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AN INTRODUCTION

TO THE

C H E M I S T R Y O F

P L A N T P R O D U C T S

VOL. II. METABOLIC PROCESJpg^*-

P A U L H A A S , D . S c ,

READER IN PLANT CHEMISTRY IN THE UNIVERSITY OF LONDON

AND

T. G. H I L L , D . S c , A R . C . S .PROFESSOR OF PLANT PHYSIOLOGY IN THE UNIVERSITY OP LONDON

SECOND EDITION

WITH DIAGRAMS

L O N G M A N S , G R E E N A N D C O .

L O N D O N • N E W YORK • T O R O N T O

1929

P R E F A C E

TO T H E SECOND E D I T I O N .

T H E expanding margin of botanical knowledge and

the trend of botanical thought have made necessary

much revision and rewrit ing; the original intention

of providing a general introduction to the problems

of plant metabol ism, adequate as a foundation on

which to base further study, has, however, been

maintained.P. H .T. G. H.

January•,

P R E F A C E

TO T H E F I R S T E D I T I O N .

I N the preparation of the present volume on the

Metabolic Processes of Plants two alternatives were

presented: the one to g ive as full an account as

possible of the literature, the other to g ive such an

account as would form a basis for further study.

The latter, and more difficult task, was chosen; for,

valuable though a digest of the relevant literature

vi PREFACE TO THE F I R S T E D I T I O N

w o u l d b e , i t would t e n d t o c o n f u s e r a t h e r t h a n t o

ass i s t t h e s t u d e n t . F o r t h i s r e a s o n w e do n o t p r o -

fess t o h a v e m e n t i o n e d all r e s e a r c h , o n t h e sub jec t

m a t t e r ; i n d e e d in some ^ i n s t a n c e s , t h e c h a p t e r on

G r o w t h for example , some c r i t i c s w i l l s a y t h a t too

m u c h has been o m i t t e d ; w e t r u s t , h o w e v e r , t h a t no

w o r k of o u t s t a n d i n g i m p o r t a n c e a n d r e q u i s i t e for

o u r t r e a t m e n t h a s been o m i t t e d . D e t a i l s r e g a r d i n g

m e t h o d s of e x p e r i m e n t h a v e b e e n o m i t t e d s ince t h i s

a s p e c t of t h e subject m o r e p r o p e r l y be longs t o a

p r a c t i c a l t r ea t i se .

P . H .T . G. H .

July, 1922.

C O N T E N T S .

PAGEPREFACE v<2HAP.

I. INTRODUCTION : THE LIVING PLANT iII. THE SYNTHESIS OF CARBOHYDRATES . . . 6

The factors 8External factors . . . . . . . 1 4

The raw materials . . . . 14Water 14Carbon dioxide . . . . . 1 6

Temperature . . . . . . . 2 5Illumination . . . . . . . 30

Chromatic adaptation . . . . 35Internal factors . . . . . . . 42

Chlorophyll . . . . . . . 42The unknown factor . . . . . . 45The products of carbon assimilation . . . 49

The organic products of carbon assimilation . . . 50Hypotheses concerning the synthesis of carbohydrates by the

green plant . . . . . . . . 56Origin of the oxygen 58Formation of formaldehyde . . . 59Polymerization of formaldehyde to sugars in vitro 60

Summary of the evidence relating to the formaldehydehypothesis . . . . . . . 62

Carbon monoxide . . . . . . 62Formaldehyde . . . . . . . 62Feeding experiments . . . . . 63Production of sugar from formaldehyde . . 63

Summary of the opinion of Willstatter and Stoll . 63Other opinions . . . . . . . 66

Formic acid 66Maquenne's hypothesis . . . . . 67

I I I . THE SYNTHESIS OF FATS . . . . . 6 9Formation from proteins . . . . . . 70Origin from carbohydrates . . . . . 71

IV. THE SYNTHESIS OF PROTEINS 81Occurrence of nitrate . . . . . . . 86Occurrence of nitrite . . . . . . - 8 7The reducing mechanism 89

" Reductase" 89Photochemical 92

Hydrolysis of protein . . . . . . . 93The origin of protein in the seed 95

viii CONTENTS

CHAP. FAGEV. RESPIRATION 99

Respiratory quotient . . . . . . 1 0 2Fermentative processes . . . . . . 1 0 6

Intensity . . . . . . . . . noStimulation . . . . • . . . . 1 1 4The action of anaesthetics 116The action of ionized air . . . . . . 1 1 9Conditioning factors 120

Temperature . . . . . . . . 1 2 0Food 125Water 129Salts 133Acidity 134Light 135

The mechanism of oxidation, and the enzyme systems in-volved . . . . . . . . 136

Dehydrase 138The Schardinger reaction . . . . . 1 4 0The nitrate-reducing enzyme of the potato . . 1 4 1

Respiratory pigments . . . . . 145Glutathione . . . . . . . . 146Oxidases . . . . . . . . 148Catalase 149

Fermentation : zymase and carboxylase . . . . 152Alcoholic fermentation . . . . . . 153Lactic acid fermentation . . . . . . 1 5 4Butyric acid fermentation . . . . . 1 5 4Factors influencing alcoholic fermentation by yeast . 155

Current ideas on the mechanism of respiration . . 159

VI. GROWTH . 169The conditioning factors 187

Temperature . . . . . . . . 187Light 194Water 202Nutrition . . . . . . . . 203Auximones . . . . . . . . 209Hormones . . . . . . . . 2 1 1

INDEX 215

C H A P T E R I.

INTRODUCTION: THE LIVING PLANT.

THE problems involved in the study of plant life m.iv be<t btordered and formulated by a consideration, mtcnti-naltyelementary, of the history of a seed plant* d ;n k

r -d ^r-ur'lThe period of rest completed—a period whicii \a.n * nvd> h induration and in different species—a s-»wed $ied bc^m- its ;tr-ruination by the imbibition of water, provided the ^'ruiitions,chiefly of moisture, temperature and aeration, are >u it able.When the. seed coat is saturated, water is absorbed by theunderlying structures both by imbibition and by osmosis., forthe seed coat, although it may be impermeable to certainsubstances,* is permeable to water. Considerable swellingcommonly results so that the volume of the seed is muchincreased and in this swelling a relatively great force is exerted :Stephen Hales in his classical experiment found that the forceexerted by swelling peas was sufficient to raise a weight of184 pounds.f

The second phase in germination now begins, growth starts :but growth is impossible without food to supply the where-withal for new structures and to make good the waste, forvital activity requires energy which is obtained by variouscatabolic processes* Thus aerobic respiration, the ordinaryoxidative process of green plants, is a marked feature con-current with growth and may be sufficiently intense to causean obvious rise in temperature. The required food, chieflyfats, carbohydrates and proteins, are stored in the embryo

* See Adrian Brown : " Ann. Bot.," 1907, 31, 790 ; " Proc, Roy.Soc.Z* B, 1909, 8i, 82. Collins: "Ann. Bot.," 1918, ja» 381. Wolf*;" Bot. Gai.," 1926, §2, 89. Kotowski: " Plant Phyvot./" 1927, a» 177,,.

f Hates: "Vegetable Staticks," 3rd Edition, London. 173s, p. 102.VOL. II.—1

2 THE LIVING PLANT

itself or in special tissues, endosperm and perisperm : andsince the food is stored in a form mostly insoluble and non-assimilable, water is the first essential and appropriate en-zymes the second, for not before it is hydrolized can food betranslocated from its storage cells and passed by osmoticprocesses to the active tissues. The enzymes may be elabo-rated in the cells or tissues containing the food, or may besecreted by specialized structures., the scutellum for example.Often the products of hydrolysis may be recognized by simplemeans, sugar for instance, in germinating barley: but some-times their assimilation may be so rapid that identification isdifficult; indeed, on occasion their presence can only be in-ferred from the results of carefully controlled test-tube ex-periments, glycerol, for example, in germinating Ricinus.The embryo thus presented with appropriate food, grows anddevelops. Growth is in some degree an understandableproblem which, on the present elementary occasion, can besufficiently indicated in a few words. Of necessity must a cellbe nourished through its surface and growth will take placeif assimilation be greater than waste by oxidative and kindredprocesses. But growth means increase, and as this increasein bulk takes place the surface area of the cell is proportion-ally lessened. A stage ultimately will be reached when thearea of the "surface is so limited in proportion to the volume ofthe cell as to permit the entry of only sufficient food to makegood the losses ; thus the surface area is a limiting factor.One of three things now is possible : the cell may remain asit is, a permanent tissue element; it may develop further,using up its own contents either entirely or in part in fittingitself for another function, water transport, for instance ; orit may divide and by so doing increase its surface area inrelation to its volume, in which event the cycle may restart.Growth thus can be interpreted in terms of physical chemistry :the first possibility mentioned hardly requires contemplation,since nothing is easier to do than nothing. The third pro-position is less easy to understand ; the second is a mystery.Thus, why should the daughter of a merismatic cell developinto a phloem element if it be cut off on the one side of its

INTRODUCTION 3

parent and into a xylem element if it be born on the other?Is it due to some subtle influence or stimulus which has itsorigin in the adjacent structural elements ; or is it due tosome quality in the cell itself, an heredital predetermination.

To these questions there are no real answers ; the factsmust be accepted, their explanation must be left to thefuture.

The embryo grows and develops into the autotrophicorganism of a form and structure determined by its conditionsof life and by its ancestry, and exhibiting those actions andreactions commonly associated with the higher plants. Theshoots and roots circumnutate and respond to various stimuli,gravity and light being the most obvious. Circumnutativeand other autonomous movements may be explained by suchconceptions as rectipetality and associated engrams ; whilstin explanation of tropisms various mechanistic hypotheseshave been formulated ; some chemical, the hormone theoryof gravitational stimulus of roots, for instance ; others physical,the statolith theory, for example.

The highly organized root_§y§tem by means of its roothairs takes up raw material by osmosis in the form of waterand its dissolved salts; in many plants, possibly in all, theosmotic strength of the cell sap of the root hairs is continu-ously adapted and is nicely adjusted to the osmotic strengthof the soil water. From the root hairs water is passed onthrough the cortex to the water-conducting elements of thevascular cylinder, and thus supplies the shoot system. Theshoot system, no less highly organized, is, in the first instance,concerned with the manufacture of food, carbohydrate, fat andprotein. In this connection the leaf, a marvel of organization—with its chlorophyll apparatus supported by the network ofveins which also are the conduits for the conveyance of fluidraw materials and for the elaborated products, and with itsmechanism for the regulation of gaseous interchange—is thegreat* synthetic factory, building up food apparently with thegreatest ease and certainly with remarkable rapidity.

In due season reproduction takes place. Of the problemshere involved the secretion of nectar, when it obtains ; the

4 THE LIVING P L A N T

facts of fertilization and the stimuli w h i c h invoke the seg-mentation of the egg; the transmission of hereditary charac-ters ; the reconstruction of the food d e s t i n e d for the use of theoffspring ; and the mechanisms of dispersal, are of fundamentalimportance.

Of the various aspects of the life of a h igher plant outlinedin the foregoing fleeting account, it is appropriate on thepresent occasion to consider those associated with metabolism,the making of food and the procurement of energy.

But hefore beginning this, it is not a l t oge the r out of placeto draw attention to certain features w h i c h , although obvious,are sometimes overlooked. The invest igat ion of the meta-bolic processes of plants is pursued by growing the plant incontrolled conditions and measuring the effect of a selectedfactor by varying that factor only. A posi t ive result will bethe resultant of the combined action of t h e factors concerned,and although it may be of the greatest value, it, obviously,can give no information of what h a p p e n e d in the livingcells. To ascertain this, other m e t h o d s , chiefly chemicaland physico-chemical, are used : a t t e m p t s may be madeto bring about in the test tube processes which occur inthe plant, or analyses may be periodically made of the con-tents of the tissues where metabolic a c t i v i t y is taking placein order to find the sequence of the chemical change. Theformer method is of value in giving informat ion of the chem-ical possibilities and has led to great a d v a n c e s in knowledge ;but it does not follow that because this o r t h a t can be accom-plished in vitro, the same events happen in vivo, for the con-ditions obtaining in the active living cell would appear to beof infinite complexity ; metabolic ac t iv i t i es , both catabolicamd anabolic, oxidative and reductive, t a k i n g place concur-rently, and thus various actions, r eac t ions and digressionsmay occur which never happen in the re la t ively simple con-ditions obtaining in the test tube.

The second method, that of analyses o f cell contents, hasyielded valuable information about final products , but so farhas thrown but little light cm the sequence i n chemical changesince the intermediate phases may be t r a n s i t o r y in the plant,

INTRODUCTION 5

or, being highly reactive, may undergo change during theprocess of extraction.

Often the presumed role of a compound can only beestablished after it has been isolated from the organismand its behaviour studied in conditions which never occurin the plant. There is sometimes a tendency to discard suchevidence as having no bearing on physiological processes ;this is not necessarily sound judgment, for a change whichcan take place in the unnatural conditions of the test tubemay be effected with ease in the living cell.

C H A P T E R I I .

T H E SYNTHESIS OF CARBOHYDRATES.

T H E term carbon assimilation, although unfortunate fromsome points of view, is employed to designate all those ac-t ivi t ies , in part physical, in part chemical, which play a partin t h e anabolism of carbon dioxide by green tissues. Theconspicuous facts of the process are that active chlorenchymao n exposure to light forms, by means of its chlorophyll,carbohydra te from the initial substances carbon dioxide andw a t e r ; oxygen, in volume roughly equivalent to the volumeof carbon dioxide consumed, is evolved during the process.*Carbohydrate is the obvious and chief end product, but proteinalso m a y be so formed, and such diverse materials as fat,t a n n i n and various organic acids have been considered, prob-a b l y on insufficient evidence, to be of direct photosyntheticorigin. The earlier phases in these synthetic processes arephotochemical, a mutation of radiant into chemical energy,a n d i t is during this phase that the oxygen, a waste product,is evolved. The presence of oxygen in the air-space systemof t h e active chlorenchyma may thus be considerably greatert h a n in normal air, and since this gas is continually excretedd u r i n g the process, it is not surprising to find that the quan-t i t y of oxygen in the surrounding atmosphere is immaterialt o t h e process and that it may be decreased to 2 per cent, or

* Bonnier and Mangin (" Ann. Sci. Nat. Bot./' 1886, 3, 1) found byvarious experimental methods that the ratio 02fCOt was always greaterthan unity for ordinary plants; the lilac gave the smallest value, 1*05, andthe liolly the largest, 1-24. A similar range was found by Aubert (" Rev.gen. Bot,/ ' 1892,4, 203) to obtain in ordinary plants, but succulents, whichhave a peculiar metabolism, gave generally a higher value, as high as7*59 in Opuntia tomentosa. Maquenne and Demoussy (" Compt, rend./'Z9*3> I5<>» 506) conclude from a large number of observations that theassimilatory quotient approximates to unity.

6

RATE OF CARBON ASSIMILATION 7

increased to 50 per cent, without adverse effect,* and, accord-ing to Harvey,f many species of marine algae growing in amedium entirely free from oxygen, but not from carbondioxide, evolve oxygen on illumination. But since the for-mation of chlorophyll is dependent upon the presence of oxy-gen, the prolonged maintenance of a low oxygen pressure willinhibit the development of more chlorophyll and this will inturn react on carbon assimilation.

Carbon assimilation is conditioned by various factors;wherefore the rate of the process will vary considerably ac-cording to the inter-relationships of these factors. For thisreason it is hardly remarkable to find that different valuesof the rate of carbon assimilation have been reported bydifferent investigators : differences ia value due not only toinappreciation of the conditioning factors, but also to differentavenues of attack. To consider a few examples: differentvalues may be expected in leaves of the same plant butof different ages, and also in sun and shade leaves owing,presumably, to their structural differences. Further, carbonassignation and respiration proceed side by side and areantagonistic with respect to oxygen and carbon dioxide;wherefore to find the true assimilation, the respiration mustbe ascertained; owing to the normal variation of daylight, abalance between these two processes must be of commonoccurrence, when, owing to the low light intensity, the rate ofevolution of oxygen during photosynthesis is so slow that itis used up in respiration as quickly as it is formed.f Finally,different methods of experiment in all probability will givedifferent values for one and the same subject,

* Friedel: " U.S. Dept. Agric.," 1901, Bull. 28.•f Harvey : " Plant Physiol.," 1928, 3, 85.X This light intensityis known as the compensation point and differs

in different plants and in the same plant grown in different conditions;also it -would appear to be lower in shade plants as the following values,taken from amongst those given by Boysen-Jensen, indicate {*' BotTidsk.," 1918, 36, 219. See also Plaetzer : " Verhand phys. med. Ges.,"Wiirzburg, 1917, 45, 31; and Harder : " Ber. deut. bot. Ges.," 1923, 4*>

-Sinapis alba, a sun plant . . . 1 -o Bunsen units x 100Sambucus nigra, sun leaves . . 0*7 >r „

shade leaves . . 03 „OxaJts acetosella, a shade plant . . 0-2 %, „

8 THE SYNTHESIS OF CARBOHYDRATES

The accompanying table gives a selection of values of therate of carbon assimilation in the open air of detached leavesof the sunflower, Helianthiis annuus^ expressed in terms ofgrams of increase in dry weight per square decimeter per hour,obtained by the authorities named :—

Sachs* . . . -01882Brown and Morris | . -00985Brown and Escombe { -00361 — -00551Thoday § . . -0169 (the average for fully turgid leaves)

•0016 (the average for quite flaccid leaves).

The figures of Sachs and of Thoday were obtained by thedirect determination of the increase in dry weight, whilst theothers were calculated from the amount of carbon dioxideabsorbed and assuming that carbohydrate only was ultimatelyformed. Since the ultimate fate of the carbon dioxide is notentirely known, the extent to which it is directly used in theelaboration of fat or protein for example, the dry weightmethod would appear to give the most accurate results.||

THE FACTORS.

It is obvious that little or no profit will accrue from thecontemplation of the above figures unless they be correlatedwith the factors which determine and control the process.

These factors are both external and internal: the externalfactors are amenable to experimental control whilst theinternal are much more evasive and thus are less understood.Of the external factors, the supply of raw materials, the degreeof temperature and the intensity and quality of the illumina-tion are the most conspicuous ; of the internal factors, thechlorophyll apparatus and the products of carbon assimilationare the least elusive.

The do trJije jpjUixnilirig^ factors, now well known, is due

* Saeks : " Arbeit, Bat. Inst," Wiirzbxurg, 1884, 3, 19.f Brown and Morris: " Joam, Cfeem. Soc.," 1S93, &3» 604.% Brown and Escombe: " iProc. Itoy, Soc.,'* B, 1905, 7&, 29.ITfcd U1 Far % critical review of Sachs's and Brown a.nd Escombe's methods,

see Tbofay: " Ftoe. Stay. Soc./' B, 190$. 8a, %*

LIMITING FACTORS 9

to F. F. Blackrnan * who enunciated the axiom that whena process is conditioned as to its rapidity by a number ofseparate factors, the rate of the process is limited by that ofthe slowest factor. The limiting factor in any definite in-stance may be identified by the experimental application ofthe principle that "when the magnitude of a function islimited by one of a set of possible factors, increase of thatfactor, and of that one alone, will be found to bring about anincrease of the function." f

The principle may be illustrated by oae of Matthaei's $many experiments on the effect of temperature on carbonassimilation in conditions constant except for temperatureand illumination. In Prunus laurocerasus there was a gradualincrease in the assimilation as the temperature was raised;at about i i ° C. a maximal assimilation of 22 rng. of carbondioxide per 50 square cm. per hour obtained and was notincreased even by raising the temperature to 25 ° C. Bydoubling the light intensity, however, the maximal assimi-lation was equivalent to 37-5 mg. of carbon dioxide per 50square cm. per hour and again there was no increase on raisingthe temperature. This means that light intensity was alimiting factor and only by its increase could a greater carbonassimilation be obtained. In his original illustration of theprinciple of limiting factors, Blackman graphically representedthe reaction as linear up to the point of limitation and thenabruptly horizontal; and it is this abruptness in the course ofthe graph which has led to some controversy and has resultedin an accretion of knowledge. But before dealing with this,it must be pointed out that the graph originally presented wasan illustration of a principle, not necessarily a precise repre-sentation of a reaction: " It is not claimed that a limitingfactor curve always adheres rigidly to a typical form with asharp angle a t the point of change of the limiting factor. It

* Blackman : " Ann. Bot.," 1905, 19, 281,f Blackman and Smith : " Proc. Roy. Soc," B, 1911, 83, 3S9.I Matthaei : "Phil. Trans. Roy. Soc," B, 1904, 197, 47. For the

application of the principle to the growth of field crops see Balls and Holton :" Phil. Trans. Roy. Soc," B, 1915, 206, 103, 403 ; and Balls: id., 1917,208, 157.

ia THE SYNTHESIS OF CARBOHYDRATES

is conceivable, and is indeed probable, tha t when two factorsare close to the limiting value a change in the one not limitingmay have some appreciable effect in assimilation. This willshow itself about the inflexion of the curve where the limitingfactor is changing. For example, when carbon dioxide islimiting, increase of temperature may cause a small increaseof assimilation by increasing the rate of diffusion of the carbondioxide. But all minor details like these apart, the hypothesisof limiting factors rests broadly on the possibility by its meansof interpreting simply and logically the greatest number ofknown facts about the rate of carbon assimilation."*

Boysen-Jensen f studied the carbon assimilation of lightand shade plants : on plotting the r a t e of photosynthesisin unit time for unit area against light intensity, he foundthat the graph approximates to a logarithmic curve; at first,*when light is limiting, the course is more or less linear ; atlast, when a factor other than light, temperature or carbondioxide for example, is limiting the course is again linear andmore or less parallel to the abscissa axis. Between theseextremes the curve is continuous and no t abrupt since it isthe resultant, not of a single factor, bu t of two or more whichcome into play when their intensity is more or less equivalentto that of the original limiting factor. Harder j : investigatedthe problem, using, for the most part , the aquatic mossFcntirudis antipyretica which can be continually used in aseries of experiments so that the ill-defined factor of variationia material, inevitable when detached leaves are used, isavoided. Using the three factors, l ight intensity, carbondioxide supply, and temperature, he found that when twofactors were kept constant and the third varied, the curveobtained was approximately logarithmic. Further, he variedlight intensity and carbon dioxide supply, using potassiumhydrogen carbonate for its source. The following; table setsforth some of his results:—

* Smith: " Ana. Bot./* 1919, 33, 535. See also Maskell: " Proc.Roy. Soe.," B> 1928, i©2, 4&8.

f Boysea-Jensea : " Bot, Tidsk./' 1918, 36, 219.% Haider: " Jahib. "wiss. Bat.," 1921, 60, 531.

INTERACTION OF FACTORS II

Per Cent, ofKHCO3.

O-OI0*04o-i60-32

Light Intensity in Metre Candles.

167

0*120*26

667

O'4IO-9II'lO1-23

2000

o-752-243*454-70

6000

0-903*456-408-6o

18,000

i-o6470

"•3515*20

36,000

1-07

16-64

It will be seen that the intensity of carbon assimilationincreases both in the vertical and the horizontal directions,i.e. with increase in carbon dioxide and with increase in illu-mination. Harder concludes that a single factor appears tobe limiting only when it is very much weaker than the otherfactors. Its action is not directly proportional at all con-centrations or intensities of the other factors, thus the higherthe light intensity, the greater is the augmenting effect of anincrease in the concentration of carbon dioxide. That is,both of these factors become interlocked and play a part,so that the rate of carbon assimilation is dependent on theconcentration of carbon dioxide as well as the intensity ofillumination.

Lundegardh * found that shade plants such as Oxalisacetosella growing in an atmosphere containing the normalamount of carbon dioxide and in varying intensities of illu-mination gave a curve of the abrupt type, for in light aboveone-tenth of full sunlight no increase in photosynthesis wasobserved unless the amount of carbon dioxide were increased.Sun plants such as Nasturtium palustre, on the other hand,behaved differently: with low light intensities photosyn-thesis was proportional to the illumination, but with increasinglight, photosynthesis was slower than the increase in carbondioxide concentration ; that is, a proportional relationshipno longer obtained. And in an atmosphere relatively richin carbon dioxide, carbon assimilation was observed even inlight of an intensity one-fortieth that of direct sunlight. Insuch instances, the curves are of the logarithmic type and

* Lundeg&rdh : " Svensk. Bot. Tidsk./' 1921, 15, 46.


Lundegardh agrees with Harder tha t t h e factors of light i n -tensity and carbon dioxide concentrat ion, when above t h enormal pressure of the natural a tmosphere, mutually influenceone another. A like difference between s u n and shade leaveshas been observed by Stalfelt * in t h e gyrnnosperms Piceaexcelsa and Pinus sylvestris ; he found t h a t the intensity ofphotosynthesis was greater in shade l e a v e s than in sun leavesa t the same light intensity as is shown b y the following t a b l ein which the light intensity is in pe rcen tages of open daylight:and the photosynthesis is in mgs. of c a r b o n dioxide assimilatedper hour per gram of fresh weight.

light.

10203°40506o708090

Carbon Assimilation.

Picea exulsa.

Sun Leaves.

•07-34•58•78"95

1-101-231-331-42

Shade Leaves.

•47•93

1-241471-64178i-8g1-972-DO

Pinus sylvestris.

Sun. Leaves.

•40•89

1-24*\54i-8o2 0 2

——

Shade Leaves.

.721251-62I-9I2152-352532692«8l

The difference in the behaviour of sun a n d shade leases p r e -sumably is due to the fact that the g r o u n d shade flora is, f r o mthe character of its habitat, sur rounded by an atmospherericher in carbon dioxide than the upper a i r arid the air of m o r eopen situations ; also shade leaves are characterized by a m o r edelicate structure, wherefore imped iments to a rapid in te r -change between the internal atmosphere of the leaf and t h a tof the surrounding air are to a greater o r lesser extent removed.This structural factor is of impor tance , possibly of g r e a timportance, in the carbon assimilation of the higher p lan t s ,wherefore comparisons of the pho tos^n the t i c ability of th .eleaves of different plants or of t h e s a m e plant grown i n

• Stalfelt: " Medd. Stat. SkogsfflrsaksEtnsV 1921, 21, 22r.

INTERACTION OF FACTORS 13

different conditions may have no real significance. This wasrecognized by Warburg * who used in his experiments Chlorella,a green alga of the simplest possible morphology, the sphere.With respect to the factors light intensity and concentrationof carbon dioxide, he found that when carbon dioxide was insmall amount, the intensity of carbon assimilation was inproportion to this amount; that is, carbon dioxide was alimiting factor. But when the amount of carbon dioxide wasprogressively increased, the photosynthesis did not corre-spondingly increase and the curve became logarithmic. War-burg's conclusion is that the intensity of photosynthesis isproportional to the concentration of carbon dioxide and tothe concentration of a second substance which enters intocombination with i t ; that is, the intensity of photosynthesisis governed by the rate of a chemical reaction. It will beobvious that the diffusion factor in his experiments is reducedto a minimum.

The balance of evidence relating to the interaction ofthe light intensity and carbon dioxide concentration factorsfavours the view that a factor is strictly limiting only when itis very weak, or " in minimum," to use Harder's terminology ;when, however, their intensity or concentration more nearlyapproach one another, an increase in either will result in anincreased photosynthesis ; that is, the reaction intensity iscontrolled by both factors. This relates only to the twofactors mentioned; to what extent it applies to combinationsof other related factors is uncertain. It is not improbablethat the complete process of carbon assimilation consists ofseparate phases,! the most obvious of which are diffusion,photochemical, and chemical which is independent of light.The intensity of each phase is conditioned by a set of factorswhich may be peculiar to itself, thus light is effective in thephotochemical but not in the chemical, in which stage tem-perature is highly important. Further, the intensity of onephase may be governed by the intensity of another, thus theaccumulation of sugar in the leaves of the beet may effect a

* Warburg : " Biochem. Zeit.," 1919, 100, 230.j See Briggs : " Proc. Roy. Soc," B, 1920, 91 > 249.


cessation of the process at ^.bout noon on a bright day not-withstanding the fact that the conditions for photosynthesisapparently are most favourable.

The importance of the conditioning factors is obvious,and it is clear that the significance of any one cannot beevaluated without reference to others.

EXTERNAL FACTORS.

THE KAW MATERIALS.

Water and carbon dioxide are the requisite raw materialsfor carbon assimilation.

WATER.—Water is essential not only as such for the fabri-cation of food, but also to keep the leaf tissues in a condition

t^for the processes to take place. With regardto the water supply, the transpiration current is the immediatesource; it is, however, not convenient on the present oc-casion to consider the problems presented by this phenomenon.

Thoday found that the rate of carbon assimilation lessenedas the leaves of Helianthus annuus lost their turgidity; inan extreme instance, when the leaves were very flaccid, thestomates were all but closed and the increase in dry weightwas very small indeed. Some determinations by Thoday ofthe average increase in dry weight of leaves in different con-ditions of turgidity have been mentioned. If cells becomeplasmolysed, constructive activities must cease; if in suchcells the turgid condition be not recovered, death supervenes.

Iljin * has obtained results similar to those of Thoday,and his work stresses the indirect action of water, for the lossof water from the leaf results in the closure of the stomates,wherefore the supply of carbon dioxide is diminished and thusthe rate of carbon assimilation is depressed. If the loss ofwater is temporary and not too great, the guard cells regaintheir turgidity and the stomates open ; but if the loss of waterhas been excessive, recovery may not tak£ place and the guardcells die, wherefore the efficiency of the leaf suffers. In suchconditions, Iljin considers that the injury to the guard cells

* njin : " Jahrb. wiss. Bot.," 1922, 61, 670 ; " Flora/\i923, 16, 360.

WAITK is

h r ] i v *|, r< p a-t in t ie ' i ' , J r u, * t * •' ,! »t»i< \ v - * *\ iit r fav »i2r> tlit h\ »!:% v- - * -\ tr * .„ *is pn - nalilv, tht active MI* **I>* -O-* t* i * *

guard ctlK ; or the * thcr Land, a d< r * i *\ v • i vv s r •} * *.ctrta ;n p* nit, t,4v ur- tht formatter »»f <ta,cl. i» it »• t : •; «r ?be pa^ul , tliere i an txeesshe n ncentrati i . ! t' i' < < 11 *,tj.which inactivates the en/vnie rt«pt>n^ibk f >r t ie t ; .*: vwhich inattivation is not reversible, thu< a rtc*»\trv t» thtnormal conditions is prevented and the guard i tils no i »ngt rfunction. It was further observed that in starch-free guardcells, severe desiccation resulted in the disappearance of thesugar, presumably owing to the increased rate of respirationwhich obtains in these circumstance. Dastur * al.-.o imd*that the decline in the photosynthetie activity of leave* isdue to the shortage of the water supply, the inadequacy ofwhich eventually terminates this particular activity of theleaf. The cessation of photosynthesis is, however, not abrupt;the marginal and intervascular regions are the first to feel thedrought and from these regions the ending of photosynthesisspreads towards the centre and the main veins of the leaf.This shortage of water is due to two causes : the increase ofthe leaf area creates an increased demand for water owing tothe increase in photosynthesis and transpiration, but thespecific conductivity of the wood f remains unchanged afterthe trachea* have attained their permanent form, hence theincreased demand is not satisfied ; secondly, as more leavesunfold, the pull exerted by the living cells of the upper leaveson the water of the conducting columns increases whilst thepull exerted by the older leaves remain the same, henct thereis a deficient supply to the older leaves.

There is, in fact, a distinct correlation between the decreasein the rate of assimilation and the fall in water content perunit of leaf area, and if the two values for a set of leaves arcplotted, the points lie very nearly on a straight line. Thisis shown by the following observations on Abutilm Dmnmmimade in carefully controlled experiments:—

• Dastur : " Ann. Bot.," 1924, 38, 779 ; 1925, 39, 769-.f See Fanner : M Proc. Roy. Soc.," B, 1919, £©» 218, aja.

*£

1 * i i • * '. - r >J- • t- :• • * i - * •• , t r / *

(A* • * i 'x j . i i i ' V\ ! i '( i • k * T l Jr ! '» •• | t 4* ' i k • * *•' j t ^3

' h ^ ^ i t l t t x t r n t , | ' r t ^ i f l u j fh*- a r ! n ^ - I • * r l » u (,» \ H K H

U ' t u f ^ l u l v i O i r i 4 A H t * l t h r o u g h f h * u p ^ r k - n i ^ t - i * r ^ l ^ t ^

r u t i c u U r p * t h if ^ M ^ i h t n t r P l u i h ^ * - f « M ^ , 4 » t r , ' a ^ , ! t b r

t h e T u r n r > i i f e , h u t i h v u ^ r k « 4 F I- I ' K i k . i - a n + 4 ^ d t l U « » * n

p A t t i ^ I \ B U f k m a u i » * u n « l I *» < i i r i « * r w ^ u r H J i r t t t t h a t

t h e * • I r v r t t < t £ - 1 * 0 ' U * i n t e r * l i u t i ^ t . ^ A ^ I r u f « » r t i t i r u f r t * t l i c

I t fcborjcsmit! condition* tt Jp|«e4n t3h*t f:4^ati a i »*lir u«f cf

horn the su*l h l W t i < Mti Iswl Bcwt I'sai f^wt

i f f*t fa«4 tltet pUst* gt»mtt m »t4i neb i» fc»*i*« *f « »»ter

tram fc:.ArtKm dMA«jk Tfie ^mumiUt^m^ Hmr%m mm n>alfar Bwnwa graNrtli

* 9k*ckm»>* ITta.1 TfJMW Eo? Sec ,' ». il^i, •••»#»5< 5»1

CARBON DIOXIDE 17

distribution of the stomates, results which Brown and Escombeconfirmed in respect to plants with stomates on but one sur-face of the leaf, but in plants in which stomates occur on bothsurfaces of the leaf, they found that in bright sunlight theintake of carbon dioxide into the upper surface is greater thanwould be expected from the ratio of distribution of the sto-mates on the two sides ; in light of a lesser intensity, however,there is a closer, but not very close, correspondence betweenthe intake of carbon dioxide and the proportional distributionof the stomates. It is suggested * that the greater infusionfound to obtain into the upper side of amphistomatous leavesmay be accounted for in that partial opening of the stomatesis likely when the incidence of illumination is on that side, andthat since the palisade parenchyma is the more active part ofthe mesophyll, there will be a steeper diffusion gradient in theupper side which will promote a more rapid flow of carbondioxide through the stomates of the upper surface.

