickstoffbindende Bakterien. Ibid. II, 10: 514-522. 1903. Lohnis, F., Beitrage zur Kenntnis der Stickstoffbakterien. Ibid. II, 14: 582-604, 713-723. 1905. Christensen, Harald, R., Ueber das Vorkommen und die Verbreitung des Azotobacter chroococcum in verschiedenen Boden. Ein BeitragzurMethodikdermikrobiologischen Bodenforschung. Ibid. II, 17: 100-119, 161-165, 378-383, 528. 1907. Bredemann, G. Regeneration der Fahigkeit zur Assimilation von freiem Stickstoff des Bacillus amylobacter A. M. et Bredemann und der zu dieser Spezies gehorenden bisher als Granulobacter, Clos- tridium usw. bezeichneten anaeroben Bakterien. Ber. Deutsch. Bot. Ges. 26: 362-367. 1908. Idem., Bacillus amylobacter A. M. et Bredemann in morphologischer, physiologischer und systematitscher Bezie- hung. Mit besonderer Berucksichtigung des StickstofTverbindungsveimogens dieser Spezies. Centralbl, Bakt. II, 23:385-568. 1909. ASSIMILATION OF NITROGEN 79 microorganisms by inoculating a grape-sugar solution with garden soil. In spite of the fact that this solution contained no nitrogenous compounds, a vigorous fermentation began immediately, with the formation of carbon dioxide, much hydrogen, and butyric and acetic acids, the process being accompanied by the fixation of atmospheric nitrogen. The amount of nitrogen combined was related to the amount of sugar used up, as is shown in the following table: Experiment number 1 2 3 4 Grams of sugar consumed 2.0 2.0 4.0 20 . o Milligrams of nitrogen fixed 3.9 5.9 9.7 2 8.o Addition of ammonium salts in very small amounts acted favorably; larger amounts retarded the fixation of nitrogen and finally stopped it altogether. The fixation of atmospheric nitrogen is possible only in substrata which are either entirely deficient in nitrogenous compounds or contain these only in very small amounts. The bacterium to which this fixation is due was named by its discoverer, Vinogradskii, Clostridium pasteurianum. It is an- aerobic, living without free oxygen. More recently Beijerinck has found another nitrogen-fixing bacterium, Azotobacter chroococum. Unlike the forms previously mentioned, this is aerobic and thrives best in the presence of air, where it also exhibits its ability to fix nitrogen. Furthermore, other investigators have found other soil microorgan- isms that possess, to a smaller degree, this power to assimilate free nitrogen. The fixation of atmospheric nitrogen is therefore a process that occurs commonlv in nature. §7. Assimilation of Nitrogen Compounds by Lower Plants. — We have seen that nitrates usually furnish the best source of nitrogen for higher plants. Of the lower plants without chlorophyll (moulds, yeasts, bacteria) not nearly all are capable of utilizing nitrates. To be sure, this property is possessed by most of the common moulds (Penicillium, Aspergillus and some species of Mucor) and one group of bacteria is sufficiently specialized to utilize nitrates as a source of nitrogen, at the same time reducing them vigorously, with elimi- nation of free nitrogen (denitrifying bacteria 1 ). Nevertheless, most lower plants require organic nitrogenous substances, or at least ammonium salts. Suitable culture media for such forms have already been referred to, and it has also been mentioned that these organisms are in great variety, as far as their nutrition is concerned. Summary 1. The Nitrogen of the Air.— By volume measurement, about four-fifths of the air is free nitrogen. Air also generally contains very small amounts of nitrogen in the form of ammonia. Free nitrogen cannot be assimilated by ordinary higher plants. Under natural conditions these plants may assimilate minute quantities of nitrogen from the ammonia of the air, but this source of nitrogen is generally quite negligible. 1 Laurent, E., Recherches sur le polymorphisme du Cladosporium herbarum. Ann. Inst. Pasteur 2 : 558-566, 581-603. 1888. Idem, Recherches sur la valeur comparee des nitrates et des sels ammomiacaux comme aliment de la levure de biere et de quelques autres plantes. Ibid. 3 : 362-374. 1889. Ritter.'G., Ammoniak und Nitrate als Stickstoffquelle fur Schimmelpilze. Ber. Deutsch. Bot. Ges. 27: 582-588. 1909. 8o PHYSIOLOGY OF NUTRITION 2. The Nitrogen of the Soil. — The soil contains free nitrogen (which cannot be assimilated by ordinary plants), ammonia and ammonium compounds, nitrates, nitrites, and organic nitrogenous substances. Nitrates in the soil are the main source of nitrogen for ordinary plants, though some forms are apparently able to assimilate some nitrogen in the form of nitrites or in the form of ammonia or ammonium salts. Organic nitrogen compounds are first decomposed (by soil microorganisms), giving nitrates, and then the resulting nitrates are assimilated by higher plants. Ammonium salts are similarly converted to nitrates in the soil, by microorganisms, as also are nitrites. Ammonium compounds and organic nitrogenous substances (arising from the decay of animal and plant tissues, etc.) are held in the soil in considerable amounts, but nitrates are readily washed out by percolating rain water, and carried away in the soil drainage. More nitrates are gradually formed, so that there is always a supply of these salts that may be absorbed through the roots of ordinary plants and assimilated. 3. Nitrification in Soils. — The production of nitrates in the soil, from other nitro- genous compounds (and from free nitrogen), occurs through the action of soil bacteria. For the activity of these nitrifying organisms a continuous supply of oxygen is neces- sary in the soil. According to Vinogradskii's work, ammonia and ammonium salts are assimilated by nitrite bacteria in the soil, which give off nitrites, and nitrites are assimil- ated by nitrate bacteria in the soil, which give off nitrates. These two groups of soil bacteria derive their carbon compounds by synthesis, from carbon dioxide or carbo- nates as source of carbon. The energy for this synthesis is derived from the oxidation of ammonia or of nitrites; the nitrites and nitrates that are produced may be considered as by-products. In the presence of organic compounds that may be readily oxidized (like sugars), these bacteria secure their carbon compounds directly, without synthesis from carbon dioxide, and they do not alter the organic nitrogenous compounds that may be present, nor does nitrification occur. Nitrogenous organic compounds are not assimilated by the nitrite and nitrate bacteria, but they are used by another group of soil bacteria, the ammonifying forms, which give off ammonia as a by-product. When bacteria of all three groups are present, the nitrogen of other nitrogenous compounds is ultimately converted, by three steps, into nitrate nitrogen. (1) The ammonifiers produce ammonium compounds (NH 4 ) from nitrogenous organic sub- stances. (2) The nitrite bacteria produce nitrites (N0 2 ) from ammonium compounds. (3) The nitrate bacteria produce nitrates (N0 3 ) from nitrites. Ammonium salts are generally not largely assimilated by ordinary plants, and ammonium nitrogen usually becomes readily assimilable only after nitrification, with formation of nitrates. Wagner found that ammonium salts were beneficial, as fertil- izer, in lime soils, but not in other soils. In lime soils the lime prevents the develop- ment of any considerable acidity. Nitrites are generally not largely assimilated by ordinary plants. 4. [6] Assimilation of Atmospheric Nitrogen by Soil Bacteria.— Still another group of soil bacteria assimilate free nitrogen, as was shown by Vinogradskii and Beijerinck, and these bacteria form organic nitrogen compounds or nitrates. The energy for this nitrogen fixation is derived from the oxidation or fermentation of organic compounds, such as sugars. Some of these nitrogen-fixing bacteria thrive in the presence of oxygen, others are inhibited by oxygen. There are also soil bacteria, thriving under special conditions, that convert nitrate nitrogen into nitrite or ammonium nitrogen, or even into free nitrogen, these being denitrifying processes. 5. Fixation of Free Nitrogen by Tubercle Bacteria. — Although ordinary plants are not able to assimilate free nitrogen, there are certain groups of them (especially the ASSIMILATION OF NITROGEN 8 1 legumes) that appear to do so. Hellriegel and Wilfarth showed that the roots of legumes are normally infected with nodule or tubercle bacteria, which remain in the soil from season to season, infecting the new plants each year. The same experimenters showed that these microorganisms carry on nitrogen fixation in the structurally characteristic root-tubercles that result from their invasion of the legume root tissues. The tubercle bacteria apparently derive carbohydrates from the host, secure utiliz- able energy through the oxidation of these substances, and use some of this energy for the formation of nitrates or nitrogenous organic compounds from carbohydrates and free nitrogen. Nitrates, or organic nitrogenous compounds, are given off by the bacteria and these substances are assimilated by the host plant. Legumes may thus grow well in soils with very small supplies of nitrates, or none at all, deriving their nitrogen from the free nitrogen of the soil, through the activities of the tubercle bacteria. In the presence of considerable supplies of soil nitrates this fixation of free nitrogen is slight or does not occur. Different legumes have different nodule bacteria; the right form of the latter must be present in the soil for any given legume. Free soil nitrogen may, therefore, be fixed (as nitrates, etc.) (i) through the action of nitrogen-fixing bacteria in the soil (see 4, above), or (2) through the action of tubercle bacteria in root nodules. Some other groups of higher plants have tubercles with nitrogen-fixing bacteria; for example, Pavetta (Rubiaceae), with lea} tubercles in which atmospheric nitrogen becomes fixed. 6. [4]. Circulation of Nitrogen in Nature. — Free nitrogen is converted into the nitrogen of nitrates and organic nitrogen compounds by the nitrogen-fixing bacteria of the soil and by tubercle bacteria. Some free nitrogen is converted into ammonia nitrogen by the action of atmospheric electricity, the ammonia finding its way into the soil, where its nitrogen is converted into nitrite nitrogen by the nitrite bacteria. Nitrites are changed to nitrates by nitrate bacteria in the soil. Nitrates (and, to some extent, ammonium compounds and nitrites) are assimilated by higher plants and disappear in the formation of complex nitrogenous organic compounds. Animals secure their nitrogen from these complex plant compounds, or from other animals. When animal and plant tissues decay, ammonia and free nitrogen result. Free atmospheric nitrogen can be combined with other elements artifically, as in the production of calcium cyanamide (CaCN 2 ). 7. Assimilation of Nitrogen Compounds by Lower Plants. — Many representatives of the moulds, yeasts, and bacteria are unable to assimilate nitrates, and must be supplied with organic nitrogenous substances, or at least with ammonium salts. Animals require organic nitrogen compounds, which they secure from plants or other animals. CHAPTER IV ABSORPTION OF ASH-CONSTITUENTS §i. Cultures in Artificial Media. — Besides the four elements, carbon, hydrogen, oxygen and nitrogen, every organ of the plant contains many other elements, the so-called ash-constituents. The four constituents just named volatilize and are lost during incineration, but more or less ash always remains. According to Knop, the average amount of ash left after burning plant tissue is about 5 per cent, of the original dry weight. The following elements have been found in the ash of plants: Sulphur Potassium Zinc Selenium Phosphorus Sodium Mercury Manganese Chlorine Lithium Aluminium Iron Bromine Rubidium Thallium Cobalt Iodine Magnesium Titanium Nickel Fluorine Calcium Tin Copper Boron Strontium Lead Silver Silicon Barium Arsenic Experiments with plant cultures in artificial media show that only a few of these elements of ash are essential to normal growth. Cultures may be prepared by using either a neutral solid medium to which various salts are added, or by dissolving the respective salts in water and employing the solu- tion thus formed. Clean quartz sand, ground pumice or ground charcoal may be used as solid media, or even finely divided platinum-wire, but the latter is very expensive. Quartz sand with various salts is most frequently used. The method of water-cultures has been well worked out in many researches dealing with the necessity of various substances for plant growth, but espe- cially in the work of Knop and Nobbe. 1 The study of artificially controlled cultures has shown that plants need the following elements in salts, for normal growth: nitrogen, sulphur, phosphorus, potassium, calcium, magnesium and iron, and sometimes chlorine also. These essential elements may be supplied to the plant as salts in water solu- tion, in the following proportions by weight: one part of KNO3, one part of KH 2 PO$, one part of MgS0 4 , and four parts of Ca(N0 3 ) 2 . A trace of ferric phosphate is also added. The addition of a nitrogen compound to the culture medium is necessary although nitrogen is not one of the ash-constituents, for plants obtain their nitrogen from the soil, as has been seen in the preceding chapter. This particular nutrient solution is known as Knop's solution. The concentration must be very low ; as long as the plants are still young, o. 1 per cent . ' Knop, Wilh., Der Kreislauf des Stoffes. Lehrbuch der Agrikulturchemie. Leipzig and St. Petersburg. 1868. P. 572-663. » 82 ABSORPTION OF ASH-CONSTITUENTS 83 suffices, but the concentration may be raised later to 0.5 per cent." The seed for the experiment may be germinated in distilled water. 6 As soon as the root has reached a suitable length, the seedling is transferred to the nutrient solution, being fixed in a perforated cork stopper with cotton packing, so that only the root reaches into the solution (Fig. 49). The culture-bottle should be protected from light, to retard or prevent the development of algae and other organisms, and the vessel is therefore covered with a paper cylinder. Care must be taken that the culture solution does not become alkaline during the growth of the plants. To prevent alkalinity a solution of phosphoric acid may be added to the culture solution so as to make it weakly acid. c Normal plants, producing flowers and fruit, can be obtained in such water cultures by observing all the necessary precautions. Salts that may be used in water-cultures are divided into two groups, those that are physiologically alkaline and those that are physiologically acid. To the first group be- long salts whose anions are absorbed by the plant more Fig. 49. — Water culture of maize seedling. This means 0.5 g. of all the salts taken together, dissolved to make 100 cc. of solution. — Various other four-salt, and some five-salt, solu- tions have been employed by various workers. For a list of these, see: Grafe, Viktor, Ernahrungsphysiologisches Praktikum der hoheren Pflanzen. Berlin, 1914, p, 56 el seq. The simplest solution yet de- vised for this sort of experiment is that of Shive, which contains but three salts (calcium nitrate, mono-potassium phosphate and magnesium sulphate) besides the iron phosphate. See: Shive, J. W., A three-salt nutrient solution for plants. Amer. jour. bot. 2: 157- 160. 1915. Idem, A study of physiological balance in nutrient media. Physiol, res. 1: 327-397. 1915. — Ed. b Distilled water is unsuitable for seed germination and for the growth of plants, because (1) it may contain small traces of toxic sub- stances — which are more influential in the absence of nutrient salts than in their presence — and (2) it acts to remove salts from the seeds and young seedlings by outward diffusion. See, in this connection: True, R. H., and Bartlett, H. H., Absorption and excretion of salts by roots, as influenced by concentration and composi- tion of culture solutions. U. S. Dept. Agric, Bur. Plant Industry. Bull. 231. 1912. True, R. H., Harmful action of distilled water. Amer. jour. bot. 1 : 255-273. 1914. Merrill, M. C, Some relations of plants to distilled water and certain dilute toxic solutions. Ann. Missouri Bot. Gard. 2: 459-506. 1915. Idem, Electrolytic determination of exosmosis from the roots of plants subjected to the action of various agents. Ibid. 2: 507-572. 1915. For earlier work on the physiological properties of distilled water, see: Livingston, B. E., Further studies on the properties of an unproductive soil. U. S. Dept. Agric, Bur. Soils. Bull. 36. 1907. It is probably best to allow germination to occur in a properly balanced nutrient solution, frequently renewed. — Ed. Frequent renewal of the solution is necessary in any case, and this avoids any need for adding acid. The salt proportions and total concentration of a nutrient solution may be maintained throughout the period of a solution-culture experiment by allowing the solution to flow continuously through the culture jar. (See: Trelease, Sam F., and Livingston, Burton E., Continuous renewal of nutrient solution for plants in water-cultures. Science n.s. 55 : 483-486. iQ22.).—Ed. 84 PHYSIOLOGY OF NUTRITION rapidly than are their kations, thereby rendering the culture solution alkaline. Potassium nitrate (KN0 3 ) is an example of these. To the second group belong those salts whose, kations are absorbed more rapidly than are the anions, thus giving the nutrient medium an acid reaction. Ammonium chloride (NH 4 C1) and ammonium sulphate [(NH 4 ) 2 S0 4 ] are physiologically acid. The injurious effects of these salts are prevented by certain reactions in complex agricultural soils, but in sand or water cultures account must be taken of these phenomena. §2. Importance of the Essential Ash-constituents. 1 — Not much is known concerning the importance of the single ash-constituents. d Of some it can be said only that their absence results in re- tardation of plant development. Two buckwheat plants are shown in Fig. 50, one of which has been grown in a solution containing all the essential elements and exhibits an entirely healthy appearance, while the other, cultivated in a nutrient solution lacking potassium, has hardly developed at all. The difference in growth is very great, although the dry substance of the normally grown buck- wheat plant contains only about 2.5 per cent, of potassium. 1 Berthelot, M., Chimie vegetale et agricole. Paris, 1899. Tome IV.* Mayer, A., 1901-1902. [See note 1, P- 33- d For modern studies on the relation between plant growth and the salt proportions and total concentration of the nutrient solution see: Totting - ham, W. E., A quantitative chemical and physio- logical study of nutrient solutions for plant cultures. Physiol, res. 1 : 133-245. 1914. (This includes a very thorough study of Knop's solution and a re- view of the literature.) Shive, 1915, 1, 2. [See note a, p. 83.] The whole subject of the necessity of the various elements for plant growth is well discussed by Russell, 1915. [See note i, p. 73.] The relations between plant growth and the supply of mineral salts may be studied also by using the solution-culture method and three or more single-salt solutions supplied separately, in rotation. This had been attempted, without suc- cess, at the Laboratory of Plant Physiology of the Johns Hopkins University, and it remained for Gericke to succeed at the University of California. (See Gericke, W. F., Water culture experimentation. Science n.s. 56:421-422. 1922.) Gericke obtained good growth of wheat with 0.0 1 volume-molecular solutions for KNO3, CaS0 4 , and MgHP0 4 , the solution rotation being four days for the first solution and one day for each of the other two. A very small amount of iron was supplied in the otherwise single-salt solutions. This method deserves further attention. B A Fig. 50. — Buckwheat plants in water- culture. A, with potassium; B, without potassium. ABSORPTION OF ASH-COXSTITUENTS 85 Sulphur is a necessary element because it is essential to the formation of proteins, which are so important in plants. It must be supplied as the sulphate of one of the essential metals; all other compounds of sulphur are injurious. It cannot be replaced by any other element. Phosphorus also is necessary. It is a constituent of nucleins (a special group of proteins), and of phosphatides. It may be introduced in the solution only as one of the phosphates of the tribasic acid (H3PO4), since other phosphorus com- pounds have been found to be harmful. It cannot be re- placed by any other element. Potassium is also abso- lutely essential. It accom- panies carbohydrates and is supposed to promote their formation. Calcium is likewise neces- sary, especially for normal leaf development. Some plants without chlorophyll (moulds) can exist without calcium, 1 and non-green phanerogams contain much less calcium than do green plants. 2 Magnesium is also neces- sary; it accompanies pro- teins and is contained in chlorophyll. Finally, plants need iron, the lack of which prevents chlorophyll formation; they become pale and chlorotic, 3 even in the light, when grown without this element. §3. Importance of the Non-essential Ash-constituents. — Plant ash contains appreciable quantities of other elements than the absolutely essential ones, and these are not to be considered as entirely without physiological effects. Each ash-constituent must be considered as exerting some slight effect in the plant, either injurious or beneficial. If plants develop apparently normally in a nutri- ent solution without a given element, it does not necessarily follow that this element, if present might not exert some beneficial influence. Silicon, for example, is abundant in many plants. Nevertheless, experi- ments with various plants in artificial media have shown that even the grasses 1 Loew, Oscar, Liming of soils from a physiological standpoint. U. S. Dept. Agric. Bull. I. 27 p. Washington, 1901. 2 Aso, K., On the lime content of phanerogamic parasites. Bull. Coll. Agric. Imp. Univ. Tokyo 4: 387-389- 1900-1002. « Molisch, 1892. [See note b, p. 51.] Pig. 51. — Portion of a cross-section through a rye stalk. At left, lodged; at right, normal. (After Koch.) 86 PHYSIOLOGY OF NUTRITION (Gramineae) can develop without this element. The lodging of grain (when the plants fail to stand erect in the field), which was earlier ascribed to a deficiency of silicic acid (H 2 Si0 3 ) in the soil, is a result of insufficient illumination. This, in turn, is due to too thick planting. Anatomical study 1 of the stalks of lodged grain shows that they have all the characteristics of etiolated stems (Fig. 51). In healthy stems we find small, thick-walled cells, while in etiolated stalks, whether lodged or not, the cells are very large and have much thinner walls. In laboratory experiments, where plants are protected from some of the unfavorable conditions of the field, silicon is not essential, but this is not true when plants develop under natural conditions. Here silicon appears to play a very important role, protecting the plant from attacks of various parasites. Fungus hyphae cannot easily penetrate cell walls that are impregnated with silica. Wheat, rye, etc., grown in nutrient solutions deficient in silicic acid often suffer so severely from rust that only great care can prevent their complete destruction. The hardness of silicated cell walls is also a very good protection against animal attack. Thus, for instance, one plant of Lithospermum arvense, grown in a nutrient solution without silicic acid, suffered severely from plant- lice even though these were removed daily, while two similar plants, standing near by and grown in similar solutions but not tended so carefully, were completely killed by these insects. The distribution of silicic acid in different parts of seeds 2 is another indi- cation of its protective action. Millet seeds without the seed-coats contain only from 4.8 to 7.1 per cent, of the total silicic acid of the seed, all the re- mainder (from 92.6 to 95.1 per cent.) being deposited in the seed-coats. Such a marked accumulation of silicic acid in the seed-coats suggests the impor- tance of this substance to plants growing under natural conditions. The investigations of Sabanin upon ripening seeds of millet show that this plant hastens, as it were, to accumulate enough silicic acid in the peripheral parts of the grain (as in the palea) to protect the increasing reserve material from unfavorable external conditions. Most plants can live without chlorine, but buckwheat deprived of this element did not attain complete development in Nobbe's experiments, and it was his opinion that chlorine favors the translocation of carbohydrates from the leaves into other organs. Knop, however, obtained normal development of buckwheat plants in a solution without chlorine, and so the question of the role of chlorine is still unsettled/ It is advisable to add chlorine to the nutrient 1 Koch, L., Welche abnorme Aenderungen werden durch Beschattung in wachsenden Pflanzenorganen hervorgerufen? Landw. Centralbl. Deutschl. 20: 202. 1872. = Sabanin, A. N., Ueber Kieselsaure in den Kornern der Hirse (Panicum miliaceum L.) [Abstract in German, pp. 295-302. Text in Russian.] Jour. exp. Landw. 2: 257-302. 1001. e Buckwheat has been repeatedly grown to maturity, with production of seed, in water- cultures without any more chlorine than might have been present in spite of all ordinary pre- cautions to exclude this element, in the Laboratory of Plant Physiology of the Johns Hopkins University; but the possibility remains that the presence of chlorine might produce more vigorous growth. Trelease's results strengthen the idea that this element is not beneficial to wheat in its early stages of growth. It exerted no injurious influence, however, in his cultures. (See: Trelease, Sam F., The relation of salt proportions and concentrations to the growth of young wheat plants in nutrient solutions containing a chloride. Philippine jour. sci. 17:527-603. 1920.). — Ed. ABSORPTION OF ASH-CONSTITUENTS 87 solution when experimenting with plants whose relation to chlorine is not under- stood; potassium chloride is best for this purpose. Observations of agri- culturists favor the idea that chlorine influences the translocation of carbo- hydrates under natural conditions. Potatoes grown in soil rich in chlorine contain less starch than those cultivated in soil deficient in this element. So, when potatoes with the highest possible starch content are desired chlorine fertilizers are to be avoided. 1 Zinc is one of the less common ash-constituents. It is contained in a variety of violet (Viola calaminaria or V. lutea var. muUicaulis), which grows exclusively in soils containing zinc. The differences by which these "calamin" violets are distinguished from the ordinary Viola tricolor are probably due to the effect of the zinc salt/ Also, Raulin used zinc in his nutrient solution (see page 46) for Aspergillus niger. Rikhter's 2 investigations showed that zinc promoted growth and the accumulation of organic substances during the early period of development of this mould, but prevented the formation of spores. Kostychev 3 also found that zinc influenced metabolism in moulds. Aluminium occurs in plant ash rather infrequently. It influences the color of the flowers in Hydrangea (II. hortensis).* Gardeners had long since noticed that the ordinary reddish-flowered hydrangea bore blue flowers when grown in certain soils, such as some forest and moor soils. Tests of many different substances showed that blue flowers always appeared if the soil con- tained soluble aluminium compounds. At first ordinary alum ( made up of aluminium and potassium sulphate, A1 2 S0 4 + K 2 SOi + 24H2O) was used, being introduced into the soil in pieces varying from the size of a pea to that of a hazel-nut, and blue flowers were always obtained. In another series of experi- ments, some plants were treated with aluminium sulphate and others with potassium sulphate. The cultures with potassium sulphate gave the usual red color, while those with aluminium sulphate always produced blue flowers, and the color appearing with this salt was more intense than that obtained by the alum treatment. The alum therefore produced the blue color because of the presence of aluminium, the potassium being without influence. This case shows clearly how the presence of a non-essential element may influence metabolism in a specific manner. Researches in recent years have shown that various elements, such as manganese, boron, rubidium, etc., are more or less favorable to plant growth. 