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STUDIES IN PLANT RESPIRATION AND 
 PHOTOSYNTHESIS 
 
 BY 
 
 Hf AV^SroEHR AND J. M. McGee 
 
 Published by the Carnegie Institution op Washington 
 Washington, February. 1923 
 
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 ^ 
 
 ^<^\ 
 
 C^V9 
 
 CARNEGIE INSTITUTION OF WASHINGTON 
 Publication No. 325 
 
 JUDD & DETWEILER, INC., 
 WASHINGTON^ D. C. 
 
N.C. 
 
 <r^-v'' 
 
 PREFACE. 
 
 The experiments on photosynthesis which were begun at the 
 Desert Laboratorj'- in Tucson, Arizona, about eight years ago 
 yielded, among other things, the conclusion that prerequisite to an 
 understanding of the nature of the energy transfer in photosynthesis 
 was a more extensive knowledge of the metabolism of chlorophyllous 
 organs. It became clear that, before attempting to follow the 
 course of the synthesis of carbohydrate material in illuminated 
 leaves, it was essential to know more about the conditions governing 
 the equilibria and mutual transformations of the various groups of 
 carbohydrates, quite independent of the photosynthetic process. 
 
 In publication No. 287 some of the more essential of these con- 
 ditions were described. A further prerequisite to an understanding 
 of the carbohydrate economy of chlorophyllous leaves is more 
 precise information regarding the nature of the carbohydrate catabo- 
 lism and the conditions governing this phenomenon. In the present 
 publication are described the results of experiments on the relation 
 of the amino-acid and carbohydrate content to the respiration of 
 leaves. The information gained from these studies, as well as from 
 those of a number of other workers in this field, helps to emphasize 
 the fact that photosynthesis is an exceedingly complex process. 
 The present status of the problem is, however, exceedingly encourag- 
 ing in that refinement of methods of observation and experimentation 
 and the utilization of the results and conceptions of allied physical 
 sciences are contributing greatly to a better understanding of the 
 phenomenon of photosynthesis. 
 
 This publication comprises the results of investigations carried 
 out during 1919-1922. It is a pleasure to acknowledge here the 
 assistance rendered in the preliminary part of this work by Dr. 
 Frances Long. 
 
 H. A. Spoehr. 
 Coastal Laboratory, 
 
 Carmel, California, June, 1922. 
 
 :^-w 
 
 
CONTENTS. 
 
 Part I. The Carbohydrate-Amino-Acid Relation in the Respiration of Leaves. 
 
 Introductory discussion 1 
 
 Methods and apparatus 21 
 
 1. The experimental material 21 
 
 2. The apparatus 23 
 
 3. The procedure of the respiration experiments 30 
 
 4. The analyses 33 
 
 Experimental results 35 
 
 1. The normal course of respiration 35 
 
 2. Glycocoll 43 
 
 3. d-glucose . . 45 
 
 4. Sucrose 55 
 
 5. d-levulose 57 
 
 6. d-mannose 64 
 
 7. Effect of the natural increase in amino-acids. Influence of light on amino-acids 
 and effect on respiration 66 
 
 8. The action of amino-acids on sugars 75 
 
 Part II. The Internal Factor in Photosynthesis. 
 
 Introductory discussion •. 76 
 
 Methods and apparatus 84 
 
 1. The experimental material 84 
 
 2. The apparatus ^ 85 
 
 Experimental results 90 
 
STUDIES IN PLANT RESPIRATION AND 
 PHOTOSYNTHESIS. 
 
 Bt H. a. Spoehr and J. M. McGee. 
 
 I. THE CARBOHYDRATE-AMINO-ACID RELATION IN 
 THE RESPIRATION OF LEAVES. 
 INTRODUCTORY DISCUSSION. 
 
 Already at the beginning of the nineteenth century it had been 
 realized that the materials which support the respiratory activity 
 in plants are carbohydrates and fats. The observations of Lavoisier 
 and of De Saussure that the respiration coefficient, CO2/O2, in 
 mature higher plants is usually very close to unity were even at that 
 time accepted as evidence of the preponderance of carbohydrates 
 as the fuel material. Since then a continually increasing number 
 of chemical compounds has been discovered in plants. When 
 derived from the higher, autotrophic plants, these multifarious 
 products must be considered as coming primarily from the carbohy- 
 drates through the various intricate channels of plant metabolism. 
 So while the substances which are used as sources of energy by the 
 lower plants and bacteria are of great variety, the higher, mature 
 chlorophyllous plants are in general consumers of carbohydrates. 
 
 It would, however, be drawing a very incomplete picture of plant 
 respiration if this were confined to the carbohydrates and fats and 
 the more elusive and complex nitrogenous components were to be 
 omitted. But unfortunately our knowledge of the function of 
 nitrogen compounds in the metaboUsm, particularly of the higher 
 plants, is most fragmentary. It is our belief that a rational con- 
 ception of the function of nitrogen in the economy of the higher 
 plants can be gained only through an analytic study of definite 
 groups of nitrogen compounds. This belief has its foundation first 
 of all in the fact that nitrogen is capable of forming such a variety 
 of compounds of basically different physiological properties, and 
 furthermore because our chemical knowledge of the nitrogen com- 
 pounds derived from protoplasmic proteins is fairly well organized 
 and related and so can serve as a most valuable experimental guide. 
 
 Even a cursory study of the carbohydrate economy of the higher 
 plants must emphasize the old conclusion that physiology is essen- 
 tially a study of dynamics. Thus far organic chemistry has dealt 
 largely with the properties and reactions of carbon compounds. 
 The recent tendency to enter into various phases of energetics and the 
 fundamental causes of chemical reactivity will make chemistry an 
 even more valuable adjunct to physiology than heretofore. 
 
 1 
 
2 STUDIES IN PLANT EESPIRATION AND PHOTOSYNTHESIS. 
 
 It would seem, then, that the true province of plant chemistry 
 should be the unraveling of the tangle of chemical reactions which 
 give rise to this mass of carbon compounds and ultimately to deter- 
 mine the function of these reactions in the economy of the living 
 plant. Historically, what is now termed organic chemistry had its 
 origin in such a task. But the chemist discovered that he could find 
 a large number of short cuts; that the intermediary action of living 
 things was not essential to the production of most of the substances 
 found in nature; that, in fact, he could greatly improve on nature in 
 the production of the multifarious substances of use in industry 
 and the arts. 
 
 In some respects the present has its points of similarity with the 
 time of Woehler and Liebig. Much sober thought is now being 
 given to the fundamental difference between the living and the 
 non-living. The first flush of success of the purely mechanistic 
 interpretation of life processes has paled as the enormous difficulties 
 of applying this conception to the highly intricate and manifold 
 manifestations of life have been more clearly recognized. The 
 methods and modes of transforming matter followed by the chemist 
 and by the living organism are perhaps not as similar as appears at 
 first glance. At first we were primarily interested in the final goal, 
 the products of material transformations; but, in chemistry as well 
 as in physiology, as we follow more closely the energetics of such 
 transformation, many almost irreconcilable differences arise which, 
 before the two can be brought together, necessitate sweeping com- 
 promises to the unknown and indefinite. And it is this question of 
 energy which is forcing us to a deeper study of living things. The 
 scientific world is awakening to the necessity of taking stock of our 
 available sources of energy. Repeatedly attention has been called 
 to the inexhaustible floods of solar energy and to the extensive use 
 of plant products to drive the machinery of our civilization. Car- 
 bohydrates are to prevent the too-rapid exhaustion of our oil and 
 coal resources. As the realization of the limits of our energy re- 
 sources has become more general, reliance on the magic accomplish- 
 ments of the chemist has increased, and the chemists, with cool 
 theoretical nonchalance, have pointed to the untold possibilities of 
 solar energy. But the living chlorophyllous plant still remains the 
 only converter of solar energy! 
 
 The chemical reaction most fundamental to all living things, 
 the photosynthetic process, the bridge between the inanimate and 
 the animate, the source of most of our energy, has stubbornly resisted 
 all attempts at solution by physico-chemical methods. Here it has 
 become plainly evident that while physiology is rife with chemical 
 possibilities, physiological experience alone is the guide to the inter- 
 pretation of biological processes. Furthermore, it has become clear 
 
STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 6 
 
 that physiology is a great deal more than applied physics and chem- 
 istry, although we must rely upon these discipUnes in order to form 
 conceptions of the various vital phenomena as operations of known 
 causes. With complete disregard of biological facts, chemists have 
 continued to evolve theories of the chemistry of photosynthesis 
 and to supplement and modify existing theories. The fundamental 
 fallacy in these speculations has been that photosynthesis has been 
 regarded — to quote a recent technical article — as ''simply a manu- 
 facture that provides material used in the process of living, "^ that 
 because a process can be suspended for hours and months it is not a 
 process or function of the Uving. The many attempts which have 
 been made to reproduce the photosynthetic process apart from the 
 Uving cell have not been successful.^ This also appHes to the experi- 
 ments in which various chlorophyll preparations have been used, 
 as well as to those in which ultra-violet Ught was employed as a 
 source of radiant energy, although the latter conditions have no 
 direct bearing on photosynthesis, because normally the plant makes 
 use of only the radiations in the visible spectrum. 
 
 That the photosynthetic process is intimately associated with 
 the protoplasmic activity of the hving cell was recognized a long time 
 ago. Boussingault,^ in his studies of the effect of gases such as 
 hydrogen, nitrogen, and methane on photosynthesis, came to this 
 conclusion. The same conclusion was reached and somewhat 
 elaborated by Pringsheim." Interesting and valuable as these 
 older investigations are, as well as the later ones of Ewart,^ Httle 
 precise information can be gained from them, because the methods 
 of experimentation and observation are naturally not in accord with 
 present standards of accuracy. 
 
 It has become evident that further insight into the complex 
 phenomenon of photosynthesis is to be gained only by means of 
 intensive study of the process under very carefully controlled external 
 conditions and the ehmination of secondary factors. Unfortunately, 
 however, in the study of photosynthesis, internal conditions, such 
 as available nutritional material, have been totally neglected and 
 httle or no regard has been paid to the previous history of the plant. 
 It is highly probable that this neglect in many instances accounts 
 
 1 Scientific American Supplement, No. 2257, 223 (1918). 
 
 * WiLLSTAETTER, R., and A. Stoll. Untersuchungen ueber die Assimilation der Kohlensaeure. 
 
 1918, 391-415. 
 Spoehh, H. a. The theories of photosynthesis in the light of some new facts. Plant World, 
 19, 1-16 (1916). 
 ' BoussiNGAULT, J. B. ^Itude sur les fonctions des feuilles. Compt. rend., 60, 608; 1865, Agro- 
 nomie, 4, 359-397 (1868). 
 
 * Pbingsheim, N. Ueber die Abhaenigkeit der Assimilation Gruener Zellen von ihrer Sauer- 
 
 stoffathmung, und den Ort, wo der im Assimilationsacte der Pflanzenzelle gebildeter Sauer- 
 stoff entsteht. Sitzber. Preus. Akad. Wiss., 763-777 (1887). 
 ' EwART, A. J. On assimilatory inhibition in chlorophyllous plants. Jour. Linnean Soc, 31, 
 364-^61 (1896); 556 (1897). 
 
4 STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 for the contradictory observations and reports which have been 
 made by different investigators on various phases of the phenomenon 
 of photosynthesis. Furthermore, wherever consideration must be 
 given to the respiratory activity of plants, it is essential that all 
 possible information be obtained regarding the internal conditions, 
 as, for instance, available carbohydrate supply. Such data can be 
 obtained only by rather laborious analytical methods. 
 
 If such an interdependence between photosynthesis and respiration 
 actually exists, a better understanding of the nature of respiratory 
 activity is an absolute prerequisite to further investigation of this 
 relationship. 
 
 WMle any consideration of the relation of photosynthesis to 
 respiration must take into account the carbohydrate economy of 
 the plant, it is evident that respiratory activity itself can not be 
 interpreted solely on the basis of carbohydrate balance and supply. 
 In fact, it has been known for some time that there is no direct 
 relation between the amount of available carbohydrates and the 
 rate at which the organism uses this material in its respiratory 
 activity. Although carbohydrates constitute the major portion of 
 the material used by the plant in its process of respiratory com- 
 bustion, the other agencies and factors which play a role in this 
 process are still but vaguely known. Our knowledge of the chemical 
 possibihties which would find application here is still very incomplete. 
 
 Plant physiologists have generally accepted the dictum that, 
 given an adequate supply of carbohydrates, water, certain inorganic 
 salts, and the proper temperature, the rate of life processes in the 
 higher plants depends upon the active mass of protoplasm. To the 
 activity of protoplasm have been ascribed all those functions and 
 reactions which could not be described in the terms and through 
 existing conceptions of physics and chemistry. The attempts of 
 MacDougal, Loeb, and others to describe the physical behavior of 
 protoplasm on the basis of colloidal phenomena have done much 
 to break away from this tendency and to determine to what extent 
 protoplasmic activity is amenable to physical simulation. Simi- 
 larly, attempts have been made by Palladin to correlate certain 
 components of the protoplasm with the respiratory activity of the 
 plants. 
 
 The carbohydrate-nitrogen relation has of late found appHcation 
 in the investigations of Kraus and Kraybill,i who have formulated 
 
 'Kraus, E. S., and H. R. Kraybill. Vegetation and reproduction with special reference to 
 
 the tomato. Oregon Agr. Exp. Sta. Bull. 149 (1918). 
 Kraus, E. S. The modification of vegetative and reproductive functions under some varying 
 
 conditions of metabolism. Amer. Jour, of Bot., 7, 400-416 (1920). 
 Harvey, E. M., and A. E. Murneck. The relations of carbohydrates and nitrogen to the 
 
 behavior of apple spurs. Oregon Agr. Exp. Sta. Bull. 176 (1921). 
 Hooker, H. D., Jr. Seasonal changes in the chemical composition of apple spurs. Misaouri 
 
 Agr. Exp. Sta. Research Bull. 40 (1920). 
 
STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 5 
 
 a general theory which has met widespread success in its practical 
 bearing on agriculture. Their investigations deal in the main with 
 the relation between vegetation and fruit setting and nitrate fer- 
 tiHzation. They have been able to differentiate the carbohydrate- 
 nitrogen relations of certain plants on the basis of their vegetative 
 and sexual activities. 
 
 There exists very httle exact information regarding the general 
 course of formation, synthesis, or fate in the general metabolism 
 of any of the organic nitrogen compounds in higher plants. The 
 number and variety of different types of nitrogenous substances 
 found in plants are enormous, and it appears an almost hopeless 
 task to unravel the tangle of chemical reactions which lead to this 
 multiplicit}^ of compounds. It seems, however, quite erroneous to 
 class together the various types of nitrogen compounds, such as the 
 proteins and related substances, the amino-acids, amides, etc., and 
 regard their phj'^siological functions as being the same or even very 
 intimately connected. An insight into this complex of chemical 
 reactions comprising metabolic energesis can, of course, be obtained 
 only through laborious experimental investigation involving much 
 observational and chemical-analytical work with living plants. 
 Chemistry can offer no short cuts, but rather supplies the instruments 
 with which to illuminate these hidden activities. Nor can the 
 conceptions of chemistry always find immediate application to reac- 
 tions in living organisms. Thus the many theoretical speculations 
 regarding the chemistry of the photosynthetic process have contrib- 
 uted very little to the solution of that problem. This is true also 
 of the chemical speculations regarding the formation of the amino 
 acids and the proteins, although there are a number of elaborate 
 theories which have been formulated to explain this complex process.^ 
 To these the warning of Pfeffer applies very aptly, which is to the 
 effect that it is a very confusing error to presume that an organism 
 must, in its metabolic economy, follow a course which seems to man, 
 under the influence of existing chemical and physical knowledge, 
 the most plausible course. All of these theories are still dealing with 
 probabilities supported by very little experimental physiological 
 evidence. Moreover, plant chemistry can hardly claim to be an 
 independent science. The great advances which have been made in 
 protein chemistry have been stimulated largely by animal physiology, 
 and progress in our understanding of plant proteins has been made, 
 in the main, by following the methods previously worked out for 
 proteins of animal origin. These studies seem to indicate that the 
 
 1 LoEB, W. Ber. d. deutsch. chem. Ges., 46, 684-697 (1913). 
 Franzen, H. Jour. Prakt. Chem., 86, 133 (1913). 
 
 Batjdish, O. Jour. Biol. Chem., 48, 489-502 (1921). Ber. d. deutsdi. chem. Gps., 46, 115-125 
 (1913); 49, 1159-1167 (1916); 50, 652-660 (1917); 51, 793-805 (1918); 52, B 35-43 (1919). 
 LoEW, O. Ibid., SO, 909-910 (1917). 
 
6 STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 proteins of plant and animal origin are very similar in composition. 
 It is, nevertheless, an open question as to what degree we are justified, 
 on the basis of this analogy, in drawing conclusions as to the function 
 of the proteins in the two types of organisms. 
 
 The extraction of proteins from leaves is associated with consider- 
 able difficulty. Winterstein^ demonstrated this by the use of dif- 
 ferent methods on a variety of plants. These difficulties arise from 
 the fact that the proteinaceous material is quite indiffusible and 
 clings tenaciously to the structural elements of the plant, and that 
 any methods of extraction or expression yield complex mixtures 
 from which the protein can be freed only by means of a variety of 
 substances which in some degree also affect the proteins. Very 
 little systematic work has been done on the proteins with a view to 
 determining which are present in green leaves. A good beginning 
 has been made by Chibnall and Schryver,^ who attempted to work 
 out methods for isolating and identifying the proteins in the leaves 
 of some of the higher plants. Osborne, Wakeman, and Leavenworth^ 
 have also outlined a procedure for the separation of various protein 
 constituents from the alfalfa plant. 
 
 Mature leaves are relatively high in proteins. The mesophyll, 
 freed from leaf-veins, often shows values for protein of over 30 per 
 cent of the dry material. Lakon,^ by means of the Molish reaction, 
 was able to demonstrate decided differences in the amount of protein 
 in variegated leaves. The chlorophyllous portions gave a very 
 intense protein reaction, while the albescent portions were poor in 
 proteins. This seems to confirm the opinion that in leaves the prin- 
 cipal masses of protein are located in or about the chloroplasts. 
 In fact, Meyer^ found that as leaves become older the reduction of 
 the green color runs parallel with a reduction in the protein-content 
 of the leaves. According to these investigations the leaf proteins 
 are located chiefly in the chloroplasts, while the nucleus and cyto- 
 plasm seem but very slightly affected by variations in protein- 
 content. Thus the size of the chloroplasts is greatly influenced by 
 the amount of protein in the leaf, in that, as the proteins disappear 
 when the leaf is kept in the dark, the chloroplasts become smaller 
 and contain less chlorophyll, while with an increase in proteins the 
 
 > WiNTERSTEiN, E. Ueber die Sticksto£fhaltigen Bestandtheile greuner Blaetter. Ber. d. deutsch. 
 
 hot. Ges., 19, 326-330 (1901). 
 Also Hamilton, T. S.. W. B. Nevens, and H. S. Grindley. The quantitative determination 
 
 of amino-acids of feeds. Jour. Biol. Chem., 48, 249-272 (1921). 
 s Chibnall, A. C, and S. B. Schryver. Investigations on the nitrogenous metabolism of 
 
 higher plants. Biochem. Jour., 15, 60-75 (1921). 
 » Osborne, T. B., A. J. Wakeman, and C. S. Leavenworth. The proteins of the alfalfa plant. 
 
 Jour. Biol. Chem., 49, 63-91 (1921). 
 * Lakon, G. Biochem. Zeitschr., 78, 145-154 (1916). 
 6 Meyer, A. Eiweisstoffwechsel und Vergilbung der Laubblaetter von Tropaeolum majua. 
 
 Flora, 11, 85-127 (1918). 
 Cf. also CaAPEK, F. Biochemie der Pflamen, 2d ed., 3, 293. Jena (1920). 
 
STUDIES IN PLANT RESPIEATION AND PHOTOSYNTHESIS. 7 
 
 reverse occurs. In this connection it is interesting to note that 
 according to the experiments of Deleano^ it is claimed that when 
 leaves {Vitis vinifera at 18° to 20°) are kept in the dark, these util- 
 ize only carbohydrates during the first hundred hours; thereafter 
 drastic disturbances in the proteinaceous components of the leaf 
 occur. The carbohydrates thus act in the nature of protein-sparers. 
 Now, Meyer points out that when leaves are kept in the dark for a 
 long time, the chloroplast apparatus becomes so greatly impaired 
 through loss of protein that, on subsequently placing the leaves in 
 the Hght, photosynthesis is no longer sufficiently active to produce 
 enough material to cover the nocturnal loss through respiration. 
 Meyer's observation of decreasing protein-content with advancing 
 age of the leaves is especially interesting when compared with the 
 studies of Nicolas,^ who found that on the basis of respiration coeffi- 
 cients the energy release in young leaves is considerably higher than 
 in old ones. 
 
 According to Emmerhng, there is with advancing age only a 
 reduction of amino nitrogen and total nitrogen but not of protein 
 nitrogen. This latter observation is in even closer accord with the 
 hypotheses of a causal connection between amino-acid content and 
 respiration. Also, the carbohydrate-content shows a general increase 
 as the leaves grow older.^ These facts indicate that carbohydrate- 
 content alone can not be taken as an index of the rate of respiration ; 
 nor, obviously, can the carbohydrate-content serve as a measure of 
 the photosynthetic activity. 
 
 The following analyses taken from Czapek {Biochemie der PHanzen, 
 1st ed., Vol. II, p. 202) indicate the amounts of protein which have 
 been found in leaves. These values have, however, only a limited 
 significance, because in the analysis neither the age of the leaves, 
 their history, nor the environic conditions were taken into consider- 
 ation. 
 
 Table 1. 
 
 Plant. 
 
 Water- 
 content. 
 
 Crude 
 protein 
 NX6.25. 
 
 Plant. 
 
 Water- 
 content. 
 
 Crude 
 protein 
 N X6.25. 
 
 Lactuca sativa 
 
 Plantago major 
 
 V.ct. 
 
 81^44 
 10.10 
 
 p.ct. 
 31.75 
 2.65 
 10.20 
 33.06 
 27.37 
 
 Ilex paraguayensis 
 
 p.ct. 
 9.41 
 
 16^29 
 
 V.ct. 
 
 4.51 
 
 6.28 
 21.23 
 38.77 
 
 5.10 
 
 Allium porrum 
 
 Cichorium endiva 
 
 Coffea arabica 
 
 Spinacia oleracea 
 
 Vicia cracca. . 
 
 
 
 1 Deleano, N. Jahrh. f. wiss. Bot., 51, 541-592 (1912). 
 » Nicolas, G. Reo. Gen. Bot., 30, 210-225 (1918). 
 MiCHEi/-DuKAND, E. lUd., 30,337-345, 377-382 (1918); 
 
 251-268, 287-317 (1919). 
 » Emmerung, E. Landw. Verauch. Stat., 34, 113 (1880). 
 
 31, 10-27, 53-60, 143-156, 196-204, 
 
8 
 
 STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 The relation of protein nitrogen to non-protein nitrogen can be 
 seen from Pigorini's* results of the analysis of Morus leaves (table 2). 
 
 The true function of the leaf proteins is a question on which there 
 exists very little definite information, although it has been considered 
 one of the most vital problems in plant physiology. In the nitrogen 
 economy of animals it is apparent that a portion of the assimilated 
 proteins serves to replace the 'Svear and tear" on the protoplasmic 
 machinery. For this Rubner introduced the conception ''repair 
 quota" of protein. Also a "growth quota" of protein is necessary 
 to supply material for the development of protoplasm in new cells 
 in growing organisms. When protein is available in such amounts 
 beyond the requirements of "repair quota" and "growth quota," 
 it can be converted into glucose and fatty acids and thus serve as 
 fuel, very much as though these substances had been ingested as 
 food. To such material Rubner gave the term "dynamic quota." 
 Under conditions of copious protein-supply the animal can to a degree 
 store or deposit the protein in the tissue cells. It seems to be an 
 undecided question whether such protein becomes a part of the 
 living tissue or is stored by the cells very much as glycogen is. The 
 
 Table 2. 
 
 
 Morning. 
 
 Evening. 
 
 1 
 Dry material. Fresh material. 
 
 Dry material. 
 
 Fresh material. 
 
 Total N 
 
 I 
 p. ct. p. ct. 
 2 445 772 
 
 p.ct. 
 
 2.534 
 
 2.368 
 
 .166 
 
 p. ct. 
 
 0.884 
 
 .824 
 
 .059 
 
 Protein N 
 
 2.309 .729 
 .1105 .043 
 
 Non-protein N 
 
 animal organism, unlike the plant, is constantly losing a certain 
 (luantity of nitrogen, and it appears that if this quantity is not re- 
 ])laced by ingested protein-food the proteinaceous body-tissue is 
 drawn upon. 
 
 In the autotrophic plant the condition seems to be quite different. 
 Here also, however, it is first of all essential that, in the discussion, 
 well-defined conditions be established and that generaUzations be 
 not made too inclusive. This has been an unfortunate feature of the 
 contributions to protein metaboUsm in plants. It should hardly 
 need emphasis that results with the fungi may not find immediate 
 application to autotrophic plants, nor that the behavior of germinat- 
 ing seeds corresponds to mature chlorophyllous leaves. In general, 
 it appears as though plants were exceedingly economical with their 
 
 ' C:zAPKK, F. Biochemie der Pfianzen, vol. ii, 294; 2d ed., 1920. 
 
STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 9 
 
 nitrogen compounds. There are no nitrogenous excreta, and the 
 plant possesses the power, to a very highly developed degree, of 
 resynthesizing the decomposition products of proteins. The excep- 
 tion to these statements should be mentioned at once. Thus there 
 are the denitrifying actions of certain bacteria and the behavior of 
 the lower non-chlorophyllous plants. These organisms exhibit a 
 behavior toward the proteins which seems to differ in many funda- 
 mental aspects from that of the higher plants.^ 
 
 In this study we are concerned primarily with the metabolism of 
 mature autotrophic leaves. A priori, the nitrogen metabolism of 
 such organs would seem to have but little similarity to that of ger- 
 minating seeds, in which it is primarily a question of the resorption 
 of stored proteinaceous material. In view of its great importance 
 it is surprising how very little work has been done on the protein 
 metabolism of leaves. 
 
 Deleano's- investigations show that the total nitrogen-content 
 of leaves of Vitis vinifera did not change appreciably during 493 
 hours of respiration in the dark. He also found but very slight 
 variation in the protein nitrogen up to 87 hours. Thereafter these 
 values decreased. These determinations were made by means of 
 Stutzer's method, so that they represent coagulable protein, and no 
 information can be gained therefrom regarding the amino acid- 
 protein relations. 
 
 Whether a sharp differentiation can be drawn between living and 
 inanimate protein, or better, perhaps, between protoplasmic and 
 non-protoplasmic protein, in the leaf appears exceedingly difficult. 
 It has also been impossible to determine in plants a factor corre- 
 sponding to the "protoplasmic mass," which has an important role 
 in the discussions of animal physiologists in considering the phe- 
 nomenon of respiration. What, finally, the function is of the total 
 protein or of the protoplasmic proteins in plants is a question on 
 which we have no direct experimental evidence. As has been stated, 
 one of the chief difficulties in this problem has been the inadequate 
 information that exists regarding the nature of the proteins of leaves. 
 
 Palladin^ has attempted to establish a relation between the rate 
 of carbon-dioxid emission and of the proteins in wheat seedlings. 
 Based upon the old observations of Reinke,* Zacharias,'^ and 
 Schwartz,^ Palladin assumed that the protoplasmic proteins are 
 
 * Mater, A. Agriculturchemie, Gaerungschemie, 138-139 (1902). 
 
 Cf. also Irving, A., and R. Hankinson. Biochemical Jour., 3, 87, 1908. Ivanoff, N. N., 
 
 Biochem. Zeitschr., 120, 1-80 (1921). 
 = Deleano, N. Jahrb. f. wiss. BoL, SI, 587 (1912). 
 ' Palladin, W. Recherches sur la correlation entre la respiration des plantes et les substances 
 
 azotees actives. Rev. Gen. de Bot., 8, 222-284 (1896). 
 
 * Reinke, J. Stiidien ueber das Protoplasma (1881). 
 
 * Zacharias, E. Bot. Zeitung (1881) 169; (1883) 209. 
 
 * Schwartz, F. Morphologische und chemische Zusammensetzung dcs Prutoplasmas. 1887. 
 
10 STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 not digested by gastric juice. He thus finds that in germinating 
 seeds the rate of carbon-dioxid emission is proportional to the 
 amount of protein indigestible in gastric juice when the supply 
 of carbohydrates is ample. While these studies are of prehminary 
 nature and the methods^ arbitrary and quite lacking in precision, 
 they are nevertheless suggestive of many interesting relations. 
 
 The fundamentally important questions in both respiration and 
 photosynthesis are those relating to the dynamic aspect of these 
 phenomena. Both of these processes are primarily of interest 
 because they represent the energy transfer of life phenomena. 
 Without such energy transfers no manifestiatons of life could be 
 possible. Just as in physics and chemistry, energetics is being 
 recognized as the key to both the structure and behavior of matter, 
 so in physiology further development awaits a more comprehensive 
 conception of energetics. For a long time physiological chemistry 
 consisted in the study of the substances which enter into this device 
 of energy transformation and the products which leave it in the form 
 of excreta; it is now more and more attempting to study the chemical 
 phenomena which underlie the activity of such energy changes. 
 By tradition the behavior of protoplasm is associated with the com- 
 plex reactions of the proteins. The most complex group of carbon 
 compounds, it is also by inference the medium for the intricate 
 reactions of life processes. Etymologically, proteins are the sub- 
 stance of "first importance." Perhaps there is here a fundamental 
 difference between plants and animals, but, at any rate, it is very 
 doubtful whether in plants a preponderant role can be ascribed to 
 the proteins. From the chemical viewpoint the conception of 
 living matter presents many insurmountable difficulties, while, on 
 the other hand, the idea of a complex of coordinated chemical reac- 
 tions taking place in a material medium falls within the domain of 
 modern physical-chemical reasoning. If "living matter" is pos- 
 sessed of properties and forms of energy sui generis, the disciplines 
 of physics and chemistry can be of very Uttle aid in interpreting the 
 behavior of Uving things. As long as we are working on a basis of 
 physics and chemistry it is of little value to introduce conceptions 
 into physiology which have no sound foundation nor direct analogues 
 in these sciences. Thus to ascribe the energy of life processes to 
 "bio tic energy" is certainly not clarifying our conceptions of these 
 processes on the basis of physical science. The fact that physiolo- 
 gists are frequently having recourse to the coining of new phrases, 
 not found in the physical sciences, to describe life processes ought 
 
 • For the action of pepsin and trypsin on plant protoplasm, cf. W. Beidebmann, Microchemische 
 Beobacthungen an den Blattzellen von Elodea. Flora, 11-12, 604 (1918). Walter, H. 
 Bioehem. Zeitachr., 122, 86-99 (1921). 
 
STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 1 1 
 
 to serve as a modulator to the mechanists and bring the realization 
 that pronouncements on the mechanistic view of life processes can be 
 made only with reserve and as partial truths. 
 
 The term ''living matter" is in a sense anthropomorphic, in that 
 we ascribe to the matter of living things attributes and qualities 
 which we recognize in our own behavior. This is in a large measure 
 responsible for the confusion in our conception of the essential nature 
 of the processes in living things, we having superimposed on the 
 matter of vegetable organisms the behavior of our own tremendously- 
 complex system, including the maze of mental and spiritual expe- 
 riences. The differences in the two conceptions at first seem insig- 
 nificant or subtle, the one of ''living matter," the other of "life in 
 matter"; but from the experimental physical-chemical viewpoint 
 the differences are fundamental. 
 
 The gap between li\dng and non-living substances in a cell has 
 never been bridged nor have the differences ever been clearly defined. 
 To the proteins has been ascribed the function of life, partly because 
 of their complex structure and reactions and also because they have 
 been found wherever vital phenomena occur. That the proteins 
 form an essential part of the medium in which these complex chemical 
 reactions, called "life," take place, and that the proteins or their 
 derivatives contribute directly or through catalytic action to these 
 reactions, embodies a conception which is compatible with our modern 
 physical-chemical thinking. This conception of a relatively inert 
 substance or medium in which the interplay of chemical reactions 
 takes place, and through which these always manifest themselves 
 as life activities, is advanced, of course, not as describing the actual 
 state of affairs, but as a hypothesis which lends itself to experimental 
 investigation. Thus in the plant-cell the carbohydrates and fats 
 serve as the fundamental sources of energy in respiration. The 
 nitrogen derivatives, with which the plant deals most economically 
 as proteinaceous compounds, constitute an essential portion of the 
 medium in which the multiplicity of reactions occur. Furthermore, 
 these proteins, of themselves inert, are of fundamental importance 
 because of their ability, through their decomposition products — 
 the amino-acids — to influence the enzymatic reactions. The dif- 
 ferences in protoplasm are thus to be ascribed to the differences in 
 the media in which the various reaction complexes occur. Pro- 
 toplasm is to be regarded as a colloidal mass of varying composition, 
 which serves as a medium for the manifold chemical reactions 
 involving the breakdown of many molecules, thus releasing energy 
 which may be used for the synthesis of new compounds, which in 
 turn may be incorporated in the colloidal mass of the substratum. 
 Nor can such a hypothesis explain or define life. It simply attempts 
 
12 STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 to ascribe more directly the final forces which induce changes in 
 living things to the same causes which activate the inorganic world. 
 This is the only hope for a mechanistic conception of life processes. 
 
 The question naturally arises whether in metabolic processes 
 which involve the liberation of energy a continual decomposition of 
 protein serves the plant (1) to contribute to the total energy release 
 of decomposition by material other than carbohydrates and fats, 
 or (2) to provide material essential to the proper functioning of 
 enzymes and catalysists, or (3) whether such a continued decompo- 
 sition is an unavoidable accompaniment of metabolic energesis. 
 Concerning the first possibility, it is evident that the amount of 
 energy obtained by the decomposition of proteins would be very 
 small, for this is usually a cleavage into amino-acids, and, further- 
 more, the leaf is capable of re-forming these amino-acids into proteins 
 without the direct utilization of an extraneous source of energy, i. e., 
 through the chemo-sjmthetic energy derived from the oxidation or 
 the breakdown of sugars. ^ The total gain in energy for the plant 
 from the decomposition of proteins would therefore be nil; and unless 
 the decomposition of proteins yields chemical energy of a certain 
 form or at a particular rate, it is difficult to see what advantage 
 would result or what the mode would be of deriving energy by a 
 continual decomposition and subsequent resynthesizing of protein 
 material. Of the possible different forms of chemical energy referred 
 to we know at present practically nothing, so that this is quite 
 beyond the ken of physical-chemical reasoning. That the rate 
 of energy release from protein decomposition differs fundamentally 
 from that of carbohydrate-oxidation can not be assumed; judging, 
 however, from the rate of accumulation of protein-splitting products, 
 it would appear that the carbohydrate breakdown proceeds con- 
 siderably faster, mol for mol. 
 