The movements of the carbon dioxide are in accordancewith the laws of gaseous diffusion; the pressure of carbondioxide in the active chlorenchyma will be very low, whilstin the atmosphere surrounding the leaf it will correspond to,say, three parts in 10,000. Thus there are set up diffusioncurrents the gradients of which vary according to the con-ditions, rate of use and degree of atmospheric motion. Theproblem of interchange between the gases contained in theleaf and in the surrounding atmosphere is not, however, sosimple as may appear from this statement. Brown and Es-combe, experimenting with leaves of Catalpa bignonioideSjfound that the rate of absorption of carbon dioxide at normaltemperature and pressure was about 0*07 c.c. per sq. cm. perhour; since the total area of the stomates was but 0-9 per cent,of the total leaf surface, it follows that carbon dioxide mustpass through the openings at the rate of 777 c.c. per sq. cm.per hour, an amount so considerable when regard is had tothe stomatal area and to the fact that this rate of absorptionis about fifty times greater than the absorption of atmosphericcarbon dioxide by a normal solution of caustic potash, that it

* Brown and Escombe: " Proc. Roy. Soc./' B, 1905, 76, 29.VOL. II.—2

' i

' f1

>, r M \ * i.

4 • t '

*' * i " * t * : 1* * «* t* • r t • « » i l l t

. \ r i " j " i j at: 1 t< I! ^ f i l

i t

?'fining ,#t tthr* ^rfwurt fr*nn

**t * artmnTi

that

t*»the t»|A«fucig t • tbey

m m 0vm imm , i ^

INFUSION OF CARBON DIOXIDE ig

stomate varying from the atmospheric density, p, to a lowerdensity, p', at the stomate cut perpendicularly by the linesof flow of carbon dioxide converging to the opening of thestomate * (Fig. i) . Thus it is that the increased flow of gasthrough the stomate is possible.

The second section in the route of the carbon dioxide isthrough the tube formed by the guard cells. Through thistube the flow is inversely proportional to the length of thetube, but the system of external shells increases the resistanceto the flow (Fig. 2). Finally, in the third section the tubeopens into the air-space system of the mesophyll bounded bythe absorbing surfaces of the chlorenchyma ; here the con-

FIG. 2.

verse of the first part of the path obtains, diffusion shells overthe lower opening of the stomate, where the density of thecarbon dioxide is //, being formed (Fig. 3). Thus in the wholesystem there is a gradient of density from p to, say, 0, with aset of shells at both ends of the stomatal tube (Fig. 4).

The obstruction to gaseous diffusion inseparable from amultiperforate septum such as -the stomatal epidermis of aleaf, varies apcording to the distance apart of the perforations :if they are placed at distances roughly equal to ten times thediameter of a perforation, each will act independently withoutinterference by its neighbours and conform to the law of di-ameters. When situated more closely together, it was found

• Q = 2k(p +

20 T H E S Y N T H E S I S O F C A R B O H Y D R A T E S

that the obstacle to diffusion w a s m u c h less t h a n the a c t u a l

obstruct ion of area b y the solid port ions of the s e p t u m . T o

quote concrete examples , B r o w n and E s c o m b e ca lcu la ted t h a t

in Helianthus the leaves could absorb 2-578 c.c. of c a r b o n

dioxide per sq. cm. per hour in m o v i n g air and 2-095 c .c . per

sq. cm. per hour in still air, as suming that the s t o m a t e s are

circular in shape. B y actual measurement it w a s f o u n d t h a t

in this same plant * in diffused l ight at a temperature of 1 9 0 C ,

0-434 c.c. of carbon d iox ide per sq . c m . per hour w a s absorbed ,

a n a m o u n t m u c h less t h a n the capaci ty of the s t o m a t e s t o

supply . S o m e of B r o w n a n d Escombe ' s conclusions h a v e b e e n

criticized b y Jeffreys f w h o has made a mathemat i ca l s t u d y of

evaporat ion, the problems of w h i c h are m u c h t h e s a m e as

those of gaseous diffusion. H e points out tha t further in -

ves t iga t ion is required and remarks t h a t " i t is n o t o b v i o u s

t h a t the surrounding s t o m a t a will not interfere w i t h the a c t i o n

of any individual to an important e x t e n t , a n d a w i n d b l o w i n g

• Brown and Escombe: " Proc. Roy. Soc./' B, 1905, 76, 29.t Jeffreys : " Pfcril. Mag./* 1918, 35, 270.

INFUSION OF CARBON DIOXIDE 21

over the surface, though unimportant for a single stoma,may be important when there are thousands spread over aconsiderable area." If there is no interference between thedensity shells of stomata ten diameters or more apart, so thateach stomate acts independently according to the law ofdiameters, an impossible situation results. Thus if there aremore than 600 stomata per sq. cm. of leaf surface, the rateof evaporation from them, which is the same thing as carbondioxide diffusion, would be greater than that from the wholeleaf surface, wherefore it must be concluded that gaseousdiffusion must be greatly impeded by the presence of otherstomata. It is not until stomata contract to one-fiftieth oftheir original diameters, that the rate of evaporation, or gaseousdiffusion, will be practically independent of the diameter.When the air is in motion and the stomata are fully open,their total evaporation, or absorption of carbon dioxide, doesnot differ much from the evaporation, or absorption, from anarea equal to the leaf surface ; * it is only when the number ofstomates per unit area is small, that each may act indepen-dently, and the law of diameters obtain.

The conditions of humidity of atmosphere, temperatureand illumination, which affect the size of the stomates, ob-viously will affect the infusion rate of carbon dioxide.f Inaddition to these movements, traceable to definite externalcauses, there is sometimes—the leaf of the cherry laurel,for instance—a diurnal rhythm of stomatal opening even whenthe leaf is grown in controlled conditions and constantlyilluminated. The result of this is that in a normal atmos-phere, when the leaf is in constant light, there is a diurnalrhythm in the carbon assimilation, the rate of which falls toa very low value during the night and rises again next morning.This diurnal, and also seasonal, variation in the assimilationrate runs parallel with the diurnal and seasonal rhythms ofstomatal openings. It is only when the external atmosphere

* Cf. Renner : " Flora/' 1910, 100, 451.t See Darwin : " Phil. Trans. Roy. Soc," B, 1898, 190, 531 ; 1916,

107, 413. Lloyd : " Carnegie Inst. Washington," No. 82, 1908. Knight:' Ann. Bot.," 1916, 30, 57.

22 THE SYNTHESIS OF CAR P.nllYJ )R VIE-

is so enriched with carbon dioxide that li^ht and not carbondioxide is limiting the rate, that a regular rate of carbonassimilation is maintained for over twenty-f^ur hours in aconstant illumination. Maskell,* to wiu.nri these observationsare due, suggests that the apparent ran* of carbon assimilation,when carbon dioxide is limiting, is determined by the externalcarbon dioxide concentration and a series of resistances whichcomprise the stomatal resistance to diffusion, the resistancein the intercellular space system and m the liquid diffusionpath froni the cell surface to the surface of the chloroplasttogether with the resistances associated with the photochemicaland dark phases of the actual process.

In natural conditions carbon assimilation is limited by thelow pressure of carbon dioxide in the atmosphere, the highvalues obtained in experiments in conditions involving anincreased supply of the gas never being attained.

In experimental work with land plants a limit is set to theincrease of carbon dioxide supply by the narcotic effect of thegas when in excess,t 25 per cent, generally will inhibit growth ;aquatic plants, on the other hand, are able to withstand arelatively high concentration. Blackrnan and Smith J foundthat Fontinalis and Elodea in water, with a carbon dioxideconcentration of 33-92 and 35-82 per cent, of saturation, andunder identical intensity of illumination and at temperaturesof 23°C. and 280 C. respectively, assimilated -0223 and '0249gram of carbon dioxide per hour per standard area of 137sq. cm. Fontinalis is less efficient, for reasons not finally deter-mined, than aquatic angiosperms such as Elodea, Fotamogetenand Ceratophyllum in utilizing carbon dioxide. Blackmail andSmith, experimenting -with Elodm and Fmtinalis in constantconditions of light and temperature and with a carbon dioxidesupply ranging from -0025 to -0540 gram per 100 c.c. of water,found that the carbon assimilation increases steadily in pro-portion to the increase in the supply of carbon dioxide. Whenthe assimilation reaches about 023 gram of carbon dioxide

• Maskell: " Proc Roy. Soc." B, 1928, wi« 467. 4**.f See C&apia : " Fkxm," 190a, t> $4!.% Blackman and Smitt*: '* Brae, Key. Soc.,'* B, 1911,

AQUATIC PLANTS 25

per hour, however, there is no further increase with an aug-mented carbon dioxide supply unless the light intensity beincreased. In other words, light intensity in this connectionis a limiting factor.

In all such experiments with aquatic plants, it is to beremembered that the conditions are not precisely the same asfor a subaerial plant, since the resistance to the din us ion ofcarbon dioxide to the chloroplast is much less in the aquatichabitat, being only the liquid diffusion phase of the land plant,i.e. the passage of carbon dioxide in solution from the solutionin contact with the surface of the plant to the chlorophyllsurface. Further, in normal conditions the amount of carbondioxide dissolved in the water surrounding the assimilatingplant depends on the rate of diffusion from the source of supply;it is, in general terms, slow and is dependent on such factorsas temperature, relative gaseous pressures, degree of agitation,and so on. The work of Blackman and Smith has been fol-lowed by others, Harder for instance, to whose observationsallusion has already been made, some of whom used a bicar-bonate solution as a source of carbon dioxide. The resultsobtained are not always comparable, and the question ariseswhether the carbon dioxide in the free state only is assimilableby the plant and not —HCO3 ions and the undissociatedfraction of such salts as sodium hydrogen carbonate. A com-parison between the two types of solution has been made byJames * working with Fontinalis a?itipyretica in controlledlaboratory conditions. He found that a flow of 40a c.c. ofsodium bicarbonate solution per hour resulted in a higher rateof carbon assimilation as compared with a solution of purecarbon dioxide of an equal partial pressure when no otherfactor was limiting ; but with a flow of 600 c.c. of bicarbonatesolution per hour and with a low intensity of light, and there-fore a slow rate of carbon assimilation, the two solutions gavethe same assimilation values. An increase in the light in-tensity resulted in the bicarbonate solution giving a greaterrate of carbon assimilation as compared with the carbondioxide solution. It thus appears tha t in bicarbonate solutions

* James: "Proc. Hoy. Soc.," B, 1928, 103* 1.


only the free carbon dioxide is available for carbon assimi-lation ; such a solution when dilute—the range used by Jamesvaried between -05 to 0*4 per cent.—is suitable for experimentalwork. Stronger solutions are to be avoided owing to theinjurious effect of the increased alkalinity.

The difficulties inseparable from critical investigations inthe field are obvious : of recent work, that of McLean * onthe carbon dioxide absorption of Cocos leaves in naturalconditions may be mentioned. He found that the rate ofabsorption is at a maximum in the morning, a depression ob-tains at mid-day, followed by an increase in the afternoon andthen a final decline towards sunset. Similar values wereobtained for detached leaves, but the curve showed a singlemaximum at about noon instead of two maxima which nor-mally obtain with attached leaves, for which difference thereis no adequate explanation. Clearly some internal limitingfactor is operating, possibly connected with the accumulationof the products of carbon assimilation (p. 49). It was alsofound that immature and old leaves absorbed carbon dioxideat a lesser rate than leaves of an intermediate age.

In view of the small amount of carbon dioxide in the normalatmosphere, it must not infrequently happen that the inten-sity of carbon assimilation is limited by this factor, wherefore,since the days of Kreusler,*f who was the first to considerthis aspect, much work has been done both on the Continentand, more recently, in this country to increase the crop yieldby the enrichment of the atmosphere by the addition ofcarbon dioxide supplied by artificial means. J The resultsobtained are in some instances contradictory, presumablyowing to the difficulty of control in field cultivation, and alsoin greenhouse culture where it is difficult to maintain a con-stant increased pressure of carbon dioxide although tempera-ture, humidity and soil conditions are amenable to regulation.

* McLean: " Ann. Bot./' 1920, 34, 367.t Kreusler : " Landw. JanrW 1885, 14, 913.% Cerigkelli ; " Ann. Sci. Agron./' 1921,38, 68. Demoussy : " Compt.

rend./' 1903, 136* 325 ; 1904, 138, 291; 1904, 139, 883. Fischer: " Beih.deat. bot. Ges.," 1927, 45, 331. Lundegardh: " Der Kreislauf derKohlensattre in der Natar/' Jena, 1924. Reinau: " Zeit. angew. Oxem./11926, 16, 495. Reidel: " Chem. Zeit./' 1921, 104, 829.

INFLUENCE OF TEMPERATURE 25

In consequence of these divergent results, Bolus and Hen-derson * have undertaken a critical investigation of the subjectin laboratory conditions, and in their preliminary communi-cation they report that the enrichment of the air with carbondioxide results in a large increase in the dry weight of plantsof Cucumis saliva as compared with the plants grown innormal air, which increase is evident within two or three daysfrom the beginning of the experiment. The following tablesummarizes their results :—

Days inc62.

1089

19X4

Mean DayTemp. °C.

27*029-029*925-636-2

Mean NightTemp. °C.

22-422-223-014-825*0

Mean CO2 Cone.Parts per 10,000.

Experimental.

25-827*542-428-93i'3

Control.

4-24-03'64-33*9

Per Cent. Increaseof Dry Weight

Experimental overControl.

50-4 ± 3-1578-7 ± 6-686-3 ± 6-242-0 ± 13-76o-6 ± 8-5

TEMPERATURE.

The statement that chemical change is profoundly in-fluenced by temperature needs no elaboration : in the majorityof instances an increased temperature accelerates a reaction;examples in which the contrary occurs are very few. The Lawof van't Hoff states that for every rise in temperature of 10° C.the reaction is increased at a definite rate, in general termsdoubled or trebled, the precise value of which is specific to thereaction, f The plant, however, is not a test tube but a verycomplex system of reacting substances, wherefore it is only inexperiments most carefully controlled and skilfully conductedthat approaches to the mathematical preciseness of well-ascertained physico-chemical laws will obtain.

Long has it been known that an increased temperatureresults in an increased carbon assimilation, but it was not

* Bolus and Henderson : " Ann. Bot./' 1928, 42, 509.f This factor is termed the temperature coefficient and is represented

by the symbol K with a number attached indicating the number of degreesconcerned, e.g. Klo.


before F. F. Blackmail's experimental researches on vegetableassimilation and respiration that the subject was really criti-cally examined. Matthaci * at the outset of her work on theeffect of temperature on carbon assimilation found that inaddition to the influential external conditions there is animportant internal condition of a plant or plant member, theresult of previous treatment such as excess of food, starvation,and change in temperature.

This is a common experience; thus Harderf found that theblue-green alga Phormidiutn foveolarutn, if cultivated in a lightof low intensity, can, in the photosynthetic process, makeuse of light of lower intensities than can plants grown in highlight intensity. Similarly plants cultivated in strong lightcan assimilate more readily in such light than can plantswhich were previously cultivated in weak light. Likewisefor temperature % : in submerged aquatic plants cultivatedfor three months at a low temperature, 4°-8° C, carbonassimilation is less at i8° C. than at 8° C , but those cultivatedat a high temperature, 2O° C, show a rise in carbon assimi-lation with increasing temperature. In higher light inten-sit ies, on the other handr both cultures show a rise in carbonassimilation with increasing temperature, but the rise is morerapid in those plants grown at higher temperatures. Warburgand Negelein§ observed that when Chlorella was cultivatedin a high light, but a small proportion of the available energywas photosyntbeticaily employed, but when grown undera low illumination the reverse obtained Stanescue || alsofound that lilac and other plants if kept in darkness for 48hours, so that the leaves are freed from starch, show a morerapid carbon assimilation than the leaves of plants grownin normal conditions, although the maximum assimilation isattained at about the same time of day.

Such facts have a most important bearing on experimentalresults, so that in order to have comparable figures it is

* JCttttei: " miL Tram. Roy. Soc," B, 1904, I97» 47-f Harder: u ftofct. Bet./' 1925,15, 305.X Htankr : " Jalirb, idss. Bet./' 1924.64* 1%§ Wstrfwirg aad Mcgeleia : " £dt* phys. Cbem,/* 1923, i©6* 191.I Sfc " Compt. itnd. Soc* bW./' 1926, $$» 132.


essential that the previous history of all the material employedshould he the same.

In Primus lanrocerasus Matthsei found that for each tem-perature to which the leaves were subjected there is a definiteamount of carbon assimilation, the maximal assimilation forthat temperature, which cannot be exceeded and cannot beattained unless the illumination be of sufficient intensity andthe carbon dioxide be adequate in amount. These maximalamounts of assimilation increase rapidly with rising tempera-tures, but at the higher temperatures the initial rate forthat temperature cannot be maintained for long but falls offregularly at a rate dependent on the temperature ; the higherthe temperature the quicker the fall, but the rate of fall isnot maintained. Thus there is a time factor for the highertemperatures. Fig. 5 summarizes the results obtained byMatthaei; it will be seen that the turning-point is 37*5° C.which was found to be within a few degrees of the tempera-ture fatal to the leaf.

The results obtained for temperatures below 25° C. con-formed to van't HofFs Law, the coefficient of increase in therate of carbon assimilation for a rise of io° C. being 2*1. Insubsequent investigations Blackrnan and Matthaei found theassimilation coefficient for the leaves of Helianthus tviberosusto be K10= 2-3, whilst for Elodea, K10 = 2-05.* The culmi-nating point of the assimilatory curve (Fig. 5) in respect toincreasing temperature is important and is paralleled in en-zyme action ; in view of the results obtained by Willstatterand Stoll, it is not unlikely that the inhibition or destructionof an enzyme at these higher temperatures may be the limitingfactor. Osterhout and Haas | obtained a coefficient of 1*81for Ulvarigida between 170 and 270 C, a figure which, accord-ing to Smith % is probably too low. Warburg § investigatedthe same phenomenon in Chlorella and thus avoided the diffi-culties inseparable from morphological complexity with avarying internal temperature and possibly an uneven rate of

* Blackmail and Smith : " Proc. Roy. Soc.," B, 1911, 83, 389.f Osterhout and A.. R. C. Haas: " Jour. Gen. PhysioU," 1919, i» 295.{ Smith: " Ann. Bot./' 1919, 33, 517.§ Warburg : " Biochem. Zeit.," 1919, 100, 230.

THE SYNTHESIS OF CARBOHYDRATES

gaseous diffusion. The values he obtained show that tem-perature has no effect on the rate of photosynthesis when theillumination is very low ; with higher light intensities, the

-238

157

uE-|°70iri•548238

I<

-149

-102

•KT & •» ' 20' 30* 4ffTemp:

FIG. 5.coefficient decreases with increasing temperature. Thus with% fthtive light intensity of 45r between 5-4o-i0Oj Qu « 4-5 ;bttwecn io* - ao#, Qlft « 2-1; md hetwrna >° - 300,


Q10 = l-6. Comparable observations \ave been made byYabusoe * and also by Lundegardh f who found the tempera-ture coefficient to be 1-03-1-45 a t 3°c-35° in "a strong lightand with an atmosphere enriched to 1-2 per cent, of carbondioxide. The maximum rate occurred at 350 C. and providedthat the initial heating was not too prolonged, the time factorwas ill-defined. Blackman observed that under strong illu-mination or in a high concentration of carbon dioxide, a riseof io° in temperature, from 15° to 250, roughly doubles thevelocity of photosynthesis ; this is termed by Warburg % the"Blackman Reaction " and it indicates that in the condi-tions specified, the rate of carbon assimilation is determinedby a chemical rather than a photochemical reaction, for, ashas been seen, with a low light intensity a rise in temperaturedoes not affect the rate of the reaction. The Blackmanreaction was originally thought by Warburg to be a prelimi-nary reaction which led to the formation of an unstable deri-vative of carbon dioxide which was decomposed by light, butlater he and Uyesugi adopted Willstatter's idea that in anearly phase of photosynthesis a peroxide is formed and thatthe Blackman reaction consists in the separation of oxygenfrom this peroxide.

CH2O[OJ -*• CH2O -f Oa.In much experimental work on photosynthesis, moderate

and higher temperatures have, for obvious reasons, been em-ployed ; it is, however, to be remembered that carbon as-similation takes place at extreme temperatures according tothe specific physiology of the plant. McGee§ found thatOpuntia can function at 55° C. aad Matthaei || found a measur-able photosynthesis in the cherry laurel leaf at — 6° C.Henrici |f similarly observed a definite photosynthesis in lichensat — 200 C. and in alpine phanerogams at — 160 C.

* Yabusoe: id., 1924, 152, 498. | I/undegardh: id., 1924, 154, 195.£ Warburg: " Biochem. Zeit.," 1919, 100, 230; 1926, i669 386.

Warburg and Uyesugi: id., 1924, 146, 486.§ McGee : " Year-Book Carnegie last. Wash./' 1921, 20, 47.|| Matthaei : lac. ciLJHenxici : " Verhand. nat. G-es. Basel," 1919, 30, 43 ; 1921, 32, 107.

See also Ewart: " Jouni. Linn. Soc, Bot./' 1896, 31, 364. Wurmser andJacquot: " Bull. Soc. chim. biol.," 1923, 5, 305.

3o THE SYNTHESIS OF CARBOHYDRATES

The internal temperature of assimilating leaves will varyconsiderably in accordance with such conditions as the inten-sity and quality of the light, the character of the leaf surfaceand so on ; Blackman and Matt had * demonstrated, bythermoelectric means, an excess of 7° C. to 160 C. in the leavesof Primus laurocerasus in bright sunlight above the adjacentshade temperature.

ILLUMINATION.

The importance of light in the formation of chlorophyllis a commonplace in. physiological experience ; chlorophyllmay, however, be formed in the dark, or in comparative dark-ness, as was shown by Schirnper f for Alg<ne and Burgerstein $for conifers, and in other plants.§ In the majority of plants,however, some exposure to light is necessary to produce thegreen pigment, presumably by its action on a precursor, termedchlorophyllogen by Monteverde and Lubimenko,!] accordingto whom plants grown in darkness for an excessive periodlose their ability to form chlorophyll when exposed to light.In passing it may be mentioned that in addition to light,other conditions are requisite for chlorophyll formation, es-pecially an adequate temperature, again a fact of commonexperience, and a supply of carbohydrate.^ Mention alsomay be made of the well-known fact that a highlight intensity,presumably owing to its destructive action on chlorophyll,may bring about a cessation of photosynthesis.**

Of the light falling on a leaf, a portion only is availablefor the chloroplasts since varying amounts, according to thecharacteristics of the leaf surface, such as the presence ofcuticle and of hairs, the thickness of the leaf and so on, will

* Blackmail and Matthaei: Ice. cti.t Sddmpcr : " Jahr. wiss. Bot./' 1&S5, 16, 1.I Biirgersteia: " Ber. dent. "bot. Ges.," 1900, iS, 168.§ See Vol. I., p. 311.ijMoatevercle aad Lubimeako: "Bull. Jard. Iinp. Bot. St. Peters-

bewarg/' 1909, J>» 27; " Ball. Acad. Sci. St. "Petersbourg," 191 -2t 609.f See Palladia: **Ber. deut. bat. Cka," 1891, ip» 229. Mansky:

"Itiodteoi. Zcfc." 1922,132, x8.**Se®Ewart; "Ajia. Bot,/* 1S97, u r 459; 1S9S, 1:2, 379. Panta-

aoili: "J&kr. wisa. Bat," 1903, 3^, 167. Urspraiig: *f Ber. d«nt. bot,& " M* 57-

INFLUENCE OF ILLUMINATION 31

be lost by reflection, absorption or transmission. Of theenergy absorbed by the leaf, many have shown that a smallproportion only, and this in varying quantity, is used in car-bon assimilation. Owing to ignorance of certain factors andthe degree of their significance in the sequence of carbonassimilation, it is not possible to give a satisfactory accountof the energy relationships of the plant. It may, however,be mentioned that Brown and Escornbe,* the first to attemptthe drawing of an energy balance sheet of the leaf, concludedthat Polygonum Weyrichii used from -42 to 1-66 per cent, ofthe available radiant energy for carbon assimilation, figuresbased partly on observation and partly on calculation:Puriewitsch,f on the other hand, found that JPolygonttmSacchalinense similarly employed from 2*5 to 7*7 per cent, ofthe radiant energy.

In no natural conditions is the full radiant energy madeuse of by plants: the photosynthetic value of the noontidesunshine at the summer solstice in these latitudes lies, accord-ing to Blackman and Matthaei,f between -04 and -05 gram ofcarbon dioxide per 50 sq. cm. of leaf surface per hour ; thehighest assimilation actually measured by these workers was•0290 gram.

The general statement that carbon assimilation varies "withthe intensity of the illumination is true only when light is thelimiting factor ; other factors, particularly temperature, areintimately associated in the process in nature. For thisreason it is impossible to consider the effect of one conditionto the exclusion of the other factors, a fact well demonstratedby Blackman and Matth.3&i.§ In one of their experiments, anabstract of which is given on p. 32, the leaves of Helicmikustuberosus were surrounded by an atmosphere containing onthe average 4 per cent, carbon dioxide and the light throughoutwas diffused and of varying intensity.

It will be seen from observations 2 to 4 and 6 and 7 thatthe assimilation was remarkably uniform in conditions of

* Brown and Escombe : " Proc. Roy. Sac./' B, 1905, 76, 29.f Puriewitsch.: " Jafcrb. wiss. Bot.," 1914, 53, 210.J Blackmail and Matthuei : " Proc. Roy. Soc.," B, 1905, 7 , 402.§ Lac. dt.

THE SYNTHESIS OF CARBOHYDRATES

constant temperature and varying illumination ; this showsthat temperature was the limiting factor. The low value inthe first observation was due to the low light intensity. Inobservation 5 in which the illumination was about the same asin 4, the temperature was raised from 1770 to 30*5° C, withthe result that the assimilation was about doubled : but sinceat this higher temperature an assimilation of at least -0289gram is possible, it follows that here the illumination was thelimiting factor.

An important generalization arrived at by F. F. Blackmanand his collaborators is that equal intensities of light incidenton equal areas of leaf produce the same amount of assimilation,

IllumioatioB.

!! 1, Very overcast .i 2. Raining, but lighter .i 3. Raining, but lighter stillI 4. Raining, heavy cloudsj 5. Raining! 6. Much brighter .

7. Sunshine, but leaf in shade .

Temperature ofLeaf °C.

I7-7181817-730-518-218-4

Real Assimilation in Gramsper 50 Sq. Cm. per Hour.

•0062•0089•OO90•0089•OI63•0089•0092

provided that light is the limiting factor and that the tempera-ture does not involve the so-called time factor; agreementwithin 5 per cent, was found to obtain in such diverse in-stances as Hdianthus, Prunus, Bomareaf Aponegeton, Elodeaand Fontinolis. Hence the conclusion is reached that " leavesin general have the same coefficient of economy in the photo-synthetic * economy/'

The amount of light required by a leaf is a specific valuefor a given temperature; thus in the examples studied byBlackman and Matthaei, Helianihus and JPrunus have at lowtemperatures similar assirailatory maxima which diverge athigher temperatures. At 29*5° C. Helianthus can assimilate

* The word " photosynthetic '* is here used in its literal sense, thatphase of carbon assimilation in which a chemical change is brought aboutby light alone. This phase is followed by a purely chemical phase,, knownas the dark p&ue, which is independent of light hut is accelerated by anincrease in temperature.

RADIANT ENERGY 33

twice as much carbon dioxide as can Prunus, but in so doing itrequires twice the amount of illumination. The difference in thetwo leaves lies in their having different coefficients of accelera-tion of their assirnilatory activity with, increased temperature.

Of the use made by the plant of specific parts of the spec-trum, it has been generally accepted that those wave-lengthsassociated with the prominent bands in the red of the absorp-tion spectrum of chlorophyll were the only ones concernedwith carbon assimilation. It is. however, to be rememberedthat the light absorbed by such a structure as a leaf is notnecessarily all employed in the photosynthetic process, that raysof different wave-lengths are not equally absorbed and thatdifferent wave-lengths have different energy values. Further,the most superficial chloroplasts are illuminated by practicallyunaltered daylight, but those more deeply situated receivelight from which certain rays have been abstracted in variousdegrees by the more superficial plastids and thus is of differentquality as compared with normal daylight. Wherefore,in relevant investigations, light intensities must be known;the quality of light, i.e. its range in wave-lengths, be ascer-tained ; and light must be, as far as is possible, the factordetermining the rate of the process. Since radiant energymust be converted into chemical energy, it is obvious that theessential requirement is the amount of energy used, or atany rate available for the use of the plant; in other words,it is profitless to consider the intensity of carbon assimilationin different coloured lights without determining their energyvalues.

Ursprung * found that radiant energy of any wave-length,including the greater par t of the ultra-violet, is capable ofinducing starch formation in green leaves, provided that asufficient time be allowed. He even found starch to be formedon exposure to infra-red rays ; it is, however, not clear whetherthis starch was a direct product of carbon assimilation sincestarch can be formed in the dark from pre-existing sugars.Ursprung found in Pkalaris arundinacea var. -picta,y which

*Ursprung : " Ber. deut. bot. Gesells.," 1917, 35, 44 ; 1918, 36, 73,86, i n , 122.

VOL. II.—3


plant was selected since its variegated leaves permit a com-parison between the green and non-green portions, that inthe red region of the spectrum the maximum absorptionobtains between the B and C lines, in the green the absorptionis general, whilst in the violet the absorption is greater thanbetween the B and C lines of the red, and beyond this thereis a rapid fall. Subsequent work on Phaseolus showed a sharprise in the assimilation curve from the outer limits of the redto a maximum situated near the C line, from which point thereis a gradual fall towards the violet. This curve does notcorrespond, more especially as regards the region of shorterwave-lengths, with the curve of absorption which rises fromE, in the green region, to the violet. In view of the fact thatcarbon assimilation takes place in this part of the spectrum,it is obvious that some new conditioning factor is operative;it is suggested that this is found in the action of the violetrays causing the stomates partially to close.

Lubimenko* found from controlled experiments that photo-synthesis is more active in the red than in the blue rays; itis only in shade plants that the process is equally intensein the two regions. Wurmser,f in his investigations on thesubject, used Ulva lactuca and from his measurements foundthat the ratio of carbon assimilation to the amount of energyabsorbed was 1-0 for red light, 4*0 for green and 2*35 for violetlight, which means that the green rays although absorbed ina much lesser degree are utilized four times more than thered. From further investigations he found that Ulva hasa photosynthetic efficiency of 59 per cent, in red light and83 per cent, in green light. The value obtained for the redrays does not, he thinks, represent maximum efficiency. Heconcludes that the efficiency is much the same for all wave-lengths and lies between 70 and 80 per cent.

Warburg and Negelein,J in most carefully controlled ex-

• Lubimenko : " Compt. rend./' 1923, 177, 606.t Wurmser : •*' Recherches sur l'assimilation chlorophyllienne/' Paris,

1921; " Bull. Soc. chim. biol./* 1923, 6, 487; " Ann. Physiol. Physiochim.Biol./' 1925, if 47.

% Warburg and Negelein: " Zeit. pnysikai, Chem.," 1922, 102, 235 ;3» l06f 191; " Natmrwiss," 1922, 10, 647.

CHROMATIC ADAPTATION 35

periments, investigated the quantity of-radiant energy ab-sorbed in relation to the work done in carbon assimilation.The ratio between these two values is a measure of the efficiencyof the organism in this particular connection ; thus if the rad-iant energy absorbed by the plant be E and the energy utilizedbe U, then the ratio U/E will give an index of efficiency. Thegreater the value of E, the smaller is the ratio ; with decreasingintensity of E the value rises until it reaches a limit, -whichlimit marks the point of maximum efficiency. Using a cultureof Chlorella, which had been grown in uniform conditionswith a low light intensity, and keeping the illumination limit-ing, a mean value of 59 per cent, and a maximum value of63*5 per cent, was obtained in a red light of a spectral range570-645 fLfx. Warburg and Ncgeiein also compared thephotosynthetic activity of the same plant illuminated withlight of different wave-lengths. The accompanying tablesummarizes their observations :—

Light. Per Cent. Efficiency.Red . 610-690 ftp 59Yellow . 578 JU/U 53-3Green . 546 fifx 44-4Blue . 436 fifji 33-8

It will be seen that the efficiency decreases with the decreasein the wave-length. Also, these values differ from thoseobtained by other investigators, mentioned above, using dif-ferent plants and different methods, a result to be expectedfor the reasons earlier given.