1 Budrin, Die kiinstlichen Dungemittel mit besonderer Berucksichtigung der Stickstoffdunger. W T arsaw, 1888. (Russian.)* [See also: Tottingham, Wm. E., A preliminary study of the influence of chlorides upon the growth of certain agricultural plants. Jour. Amer. Soc. Agron. 11: 1-32. 1019.] - Richter, Andreas, Zur Frage der chemischen Reizmittel. Die Rolle des Zn and Cu bei der Ernahrung von Aspergillus niger. Centralbl. Bakt. //. 7- 417-429. iQOi. 3 Kostytschew, S., Der Einfluss des Substrates auf die anaerobe Athmung der Schimmelpilze. Ber. Deutscb,. Bot. Ges. 20: 327-334- 1902. 1 Molisch, Hans, Der Einfluss des Bodens auf die Bluthenfarbe der Hortensien. Bot. Zeitg. 55 : 40-61. 1897. 'But the studies of Hoffmann appear to controvert this statement. According to this author the calamin violet is the same whether grown with or without zinc, and Viola tricolor does not take the calamin form when supplied with zinc. See: Hoffmann, H., Culturver- suche. Bot. Zeitg. 33: 601-605, 617-628. 1875. Idem, Untersuchungen iiber Variation. Ber. Oberhess. Ges. Giessen 16: 1-37. 1877. — Ed. 88 PHYSIOLOGY OF NUTRITION These elements act like catalyzers, 1 while the plastic ash-constituents (phos- phorus, sulphur, potassium, magnesium, calcium) have to do with the structure of the cell and its parts; these latter may also act as catalyzers, however. §4. Ash-analysis of Plants.- — Besides the growing of plants in artificial media, the analysis of plants grown under natural conditions is also useful in the determination of the relative importance of the various mineral elements. Large numbers of such analysis have been carried out, and the results obtained up to 1880 have been assembled and arranged in a very helpful way by Wolff. 2 The ash-analyses of entire plants show that the amount of each individual ash-constituent is different with different plants. The agriculturist, for ex- ample, recognizes three groups of cultivated forms, silicon plants, calcium plants and potassium plants, according to which one of these three elements is most abundant in the ash. The following table (after Liebig) contains the results of ash-analyses of some of the plants belonging to these three classes. Salts of K and Na Silicon plants Calcium plants Potassium plants f Oat straw and grain . . . \ Rye straw Havanna tobacco Stems and leaves of pea Sugar cane Artichoke per cent. 34.00 18.65 24-34 27.82 88.80 84.30 Salts of Ca and Mg per cent. 4.00 16.52 67.44 63-74 12.00 i5-7o Silicic Acid per cent. 62.08 63.89 S.30 7.81 The total amount of ash is also known to be different in different species. Water plants are richest, woody plants are among the poorest, and herbs take a middle place, with reference to the amount of ash they contain. A comparison of the ash-analyses of the alga Chara and the tree Fagus (beech) is shown in the next table. Entire Ash Content, Per Cent, of Dry Weight Amounts of Various Elements in Ash Calculated as Oxides, Per Cent, of Total Ash K 2 CaO MgO Fe 2 2^3 P 2 2^5 SO3 SlOo Chara fcetida . . . Fagus sylvalica Wood Bark Leaves 39.080 0-355 5.860 5-i4o 0.40 96.23 1-39 0.28 0.28 0.49 14.40 60.20 4-5o 2.30 2.70 3-50 5.10 83.40 3.60 0.70 2.10 1 .00 21.80 44-30 7.20 2.30 7.80 2.40 0.58 10.00 3 -7o 10.50 1 Agulhon, H., Recherches sur la presence et le role du bore chez les v6g6taux. Paris, 1910. * Wolff, Emil, Aschen-Analysen von landwirthshaftlichen Producten, Fabrik-abfallen und wildwach- senden Pflanzen. I Theil. Berlin, 1871. Idem, Aschen-Analysen von land- and forstwirtschaftlichen Producten II Theil. Berlin, 1880. ABSORPTION OF ASH-CONSTITUENTS 8 9 This distribution of the ash shows that the tissues richest in ash are those in which living cells are most numerous, such as those of algae and the leaves and cortex of the beeech. Dead cells contain much less ash, since the salts begin to pass out at about the time death occurs; thus, the hard wood of the beech contains much less than does the dry substance of the living leaf tissue. Different amounts of ash occur in different organs of the same plant. Leaves are richer in ash than stems and roots. The amounts of the different chemical elements likewise vary; calcium, for instance, predominates in leaves. The ash content of each organ changes during the course of its development; in leaves it increases with age, while in roots and stems it decreases. In the case of roots and stems the number of dead cells, poor in ash, increases with age. The following table gives the total ash content and the proportions of the vari- ous elements in the ash, for beech leaves (Fagus sylvatica) at three different stages of their development. Date Total Ash, Per Cent, of Dry Weight May 16 July 18. Oct. 15. 41 4-7 7-1 Amounts of Various Elements in Ash, Cal- culated as Oxides, Per Cent, of Total Ash K 2 42.1 17. 1 7-i CaO 13-8 42.3 50.6 MgO Fe 2 3 P 2 5 j 4-3 0.8 32-4 5-6 1.4 8.2 4-i i-3 5-i Si0 2 1.6 21.3 30-5 These analyses of beech leaves show how strikingly the amount of the differ- ent ash-constituents alter with the age of the leaves. Calcium and silicon show a marked increase in amount while potassium and phosphorus decrease as the leaves become older. But, as has been well pointed out by Wehmer, 1 it is not to be concluded from these analyses that the absolute amounts of potassium and phosphoric acid diminish in such leaves. For example, if 50 g. of potassium and 50 g. of other elements were present in a certain quantity of young leaves, we should then find 50 per cent, of potassium in the ash. If we suppose that the leaves take up 100 g. more of the other elements but that the amount of potassium remains unchanged, then we should expect to find only 25 per cent, of potassium in the ash of the older leaves. According to Riesmuller's anal- yses, the ash of 1000 beech leaves contained, at different times of the year, the percentages and absolute amounts of potassium shown in the following table. 1 Wehmer, C, Zur Frage nach der Entleerung absterbender Organe, insbesondere der Laubblatter. Unter Beruchsichtigung der vorliegenden Aschenanalysen vom kritischen Standpunkte beleuchtet. Landw. Jahrb. 21 : 513-569- 1892. 9° PHYSIOLOGY OF NUTRITION Time of Analysis May June July August. . . October. . . November Absolute Amount of Potassium grams 0.7 1.2 1 .2 1 . 1 0.8 0.7 The percentage content of potassium in the ash underwent a marked decrease during the course of the summer, but no corresponding decrease in the absolute amount of potassium is apparent. The absolute amount is maintained fairly- constant during the growing period, and undergoes a marked decrease only in late autumn. Similar results were also obtained for phosphoric acid (PO4). §5. Microchemical Ash-analysis. 3 — Ash-analyses of the kind just referred to can be carried out only with large amounts of material, but in exact studies of the distribution and translocation of ash-constituents small quantities must suffice, and microchemical analysis is resorted to in such cases. 1 Platinic chloride is used for the identification of potassium, beautiful crystals of potas- sium chloroplatinate being formed (Fig. 52). To identify calcium, dilute sul- 1 Haushofer, K., Mikroskopische Reaktionen. Braunschweig, 1885. Kle merit, Constantin, and Renard, A., R6actions microchimiques a cristaux et leur application en analyse qualitative. 132 p. Brux- elles, 1886. Schimper, A. F. W., Zur Frage der Assimilation der Mineralsalze durch die gnine Pflanze. Flora 73: 207-261. 1890. P. 207. [Zimmerman, A., Die botanischen Mikrotechnik. Tubingen, 1892. Idem. Botanical microtechnique, a handbook of methods for the preparation, staining, and microscopical investigation of vegetable structures. Translated by J. E. Humphrey. XII + 296 p. New York, 1893. Richter, O., Die Fortschritte der botanischen Mikrochemie seit Zimmermann's Botanische Mikrotechnik. Sammelreferat Zeitsch wiss. Mikroskopie 22 : 1904-261. 1905. Emich, F., Lehrbuch der Mikrochemie. Wiesbaden, 1911. Molisch, Hans, Mikrochemie der Pflanze. Jena, 1913.] On these methods for ash-analysis the reader is referred to Molisch, 1913, cited just below. The following points may be of value in connection with the discussion given in the text. The reaction given for potassium fails to distinguish between potassium and ammonium. (On this difficulty see: Weevers, Th. I., Untersuchungen iiber die Lokalization und Funktion des Kaliums in der Pflanze. Recueil trav. bot. neerland. 8: 289. 1911.) When calcium is plentiful the crystals mentioned occur in dense masses, so that their individual form is seen only at the periphery of the mass. The reaction here given for iron serves only to identify it when in the ferrous condition. For other tests for this element in inorganic compounds see Molisch, 1913. In organic compounds {masked iron) it cannot be identified by any known microchemical methods. (See: Wiener, Adele, Microchemical proof of iron, especially masked, in plants. Rev. in: Chem. abstracts 11: 615-616. 1917. [Original not seen; cited as: Biochem. Zeitsch. 77: 27-50. 1916].) To identify phosphorus in organic com- pounds it is necessary first to incinerate the material, after which the test given may be applied. The precipitation of the phosphate ion as ammonium-magnesium phosphate (see under magnesium) offers a more sensitive method, not affected by the presence of organic substances. (See Molisch, 1913.) The tests for sulphur given in the text apply only to sulphates and are, moreover, not reliable for plant tissues. There is no microchemical test available for sulphur as it is usually encountered in plant cells. A more reliable test for chlorides is that of Macallum. (See: Macallum, A. B., On the nature of the silver reaction in animal and vegetable tissues. Proc. Roy. Soc, London B 76: 217-229. 1905.) — Ed. ABSORPTION OF ASH-CONSTITUENTS 91 phuric acid is added, which forms needle-like crystals of calcium sulphate (gyp- sum) in the presence of this element (Fig. 53). Magnesium crystallizes, as ammonium-magnesium phosphate (in a great variety of forms), upon the Fig. 52.— Crystals of potassium chloroplatinate. Fig. 53.— Crystals of calcium sulphate. addition of sodium phosphate and ammonia (Fig. 54) . Iron is identified by the blue color produced with potassium ferrocyanide. Phosphates are identified by treatment with a solution of ammonium molybdate in nitric acid, greenish- Fig. 54. — Crystals of ammonium magnesium phosphate. Fig. 55. — Crystals of ammonium phospho- molybdate. yellow crystals of ammonium phospho-molybdate being formed and gradually becoming bright green (Fig. 55). Upon addition of strontium nitrate, sulphur separates out as small rounded crystals of strontium sulphate (Fig. 56). An- ^0 0°^ Fig. 56— Crystals of strontium sulphate. Fig. 57.— Crystals of thallium chloride. other test for sulphuric acid is the addition of caesium chloride and aluminium chloride, which leads to the formation of large crystals of caesium-alum. Chlor- ides may be identified by adding thallium sulphate, with the formation of characteristic crystals of thallium chloride (Fig. 57). 9 2 PHYSIOLOGY OF NUTRITION §6. The Plant and the Soil.* — Plants obtain all their essential ash-constitu- ents from the soil. The following table gives an idea of the compositions of several different kinds of soil, the numbers representing the amounts of usual soil bases, calculated as oxides and expressed as percentages of the total dry weight of the soil. Loam Loamy Marl Lime Marl 40.70 11.80 32.00 10.60 8.90 1.50 6.00 47.00 1 .20 0.20 0.05 O.IO Si0 2 . Al 2 O a Fe 2 0; CaO. MgO K 2 0. 51-52 17-93 7.42 i-57 7.27 4.10 Every soil covered with vegetation contains organic as well as mineral sub- stances. Bog soils are particularly rich in organic materials, as is evident from the following table, which again presents percentages on the basis of the dry weight of the soil. P 2 5 N Humus Black soil, Government of Orlov, Russia Black soil, Government of Saratov, Russia Soil of low moor Soil of high moor 0.128 0.223 0.250 0.090 0.268 0.607 3-230 1 .060 13.080 14.580 82.560 91.470 The chemical analysis of a soil can give no definite idea of its properties, however.* In order to predict a good crop from a given soil, it is not enough to know that it contains potassium, phosphorus and the other essential elements; it must also be known whether these elements occur in compounds that plants can assimilate. Nile silt, famous for its fertility, contains only 0.5 per cent, of potassium and needs no further addition of this element, but mica-schist soil contains 3 per cent, of potassium and remains unproductive unless a potassium fertilizer is added. To obtain a better idea of the productiveness of a soil, the analysis of its water or hydrochloric acid extract is carried out, in addition to determining the essential minerals present. The necessary elements for plant growth are con- tained in very small quantities in the extract, but it must be borne in mind that * An excellent treatise on the soil is: Mitscherlich, E. A., Bodenkunde fur Land- und Forstwirte. 2 Aufl. 317 p. Berlin, 1913. A less scientific treatise is: Hilgard, E. W., Soils, their formation, properties, composition, and relations to climate and plant growth in the humid and arid regions. 593 p. New York, 1912. Best of all presentations of the soil, from the standpoint of plant physiology, is that of Russell (1921). [See note i, p. 73I. — Ed. 1 Cameron, F. K., The soil solution. 136 p. Easton, Pa. 191 1. — Ed. ABSORPTION OF ASH-CONSTITUENTS 93 not nearly all of the materials thus extracted from the soil can be assimilated by the plant, and also that much material that the plant might eventually absorb is not thus extracted. It must also be emphasized that plant species differ very greatly in their power to absorb salts from the soil. If the soil does not contain the essential elements in a sufficient amount and in the proper form for assimilation by plants, its productiveness may be in- creased by the addition of suitable fertilizers. The gain that may be obtained from the use of the fertilizer depends not only upon the properties of the latter but also upon those of the soil and upon the plant species that is to be culti- 63 39 36 2 Fig. 58. — Effect of fertilizing oats with different kinds of Thomas slag (1-3) and with phosphorite (4), all showing different solubilities of their phosphates in ammonium citrate solution. The relative solubilities of the phosphates are shown by the numbers below the pots. Culture 5 received no addition. (After P. Wagner.) vated. For example, let us consider phosphatic fertilizers. Thomas slag is one of the best of these. It is a by-product derived from the manufacture of steel out of pig iron. The latter contains silicic acid, sulphur and phosphorus, which are oxidized, through the addition of lime in the process, to calcium salts, and these rise to the surface of the molten steel as slag. Such slag varies accord- ing to the solubility of its phosphoric acid in an acid solution of ammonium citrate. The varieties with large amounts of phosphates that are soluble in ammonium citrate are good fertilizers, while other varieties are not useful in this way. 94 PHYSIOLOGY OF NUTRITION This is shown by Wagner's experiments 1 with oats (Fig. 58). Three culture vessels received equal amounts of phosphoric acid (0.5 g.) as pulverized Thomas slag; but different kinds of slag were used, showing different solubilities of their phosphates in ammonium citrate solution. The fourth vessel received twice as much phosphoric acid (1.0 g.), in the form of pulverized phosphate rock (phosphorite), and the fifth received no phosphorus fertilizer at all. The fol- lowing table shows the effects of these fertilizers upon the growth of the plants. Culture No. 1 2 3 4 5 Phosphoric Acid Added grams 0.5 0.5 1 .0 Kind of Fertilizer Thomas slag Thomas slag Thomas slag Phosphorite . No fertilizer. Solubility in Ammonium Citrate per cent. 65 39 36 Yield grams 416.7 306.9 281. 1 1590 144.0 Gain Due to Fertilizer per cent. 272.7 162.9 137.1 15.0 This experiment shows very clearly how fertilizers may differ in quality. Although the fourth culture contained more phosphoric acid than any of the others, its yield exceeded that of the unfertilized plants by only about 15 g. ; the plants could not assimilate this particular phosphorus compound. It appears that the greater the amount of phosphorus compounds that can be dissolved out of the fertilizer by ammonium citrate solution, the better can the fertilizer be utilized by the plant and the greater is the yield. Not only the properties of the fertilizer, but also the peculiarities of the plants under cultivation must receive attention. The same fertilizer, added to a gTven soil, may be beneficial to one plant and entirely useless to another. In Prianishnikov's experiments, 2 for instance, various plants were cultivated in sand supplied with the necessary nutrient salts. In one series of experiments phosphorus was supplied as mono-sodium phosphate (NaEUPOO, in the other as phosphate rock (phosphorite), which contains calcium phosphate, calcium carbonate, sand, loam, iron oxide, and aluminium. Millet grown in these two media gave a yield of 29.07 g. with the soluble phosphate and one of only 0.57 g. with phosphate rock (Fig. 59). Millet and other grains either cannot utilize phosphorite in sand cultures at all, or else they can utilize it only to a very slight degree. The Papilionaceag (peas, beans, etc.), however, show an entirely dif- ferent behavior toward phosphate fertilizers. Scarcely any difference can be discovered between pea cultures supplied with soluble phosphates and those supplied with phosphorite (Fig. 59). The value of phosphate rock as a fertilizer depends not only upon the nature 1 Wagner, Paul, Dungungsfragen unter Beriicksichtigung neuer Forschungsergebnisse. Heft III. 56 p. Berlin, 1896. 2 Prianishnikov, D. N., 1st die Phosphorsaure der Mineralphosphate der Kulturpflanzen zuganglich? [Russian, with German abstract.] Ann. Inst. Agron. Moscou 5 (Partie non officielle): 90-110. 1899. ABSORPTION OF ASH-CONSTITUENTS 95 of the plant but also upon that of the soil. That the small grains fail to assimi- late phosphorite in sand cultures does not necessarily mean that they behave in the same way in cultures with other kinds of soil. In Prianishnikov's experi- ments summer-rye was grown in black soil from the Government of Voronezh, in light sandy loam from the Government of Minsk, and in two light-colored, uncultivated sands ("Podsol") from the vicinity of Moscow, all four soils being fertilized with phosphate rock. His results are presented in the following table. NaH 2 P04 Phosphorite NaH 2 PO* Phosphorite Fig. 59. — Comparative effects of sodium phosphate and of phosphorite upon millet and pea in sand cultures. (After Prianishnikov.) Soil Yield of Grain Unferti- lized Fertilized with Phosphorite Total Weight of Grain and Straw Unferti- lized Fertilized with Phosphorite grams grams grams grams Black soil 1-95 2.30 5.6s 5-8o Sandy loam .... 1-25 1 -5° 3-55 4.40 Sand No. 1 0.40 4-75 3-3° 10.75 Sand No. 2 1 .40 3-3° 2-35 11 .10 Increase in Yield Due to Fertilizer per cent. 3 24 226 372 9 6 PHYSIOLOGY OF NUTRITION Phosphorite fertilizers had very good effects upon the uncultivated sands (Podsol), but no effect at all upon the black soil. The sands apparently in- creased the solubility of phosphate rock, since summer-rye cannot assimilate phosphoric acid in the form in which it occurs in this fertilizer, and the black soil appears to have had no such effect. In sand cultures phosphorite can be made available for the small grains by supplying them with a complementary fertilizer, such as ammonium salts, which are physiologically acid. Since adequate amounts of ammonium salts are usually injurious to plants in water and sand cultures, Prianishnikov 1 re- placed only a part of the requisite sodium nitrate in his sand cultures by an equivalent amount of ammonium sulphate. This gives a medium that tends Fig. 60. — Effect of ammonium salts upon the availability of phosphorite for oats in sand cultures. {After Prianishnikov.) See text for explanation. to become more acid with increase in its content of the ammonium salt, and so phosphate rock supplied to such cultures might be expected to become soluble and thus available to the plants. This expectation was realized in experiments with oats. The results of such an experiment are given in the table below. The appearance of the first six cultures, in the order followed in the table, is shown in Fig. 60. Culture Weight of Tops No. 1 2 3 4 5 6 Treatment grams Control, KH2PO4 + NaN0 3 19- 7 Phosphorite + NaN0 3 6.9 Phosphorite + K(NH 4 ) 2 S0 4 + %NaN0 3 22.0 Phosphorite + K(NH 4 ) 2 S0 4 + KNaN0 3 20.5 Phosphorite + M(NH 4 ) 2 S0 4 + MNaN0 3 19.2 Phosphorite + (NH 4 ) 2 S0 4 1.6 1 Prianishnikov, T>. N., Results of vegetation experiments for 1899 and 1900. Moscow Agric. Inst. 7 (non-official part): 85-129. 1901. [Russian.] Bull. ABSORPTION OF ASH-CONSTITUENTS 97 These results support the idea that partial replacement of sodium nitrate by ammonium salts renders the phosphoric acid of the phosphate rock available for oats; when one-fourth or one-half of the NaN0 3 was replaced by (NH 4 ) 2 S0 4 the yield did not fall below that of the control, as it did in the other cases. It is clear that the nutrient materials in the soil are utilized to unequal degrees by different plants. As we shall see later, roots excrete acid substances that favor the solution of soil materials otherwise practically insoluble in water. Further- more, many plants are characterized by having their roots covered with fungus hyphae, a fact discovered by Kami- enski. 1 Frank 2 gave the name myco- rhiza to this weft of fungus hyphae growing upon roots, and emphasized the impor- tance of this whole phenomenon in the physiology of nutrition. Plants that have mycorhiza are said to be mycotrophic. We owe extended investigations upon the physiological importance of mycorhiza to Stahl. 3 In some cases the fungus hyphae cover the surface of the roots (ectotrophic mycorhiza), as is shown in the case of beech roots (Fig. 61). The tip region of Pig. 6i. — Ectotrophic mycorhiza of the beech; a, humus particles; b, strands of fungus hyphae penetrating the soil. Pig. 62. — Endotrophic mycorhiza in epider- mal cells of the root of Andromeda poll folia, the root shown in cross-section. the root is covered with hyphae some of which branch out into the soil and attach themselves to particles of humus. In other cases the fungus hyphas are found within the cells of the root (endotrophic mycorhiza), as in the case of Andromeda polifolia (Fig. 62). Here the hypas occur in the large cells of the root epidermis. Mycorhiza is of common occurrence, being found on the majority of vascular plants, not only trees, shrubs and herbs, but even mosses. Some plants cannot 1 Kamienski, Fr., Die Vegetationsorgane der Monotropa hypopitys L. Vorlauf. Mitth. Bot. Zeitg. 39= 4S7-46I. 1881. - Frank, B., Ueber die auf Wurzelsymbiose beruhende Ernahrung gewisser Baume durch unterirdische Pilze. Ber. Deutsch. Bot. Ges. 3: 128-145. 1885. 3 Stahl, E., Der Sinn der Mycorhizenbildung. Ein vergleichend-biologische Studie. Jahrb. wiss. Bot. 34: 539-668. 1900. 7 9 8 PHYSIOLOGY OF NUTRITION thrive without mycorhiza, others are never found with it, and still others occur sometimes with and sometimes without. The non-green seed-plants appear generally to belong to the first group. Mycorhiza develops mainly in soils rich in humus, where the fungus hyphae facilitate the entrance of nutrient substances into the plant. Non-green seed-plants draw organic as well as inorganic substances from the soil by means of their mycorhiza. The importance of mycorhiza to green plants is probably most pronounced in connection with the absorption of the ash-con- stituents, although these may be taken up first in organic compounds. The properties of humus soils are not by any means to be considered only from a purely chemical standpoint. The abundance of bacterial and fungous organisms in the soil makes it almost like a living thing, and all the microorganisms of the soil require large amounts of mineral substances. If a higher green plant grows in humus soil it must compete with these microorganisms for its nutrition, and this competition is especially active since the nutrient materials in humus are not as well suited to the needs of green plants as are those in mineral soils. p IG £., Cultures of Lepidium sativum in humus soil. On the left, two vessels with sterilized • soil - on the right, two vessels with unsterilized soil. {After Stahl.) It appears that plants with an associated fungus, forming mycorhiza, are thus enabled to compete with soil microorganisms not associated with them much more successfully than can plants without mycorhiza. How difficult the growth of these latter may be in humus soil is shown by the following experiment of Stahl. Humus soil from a beech forest was placed in four vessels, two of which were sterilized with ether and chloroform vapor, thus killing all the micro- organisms of the soil without otherwise altering it. Seeds of Lepidium sativum, a plant without mycorhiza, were then planted in all four vessels. Healthy plants developed in the sterilized vessels, while the plants grew but poorly in those that were not sterilized (Fig. 63). The microorganisms of the soil are thus seen to have retarded the growth of Lepidium to a very marked degree. No trace of nitric acid or nitrates can be found in the mycorhiza, nor is any usually found in soils in which mycotrophic plants are growing. This fact con- firms the opinion that mycotrophic plants differ from those without mycorhiza in their manner of nutrition. If fact, the experiment with ammonium fertilizers, mentioned above, shows that such fertilizers have no effect in soils rich in humus and poor in lime (which are usually occupied by mycotrophic plants), and that intrification progresses with great difficulty in these soils. ABSORPTION OF ASH-CONTITUENTS 99 If a particular kind of plant is grown for several years in succession upon the same soil, the crop gradually decreases, in spite of the addition of plenty of fertilizers. This is the well-known phenomenon of "soil sickness." In this case we do not have to deal with an inadequate supply of mineral nutrients, but with something entirely different. The work of Whitney and Cameron, and that of Livingston, Schreiner, and other American investigators, 1 has indi- cated that plants produce poisonous substances (toxins) in the soil. 7 ' These toxins appear, in many cases, to be poisonous only to the particular kind of plant in connection with which they were produced, and this may explain the fact that » Whitney, Milton, and Cameron, F. K., Investigations in soil fertility. U. S., Dept. Agric. Bur. Soils- Bull. 23. 48 p. Washington, 1904. Livingston, B. E., Britten J. C, and Reid, F. R., Studies on the prop- erties of an unproductive soil. Ibid. Bull. 28. 39 p. Washington, 1905. Livingston, 1907. [See note 6, p. 83.] Schreiner, Oswald, Reed, Howard S., and Skinner, J. J., Certain organic constituents of soils in relation to soil fertility. Ibid. Bull. 47. 52 p. Washington, 1907. Schreiner, Oswald, and Shorey, Edmond C, The isolation of picoline carboxylic acid from soils and its relation to soil fertility. Jour. Amor. Chem. Soc. 30: 1295-1307. 