 As to the second possibility of the function of protein decom- 
 position — the providing of material essential to the functioning of 
 enzymes — the experiments hereinafter described may contribute some 
 information. This aspect is of interest on account of the stimulating 
 action which amino-acids exert on certain enzymes, as well as on 
 account of the fact that amino-acids, being amphoteric electrolytes, 
 i. e., capable of uniting with both acids and bases, have the power of 
 maintaining the hydrogen-ion concentration within definite limits. 
 
 That amino-acids accumulate in seedlings left in the dark has 
 been known since the time of Hartig and Boussingault. This 
 
 ' It is quite well established that leaves are capable of synthesizing proteins from amino-acids 
 and even nitrates in the dark in the presence of an abundant carbohydrate supply. Protein 
 decomposition in the presence or absence of oxygen differs primarily in the proportion of 
 the various amino-acids, principally asparagine, tyrosine, and leucine. Suzuki, Bot. Centbl., 
 75, 289 (1898); Zalbski. W., Ber. d. deutsch. bot. Ges., 15, 536-542 (1897); Sapoznikow, W.. 
 Bot. Centbl., 63 (1893); Palladin, W., Ber. d. deutsch. bot. Ges., 6, 205-212, 296-304 (1888). 
 
STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 13 
 
 accumulation of amino-acids has been the subject of numerous inves- 
 tigations and the basis of much speculation regarding the function 
 and fate of proteins in plant respiration. ^ Suzuki^ is also of the 
 opinion that during the night the proteins of the leaf are broken 
 down to amino-acids and that these migrate to other parts of the 
 plant. This seems to be very near to the actual state of affairs, 
 although the extent to which this takes place is still an undecided 
 question. The solution thereof is made very difficult because other 
 processes, such as photosynthesis during the day and respiration 
 at night, may often mask any definite results observable in the 
 analytical data. 
 
 Since the development of protein chemistry the improved methods 
 of isolating and determining the various amino-acids have made it 
 possible to establish what compounds are formed in the decomposition 
 of the proteins of plants when these are left in the dark. Most of 
 the investigations have been carried out on germinating seeds, so 
 that many of the results are not altogether applicable to mature 
 leaves. However, Schulze for many years has been working on the 
 amino-acid content of plants and he has obtained some very valuable 
 results. Seedlings of Lupinus grown for 14 days in the open were 
 reported by Schulze and Castoro^ to contain nitrogen compounds, 
 as follows : 
 
 Table 3. 
 
 
 Total nitrogen. 
 
 Proteins. 
 
 Asparagine. 
 
 Arginine. 
 
 Cotyledons (dry material) 
 
 Stems (dry material) 
 
 p.ct, 
 7.83 
 6.77 
 5.40 
 6.6 
 
 p.ct. 
 14.64 
 
 9.56 
 11.22 
 24.66 
 
 p.ct. 
 17.59 
 21.12 
 10.23 
 6.65 
 
 p. ct. 
 0.14 
 
 '!006 
 
 Roots (dry material) 
 
 Leaflets (dry material) 
 
 
 Schulze attributes the great difference in the asparagine content 
 between the stems (21.12 per cent) and the leaves (6.65 per cent) 
 to the photosynthetic action of the leaves, which in the presence of 
 carbohydrates convert the asparagine into proteins. In harmony 
 with this it is pointed out that the protein-content of the leaves is 
 2.5 times as great as in the stems. 
 
 1 Pfeffer, W. Untersuchungen ueber die Bedeutung des Asparagins beim Keimen der Samen. 
 Jahrb. f. wiss. BoL, 8, 429-574 (1880). 
 Meuniek, F. fitude sur I'asparagine. Ann. Agron., 6, 275-281 (1880). 
 
 Borodin, J. Ueber die physiologische Rolle und die Verbreitung des Asparagins im Pflanzen- 
 reiche. Bot. Zeit., 36, 801-832 (1878). 
 » Suzuki, W. Bull. Coll. Agr. Tokyo 2, 458 (1896). 
 
 ' Schulze, E., and N. Castoro. Beitrege zur Kenntniss der Zusammensetzung und des Stoff- 
 wechsels der Keimpflanzen. Zeit. physiol. chem., 38, 244 (1903). 
 Also KiESEL, A., Zeit. physiol chem., 49, 72-80 (1906) ; Andr6, G., Compt. rend., 148, 1685-1687; 
 Thompson, S. G., Jour. Amer. Chem. Soc., 37 230-235 (1915). 
 
14 
 
 STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 In our own experiments with mature leaves of Helianthus annuus 
 we found that the amino-acid content of the leaves was higher than 
 that of the stems, while the carbohydrate-content was considerably 
 higher in the stems. 
 
 Comparing seedUngs which had developed in the light with those 
 grown in the dark, Schulze and Castoro found that the amount of 
 amino-acids in etiolated seedlings 7 days old was higher than in the 
 green plants twice as old, while the amount of asparagine was about 
 the same in both. In general, it would seem from these investigations 
 that the amount of amino-acid formation and protein breakdown is 
 considerably reduced when there are carbohydrates present or the 
 power of carbohydrate formation through photosynthesis is available 
 to the seedlings. This substantiates the general principle that 
 carbohydrates are protein-sparers, which has been found by other 
 
 Table 4. — Distribution of nitrogen compounds in 
 leaves and stems according to Schidze's analysis. 
 
 
 In leaves. 
 
 In stems. 
 
 
 62.56 
 
 8.06 
 
 21.46 
 
 7.92 
 
 23.04 
 
 6.20 
 66.17 
 4.59 
 
 Phosphotungstic-acid 
 
 precipitate 
 
 Asparagine 
 
 Other compounds 
 
 investigators. 1 These studies by Schulze have, however, only an 
 indirect bearing on our investigations, because Schulze was dealing 
 largely with the germination of seeds in which reserve proteinaceous 
 material was in the course of the metabolic activity of the plant 
 resolved into simpler compounds, and the plants were not mature 
 self-supporting organisms. Schulze^ has made some extensive 
 investigations of the amino-acid transformations in young plants. 
 In seedlings of the Vicia saliva, Pisum sativum, Lwpinus luteus, 
 and Lupinus alhus grown in the dark there occurs a marked accumu- 
 lation of amino-acids. The nature of the amino-acids changes with 
 the length of time the plants are allowed to remain in the dark. 
 Seedlings left in the dark 6 to 7 days contain leucin, asparagine, 
 arginine, histidine, lysine, and tyrosine in small quantities. After 
 remaining in the dark for longer periods, 2 to 3 weeks, leucine, tyro- 
 sine, and arginine decrease, while the amount of asparagine is greatly 
 increased. Schulze considers leucine, arginine, histidine, and lysine 
 as the primary products of the protein breakdown in leaves, and that 
 these substances in turn are converted into asparagine and glutamine 
 
 » Deleano, N. T. Jahr. f. wiss. BoL, SI, 541-592 (1912). 
 
 * Schulze, E. Ueber den Umsatz der Eiweissstoffe in der lebenden Pflanze. 
 chem., 30, 241-312 (1900). 
 
 Zeit. physio. 
 
STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 15 
 
 after longer periods of darkness. He considers that asparagine is 
 used in the leaves for protein synthesis. This is concluded from the 
 smaller quantities of asparagine found in the leaves. 
 
 In our own experiments with germinating beans (see page 40), 
 some in the dark and some in the light, we found but slight difference 
 between the "light" and ''dark" plants. However, these experi- 
 ments were run only 120 hours, while in the investigations of others, 
 already referred to, the analyses were made after periods of 1 to 4 
 weeks. 
 
 The experiments with mature leaves which are described hereafter 
 show clearly that in the dark there is an accumulation of amino 
 acids. This is true not only of the excised leaves but also of those 
 attached to the plant. How intimate a causal interrelationship 
 exists between amino-acid accumulation and carbohydrate depletion 
 it is impossible to state from the experimental data as yet available. 
 This is, of course, an extremely complicated problem, and requires 
 for solution much deeper knowledge of the nature and function of 
 plant proteins than we are now in possession of. This point, how- 
 ever, does become evident, that the amino-acids have a profound 
 influence on the rate of respiratory activity of the leaves, in the 
 sense that an increase in amino-acid content is accompanied by an 
 increase in carbon-dioxid emission and carbohydrate consumption. 
 
 In compendium, then, it appears that in mature leaves: 
 
 1. Amino-acids accumulate in the dark as carbohydrates are consumed. There 
 is some evidence that proteins also diminish in this process. 
 
 2. The rate of carbohydrate consumption is accelerated by amino-acids. At the 
 same time the fact must not be neglected that respiratory activity is a product 
 (within limits) of mass action, the rate depending as one factor upon the supply of 
 available carbohydrates. 
 
 The questions then presented are: In what manner can amino 
 acids influence the rate of respiratory activity? What is the influence 
 of these substances on the general carbohydrate metabolism? Are 
 there any chemical relations between amino-acids and sugar which 
 can account for this behavior, or is this influence exercised through 
 the operation of enzymes? Contributing to the elucidation of these 
 questions are a number of facts which can be drawn from allied 
 sciences. 
 
 Of direct bearing on the question of the influence of amino-acids 
 on the respiratory activity is the phenomenon of the specific dynamic 
 effect of proteins, which, observed in animals, has been the subject 
 of extensive experimental investigation and much controversy. 
 Rubner and others established that, in the diet of animals, fats and 
 carbohydrates are mutually interchangeable on a calorific basis. 
 Thus 100 grams of fat can be replaced by 232 grams of starch and 
 by 234 grams of saccharose. In attempting to apply this principle of 
 
16 
 
 STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 isodynamic values to proteins, a number of difficulties were encoun- 
 tered. It was found that an increase of protein in the diet stimulates 
 the total metaboUsm, so that actually more food is utilized and more 
 heat emitted from a diet rich in protein than from one containing 
 little protein. Rubner^ subjected this phenomenon to a very 
 thorough and painstaking investigation and gave it the name of 
 specific dynamic action of proteins. He found that the increase in 
 metabolic activity was greater with proteins than with any other 
 class of foods and that dogs fed on meat respired more actively 
 without doing any external work. After establishing the relation 
 of environmental temperature to metaboUc rate and excluding the 
 reflex increase in metabolism through chemical regulation, Rubner 
 was able to determine the true values of the specific dynamic action 
 of proteins. The ingestion of the starvation requirement for the 
 various forms of food raised the metabolism as shown in table S.^ 
 
 Table 5. 
 
 
 Diet. 
 
 Heat 
 increase. 
 
 Meat 
 
 p.ct. 
 
 36.7 
 
 12.7 
 5.0 
 
 13.4 
 4.0 
 4.0 
 
 21.5 
 
 Fat 
 
 
 10 per cent meat, 90 per cent fat. . . 
 Meat, fat, sugar 
 
 Meat, fat, starch 
 
 20 per cent meat, 80 per cent fat. . . 
 
 A great advance in the understanding of the specific dynamic 
 action of proteins was made by the investigations of Lusk.^ Instead 
 of using proteins Lusk fed amino-acids. The same action is observ- 
 able with these splitting products of the proteins. It was found 
 that glycocoU and alanine greatly increase the metabolic activity, that 
 leucin and tyrosine exert but a slight effect, and that glutamic acid 
 is without effect. These findings are especially interesting in view 
 of the following facts: It is known that glycocoU and alanine are 
 completely convertible into glucose in the diabetic organism, whereas 
 glutamic acid is so converted that three of its carbons go to form 
 glucose, while the other two carbon atoms are oxidized. When 
 glycosuria is artificially produced by giving phlorihizin, and the 
 
 ' Rubner, Max. Die Gesetze des Energieverbraucha bei der Ernaehrung (1902). 
 
 * Rubner, Max. I. c, 325. 
 
 ' Lusk, Graham, and S. A. Riche. Animal calorimetry. V, Influence of the ingestion of 
 amino-acids upon metabolism. Jour. Biol. Chem., 13, 155-183 (1912). 
 Lusk, Graham. The cause of the specific dynamic action of protein. Arch. Intern. Med., 
 12, 485-487 (1914). Animal calorimetry: XI. An investigation into the causes of the 
 specific dynamic action of the foodstuffs. Jour. Biol. Chem., 20, 555-617 (1915). 
 
STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 17 
 
 dog is given glycocoll, there is no oxidation of this material, the energy- 
 content of the glycocoll is eliminated in the urine as glucose and urea, 
 nevertheless the metabolism is greatly increased. When corre- 
 sponding or even larger quantities of glucose are given there is no 
 stimulation of metabolism. From this it follows that the immediate 
 cause of the specific dynamic action is not in the oxidation of the 
 amino-acids and liberation of energy therefrom, but that the amino- 
 acids in some manner stimulate the activity of the cells and thus 
 cause them to metabolize more food material. 
 
 The specific dynamic action of proteins, especially with the insight 
 gained through Lusk's investigations, throws considerable light on 
 the general problem of the function of amino-acids in energesis. 
 It does not, however, enlighten us on the fundamental causes of this 
 stimulating action of the amino-acids. 
 
 Our first inclination was to search for a purely chemical explanation 
 which could account for this remarkable behavior. A natural 
 supposition seemed to be that the amino-acids affected the sugars in 
 such a manner as to make them more easily broken down and oxi- 
 dized. As Nef^ has shown, glucose is a relatively stable sugar, 
 while fructose is more completely oxidized. Thus, in order to reduce 
 completely a mixture of 382.5 grams CUSO4 and 163.8 grams NaOH 
 (Fehling's solution), the amounts of the various sugars shown in 
 table 6 were required: 
 
 Table 6. 
 
 Sugar. 
 
 Grams required 
 for reduction. 
 
 Corresponding to 
 atoms of oxygen. 
 
 Arabinose .... 
 
 Mannose 
 
 Dextrose 
 
 Levulose 
 
 55.0 
 66.4 
 68.0 
 64.0 
 
 2.13 
 2.45 
 2.39 
 2.16 
 
 Similarly, Lusk^ demonstrated that fructose was metabolized 
 more actively than glucose, so that the percentage of increase over 
 the indirect basal metabolism for the various sugars was found to be : 
 glucose 30, fructose 37, sucros6 34, galactose 22, and lactose 3. 
 In view of these facts it was possible that the amino-acids acted as 
 isomerizing agents, converting the glucose into the more active 
 fructose, just as had been shown to happen with a great variety 
 of substances, e. g., Ca(0H)2, Pb(0H)2, NaaCOs, etc.^ All of our 
 experiments directed to estabHsh such an isomerizing action of the 
 amino-acids on various hexose sugars yielded negative results. 
 
 1 Nep, J. U. Annalen der Chemie (Leibig), 357, 219 (1908). 
 
 « LuBK, Graham. Jour. Biol. Chem., 20, 656 (1915). 
 
 » Nef, J. U. Annalen der Chemie (Liebig), 357, 294-312 (1908); 403, 240-383 (1914). 
 
18 STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 Furthermore, while fructose fed to mature leaves showed relatively 
 high respiratory activity, we were surprised to find that the respir- 
 atory activity of the leaves fed fructose was apparently depressed 
 when amino-acids were also given. 
 
 A more encouraging possibility for an explanation of the stimula- 
 tory action of amino-acids was offered by the consideration of the 
 influence of these substances on enzyme activity. That some amino 
 acids have a decided effect on the rate of activity of certain enzymes 
 has been known for a long time. Effront^ in 1893 reported that 
 asparagine accelerates the hydrolysis of starch by takadiastase and 
 later^ showed that this accelerating action was also accomplished 
 by other amino-acids, while amines and their salts and acid amides 
 and their salts showed no such action. 
 
 The observation that the saccharification of starch by pancreatic 
 juice was accelerated by amino-acids was reported by Terroine 
 and Weill. ^ Even very low concentration of gylcocoll, 1:10,000, 
 increases the velocity of the saccharification 30 to 40 times. Dakin^ 
 observed that many condensations which have their analogues in 
 the metabolism of living organisms are induced by amino-acids, 
 peptones, albuminose, and some proteins, while the same reactions 
 do not take place without these substances. Jacoby and Umeda^ 
 also observed that the activity of soja-urease is greatly accelerated 
 by glycocoll, anahne, tyrosine, leucin, and glutamic acid. The 
 activity of pine diastase was investigated by Ujhara," who found 
 that this action was increased 48.5 per cent by very small quantities 
 of glutamic acid and by glycocoll 45.1 per cent. Similar results were 
 obtained with dog-serum diastase. Sherman and Walker^ have 
 subjected the accelerating action of amino-acids on diastase to thor- 
 ough investigation. They have found the acceleration to take place 
 with diastase derived from a variety of sources as well as with starches 
 of widely different origin. Also, the acceleration is not due to any 
 change in the hydrogen-ion concentration nor to a more favorable 
 concentration of the electrolytes. 
 
 Finally, Burge^ has investigated the influence of introducing 
 various amino-acids into the intestine and stomach on the catalase 
 activity of the blood. While this is stimulated apparently by a 
 large variety of substances, it would seem that the amino-acids 
 
 » Effront, I. Mon. Sci., 41, 266 (1893). 
 
 * Idem. Ibid., (4), 18, 561 (1904). 
 
 » Terroine, E., and J. Weill. Chem. Abstracts, 4, 1535 (1913). 
 
 * Dakin, H. D. Jour. Biol. Chem., 7, 49-55 (1910). 
 
 * Jacoby, M., and N. Umeda. Biochem. Zeitschr., 68, 23-47 (1918). 
 « Ujhara, K. Chem. Abstracts, 12, 1971 (1918). 
 
 ' Sherman, H. C, and Florence Walker. Influence of aspartic acid and asparagine upon the 
 enzymatic hydrolysis of starch. Jour. Amer. Chem. Soc, 41, 1866-1873 (1919); 43, 2461- 
 2476 (1921). 
 
 «Burqe, W. E. Amer. Jour. Physiol., 47, 351-355 (1918); 48, 133-140 (1919); Science, n. s., 
 49, 594-595 (1919). 
 
STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 19 
 
 are more effective in this respect than any of the other substances 
 tried, i. e., sodium acetate, acetamide, glycerine, oleine, and dextrose. 
 
 It must be reaUzed, of course, that the evidence regarding the 
 behavior of amino-acids toward enzymes, here introduced, does not 
 serve as a direct explanation of the influence of amino-acids on the 
 rate of respiratory activity nor of the specific dynamic action of 
 proteins. However, in the present very incomplete state of our 
 knowledge of this subject, the information of these relations may 
 serve as a valuable guide in developing further our conceptions of 
 the respiratory processes. So, while there is as yet no simple expla- 
 nation of the role of amino-acids in respiration, it seems also at 
 present exceedingly doubtful whether such a process is amenable 
 to simple treatment. 
 
 We are obliged to recognize more and more that not only are the 
 various activities of a living organism intimately interrelated, 
 but that the proper functioning of any one process depends upon 
 the coordination of various enzymatic activities. Thus in the oxy- 
 biosis of carbohydrates there are naturally a great many steps be- 
 tween the stored starch and the formation of carbon dioxid and 
 water. Plant physiologists have very generally fallen into the 
 habit of overemphasizing and reasoning as to the modus vivendi 
 of reactions in living things from evidence of the substances found in 
 such organisms. It must be borne in mind that, applying the prin- 
 ciples of the kinetics of step reactions, the rate of the whole reaction 
 is determined by the rate of the step progressing with the lowest 
 speed, and the more rapidly a total reaction progresses the fewer 
 intermediate products are there possible to detect. Thus, often 
 the most important and reactive products are not detectable by our 
 present chemical methods. 
 
 Fundamentally a better understanding of respiration depends 
 upon a knowledge of the nature of enzyme activity, and this, of 
 course, brings us to the very frontier of scientific thought. While 
 the activity or mode of action of enzymes has its analogy in more or 
 less well-defined behavior of catalysis, the composition of the enzymes 
 themselves is an open question. Briefly, there appear to be two 
 schools — the one conceiving of an enzyme as a definite complex 
 substance with catalytic properties, the other regarding enzymes 
 not as chemical individuals but rather as mixtures or complex 
 systems. Although the former conception has received very little 
 advancement through the efforts of synthetic chemistry, it has its 
 strong adherents, as, for instance, Willstaetter,^ who hopes by means 
 of preparation work to obtain constantly purer products until their 
 chemical nature can be established. On the other hand, following 
 the idea of enzymes as complex mixtures, some very suggestive 
 
 » WiLLSTAETTEB, R. Zeitsch. f. atigew. chem., 33, 209 (1920). 
 
20 STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 results have recently been obtained which are pertinent to the 
 general subject of the role of amino-acids in respiration. 
 
 Various claims have been advanced from time to time regarding 
 the artificial production of enzymes, and a number of catalytic 
 reactions have been produced by means of these products — reactions 
 which are very closely analogous to those produced by enzymes. 
 In the extensive literature on this subject a very serious error has 
 been allowed to enter — a matter of inference originally rather than 
 of direct statement. It is a very debatable question whether the 
 results with these "artificial enzymes," i. e., mixtures of substances 
 which induce certain reactions, which are also produced by true 
 enzymes, are of such great value in revealing the composition of the 
 enzymes as has been claimed. It appears rather that these reactions 
 can be induced by a variety of catalyses, as has been found to be 
 the case for many inorganic and organic reactions, and simply that 
 it has been possible for chemists to produce one of these catalysts. 
 However, it does not now follow that this is necessarily the same 
 catalyst which is produced and used by the organism in the form of 
 an enzyme. The point is essentially one of interpretation of experi- 
 mental results. 
 
 Nevertheless, the results obtained with these various "artificial 
 enzymes" are of great value in explaining catalytic action, and 
 therefore are of direct bearing on enzyme activity. Herzfeld* has 
 shown that the action of the proteolytic enzymes, pepsin and 
 trypsin, can be simulated by a prepared mixture of amino-acids and 
 polypeptides. According to this author's views, autolysis is simply 
 an autocatalysis which is started by the introduction into the system 
 of some of the products of decomposition. Baur and Herzfeld^ 
 have carried these researches further, and announce a splitting of 
 glucose into alcohol and carbon dioxid by means of carefully prepared 
 mixtures containing peptone, bile salts, dextrin, and sodium bicar- 
 bonate. Although the proportion of carbon dioxid and alcohol 
 formed in these reactions does not correspond to that obtained in 
 zymase fermentation, the authors explain this by the fact that their 
 catalyst also produces other sugar decompositions. Provided the 
 toluene employed to prevent infection by living organism was 
 thoroughly efficacious, which is open to some doubt, these experiments 
 point the way to a very fruitful field in the investigation of enzyme 
 activity. 
 
 >Herzfeld, E., Biochem. ZeUschr., 64, 103-105 (1914); 68, 402-435 (1915); 88, 260 (1918). 
 * Baur, E.. and E. Herzfeld. Biochem. Zeitschr., 117, 96-112 (1921). 
 
STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 21 
 
 METHODS AND APPARATUS. 
 
 1. The Experimeiital Material. 
 
 For the study of respiration and photosynthesis the leaves of all 
 plants are not equally well suited. That is, in some plants, due to 
 structural peculiarities, these processes exhibit complications and 
 complexities which make the interpretation of observational data 
 extremely difficult. The fundamental feature of both processes 
 is that they depend upon the ingress and egress of gases. The most 
 satisfactory methods of measuring the rate of respiration and photo- 
 synthesis are based upon the gaseous exchange, and any factor 
 which at all influences the easy diffusion of these gases interferes 
 with the accuracy of the determinations. For this reason, thin 
 leaves are more easily worked with than those which are succulent 
 or possess protective devices against the loss of water. 
 
 Moreover, in a study of respiration, analytical data are of great 
 importance and the leaf material should not present undue diffi- 
 culties for the chemical determination of the materials intimately 
 associated with the respiratory activity. The plant material should 
 also be capable of being easily grown in quantity and should not be 
 susceptible to infection or disease. Further insight into the mechan- 
 ism of the processes of respiration and photosynthesis can be gained 
 only by means of experimentation, that is, by subjecting the plant 
 to a variety of external conditions and at will altering some of the 
 internal constituents. For such a procedure the plant must not be 
 too sensitive nor easily affected by shght variations of external 
 conditions. In view of the fact that these studies were directed 
 primarily toward an attack on the problem of photosynthesis, it 
 was desirable to work with leaves in which the chlorophyll apparatus 
 was well developed, and that these leaves should be autotrophic in 
 the sense that, aside from mineral nutrients, they should be entirely 
 capable of producing their own food material. 
 
 It would, of course, be desirable to gain information relative to 
 the respiratory and photosynthetic processes of a large variety of 
 species. We are fully aware that different species exhibit great 
 variation in their behavior. Similarly, a knowledge of the behavior 
 of these plants in the field, under natural conditions, would probably 
 be of greater significance than the information gained under such 
 highly artificial conditions. However, these ideals are not amenable 
 to the experimental method and can be attempted only at too great 
 a sacrifice of precision and intensity of investigation. 
 
 We were therefore constrained to confine our studies to a very 
 limited number of plants which fulfilled the requirements mentioned. 
 The wild sunflower of southern Arizona, Helianthus annuus, and 
 
22 STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 the "Canada Wonder" bean have answered the requirements 
 admirably, and we have been able to grow this material in the green- 
 house during most of the year. Naturally, care was exercised to 
 use only perfect leaves, free from infections and other imperfections. 
 By thus concentrating our efforts we hoped to be able to gain deeper 
 and more detailed information which might serve to elaborate some 
 general principles of plant metabolism. 
 
 For most of the experiments leaves which had been cut from the 
 plant were used. The method of cutting is described later. 
 
 Although experiments with excised leaves apparently represent 
 highly artificial conditions, this method possesses many distinct 
 advantages. An entire plant usually requires so much space that 
 it is exceedingly difficult and cumbersome to construct respiration 
 chambers which will accommodate the plant and at the same time 
 permit accurate control of the temperature. By using excised 
 leaves the space can be very materially reduced and the requirements 
 of temperature control adequately met. A relatively small volume 
 of the respiration chamber has another advantage in that with a given 
 rate of the air-stream it permits the more perfect removal of the 
 gases. By cutting the leaf from the plant the factor of translocation 
 is also eliminated. Thus materials which are formed in the leaf 
 during the course of respiration do not migrate to other parts of the 
 plant, and the only source of supply of food material is that which is 
 stored in the leaf itself. The general effect of these conditions as 
 compared with the attached leaves is, as it were, to shorten the time 
 of certain reactions or accentuate their intensity. By removal from 
 the rest of the plant, the leaf has thus been severed from its base 
 of supplies as well as from the receiver of its surplus products. 
 
 The use of excised leaves also offers the only satisfactory method 
 of feeding to leaves substances the behavior of which it is desired to 
 study. In comparison with animals, physiological work with plants 
 is at a greater disadvantage in this respect. It is not possible to 
 ingest into plants a definite quantity or kind of food material, as 
 can be done with animals. However, it has been established that 
 many organic substances are taken up into the leaves in a short time 
 when the petioles are placed in solutions of these substances. On 
 the other hand, under the conditions of our experiments there was 
 no migration of materials, such as sugars, from the leaf into the 
 nutrient solution. 
 
 The plants used in these investigations were grown in a green- 
 house in a loam soil to which no fertilizer had been added. Care 
 was taken to use leaves of about the same age, size, and development. 
 It was endeavored to study the behavior of mature, well-functioning 
 leaves; so both rapidly growing and old leaves were discarded. 
 
STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 23 
 
 2. The Apparatus. 
 
 The determinations of the rate of respiration were made on the 
 principle of drawing air free from carbon dioxid over the leaves 
 and absorbing the carbon dioxid given off by the leaves in a stand- 
 ardized solution of barium 
 hydroxide. The appara- 
 tus was so arranged that 
 the leaves were always 
 under the pressure of the 
 atmosphere. A small pis- 
 ton-pump driven by an 
 electric motor drew the 
 air through the entire ap- 
 paratus; a large reservoir 
 and a pressure regulator 
 assured the regularity of 
 the stream. The air was 
 drawn from out of doors 
 in order to avoid the pos- 
 sibility of deleterious ef- 
 fects from impurities in 
 the air of the laboratory. 
 It was passed first through 
 a train of soda-lime tubes 
 to remove the carbon di- 
 oxid and then through a 
 coil of metal tube, 10 feet 
 long, which was immersed 
 in the water thermostat. 
 A large Freas electric ther- 
 mostat was employed and 
 set at exactly 25°. From 
 the metal tube the air 
 passed to the upper open- 
 ing of the respiration 
 chamber. 
 
 A drawing of the respi- 
 ration chamber is given 
 in figure 1. This con- 
 sists essentially of a gal- 
 
 vanized-iron can 40 cm. deep and 18 cm. in diameter. A heavy 
 lead base permits the entire chamber to be submerged in the 
 water of the thermostat. To the top of the chamber is sol- 
 dered a collar which forms a trough. Into this trough fits loosely 
 
 Figure 1. 
 Respiration chamber with mercury seal and cover, with 
 broken out section showing container for nutrient solu- 
 tion and device for holding petioles of leaves. 
 
CSi«^ 
 
 24 
 
 If- 
 
 STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 an inverted glass crystallizing dish and over this a galvanized- 
 iron cover. As a seal, the trough contains mercury about 3 cm. 
 deep and over this is placed water. In order to protect the 
 trough and metal cover against the action of the mercury, these 
 were first electroplated with copper, then covered with nickel, and 
 finally given a coat of waterproof lacquer. When the respiration 
 chamber is in use it is entirely covered by the water of the thermostat 
 and quickly attains the temperature of the bath. The metal cover 
 
 Figure 2. 
 Apparatus for automatically changing the course of the air-stream from one absorbing tube 
 to another. The bell-valve with mercury seal is opened by means of the electromagnetic coil 
 and closed when the current is cut out of the coil. One valve is attached to each coil. In the 
 cut, one complete valve connected with the electromagnet which controls it is shown; the three 
 other coils are indicated. 
 
 can be removed during the course of an experiment to observe the 
 leaves in the chamber or to admit light, as the glass cover in the 
 mercury makes an absolutely tight seal. 
 
 On the bottom of the chamber is a glass dish containing a large 
 number of short pieces of glass tubing set on end. This dish contains 
 the nutrient solution and the glass tubes are of a size to admit easily 
 
STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 25 
 
 the petioles of the leaves; one leaf in position is shown in the drawing. 
 Fresh sterilized solutions are of course used for each experiment, 
 the entire chamber and dish being sterilized each time. 
 
 The air-stream enters the chamber through the upper tube and 
 leaves through the lower one. To the latter a trap is attached to 
 catch the water-vapor which is carried out and condenses in the 
 tubes. By means of glass tubing the air-stream is distributed 
 through four glass stopcocks to the four 
 mercury bell-valves. These valves are 
 electrically controlled by means of a 
 clock and require detailed description. 
 
 Our preliminary studies on the res- 
 piration of leaves soon revealed the 
 necessity of making carbon-dioxid deter- 
 minations over a longer period of time 
 than had usually been done. From ob- 
 servations of a few hours or a whole day 
 only limited information can be gained 
 as to the true nature of this process, 
 especially if the leaves are to be fed 
 various substances. Moreover, ^5 too 
 long periods, as during an entire night, 
 are for obvious reasons to be avoided. 
 These requirements necessitated atten- 
 tion being given to the experiments 
 during the entire 24 hours of the day. 
 Therefore, in order to obviate the night 
 work, an apparatus was devised which 
 automatically changed the course of the 
 air-stream from one absorbing tube to 
 another at definite times. 
 
 As was stated, the air-stream passes 
 from the distributing tubes to the mer- 
 cury valves. The construction of these 
 valves, together with the electric coils 
 which control them, is pictured in figure 
 2; a detail of the mercury valve is shown in figure 3. In figure 2 
 the valve is show^n open, giving free passage of the air-stream from 
 the distributing tube through the valve to the absorbing tubes. The 
 valve is connected by means of a heavy wire to a piece of laminated 
 iron which forms the core of an electric magnet. When the current 
 passes through the wire coil, the core is drawn up and the valve 
 remains open as long as the current passes through this coil. As soon 
 as the current stops passing through the coil, the iron core drops 
 and the valve is closed by means of the glass tube which is immersed 
 
 Figure 3. 
 Detail of bell- 
 valve, made of glass 
 and sealed with mer- 
 cury. The upper 
 portion of the glass 
 tube is attached to 
 the core of the elec- 
 tromagnet; the lower 
 end is immersed in 
 mercury when the 
 valve is closed and 
 drawn out of the 
 mercury by a magnet 
 to permit the air- 
 stream to pass into 
 the absorbing tubes. 
 
26 
 
 STUDIES IN PLANT RESPIKATION AND PHOTOSYNTHESIS. 
 
 in the mercury in the small bottle. The detail in figure 3 shows what 
 is essentially a bell-valve made of glass and sealed with mercury. 
 
 As is shown in figure 2, the apparatus contains four of these 
 valves, each with its own electromagnet and each connecting with 
 a separate absorption tube. The time during which each valve 
 was held open represents one period of carbon-dioxid determination. 
 
 The manner in which the electromagnets are controlled can be 
 seen by referring to figure 4. This apparatus consists essentially 
 
 Figure 4. 
 The clock which makes electrical contact through the mercury trough and 
 thus controls the valves by means of the electromagnets. 
 
 of a circular piece of wood 5 cm. thick and 30 cm. in diameter. 
 A circular trough has been cut in the upper face. The whole is 
 mounted on three wooden legs. In the center hole is a 24-hour 
 cylinder^clock, C. This carries a metal arm, on one end of which 
 is a mercury cup,*'and through this cup connection is made with the 
 electric current: of the^line through the wire and binding post L. 
 From the otherend of the arm is suspended a looped piece of copper 
 
STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 27 
 
 wire. This copper wire floats on the mercury in the trough. The 
 sides of the trough and the rest of the wood were covered with several 
 coats of Bakelite paint to insure insulation. The trough is parti- 
 tioned off with thin pieces of mica, dividing the whole trough into 
 quadrants which are completely insulated from each other. If 
 necessary the trough can be divided into a larger nuniber of sections. 
 The mercury in each section (in fig. 4 there are four) is connected 
 by means of a short wire to a binding post V. A wire from each one 
 of these binding posts connects to one of the electromagnetic coils, 
 the other end of the coil connecting to the line. Thus the circuit 
 is completed, the current running from the line to binding post 
 L, in turn through the mercury cup on the clock, the arm (not through 
 the clock !) , through a portion of the mercury in the trough, through 
 a sectional binding post V, to a coil, and back to the line. 
 
 As the clock moves, the arm is carried over the trough, making 
 contact in one section of the mercury. When the arm reaches a 
 mica intersection it breaks the connection with one section and makes 
 contact with another. Thus the current is broken from one coil 
 and run through another coil, which process closes one valve and 
 opens another. In this manner a 24-hour clock with four intersec- 
 tions makes possible four 6-hour periods. The periods can be in- 
 creased or shortened at will by changing the number of intersections 
 or by using a clock of different rotation. For this work commercial 
 110-volt A. C. current was used; the coils were constructed with a 
 resistance of about 80 ohms and heated very little. Sparking can 
 be avoided by insertion in the circuit of suitable condensers, although 
 no trouble was experienced from this source. 
 