CHROMATIC ADAPTATION.—This consideration of the quality

of light naturally introduces the problem of chromatic adap-tation. As is well known, the colour of deep water is greenowing to the greater absorption by the water of the long rays,which are strikingly absorbed and utilized by the chloroplasts.A plant growing in deep water, therefore, is handicapped bythe different quality of light. Many marine algae have, inaddition to their chlorophyll, water-soluble pigments—-phyco-erythrin for example *—-which are complementary in colourto that of the surroundings and thus are able to increase the

* See Vol. I, p. 353.3*


amount of radiant "energy absorbed. This, in brief, is Engle-mann's * theory of chromatic adaptation, a theory whichhas been extended to include many plants, Phaeophyceae,Bacilliariales, Bacteria, and terrestrial plants which possessthese extra pigments. Gaidukov f in his experiments withOscillaria found that the cultures changed their colour, violetin dim light, to the complementary colour to that in whichthey were growing ; thus if grown in red light, the new cellswould be green, and so on. Whether this is a true exampleof chromatic adaptation is, perhaps, doubtful, for otherauthors have found like colour changes to be effected by theconditions of nutrition, a deficiency of iron, by wounding, andso on.

Richter $ considers that the determining factor in thecolour of red algae is the intensity rather than the qualityof the light, the water soluble pigments playing no part inthe photosynthetic process. Wurmser,§ on the other hand,whilst agreeing that the presence of phycoerythrin is an adap-tation to weak light, concludes that it also is an optical sen-sitizer which aids the chloroplasts to absorb green light.

Harder || studied Phormidium foveolarum, which is purplein blue light, owing to the development of phycoerythrin, andgreen in red light, owing to the development of phycocyanin.He found that the purple plants have a higher rate of photo-synthesis in blue light than in red, whilst the green forms showa higher rate in red than in blue light. He thus supportsEnglemann's hypothesis, but his experiments also show thatlight intensity is of great importance, but even so there isalways a higher rate of photosynthesis in the plants whichhave a complementary pigment. On an earlier page mentionhas been made of Harder's observation that Phormidiumwhen cultivated in a strong light shows a higher rate of carbon

* Englemann: " Bot. Ztg./' 1883, 41, 1, 17 ; 1884, 4a, 81, 97.f Gaidukov: " Preuss. Akad. Wiss./' 1902, 5, 1 ; " Ber. deut. bot.

Ges.," 1903, aif 484, 517, 535.t Eickter: " Ber. deut. bot. Ges.," 1912, 30, 280.§ Wurmser : " Recherches sur l'assimilation chlorophyllienne," Paris,

1921.|| Harder : " Ber. deut. bot. Ges.," 1922, 40, 26 ; ." Zeit. Bot./' 1923,

*5> 305-

ULTRA-VIOLET RADIATION 37

assimilation in a high light intensity than do plants grown ina weak light and vice versa.

The balance of evidence favours the view that comple-mentary chromatic adaptation is a natural phenomenonalthough not so widely spread as has been supposed, and thatit is of little value to study the effect of light quality withoutconsidering light intensity.

To return to the main thesis: in all these observations thesignificant fact is that the longer rays^bring about ajrofoujidcherjaical.^change, whereas in inorganic photochemistry theshort rays are by far the most efficient, the longer having littleor no action. For this reason not a little work has been doneon the action of the short rays, the ultra-violet, in bringingabout the formation of organic compounds from carbondioxide and water. All such work, interesting though itbe, throws no light on the fundamental problem of carbonassimilation, since the green plant can effect the transformationin conditions which preclude the presence of ultra-violetillumination. With this caution a brief r6sum6 of suchinvestigations may be given.

Stoklasa and Zdobnicky * brought about the synthesisof carbohydrate in the absence of chlorophyll by passing lightfrom a quartz mercury lamp through a mica window into avessel containing a mixture of carbon dioxide and nascenthydrogen. Formaldehyde was slowly produced, and this, inthe presence of caustic potash, was polymerized with theformation of a sugar or a mixture of sugars which was opticallyinactive and not fermentable by yeast. The authors suggestedthat the chlorophyll in plants acts as a means of absorbingultra-violet rays, a suggestion which has since been found tobe true. Bertholet and Gaudechon f found that formalde-hyde is produced by the action of ultra-violet rays on carbondioxide in the presence of a reducing agent, and, reversely, thatcarbohydrates are decomposed by sunlight and by ultra-violet

* Stoklasa and Zdobnicky : " Chem. Zeit.," 1910, 945.f Bertholet and Gaudechon : " Compt. rend./' 1910, 150, 1690, 151,

395 ; 1912, I55> 401, 831.


light from a mercury-vapour lamp. The products of decom-position are carbon monoxide, carbon dioxide, methane andhydrogen; aldehydic sugars differ from ketonic sugars both inthe readiness with which they are decomposed and in thecomposition of the gaseous mixtures produced.

Usher and Priestley * found that ultra-violet light canbring about the decomposition of aqueous carbon dioxide with-out the intervention of an optical or chemical sensitizer, a resultcontrary to that of Stoklasa and Zdobnicky, who found thatformaldehyde was not produced by the action of ultra-violetlight on carbon dioxide and water. In view of these contraryresults, Spoehr f tried the effect of ultra-violet radiations oncarbonic acid and its salts ; in no experiment was a sufficiencyof formaldehyde produced to give a positive reaction with thereagents employed. In all experiments Spoehr found thatformic acid was the only reduction product. Attempts toreduce formic acid to formaldehyde by sun or by ultra-violetlight failed, but after ten to fifty hours exposure there remainedon evaporation a non-volatile yellow syrup, of a compositionnot yet determined, which reduced Fehling solution.

A further study of the artificial photosynthesis of carbo-hydrates was undertaken by Baly, Heilbron and Barker J whoby exposing a solution of carbon dioxide in water to ultra-violet light of different wave-lengths obtained evidence ofthe formation of formaldehyde and also prepared a syrupwhich, according to Irvine and Francis § consisted of 80 percent, non-sugars and not more than 10 per cent, of a hexose-like material from which ketoses were absent.

The views put forward by Baly and his collaboratorsconcerning the origin and mode of formation of the formal-dehyde produced in the experiments mentioned above, werecriticized by subsequent workers || and in later papers Baly,

* Usher and Priestley: " Proc. Roy. Soc.," B, 1911, 84, 101.t Spoehr: " Plant World/' 1916, 19, 1.t Baly, Heilbron and Barker : " J. Chem. Soc./' 1921, 119, 1025.§ Irvine and Francis : " Ind. Eng. Chem./* 1924, 16, 1019.II Spoehr : ." J. Amer. Chem. Soc./' 1923, 45, 1184. Bauer and Reb-

mann ; " Helv. Chim. Acta,/' 1922, 5, 828. Bauer and Buchi: id., 1923,•6» 959- Porter and Ramsperger : " J. Amer. Chem. Soc," 1925,47, 79.

] : i U\ YhA.V.i K \P IAi h »\ $Q

willi .< naniP:;r -of f'/il v.--\\ • «rki r>.* mÎif;;/- ];> ^ri^nia.1 viewsan-I >u^- . '^ tiit!- »;,r f< ••rmald>'hy.iv .H>T.emed :n the SillierrxprrnnrnN .*!-.•-'• trî.; tee M' îuLiry deO'nipîT.i.'M vi thej ' h; > t. > > v n t n v - •. •'. • d i. a r b <« 11 y d r a t e.

"1 hev t';:vi vj-n^vcr, that if carbon dmxide be. e\|>>-..d lothe artiMfi r.f nltra-\'inlct light in the presence 3~>f sue!: sub-stances as hamini sulphate, aluminium powder, the basicrarboikites of aluminium, zinc or magnesium, which present asurface upon which the gas may be absurbed, a condensationcompound is produced which is charred by sulphuric acid andreduces Bem-dut solution afler hydr»>!ysis ; the exact natureof this subst,-tTire has yet to be di'terminvd, but it possiblycontains carbohydrate.

Of more ininiediate interest in the problem ui naturalphotosynthesis are the results obtained with the use of visiblelight, from an ordinary tungsten .filament lamp or a ioo-watthalf-watt lamp, in place of ultra-violet radiation; usingcoloured absorbing surfaces such as the basic carbonates ofnickel or cobalt, these authors were able to obtain a largeryield of organic material than with the colourless substancesand ultra-violet light mentioned above. This material is amixture one component of which is a carbohydrate as isindicated by the facts that it gives the reactions of Molischand Rubncr, it reduces Benedict's solution and also forms asolid nzazone. The material also contains a more complexsubstance which reduces Benedict's solution after hydrolysiswith acid. In these experiments no free oxygen is given off;it is suggested that it is absorbed on the surface of the powder,possibly forming a peroxide, and this in time may poison thesurface and stop the photosynthetic action. Thus with alight of medium intensity^ photosynthesis stops after twohours ; in a stronger illumination the organic material firstformed may be decomposed ; with a decreasing intensity oflight, the amount of material synthesized by unit quantity oflight increases.

* BaJy, Da vies, Johnson and Shanaasy : " Proc. Roy. Soc.," 1927, A116, 197. IlAly, Strphen and Hood, id.f 212 ; Baly and Davies, id., 219,


The results obtained by the use of visible light and acoloured surface are of interest in that it would appear that acloser imitation for the first time has been made in the testtube of what occurs in the green cell. In each the process isconditioned by visible light and a pigmented surface ; thepresumed poisoning effect of the cobalt surface by the oxygenis paralleled by the fatigue effect exhibited by leaves in strongillumination, and in both there is a recovery. The maximumyield of organic material obtained by the authors was 0-075gram in two hours, and assuming that the product be glucose,it is calculated that the gain in calorific value is 0-48 calories persq. cm. of surface per hour, which just falls without the rangegiven by Puriewitsch,* 0-27-4-1 calories, for a number of plants.But this for the time being has no significance ; the intensityof carbon assimilation in the plant varies much with the condi-tions, and, on the other hand, the material obtained by Balyand his fellow-workers is not all glucose. The authors alsoargue that the constant ratio of chlorophyll A to chlorophyllB, observed by Willstatter and Stoll, is maintained by thecarotin which becomes oxidized to xanthophyll. Since theratio of xanthophyll to carotin tends to increase during photo-synthesis, it is suggested that the slow recovery process in theleaf is that in which the xanthophyll is again reduced to carotin.

Chemical change may be brought about by electricalenergy; indeed, in connection with plants, the effect of elec-trical currents on vegetable growth is a not unimportantbranch of applied botany.

Royerf by electrical means brought about the reductionof carbon dioxide, and similarly Coehn,J in 1904, producedformic acid from this same compound. Brodie § found thatby means of a silent discharge formaldehyde, together withmarsh gas, was produced from a mixture of hydrogen andcarbon dioxide ; and L6b,|j in 1906, found that formaldehydemay be produced by the action of a silent discharge of elec-

* Puriewitsch : " Jahrb. wiss. Bot.," 1914, 53, 229.t Royer: " Compt. rend./' 1870, 70, 731.j Coehn: " Ber. deut. chem. Gesells.," 1904, 34, 2836, 3593.§ Brodie : " Proc. Roy. Soc,/' 1874, 22, 171.II Ldb : ' ' Zeit Electrochem.," 1906, 12, 282.

ELECTRICAL ENERGY 41

tricity through a solution of carbon dioxide in water. Fenton *also has pointed out that the synthetic action of light and ofthe silent electrical discharge are practically identical. Thusthere is evidence which suggests that electric energy may playa part in the earlier processes of photosynthesis ; a suggestionwhich is supported by the fact that, according to Polacci,fthe formation of carbohydrates is promoted in leaves byelectrical energy, provided it be not too intense, especiallywhen a continuous current is made to pass directly into thetissues.

As a result of a number of experiments, Gibson % comes tothe conclusion that the light rays which are absorbed by thechlorophyll are transformed into electrical energy, and it isthis transformed energy which brings about the decompositionof carbonic acid to formaldehyde and oxygen. This opinionis based on evidence the details of which apparently have notbeen published.

Waller § and others have made many observations on thephoto-electric responses of plants, but much further investi-gation is necessary before the correlation of these phenomenawith the photosynthetic process is possible. Attention mayalso be drawn to the work of Kernbaum,|| who found that waterexposed to the influence of jS-rays and of ultra-violet rays ledto the production of hydrogen and hydrogen peroxide. Usherand Priestley also found that an aqueous solution of carbondioxide could be decomposed by the a and j8-rays from radiumemanation. The action of #oooi c.c. of radium emanation on200 c.c. of water saturated with carbon dioxide resulted infour weeks in the production of hydrogen peroxide and for-maldehyde. Most of the latter was in a polymerized state,but the solution contained no sugar.

The effect of salts of radio-active metals such as potassiumand rubidium has been the subject of many investigationsbut it is impossible as yet to draw any definite conclusions.

* Fenton : " Journ. Chem. Soc.," 1907, 91, 687.t Polacci: " Atti. Inst. Bot.," Pavia, 1905, II., 11,7.{ Gibson : " Ann. Bot./' 1908, 22, 117.§ Waller : id., 1925, 39, 515.[| Kernbaum : " Compt. rend.," 1909, 148, 755, 149, 273.


In this connection it may be mentioned that Henrici *concluded that ionized air accelerates carbon assimilation oflowland plants, when the illumination is low, but not when thelight is of medium intensity; in strong light the ionized airretards the process. In alpine plants, an acceleration wasobserved both in weak and in medium light, whilst in strongillumination neither increase nor decrease obtained. In someexamples the ratio of the rate of carbon assimilation in ionizedair to the rate in de-ionized air was as 5 is to I. This work iscriticized by Whimster f who points out that the results arenot based on the means of a number of experiments, whereforeit is impossible to calculate the probable error. Further, thefigures given by Henrici represent apparent assimilation, noallowance being made for respiration which, as will be seenlater, is markedly affected by an ionized atmosphere. Usingthe leaf of Pelargonium zonale, Whimster found no furthereffect on the rate of carbon" assimilation under the action ofionized air than a 3 per cent, depression, a value of no sig-nificance ; the real assimilation, in which the increased rate ofrespiration is taken into consideration, showed a 5 per cent,increase which again is below the significant level.

INTERNAL FACTORS.

CHLOROPHYLL. %—Plant physiologists for long have recog-nized that the intensity of carbon assimilation must be de-pendent on the chlorophyll and its amount; it is, however,but recently that the problems involved have been criticallyexamined. Irving,§ who used the leaf's carbon dioxide ofrespiration in her experiments, found by gasometric methodsthat etiolated leaves, either when they are orange-yellow orwhen they have attained a considerable degree of greenness,do not possess any appreciable power of synthesizing carbondioxide. If there be any photosynthetic activity, it cannotbe greater than one-tenth part of respiration nor come within

* Henrici: " Arch. Sci. Phys. Nat./' 1921, 3, 276.f Whimster : " Ann. Bot.," 1927, 41, 357.% A general account of chlorophyll, its chemistry and constitution, will

be found in Vol. I.§ Irving : " Ann. Bot.," 1910, 34,, 805.

CHLOROPHYLL 43

I per cent, of the activity subsequently developed. Carbonassimilation thus begins only when the leaves are fully greenand develops very quickly ; wherefore it follows that the firstorigin of this function is not correlative to the amount ofchlorophyll produced, or, in other words, that the amount ofchlorophyll is not a conditioning factor in the early stages ofcarbon assimilation.

Willstatter and Stoll * were the first to make quantitativeestimations of the amount of chlorophyll in leaves, by themethods already outlined.! Also they measured the amount ofcarbon assimilation of the leaves of different plants and of thesame plant in different conditions—normal, etiolated, autum-nal, and so on—and thus arrived at the assimilation numberwhich is the ratio between the amount of carbon dioxide as-similated per hour and the chlorophyll content, both expressedin milligrams. A selection of the values obtained are tabulatedbelow.

Willstatter and Stoll, whose experimental methods weresimilar to Irving's, with the chief exception that they useda 5 per cent, concentration of carbon dioxide, found tha tleaves with but a small portion of their full chlorophyll contentdeveloped can assimilate to a measurable degree.

WILLSTATTER AND STOLL'S ASSIMILATION NUMBERS.

Plant.

Primula .RubusTUia {

PopulU3 \

Elm {

Kind of Leaf.

NormalNormalYoungOlder

Dark green autumn leavesYellow-green autumn

leavesYellow varietyGreen variety

ChlorophyllContent in

Mgms.

n-416-25*2

22*515-23*9

9*5I3*o

CO, Assimilatedper Hour,Mgms.

IO59474

1481523i

7580

AssimOa-tioa

Number.

9*i5.8

14-26-6

IO*O7-9

7-6

This conclusion is contrary to that of Irving, a differenceprobably due to the fact that Irving used young leaves whilst

* Willstatter and Stoll: " Ber. deut. chem. Gesells.," 1915, 48, 1540.f See Vole I., Section on Pigments.


Willstatter and Stoll employed older and sometimes mucholder material. This explanation is due to Briggs,* in whosememoir a critical examination of the work of the above-men-tioned authors will be found ; this author demonstrates thatthe age of a leaf and the lapse of time from the greening tothe measurement of photosynthetic activity are all important.If a leaf is cut from a seedling in the dark at an early stagein its development and partly greened by exposure to light,its photosynthetic activity will be zero or very small; if, onthe other hand, the same procedure is repeated with a similarleaf from the same plant after an interval of a few days,the photosynthetic activity will be strongly marked. Briggsconfirms Irving1 s main conclusions : a young green leaf mayshow no or very little carbon assimilation and the power ofphotosynthesis lags behind the development of chlorophyll.This power increases with age whether the leaf be in the darkor in the light, even though there be no concurrent increase inthe chlorophyll content. But the same phenomenon does notnecessarily occur in seedlings. In a further communication,Briggs j* has shown that in Helianthus, Acer and Cucurbita, forexample, where the cotyledons serve as organs of storage and,on becoming green, are the first organs of carbon assimilation,there is no lag between greening and photosynthesis whenlight or temperature is limiting. On the other hand, inPhaseolus, Ricinus and Zea, plants in which there is formed acarbon-assimilating organ distinct from the storage organ, thephotosynthetic power is not developed until some time aftergermination, and in natural growth conditions the lag ob-tains. It will be remembered that the cotyledons of Phaseolusare entirely organs of storage, the first foliage leaves being thefirst organs of assimilation; similarly in Zea, the first foliage leafis concerned with photosynthesis, the cotyledon being occupiedwith the absorption of food from the adjacent endosperm ;in Ricinus, on the other hand, the cotyledons are embedded inthe endosperm and afterwards expand to form the first carbonassimilating organs. In these so specialized plants, the com-

* Briggs : " Proc. Roy. Soc," B, 1920, 91, 249.f Ibid., 1922, 94, 12.

r \ i \ \ o \ Y N FAC i "*k-* 4;

mon fact that the n ^ r v c U">d îpph1 1- r,-? '.- ••'a!..s;:v. ] \ :' v:\the organ- •>• i?iV ^.-ruhn,; which an- the iir.-T ?••• -::• -v\ :•:, ::••-syntliesis, -n^ê-t tliat ike tr,ii>l'u;ati.;:i ;.f 1.•••>,! ^lit.n.iNto tlie assnuilaun^ structure may be a « au-al Javt-r ir. !«.' Lidescribed ; \X i>, Ji.nvt-vtr, wry dûl.)ttiil it it ]•>;• x\-v v\\ factorThe accompanying table gives some of Bri^s 's results, inwhich the carl>on assimilation is represented in cubic centi-meters of oxygen evolved per hour per 5*.) sq. cm. of surfacein a light intensity of 9000 lux.

Helumthus . . -' . *•» • 3s#"r> 3-00

« — • • ' :?. - : ^ . I S

14

10178

20

; JO-7

• 13311-9

; lO'O

i I2'6

O-CK>

Z # a . . { ! 1 9 : 1 3 ) 0 - 3 6

o ° 6 i

Dastur * suggests that the variation in the assimilationnumbers obtained by Willstatter and Stoll is due to loss ofwater, a factor of great importance in photosynthesis. Further,the irregularities observed in autumn leaves and also in normalleaves may be explained by the difficulty in selecting leaves ofthe same photosynthetic power, for this power is not neces-sarily the same in leaves of apparently the same age or col-lected at the same distance from the apices of the shoots,notwithstanding the fact that they may appear equally healthyand fresh.

T H E UNKNOWN FACTOR.—The fact that the temperature

relations of carbon assimilation are those of a chemical ratherthan a photochemical reaction indicates the presence of aninternal factor, independent of the chlorophyll and associatedrather with the protoplasm, which controls the rate of carbonassimilation. Irving concludes that this factor controls the

: " Ann. Bot.»" 1925, 3&, 769*


beginning of the process since it is not developed so quicklyas the chlorophyll, wherefore the rest of the mechanism mustawait its appearance.

The assimilation numbers arrived at by Willstatter andStoll * are inconstant, which is indicative of there being someother operating factor : if they were constants, strong evidencethat chlorophyll was the all-important conditioning factorwould be provided. According to Willstatter and Stoll fit is an enzyme which thus limits carbon assimilation. Theyfind that in leaves rich in chlorophyll, increased illuminationhas but little effect upon assimilation nor is it diminished ifthe illumination is decreased to one quarter. This indicatesthat the chlorophyll is present in excess compared with theassimilatory enzyme. The increase in carbon assimilationfollowing an increase in temperature they consider to be dueto the stimulation of the enzymatic process.

In leaves containing little chlorophyll and in yellow varietiesthe conditions are reversed ; the enzyme here being in excess,increased temperature has little effect in stimulating assimi-lation. On the other hand, increased illumination has a very-marked effect.

The remarkable phenomena accompanying autumnalchanges in leaves are due to the fact that either the chloro-phyll suffers more than the enzyme, resulting in increase ofassimilation number, or conversely the enzyme suffers most,in which case the assimilation number falls.

The failure to bring about carbon assimilation by meansof chlorophyll isolated from the leaf may be attributed, accord-ing to the authors, to the absence of the enzyme.

This belief in the existence of a controlling enzyme alsois shared by Osterhout and Haas $ who experimented withvarious plants, both fresh water and marine, grown in a culturesolution containing gaseous carbon dioxide. The removal ofcarbon dioxide was followed by the method of determinationof the hydrogen ion concentration in the culture medium. It

* Willstatter and Stoll: "Ber. dent. chem. Gesells./' 1915, 48, 1540.f Ibid,, p. 1552.t Osterhout and A. R. C. Haas : " Proa Nat. Acad. Sci.," 1918, 4, 85.

WN K V T n R S 47

was found that pluit nsynllu'sxs l><* ;tu iittittf*<Iia(:i*Iy on exposureto li^'ht ; af first (hr rrnmval '4 rarhon din.Kidr was rapid,then nun'** slnwly until a m;*\immn vv;t% rraclit-d when the rateroinainrcl constant. Thr^r ;nit IKn*s nmsidrr that tiiultr tin;mihimw ni li^ht smnr r;it.i!\Nl is tlrvrluprd which facilitatesthe jihotosyiithetk pr<>*v^. B r i ^ s r«»itrlu<Ii\s that Hit? ac-tivity of the jihot i>< hemiral plia^r uf tin* carbon assimilationiMri'liauisin in a milling, as rnni |anul with that of more matureleaves, is in some way limitc*I : the photochemical phasedepends for its intensity n<»f ttn\y tm the chlorophyll but alsoupon some other far tor vvlii«ij itirrvascs with age during tin?early stairs <>{ lc;if ili'vel»»|»iu«-nt.

Tin; <>hsfrvati»u»s of M*4iv.rh * that lcavfes after slow dryingor fret/zing, treatment H that it*» nut first n>y eu/,yme,s, rt'.taiusome; [>(IW«T *»f rarhon ir^-iriulation and ran evolve; oxy^t'ii,wfhcreas the s;tim« leaves killed by rapid drying or innnersicmin hot vtatvr xhow no mirli \mw*rt :i|(ain iiuiirattrs the partici-pation at an fnxyiiui in the pnm*?w.

There is llaw Htrung evidrnci: of the exmtcirieu of a control-ling factor, pmhably fiizyniic, iHwtfiti;il in the: earlier phasesof carbon assimilation. Brigg^f ulsn draw» attention to theprobability that the amount *»f "* naci ive chlorophyll surface/1

whk:It i$ nctt neccHâriiy flit; sunn*. A*m tlie total ciiloroplastsurface, k an importattt iftlcntal fa«rt«r ancl anything whichmilm'm.%"* thr atnnunt *»f tliin mrUitv^ surh as age, iron chlorosisand lark of rî'iithtl rlrni* nt^ in the raw fowl matcriul, isindirectly it factor rontmlhnj; th«? ratt! of <:arbc»n UMsiiutilation,An inercuHc (*r dt'^rcaM: in thi: r«;.«i.iv« chlorophyll surface willcorrespondingly $ncrca*« «#r thwnta*t* t\w mtlmn mnhml&titmwhen a given tcmpt'raturi: in limit ing and also, in certainccmclition^ when a definite ligtit in t imi ty &nd partial premmireof carbon dioxide are limiting. This conception in un tmtcxmwof mvamnimcnlH *>f th« rarhon •iîrnilation of Phmealus grownin culture Htilutions lsickini; »udt e**«t*ntial clemt:fit» assium, magnc2»itirn9 iron nr phoi|>linrii«*; in th«is«.j ctrcthe carbon assintiiatbn in always \vm tiiati in plantu grown in

THE SYNTHESIS OF C A R B O H Y E ) ! ^

a complete culture medium whether light o rlimiting. This is shown in the accompanyingBriggs's results which gives the real a s s imi l ab l ecm. of surface expressed in percentages of the r ^ - tplants growing in the normal culture solution of iwhen light and temperature were limiting.

Leaves.

First fsimple IFirst fpinnate \

FactorLimiting.

LightTemp.LightTemp.

PotPlant.

IOOIOO

NormalSolution.

IOOIOO7779

-Mg.

82826464

- P-

10397

65

It will be seen that photosynthesis is subnormaltemperature are limiting, which means that s u b ]the photochemical phase, which is more i n f i u o nmination, is accompanied by a subnormality i n . 1phase which is mostly influenced by temperafct-i]both phases generally show the same degree of s i

In passing it may be observed that the p a r t I cwhich an element is presented to the plant m a y \tance. This is shown by the following table t a t c ekell's observations on starch production in t h e lcpotato,* from which he concluded that the a m o u .varies not only with the ordinary c o n d i t i o n i n galso with the nature of the available potash. T l n usulphate when added to the soil was found to b e raxin starch production than treatment with p o t a s s i iand other potassic manures. The figures given a tvalues of colorimetric estimations made o v e r s

Treatment.No potash, (control)Potassium sulphatePotassium chloride" Potash, manure salts *'

Starch Product ioi2-0132*6672-1391-893

seventeen days in September ; the quantities of a,<i<manure were so controlled that the culture plots w e

•Maskell: "Ann. Bot." 1927, 41, 327.

INTERNAL FACTORS 49

amount of potash equivalent to two hundredweight of potas-sium sulphate per acre.

The superiority of the plants treated with potassium sul-phate is associated with a more rapid translocation of starchfrom the leaflets.

THE PRODUCTS OF CARBON ASSIMILATION.—Much investi-

gation is necessary before the precise significance of this factorcan be determined: of the products of carbon assimilation,^rbpjhy^drate and .oxygen are the most conspicuous.

It is well known that, according to the law of mass action,chemical action diminishes as the products of the activityaccumulate ; the initial rate can only be maintained providedthe initial proportions of the reacting substances are preservedand the products of the reaction removed. In the leaf a furthercomplication arises since sugar is an osmotic substance and itsundue accumulation may lead to a physical disturbance in thesynthesizing machine such, for example, as the closure of thestomates. This may be averted by the conversion of the sugarinto search, a marked and well-known phenomenon in manyplants, which temporary reserve is translocated at nightfallafter hydrolysis. But a great accumulation of starch mayinhibit photosynthesis in that, presumably, it reduces theeffective surface of the chloroplasts which, according to Briggs,Warburg, Wurmser and others, is the seat of the primaryphotochemical action. Translocation is most apparent in thedark, but it takes place also by day and it is only in sugar leavesthat the undue concentration of soluble carbohydrates, owing tohindrances to rapid translocation, or to a high assimilatory effi-ciency, is likely to prove an important factor in carbon assimila-tion. Broock * found that the leaves of the sugar beet onbright sunny days showed a rapid increase in their dry weightup to midday, at which hour there was a sudden decrease : thisloss in weight was more or less uniform until about midnight.The sudden drop in the carbon assimilation at noon indicatesthat the process was brought to a standstill owing, in all

* Broock : " tJber tagliche und sttindliche Assimilation einiger Kultur-pflanzen," Halle, 1892. See Thoday: " Proc. Roy. Soc," B, 1910,&*, 443-

VOL. II.—4

50 THE SYNTHESIS OF CARIBOUVI >RA'I !•>

probability, to the closure of the stonuites rt-Mihn^ imn; theaccumulation of materials which wen* produm! at a ^rvaUrrate than that at which they could he translncatnl.

Kostychew and his collaborators • t'nund :h.it the phut**,synthesis of Arctium and Phragmites on bright warm daysreaches a maximum several hours before •runst.-T, UM.;a)]y inthe forenoon; which maximum is followed by a marked re-tardation owing to the removal of the products of the iterationnot keeping pace with the formation of new m.'Urri.il, Anincrease in weight of approximately 0*13 gram prr ;t>u >q. cm.of leaf surface is enough to end photosynthesis When theconditions of photosynthesis are not so favourable, fur examplein dull weather, the sudden retardation of the process is notobservable. Similar results were obtained in the study ofvarious aquatic plants.f It was also found that variousalgae, e.g. Mougeotia, Spirogyra, Cladophara and Rhizoclonium,exhibit different assimilation intensities which are invariablygreater than those of aquatic phanerogams, e.g. Ceratophyllum,in spite of the fact that the latter generally contain a largerquantity of chlorophyll per gram of dry weight. These ob-servations, together with those of McLean alluded to on anearlier page (p. 24), indicate that the phenomenon is widelyspread.

With regard to the oxygen evolved, since carbon assimi-lation by green leaves is unaffected by various concentrationsof oxygen in artificial atmospheres, and since the oxygenpressure of the internal atmosphere of plants is a very variablequantity, it would not appear that the amount of oxygen isa limiting factor of any great significance.

THE ORGANIC PRODUCTS OF CARBON ASSIMILATION.

The ultimate products of carbon assimilation are carbo-hydrates ; Brown and Morris,% in their classical work on thechemistry and physiology of foliage leaves, identified sucrose,

* Kostycbew, Kudriviewa, Moisscgewa awl Sm&mow*; '* Zeit. wiss.Bc^.," 1926, 1, 679.

f Kostychew and Soklatenkow : id.. 1926, 1.} Brown aad Morris : " Joura. Chem. Soc,," 1S93. 63, 604.

PRODUCTS OF CARBON ASSIMILATION 51

dextrose, levulose and maltose in the leaves of Tropceolum,the sucrose being in greatest abundance. These results weregenerally accepted, reinvestigations of the subject being ofrecent date. Parkin,* in his investigation of the sugars of theleaf of Galanthus nivalis) selected for its convenience as asugar leaf, found that a considerable quantity of sugars, 20to 30 per cent, of the dry weight, occurred in the active leaves ;sucrose, dextrose and levulose were recognized, but maltosewas never found, a result which was to be expected, since starchis not formed in the leaves in any appreciable quantity. Theamount of sugar increases towards the base of a leaf and con-currently the ratio of the sucrose to the hexoses diminishes.The sucrose increases rapidly in amount after sunrise, reachesa maximum and diminishes during the night; the percentageamounts of the hexoses of the leaf, however, remain fairlyconstant. As the season advances, the proportion of sucroseto the hexoses decreases, the latter being formed at the expenseof the former. Parkin confirms Brown and Morris in theobservation that the levulose is, as a rule, more abundantthan the dextrose and supports the contention that sucrose isthe Jirst recognizableL .sugar.Jaxarbon assimilation, the hexosesbeing formed from it by inversion. It has been suggestedthat the reason for levulose being more abundant than dex-trose is that the latter is more used by the leaf in its respiratoryprocesses, the fructose being more suitable for tissue formation.The observations of Robbins j* and of Brannon { however,indicate that fructose is not better than glucose for tissue for-mation. In water culture it was found that glucose was thebetter, whilst fructose may have a temporary toxic effect,especially in the absence of glucose.

Davis, Daish and Sawyer, § in the instances of the potato andthe mangold, agree that sucro§£ is a primary sugar of carbonassimilation and from it the hexoses arise. The cane sugar andhexose are at their maximum at mid-day and the amountsincrease with the season. Unlike Brown and Morris they

• Parkin : " Biochem. Journ.," 1912, 6, 1.f Robbins: "Bot. Gaz.," 1922, 73, 376.t Brannon : id., 1923, 75, 370.§ Davis, Daish and Sawyer : " Journ. Agric, Sci.," 1916, 7, 225.


found pentose to be present whilst maltose was confiMt-M.yabsent, both by day and by night, from the Iravt^ mentioned.In view of the presence of starch in the potato k-af, it is Mjr-prising that the presence of maltose was not eMahhshed :there is, however, good reason for supposing that maltaseis commonly present in plants in which starch occurs andis digested ; this enzyme is rather indiffusible, is destroyedby alcohol and chloroform in ordinary conditions, and isvery susceptible to heat, a temperature of 50° C. having amarked adverse eifect upon its activity, whilst at $zc C. itmay be destroyed. It is these properties of maltase whichaccount for the varying statements regarding the occurrenceof maltose in green leaves: if the procedure of experiment issuch as to destroy the maltase, maltose will be found ; if, onthe other hand, the enzyme is not destroyed, the maltose isconverted into hexose sugars. * Davis, Daish, and Sawyerdropped their material of experiment into boiling alcohol andthus destroyed any maltase that might have been present ;their inability to detect maltose was therefore not due to theactivity of maltase- The conclusions of these authors regard-ing the presence of pentose—--they estimated that pentoserepresented from 5 to 25 per cent, of the total sugars—is ad-versely criticized by Colin and Franquet f who found that theamount of this sugar in the leaves of the potato and of thebeetroot is at most less than 0*03 per cent, of the total sugars.