1908. Idem, The isolation of dihydroxy-stearic acid from soils. Ibid. 30: 1590-1607. 1908. Idem, The isolation of harmful organic substances from soils. U. S. Dept. Agric, Bur. Soils. Bull. 53. 53 p. Washington, 1909. ' A discussion of some of the earlier literature regarding this general idea of soil toxins is given by Livingston, 1907. [See note b, p. 83.] This earlier literature (not considered by Whitney and Cameron, 1904, nor by Livingston ei al., 1905 [note 1, just above]) is rather extensive. The idea that plants may excrete into the soil substances that may be poisonous to other plants, appears to have originated with A. P. DeCandolle (Physiologie vegetale. Paris, 1832), but the experimentation invoked by this writer's suggestion seemed to dis- prove the hypothesis, and the whole matter was laid aside until it was taken up again, in a modern way, by the Duke of Bedford and S. U. Pickering (at the Woburn Experimental Fruit Farm, near Bedford, England) and by the American students mentioned above. On the Woburn work see : Pickering, Spencer U., The effect of grass on apple trees. Jour. Roy. Agric. Soc. England 64 (of entire series): 365-376. London, 1903. Also see the Reports of the Woburn Experimental Fruit Farm after 1897. In later years the general hypothesis that unproductiveness in agricultural soils is frequently due to soil toxins has been well established by workers in various parts of the world, and it is now generally accepted. Evidence that agricultural plants do actually excrete toxic substances into the soil is not very strong in any of this work, however. Better than to assert that they are so excreted is to state that there is evidence that the soil frequently contains toxins and that these sometimes result, directly or indirectly, from the growth of higher plants. As to the manner in which these poison substances arise in the soil, no definite statements can yet be made, but they are surely not generally excreted as such from plant roots.. There is physio- logical evidence, however, that such substances are given off by living roots when the latter are practically deprived of oxygen. (See p. 1 26.) It seems highly probable that soil microorgan- isms play an important part in the production of the toxic substances here considered. Ex- creted substances, the materials of dead root-cap cells, root-hairs, roots, etc., or even substances carried down into the soil by rain (as from the bark of trees and fallen leaves) may become altered by the action of microorganisms so as to produce poisons. That such poisons are present in many soils has now been established without question by Schreiner and his co- workers, and also that their deleterious effect upon plants may often be removed by oxidation, or by the addition of proper substances. The general acceptance of the hypothesis of toxic soil constituents as a frequent cause of unproductiveness was much retarded by the form of its original statement, by Whitney and Cameron (1904), which emphasized actual root excretion at the expense of all the other logical possibilities. It was of course to be expected that such poisons might arise in the soil in a great variety of ways, and the theory of soil toxins is not to be considered without continual reference to the microbiology of the soil. Russell (1921. [see note i, p. 73.]) gives a clear dis- cussion of this whole matter, from the standpoint of field experiments. — Ed. IOO PHYSIOLOGY OF NUTRITION p IG 6 4- — Wheat plants grown in extract of toxic soil. I and 2, undiluted extract; 3 and 4, equal parts of extract and distilled water; 5 and 6, one part extract diluted with nine parts of distilled water. (After Schreiner and Shorey. Reproduced by permission of U. S. Dept. Agric, 1909.) Fig. 65. — Vicia faba (Windsor bean) plants grown in water extract of bog-soil and in bog water. 1, extract; 2, bog water; 4, bog water neutralized with calcium carbonate; 5, bog water treated with carbon-black and filtered. (After Dachnowski.) .ABSORPTION OF ASH-CONSTITUENTS IOI a soil that is unproductive for tomatoes may still produce a good crop of grain. Cultures in water extracts of unproductive soil give but poor growth, but growth is improved proportionally with the dilution of the extract with distilled water (Fig. 64). Addition of lime frequently neutralizes the toxic effect. To secure a good crop in an unproductive soil that contains toxins, it is necessary to find substances or treatments that render the soil toxins harmless. The effects of water extract from bog-soil and those of bog water, upon the development of Vicia faba 1 (Windsor or broad bean), are shown in Fig. 65. The addition of calcium carbonate and the adsorptive action of carbon- black have been very effective here. In this case the toxic action of the bog water was probably due to toxins arising from the microorganisms of the soil, 2 rather than to toxins emanating from the bog plants. fc Toxins of some agricultural soils are organic in nature, as is indicated by the following experiment. 3 Water extract of a soil that had become alfalfa-sick was toxic to this plant, but if the soil was brought to a red heat before making the extract the latter was not toxic. Water extracts of other soils, which had not been in alfalfa culture, had no injurious effect upon the growth of this plant. Experiments have also been made to determine the effects of various plant substances upon plant growth. Such substances are sometimes injurious and sometimes beneficial. Watering with a 3-per cent, solution of nicotin, for instance, produces good growth in tobacco, and is likewise beneficial to potatoes. 4 1 Dachnowski, Alfred, The toxic properties of bog water and bog soil. Bot. gaz. 46: 130-143. 1908. • Lohnis, F., Handbuch der landwirtschaftlichen Bakteriologie. Berlin, 1910. ' Pouget, I., and Chouchak, D., Sur la fatigue des terres. Compt. rend. Paris 14s: 1200-1203. 1907. « Otto, R., and Kooper, W. D., Untersuchungen iiber der Einfluss giftiger, alkaloidfuhrender Losungen auf Boden und Pflanzen. Landw. Jahrb. 39: 397-407. 1910. k That bog waters are toxic to ordinary plants (at least, in that they have an acid reaction), has long been suspected. Schimper (Schimper, A. F. W., Plant geography upon a physiologi- cal basis. Translated by W. R. Fisher. Oxford, 1903) considers bogs as physiologically dry, but is not clear as to just what physiological dryness may be due to. Livingston tested the two logical possibilities in this case. He found (Livingston, B. E., Physical properties of bog water. Bot. gaz. 37: 383-385. 1904) that high osmotic concentration of bog water is not a possible explanation of physiological dryness; bog water has a freezing-point no lower than that of water from drained swamps and rivers of the vicinity. By the use of an alga as a physiological indicator, the same author showed very clearly that bog waters usually contain toxic substances. (Livingston, B. E., Physiological properties of bog water. Bot. gaz. 39: 348-355. 1905.) It appeared also that this toxicity (for the alga used) was surely not directly related to acidity, the degree of acidity being measured with phenolphthalein as indicator. It is interesting to note that this first step toward an analysis of the bog-water problem occurred at almost exactly the same time as the general problem of toxic substances in arable soils was opened up (in its modern sense) by Whitney and Cameron (1904) [see note 1, p. 99] and by Belford and Pickering (1903) [see note j, p. 99]. The three lines of work were entirely independent. Transeau also (Transeau, E. N., On the development of palisade tissue and resinous deposits in leaves. Science, n. s. 19: 866-867. i*) 1 ^) bad shown that bog water is toxic, to Rumex at least, before the excellent studies of Dachnow- ski (cited here in text), and those of Rigg were published. (Rigg, G. B., The effect of some Puget Sound bog waters on the root hairs of Tradescantia. Bot. gaz. 55: 314-326. 1913- Idem, The toxicity of bog water. Araer. jour. bot. 3: 436-437. 1916. Idem, A summary of bog theories. Plant world 10 : 310-325. 1916.) It seems probable that microorganisms and lack of oxygen have to do with the production of these bog toxins. — Ed. 102 PHYSIOLOGY OF NUTRITION Summary i. Cultures in Artificial Media. — The essential chemical elements for plants in general are: Carbon, hydrogen, oxygen, nitrogen, sulphur, phosphorus, potassium, calcium, magnesium, and iron (C, H, O, N, S, P, K, Ca, Mg, Fe). Carbon and oxygen enter ordinary green plants as carbon dioxide (C0 2 ), while hydrogen and oxygen enter as water (H 2 0) . As already seen, these two compounds are decomposed in chlo- rophyll-bearing cells, by the action of sunlight, forming carbohydrates ([CH 2 0]„) and free oxygen. From carbohydrates and other substances the plant cells form the many different organic compounds found in the plant body. Tissues without chloro- phyll must absorb their carbohydrates (and often many other organic compounds from their surroundings, including the green tissues of the same plant. As has also been, seen, nitrogen enters the ordinary plant mainly as nitrates (sometimes as nitrites, ammonium salts, or organic nitrogenous substances), and these become combined with carbohydrates, etc., to form many of the most complex substances occurring in plants. When plants are completely burned all of the carbon, hydrogen, oxygen, and nitro- gen are given off as gases, but there remain small amounts of many other essential and non-essential elements in the form of incombustible ash. The total ash of ordinary plants constitutes only about 5 per cent, of the total dry weight, or about 0.02 per cent, of the green weight. The other essential elements (S, P, K, Ca, Mg, Fe) of the ash are absorbed by ordinary plants from the soil, just as are water and nitrates, and the supply is in the form of inorganic salts: mainly nitrates (N0 3 ), sulphates (S0 4 ), and phosphates (P0 4 ), of potassium, calcium, magnesium, and iron. The elements absorbed through the roots may be studied by artificially controlled cultures in water solutions or in pure quartz sand, etc., the latter of course containing water solution in its interstices. Numerous different solutions have been tested by many workers. A very good medium for solution cultures may be prepared with cal- cium nitrate [Ca(N0 3 ) 2 ], mono- or di-potassium phosphate, (KH 2 P0 4 or K 2 HP0 4 ), and magnesium sulphate (MgS0 4 ), about seven thousandths of a gram-molecule (the molecular weight expressed in grams) of each salt, all dissolved together in a liter of water, with addition of a very small amount (about 3 mg.) of an iron salt such as ferrous sulphate (FeS0 4 .7H 2 0). This is one of Shive's nutrient solutions. Three single-salt solutions (with trace of an iron salt) may be used in rotation, if concen- trations and time periods are properly chosen. 2. Importance of Essential Ash-constituents.— Little is known as to just how the small amounts of essential ash constituents are used in the plant, but all must be supplied. Sulphur occurs in proteins, phosphorus in nucleins (a special group of protein-like substances), magnesium occurs in chlorophyll, and iron is essential for the formation of chlorophyll. 3. Importance of Non-essential Ash-constituents. — Although plants grow well with only the essential elements supplied, yet they generally contain many non-essen- tial elements, and these are not without influence upon growth and development when they are present in the right amounts. Grasses accumulate silica in the epidermis and are thus more or less protected, from fungi, etc., by a glassy layer on the exterior. 4. Ash Analysis of Plants. — The chemical analysis of the ash of a plant shows what elements are present and in what proportions they occur. Different species differ in these respects, and also in the amount of total ash per unit of weight, etc. The nature of the soil influences the ash content of the plant. Different parts of the same indi- vidual plant differ in ash content. Leaves are generally richer in ash than stems and roots. The ash content alters with the age of the organ or tissue. ABSORPTION OF ASH-CONSTITUENTS 103 5. Microchemical Ash Analysis. — Small amounts of plant tissue may be studied by microchemical methods, to determine what chemical elements are present. 6. The Plant and the Soil. — Ordinary plants obtain all their ash constituents from the soil, but a chemical analysis of the soil is of little value in determining whether a plant can thrive in any given soil. The essential elements must be present as the proper salts, and these must be supplied to the obsorbing roots at proper rates. Soils may generally be much improved for growing plants by the addition of certain inorganic salts, or of material that will produce these when it is decomposed by soil microorganisms. To determine the value of a fertilizer, it must generally be actually tested with the given soil and with the kind of plant that is under consideration. Many plant roots are normally accompanied by fungus hyphae as mycorhiza, these hyphae either forming a weft about the root or occurring in the cavities of the superficial cells. Mycorhiza is necessary for many plants, especially when growing in humus soils. It appears that the fungus hyphae facilitate the movement of substances from the soil into the roots. There is little or no nitrification in humus soils, and it is possible that the mycorhiza in such soils may furnish the roots with some nitrogenous substances other than nitrates. A soil may be unproductive because it contains too much (or too little) of the soluble mineral salts, or because it contains very injurious substances in toxic amounts. "Soil sickness," often resulting from repeatedly growing the same crop on the same soil, appears to furnish an example of this, the toxic materials being probably organic in such cases. They seem to be produced from the decay of plant roots, etc., or from substances emanating from the roots, and they appear often to be related to the activi- ties of microorganisms in the soil. Such a " toxic " soil may produce good growth of one kind of plant (as wheat) while it is very injurious to another kind (as tomato). Bog soils are toxic to many forms of plants, although characteristic bog plants thrive in them. CHAPTER V ABSORPTION OF MATERIALS IN GENERAL §i. Materials Absorbed by Plants. — We have seen in the preceding chapter that only a few inorganic materials are needed in the construction of the plant body. These essential substances are carbon dioxide, water, and certain salts containing the elements N, S, P, K, Ca, Mg, and Fe, these salts being dissolved in the soil water. From these substances [including the ten essential elements, C, H, O, N, S, P, K, Ca, Mg, and Fe] various kinds of organic compounds are built up by the green plants. Atmospheric oxygen is also absorbed by plants. Absorption of free oxygen does not generally result in an increase in dry weight, however, but is generally accompanied by the elimination of water and carbon dioxide, and thus results in a loss of plant material. Some of the organic compounds thus undergo oxidation through the respiratory process, which will be discussed later. Some of the materials that enter the plant are commonly met with in the gaseous form (carbon dioxide and oxygen), others are generally encountered as solids (the salts of the soil, including nitrogen compounds), but they all enter plant cells as substances dissolved in water. In entering, they must all pass through the cell wall, as well as the outer layer of the protoplasm. The mechanics of the absorption of materials by plant cells is thus based upon the laws of controlling the migration of substances dissolved in other substances." §2. Diffusion of Gases.— If two gases are separated by a membrane per- meable to them they pass through the spectum and mix. Whether there is a septum between them or not, this mixing process is termed diffusion. Two cases may be differentiated here. The first case refers to septa in which the gases are not dissolved (e.g., a dry porous clay plate). The other case relates to septa in which the gases are dissolved (e.g., moist animal bladder). 6 The ° Of course the oxygen of the air and of the soil and the carbon dioxide of the air cannot enter plant cells without being first dissolved in water; if not dissolved at a greater distance they go into solution in the water of the cell, which extends to the exterior surface of each exposed cell wall, these walls being impregnated with water of imbibition. The distinctions between solids, liquids and gases have nothing to do, primarily, with the kind of matter considered, but only with its state, which generally depends upon temperature. The author's presentation is here departed from to a certain extent, to avoid his apparent implication that gases enter plant cells in a manner different from that by which substances that are usually solid or liquid make their entrance. — Ed. b The term dialysis refers to the process of separating two dissolved substances by letting one diffuse through a septum that is impermeable to the other — a common laboratory opera- tion — and follows the same principles, whether the diffusing substance is commonly met with in the gas, liquid, or solid form. The word osmosis, frequently encountered in connection with the diffusion of substances through membranes, should be dropped, for it does not add to 104 AB SORPTION OF MATERIALS IN GENERAL 105 velocity of diffusion of undissolved gases depends upon the density of the dif- fusing gas (temperature and pressure being the same) and is inversely propor- tional to the square root of this density. For instance, the density of hydrogen is approximately 1, while that of oxygen is 16, and the velocities of diffusion of these two gases are to each other as 1 is to 4; i.e., hydrogen passes through a dry porous clay septum four times as rapidly as does oxygen when the t w< 1 gases have the same temperature and pressure. In the diffusion of dissolved gases the density of the gas plays no direct part. Here the velocity of the movement is directly proportional to the co- efficient of solubility of the gas in the solvent contained in the septum. In the absorption of gases by plant cells, it is diffusion of dissolved gases that is encountered, since the cell walls are impregnated with water. According to the law of gas diffusion, carbon dioxide should enter plant cells more slowly than do any of the other gases encountered; on the basis of the principle of diffusion of dissolved gases, it should enter more quickly that the others, since it possesses the greatest solubility in water (and in water-impregnated mem- branes). Thus it happens that carbon dioxide, in spite of the small amount of it in the air, is still absorbed by plant cells in adequate amounts/ §3. Absorption of Gases.— Plants possess various structures that favor gas absorption and gas movement, among which are stomata, lenticels, and numer- ous intercellular passages traversing the plant body in all directions. The clearness and is frequently confusing. We have two kinds of diffusion with which to deal here, one being the intermingling of gases as such and the other that of substances (such as carbon dioxide, alcohol, potassium nitrate, etc.) while dispersed (dissolved) in a solvent; the solvent is usually liquid (water), but substances may dissolve in solid material — as carbon dioxide in the wax-like, cuticular material of many exterior cell walls. Diffusion of undissolved gases is met with in the inward and outward movement of water vapor, carbon dioxide and oxygen through stomatal openings and from place to place in the plant body through gas-filled intercellular spaces, but gases do not pass through the cell walls or protoplasm of active cells, and therefore cannot get inside the cells, unless they are first dissolved, usually in water. (See below, in text.) Of course, when water vapor is dissolved in liquid water it simply becomes a part of the liquid, being condensed from the gaseous to the liquid state. This and the following para- graphs have been subjected to some modification, in accordance with these principles. It may be added at this point that, besides the diffusion of gases and that of dissolved sub- stances, there is another kind of movement met with in plants, namely that of molar stream- ing. This occurs with gases and liquids and also (but not commonly in the plant) with suitably sub-divided solids (as sand). When a gas or liquid is forced through openings, by pressure, it is this molar movement that has to be considered. Diffusion may go on at the same time, in the liquid or gas stream, its direction being independent of the direction of the streaming. If diffusion and streaming are in the same direction, the rate of movement is the sum of the rates of diffusion and streaming; if they are in opposite directions the difference is the rate of movement. — Ed. c Carbon dioxide is about three times as soluble in water as is oxygen. It is as a gas, however (undissolved in either liquid or solid), that carbon dioxide first enters the ordinary green plant through stomatal openings. See: Blackman, 1895. [See note 2, p. 36.] Brown, 1899. [See note 1, p. 34.] Brown and Escombe, 1900. [See note 1, p. 34.] Having entered by gas diffusion, carbon dioxide soon passes into solution in the water with which the cell walls abutting on the sub-stomatal intercellular spaces are impregnated, and it diffuses as a dissolved substance through these walls and into the cells. — Ed. 106 PHYSIOLOGY OF NUTRITION • migration of gases through different kinds of plant septa has been investigated by many authors. The most recent and extensive studies on the molar or streaming movement and the diffusion of gases through plant cell walls are due to Wiesner and Molisch. 1 In these experiments a piece of dry plant tissue was fastened over one end of a straight glass tube (6 mm. in internal diameter and 50 to 100 cm. long) with sealing wax, and the joint was then covered with a mixture of equal parts of resin and beeswax. When soft, succulent tissues were employed, the tissue was kept in place by a perforated metal cap, and was kept from being crushed by rubber rings, the openings of which just fitted the end of the glass tube. The tube was partly or entirely filled with mercury and the open end was closed with the finger while the tube was inverted, the open end being then placed in a vessel of mercury. The tube was finally ar- ranged in an upright position, with the mercury below. After a number of days the height of the mercury column in the tube was measured. An experiment with birch bark may serve as an example. A piece of white periderm, 0.09 mm. thick, was used. The height of the mercury column in the tube was 400 mm. at the beginning of the experiment and remained the same, after fourteen days, the usual corrections for temperature and pressure having been applied. Wiesner and Molisch came to the following conclusions from the result of these experiments. 1. Plant cell walls, either wet or dry, whether the cells are alive or dead, are impermeable to the molar movement of gases under ordinary pressures. 2. Protoplasm and cell sap are likewise impermeable to this kind of gas movement, so that there is no movement of air as such through tissue without intercellular passages. This experiment explains how negative gas pressure (i.e., pressure less than that of the surrounding atmosphere) in wood may be maintained, which will be discussed later. Similar tubes filled partly with mercury and partly with various gases were employed in experiments upon the diffusion of gases through dry and moist plant membranes. The velocity of outward diffusion was indicated by the rate of rise of the mercury column in the tube. An experiment with periderm of the potato tuber may be taken as an example. Two tubes were filled with carbon, dioxide, one being closed with a dry, the other with a moist piece of periderm. In the tube with the dry membrane the mercury rose only 5 mm. during a period of thirty days, while the tube with moist membrane showed a corresponding rise of about 40 mm. This experiment shows that the interchange of gases through the wet membrane occurred according to the principles of diffusion of dissolved gases; carbon dioxide passed outward through the membrane more rapidly than- air passed inward, thus causing the mercury to rise in the tube. If the septa to be studied were permeable to, but did not dissolve the gas (as in the case of a dry porous clay plate), then, according to the law of gas diffusion, the mercury column should fall. From a series of experiments similar to this, these authors came to the following conclusions: 1 Wiesner, J., and Molisch, H., Untersuchungen uber die Gasbewegung in der Pflanze. Sitzungsber. (math.-naturw. Kl.) K. Akad. Wiss. Wien. o8^ : 670-713. 1890. ABSORPTION OF MATERIALS IN GENERAL I07 1. Gases move through cell walls only in solution in the water imbibed in the wall; when intercellular spaces are present, they of course facilitate the move- ment through the tissue. 2. Gases pass through cell walls the more easily, the more thoroughly the latter are impregnated with water. Diffusion is most rapid through cell walls of algae and, in general, through those of submerged plant parts. 3. Cell walls that are neither lignified nor suberized do not permit the passage of some gases when the walls are dry, but carbon dioxide and oxygen pass through practically dry walls if the latter are lignified or ssuberized. d These Experiments suggest an important ecological consideration as regards suberization and cutinization in plant tissues. If the entire surface of the plant were covered by a dry membrane of pure cellulose, then the interior cells would be suffocated, but the presence of cork and cutin, in the absence of lenticels and while the stomata are closed, protects plants from desiccation without at the same time preventing gaseous exchange. 