 From the mercury valves the air-stream passes to the absorbing 
 tubes. These were 10-bulb Meyer tubes which were supported 
 by wooden racks. The glass tubes were connected by means of 
 heavy rubber tubing and all rubber connections were wired and 
 covered with Bakelite paint. Beyond the absorbing tubes a pressure 
 regulator was inserted. This was patterned after the well-known 
 Palladin regulator^ and modified so that a cylinder containing a 
 saturated solution of calcium chloride was used as the liquid. This 
 permitted more accurate adjustment of the pressure than mercury. 
 A water manometer was also inserted through the rubber stopper 
 in the cylinder, so that any change in the pressure in the system 
 could be detected at once. The pressure regulator was immersed 
 in the water of the thermostat to avoid the influence of changes of 
 temperature. With this device the pressure was kept quite con- 
 stant, never exceeding 2 cm. of water, and the air-stream was per- 
 fectly regular. Connection was made from the pressure regulator 
 
 1 Abdebhalden, E. Handbuch der Biochemischen Arbeitsmethoden, vol. iii, 1, 481 (1910). 
 
28 STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 to a large carboy and from this to the electric pump. The rate at 
 which air is drawn through the apparatus is of minor importance 
 as long as it allows no accumulation of carbon dioxid in the respiration 
 chamber and as long as the carbon dioxid is completely absorbed 
 in the barium-hydroxide solution. 
 
 The carbon dioxid absorbed by the barium-hydroxide solutions 
 was calculated from determinations of the changes in concentration 
 of this solution. At first this was done by titration with standard 
 acid; later this method was supplanted by the more rapid and more 
 accurate method of determining the electrolytic conductivity of the 
 solution. 
 
 For the titration method a solution of about 0.1 normal strength 
 was used. This was made Up in 18-liter lots, and to each 1,000 c. c. 
 1 gram of barium chloride was added. The barium-hydroxide 
 solutions, through which carbon dioxid had passed, were transferred 
 to narrow bottles, the barium carbonate was allowed to settle, and 
 three aliquot portions of the clear supernatant solution were used for 
 titration with 0.1 normal hydrochloric acid. In order to attain very 
 accurate results with this method it is necessary to realize that 
 barium carbonate is slightly soluble in the barium-hydroxide solution. 
 From the work of Vesterburg^ and of Weissenberger^ it is evident 
 that the addition of barium chloride to suppress the hydrolysis of 
 the barium carbonate is essential in order to attain satisfactory 
 results with this method. This is a precaution frequently neglected 
 in determining rates of respiration by this procedure. 
 
 On account of the labor and time involved in making the many ti- 
 trations required by the foregoing method, an electrolytic method 
 was devised which gave exceedingly satisfactory results. For this 
 purpose a pure barium-hydroxide solution was used, prepared with 
 C02-free water. Considerable preliminary experimentation was 
 necessary to work out a satisfactory and simple apparatus for the 
 electrolytic determinations. It was found quite impracticable to 
 incorporate the electrodes for the conductivity determinations within 
 the absorption tubes. The chief difficulty encountered here was, 
 in brief, that the electrodes became covered with barium carbonate 
 and thus greatly increased the resistance of the cell. Hence the 
 cell-constant at the beginning and end of the determination was not 
 the same; even a very slight deposit of barium carbonate, such as is 
 formed by wetting the electrodes with a barium-hydroxide solution 
 and allowing this to remain in the air for a few minutes, gave spurious 
 results. The procedure which was followed, therefore, was to transfer 
 the entire contents of the absorption tube into a glass vessel with a 
 wide mouth. This was carefully stoppered and the barium car- 
 
 > Vesterburo, A. Zeit. physik. chem., 70, 550-568 (1910). 
 » Weissenberger, G. Ibid., 88, 257-270 (1914). 
 
STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 29 
 
 bonate allowed to settle out completely. When the supernatant 
 liquid was perfectly clear, this vessel was placed in the water ther- 
 mostat kept at 25°. The stopper was replaced by a dip-conductivity 
 arrangement, as shown in figure 5. The electrodes are essentially 
 the same as those used in the Cain method^ for the electrometric 
 determination of carbon in steel. A Leeds & Northrup combination 
 bridge and galvanometer operating on a 110-volt A. C. 60-cycle 
 current gave highly satisfactory results. 
 For the successful operation of this 
 method a number of jfacts had to be 
 determined which were not available in 
 satisfactory form in the chemical liter- 
 ature. It was necessary to choose a 
 barium-hydroxide solution of a con- 
 centration such as would completely 
 absorb the carbon dioxid in the air- 
 stream at the maximum rate emitted 
 by the leaves and for the longest period 
 of observation. Such a solution should 
 also show the maximum change of re- 
 sistance for a given change in concentra- 
 tion. After preliminary experiments in 
 which these limits were established, a 
 solution was decided upon with an initial 
 concentration of 0.12 normal, which 
 could wdth safety be decreased, through 
 the precipitation of barium carbon- 
 
 FlGUKE 5. 
 
 Conductivity cell used to determine the strength of 
 the barium-hydroxide solution. The barium carbon- 
 ate is allowed to settle and the resistance of the clear 
 supernatant solution is determined at 25°. 
 
 ate, to 0.05 normal. It was necessary then to obtain experimental 
 data from which a curve of specific resistance could be drawn for 
 definite concentrations of the barium-hydroxide solution. The 
 concentrations of the solutions used for these determinations were 
 obtained by means of titration with hydrochloric acid, and the 
 resistances were determined at 25° by means of the cell shown in 
 figure 5. The values thus obtained are given in table 7. Fortu- 
 nately there were available two determinations of the specific con- 
 ductivity of barium hydroxide by A. A. Noyes^ at concentrations 
 very close to both extremes of the concentrations used by us. Cal- 
 
 1 Cain, J. R., and L., C. Maxwell. Jour. Ind. and Eng. Chem., 11, 852 (1919). 
 
 2 NoYEs, A. A. Tables annuellea constantes et donnees num^riques de chimie, de physique et 
 
 de technologie, vol. i, p. 463 (1912). 
 
30 
 
 STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 dilating the specific resistance from these values, those of Noyes 
 are for 0.10 normal 48.97 and for 0.05 normal 92.98, while our results 
 were respectively 49.80 and 92.20. 
 
 In view of the fact that our determinations were not made for 
 the purpose of obtaining physical-chemical constants, and we 
 required only relative values for the determination of respiration- 
 rates, the agreement is quite satisfactory. 
 
 Table 7. — Specific resistance of various concentrations of barium-hydroxide solution, 
 determined at 25°. 
 
 Cell constant = 1 .2255. 
 
 No. 
 
 Normal 
 
 Observed 
 
 Specific 
 
 No. 
 
 Normal 
 
 Observed 
 
 Specific 
 
 concentration. 
 
 resistance. 
 
 resistance. 
 
 concentration. 
 
 resistance. 
 
 resistance. 
 
 Stock 
 
 0.11690 
 
 53.2 
 
 43.40 
 
 6 
 
 0.08297 
 
 72.3 
 
 59.00 
 
 1 
 
 0.11028 
 
 55.8 
 
 45.53 
 
 7 
 
 0.07771 
 
 76.5 
 
 62.42 
 
 2 
 
 0.10758 
 
 57.0 
 
 46.51 
 
 8 
 9 
 
 0.06940 
 
 84.6 
 
 69.03 
 
 3 
 
 0.10337 
 
 59.2 
 
 48.30 
 
 0.06111 
 
 95.1 
 
 77.60 
 
 4 
 
 0.09787 
 
 62.4 
 
 50.92 
 
 10 
 
 0.05717 
 
 100.7 
 
 82.17 
 
 5 
 
 0.08899 
 
 67.8 
 
 65.32 
 
 11 
 
 0.05221 
 
 108.9 
 
 88.86 
 
 The curve plotted from these determinations is given in figure 6. 
 On the basis of these values it was possible to calculate the amount of 
 carbon dioxid represented by any given reduction in the concen- 
 tration of the absorbing barium-hydroxide solution. The manner 
 in which this was employed is given in the following section. 
 
 3. The Procedure of the Respiration Experiments. 
 
 Before using the apparatus just described, it was of course tested 
 for small leaks, the absorption capacity of the soda-lime train at the 
 required rate of the air-stream was determined, and the limits of 
 complete absorption by the barium-hydroxide solution were estab- 
 lished. While the apparatus seems to be rather complex, for con- 
 tinuous use for an extended series of determinations the various 
 appliances proved very reliable and efficient. 
 
 Before each experiment the respiration chamber was washed out 
 with a solution of formaldehyde and then with distilled water. 
 Thereafter filtered air was drawn through the chamber until the 
 last trace of formaldehyde was removed. All nutrient solutions 
 were also steriUzed by heating twice in an autoclave. 
 
 Only the purest chemicals available were used. The sugars 
 were Pfanstiehl brand. Some inconvenience was occasioned by the 
 difficulty of procuring reliable amino-acid. At the time these 
 experiments were begun amino-acids were not procurable or only at 
 an exorbitant price and of a quality utterly unfit for physiological 
 work. We were therefore obliged to devote about three months 
 
STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 31 
 
 to the preparation of amino-acids. The laboratory at Carmel, 
 California, had not yet been completed, and we are greatly indebted 
 to Professor G. N. Lewis for extending to us the opportunity of 
 carrying out a large part of this preparation work in the chemical 
 laboratories of the University of California. We were thus enabled 
 to prepare adequate quantities of a variety of amino-acids of the 
 highest purity. 
 
 The mineral nutrient solution, recommended by Dr. B. M. Duggar, 
 was the following: Solution No. 1: 3 grams CaS04 in 3,000 c. c. 
 HaO. Solution No. 2: 6 grams MgS047H20 in 1,000 c. c. H2O. 
 Solution No. 3: 3 grams Merck's ''soluble ferric phosphate" in 
 1,000 c. c. H2O. Solution No. 4: 12 grams KCl in 1,000 c. c. H2O. 
 The complete nutrient solution was made by mixing in the following 
 proportions: 30 c. c. No. 1; 10 c. c. No. 2; 10 c. c. No. 4; diluted to 200 
 c. c. ; and then 40 c. c. of No. 3 was added. This mixture gave excellent 
 results. A number of other commonly used nutrient solutions 
 employed in the preliminary experiments were unsatisfactory 
 because the leaves, after being in the dark for some time, were 
 wilted at the tips, had dark spots, appeared rather yellow, or showed 
 other abnormal conditions. 
 
 All the solutions were thoroughly sterilized before using by twice 
 heating in the autoclave to 15 pounds pressure. The mineral 
 nutrient, sugar, and amino-acid solutions were heated separately 
 before mixing. The dish and the glass tubes which held the leaf 
 petioles were also sterilized. 
 
 Great care was exercised in selecting the leaves in order to use 
 those of the same age and development. Consideration was also 
 given to the conditions to which the plants had been exposed previous 
 to the cutting of the leaves, viz, temperature and illumination. 
 Where this was of importance the leaves were taken only when these 
 conditions were as nearly the same as can be obtained in a green- 
 house. The petioles of the leaves were cut under distilled water. 
 They were immediately taken to the laboratory and about an inch 
 of the petiole cut off under water. In the meantime the nutrient 
 solution had been prepared and placed in the respiration chamber. 
 The leaves were then quickly transferred to the solution in the 
 chamber, care being taken that a drop of water adhered to the end 
 of the petiole. The leaves were placed as loosely as possible, with 
 the petioles well submerged in the nutrient solution. The glass 
 cover was then placed over the chamber and then the metal cover. 
 The level of the water in the thermostat was raised so that it covered 
 the entire chamber. Before cutting the leaves the absorption tubes 
 had been filled and connected, so that as soon as the chamber was 
 closed the air-stream could be started through the apparatus. For 
 about the first hour no account was kept of the carbon-dioxid emis- 
 
32 STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 sion, as results obtained during this period would be spurious, owing 
 to the carbon dioxid of the air in the chamber, etc. Thereafter, 
 however, the determinations of the rates appear to be quite concor- 
 dant, depending upon nutritional conditions, and no evidence of 
 traumatic effects was noticeable. We did not find it advisable to 
 run these experiments over 100 hours, as the danger of infection 
 developing after this time became great, and this naturally would 
 introduce serious error. 
 
 To return to the barium-hydroxide solution: 125 c. c. of this 
 solution was introduced into each absorption tube by means of a 
 pipette. After the air-stream had passed through one of these 
 tubes for a definite period, it was disconnected from the apparatus, 
 and the solution, containing the suspended barium carbonate, was 
 transferred to a glass vessel (see fig. 5) and tightly stoppered. By 
 
 .16 
 
 Figure 6. 
 Specific-resistance curve of barium hydroxide at concentrations from 0.05 to 0.12 
 normal. The ordinate represents the specific resistance, while on the abscissa the CO2 
 gram equivalents of 125 c. c. barium-hydroxide solution are plotted for concentrations of 
 0.05 to 0.12 normal. 
 
 rapid manipulation there is no appreciable error introduced from 
 the carbon dioxid of the air. The barium carbonate was allowed 
 to settle until the supernatant liquid was perfectly clear. The 
 vessel was placed in the thermostat kept at 2.5° and the resistance 
 determined by means of the apparatus already described. At 
 frequent intervals the electrodes were dipped in dilute hydrochloric 
 acid and while not in use kept in distilled water. With these pre- 
 cautions the cell-constant remained unchanged for a long time. 
 
 As was described in the previous section, a curve was drawn with 
 the ordinate as specific resistances and on the abscissa the normal 
 concentration. From this data the carbon-dioxid equivalent for 
 
STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 33 
 
 125 c. c. of solution (the amount used in the absorption tubes) can 
 easily be calculated and used as the abscissa of the curve just men- 
 tioned. Thus, after the observed resistance value of a barium- 
 hydroxide solution had been converted into the correct value of 
 specific resistance by correcting for the cell-constant, the CO2 
 equivalent could be directly read from the chart. The difference 
 between this, and the CO2 equivalent of the original solution gives 
 the amount of carbon dioxid emitted during the period the air passed 
 through the barium-hydroxide solution. This chart is reproduced 
 in figure 6. By this means the respiration rates were determined 
 with very satisfactory accuracy and consumption of a minimum 
 amount of time. 
 
 4. The Analyses. 
 
 For each experiment 15 to 20 mature leaves were cut. One-half 
 of these were taken for immediate analysis; the rest of the leaves 
 were analyzed after the respiration experiment. Both sets were of 
 course treated exactly alike. Dry-weight determinations were made 
 in duplicate. The leaves were folded up and placed in wide-mouth 
 weighing bottles, the covers placed on the bottles, and weighed. 
 The weighing bottles, with the covers on, were then placed in an 
 electric oven at 98° to insure rapid kiUing. After about 30 minutes 
 the covers were removed and the material was dried at the same 
 temperature for 24 hours. The weighing bottles were then placed 
 in a desiccator and weighed. This dry material was then ground in 
 an agate mortar to a fine powder, after which it was ready for analysis. 
 
 For the determination of the total sugars, 0.5 gram of the leaf 
 material was hydrolyzed for 3 hours in 25 c. c. of 1 per cent hydro- 
 chloric acid, filtered, neutralized with sodium bicarbonate made up 
 to 100 c. c, and the sugars determined with an alkaline copper- 
 sulphate solution by a method the details of which have already 
 been described. ^ Although this method entails more work than some 
 of the other methods in common use, it was found that these pre- 
 cautions were justifiable in order to obtain accurate results with 
 plant material. 
 
 The determination of total sugars seemed to yield sufficient infor- 
 mation relative to the carbohydrate-supplj^ of the leaf, especially 
 in the light of previous investigations which showed that with ample 
 water-supply the carbohydrate balance was in favor of the simpler 
 sugars, hexoses and disaccharides. Special experiments showed 
 that the method of hydrolysis used (1 per cent hydrochloric acid) 
 affected only the reserve starch and not the structural elements, the 
 cellulose of the leaf. It could therefore be assumed that the values 
 for the total sugars give an indication of the amount of carbohydrate 
 material which was available to the leaf for respiratory purposes. 
 
 1 Spoehr, H. A. Carnegie Inst. Wash. Pub. No. 287, 31 (1919). 
 
34 STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 For the determination of amino-acids, 1 gram of the dry material 
 was heated for 3 hours with 25 c. c. of water on the boiling water-bath. 
 This was filtered and thoroughly washed with hot water. The 
 filtrate and washings were concentrated on the water-bath to 25 c. c. 
 It was found inadvisable to try to remove the small amount of soluble 
 protein from this extract, because any procedure designed to accom- 
 plish this also affected the amino-acid content, and this amount of 
 protein exerted an exceedingly slight influence on the amino-acid 
 determinations. These determinations were made by means of 
 the Van Slyke micro apparatus. Deleano^ stated that the nitrous- 
 acid method of determining amino groups was unreliable when there 
 were carbohydrates and nitrates present in the mixture. Special 
 tests made with the Van Slyke apparatus with pure amino-acids to 
 which nitrates and sugars were added did not confirm this statement 
 of Deleano. ' With some of the plant extracts there was considerable 
 frothing, but this was obviated by the use of a drop of caprylic 
 alcohol added to the reaction mixture in Van Slyke apparatus. 
 
 The results of the respiration-rate determinations are reported 
 on the basis of the amount of carbon dioxid emitted per gram 
 of dry material. This is of course an arbitrary method and of only 
 relative value. However, there exists as yet no rational basis in 
 plant physiology of connecting the respiratory activity of vegetable 
 organisms, and the establishment of such a basis must await a 
 clearer understanding of the nature of the process itself. The 
 basis of fresh weight has the great disadvantage that the alterations 
 in water-content, which are easily effected in plants, very materially 
 change the results. The use of a surface standard, as has been done 
 in some work in animal physiology, has Uttle to recommend it in 
 work with plants. 
 
 In the interpretation of analytical data obtained from living 
 material great care must be exercised to refrain from accepting the 
 apparent results as the true state of affairs. Very rarely is a single 
 chemical change unaccompanied by other changes which may run in 
 the same or opposite directions. Especially is this difficulty encoun- 
 tered in the interpretation of analytical data of leaves calculated 
 on the basis of percentage of original dry or fresh material. Thus 
 the increase in one component very often simply means the decrease 
 of another component which goes to make up the total mass of the 
 original material. 
 
 In the tabulation of the experimental data some liberty was also 
 taken in expressing the time in decimals of hours instead of in hours 
 and minutes. However, this method facilitates greatly the work 
 of calculating and avoids a common source of error. 
 
 J Deleano, N. Jahrb. wisa. Bot., 51, 552 (1921). 
 
STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 35 
 
 EXPERIMENTAL RESULTS. 
 
 1. The Normal Course of Respiration. 
 
 It has been known for a long time that as the supply of carbohy- 
 drates in a plant diminishes the rate of carbon-dioxid emission also 
 decreases. This in general may be conceived of as a matter of mass 
 action, in that as the concentration of the available material to be 
 oxidized becomes less the rate of the reaction is decreased. It has 
 also been recognized, however, that these relations are not as direct 
 and as simple as was supposed at first, for there are upper and lower 
 limits where other complications or limiting factors enter and where 
 the general complex of reactions takes other courses and makes use 
 of different material.^ But in order to form some conception of the 
 behavior of chlorophyllous leaves apart from the photosynthetic 
 process it is necessary to know what might be termed the normal 
 course of respiration, meaning thereby the rate at which the stored 
 material is converted into carbon dioxid and water with the liberation 
 of energy. In many investigations on the photosynthetic process 
 the assumption that respiration proceeds at the same rate in the 
 light as in the dark has been accepted as definitely established, or 
 the possibility of any variation of this factor has been totally neg- 
 lected. In a practical sense we are interested in photosynthesis 
 as a process in which the amount of energy stored exceeds that 
 liberated by the organism. Nevertheless, if we are to formulate 
 any sort of conception of the synthetic processes or make a quanti- 
 tative measure thereof, the reverse action, going on simultaneously, 
 can not be neglected. 
 
 No conception such as the basal metabolism in animals has been 
 introduced into plant physiology, and, on account of fundamental 
 differences in the two organisms, it is improbable that such relations 
 can be worked out. But the normal course of respiration, although 
 of necessity a rather arbitrary value, offers a base-line, as it were, 
 on which subsequent respiratory activity can in a sense be super- 
 imposed. Another feature of such respiration rates is that with 
 sufficiently short periods and accuracy of CO2 determinations it 
 becomes evident that these rates show irregularities which emanate 
 from the internal w^orkings of the leaves and are independent of any 
 external conditions. We shall call attention to one such irregularity 
 in the form of a sudden rise in the respiration rate after the leaves 
 had been in the dark for about 40 hours. 
 
 In order to give a comparison of excised leaves with the normal 
 plant, the rate of respiration of an entire plant was determined. 
 As the analytical data in table 13 indicate, the plant has in the stems 
 
 > Spoehh, H. a. The carbohydrate economy of cacti. Carnegie Inst. Wash. Pub. No. 287 
 (1919). 
 
36 
 
 STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 and roots a large reserve of carbohydrates on which it can draw for 
 the maintenance of its respiratory activity. Any changes in respira- 
 tion due to carbohydrate exhaustion do not, therefore, in the case 
 of the attached leaves, appear for a considerable time, while in the 
 case of excised leaves such changes become apparent after relatively 
 short periods of darkness. For this experiment an entire Helianthus 
 annuus plant was used. The plant was growing in soil in a glass jar. 
 Before the respiration rates were determined, the soil was covered 
 with tinfoil and the edges and portions around the stem of the plant 
 were sealed with cocoa-butter. The experiment was allowed to run 
 for 94.25 hours, after which the plant was still in perfectly healthy 
 and vigorous condition. The results of the determination are given 
 in table 8. 
 
 Table 8. — Rate of emission of CO2 by a small potted plant of Helianthus annuus. 
 Soil covered with tinfoil and sealed with cocoa-butter. CO2 absorbed in Ba(0H)2 solution, 
 0.11835 normal, 125 c. c. of which haa the equivalent of 0.3254 gram CO2. 
 
 No. 
 
 Time. 
 
 
 
 Observed 
 
 Grams CO2 
 
 
 
 Mg. CO2 
 
 
 Total 
 
 resist- 
 
 equivalent 
 
 Grams 
 
 Mg. CO2 
 
 per hour 
 
 Hrs. 
 
 hours. 
 
 ance m 
 
 of 125 c.c. 
 
 CO2 ab- 
 
 per 
 
 per gram 
 
 
 
 ohms. 
 
 Ba(0H)2 
 solution. 
 
 sorbed. 
 
 hour. 
 
 dry 
 weight. 
 
 1.25 
 3.00 
 
 1.25 
 4.25 
 
 
 
 
 
 
 54.85 
 
 0.3140 
 
 0.0114 
 
 3.80 
 
 4.61 
 
 6.5 
 
 10.75 
 
 56.6 
 
 .3024 
 
 .0230 
 
 3.53 
 
 4.19 
 
 G.5 
 
 17.25 
 
 57.5 
 
 .2970 
 
 .0284 
 
 4.37 
 
 5.18 
 
 5.0 
 
 22.25 
 
 56.6 
 
 .3024 
 
 .0230 
 
 4.60 
 
 5.46 
 
 6.0 
 
 28.25 
 
 57.3 
 
 .2982 
 
 .0272 
 
 4.53 
 
 5.37 
 
 6.5 
 
 34.75 
 
 58.0 
 
 .2942 
 
 .0312 
 
 4.80 
 
 5.69 
 
 6.5 
 
 41.25 
 
 57.9 
 
 .2950 
 
 .0304 
 
 4.67 
 
 5.54 
 
 5.0 
 
 46.25 
 
 56.9 
 
 .3008 
 
 .0246 
 
 4.92 
 
 5.84 
 
 6.0 
 
 52.25 
 
 57.5 
 
 .2970 
 
 .0284 
 
 4.73 
 
 5.61 
 
 6.5 
 
 58.75 
 
 57.8 
 
 .2954 
 
 .0300 
 
 4.61 
 
 5.47 
 
 6.5 
 
 65.25 
 
 57.5 
 
 .2970 
 
 .0284 
 
 4.36 
 
 5.17 
 
 5.0 
 
 70.25 
 
 56.3 
 
 .3040 
 
 .0214 
 
 4.28 
 
 5.08 
 
 6.0 
 
 76.25 
 
 56.7 
 
 .3016 
 
 .0238 
 
 3.96 
 
 4.70 
 
 6.5 
 
 82.75 
 
 57.1 
 
 .2992 
 
 .0262 
 
 4.03 
 
 4.78 
 
 6.5 
 
 89.25 
 
 56.9 
 
 .3004 
 
 .0250 
 
 3.84 
 
 4.55 
 
 5.0 
 
 94.25 
 
 55.9 
 
 .3070 
 
 .0184 
 
 3.68 
 
 4.36 
 
 ll''45'°a.m. to 1 p.m. . 
 1 p. m. to 4 p.m 
 
 4 p. m. to lOi'SO" p.m 
 10''30°> p.m. to 5 a.m . . 
 
 5 a.m. to 10 a.m .... 
 10 a.m. to 4 p.m 
 
 4 p.m. to lO'^SO'" p.m 
 10''30™p.m. to 5 a.m. . 
 
 5 a.m. to 10 a.m 
 
 10 a.m. to 4 p.m 
 
 4 p.m. to 10''30'° p.m 
 itf'SO'^p.m. to 5 a.m. . 
 
 5 a.m. to 10 a.m 
 
 10 a.m. to 4 p.m 
 
 4 p.m. to 10*^30" p.m 
 10'>30'" p.m. to 5 a.m . . 
 
 5 a.m. to 10 a.m 
 
 These results are graphically represented by the curve in figure 7. 
 An initial drop in the respiratory rate is noticeable here; it is very 
 much more prominent in the experiments with excised leaves. 
 The striking feature of this curve is that during about the first 48 
 hours there is a gradual increase in the rate of respiration. During 
 all this time the available supply of fuel material is diminishing and 
 there are no alterations in temperature or other external conditions. 
 It therefore seems justifiable to conclude that some internal factor 
 comes into play which acts in a stimulatory manner. From the 
 experiments hereinafter recorded it would seem that the amino-acids 
 
STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 37 
 
 are themselves this factor, or are very intimately associated therewith. 
 The amino-acids exert this stimulating action on the respiratory 
 activity; they behave very much like a catalyst, accelerating the 
 rate of carbohj^drate catabolism- and resulting in an increased release 
 of energy when the supply of material is decreasing. Thus the 
 
 Si GO 65~ 
 FiGUHE 7. 
 
 Rate of respiration of an entire plant of Helianthus annuus. Soil covered with tinfoil 
 and sealed with cocoa-butter. The ordinate represents rng. COo per hour per gram dry 
 material, and the abscissa the time in hours. 
 
 catabolic activity of the plant tends to be maintained, though the 
 store of fuel is ebbing, until photosynthetic activity again replenishes 
 the fuel supply and the light diminishes the amino-acid content. 
 True to the nature of catalyst, the amino-acids do not yield the 
 energy, but when the concentration of the subsirat (the carbohydrates) 
 is sufficiently decreased, the rate of the general reaction also decreases. 
 This accounts for the dropping in the latter portion of the curve. 
 
 Table 9. 
 
 
 Dry 
 
 weight. 
 
 Total sugars 
 in dry material. 
 
 Amino nitrogen 
 in dry material. 
 
 Original condition 
 
 After 71.25 hours 
 
 in daik 
 
 p.cl. 
 10.59 
 
 12.45 
 
 p. ct. 
 5.93 
 
 2.39 
 
 p.cl. 
 0.102 
 
 0.380 
 
 These respiration curves of necessity show in but a gross manner 
 the course of respiratory activity. They represent the resultant 
 of forces. The reversal of the rate (a drop in the curve) simply 
 indicates when an influence opposed to the stimulatory one is 
 the more potent; it does not show when these various forces 
 started. As the analytical data given in tables 12 and 13 show, 
 leaves attached to the plant draw heavily upon the carbohydrate 
 
38 
 
 STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 supply in the rest of the plant. This undoubtedly accounts for the 
 fact that the plant is able to continue its initial respiratory rate for 
 so long a time, which is in decided contradistinction to the behavior 
 of the excised leaves when these are not suppHed with carbohydrates. 
 
 
 Table 10. — Rate of CO2 emission of 8 leaves of Helianthus annmts 
 Petioles in nutrient solution containing no organic substances. 
 
 at U°. 
 
 No. 
 
 Time. 
 
 Hours. 
 
 Total 
 hours. 
 
 Cubic 
 centimeters 
 0.1/NHCl. 
 
 Mg. CO2 
 per hour. 
 
 Mg. CO2 per 
 hour per gram 
 dry material. 
 
 
 1 
 2 
 3 
 4 
 5 
 6 
 7 
 8 
 9 
 
 SMo-" p.m. to 4'>3(r p.m 
 
 4''30-" p.m. to 8''45» p.m 
 
 8''45'>" p.m. to 9''15" a.m 
 
 9''15™ a.m. to 3 p.m 
 
 0.75 
 4.25 
 
 12.50 
 5.75 
 6.25 
 
 11.75 
 6.75 
 5.0 
 
 12.25 
 6.00 
 
 0.75 
 5.00 
 17.50 
 23.25 
 29.50 
 41.25 
 48.00 
 53.00 
 65.25 
 71 25 
 
 
 
 
 27.40 
 60.30 
 20.80 
 18.50 
 30.80 
 19.60 
 13.15 
 29.60 
 14 65 
 
 14.190 
 11.088 
 7.964 
 6.512 
 5.764 
 6.380 
 5.786 
 5.224 
 5.268 
 
 3.337 
 2.607 
 1.873 
 1.531 
 1.355 
 1.500 
 1.360 
 1.229 
 1.239 
 
 3 p.m. to 9''15°'p.m 
 
 9''15"' p.m. to 9 a.m 
 
 9 a.m. to SHS"" p.m 
 
 3''45'" p.m. to 8''45°' p.m 
 
 8^ 45" p.m. to 9 a.m 
 
 9 a.m. to 3 p.m. .. 
 
 
 
 
 In table 10 are given the results of the rate of carbon-dioxid emis- 
 sion from sunflower leaves which had been cut from the plant. 
 The petioles of the leaves were immersed in a sterilized nutrient 
 solution which contained only inorganic salts. The following analyti- 
 cal data give an idea of the changes of material in the leaves during 
 the period of respiration. 
 
 Figure 8. 
 Solid line represents rate of 
 respiration of 8 leaves of Heli- 
 anthus annuua at 24°; petioles 
 in a nutrient solution contain- 
 ing no organic substances. 
 Values taken from table 10. 
 Broken line represents rate of 
 respiration of 15 leaves of Can- 
 ada Wonder bean; petioles in 
 nutrient solution containing no 
 organic substance. Values tak- 
 en from table 11. The ordinate 
 gives mg. CO2 per hour per 
 gram dry material, the abscissa 
 the time in hours. 
 
 In table 11 similar results are given for excised bean leaves. 
 From the curves of these respiratory rates, given in figure 8, it is 
 evident that in the excised leaves the rate of CO2 emission declines 
 rapidly when the only supply of carbohydrates is the material stored 
 in the leaf, and that these respiratory rates decrease with but a slight 
 variation, except at about the forty-eighth hour. These graphs 
 represent the normal course of respiratory activity of excised leaves. 
 
STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 39 
 
 The course of the changes of material was followed by placing 
 five vigorous potted Helianthus plants in the dark at 20° and every 
 24 hours cutting 10 to 12 leaves, which were then analyzed. After 
 the plants had been in the dark for 96 hours they were again placed 
 in sunlight in the greenhouse for 4 hours and another sample of 
 leaves analyzed. The results are given in table 12. 
 
 Table 11. — Rate of CO2 emission of 15 leaves of "Canada Wonder'^ bean at S4°. 
 Petioles in nutrient solution containing no organic substances. 
 
 
 
 
 Total 
 hours. 
 
 Cubic 
 
 Mg. CO2 
 
 Mg. CO2 per 
 
 No. 
 
 Time. 
 
 Hours. 
 
 centimeters 
 
 per hour. 
 
 hour per gram 
 
 
 
 
 0.1 /N HCl. 
 
 
 dry material. 
 
 1 
 
 3''30" p.m. to 9''30" p.m 
 
 6 
 
 6 
 
 33.95 
 
 12.40 
 
 2.793 
 
 2 
 
 0^30^ p.m. to 3''30™ a.m 
 
 6 
 
 12 
 
 23.10 
 
 8.40 
 
 1.892 
 
 3 
 
 3''30" a.m. to 9''30°" a.m 
 
 6 
 
 18 
 
 25.10 
 
 9.10 
 
 2.049 
 
 4 
 
 9''30™ a.m. to 3''30°' p.m 
 
 6 
 
 24 
 
 24.10 
 
 8.70 
 
 1.959 
 
 5 
 
 3''3CF> p.m. to 9''30'" p.m 
 
 6 
 
 30 
 
 22.60 
 
 8.30 
 
 1.869 
 
 6 
 
 9^30°^ p.m. to 9''30™ a.m 
 
 12 
 
 42 
 
 35.60 
 
 6.50 
 
 1.464 
 
 7 
 
 g'^SO™ a.m. to 3''3Cr p.m 
 
 6 
 
 48 
 
 19.10 
 
 7.00 
 
 1.576 
 
 8 
 
 3''30™ p.m. to 9'^30"' p.m 
 
 6 
 
 54 
 
 18.10 
 
 6.63 
 
 1.495 
 
 9 
 
 9''30'" p.m. to 9'>30"' a.m 
 
 12 
 
 66 
 
 30.25 
 
 5.50 
 
 1.238 
 
 The analyses given in table 12 indicate a gradual depletion of the 
 carbohydrates available to the leaves when the plants are left in the 
 dark. This depletion can not be made up entirely by drawing upon 
 the reserve material in other parts of the plant. In spite of this 
 depletion it is noteworthy, as has been pointed out, that the rate of 
 respiration of the entire plant rises and then decreases slowly, as is 
 
 Table 12. — Analysis of leaves of Helianthus annuus, after exposure to 
 light, then in dark for 96 hours, and again in light for 4 hours. 
 
 
 
 Amino 
 
 Total 
 
 Leaf samplea taken. 
 
 Dry weight. 
 
 nitrogen in 
 
 sugars m 
 
 
 
 dry material. 
 
 dry material. 
 
 
 p.ct. 
 
 p.ct. 
 
 p.ct. 
 
 After 5 hours sunlight . . 
 
 17.45 
 
 0.230 
 
 1.824 
 
 24 hovu-s in dark 
 
 16.61 
 
 0.200 
 
 0.983 
 
 48 hours in dark 
 
 15.15 
 
 0.271 
 
 0.736 
 
 72 hours in dark 
 
 15.47 
 
 0.254 
 
 0.921 
 
 96 hours in dark 
 
 15.21 
 
 0.286 
 
 0.777 
 
 4 hours in sunlight .... 
 
 16.52 
 
 0.203 
 
 1.373 
 
 shown in figure 7. After 4 hours of photosynthetic work the leaves 
 have again accumulated considerable sugar. The amino-acids, 
 however, increase in the dark and after subsequent exposure of the 
 plant to light again decrease. 
 