Gast % examined the carbohydrate content of the leaves ofTropaolum, Vitis, Mas a and other plants collected in the earlyafternoon and early morning: he found that sucrose alwayswas more abundant than maltose and the other sugars identi-fied ; he is in accordance with Brown and Morris regarding*the absence of pentose. Kylin § found that in general termstbe amount of glucose varies inversely as the amount of starch.Sticrae Is generally the most abundant sugar, especially i nsugar teavtst but Gentiana hrewidms, a sugar leaf, does not

* Starts: - ttacfcML Jem*.," 191% *% n , 49, 36.f GaD* and Franquet: ** Bull. Sac* cite. Httt," 1917. % 114.t O t * : " ft*. fiiiysM. Qm*V 1*19, ffe 1.I Kytfcn: «&» tfiS, 101, 77.


contain any sucrose, whilst Tilia europcea, a starch leaf, con-tains much sucrose.

These authors, together with others, conclu.de that sucroseis the first sugar of photosynthesis, but, on the other hand,there is evidence which may indicate that hexose, the sugarrequired on current theoretical grounds, is the first sugar tobe formed, and various arguments have been formulated toshow that the results of the work outlined above really in-dicate that hexose and not sucrose is the first sugar formed inthe photosynthetic process.

Lepovsky and others * from their study on the sugars ofthe sugar beet, conclude that reducing sugars are amongst thefirst products of carbon assimilation. Weevers f comparedthe nature of the sugars in the green and non-green parts ofa number of variegated leaves. In ten out of the twelveexamples examined, sucrose was the only sugar identified inthe non-green parts, whilst the green portions contained bothsucrose and glucose. Further, hexoses were the first sugarsto appear and then sucrose in the chlorophyllous parts ofthese leaves on exposure to light after a period of darknesssufficient to ensure depletion of carbohydrate. Invertase waspresent both in the green and in the non-green regions, whichsuggests that this enzyme by a reverse action condenses mono-saccharides to disaccharide. Priestley,% in his review of theevidence, points out that the sugars formed in the green leafare intermediate steps in the formation of more complex bodies,such as polysaccharides and proteins, for the prompt formationof which the intermediate steps must be rapidly accomplished.Wherefore it is reasonable to conclude that the substancesformed in the intermediate stages, including the first sugarof photosynthesis, would show but little change in their con-centration ; they would not accumulate in the light and thuswould not show fluctuation in amount. In other words, thefact that of the sugars of the green assimilating leaf, sucroseshows the greatest fluctuation in amount during the day

• Lepovsky, Schulz and Link : " Journ. Agric. Res./' 1926, 33, 59.f Weevers : " Proc. Kon. Akad. Wetens., Amsterdam," 1923, 27, 1.% Priestley: " New Phyt.," 1924, 23, 255.

i4 TUT. -YNTHKSB OF f A t t i u i ]YJ>R.\ rh^

.- •• i- - not ill it u i- tin }>-i - . t * ' ; t ^ j , : ! . i - '^ .»*

S?t u% j ,H t . '>n«t 1 >\u\»r, i! - : M \* * "' : "u r *•'

. r ,' * a- a •* 'I*. ! ir\ ;r <luil In * » ;>' i*\ 1 ! *.» I roi u-

Mr * % ^ . r-T. 1 uit'i 1 °KI •A ntK - t- - , s <lr»»h -.- ^ f *Uif' h\ 'ID-** fa. I'Jat ?tar« h •mil olLi r iri ! \ikatt1 j»ri *luc!^t I }, : sv^iu^h arc i>td up in tin* a <!> »h, ]>r«'» «*c«=; nf the

I>rji **tlt \ ion>hults that neither phv-i >l ^1 ,1! n r ihcmicaldata >upi »>rt the \ii\v that cane suj;ar ii.i* it- nn^in in >tareL.If starch he a g]uc«»e anhydride, it ft»l! »ws Jiat if sucrose isit>TTPX\l from starch iome of the glur* -i must be convertedinto kvulose. There is, however, some evidence that starchis converted in sucrose. Schroeder and Horn * found that therane sugar content in detached starch containing leaves ofTrop&olunt varies with the water content, an increase occurringwith a falling water supply and a decrca.se with a rising watercontent, It was also observed that in the detached leaves ofmany plants the starch disappears more rapidly if the leavesare allowed to wilt. Ahrens f also observed that in the wiltinghad starch was converted into sucrose and hexose, presumablyby hydrolysis ; but from what is known of enzyme action,the hydrolysis of starch should accompany an excess ofwater whilst a lack of water should promote synthesis.For this reason Priestley discounts t h e above work, in so farm it affects the present problem ; it has., however, been sup-ported by the observations of de Wolff, J who found that sucrosewas produced from starch grains containing not more than53 per cent, of dry starch when slowly dried, but if the grainscoii'taintd mote than the stated amount of dry starch, no•derate was formed on similar t reatment . This indicates that\&m ft&tkM»bft|» between the amount of water and starch is afactor of faufxwtft&ce, amd presumably in Ahrciufs wilting leaves

• Sfkm^m ami Hora : " Biocfcem, Z*-itV iff2» lJ®» 165.f mmm: m Smt ***** *P4> 5*


there was a sufficiency of water for the reaction to take place.Priestley's thesis is examined by Parkin * and by Chapman fwhose papers may be consulted in the original. Attentionmay be drawn to two facts : that all sorts of mutations,sometimes chemically obscure, obtain between various car-bohydrates both in the plant and in the animal; and that themethods of chemical analysis are in the main too gross for theidentification of the subtle changes which rapidly occur inthe green leaf in which in the light anabolic and katabolicchanges are taking place together with others concerned intranslocation. For these reasons it is not illogical to assumethat the results so far obtained have no significant bearingon the point at issue. As Spoehr J justly remarks, " I n acomplex system such as that with which we. are dealing here,in which the synthesis and hydrolysis of complex compoundsare affected by a variety of conditions, the quantity of anyparticular substance gives little information regarding therole of that substance in a series of reactions. All of the car-bohydrates, monosaccharides, disaccharides, and starch arecapable of conversion into each other quite independentlyof the photosynthetic process, and even the identity of thehexoses is not fixed, but they may undergo stereoisomericchange. Unquestionably it would be very helpful if it wereknown just what the first sugar is that is produced in photo-synthesis. In the present state of our knowledge glucosefits the theoretical requirements most adequately. Yet thefact cannot be entirely disregarded that the demonstration ofglucose actually being the first sugar formed is still wanting."

Fats sometimes have been described as direct products ofcarbon assimilation, more especially in plants, such as Van-cheria, which have an abundance of fat-like substance in theirgreen tissues. The evidence for the contention is anythingbut satisfactory, especially in view of the transmutation ofcarbohydrate into fat in various plants and under the influence

* Parkin : " New Phyt.," 1925, 24, 57.f Chapman : id., 1925, 25, 308..J Spoehx : " Photosynthesis/' New York, 1926, p. 219.


of certain external conditions such as low temperature.* Theproblem has been considered by Meyer j* who describes thepresence of a colourless fluid of unknown composition andhaving the physical properties of an oil which occurs in thechloroplasts of Tropceolum. This substance, which is not afat, since it gives on distillation hexylenealdehyde, increaseswith assimilation, and is considered by Meyer to be a definiteproduct of carbon assimilation.

HYPOTHESES CONCERNING THE SYNTHESIS OF CARBO-HYDRATE BY THE GREEN PLANT.

With the problems associated with the march of eventsfrom the initial carbon dioxide and water to the final carbo-hydrate, the realms of theory are entered. The hypothesesare many and have this in common—none entirely satisfy theplant physiologist; for which reason it is proposed to considerbut few in any detail on the present occasion, not that thosediscarded are barren of ideas—indeed, some contain valuablesuggestions—but that their examination, more especiallywhen unsupported by experimental evidence, would tend ratherto obscure than to clarify the problem.

In 1870 Baeyer put forward the hypothesis that the carbondioxide is split up by the plant into carbon monoxide andoxygen, and that the water is concurrently resolved into itsconstituent elements. The carbon monoxide and hydrogenthus produced then combine to produce formaldehyde, whichundergoes polymerization, and so forms a hexose.

These changes may be represented in the following equa-tions :—

ri. CO2 = CO -f oU. H2O = H2 + 03. CO -h H, = CHaO4. 6(CH2O) =

Thus, according to the theory, there are two distinct actions,the first leading to the formation of formaldehyde, and thesecond to the production of sugar.

* See Vol. I.tMeyer: "Ber. dent. bot. Geselis.," 1917, 35, 586; 1918, 36, 235,

674.

BAEYER'S THEORY 57

Considering the first part of Baeyer's theory, it is seen thatboth carbon monoxide and hydrogen are supposed to be pro-duced, but carbon monoxide has been found but once in afree state in the living plant (p. 105), and it is a substance whichdoes not lend itself at all readily to constructive metabolism,the evidence as to whether plants are able to make use of itfor synthetic purposes being contradictory. Bottomley andJackson * state that if the carbon dioxide normally presentin the atmosphere be replaced by about twenty times as muchcarbon monoxide—the increase in the amount of the latterbeing necessary on account of its lesser solubility in wateras compared with carbon dioxide—plants of Tropceolumformed starch and flourished. Preliminary analyses alsoshowed tha t seeds, when germinated in an atmosphere in whichthe carbon dioxide had been replaced by carbon monoxide,there was an increase of organic carbon in the seedlings.Further, they found that a negative pressure obtained in thevessels containing the plants assimilating carbon monoxide.This was to be expected if the hypothesis be accepted, for ifthe carbon monoxide be used up in photosynthesis, then theamount of oxygen set free would be half that evolved in normalphotolysis. On the other hand, Krash&iinnikoff f has cometo the conclusion, based on a number of experiments, thatgreen plants cannot make use of carbon monoxide ; he pointsout, however, that his evidence does not prove that carbonmonoxide is not formed in the early stages of photosynthesis.It may also be remarked that according to the investigationsof Sulander,$ carbon monoxide acts as an anaesthetic, but ismuch weaker in its action than chloroform. He found that0-5 per cent, of this gas was sufficient to inhibit the growthof seedlings of the lupin and the germination of the spores ofmany Fungi. On the other hand, Wehrner§ found that theseedlings of cress arid barley are not injured by exposureto an atmosphere containing 50 per cent, of carbon monoxide.

* Bottomley and Jackson : 0t Proc. Roy. Soc.," B» 1903.7*, 13a*I Kra^L^nitmikofi : " Rev, g6n. Bat./* 1909,31»177.% Salan.de* : " Beth, bot. Centrbl./* 1909, 24, I., 357.§ Wefcsaer: " Ber. deut. bat. Ges.," 1925, 43* 184,


It does not therefore follow that carbon monoxide is not formedin plants. As is well known, carbon dioxide is itself an anaes-thetic if present in a sufficient amount, and possibly it is morepotent in this respect than is carbon monoxide, for Sulanderfound that often the streaming movements of protoplasm wasnot affected even after several hours' exposure to the last-named gas.

According to Baeyer's hypothesis oxygen and formalde-hyde are early products of the process and these may now beconsidered.

ORIGIN OF THE OXYGEN.

On the thesis that there are two phases in the photo-synthetic process, one a light reaction and the other a dark,or chemical, reaction, the oxygen may be split off in either theone or the other. Accordingly Warburg * and others tried theeffect of increased temperature, poisons and narcotics onCkforella under high illumination, the Blackman reaction, andunder low light intensity. In both conditions carbon assimila-tion was inhibited by narcotics and the Blackman reaction alsoby hydrocyanic acid, but not that in low illumination. Further,the former was accelerated by a rise in temperature, the curveshowing a linear function of temperature between IO°~3O° C.;but when light was limiting, an increased temperature waswithout effect on the photochemical reaction. The alga wasthtti grown in the dark with hydrogen peroxide added to theculture solution and the rate of the evolution of oxygen, bythe action of the Ckhrella on the peroxide, measured aftertreatment with narcotics, poisons and varying temperatures.With regard to temperature the same linear function was foundas in the Blackman reaction, and the action of hydrocyanicacid and narcotics was the same. In view of this parallelism,tltese ftuthws support'Willstatter's hypothesis that the oxygentvolvtd during carbon assimilation owes its origin to the split-ting irf the chlorophyll peroxide combination (p. 65).

230; 1920, i©& x8S| « 9 4 4 ^ 4 Yabusoe: id,, 1924,

ASSIMILATION OF FORMALDEHYDE 59

FORMATION OF FORMALDEHYDE.

Formaldehyde, as is well known, is poisonous, so that, ifformed, it must be polymerized before it has time to injurethe protoplasm, and experiments have shown that, in certainconditions, formaldehyde may be made use of by the plant.Thus Bokorny * showed that Spirogyra in the absence ofcarbon dioxide can make starch when supplied with a compoundof formaldehyde and sodium hydrogen sulphite ; also Tre-bouxf and Bouilhac have stated that Elodea, Sinapis, andcertain Algae can form starch in the dark when supplied withdilute (-0005 per cent.) solutions of formaldehyde.

Grafe J found that green seedlings grown in the light, in anatmosphere free from carbon dioxide and containing not morethan 1*3 per cent, of formaldehyde vapour showed a greaterincrease in growth and in dry weight as compared with thecontrols. These results were corroborated by Baker,§ whoconcluded from most carefully controlled experiments thatformaldehyde could be assimilated to a certain extent in thelight, but not in the dark, in an atmosphere void of carbondioxide.

Jacoby || compared the dry weights of starch-free leavesof Tropceolwn, grown in an atmosphere lacking carbon di-oxide, with and without the addition of gaseous formaldehyde.Those treated with formaldehyde were more green and showedan increase in dry weight varying from 17 to 5-4 per cent,over the controls. He concluded that in all probability notonly was the formaldehyde fixed, but underwent a conden-sation similar to that formulated by Willstatter and Stoll;in other words, formaldehyde is an intermediate product incarbon assimilation.

Bodna'r, Roth and Bernauer f similarly found an increasein the dry weight and carbohydrate content of leaves exposed

* Bokorny : " Biol. Zentrbl.," 1897, 17, 1; 1916,36, 385 ; " Ber. deut.chem. Gesells./' 1891, 24,, 103.

f Treboux : " Flora/' 1903, 92, 49.X Grafe : " Ber. deut. bot. Gesells./' 1911, 29, 19.§ Baker: " Ana. Bot./' 1913, 27, 411.|| Jacoby : " Biochem. Zeit./' 1919, 101,1.f Bodnar, R6th and Bernauer : " Biochem. Zeit.," 1927, 190, 304.


to the vapour of formaldehyde, and Sabalitschka and Weidling *observed that El ode a is able to build carbohydrate from solu-tions containing 0*024 per cent, of formaldehyde or 0*032 percent, of acetic aldehyde in darkness as well as in light.

There is much positive evidence of the presence of formal-dehyde in green leaves. Tims Klein and Werner f from theirexperiments conclude that formaldehyde and acetic aldehydethus occur; the former was only found in leaves in whichcarbon assimilation had taken place; it was absent in leaveskept in an atmosphere free from carbon dioxide, or grown indarkness, and in leaves narcotized by phenyl urethane or byhydrocyanic acid. Acetic aldehyde, on the other hand, wasfound in narcotized leaves, and also in leaves kept in darkness ;it is regarded by the authors as a product of respiration.

Mention has above been made of the formatioa of formal-dehyde and of formic acid from carbon dioxide and waterunder the influence of different forms of energy.

Willstatter and Stoll J argue that the "assimilatory ratio,"COr ( \ should provide evidence regarding the possible inter-mediate products; they point out that oxalic, formic andglyoxylic acids and formaldehyde are possible intermediateproducts between the carbon dioxide and carbohydrate. Theassimilatory ratios of these substances are respectively, 4, 2,1*33 and 1. In their experiments, they found the assimilatoryratio to be I and it remained constant, even in succulents, invarying amounts of carbon dioxide and oxygen and with thetemperature ranging from 10° C to 350 C; it is concludedtherefore that the carbon dioxide is reduced to formaldehyde,this bung the only product having a single carbon atom.

POLYMERIZATION OF TORMALBEHYDE TO SUGARSIN VITRO.

Laboratory experiments on the polymerization of formal-dehyde to sugars not infrequently are quoted in support oftb* formaldehyde hypothesis.

t Sabalitschka: #i JL aagew. €H«m.r" 193a. £& 684. Sabalitschlca

•JEfcto *ttt Wwmmi * MKtaB. &*L0n 19a*, i&S, 3*1. See also

t taH 00 A14hd

POLYMERI2ATION OF FORMALDEHYDE 61

The first successful attempt to bring about such a poly-merization was made by Butlerow in 1861, who, by thecatalytic action of lime water, at ordinary temperature, ontrioxymethylene (itself a polymer of formaldehyde^, obtaineda syrup with a somewhat bitter taste, which he called methyl-enitan. Subsequently Loew undertook an investigation ofthe action of milk of lime on formaldehyde, and devised thefollowing experiment. A 4 per cent, solution of formaldehydeis mixed with an excess of milk of lime and repeatedly shaken.for about half an hour ; after filtering, the mixture is set asidefor some days unti l the pungent smell of formaldehyde hasdisappeared. The solution, which will now reduce Fehling'ssolution, yields a colourless syrup with a very sweet taste,*This substance, which is known as crude forrnose, was exam-ined by Emil Fischer, who found it to consist of a mixture ofvarious hexoses and succeeded in isolating from it a smallquantity of a sugar—acrose. Acrose can also be obtained bythe action of dilute caustic soda on glycerose, a substanceobtained by the oxidation of glycerol. From the acrose thusformed Fischer was able by an elaborate series of reactions toprepare ordinary fructose or levulose. In respect to the actionof weak alkalis, Spoehr found that sunlight had no action ona 3 per cent, solution of formaldehyde in the presence of suchsalts as calcium carbonate, potassium carbonate, and potas-sium nitrate in decinormal concentration, no trace of sugarbeing found after a n insolation period of four months.

With, regard to the formation of the higher carbohydrates.Fisher aad Armstrong f were able to synthesize a disac-charide—isolactose—by the action of an enzyme, Kefir lactase,on a mixture of glucose and galactose; the same authorsalso syn thes ize qjelibiose ; similarly isotnaltose has been ob-tained by Croft-rlill £ by the action of maltase on glucose;

•More recently Ewart (" Proc. Roy. Soc.," Victoria, 1919, 31, 32S)has worked out the best conditions for the polymerizatioa of formaldehydeto sugar, aad finds that the most rapid reaction is produced by sodiumhydroxide in the presence of a neutral calcium salt.

t Fisher and Armstrong : " Ber. deut. chenu Gesells.,11 1902, 35* 3*44-$ Croft-Hill: " Jcrora. Chern. Soc.," 1898, 73, 634 ; lee also Earner-

ling : ** B©r. dent, chenu Gesells./' 1901, 34,* 600, 2206.


and Pictet and Vogel * have synthesized sucrose from glucoseand fructose.

SUMMARY OF THE EVIDENCE RELATING TO THEFORMALDEHYDE HYPOTHESIS.

It is obvious from this survey that the hypothesis of Baeyerhas influenced to a remarkable degree investigations on thesynthetic aspects of carbon assimilation ; this to a large extentis psychological, for the hypothesis is attractive in that it isplausible and lends itself readily to test-tube investigation.The evidence readily centres around distinct nuclei—theidentification in the plant of the critical initial intermediateproducts; the effect of presenting the plant with some ofthese initial substances in place of natural raw material; andthe elaboration of the final from the supposed initial productsin the laboratory—and may be briefly considered under theseheads :—

Carbon Monoxide.—This gas, if produced, must be fixedwith extreme rapidity, for there is no evidence of its occur-rence as such in assimilating tissues, which is remarkable fora compound so inert. The evidence for its use by the plantwhen substituted for carbon dioxide is inconclusive.

Formaldehyde.—There is no doubt that formaldehyde canbe obtained from green leaves; there being many compoundswhich yield it more or less readily on decomposition. Theproduction of formaldehyde in artificial chlorophyll systemswas once considered strongly to support Baeyer's hypothesis,!but the work of J6rgensen and Kidd, who were the first to usepure chlorophyll in this connection, seriously discounts thesignificance of the reactions since the formaldehyde has itsorigin in the oxidation of the chlorophyll; it is not producedin such systems if oxygen be absent. Much of the evidencerelating to the production of formaldehyde from carbon dioxideand water under the influence of radiant energy is contradictory.

Its balance strongly supports the view that free formalde-

* Pictet and Vogel: " Compt. rend./' 1928, 186, 727. See Vol. I.,p. 113-

f See, for example, Usher and Priestley: " Proe. Roy. Soc," B, 1906,77»369; 1906,78,318; 1911,84,101.

<\'

1 v IT I> :• t t. n t- 1 * r M I * * * i / ' r ^ - *t \-.'jn \ » a'i ^ ! r1,} ur1 ' ^ If * 4 : r i * ,„tl.t } h »f s v*. M | r - ' i- ii .. . ;, t* ,« -i :>«<m »t i ^ il-\ih» > } ' - ?f U -j^ar r *> * , J1 *other wav

I lit * :>*i nti m "1 \\i"l>* i*l* •• nl ^ t 11 1 t* ' T *simJit( r\ rttn I- m^ niouN i it >i:u * t\ f n , I'it,/* ^ ^ tni t at tuUK di niim^trattd the rvnim e is n »t i »n%*n r»to.

beednt^ bxpeimr>L 111at } Lints in aln^m.a] tunti »n- d i j m t i l »t tl < ir nat ir A! raw t«m«l r.,iTcrKtK rv ivmake UM" »t (itlitr sUhsMnu^ n i h i ial rat >r ! t ] »*" : 'fcht \\^ that tin plant i a !*•.!] - n»; n*al * i * *v t i41 i *virtue of it- j DWH- I in t i a * rtain *i* TLf ^ xk4 - : *sub*»titutt>. ILt idi,^ i\ i )t | i i \ t ti at tl« - - j . -f* «r -are normal intermediate product in t u natural pr t ^ If

on the other hand, it s-* htld tl at thL kin 1 f i.\is]L*i J w rclireet value, then the ui%ht of tvidince io tn t ie >:dt «tformic acid,

The Production of Sugar from Formaldehyde.—The evi-dence for the condensation of formaldehyde to hexose sugarsin the laboratory under the influence of various catalysts isconvincing : but valuable though such evidence be, especiallyin providing points d'appui, it does not follow that such asequence obtains in the plant. The formaldehyde hypothesisrequires a hexose as the initial sugar: the general opinionof those who have concerned themselves with the final productsof carbon assimilation is that sucrose, not a hexose, is thefirst recognizable sugar, but, as has been pointed out on anearlier page, the known facts may equally well indicate that ahexose is the first formed sugar.

SUMMARY OF THE OPINION OF WILLSTlTTER AND STOLJL

Willstiitter and Stoil agree with Baeyer that formalde-hyde is the intermediate link between the carbon dioxidesupplied to the plant and the carbohydrate synthesized withthe help of chlorophyll. It has already been pointed out(p. 6o) that their chief argument in support of this view isbased upon the constant value, i t obtained for the so-called

f.4 1 UK ^ Y \ THF.?IS OF * U; ] ;i iliY! <K \11-

.i-- -,.:! ,! r\ r.it M «j '. M« , V •,! -, , A I . r*, fL\ >V r « tf'• n •! \ ' - <.} * * i * ji% * . \ <\n r

:* 't I . at J, ! \ !' 1 t r * r > ! > ' , ( \ \ ^ ,a ! !1>,ir,^ t t ^ f* r .^ - .< - : \ -• i - ^ \ A i ^!\ r i an 1* r- ti < <-. ! ^ ' , » I'-' i r* J! l f

f >• ^ » it in thati i- t . i -^ % «' r \ r a * i ' \ \ u"'l * r, i ! I \ , A ; 2 i>r l \ Î r u : *' - a * > > nt i ii' <! \\ - r, t< t« I?5M!«1* ]i\dr al.îr

^^.. 'r,- ?Ki \ il'ji I >r\ijf h :> thf ' ^ j , 1 a M iliv >hfaiiu'il ina :,ur;iir <£ t\}»rniMit< m \ **: ! v>hu i^ Ihi^ arû-nant i- r<t IU^MI up»»n tlu attuai \ ; inr i^nt i l iinr.on^tra-ti*«n <i fonnaldchyde iM^lattd fr«iTi- a-^rnilatin^; k a \ t \ and.wit»i nâni to Thi> \ annular qu^^ti n, t\iM authors ha\t»re\N.i many oi tin ^tatuiitnt^ n\ul* i>v piwi* ih \M rkersIt mill be rtiiicirsbiTid that repeatv d afUript ha\e beon madeto demonstrate the ft rmatii n i»f f -rmaldthyde by the actioni»f v\\h ruphyll upon carbon dinxid*1 outride the plant. Theearhtr wurk of Usher and Ine^tlcv, dtni»>n»tratmg thi for-mation of tfus hubi-tanee in tilnb at chlorophyll exposed toan atmosphere of carbon ditxide in ^ttnlight, was s>hown byWager* and Warnerf to be faulty, inasmuch, as Wagershowed, that no formaldehyde was produced if oxygen wasexcluded, whilst Warner showed that carbon dioxide wasunnecessary and took no paxt in the production of the for-maldehyde. Warner concluded that the formaldehyde was infact an oxidation product of the chlorophyll since oxygenwas actually absorbed in the process. The experiments of Will-statter and Stoll, show that no formaldehyde at all was formedif pure chlorophyll in colloidal solution was employed, thecolloidal solution being considered to approximate most closelyto the condition of the chlorophyll in the chloroplast. Theforautktehyde described by the earlier workers is attributedt# the oxidation of impurities accomfianying the samples ofcMomphyli lined by them.

The failure to obtain any trace of formaldehyde from purem mim m attributed by WtUttltter and Stoil to

f Wmmm 1 ML, &k

VIEWS OF WILLSTATTER AND STOLL 65

the absence of the essential enzyme which these authors pos-tulate in the green leaf, and this brings us to the considerationof the mechanism of the action of chlorophyll upon carbondioxide as visualized by these authors.

Experiments in vitro have shown that carbon dioxide canform with chlorophyll (I.) in colloidal solution an additivecompound of the type of a bicarbonate (II.) as expressed bythe equation

1 > V 1>NH n ° H

I n

This compound (II.) cannot be imagined to be capable ofparting with two atoms of oxygen with regeneration of chloro-phyll, so that some intramolecular rearrangement must firsttake place, and this, according to Willstatter and Stoll, in-volves the absorption of energy which is supplied by the sun-light. This change to a formaldehyde peroxide compound(III.) may be illustrated as follows :—

It is true that experiments in vitro have entirely failed todemonstrate the formation of a peroxide compound by means ofhorse-radish peroxidase, but this is considered to demonstratethe essential difference between test-tube experiments andthe activity of the living cell. Further, there is no doubt thatchlorophyll in the chloroplast is protected from photo-oxidationor decomposition by carbon dioxide in a way that chlorophyllin colloidal solution in vitro is not, since the chloroplast willtolerate concentrations of carbon dioxide which decomposechlorophyll in colloidal solution to the magnesium-free com-pound phaeophytin (V.) with precipitation of magnesiumcarbonate as illustrated by the equation

C65H?tO6N4Mg + H20 -1- CO2 - C65H74O6N4 + MgCO3

V.VOL. II.—5

fr, m i >\ \ i m - ; - m * w iv ' ; : \ ; i -

R I .' Ma O.CVi — R I " Mq 0 C* • 0" ° 1>NH ' ^ill

( > N n |>N.R I ;:::-..-Mg.OX^ - R Nfg*O*HCHO

1>HH i>N1 IV

Within the living cell the dt/conjposition of the piformaldehyde compound till/; is a^umi'd to be brought aboutby an enzyme, the existence of which enzyme is supported bythe experimental evidence relating to the assimilation numberwhich has already been considered [p. 45>

Wills tatter and StoIFs views are founded on Baeyer'stheory ; this formaldehyde hypothesis is certainly attractiveand is supported by many and various observations; itsdegree of probability is a matter of personal opinion, beingdependent on the relative values of the known facts. Butthe attractiveness of a hypothesis, or of a witness, or of anitem of evidence must not outweigh the many deficiencies,the fulfilment of which is requisite for a relative scientificproof, an absolute proof being hardly possible ; wherefore theconclusion is reached that the formaldehyde hypothesis hnot proven,

OTHER OF IN IONS.

FOEM1C ACID,

Erlenmeyer was the first to suggest that formic acid istltt mmt likely intermediate product af early origin, but it is$®ly df recent yean- that renewed attention has been given totilt, poatibility. Tlie irork of Sp#ehr has already been men-tioned ; ht demonttfftted that ctrtKin dkracide and water aremmify raiweed to tmrnk acid bf mentis of radiant energy;

MAQUENNE'S HYPOTHESIS 67

from formic acid a sugar-like substance is formed, in con-ditions such as obtain in an active leaf, which the plantcan utilize as a foodstuff. He points out that in Grafe's andBaker's work the plants were in reality supplied with formicacid, since formaldehyde is oxidized in the light, and he him-self found that plants increased in dry weight in an atmosphereof formic acid. Wislicenus * also supports the formic acidhypothesis. The thesis, however, has attracted but littleattention, for which reason there is not enough evidence towarrant a conclusion.

MAQUENNE'S HYPOTHESIS.

Maquenne f has formulated a theory of photosynthesiswhich is independent of the formation of formaldehyde orformic acid as intermediate products.

According to current ideas, the conversion of n moleculesof carbon dioxide into a compound containing n carbon atomsinvolves a polymerization which, according to the formalde-hyde hypothesis, is subsequent to the formation of the inter-mediate compound, formaldehyde. On Maquenne's hypothe-sis, polymerization may precede or merely accompany thedecomposition of carbon dioxide.

Willstatter and Stoll consider that chlorophyll may existin a true or in a colloidal solution ; J in' the former state it isunaffected by carbon dioxide but in the latter state carbondioxide and chlorophyll combine to form a compound of abicarbonate type.

Maquenne suggests that colloidal chlorophyll is either asimple hydrate of ordinary molecular chlorophyll or a polymerin which the molecules are united by loose supplementaryvalencies due, say, to the magnesium becoming tetravalentas in formula III.

On the addition of carbon dioxide and water, a substanceof formula I. results which by photochemical action yieldsa compound of formula II. which, being unstable, gives off

•Wislicenus : " Ber. deut. chem. Ges.,M 1918, 51, 942.f Cf. Maquenne : " Compt. rend.," 1923, 177,, 853.J See Vol. I., p. 307.

5*

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> N ^ |

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> N H !M g - O - C H ' .

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o

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g e n i o u s s p e c u l a t i o n c a n b e a c c e p t e d ; o n e p o i n t , a n d t h a t

e s s e n t i a l , o n l y w i l l h e r e b e m a d e : t h e t h e s i s i s f o u n d e d o n t h e

c o l l o i d a l s t a t e of c h l o r o p h y l l , a f o u n d a t i o n w h i c h , f r o m t h e

w o r k of S t e r n * a n d N o a c k , f i s n o t c e r t a i n ,

• S t e r n : *' liter, den t . ho t . Ges.,1* I Q J O , 3 8 , 2 8 ; t l Zeit , b o t . / '

f Noack ; " Bioc-hem, Zei t . , " 1927, 183, 133 , 153.

F U R T H E R R E F E R E N X E S

S t i k t : " F b c t o s y n t h M i s / ' L o n d o n , 1^25."•' FhoiosyBthes is ," New Y o r k ,

C H A P T E R I I I .

THE SYNTHESIS OF FATS.

A CONSIDERATION of the salient features of the physiologicalsignificance of fats has been given in the first volume,* inwhich place the consideration was retained for the sake of acloser association of the characterization and ni'hvr pr^MTtirsof fats with the physiological problems involved. F^r thisreason a general survey only, with such departures intu detailas necessity demands, will be given on the present occasion.

In the plant fats are commonly associated with the reservefood in seeds, spores, and vegetative perennating organs,but in smaller quantity they are probably present in all livingcells. As a food they have considerable value in that on physio-logical combustion they yield more energy weight for weightthan either protein or carbohydrate, the relative energy-producing values being roughly 5 : 3 : 2 respectively.f

The immediate question is the origin of fats : Have they aphotosynthetic origin from raw material in much the sameway as carbohydrates, or have they their origin in the trans-formation of carbohydrate or of protein ?

It was once thought that in some plants fats were directphotosynthetic products; Vaucheria was the prominentexample amongst the lower plants and of the higher plantsthose showing elafoplasts were quoted. As has been mentioned,there is some doubt regarding the composition of the fat-likebody occurring in Vaucheria f and on the available evidenceit is impossible to draw a definite conclusion.

* Vol. I., p. 34. See also Terroine : " Ann. Sci. Nat. Bot.," 1919,s6r. x., 1, i.

f See Terroine, Trautmann and Bonnet : " Compt. rend.," 1Q25,18a, 1181; " Bull. Soc. chim. biol.," 1925, 7, 461.

t See Vol. I., p. 35.69

H i - V M 1 I 1 - . - < ! ; \ >

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t .»*' -. i

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tHO-

CHjO

i ! *

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-CO ,

-IV--'\

\Ml' » ,

. k

ROOHOH

t *• •) !\ t I \)

\ a • ir ! *"

T.

4,\< r i p:

!i ^ • tin. ' ill .11

! m h<• t \ i ra

i.'^j h,*»r.