4. Carbon dioxide passes out of plant cells more rapidly into air than into water. Since Wiesner's experiments indicate that gases may pass through the cuticle, the question arises, to what extent do open stomata increase the rate of gaseous exchange through the epidermis? To answer this question F. F. Blackman 1 constructed a special apparatus described below (Fig. 66). Two brass rings, each prolonged into two tubes at opposite points and each with a glass plate attached to one side, were used as gas chambers, each chamber being about 5 mm. deep and 36 mm. broad. A leaf was clamped between two chambers of this kind and the joints were sealed with wax. Oblong chambers were used for narrow leaves (Fig. 66, A). Gas of known composition was passed simulta- neously, but separately, through both chambers and then analyzed. Experi- ments with leaves having stomata only on the lower surface showed that the respiratory gas exchange occurred almost entirely through these openings. For example, a leaf of Nerium oleander gave out 0.002 g. of CO2 from its upper surface while 0.065 g- escaped from the lower; thus the two sides gave off this gas in the ratio of 3 to 100. Further experiments upon the assimilation of carbon dioxide in light showed that leaves absorb this gas from the air almost exclusively through the stomata. Leaf surfaces without stomata practically fail to absorb carbon dioxide. When the lower surface alone is provided with stomata, coating this surface with petrolatum greatly decreases gaseous exchange without wholly stopping it, as • Mangin has shown (see page 35). When stomata occur on both sides of the 1 Blackman, F. F., 1895, No. II. [See note 2, p. 36.] d Molar movement of gases can occur only through intercellular spaces and relatively large openings in plant membranes (stomatal openings, etc.), and gas diffusion can occur through such openings and through dry membranes with relatively large pores (porous porcelain, etc.). The diffusion of dissolved gases is possible if the gas is soluble in the membrane. When the latter contains water this kind of diffusion can occur, for the gas dissolves in the water. When the membrane contains little or no water, but contains suberin, etc., the action is similar to that of a wet membrane, if the gas dissolves in the wax-like material as it does in water. — Ed. io8 PHYSIOLOGY OF NUTRITION leaf, the amount of carbon dioxide absorbed is greater on the side where these openings are most abundant. In the case of Alisma plantago, the number of stomata on the upper is to the number on the lower surface as 135 is to 100. While the upper surface was absorbing 0.10 or 0.15 g. of the gas the lower sur- face absorbed 0.06 or o.n g. These experiments led Brown and Escombe 1 to carry out the following inter- esting investigations. The Catalpa leaf has stomata only on the lower surface, through which carbon dioxide is absorbed in the presence of light. Under the most favorable conditions 700 cc. of this gas is absorbed per hour, per square meter of leaf surface. If it is assumed that absorption proceeds equally over the entire leaf surface, then each molecule of carbon dioxide enters the leaf with an average velocity of 3.8 cm. per minute. This velocity is only half of that with which carbon dioxide is absorbed by the exposed surface of a sodium Fig. 66. — Apparatus for the study of gaseous exchange through the upper and lower surfaces of leaves. (After Blackmail.) hydroxide solution. But since the gas is absorbed only through the stomata, and since the total area of the stomatal openings is not greater than one-one- hundredth of the entire leaf surface, then a surprisingly large number (380 cm.) is obtained as the average velocity of absorption of carbon dioxide through the stomata. This number is fifty times as great as that representing the absorp- tion of CO2 by the free surface of sodium hydroxide solution. These results led to the following experiment. Test-tubes were filled with aqueous solution of sodium hydroxide and covered with thin, perforated plates, different plates 1 Brown, 1899. [Brown and Escombe, 1900.] [See note 1, p. 34.] ABSORPTION OF MATERIALS IX GENERAL IO9 having openings of different diameters. Some of the results are tabulated below. The velocity of carbon dioxide diffusion was found to be proportional, not to the area of the opening in the plate, but to its diameter. Diffusion of CO2 Ratio of Areas of Openings Ratio of Diameters of Openings Ratio of Amounts of C0 2 Diameter of Opening Per Hour Per Hour, per Square Centimeter mm. cc. cc. 22 . 70 0. 2380 0.0588 1 .000 1 .000 1 .00 6.03 0.0625 0.2186 0.070 0. 260 0. 26 3- 2 5 0.0399 0.48SS 0.023 0. 140 0. 16 2.12 0.0261 0.8253 0.008 0.093 0. 10 While the area of the smallest opening (diameter 2.12 mm.) was less than a hundredth of that of the largest (diameter 22.7 mm.), the amount of gas passing the former was one-tenth, rather than one-hundredth, of the amount passing the latter. From this it follows that if a vessel of sodium hydroxide solution is covered with a thin plate perforated with very small openings, the quantity of carbon dioxide absorbed may be as great as though no cover were present at all. The total area of all the openings may be only a small fraction of the total surface of the plate, however. It was found that diffusion was most rapid when the distances between the openings were each ten times as great as the diameter. This proportion holds approximately for the distribution of stomata in most leaves. Therefore the velocity of gas absorption is as great when the stomata are open as it would be if no cuticle were present and if the whole leaf were cov- ered with a wet membrane of pure cellulose. Investigations of movements of gases in water plants 1 have shown that the air of the intercellular spaces has about the same composition as that of the ex- ternal atmosphere. §4. Diffusion of Dissolved Substances. 2 — Many substances that are not gases at ordinary temperatures are soluble in water, but not all substances are appreciably so; oils, for example, are generally practically insoluble in water. 6 1 Devaux, Henri, Du mecanisme des 6changes gazeux chez les plantes aquatiques submergdes. Ann ■ sci. nat. Bot. VII 9: 35-179- 1889. - Dastre, M. A., Traite de physique biologique 1 : 466. Paris, 1901. e The following discussion of osmotic pressure and related phenomena is largely due to the editor, but the spirit and apparent intent of the author is followed as closely as possible, at the same time avoiding the author's curious conceptions that dissolved substances are liquids and that "osmosis" and diffusion are essentially different. For another attempt at presenting these phenomena to the student of physiology, see: Livingston, B. E., The role of diffusion and osmotic pressure in plants. Chicago, 1903. Also see: Findlay, Alexander, Osmotic pres- sure. London, 1913. Washburn, Edward W., An introduction to the principles of physical chemistry. 2d ed. 516 p. New York, 19 21. The last-named discussion is the most thorough from the physical and mathematical point of view. — Ed. I IO PHYSIOLOGY OF NUTRITION Whether the dissolved substance is a gas, liquid or solid under ordinary conditions, it forms an aqueous solution when it is dissolved in water. The dissolved substance is usually called the solute and the water in which it dissolves is the solvent. A solution may contain many different kinds of solutes, all dissolved in the common solvent. All dissolved substances diffuse in all directions within the limits of the solution or solvent, and tend to become equally distributed throughout its volume. If two solutions having a common solvent but different solutes be brought into contact, the two solutes diffuse into each other's region and they eventually become completely mixed, so as to form a single solution of two solutes. The solvent itself exhibits a corresponding tendency to diffuse in all directions; if a mass of pure water be brought into contact with an aqueous solution, water enters the solution and dilutes it, while the solute or solutes enter the water and convert it into a solution, this process continuing until the resulting solution becomes uniform throughout. (If the solute be another liquid — as alcohol, glycerine, etc. — the solute may become the solvent when it predominates. Thus we may have a solution of glycerine in water or a solution of water in glycerine, etc.). It appears that the solute and solvent attract each other and that the latter enters between the particles of the former, thus hastening their outward diffusion. If a membrane that is permeable to water but relatively impermeable to the solute be placed around the solution and be, in turn, surrounded by the pure solvent, a pressure, called osmotic pressure, is developed, which tends to drive the membrane outward before the outwardly diffusing solute, thus stretching — or even rupturing — the membrane. This phenomenon of osmotic pressure was discovered by Dut- rochet, 1 as early as 1827, who observed the escape of zoospores from an alga and tried to arrive at an explanation for the bursting of the sporangium. He supposed that an increased absorption of water by the sporangium was brought about by water-attracting substances within, and that this caused the rupture. If an animal bladder filled with aqueous sugar or salt solution is placed in water, the solvent enters, and the outwardly directed osmotic pressure simultaneously developed may become so great as to rupture the membrane itself. The rupture of the alga sporangium as observed by Dutrochet, was caused in a similar way f [ l Dutrochet, Rene Joachim Henri, Nouvelles observation sur l'endosmose et l'exosmose, et sur la cause de ce double phenomene. Ann. chim. et phys. 35: 393-400. 1827.] { It is still commonly stated or implied that the entering water turns on itself after entrance, and, thus tending to return, presses outwardly upon the membrane and causes the rupture. But the bladder membrane is, in itself, as permeable to water diffusing in one direction as to the same substance diffusing in the other, and more water enters than passes out, so that if there is a pressure of water in either direction it should tend to collapse the bladder, not to explode it from within. A logical picture may represent the osmotic pressure causing the rupture as directly due to a tendency of the solute particles (as of sugar or salt, or ions), or of any combina- tions of solute particles with water particles (in so far as these are unable to pass the septum), to diffuse outward into the surrounding solvent. This, in turn, may be considered as brought about or made possible by the entrance of water (at least it cannot occur without this entrance), which, finally, may be due to an attraction exerted upon the water by the solute. Such a simple picture may still serve the purposes of physiology, although serious complications appear to arise sometimes when a complete appreciation of osmotic and related phenomena is attempted. The most thorough discussion of osmotic pressure so far available is that given by Washburn [see note e, p. 109]. — Ed. .ABSORPTION OF MATERIALS IN GENERAL III Briicke (1843) advanced a theory of diffusion through septa, based upon the observation that if two liquids are separated by a membrane, the one that wets the membrane more thoroughly (i.e., in which the latter swells more rapidly) penetrates more rapidly. For example, if a membrane of rubber or collodion be employed, then alcohol passes through more rapidly than water, but with a membrane of animal bladder the opposite is true. Rubber and collodion membranes imbibe alcohol more rapidly than water and they also swell more in alcohol. Thus alcohol passes through such septa more rapidly. But animal bladder swells in water and shrinks in alcohol, and water passes through such a membrane more quickly than does alcohol. Animal bladder swells more in pure water than in salt solution, and the former passes through such a septum more rapidly than does the latter. These facts indicate that the water is more forcibly attracted by the membrane substance than are the salts, so that the concentration of the imbibed solution in the pores of the membrane increases as the distance from the pore walls becomes greater. Ludwig 1 has shown further that if dry pieces of animal bladder are placed in a solution of sodium sulphate or sodium chloride, the solution that is imbibed is less concen- trated than what remains. By means of a hand press he pressed some of the liquid out of such impregnated pieces of bladder, and found that the expressed solution possessed a concentration higher than the average concentration of the solution originally within the pores of the bladder." Osmotic pressure is studied by various kinds of osmometers. BaranetskiiV 2 osmometer consists of two chambers separated by a membrane, one containing a salt solution, which is to increase in volume, while the other contains water introduced through a funnel that is attached by a rubber tube. As the solution increases in volume a rubber-tube outlet from the solution chamber allows the overflow to be caught in a graduated flask. The surface of the water in the funnel must be kept at the same height as that of the solution in the exit tube. The movement of liquids through the membrane continues until the concentration of the two solutions is the same on both sides. Experiments upon diffusion of dissolved substances through membranes have shown that all water-soluble substances may be classified into two groups according to their relation to the membrane, those which can pass through the membrane (crystalloids) and those which cannot (colloids). Upon these dif- ferent properties of colloids and crystalloids depends the method of dialysis, by which colloid material may be separated from crystalloids. Many plant substances are colloids and they cannot, therefore, diffuse out of the cells.* 1 Ludwig, C, Ueber die endosmotischen Aequivalente und die endosmotische Theorie. Poggendorff's Ann. Phys. u. Chem. 154 ("der ganzen Folge") : 307-326. 1849. 2 Baranetskii, I. Investigations on diosmosis as related to plants. [Russian.] Inaug. Dissertation. St. Petersburg, 1870. Barenetzky, J., Diosmotische Untersuchungen, Poggendorff's Ann. Phys. u. Chem. 223 ("der ganzen Folge") : 195-245. 1872. These considerations give the reason why the membrane is more permeable to one sub- stance than to the other, or, they merely state this fact in other terms. — Ed. h But the matter is not so simple as this. Many water-soluble crystalloids fail to pass cer- tain membranes that are permeable to water, and some colloids do pass them. Colloids and crystalloids are difficult to distinguish accurately, these terms referring to the state rather than 112 PHYSIOLOGY OF NUTRITION Membranes of animal bladder, parchment paper and collodion, as well as the so-called precipitation-membranes, are all used for osmotic experiments. Cellulose membranes, giving the cellulose reaction with zinc chloride and iodine (Baranetskii, 1870) can be produced by treatment of collodion membranes with ferric chloride. Of the above-mentioned membranes, animal bladder is much like the plant cell wall in its osmotic properties, while precipitation membranes are only very slightly permeable to many substances and can give rise to high osmotic pressures. Suitable supports must be pro- vided for these delicate membranes. Pfeffer 1 employed porous clay cylinders such as are used in electric batteries. When such a porous cell is filled with a copper sulphate (CuS0 4 ) solution and placed in a solution of potassium ferrocyanide (K 4 Fe(CN)6), a membrane of copper ferrocyanide (Cu- 2 Fe- (CN) 6 ) is precipitated in the porous wall. Similar precipitation membranes may be ob- tained with other substances, such as iron silicate. To measure osmotic pressure the porous cylinder, with its membrane, is filled with the solution to be studied and is con- nected with a mercury manometer, the cylinder being submerged in water (Fig. 67). The magnitude of the pressure exerted at - equilibrium is then read upon the manometer. % to the nature of the substance considered. In this connection see: Weimarn, P. P. von, Grundziige der Dispersoidcheme. 12 7 p. Dresden,, 1915. For a clear and very readable discussion of colloids in general, see- Ostwald, Wolfgang, Die Welt der vernachlassigten Dimensionen. x + 219 p. Dresden and Leipzig 191 5. Also see: Hatschek, Emil. An Pig. 67. — Pfeffer osmometer (z), introduction to the physics and chemistry of colloids. with closed mercury manometer. 4th ed. i7<; p. London, 1922. Other books on (After Pfeffer.) this subjec 1 are mentioned in the List of Books, p. xix. — Ed 1 Pfefier, W., Osmotische Untersuchungen. Leipzig 1877. i The most perfect precipitation membranes yet made are those of Morse and his coworkers, who have been engaged for many years in very thorough studies on the osmotic pressures developed by concentrated solutions. This work has been carried out in the Chemical Laboratory of the Johns Hopkins University. Much improved forms of the Pfeffer cell have been employed and the copper ferrocyanide membranes of these writers have proved quite impermeable to cane sugar for many days, even with very high pressures. For accounts of this work see: Morse, H. N., and Horn, D. W., The preparation of osmotic membranes by electrolysis. Amer. chem. jour. 26: 80-86. 1901. ABSORPTION OF MATERIALS IN GENERAL 113 Walden 1 obtained semi-permeable precipitation membranes in the following manner. The upper end of a glass tube 5 cm. long and 1 cm. wide is closed by the finger and the lower end is dipped into a solution containing 50 g. of water, 10 g. of gelatine, and 1 g. of ammonium chromate. When the tube is lifted from the solution, the lower end remains closed by a thin membrane, which is rendered insoluble in water by the action of light. A precipitation membrane of copper ferrocyanide is then deposited in the hardened gelatine film, according to the method employed by Pfeffer. Experiments with precipitation membranes have given the general results summarized below. Other conditions remaining the same: — 1. Osmotic pressure is proportional to the concentration of the solution. Thus 1-, 2- and 4-per cent, solutions of cane sugar developed osmotic pressures equivalent to 53.2 cm., 1 01. 6 cm. and 208.2 cm. of a mercury column, respectively. 2. Osmotic pressure increases with rise in temperature. A i-per cent, saccharose solution at temperatures 6.8°, 13. 7 and 2 2°C. gave osmotic pressures of 50.5 cm., 52.5 cm. and 56.7 cm. of a mercury column, respectively. 3. Osmotic pressure depends upon the nature of the dissolved substance. Six-per cent, solutions of (1) gum arabic, (2) gelatine, (3) saccharose and (4) potassium nitrate gave osmotic pressures of (1) 25.9 cm., (2) 23.8 cm., (3) 287.7 cm. and (4) 700 cm. of a mercury column, respectively. Colloids (such as gum arabic and gelatine) thus produce much lower osmotic pressures than do crystalloids. » 4. Osmotic pressure depends upon the nature of the membrane. Six-per cent, solutions of the four substances named above gave the following osmotic pressures (in centimeters of a mercury column) with membranes of copper ferrocyanide, parchment paper and animal bladder, respectively. Morse, H. N., The osmotic pressure of cane sugar solutions at high temperatures. Ibid. 48: 29-94. 191 2. Idem, The osmotic pressure of aqueous solutions. Carnegie Inst. Wash. Pub. 198. 222 p. 1914. During the same period other very important experimental studies on the osmotic pressure developed by concentrated solutions have been prosecuted by Berkeley and Hartley, in England. See: Berkeley, Earl of, and Hartley, E. G. J., On the osmotic pressure of some concentrated solutions. Phil, trans. Roy Soc. London A206 : 481-507. 1906. For a general discussion, see Findlay, 1913, also Washburn, 1921. (See note e, p. 109.)— Ed. 1 Walden, Paul, Ueber Diffusionserscheinungen an Niederschlagsmembranen. Zeitsch. physik. Chem. 10:699-732. 1892. ' As is brought out a little farther on, the concentration of the solutions should not be stated in terms of percentage for such comparisons; they should be given in terms of a volume- molecular, or still better, of a weight-molecular solution. The former gives the number of gram-molecules of solute dissolved in a liter of solution (at a stated temperature) and the latter gives the number of gram-molecules of solute dissolved in 1000 g. ( —5— = 55.56 g.-mol.) of Io water taken as H 2 0. For a valuable discussion of the relation of volume-molecular and weight-molecular solutions to physiological considerations, see: Renner, O., Ueber die Berech- nung des osmotischen Druckes. Biol. Centralbl. 32 : 486-504. 1912. The general principle holds, as stated in the text, however. See also note n, below (p. 123). — Ed. 8 114 PHYSIOLOGY OF NUTRITION Substance, in 6-Per Cent. Solution Kind of Membrane Copper Ferrocyanide Parchment Paper Animal Bladder Gum arabic cm. Hg 25-9 23.8 287.7 700.0 cm. Hg 17.7 21.3 29.0 20.4 cm. Hg 14. 2 Gelatine Saccharose Potassium nitrate 15-4 14-5 8.0 The crystalloids, saccharose and potassium nitrate, produced lower pressures than did the colloids, gum arabic and gelatine, when plant or animal membranes were used. This seems to be in disagreement with statement 3, above, but it is explained by the fact that these two crystalloids readily pass through such membranes, while the precipitation membranes are almost impermeable to them. IV. N- 1 2 Pig. 68. — Successive stages of plasmolysis. N, nucleus; V, vacuole. (After deVries.) Pfeffer's experiments indicated that, other conditions remaining the same, the magnitude of the osmotic pressure differed according to the nature of the dissolved substance, and the question arose whether this phenomenon obeyed any law. This question was answered by deVries, 1 who used living plant cells instead of the artificial cells employed by Pfeffer. He determined the isosmotic (or isotonic) coefficients of various substances by means of the plasmolytic method. As is well known, plasmolysis occurs when a living plant cell is placed in a sufficiently strong (10-per cent.) solution of such substances as cane sugar, sodium chloride, etc. At first there is a decrease in cell volume, to a certain point, after which the protoplasm separates from the cell wall and withdraws inward (Fig. 68). The cell gradually regains its earlier form if the salt solution is 1 Vries, Hugo de, Eine Methode zur Analyze der Turgorkraft. Jahrb. wiss. Bot. 14: 427-601. 1884. ABSORPTION OF MATERIALS IN GENERAL 115 replaced by water. Cells with colored sap are very good for plasmolytic ex- periments, since the coloring matter is retained within the shrinking vacuole, leaving the space between the protoplasm and the cell wall filled with colorless solution. By the use of such cells plasmolysis may be readily detected, even in its incipient stages. DeVries used mature cells with colored sap and determined the concentration of the plasmolyzing solution when the latter was just strong enough to cause separation of the protoplasm from the wall at the corners of the cell (Fig. 68, 3). If no further contraction of the protoplasm occurs it follows that the osmotic pressure within the vacuole just equals that of the external solution. The same experiment was repeated with various substances, and the limiting concentration (i.e., that concentration which is just strong enough to cause incipient plasmolysis) was determined for each. In this way concentrations of various substances were found that produced the same osmotic pressure with the same membrane. Such solutions are termed isosmotic or isotonic. The colored epidermal cells of the leaf sheath of Curcuma rubricaidis, of the leaves of Tradescantia discolor, and of the petiolaf scales of Begonia manicata, are all very well suited to such experiments as that just described. Twelve preparations may be made for each experiment, six being placed in various concentrations of the substance to be studied, and the other six in corresponding concentrations of potassium nitrate. All preparations must be taken from the same region of the leaf or other plant organ. To accomplish this, a narrow rectangle is marked on the leaf, and divided longitudinally into halves and transversely into six divisions, the area of each of the resulting sections being about 1 sq. mm. Each piece of epidermis is removed with a razor and placed in a glass cylinder (about 10 cm. tall and 2 cm. in diameter^') containing the solution to be tested. The cylinders are loosely stoppered to prevent evapora- tion, and the preparations are left in the solutions about two hours. Volume-molecular solutions were employed, containing the molecular weight of the solute in grams (called a gram-moleCule or a mol) 1 per liter of solution. [See note j. p. 113.] A volume-molecular solution (m) of potassium nitrate contains, for example, 1 g.-mol. (101.1 g.) of the salt in a liter of solution, and a tenth-molecular solution (0.1 ' m.) contains 10. n g. of the salt per liter. In physiological studies it is generally more convenient to calculate solution con- centrations as gram-molecules per liter than to consider them in terms of percentage. DeVries compared the osmotic pressures developed by equimolecular solu- tions of various substances, and found that the substances tested fell into four groups according to the amount of pressure developed, the four different pres- sures obtained being, relatively, 0.066, o. 100, 0.133, and 0.166. The second group represents the pressure caused by potassium nitrate. These numbers are approximately in the proportion of 2 : 3: 4: 5, so that if the pressure produced 1 Ostwald, Wilhelm, Lehrhuch der allgemeinen Chemie. 2te Aufl. 2: 212. Leipzig, 1906. [Idem, Outlines of general chemistry. Translated by James Walker. London, 1895.] k Much shorter vials are more convenient, about 1 cm. in diameter and 2 cm. high. — Ed. u6 PHYSIOLOGY OF NUTRITION by a volume-molecular solution of potassium nitrate be considered as 3, then the pressure developed by a volume-molecular solution of any other substance not in the same group is 2, 4, or 5, according to the group in which the given substance belongs. On this account deVries adopted as his unit of osmotic pressure one-third of the pressure produced by a volume-molecular solution of potassium nitrate, so that a volume-molecular solution of this salt, or of any other salt belonging to the same group, always produced a pressure of 3, and the three other groups of substances gave pressure of 2, 4 and 5, respectively. The numbers 2, 3, 4 and 5 were termed isosmotic coefficients; they represent the relative osmotic pressures developed by equimolecular solutions of the various substances. The isosmotic coefficients were determined in the following manner. Three cane sugar solutions, 0.20-, 0.22- and 0.24-volume-molecular, and three solu- tions of potassium nitrate, 0.12-, 0.13- and 0.14- volume-molecular, were em- ployed, for plasmolytic experiments with epidermal cells of Curcuma rubricaulis. Each experiment lasted seven hours. The results obtained in three such tests are given in the following table, where n denotes that no plasmolysis occurred, hp denotes that about half of the cells were plasmolyzed and p denotes that most of the cells were plasmolyzed. IC denotes the isosmotic concentration, taken to be osmotically equal to the cell sap. Volume-molecular concentration is denoted by m. Saccharose Potassium Nitrate Experiment Ratio of no. ICi TO ICi 0.2OWJ 0.22m 0.24m Id 0.12/K 0.13m 0.14m IC 2 m m 1 n hp P 0.22 n hp P 0.130 0.591 2 n P P 0.21 n P P 0.125 o.595 3 n P P 0.21 n P P 0.130 0.610 0. 602 Since the osmotic pressure produced by a volume-molecular potassium nitrate solution is taken as 3, the numbers in the last column are to be multi- plied by 3, and the average ratio thus becomes 1.81, which is the isosmotic coefficient of saccharose when that of potassium nitrate is considered as 3. A list of substances thus tested by deVries is given in the next table, together with their isosmotic coefficients, as actually derived from experiment and also in round numbers. The next to the last column gives the percentage concen- trations thus found to be isosmotic with a one-tenth volume-molecular solution of KNO3, and the last column gives the osmotic pressure produced by a i-per cent, solution of each substance. ABSORPTION OF MATERIALS IN GENERAL 117 Substance Chemical Formula Molecular Weight Isosmotic Coefficient Ob- served In Round Numbers Concentration Isosmotic with 0.1 m KXO3 Osmotic Pressure Produced by i.o-Per Cent. Solution Saccharose Glucose Glycerine Citric acid Oxalic acid Potassium nitrate Ammonium chloride . . Potassium sulphate . . . Magnesium sulphate . . Magnesium chloride . . Potassium citrate C12H22O11 C 6 Hi 2 06 C3H8O3 C 6 H s 7 C2H2O4 KNOa NH4CI K 2 S0 4 MgS04 MgCh X3C6H5O7 342.0 180.0 92.0 192.0 90.0 101 .0 53- S 174-0 120.0 95-0 306.0 1.88 2 1.88 2 1.78 2 2.02 2 2 3-00 3 3-00 3 3-90 4 1.96 2 4-33 4 5. 01 5 per cent. 5. 13 2.70 1.39 2.88 35 01 S3 30 80 0.71 1.84 atmospheres 0.69 . 1. 