 In table 13 are given the results of plants similarly treated in 
 which the leaves, stems, and roots were analyzed separately. Two 
 
40 
 
 STUDIES IN PLANT EESPIRATION AND PHOTOSYNTHESIS. 
 
 similar Helianthus plants of the same age and size, growing in large 
 pots, were used; the one was analyzed at once, the other after being 
 in the dark at 20° for 96 hours. 
 
 Table 13. — Analyses of Helianthus annuus plants, leaves, stems, and roots separately after 
 exposure to light for 5 hours and after being in the dark for 96 hours. 
 
 
 After exposure to light 
 for 5 hours. 
 
 After being in dark for 
 96 hours. 
 
 Leaves. 
 
 Stems. 
 
 Roots. 
 
 Leaves. 
 
 Stems. 
 
 Roots. 
 
 Dry weight 
 
 Amino nitrogen in dry material .... 
 Total sugar in dry material 
 
 p.ct. 
 18.10 
 .219 
 1.61 
 
 p. ct. 
 
 20.24 
 
 .101 
 
 12.17 
 
 p.ct. 
 
 " ^116 
 
 5.28 
 
 p.ct. 
 15.93 
 .294 
 .81 
 
 p.ct. 
 17.70 
 
 .075 
 11.36 
 
 p.ct. 
 
 ";i04' 
 3.92 
 
 The analyses in table 13 show a general decrease in the carbo- 
 hydrate-content of the plant kept in the dark. Here also the leaves 
 of the plant kept in the dark show an increase in amino-acids. The 
 stems and roots, however, show a very slight decrease in amino 
 compounds. 
 
 The experiments of Schulze and Castoro^ on the accumulation of 
 amino-acids have already been mentioned. In this work the seedlings 
 were kept in the dark for 2 weeks and the amino-acid content in 
 these plants was very much higher than in the ones exposed to the 
 
 Table 14. — Amino nitrogen in beans (Canada 
 Wonder) sprouted on sawdust. 
 
 No. of 
 hours. 
 
 In light. 
 
 In dark. 
 
 Amino 
 nitrogen. 
 
 Dry 
 
 weight. 
 
 Amino 
 nitrogen. 
 
 Dry 
 weight. 
 
 
 
 24 
 
 72 
 
 96 
 
 144 
 
 192 
 
 p.ct. 
 0.252 
 
 p.ct. 
 
 p.ct. 
 
 0.252 
 .146 
 .274 
 .399 
 .372 
 .403 
 
 p.ct. 
 
 
 
 41.40 
 39.81 
 36.42 
 29.22 
 26.04 
 
 .357 
 .343 
 
 .368 
 .411 
 
 36.41 
 35.90 
 
 32.89 
 30.13 
 
 light. During this length of time the seedlings were capable of 
 developing well-formed chlorophyllous leaves. We carried out 
 comparative experiments in which periodic analyses were made on 
 germinating seeds, in the light and in the dark, for a shorter time, 
 the seedlings having still available some stored proteinaceous matter. 
 In these experiments no differences of consequence were observable 
 between the seedlings grown in the dark and those grown in the light. 
 
 1 Schulze, E., and N. Castobo. Zeitschr. physiol. chem., 38, 244 (1903); 49, 72 (1906). 
 
STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 41 
 
 With mature excised leaves kept in the dark the change in amino- 
 acid and carbohydrate content naturally becomes much more 
 marked. Thus in table 16 are given the results of analyses of an 
 experiment in which 40 similar leaves of about the same age and size 
 were cut from plants. These plants had been exposed to sunUght 
 in the greenhouse for about 9 hours. The cut leaves were placed 
 in battery jars, with petioles in nitrogen-free nutrient solution, and 
 kept in the dark; every 24 hours 8 leaves were removed and analyzed. 
 
 Table 15. — Amino nitrogen in beans (Canada 
 Wojider) sprouted on filter-paper. 
 
 No. of 
 hours. 
 
 In light. 
 
 In dark. 
 
 Amino 
 nitrogen. 
 
 Dry 
 weight. 
 
 Amino 
 nitrogen. 
 
 Dry 
 weight. 
 
 
 
 24 
 
 48 
 
 96 
 
 120 
 
 p.cl. 
 0.252 
 
 p.ct. 
 
 p.ct. 
 
 0.252 
 .146 
 .232 
 .248 
 .342 
 
 p.ct. 
 
 41.40 
 39.43 
 31.04 
 30.59 
 
 
 .195 
 .264 
 .373 
 
 38.18 
 32.07 
 32.32 
 
 The results given in table 16 show a decided and regular decrease 
 in the carbohydrate-content of the leaves kept in the dark. Also, 
 the amino-acids in these leaves increased with continued time in 
 the dark, although there was no inorganic nitrogen given in the 
 solution. It would appear, therefore, that there is a continuous 
 formation of amino-acids, presumably from proteins, and that under 
 these conditions the rate of protein decomposition exceeds the rate 
 of protein synthesis, with the result that the splitting products in 
 form of amino-acids accumulate in the leaves. The effect of this 
 process on the rate of respiration and its relation to the carbohydrate 
 economy of the plant can in part be gathered from the following 
 experiments. 
 
 In table 16 were also given the results of analyses of leaves kept 
 in the dark and showing the gradual depletion of the carbohydrate 
 material used by the plant as material from which it derives its 
 energy. The course which the rate of respiration follows under 
 these circumstances has been described in figure 8. These phenom- 
 ena are of fundamental importance and appear relatively simple 
 and well known. The fate and behavior of the proteins under Uke 
 circumstances are, however, quite obscure. Furthermore, it is 
 impossible and irrational to try to follow the fate of these substances 
 in plant respiration without simultaneously considering the car- 
 bohydrate economy. As a counterpart to the experiment sum- 
 
42 
 
 STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 marized in table 16, the results of a similar experiment are given in 
 table 17, in which the petioles of the leaves are placed, instead of in 
 nitrogen-free nutrient solution only, in such a solution containing 
 also 7 per cent of d-glucose. Previous to cutting the leaves the 
 plants had been in the sunlight in the greenhouse for about 9 hours. 
 
 Table 16. 
 
 -Analysis of excised leaves of Helianthus annuus kept in the dark ai 
 20°, with petioles in nitrogen-free nutrient solution. 
 
 No. of hours 
 
 
 Amino 
 
 Total 
 
 leaveB were 
 
 Dry weight. 
 
 nitrogen in 
 
 sugars m 
 
 kept in dark. 
 
 
 dry material. 
 
 dry material. 
 
 
 p.ct. 
 
 p.et. 
 
 p.ct. 
 
 
 
 13.48 
 
 0.149 
 
 2.49 
 
 24 
 
 10.76 
 
 .187 
 
 1.51 
 
 48 
 
 10.10 
 
 .268 
 
 0.90 
 
 72 
 
 13.46 
 
 .272 
 
 0.69 
 
 96 
 
 10.33 
 
 .422 
 
 0.44 
 
 From the results given in table 17 it is evident, as was shown many 
 years ago by Boehm, that when excised leaves are placed with the 
 petioles in a nitrogen-free nutrient solution containing 7 per cent 
 d-glucose the sugar is taken up into the leaves and accumulates 
 there. As will be seen later at higher temperatures, the sugar under 
 these circumstances accumulates but very little, if at all. However, 
 
 Table 17. — Analysis of excised leaves of Helianthus annuus 
 kept in the dark at 20°, tcith petioles in nitrogen-free 
 nutrient solution plus 7 per cent d-glucose. 
 
 Number of 
 
 
 Amino 
 
 Total 
 
 hours leaves 
 
 Dry weight. 
 
 nitrogen in 
 
 sugars in 
 
 kept in dark. 
 
 
 dry material. 
 
 dry material. 
 
 
 V.ct. 
 
 p.ct. 
 
 p.ct. 
 
 
 
 10.73 
 
 0.164 
 
 1.86 
 
 24 
 
 13.24 
 
 .249 
 
 1.83 
 
 48 
 
 13.12 
 
 .176 
 
 2.42 
 
 72 
 
 13.29 
 
 .268 
 
 2.75 
 
 96 
 
 12.69 
 
 .183 
 
 3.91 
 
 when there is thus an abundant supply of carbohydrates, the amino- 
 acids increase less than in the absence of sugar. The questions 
 which arise, then, are : Do the carbohydrates in any manner influence 
 amino-acid formation, and do the amino-acids affect the rate of 
 carbohydrate consumption by the leaf-cells? Also, what is the 
 influence of light and dark in the formation of amino-acids? The 
 experiments hereinafter described may throw some light on these 
 questions. 
 
STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 43 
 
 2. GlycocolL 
 
 That amino-acids accelerate the rate of carbohydrate consumption 
 in the leaves becomes evident from the following experiment: A 
 number of healthy leaves of about the same age were cut from the 
 plants in the manner already described and placed in battery jars. 
 In one set the petioles were put in 100 c. c. nitrogen-free nutrient 
 
 Table 18. 
 
 Leaves in — 
 
 Dry weight. 
 
 Amino 
 
 nitrogen in 
 
 dry material. 
 
 Total 
 
 sugars in 
 
 dry material. 
 
 Original condition 
 
 p.ct. 
 14.36 
 14.31 
 11.36 
 
 p.ct. 
 
 0.089 
 .238 
 .293 
 
 p.ct. 
 
 11.48 
 3.34 
 2.75 
 
 Nutrient solution only 
 
 Nutrient solution plus glycocoU. . 
 
 solution plus 100 c. c. of water; in the other set the solution consisted 
 of 100 c. c. of the same nutrient solution plus a solution of 0.5 gram 
 glycocoU in 100 c. c. of water. These solutions were steriHzed as 
 usual and the experiments carried out in duplicate. After remaining 
 in the dark for 72 hours at 20°, all the leaves were removed from the 
 jars and analyzed. The results of the analysis are given in table 18. 
 
 Table 19. — Rate of €0% emission of 8 leaves of Helianthus annuris at 24°. 
 Petioles in nutrient solution containing 0.11 per cent of glycocoU. 
 
 No. 
 
 
 1 
 2 
 3 
 4 
 5 
 6 
 
 8 
 9 
 10 
 
 Time. 
 
 Hours. 
 
 Total 
 hours. 
 
 Cubic 
 centimeters 
 0.1 N HCl 
 
 Mg. CO2 
 per hour. 
 
 Mg. CO2 per 
 hour per gram 
 dry material. 
 
 4''30°^ p.m. to 5 p.m 
 
 5 p.m. to 10''15™ p.m 
 
 10''15™ p.m. to 9 a.m 
 
 0.50 
 5.25 
 
 10.75 
 6.50 
 5.25 
 
 12.50 
 6.00 
 8.00 
 9.75 
 7.00 
 5.25 
 
 
 
 
 
 5.75 
 16.50 
 23.00 
 28.25 
 40.75 
 46.75 
 54.75 
 64.50 
 71.50 
 76.75 
 
 49.00 
 80.10 
 39.00 
 26.50 
 55.95 
 26.35 
 30.50 
 36.30 
 28.00 
 20.35 
 
 20.548 
 16.412 
 13.200 
 11.088 
 9.864 
 9.658 
 8.382 
 8.184 
 8.800 
 8.492 
 
 3.570 
 2.852 
 2.293 
 1.926 
 1.748 
 1.678 
 1.456 
 1.422 
 1.529 
 1.478 
 
 3''30™ p.m. to 8M5" p.m 
 
 gh45m p jji_ to 9''15" a.m 
 
 g'^lS'" a.m to SHS'^ p.m 
 
 3''15'" p.m. to lli'lS^p.m.... 
 ll^'lS^'p.m. to 9 a.m 
 
 
 
 From the results given in table 18 it appears that the leaves 
 placed in nutrient solution gained only in amino-acid content and 
 that the leaves placed in a glycocoU solution showed an even greater 
 gain in amino-acids. It is also evident that the leaves fed glycocoU 
 consumed more carbohydrates than did the ones which had been 
 placed in nutrient solution only. 
 
44 STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 In tables 10 and 11 and figure 8 are given the results of what was 
 termed the normal course of respiration. This represents the rate 
 of carbon-dioxid emission from Helianthus leaves in the dark at 24° 
 in nutrient solution containing no organic material, and indicates 
 the rate at which the carbohydrates in the leaves are consumed. 
 
 40 45 
 
 Figure 9. 
 Rates of respiration with and without glycocoll. The broken line represents the 
 rate of respiration of 8 leaves of Helianthus annuus at 24°; petioles in a nutrient solu- 
 tion containing no organic substances. Values taken from table 10. Solid line repre- 
 sents the rate of respiration of 8 leaves of Helianthus anmms at 24°, petioles in nutrient 
 solution containing 0.11 per cent of glycocoll. Values taken from table 19. The ordi- 
 nate represents mg. CO2 per hour per gram dry material, the abscissa the time in hours. 
 
 As a counterpart to this, an experiment was carried out in which 
 the nutrient solution contained an amino-acid in order to determine 
 w^hat the influence of this is on the rate of respiration. The Helian- 
 thus leaves were taken from the same plant, a very large and strong 
 one, and were treated in precisely the same manner. The results 
 of this respiration experiment are given in table 19 and in figure 9, 
 the graphs of the two experiments, the one with and the other without 
 amino-acid. 
 
 The analytical data representing the original condition of the 
 leaves and that at the end of the experiment, after having been 
 in the dark for 76.75 hours at 24°, with the petioles of the leaves 
 in nutrient solution containing 0.11 per cent glycocoll, are given in 
 table 20. 
 
STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 45 
 
 In order to compare the respiratory activity of the two sets of 
 leaves, those with and those without glycocoll in the nutrient 
 solution, the determinations of carbon dioxid are summarized in 
 table 21. 
 
 From the foregoing it is evident that the leaves which had been 
 fed glycocoll respired more actively than those without the amino- 
 acid. On the basis of fresh material, the former produced 22.43 
 
 Table 20. 
 
 
 Dry weight. 
 
 Amino 
 
 nitrogen in 
 
 dry material. 
 
 Total 
 
 sugars in 
 
 dry material. 
 
 
 p. ct. 
 14.45 
 13.12 
 
 p.ct. 
 
 0.100 
 
 .459 
 
 p.ct. 
 9.28 
 2.36 
 
 After 76.75 hours in dark. . . 
 
 per cent more carbon dioxid than the latter, and on the basis of 
 dry material the respective difference was 15.09 per cent in favor 
 of the leaves given glycocoll. It is also evident that the leaves which 
 had had glycocoll showed a much greater reduction in the amount 
 of carbohydrates; the slight difference in the length of time the two 
 experiments ran would have but slight effect on these results. It 
 
 Table 21. 
 
 
 Total mg. 
 CO2 formed. 
 
 Total mg. CO2 
 formed per gram 
 fresh material. 
 
 Total mg. CO2 
 
 formed per gram 
 
 dry material. 
 
 Without glycocoll. 
 WithglycocoU.... 
 
 549.56 
 862.51 
 
 16.082 
 19.690 
 
 129.20 
 149.80 
 
 might be argued that the leaves given glycocoll had an original 
 higher content of carbohydrates and that this might account for 
 the greater respiratory activity. However, it has been our experi- 
 ence in a great many cases that within certain upper and lower 
 limits the initial carbohydrate-content does not influence the rate 
 of respiration under controlled conditions of temperature and water- 
 supply. Finally, it is also noticeable that the leaves given glycocoll 
 have a higher amino-acid content at the end of the experiment. 
 
 3. d-Glucose. 
 
 From the foregoing experiments the simple facts become evident 
 that when excised leaves are kept in the dark at 25° the amino- 
 acid content rises, and that when the leaves are given glycocoll 
 
46 
 
 STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 this is taken up in the leaves and stimulates the rate of respiration 
 and carbohydrate consumption. However, the excised leaves have 
 as their only supply of carbohydrates the material stored in the 
 leaves, which is depleted in a relatively short time. It has been 
 shown, though, that sugars are easily taken into the leaf through 
 the petioles from a nutrient solution. Under these circumstances 
 the respiratory rate exhibits some interesting variations. In table 23 
 are given the results on an experiment in which the excised Helianthus 
 leaves were fed d-glucose, so that the carbohydrate-content was 
 practically the same at the beginning and end of the experiment. 
 During the course of the experiment, 80 hours at 24°, the amino- 
 acid content increased about threefold. The analytical data are 
 given in table 22. 
 
 Table 22. 
 
 
 Dry weight. 
 
 Amino 
 
 nitrogen in 
 
 dry material. 
 
 Total 
 
 sugars in 
 
 dry material. 
 
 Initial condition 
 
 After 80 hours in dark. . 
 
 p.ct. 
 13.42 
 12.02 
 
 p.ct. 
 
 0.101 
 
 .312 
 
 p.ct. 
 7.55 
 7.03 
 
 The determinations of the rate of respiration are given in table 23 
 and figure 10. These show that although the leaves were constantly 
 receiving sugar, the rate of CO2 emission decreased regularly during 
 the first 30 hours. Thereafter the respiration rate increased so 
 that after 80 hours this was slightly above the initial rate. It should 
 be noted that at this time the carbohydrate-content was slightly 
 below the initial condition, while the amino-acids had increased 
 considerably. 
 
 Table 23. — Rate of CO2 emission of 9 leaves of Helianthus annuus at 24°- 
 Petioles in nitrogen-free nutrient solution containing 7 per cent of d-glucose. 
 
 No. 
 
 Time. 
 
 Hours. 
 
 Total 
 hours. 
 
 Cubic 
 centimeters. 
 0.1/N HCl. 
 
 Mg. CO2 
 per hour. 
 
 Mg. CO2 per 
 hour per gram 
 dry material. 
 
 
 1 
 2 
 3 
 4 
 5 
 6 
 7 
 8 
 9 
 10 
 
 12'»15°' p.m. to 2''30° p.m 
 
 2»>30° p.m. to 9''30" p.m 
 
 9''30°'p.m. to 9 a.m 
 
 9 a.m. to 3 p.m 
 
 2.25 
 7.00 
 
 11.50 
 6.00 
 6.00 
 
 12.25 
 6.50 
 6.50 
 
 11.00 
 7.50 
 4.25 
 
 2.25 
 9.25 
 20.75 
 26.75 
 32.75 
 45.00 
 51.50 
 58.00 
 69.00 
 76.50 
 80.75 
 
 
 
 
 43.10 
 64.85 
 32.05 
 31.25 
 71.25 
 41.45 
 39.65 
 66.85 
 48.40 
 27.00 
 
 13.530 
 12.409 
 11.748 
 11.440 
 12.804 
 14.036 
 13.420 
 13.354 
 14.190 
 13.970 
 
 3.083 
 2.828 
 2.677 
 2.607 
 2.918 
 3.198 
 3.058 
 3.043 
 3.234 
 3.183 
 
 3 p-ro- tf> 9 p-Tn 
 
 9 p.m. to 9»'15°' a.m 
 
 9»'15'° a.m. to 3''45°' p.m 
 
 3*'45» p.m. to 10''15'° p.m 
 
 10''15" p.m. to 9''15"' a.m 
 
 9''15" a.m. to 4''45" p.m 
 
 
STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 47 
 
 In the following experiment the conditions were the same as in 
 the previous one, with the exception that the initial carbohydrate- 
 content was lower and the leaves showed a greater gain in amino 
 acids. The petioles of the excised leaves were also placed in a nitro- 
 
 FlQUBB 10. 
 
 Rate of respiration of 9 leaves of Helianthus annuus at 24°; petiole in 
 nitrogen-free nutrient solution containing 7 per cent of d-glucose, from table 
 23. The ordinate represents mg. CO2 per hour per gram dry material, the 
 abscissa the time in hours. 
 
 gen-free nutrient solution containing 7 per cent of d-glucose, but 
 the leaves also showed no gain in carbohydrate-content. There 
 was, however, a very decided increase in the amino-acids after 
 94.25 hours in the dark. The analytical results are presented in 
 table 24 and the course of respiration in table 25 and figure 11. 
 
 Table 24. 
 
 
 Dry weight. 
 
 Amino 
 
 nitrogen in 
 
 dry material. 
 
 Total carbo- 
 hydrates in 
 dry material. 
 
 Original condition 
 
 p.ct. 
 14.19 
 11.74 
 
 p.ct. 
 
 0.132 
 
 .697 
 
 p.ct. 
 1.64 
 1.14 
 
 After 94.25 hours in dark. . . 
 
 The noteworthy feature of the foregoing experiment is the very 
 marked drop in the respiration rate during the first 35 hours, and 
 thereafter the decided increase, so that the rate was higher at the 
 end of the experiment than at the beginning. From previous experi- 
 ments, as indicated in table 17, it seems evident that when leaves 
 are placed with the petioles in a 7 per cent glucose solution the carbo- 
 hydrate-content is maintained after the first 24 hours, so that it 
 may be assumed that these leaves, with their initial relatively low 
 carbohydrate-content, did not suffer a decided reduction of this 
 material. It is evident that after about 35 hours the rate of CO2 
 emission increased decidedly, so that at the end of 94.25 hours, in 
 
48 
 
 STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 spite of the slightly lower carbohydrate-content, the leaves showed 
 a higher respiration rate than at the beginning of the experiment. 
 This indicates that after about 35 hours there are produced within 
 the leaf conditions which favor a more active catabolism of carbo- 
 hydrates. From the experiments already described and others 
 which are to follow, it appears certain that, when leaves are kept in 
 the dark, synchronous with the reduction of the carbohydrates 
 
 Table 25. — Rate of emission of CO2 by 6 leaves of Helianthus annuus at 25°. 
 
 Petioles in nitrogen-free nutrient solution containing 7 per cent d-glucose. CO2 absorbed 
 in Ba(0H)2 solution 0.1169 normal, 125 c.c. of which has the equivalent of 0.3214 gram CO2. 
 
 No. 
 
 Time. 
 
 Hrs. 
 
 Total 
 hours. 
 
 Observed 
 resist- 
 ance in 
 ohms. 
 
 Gram CO, 
 equivalent 
 of 125 c. c. 
 Ba(0H)2 
 solution. 
 
 Gram 
 CO2 ab- 
 sorbed. 
 
 Mg. CO2 
 per 
 hour. 
 
 Mg. CO2 
 per hour 
 per gram 
 
 dry 
 weight. 
 
 ■ 
 
 5 
 6 
 7 
 8 
 9 
 10 
 11 
 12 
 13 
 14 
 15 
 16 
 
 llM5" a.m. to 1 p.m.. . 
 
 I p.m. to 4 p.m 
 
 4 p.m. to 10''30" p.m . . 
 10^30°" p.m. to 5 a.m . . 
 
 5 a.m. to 11 a.m 
 
 II a.m. to 4 p.m 
 
 4 p.m. to 10''30» p.m. . 
 10''30" p.m. to 5 a.m . . 
 
 5 a.m. to 10 a.m 
 
 10 a.m. to 4 p.m 
 
 4 p.m. to lO'^SO™ p.m. . 
 10''30" p.m. to 5 a.m. . . 
 
 5 a.m. to 10 a.m 
 
 10 a.m. to 4 p.m 
 
 4 p.m. to 10''30" p.m. . . 
 10''30™ p.m. to 5 a.m. . . 
 
 5 a.m. to 10 a.m 
 
 1.25 
 3.00 
 6.50 
 6.50 
 6.00 
 5.00 
 6.50 
 6.50 
 5.00 
 6.00 
 6.50 
 6.50 
 5.00 
 6.00 
 6.50 
 6.50 
 5.00 
 
 1.25 
 4.25 
 10.75 
 17.25 
 23.25 
 28.25 
 34.75 
 41.25 
 46.25 
 52.25 
 58.75 
 65.25 
 70.25 
 76.25 
 82.75 
 89.25 
 94.25 
 
 
 
 
 
 
 66.6 
 83.1 
 93.7 
 69.8 
 72.6 
 74.9 
 76.9 
 71.5 
 76.7 
 77.3 
 82.3 
 77.4 
 89.8 
 99.6 
 
 0.2522 
 .1968 
 .1730 
 .2400 
 .2298 
 .2216 
 .2154 
 .2336 
 .2160 
 .2140 
 .1988 
 .2134 
 .1812 
 .1612 
 .1340 
 .1740 
 
 0.0692 
 .1246 
 .1484 
 .0814 
 .0916 
 .0998 
 .1060 
 .0878 
 .1054 
 .1074 
 .1226 
 .1080 
 .1402 
 .1602 
 .1874 
 .1474 
 
 23.06 
 19.16 
 18.38 
 
 is! 32 
 15.35 
 16.30 
 17.56 
 17.56 
 16.52 
 18.86 
 21.60 
 23.36 
 24.64 
 28.83 
 29.48 
 
 6.80 
 5.65 
 5.42 
 
 5^40 
 4.23 
 4.81 
 5.18 
 5.18 
 4.87 
 5.56 
 6.37 
 6.89 
 7.27 
 8.50 
 8.69 
 
 93.1 
 
 there takes place an increase in the amino-acids. Moreover, an 
 increase in the amino-acids stimulates the rate of carbohydrate 
 catabolism. Under ordinary circumstances, when the stores of 
 carbohydrates are being depleted rapidly, the effect of the natural 
 increase in amino-acids is but slightly noticeable, as was seen in 
 figure 8. When, however, the leaf is fed sugar and the amino-acids 
 increase appreciably, the result is a decided stimulation of CO2 
 emission. These relations are, however, of a more complex nature 
 than would appear at first glance. They of course involve the 
 metabolism of the proteins as a source of the amino-acids, and the 
 converse question arises as to what extent the formation of amino 
 acids from proteins is affected by a decreasing supply of carbohy- 
 drates. These questions will be referred to later in this paper. 
 
 Before taking up the effect of amino-acids on respiration in more 
 detail, there remains to be described the course of respiration under 
 
STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 49 
 
 conditions in which there is a decided increase in the carbohydrate- 
 content of the leaves and but a shght increase in amino-acids. In 
 the following experiment, carried out with Canada Wonder bean 
 leaves, the experimental conditions were the same as in the two 
 
 50 35^ 40 4S SO 5S 60 6S 70 75 80 85 90 95 
 
 Figure 11. 
 Rate of respiration of 6 leaves of Helianthus annuus at 25°; petioles in nitrogen- 
 free nutrient solution containing 7 per cent d-glucose. The ordinate represents mg. 
 CO2 per hour per gram dry material, the abscissa the time in hours. 
 
 previous experiments, i. e., the petioles of the leaves were in a 
 nitrogen-free nutrient solution containing 7 per cent glucose, and the 
 CO2 emission was determined in the dark at 25°. From the analytical 
 
 Table 26. 
 
 
 Dry weight. 
 
 Amino 
 
 nitrogen in 
 
 dry material. 
 
 Total 
 
 sugars in 
 
 dry material. 
 
 
 p.ct. 
 16.82 
 14.70 
 
 p.ct. 
 
 0.335 
 
 .469 
 
 p.ct. 
 2.53 
 3.45 
 
 After 58.35 hours in dark. . . 
 
 data in table 26 it is evident that there was a decided increase in 
 the carbohydrate-content, but only a slight increase in the amino 
 acids. The rates of respiration are given in table 27 and figure 12. 
 
50 
 
 STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 It should be noted that in the foregoing experiment, unlike the 
 two previous ones, there is an increase in the carbohydrate-content 
 and the increase in the amino-acids is very small. With these facts 
 in mind, the results of the rates of respiration as plotted in figure 12 
 are noteworthy. The rapid drop between the fifth and tenth hours 
 
 Table 27. — Rate of COi emission of 15 leaves Canada Wonder hean at 25°; -petioles in 
 nitrogeiv-free nutrient solution containing 7 per cent d-glucose. 
 CO2 absorbed in Ba(0H)2 solution 0.1169 normal, showing resistance of 53.2 ohms in cell with 
 constant of 1.2255 at 25°. 
 
 
 
 
 Total 
 hours. 
 
 Observed 
 
 resist- 
 
 Gram CO2 
 equivalent 
 
 Gram 
 
 Mg. CO2 
 
 Mg. CO2 
 per hour 
 
 No. 
 
 Time. 
 
 Hrs. 
 
 of 125 c. c. 
 
 CO2 ab- 
 
 per 
 
 per gram 
 
 
 
 
 ohms. 
 
 Ba(0H)2 
 
 sorbed. 
 
 hour. 
 
 dry 
 
 
 
 
 
 solution. 
 
 
 
 weight. 
 
 
 
 1 
 
 ll''55">a.m. to i^2'^ p.m. . 
 l'>2'" p.m. to 5''2°' p.m 
 
 1.10 
 4.00 
 
 1.10 
 5.10 
 
 
 
 
 
 
 68.3 
 
 0.2422 
 
 0.0792 
 
 19.8 
 
 5.13 
 
 2 
 
 5»'2'° p.m. to ltf'32™ p.m. . 
 
 5.50 
 
 10.60 
 
 71.7 
 
 .2298 
 
 .0916 
 
 16.6 
 
 4.30 
 
 3 
 
 10^32'° p.m. to3h32'"a.m. 
 
 5.00 
 
 15.60 
 
 73.6 
 
 .2232 
 
 .0982 
 
 19.6 
 
 5.08 
 
 4 
 
 3»'32-"a.m. to 8i'32°' a.m . . 
 
 5.00 
 
 20.60 
 
 68.1 
 
 .2428 
 
 .0786 
 
 15.7 
 
 4.07 
 
 5 
 
 8''32°'a.m. to2''32°'p.m.. 
 
 6.00 
 
 26.60 
 
 70.9 
 
 .2328 
 
 .0886 
 
 14.7 
 
 3.81 
 
 6 
 
 2''32°'p.m. to ll''17'"p.m. 
 
 8.75 
 
 35.35 
 
 77.0 
 
 .2120 
 
 .1094 
 
 12.5 
 
 3.24 
 
 7 
 
 ll''17'»p.m. to 4''17'°a.m. 
 
 5.00 
 
 40.35 
 
 64.3 
 
 .2592 
 
 .0322 
 
 12.4 
 
 3.21 
 
 8 
 
 4''17'"a.m. to 9''17'"a.m. . 
 
 5.00 
 
 45.35 
 
 67.6 
 
 .2450 
 
 .0764 
 
 15.3 
 
 3.97 
 
 9 
 
 9''17»a.m. to 3'>17'"p.m. . 
 
 6.00 
 
 51.35 
 
 71.4 
 
 .2310 
 
 .0904 
 
 15.1 
 
 3.91 
 
 10 
 
 Si^lT"" p.m. to 10''17'°p.m. 
 
 7.00 
 
 58.35 
 
 71.0 
 
 .2326 
 
 .0888 
 
 12.7 
 
 3.29 
 
 Figure 12. 
 Rate of respiration of 15 
 leaves of Canada Wonder bean 
 at 24°; petioles in nitrogen-free 
 solution containing 7 per cent 
 d-glucose. Values taken from 
 table 27. The ordinate repre- 
 sents mg. CO2 per hour per 
 gram dry material, the abscissa 
 the time in hours. 
 
 of the experiment is probably due to the fact that the sugar from 
 the nutrient solution had not yet got into the leaf in sufficient 
 quantity to cover the loss through respiration. Thereafter the 
 rate of CO2 emission shows a rapid rise, soon followed by a regular 
 decline far below the initial rate, until the fortieth hour, when there 
 
STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 51 
 
 again occurs the characteristic rise, which, however, is not main- 
 tained and the rate again falls, so that after about 60 hours the rate 
 of respiration is far below the initial rate, in spite of the fact that 
 the leaves have actually increased in carbohydrate-content. 
 
 Table 28. 
 
 
 Dry weight. 
 
 Amino 
 
 nitrogen in 
 
 dry material. 
 
 Total 
 
 sugars in 
 
 dry material. 
 
 Initial condition 
 
 After 74 hours in dark. . 
 
 p.ct. 
 12.72 
 12.78 
 
 p.ct. 
 0.124 
 
 .888 
 
 p.ct. 
 1.33 
 2.07 
 
 Undoubtedly the carbohydrate supply alone does not determine 
 the rate of respiration, but there is necessary an accessory factor 
 which aids the successive chemical reactions constituting this process. 
 That amino-acids act in this stimulating manner is established by 
 
 Table 29. — Rate of emission oj CO2 by 6 leaves of Helianthus annuus at 25°. 
 Petrioles in nitrogen-free nutrient solution containing 7 per cent d-glucose and 0.11 per cent 
 glycocoll. CO2 absorbed in Ba(0H)2 solution 0.1169 normal, 125 c. c. of which has the equivalent 
 of 0.3214 gram CO2. 
 
 No. 
 
 Time. 
 
 Hrs. 
 
 Total 
 hours. 
 
 Observed 
 resist- 
 ance in 
 ohms. 
 
 Gram CO2 
 equivalent 
 of 125 c. c. 
 Ba(0H)2 
 solution. 
 
 Gram 
 CO2 ab- 
 sorbed. 
 
 Mg. CO2 
 per 
 hour. 
 
 Mg. CO2 
 per hour 
 per gram 
 
 dry 
 weight. 
 
 
 
 1 
 
 2 
 
 3 
 
 4 
 
 5 
 
 6 
 
 7 
 
 8 
 
 9 
 
 10 
 
 11 
 
 12 
 
 13 
 
 14 
 
 
 1.0 
 2.0 
 6.5 
 6.5 
 5.0 
 5.0 
 6.5 
 6.5 
 5.0 
 2.0 
 4.0 
 6.5 
 6.5 
 5.0 
 6.0 
 
 1.0 
 3.0 
 9.5 
 16.0 
 21.0 
 26.0 
 32.5 
 39.0 
 44.0 
 46.0 
 50.0 
 56.5 
 63.0 
 68.0 
 74.0 
 
 
 
 
 
 
 
 58.2 
 74.6 
 72.6 
 70.8 
 69.3 
 77.2 
 80.4 
 72.2 
 60.7 
 68.0 
 81.8 
 84.6 
 74.6 
 81.4 
 
 0.2930 
 .2226 
 .2294 
 .2360 
 .2412 
 .2140 
 .2040 
 .2312 
 .2798 
 .2466 
 .2000 
 .1932 
 .2228 
 .2014 
 
 0.0284 
 .0988 
 .0920 
 .0854 
 .0802 
 .1074 
 .1174 
 .0902 
 .0416 
 .0748 
 .1214 
 .1282 
 .0986 
 .1200 
 
 14.20 
 15.20 
 14.15 
 17.08 
 16.04 
 16.52 
 18.06 
 18.82 
 
 4.45 
 4.77 
 4.44 
 5.36 
 5.03 
 5.18 
 5.67 
 5.90 
 
 5 p.m. to lli'SO^^ p.m 
 
 11''30" p.m. to 6 a.m 
 
 6 a.m. to 11 a.m 
 
 11 a.m. to 4 p.m 
 
 4 p.m. to ICSO'" p.m.... 
 10''30" p.m. to 5 a.m 
 
 5 a.m. to 10 a.m .... 
 