" rATItt

To ink substance the authors give the name of pliosphalidiearid and suggest that it may be the precursor of lecithin andkephalm in the animal body. These facts, however, do notprovide evidence of the photosynthetie origin of fats, nordo the presence of elaioplasts, which apparently are degradedchloroplasts. It must, therefore, be concluded that the factsrelating to the occurrence of fats and kindred substances inchlorophyll-containing cells do not permit the drawing of adefinite conclusion in regard to the present problem, and, infact, there would appear to be no evidence to warrant theconclusion that fats have a synthetic origin from raw materialsin the same sense as carbohydrates. For this reason theirorigin must be sought out elsewhere : the proteins and thecarbohydrates are the obvious sources.

Fmnaiian from Proteins.—Of the formation of fats fromproteins, little is known : the known facts indicate a relation-ship between these two classes of compounds, but the signi-ficance of this relationship is not clear. Evidence is notwanting that proteins, in certain circumstances, may be con-verted into fats ; but this evidence relates to the animal ratherthan to the plant.

In the plant there would appear to be some correlation,

. 31, 335, 233,

VRn\\ fWRBt MiYI^A i K- 71

although r>]»M'iit'i\ between proiiin dr.-] • .,'• ]; > ^f ..r\ •f«»und that the "il and protein content --if • ] , - r v : - ; i n :--;-correlated, in that ciiltura! ronditi-ns \\hi=.'h m;-'..:•;•-•• •!>•• ;:•.*also influence the other in the opposite diro..ti<--n ; :v:j'r,:,rw.zwith lime, phosphate and organic substances result- :u l3:iincrease of protein and a decrease in fat ; p-utash, -,n "I;,- -.-tixrhand, has an opposite effect.

Origin from Carbohydrates.—The weight of i;\i>krxefavours the view that in the carbohydrates is to be found theorigin of fats in most instances.

The conversion of carbohydrate t<> idt m plant-., r ii\ir

t

of plants, exposed to a low ternperatun . the <\^ >] * T. :;tfats in immature seed separated from t . j . par».^/ ' ..4.,* : ..*.'the fattening of animals on a carbohydrate ai« i :r I:- .t. *..close physiological connection betvutn ti^ tu,. \ ^ tcompounds.

The evidence given by the facts relating to the developpienof fats in storage organs such as the seed and structures ofvegetative propagation and to the changes which take placein the germination of a fat-containing seed, may here begiven.

Fat makes its appearance in the storage organ as that mem-ber approaches maturity. There can be but little doubt thatit is formed in situ from materials in the immediate neighbour-hood, a fact which is emphasized by many observations on thedevelopment of fats in seeds isolated from the plant whilst inan immature condition and originally containing little or nofat.

Quantitive results are not wanting. Numerous obser-vations, amongst which those of Schmidt, Le Clerc du Sablon,de Luca, Funaro, and Ivanow may be mentioned, show thatin the maturation of the seed, the increase in the amount offat and the decrease in the amount of carbohydrate are con-current. This is illustrated in the accompanying tablesrepresenting the results of two experiments by Le Clere duSablon:—

* Stark : " Journ. Amer. Soc. Agron./* 1924, 16, 636.

J2 THE

I! Datr of Gatfecnrg.i; r> July .

i August' 15 Augusti 1 September4 October

;Date nf Gathering,

9 June4 July1 August .r September4 October .

SYNTHESIS OF

J.NVT.

1

3it-*425Q6a

ALMOXI>.

Per Cent.Fat.

210374446

; PerCent.j lilllCOSf.

6 I4-2 ;0 ;00 1

FAT-

}>ru;:t. 1

*'4 ••0 ;00

FVr trnt.

4*M2-8 !2't\ ':r5 |

00-5o-bo-1 -6

Per Cent.Starch aiulpextrius.

14* 16-25-45'3

The carbohydrates which serve as raw material for theelaboration of fats vary with the p lan t ; glucose, sucrose andstarch would appear to be most commonly employed in thisconnection, the last two being initially hydrolysed.

It is a well-known fact that during the germination of fat-containing seeds, carbohydrates appear and fats disappear.Thus Maie * found that the isolated cotyledons of arachisseeds increased in their dry weight from 2*2613 to 2-6153grams, an increase due to the fixation of oxygen, whilst theirsugar content increased from *34l6 to *46$4 gram.

The disappearance of the fats is due, in the main, to theirtranslocation to the rneristematic and growing regions of thedeveloping embryo. This conveyance may be effected by themovement of fats as such in addition to the transport of theproducts of their hydrolysis, which change takes place in thestorage organ.

Schmidt f studied the movement of oils in the intercellularspace system of the axis of pea seedlings and found that therate of movement depended on the amount of fatty acid present;

f Sdwldt; " Fim," ttga* 74* 3****

ORIGIN FROM CARBOHYDRATES 73

he also found that a neutral oil would not enter the edl unlessthere were present a minimum of 10 per cent, of free fattyacid. It was therefore concluded that the free acid formed asoap which emulsified the fat, in which condition it could movefrom cell to cell, a conclusion which Schmidt supported byhis inability to detect either sugar or starch in the endospermof germinating fat-containing seeds. Rhine * has repeatedSchmidt's work and disagrees with his observations and con-clusions. Rhine found that the rise of oils in the intercellularspace system was not dependent on the presence of free acid,and that neutral fats do enter the ceil from the intercellularspaces, but this is dependent, not on the presence of free acid,but on the water deficiency of the cell wall ; if the cell wall isnot allowed to lose water, which, from the experimental pro-cedure, is likely to happen, there is no intake of fat. Thepresence of relatively large quantities of sugar was establishedin the endosperm of germinating seeds of the castor oil plant,hemp and flax ; Schmidt found none. Rhine also points outthat the soap formation would appear to be improbable inview of the pH values which occur. The formation of a soapis dependent on a neutral or alkaline medium, an acid reactionis inhibitive; in the plants examined, the reaction was alwayson the acid side ; in most tissues the pH was 6-2, the highestvalue observed being 6-8.

As may be read in the chapter on Respiration, the respi-ratory ratios differ in accordance with the material consumed ;from his comparison of these ratios in the hypocutyls of seed-lings of fat-containing seeds and of starchy seeds, Rhine con-cludes that the material respired by the former could not befat. Thus the general conclusion is reached that in normalconditions fats are not translocated as such, and that the fatcontained in a seed undergoes hydrolysis and is convertedinto sugar before transport takes place.

Le Clerc du Sablon, Ivanow, Miller and others have thrownmuch light on the hydrolysis of fats in their studies on thegermination of various fat-containing fruits and seeds. Lipase

* Rhine : " Bot. Gaz.," 1926, 82, 154,

THE SYNTHESIS OF FATS

is the enzyme concerned, and by its activity the fat is hydro-• lysed into glycerol and fatty acid, which products are usedeither immediately for the needs of the ^tvdings or are firstconverted into carbohydrate. Agreement is general that asgermination proceeds, the acid radicals becomes saturated,as is indicated by the decrease in the iodine value, and therate of consumption of these acids is inversely proportionalto their degree oi saturation.

As the fats decrease, the carbohydrates increase, a factbrought out in the following analysis of arachis seedlings byMaquenne:—*

Age in Days. Per Cent.Fat.

Per Ccnit.Carbo-l&ydrate other than

CelluloseCellulose andother InsolubleCarbohydrates.

06

10121828

5*'3949-8136-1929-0020-4512-16

n-55S-35

12-5212-349*4°

2-513-46

5-227.299-4$

The nature of the carbohydrate formed differs in differentplants; glucose would appear to be the most common, butsucrose, starch and dextrin^ also are described. It is notunlikely, as Maquenne has pointed out, that the nature of thecarbohydrate depends upon the Iqgdjqf fat and its degree ofsaturation. He considers that the saturated fatty acids con-tribute in a much lesser degree to the formation of sugars thando the unsaturated acids, and that the saturated fatty acidsare principally used for respiratory purposes. Agreement,however, between the authorities in respect to this aspect ofthe subject does not obtain. Ivanow considers that there isno real difference between the saturated and unsaturatedfatty acids in their power to give origin to carbohydrates,the difference in their amounts being due to the more rapid con-version of the unsaturated variety. However this may be,the salient feature in the germination of a fat-containing seed

* MaquenDe: " Qwapfc. road-/*

LIPASE ;5

is the conversion of the fat into carbohydrate, the reverseto what obtains during the maturation of the seed. Thechange is effected by the activity oflipase which hydrolvsesthe fat into glycerol and fatty acid.

The work of Ivanow has shown that lipase has a reversibleaction, and whether it hydrolyses or synthesizes fats is merelya question of conditions, mainly the presence or absence ofwater. The glycerol extract of a fat-containing seed, whichextract contains the lipase, mixed with oleic acid will synthe-size a fat : the addition of water will result in the hydrolysisof this fat into glycerol and fatty acid.

In the synthesis of fats, Ivanow considers that highersaturated acids of the fatty series are the first to be formedfrom the sugar, and these are converted into unsaturated acidswhich combine with the glycerol to form the fat.

With regard to the origin of glycerol, the chemical relation-ship between this substance and glucose is so close as to suggestat once the possible inter-relation of the two; further, glycerolmay have an origin in respiratory processes as is shown by theproduction of this substance during the alcoholic fermentationof sugar.

Maclean and Hoffert * have studied the formation of fatin Saccharomyces. When yeast is incubated in oxygenatedwater, part of the reserve carbohydrate disappears and theamount of fat increases. This change does not take place ifthe yeast is grown in M/io oxygenated solutions of propyl,butyl and isoamyl alcohols, but does happen when the plantis incubated in M/io solutions of sodium formate, sodiumpropionate, sodium butyrate, glycerol and acetone. Whencultivated in 5 per cent, solutions of ethyl alcohol, sodiumacetate, sodium lactate or sodium pyruvate, an increase inthe amount of fat occurs similar to that effected by a 0-5per cent, solution of glucose, but this amount of fat is notfurther increased by the higher concentration of the substancesmentioned. In sugar solutions, on the other hand, the cellsincubated in oxygenated solutions store up fat, in addition

* Maclean and Hoffert: " Biochem. Journ.," 1923, 17, 720. See alsoMaclean: id., 1922, 16, 370.

; 6 THE SYNTHESIS OF FATS

to carbohydrate, in amounts dt*pendin£ ^n the wû^ni ration ofthe sugar and thi> is independent of the natun1 *»! the suâr.

Maltose is rn^re effective in prom>>nn^ the -t^raê of car-bohydrate and less effective in bringing about flic formationof fat as compared with fructo-c, ^luaê and sucf'ê: thepresence of phosphate increases the amount *>f fat antl de-creases the amount of carbohydrate stored. The presenceof oxygen is an essential condition for this fat production.

In the present consideration, the salient points of Mac-lean and Hotfert/s investigation arc the relationship betweencarbohydrate and Cat and the possible substitution of carbo-hydrate in the culture medium by sails of acetic, lactic andpyruvic acids.

Haehn and Kinttof * showed that the fungus Endomycesvernalis was able to build up fat "when grown on a mediumcontaining acetic aldehyde and even ethyl alcohol as the onlysource of carbon, which possibly indicates that acetic alde-hyde may be a stage in the conversion of sugar into fats. Thesequence they suggest is glucose -> pyro-racemic acid -*acetaldehyde -> aldol -> glycerol esters.

The evidence outlined above gives strong support to thethesis that fats have their origin in carbohydrates: thechemical aspect of this probability may now be considered.

The striking recurrence of the eighteenj:art>on atom fattyacids belonging to the various series exemplified by stearicacid C18HMGj, oleic acid C18H8#Ot, linoleic acid ClsHaaOft,linolenic acid QgH^Qagave rise to the suggestion that suchacids were produced by the direct union of tliree six-carbonatom sugars; there are, however, several reasons for rejectingthis hypothesis. In the first place, there is no particular reasonto exclude the association of hexoses withpentoses; thus sup-posing one hexose and two pentoses to be involved, an acidcontaining sixteen carbon atoms would result ; a difficulty,however, arises in th€ fact that a single molecule of a hexoseand a patios® respectively would result in a fatty acid con-taining eleven carbon atoms, but it is a well-known fact that

* Raehn and Ktattof: " Her. devit. chem Ck*./' 1925, 56, 439.

ORIGIN OF FATTY ACIDS 77

the acids entering into the composition of the vegetable fatsinvariably contain an even number of carbon atoms. On thesegrounds this theory of the formation of the fatty acids mustbe rejected.

Another question of importance in connection with thefatty acids is whether the acids of lower molecular weight havebeen derived from those of higher molecular weight; orwhether, on the contrary, they represent stages in the buildingup of acids of high molecular weight. The discovery byWindaus and Knoop that in nature the point of attack upona fatty acid is the Jj-.carbon atom, has proved of considerableimportance. The process may be illustrated by a concreteexample: butyric acid is oxidised in the animal to /3-hydroxy-butyric acid

CH3CH2CH2COOH -f-O-^ CHaCHOH . CH2. COOH/ttiydroxybutyric acid

a reaction which was first effected in vitro by Dakin with theuse of hydrogen peroxide. In general terms, an acid of theformula R—CH2 . CH2COOH is thus converted into an acidR . CHOH. CH2COOH, and this, on further oxidation, yields anacid RCOOH which, as will be seen, contains two atoms ofcarbon less than the original acid. "This method of oxidationthus accounts for the production by metabolic oxidation onlyof acids with an even number of carbon^atoinSj assuming theoriginal acids to have contained an even number.

The alternative suggestion that acids of high molecularweight are built up from those of lower molecular weight isone which is more acceptable in explaining the production offatty acids from carbohydrates.

It was first suggested by Nencki * that in the productionof butyric^acid from lactic_acid by fermentation, acgtic.alde-^yde.was an intermediate stage; the changes involving theformation of a four-carbon fatty acid (butyric acid) from athree-carbon acid (lactic acid) could be accounted for asfollows :—

CHaCHOHCOOH = CH8CHO + COa + H2.Lactic acid Acetic aldehyde

•Nencki: " J. prakt. Chem.," 1878, 17, 105.

-S THE SYNTHESI- n | - F \ F -

Two inn!emlc- «->f nrc!ic aklehynr ?;;<-p. ijnjkrô an acondensation •••••

i'H3i.HO •- C"H3CHO ::••• ij-f , H<*HiH/H*>

ami the ranking akiol by muUviiUr svarrun^vmc-ni ^butync acid--

CHaCHOH i HaCHO - CB,t HXHXOoB.

The ^nrqstion *»f making u>c *-.f a!<lo] condensations forthe sumsîve building up of morr and more complex aldolsby the serial addition of two atoms of car!>'>n was developedby Magnus Levy * and subsequently by Leathe*vt Thus theformation of an eight-carbon fatty acid can be explained bythe following changes :—

4CHaCHO •--.= CH,CHOH CH,CHOH l..'HaCHuH CHXHOwhich, by loss of four molecules of water, would give the un-saturated aldehyde CH3. CH : C1I . Cli : CH . CII: CH . CHO,from which, by the addition of six atoms of hydrogen andone of oxygen, would be obtained the eight-carbon atom fattyacid CHjCH.CHjCH.CHtCHjCHjCOOH, octoic or caprylicacid. An extension of this suggestion is that due to Smedleyand Lubrzyrj^ka J according to whom pyruvic acid producedby the decomposition of carbohydrate is the starting-pointfor the formation of fatty acids ; pyruvic acid is knownto yield acetic aldehyde by the action of the enzyme car-boxylase, and the aldol condensation of this acetic aldehydewith another molecule of pyruvic acid would yield a five-earbon compound according to the following equations:—

|i} CHgCOCOOH - CHt€HO f CO,Pymvic acid AcrtakWhyde

(2) CHtCHO -f CHt CO COOH - CHS€HOII CHtCO COOH.The latter substance by loss of water would give an un-

saturattd ketonic acidCHJCH : CH CO COOH

which could react in cither of the two following ways to give acompound.

HMi Levy: * * £wgtta*&8*ft Ai«IUv/* tôtt 363,.. f Ijmtbm: " Problems m Xnktmd Meta-ho^m,1' L<>«don, 1906.

ORIGIN OF FATTY ACIDS 79

(3) CH3CH : CH CO COOH + O = CH3CH : CH COOH + CO2Crotonic acid

CH3CH : CH COOH + 2H = CH3CH2CH2COOHButyric acid

or(4) CH3CH : CH CO COOH—CO2 = CH3CH : CH CHO.

Crotonic aldehydeCH3CH : CH CHO+CH3 CO CO0H=CH3CH : CH . CHOHCHa. CO . COOH

yielding a seven-carbon compound which by a series ofchanges similar to those undergone by the five-carbon com-pound would give a six carbon acid—

CH8CH2CHaCH2CH2COOHCaproic acid

While there may be some difficulty in accepting this theoryof the formation of fatty acids in the animal body, owing touncertainty of the production of pyruvic acid as a normalproduct of carbohydrate metabolism, as is pointed out byLeathes, this difficulty would not apply to the decompositionof carbohydrate by micro-organisms.

It has already been pointed out in Volume I. that Neuberghas established the production of pyruvic acid in aloollQlicfermentation and has shown it to be the p££cursor of...theacetic aldehyde formed in this process; Neuberg and Arinstein,*in an investigation of butyric acid and butyl alcohol fermen-tation of glucose by Bac. butylicus Fitzianus, have establishedthe fact that here also pyruvic acid is the intermediatesubstance; this may give rise either to acetic aldehyde or tobutyric acid and butyl alcohol, but these products are mutuallyexclusive, showing that butyric acid and butyl alcohol arenot formed by way of acetic aldehyde. This the authorsproved as follows: the bacillus when grown on either aceticaldehyde, or its aldol CH3CHOH CH2CHO, produced no butyricacid, but when grown on the aldol of pyruvic acid, it producedbutyric acid in quantity. The mechanism of this reaction isexplained as follows :—

CH3CO COOH + CH3CO COOH = COOHPyruvic acid |

CH3C OH CH2CO COOHPyruvic acid aldol

•Neuberg and Arinstein: "Biochem. Zeit.," 1921, 117, 269.

So THE SYNTIIKSI-" <*F F.\T>

and the latter by l^ssof two m,V r y k , .,:f * dr)u,\-, <]i-'\i«le £ave

i'i 1 ,C!!(>1111!,CHO - -* ( II Ht 11,,t } 1 ,i i. M ÎHat Jr,v -I v.l

In addition, ^malltT quaDtitit^ i j *. ,ipn>: anil i apr\'hr ands,containing six aiul ti^ht faring] atoir*. n^r-t ? tivtiy, u r reobtaintd. Thrse invTritigati<->Ti>. ti\i;t.it i!».ir u'ith- th*»si' mentionedon an tamer pagt\ fir^vidt' a fair ani-unt r»f txptriinentalevidence in ••upp^rt of tise virw that. >nârs îve nî1 to fatsby an initial breaking down to ômpnuiul* rr>ntainin^ a smallnumber of carbon atoms, and then combining the^r to formcompounds of higher molecular weight. The methods out-lined above also account for the Io>s of uxvên necessitatedin passing from a carbohydrate containing six carbon af(.»rnsto a fatty acid of the same number of carbon atoms as isshown by the formula? C6Hl2Os ~Cg!IuOj, an np-eratioii whichwould be very difficult by direct reduction, but is overcomeby the removal of hydroxyl groups ab water.

C H A P T E R IV.

THE SYNTHESIS OF PROTEINS.

IN view of our limited knowledge of the chemistry of proteins,the degree of our ignorance respecting their synthesis in plantsis not surprising.

It is generally agreed that the leaves are the importantcentres of protein formation, and they show a periodicity intheir nitrogen content. Otto and Kooper,* Le Clerc duSablon f and others J found that there is a gradually decreas-ing amount of nitrogen from the spring to the autumn, andthat leaves of several different plants, even in different stagesof development, exhibit a greater nitrogen content in themorning than in the evening. These observations are con-firmed by Chibnall, § who found that a definite fall in the pro-tein content of the leaves of the runner bean occurs during thenight, a fall due to the translocation of the dissociation pro-ducts of the proteins effected by the presence in the leaves ofproteolytic enzymes.||

This same author f also has investigated the relationshipbetween the various combinations of nitrogen in the leavesof Phaseolus vulgaris var. multiflorus at different times of theday and in different stages of development.

With respect to age, Rippel ** found that the proteincontent of the leaves of Populus canadensis suddenly dimin-ishes as the yellow autumnal colour is assumed, a diminutiondue to the cessation of protein synthesis rather than to anincreased rate of transportation.

* Otto and Kooper : " Landwirthsch. Jahrb.," 1910, 39, 167.t Le Clerc du Sablon : " Rev. Gen. Bot.," 1904, 16, 341.J See Chibnall: " Ann. Bot./' 1923, 37, 511.§ Chibnall: "Biochem. Journ./' 1924, 18, 387, 395, 405.j| Ibid., 1922, 16, 599, 608. ^Ibid., 1922, 16, 344.•* Rippel: " Biol. ZentrlbL," 1921,.41, 508.

VOL. II.—6 8l

82 THE SYNTHESIS OF P K M I T J X -

It must not be concluded from tJuv,* .K • r- s. i ? i •.- J > - fLi! fhrmaking of proteins by the green plan! j> ::*•.- — ,mU ,1 r^ii*;!-<synthesis. Fnr the process to take p! i>v ?^T. - n-i-f he Ji\di}~able the appropriate raw material* ar.d *>f tL- -r > ,trand various element?, especially i m r ^ i n , m -nifbinations, are the rn«»st signilieant. Hie < .iri'^hvdr.ite ]>rr-sumably must be a sugar, and provided if in: a\,.ubhk\ proteinsynthesis is independent of light. * and ran take place in :illparts of the plants although in dirirrent drgm-s.f Sina.-the photosynthesis of carbohydrates is a divlight. proi:e»,it is not surprising to tind that the production *>f }»n»-teins may be four or live times as great m xhv light as 211darkness. {

Schirnper,§ experimenting with cut leaves uf Safnlnwusand with potted plants of Pelargonium zonxle, found thatnitrates were destroyed in green leaves exposed to daylightbut were not so destroyed if the leaves were kept in thedark, and in agreement with this, shade leaves were foundto be richer in nitrates than sun leaves, Furthermore, nodestruction of nitrate occurs in etiolated leaves exposed tosunlight.

The investigations of Ullrich jj indicate that the chloro-glasts, the centres of carbohydrate formation, play a partin protein synthesis. Detached leaves of Bmssica, Cueurbita,Lactuca, Phaseolus, Tropatolum and Zea, grown in the lightand supplied with nitrate, produced protein which probablyhad its origin in the plastids, for these showed an increasein size in comparison with the plastids of leaves grown insimilar conditions but deprived of nitrogen. When protein-containing plastids were digested with pepsin, the appearanceof the remaining structure was like the chloropla&ts of thenitrogen starved leaves.

As has been indicated above, protein synthesis can takeplace in darkness provided that an adequate supply of carho*

©, JM» 623.Rend. Ace. Urtcei Roma," 1^3 J3, n.,f

I Montemartini: w AtH R. last. Bot. Bavin/' i^s- l f >*§ Sckimper : " Ik>t. Zeit . ' 188S, 46, 12H.(i Ullrich: "Zeit. Bot./* 1914, 16,

! v ]ra' S i * '

<arhi«:.\dr.it< . f 1, , , p:r n C ;. ' M • :i i^ i tisrthj a-, i M4'»>» t t*T i' %. -! ^ i'i •" Mi - " ;

thit it * ,\v .\ nf lit Ht/t' } r. t* m r- r !.J it"s n •<«ilt- . t n'ff«4?'; IT. ^JI, ; U' 1 ^ • " ^ ^ * «*

carbnhv»Ir»»tr in ,i.i» »JI it ar '.? * ^ i\ *i'i ' !»no d* ubt li it ]• J t . " t - . ! * , ! • r * * ^ -tein> but Hi:- !i- I ! i ^ l if ! r * • / ' ,»« --is abnnrnul *n A ^r »\;*i - ^ ; t *• i'a phottu hi niii al uu jjnijt r r >rn\i' * v,h1* "^ - . ^ ^that the s\ nth -K in cLirhiii^M^ du t ^ ^ I M ' I ; r-- i, , - aprncesbcsurichridurt'tht* iutrat»s.in>i*«> ai i I'M' }^ . . + i;of proteins. It ha» also been stated that if but - u,\? q.T,-tities of carbohydrate are available, the 5yntlu>i* < i ^ it ttm*in darkness, may *toj> at the formation nf ainidt-*f ^hi hsome plants eg, Alga* such as PhuroiMiH* and kapmJiumand the Fungi Eurotium and Penicillin**, can directh jL^tmi-late.**

There is available but. little exact knowledge regardingthe specific action of invisible wave-lengths of radiant energywhich may be concerned in the different phase* of proteinmetabolism. Sehanz tf iinds that, ultra-violet light rendersproteins less soluble, possibly on account of its detetertrmsaction on cnzymes.JJ and in this is found a reason for the duarfhabit of alpine plants "which are subjected to a high degree of

* Zaleski &nd Sufuki: " Ber, dtnt, bot. G«.," i$ 7* if, 536; " ©ot.Centrlbl,,'1 igoi, 87, 281. Sutmki: 'l Bull. Coll Ag. Tokyo," iB«A a, 409;

f Zaleaki : " Her, d«it. Wt. C»e«.»1' I9CK|? 37» 56.J Loew : id., igtj, 90, 90*;*.§ MucsKcher : " Iteit, Ga«.,r* 1923, 7s, 249-fj Baudisch . " Zentr, lUkt. Parasit./' 1 12, ja, 520f Jakob*. " Biol. Oistrlbi," 1S98. 18 5 .?-#* Luti : '' Ball. Soc. Bol. France/1 igo » 4», 119.ff SchatuE: " H«r. deut. bot* i>s./* 101*. » » f>iQ,XttimVoLL

8 4 THE SYNTHK-IS O F rkoTI . ; i \>

insulation. Bovie * draws attention to tin- tihnriKy of theextreme ultra-violet ray?5, the.1 *u-*/a!]» ..i Schumann rays, andpoints out that the photolysis of proton and *»f protoplasmincreases with the diminution in Un w .tve-kn^th <>f the in-cident li£hl : in tlie extreme ultra- v-^ki repnn, tlie amountof chemical change is proportional t«.» the product of the lightintensity and the time of exposure.

It will be remarked that these ob^.trvation* refer not directlyto protein synthesis but rather to the action of these qualitiesof radiant energy on the general growth of the plant, a subjectwhich will be considered later.

The supply of nitrogen is an essential factor, and thiselement must be presented in a form suitable for the nu-tritive processes of the plant. Thus amongst the non-greenplants, Sacckaromyces is unable to make use of nitrates andbut little use of simple amines ; urea can be assimilated incertain conditions, but the best results follow the employ-ment of peptone, a culture solution of peptone and sucrosegiving 177-4 per cent, increase in dry weight after the com-pletion of fermentation.f

There is, however, much variation shown by the lowernon-green plants in this respect; Bacillus coli will flourish on adiet containing salts either of ammonia, of simple amides orof ami no acids, whilst the cholera bacillus can apparently makeno use of ammonium salts but is able to utilize ammo acids.For the Fungi, Boas % considers tha t in general ammoniumsalts, and especially the ammonium salts of organic acids suchas quinic acid, are better than amino acids as a source of nitro-gen in. protein synthesis. He points out that such feedingexperiments should be brief, in order to avoid secondary,especially proteolytic, changes, and that the reaction of theculture medium is all-important. Acidity may inhibit thegrowth of many fungi, &o that if ammonium salts of inorganicacids be used as a source of nitrogen, the observed resultsmxy be due not to ths inability of the plant to employ the

t Bokmmy : *' €$mm. Sell./* 4{ ® " Biochem. Zcit.," 1918, 8*, 110,

particular s.ilt. but fn thv .KIJ.;-V u; ,\ h .•;-, \ : \ • . .. > • . - ,g*-v> is a—innlated.

w svr and !<>imd that the niU'Xiii n.r.f.T? •. f ?;:..- <•.•,, . .,.-..decreases to a steady value uhn'h ;*• ;;r:.tJV: v ••; ••••.. ": .";variations in the amount of ammonium >ulpkiK n: v.. . .;:*.,:-'medium, If the ammonium salt be replaced by -'-ip.i-' -•-,:vr.iT :or by urea, the nitrogen rontent is una^j'.;tr.'d. h\ii J?.» --;l -t=-tutitm by peptone lowers it slightly, while ônÎr i r !'--^-.T> itniueh. A eonsiderable irurrease in 1 hi: i-.u]^'ih\ <]r.:%^- r -ri* ••?::T.glucose or surn»se, of tlw eulture nk-*]iinn *• ai*.-.*.> ;\n rv r- i.-; uithe nitrogen content of \vun^ ciHtnri-- hut ;,.t.- t'-.-' r-,-.r,,effect on < 1 eultures. Ihe substitu?j,.n •-f ^!u-• — ;A .j.r.-i-binosc or by xylose lias no etlect, but âl.i^*-î' •> :',,.•> :.:, .,; •preeiabie decrease in the nitrogen contm? l;u-:. ,iu'L-r^consider that the nitrogen metabolism of As*cr?iûs i- *:ri;i!arto tiiat of higher animals.

For ordinary green plants tiv -uj]'l\ »t r./1* , - : . Iin the simple nitrogen-containiny "-alt-< f t!.i ^ •! *J* " I ^,-the fertility of the soil, not only \uth re**]: eet to ratrat^ 1 talso in regard to other substances,t i> ,\n u.ij, ortaru ta« t^rconditioning the amount of protein found in the par.f Inaddition to nitrates, some plants can n;akt ti-e f arrnj^ni^îsalts. Hutchinson and Miller J tour 1 tin-, t» I tn . i,conditions of eulture which prtelutud tl -. » rc^v t, \ > :tr it în the soil. In this respect, howiwr all ila^*- d T * ^have alike; whilst some will p%*w cvjuallv w.lt w •.*' r

supplied with nitrates or ammonium -alt- otKr*> f; 'un>:.best when supplied with the former, ami t»tlier> ^ttr,:n^lvprefer ammonium salts to begin with and then nitrate?*

Mention has just been made of the importance of soil substances other than nitrates in the protein synthesis of plants :potassium may be taken in illustration. Species of bacteriagrown in the dark in eulture media containing the requisite

* Terroinc, Wurmser and Montane: " Bull. Soc. chim. bioi,"

f Whitson and Stoddart : " Ann. Rep. Wisconsin Kxp. Sta../* 1904,

tHutthiwon and Miller: «• Journ. Agric. Sci.," 1909. 3» 1 ^

8 6 7 1 I f < V \ T I I F < F <>r

o r g a n i c f*> >d i r a t t r i a l - a n d - i ! * * s * * ,

s h o w b u t j . t»»r d i \ t l < ' p n u n t . r . i 1 ' * - 1 * ]

s i m i l a r K u l t u r a l c m l i t i n> « n . i l h : ^ * * * , i

t i n i v s K<i- ] n t f i n a n d u i ; h U t T , t i r - v , s j * . , u

w i t h o u t | * t a w u m , d - r a n p a r j * w, * * <*» ! A

g r o w n i n > U r i l e c o m h t m n ^ i i u i . ' ; * * * * : , ^ i i ^ n ,»

a s u g a r a n d >jf p l i e d w i t h kn« w n , u * t •* i r i n «'i >\ ' !• ,

i t w a s l \ u n d t h a t t h o - c j ^ r u ^ n i n t 1 f w i " »' ; ' r 1 ' t

o f p o t a s s i u m a s r t g a r d s t h e *>vnt*;t - , - t i r * »*, - ! t h «t *' ^

a d d i t i o n o f s u ^ a r t«> t h e c u l t u r e i n t < l i i t ) j itT» ; , * U M : I is i f

o f p r o t e i n . I n d a r k n e s s , o n t h e < t ^ r ! m I i l i s ^ v ^ i r t i l -

d e v e l o p m e n t o c c u r r e d a n d o n l y t , - ^ ' ^ - ; ' • i w / •

p o t a s s i u m s a l t s s h o w e d p r o t e i n fi r . - s " *i

I t w o u l d t h e r e f o r e a p p e a r t h a t t 1 a* ! : t t *• * j -Mi T ' . *

i s p a r t l y i n d i r e c t , a n d o n l y i n d a r k r u « < - .? »- «f * l.iv 4 n s v ; i • i t o , *

r o l e i n t h e p r o d u c t i o n o f p r o t e i n ? . r - i i ^ 1 p * ) t a - ^ n : r ^ > i r , i ! d ' \

r a d i o a c t i v e , i t m a y p o s s i b l y < e r v e a - a *- i ; r« •/ »! c m r ^ v n i

p r o m o t i n g m e t a b o l i c a c t i v i t y , b u t u - j . is b - - . - i n ^ a r ^ d * ^

a p r e v i o u s p a g e , n o d e f i n i t e c o n c l u s i * » r . a i . b> -iravHn t r -m t l . c

a v a i l a b l e e v i d e n c e . *

O C C U R R E N C E O F T I T R A T E ,

T h e o b v i o u s e s s e n t i a l i t y o f n i t r o g e n i n t h e b u i l d i n g o f

p r o t e i n s a n d t h e f a c t t h a t i n g e n e r a l t h e e l e m e n t i s a b s o r b e d

b y t h e p l a n t i n t h e f o r m of n i t r a t e * h a s b e e n r e m a r k e d u p o n .