25 2.54 1.23 2.62 3-50 6.67 2.72 1.93 4.98 1.92 In the above table the isosmotic coefficients are seen to be about 2, 3, 4 and 5. If the coefficient for saccharose and the other organic compounds be taken as unity, then the remaining ones become %, 2, and %. It is also evident from this table that the osmotic pressures produced by the non-electrolytes (saccharose, glycerine and the other organic compounds) are re- lated to their molecular weights. A solution containing 92 g. of glycerine per liter produces the same osmotic pressure as one of cane sugar containing 342 g. per liter. These two solutions contain very different amounts of substance by weight, but they contain equal numbers of molecules (i.e., they are equi- molecular). Here all molecules produce the same osmotic pressure, and the osmotic pressure of a solution is thus proportional to its molecular concentra- tion. This agrees with Avogadro's law for gases, which states that gas pressure is proportional to the number of molecules occurring in a given volume. Van't Hoff compared solutions of solid bodies in liquids, with gases, and concluded that osmotic pressure follows the same law as does gas pressure. One gram- molecule of any gas (e.g., 44 g. of C0 2 ) occupies a volume of 22.4 1., with a pres- sure of 760 mm. and at a temperature of o°C. When this volume of gas is re- duced to 1 1., the pressure becomes 22.4 atmospheres. If the van't Hoff theory is correct, a molecular solution of cane sugar containing 342 g. per liter, should produce 22.4 atmospheres of osmotic pressure, and a i-per cent, solution of the same substance should give an osmotic pressure of 0.69 atmospheres at i5°C. The pressure actually produced by a i-per cent, solution of cane sugar lies between 0.62 and 0.71 atmospheres according to Pfeffer's measurements, which constitutes a brilliant confirmation of the theory. The following table gives a summary of other osmotic values for cane- sugar solutions, as observed by Pfeffer and as calculated by the van't Hoff theory. n8 PHYSIOLOGY OF NUTRITION Concentration of Cane Sugar Osmotic Value Observed Calculated per cent. atmospheres atmospheres I.O 0.664 0.665 2.0 I-336 I-336 2-5 1.997 1.639 4.0 2-739 2.742 6.0 4.046 4.050 It is different with electrolytes; from the table given on page 117 it is clear that, of the crystalloids, isosmotic solutions of electrolytes (metallic salts) and non-electrolytes are not equimolecular, the molecular concentrations of the former being much lower. Furthermore, there is no constant relation between the isosmotic concentrations of solutions of electrolytes on the one hand and of non- electrolytes on the other, so that electrolytes do not agree with the gas-pressure theory of osmotic pressure. For example, a 0.1-volume-molecular solution of KNO3 ought, according to this theory, to give a pressure of 0.235 atmos- pheres, but it actually gives one of 0.352 atmospheres. If the value derived directly from the van't Ffoff theory be multiplied by %, the isosmotic coeffi- cient of this salt (considering the coefficient of cane sugar as unity), the value 0.352 is obtained, which is the same as that found experimentally. Equimole- cular solutions of potassium nitrate and of organic substances are thus not isosmotic. To obtain a solution of potassium nitrate that shall produce the same osmotic pressure as does a 0.1-molecular cane-sugar solution it is necessary to prepare a Ms (% X Mo) molecular solution of the salt. Salts with other isosmotic coefficients must be employed in corresponding concentrations. Thus, a 0.05-molecular solution of potassium sulphate is isosmotic with a 0.1-molecular solution of cane sugar. The osmotic pressure of a weak solution of an electrolyte is thus equal to the theoretical pressure multiplied by the isosmotic coefficient of the electrolyte in question. This departure from the theory is explained by Arrhenius' hypothesis, which supposes that electrolytes in solution dissociate into ions. In a sodium chloride solution, for example, sodium and chlorine ions are both present as well as molecules of sodium chloride. The more dilute the solution, the greater is the degree of dissociation. According to the Arrhenius theory of electrolytic dissociation, the isosmotic coefficient of potassium nitrate indicates that the number of particles in a solu- tion of this salt is increased by dissociation, and if half of the molecules be con- sidered as dissociated the total number of particles ought to be % of what it would be without dissociation, and the osmotic pressure should be correspond- ingly increased. A dissociated molecule of KNO ;i , in the form of two ions, K and NO3, produces twice as much osmotic pressure as does an undissociated molecule. ABSORPTION OF MATERIALS IN GENERAL Iig Potassium sulphate has an isosmotic coefficient of 2 at the concentrations employed by de Vries, the molecule of this electrolyte dissociates into three ions, K, K and S0 4 , and the coefficient 2 indicates, in this case also, that half the total number of molecules are to be considered as dissociated. The number of par- ticles in solution would thus be about doubled, for K + 3 X H = 2 - 1 DeVries used salt solutions of about 0.1 -volume-molecular concentration, these being about half dissociated. The degree of dissociation varies with the concentration, and so the osmotic coefficients obtained by deVries cannot be used for solutions of other concentrations, the coefficients for which must be obtained through the use of isosmotic solutions, 1 employing a solution of an undissociated and unhydrated substance as a standard. Errera 2 proposed the myriotonie as a unit for the measurement of osmotic pressure, to replace the arbitrary one of an atmosphere. A tonie is the pressure exerted upon a surface of 1 sq. cm. by 1 dyne (the well-known unit representing the force necessary to give a velocity-acceleration of 1 cm. per second to a mass of 1 g.). The terms dekatonie, hectotonie, kilotonie and myriotonie (10,000 tonies) are employed for greater pressures. A myriotonie (M) is about one one-hundredth of an atmosphere." 1 §5. Absorption of Dissolved Substances. — Only a few direct experiments upon the entrance of dissolved substances into the cell are available. Some con- clusions concerning the mechanism of absorption may be drawn from plasmoly tic experiments with salt solutions. Every substance entering the cell must pass through two membranes, the cell wall and the protoplasmic membrane. Most dissolved substances easily penetrate the cell wall, but the protoplasm is imper- meable, or nearly so, to many of these. The osmotic properties of the protoplasmic membrane are similar to those of Pfeffer's precipitation membranes. Only the living protoplasm is here meant, however; dead protoplasmic membranes have entirely different proper- ties. Thus pigments are persistently retained within the cell sap by the living protoplast, but these and other dissolved substances diffuse out very rapidly after the cell is dead. Like precipitation membranes, the protoplasmic mem- brane is not completely impermeable to most substances. For example, Pfeffer 3 1 Hamburger, H. J., Osmotischer Druck und Ionenlehre in den medicinisctun Wissenschaften. 3 v. Wiesbaden, 1902-1904. Hober, Rudolf, Physikalische Chemie derZelleund der Gewebe. 2 Aufl. Leipzig.. 1906. [4 Aufl. Leipzig, 1914-] Brasch, Richard, Die Anwendung der physikalischen Chemie auf die Phy- siologic und Pathologie. Wiesbaden, 1901. 2 Errera, L., Sur la myriotonie comme unite dans les mesures osmotiques. Recueil Inst. Bot. Bruxelles 5: 193-208. 1902. 3 Pfeffer, W., Ueber Aufnahme von Anilinfarben in lebenden Zellen. Untersuch. Bot. Inst. Tubingen 2: 179-331. 1886-1888. 1 The degrees of dissociation are actually much greater, however, than are assumed in this discussion. DeVries's isosmotic coefficients are now to be regarded as of historical interest only. The best discussion of the calculation of osmotic values of solutions is that of Washburn, 1921. [See note e, p. ioo.| — Ed. m This unit has never come into general use and it is now highly improbable that it ever will. Pressures are generally stated in terms of millimeters or centimeters of a mercury column or in atmospheres, an atmosphere being 760 cm. of mercury. It seems undesirable to state osmotic pressure in any other terms than those already used for other kinds of pressure. — Ed. 120 PHYSIOLOGY OF NUTRITION ■ '' ,, ' '_' ' I' ' i i i i j _ Fig. 69. — Cell of Zygnema with crystals formed by methylene blue. succeeded in introducing useless and even injurious substances (such as aniline dyes) into the living cell. He found that the following pigments penetrated: methylene blue, methyl violet, bismarck brown, fuchsin, cyanin, safranin, methyl green, methyl orange, tropaeolin 00 and rosolic acid. The concentra- tions of the solutions employed were very low (from 0.001 to 0.00001 per cent.). Some of the dyes, (e.g. methylene blue) first enter the cell sap and color it, but form crystals after a time; Fig. 69 shows an alga cell (Zygnema) with crystals formed by methylene blue. Other dyes (e.g., methyl violet) stain the proto- plasm itself. In neither case is the cell fatally injured. Overton 1 studied a number of different dyes and found that the permeability of the protoplasm to these varied according to their chemical constitution. Basic aniline dyes readily enter the cell, but most of their sulphuric acid derivatives penetrate either not at all or very slowly. Dyes that have accumulated in the cells diffuse out when the cells are placed in water, this outward passage being accelerated by the addition of 0.01 per cent, of citric acid to the water. 2 Citric acid thus appears to change the osmotic properties of the protoplasm. No dye accumulates in the cell if the solution contains 0.01 per cent, of citric acid, but the dye is absorbed from the surrounding solution in the absence of the acid. It is thus possible to alter at will the osmotic properties of cells. It is well known that plants can absorb and accumulate the essential chemical elements from very dilute solutions. Some non-essential elements enter the plant cell only until their effective concentration becomes the same within and without, but some others, as well as the essential elements, continue to enter and accumulate in the cell, even from a weak- solution, since they are converted into new com- pounds after entrance and so the internal con- centration never becomes equal to the external. An illustration of continued absorption may be found in the accumulation of iron tannate in an artificial cell of collodion or animal bladder filled with tannin solution and surrounded by one of ferric chloride. Tannin does not escape through the membrane, but ferric chloride diffuses into the cell and there enters into combi- nation with the tannin to form iron tannate, which also remains in the cell. Ferric chloride is continually consumed in the formation of the iron tannate, and its concentration within the cell never becomes the same as that outside. If the tannin solution is sufficiently concentrated 1 Overton, E., Studien uber die Aufnahme der Anilinfarbe durch die lebende Zelle. Jahrb. wiss. Bot. Fig. 70. — Apparatus for show- ing diffusion of copper sulphate through a membrane into a tube containing zinc. 34: 669-701. 1900. » Pfeffer, 1886-88. [See note 1, page 121.] ABSORPTION OF MATERIALS IN GENERAL 121 all of the ferric chloride will pass from the outer solution into the cell. In a similar way plant roots appear to absorb the essential elements, as well as other substances, from the surrounding solution. The following experiment also illustrates this phenomenon of continued absorption (Fig. 70). A roll of sheet zinc is placed in a short glass tube of large diameter, the tube being filled with water and having both ends closed with animal bladder or parchment paper. The tube is placed in a dilute solution of copper sulphate, which passes through the membranes into the tube. Here the copper of the salt is replaced by zinc, and the zinc sulphate thus formed diffuses into the outer solution. Copper sulphate continues to enter until all of it, or all of the zinc, has been used up. The same phenomenon occurs in the growth of bacteria and moulds on various organic compounds. Of two substances having different nutritive values, the cells take up mostly the one with the higher value, frequently leaving the other entirely untouched. For instance, Aspergillus niger absorbs only glucose from a mixture of this substance and glycerine, so long as the former is present in the solution. 1 Outward diffusion through the cell membranes is also subject to regulation. Nathansohn's experiments 2 indicate that sodium chloride easily penetrates the cells of Codium tomentosum (a marine alga) but that this salt cannot be com- pletely withdrawn from the cells after it has once entered. When the alga is placed in an isosmotic solution (4 per cent.) of sodium nitrate, the chloride con- tent of the cell sap rapidly decreases at first, but the outward diffusion of chloride ceases after a time, as is clear from the following table. The figures denote chlorine content, calculated as per cent, of HC1. Original Chlorine Content after a Period of Chlorine Content I DAY 3 DAYS 8 DAYS 15 DAYS 25 DAYS 2.24 0.92 0.93 0.90 O.84 O.76 Plasmolysis of cells has already been described (Fig. 68). DeVries 3 plas- molyzed whole plant organs as well as cells, and showed that growing parts (such as stems, roots and flower stalks) are noticeably shortened after immer- sion in a plasmolyzing solution, but regain their original stiffness and elas- ticity when returned to pure water. This rigidity, which is a result of osmostic pressure, is called turgidity. The rate at which water and dissolved substances penetrate the protoplasm > Pfeffer, W., Ueber Election organischer Nahrstoffe. Jahrb. wiss. Bot. 28: 206-268. 1895. 2 Nathansohn, Alexander, ZurLehre vom StoSaustausch. Ber. Qputsch, Bot Ges. 19: 500-513. 1901. 3 Vries, Hugo de, Untersuchungen uber die mechanischen Ursachen der Zellstreckung. ausgehend von der Einwirkung von Salzlosungen auf den Turgor wachsender Pflanzenzellen. Leipzig, 1877. Idem, Untersuchungen uber die mechanischen Ursachen der Zellstreckung. Halle, 1877. 122 PHYSIOLOGY OF NUTRITION is influenced by external conditions. Van Rysselberghe 1 studied the effect of temperature upon this rate. In one series of experiments pieces of pith from young twigs of Sambucus nigra (elder) were placed in water and then trans- ferred to 26-per cent, solutions of cane sugar at different temperatures. Each piece was 114 mm. in length at the outset, and their lengths were redetermined at stated intervals. The lower the temperature, the more slowly did plasmolysis occur. The amounts of shrinkage observed for such pieces of Sambucus pith, with different temperatures and after different periods of time, are shown in the following table. Temperature. deg. C. 6 12 16 20 25 Time Period hours mm. mm. mm. mm. mm. mm. 2 4-5 8.5 20.0 33-0 40.5 40.5* 4 7-5 135 25.0 38.0 42.0* 6 10. 17.0 28.0 42.0* 8 12.5 20.0 30.0 10 14.0 21-5 3i-5 24 21 .0 310 40.0 *No further shrinkage. * In another series of experiments plasmolyzed pieces of Sambucus pith were placed in water at various temperatures, with the same result; the return of turgidity was more rapid as the temperature of the water was higher. These results are shown graphically in the curve of Fig. 71, where the abscissas are the temperatures and the ordinates are the velocities of the movement of water through the protoplasmic membrane (both inward and outward.) 8 7 n .5 7 J 2 z? c 16 c SO c 26' 30* Fig. 71. — Graph representing relation of temperature to velocity of penetration of water through the protoplasmic membrane. The rates at which dissolved substances diffuse through the protoplasm also depend on temperature. If the velocity of movement at o°C, be taken as unity, then the following relative velocities are obtained for potassium nitrate, glycerine and urea, for various higher temperatures. 1 Van Rysselberghe, Fr., Influence de la temperature sur la permeabilite du protoplasme vivant pour l'eau et les substances dissoutes. Recueil Inst. Bot. Bruxelles 5: 200-249. 1902. [Idem, Reaction osmotique des cellules vegetales a la concentration du milieu. Mdm. cour. Acad. Roy. Belgique 58: 1-101. 1898.] ABSORPTION OF MATERIALS IX GENERAL 123 Temperature, deg. C. Substance 6 12 16 20 25 1 .0 1 .0 1 .0 1.8 1.9 2.1 4-4 4.2 4-5 6.0 5-6 5-3 7-3 7.0 7.0 7-3 7.0 7.6 The cell sap frequently exhibits high osmotic values. 71 DeVries found that sap expressed from young plant organs showed the osmotic values given in the table below. Source of Expressed Sap Csmotic Value Gunnera scabra (petioles) . . . Solatium tuberosum (leaves) Sorbtis arcuparia (berries) . . Beta vulgaris (roots) atmospheres 3-5 5-5 9.0 21 .0 The moulds Aspergillus niger and Penicillium may develop osmotic pres- sures as great as 157 atmospheres, when they are grown in concentrated sugar or salt solutions. n A solution alone has no osmotic pressure, this being produced by two solutions (or a solu- tion and the pure solvent) and a membrane, all acting together. When the "osmotic pres sure" of a solution is spoken of, the maximum osmotic pressure that might be obtained with that solution, at the given temperature, is meant. To obtain this maximum the membrane em- ployed must be quite impermeable to all the solutes (dissolved substances) of the solution, and the membrane must be in contact with the solution on one side and with the pure solvent (water) on the other. These conditions are probably never actually fulfilled in the case of plant cells. If we employ the term osmotic value for the maximum pressure, then the actual pressure developed in any cell is usually of somewhat lower magnitude than is the osmotic value of the cell sap. Diffusion tension of the solute is another term that may be employed for the osmotic value, with reference to the solution itself, but this is not without objection. These measurements of deVries' were made by means of cell membranes (plasmolytic method), so that the nature and condition of the cells used as indicators enter into the argument here, and he was not really measuring the osmotic values of these expressed solutions. — Ed. Fitting has studied the osmotic pressures of the cells of plant leaves, by the plasmolytic method, using potassium nitrate solutions, in a very thorough way. He dealt especially with desert plants. See: Fitting, Hans, Die Wasserversorgung und die osmotischen Druckver- haltnisse der Wustenpflanzen. Zeitsch. Bot. 3 : 209-275. 1911. Livingston, B. E., The rela- tion of the osmotic pressure of the cell sap in plants to arid habitats. Plant world 14: 153-164. 1911. (This is a somewhat critical review of Fitting's paper.) While plant cells in general have osmotic pressures of from 5 to n atmospheres, Fitting found pressures much exceeding 100 atmospheres in the leaves of some desert plants. This value is greater for plants growing in 124 PHYSIOLOGY OF NUTRITION DeVries determined the partial osmotic pressure developed by some of the constituents of the cell sap. The following table gives an idea as to what sub- stances are instrumental in the production of osmotic pressure in plants. The figures denote percentage of the total pressure. Source of Expressed Sap Potassium. Salts of Or- ganic Acids Malic Acid Glucose Sodium Chloride Other Sub- stances Heracleum spondilium (petioles) 5-9 9.1 69.1 6.4- 9-5 Rochea falcata (leaves) 3-i 42-3 23.1 n-5 20.0 Diffusion in solution is very important in the absorption of materials by plants but it cannot account for the transfer of absorbed substances within the plant, for movement by diffusion alone is much too slow. 1 For example, it would take 319 days for 1 mg. of sodium chloride, a rapidly diffusing substance, to diffuse 1 m. out of a 10 per cent, solution of that salt. A period of fourteen years would be required for the same amount of albumin to migrate the same dis- tance. Since diffusion progresses rapidly in gelatine and agar as well as in water, these substances may be employed in diffusion experiments, being poured into a glass cylinder while hot and then covered, after cooling, with a solution of the substance to be studied {e.g., indigo). Intercellular protoplasmic connections, like thin threads reaching through the cell walls, are now known to be of common occurrence in plants (Fig. 72). How these structures may influence exchange of materials between the cells is still unknown, however. very dry habitats than for those growing in more moist situations. For further studies bearing on this and related matters, see: Dixon, H. H., and Atkins, W. R. G., On osmotic pressures in plants and on a thermo-electric method of determining freezing points. Proc. Roy. Dublin Soc, n.s. 12: 275-311. 1910. Idem, Osmotic pressures in plants. I. Methods of extracting sap from plant organs. Ibid, n.s 13: 422-433. 1913. (Reprinted in: Notes from Bot. Sch., Trinity Coll., Dublin 2 : 154-165. 1913.) Idem, same title, II. Cryoscopic and conductivity measurements on some vegetable saps. Ibid. n.s. 13: 434-440. 1913. (Reprinted in: Notes from Bot. Sch., Trinity Coll., Dublin 2: 166-172. 1913.) Harris, J. Arthur, and Lawrence, John V., assisted by Gortner, R. A., The cryoscopic constants of expressed vegetable saps as related to local environmental conditions in the Arizona deserts. Physiol, res. 2: 1-49. 1916. (Other papers are there referred to.) Hibbard, R. P., and Harrington, O. E. , De- pression of the freezing-point in triturated plant tissues and the magnitude of this depression as related to soil moisture. Ibid. 1 : 441-454. 1916. For a general discussion of the osmotic relations of cells see: Atkins, W. R. G., Some recent researches in plant physiology, xi + 328 p. London, 1916. — Ed. 1 Stefan, J., Ueber die Diffusion der Fliissigkeiten. II. Berechnung der Grahamschen Versuche. Sitzungsber. (math.-naturw. Kl.) K. Akad. Wiss. Wien 79II : 161-214. 1879. Vries, Hugo de, Ueber die Bedeutung der Circulation und der Rotation des Protoplasma fur den Stofftransport in der Pflanze. Bot. Zeitg. 43: 1-6, 17-26. 1885. ABSORPTION OF MATERIALS IN GENERAL 125 Plants can absorb solid soil constituents but these must first be dissolved in water. If a polished marble plate is placed in the bottom of a box in which seedlings are grown, many of the roots come into close contact with the plate, Pig. 73. a, thick cell wall; b, canals piercing cell Fig. 72. Fig. 72. — Cells of endosperm of Areca oleracea. walls and containing protoplasmic strands. Fig. 73. — A piece of calcium carbonate dissolving in hydrochloric acid as this diffuses upward through the bladder membrane M. and if the latter is removed after a time the imprint of the roots may be seen on the polished surface, etched by acid root excretion. The acid character of root excretion may also be shown by the reddening of blue litmus paper against which the roots are induced to grow. The following experiment illustrates the solution of soil particles and their absorption after being dissolved. A broad glass tube with its lower end firmly bound with animal bladder (Fig. 73) is filled with and inverted over a weak solu- tion of hydrochloric acid, so that the cylinder remains filled. A piece of marble is placed upon the smooth surface of the bladder. The marble gradually becomes smaller and smaller as it is dissolved by the acid imbibed in the membrane. Calcium chloride is formed during the process and diffuses slowly through the membrane into the solution below, where it can be identified with suitable chemical reagents. Czapek 1 studied the nature of root excretions. He employed plates made of a mixture of aluminium phosphate and plaster of Paris. These are soluble in many acids (hydrochloric, nitric, sulphuric, phosphoric, formic, oxalic, suc- cinic, lactic, malic, citric, and tartaric) but they are insoluble in carbonic, acetic, propionic and butyric acids. Various kinds of roots produced no effect 1 Czapek, Friedrich, Zur Lehre von den Wurzelausscheidungen. Jahrb. wiss. Bot. 29: 321-390. 1896. 126 PHYSIOLOGY OF NUTRITION upon these plates, when they were exposed to the roots as was the marble men- tioned above, and it therefore follows that acids belonging to the first list just given are not noticeably present in root excretions. In other experiments by the same writer Congo red was employed, which becomes brownish-red through the action of carbonic acid and bright blue through the action of acetic, pro- pionic and butyric acid. The roots turned the Congo red only brownish-red, without any tendency toward blue, from which it appears that the corrosion of the marble (in the experiment described above) and of soil particles, is to be attributed to the action of carbonic acid excreted by the roots. According to Stoklasa and Ernest 1 roots excrete organic acids only when inadequately supplied with oxygen. The following examples indicate how much may be accomplished by plants in dissolving soil particles. Lind 2 showed that the hyphae of certain fungi in pure culture were able to penetrate through marble plates and bones. Nadson 3 described a considerable number of algae that penetrate somewhat deeply into limestone and shells, dissolving the material. These forms experience severe competition with many other algae on the surface of the substratum, but their ability to grow in solid limestone, which is impenetrable to their competitors, gives them a definite advantage in the struggle for existence. Nadson found that these algae excrete oxalic acid. p It is also well known that parasitic fungi penetrate the cell walls of their host plants. Miyoshi 4 found that fungus hyphae can pierce membranes of very different kinds. The membranes to be studied were placed over nutrient gela- tine and inoculated with spores. As germination took place the hyphae bored through the membranes and reached the nutrient media below. Summary i. Materials Absorbed by Plants. — From the air the ordinary plant absorbs carbon dioxide (and also oxygen sometimes, especially at night). From the soil it absorbs water, and inorganic salts that contain nitrogen and the six essential ash constituents (S, P, K, Ca, Mg, Fe) . As stated in Chapter III, free nitrogen is absorbed by some lower forms and by the nodule bacteria in the tubercles of legume roots, etc. Small amounts of oxygen appear to be absorbed from the soil by active roots. All these substances, supplying the ten essential elements, and also many that supply non-essential elements, are absorbed by diffusion in solution, generally in aqueous solution. (When the transpiration rate is high, however, it appears that these sub- i Stoklasa, Julius, and Ernest, Adolf, Beitrage zur Losung der Frage der chemischen Natur des Wur- zelsekretes. Jahrb. wiss. Bot. 46 : 55-102. 1909. 2 Lind, K., Ueber das Eindringen von Pilzen in Kalkgesteine und Knochen. Jahrb. wiss. Bot. 32 : 603-634. 1898. 3 Nadson, G., Die perforierenden (kalkbohrenden) Algen und ihre Bedeutung in der Natur. [Abstract in German, pp. 35-40. Text in Russian.] Scripta Botanica Hort. Univ. Imp. St. Petersburg 18: 1-40. 1900-1902. * Miyoshi, Manabu, Die Durchbohrung von Membranen durch Pilzfaden. Jahrb. wiss. Bot. 28: 260-289. 1895. p Also sec: Diels, L., Die Algen-Vegetation der Siidtyroler Dolomitenriffe. Ber. Deutsch. Bot. Ges. 32 : 502-526. 1914. — Ed. ABSORPTION OF MATERIALS IN GENERAL 12 7 stances may enter roots from the soil by a mass streaming a|id nitration of the soil solution through the peripheral cells, to the xylem vessels.) To enter plant cells, these substances must be dissolved in water (or some other substance in the cell wall). They diffuse through the peripheral, water-impregnated cell walls, into the proto- plasm. Carbon dioxide and oxygen diffuse through the suberin or lignin of cell walls that are impregnated with one of these substances, as well as through the imbibed water. 