 10 a.m. to 12 m 
 
 12 m. to 4 p.m 
 
 18.70 
 18.60 
 19.70 
 19.70 
 20.00 
 
 5.87 
 5.86 
 6.19 
 6.19 
 6.28 
 
 4 p.m. to \(^ZQi^ p.m 
 
 10'>30'" p.m. to 5 a.m 
 
 5 a.m. to 10 a.m 
 
 10 a.m. to 4 p.m 
 
 the experiments here described, and the point naturally suggests 
 itself that under normal conditions in the dark, when the carbohy- 
 drate-content decreases and the amino-acids increase, there is a 
 similar stimulating action which would tend to maintain a relatively 
 higher respiration rate. From such data as are available it appears 
 that the accumulation of amino-acids is a relatively slow process, and 
 
52 
 
 STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 it seems highly probable that the rise in the respiration rate observed 
 after 30 to 40 hours represents the time when the amino-acids have 
 accumulated sufficientlj^ and their influence on the rate of respiration 
 becomes noticeable. The experiments on this point will be taken 
 up later. 
 
 40 50 60 70 
 
 FlGtTBB 13. 
 
 Rate of respiration of 6 leaves of Helianthus anmms at 25°; petioles in nitrogen-free 
 nutrient solution containing 7 per cent d-glucose and 0.11 per cent glycocoll. Values 
 taken from table 29. The ordinate represents mg. CO2 per hour per gram dry material, 
 the abscissa the time in hours. 
 
 Turning now to the experiments in which amino-acids were fed 
 the leaves, it should be noted that the conditions here were precisely 
 the same as in the foregoing experiments, with the exception that 
 the nutrient solution contained, besides the inorganic salts and the 
 specific sugar, a definite amount of amino-acid. In tables 23, 25, 
 
 Table 30. 
 
 
 Dry weight. 
 
 Amino 
 
 nitrogen in 
 
 dry material. 
 
 Total 
 
 sugars in 
 
 dry material. 
 
 
 p.ct. 
 14.92 
 12.71 
 
 p.ct. 
 
 0.142 
 
 .653 
 
 p. ct. 
 1.27 
 1.07 
 
 After 76.25 hours in dark . . . 
 
 and 27 were presented the results of respiration determinations of 
 leaves which had been given d-glucose. In the following experi- 
 ment with Helianthus leaves 0.11 per cent of glycocoll was added to 
 
STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 53 
 
 the nutrient solution. The analytical data show that during the 
 course of the experiment the leaves gained both in amino-acids and 
 in carbohydrate-content. 
 
 Table 31. — Rate of emission of CO2 by 6 leaves of Helianthus annuus at 25°. 
 Petioles in nitrogen-free nutrient solution containing 7 per cent d-glucose and 0.16 per cent 
 asparagine. CO2 absorbed in Ba(0H)2 solution 0.11835 normal, 125 c. c. of which has the COj 
 equivalent of 0.3254 gram CO2. 
 
 No. 
 
 Time. 
 
 Hrs. 
 
 Total 
 hours. 
 
 Observed 
 resist- 
 ance in 
 ohms. 
 
 Gram CO? 
 equivalent 
 of 125 CO. 
 Ba(0H)2 
 solution. 
 
 Gram 
 CO2 ab- 
 sorbed. 
 
 Mg. CO2 
 per 
 hour. 
 
 Mg. CO2 
 per hour 
 per gram 
 
 dry 
 weight. 
 
 
 
 1 
 
 2 
 
 3 
 
 4 
 
 5 
 
 6 
 
 7 
 
 8 
 
 9 
 
 10 
 
 11 
 
 12 
 
 13 
 
 lli'45'"a.m. to 1 p.m.... 
 1 p.m. to 4 p.m .... 
 
 1.25 
 
 3.0 
 
 6.5 
 
 6.5 
 
 5.0 
 
 6.0 
 
 6.5 
 
 6.5 
 
 5.0 
 
 6.25 
 
 6.5 
 
 6.5 
 
 4.75 
 
 6.0 
 
 1.25 
 4.25 
 10.75 
 17.25 
 22.25 
 28.25 
 34.75 
 41.25 
 46.25 
 52.5 
 59.0 
 65.5 
 70.25 
 76.25 
 
 
 
 
 
 
 60.7 
 69.0 
 71.4 
 67.8 
 72.2 
 74.0 
 75.3 
 70.3 
 80.2 
 80.9 
 82.4 
 72.2 
 81.5 
 
 0.2800 
 .2428 
 .2340 
 .2476 
 .2312 
 .2250 
 .2202 
 .2376 
 .2046 
 .2026 
 .1986 
 .2312 
 .2010 
 
 0.0454 
 .0826 
 .0914 
 .0778 
 .0942 
 .1004 
 .1052 
 .0878 
 . 1208 
 .1228 
 .1268 
 .0942 
 .1244 
 
 15.13 
 
 12.7 
 
 14.06 
 
 15.56 
 
 15.70 
 
 15.44 
 
 16.18 
 
 17.56 
 
 19.32 
 
 18.89 
 
 19.50 
 
 19.83 
 
 20.73 
 
 4.22 
 3.54 
 3.92 
 4.34 
 4.38 
 4.30 
 4.51 
 4.90 
 5.39 
 5.27 
 5.44 
 5.53 
 5.78 
 
 4 p.m. to lO'^SO'" p.m 
 
 10i'30"' p.m. to 5 a.m.... 
 
 5 a.m. to 10 a.m. 
 
 10 a.m. to 4 p.m 
 
 4 p.m. to 10'^30™ p.m 
 
 IQi'SO™ p.m. to 5 a.m 
 
 10 a.m. to 4''15" p.m 
 
 4''15" p.m. to 10*^45™ p.m. 
 10'>45'» p.m. to bHo"" a.m . 
 
 5''15'» a.m. to 10 a.m 
 
 10 a.m. to 4 p.m 
 
 From the determinations of the rates of CO2 emission, given in 
 table 29 and figure 13, it is apparent that when the leaves are given 
 both d-glucose and glycocoU the rate of respiration rises at once 
 and, with slight irregularities, maintains this increased rate for at 
 least 74 hours. The stimulating action of glycocoll becomes evident 
 when the graph of the experiment is compared with the three pre- 
 ceding ones. This is especially apparent during the first 35 hours, 
 that is, before the effect of the amino-acids accumulating in the 
 leaves becomes noticeable. 
 
 Table 32. 
 
 
 Dry weight. 
 
 Amino 
 
 nitrogen in 
 
 dry material. 
 
 Total 
 
 sugars in 
 
 dry material. 
 
 Initial condition 
 
 After 76 hours in dark. . 
 
 V.ct. 
 15.33 
 12.94 
 
 V.ct. 
 
 0.121 
 
 .569 
 
 V.ct. 
 1.49 
 1.03 
 
 That other amino-acids have a similar effect is shown by the fol- 
 lowing experiment with asparagine and Helianthus leaves. All con- 
 ditions were kept precisely the same and the nutrient solution 
 contained 0.16 per cent of asparagine. The analytical data in table 30 
 
54 
 
 STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 show that while the initial carbohydrate-content was not quite 
 maintained at the end of the experiment, there was a decided gain 
 in amino-acids. 
 
 The determinations of the rates of carbon-dioxid emission are 
 given in table 31 and figure 14. A noteworthy feature of these 
 results is the rather rapid initial drop in the respiration rate before 
 the subsequent rise. This is probably due to the time required for 
 the asparagine to pass through the petiole and penetrate into the 
 cells. GlycocoU, on the other hand, has notable penetrating 
 qualities, and the effect of this amino-acid becomes noticeable very 
 quickly in the respiration-rate. 
 
 20 30 40 
 
 Figure 14. 
 Broken line indicates rate of respiration of 6 leaves of Helianthus annuus at 25°; 
 . petioles in nitrogen-free nutrient solution containing 7 per cent d-glucose and 0.16 per 
 cent asparagine; from table 31. The solid line indicates the rate of respiration of leaves 
 of Helianthus annuus at 25°, petioles in nitrogen-free nutrient solution containing 7 per 
 cent d-glucose and 0.11 per cent of alanine; from table 33. The ordinate represents 
 mg. CO2 emitted per hour per gram dry material, the abscissa the time in hours. 
 
 Alanine seems to be taken up very slowly. The same conditions 
 were maintained as in the preceding experiments and pure alanine 
 was given as the amino-acid. The analytical data in table 32 show 
 a relatively slight increase in amino-acids after the leaves had been 
 
STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 55 
 
 in the dark for 76 hours. The rate of respiration as shown in table 33 
 and figure 14 is also correspondingly low and the rise in the rate 
 comes quite late. 
 
 Table 33. — Rate of emission of CO2 by 6 leaves of Helianthtis annuus at 25°. 
 Petioles in nitrogen-free nutrient solution containing 7 per cent glucose and 0.11 per cent 
 alanine. CO2 absorbed in Ba(0H)2 solution 0.11835 normal, 125 c. c. of which has the CO2 equiva- 
 lent of 0.3254 gram CO2. 
 
 No. 
 
 Time. 
 
 Hrs. 
 
 Total 
 hours. 
 
 Observed 
 resist- 
 ance in 
 ohms. 
 
 Gram CO2 
 equivalent 
 of 125 c. c. 
 Ba(0H)2 
 solution. 
 
 Gram 
 CO2 ab- 
 sorbed. 
 
 Mg. CO2 
 per 
 hour. 
 
 Mg. CO2 
 per hour 
 per gram 
 
 dry 
 weight. 
 
 
 
 1 
 
 \ 
 
 4 
 5 
 6 
 7 
 8 
 9 
 10 
 11 
 12 
 13 
 
 
 1.0 
 3.0 
 6.5 
 6.5 
 5.0 
 6.0 
 6.5 
 6.5 
 5.0 
 6.0 
 6.5 
 6.5 
 5.0 
 6.0 
 
 1.0 
 4.0 
 10.5 
 17.0 
 22.0 
 28.0 
 34.5 
 41.0 
 46.0 
 52.0 
 58.5 
 65.0 
 70.0 
 76.0 
 
 
 
 
 
 
 1 p m. to 4 p.m 
 
 65.5 
 77.8 
 77.9 
 70.7 
 72.5 
 73.6 
 73.9 
 67.0 
 78.9 
 76.4 
 77.2 
 71.6 
 78.3 
 
 0.2570 
 .2122 
 .2118 
 .2360 
 .2300 
 .2264 
 .2252 
 .2504 
 .2086 
 .2166 
 .2140 
 .2332 
 .2106 
 
 0.0684 
 .1132 
 .1136 
 .0894 
 .0954 
 .0990 
 .1002 
 .0750 
 .1168 
 .1088 
 .1114 
 .0922 
 .1148 
 
 22.80 
 17.41 
 17.47 
 17.88 
 15.90 
 15.23 
 15.41 
 17.43 
 
 6.027 
 4.602 
 4.618 
 4.726 
 4.203 
 4.026 
 4.073 
 4.607 
 
 4 p.m. to 10*'30™ p.m 
 
 10''30™ p.m. to 5 a.m 
 
 10 a.m. to 4 p.m 
 
 4 p.m. to 10''30°' p.m 
 
 10''30™ p.m. to 5 a.m 
 
 10 a.m. to 4 p.m 
 
 4 p.m. to 10*'30" p.m 
 
 10''30" p.m. to 5 a.m 
 
 5 H Tn. t-O 10 p m , . 
 
 16.73 
 17.13 
 18.44 
 19.13 
 
 4.422 
 4.528 
 4.874 
 6.056 
 
 10 a.m. to 4 p.m 
 
 4. Sucrose. 
 
 Some attention was also given to the influence of sugars other 
 than d-glucose. Thus the rate of respiration was determined of 
 leaves which were given sucrose as well as a mixture of this sugar 
 and an amino-acid. In these experiments the conditions as to 
 temperature, etc., were the same as in the preceding determinations. 
 
 Table 34 gives the analytical data of the leaves in the initial 
 condition and after having stood in the dark with the petioles in a 
 nitrogen-free nutrient solution containing 7 per cent of sucrose. 
 
 Table 34. 
 
 
 Dry weight. 
 
 Amino 
 
 nitrogen in 
 
 dry material. 
 
 Total 
 
 sugars in 
 
 dry material. 
 
 
 p.ct. 
 15.00 
 14.00 
 
 p.ct. 
 
 0.126 
 
 .335 
 
 p.ct. 
 2.14 
 
 2.28 
 
 After 76.5 hours in dark 
 
 The rates of respiration of this experiment are given in table 35 
 and figure 15. It will be noted that although there is a slight increase 
 in both the amino-acid and carbohydrate-content, the leaves do 
 
56 
 
 STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 not maintain the initial rate of CO2 emission and the general course 
 of the rate is down. The rise after 35 hours is very noticeable; this, 
 however, is not maintained and the rate gradually drops. 
 
 Table 35. — Rate of ennssion of CO2 by 6 leaves of Helianihus annuus at 25°. 
 Petioles in nitrogen-free nutrient solution containing 7 per cent sucrose. CO2 absorbed in 
 Ba(0H)2 solution 0.11835 normal, 125 c. c. of which has the CO2 equivalent of 0.3264 gram CO2. 
 
 No. 
 
 Time. 
 
 Hrs. 
 
 Total 
 hours. 
 
 Observed 
 resist- 
 ance in 
 ohms. 
 
 Gram CO2 
 equivalent 
 of 125 c. c. 
 Ba(0H)2 
 solution. 
 
 Gram 
 CO2 ab- 
 sorbed. 
 
 Mg. CO2 
 per 
 hour. 
 
 Mg. CO2 
 per hour 
 per gram 
 
 dry 
 weight. 
 
 
 
 2 
 
 3 
 
 4 
 
 5 
 
 6 
 
 7 
 
 8 
 
 9 
 
 10 
 
 11 
 
 12 
 
 13 
 
 11''30'" a.m. to 12»'30™ p.m. 
 
 12''30° p.m. to 4 p.m 
 
 4 p.m. to 10''30'" p.m 
 
 10''30" p.m. to 5 a.m .... 
 
 1.0 
 3.5 
 6.5 
 6.5 
 5.0 
 6.0 
 6.5 
 6.5 
 5.0 
 6.0 
 6.5 
 10.5 
 1.5 
 5.5 
 
 1.0 
 4.5 
 11.0 
 17.5 
 22.5 
 28.5 
 35.0 
 41.5 
 46.5 
 52.5 
 59.0 
 69.5 
 71.0 
 76.5 
 
 
 
 
 
 
 64.6 
 75.9 
 76.1 
 69.6 
 73.6 
 75.5 
 78.9 
 70.7 
 75.4 
 75.7 
 102.7 
 56.7 
 72.6 
 
 0.2608 
 .2184 
 .2172 
 .2402 
 .2264 
 .2198 
 .2088 
 .2360 
 .2198 
 .2188 
 .1554 
 .3020 
 .2298 
 
 0.0646 
 .1070 
 .1082 
 .0852 
 .0990 
 .1056 
 .1166 
 .0894 
 .1056 
 .1066 
 .1700 
 .0234 
 .0956 
 
 18.45 
 16.46 
 16.64 
 17.04 
 16.50 
 16.24 
 17.93 
 17.88 
 17.60 
 16.40 
 16.19 
 15.60 
 17.38 
 
 5.42 
 4.84 
 4.89 
 5.00 
 4.84 
 4.77 
 5.26 
 5.25 
 5.17 
 4.81 
 4.75 
 4.58 
 5.10 
 
 10 a.m. to 4 p.m 
 
 4 p.m. to 10''30" p.m .... 
 10''30™ p.m. to 5 a.m .... 
 
 5 a.m. to 10 a.m . 
 
 10 a.m. to 4 p.m 
 
 4 p.m. to lOHOP" p.m 
 
 10''30°' p.m. to 9 a.m 
 
 9 a.m. to 10'>30'" a.m 
 
 10''30°^ a.m. to 4 p.m 
 
 When, on the other hand, the leaves are given, besides sucrose, 
 a small amount of glycocoU, the course of the respiration rates is a 
 gradual rise. The changes in material are given in the analytical 
 data in table 36. 
 
 Table 36. 
 
 
 Dry weight. 
 
 Amino 
 
 nitrogen in 
 
 drj' material. 
 
 Total 
 
 sugars in 
 
 dry material. 
 
 Initial condition 
 
 After 76 hours in dark. . 
 
 V.ct. 
 15.49 
 14.11 
 
 p.ct. 
 
 0.086 
 
 .310 
 
 p.ct. 
 2.11 
 2.70 
 
 The rates of respiration are given in table 37 and figure 15. From 
 these it is apparent that after an initial drop in the rate of CO2 
 emission, which probably represents the time required for the 
 material in the nutrient solution to migrate into the leaves, there is 
 an irregular rise which becomes pronounced after about 35 hours. 
 The principal difference between the leaves which have been given 
 glycocoU and those without is that in the former there is a marked 
 rise in the rate immediately after the initial drop. This is probably 
 
STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 57 
 
 attributable to the influence of the glycocoll from the nutrient 
 solution, while the rise after 35 hours, which is noticeable in both 
 experiments, represents the effect of the natural accumulation of 
 amino-acids in the leaves, which becomes noticeable even in those 
 leaves which have been given no sugar in the nutrient solution. 
 
 AO 50 60 70 90 
 
 Figure 15. 
 Rates of respiration with sucrose. The broken line indicates the rate of respiration 
 of 6 leaves of Helianihiis annuus at 25°; petioles in nitrogen-free nutrient solution con- 
 taining 7 per cent sucrose. Values taken from table 35. The soUd line indicates the 
 rate of respiraton of 6 leaves of the same plant at 25°; petioles in nitrogen-free nutrient 
 solution containing 7 per cent sucrose and 0.11 per cent glycocoll. Values taken from 
 table 37. The ordinate represents mg. CO2 per hour per gram dry material, the abscissa 
 time in hours. 
 
 5. d-levulose. 
 
 As was pointed out in the introductory discussion, the purely 
 chemical experiments of Nef, as well as the physiological studies of 
 Lusk, point to the conclusion that d-levulose is more easily oxidized 
 than any of the other hexose sugars. Among plants, particularly 
 the lower ones, there is a great diversity in the capacity for using 
 different sugars. There exists little information on the relative 
 value of the various sugars in respect to respiratory activity of 
 higher plants. Palladin^ attempted to determine the influence of 
 various sugars on the rate of respiration. He found that d-levulose 
 produced a greater carbon-dioxid emission than either d-glucose, 
 sucrose, maltose, raffinose, glycerine, or mannit. However, Palladin 
 worked with etiolated bean shoots. These were placed in solutions 
 of the various sugars for 2 to 4 days and then the rate of respiration 
 
 1 Palladin, W. Rev. Gen. de BoL, 13, 19, 93, 127 (1901). 
 
58 
 
 STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 was determined during a few hours. The results from these experi- 
 ments can, of course, not be taken as giving definite results as to 
 the use in respiration of the various sugars employed. First of all, 
 no account is taken of the possible conversion of the sugars in the 
 leaf during the time the sprouts were in the sugar solution. Also, 
 the length of time during which respiration determinations were 
 made was entirely too short to gain any conclusive idea as to the 
 true rate of this process, and it is very questionable whether it is 
 justifiable to draw conclusions as to the normal behavior of plants 
 from a study of etiblated shoots. Finally, Palladin carried out his 
 respiration determinations with levulose in diffuse light, which is 
 another disturbing factor. 
 
 Such evidence as is now available seems to point to the conclusion 
 that in the plant, unlike the animal, d-levulose is not the most 
 easily oxidized sugar. Brown and Morris^ made comparative 
 analyses of the sugars in Troposolum majus. Two sets of excised 
 leaves were used; the one was analyzed immediately, the other 
 after having remained in the dark for 24 hours with the petioles 
 standing in water. Thus they found: 
 
 Table 37. — Rate of emission of CO2 by 6 leaves of Helianihus annuus at 25°. 
 Petioles in nitrogen-free nutrient solution containing 7 per cent sucrose and 0.11 per cent 
 glycocoU. CO2 absorbed in Ba(0H)2 solution 0.11835 normal, 125 c. c. of which has the equiva- 
 lent of 0.3254 gram CO2. 
 
 No. 
 
 Time. 
 
 HrB. 
 
 Total 
 hours. 
 
 Observed 
 resist- 
 ance in 
 ohms. 
 
 Gram CO2 
 
 equivalent 
 of 125 c. c. 
 Ba(0H)2 
 solution. 
 
 Gram 
 CO2 ab- 
 sorbed. 
 
 Mg. CO2 
 per 
 hour. 
 
 Mg. CO2 
 
 per hour 
 per gram 
 
 dry 
 weight. 
 
 
 
 1 
 
 2 
 
 3 
 
 4 
 
 5 
 
 6 
 
 7 
 
 8 
 
 9 
 
 10 
 
 11 
 
 12 
 
 13 
 
 
 1.0 
 3.0 
 6.5 
 6.5 
 5.0 
 6.0 
 6.5 
 6.5 
 5.0 
 6.0 
 6.5 
 6.5 
 5.0 
 6.0 
 
 1.0 
 4.0 
 10.5 
 17.0 
 22.0 
 28.0 
 34.5 
 41.0 
 46.0 
 52.0 
 58.5 
 65.0 
 70.0 
 76.0 
 
 
 
 
 
 
 1 p.m. to 4 p.m 
 
 59.6 
 67.1 
 69.8 
 66.3 
 70.1 
 70.6 
 71.5 
 68.3 
 73.8 
 73.8 
 74.7 
 69.5 
 73.9 
 
 0.2856 
 .2500 
 .2400 
 .2534 
 .2384 
 .2366 
 .2334 
 .2456 
 .2256 
 .2256 
 .2224 
 .2406 
 .2250 
 
 0.0398 
 .0754 
 .0854 
 .0720 
 .0870 
 .0888 
 .0920 
 .0798 
 .0998 
 .0998 
 .1030 
 .1848 
 .1004 
 
 13.26 
 11.60 
 13.13 
 14.40 
 14.50 
 13.66 
 14.15 
 15.96 
 16.63 
 15.35 
 15.84 
 16.96 
 16.73 
 
 3.84 
 3.36 
 3.80 
 4.17 
 4.20 
 3.95 
 4.10 
 4.62 
 4.81 
 4.44 
 4.58 
 4.91 
 4.84 
 
 4 p.m. to lOi'SO" p.m 
 
 lO'^aO™ p.m. to 5 a.m 
 
 5am to 10 a.m 
 
 10 a.m. to 4 p.m 
 
 4 p.m. to lO'^SO™ p.m 
 
 10'»30° p.m. to 5 a.m 
 
 10 a.m. to 4 p.m 
 
 4 p.m. to 10''30" p.m 
 
 10^30" p.m. to 5 a.m 
 
 5 a.m. to 10 a.m 
 
 10 a.m. to 4 p.m 
 
 Evidently there is a considerable loss of carbohydrates due to 
 respiration, as well as a decided change in the relative amounts of 
 the various sugars. In all probability the starch was converted into 
 maltose, which yields dextrose, and the cane sugar was inverted to 
 
 > Brown, H. T., and G. H. Morris. Jour. Chem. Soc. London, 63, 671 (1893). 
 
STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 59 
 
 equal quantities of dextrose and levulose. There was thus un- 
 doubtedly a preponderance of dextrose. Nevertheless, there was a 
 much greater increase of levulose than of dextrose during the 24 
 
 Table 38. 
 
 
 Leaves analyzed 
 at once. 
 
 Leaves kept in 
 dark 24 hours. 
 
 Experiment No . . 
 Starch 
 
 III. 
 3.693 
 9.98 
 0.00 
 1.41 
 2.25 
 
 IV. 
 5.425 
 7.33 
 0.00 
 2.11 
 2.71 
 
 III. 
 
 2.980 
 
 3.49 
 
 0.53 
 
 3.46 
 
 1.86 
 
 IV. 
 0.906 
 3.35 
 1.34 
 3.76 
 1.28 
 
 Cane sugar 
 
 Dextrose 
 
 Levulose 
 
 Maltose 
 
 hours the leaves were in the dark. Brown and Morris calculate 
 from these results that far more dextrose than levulose is used in 
 the respiratory process, as is shown in table 39. 
 
 Table 39. — Stigars used up in respiration 
 expressed as percentages of the dry-leaf 
 material. 
 
 
 Expt. III. 
 
 Expt. IV. 
 
 Dextrose 
 
 Levulose 
 
 Maltose 
 
 2.66 
 1.19 
 1.10 
 
 0.65 
 0.34 
 5.94 
 
 The work of Parkin* substantiates the conclusions of Brown and 
 Morris. Working with Galanthus nivalis, the leaves of which do 
 not store starch, Parkin found that levulose, as a rule, is in excess 
 of glucose, irrespective of the time of day the leaves are taken for 
 analysis. 
 
 "Out of 52 duplicate leaf analyses made, 47 had the fructose in excess of the glucose, 
 and only 7 the reverse. Representing fructose as unity, in the former cases the ratio 
 varied from 1 : 0.4 to 1 : 0.76, and in the latter from 1 : 1.01 to 1 : 1.06. The calculation 
 of the separate amounts of glucose and fructose depends chiefly upon one observation, 
 viz, the optical angle of rotation. A slight error in the reading of this will affect 
 the results considerably; consequently it might hardly be expected that any further 
 conclusion beyond the bare fact of the excess of one hexose sugar over the other 
 could be reached." 
 
 A later investigation of the carbohydrates of foUage leaves by 
 Gast^ yielded very similar results. Tropceolum niojus and Vitis 
 vinifera during 24 hours of respiration in the dark showed a rela- 
 
 1 Parkin, J. Biochem. Jour., 6, 1-47 (1911). 
 
 2 Gast, W. ZeU. physiol. chem., 99, 1-53 (1917). 
 
60 
 
 STUDIES IN PLANT EESPIRATION AND PHOTOSYNTHESIS. 
 
 lively greater decrease of glucose than of levulose. While Musa 
 ensente Gruel, which forms very little starch, showed a decrease in 
 cane sugar and maltose during the period of darkness, glucose in- 
 creased and levulose remained the same. The results with Cucurbita 
 ficifolia Bet6 and with Canna viridiflora R. and Pav. are not as definite, 
 
 Table 40. 
 
 
 Dry weight. 
 
 Amino 
 
 nitrogen in 
 
 dry material. 
 
 Total 
 
 sugars in 
 
 dry material. 
 
 Initial condition 
 
 p.ct. 
 16.21 
 14.95 
 
 p.ct. 
 
 0.144 
 
 .552 
 
 p.ct. 
 1.88 
 1.60 
 
 After 76.75 hours in dark . . . 
 
 but seem also to indicate that in the course of respiration more of 
 the glucose is utilized. The expression of analytical data in terms 
 of percentages of the dry-leaf material makes uncertain the calcula- 
 tion of the amounts of the various sugars used up. 
 
 Table 41. — Rate of emission of CO2 by 9 leaves of Helianihus annuity at 25°. 
 Petioles in nitrogen-free nutrient solution containing 7 per cent levulose. CO2 absorbed in 
 Ba(0H)2 solution, 0.12153 normal, 125 c. c. of which has CO2 equivalent of 0.3343 gram CO2. 
 
 No. 
 
 Time. 
 
 Hrs. 
 
 Total 
 hours. 
 
 Observed 
 resist- 
 ance in 
 ohms. 
 
 Gram CO2 
 equivalent 
 of 125 c. c. 
 Ba(0H)2 
 solution. 
 
 Gram 
 CO2 ab- 
 sorbed. 
 
 Mg. CO2 
 
 per 
 
 hour. 
 
 Mg. CO2 
 per hour 
 per gram 
 
 dry 
 weight. 
 
 
 
 1 
 2 
 3 
 4 
 5 
 6 
 7 
 
 I 
 
 10 
 11 
 12 
 13 
 
 11^15^ a.m. to 12i'15°> p.m. 
 
 12»'15°' p.m. to 4 p.m 
 
 4 p.m. to 10''30°^ p.m 
 
 IQi^SO" p.m. to 5 a.m 
 
 1.0 
 
 3.75 
 
 6.5 
 
 6.5 
 
 5.0 
 
 6.0 
 
 6.5 
 
 6.5 
 
 5.0 
 
 6.0 
 
 6.5 
 
 6.5 
 
 5.0 
 
 6.0 
 
 1.0 
 4.75 
 11.25 
 17.75 
 22.75 
 28.75 
 35.25 
 41.75 
 46.75 
 52.75 
 59.25 
 65.75 
 70.75 
 76.75 
 
 
 
 
 
 
 65.3 
 74.1 
 76.9 
 68.2 
 73.2 
 74.0 
 76.3 
 69.0 
 75.0 
 75.6 
 75.8 
 68.3 
 74.4 
 
 0.2548 
 .2214 
 .2122 
 .2426 
 .2246 
 .2218 
 .2140 
 .2396 
 .2184 
 .2164 
 .2160 
 .2422 
 .2238 
 
 0.0795 
 .1129 
 .1221 
 .0917 
 .1097 
 .1125 
 .1203 
 .0947 
 .1159 
 .1179 
 .1183 
 .0921 
 .1105 
 
 21.20 
 17.36 
 18.78 
 18.35 
 18.30 
 17.30 
 18.50 
 18.94 
 19.31 
 18.13 
 18.20 
 18.42 
 18.41 
 
 5.500 
 4.503 
 4.871 
 4.760 
 4.747 
 4.487 
 4.799 
 4.913 
 5.009 
 4.703 
 4.721 
 4.778 
 4.775 
 
 10 a.m. to 4 p.m 
 
 4 p.m. to 10^30°^ p.m 
 
 10''30'° p.m. to 5 a.m. . . . 
 
 5 a.m. to 10 a.m 
 
 10 a.m. to 4 p.m 
 
 4 p.m. to lO'^SO" p.m. . . . 
 10''30°> p.m. to 5 a.m 
 
 10 a.m. to 4 p.m 
 
 While the evidence is as yet by no means conclusive, it appears 
 that levulose in leaves is more stable than glucose. The question 
 resolves itself into whether d-glucose is actually more easily con- 
 sumed in the leaf or whether d-levulose is, under certain conditions, 
 converted into d-glucose. The problem of the glucose-fructose 
 relation is a very fundamental one in the carbohydrate economy of 
 
STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 61 
 
 plants. Its solution is associated with the extraordinary difficulty 
 of quantitatively determining the various sugars in leaves which 
 have been subjected to a series of experimental conditions. 
 
 Table 42. 
 
 
 Dry weight. 
 
 Amino 
 
 nitrogen in 
 
 dry material. 
 
 Total 
 
 sugars in 
 
 dry material. 
 
 Initial condition 
 
 After 76.25 hours in dark. . . 
 
 p.ct. 
 15.91 
 13.85 
 
 p.ct. 
 
 0.190 
 
 .615 
 
 p.ct. 
 1.969 
 1.563 
 
 Unfortunately the results of experiments which we have carried 
 out with this problem in view had to be discarded on account of 
 some uncertainties which developed later in our analytical methods. 
 However, the investigation of this phase of the carbohydrate econ- 
 omy problem is to be continued. 
 
 Table 43. — Rate of emission of CO2 hy 7 leaves of Helianihus annuus nl 25°. 
 Petioles in nitrogen-free nutrient solution containing 7 per cent d-levulose and 0. 1 1 per cent 
 glycocoll. CO2 absorbed in Ba(0H)2 solution, 0.11835 normal, 125 c. c. of which has the CO2 
 equivalent of 0.3254 gram CO2. 
 
 No. 
 
 Time. 
 
 Hrs. 
 
 Total 
 hours. 
 
 Observed 
 resist- 
 ance in 
 ohms. 
 
 Gram CO2 
 equivalent 
 of 125 c. c. 
 Ba(0H)2 
 solution. 
 
 Gram 
 CO2 ab- 
 sorbed. 
 
 Mg. CO2 
 per 
 hour. 
 
 Mg. CO2 
 per hour 
 per gram 
 
 dry 
 weight. 
 
 
 
 1 
 
 2 
 
 3 
 
 4 
 
 5 
 
 6 
 
 7 
 
 8 
 
 9 
 
 10 
 
 11 
 
 12 
 
 13 
 
 11>'45"' a.m. to 1 p.m... 
 
 . 1.25 
 . 3.0 
 . 6.5 
 . 6.5 
 . 5.0 
 . 6.0 
 . 6.5 
 . 6.5 
 . 5.0 
 . 6.0 
 . 6.5 
 . 6.5 
 . 5.0 
 . 6.0 
 
 1 25 
 
 
 
 
 
 
 4.25 
 10.75 
 17.25 
 22.25 
 28.25 
 34.75 
 41.25 
 46.25 
 52.25 
 58.75 
 65.25 
 70.25 
 76.25 
 
 64.4 
 74.3 
 76.6 
 68.7 
 71.7 
 72.8 
 74.0 
 69.0 
 73.9 
 73.6 
 74.3 
 69.0 
 74.2 
 
 0.2620 
 .2238 
 .2160 
 .2438 
 .2332 
 .2290 
 .2246 
 .2426 
 .2250 
 .2264 
 .22.38 
 .2426 
 .2242 
 
 . 0634 
 .1016 
 .1094 
 .0816 
 .0922 
 .0964 
 .1008 
 .0828 
 .1004 
 .0990 
 .1016 
 .0828 
 .1012 
 
 21.13 
 15.63 
 16.83 
 16.32 
 15.36 
 14.83 
 15.50 
 16.56 
 16.73 
 15.23 
 15.63 
 16.56 
 16.86 
 
 6.00 
 4.44 
 4.78 
 4.64 
 4.36 
 4.21 
 4.40 
 4.71 
 4.75 
 4.33 
 4.44 
 4.71 
 4.79 
 
 4 p.m. to 10^30™ p.m . . . 
 10^30'" p.m. to 5 a.m. . . 
 
 10 a.m. to 4 p.m 
 
 4 p.m. to 10''30'° p.m . . . 
 10'»30" p.m. to 5 a.m . . . 
 
 5 a.m. to 10 a.m . ... 
 
 10 a.m. to 4 p.m 
 
 4 p.m. to 10'^30°' p.m. . . 
 10^30"' p.m. to 5 a.m... 
 
 5 a.m. to 10 a.m 
 
 10 a.m. to 4 p.m 
 
 In the three following experiments d-levulose was used as the 
 sugar in the nutrient solution. The first of these was carried out 
 with d-levulose as the only organic substance in the nutrient solu- 
 tion. The analytical data of the changes of materials in the leaves 
 are given in table 40. 
 
 These leaves also showed the usual gain in amino-acids after 
 remaining 76.75 hours in the dark. There was, however, a decrease 
 
62 
 
 STUDIES IN PLANT RESPIBATION AND PHOTOSYNTHESIS. 
 
 in the carbohydrate-content, although d-levulose is notably a good 
 nutrient for starch-forming plants. The rates of respiration are 
 given in table 41 and figure 16. 
 
 Table 44. 
 
 
 Dry weight. 
 
 Amino 
 
 nitrogen in 
 
 dry material. 
 
 Total 
 
 sugars in 
 
 dry material. 
 