T h e w o r k of A n d e r s o n f a n d e a r l i e r a u t h o r s h a s d e m o n -

s t r a t e d t h e p r e s e n c e of n i t r a t e i n a . l a r g e n u m b e r of p l a n t s ;

b u t a r e v i e w of t h e l i t e r a t u r e s h o w s s o m e d i s c o r d a n c e , w h i c h

i s p r o b a b l y d u e t o t h e f a c t t h a t t h e a m o u n t of d i s c e r n i b l e

n i t r a t e i n l e a v e s v a r i e s a c c o r d i n g t o t h e s e a s o n of t h e y e a r ,

t h e richness of t h e s o i l , a n d t h e c o n d i t i o n s of g r o w t h , m o r e

e s p t c i a l l y t h e d e g r e e o f i n s o l a t i o n . T o c i t e b u t t w o e x a m p l e s :

.$ms*d0jimHe0M g<ccmvn%in its n a t u r a l h a b i t a t , a s h i n g l e b e a c h

f r o m " s f h k h s a l t s a r e r e a d i l y S e a c h e d l a n d e x p o s e d t o m a x i m u m

i m o t a t i k m , g a v e n e g a t i v e r e s u l t s w h e n t e s t e d f o r n i t r a t e ; t h e

s a m e p l a n t gmrnng in g a r d e n s o i l a n d i n a c e r t a i n a m o u n t

J» 107.

OCCURRENCE OF NITRATE 87

of shade gave strong positive reactions. Rumex conglomeratusgrowing on an old manure heap gave much stronger reactionsfor nitrate than another plant of the same species growing afew yards away in a hedgerow. From these and many similarobservations, the conclusion is warranted that the presence ofdetectable free nitrogen only is to be expected when the uptakefrom the soil is greater than its rate of elaboration, and thisrelationship in vigorous vegetation is determined by the qualityof the soil and the habitat of the plant particularly with regardto illumination. With respect to diurnal and seasonal vari-ation, Anderson observed that in Solarium dulcamara theamount of nitrate in the morning may be considerably lessthan in the late evening ; and Mercurialis perennis gave posi-tive reactions in October and negative in June. Further,there is a localized distribution in the plant; thus althoughnitrate was found in the leaves of various Solanaceae, none wasfound in the young green berries ; similarly although nitrateoccurred in the etiolated inner leaves of a cabbage, none wasfound in the outer green leaves.

Nitrate as such is a comparatively inert substance and doesnot readily lend itself to chemical change, wherefore in theplant it must presumably be changed into a more labile sub-stance before it is elaborated into proteins ; nitrite immediatelyis suggested on chemical grounds, so that two problems arise :the occurrence of nitrite in the plant and the mechanismwhich effects the reduction of nitrate to nitrite.

OCCURRENCE OF NITRITE.

The literature of the subject shows some disagreementabout the presence of nitrite in the green plant. This is dueto the following facts. The amount of nitrite is always small,so that its presence must be detected by delicate colour reac-tions ; Griess Ilosvay reagent is commonly used; it givesa pink colour with dilute solutions of free nitrite, but it is acidin reaction, and thus a pink colour also is given if traces ofanthocyanins are present.

The expressed juice of the plant must be used ; this is asolution of various substances which may react with the

88 THE SYNTHESIS O F PROTEINS

nitrite on the contact inevitable after expression; thus if tan-nins be present they and the nitrite would interact so thatno reaction for nitrite would be given on testing. Also amechanism may occur in some plants , e.g. Allintn, whichprevents the detection of nitrite added to the plant juice.

The sap must be examined immediately after expression ;a plant juice, e.g. Sambucus, may give a positive reactionwhen first extracted, but a negative reaction after the lapseof a few hours.

There is, however, no doubt t h a t free nitrites are of com-mon occurrence in the green plant. Aso * established theiroccurrence in etiolated potato shoots, and Klein "j* detectedthem in Erythrina and other plants, bu t he points out that insome plants, more especially those with root tubercles, thepresence of nitrite may be due to the activity of bacteria.Taking such precautions as are indicated above, Andersonfound that more than 2O per cent, of the angiosperrns of di-verse affinity J examined, gave positive reactions for nitrite.

It is sometimes assumed that ni t r i tes are toxic to the plant,and that they must be quickly elaborated in a healthy plantand not allowed to accumulate. F rom culture experiments,it would appear that nitrite is poisonous if supplied to theplant above a certain limit, but this limit apparently varieswith the conditions of the habitat of the plant. Feh6r andYagi § found that no ill-effect followed ^when nitrites wereadded to the soil in amounts up to I gram per kilogram. Theylater observed that o-88 per cent, of nitrite, calculated as N2O3)

in clay and sandy soils, and -95 to I -OI per cent, in gardensails rich in humus poisoned the plant . When grown inwater culture a greater amount of nitr i te is tolerated ; I -25 to3*73 P e r cent, was found to be injurious to growth but notso far as to cause death.

* Iso : " Beth. Bat. Zeatr./' 1 03, 15, 208 ; 1914,32, r46.f Klein : id., $©> 141.t Araideae, Caryopliyllaceae, Compositae, Cucutrbitacea,. Leguininosae,

Lythraoeae, Resedaee«, Rosaceae, Salicaceae, Scrophi3iariacea&, Solanaceae.TJImaceae and TJrticace».

§ Fefadr and V£gi: " Biochem. Zeit.," 1924, 153, 156; 1926,174*262.

REDUCTASE 89

THE REDUCING MECHANISM.

If the formation of nitrite is the first stage in the synthesisof proteins, the question arises as to the mechanism in theplant which effects the reduction of nitrate to nitrite. Thereduction may be due to the action of an organic " reductase,"or it may be a photochemkal^reaction. If it be the latter,the process must take place in the subaerial parts, essentiallyin the leaves, and since proteins are formed in structures farremoved from light, it follows that the nitrite must be removedto those places where the elaboration is taking place. Thispresents certain difficulties, but is not altogether to be dis-missed since nitrite has been detected in stem and roots.Also nitrite has been found in the sap exuded by root pressurefrom the cut ends of the fuchsia and the vine,* but since theflow of sap is upwards, the observations have, in all probability,no bearing on the immediate problem, although they indicatethat the reduction of nitrate to nitrite occurs in the root, whereit is dark, as has been found by Thomas f in Pyrus mains.

Considering now the evidence.11 Reductase."—The ability of the plant to convert nitrate

into nitrite was first observed by Laurent,J and later, Irvingand Hankinson,§ working on Elodea, came to the conclusionthat nitrite must be an intermediate compound in the meta-bolism of nitrates.

Kastle and Elvove || were the first to show that the ex-pressed sap of potato tubers and etiolated shoots, and thesap of other plants, was able to effect the change from nitrate tonitrite, especially in the presence of acetaldehyde and otherlike bodies. Their observations were confirmed by Bach f whoworked on potato tubers. Anderson ** following the work ofBach ff and of Haas and Hill $ J on milk, found in twenty-three

* Kastle and Elvove : " Amer. Chem. Journ.," 1904, 31, 606; Priestleyand Armstead : " New Phyt.," 1922, 21, 62.

f Thomas : " Science," 1927, 66, 115.I Laurent: " Ann. Inst. Pasteur/' 1890, 4, No. n .§ Irving and Hankinson : " Biochem. Journ./' 1908, 3, 87.|| Loc. cit. % Bach : " Biochem. Zeit./' 1913, 52, 412.** Loc. cit. ft Bach : loc. cit.Xt Haas and Hill: " Biochem. Journ." 1923, 17, 671. Also Haas and

Lee: id., 1924, 18, 614.

po THE SYNTHESIS OF PROTEINS

angiosperms of varied affinity the power to bring about t h ereduction, the best results being given by the potato, tubersand shoots, and shoots of Solanum dulcamara. There is insome instances a seasonal variation, thus Solanum dulcamarawas just active in October, but in June the same species, ga th -ered from various localities and at all times, gave good positiveresults. The '* reductase " is a thennolabile oxidizable s u b -stance precipitated from its solution by saturation with a m -monium sulphate. The best conditions for the manifestationof its ability are a temperature of 45° C* and the presence ofan accelerator, such as acetaldehyde, which, obviously, a reartificial conditions; at ordinary temperatures in the absenceof an accelerator the reaction is very slow. In the majorityof its properties it resembles the aldehydase of animalorigin, e.g. that of milk. If this be the mechanism for t h ereduction of nitrate to nitrite in the plant, it must be assumedthat the conditions obtaining in the living cell are much morefavourable for the reaction to take place. The elucidationof the problem was carried further by the concurrent work ofEckerson f on tomato plants ; he found that the plant juicein the presence of fructose, or glucose, and free oxygen easilyreduced nitrate to nitrite in a slightly alkaline medium, t h ebest reaction occurring at pH 7-6 ; the juice of nitrogen highplants, containing nitrates and sugars in abundance, rapidlyreduced nitrate at 500 C. at pH 7-6, and in these conditionsboiled juice was as effective as unboiled. In carbohydratehigh plants, containing much sugar but no nitrate, the re-duction of added nitrates was localized to those regions whichgave a slightly alkaline reaction, i.e. the stem tip just behindthe growing region, cells of the leaves especially near t h ephloem, cortical cells of the petioles, phloem parenchymaof the stem bundles and adjacent cortical cells in the nodalregions. It was also observed that newly formed aminesand amiao acids, aspartic acid, asparagine, alanine, etc., a p -peared in the regions of the nodes ; and in the petioles, in t h e

* In experiments with, plant juices, especially if continued over rela-tively kmg periods of time, due precaution must be taken against bacterialactivity wkidi may ghre rise to nitrite.

t Ecketscm: •• B«fc. GaaL," 1924,77, 377.

REDUCTASE 91

blades of young leaves and immediately behind the stem tipsin carbohydrate Iiigh plants three or four days after nitratehad been supplied to the soil. Never was the process ofreduction affected by the presence or absence of light, and inthese experiments an activator such as acetaldehyde was notused. From these observations it would appear that thedegree of activity of the " reductase" is much affected by thereaction of the medium and that possibly sugar takes the placeof acetaldehyde. The process is thus brought closer to naturalconditions, but the best results were obtained at unnaturallyhigh temperatures.

In an attempt to correlate the action of the potato tft re-ductase " with the aldehydase responsible for the reductionof methylene blue to theleuco compound (see p. 139), Michlin *and Bernheirn j* made preparations of the " reductase " of thepotato, the former by the precipitation by acetone from anaqueous extraction and the latter by precipitation by ammon-ium sulphate, dissolving in water, and treatment with charcoal;after which the enzyme was precipitated by saturation withammonium sulphate, centrifuged, dried in a vacuum desic-cator and dissolved in water as required. Bernheim foundhis preparation to have a wide pH range, 3 to 8-6, with anoptimum at 5-5, with respect to nitrate reduction ; at pH 7 to7-5 there was a good nitrate reduction, in the presence of acet-aldehyde, which corresponds sufficiently closely with Eckerson'sobservations.f The action of the enzyme is independent oftraces of iron, as is indicated by the addition of KC!N. Bern-heim finds that there is a close relationship between thispotato aldehydase and typical aldehydase of animal origin,the hitherto assumed difference between them disappearingwhen the pH is adjusted.

In the reaction, on Wieland's thesis (see p. 138), nitrateis the hydrogen acceptor, and in these test-tube experimentsaldehyde is the hydrogen donator ; in the plant some other

* Michlin : *' Biochem. Zeit.," 1927, 185, 216.f Bernheirn : " Biochem. Journ.," 1928, 32, 344.j It is to be remembered that the optimum reaction varies with the

degree of purity of the enzyme and upon the nature of the substrate (seeVoL I., p. 468).

92 THE SYNTHESIS OF PROTEINS

donator must be found ; from Eckerson's work and on t h e o -retical grounds sugar is indicated, but Bernheirn found t h a t ,in vitro, both glucose and sucrose gave negative results .To summarize these observations : there occur in a number ofplants a reducing mechanism, generic with aldehydase of animalorigin, capable of reducing nitrate to nitrite, provided a nhydrogen donator is present. There are, however, certaindifficulties to its unqualified acceptance as the mechanism usedby the plant in the first stage of protein synthesis : in vitrothe action is very slow at air temperature and in the absenceof acetaldehyde, but at higher temperatures (Anderson, 45° ;Eckerson, 500 ; and Bernheim, 27° C.) the reaction is relativelyrapid, especially in the presence of acetaldehyde. Further,in the test tube, the activity of the mechanism falls off in.time fora reason unknown, but this may not occur in the very"different conditions obtaining in an active plant cell.

Photochemical.—The evidence in support of the thesis tha tthe reduction of nitrate to nitrite is a photochemical reductioais not extensive.

It was first shown by Thiele * that potassium nitrate, onexposure to the rays from ~a quartz mercury vapour lamp,was reduced to potassium nitrite with evolution of oxygen.With a view to the further study of the photochemical decom-position of nitrites, Baudisch f exposed mixtures of potassiumnitrite and methyl alcohol in aqueous solution to diffuseddaylight and to ultra-violet light and found that the methylalcohol became oxidized to formaldehyde at the expense ofthe nitrite which was reduced to hyponitrite, and this latter,at its moment of formation, reacted with the formaldehydeto produce the potassium salt of formhydroxarnic acid—

KNOt + CH»OH *. KNO -f HCHO -f HtOKNO -f HCHO = H .C .OH

i!NOK

The reduction of nitrate or nitrite in the presence of alcohol is

* Thiele : " Ber. deut. chena. Gtsdte.," 1907,40* 4914. See also Mooreand Webster: " Proc. Roy. Soc,/' B, 1919,<x>, 158.

f Baadisdh: "Bar. dtnt. cktm. Gesells./' 1911, 44, 1009. S&e alsokx. c&.# 1916, 4% 1176, asHi 1918, 51, 793. BUy, Hcilbrou and Hudson," Jcnara. Ckem. Soc./' 1922, 121,1078.

HYDROLYSIS OF PROTEIN! 93

a purely photochemical reaction since no such change couldbe produced in the dark even if the solutions were boiled-

It will be observed that the evidence mainly relates toultra-violet radiation, in the absence of which the plant canflourish and form proteins. For this reason, and especially inview of the facts relating to the occurrence of nitrites and theformation of proteins in the dark places of the plant, the con-clusion is justified that in the plant the photochemical re-duction of nitrate is without significance.

Assuming that the reduction of nitrate is the beginning ofprotein synthesis, the further sequence of reactions is a matterof speculation. If the doctrine of the reversibility of enzymeaction be held, as it generally, if not always is, the reversal ofthe changes effected by the plant, by means of its enzymemechanisms, in the dissociation of the protein molecule, maythrow light on the formation of proteins ; for this reason thesechanges may first be considered.

HYDROLYSIS OF PROTEIN.

The protein content of a seed or other storage organ,whether temporary, as the leaf, or more permanent, is areserve food which must be hydrolysed before it can be trans-located and made available for the growing parts. The cleav-age products of proteins yield a large number of amino acids(Vol. L, p. 443) and a number of amides. The following aminoacids have been identified in the plant : arginine, glyciae,histidine, leucine, lysine, phenyl alanine, tryptophane, andtyrosine. Of the amides, asparagine and glutarnme are im-portant members, from the point of view of plant physiology,and of these asparagine is the more conspicuous.

This dissociation of protein takes place with some degreeof rapidity; Jodidi * found that after eight days7 germinationin darkness, 48 per cent, of the proteins originally present in thefruit of the maize was hydrolysed to water-soluble diffusiblecompounds. The evidence of his analyses indicate that pro-teoses and polypeptides are amongst the first degradation

* Jodidi : " J. Agric. JRes.," 1925, 31, 1149.


products, and that the amino acids and acid amides are formedfrom the polypeptides.

In Cicer arietinum, there is a marked increase in t h eamount of amino acids and their amides during germi-nation, which increase is at the expense of the proteins. **Similarly there is an accumulation of amides in the shoots o fgerminating peas ; the amount of amide nitrogen in the s e e dleaves fluctuates much at first but in the later stages of ge r -mination there is a marked increase, whilst, concurrently,the tf-amino acids decrease in amount and finally disappear-From these relationships between the amides and amino acids,Sure and Tottingham f consider that arnino acids serve for t h eproduction of amides.

Of the cleavage products of proteins, asparagine is amongstthe more conspicuous in the plant. It occurs in the d e -veloping parts | in greater abundance than in the memberswhere the reserve proteins are stored; Schulze § found t h a tonly 7*62 per cent, of asparagine occurred in the cotyledons,whilst 31*81 per cent, occurred in the axis of the lupin. Alsothe relative amounts of asparagine and aspartic acid s h o wconsiderable variation during germination and, in the l a s tstages, the amount of asparagine formed is in a proportiongreater than the amount of protein decomposed.

Asparagine is possibly a secondary product.|| Accordingto Prianischnikow,lf in the germination in darkness of legu-minous seeds, its origin is due to the oxidation of the aminoacids derived from the protein reserves, ammonia being formedand then elaborated into asparagine. When etiolated seed-lings are illuminated the asparagine disappears, being used i nthe construction of new protein.

The above facts relate to reproductive organs; on t h e

• Zlataro-flE : " Biochem. Zeitseh.," 1916, 75, 200.t Sure and Tottingham : " Journ. Biol. Chem./' 19T6, 26, 535.$ This may easily be shown by germinating a lupin seed in the dark:

until the hypocotyl is a few inches in length. On mounting a section oJfthe hypocotyl in strong alcohol and examining under the microscope, a,large Bmnber of crystals of asparagine will be seen.

§ Schulze: " Landw. Jahrb./' 1878, 411.|f See Choate : ** Bot. Gass./* 1921, 71, 409,Tj Prianischnikow ; " Biochem. Eeit./* 1924, 15a, 406.

ORIGIN IN THE SEED 95

vegetative side similar changes occur. Chibnall,* workingon the protein metabolism of the runner bean, found that thedecomposition products of the proteins of the leaf consist ofasparagine and other bodies containing free amino nitrogen.He considers that asparagine is the chief form in which nitro-gen in a state capable of resynthesis is translocated throughthe plant. Detached leaves kept with their petioles in water,translocation thereby being excluded, contain, after a fewdays, little or no asparagine, whilst other amide nitrogen ismore abundant than in the leaves attached to the plant.

The consideration of these facts fully warrants the con-clusion that in the plant proteolysis yields amino acids withamides, especially in the form of asparagine, of, possibly,secondary origin. Does a reversal of this sequence occurwhen proteins are laid down as reserves in storage organs ?

THE ORIGIN OF PROTEIN IN THE SEED.From the facts of animal physiology there is no doubt that

amino acids, the products of the hydrolysis of protein food inthe alimentary tract, are reconstructed to form protein in thevarious tissues after their passage through the walls of theintestine into the blood stream. There is reason to supposethat the same sequence obtains in the plant. Thus Zaleski ffound that during the ripening of pea seeds there is an in-crease in the amount of the protein at the expense of the aminoacids and organic bases, as is indicated by nitrogen determina-tion of these compounds.

N of proteinsN of amino acids .N of organic bases .N of other compounds

Control.

79-2 per cent, of total NS'7 „

108i'4

After Five Days.

89*2 per cent, of total N4-6 „5-t :: ::

The results were not so well marked for all seeds ; thusin similar conditions but little protein synthesis took place

* Chibnall: " Biochem. Journ./' 1924, 18, 387, 395, 405.f Zaleski: " Ber. deut. bot. Gesells./' 1905, 23, 126 ; " Beih. bot.

Zentrbl.," 1911, 27, 63.

96 THE SYNTHESIS O F PROTEINS

in the maize, whilst in the sunflower there was a diminutionof protein. Woodman and Engledow,* in their detailedbiochemical study of the development of the wheat grain,found that there was a regular increase in total nitrogen andin the percentage of protein n i t rogen during development.The nitrogen enters the developing grain, which contains nonitrate, in the form of asparagine, a n d they conclude that theasparagine is oxidized to ammonia which is the starting-point of the protein synthesis. T h e y also observed thatgliadine was greater in amount than glutenine, and that thesetwo substances increased at much t h e same rate until the be-ginning of the desiccation of the g ra in , after which the glute-nine remained constant and g l u t e n appeared. Smirnow fcompared the nitrogen metabolism of etiolated seedlings ofhigh carbohydrate content, e.g. ba r ley , and of low carbohy-drate content, e.g. lupin, which w e r e grown in sterile culturesolutions in controlled conditions. It was found that thebarley seedlings readily absorb ammonia , which is followed byan increase in the amount of asparagine. The rate of absorp-tion depends on the amount of s u g a r contained in the plant ;at first it is rapid and then falls off and stops as the sugarcontent diminishes to a certain limit. The presence of calciumsalts promotes the formation of asparagine, but the processends sooner since the sugar is more rapidly used up.

The degree of uptake of a m m o n i a by lupin seedlings isdependent on the presence of suga r in the culture medium.Seedlings given ammonium salts a n d glucose show a higherasparagine content, a higher p r o t e i n content, and a lesseraccumulation of ammonia in the t issues than seedlings givenammonium salts but no sugar. Fur ther , the ratio of aspara-gine nitrogen to ammonium ni t rogen is twice as great in theseedlings given sugar in addition t o the ammonium salts.Younger seedlings after being g iven ammonium and glucosealso showed a higher amino acid content , but the amount felloff with age as the proteins are regenerated.

These works indicate that in t h e plant amides and amino

* Woodman and Eagledow : " Jourru Agric. Sci," 1924, 14, 563.f Smirnow : " Bioc&em. Zeit.," 1923, 137, 1.

ASPARAGINE 07

acids are reconstructed into proteins ; that carbohydrateis essential to the process, and corroborate the conclusionarrived at on an earlier page, that light as such is of no directconsequence ; and that asparagine plays a prominent part.

As has been seen, asparagine may be produced from aminoacids, possibly by the combination of ammonia and asparticacid * to form ammonium aspartate which gives origin to theasparagine by the loss of a molecule of water. Prianischnikov,fwhilst recognizing in ammonia the end product of proteindissociation, considers that it also is an initial stage in theformation of proteins, and, according to him, asparagine isthe form in which it is stored.

Asparagine lends itself to condensation more or less readily,and by simply boiling a solution, the dipeptide of asparticacid is formed.J

There is no definite information of the details of aminoacid synthesis in the plant. In the laboratory the followingsynthesis have been effected (see Vol. I , p. 450) :—

Ammonia -j- glyoxylic acid-> fonnylglycine and acetyl alanineAmmonia 4- J3-vinyl acrylic acid~> diamino valeric acid.Ammonia -f- fumaric acid -> aspartic acid.

If like synthesis occur in the plant, ammonia and unsaturatedacids are requisite.

Nothing is known of the combination of amino acids intothe final protein; it will, however, be remembered thatFischer synthesized a number of polypeptides, some ofconsiderable complexity, from amino acids in the laboratory(see Vol. I , p. 447).

To summarize our little knowledge of the synthesis ofprotein in the plant: nitrate is reduced to nitrite and possiblyto ammonia, which reacts with sugar, or a degradation pro-duct of sugar, possibly an unsaturated acid, to form simpleamino acids which may give rise to asparagine, or otheramines, a temporary phase which can easily be decomposed

* Schulze: loc. cit. Prianischnikov: " Ber. deut. bot. Gesells./' 1904,22, 35. Treboux : id., 570.

f Prianischnikov: " Bull. Agric. Tntell.," 1917, 8, 204.t Ravenna and Bosinelli: " Rend. R. Acad. Lincei./' 1919, 28, ii, 113.

VOL. 11.—7


again to amino acid. The amino acids then combine to formpolypeptides which in turn give rise to the proteins.

With regard to other views concerning the synthesis ofproteins, Treub,* from his investigations on the distributionof, the periodic variation in the amount of, and cognate ob-servations on the cyanogenetic glucosides,f concluded thathydrocyanic acid is the first recognizable product of nitrogenassimilation, and possibly is the first organic nitrogen com-pound formed. Whilst on purely chemical grounds it is notimpossible that, acetone cyanhydrin, (CH3)2: C(OH)CN, maybe a stage in protein syntheis, Treub's conclusions are notconvincing: free hydrocyanic acid has not been identifiedin plants, and its compounds may equally well result from theoxidation of amino acids : in other words, the known factsregarding compounds of hydrocyanic acid in the plant neitherprove nor disprove Treub's hypothesis.^

* Treub: " Ann. Jard. Bot. Buitenzorg," 1895, 13, 1 ; 1904, 19,86 ; 1907, 21, 107.

f See Vol. I., p. 253.t Rosenthaler: " Schweiz. Apoth. Ztg." 1920, 58, 137; 1921, 59,

10, 22 ; " Biochem. Zeit./' 1923, 134, 21,5, 321.

C H A P T E R V.

RESPIRATION.

THE maintenance of life is impossible without a sugpjy^pfenergy. This energy is acquired by various chemical reactionsof an exothermic character whereby substances of differentnature are converted into compounds of lower thermal energy,and thus energyjs released. This is more obvious in an animalthan in a plant, which generally is less obtrusive in its move-ments and various activities and may make_use of radiantenergy, more particularly in the production of food.

That energy may be produced by the combustion of asuitable fuel is a commonplace and is illustrated in the steamengine, the boilers of which are heated by the thermal energyliberated by the combustion of fuel which varies accordingto local circumstances. The heat-producing power, or calorificvalue, of fuels varies, the most efficient material is that whichproduces the maximum number of heat units or calories fora given weight of substance.

The heat produced in the combustion of a fuel is due tothe chemical reaction of oxidation, or, in other words, the heatgiven out when the constituent elements of the fuel severallycombine with oxygen to form the corresponding oxides. Theheat of combustion of a compound will, therefore, dependupon the heat of combustion of its constituent elements andis greater the richer the compound is in elements possessinga high heat of combustion. The most economic fuel is, there-fore, that which contains a minimum of constituents whichcannot yield heat on combustion with oxygen ; of these wateris the most significant, followed by various metallic elementswhich form the so-called ash constituents. It is obvious thatthe more complete the combustion, the greater is the energy

99 7*

IOO RESPIRATION

set free, wherefore the heat engineer devises the completeoxidation of available fuel, wood, coal, coke, and mineral oil,to carbon dioxide and water. In the living organism, on theother hand, this combustion may not be so complete and may-stop at a phase several stages short of the ultimate, a portionof the total potential energy of the material physiologicallyconsumed being sufficient for its needs. For instance, Bacilluscoli and B. pyocyaneus in the resting condition obtain sufficientenergy in oxidizing succinic acid to fumaric acid, but if thesebacteria are proliferating the fumaric acid is also oxidized assoon as it is produced.*

It is a principle in physical chemistry that the heat ofany chemical reaction depends solely upon the initial and finalproducts, and the total heat evolved is the same by whatevermethod the final products are obtained, i.e. whether in onesingle process or by a series of intermediate stages, and alsowhether the reaction proceeds rapidly or so slowly that thereis no perceptible rise in temperature. In view of these factsit will be clear that the same laws hold for the low temperatureof oxidation of various substances in the living cell as for thecombustion of these substances in air or oxygen.

The heat of combustion of an element is determined bycausing it to combine with oxygen in a closed chamber andmeasuring the heat evolved calorimetrically. The two equa-tions—

C + 02 = C02 + 94'8KH2 -f O - H,0 + 69K

indicate that by the complete oxidation of 12 grams of carbonor 2 grams of hydrogen 94-8 and 69 kg. calories f are evolved.

These equations may be expressed in another form:—

1 gram of hydrogen on combustion yields 34*5K1 ,, ,, carbon ,, ,, „ 7*9K

* Quastel: " Biochem. Journ.," 1924,18,365. Quastel and Whetham:id., 1924, 18, 519; 1925,19, 5i9, 645.

t A kilogram calorie is the amount of heat required to raise the tem-perature of 1 kg. of water through i° C. A statement to the effect that thecomplete oxidation of glucose, for example, liberates 709 kg. calories there-fore means that the heat energy liberated during the combustion of 180grams of glucose is sufficient to heat 709 kgs. of water through i° C.

CALORIFIC VALUE 101

From which it will be seen that hydrogen on combustionyields relatively much more heat than carbon ; consequentlycompounds rich in hydrogen have a high calorific value.Moreover, since oxygen and nitrogen have no calorific value,it follows that the presence of these elements in compoundsreduces their calorific value. Thus it comes about that fatswhich contain only about 11'per cent, of oxygen have a con-siderably higher heat of combustion than carbohydrates whichcontain as much as 53 per cent, of this element. The actualvalues of the heat obtainable by the combustion of some ofthe more important fuel substances of the living cell are asfollows :—

1 gram of carbohydrate — 4-1 calories1 ,, ,, alcohol = 7-11 ,, ,, fat == 9-1 ,,1 ,, ,, protein = 5-8 ,,

In the procurement of energy the plant exhibits a widerrange than does the animal, and this to a larger extent than isoften thought; thus Ramann and Bauer * have estimatedthat young saplings of deciduous trees may show a loss of 20to 45 per cent, of their dry weight during the burst of activitywhich follows the winter sleep.

The term resrjjration here is used tqjnclude all^thpsejgro-cesses which^ involve a liberation of energy_ejjipj.oyaWe bythe organism in its various activities. Respiration is notmerely the absorption of oxygen and the excretion of carbondioxide, as is too often supposed, an idea having its origin inthe lungs of an animal being termed the organs of respiration.Respiration is essentially a catabglicprocess, and anyj^rganof a plant or of an animal which is doing work is an organ ofrespiration in that it cannot accomplish its task without theenergy obtained by the exertion of appropriate mechanisms.The lungs and the respiratory tract, on the one hand, and thestomates, lenticels, " respiratory chamber," and the intercel-lular space system on the other, are strictly comparable : theyare organs of breathing ; structures, reservoirs, and surfacesfor the conveyance and initial absorption of oxygen, and for

* Ramann and Bauer: " Jahrb. wiss. Bot.," 1911, 50, 67.

102 RESPIRATION

the ultimate elimination of the gaseous waste ofcombustion.

This motive power commonly is obtained blogical^combustion of carbohydrate, by which,a molecule of sugar is completely oxidized by toxygen giving origin to 6 molecules each of Ccand water with the liberation of a considerateenergy :—

C«H12O, -f 6Oj = 6CO, 4- 6H2O -f 674K.

The liberation of these 674 calories by 1oxidative degradation of a molecule of carbcinvolve the production of temperatures far ab(served in the living plant, but, as has been indthe same amount of thermal energy will be set iprocess takes place in slow stages with a hard!rise in temperature. This oxidation is effectedby means of enzymes, and when the respirati<a considerable amount of heat is evolved and dithese aspects will be considered later.

RESPIRATORY QUOTIENT.

The above equation shows that the volundioxide evolved is equal to that of the jpxygenthe plant this correlation provides a well-knowinvestigating certain aspects of the respiratorThis fact, first appreciated by de Saussure, is conthe Respiratory Quotient, in the consideration cwell to realize that it has but little value in hessential parts of the process, correlating as it dfinal product, carbon dioxide, of a long series ofthe absorbed oxygen, the two being but remcThe ratio CO2/O2 is variable not only in differeialso in the same plant at different phases of iin other words, the value of the ratio is subjecditioning factors. Puriewicz,* for instance, foi

• See Bonnier and Mangin : " Ann. Sci. Nat. Bot.," xl1885, vii., 2, 315 ; 1886, vii., 3, 5.

•f Puriewicz: ** Jahrb. wiss. Bot./' 1900, 33, 573.

RESPIRATORY QUOTIENT 103

amount of carbon dioxide evolved showed a much greaterrange in variation than did the absorbed oxygen in the sameplant in different conditions, the figures obtained in a seriesof experiments showing a variation of — 14 to 120 per cent,of the average for carbon dioxide, whilst the oxygen variedfrom o to 48 per cent *of the average. Ruby * observedin the olive that the ratio remained practically constant andwas but little affected by the age of the plant or of the organexamined ; generally it is rather higher in leaves from fruitingbranches, a fact possibly connected with the greater abundanceof available food, and in the early season of the year it is lessthan unity but later rises to unity.

In the germination of the white mustard, Brassica alba,Stalfelt f observed that an increase in the partial pressureof oxygen from 20 to 50 per cent, led to an increase in oxygenabsorption. In normal conditions, he found that the maximumabsorption of oxygen occurred on the fourth day of germinationwhen the ratio CO2/O2 was about 0-50.

When the carbon dioxide evolved is due to the immediatephysiological combustion of available substances, it will beapparent that the respiratory quotient will vary accordingto the nature of this food.

Thus, in general terms, if carbohydrate is the immediaterespirable_substance, the respiratory quotient will be in theneighbourhood of unity ; on the other hand, if fat is con-sumed the ratio will be less than unity owing to the fixationof oxygen by unsaturated fatty acids. Thus in germinatingflax, which is fat containing, figures varying from 0*3 to 0*64were obtained by Bonnier and Mangin.

These ratios are applicable only in those instances wherethe physiological combustion is complete. But the processmay stop at an intermediate stage; thus the following acids,

CH2OH COOH COOH COOH CH2COOH

(CHOH)4 (CHOH)4 (CHOH), COOH C(OH) . COOH

COOH COOH COOH CH2COOHGluconic Saccharic Tartaric Oxalic Citric

* Ruby : " Ann. Sci. Nat. Bot.," 1917, 20, 1.f Stalfelt: " Biol. Zentrlbl.," 1926, 46, 1.

104 RESPIRATION

can be produced from sugar both in vitro and in vivo, for allhave been identified in the degradation products of var iousstrains of Aspergillus niger* grown on carbohydrate sub-strates.f It is obvious that in all such examples the re -spiratory quotient will be less than unity. Oxalic, malic,citric and tartaric acids are of fr^uent occurrence in t h ehigher plants. True it is, that it is difficult to decide whe the rthese, in common with other products isolated from the p lan t ,are of catabolic or anabolic origin. In the ripening of fruit,the disappearance of organic acids is concurrent vrith t h eappearance of sugar; it is an open question whether t h eacids give origin to the sugar or are used up in respiration.

If these acids are used in respiration, it is worthy of n o t ethat malic acid is the most easy to oxidize and citric acid t h emost difficult, tartaric acid being intermediate ; the quest ionarises whether this circumstance is in any way connectedwith the fact that apples and pears, which chiefly con ta inmalic acid, can ripen in northerly regions; that grapes, wh ichcontain tartaric acid, require a warmer climate and do n o tripen so effectively as the apple in the northern t empera t ezone, whilst the orange and lemon, which, contain citric acid,are confined to tropical or sub-tropical countries.