2. Diffusion of Gases. — The ultimate particles of every gas, and of every mixture of gases, are considered as always in motion (somewhat as the individuals of a swarm of gnats in the air) and as always tending to spread outward in all directions, until some impermeable wall is encountered. They tend to distribute themselves uni- formly throughout all the space that is available. This spreading movement of the individual gas particles is called diffusion of the gas ; it is not to be confused with mass flow and convection, by which the gas streams or flows as a whole, like wind. If two masses of different gases are brought into contact (as in the two halves of a closed chamber) and if no convection or stirring motion is present, the particles of both kinds of gas diffuse outward, each kind into the space of the other kind, as though the other kind were not present, and they eventually become uniformly mixed. Rates of diffusion of different kinds of gases are proportional to the square roots of their respect- ive densities; hydrogen diffuses four times as rapidly as oxygen (densities, 1:16), temperature and pressure being the same for both. If septum or wall separates the two original gas masses, diffusion takes place in both directions through the septum if that is permeable to both gases; if the septum is permeable to but one of the gases, diffusion occurs in one direction only. If the material of the septum is such that the gas dissolves in it, then the gas diffuses through this material in the dissolved state (as a solute) , Solutes (whether they are gases, liquids, or solids under ordinary con- ditions) diffuse through the solvent in a manner analogous to that of gas diffusion, but the rate of diffusion here is proportional to the concentration gradient in the liquid. With a liquid-water septum separating two different gases (which are at the same pressure and temperature), the rates of diffusion through the septum are proportional to the solubilities of the two gases in water. There may also be mass streaming of the septum material itself, which would apparently alter the rate of this diffusion, the solute being carried by, rather than diffusing in, the solvent. 3. Entrance of Gases into Plants. — Gases, as such, diffuse into (and out of) ordinary plants through stomata and lenticels (openings in the peripheral layer of cells, con- necting directly with gas-filled, irregular, intercellular channels in the tissues). Gas diffusion continues in the intercellular spaces. There is also some mass streaming of gases through intercellular spaces and their external openings. But beyond the cell walls bounding these channels gas diffusion and gas streaming do not reach. Through suberized, lignified, or cutinized cell walls, substances that are ordinarily gases diffuse in solution in the substance of the walls, as well as in the small amounts of water held by imbibition. Through ordinary, water-impregnated walls (and also through the cell contents) they diffuse as solutes in the water. Most of the carbon dioxide and oxygen exchange of ordinary plants occurs through the stomata or lenticels, the true absorption (or elimination) occurring, however, at the peripheries of the intercellular channels, where the gases pass into (or out of) solution in the imbibed water of the cell walls that bound these channels. Gas diffu- sion through stomata occurs at a rate proportional to the linear dimensions of the openings or pores, other conditions being constant; the rate is therefore relatively 128 PHYSIOLOGY OF NUTRITION much greater for these small openings than would be the case if it were proportional to the areas of the cross sections of the openings. 4. Diffusion of Dissolved Substances. — Solute particles diffuse outward in the solvent, much as do gas particles in space, and tend to become equally distributed throughout its volume Solute diffusion does not extend beyond the spatial limits of the solvent. Mass streaming or convection accelerates or retards the apparent rate of diffusion, just as is true for gases. Solute and solvent particles attract each other. If pure water is separated from an aqueous solution by a septum permeable to both solvent and solute, diffusion of both substances occurs through the septum and a uniform solution on both sides finally results. If the septum is permeable only to the solvent (water), then diffusion takes place only in the direction from solvent to solu- tion, and osmotic pressure is developed in the latter. This is like gas pressure in many respects, being proportional to the outward-diffusing tendency of the solute particles. Salts dissociate or ionize to some extent in solution, and the osmotic pressure that can be developed by a given solution (its osmotic value) is nearly proportional to the total number of particles contained in a unit of volume; more precisely, it is proportional to the quotient of the number of solute particles present divided by the total number of particles (solvent and solute). When a septum — such as the outer surface of the protoplasm of a cell — separates, two different aqueous solutions, each containing many kinds of solutes as well as water, the septum may retard the diffusion of water or that of any of the solutes, but its presence does not render diffusion any more rapid than it would be if the septum were not present. Retardation may be greater for some substances than for others. Plasmolysis is the tearing of the protoplasmic lining away from the cell wall, frequently due to the presence of more non-permeating solute particles, per unit of volume, on the outside of the protoplasmic periphery than on the inside. Turgor results largely from the reverse condition, being generally due to osmotic pressure developed within the cell, by solutes to which the protoplasm is impermeable. This results in the protoplasm being pushed outward against the cell wall, which becomes stretched. 5. Absorption of Dissolved Substances. — Most dissolved substances diffuse through cell walls rather rapidly, but the protoplasm is frequently impermeable to many solutes that are present and it retards the inward (or outward) diffusion of others. The permeability of the protoplasm of a cell to the various solutes within and without, alters from time to time, according to conditions in the surroundings and within the cell. Carbon dioxide and oxygen (and other gases in the air) pass into solution in the water, etc., of cell walls and then diffuse as other dissolved substances. These materials, and also salts, etc., dissolved in the soil solution, diffuse through the cell walls and protoplasm of roots. (It appears that they may also be carried in by mass streaming when the transpiration rate is high ; if this occurs, the peripheral cells of the roots may act somewhat as filters, allowing the soil water and some of its solutes to enter with the stream but causing other solutes to remain outside or to enter more slowly than do water and the solutes that penetrate the membranes readily.) A solute may accumulate in the interior of a living cell until its concentration there is higher than that in the solution from which it diffuses. This phenomenon is some- times to be explained on the ground that the accumulating solute is chemically altered upon passing into the cell, in which case it only apparently surpasses the concentration of the solution from which it comes. In other cases the physical explanation is still uncertain. The osmotic value of cell sap is generally between two and six atmos- ABSORPTION OF MATERIALS IN GENERAL 1 29 pheres, but it may be much higher, as much as 157 atmospheres having been reported for moulds. The actual osmotic pressure in a cell is generally much lower than the osmotic value of its sap. The rate of diffusion of water and solutes through protoplasm is influenced by temperature, by the condition of the protoplasm, etc., as well as by the concentration difference (or gradient) between the interior and exterior of the cell. Carbon dioxide continually diffuses out of roots into the soil solution (excepting when the transpira- tion rate is so high that the flow of water into the root's is more rapid than the diffusion rate of carbon dioxide). This substance (forming carbonic acid when dissolved in water) acts as a solvent on many solid soil constituents. Organic acids appear to diffuse out of roots when the latter are poorly supplied with oxygen, and these acids may have a similar action on solid materials in the soil. Some fungi and algae normally give off organic acids, as do many bacteria also. CHAPTER VI MOVEMENT OF MATERIALS IN THE PLANT §i. General Occurrence of Movement of Materials. — From previous statements it is clear that the essential materials are not always directly ab- sorbed by the plant organs in which they are ultimately used. Organic materials are produced from inorganic substances in the green leaf, but the leaf itself can absorb only carbon dioxide. The other materials (water and mineral constitu- ents) that are necessary in the formation of organic compounds are absorbed by the roots, and usually travel long distances before finally reaching the leaves. Similarly, organic materials are frequently used in large quantities in organs where they are not produced; for instance, in all growing parts that lack chloro- phyll. This is especially true of organic materials that are elaborated from inorganic compounds; new kinds of organic substances may of course be pro- duced in any region of the plant, from other organic substances that have been previously formed there, or that come from elsewhere. The organic substances that are requisite for the formation of new cells come to these cells from the leaves, and they also frequently travel long distances before reaching the point where they are used, as in the case of growing root-tips. It is clear, therefore, that there is a general movement of materials within the plant. The compounds occurring in plants may be in the solid as well as in the liquid or gaseous condition. Solid substances, however, must first pass into solution before translocation can occur, since otherwise they cannot pass through cell walls. The study of the movement of materials in plants may, accordingly, be reduced to a consideration of the movement of gases and of water and dissolved substances. §2. Movement of Gases. — Many air passages (intercellular spaces) are always present in the cortex of stems and roots as well as in the parenchymatous tissues of leaves. The lenticels, small openings in the bark, and the stomata also, bring these passages into direct connection with the external air, and the internal atmosphere is thus always under the same pressure as that of the air outside, while renewal of the internal air may readily occur through openings to the outside. Gas exchange through the cortex of water plants is greatly hastened by differential diffusion of air, which was first observed in the leaves oiNelumbium speciosum. 1 The leaf of this plant consists of a round leaf-blade, from the center of the lower surface of which the petiole projects. Stomata occur only 1 Barthelemy, A., De la respiration et de la circulation des gaz dans les vegetaux. Ann. sci. nat. Bot. V . 19: 131-175. 1874. [See also, for observations and a better explanation: Ohno, N., Ueber lebhafte Gasausscheidung aus den Blattern von Nelumbo nucifera. Zeitschr. Bot. 2: 641-664. 1010. [Rev. by Livingston in: Plant world 14: 7-2-73- ion) 130 MOVEMENT OF MATERIALS IN THE PLANT 13I on the upper surface. If water happens to lie upon the upper leaf surface gas bubbles are observed to be given off rapidly on sunny days, these bubbles arising from the stomata and from any chance openings in the surface of the petiole. This evolution of gas is so violent at times that the water appears to be boiling. This phenomenon is unrelated to life processes, since it occurs also with dead leaves. A similar elimination of gas may be artificially produced by a special arrangement. This consists of a cylindrical porous clay cell rilled with finely powdered chalk, or simply with air. A glass tube is inserted through a stopper closing the open end; this tube corresponds to the petiole of the Nelumbium leaf, while the cell corresponds to the leaf-blade. The porous cell is first dipped in water and is then supported obliquely, the tube ending in a vessel of water below. When the clay cell is heated, gas is given out in large quantities from the open end of the glass tube. This gas is air, practically saturated with water vapor. Frequently the volume of gas thus eliminated is as much as forty times as great as that of the cell itself, so that gas must enter the cell through the porous wall during the experiment. This phenomenon is caused by unequal heating, both in the case of the porous clay cell and in that of the Nelumbium leaf. a The underground portions of many plants growing in submerged, swampy, or poorly aerated soils, 6 possess root outgrowths that grow upward into the air Ohno found the pressure under which gas escapes from Nelumbo leaves to rise sometimes to more than 40 mm. of a mercury column. The explanation is somewhat complicated. The gas pressure outside the clay chamber is due to a large partial pressure of oxygen and nitrogen and a very much smaller one of water vapor, the magnitude of the latter depending upon the humidity of the air. The conditions are reversed on the inside, where the larger partial pressure is due to water vapor and that due to the other gases of the air is smaller. The wet porous clay wall, being permeable to the other gases as well as water, movement takes place in both directions; water moves outward and evaporates, and nitrogen and oxygen diffuse inward. Since there is an excess of liquid water, the partial pressure of water vapor on the inside remains constant in spite of the outward movement. Also, the water vapor that evap- orates from the external surface of the porous clay is quickly removed from the vicinity by air currents, so that the partial gas pressure due to water vapor on the outside also remains nearly constant. The external partial pressure of nitrogen and oxygen is also constant, in spite of the inward diffusion, for there is here an excess of these gases and the whole atmosphere is available. But, as these gases diffuse into the chamber they raise the partial pressure of non-aqueous gases within, and so increase the total gas pressure on the inside. Since the cham- ber opens to the outside through the tube, this internal gas pressure can never rise much above what it was at the start, for bubbles escape from the open end of the tube. The arrangement is a sort of osmometer, with a concentrated solution of water vapor in the other gases on the inside and a very dilute solution of the same sort on the outside, the wet wall being more permeable to nitrogen and oxygen than to water vapor. A relatively large amount of water vapor is contained in the gas that exudes from the tube. The heating of the tube seems to accelerate the process partly because it tends to remove the water vapor as it evaporates from the tube, so as to keep the external partial pressure of the other air gases near its original high value. It thus acts like a stirrer in an osmometer cell, which keeps the internal solution from becoming too much diluted next to the membrane. Also, at higher temperature the vapor pressure of water inside the chamber is higher. — Ed. b These structures (called "knees") are characteristic of Taxodium distichum (bald cypress), of the swamps of the southeastern United States. For an excellent photograph showing these see: Schimper-Fisher, 1903. [See note k, p. ior.] Fig. 48, facing p. 74. — Ed. 132 PHYSIOLOGY OF NUTRITION (Fig. 74). The tips of these are composed of spongy tissue and readily allow the entrance of air. These outgrowths are like ventilation pipes in that they promote the movement of air to the roots below. The air spaces of the cortex are thus always directly or indirectly in communication with the external atmosphere. 1 The central woody cylinder of the stem also contains air. HohnePs 2 ex- periments indicate that the air in the wood and that in the cortex are not at all continuous. In these experiments (Fig. 75) a leaf is fastened, by means of a rubber stopper, in a wide-mouth bottle (g), which has a lateral opening below, the latter fitted with a tube and funnel (/) through which mercury is introduced. The air in the cylinder, compressed by the mercury, is forced through the sus- pended leaf and rises in bubbles through the water in the glass vessel above (/). Fig. 74. — Part of stem of Jussicea repens, with ventilation roots (w); surface of water, O. Fig. 75. — Hohnel's apparatus. Pfeffer.) (After Microscopic study shows that bubbles are extruded only from the cortex, not from the central cylinder. Air enters the leaf through the stomata and air spaces of the leaf cortex and is given out from the cortical region of the stem, without entering the wood. The aeration system of the wood is a closed system and does not communicate at all with that of the cortex. Hbhnel demonstrated the existence of negative gas pressure in the wood of stems. If a twig, or the petiole of a leaf, is cut under mercury on a sunny day in summer, mercury rises very rapidly through the cut surface into the vessels (Fig. 76), which then appear gray in cross-section, from the presence of the 1 Goebel, K., Ueber die Luftwurzeln von Sonneratia. Ber. Deutsch. Bot. Ges. 4: 240-255. 1886. Jost, Ludwig, Ein Beitrag zur Kenntniss der Athmungsorgane der Pflanzen. Bot. Zeitg. 45 : 601-606, 617-627, 633-642. 1887. * Hohnel, Franz Xavier R. von, Einige anatomisehe Bemerkungen uber das raumliche Verhaltniss der Intercellularraume zu den Gefassen. Bot. Zeitg. 37: 541-545. 1879. Idem, Beitrage zur Kenntniss der Luft- und Saftbewegung in der Pflanze. Jahrb. wiss. Bot. 12: 47-131. 1870-1881. MOVEMENT OF MATERIALS IN THE PLANT 133 metal. Occasionally vessels may thus be injected with mercury for a distance of from 50 to 60 cm. above the cut surface. Experiments of this kind show that the attenuation of the air in the vessels may be very considerable. Negative pressure in wood may be demonstrated in still another way. A leafy branch with two or more twigs (Fig. 77) is placed with its cut end in water. One of the twigs is cut off and the cut end (b) is connected with rubber tubing to a glass tube (a), the lower end of which dips into mercury. After some time the mer- cury rises in the tube indicating that the air in the wood is rarefied. The air of the stem is most attenuated when the activity of the plant is greatest. As will be seen later, this phenomenon of negative gas pressure bears an important relation to the movement of water in the stem. Fig. 76. — The cutting of a stem under mercury §3. Movement of Water and Dissolved Substances.— The first experiments upon the movement of water and solutes in plants were carried out by Malpighi in 1 67 1. He removed a ring of bark from a woody stem and found that the region above the wound continued alive and grew even more rapidly than be- fore, producing an annular swelling (Fig. 78), while the region below the wound failed to develop further. The girdling operation is thus seen to have no effect at all upon the movement of water from the soil into the upper portion of the plant, although it stops the movement of organic materials into the lower regions. Malpighi concluded from this experiment that the soil solution moves upward through the wood, while the organic substances produced in the leaves pass downward through the cortex. The movement of water is sometimes spoken of as the ascending current, and that of organic (or plastic) substances as the descending current. The expressions ascending and descending are not to be interpreted literally, however; in the drooping branches of the weeping willow, for example, the ascending stream descends and the descending one c The phenomenon is mainly dependent upon the rate of loss of water by transpiration from the leaves and upon the rate at which water reaches the leaves from below. The word activity, as used in the text, is rather indefinite, but it may be taken to refer to conditions promoting high transpiration rates. — Ed. 134 PHYSIOLOGY OF NUTRITION ascends. If a ring of bark is removed from a drooping branch of this willow, the swelling develops not above but below the wound. §4. The Transpiration Stream. — The upward movement of the soil solution in the plant depends upon a large number of conditions. Water can enter the plant only if a part of the water already present be lost. d Water is removed from the plant by evaporation from the leaves, the process being called trans- piration, and this is the main condition determining the movement of water. (a) Transpiration. e — Transpiration may be studied in a number of ways, some of which will now receive attention. »-JP Fig. 77. — Apparatus for showing negative gas pressure in wood. {After Pfeffer.) Fig. 78. — Malpighi's girdling experi- ment; the twig is immersed in water to the line h. i. The quantity of water transpired may be found by determining the loss in weight of the plant and its container. The pot in which the plant is rooted is hermetically sealed in a sheet-metal container. The seal (which may be of plastiline or of a mixture of paraffine and petrolatum, etc.) should have three d While this is the main consideration, it may be remembered that enlargement alone, with- out any loss of water, must necessitate water entrance into the enlarging cells. Also, water may be removed from a cell and still not pass out of it, as when it becomes chemically combined within (formation of carbohydrate from water and carbon dioxide, formation of glucose from starch, etc.). — Ed. e For an excellent review of the literature of transpiration, see: Burgerstein, A., Die Transpiration der Pflanzen. Jena, 1904. Also: Zweiter Teil (Erganzungsband). Jena, 1920. — Ed. MOVEMENT OF MATERIALS IN THE PLANT 135 openings, through one of which the stem of the plant projects. The second opening is usually closed and bears a tube through which water may be added to the pot, and the third bears a small glass tube drawn to a fine, open point above. Through the capillary opening of this tube the air in the apparatus remains in equilibriuin with that of the external atmosphere. The loss in weight of the apparatus is due almost entirely to the loss of water from the plant by evaporation. 1 A tall cylindrical vessel of water may be used for small plants in experiments of short duration. The plants are fastened, by. means of silk- wrapped wire, with their roots in the water and their green parts projecting into the air, a thin layer of oil being placed over the water surface to prevent evapora- tion/ The loss in weight of the apparatus, in this case also, is due almost Fig. 70. — Kohl's apparatus for the study of plant transpiration. wholly to evaporation of water from the plant. 2 2. The amount of water absorbed by the plant may be measured, Kohl's 3 1 Hales, Stephen, Vegetable Staticks. London, 1727. - Wiesner, Julius, Untersuchungen iiber den Einfluss des Lichtes und der strahlenden Warme auf die Transspiration der Pflanze. Sitzungsber. (math.-naturw. Kl.) K. Akad. Wiss. Wien 74 1 : 477-531- 1877. 3 Kohl, F. G., Die Transpiration der Pflanzen and ihre Einwirkung auf die Ausbildung pflanzlicher Gewebe. Braunschweig, 1886. * Oil is apt to penetrate into the stem, and the wax seal is muck to be preferred. For a short distance above and below the water surface, the stem may be covered with some material (as plastiline, chicle — the base of the common chewing-gum of the American market — etc.) that does not absorb water and prevents the oil from coming into contact with the plant, in which case the oil-seal method may be satisfactory. Some of the plastilineon the American market is unsuitable, however, for it injures some plants. — Ed. 136 PHYSIOLOGY OF NUTRITION apparatus, shown in Fig. 79, being well suited to such studies. The roots of the plant, together with a thermometer, are placed in a tube of water (r), which communicates below with a long capillary glass tube and also with a rubber tube closed with a glass plug (gl). As transpiration proceeds, the water menis- cus advances along the capillary tube. To refill the latter, the glass plug is simply inserted somewhat farther into the rubber tube. By placing a bell-jar over the plant the atmosphere surrounding the latter may be kept either moist or dry. To keep it moist a sponge saturated with water may be placed under the bell-jar, the walls of which may also be moistened. To keep the atmosphere dry, air may be drawn by an aspirator through a series of wash bottles filled with concentrated sulphuric acid or with pieces of pumice saturated with this acid. The plant may be kept in darkness by covering the bell-jar with an opaque paper cylinder. 3. Finally, the amount of liquid water absorbed and the amount of water vapor lost at the same time may be determined. In this connection, Vesque's 1 apparatus may be used, which consists of a U-shaped tube, one arm of which is broad and the other narrow. This is filled with water and the roots of the plant are placed in the broad arm with a tightly fitting stopper about the stem. Loss in weight of the entire apparatus gives the quantity of water evaporated, while the depression of the water in the narrow arm indicates the amount of water absorbed by the plant." In addition to the apparatus already described, cobalt paper was employed by Stahl 2 to study transpiration. Swedish filter paper is dipped in a 5-per cent. solution 3 of cobalt chloride, and is then dried in the sun or in an oven. It should be stored in a dry place. This paper is intensely blue when dry but the color changes to a bright pink as water is absorbed. The paper is placed upon the leaf surface that is to be studied, and is covered with a small glass or mica plate. For example, a slip of dry cobalt paper, placed against the lower sur- face of a leaf with stomata on this side only, turns pink in a few seconds on a sunny day, but may remain blue for several hours when placed against the upper leaf surface, where stomata are lacking. This experiment shows clearly the influence of stomata upon transpiration.* 1 Vesque, Julien, L'Absorption comparee directement a. la transpiration. Ann. sci. nat. Bot. VI, 6 : 201-222. 1877. 2 Stahl, 1894. [See note 1, p. 36.] 3 Weaker solutions (1- or 2-per cent.) are more suitable in delicate tests, where the differences in trans- piration are small. It is not strictly true that loss of weight in these experiments is to be interpreted solely as loss of water, though other losses are generally negligible. Perhaps the only case where significant errors may be involved on account of this assumption is that in which leaves, etc., fall from the plant during an experiment. For a complete picture of the meaning of loss of weight, however, aside from such obvious accidents as the fall of leaves, it should be remembered that carbon dioxide and oxygen leave the plant in the same way as does water vapor, that absorption of these two gases also occurs, and that many vola- tile oils, etc., also evaporate into the air to some extent. — Ed. h The cobalt-chloride method really furnishes a means for measuring only the power of the leaf to retard water loss by transpiration, the transpiration rate itself depending upon the evaporating power of the air and upon the intensity of absorbed radiant energy as well as upon this power. On various improvements upon Stahl 's method and upon the transpiring MOVEMENT OF MATERIALS IN THE PLANT 137 The amount of water lost from plants by evaporation is very large; in Wiesner's experiments, for instance, three maize seedlings weighing 1.6 g. lost 0.198 g. of water during a single hour in sunlight. Wollny 1 measured the amount of water lost by evaporation from several plants during their entire vegetative period and also determined the dry weights of the harvested plants and the amounts of water evaporated for each gram of dry material for the entire period of growth.* These values, in grams, appear in the table below. Kind Loss from Plants and Soil Together Total Loss For Whole Period Plant Loss, OF Plant June July Aug. Sept. Oct. Total From Soil From Plants per Gram of Dry Weight Maize Oats Pea 647 482 773 3"3 2095 978 576i 2733 917 2754 2008 941 801 12,275 7,3i8 4,410 1063 178 234 11. 212 7,140 4,176 grams 233 665 416 Although plants evaporate large amounts of water, as is evident from the data just given, the amount of water lost from a certain area of leaf is con- siderably less than that lost from an equal area of a free water surface. Ac- cording to Hartig, 1 sq. m. of free water surface lost 2000 cc. of water in twenty-four hours, while an equal area of beech leaves lost only 210 cc. 1 ' power of leaves, see: Livingston, B. E., The resistance offered by leaves to transpirational water loss. Plant world 16 : 1-35. 