 Original condition 
 
 After 77 hours in dark. 
 
 p.ct. 
 18.31 
 14.79 
 
 p. ct. 
 0.157 
 
 .480 
 
 p.ct. 
 2.16 
 1.69 
 
 The preceding experiment was repeated, all conditions remaining 
 the same, except that the nutrient solution contained, besides the 
 d-levulose, 0.11 per cent of glycocoll. The analyses of the leaves 
 are compiled in table 42. 
 
 Table 45. — Rate of emission of CO2 hy 7 leaves of Helianthus annuus at 25°. 
 Petioles in nitrogen-free nutrient solution containing 7 per cent d-levulose and 0.138 per cent 
 asparagine. CO2 absorbed in Ba(0H)2 solution. Nos. 1 to 8, inclusive, in solution 0.11835 
 normal, 125 c. 0. of which has the CO2 equivalent of 0.3254 gram CO2. Nos. 9 to 13 in solution 
 0.12153 normal, 125 c. c. of which has the CO2 equivalent of 0.3343 gram, CO2. 
 
 No. 
 
 Time. 
 
 Hrs. 
 
 Total 
 hours. 
 
 Observed 
 resist- 
 ance in 
 ohms. 
 
 Gram CO2 
 equivalent 
 of 125 c. c. 
 Ba(0H)2 
 solution. 
 
 Gram 
 CO2 ab- 
 sorbed. 
 
 Mg. CO2 
 per 
 hour. 
 
 Mg. CO2 
 per hour 
 per gram 
 
 dry 
 weight. 
 
 
 
 1 
 2 
 3 
 4 
 5 
 6 
 7 
 8 
 9 
 10 
 11 
 12 
 13 
 
 lO'^SO™ a.m. to 12 m 
 
 1.5 
 4.0 
 6.5 
 6.5 
 5.0 
 6.0 
 6.5 
 6.5 
 5.0 
 6.0 
 6.5 
 6.5 
 5.0 
 6.0 
 
 1.5 
 5.5 
 12.0 
 18.5 
 23.5 
 29.5 
 36.0 
 42.5 
 47.0 
 53.0 
 59.5 
 66.0 
 71.0 
 77.0 
 
 
 
 
 
 
 73.0 
 79.9 
 81.5 
 70.5 
 74.5 
 74.6 
 73.9 
 68.7 
 70.5 
 71.2 
 73.1 
 69.3 
 76.0 
 
 0.2282 
 .2056 
 .2010 
 .2368 
 .2232 
 .2226 
 .2250 
 .2438 
 .2366 
 .2346 
 .2280 
 .2414 
 .2182 
 
 0.0972 
 .1198 
 .1244 
 .0886 
 .1022 
 .1028 
 .1004 
 .0816 
 .0977 
 .0997 
 .1063 
 .0929 
 .1161 
 
 24.30 
 18.43 
 19.13 
 17.72 
 17.03 
 15.81 
 15.44 
 16.32 
 16.28 
 15.33 
 16.35 
 18.58 
 19.35 
 
 5.521 
 
 4.187 
 4.346 
 4.026 
 3.869 
 3.592 
 3.508 
 3.708 
 3.700 
 3.483 
 3.715 
 4.222 
 4.396 
 
 4 p.m. to 10''30™ p.m. . . . 
 IQi'SO™ p.m. to 5 a.m 
 
 5 a.m. to 10 a.m. . .... 
 
 10 a.m. to 4 p.m 
 
 4 p.m. to lO^ZQT p.m 
 
 10''30°^ p.m. to 5 a.m 
 
 10 a.m. to 4 p.m 
 
 4 p.m. to 10'»30" p.m 
 
 10''30" p.m. to 5 a.m 
 
 5 a.m. to 10 a.m 
 
 10 a.m. to 4 p.m 
 
 The rates of respiration for this experiment are given in table 43 
 and in the graphs in figur.e 16. 
 
 The experiment was again repeated, the glycocoll being replaced 
 in this instance by asparagine. The analytical data are presented 
 in table 44. 
 
 The rates of respiration are shown in table 45 and in the graphs 
 in figure 16. 
 
STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 63 
 
 A comparison of the results of the foregoing three experiments 
 as presented in jfigure 16 shows at once that apparently d-levulose 
 does not produce an increased respiratory activity in Helianthus 
 leaves. Also, the graphs of the rates of respiration differ considera- 
 bly from the experiments in which d-glucose and sucrose were fed 
 the leaves. Of these sugars, d-levulose seems to produce the least 
 active respiratory activity and d-glucose the most active, while 
 sucrose lies midway between the two. In none of the experiments 
 
 J I L 
 
 to 20 30 4C SO CO 70 80 
 
 Figure 16. 
 Rates of respiration with d-levulose. The dotted line indicates the rate of respira- 
 tion of leaves of Helianthus annuus at 25°; petioles in a nitrogen-free nutrient solution 
 containing 7 per cent d-levulose; values from table 41. The broken line indicates the 
 rate of respiration at 25° with a nitrogen-free solution containing 7 per cent d-levulose 
 and 0.11 per cent glycocoU, as per table 43. The soHd line indicates the rate of respi- 
 ration at 25° with a nitrogen-free nutrient solution containing 7 per cent d-levulose and 
 0.138 per cent asparagine. The ordinates represent mg. CO2 per hour per gram dry 
 material , and the abscissa the time in hours. 
 
 with Helianthus leaves in which these sugars alone were given was 
 the initial sugar-content maintained. However, the reduction in 
 all these cases was about the same, as was also the approximate 
 increase in relative water-content. Nevertheless, these sugars show 
 decided variation in their effect on respiratory activity. Moreover, 
 the influence of adding amino-acids to the nutrient solutions con- 
 taining either d-glucose, sucrose, or d-levulose is very different. 
 With d-glucose the effect is marked, particularly before the fortieth 
 
64 
 
 STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 hour of the experiment with the more easily penetrating glj^cocoll 
 and asparagine. With d-levulose no stimulating effect can be 
 noticed in our experiments; in fact the respiration rates in the 
 experiments with glycocoU or asparagine and d-levulose are relatively 
 below those with this sugar alone during the first half of the time. 
 
 6. d-mannose. 
 
 A sugar which is closely related in structure to both d-glucose 
 and d-levulose is d-mannose. These three sugars have a common 
 enol and are easily converted into each other. ^ Aside from its 
 rather common occurrence as an anhydride, mannan, in seed coats 
 and similar organs, the physiological behavior of d-mannose in 
 plants has been investigated very little. Knudson^ found that 
 d-mannose has a decidedly toxic action on the root tips of wheat 
 and peas grown in sterile agar cultures. 
 
 We carried out but two complete experiments with d-mannose, 
 one without and the other with glycocoU in the nutrient solution. 
 The results were quite surprising in that there was no toxic action 
 apparent and that the rates of respiration developed a very great 
 increase over the initial rate. 
 
 In table 46 the analytical data are given representing the changes 
 in material in leaves which have been given d-mannose. 
 
 Table 46. 
 
 
 Dry weight. 
 
 Amino 
 
 nitrogen in 
 
 dry material. 
 
 Total 
 
 sugars in 
 
 dry material. 
 
 
 p. a. 
 
 14.68 
 13.64 
 
 p.ct. 
 0.153 
 0.788 
 
 p.ct. 
 1.24 
 0.95 
 
 After 77.5 hours in dark .... 
 
 The results of the determination of respiration-rates for this experi- 
 ment are given in table 47 and figure 17. 
 
 The foregoing experiment was repeated with the addition of 0.11 
 per cent of glycocoU to the nutrient solution. The analytical data 
 are given in table 48. 
 
 The results of the determination of the respiration rates are com- 
 piled in table 49 and figure 17. 
 
 The experiments with d-mannose are noteworthy on account of 
 the great increase in amino-acids in both cases. Although the 
 petioles of the leaves were standing in a 7 per cent solution of 
 d-mannose, there was a very appreciable decrease in the total sugar- 
 
 1 Spoehh, H. a. The carbohydrate economy of cacti. Carnegie Inst. Wash. Pub. No. 287 
 
 (1919). 
 
 2 Knudbon, L. Amer. Jour, of BoL, 4, 430-437 (1917). 
 
STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 65 
 
 content of the leaves. Nevertheless, the respiration rates at the 
 end of the experiment show a remarkable increase over the initial 
 rates, although the latter were relatively not low. There is here 
 the same phenomenon, exhibited by d-glucose in figure 11, of an 
 
 Table 47. — Rate of emission of CO2 by 8 leaves of Helianthus annum at 25°. 
 Petioles in nitrogan-free nutrient solution containing 7 per cent mannose. CO 2 absorbed 
 in Ba(0H)2 solution 0.12153 normal, 125 c. c. of which has the CO2 equivalent of 0.3343 g. CO2. 
 
 No. 
 
 Time. 
 
 Hrs. 
 
 Total 
 hours. 
 
 Observed 
 resist- 
 ance in 
 ohms. 
 
 Gram CO2 
 equivalent 
 of 125 c. c. 
 Ba(0H)2 
 solution. 
 
 Gram 
 CO2 ab- 
 sorbed. 
 
 Mg. CO2 
 per 
 hour. 
 
 Mg. CO2 
 per hour 
 per gram 
 
 dry 
 weight. 
 
 
 
 1 
 
 2 
 
 3 
 
 4 
 
 5 
 
 6 
 
 7 
 
 8 
 
 9 
 
 10 
 
 11 
 
 12 
 
 13 
 
 10*^30™ a.m. to 12 m 
 
 1.5 
 4.0 
 6.5 
 6.5 
 5.0 
 6.0 
 6.5 
 6.5 
 5.0 
 6.0 
 6.5 
 6.5 
 5.0 
 6.0 
 
 1.5 
 5.5 
 12.0 
 18.5 
 23.5 
 29.5 
 36.0 
 42.5 
 47.5 
 53.5 
 60.0 
 66.5 
 71.5 
 77.5 
 
 
 
 
 
 
 
 
 64.1 
 72.7 
 76.4 
 68.1 
 71.9 
 75.1 
 76.1 
 71.0 
 79.6 
 83.2 
 87.0 
 79.3 
 99.0 
 
 0.2598 
 .2262 
 .2140 
 .2428 
 .2294 
 .2180 
 .2148 
 .2326 
 .2040 
 .1944 
 .1854 
 .2048 
 .1622 
 
 0.0745 
 .1081 
 .1203 
 .0915 
 .1049 
 .1163 
 .1195 
 .1017 
 .1303 
 .1399 
 .1489 
 .1295 
 .1721 
 
 18.62 
 16.63 
 18.50 
 18.30 
 17.48 
 17.89 
 18.38 
 20.32 
 21.71 
 21.52 
 22.90 
 25.90 
 28.08 
 
 5.540 
 4.947 
 5.504 
 5.444 
 5.200 
 5.322 
 5.468 
 6.045 
 6.459 
 6.402 
 6.813 
 7.706 
 8.533 
 
 4 p.m. to 1U''30™ p.m 
 10''30'" p.m. to 5 a.m 
 
 
 
 
 4 p.m. to 10^30" p.m 
 lO^'SO" p.m. to 5 a.m 
 
 5 a.m. to 10 a.m . . 
 
 
 10 a.m. to 4 p.m .... 
 
 
 4 p.m. to 10''30'" p.m 
 10''30™ p.m. to 5 a.m 
 5 a.m. to 10 a.m 
 
 
 10 a.m. to 4 p.m .... 
 
 
 
 increased respiration rate with decreased total sugar-content and 
 increased amino-acids. The sugar-content alone is no index of the 
 rate at which the leaves utilize this material. Moreover, under the 
 conditions of the experiment, where sugar is being constantly sup- 
 
 Table 48. 
 
 
 Dry weight. 
 
 Amino 
 
 nitrogen in 
 
 dry material. 
 
 Total 
 
 sugars in 
 
 dry material. 
 
 Initial condition 
 
 p.ct. 
 15.00 
 13.29 
 
 p.ct. 
 0.252 
 0.741 
 
 1.18 
 0.72 
 
 After 76.75 hours in dark . . . 
 
 plied, it is quite conceivable that this is oxidized as rapidly as it is 
 taken up. This state of affairs must, of course, also frequently 
 exist under natural conditions. But the increased rate of respiration 
 at the end of the experiment can not be due to a greater supply of 
 sugar, but to the cooperation of some other factor which increases 
 its activity with time. The increase in amino-acids under the 
 circumstances is more than a coincidence and plays an important 
 
60 
 
 STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 role in the respiratory activity. In the experiments with d-mannose 
 the glycocoU which was given in the nutrient solution had apparently 
 very little effect, as the normal accumulation of amino-acids in the 
 leaf in this case was so very great. There must, of course, also be 
 Umits beyond which there is little increase in amino-acids and above 
 which the rate of respiration does not rise, so that further addition 
 of an amino-acid would be of little consequence. 
 
 Table 49. — Rate of emission of 8 leaves of Helianthus annuus at 25°. 
 Petioles in nitrogen-free nutrient solution containing 7 per cent d-mannose -f- 0.11 per cent 
 glycocoU. CO2 absorbed in Ba(0H)2 solution 0.12153 normal, 125 c. c. of which has the CO2 
 equivalent of 0.3343 gram CO2. 
 
 No. 
 
 Time. 
 
 Hrs. 
 
 Total 
 hours. 
 
 Observed 
 resist- 
 ance in 
 ohms. 
 
 Gram CO2 
 equivalent 
 of 125 c. c. 
 Ba(0H)2 
 solution. 
 
 Gram 
 CO2 ab- 
 sorbed. 
 
 Mg. CO2 
 per 
 hour. 
 
 Mg. CO2 
 per hour 
 per gram 
 
 dry 
 weight. 
 
 
 
 1 
 
 2 
 
 3 
 
 4 
 
 6 
 
 6 
 
 7 
 
 8 
 
 9 
 
 10 
 
 11 
 
 12 
 
 13 
 
 11>'15'" a.m. to 12i'15°> p.m. 
 12''15" p.m. to 4 p.m. . . . 
 
 4 p.m. to 10''30°' p.m 
 
 10*'30'" p.m. to 5 a.m .... 
 
 1.0 
 
 3.75 
 
 6.5 
 
 6.5 
 
 5.0 
 
 6.0 
 
 6.5 
 
 6.5 
 
 5.0 
 
 6.0 
 
 6.5 
 
 6.6 
 
 5.0 
 
 6.0 
 
 1.0 
 4.75 
 11.25 
 17.75 
 22.75 
 28.75 
 35.25 
 41.75 
 46.75 
 52.75 
 59.25 
 65.75 
 70.75 
 76.75 
 
 
 
 
 
 
 61.3 
 67.1 
 71.1 
 66.3 
 68.0 
 70.0 
 69.7 
 65.2 
 69.4 
 72.0 
 76.4 
 73.0 
 90.0 
 
 0.2730 
 .2468 
 .2320 
 .2493 
 .2432 
 .2358 
 .2370 
 .2552 
 .2380 
 .2288 
 .2138 
 ,2254 
 .1786 
 
 0.0613 
 .0875 
 .1023 
 .0850 
 .0911 
 .0985 
 .0972 
 .0791 
 .0963 
 .1055 
 .1205 
 .1089 
 .1557 
 
 16.34 
 13.46 
 15.73 
 17.00 
 15.18 
 15.15 
 15.00 
 15.82 
 16.05 
 16.23 
 18.53 
 21.78 
 25.95 
 
 4.932 
 4.036 
 4.748 
 5.131 
 4.582 
 4.573 
 4.528 
 4.775 
 4.845 
 4.899 
 5.593 
 6.574 
 7.833 
 
 10 a.m. to 4 p.m 
 
 4 p.m. to 10''30°' p.m 
 
 10*^30" p.m. to 5 a.m 
 
 5am to 10 a m 
 
 10 a.m. to 4 p.m 
 
 4 p.m. to 10''30'° p.m 
 
 10''30™ p.m. to 5 a.m .... 
 
 5 a.m. to 10 a.m 
 
 10 a.m. to 4 p.m 
 
 7. Effect of the Natural Increase in Amino- Acids. Influence of Light 
 on Amino-Acids and Effect on Respiration. 
 
 In the experiments which have been described attention has 
 repeatedly been called to the rise in the rates of respiration after 
 about 35 or 40 hours. An examination of the graphs shows this 
 rise in practically all of the experiments, and particularly is it 
 noticeable in the cases in which no amino-acid was contained in 
 the solution. It is perhaps unnecessary to point out that these 
 graphs do not represent the true course of respiratory activity, for 
 the points in the curves represent the rates of carbon-dioxid emission 
 during a certain period, usually 6 hours. Naturally such an experi- 
 mental procedure makes the variations appear much more abrupt 
 than in all probability they are, so that the rise in respiration rate 
 between the thirty-fifth and fortieth hour must be regarded with the 
 foregoing in view. 
 
 When excised Helianthus leaves are kept in the dark, with the 
 petioles in nitrogen-free nutrient solution (table 16), there is a gradual 
 
STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 67 
 
 rise in the amino-acid content of the leaves. Our experiments indi- 
 cate that amino-acids have a stimulating effect on the respiratory- 
 activity of leaves containing the natural sugars, or when they are fed 
 d-glucose or sucrose. It seems permissible to assume that the amino 
 acids which accumulate in the leaves when kept in the dark would 
 exert a similar stimulating effect if the leaves contained sufficient 
 
 40 50 60 70 80 
 
 Figure 17. 
 Rates of respiration with d-mannose. The broken line indicates the rate of res- 
 piration of 8 leaves of Helianthus at 25°; petioles in a nitrogen-free nutrient solution 
 containing 7 per cent d-mannose, values taken from table 47. The solid line indicates 
 the rate of respiration of 8 leaves of Helianthus at 25° with petioles in a nitrogen-free 
 nutrient solution containing 7 per cent d-mannose and 0.11 per cent glycocoU, values 
 taken from table 49. The ordinate represents mg. CO2 per hour per gram dry material, 
 the abscissa the time in hours. 
 
 sugar. Normally, of course, when excised leaves are kept in the 
 dark, besides an accumulation of amino-acids there is a rapid reduc- 
 tion of the sugar-content, so that any stimulating effect of the 
 accumulating amino-acids could not be exerted on account of the 
 reduced fuel material. 
 
 If, however, after the leaves have remained in the dark for a 
 time and the amino-acids have accumulated, glucose is then fed, the 
 
68 
 
 STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 effect of this increased amino-acid content becomes noticeable at 
 once. Thus excised leaves which had been kept in the dark for about 
 40 hours and then given d-glucose exhibited an immediate increase 
 in the rate of carbon-dioxid emission. This is not the case when 
 leaves are given d-glucose immediately after cutting from the plant; 
 there is then a primary drop in the CO2 curve and a rise after about 40 
 hours (see figs. 10 and 11). The combination, then, of the accumu- 
 lated amino-acids after 40 hours of darkness and an abundant sugar 
 supply results in an accelerated rate of respiration. 
 
 Table 50. — Rate of CO2 emission, at 24°, of 8 leaves of Helianthus annuus. 
 The excised leaves were placed in a nutrient solution free from organic naaterial and nitrogen 
 
 and kept in the dark for 43.5 hours. They were then transferred to a nitrogen-free nutrient 
 solution containing 7 per cent d-glucose and the rates of respiration determined. 
 
 No. 
 
 Time. 
 
 Hours. 
 
 Total 
 hours. 
 
 Cubic 
 centimeters 
 0.1/NHCl. 
 
 Mg. COo 
 per hour. 
 
 Mg. CO, per 
 hour per gram 
 diy material. 
 
 1 
 2 
 3 
 4 
 5 
 6 
 7 
 8 
 9 
 10 
 11 
 
 11''30'" a.m. to 12''12" p.m. . 
 
 12''15'" p.m. to 5 p.m 
 
 5 p.m. to 8''3CF' p.m 
 
 8^30"^ p.m. to 9 a.m 
 
 0.75 
 4.75 
 3.50 
 
 12.50 
 6.00 
 6.75 
 
 11.75 
 6.75 
 5.50 
 
 11.50 
 6.00 
 
 0.75 
 5.50 
 9.00 
 21.50 
 27.50 
 34.25 
 46.00 
 52.75 
 58.25 
 69.75 
 75.75 
 
 
 
 
 27.65 
 22.25 
 84.50 
 51.00 
 56.20 
 85.80 
 54.65 
 43.20 
 87.85 
 56.45 
 
 12.80 
 13.99 
 14.85 
 18.70 
 18.26 
 16.19 
 17.60 
 17.18 
 17.01 
 20.68 
 
 2.387 
 2.608 
 2.768 
 3.486 
 3.404 
 3.018 
 3.283 
 3.203 
 3.171 
 3.855 
 
 3 p.m. to 9^45'" p.m 
 
 9''45-" p.m. to 9^30™ a.m 
 
 9''30™ a.m. to 4^15°" p.m 
 
 4'>15°' p.m. to 9'^45'" p.m 
 
 gMS-" p.m. to 9''15'" a.m 
 
 gMS'^a.m. to3''15«'p.m 
 
 This fact becomes evident from the following experiment, in which 
 8 Helianthus leaves were cut from the plant, placed immediately 
 in nitrogen-free mineral nutrient solution, and kept in the dark. 
 After 43.5 hours the leaves were put in the respiration chamber 
 with fresh nutrient solution containing 7 per cent of d-glucose. 
 From the results of the determinations of the rates of carbon- 
 dioxid emission in table 50 and figure 18 it is apparent that the rate 
 of respiration rises immediately and, with some irregularities, then 
 attains its maximum. 
 
 It is, moreover, a noteworthy fact that when the foregoing experi- 
 ment is repeated in such a manner that the nutrient solution con- 
 tains, besides d-glucose, 0.11 per cent of glycocoll, there is practically 
 no difference in the rate of carbon-dioxid emission. In other words, 
 it appears that the natural accumulation of amino-acids is just as 
 effective in stimulating respiratory activity as when amino-acids 
 are fed to the leaves. It was shown in the previous experiments 
 that the influence of amino-acids when fed to the leaves was espe- 
 cially noticeable in the first 40 hours of the experiment, that is, before 
 the natural accumulation of amino-acids in the dark becomes eft"ective. 
 
STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 69 
 
 It might be argued that this rapid increase in the rate of respira- 
 tion of leaves, which had been previously left in the dark and then 
 given d-glucose, was due simply to the greater available supply of 
 
 Table 51. — Rate of CO2 einission, at S4°, of S leaves of Helianlhus annuus. 
 The excised leaves were placed in a nutrient solution free from organic material and nitrogen 
 and kept in the dark for 42.75 hours. They were then transferred to a nutiient solution con- 
 taining 7 per cent d-glucose and 0.11 per cent glycocoll and the rates of respiration determined. 
 
 No. 
 
 Time. 
 
 Hours. 
 
 Total 
 hours. 
 
 Cubic 
 centimeters 
 0.1/N NCI. 
 
 Mg. CO2 
 per hour. 
 
 Mg. CO2 per 
 hour per gram 
 dry material. 
 
 1 
 
 2 
 3 
 4 
 5 
 6 
 7 
 8 
 9 
 10 
 
 11''45"' a.m. to 12''30"' p.m. . . 
 
 12''30'" p.m. to 5 p.m 
 
 5 p.m. to 9*'45™ p.m 
 
 QMS'" p.m. to 11 a.m 
 
 11 a.m. to 9''30'" p.m 
 
 Qi'SO"" p.m. to 9 a.m 
 
 9 a.m. to 3''30™ p.m 
 
 3*'30'° p.m. to ll'>30"' p.m. . . 
 
 ll''30'° p.m. to 9'' IS'" a.m 
 
 ^HS^ a.m. to 3''15'" p.m 
 
 0.75 
 4.50 
 4.75 
 13.25 
 10.50 
 11.50 
 6.50 
 8.00 
 9.75 
 6.00 
 
 0.75 
 5.25 
 10.00 
 23.25 
 33.75 
 45.25 
 51.75 
 59.75 
 69.50 
 75.50 
 
 
 
 
 27.60 
 34.10 
 96.00 
 92.30 
 87.40 
 58.40 
 68.45 
 85.55 
 61.25 
 
 13.49 
 15.80 
 15.97 
 19.36 
 17.63 
 19.76 
 18.81 
 19.34 
 22.44 
 
 2.389 
 2.798 
 2.829 
 3.429 
 3.087 
 3.500 
 3. 332 
 3.408 
 3.975 
 
 sugar. In the experiments already described it has been pointed 
 out that the rate of respiration bears no direct relation to the 
 carbohydrate-supply. There are numerous instances in which an 
 increase of carbon-dioxid emission was obtained with a decreased 
 total sugar-content. In these cases the amino-acids had consistently 
 
 50 
 
 40 
 
 Figure 18. 
 The solid line indicates the rate of re.spiration at 25° of 8 leaves of Helianthus which 
 were kept in the dark for 43.5 hours previous to putting in a nitrogen-free nutrient solu- 
 tion containing 7 per cent d-glucose, as per table 49. The" broken line indicates the rate 
 of respiration at 25° of 8 similar leaves which were kept in the dark for 42.75 hours pre- 
 vious to putting in a nitrogen-free nutrient solution containing 7 per cent glucose and 
 0.11 per cent glycocoll, as per table 50. The ordinate represents mg. CO2 per hour per 
 gram dry material and the absci.ssa the time in hours. 
 
70 STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 increased. In table 27 and figure 12 is also given a case of decreased 
 respiration rate accompanied by increased sugar-content and de- 
 creased amino-acids. However, in table 12 were given the results 
 of the carbohydrate and amino-acid determinations of leaves from 
 plants which were kept in the dark, showing the gradual decrease 
 in carbohydrates and increase in amino-acids. When these plants 
 were again placed in the light the reverse process occurred; there 
 was an increase in sugars and a decrease in amino-acids. 
 
 Figure 19. 
 Rate of respiration subsequent to periods of illumination. Six leaves of Helianthus 
 at 25°; petioles in a nitrogen-free nutrient solution containing 7 per cent d-glucose. The 
 solid lines indicate the CO2 emission in the dark, the dotted lines indicate the periods 
 during which the leaves are illuminated. The ordinate represents mg. CO2 per hour 
 per gram dry material, the abscissa the time in hours. 
 
 There remains, therefore, to show the effect on the rate of respira- 
 tion of temporarily decreasing the amino-acids while the carbohy- 
 drate-content remains high. It has been shown that the effect of 
 Ught is to decrease the amino-acids. The following experiment was 
 carried out so that the leaves which from the beginning of the experi- 
 ment were fed d-glucose were illuminated for 6 to 7 hours after 
 having been in the dark for about 25 hours. Thus during the course 
 of the experiment there was a period of illumination of 7 hours 
 after the twenty-fifth hour, another period of illumination of 6 hours 
 after the fiftieth hour, and a third period of illumination after the 
 seventy-fourth hour. As a source of illumination there was used a 
 750-watt tungsten filament lamp at 40 cm. distance. The glass 
 cover of the respiration chamber was below the level of the water 
 in the thermostat. The results of this experiment are given in 
 table 52 and figure 19. 
 
 During the periods of illumination the amount of carbon dioxid 
 absorbed was very low. This would indicate that a large part of 
 
STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 71 
 
 the carbon dioxid of respiration was fixed through photosynthesis. 
 The carbohydrate-content of the leaves, at the end of the periods 
 of illumination, was therefore, in all probability, higher than at the 
 beginning of illumination. It must also be borne in mind that the 
 petioles of the leaves were in a 7 per cent d-glucose solution during 
 the entire experiment. 
 
 Table 52. — Rate of emission of CO2 by 6 leaves of Helianthus annuus at 25°; petioles in 
 nitrogen-free nutrient solviion containing 7 per cent d-glucose. 
 The leaves weie illuminated during periods Nos. 5, 10, and 15. CO2 absorbed in Ba(0H)2 
 solution 0.12170 normal, 125 c. c. of which has the equivalent of 0.3346 gram CO2. 
 
 No. 
 
 Time. 
 
 Hrs. 
 
 Total 
 hours. 
 
 Observed 
 resist- 
 
 Gram CO2 
 equivalent 
 of 125 c. c. 
 
 Gram 
 CO2 
 
 Mg. 
 CO2 
 
 Mg. CO2 
 per hour 
 per gram 
 
 
 
 
 ance in 
 ohms. 
 
 Ba(0H)2 
 
 absorbed. 
 
 per 
 hour. 
 
 dry 
 
 
 
 
 
 solution. 
 
 
 weight. 
 
 
 
 2''30'" p.m. to 3''30°> p.m. 
 
 1 
 
 1.0 
 
 
 
 
 
 
 1 
 
 3i'30'- p.m. to 9''30'" p.m. 
 
 6 
 
 7.0 
 
 71.3 
 
 . 2436 
 
 oiogio 
 
 i5!l6 
 
 7!38 
 
 2 
 
 Qi-SO^ p.m. to 3''30"' a.m. 
 
 6 
 
 13.0 
 
 72.9 
 
 .2376 
 
 .0970 
 
 16.16 
 
 7.87 
 
 3 
 
 3^3(y a.m. to 9''30'" a.m. 
 
 6 
 
 19.0 
 
 73.2 
 
 .2366 
 
 .0980 
 
 16.33 
 
 7.95 
 
 4 
 
 g'^SO'^a.m. to 3''30"' p.m. 
 
 6 
 
 25.0 
 
 70.7 
 
 .2456 
 
 .0890 
 
 14.83 
 
 7.22 
 
 5 
 
 3^30"' p.m. to 10''30°'p.m. 
 
 
 32.0 
 
 60.4 
 
 .2920 
 
 .0426 
 
 6.08 
 
 2.96 
 
 6 
 
 10''30'" p.m. to l^'SO'" a.m. 
 
 3 
 
 35.0 
 
 59.5 
 
 .2978 
 
 .0368 
 
 12.26 
 
 5.97 
 
 7 
 
 l^3(y^ a.m. to 7i'30°' a.m. 
 
 6 
 
 41.0 
 
 67.9 
 
 .2570 
 
 .0776 
 
 12.93 
 
 6.29 
 
 8 
 
 7''30"» a.m. to 10^30" a.m . 
 
 3 
 
 44.0 
 
 60.0 
 
 .2948 
 
 .0398 
 
 13.23 
 
 6.44 
 
 9 
 
 lQl^3(y^ a.m. to 4''30°' p.m. 
 
 6 
 
 50.0 
 
 69.1 
 
 .2524 
 
 .0822 
 
 13.70 
 
 6.67 
 
 10 
 
 4''30'° p.m. to 10''30"p.m. 
 
 6 
 
 56 
 
 60.3 
 
 .2930 
 
 .0416 
 
 6.93 
 
 3.37 
 
 11 
 
 10''30"' p.m. to l»'30°'a.m. 
 
 3 
 
 59.0 
 
 60.1 
 
 .2938 
 
 .0408 
 
 13 . 60 
 
 6.62 
 
 12 
 
 1''30'" a.m. to 7'»30"' a.m. 
 
 6 
 
 65.0 
 
 68.5 
 
 .2550 
 
 .0796 
 
 13.26 
 
 6.45 
 
 13 
 
 7'»30'"a.m. to 10''30"a.m. 
 
 3 
 
 68.0 
 
 60.4 
 
 .2920 
 
 .0426 
 
 14.20 
 
 6.91 
 
 14 
 
 10''30™a.m. to 4'^30™p.m. 
 
 6 
 
 74.0 
 
 70.5 
 
 .2466 
 
 .0880 
 
 14.66 
 
 7.14 
 
 15 
 
 4'>30^ p.m. to 10''30™ p.m. 
 
 6 
 
 80.0 
 
 59.5 
 
 .2978 
 
 .0368 
 
 6.13 
 
 2.98 
 
 16 
 
 10^30™ p.m. to li'30°' a.m. 
 
 3 
 
 83.0 
 
 59.8 
 
 .2960 
 
 .0386 
 
 12.86 
 
 6.26 
 
 17 
 
 l^SO"' a.m. to 7i»30° a.m . 
 
 6 
 
 89.0 
 
 68.0 
 
 .2570 
 
 .0776 
 
 12.93 
 
 6.29 
 
 18 
 
 7''30°' a.m. to l''30°' p.m. 
 
 6 
 
 95.0 
 
 68.0 
 
 .2570 
 
 .0776 
 
 12.93 
 
 6.29 
 
 The experiment shows clearly that following a period of illumi- 
 nation there is a decrease in the rate of respiration; with continued 
 darkness the rate rises slowly again until the next period of illumi- 
 nation. It must be remembered that under the conditions of the 
 experiment the leaves had a large supply of available sugar in the 
 form of d-glucose. From the experiments which have been described 
 it becomes evident that there are at least two factors affecting the 
 rate of the respiratory process. One of these is the supply of 
 carbohydrates and the other is the amount of amino-acids. A change 
 in either of these factors alters the rate of respiration. Under most 
 circumstances light seems to affect these two factors in an opposite 
 manner: the carbohydrates are increased by the photosynthetic 
 activity of light, while under these conditions the amino-acids 
 decrease. It is conceivable that in this manner the rate of respiration 
 tends to be equalized under varying external conditions. 
 
72 STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 The increased rate of growth in the dark, which has been very 
 commonly observed, can also in a measure be explained on the basis 
 of the foregoing experiments. In the dark there is an accumulation 
 of amino-acids in most plant parts. Given an adequate suppl}'^ of 
 carbohydrates, stored in the stems and other parts of the plant, 
 an increase in amino-acids resulting from the absence of light would 
 cause an increased respiratory rate. The behavior of an entire 
 plant has been described in the first experiment, under table 8 and 
 figure 7. It is therefore suggested that the augmented respiratory 
 activity is at least one important factor contributing to the increased 
 rate of growth during periods of darkness. 
 
 There are many points regarding the relation of carbohydrates 
 and amino-acids to light which require further study. These ques- 
 tions seem to us to be of great importance to the problems of photo- 
 synthesis and are now being subjected to investigation. 
 
 It would, of course, have been highly desirable if the proteins as 
 well as the amino-acids could have been determined in the leaf 
 material. This, however, was impossible, largely because of the 
 relatively small amount of material remaining from the respiration 
 determinations and from the analyses for amino-acids and sugars. 
 In view of the fact that the leaves were kept in a nutrient solution 
 containing no nitrogen, it seems highly probable that the amino- 
 acid increase came from the breakdown of the proteins. That the 
 leaves contained ample proteinaceous material to supply the increase 
 in amino-acids becomes evident from the following analyses. A 
 typical dry-leaf material of Heliarithus contained 5.50 per cent of 
 nitrogen as NH2, determined by the Folin micro-Kjeldahl method. 
 The water extract yielded 0.119 per cent nitrogen as NH2 by the 
 Van Slyke method, representing free amino-acids, and 0.463 per 
 cent nitrogen as NH2 by the Folin micro-Kjeldahl method. Evi- 
 dently some nitrogenous material other than amino-acids is extracted 
 by means of water. This, however, does not affect the Van Slyke 
 results. Some idea of the amount of protein in the dry-leaf 
 material can be gained from the results of the hydrolysis with 20 
 per cent hydrochloric acid. This hydrolyzed material contains total 
 nitrogen as NH2 of 5.03 per cent by the FoUn micro-Kjeldahl method. 
 The discrepancy between this figure and the 5.50 per cent represent- 
 ing the total nitrogen of the material which had not been previously 
 treated with 20 per cent acid must represent the humin formation 
 resulting from the reaction of the amino-acids with the carbohydrate. 
 The hydrolyzed material yielded amino nitrogen by the Van Slyke 
 method of 2.88 per cent. Although the latter value is undoubtedly 
 a little low on account of the humin formation during hydrolysis, 
 it represents a considerable reserve of protein when compared with 
 the original amino nitrogen in the water extract of 0.119 per cent. 
 Thus the hydrolyzed material contains about twenty times more 
 
STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 73 
 
 amino-acids than the water extract. This gives an indication of the 
 amount of protein in the leaf as compared with the amino-acids. A 
 considerable number of analyses such as the one just described were 
 carried out, all of which represented about the same proportions, but 
 as the results have not a real quantitative value they are omitted here. 
 