Succulent plants provide examples of a normal s toppageof the respiratory process at an intermediate stage. Membersof the Crassulaceae do not give off carbon dioxide when firstplaced in darkness, although the absorption of oxygen is act ive ;there is, however, an accumulation of organic acids, malicand oxalic, and it is not until these have accumulated inrelatively large quantities that carbon dioxide is evolved asin a normal plant.] The peculiar metabolism of these p lan t sis generally associated with and explained by their massivestructure rendering the movement of gases a relatively slowprocess, the catabolism stops at an intermediate stage a n d

*The respiratory activities of tbis and other moulds are commerciallyexploited.

tFranzen and Schmidt: " Ber. deut. chem. Ges.," 1925, 58, 222.Amehing: " Zeit. physiol. Chein.," 1927, i6<$, 161. Butkewitsch : " Bio-chem. Zeit./' 1927/ 1&2, 99.

tSeeMcolas: " Compt. read.*" 19x8,167, 131.

CARBON MONOXIDE 105

thus forms a reserve of carbon which is converted into carbondioxide, available for carbon assimilation, by the decomposi-tion of the acids when photosynthetic conditions supervene.Comparable phenomena occur in bacteria in certain conditions,the formation of acids being a well-known occurrence in theiroxidative activities.

Of other products of respiration, carbon monoxide has beendescribed by Langdon and Gailey in the pneumatocysts ofNereocystis Luetkeana,* a unique example as far as is known.The bladders contain a mixture of nitrogen, oxygen, car-bon monoxide, but no carbon dioxide. An analysis of thegaseous contents of over a thousand of these floats showed thecarbon monoxide to vary from 1 to 12 per cent, by volume,whilst the oxygen ranged from 15 to 25 per cent. Only whenoxygen is present does carbon monoxide form in the bladders ;if the oxygen be replaced by nitrogen or hydrogen, no carbonmonoxide results. The gas is produced naturally both by dayand by night but it is not formed when the plant is groundand allowed to undergo autolysis or decay, in which circum-stances carbon dioxide and hydrogen are produced. Fromthese facts it is concluded that the gas is a respiratory productand has no connection with carbon assimilation.

In further illustration of the respiration of fats, the workof Stephenson and Whetham j* on the Timothy grass baccillus,which is characterized by a high fat content, may be mentioned.It was cultivated at 3J0 on a medium containing ammoniumphosphate as the sole source of nitrogen, and glucose as theonly source of carbon, the pH being maintained at 8*0. Theperiodic analyses of the proteins and fats of the plants andanalyses of the medium showed that the sugar was completelyused in about three weeks, during which period the amountsof fat "and protein synthesized gradually increased to a maxi-mum until no sugar remained in the culture medium. Thenthe amount of fat contained in the organism rapidly decreased,

•Langdon: " Journ. Amer. Chem. Soc," 1917, 39, 149. Langdonand Gailey : " Bot. Gaz.," 1920, 70, 230.

f Stephenson and Whetham : " Proc. Roy. Soc," B, 1922, 93, 262 ;1923* 95> 200.

106 RESPIRATION

but not so the protein. Carbon dioxide was continually giyeraoff, but no intermediate products were detected. In the earlier*phases, when the sugar is consumed, and fat and protein syn -thesized, the respiratory quotient is high : but in the later"phases, after the sugar has been used up, the amount of ca r -bon dioxide emitted falls off and the respiratory quotient drops-This change in the respiratory quotient supports the view,based on chemical analyses, that fat is the respirable materialof the plant.

De Boer,* in his investigation of the respiration of Phyc-otnyces, found that the respiratory quotient varied accord-ing to the food consumed ; on a linseed substratum, on whichthe respiration is more intense than on a carbohydrate medium,it ranged from 0-65 to about 075, while on bread, an essentiallycarbohydrate diet, its average was 1 rising to 1*20 at the peak:of respiratory activity.

With regard to the physiological consumption of proteinsin lower organisms, Terroine, Wurmser and Montane* j - foundthat Aspergillus niger in conditions of starvation lost relativelymuch protein; this fact, taken in conjunction with the ob-servations that on a medium containing glucose the respiratoryquotient is in the neighbourhood of unity and falls to 0-8during starvation, % leads the authors to conclude that t h eplant uses protein rather than carbohydrate as a reservematerial. Further, when Aspergillus is grown on a mediumcontaining proteins as the sole source of food, the productsof respiration are carbon dioxide, ammonia and water.

FERMENTATIVE PROCESSES.

The equation given on page 102 represents the result of ex-haustive^ajgrqbic consumption of carbohydrate. Less economicmethods of obtaining energy from carbohydrate are also em-ployed by the plant, the carbohydrate being broken down t oproducts of lower heat energy, but still having considerablepotential energy which is possible of exploitation by further

*De Boer: " Rec. Trav. bot. Norland.," 1928, 25, 117.t Terroine, Wurmser and Montan6: " Bull. Soc. Chim. BioL," 1922,

4* 625.X See Kosinski: " JaarbCLch. wiss. Bot./' 1902, 37, 137.

FERMENTATION 107

oxidation. Such processes may be effected without the inter-ventiori_of atmospheric oxygen and are commonly known asfexmentations. Alcoholic, lactic and butyric fermentation ofsugar are well-known examples ; in all the amount of energyreleased is much smaller^ than that from complete oxidation ;this is shown in the following equations :—

C8H12O6 = 2C2HftO + 2CO2 -f 28JCAlcohol

C«H12O6 = 2CH3CHOHCOOH + 18KLactic acid

C6Hl2O6 = CH3CH2CH2COOH -f 2CO2 + 2H2 + 15KBuytric acid ""*

In illustration, the fermentation of sugar by yeast may bebriefly considered.

As in normal oxidation of sugar, the fermentation processis accompanied by the evolution of heat, although to a muchtesser extent, a gram molecule of maltose yielding 42*7 kg.calories, taking Brown's * value of the heat of fermentationof 1 gram of maltose as being 125 calories. The alcoholicfermentation of sugar is therefore a wasteful process forobtaining energy when compared with oxidation, since byits means nearly thirty-one times as much sugar must be con-sumed to obtain as much energy as is yielded by the directoxidation of sugar. The significance of this figure of the heatof fermentation of maltose may be realized by Brown's ob-servations that between the temperatures of 140 and 160 C.the time required by a yeast cell to ferment its own weight ofsugar varies from eighteen and a half to nineteen and a halfhours and that the heat generated during one hour is sufficientto raise the temperature of the cell by 150 or 160 C. Fromsuch observations Brown estimates that at 300 C. yeast canferment its own weight of maltose in 2-2 hours and the poten-tial rise in temperature in the cell in one hour will be 75-5° C.under adiabatic conditions, figures indicative of an intensemetabolism and, apparently, a great waste of energy. Brownsuggests that the explanation for this waste is to be foundin the fact that brewers' yeast is a cultivated plant grown inunnatural conditions. The wild yeasts lead a quiet life on the

* Brown : " Ann. Bot.," 1914, 28, 197.

108 RESPIRATION

skin of, say, the grape: rupture of the skin of the fruit providesa nutrient medium eminently suitable for growth and repro-duction accompanied by a free access of oxygen. A periodof intense activity immediately supervenes and budding takesplace, under the continued action of the oxygen, with remark-able rapidity : this activity necessitates a continuous supplyof energy, which is provided by fermentation. Reproductionand fermentation thus are correlated. " That we can bymeans more or less artificial keep the reproductive power ofa yeast in abeyance, whilst still availing ourselves of its fer-mentative power, has hitherto obscured the relation of thetwo functions, and hence has given rise to the somewhatexaggerated idea of the purposeless and prodigal waste of theyeast cell regarded as a living unit."

In view of the definition given above, it is obvious thatany process, oxidative or reductive, which liberates energyavailable for use by the plant is to be included amongst res-piratory processes, irrespective of the initial products con-sumed and the final products evolved. Thus, in addition tothe oxidative processes of the higher plants in which fats,carbohydrates, proteins, and, in extreme circumstances, evenprotoplasm may be physiologically consumed, the diverse meta-bolic processes of bacteria and comparable organisms in reduc-ing sugar to alcohol, sulphate to sulphide, or oxidizing alcohol toacetic acid, lactose, to lactic acid, ammonium salts to nitrites,nitrites to nitrates, and so on, are all processes of respiration,notwithstanding the fact that many of these activities may

be extra-cellular. For green^plants, oxygen is a a}mriiQnessential, although, as is well known, certain organisms, suchas the lactic bacteria, can only flourish in the absence of oxygen,whilst others, although oxygen is essential normally, have thefaculty of tiding over a period of its absence. Hence respi-ration may be distinguished as aerobic, anaerobic,* and fs£uJUtative anaerobic. The final waste products are diverse anddepend upon the chemical nature of the material consumed

•The use of the term, "intra-molecular respiration" for anaerobicrespiration is wrong since all forms of respiration are essentially intra-molecular.

EFFECT OF CARBON DIOXIDE 109

and the method of its physiological combustion, whether byaerobic or anaerobic means.

The ordinary green plant, and certain non-green plants,in their respiration absorb oxygen and ultimately give offcarbon dioxide and water. This respiration is unceasing andis continued in all living members whether active or passiveuntil death ensues. If oxygen be entirely withheld, growth,movement, irritability and activity in general ultimately willcome to an end.*

The accumulation of the products of physiological com-bustion will bring about a modification, if not a completecessation of the process, which will lead to the termination ofother activities. Thus rotation of protoplasm in the cells ofElodea will come to an end in the presence of an undue amountof carbon dioxide. The germination of seeds is retarded orinhibited by high partial pressures of carbon dioxide in theatmosphere : this inhibition may remain in force only so longas the seeds are exposed to the enriched atmosphere, germi-nation taking place after removal to a normal atmosphereas in the bean, cabbage, barley, pea, and onion; or, the in-hibition may continue indefinitely after removal to normalsurroundings, germination only taking place after completedrying and re-wetting or by the removal of the testa as inBrassica alba. The degree of increase in the partial pressureof carbon dioxide required to effect inhibition of germinationvaries for different plants, and the retardation of germinationdepends on the time of exposure and the character of theseed. Similarly the sprouting of a potato is inhibited by anincrease of 20 per cent, in the carbon dioxide of the atmosphere.A higher concentration causes marked injury and ultimatelydeath, f

• The irritability of plants is outside our present consideration : anintroduction to the problems regarding the minimum pressure of oxygennecessary to maintain movements, the streaming of protoplasm in chloro-phyll-containing cells in darkness and in an atmosphere free from oxygen,and similar subjects will be found in the larger text-books on general plantphysiology.

t Kidd ; " Proc. Roy. Soc.," B, 1914, 87, 408; " New Phytol., 1919,18, 248.

i io R E S P I R A T I O N

Spoehr and McGee * examined the effect of varying tinepartial pressure of carbon dioxide o n the respiration of l e a v e s :they found that when the amount of carbon dioxide is i n c r e a s e d ,the amount of carbon dioxide emi t t ed from the leaf is reduce=dfor a while and then rises to t h e original rate. C o n v e r s e l ywhen the partial pressure of the carbon dioxide of the s u r r o u n d -ing atmosphere is reduced, the ra te of evolution of c a r t o o ndioxide is increased for a time a n d then falls to the o r i g i n a lrate.

These facts are of considerable importance, not only a sregards the economic aspect b u t also in their bearing on e x -perimental work: results obta ined for subjects contained I nclosed vessels, as is not infrequent in experiments on r e s p i r a -tion, in which the products of t h e oxidative processes a c c u m u -late, may be an expression of the p lan t ' s activity not in a norrxisdbut in a pathological condition a n d , therefore, may be v a l u e -less. Further, a state of carbon dioxide anaerobosis may t > eset up.

I N T E N S I T Y .

In general terms, the more ac t ive the body the more i n -tense the respiration, provided t h a t the conditioning f a c t o r s ,such as temperature, food, facility of gaseous exchange a n dcirculation in the plant, and so on, are favourable. C o n s i d e r -ing the plant as a whole, Bonnier and Mangin f r e c o g n i z etwo respiratoTyjo^xiiiia in its seasonal development, the f i r s tat ^^jrnir^tiony or on the unfolding of the leaf buds, and t r x esecond at the^pînôfjthte flowery buds. Ruby J found t r i a tthe intensity of respiration was greater in leaves of y o u . r i gthan of old plants ; thus the a m o u n t of carbon dioxide evo lvê^per hour per gram of fresh weight of leaves from trees o r x eyear old, three years old, and m a n y years old was r e spec t ive ly•200 c.c, -150 c c , and -ioo c.c. I n all instances the growtJn.period showed a one-and-a-half t o two-fold increase in r e sp i r a . -tion as compared with the non-grcrwing periods. Nicolas § cc* m .

* Spodir and McGee: "Amer. Journ. Bot.r" 1924, 11, 4^5.f Bonnier and Mangia: '* Aaa. Set* 2$at. Bot./* 1S85, 5, 315.I Ruby: stf., 1917, 3D, 1.§ Nicolas: " Rev. gâ, Bot./* 19 iS, 3**, aog.

INTENSITY m

p a r e d the respiration of the vegetative parts of annual, biennial,&nd perennial plants and found that leaves and portions ofs t e m s of the same branch varied according to their age, thosef rom the apiqal regions showing a three to seven-fold intensityof respiration as compared with similar structures from theb a s a l parts .

Kidd, West, and Briggs * studied the respiration of Helian-thus annuus both in the laboratory and in the field. Theyp o i n t out that the factors which may affect the rate of res-p i ra t ion per unit of dry weight of tissue are the concentrationof the respirable material, the concentration of oxygen, thetempera ture , and the effective amount of respiring cell matterp e r unit of dry weight. This last is the internal factor, there su l t an t of many factors, none of which as yet fully under-s t o o d and some of which probably not yet formulated. Thein te rna l factor can only be accurately measured when the otherfac tors are not conditioning respiration. For purposes ofmeasur ing its effect, Kidd, West, and Briggs employ a respira-t o r y index which is the respiration, measured by the rate ofc a r b o n dioxide produced, per gram of dry weight at 10° C.w h e n the amount of respirable material is not limiting andw h e n the external concentration of oxygen is that of thea tmosphere . From a large number of observations they con-c lude tha t the respiratory index of the entire plant continu-ous ly declines with increasing age. For example, entirep l a n t s 2 days from germination gave 3 mg. carbon dioxidep e r gram of dry weight per hour, whilst plants 136 days fromgerminat ion yielded but -39 mg- carbon dioxide per gram ofd r y weight per hour. A similar decrease in the respiratoryi n d e x is exhibited by the stem, leaves aad flowers. Int h e stem the ^value fell from -8 mg. on the 36th day fromgerminat ion to -08 mg. on the 1:36th day from germination;d u r i n g the same period the measure for the leaves decreasedf r o m 1*56 to -44 mg. The fact that the initial respiratoryi n d e x of successive leaves decreases with the age of the plantind ica tes a respiratory decrement of the rnerismatic tissuew i t h age and from this it fallows tha t the fall in the respiratory

• KiM, West and Briggs: " Prac. Roy. Soc./' B, 1921,9*» 368.

H2 RESPIRATION

index of the whole plant is not due to the proportionateincrease with age of dead tissue, sclerenchyma and tracheae,for instance.

Hover and Gustafson * have made the interesting obser-vation that not only does the rate of respiration of the leavesof maize, wheat, oat and sorghum fall off with age, but thatthere is a gradual increase with increasing age after middle age.This, however, is not shown by the leaves of the sunflower andby the stems of maize.

Blackman and Parija f have studied the respiration ofstored apples. When maturity has been passed the organism,or a part of the organism such as an apple, enters the phaseof senescence in which the general tone becomes graduallyless owing to the increasing relaxation of the control of whatmay be termed the protoplasmic factor. One result of thisspontaneous change is an increased rate in the hydrolysisof reserve food whereby there is formed more material availablefor respiration and this leads to a greater production of carbondioxide. But stored apples are isolated structures, whereforethe only material available for their respiration is that whichthey themselves contain. It follows that a time will comewhen starvation begins and this results in a fall in the rateof respiration. This rate at any moment is thus the re_-syLtML.of two og2osed_ factors, increased hydrolysis whichaccelerates and starvajj^n^which hinders. This generalizationis based on many experimental observations on apples at atemperature of 22° C.: these show that there is at first aslow increase in the rate of respiration which progressesquickly and then more slowly to the maximum value, afterwhich there is a falling off, slow at first and then faster,towards zero. This sequence is comparable to that observedby Hover and Gustafson mentioned above. Blackman andParija do not claim that their generalization is fully estab-lished by the evidence produced ; it is, however, a hypothesiswhich explains the observed facts.

* Hover and Gustafson.: " Journ. Gen. Phys.," 1926, 10, 33.f Blackman and Parija: " Proc. Roy. Soc./* B, 1928, 103, 412.

INTENSITY I I 3

Nicolas * found that the presence of anthocyanins was afactor, or rather the symptom of an internal factor, of im-portance in determining the intensity of respiration. Leavescontaining a redjMgment either as a youth form or as a per-manent character absorbed more oxygen and exhibited anincreased respiration as compared with green leaves, whilstleaves turned red either by accident or by stress of conditions fshowed a decreased respiration as compared with green leaves,the amount of carbon dioxide being smaller.

The intensity of respiration commonly is measured by theamount of an end product given off in unit t ime: in aerobicrespiration the final product measured is carbon_diqxide,or, in special cases, temperature is measured; in anaerobicrespiration, the production of alcohol may be estimated.

It is clear that the results obtained by the measurement ofcarbon dioxide may not be a true expression of the respiratoryactivity since the exhalation of the gas may be greatly hinderedby various circumstances. The surface of the respiring organis one such: Hoffmann J found that the amount of carbondioxide evolved in twenty-four hours per kilogram of large,medium and small-sized potatoes was 259, 314 and 326 mg.respectively; barley gave confirmatory results. He also foundthat the nitrogen content was important. Further, a depressionof the carbon dioxide output is associated with the succulenthabit, hence in order to obtain a figure representing a truemeasure of the activity of respiration, the increase in organicacid content in addition to the gaseous carbon dioxide should bedetermined. This aspect is considered by Maige and Nicolas,§who point out that in the flowers they examined, the respi-ration intensity increased with age when stated in terms ofgaseous exchange, but showed a decrease with age whenmeasured in terms of wet and dry weight. The gynaeciumshows, as might be expected from the activity of the contained

* Nicolas : " Compt. rend.," 1918, 167, 131.t See Vol. I., p. 336.X Hoffmann : " Journ. f. Landw.," 1916, 64, 289.§ Maige : " Rev. gen. Bot.," 1907, 19, 1 ; " Ann. Sci. Nat. Bot./' 1911,

14, 1. Maige and Nicolas : " Rev. g6n. Bot.,'* 1910, 22, 409.VOL. II.—8

*-* 4 t'iri * dt',C VnT i- '\I*J , > x , ' J t Jt . tL»T parts «>f

It i- M A * • 4, v J.-*^*, ' \ r I ' S I- -in*p]' iruins, dueI n i u f; '•- ! . '^ M \ n L I :>< 1 — ii t* t!a \»«lulu»a i>f! tal i* a <«:,•>: il till M n- ; ^a^ >n I'' t tiiiipt'-ratureat*uir'd i*> * nvi V v* ,i 4 a T ", kal>l« n-f ni.iy resultwi i. !: i »\ i" r« tl\* i In iKfj-tn^ ! * •iakc-3 anil into a bar-r A ) A i 1 in-<, LiwVi < «:ttiu^-. -,*< iahy if tht*rt' i> a goodu'l'i *\*ur ' it t,<»\,r i ' \a i t tut ^,*r« rn'n!^ have been madet«\ van »u> iu\t-tJ Ml «r* : M^h-t h * Ini u] that the bulkedlf.i\f» "t (\irpiuus bfiUiUj rtMo'ud a Us nptTature of 5l°C.in t.fteti) hi»u^, a fall then t*n k |>LHT M» that at the end ofinrtV'ti^hi hour^ tin* ttnipvraturv \va^ 5 f C After tlie lapseof unt* hundred and f« ur ]i?»ur> a, secondary maximum at4"" t\ was atta'iud, the tcr lpcraturt again sin wed a fall to31° C. alter one hundred and eighty hours. Of these two maxi-ma, the first is an expression of the true respiration intensityof the leaves, whilst the cause for the second is to be found inbacterial activity. Pierce f found that in germinating peasthe greatest average gain in heat was 923*9 calories accumu-lated in 23*5 hours, which is about equivalent to 8-55 caloriesper minute per kilogram of peas, a measure roughly onequarter less than the amount of lieat given off by a mouseunder similar experimental conditions. It was further foundthat the amount of heat liberated by germinating peas de-creased with age.

Since respiration is a means of obtaining energy for theneeds of the plant, the evolution of heat represents excess ofenergy and is a waste product, for which reason the temperatureof a normally respiring plant is not by itself a sure guide to theamount or intensity of physiological combustion but rathera measure of the inefficiency of the organism.

The sfcimulatiiMt of the plant accelerates respiration, whichmarked by a rise in the output of carbon dioxide

'• Bot. Z*$.r 1**. Mf ai I.f nmm: *' tot GML." i«it, 53* • * $m »1M> Bemfiiw: " Ana. Sci.

Mai. rk*., 1*93.1 s, 11.

STIMULATION 113

and generally also by a rise in temperature. The intensifica-tion *>f respiration resulting from traumatic stimulation longlias been known and now is not an uncommon laboratoryexercise. Richards * found a gradual rise in temperaturefollowing the stimulus, attaining its maximum about twenty-lour hours after the infliction of the injury. In massivetissues the effect is local, but in less compact structures, anonion bulb for example, the rise in temperature may be demon-strated over a more extensive area. The rise may be two orthree times as large as the difference between the normaltemperature of a potato and that of the surrounding air.The precise increase in respiration depends on the extent ofthe injury and the nature of the tissue operated on. Thereis an initial outburst of carbon dioxide, followed by a fall, afeature not at all uncommon after drastic stimulation, andthe absorption of oxygen is rather greater than the amounttheoretically required for the quantity of carbon dioxideevolved. Richards considers that the initial outburst ofcarbon dioxiHe In part is due to the release of the gas normallyenclosed within or absorbed by the tissues, the *:i residual"carbon dioxide which normally is not exhaled.

In the potato, Hopkins f demonstrated an increase in tlie-sugar content of the tuber after wounding, it gradually risesto a maximum and then falls. He considers that the increasedintensity of the respiration is explained, at any rate in part,by the increase in the sugar content.

Mulltr $ investigated the respiration of trees after lopping,and found a seasonal variation in the traumatic reaction:the reaction of Puea is about the same all the year round,whilst Fagus and Fraxinus show the greatest reaction inspring and autumn, and the least in summer, as estimatedby the output of carbon dioxide at the mean temperatureof the month.

White § found that pollination produces a rapid increase inrespiratory activity and affects the COt.Ot ratio which generally

* Richards : " Ann. Bot," 1897, 11, 49.t Hopkins • " Bot. Gai.," 1927, &4» 75-} Mailer : " Dansk. Botaaisk Ark./* 1924, 4, 1.§ White : "Ann. Bot./' 1907, ai» 487.

8 *

nh RESPIRATION

is greater in pollinated carpels a> o.>mpared \vitli unpollinatedgyna'cia. The most striking instance was afiunled hy tin*IVlargnnium. the pollinated varju/k • »f which evolved from hveto eight times as much carton ili^xule as the unpnllmated.

Mention ako may lie made of N-hW's * observations, whofuund that the respiration of a £n-tropically stimulated rootis greater than that of an unstimulated root, the respirationrate of the convex side he ing greater than that of the concaveside during the period of perception and response.

Products of the plant's own metabolism may stimulateits respiration. Thus Spuehr and MeGee f observed thatwhen detached leaves of the sunflower and the bean weregrown in a nutrient solution containing sugar and aniino acid,the rate of respiration increased with that of the amino acidin the culture medium even when the leaves contained suppliesof carbohydrate. When grown in darkness, when carbohy-drate decreases and amino acid increases, there is a likestimulation which tends to maintain a relatively higher rateof respiration, From such observations they conclude thatcarbohydrate alone does not determine the intensity of res-piration, but that there is an accessory stimulating factor,amino acid, in the plants examined. On the other hand,Hafenrichter $ concludes that in the soy bean grown indarkness, the accumulation of amino acids is incidentalrather than necessary to respiration ; he obtained no evidencethat ainino acids stimulate respiration.

Although vegetable pathology is without our presentprovince, mention may be made of the fact that, as in theanimal, disease is frequently marked by an increased respira-tion intensity. §

THE ACTION OF \NJESTHET1CS.

Tht action of anaesthetics on the output of carbon dioxideis partly stitntdatoiy and partly narcotic. Irving || found that

• Sefclty : ** Bat. Gas.," i®m, 7®, 69.f Sfw lur ai*4 McGe*; " C&ra*gi« latfc. Wis|.,w N«x 325,1925.I Hafenric&ter: " Bat. Gax," 1928. S& 271.§ Stoft, fee csaaqpto, Wfcfaaor m®& Hfttiw : *' Jacra* Agrk. Bm."6 X> " Bot, Gax./* i^26f $1, 323.

An. B ..»

ACTION OF AN.ESTHETIC> n ~

tho rfleet <>f a single dose depended en :N -tr : „;*' .; »!• -•/nf c> | r.r, of rhlopiform ill y7(> c-r- '^' t i j r ^r:!^- ,*; ».*•an immediate rise in. the output »>f . arK n »Ii «v. L * ••etuvt. subsequently disappears and the ha\r* linn i\ \ , amuch carbon diuxkle as in normal respiration. In rv.^.i,*1.druses, v2 e..t\ <»f chloroform in 97*-* c-^- *'f t4^r' -^'.rt' - !it *"'-tlit? same initial outburst of carbon tiioxith*. Mi:i li LCU AV%JVmore quickly than after a small single tlf-c J :M f. r a timeremains below normal ; after about cix hours nr>vtry i>cninplete and the evolution nf carhun dio\id-.' !•* r.- TÂA)With a (loêt)f i r.r. of chloroform in 0,7*u\c. r>f air, t! t iTiifj.iloutburst of carbon dioxide is earlier and it* run , 1- -r». •.: t rthe production of rarbon dioxi»k» <lo\&K dt:u;:;i-] ,- ..r, 1 : \ . nis no recovery. Following a strong <io-i nf r'r.l-.r !.»::: , ! ' • * 'in Q70C.C of air, there i» no detectable initial ourbur-! **T; i ! .ecarbon dioxide output quickly falls to zt r< • Sr.« .ti»y;;..--tratian of a continuous duse nf chl *ri *f*»rn: )>r \\c-\ - : !.»• * a ::.*>*effect as a single dose two or three times a*« *tr :i£. i i; -.Li\ *likewise found tliat m Prunuslanroifrasus, lieluimhus :ute+sz%sand Troptfolum tnajus a small dose »>£ ehUTot Mi: liad?* t<*an inimediate stimulation i»f respiration, the ev«uut:'. r. vtcarbon dioxide arul the absor]>tiun nf uxygin i inria^r^ ir.like pr»»jK*rtion, v, inch indieat^ that the two art <: * > rd;ruu»i.If, however, the I1I-M* IS ûtiitiently \dr^: t*) t'-'j't a \>i^L-disorganizati'»Ti, MI-'II a^ changein t\;l--ur r lîrr > a!i r: . :^!outburst .«f carbnii tii»•siitle tLtn a fall t«» a \ n , \v !• v./ a*; :the ab-st»r])ti«^n *»f ii.xyên n^ 1-m tr ?t %s ^:.\ ._ : IJM*. •:with the amount nf carbon di«'\»ie ev^lvcJi. I,,-; a!~- rt*^ !-*of oxygen in some ^vay is connected with the a l l L-ntt.n:$,especially tannin. la the Tropuolum leaf. v^Kul' > triefrom tannin, the absorption of oxygen i* niu^'h I» *vwt r thanthe output of carbon dioxide, wherea* AI the riurry Lmn Iand the artichoke, both of which contain taunt:). t;,i uululup-take of oxygen is very rapid, then it tJeelums byt

* Thalay : "Aon. Bot.," 1913, 27, (yqj.f The change in colour is due tothe oxidation of tannins and giiicaftiti f

hy &pjpropnuLte enxymes, oxidises for example (see Vol. 1., p. 40 ). It maybe conveaieatly tecifc bv exp<Miug tiie fton-ers of the wtiite like to the IUISMRIof phenol nader a bell gla&s.

I i8 RESPIRATION

remains at a level much higher than the output of carbondioxide.

Similar observations have been made by others, thusThomas * found that wheat subjected to the action of ethershows an increase of respiration followed by a decrease.

Exposure to 7*3 per cent, of ether is only stimulatory pro-vided it be of short duration, an exposure of more than thirtyminutes resulting in death. Similarly the main observationsof Thomas have been corroborated and extended by Smith fwho used the hydrogen ion concentration method in findingthe rate of production of carbon dioxide in wheat seedlings.It was found that the first effect of ether, used in concen-trations of I per cent., 3-65 per cent, and 7-3 per cent., wasto depress the rate of respiration ; this was followed by a rapidincrease to above the normal rate, which was in turn followedby a decline to much the same level in all concentrations ofanaesthetic, in times varying with the dose employed ; thestronger the solution of ether used, the quicker the fall.

Irwin, X using the corolla of Salvia, found that the effectof a high concentration of ether is an increased intake of oxygenand output of carbon dioxide, whilst at the same time theacidity of the cell sap is reduced. The action of anaestheticson the lower plants results in reactions similar to those ex-hibited by higher plants under like treatment.

Gustafson § found that Aspergillus niger, subjected to theaction of ether, acetone, or formaldehyde, exhibits an increasein its respiratory activity followed by a decrease. The use ofcaffeine in saturated solution similarly brings about an increasefollowed by a decrease, whilst a 0-5 per cent, solution resultsin a decrease of respiration intensity.

Similar results were obtained by Brooks || in his experimentswith Bacillus subtilis, for which plant ether is toxic in low,0037 t o I # I P e r cent., and in high, 3-65 to 7*3 per cent., con-centration. In strengths intermediate between these, the drugacts as a stimulant to growth. Further, the action of ether

* Thomas : " Journ. Gen. PhysioL," 1918, 1, 203.t Smith : id,, 1921, 4, 157. % Irwin : id., 1919, I, 399.§;Gustafson : " Journ. Gen. PhysioL," 1919, 1, 181.11 Brooks : id., 1919, 1, 193.

ACTION" OF IONIZED AIR m}

varies according to the presence or absence of ntl >r ^u!>stances: thus whilst the respiratory aethitv ma\ l«c t tt\times as great as the normal under the u< tn*n <»i a 7-5 |.«.rcent, solution of ether, this high intensity is o-n>i«Lnu>Ivreduced if 085 per cent, of sodium chloride ha added.

According to A. C. R. Haas,* small do^es of ana^tLtti-fare without result on the respiration of Lammana, dr^t>sufficiently large to produce a measurable result etrtnt aprolonged increase in respiration, whilst strong doses resultin an initial increase followed by a decrease ultimately tr>zero. la no experiment was it found that the initial tricei <>fan anaesthetic was a lowering of the respiration intensity fromwhich it is concluded that the state of anaesthesia is n^)t hrnu^Jitabout by the lowering of the respiration. It is dear from tLtseresults that in general anaesthetics have a twofold action, Asmall dose is a stimulant, a large dose is a narcntie. And tla-<is true not only for respiration but also tor other LXjifo^R-rwof plant activity such as the circulation of protoplasm wit funthe cell.

THE ACTION OF IONI2ED AIR

Spochr f was the first to observe that the respiration **f theplaat is regularly higher in conditions whi,:h \<LYV »»n!y a*regards daylight, and darkness, differing, in other ^ ^ r l s ^nlvin the degree of ionization. The difierL'nct> ol's-ervt-i -Atrehowever, not considerable; in the wheat plant the riti-> 4\>rate/night rate was found tu be 1-042 m nrrniai .nr a\dl*oio in dcioniared air.

StoppelJ found a 0*3 to 1*13 per ctnt. increase m tixrespiration of cut shoots when supplied with air i^nucilby passing through a tube containing carnotite, a radio-activemineral. In the carefully controlled experiment * vi Muldle-ton,§ barley seedlings were grown on sterilized gla<s WIHA APAglass beads watered with a culture solutiiiii. Tl:c rulturewas made in a glass box and supplied with a current of air

* A. C. R. Haas : " Bot. Oai./* 1919, 67, 577.

J StofHHsl: Bot, Zeit./ 1920, n» i<>2o.§ MkJdleton: ' * Ana. Bat. * * 192jt 41, j.#5.