1913. Bakke, A. L., Studies on the transpiring power of plants as indicated by the method of standardized hygrometric paper. Jour. ecol. 2 : 145- 173. 1914. Livingston, B. E., and Shreve, Edith B., Improvements in the method for determining the transpiring power of plant surfaces by hygrometric paper. Plant world 19 : 287-309. 1 91 6. — Ed. 1 Sachsse, Robert, Lehrbuch der Agriculturchemie. Leipzig, 1888. P. 423. [Whollny, E., Der Einfluss der Pflanzendecke und der Beschattung auf die physikalischen Eigenschaften und die Fruchtbarkeit des Bodens. 197 p. Berlin, 1877. P. 126.] * This ratio has been called the water requirement. For an excellent review of the literature of this subject see: Briggs, L. J., and Shantz, H. L., The water requirement of plants. II. A review of the literature. U. S. Dept. Agric, Bur. Plant Ind., Bull. 285. 1913. — Ed. ' Such comparisons are without very much significance unless the two surfaces that are com- pared have the same shape and the same exposure. In such studies as that here referred to it has frequently been the practice to compare evaporation rates from circular, horizontally exposed, free water surfaces with the corresponding rates of transpiration from an equivalent area of plant leaves. The form and exposure of the latter surface is generally exceedingly com- plex, while these characters of the water surface are relatively simple, and no very useful com- parison is possible by such methods. The evaporating surface of the physical apparatus must resemble the plant surface, in form, size, color, etc., as nearly as is practicable. In this connec- tion, see Renner, O., Experimentelle Beitrage zud Kenntnisder Wasserbewegung. Flora 103 : 171-247. 1911. Idem, Zur Physik der Transpiration. Ber. Deutsch. Bot. Ges. 29 : 125-132. 1911. Idem, Zur Physik der Transpiration II. Ibid. 30: 572-575. 1912. Perhaps the Livingston spherical porous-cup atmometer furnishes the best evaporating surface for com- parison with plants in general, but for detailed study a special atmometer constructed after the pattern of the particular plant used should be employed. On the porous-cup atmometer see: Livingston, B. E., Atmometry and the porous-cup atmometer. Plant world 18 : 21-30, 51-74, 95-111, 143-149. 1915. Livingston, B. E. and Thone, Frank, A simple non-absorb- ing mounting for porous porcelain atmometers. Science, n. s. 52: 85-86. 1920. — Ed. 138 PHYSIOLOGY OF NUTRITION Leaves removed from the plant lose much more water than those still attached to the plant. Krutizky 1 found that a single leaf of Cyssus antarcticus lost 10.6 cc. of water in one day, while a branch of the same plant with six leaves, lost only 10.8 cc. Results obtained from studies with cut leaves are thus not to be applied directly to entire plants. After the foregoing introductory remarks, the influence of external conditions upon the rate of transpiration will now be considered. Light exerts a pronounced influence upon the amount of water evaporated. 2 For instance, three maize seedlings weighing 1.6 g. transpired in one day, 198 mg. in sunlight, 68 mg. in diffuse light and 27 mg. in darkness. Plants trans- pire much more actively in light than in darkness. If they are transferred from darkness to light, or the reverse, the rate of transpiration is not suddenly increased or decreased, but the change in rate takes place gradually. The daily periodicity of transpiration also depends upon light. 3 The amount of water absorbed during the whole period of twenty-four hours is practically equal to that lost by transpiration in the same period, but there is no such agree- ment between the rates of absorption and transpiration for the various hours of the day; plants are generally nearly saturated with water at night but during the day there is a saturation deficit/" All rays of the spectrum are not equally effective in promoting transpiration from green plants, the maximum effect is produced in the blue and violet regions. The red rays between the Fraunhofer lines B and C are next, in order of their influence. The same wave-lengths of light that are most absorbed by chloro- phyll are thus also most effective in promoting transpiration. Of all the external factors influencing transpiration, light is undoubtedly the most important. The question arises as to how much light is used in this proc- ess. An experiment 4 showed that sunflower leaves transpired on a sunny day 275 cc. per square meter of leaf surface per hour. To evaporate this amount of water requires 166,800 gram-calories of heat per hour. This leaf area received 600,000 calories per hour, so that it appears that 27.5 per cent, of the total amount of radiant energy received was used up in transpiration; as will be remembered, only about 0.5 per cent, is used up in the assimilation of carbon. 1 Famintsyn, A., Exchange of materials and transformation of energy in plants. [Russian.] Zapiski Akad. Sci. St. Petersburg 46, Appendix, xvi + 816 p. 1883. 2 Baranetsky, J., Ueber den Einfluss einiger Bedingungen auf die Transpiration der Pflanzen. Bot. Zeitg. 30: 65-73, 8ib-8ob, 97-109. 1872. Wiesner, 1877. [See note 2, p. 135-] Kohl, 1886. [See note 3. P. 135] 3 Eberdt, O., Die Transpiration der Pflazen und ihre Abhangigkeit von ausseren Bedingungen. Mar- burg, 1889. 4 Brown and Escombe, 1900. [See note 1, p. 34.] * Renner, O., Beitrage zur Physik der Transpiration. Flora 100 : 451-547. 1910. Idem Versuche zur Mechanik der Wasserversorgung. 1. Der Druck in den Leitungsbahnen von Freilandpflanzen. Ber. Deutsch. Bot. Ges. 30: 576-580. 1912. Idem, same title. 2. Ueber Wurzeltatigkeit. Ibid. 30: 642-648. 191 2. Livingston, B. E., and Brown, W. H., Relation of the daily march of transpiration to variations in the water content of foliage leaves. Bot. gaz. 53 : 309-330. 191 2. Lloyd, F. E., Leaf water and stomatal movement in Gossy- pium and a method of direct visual observation of stomata in situ. Bull. Torrey Bot. Club. 40: 1-26. 1913. Shreve, Edith B., The daily march of transpiration in a desert perennial. Carnegie Inst. Wash. Pub. 194. 1914. — Ed. MOVEMENT OF MATERIALS IN THE PLANT 139 Although leaves removed from the plant evaporate much more water than do apparently similar attached leaves, nevertheless this experiment shows that con- siderably more solar energy disappears in the process of transpiration than in the decomposition of carbon dioxide. The humidity of the surrounding air is a second condition markedly influenc- ing the rate of transpiration. The less water vapor the air contains, the more rapid is transpiration, and the transpiration rate decreases as the water-vapor content of the air increases. l Temperature also influences transpiration, but the relation here is compli- cated by the fact that life-processes in general are greatly affected by tempera- ture. Movement of the air also increases transpiration. Finally, the chemical properties of the soil exert a marked influence upon the amount of water evapo- rated from leaves. Experiments with water cultures show that transpiration is controlled both by the concentration of the solution and by the presence or absence of certain substances. Thus, acids may accelerate, while alkalies may retard transpiration. Addition of a small amount of some salt to distilled water in which plants are rooted produces an increased rate of transpiration, but addition of larger amounts causes a gradual decrease in the rate. The transpiration of plants grown in solution containing the essential mineral elements becomes less as the concentration of the solution is increased.'" Besides the external factors mentioned above, there are also internal condi- tions that control transpiration, these being related to the organization of the plant. First, the age of the plant is important. During the period of greatest activity of the leaf, while it is still growing, the rate of transpiration is highest. The reason for this is that the epidermis of young leaves is very permeable to water; transpiration decreases later, but a second maximum is reached when the stomata begin to function. Thereafter the rate of transpiration gradually decreases as the epidermis hardens, in spite of the influence of the stomata. The rate of transpirational water loss from leaves is also correlated with the form and character of their anatomical structures (e.g., number of stomata, thickness or permeability of the epidermis, etc.). A discussion of the resistance offered by plants to transpiration will be presented later, in Part II, Chapter III. Liquid water, as well as water vapor, is given out from many plants, through hydathodes.' 1 The exudation of liquid water may partly replace transpiration, 1 The best study of the influence of air humidity as such is: Darwin, F., On a method of studying transpiration. Proc. Roy. Soc. London B87 : 269-280. 1914. Reviewed by Liv- ingston in: Plant world 17: 216-219. 1914. — Ed. m On the influence of chemicals upon transpiration see: Reed, Howard S., The effect of certain chemical agents upon the transpiration and growth of wheat seedlings. Bot. gaz. 49 : 81-109. 1910. On the influence of the osmotic concentration of the medium see: Briggs and Shantz, 1913 [see note i, p. 137]; Tottingham, 1914, [see note d, p. 84]; Shive, 1915, 2 [see note a, p. 83]; Trelease, 1920, [see note c, p. 86]. — Ed. " Moll, J. W., Ueber Tropfenausscheidung und Injection bei Blattern. Bot. Zeitg. 38 : 49-54. 1S80. Idem, Untersuchungen liber Tropfenausscheidung und Injection von Blat- tern. Verslag. en Meded. K. Akad. Wettensch. Naturk. Amsterdam 2 R., 15: 237-337. 140 PHYSIOLOGY OF NUTRITION occurring mostly when transpiration is retarded for some reason, as for example, in the case of the Aroidea? and other plants living in moist places (Fig. 80) ." (b) Exudation Pressure; — The second condition determining the movement of water in stems is the so-called root pressure, sap pressure, or exudation pres- sure, which produces bleeding. This phenomenon was first investigated bv Hales. 1 If a branch is cut from a grapevine in the spring, before the buds open, a watery fluid is extruded from the wound. Hales bound a piece of animal bladder over the cut end and found that the sap was excreted with such force that the bladder was much swollen at first and was finally broken. To measure the force with which the sap was extruded, Hales connected the cut end of a branch with Fig. 80. — Guttation from hydathodes at the edge of a leaf of Impatiens sultani. (After Pfeffer.) Fig. 81. — Arrangement for measuring exu- dation pressure. (After Pfeffer.) 1880. Volkens, G., Ueber Wasserausscheidung in liquider Form an den Blattern hoherer Pflanzen. Jahrb. K. Bot. Gart. u. Bot. Mus. Berlin 2 : 166-209. 1883. Gardiner, Walter, On the physiological significance of waterglands and nectaries. Proc. Cambridge Phil. Soc 5: 35-50. 1883. Wieler, A., Das Bluten der Pflanzen. Cohn's Beitrage zur Biol. d. Pflanzen 6: 1-211. 1893. Haberlandt, G., Anatomisch-physiologische Untersuchungen iiber das tropische Laubblatt. II. Ueber wassersecernirende und-absorbirende Organe. (I. Abhandlung.) Sitzungsber. (math.-naturw. Kl.) K. Akad. Wiss. Wien I03 r . 489-538. 1894. Idem, same title (II. Abhandlung.) Ibid. 1 04/: 55-116. 1895. Idem, Zur Kenntniss der Hydathoden. Jahrb. wiss. Bot. 30 : 511-528. 1897. — Ed. "Hales, 1735- [See note i, p. 135.] Guttation, as Burgerstein terms this excretion of liquid water [see note e, p. 134], may be in- duced in many plants by injecting the cut stem or petiole with water under pressure. A simple way is to attach a cut branch, by a rubber tube (properly reinforced with cloth wrapping), to the water-tap, having first filled the tube with water, and then to open the tap. Fuchsia, .Impatiens sultani, and Tropaolum majus (garden nasturtium) serve very well. This phe- nomenon was first described by deBary (Bot. Zeitg. 27: 883. 1869). See also, for another early description: Prantl, K., Die Ergebnisse der neueren Untersuchungen iiber die Spal- toffnungen. Flora 55: 305-312, 321-328, 337-346, 369-382. 1872.— Ed. MOVEMENT OF MATERIALS IN THE PLANT 1 4 1 a mercury manometer (Fig. 81). The mercury is forced up in the free arm of the tube by the pressure of the exuding sap, attaining a height, in one of Hales' experiments, of 103 cm. or about 1.5 atmospheres. Instead of removing a branch, an incision may be made in the stem. Bleeding is characteristic of many woody plants in the spring; this is called spring bleeding, since it occurs only in the spring before the leaves expand. After the leaves expand an incision in the stem or the removal of a branch usually fails to produce bleeding; water is then being lost from the leaves by transpiration. Under these conditions bleeding may be induced at the surface of the stump of a cut stem, the leafy portion having been entirely removed. Bleeding may be demonstrated in this way throughout the entire vegetative period, in both woody and herbaceous plants, but the same plant may not show it at all times during its period. To measure the force with which sap is extruded, a mercury manometer is connected to the cut stump of the plant. To measure the amount of liquid ex- creted the manometer may be replaced by a glass tube connecting with a graduate. The recording apparatus of Baranetskii serves the same purpose. Here the liquid flows into a U-shaped tube, lifting a float in the free arm. The float is fastened to one end of a silk thread that passes over a pulley, and a pointer attached to the other end of the thread traces a curve on a smoked, rotat- ing drum. In another apparatus constructed by Baranetskii, the excreted liquid is caught in separate tubes, each tube remaining beneath the outlet tube from the plant for a single hour. The tubes are arranged on the rim of a wooden disk with vertical axis, and this is rotated, by clockwork, just far enough every hour to place a fresh tube under the outlet. Exudation pressure, as indicated by the height of a mercury column, varies in different plants, being less in herbaceous than in woody forms. Thus, in Hofmeister's 1 experiments the height attained by the mercury column was 66 mm. with Atriplex hortensis, and 461 mm. with Digitalis media. The amount of sap excreted by herbaceous plants greatly exceeds the total volume of their roots. Much of the excreted liquid must therefore enter the roots after the cut is made. A plant of Urtica urens excreted 3025 cc. of sap, and the total volume of its root system proved to be only 1350 cc. Similarly, the root volume of a plant of Helianthus animus was only 3370 cc, and yet this plant excreted from its cut stump 5830 cc. of liquid. p There is a daily periodicity in the rate of bleeding 2 and this has no relation to temperature. The time of occurrence of the maximum and of the minimum rate of liquid excretion is not the same for different plants. Etiolated plants exhibit no periodicity. Analyses of the sap extruded by bleeding stems are 1 Hofmeister, W., Ueber das Steigen des Saftes der Pflanzen. Flora, n. R. 16: 1-12. 1858. Idem, Ueber Spannung, Ausflussmenge und Ausflussgeschwindigkeit von Saften lebender Pflanzen. Ibid. n. R. 20: 97-108. 1862. 2 Baranetzky, J., Untersuchungen uber die Periodicitat des Blutens der Krautigen Pflanzen und deren Ursachen. (Besonders abgedruckt aus den Abhandl. Naturf. Ges. Halle 13/) 63 p. Halle, 1873. p In this connection see: Hofmeister, W., Ueber Spannung, Ausflussmenge und ausfluss- geschwindigkeit von Saften lebender Pflanzen. Flora 45 : 97-108, 113-120, 138-144, 145- 152, 170-175. 1862. The numbers given in the text are from this paper. — Ed. 142 PHYSIOLOGY OF NUTRITION very interesting. In Ulbricht's 1 experiments, potato tubers planted on April ii, produced stems that bloomed on July 5. On July 9 the stems were cut off at from 4 to 6 cm. above the soil. The sap that exuded during the next five days was collected, the exudation for each day being kept separate, so that five portions of sap were available for analysis, the results of which are shown in the following table. The quantities (stated in milligrams) refer to a liter of sap in all cases. First Day Second Day Third Day Fourth Day Fifth Day Combustible material ', 450 Ash I 1160 Total dry weight j 1610 310 980 1290 220 960 1180 280 910 1 190 295 945 1240 These numbers show plainly that the total solids consisted mainly of mineral substances, but this statement is still further emphasized by the fact that the combustible material does not represent organic matter alone, for nitric acid and some other inorganic substances are vaporized during incineration, so that it is certain that the sap always contained more inorganic substances than the data show. This result was to be expected, since the ascending water current distributes absorbed soil solution throughout the plant. The presence of organic substances in sap extruded from the xylem may be explained by the fact that the soil solution does not enter this tissue directly, but is transferred into the wood soon after its entrance. It can hardly be supposed that parenchymatous cells, which are so rich in organic substances, should excrete nothing but inor- ganic materials into the vessels. The composition of sap excreted in early spring is very different from that of sap excreted in summer. Birch sap was collected from an opening in the tree trunk just above the soil surface, on each of six different days, between April 5 and May 2 2. 2 The sugar, protein, malic acid, and ash contents per liter of sap are given below, in milligrams, together with the dates on which the sap April 5. April 1 1 . April 17. May 2. May 19. May 22 . Date of Flow Sugar 12,500 13,500 10,900 IO,IOO 9,400 6,900 Protein 21 6 6 Malic Acid Ash 33? 437 500 640 1080 >Ulbricht, R., Ein Beitrag zur Kenntniss der Blutungssafte einjahriger Pflanzen. Landw. Versuchsst. 6:468-474. 1864. [Idem, same title. Ibid. 7 : 185-192. 1865] *Schroeder, Julius, Die Fruhjahrsperiode der Birke (Belula alba L.) und des Ahorn (Acer plalanoides L.) Landw. Versuchsst. 14: 1 18-146. 187 1. MOVEMENT OF MATERIALS IN THE PLANT 1 43 was collected. It is apparent from these analyses that the sap contains less in- organic substances and more organic materials in the earlier part of the season than at the later dates. This is explained by the facts that organic materials accumulate in the woody tissue of perennial plants during the summer, and that they are rapidly removed to the growing regions with the opening of the follow- ing spring; it is at the expense of this accumulated food that spring leaves are formed. After the leaves develop, the sap contains inorganic substances mainly, and spring bleeding thus becomes transformed into summer bleeding. The term bleeding thus denotes the exudation of sap from the woody tissues of wounded plants, brought about by the absorption of water and dissolved mineral substances by the parenchymatous cells of the root, and the movement of this solution into the vessels of the xylem, in which it is carried upward to the wound. The causes upon which this phenomenon is dependent have not yet been found out.'' (c) Movement of water in the stem 1 depends upon a number of conditions. Water moves upward in the xylem, as was shown in Malpighi's girdling experi- ment. Sach's theory, which supposed that it traverses the vessel walls, was proved untenable and is no longer upheld. The ascending current moves in the lumina of the vesssels and tracheides, as is shown by the fact that wilting promptly occurs when these are plugged. The following experiment demon- strates this. A mixture of 20 parts of gelatine in 100 parts of water, melting at 33 and still liquid at 28°C. (at which temperatures plant tissues is not at all in- jured) is prepared, and enough India ink is added to make the preparation readily visible in the vessels. The stem of a leafy shoot is cut under this prepa- ration, the latter having been warmed to 33°C. The liquid rises in the vessels and is allowed to harden by cooling. A small piece is then cut from the base of the stem, to give a fresh absorbing surface, and the cut end is placed in water. After several hours wilting occurs in the leaves, while the leaves of a similar 1 Votchal, Ueber die Bewegung des Wassers in den Pflanzen. Moscow, 1897 (Russian).* Bohm, Joseph, Ueber die Ursache des Saftsteigens in den Pflanzen. Sitzungsber. (math.-naturw. Kl.) K. Akad. Wiss. Wien. 48*: 10-24. 1863. Hartig, R., Die Gasdrucktheorie und die Sachs'sche Imbibitions-Theorie. Berlin, 1883. Strasburger, Eduard, Ueber den Bau und die Verrichtungen der Leitungsbahnen in den Pflanzen. (Histologische Beitrage, Heft 3.) Jena, 1891. Askenasy, E., Ueber das Saftsteigen. Verhandl. Naturhist.-Med. Ver. Heidelberg, n. F. 5 : 325-345- [Gesammtsitzung vom 7- Dez., 1894, und 1. Febr., 1895. Heft 4, dated 1896.] Idem, Beitrage zur Erklarung des Saftsteigens. Ibid, 5: 429-448. [Ge- sammtsitzung vom 6. Marz, 1896. Heft 4. Vol. dated Heidelberg, 1897.] Godlewski, E., Zur Theorie der Wasserbewegung in den Pflanzen. Jahrb. wiss Bot. 15; 5 69-630. 1884. [Schwendener, S., Unter- suchungen uber das Saftsteigen. Sitzungsber. (math.-naturw. Mitth.) K. Preuss. Akad. Wiss. Berlin 1886; 355-396. 1886. Idem, Vorlesungen uber mechanischen Probleme der Botanik. Leipzig, 1909.] « Molisch showed that the phenomenon of sap exudation from holes and cuts in the upper regions of palm stems is not due to a pressure normally present in the plant, but that the pres- sure here indicated is brought about as a result of wounding. The cells near the cut surface undergo an alteration, and bleeding begins only after enough time has elapsed to allow this alteration to occur. The altered cells resemble gland cells and secrete the liquid. But it appears improbable that all the cases of bleeding are to be thus explained. See: Molisch, Hans, Botanische Beobachtungen auf Java. (III. Abhandlung.) Die Secretion des Palm- weins und ihre Ursachen. Sitzungsber. (math.-naturw. Kl.) K. Akad. Wiss. Wien I07 z : 1247- 1271. 1898. Idem, Ueber localen Blutungsdruck und seine Ursachen. Bot. Zeitg. 60: 45-63. 1902. 144 PHYSIOLOGY OF NUTRITION branch, the vesesls of which are not thus plugged, may remain turgid for a number of days. 1 Air as well as water is present in the vessels and is very much rarefied at times, as was shown by Hohnel's experiments. To show the presence of water in the vessels, a piece is removed from a young stem by means of a double pair of shears, so arranged that the two cuts are made at the same time. From the piece thus obtained, longitudinal sections are prepared and examined under the microscope, of course without any addition of water. If the two cuts are not made simultaneously no water is observed in the vessels, for, because of nega- tive pressure in the gases of the wood, air rushes into the vessels at the cut surface as soon as the incision is made, driving the water before it into other regions of the plant. The water columns in the vessels are frequently interrupted by air bubbles and these may be demonstrated under the microscope. To accomplish this the parenchymatous tissue is carefully removed from one of the woody bundles of a young stem with but little wood (e.g., Begonia or Dahlia). Thus the bundle is exposed, but is uninjured and is still in connection with the rest of the plant at both ends of the preparation. Study of such preparations shows that the vessels are nearly filled with water and contain but few air bub- bles in moist, cloudy weather, but that they contain less water and consequently a greater amount of air 2 on sunny days. All the investigations that have so far been made indicate that the water columns in the vessels are not completely broken by air bubbles. Cross-sec- tions of the vessels show that they are not perfectly cylindrical but are more or less prismatic and many-sided and that this irregularity in form is further in- creased by circular, spiral and other secondary thickenings of the walls. Air bubbles tend to assume a spherical form and the irregularly shaped portions of the vessels are thus not completely filled with air, so that a continuous water column results, the air bubbles being wholly surrounded by water/ 1 Errera, Leo, Ein Transpirationsversuch. Ber. Deutsch. Bot. Ges. 4: 16-18. 1886. 2 Capus, Guillaume, Sur l'observation directe du mouvement de l'eau dans les plantes. Compt. rend. Paris 97: 1087-1080. 1883. r It is doubtful whether this is true when the transpiration rate is considerable and the soil fairly dry. Wherever a gas bubble occurs in a vessel it should enlarge, under these conditions, until it fills that entire vessel segment from the cross-wall below to the one above. The gas- liquid surface tension in such a case as is postulated in the text would have to be as great as the sum of the gas pressure in the enlarged bubble and the tensile stress exerted upon the water by the transpiration process going on above. The gas pressure in the bubble must be less than a single atmosphere, but the magnitude of the tensile stress is at least more than equivalent to an atmosphere. Thus the sum just mentioned is frequently of the order of several atmos- pheres and is surely of greater magnitude than the gas-liquid surface tension. It follows that the bubble must enlarge until its surface film comes into contact with the surrounding vessel walls at every point; thus reinforced, the surface layer of the liquid can withstand the great attraction exerted by the stressed water-mass, and the gas bubble does not expand farther. When the water has been under stress for a sufficient time there should be no free water between cell walls and gas at any point in the entire plant body; all such surfaces should be cell-wall surfaces, at which the liquid surface is held by the force of imbibition. Indeed, this condition would probably be attained by the action of the gas pressure within the bubble, before any stress developed in the liquid at all. The picture presented in the text at this MOVEMENT OF MATERIALS IN THE PLANT 145 Votchal has carried out a thorough investigation upon the transmission of pressure by wood containing both air and water. [See note 1, p. 131 for reference]. Portions about 2 m. in length, from saplings or branches, were placed in a horizontal position and water was forced through them from one end to the other, by means of water or mercury pressure applied through glass tubes suitably attached. The rate of entrance of water at one end and that of exit at the other vary in a regular manner for a time after pressure is first applied. VotchaFs representation of these variations is reproduced in the diagram of Fig. 82. The variation in the entrance rate, at the end where pressure is applied, is shown by the line a. This rate first increases with remarkable rapidity and soon attains a rather high value (a), but this high rate is maintained only during several hundredths of a second after the pressure is applied. The next stage (a(S) shows a decreasing rate and is of longer duration, continuing for from one-half to two minutes. In the third stage (fiy) the velocity continues to fall, but more slowly and gradually, and it finally assumes a constant value. In short pieces of stem the final constant rate is attained after five minutes, but Fig. 82. — Diagram showing variations in rates of entrance and exit of water moving under pressure through a section of woody stem. (After Votchal.) with longer pieces this period may be prolonged. The simultaneous variation in the rate of exit, at the opposite end of the piece of stem, is shown by the line a! &'. The velocity of movement here increases very slowly, gradually attaining a value equal to that of the rate of entrance at the other end. When the two rates become equal, the two curves become coincident, and water point can be true only with comparatively low transpiration rates, and with comparatively ready entrance of water into the vessels below. The compound water column of the stem is not broken in all vessels at the same level, however, and the transpiration stress is trans- mitted laterally from the water of one vessel to that of adjoining ones, around the gas-filled vessel segments. These matters have been very thoroughly treated by Dixon, and Overton and Renner have each brought forward additional convincing arguments in favor of the general interpretation adopted in the present note. See: Dixon, H. H., Transpiration and the ascent of sap. Prog, rei bot. 3 : 1-66. 