 Special tests were also made to determine whether there was an 
 accumulation of nitrates in the leaves which had been kept in the 
 dark. However, no indication could be found that this was the 
 case. In fact, no tests for nitrates could be obtained in the water 
 extracts. Similarly, the amount of ammonia was so small that it can 
 be considered as insignificant. The ammonia was determined by 
 treating the leaf powder, suspended in water, with magnesium oxide 
 and distilling in a stream of air at 35° to 40° under reduced pressure, 
 15 mm. The distillate was taken up in standard acid solution. 
 
 It is well known that with an ample supply of carbohj^drates, 
 protein synthesis can take place in the leaf in the dark from in- 
 organic nitrates.^ The observation that light acts favorably on 
 protein synthesis — more than three times the amount which is 
 formed in the dark — has been made by a number of workers, ^ 
 Since it had been shown that protein synthesis from inorganic nitrogen 
 compounds occurs only in the presence of an abundance of carbo- 
 hydrates, the beneficial influence of light was attributed to photo- 
 synthetic elaboration of carbohydrates by means of light. That this 
 was not the only interpretation to be given to experiments showing 
 the favorable action of light on protein syntheses was made evident 
 by the experiments of Godlewski.^ He showed that protein synthesis 
 from nitrates was very much greater in the light, even when photo- 
 synthesis was largely excluded by keeping the plants in a carbon- 
 dioxid-free atm.osphere, and that his wheat seedling grown under 
 these conditions contained not only as much protein as was in the 
 original seeds, but had formed a certain amount over and above 
 this. Such was not the case with the seedHngs grown in the dark. 
 Evidently, then, light exercises a very direct influence on protein 
 synthesis. These observations have led to much experimentation 
 and extensive speculation in an endeavor to ascertain the mode of 
 synthesis of the complex proteins. 
 
 Emmerling^ and others consider that amino-acids are the first 
 products of nitrogen assimilation in the leaves. However, this part 
 of the problem is still largely in the speculative stage and little 
 direct experimxcntal evidence is available. That leaves take up 
 amino-acids and, in the presence of sugar, increase in protein- 
 content was demonstrated in a qualitative m anner by Hansteen.^ 
 
 ' Zaleski.W. Ber. deutsch. hot. Ges., 15, 536-542 (1897). Prianischn'ikow, D. lbid.,17,151 (1899). 
 - Laurent, E., and M. Mahchal. Bull, de I'Acad. Roy. de Belgique, 32, 55 (1903). 
 » GoDLEWSKi, E. Bull. Acad. Science de Cracovie, 313 (1903). 
 
 * Emmerling, E. Landw. Versuch. Stat., 34, 113 (1880). 
 
 * Hansteen, B. Jahrb. wiss. Bat, 33 417 (1899). 
 
74 
 
 STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 From the experiments of Saposchnikowi it appears that excised 
 leaves rapidly take up asparagine and in the light show an increase 
 in protein-content. 
 
 The increase of amino-acids in leaves kept in the dark offers the 
 key to the interpretation of some observations of long standing 
 which have never been adequatelj^ explained. In an excellent paper 
 on the chemistry and physiology of foliage leaves, published by 
 Brown and Morris^ in 1893, determinations are reported of the 
 increase of diastatic activity of leaves kept in the dark. Thus, for 
 example, in experiments with Hydrocharis morsusrance they found : 
 
 Table 53. 
 
 
 Diastatic 
 activity. 
 
 Increase in 
 diastase. 
 
 After full insolation 
 
 In darkness 47 hours 
 
 In darkness 96 hours 
 
 p.ct. 
 0.267 
 0.476 
 0.676 
 
 p.d. 
 
 "78.2 
 153.1 
 
 The explanation given for this phenomenon by Brown and Morris 
 is, in brief, that the protoplasm elaborates diastase according to 
 the requirements of the leaf. 
 
 "As long as conditions are favorable for assimilation, the leaf-cells are supplied 
 with an abundance of newly assimilated materials and so plentifully that the supply 
 exceeds their powers of metabolism and translocation. The excess of nutritive 
 material is in part at least deposited as starch. At this period there is little or no 
 elaboration of diastase of the protoplasm, probably none at all in those cells in which 
 starch deposition is in active progress. When the light fails, and assimilation falls 
 off, the living cells speedily use up or translocate the excess of the soluble assimilative 
 products, e. g., cane sugar, and begin to draw their supplies from the reserve of starch. 
 To enable them to do this effectually, the somewhat starved protoplasm now com- 
 mences to elaborate the needed diastase more rapidly, and this secretion becomes 
 still more marked as the starvation point of the cell is neared." 
 
 It seems to us that this argument ascribes to protoplasm final 
 causes beyond the justification of the experiments, in a manner 
 already briefly considered in the introductory discussion of this 
 paper. The fact that amino-acids increase in the leaves kept 
 in darkness has been established repeatedly. The accelerating 
 influence of amino-acids on the diastatic activity as determined by 
 Sherman and others offers a more direct explanation of the periodic 
 variations of diastase in leaves than the one originally advanced 
 by Brown and Morris. Thus an increase in diastatic activity of 
 leaves which had been kept in the dark would simply mean that the 
 amino-acids in the leaves had increased and therebj^ produced 
 conditions which are favorable to diastatic activity. 
 
 ' Saposchnikow, W. Bot. Zentrbl., 63, 246 (1895). 
 
 ' Bbown, H. T., and G. H. Morris. Jour. Chem. Soc. London, 63, 644 (1893). 
 
STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 75 
 
 8. The Action of Animo-Acids on Sugars. 
 
 In searching for a possible chemical explanation of the stimulating 
 effect of amino-acids on the respiration of leaves, the action of amino 
 acids on sugars deserves some consideration. In this regard the 
 work of Maillard^ has received considerable notice as offering the 
 key to the amino-acid-carbohydrate and protein relation in living 
 organisms. By the action of amino-acids on glj^cerine, Maillard 
 obtained substances to which he ascribed the properties of poly- 
 peptides. Subsequ:ently he studied the action of amino-acids 
 on a variety of sugars and obtained the well-known reactions which 
 lead to the formation of humin compounds. It is very questionable 
 whether these reactions can find application to living organisms 
 such as leaves. Maillard used very concentrated solutions, e. g., 
 4 grams of glucose in 3 to 4 c. c. of water and 1 gram of glycocoll. 
 Moreover, the reaction proceeds only very slowly at ordinary tem- 
 perature, so that his conditions can not be considered as having 
 direct biological significance. 
 
 A solution of d-glucose, 5.6853 grams, in 100 c. c. water in a 2-dm. 
 tube, gave a rotation a= -H5.50 at 10°. To 50 c. c. of this solution 
 0.2401 gram of glycocoll was added; the rotation of this solution was 
 a= +5.47°. After 24 hours both solutions gave the same rotation. 
 The d-glucose solution containing the glycocoll was heated for 4 
 hours at 90° with a reflux condenser. After making up carefully to 
 volume, the rotation was practically unchanged, a =+5.52°. 
 
 The experiment was repeated with d-levulose. To a solution 
 of 8.9675 grams d-levulose in 100 c. c. water was added 0.5002 gram 
 glycocoll. This gave a rotation in a 2-dm. tube of a =—16.50°. 
 After heating on a boiling water-bath for 5 hours the solution was 
 very slightly yellow and gave a corresponding rotation of a = — 16.40°. 
 
 It is therefore very doubtful whether the amino-acids in this 
 dilution have any influence toward the mutual transformation of the 
 hexoses such as is exerted by the weak alkalies. Nor does there 
 seem to be, under the conditions of dilution and temperature em- 
 ployed in these experiments, any other profound action on the sugars. 
 
 The foregoing experiment with solutions of glycocoll and d-levulose 
 were repeated, using instead of water the nitrogen-free mineral 
 nutrient solution employed in the respiration. Also under these 
 conditions the rotation remained unchanged in the solution kept at 
 ordinary temperature as well as in one heated on the boiling water- 
 bath for 2 hours. Similarly, only negative results were obtained in 
 experiments with solutions of d-glucose and glycocoll to which was 
 added the juice obtained by thoroughly grinding Helianthus leaves 
 with quartz sand. 
 
 1 Maillard, L. C. Ann. de Chimie. (9 s^rie.), 5, 258-317 (1918); 2, 210-268 (1914). Ivanoff, 
 N. N. Biochem. Zeitschr., 120. 1-80 (1921). 
 
11. THE INTERNAL FACTOR IN PHOTOSYNTHESIS. 
 INTRODUCTORY DISCUSSION. 
 
 It has been realized for some time that in the photosynthetic 
 process taking place in chlorophyllous leaves there is an essential 
 internal factor the nature of which has thus far not been discovered. 
 That such a factor exists is concluded not only from the failure of all 
 attempts which have been made to reproduce photosynthesis outside 
 of the living cell, but recently also from direct experiments with living 
 leaves. The existence of such a factor is especially noticeable under 
 circumstances where the rate of the photosynthetic activity varies 
 quite independently of external conditions. The nature of this 
 factor and the seat of its activity have been given a large variety 
 of purely hypothetical explanations. 
 
 In the course of investigations on certain phases of the problem 
 of photosynthesis which have been in progress for a number of years, 
 it was recognized that a better understanding of the internal factors 
 affecting respiration was a prerequisite to a rational interpretation 
 of photosynthesis. Considerable information on the nature of car- 
 bohydrate consumption in leaves has been gained and has aided 
 materially in the experimental work on photosynthesis. It now 
 seems highly probable that a solution of the problem of the internal 
 factor in photosynthesis can be found in the intimate interrelation 
 between photosynthesis and respiration. 
 
 Pantaneli^ maintained that in photosynthesis the major role is 
 to be ascribed to the protoplasmic function of the colorless com- 
 ponents of the chloroplasts. Willstaetter and Stoll,^ in their splendid 
 and thorough investigation of the relation of the chlorojihyll com- 
 ponents to photosynthesis, attempt to determine whether the 
 differences in the photosynthetic activity of a leaf can be explained 
 by ascribing a dual function to a single chemical component of the 
 chlorophyll. That this factor is not to be sought in the chlorophyll 
 components or in such physical conditions as the degree of dispersion 
 of the chlorophyll pigments follows from the great and irregular 
 disproportionality which has been found to exist between the chloro- 
 phyll-content and photosynthetic activity. Willstaetter and Stoll 
 are of the opinion that besides the chlorophyll there is another 
 chemical agent essential to the photosynthetic. process. The func- 
 tion of this agent they believe can be ascribed either to the general 
 behavior of the plant protoplasm or, in attempting to enter more 
 deeply into its chemical nature, it must be ascribed to a definite 
 
 • Pantaneli, E. Jahrh. f. wiss. BoL, 39, 165 (1903). 
 
 = Willstaetter, R., and A. Stoll. Untersuchungen uebor die Assimilation der Kohlensaeure, 
 41-166 (1919). 
 76 
 
STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 77 
 
 component of the protoplasm. This component they assume to be a 
 specific enzyme contained in the stroma. 
 
 So far as these conclusions go they are apparently in undeniable 
 agreement with observational facts, for it is well known that even 
 mild disturbances of the structure of the chlorophyllous cell result 
 in inhibition of photosynthesis. Of great interest also is their 
 observation that the internal factor is affected by temperature in 
 much the same manner as most enzyme reactions. 
 
 In Willstaetter and Stoll's experiments on the effects of variations 
 in temperature and light intensity with leaves rich and poor in 
 chlorophyll, the disproportionahty between chlorophyll-content 
 and photosynthetic activity becomes clearly evident. Moreover, 
 [leaves poor in chlorophyll are more dependent upon variations in 
 light intensity, while those rich in chlorophyll show greater variations 
 with temperature. The two components of the photosynthetic 
 apparatus thus operate in such a manner that the pigment reacts to 
 variations in light intensity more in the case of the leaf with low 
 chlorophyll-content than the one with high chlorophyll-content, 
 while the internal factor reacts more directly to temperature and 
 manifests itself to a larger degree in the leaves of high chlorophyll- 
 content. This fact can be expressed in terms of limiting factors in 
 such a way that in leaves poor in chlorophyll this component is the 
 limiting factor, while in leaves rich in chlorophyll the internal factor 
 is the limiting one, and complete utilization can not be made of the 
 higher chlorophyll-content on account of the relatively low activity 
 of the internal factor. 
 
 The most valuable experimental data on this subject are those 
 pertaining to the temperature coefficients. Unfortunately, however, 
 while there have been made a number of careful determinations of 
 this nature, these represent a variety of rather isolated observations 
 on different plant material, with varying methods, and the nutritional 
 conditions of the plant have not been taken into consideration suffi- 
 ciently. We are at present engaged in a more exhaustive study of 
 the subject with mature leaves. 
 
 Warburg^ found that the temperature coefficient with high light 
 intensity and high carbon-dioxid concentration decreases with in- 
 creasing temperature, so that at 5° it is 4.3 and at 32° it is 1.6. With 
 low light intensity the temperature coefficient is about unity. These 
 observations can be explained on the basis of Blackman's theory of 
 limiting factors; but on careful consideration the question is also 
 raised whether this theory is not due for a revision or extension, not 
 as to its observational basis, but rather regarding the interpretation 
 of the dynamics of the factors involved. Temperature relations 
 
 1 Wahburg, O. Biochem. Zeitschr., 100, 230-370 (1919). 
 
78 STUDIES IN PLANT RESPIKATION AND PHOTOSYNTHESIS. 
 
 such as were found by Warburg are not at all uncommon for phys- 
 iological processes.^ Becking^ has recently published a theoretical 
 discussion of the physical factors involved in the temperature 
 coefficients of vital phenomena. Osterhaut and Haas,^ on the basis 
 of determinations of the temperature coefficient of photosynthesis 
 of Ulva rigida, conclude that this process involves two reactions — 
 one a light reaction with a low temperature coefficient and an ordi- 
 nary chemical reaction with a high Qio. 
 
 A detailed discussion of temperature coefficients can not be entered 
 upon here. Suffice it to say, however, that in a process such as 
 photosynthesis there enters such a large number of factors that 
 the question naturally arises whether the very small number of 
 really concordant results is not due to chance or more probably to 
 the choice of limited conditions to the exclusion of the broader 
 aspect of the question. The many factors and steps which con- 
 tribute to the complete process naturally produce a complicated 
 situation, and determinations of temperature coefficients must 
 represent the mean of a number of reactions. Thus, while the 
 rate of photosynthesis is reduced with a decrease in temperature, 
 the absorption of carbon dioxid at 25° is about half that at 5°.* 
 
 Recent investigations in the field of photochemistry emphasize 
 the complex nature of these reactions, and it will require an enormous 
 amount of experimental data before the kinetics of the photosyn- 
 thetic process are made clear. Stokalasa's^ attempt to explain the 
 internal factor in photosynthesis on the basis of the radioactivity 
 of potassium, which is found in higher concentration near the chloro- 
 plasts, still requires much experimental evidence before it is beyond 
 the realm of the purely hypothetical. 
 
 In the consideration of the relation between respiration and 
 photosynthesis it is primarily the mature leaf which must be studied. 
 The principal interest in photosynthesis centers about the leaf which 
 is producing carbohydrate material above its immediate needs. 
 For this reason also the behavior of germinating seeds or seedlings 
 which have available reserve food material is of rather secondary 
 importance in the study of photosynthesis. 
 
 The problem of photosynthesis is essentially one of energy transfer. 
 Unfortunately, however, our knowledge of the energy relations in 
 photosynthesis, as well as of plant respiration, is most rudimentary. 
 The former is practically confined to the observations of Brown 
 
 > Fawcett, H. S. University of California Publications in Agricultural Sciences, 4, No. 8, 217 
 
 (1921). 
 2 Becking, L. B. Dissertation, University of Utrecht (1921). 
 
 sQsTEBHAUT, W. J. V., and A. R. C. Haas. Jour. Gen. Physiol, 1, 295-298 (1919). 
 « WiLLSTAETTEK, R., and A. Stoll. I. c, p. 181. 
 sStokalasa, J. Biochem. Zeitschr., 108, 159-184 (1920). 
 
STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 79 
 
 and Escombe^ and of Puriewitsch ;- thus far no measurements have 
 been made which determine directly the amount of radiant energy 
 used in photosynthesis. Such data as we now possess have been 
 obtained from calculations based either on the amount of carbon 
 dioxid absorbed (Brown and Escombe) or on the heat of combustion 
 of the photosynthate (Puriewitsch). Fundamentally the measure- 
 ments of Brown and Escombe and of Puriewitsch are based upon the 
 same principle, i. e., taking the heat of combustion of the photosyn- 
 thate or of glucose, or its equivalent in CO2, as a measure of radiant 
 energy utilized. The notable facts in Brown and Escombe's studies 
 are that only a small proportion of the energy absorbed by the leaf 
 is used in photosynthesis and that the amount actually used is a 
 variable quantity. 
 
 Puriewitsch's figures exhibit similar variations in the percentage 
 of radiant energy used in photosynthesis, ranging from 0.6 per cent 
 to 7.7 per cent. In these experiments the intensity of illumination 
 was high and normal air was used, so that the carbon dioxid was 
 probably the limiting factor. Under such conditions it is to be 
 expected that the percentage of radiant energy used in photosyn- 
 thesis would vary inversely with the intensity of illumination. This, 
 however, is not the case. He finds that the longer the period of 
 illumination the lower is the percentage of radiant energy utilized 
 in photosynthesis. Thus for Polygonum sacchalinense the time and 
 percentage of energy used are as follows: 1 hr. 20 min. 7.7 per cent; 
 2 hrs. 20 min. 3.7 per cent; 5 hrs. 1.1 per cent and 2.5 per cent. 
 Since the ''time factor" in photosynthesis does not become evident 
 under 25°, these variations can probably not be ascribed to it, and 
 we must assume that as the plant accumulates carbohydrates less 
 radiant energy is utilized. It can not be assumed, as Puriewitsch 
 does, that in his experiments this decrease is due to the accumulation 
 of the photosynthate, which would mean the interference by the 
 well-known Saposchnikoff effect — in brief, an inhibition of the 
 photosynthetic activity, probablj'- due to the accumulation of car- 
 bohydrates.3 This effect seems to become apparent in land plants 
 only after more prolonged exposure than in the experiments of 
 Puriewitsch. Unfortunately, Puriewitsch gives no data as to the 
 rate of respiration; but it is safe to assume, from the experiments 
 of Matthaei'' and our own on the relation of carbohydrate-content 
 
 > Brown, H. T., and F. Escombe. Proc. Roy. Soc. London, B. 76, 29-111 (1905). 
 
 2 Puriewitsch, K. Jahrb. f. wisa. BoL, 53, 210-254 (1914). 
 
 » Saposchnikoff, W. Ber. d. deut. bot. Ges., 11, 391-393 (1893). 
 
 EwART, A. J. Jour. Linnean Soc, 30, 439-443 (1896); 31, 573 (1897); Ann. of Bot., 11, 439- 
 480 (1897). 
 «Matthaei, G. Phil. Trans. Roy. Soc. London, B. 197, 50 (1904). 
 
 Borodin, J. Mem. de I'acad. imp. des Sciences de St. Petersburg (Serie 7), 28, 4 (1881). 
 
80 STUDIES IN PLANT BESPIRATION AND PHOTOSYNTHESIS. 
 
 to respiration, that as photosynthesis proceeds the rate of respiration 
 increases. It would appear, then, that the amount of radiant energy 
 utiHzed in the photosynthetic process decreases as photosynthesis 
 proceeds, and the plant's internal-energy release presumably increases, 
 due to higher respiratory activity from the greater available car- 
 bohydrate-supply. 
 
 That a general relationship exists in leaves between the rate of 
 carbohydrate synthesis and consumption has been recognized for 
 a long time.^ Boysen-Jensen^ summarizes his studies as follows: 
 
 "In Sinapis the intensity of the CO2 assimilation is very great, rising to at least 
 6 mg. CO2 per 50 cm.^ per hour at 20°. Also respiration in the leaves is great, about 
 8 mg. CO2 per 50 cm." per hour at 20°. The point of equilibrium between CO2 
 assimilation and respiration lies at a light intensity of 1.0 (Bunsen units XlOO). 
 The development of a Sinapis plant is very weak. In 44 weeks the dry-matter con- 
 tent rises from 0.5 gram to 38 grams per 100 of plant. In favorable conditions the 
 daily percentage production of dry matter can be estimated as about 15. 
 
 "In Oxalis the maximal intensity of CO2 assimilation is very small, about 0.8 
 mg. CO2 per cm.^ per hour at 20°. Also respiration of the leaves is very small, about 
 0.1 to 0.2 mg. CO2 per 50 cm.^ per hour at 20°. The point of equilibrium between 
 CO2 assimilation and respiration lies at a light intensity of 0.2. The daily produc- 
 tion of dry matter is at 2.1." 
 
 If an actual chemical or energetic relationship exists between 
 the photosynthetic and respiratory activities of the leaf it should be 
 expected that any disturbance in the respiratory activity would be 
 reflected in photosynthesis. The observations on the effect of re- 
 duced oxygen pressure and of narcotics on photosynthesis are of 
 special interest in this connection, Boussingault^ and Pringsheim* 
 investigated the effect of lack of oxygen on the rate of photosynthesis 
 and concluded that it is inhibited thereby. Willstaetter and Stoll,^ 
 from their extensive experiments decide that oxygen is absolutely 
 necessary for the photosynthetic reaction, but that very small 
 amounts of oxygen suffice. This small amount of oxygen, they 
 claim, need not be present as free oxygen, but as loosely bound, 
 "dissoziabel gebundener." Although their experiments were appar- 
 ently carried out with great care, it is a question whether they 
 succeeded in removing all the oxygen which is occluded in the leaves. 
 This fact, however, stands out clearly in their experiments, that the 
 more perfectly the oxygen has been removed the greater is the 
 inhibition of the photosynthetic activity. Also, leaves which had 
 been previously kept in the dark and their carbohydrate-content 
 thus reduced showed no photosynthesis in an atmosphere freed from 
 oxygen. From these experiments it appears that leaves with low 
 
 1 EcKERSON, S. Bot. Gaz., 48, 224-228 (1909). 
 
 2 Botsen-Jensen, p. Botanisk Tidsskrift, 36, 21&-259 (1918). 
 ' B0US8INGAULT, J. B. Compt. rend., 61, 608 (1865). 
 
 * Pringsheim, N. Sitzber. der Preus. Akad. der Wias., 763 (1887). 
 
 • Willstaetter, R., and A. Stoll. I. c, 344r-370. 
 
STUDIES IN PLANT -RESPIRATION AND PHOTOSYNTHESIS. 81 
 
 carbohydrate-content, i. e., in which the respiratory energy release 
 is very low, can stand the absence of oxygen less easily than can 
 leaves with higher carbohydrate-content. Also, an anaerobic form 
 of respiration, in which the course of the reaction is different and the 
 energy of release quite low, does not suffice. 
 
 In harmony with the conception of the dependence of photo- 
 synthesis on respiration are the observations of the effects of anes- 
 thetics or narcotics on photosynthesis. Claude Bernard discovered 
 the inhibiting effect on photosynthesis of chloroform, and this fact 
 has been repeatedly confirmed. ^ In fact, photosynthesis is far more 
 sensitive to the action of chloroform and ether than is respiration, 
 so that amounts of these anesthetics, which hardly affect the rate 
 of CO2 emission, exert a decidedly inhibiting effect on photosynthesis, 
 and with higher concentrations the capability for photosynthetic 
 work is entirely lost. Willstaetter and Stoll- have shown that in 
 leaves which have lost their photosynthetic power the four chloro- 
 phyll components are unchanged. Unfortunately, we have not been 
 able to gather sufficient data on the nature of carbohydrate metabo- 
 lism in leaves during narcosis to show definitely how the energy 
 release is affected. It would be using very unreliable evidence to 
 draw conclusions as to this point from the tropic and general move- 
 ment phenomena or other responses of plants under the influence of 
 narcotics. 
 
 The Very interesting experiments of Pelster' bear directly on the 
 relation of photosynthesis and respiration. He found that while 
 photosynthesis is much lower in the varieties of the same species 
 containing Uttle chlorophyll, there is no direct ratio between photo- 
 synthetic activity and chlorophyll-content. Furthermore, the fight- 
 green or aurea varieties, with low chlorophyll-content, also have a 
 low respiratory activity as compared with the normal varieties. 
 However, here also there is no direct relation between respiration 
 and chlorophyll-content. But Pelster's results seem to show a 
 relation between respiratory and photosynthetic activity. The 
 quotient of the respiration and photosynthesis values of the light- 
 green types are: Ptelea 84.1/47.5 = 1.77, Catalpa 58.8/34.2 = 1.72, 
 Mirabilis = 2.0, Ulmus = 2.0, Populus = 2.1, while Atiflex showed 
 the very low quotient 1.3. 
 
 The findings of Pelster can be explained, of course, in a large 
 measure also, on the ground that due to the low photosynthetic 
 
 1 Bernard, Cl. Lecons sur les phfenom^nea de la vie, 278 (1878). 
 
 Irving, A. A. Ann. of Bot., 25, 1077-1099 (1911). 
 
 Kegel, W. Inaug. Diss., Goettingen (1905). 
 
 EwART, A. J. Jour. Linnean Soc, 31, 439 (1895). 
 
 Bonnier, G., and L. Magin. Ann. d. Sci. Nat. Bot.. (7), 3, 14 (1886). 
 s Willstaetter, R., and A. Stoll. I. c, 39. 
 • Pelster, W. Beitrdge zur Biologic der Pflamen, 11, 249-304 (1912). 
 
82 STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 activity the leaves have a relatively low supply of oxidizable material, 
 resulting in correspondingly low respiration rates. Therefore, the 
 experimental conditions in the investigations of Willstaetter and 
 StoU were destined to give more definite results. 
 
 In just what manner respiration can participate in the photo- 
 synthetic process it is as yet impossible to say with any degree of 
 certainty. That the heat liberated in the process of plant respiration 
 is to be regarded largely as an energetic waste product now seems 
 highly probable. 
 
 If respiration is to be considered as a producer of energy for the 
 maintainance of the so-called life processes, the potential energy of 
 the food materials can not be converted entirely into heat, for if 
 that were the case the heat of respiration could be substituted by heat 
 applied from without. This, of course, is not the case. There must 
 be produced in the course of respiration other forms of energy which 
 are used in the metabolic processes of the plant. That such is the 
 case was indicated in the old experiments of Bonnier,^ who found 
 that considerably less heat was evolved than could be expected from 
 the respiratory coefficient. Further calorimetric studies of this 
 kind would be highly desirable, carried out on the basis of knowledge 
 obtainable regarding the transformation of the carbohydrates and 
 other food material and with the application of modern temperature- 
 measuring devices of high accuracy. While such calorimetric 
 investigations would serve as excellent guides and checks, it is 
 evident that they can tell us little of the more intricate details of 
 the energy release in respiration. 
 
 Besides heat, then, it would appear that the plant has available 
 and uses a considerable amount of energy derived from the breaking 
 down and combustion of carbohydrates and other food material. 
 Plant physiologists have widely accepted the dictum that this energy 
 is converted into work by the plant. The nature of the work which 
 is thus performed by the combustion of organic substances has been 
 described in only vague and indefinite terms, although it is the basic 
 problem of plant life. In the case of animals, where the factors of 
 locomotion, balance, and other purely mechanical movements are 
 involved, the work done is of a far more evident nature.^ In the 
 plant, however, it requires rather more searching perception to 
 determine in what manner this release of energy in respiration takes 
 form, for it is a strictly chemical phenomenon. The discussion here 
 is limited to the energy liberated by the breakdown and oxidation 
 of food material. The tensions and pressures related to turgor or 
 the possible mechanical work involved in the activity of the so-called 
 
 1 BoNNiEE, G. Ann. Sci. Nat. Bot., (7), 18, 1 (1893). 
 
 Spoehr. H. A. Carnegie Inst. Wash. Pub. No. 287, 21 (1919). 
 « Oppenheimek, C. Der Mensch als Kraftmachine. Die Naturwiesenschaften, 23, 64-72 (1920). 
 
STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 83 
 
 osmotic energy are, of course, of enormous importance in the life 
 of the plant. Similarly, the phenomena dependent upon inhibition 
 of water by the colloidal material and surface-tension energy are 
 of great significance to the economy of the plant and are capable of 
 many external manifestations. Nevertheless, their relation to the 
 energy transformations of food material can be considered at most 
 to be only an indirect one. 
 
 In general, photosynthesis and respiration bear an intimate 
 association, not only on the basis of direct observation, but because 
 oxidation and reduction actions in the living organism are intimately 
 connected and apparently dependent upon the same or very closely 
 allied agents. The two processes of photosynthesis and respiration, 
 proceeding in opposite directions, may be related either on the basis 
 that the energy released in respiration actually aids or is essential 
 for one of the steps of the reduction process, or the relation may be 
 based upon the action of an enzyme which functions in both reactions. 
 Under no circumstances, of course, can all the energy for the reduc- 
 tion of the carbon-dioxid come from the oxidation of the carbohy- 
 drates. An extraneous source of energy is essential. 
 
 Thermodynamically, the contribution which respiration could 
 make in the photosynthetic process would naturally be relatively 
 small, so that this amount of energy could at best serve only as a 
 partial source. 
 
 For a molecular relationship recourse must be taken to our modern 
 conceptions of the nature of carbohydrate breakdown or glycosis. 
 According to this view, as has been elaborated in an earher publi- 
 cation,^ precursory to oxidation there must take place a cleavage 
 or dissociation of the molecule. This action has as its result the 
 formation of a very large number of enormously reactive substances. 
 These pieces, the products of dissociation, either rearrange, react 
 with each other, or react with some other substance present in the 
 medium. Now, these pieces, on account of their enormous reactivity, 
 can not be isolated. It is, in fact, only from the products formed 
 by their reaction or rearrangement that they can be known. They 
 are, nevertheless, of foremost importance in the chemical reactions 
 involved in carbohydrate breakdown. A molecular relation of 
 photosynthesis and respiration would depend upon the activity 
 of these intermediary products of sugar catabolism. Just as these 
 products serve as the building-blocks from which other compounds 
 of higher potential energy may be formed in the cell, it is conceiv- 
 able that they also may react with carbon dioxid or with some of 
 the primary products of the photochemical breakdown of carbon 
 dioxid.^ 
 
 » Spoehr, H. A. Carnegie Inst. Wash. Pub. No. 287, 5-24 (1919). 
 "Siegfried, M., and S. Howwjanz. Zeit. f, Physiol. Chem, 59, 376 (1909). 
 
84 STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 One of the most remarkable and puzzling features of the carbohy- 
 drate synthesis in the plant is the fact that the sugars found in nature 
 are confined to a small number of those which are theoretically 
 possible on the basis of stereoisomeric constitution. A conception 
 of the manner in which the plant-cell is able to synthesize carbohy- 
 drates from compounds which are already extant in the cell has been 
 suggested by E. Fischer. This is based upon the fact that in the 
 artificial synthesis of sugars from compounds containing a smaller 
 number of carbon atoms, by means of the cyanhydrine reaction, the 
 influence of existing stereoasymmetry of these carbon compounds is 
 exerted on the final product. In other words, an asymmetric carbon 
 compound yields an asymmetric product. In the same way, the 
 reactive products of the primary carbohydrate dissociation would 
 also be asymmetric substances, and these, by reaction roughly anal- 
 ogous to the cyanhydrine reaction, have the power of uniting with 
 the products of photochemical decomposition of carbon dioxid and 
 thereby yield an asymmetric product in the form of d-glucose, d-fruc- 
 tose, or its condensation product, sucrose. As these substances in 
 turn are used for the formation of the large variety of other com- 
 pounds found in plants, they would serve as the basis of the asym- 
 metry of these. 
 
 The most noteworthy result of the energy release of respiration in 
 plants is the formation of new compounds. These compounds may 
 be of higher energy content, although they may not again serve the 
 plant as food material, but enter only into the structural or plasmic 
 elements of the organism. Or respiratory glycosis may serve the 
 plant in such a way that the primary products thereof are substances 
 on to which carbon dioxid or its primary splitting product can be 
 added to form a further supply of hexose sugars. Given, then, a 
 properly functioning respiratory system, together with the necessary 
 simpler materials and an apparatus capable of utilizing radiant 
 energy, the plant is able to manufacture not only the large variety 
 of substances found in its structural elements, but also to add organic 
 material derived from the carbon dioxid of the air. 
 
 METHODS AND APPARATUS. 
 1. The Experimental Material. 
 
 The experiments on the photosynthetic activity were carried out 
 with single excised leaves. For this work leaves of the Canada 
 Wonder bean and of the sunflower were used. The methods of 
 culture were the same as those employed for the material used in 
 the respiration experiments and the same precautions were observed 
 in regard to using only perfect leaves and in the method of cutting 
 and handling the leaves. These always remained in perfectly 
 
STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 85 
 
 healthy condition during the course of the experiment. A consider- 
 able number of experiments was carried out; but as these in each 
 case yielded consistent results, only one, representative of the altered 
 conditions, is described here. 
 
 2. The Apparatus. 
 
 The method employed for determining the rate of photosynthetic 
 activity was that based upon the differential determination of 
 carbon dioxid. A stream of air containing a known concentration 
 of carbon dioxid was passed over the leaf. The rate of carbon- 
 dioxid emission from the leaf when this was in the dark was first 
 determined; this gave the value for the rate of respiratory activity. 
 The leaf was then illuminated and the amount of carbon dioxid in 
 the gas-stream after it had passed over the leaf was determined. 
 
 Figure 20. 
 Arrangement of apparatus for study of the rates of photosynthesis. The entire apparatus 
 is in a constant-temperature room. The leaf-container is in a water-thermostat, which is elec- 
 trically heated, controlled, and stirred. 
 
 Thus the total amount of carbon dioxid fixed by the leaf could be 
 calculated. The rate of the gas-stream was maintained absolutely 
 constant throughout the course of the experiment. The entire 
 apparatus was in a constant-temperature room which was ther- 
 mostatically controlled. The leaf was in a container which was 
 placed in an electrically controlled water-thermostat, usually at 24° ; 
 the room was kept at 20°. 
 
 For these experiments a single excised sunflower or bean leaf was 
 used. The same advantages and disadvantages of working with 
 excised leaves for determining rates of respiration which have already 
 
86 
 
 STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 been discussed apply to the work on photosynthesis. With a single 
 leaf the illumination in relation to the surface of the leaf can be more 
 accurately controlled than when there is a larger number of leaves. 
 In this way the leaf can at all times be kept at right angles to the 
 source of hght, and the shading by other leaves, or changing of the 
 angle of incidence of the light, can be absolutely avoided. 
 
 The same precautions of culture, cutting, and handling of the 
 leaves which were observed in the work on respiration, already 
 described, apply to the material used for this work. The experience 
 and knowledge gained from a study of the rates of respiration and 
 the changes of material under varying conditions were naturally of 
 great value in a study of the photosynthetic activity. 
 
 N- 
 
 
 S 
 
 Figure 21. 
 Container for leaf used in photosynthesis experiments. E shows the elevation 
 and a leaf through the glass plates with tube A'^, containing the nutrient solution. 
 <S is the cross section of the container. T is the transverse section showing the 
 glass plates G and the mercury seal. 
 
 A diagram of the apparatus used in these experiments is shown 
 in figure 20. As has been stated, the experiments were carried out 
 in a dark-room which was thermostatically controlled and kept 
 at 20°. The air was taken from a large gasometer. This was first 
 partially filled with carbon dioxid in amounts as required. The 
 carbon dioxid was prepared from white marble and hydrochloric 
 acid and was washed through a solution of sodium carbonate and 
 
STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 87 
 
 one of potassium permanganate. The gasometer was then filled 
 with air drawn from out of doors. Special experiments were carried 
 out to insure that there was no stratification of carbon dioxid in the 
 gasometer and that this gas was of the same concentration at the 
 beginning and end of the experiment. The gasometer was sealed 
 by means of heavy mineral oil. 
 
 By means of glass tubing, the gasometer was connected to a spiral 
 of metal tubing which stood in the water of the thermostat, so that 
 the air attained the temperature of the bath before entering the 
 leaf-container. The connections made by means of heavy wall 
 rubber tubing were wired and covered with several coats of Bakelite 
 paint. 
 
 For these experiments a single leaf was placed in a chamber or 
 cell of special construction. This consisted, essentially, of a metal 
 frame, 15 by 25 cm., two sides of which were glass plates, 5 mm. 
 apart. This leaf-cell is shown in figure 21, in elevation (E) in section 
 (S) and in transverse section ( T) . The upper part of the metal frame 
 carries a metal trough, similar to that of the respiration chamber, 
 already described. This trough was also electroplated with copper 
 and nickel and covered with lacquer, so that it could hold mercury. 
 Into this trough fits a metal cover (C in fig. 21). Distilled water 
 was poured over the surface of the mercury, and when the cover was 
 placed in the trough a completely air-tight seal was made. Through 
 the cover passes a metal tube by means of which the air-stream 
 enters into the leaf-container. A narrow-bore tube through the 
 lower part of one edge of the container provides the means for 
 carrying off the air-stream. At the bottom of the leaf-container is a 
 glass tube with a flared opening. Into this was placed the petiole 
 of the leaf, which was suppUed with nutrient solution through the 
 tube N. This tube was filled with nutrient solution and sealed at 
 the upper end with a piece of rubber tubing and a screw-clamp. 
 During the course of an experiment, the level of the solution in which 
 the petiole stood went down; this was adjusted from time to time 
 by opening the screw-clamp at the upper end of the tube. 
 
 To the exit-tube of the leaf-container is attached a water-trap con- 
 taining phosphorus pentoxide. The experiments were carried out 
 in a saturated atmosphere. This was assured by filling the gasometer 
 with moist air; moreover, the water over the mercury seal and the 
 nutrition solution provided ample water-vapor. The tubes through 
 which the air-stream passes after leaving the leaf-container are all of 
 small bore, 2 mm. It was therefore essential to avoid the formation 
 of water-drops by condensation in these tubes. This was accom- 
 plished by means of the water-trap filled with phosphorus pentoxide. 
 
 From the leaf-container the air-stream passed to the control- 
 valve. This is shown in figure 22, and consists of a very fine needle- 
 
88 
 
 STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 valve which pierces just through a piece of lead L. By means of 
 this valve the rate at which the air-stream passes through the appa- 
 ratus could be very accurately regulated. The valve is attached to 
 a small vessel containing a little phosphorus pentoxide. 
 
 From the control-valve the air-stream passes through narrow-bore 
 glass tubes to the distributing tube. This consists of glass stopcocks 
 by means of which the air-stream can be directed to the absorption 
 tubes. Each stopcock is connected to a narrow glass tube slightly 
 bent up at the end. Over these tubes are fitted the absorption tubes 
 by means of a small rubber stopper. The air-stream can thus be 
 directed from one absorption tube to another 
 without interrupting the rate of flow. 
 
 The absorption tubes consist of 10-bulb glass 
 tubes, the lower end of which is slightly bent. 
 They are filled by means of a pipette, carefully 
 graduated. For these experiments 68.12 c. c. 
 of barium hydroxide was used for each determina- 
 tion. It was found that the bulb-tubes break up 
 the air-bubbles more thoroughly and thus assure 
 more complete absorption than the straight-walled 
 Pettenkoffer tubes. 
 
 Fig. 22. — Needle control valve by means of which the rate of the 
 au--stream passing through the apparatus is regulated. The valve is 
 shown in section; a fine needle pierces the sheet of soft lead L, and 
 is raised and lowered by means of a packed screw. 
 
 From the absorption tubes connection is made to a bottle which 
 served to determine the rate of the gas-stream. This bottle is 
 partly filled with water, over which is a layer of mineral oil. The 
 glass tube from the absorption tubes extend to the bottom of this 
 bottle. The rate of the gas-stream is determined by counting 
 the gas-bubbles passing through the water in the bottle during a 
 certain period. The time measurements were made with a stop- 
 watch, and by means of the control-valve the rate of flow could be 
 very accurately adjusted. A rate of 70 bubbles per minute repre- 
 sented a flow of 310 c. c. per hour. Care was taken to maintain 
 constant the pressure in the aspirator. This was indicated by a 
 manometer filled with aniline oil. Blank experiments showed that 
 with the constant temperature of the room and in the water ther- 
 mostat a very regular flow of air, and consequently of carbon dioxid, 
 is obtainable and obviates the use of cumbersome and inaccurate 
 gasometers. 
 
 The aspirator consists of an inverted galvanized-iron cylinder 
 which is drawn out of a vessel of water. The cyhnder is attached 
 to a weight by means of a light wire cable. This passes over the rim 
 
STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 89 
 
 of a bicycle wheel which is suspended from the ceiUng of the room. 
 The wheel has excellent ball-bearings and is fastened, so that there 
 is no play and very little friction. As the weight descends it 
 lifts the cylinder out of the water, which causes the air-stream to 
 be drawn through the entire apparatus. It is, however, necessary 
 to make an adjustment for the increasing weight of the cylinder, as a 
 larger portion of this comes out of the water. This is attained by 
 increasing the pull on the other end of the cable through a supple- 
 mentary weight placed on the wheel. When 
 the cylinder is completely down this weight is 
 at the top of the wheel, exerting no pull. As 
 the cylinder is drawn out of the water, the 
 wheel turns and the pull of the supplemen- 
 tary weight increases as the horizontal dis- 
 tance between it and the center of the wheel 
 increases. Thus the increasing weight of the 
 cylinder is adjusted; and while the two phe- 
 nomena do not mathematically balance each 
 other, the adjustment can be made sufficiently 
 accurate so that no differences in the rate of 
 the air-stream are noticeable. 
 
 The length of time for each observation 
 was usually two hours. The amount of car- 
 bon dioxid fixed by a single leaf in this length 
 of time is very small. It was therefore essen- , 
 tial that the accuracy of the method of de- ^ 
 termining carbon dioxid be correspondingly 
 high. For these experiments the same princi- 
 ple of carbon-dioxid determination was em- 
 ployed as in the respiration experiments. On 
 account of the shorter periods and smaller 
 amounts of carbon dioxid to be absorbed, a 
 smaller range in the specific resistance curve 
 of barium-hydroxide solution was used. 
 Greater accuracy was attained by using an 
 electrolytic cell of higher resistance. The 
 original barium-hydroxide solution was 0.1001 
 normal, and in the absorption of the carbon 
 dioxid this was reduced to about 0.086 nor- 
 mal. For each determination 68.12 c. c. of 
 solution was introduced by means of a pipette 
 
 Fig. 23. — Electrolytic cell for determining the conductivity 
 of the barium hj'dioxide solutions used to absorb the CO2 in the 
 photosynthesis experiments. The solution is protected from the 
 air by a soda-lime tube. 
 
90 STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 into the absorbing tube, and the method of calculating the carbon 
 dioxid for each period was essentially the same as in the respira- 
 tion experiments. A curve of the specific resistance for concentra- 
 tions of 0.1001 to 0.0860 normal, similar to the one used for the 
 respiration work, was determined by means of the high-resistance 
 cell, and was used for establishing the amount of carbon dioxid 
 absorbed. 
 
 The barium-hydroxide solution, after absorption of the carbon 
 dioxid, was transferred rapidly to narrow bottles; these were then 
 stoppered and sealed with paraffine. After all the barium carbonate 
 had settled out, the conductivity of the clear supernatant liquid 
 was determined bj^ means of the electrolytic cell shown in figure 23. 
 This is essentially a pipette form of cell with a glass stopcock, and 
 the solution is protected from the air by means of a soda-Hme tube. 
 The cell had a resistance of 1,950 ohms when filled with 0.1 normal 
 potassium-chloride solution. For making the conductivity deter- 
 minations, the cell was submerged slightly above the upper electrode 
 in a water thermostat kept at 25°. A difference of 1 ohm in observed 
 resistance represented about 0.00014 gram of carbon dioxid when 
 using 68.12 c. c. of the barium-hydroxide solution. For the deter- 
 mination of the carbon dioxid in the different periods of a single 
 experiment, differences of at least several ohms were observed, 
 which gives an indication of the degree of accuracy of the method. 
 
 As a source of Ught there was used a 500 or 750 watt tungsten 
 Mazda lamp. The distance of the filament from the leaf was 35 
 cm., and the light traversed about 8 cm. of water in the thermostat. 
 The electric lamp was so placed that the light fell on the leaf at right 
 angles. Immediately above and fitting over the top of the glass 
 bulb of the lamp was a metal hood in the form of an inverted funnel. 
 This hood was connected by means of a 5-cm. pipe to an electric 
 suction fan. In this way the hot air surrounding the electric lamp 
 was drawn off. 
 
 After each experiment the leaf was removed from the leaf-container 
 and placed in a photographic printing-frame with blue-print paper. 
 From the print thus produced the area of the leaf was determined 
 by means of a planimeter. 
 
 EXPERIMENTAL RESULTS. 
 
 The problems of photosynthesis must be met in succession. It is 
 questionable whether the ultimate or final causes can be discovered 
 before a clearer understanding is gained of the workings of the 
 immediate causes and relationships. The introduction by Blackman 
 of the conception of Hmiting factors into the experimentation on this 
 phenomenon, while it has contributed little to the ultimate causes 
 involved, has aided very materially in the experimental investigation. 
 
STUDIf:S IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 91 
 
 No. 
 
 On the other hand, the many claims of simple and complete explana- 
 tion of the process, based usually on very limited and inadequate 
 experimentation, have done more to befog the issues than to clarify 
 them. The factors of carbon-dioxid supply, light intensity, the 
 quantity of chlorophyll, temperature, and water-supply have all 
 received more or less exhaustive treatment. Practically all of these 
 investigations have revealed the fact that there are other internal 
 factors operative. The nature of these internal factors is unques- 
 tionably complex, though there is no reason for supposing that they 
 are not amenable to physical and chemical investigation. 
 
 Table 54. — Rates of respiration and synthesis of an excised bean leaf {Canada Wonder) 
 kept in the dark 64 hours previous. 
 In atmospheric air at the constant rate of the air-stream equal to 0.64 mg. CO2 per 2-hour 
 period. Observations made at 23.8°. Area of leafj = 111.84 sq. cm. Light intensity 7,140 lux. 
 
 Time. 
 
 ai'lS" 
 5'>30'' 
 
 7h45m 
 
 10 p.n 
 
 12»'15" 
 2''30" 
 
 4h45m 
 
 7 a.m 
 
 9h20n) 
 
 llMO" 
 1''55'" 
 
 4h05m 
 
 6''20'° 
 8''35'" 
 10''45" 
 
 Dec. 13: 
 
 p.m. to 5''15™ 
 
 p.m. to 7'^30™ 
 
 p.m. to 9H5"' 
 
 a to 12 n 
 
 Dec. 14: 
 "a.m. to 2'' 1.5" 
 a.m. to 4'>30" 
 a.m. to 6''45" 
 
 . to 9 a.m 
 
 a.m. to ll'^20'» 
 ° a.m. to l'>40" 
 p.m. to 3*^55'" 
 p.m. to 6''05'" 
 p.m. to 8''20°> 
 p.m. to 10'»35™ 
 '»p.m.tol2''45'» 
 
 a.m. 
 p.m 
 p.m 
 p.m 
 p.m 
 p.m 
 a.m 
 
 Dark 
 Do. 
 Do. 
 Do. 
 
 Do. 
 Light 
 
 Do. 
 
 Do. 
 
 Do. 
 Dark 
 Light 
 
 Do. 
 
 Do. 
 
 Do. 
 Dark 
 
 Hrs. 
 
 Total 
 hours. 
 
 66.00 
 68.25 
 70.50 
 72.75 
 
 75.00 
 77.25 
 79.50 
 81.75 
 84.08 
 86.41 
 88.66 
 90.82 
 93.07 
 95.32 
 95.48 
 
 Mg. 
 CO2 
 ab- 
 sorbed. 
 
 1.34 
 1.22 
 1.12 
 0.98 
 
 0.89 
 0.27 
 0.10 
 0.04 
 0.05 
 1.01 
 0.17 
 0.10 
 0.09 
 0.23 
 1.38 
 
 Respira- 
 tion, mg. 
 
 COj 
 emitted. 
 
 0.70 
 0.58 
 0.48 
 0.34 
 
 0.25 
 
 0.37 
 
 Photo- 
 ; synthesis, 
 mg. CO2 
 fixed. . 
 
 0.62 
 0.79 
 0.85 
 0.84 
 
 0.84 
 0.91 
 0.92 
 0.78 
 
 Respira- 
 tion per 
 sq. cm. per 
 hour in 
 mg. CO2. 
 
 0.0063 
 0.0051 
 0.0042 
 0.0033 
 
 0.0022 
 
 0.0034 
 
 0.0066 
 
 Photo- 
 synthesis 
 
 per sq. cm. 
 
 per hour in 
 mg. CO2. 
 
 0054 
 0072 
 0076 
 0076 
 
 0076 
 0080 
 0082 
 0072 
 
 The course of carbon-dioxid emission by leaves as the supply of 
 available carbohydrates diminishes has been described in the first 
 section of this publication. The decrease in the rate of carbon- 
 dioxid emission is a well-known feature of these conditions. How- 
 ever, the course of the photosynthetic process under these circum- 
 stances has received little attention. 
 
 1. In table 53 the results are given of the rates of respiration 
 and subsequent photosynthesis of a bean leaf which had previously 
 been kept in the dark for 64 hours. The carbohydrate supply had 
 thus been very appreciably diminished and the decreasing rate of 
 carbon-dioxid emission is evident. For the experiment atmospheric 
 air was employed and throughout the experiment the temperature 
 of the thermostat was kept at 23.8°. 
 
92 
 
 STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 After the leaf had been in the dark for 75 hours, the rate of respi- 
 ration was 0.0022 mg. CO2 per square centimeter per hour. During 
 the subsequent 2 hours of photosynthesis the leaf fixed 0.0054 mg. 
 CO2 per square centimeter per hour. Thereafter the rate of photo- 
 synthesis rose, and after 86.41 hours the rate of respiration had also 
 
 il 
 
 _r 
 
 Figure 24. 
 Rates of respiration and photosynthesis of an excised leaf of Canada 
 Wonder bean kept in the dark 64 hours previously, as per data given in table 53. 
 The values above the zero line indicate rates of photosynthesis; those below, 
 the rates of respiration. The ordinates represent mg. CO2 fixed and emitted 
 per sq. cm. per hour X 10; the numbers of each square correspond to the periods 
 as given in table 54. 
 
 risen to 0.0034 mg. CO2 per square centimeter per hour. Apparently 
 the carbohydrate-supply had been increased through the photosyn- 
 thetic activity. During the next periods of exposure to light the 
 photosynthetic rate continued to rise, and the final period of respi- 
 ration shows a considerably higher rate. The photosynthetic rates 
 are in each case calculated by subtraction from the last preceding 
 respiration period. This undoubtedly accounts for the relatively 
 low values of the last photosynthetic period (Nos. 9 and 14), for 
 the leaf was at this time emitting carbon dioxid at a higher rate 
 than during the last respiration period. If the last photosynthetic 
 rates were calculated from the first succeeding respiration period, 
 the rate of the former would result considerably higher. This fact 
 
STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 93 
 
 stands out and has been observed repeatedly, that under the con- 
 ditions of the experiment, the rate of photosynthesis of a leaf, the 
 store of carbohydrates of which has been greatly reduced, is initially 
 low and rises with continued exposure to light. Thereby the respi- 
 
 Table 55. — Rates of respiration and photosynthesis of leaves of Helianthus annuus at 34°. 
 
 Taken after plant had been exposed to sunlight and after increasing periods of darkness. 
 In atmospheric air, light intensity 7.140 lux. 
 
 fFirst leaf, 106.55 sq. cm.] 
 
 No. 
 
 1 
 2 
 3 
 4 
 5 
 6 
 7 
 
 Time. 
 
 Leaf 
 ex- 
 posed 
 to- 
 
 Hrs. 
 
 Total 
 hours. 
 
 Mg. 
 CO2 
 ab- 
 sorbed. 
 
 Respira- 
 bion, mg. 
 
 CO2 
 emitted. 
 
 Photo- 
 synthesis 
 mg. CO2 
 fixed. 
 
 Respira- 
 tion per 
 sq. cm. per 
 hour in 
 mg. CO2. 
 
 Photo- 
 
 synthesis 
 per sq. cm. 
 per hour in 
 
 mg. CO2. 
 
 March 5. 
 6''15'" p.m. to 7''15'"p.m. 
 7''27" p.m. to 9''27°' p.m. 
 qHO™ p.m. to ll''40°' p.m. 
 11''55"' p.m. to li'SS^a.m. 
 2''05'» a.m. to 4''05" a.m . 
 4''15°' a.m. to 6''15'"a.m. 
 6''30" a.m. to S^'SO™ a.m . 
 
 Dark 
 
 Do. 
 
 Do. 
 Light 
 
 Do. 
 Dark 
 
 Do. 
 
 2 
 2 
 2 
 2 
 2 
 2 
 2 
 
 2.00 
 4.20 
 6.40 
 8.65 
 11.15 
 13.31 
 15.56 
 
 1.82 
 1.41 
 1.39 
 0.26 
 0.24 
 1.27 
 1.22 
 
 1.180 
 0.770 
 0.750 
 
 0.63 
 0.58 
 
 1.130 
 1.150 
 
 0.0111 
 0.0073 
 0.0071 
 
 
 
 
 0.0107 
 0.0109 
 
 
 0.0059 
 0.0054 
 
 
 
 " 
 
 
 [Second leaf, 93.97 sq. cm.] 
 
 
 
 
 8 
 9 
 10 
 11 
 12 
 13 
 
 March 6. 
 7''30°' p.m. to 9 ''30°' p.m. 
 9''45'" p.m. to ll''45°' p.m. 
 ll''55° p.m. to 1''55'" a.m. 
 2''05"a.m. to 4''05°'a.m. 
 4^15" a.m. to 6''15°'a.m. 
 6''25'" a.m. to 8''25°' a.m . 
 
 Dark 
 
 Do. 
 
 Light 
 
 Do. 
 Dark 
 
 Do. 
 
 2 
 
 2 
 2 
 2 
 
 2 
 2 
 
 28.56 
 30.81 
 32.97 
 35.13 
 37.29 
 39.45 
 
 1.02 
 1.03 
 0.27 
 0.23 
 0.92 
 0.90 
 
 0.38 
 0.39 
 
 "o!28'" 
 0.26 
 
 "o!76 
 0.80 
 
 0.0040 
 0.0041 
 
 
 
 0.0080 
 0.0085 
 
 
 0.0029 
 0.0028 
 
 
 
 
 
 [Third leaf. 98.29 sq. cm.] 
 
 
 
 
 14 
 15 
 16 
 17 
 
 March 10. 
 4''05°' p.m. to 6''05'^ p.m . 
 6''15°' p.m. to 8''15'»p.m. 
 8''25°' p.m. to 10''25°' p.m . 
 10''35'" p.m. to 12''35°' a.m . 
 
 Dark 
 Do. 
 
 Light 
 Do. 
 
 2 
 2 
 2 
 
 2 
 
 119.11 
 121.27 
 123.43 
 125.59 
 
 1.07 
 1.06 
 0.35 
 0.43 
 
 0.43 
 0.43 
 
 "o!7l' 
 0.63 
 
 0.0043 
 0.0042 
 
 
 
 0.0071 
 0.0064 
 
 
 
 [Fourth leaf, 91.91 sq. cm.] 
 
 18 
 19 
 20 
 21 
 
 March 12. 
 8''10'" a.m. to 10''10°' a.m . 
 10''25'" a.m. to 12''25'" p.m . 
 12»'35°'p.m.to2''35°'p.m. 
 2h45m pua. to 4''45°' p.m . 
 
 Dark 
 Light 
 Do. 
 Dark 
 
 2 
 
 2 
 2 
 
 2 
 
 179.17 
 181.42 
 183.58 
 185.74 
 
 0.82 
 0.52 
 0.79 
 1.77 
 
 0.18 
 
 "i^is" 
 
 "0.30 
 0.03 
 
 0.0019 
 
 
 0.0032 
 0.0003 
 
 
 0.0159 
 
 
 ration rates also show an increased rate and subsequent periods of 
 photosynthesis exhibit correspondingly higher rates. The results 
 of this experiment are shown graphically in figure 24. 
 
 2 The following experiment was carried out in a different manner. 
 A mature leaf was cut from a large, healthy sunflower plant which 
 
94 
 
 STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 had been exposed to bright sunHght in the greenhouse during the 
 two previous days. The leaf was taken in the afternoon and placed 
 immediately in the apparatus for determination of the respiration 
 and photosynthesis rates. The entire plant was covered with a 
 large hood of black paper, so as to exclude all light without altering 
 other conditions. On succeeding days leaves were cut from this 
 
 4)S_ 
 
 ffi 
 
 u 
 
 Figure 25. 
 Rates of respiration and photosynthesis of leaves of Helianfhus annuua at 24°, after increas- 
 ing periods of darkness. The values above the zero line indicate rates of photosynthesis, those 
 below, rates of respiration, expressed on the ordinates in mg. CO2 fixed, or emitted, per sq. cm. 
 per hour X 10. Data taken from table 65. 
 
 plant and their respiration and photosynthesis rates determined. 
 In this manner leaves were used which had been attached to the 
 plant during the period of starvation and their store of carbohydrates 
 was not depleted as rapidly as when they were cut from the plant. 
 Moreover, the temperature of the greenhouse in which the covered 
 plant stood did not rise above 22°, while the determinations were 
 carried out at 24°. For these reasons some of the leaves at first 
 
STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 95 
 
 show a slightly higher initial rate of respiration than the ones which 
 had been cut and placed in the apparatus in the preceding period. 
 Thus the first leaf was taken after an entire day of insolation, and 
 the succeeding ones after 26.56, 117.11, and 177.17 hours of darkness. 
 The results are given in table 55. 
 
 From the graph (fig. 25) in which these determinations are plotted, 
 it is evident that the leaves which had been kept in the dark show a 
 continual decrease in respiratory activity. Moreover, the amount of 
 carbon dioxid fixed also shows a decline with decreasing respiration. 
 In the last two periods of the fourth leaf there are signs of internal 
 
 Table 56.— Rates of respiration and photosynthesis on three successive days of an 
 excised leaf of Helianthus annuus kept in the dark. 
 
 
 CO2 concentration was nine times that of atmospheric air; temperature 24°; light 7,140 
 lux; area of leaf, 86.43 sq. cm. The air-stream equals 5.30 mg. CO2 per hour. 
 
 No. 
 
 Leaf 
 ex- 
 posed 
 to- 
 
 Time 
 
 Hrs. 
 
 Total 
 hours. 
 
 Mg. 
 CO2 
 ab- 
 sorbed. 
 
 Respira- 
 tion, mg. 
 
 CO2 
 emitted 
 per hour. 
 
 Photo- 
 synthesis, 
 mg. CO2 
 fixed per 
 hour. 
 
 Respira- 
 tion per 
 sq. cm. per 
 hour in 
 mg. CO2. 
 
 Photo- 
 synthesis 
 
 per sq. cm. 
 
 per hour in 
 mg. CO2 
 
 1 
 2 
 3 
 
 4 
 5 
 6 
 
 7 
 8 
 9 
 
 Dark 
 Light 
 Dark 
 
 Dark 
 T,ight 
 Dark 
 
 Dark 
 Light 
 Dark 
 
 March 17. 
 1H5" p.m. to 3''45" p.m. . 
 3''50™ p.m. to 5''50" p.m. . 
 5''55™ p.m. to 7*'55™ p.m. . 
 
 March 18. 
 gJ^OS-^a.m. toll''05"a.m. 
 ll*»10^a.m. to l^'lO^p.m. 
 l''15'"p.m. to3''15'"p.m.. 
 
 March 19. 
 3''15° p.m. to Sills'" p.m. . 
 5^2(y^ p.m. to 7»'20°" p.m. . 
 7''25°' p.m. to 9^25-" p.m. . 
 
 2 
 2 
 2 
 
 2 
 2 
 2 
 
 2 
 2 
 2 
 
 2.000 
 4.082 
 6.166 
 
 21.533 
 23.616 
 25.700 
 
 49.70C 
 51.783 
 53.866 
 
 20.76 
 11.16 
 20.61 
 
 19.18 
 13.31 
 20.21 
 
 16.61 
 15.86 
 16.26 
 
 5.08 
 
 
 0.0587 
 
 
 4.80 
 
 0.0554 
 
 5.00 
 4.60 
 
 0.0578 
 0.0531 
 
 
 
 3.25 
 
 0.0376 
 
 4.81 
 3.01 
 
 0.0556 
 0.0348 
 
 
 
 0.38 
 
 0.0044 
 
 2.83 
 
 0.0327 
 
 
 
 disturbance. The leaf had a sUghtly mottled appearance and was 
 curled in on the edges, so that the very high rate of carbon-dioxid 
 emission in the final period, No. 21, was probably due to protoplasmic 
 disturbances. In two subsequent 2-hour periods the rate of carbon- 
 dioxid emission gave even sUghtly higher values and the leaf showed 
 unmistakable signs of injury, so that these results are not included. 
 In the experiment described under 1, the longer periods of illumina- 
 tion are followed by an increase in respiratory activity. It is 
 apparent that in the present experiment, however, there apparently 
 has not been sufficient carbohydrate synthesis during the periods of 
 illumination to produce an increase in respiration when the leaf is 
 again put in the dark. 
 
 3. In the two preceding experiments the concentration of carbon 
 dioxid was relatively low, that of atmospheric air. In the following 
 experiment this was increased ninefold. The leaf was cut from the 
 
96 
 
 STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 plant after the latter had been exposed to the sunlight in the green- 
 house for 7 hours. The respiration and photosynthesis rates were 
 then determined. The leaf remained in the frame in the dark with 
 a constant air-stream passing over it. After about 24 hours the 
 respiration and photosynthesis rates were again determined, and 
 likewise after about 48 hours. The results of this experiment are 
 given in table 56 and in figure 26. 
 
 Figure 26. 
 Rates of photosynthesis and 
 respiration, on three successive 
 days, of a leaf of Helianthus 
 annuus kept in the dark. The 
 values above the zero line indi- 
 cate rates of photosynthesis, 
 those below, rates of respira- 
 tion, expressed on the ordinates 
 in mg. CO 2 fixed, or emitted, 
 per sq. cm. per hour. Data and 
 conditions as per table 56. 
 
 
 In this experiment, in which the carbon-dioxid content of the air 
 was greatly increased, the rate of photosynthesis shows, as in the pre- 
 vious experiments, a course which in general parallels that of the res- 
 piratory activity of the leaf. In all of these experiments the same 
 source and intensity of illumination was employed. 
 
 4. The carbon-dioxid content of the air-stream was increased 
 further, so that it was 15.5 times that of atmospheric air. In this 
 experiment a sunflower leaf was also used. It is doubtful whether 
 with the intensity of Ught employed the carbon dioxid was the limiting 
 factor, because the photosynthesis per square centimeter per hour 
 in this experiment is slightly less than in the previous one, in which 
 the carbon-dioxid concentration was almost half of that used in the 
 present experiment. In table 57 and figure 27 the results of this 
 experiment are given. The general decline of the rate of photosyn- 
 thesis, with decreasing respiratory activity, also holds for these 
 conditions. The photosynthetic rates are calculated from the last 
 
STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 97 
 
 preceding respiration rate. For this reason the last photosynthesis 
 period, No. 7, appears low. Calculated on the basis of the next 
 following respiration rate, which represents more nearly the true 
 condition, the photosynthesis rate would be 0.0523 instead of 0.0413 
 mg. CO2 per square centimeter per hour. 
 
 Table 57. — Rates of respiration and photosynthesis of a leaf of Helianthus anyiuus. 
 The CO2 concentration was 15.5 times that of atmospheric air, temperature 24°; light 1,740 
 lux; area of leaf, 120.81 sq. cm. The air-stream equals 9.98 mg. CO2 per hour. 
 
 No. 
 
 Leaf 
 ex- 
 posed 
 to- 
 
 Time. 
 
 Hrs. 
 
 Total 
 hours. 
 
 Mg. 
 CO2 
 ab- 
 sorbed. 
 
 Mg. 
 
 CO2 
 
 ab- 
 sorbed 
 
 per 
 hour. 
 
 Respira- 
 tion, mg. 
 
 CO2 
 emitted. 
 
 Photo- 
 synthesis, 
 mg. CO2 
 fixed. 
 
 Respira- 
 tion per 
 
 sq. cm. 
 per hour 
 
 in mg. 
 CO2. 
 
 Photo- 
 synthesis 
 
 per sq. 
 
 cm. per 
 
 hour in 
 mg. CO2 
 
 2 
 3 
 
 4 
 5 
 6 
 
 7 
 8 
 
 Dark 
 Light 
 Dark 
 
 Dark 
 
 Light 
 Light 
 Light 
 Dark 
 
 March 30. 
 
 4 p.m. to 5''30" p.m 
 
 3''45°'p.m. to 7''15'°p.m. . 
 7''30™p.m. to 9 p.m 
 
 March 31, 
 10''40°' a.m. to 12»'10°» p.m. 
 12''25"> p.m. to l''55°'p.m. 
 1''55™ p.m. to 3''25" p.m. . 
 3''25'" p.m. to 5''25'" p.m. . 
 5''35'" p.m. to 7''35° p.m. . 
 
 1.5 
 1.5 
 1.5 
 
 1.5 
 1.5 
 1.5 
 2.0 
 2.0 
 
 1.50 
 3.00 
 4.50 
 
 19.66 
 21.16 
 22.66 
 24.66 
 26.66 
 
 18.66 
 8.81 
 18.66 
 
 16.91 
 
 9.56 
 
 9.21 
 
 12.56 
 
 22.81 
 
 12.44 
 5.87 
 12.44 
 
 11.27 
 6.57 
 6.14 
 6.28 
 
 11.40 
 
 2.46 
 
 
 0.0204 
 
 
 6.57 
 
 0.0543 
 
 2.46 
 1.29 
 
 0.0204 
 0.0106 
 
 
 
 4.90 
 5.13 
 4.99 
 
 0.0405 
 0.0424 
 0.0413 
 
 
 
 
 
 1.32 
 
 0.0109 
 
 
 
 In the experiments described under 2, 3, and 4, the decrease in 
 photosynthetic activity occurs after the leaves had remained in the 
 dark for more or less prolonged periods. The possibiUty naturally 
 suggests itself that the reduction might be due to a decrease in the 
 chlorophyll of the leaves, caused by keeping the plants in the dark. 
 On the basis of the investigations of Willstaetter and Stoll,» it is highly 
 improbable that this is the case. They kept leaves in the dark at 
 34° to 37° for 48 hours, and although, toward the end of this period, 
 there were evidences of protoplasmic disturbances (abnormally high 
 CO2 evolution), the determinations of the chlorophyll components 
 at the end of the period gave the same results as normal leaves. 
 
 It is doubtful whether there exists between the respiratory and 
 photosynthetic activities anything like a fixed or quantitative ratio. 
 The quotients determined by Pelster, after all, represent rather con- 
 fined conditions. Similarly, we found that with atmospheric air the 
 quotients are about of the same magnitude as those found by Pel- 
 ster. With increasing carbon-dioxid content, however, the quanti- 
 tative relation of these values changes, although the relative magni- 
 tude seems to indicate an interrelationship. Moreover, this inter- 
 relationship appears to be of a more intricate nature than if it were 
 
 Willstaetter, 
 p. 36. 
 
 R., and A. Stoll. Untersuchungen ueber die Assimilation der Kohlensaeure, 
 
98 
 
 STUDIES IN PLANT RESPIRATION AND PHOTOSYNTHESIS. 
 
 simply a case of high photosynthesis supplying carbohydrates in 
 such large amounts that the leaf is at all times well supplied and thus 
 burns this sugar at a correspondingly high rate. 
 
 The experiments on the relation of respiration and photosynthesis 
 are being extended, especially with a view of determining the temper- 
 ature coefficients under a variety of conditions. There is considerable 
 evidence for believing that the photosynthetic process is of a dual 
 
 Figure 27. 
 Rates of photosynthesis and respira- 
 tion, on successive days, of a leaf of Hdi- 
 anthus annuus. The values above the 
 zero line indicate rates of photosynthesis, 
 those below, rates of respiration, ex- 
 pressed on the ordinates in mg. CO2 
 fixed, or emitted, per sq. cm. per hour. 
 Data and conditions as per table 57. 
 
 UD 
 
 n 
 
 u 
 
 nature or even more complex, involving the general principle of 
 coupled reactions. That one of these, or one group of these, reactions 
 is photochemical there seems to be little doubt. The nature of the 
 other reaction (Willstaetter's enzymatic reaction), or that associated 
 with the respiratory activity, is still very obscure. In what manner 
 these two sets of reactions complement each other or how they are 
 coupled is one of the most vital points of the photosynthesis problem. 
 Some speculations hereon were given in the introductory discussion 
 of this section. However, at this stage of development the only 
 method which promises any real advance is the experimental one. 
 In view of the importance and the insight which can be gained from 
 the interpretation of temperature coefficients on the basis of recent 
 physical-chemical investigations, an extension of these determinations 
 seems highly desirable and offers another means of analyzing the 
 phenomenon of photosynthesis. 
 
 *