1909. Idem, Transpiration and the ascent of sap in plants. London, 1914. Renner, 1910. [See note k, p. 138.] Idem, 1911,^,^. [Seenotej, p. 137-] Idem, 191 2, 1, 2. [See note k, p. 138.] Idem, Theoretisches und Experimentelles zur Kohasions-theorie der Wasserbewegung. Jahrb. wiss. Bot. 56 : 617-667. 1915. Holle, H., Untersuchungen iiber Welken, Vertrocknen und Wider-straff werden. Flora 108 : 73-126. 1915. Overton, J. B., Studies on the relation of the living cells to the transpiration and sap-flow in Cyperus. Bot. gaz. 51 : 2S-63, 102-120. 1911. — Ed. 10 146 PHYSIOLOGY OF NUTRITION is then moving through the piece at a uniform rate throughout. Similar experiments with tubes filled with sand containing air and impregnated with water gave concordant results with those obtained with the pieces of stem. Votchal conceives that air bubbles in the wood act simply as resilient springs that transmit and distribute the thrust imparted to them more slowly and evenly than would a continuous, homogeneous water column. The effective forces applied at the ends of the conducting channels — i.e., the force of foliar transpiration and that of root pressure — furnish energy to account for the ascending water current in plants. Root pressure, produced by osmotic forces, exerts a pressure upon one end of the water column in the wood, while evapo- ration of water from the leaves establishes traction at the opposite end. 8 A simple experiment (Fig. 83) indicates the magnitude of the force that draws water into the leaves to replace that lost by evaporation. If the cut end of a leafy branch or stem is carefully sealed to the upper end of a glass tube filled with water, and if the lower end of the tube dips into mercury, then mercury is drawn up into the tube, replacing the water absorbed at the cut surface, which in turn replaces that lost by evaporation from the leaves. In Bohm's 1 experiments the mercury column rose 86 and even 90 cm. in the tube, thus considerably exceeding the height of mercury column supported by atmospheric pressure upon the free mercury surface below. AskenasyV experiments indi- cate that the rise of the mercury column here shown has a simple physical cause. In these experiments the upper, broad portion of a glass funnel, the neck of which was fused to a long glass tube, was filled with a thick layer of plaster of Paris; when the plaster hardened the apparatus was filled with water, the glass tube dipping into mercury below. As water evaporated from the plaster surface the mercury rose in the tube and attained a height of 82 cm., which is, here also, noticeably greater than that attained under the action of atmospheric pressure. The funnel may be covered with animal bladder instead of being filled with plaster (Fig. 84)." These experiments indicate the great magnitude of the force of cohesion existing between the molecules of water; the water column is not broken even when it is subjected to a considerable stress. These experiments also give some idea of the magnitude of the imbibition force resident in 1 [Bohm, J., Capillaritat und Saftsteigen. Ber. Deutsch. Bot. Ges. n : 203-212, 1893-! 8 Root pressure is not to be considered as generally important in the ascent of water through plant stems. The mere existence of "negative gas pressure" in the vessels shows that the liquid above the gas bubbles is not being forced upward by a pressure applied below. Perhaps the simplest argument in favor of dismissing root pressure from consideration in the general problem of rise of sap lies in the fact that this pressure is found to be highest when water movement is slowest and lowest when movement is most rapid. — Ed. 1 Askenasy, 1896, 1897 [See note 1, p. 143.] Dixon, 1914- [See note r, 144-1- — Ed. " But the bladder membrane has not been recorded as ever showing a rise of the mercury column above the height of the barometer. The experiment usually fails to demonstrate this important point, even with porous porcelain or plaster of Paris; the water column almost always breaks before a stress of one atmosphere is developed. In this connection see: Ur- sprung, A., Zur Demonstration der Fliissigkeits-Kohasion. Ber. Deutsch. Bot. Ges. 31 : 388- 400. 1913. Idem, Ueber die Blasenbildung in Tonometern. Ibid. 33: 140-153. 1915. Idem, Ueber die Kohiision des Wassers im Farnannulus. Ibid. 33: 153-162. 1915. — Ed. MOVEMENT OF MATERIALS IN THE PLANT 147 cell walls of plants and also in plaster of Paris; this force is so great that when water is removed from the cell wall by evaporation more water is immediately withdrawn from the interior of the cell in spite of the osmotic force that opposes such movement. Transpiration from the leaves, the force of imbibition in the cell walls, and the cohesion of liquid water, are therefore the main causes underlying the movement of water in plant stems. The so-called root pres- sure, which causes bleeding in plants, may also be involved here to some extent/ The amount of water passing through the plant is important in the distribu- . tion of mineral substances throughout the organism, as well as in their absorp- tion. Schlosing's studies with tobacco plants may serve as an illustration 1 Pig. 83. — Arrangement to show rise of a mercury column caused by evaporation of water from the leaves of a cut twig. Fig. 84. — Evaporation of water through a membrane, causing rise of mercury in tube below. Compt. rend. Paris 69: 1 Schloesing, Th., Vegetation comparee du tabac sous glocke et a. l'air libre. 353-356. 1869. " The discussion here given of the physics of the rise of the transpiration stream is fragmen- tary and incomplete, but it has not seemed advisable to attempt to render it much more thorough in the limited space to which editorial notes should be restricted in a translation such as the present volume. The notes that have been added to this section aim to place before the student the main points omitted by the author, and to give references to the literature, so that the best treatments of the modern phase of this much-discussed problem may be read. The writings of Dixon, Renner, and J. B. Overton, cited in note r, p. 144, should be referred to, at any rate. The existing text-books are all unsatisfactory in regard to this subject, the Dixon theory not yet having been adequately incorporated into any of them. — Ed. 148 PHYSIOLOGY OF NUTRITION of this. A portion of a plant was allowed to grow in a water-saturated atmos- phere, under a bell-jar, while the remainder was exposed to natural condi- tions. The ash content in the leaves grown in the moist atmosphere was lower than that of the other leaves, the former being only 13 per cent., while the latter was 21.8 per cent., of the total dry weight. 1 " §5. Movement of Organic Substances. — Malpighi's girdling experiment, already described (page 133), indicates that organic substances move through plant stems only in the cortex. This region, however, includes many different kinds of tissue and the question arises whether the movement here considered occurs equally throughout the cortex or only through special parts of it. Han- stein 1 carried out a series of experiments in this connection and found that the removal of a ring of cortex did not always stop growth in the region below the lesion. Anatomical study of the plants that were not injured showed that some of these possessed vascular bundles in the pith as well as in the ring of vessels always found in dicotyledonous plants, while others possessed no collateral bundles and had only bicollateral ones. Girdling had no effect upon the growth of monocotyledonous plants. Hanstein concluded, therefore, that this dif- ference between different plants, in regard to the effect of girdling, is due to the fact that all the sieve-tubes are removed in the girdling of most dicotyledonous plants, while only a part of them are removed in those dicotyledons that have vascular bundles in the pith, and in monocotyledons with bicollateral bundles. Sieve-tubes are therefore the main channels through which the movement of or- ganic material occurs. By virtue of their anatomical structure these tubes are better suited for this movement than are any of the other tissues of the cortex. This conclusion does not at all exclude the possibility that organic substances may move by diffusion through any other living cells, especially through the very small pores by which many cell walls are perforated. A peculiarity of the move- ment of organic materials is that it is regulated exclusively by the activity of living cells and that it is a result of this activity. In other words, this move- ment is controlled by internal conditions. External conditions affect transloca- tion only as they affect the life-processes of the cells in general. With the upward movement of the soil solution it is quite different, for, as has been seen, this is very largely dependent upon such external conditions as light, humidity, wind, etc. The movement of the soil solution has been somewhat thoroughly investi- gated in its general aspects, but our knowledge of the translocation of organic materials rests upon only a few well-known facts and is largely hypothetical. The movement of organic materials has been extensively studied in connec- 1 Hanstein, Johannes, Versuche iiber die Leitung des Saftes durch die Rinde und Folgerungen daraus. Jahrb. wiss. Bot. 2: 392-467. i860. " But see: Hasselbring, Heinrich, The relation between the transpiration stream and the absorption of salts. Bot. gaz. 57: 72-73. 1914. Hasselbring's conclusion is the direct opposite of the one reached by Schlosing. The question as to what rates of transpiration are necessary to elevate the requisite amount of salts in tall plants deserves further atten- tion at the hands of experimenters. It appears clear enough, on a priori grounds, that some transpiration must generally give better growth than none at all, but the rates generally experienced by ordinary plants are probably much higher than the optimum. — Ed. MOVEMENT OF MATERIALS IN THE PLANT 1 49 tion with seed germination. The most important work in this field was done by Sachs. 1 By means of microchemical tests applied to hand sections of seeds and seedlings, he investigated the most important organic substances (such as proteins, sugars, fats, acids, tannins), with regard to their distribution in the tissues. By comparing the distribution of these substances as shown in the seed with that exhibited in the seedling and in different regions of the older plant, Sachs reached his conclusions as to the paths of translocation. He found, for example, that the cortex contains cells that are filled with starch grains during germination, and that these cells form a continuous series (which he called the starch sheath) reaching outward from the cotyledons into all parts of the plantlet. From these observations he concluded that it is in this sheath that starch moves from the cotyledons into other regions, as growth proceeds. The sort of observations on which this conclusion was based bear, however, only upon the distribution and accumulation of the substances in question, in the various organs of the plant; the fact that a continuous series of cells all contain a certain substance does not indicate that the substance in question is moving through those cells. In the case of the starch-filled cells above mentioned, the subsequent experiments of Heine 2 showed that this material is not there in process of translocation, but that the contents of these cells represent merely local accumulations. This author removed rings of tissue from stems of young seedlings, so as to remove the starch sheath at the region of girdling, and found that such treatment neither hindered the development of the plants nor lessened the amount of starch in those regions of the sheath beyond the wound. There- fore, in this case also, the organic materials must have moved through the phloem (leptome) of the bundles, which was not injured by the girdling operation. Some of the plastic material passing through the uninjured phloem found its way to the sheath cells and there accumulated locally as starch. There are also available some studies, by Sachs, Sapozhnikov, 3 and others, bearing upon the translocation of organic substances from the leaves, where they are formed, to other portions of the plant. As carbohydrates are produced in the leaves they continually move into the stem. Comparison of the loss of carbohydrates from attached leaves with the loss, in the same time, from similar leaves that have been detached from the plant, shows that this rate of loss is more than five times as great in the first case as it is in the second. This obser- vation indicates clearly that translocation of carbohydrates from leaves to stem actually occurs. Carbohydrates disappear from the detached leaves only through local consumption, and the rate of its disappearance is much lower than in the case of leaves that remain attached to the plant. This movement 1 Sachs, J., Uebersicht der Ergebnisse der neueren Untersuchungen uber das Chlorophyll. Flora, n. R. 20: 120-137. 1862. Idem, Mikrochemische Untersuchungen. Ibid., n. R. 20: 289-301. 1862. Idem. Ueber die Stoffe, welche das Material zum Wachsthum der Zellhaute liefern. Jahrb. wiss. Bot. 3 : 186-188. 1863. Idem, Ueber die Leitung der plastischen Stoffe durch verschiedene Gewebeformen. Flora, n. R. 21: 33-42. 1863. Idem, Beitrage zur Physiologie des Chlorophylls. Ibid., n. R. ax: 193-204. 1863. 2 Heine, H., Die physiologische Bedeutung der sogenannten Starkescheide. Landw. Versuchsst. 35 : 161-193. 1888. s Sapozhnikov, Die Bildung der Kohlehydrate in den Blattern and ihre Bewegung in der Pflanze. Moscow , 1890. (Russian.)* Idem, 1890. [See note 4, p. 31.] Idem, 1891. [See note 3, p. 38. | Idem, 1893- [See note 4, p. 31.] 150 PHYSIOLOGY OF NUTRITION of carbohydrates takes place through the phloem. 1 There is a daily periodicity in the movement of carbohydrates out of the leaf; the maximum rate of movement occurs, according to Sapozhnikov, during the early hours of the night, between 7 :?,o and 1 1 :3c In perennial plants the accumulated material formed during the summer in never wholly consumed in the same season; a large part is accumulated and remains in the plant until the following spring. The renewed activity of early spring and the development of new shoots and leaves occurs at the expense of organic material accumulated in the preceding year. Accumulation begins very early in the season in some plants — in May, for instance, in the case of the maple; in other plants it begins later — in the oak, for example, in July, and in the Scotch pine, in September. The material first accumulates in the young twigs, from which it gradually moves down the stem until the roots also are rilled. Accumulation ceases at the end of the summer or in the autumn — not until the middle of October in the case of the pine, for example. In winter the accumulated material, consisting mainly of oil and starch, fills all the pith, the medullary rays, the cortex and some parts of the xylem. The solution of the accumulated material begins in early spring. As it dissolves it passes through the medullary rays into the vessels of the xylem, in which it moves to the growing regions, as has been pointed out. If the young twigs are killed by a late spring frost, after the winter reserve has been used up, the death of the tree may follow. Organic materials are removed from storage tissues into other tissues only when they are being consumed in the latter or are moving through these tissues into still more distant regions. 2 If the embryo is removed from a seed of maize or barley, for example, and if the remaining endosperm is planted in moist soil, then the starch of the endosperm is neither removed nor converted into sugar. If, however, the endosperm is placed on the point of a little cone of plaster of Paris, the lower end of which dips into water, the starch is then dis- solved and the resulting sugar diffuses into the water below. Maize endo- sperm is thus completely emptied of starch in from thirteen to eighteen days and a considerable quantity of carbohydrates appears in the water. Similar experi- ments may be performed with bulbs, roots, rhizomes and branches. Lack of oxygen in the atmosphere about the endosperm, or the presence of ether or chloroform vapor, terminates this process. Summary 1. Movement of Materials in General. — Substances enter the plant body at certain parts of its periphery, and then move to distant regions, being in many cases decom- posed and their elements being recombined in various ways during their stay in the plant. Some materials remain in the plant until its death, while others are continuously or intermittently given off to the surroundings. 1 Czapek, Friedrich, Ueber die Leitungswege der organischen Baustoffe im Pflanzenkorper. Sitzungsber. (math, naturw. Kl.) K, Akad. Wiss. Wien 106: 117-170. 1897. 2 Puriewitsch, K., Physiologsche Untersuchungen iiber die Entleerung der Reservestoffbehalter. lahrb. wiss. Bot. 31 : 1-76. 1898. MOVEMENT OF MATERIALS IN THE PLANT 151 2. Movement of Gases. — The internal gas spaces (the intercellular channels mentioned in Chapter V, Section 3) are continuous with the external atmosphere (through stomata and lenticels) and gas streaming as well as gas diffusion may occur through these channels. Gases enter into solution and diffuse through cell walls, protoplasm, etc., just as do other dissolved substances. Dissolved gas may go out of solution and enter the gas spaces in any region of the plant. Thus, dissolved nitrogen, oxygen, etc., may diffuse from the soil into the roots and may subsequently pass out of solution into an intercellular channel. Dissolved oxygen, produced in the chlorophyll- bearing cells of a leaf during a period of sunlight, diffuses as a solute, mainly to the periphery of a sub-stomatal gas space, where it passes out of solution and then diffuses as a gas through the stomatal pore into the surrounding atmosphere. Of course it may also diffuse in other directions through the tissues. The gas spaces of the xylem are not continuous with those of the cortex, but gases may move from one system of channels to the other, first passing into solution, then diffusing as solutes, and finally passing out of solution again. These gas spaces of the xylem are generally not inter- cellular; they occupy portions of the vascular channels (that is, the interiors of much elongated cells that are dead and without protoplasm and that have lost their end walls in many cases where adjacent cells were originally in contact). The pressure of the gas in the vessels is frequently much lower (especially when the transpiration rate is high) than that of the environmental atmosphere and of the intercellular cortical channels. On the other hand, the gas pressure in the xylem may sometimes be higher than that in the cortical channels (when there is sap pressure). 3. Movement of Water and Dissolved Substances. — Girdling experiments show (1) that water and soil solutes move from the roots to other parts of the plant body through the xylem vessels that are not blocked with gas; and (2) that organic solutes (such as sugar) move from the leaves, and from regions where such substances have been accumulated, to other regions, through the phloem of the vascular tissue. 4. The Transpiration Stream. — Water evaporates from the water-impregnated cell walls that bound the sub-stomatal gas spaces of leaves, and it then diffuses, as water vapor, through the stomatal openings into the external atmosphere. This process is called stomatal transpiration. Water also evaporates directly into the atmosphere, but at a slower rate, from the cutinized epidermal cell walls which always contain some imbibed water. This process is cuticular transpiration. Transpiration tends to dry the cell walls from which the water evaporates, and thus to increase the imbibitional attraction they exert on the liquid water within the cells and farther back in the tissues. Equilibrium tends to be reestablished by movement of water out of the xylem vessels, through intervening cells, to the evaporating surfaces. The forces drawing water out of the vessels are very great, and they tend to stretch the whole -water mass of the plant body. The vessels are sufficiently rigid to prevent their being collapsed by this inward pull on their walls, and the strain (by virtue of the cohesion of water) is transmitted to all parts, tending to remove some water from all cell walls whose outer surfaces are in contact with gas. At root surfaces this tendency results in the drawing in of water from the soil (probably carrying dissolved salts with it, in so far as the cell membranes are permeable to these solutes). As transpiration pro- ceeds, so long as the water supply is maintained at the absorbing root surfaces (espe- cially root-hairs), there is a flow of water into the roots, through the xylem vessels, and into the cell walls from which evaporation is occurring. This mass flow of water (carrying solutes) through the plant is called the transpiration stream. Some of the water drawn into the roots is used in tissue enlargement (which is primarily imbibi- 152 PHYSIOLOGY OF NUTRITION tional and osmotic swelling), in the photosynthesis of carbohydrates, etc., and con- sequently the rate of water absorption by the root system is, on the whole, for long periods, a little greater than the rate of transpiration. Also, some water is lost, in some plants, by being excreted to the exterior in the liquid form, as from hydathodes and nectaries, which excrete aqueous solution at leaf margins, on flower parts, etc. This glandular excretion of aqueous solution by hydathodes is termed guttation. Compared with transpiration, guttation is a slow and not very important process; it is encountered in comparatively few plants and is not maintained for long periods. Water loss through nectaries is still less significant in this connection. Sap pressure, by which the water solution of the vessels is sometimes under pressure instead of under tension (it is under pressure only when the transpiration rate is very low and the soil water supply is plentiful), appears to be due to a sort of gland action (some- what like that of hydathodes on leaf margins) in the tissues of the roots, etc., resulting in the active forcing of solution from the cortex into thexylem vessels; the water thus forced into the xylem is derived from the surrounding tissue, and ultimately from the soil. Bleeding, as of cut grape shoots in early spring, is partly or wholly due to sap pressure. Sap pressure does not occur when the transpiration stream is rapid; at such times the solution in the xylem vessels is under tension; therefore this pheno- menon cannot generally be the cause of the rise of sap in stems. This rise is directly due to the removal of water from the xylem above, to the tensile or stretching strain transmitted through the water of walls, protoplasm, vacuoles, etc., in all directions, and to the inward flow from the soil adjacent to the root surfaces. The molecular physics of sap pressure, gland secretion, etc., is not yet understood. The rate of transpiration nearly controls the rate of water absorption in ordinary plants with a plentiful water supply. When plants are well supplied with water at the absorbing surfaces of the roots, the rate at which water is evaporated from leaves and stems is dependent on several conditions, which may be grouped as internal and external. Among the internal conditions are: The structure of the plant, the kind of epidermis, the distribution, size, and open or closed condition of stomata, the degree of water saturation of the tissues, the power of the foliage to absorb solar radia- tion, the rate of water movement from roots to transpiring surfaces, etc. Many (but not nearly all) stomata open and close according to conditions. Such stomata usually open when the light intensity increases about dawn, and close more or less completely with the diminution of light intensity in the evening. They also usually close when wilting approaches. Generally the guard cells are more turgid when the pores are open. Stomatal movement is due to changes in the turgor relations (ten- sions) between the guard cells and the other epidermal cells. External conditions influencing the transpiration rate, when the root surfaces are well supplied with water, are the evaporating power of the air (air temperature, air humidity, air movement) and the intensity of absorbed sunlight. Over 25 per cent, of the radiant energy absorbed may be converted into the latent heat of water vapor in this way, without considerable change in the temperature of the foliage. When the supply of water to the absorbing roots is not adequate, the rate of this supply greatly influences the transpiration rate by limiting the rate of absorption by the roots. Plants usually transpire more than they absorb during the day and absorb more than they transpire during the night. There are usually several hundred stomata per square millimeter of leaf surface, the stomata being frequently more numerous on one leaf surface than on the opposite one. For plants of the same kind, all with the same environment and all having MOVEMENT OF MATERIALS IN THE PLANT 1 53 been grown under the same condition complex, the amount of water lost per day is about proportional to the extent of the leaf surface. According to Wollny, a maize plant transpired nearly 13 liters of water during its entire growing season. A pea plant correspondingly gave off nearly 4.5 liters. The transpiration rate is very important in determining the rate at which dissolved salts, as well as water, enter the roots from the soil, also in determining the rate at which dissolved salts are earried to the leaves. The dissolved material is left in the leaves when the water evapor- ates, and old leaves generally have a large salt content. 5. Movement of Organic Substances. — Organic materials (such as sugars, etc.) must be in aqueous solution to move from one region of the plant to another. Evi- dence points to the sieve-tubes of the phloem as the main path of movement of these solutes. They may diffuse, however, in all directions, so far as the protoplasmic mem- branes and cell walls are permeable to them. Also, some organic materials move in the transpiration stream, through the xylem vessels. Carbohydrates produced by photosynthesis in the cells of green leaves, in sunlight, diffuse outward and move to other parts of the plant through the phloem. They, and other organic materials, frequently accumulate in storage tissues, often going out of solution there (e.g., starch). Such accumulations usually dissolve again later, and move once more when new growth begins. The molecular physics of this movement through the phloem is not understood; the rate of the movement is too great to be accounted for by simple diffusion. Materials enter the plant body and move about therein according to the principles and considerations that have been briefly stated. It remains to consider their exit from the plant body. As has been shown, water is almost continually being given off to the surrounding air by ordinary plants (transpiration, guttation). It has also been mentioned that salts, sugars, etc., are given off to a small degree through gut- tation and gland action. Oxygen passes from green leaves into the air during sunlight periods, and some carbon dioxide escapes in a similar way during periods of darkness. Very small amounts of volatile materials beside