••*. 1 * T; r« }•] \ r f «il. T i n i m i i z e d a i r

<,*, ni t t r u t ^ - l ! »r « -m1 h o u r a n d t h e n; -.ir 1 M T I 1 j h \ a \ - o b t a i n e d a n i n -

" . a r ' - > < . r - i j ' ^ t ' ! h . *! ^ r t r *>i i ^ i i u a t u n : w i t h a i r i o n i s e d

2' * - ^ - - t • • i L i f 'f t ' i e Mirr > u n d i n g a i r , a n i n -

u . r.i! ! s 'l%\< ,i f t n h .i]>;>|j a t i i»n »»f t h e pol< n i u m ; w i t h

i ^ ^ ! " - M > t Mt «'i i i ' >n r , i i a i r A m a r k e d l y s i g n i f i c a n t i n -

t n . i " ,!*.»;:.•, 1 in I 'M - K M I U ! p e r i o d «»nly, a n d w h e n t h e a i r

%.*•* i« ' i ; /**! a t r i i l i -«n l i n u > t h e r e w , b im h i ^ n i n c a n t i n c r e a s e

ui n - | i r a . i M M n i i l i r r r - i i l t ^ w e r e o b t a i n e d b y W h i m s t e r *

u i f h tn« ]f , i i nf Pdar^oniwn ^ntah\ w h o f o u n d a p e r c e n t a g e

i r u T f . i s r of 85*7 :i: 7*1 i n t h e r e s p i r a t i o n r a t e a t 2 5 ° C. i n a i r

R*mzed f r o m 5C4 t o 7 2 S t i m e s t h a t of n o r m a l a i r . T h e r e a s o n

fo r t h e d i f f e r e n c e is n o t o b v i o u s : i o n i z e d o x y g e n is m o r e

p o t e n t t h a n d e i o n i z e d o x y g e n s o t h a t t h e i o n i z e d o x y g e n o f

t h e d a y t i m e a i r , a c r o r d i n g t o S p o e h r , p o s s i b l y a c c e l e r a t e s

t h e p u r e l y o x i d a t i v e p r o c e s s of r e s p i r a t i o n , n o t t h e i n i t i a l

d i s r u p t ion vi t h e r e s p i r a b l e i n a t e r i a l . B u t u n t i l i t c a n b e

s a i d u' i th c e r t a i n t y a t w h a t s t a g e i n r e s p i r a t i o n o x y g e n b e c o m e s

• o p e r a t i v e , a n a d e q u a t e e x p l a n a t i o n i s n o t p o s s i b l e .

C O N D I T I O N I N G F A C T O R S .

T E M P E R A T U R E .

I n g e n e r a l t e r m s a n i n c r e a s e i n t e m p e r a t u r e r e s u l t s i n a n

i n c r e a s e i n t h e r e s p i r a t o r y a c t i v i t y u n t i l d e a t h t a k e s p l a c e .

T h e f o l l o w i n g f i g u r e s , g i v e n b y M a t t h a ? i , f r e p r e s e n t t h e m e a n

v a l u e of c a r b o n d i o x i d e e v o l v e d b y 2 g r a m s of c h e r r y l a u r e l

leaf p e r h o u r a t t h e t e m p e r a t u r e s p e c i f i e d : —

4! 4 . 2 C , . . *ecMtti<X>a 25-7° C. . . <ooo6COai f J # C . . -^oojCOt 29'a* C. . . -oooSJCO,oooSJCO,

•ooijCO,

T V iiMqpectioii ©f t h e s e fipires i h o w » t h a t f o r t h e l o w a n d

tc t f tperat t t i c* e s n p b j r e d tbtm i s a g r a d u a l i n c r e a s e i n

.," B,. I9O#.» t f 7 , 47.

TKMPKRATl/UK ui

tlsiv n^pirati^n tirrivity. At higher temperaturis venationt>l»tani- and it \v«i> i«>und that the respiration inrt-nsiTv r.tpi«Iivi-lui-n^rtj wit!i the tune * »f explore and that k-tt\L> ::\ prv-ei>.t.ilv the same external conditions v;ave varying results duottt snme internal factor. This is illustrated by the foll^win^result* <»f ctrtain experiments «»n isolated cherry laurel k'uvc?t-x posed to light and carbon assimilating, The respirationvalues represent the carbon dioxide given oil by 2 grams of lea!per hour :-•••

3-S-yc, . . -ooioJCO, , 40•*)'(.'. . . -*x>i4O.>t

4 2-i> i* . •t-»i5("e>j

Also it was found * that, the leaves of tins plant in theirrespiration exhibit the same relation to temperature a< theydo in their carbon assimilation in that t)i^ initial value athigher temperatures is not maintained but. fail- away, thehigher the temperature the steeper the fall,

An increased temperature must be operative for some timebefore the stimulation of the respiratory process i* noticeable,a sliarp variation in temperature is without effect and a changein the respiration intensity after transference from one tem-perature to an* th<T l* gradual.*

MulltT-Thur^au ami Srhnuder Oreih % in thur m\t*ti-gatiuii on the rr^jiration «»f p»ta!».t> iuur»l n i r h tl;c ^aauphenomena: merv \ ariaf hm m it n.j mature irititrd t>\heating and co'dnii; i> with, ut ri-ult UHILMS tin ».\p ^ jrc f >the IiigiuT tvmpL raturt* he *uliii 11 i.i'v pr« 1. 1 ul. F. ta^tubers, after an t i\| i»j.ure to a ttmj»\.raturc »•! 4(' C t«»rseveral hours and then transferred t*> riwm temperJiturt,show a gradual increase in respiration intensity which reachesits maxinmrn afttr a lapst <f twtnty-f»ur h<»urs.to higher temperatures, e o. 44 c\, results in aincrease in the intenMty uf re&\ irati»>n which n\4nmanent change in the organism. T» « htain 111 -the temperature must be sufficiently high, no etlect being

* See Bi&cknun ; " Ann., Bot.," 1905, »9» i&i •>f BUnc : " Rev. f n. Bot.," i xb, aH» 65.I Mttlkr-Thurgaa and Schncndei-Orelk ; " Flora,-' 1910, l@l» JCHJ.

122 RESPIRATION

observed for temperatures below 380 C. This is the minimaltemperature for the phenomenon described in the potato, butthe value probably varies in different plants. In the potato,the difference between the maximum intensity of respirationof heated tubers and that of unheated tubers is propor-tional to the difference between the minimal temperature andthe increased temperature to which the tubers were exposed.Thus it was observed that after exposure to 440 C. the res-piration was two and a third times as great as after anexposure to 400 C. These authors consider that the effect ofthe high temperature is adversely to affect the protoplasm inmuch the same way as does old age : as a result of this de-cadence, less starch is formed by the leucoplasts ; also theenzymes are less active so that there is less invert sugar forthe leucoplasts to fix. For instance, at 180 C. previouslyheated potatoes contain a relatively large amount of sugar;at 0° C, which temperature inhibits starch formation in normaltubers,* the much slower formation of sugar indicates that theincreased temperature also has had an injurious effect onsugar formation although the amount of diastase apparentlyis unaltered. The weakening due to high temperatures alsois seen in the respiratory activity of wounded potatoes : thegeneral effect of wounding (see p. 115) is to increase the in-tensity of respiration, which increase is maintained ; afterwounding there is no immediate respiratory response in pre-viously heated tubers, and even when recovery from theheating had apparently been made, the maximum intensityof respiration due to the traumatic stimulus never reachedso high a degree as in unheated wounded tubers.

Dormant structures were the subjects of this investigation ;the subjects for the work of Kuijper,f on the other hand, wereactive seedlings of the lupin, pea, and wheat. The seeds weregerminated at about 20° C. and then exposed to varioustemperatures, the carbon dioxide being estimated hourly forsix hours (Fig. 6). At temperatures not exceeding io° C. theoutput of carbon dioxide is constant; from 10° C. to 20° C.

* See Vol. I., p. 177.f Kuijper: " Extr. Trav. Bot. Norland.," 1910, 7, 130.

TEMPERATURE

there is a continuous rise in the respiration of seedlings of allages, which rise is followed by a fall; at temperatures above20° C. and up to 400 C. the amount of carbon dioxide evolved

35"

1 2HOURS

j?IG# 6#—Respiration of the pea at various temperatures.

fluctuates : a rapid fall occurs in the first two hours, then arise for one or two hours, followed by a continuous fall. Atstill higher temperatures there is a continuous reduction in the

124 RESPIRATION

evolution of carbon dioxide, a result which is in accordancewith the observations of Blackman mentioned above.

These observations are different from those made by Mtiller-Thurgau and Schneider-Orelli who observed a permanent in-crease in respiration after due exposure to high temperatures,whilst Kuijper found a decrease to obtain. This discrepancymay be due to the fact that Kuijper's seedlings were subjected tothe increased temperature for a lesser time than the potatoes ofMtiller-Thurgau and his collaborator, and, as has already beenremarked, the material employed was in active growth on theone hand whilst on the other it was resting. Kuijper explainshis results on the hypothesis that there are two distinct pro-cesses concerned in respiration which are affected differentlyby a continuance of high temperature, one phase being earlydepressed whilst the other is stimulated ; there is here a pos-sible correlation with the dual origin of the carbon dioxideof respiration, which, according to Palladin, arises from theactivity of an oxidase and of a carboxylase. Like Miiller-Thurgau and Schneider-Orelli, Kuijper recognizes the inter-relation of temperature and the nature of the available res-pirable material. Thus of the seedlings examined, the lupinshowed a rise in the respiration intensity from 150 C. to 20° C,the pea from 20° C. to 25° C, and the wheat at 300 C. Thefluctuation period was well marked at 250 C, 300 C. and 350 C.respectively, and the continual faTl in carbon dioxide outputobtained at 250 C, 350 C. and 400 C. respectively. Of thefood available, the lupin has 37 per cent., the pea 22 per cent.,and the wheat 12 per cent, of protein, whilst of starch thelupin has none, the pea 54 per cent, and the wheat 74 per cent.

If respiration be a purely chemical process, a combustionof, say, sugar, van't HofFs law should apply throughout theprocess : the evidence shows, however, that the law is followedonly for lower temperatures ; at higher temperatures, as hasbeen seen, fluctuations occur and correlation comes to an end,which is indicative of the intervention of internal factors, thenature of which is not clearly understood. Clausen * found

* Clausen : " Landw. Jahrb.," 1890, 19, 893.

FOOD 125

the rate of evolution of carbon dioxide from seedlings and budsto be about doubled with an increase of io° C, and Kuijperfound the law to apply at temperatures between 0° C. and 20° C.in. the pea and wheat, and between 0° C. and 250 C. for thelupin, the coefficient lying between 2 and 3 for a difference intemperature of 10° C. According to Lundegardh,* the res-piratory curve falls into four regions : there is at first a gradualincrease with rising temperature from the minimum to thefirst optimum at 400 C, in this phase Q10 varies from 1-9 to3'2] there next follows a rapid increase from 400 to 460 C. ;the third phase is characterized by a marked increase to thesecond optimum at 500 C.; finally at temperatures above50° there is a rapid fall in the respiration intensity. Lunde-gardh explains the results obtained at temperatures above400 C. on the assumption that at about this point the proto-plasm enters into a more labile phase comparable to that ofa colloid whose viscosity suddenly changes at a high tempera-ture.

In passing, it may be mentioned that the maintenanceof a relatively high temperature is one of the factors whichis responsible for physiological diseases, e.g. blackheart inpotato tuber.f

With regard to low temperatures, it is a well-known factthat seeds, lichens, mosses, etc., especially when dry, retaintheir vitality on exposure to intense cold ; even so low atemperature as — 2500 C. will not cause death. Since res-piration is in its complete expression a concomitant of life,the process must continue in an attenuated form even at solow a temperature as that of liquid air.

FOOD.

The continuance of respiration ultimately depends uponadequate food supplies, and the intensity of respiration maybe much influenced by the presence of substances immediatelyavailable as respirable material. Formerly it was considered

* LundegaYdh : " Biochem. Zeit.," 1924, 154, 195.f See, for example, Bennett and Bartholomew: "Univ. California

Publications," Technical Paper, No. 14, 1924.

126 RESPIRATION

that sugar, more especially dextrose, was essentially the res-pirable material, but no statement of general application canbe made. The action of foods in this connection varies withdifferent plants and their physiological condition, and is in-fluenced by the operation of various natural internal factors,such as acidity, which are not directly controllable in ex-perimental work, together with external factors such as tem-perature; thus in Phycomyces the normal combustion offats changes to the consumption of carbohydrates at hightemperature.*

But notwithstanding this, general physiological experienceshows that carbohydrates are highly important in aerobicrespiration. Palladin,f for instance, found that 100 gramsof etiolated bean leaves, with depleted carbohydrate, gave offIO2-8, 95*9 and 70-2 mg. of carbon dioxide respectively forthree successive hours ; but after these leaves had been grownin darkness on a solution of cane sugar for two days, by whichmeans their carbohydrate content was increased, the rate ofevolution of carbon dioxide was 152-6, 147*5, 146-8 and 144*5mgs. respectively for four successive hours.

A more prolonged culture on cane sugar does not result ina corresponding increase in the rate of carbon dioxide pro-duction although the carbohydrate reserve is greater; thusafter a treatment over a period of forty hours, the leavesevolved no more carbon dioxide than they did after fourhours. Such observations indicate that a certain content ofcarbohydrate is necessary, but an increase above this value,which is a variable figure, is merely increasing the respirablecapital of the organ.

Mliller-Thurgau and Schneider-Orelli % found that therespiration of sweet potatoes is very high compared withnormal tubers ; in the autumn, when little if any sugar ispresent, the respiration is low, but as the tubers increase inage and sugar accumulates there is concurrently a more intenserespiration. According to Knudston § the respiration of the

• See De Boer: " Rec. Trav. bot. neerland./' 1928, 25, 117.•f Palladin: " Rev. gen. Bot./' 1893, 5, 449.J MaHer-Thurgau and Schneider-Orelli: loc. dt.§ Kmtdston: " Cornell Agric. Exp. Sta. Mem./' 1916,9, i*

FOOD 127

vetch is intensified to different degrees by saccharose, glucoseand maltose, the last mentioned being less effective than theothers. Spoehr,* from his investigations on the carbohydrateeconomy of the Cactaceae, concludes that the rate of respirationis not controlled by any one group of sugars, thus hexosesugars, which often are considered to be of exceptional im-portance in respiration, occur in the Cactaceae in variableamounts in varying conditions of existence and sometimesmay be almost absent, but the respiration intensity is notthereby reduced in any marked degree. In conditions whichinvolve a poor supply of hexose sugars, the polysaccharidesare consumed in the respiratory processes and such con-ditions a r e possibly connected with the formation of pento-sanes. T h e conditions referred to are temperature and water :a low wa te r content accompanied by a high temperaturebring a b o u t a decrease of monosaccharides and an increase ofpolysaccharides and of pentosanes, whilst the contrary con-ditions, a high water content and low temperature, are as-sociated with an increase of monosaccharides and a decreaseof polysaccharides and of pentosanes.

Wi th regard to the proteins, Palladinf has shown thatduring t h e germination of wheat in darkness the total proteincontent and the soluble carbohydrates diminish, whilst theevolution of carbon dioxide and proteins insoluble $ in pepsin,increase in amount. As development proceeds, the carbo-hydrates, which originally were abundant, become depletedand the decomposition of protein is less vigorous and may evencease. Hence in the later stages of germination there is amore vigorous respiration in conditions of low protein con-tent t h a n in the early phases of germination when moreprotein occurs. The proteins which disappear are those ofthe reserve food, not those which are presumably membersof the protoplasmic complex and which show a direct relationto the amount of carbon dioxide evolved. This is shown inthe following table which relates to wheat germinating at

* Spoehr : " Carnegie Inst. Pub./' 1919, No. 287.f Palladin : " Rev. ge*n. Bot.," 1896, 8, 225.j The solubility being determined in vitro.

128 RESPIRATION

20° to 21° C. and gives the ratio of the carbon dioxide evolvedper hour to the amount of protein unacted upon by pepsin :—

Age of Seedling CO..in Days. ^ ; -

4 . . . . . . . . i-o66 i-o57 I-I89 . . . . . . . . i-i5

Palladin found a similar ratio to obtain in other plants,the following being some of the values obtained:—

Wheat seedlings . . . . 1-05, 1-15, 1-07, 1-18Lupin seedlings . . . .1 -12Etiolated leaves of the broad bean after

cultivation on sugar solution . 110

The differences between these observed ratios are notconsidered to be significant by Palladin in view of the experi-mental difficulties. From his observations he draws thegeneral conclusion that for a given temperature and with anadequate amount of carbohydrate, the relation between thequantity of carbon dioxide evolved by different plants in onehour and the quantity of protein unacted upon by gastricsecretion, in vitro, is a constant. For a temperature of 190

to 200 C, CO2/N2=-- 1-i.The amount of the protein referred to, as Kidd, West and

Briggs have pointed out, is a measure of the amount of respiringcell matter.

With regard to the fats, there is but little precise infor-mation available from the point of view of respiration. Theirconversion to carbohydrate may be one phase of the involvedproblems, and necessitates a much greater up-take of oxygenwhere fats are the.predominating respirable material as com-pared with those in which carbohydrate is the prime respirablesubstance, that is, the respiratory quotient of a fat-containingseed is of a comparatively low value. To what extent theproducts of the hydrolysis of fats are immediately respirable,is another aspect of the subject awaiting investigation.

The observed differences in the respiratory activity issometimes ascribed to the difference in the quantity of thematerial available for physiological combustion. According

INFLUENCE OF WATER 129

to Hafenrichter,* this is not so in the two varieties, Manchuand Midwest, of the soy bean examined by him. The man-chu variety has a high oil and low protein content, and theMidwest variety has a low oil and a high protein content.

It was found that the intensity of respiration of these twovarieties was not the same at the same temperature, northrough a range of temperatures; further, the effect of tempera-ture varied at different stages of development, and is not thesame for each variety. According to the author, his resultssupport the view that in respiration the material to be con-sumed is selected from those available for physiological com-bustion. It was also observed that the seedlings when starved,by continuous growth in darkness, showed a marked increasein respiration prior to the complete exhaustion of the plant.

WATER.

Since an ordinary living plant is mostly water, it is obviousthat any circumstance which leads to a marked increase ordecrease of water may alter profoundly the expressions oflffe ; thus if water be removed from a plant to an extent suf-ficient to make the cells flaccid, growth will come to an end,and therefore the respiratory activity will be ultimately con-siderably lessened. This statement is intentionally qualified,for an increase and also a decrease in the water supply maylead to an immediate amplification in the intensity of respira-tion. Maige and Nicolas f found in several instances that arise in turgescence is followed by an increase in respiratoryactivity, and a similar result obtains, although to a lesserdegree, when a decrease in turgescence is brought about insimilar material either by natural evaporation or by the actionof a mildly plasmolyzing sugar solution. If a further removalof water be effected by the use of a 10 to 20 per cent, solutionof glucose, a decrease in respiration, as indicated by a smallerintake of oxygen and output of carbon dioxide, results. Smith Jsimilarly observed that vegetative organs of the snowdrop, thebean, and Tropceolum, after drying in a desiccator until a

* Hafenrichter: " Bot. Gaz.," 1928, 85, 271.f Maige and Nicolas : " Rev. g£n. Bot./' 1910, 22, 409.t Smith : " British Ass. Rep.," 1916, 85, 725.

VOL. II.—9

i3o RESPIRATION

third to a half of the water" had been removed, showed a greaterrespiration as compared with normal members of the plantsmentioned. Up to a loss of 30 per cent, of water the respiratoryactivity increased in proportion to the amount of water lost;from 30 to 60 per cent, loss of water the respiration was uniformat the increased rate attained with the 30 per cent, loss ; finally,with a loss of from 60 to 100 per cent, of water the respirationdecreased proportionately to the amount of water removed.Iljin,* in a large number of observations on various plants,both mesophytic and xerophytic, also observed that a lossof water from the shoots stimulates respiration, which ismore considerable in mesophytes than in xerophytes. In thelatter plants, which from their habitat have a relatively lowreserve of food, the increase in respiration may cause a 20per cent, depletion in the reserve food in the course of a day.All plants do not behave alike in these respects; thepaeony and the asparagus show no increase in respirationfollowing removal of water, and Palladin and Shelournovaffound that potato tubers exhibited a lowering of respirationon dehydration in the conditions of their experiments whichwere not the parallels of those of the authors just quoted.

There is no agreed explanation of these facts: Smithsuggests that the phenomenon is due to changes effected inthe activity of the enzymes involved, whilst Maige and Nicolasconsider that the increased turgescence increases growthand consequently respiration whilst the decreased turgescencestimulates respiration since the respirable materials are con-centrated. If this be so, it is reasonable to suppose that thereis, mutatis mutandis, an optimal concentration of these sub-stances, and it is the passing of this strength which accountsfor the diminution in respiration in tissues subjected to theplasmolyzing action of strong sugar solutions.

The long periods of time through which certain plants cansustain life on a diminished water supply are remarkable:Long % found that a shoot of Echinocactus retained life and

* Iljin : " Flora/' 1925,16, 379.t Palladin and Shelouniora : " Bull. Aca4. ScL Petrograd," 1918, Soi,{Long: "Bot. Gaz.," 1918, 65, 354.


exhibited respiration after a period of desiccation of thirtymonths, the first eight in full sunshine and the remainder ina dark room at air temperature. Seeds, however, provide themost striking instances and the importance of a knowledge ofthe conditions affecting respiration, and consequently heating,of stored seeds is great. It is not unlikely that the respira-tion of really dry seeds is, to a large extent, anaerobic; in air-dry seeds there is a small evolution of carbon dioxide whichmay have its origin in the testa only.*

Jacquot and Mayer f observed the respiration of the driedseeds of the bean, maize, etc., and found that, as in plantsperiodically subject to desiccation (see below), respirationprogressively increases with the increase in the amount ofwater to a maximum which is characteristic of the plant-A further increase of water leads to a diminution of therespiratory rate. They consider that the intensity of res-piration depends not only on the contents, including water,of the cell, but also on their relative properties. Baileyand Gurjar £ find that the spontaneous heating of damp grainwhen bulked is chiefly due to the activity of the embryoin oxidizing sugars, the degree of respiration increasingwith the amount of available water. Up to 14*5 per cent, ofwater there is a uniform aad gradual rise in the intensityof respiration; in greater proportions, water will produce amarked respiratory acceleration. In this respect gluteaousmaterial is important in that its degree of viscosity is loweredas it takes up water ; this permits a more rapid diffusion andhence a greater respiration. This is illustrated by the factthat soft starchy varieties of wheat exhibit a higher respira-tion intensity than hard vitreous wheats with the same per-centage of water. The embryo is the seat of enzyme secretion ;shrivelled wheat, which contains less endosperm and the sameamount of embryo as compared with plump wheat, shows arespiratory activity twice or thrice as much as plump with the

* Becquerel: " Ann. Sci. Nat. Bot," 1907, 5, 193.f Jacquot and Mayer: "Ann. physiol. ptiysicttim. biol.," 1926, 2,

408.I Bailey and Gurjar: " Joiirn. Agric! Res.," 1918, 12, 685.

132 RESPIRATION

same water content, 14 per cent. ; with less water than 14per cent., there is little or no difference between the two kindsof grain. The effect of respiration in bulked grain is a risein temperature ; this is cumulative and causes an accelerationin respiration up to about 55° C., which temperature also hasthe effect of stimulating diastatic activity and hence renderingavailable more immediately respirable material. On the otherhand, the resulting accumulation of carbon dioxide has a nar-cotic effect and reduces the rate of respiration.

Some reference may now be made to lower plants, many ofwhich, from the nature of their habitat, have to withstandlong periods of desiccation. Long has it been known thatdry mosses show a very low rate of respiration, which riseswith the increase of the water content of the plant.* Accord-ing to Mayer and Plantefol,t the respiratory quotient of mossesvaries according to the degree of imbibed water, and may behigher than unity when the water content is low. They alsocame to the interesting conclusion that the respiration ofdry moss is anaerobic, as is possible also in dry seeds, since theyield of carbon dioxide in vacuo is as much as it is in air.

In the lichens, the increase in respiration also depends onthe amount of water contained in the plant. Jumelle % foundthat on addition of equal amounts of water to material havingrespectively a low water content and a high water content,the rate of respiration was greater in the former than in thelatter. The addition of water beyond a certain point is with-out effect on the respiration rate ; there is, moreover, a periodof lag shown by lichens subjected to prolonged drought,some time elapsing after the addition of water before the res-piration rate is equal to that of fresh material of equal watercontent. Fraymouth § likewise observed that the lichenParmelia physodes showed no great increase in the rate ofrespiration until a water content of 60 per cent, was reached ;

*Bastit: " Rev. ge"n. Bot.f" 1891, 3, 255. Jonsson: " Comp. rend./'1894, 69, 440.

f Mayer and Plantefol: "Ann. physiol. physichim. biol./' 1925, I,361.

% Jnmelle : " Rev. g6n. Bot.," 1892, 4, 359.I Ftaymouth : " Ann. Bet./' 1928, 42, 75.

ACTION OF SALTS 133

with continued absorption of water, there is an accelerationin the respiration until a water content of 200 per cent, isreached, after which the respiration rate is slower. The algaconstituent of Parmelia physodes is a species of Trebouxiawhich Fraymouth found to be less capable of responding todrought conditions than the free growing Trebouxia whichshowed a maximum rate of respiration when the water contentwas about 30 per cent. The alga Prasiola crispa behavedsimilarly ; as in Trebouxia, after severe and prolonged drought,the respiration is so feeble as to be hardly determinable, butimmediately on the access of water the respiration rate risesto a value equal to that of the fresh plant with an equivalentwater content. Comparable results were obtained with themoss Hypnum.

SALTS.

The action of salts on the intensity of respiration variesaccording to their chemical nature, their concentration andtheir association with other salts ; thus salts of the heavymetals are very toxic whilst those of the alkali metals andalkaline earths accelerate or retard according to their con-centration and association. Lyon * found that phosphateaccelerates both the aerobic and anaerobic respiration ofElodea. Nitrosomonas shows a maximum respiration in N/200ammonia, whilst a strength of N/io depresses respiration in amarked degree.-j* Similarly the respiration of Bacillus sub-tilts is constant in the presence of low concentrations of thechlorides of sodium, potassium and calcium ; an increase inconcentration leads to a reduction in respiration. Comparedwith cultures lacking these salts, the respiration intensityis increased by the presence of sodium chloride, potassiumchloride, calcium chloride or magnesium chloride in concen-trations of -2M, -I5M, -O5M, and -O3M respectively. Inhigher concentrations the rate of respiration is decreased.:{:With regard to concentration, Inman § studied the behaviourof Laminaria and Chlorella.

* Lyon : " Journ. Gen. Physiol.," 1924, 6, 299.f Meyerhof : " Pfluger's Archiv," 1917, 166, 240.\ Brooks : " Journ. Gen. Physiol.," 1919, 2, 5 ; 1920, 2, 331.§ Inman : id., 1921, 3, 533 ; 4, 171.

134 RESPIRATION

The respiration of Laminaria is depressed after treatmentwith either hypotonic or hypertonic solutions, the depressionbeing greater in the latter concentration and shows a linearrelationship. With hypotonic solutions the relation betweenconcentration and respiration is more complex. In Chlorellathe respiration is depressed on exposure to hypertonic solutions:if the period of treatment is short, the original rate of respira-tion is attained on removal from the solution ; if the exposureis prolonged, the respiration rate does not rise to the originallevel after removal from the solution, a permanent change inthe organism has been effected, a lowering of the rate of meta-bolism. Since the concentration of the surrounding solutionhas effect on the water content of the tissues, this aspect isclearly connected with the relationship of respiration andwater content.

The use of mixtures of salts also accelerate or retardaccording to the degree of their antagonism: thus the normal res-piration rate of Aspergillus niger in a culture medium containing19 c.c. of «5M sodium chloride and 1 c.c. of «5M calcium chlorideis maintained, although each of these salts in these concen-trations and acting alone will accelerate respiration.* Thechlorides of magnesium and sodium, of sodium or potassiumand calcium exhibit a conspicuous antagonism, whilst thechlorides of magnesium and calcium and of sodium and potas-sium are antagonistic but slightly in their effect on respiration.

ACIDITY.

The degree of acidity of the cell sap shows much variationand is an expression of the physiological condition and ofparticular metabolism. Thus the acidity of anthocyanincontaining leaves may be double that of green leaves of thesame species, and the acidity of fleshy plants is greater bynight than by day.

In some plants the hydrogen ion concentration mayprofoundly modify the respiration intensity; Nitrosomonas,for example, shows the greatest rate of respiration in a mediumin which the^H value is between 8*4 and 8*8, beyond the limits

* Gustafson: '* Jotirn. Gen. Physiol.," 1919, 2, 217.

ACIDITY 135

of pK Q'4 — j-6 the process comes to a standstill.* In Penicil-Hum chrysogenum variations in the value of pH between 4 and8 do not affect the normal rate of respiration, that is, the rateat neutrality, pH = 7 ; an increase of the value to 8-8 resultsin the respiration decreasing to 60 per cent, of the normal, atwhich level it remains ; a decrease, on the other hand, in thevalue of pH to 2*65 causes a gradual rise in the respiration ratefollowed by a gradual fall to the normal. At pH I - IO to 1-95the preliminary rise, amounting to 20 per cent., is followed bya fall to below normal. The depression brought about by aconcentration pH = 1-95 or less is irreversible, whilst the simi-lar decrease effected by a pH value of 8-8 is reversible, therespiration rate returning to normal after the plant is placedin a neutral solution. In acid solutions there is an increasein the production of carbon dioxide and a decrease in alkalinesolutions, a phenomenon which may be paralleled in the testtube : a neutral solution of dextrose and hydrogen peroxideshows an increase in the evolution of carbon dioxide on theaddition of acid but not on the addition of alkali.f Witze-mann % also has demonstrated the oxidation of sugar by hy-drogen peroxide in the presence of disodium hydrogen phos-phate. Whether the phosphate here plays any part in theformation of a hexose phosphate such as is known to occur inalcoholic fermentation § has yet to be demonstrated.

LIGHT.

That plants respire both by day and by night is a well-known fact from which it would appear that radiant energy assuch is not a conditioning factor in respiratory activity ; itsaction is indirect in providing through its photosyntheticactivity a supply of respirable material. Thus an isolatedgreen leaf in darkness shows a continuous fall in respiration ;in the light, on the other hand, the respiratory values during acarbon assimilation experiment are continually changing byvirtue of the carbon assimilation and may be doubled by an

• Meyerhof : loc. cit.I Gustafson : " Journ. Gen. Physiol.," 1920, 2, 617 ; 3, 35.t Witzemann : " Journ. Biol. Chem.," 1920, 45, 1.§ Vol. I., p. 494-

136 RESPIRATION

exposure of four or five hours to light and carbon dioxide.This is possibly due to the production of new carbohydrate,although the observed increase is not proportional to theamount of carbon dioxide decomposed.* The high intensityof respiration sometimes observed in green plants in brightsunshine f is due, at any rate in part, to the copious supplyof sugar. In such conditions, however, other factors areoperative : the higher temperature, for instance, would in-crease the respiration intensity; whilst if the illumination,the temperature and humidity conditions of the atmospherewere operative all in the same direction to cause undue loss ofwater, the flaccid leaves would ultimately show a respiratorydepression.

Lundegsrdh $ has shown that the respiration of shadeplants, e.g. Oxalis, is lower than in sun plants, e.g. Nasturtiumand Atriplex. In the former an equilibrium between respira-tion and carbon assimilation, the compensation point (seep. 7), occurs in normal air at a light intensity of 1/120 to 1/140that of direct sunshine, whilst in sun plants the equilibriumis reached in a light intensity of 1/40 to 1/60. In order thatthe carbon assimilation of shade plants may compensate therespiration during the night, an average intensity of illumin-ation of 1/93 that of sunshine at a temperature of i8°C. isnecessary and an increase of this light intensity is requisitefor the maintenance of growth. Of shade plants in general,Oxalis has a great power of carbon assimilation and this helpsto explain why it can grow in places where the light intensityis very low.

THE MECHANISM OF OXIDATION, AND THE ENZYMESYSTEMS INVOLVED.

The catabolic processes of plants may be directly referableto specific enzymes, zymase for instance in alcoholic fermenta-tion ; but in the respiratory activities of higher plants, their roleis not defined with that precision and degree of completeness

f See Ros£ : " Hev. g&eu Bot./* igio, 22, 385.tm Sveask. fcot. Tidskrift.," 1921, 15, 46.

MECHANISMS OF OXIDATION 137

which is desirable, although it is generally agreed that enzymeaction plays an important part in the process. Enzymes as-sociated with the common end products of respiration _ aresubjects for first consideration and of them most attentionhas been given to dehydrase, oxidase, catalase, zymase, andcarboxylase.

In considering the possible mechanisms of oxidation itmust be borne in mind that they have a physical as well as achemical aspect. The former aspect has been specially em-phasized by Warburg, who considers oxidation to be largely aquestion of surface catalysis in which some form of iron playsan important part. From experiments on pigeon's blood,Warburg * showed that when the corpuscles are hgemolysedby alternate freezing and thawing, and the blood then centri-fuged, respiration was continued on the tissue residues, nuclei,etc., of the lower layer, while the supernatant serum, containingthe haemoglobin, showed no respiration ; this difference inactivity he considered to indicate the significance of solidsurfaces ; similar conclusions were arrived at from experimentson sea-urchin's eggs.

Warburg also showed that the oxidation of amino acidscould be carried out at ordinary temperatures by adsorbingthem upon charcoal and exposing to air, whereby they yieldedthe same oxidation products as when oxidized in the livingcell, namely, carbon dioxide, water and ammonia.

The well-known depression of respiratory activity in theliving cell produced by narcotics Warburg attributes to th^fact that these substances are readily adsorbed upon the oxi-dizing surface and so reduce their oxidative efficiency. Thisview he supported experimentally on the carbon model byshowing that dimethyl urea, and similar narcotic substances,which were readily adsorbed by the charcoal, greatly reducedits oxidizing powers for amino acids.

Warburg, moreover, showed that iron played an importantpart in this charcoal model, since if chemically pure carbon,obtained by igniting benzoic acid, be employed, practically

* Warburg : " Biochem. Zeit.," 1921, 119, 134; " Zeit. Electrochem.,"1922, 28, 7a. See also Vol. I., p. 411.

138 RESPIRATION

no oxidation is effected, whereas blood charcoal containingiron was very active. Further, the activity of a pure charcoalcould be greatly increased by steeping it in an iron salt andthen heating to a red heat; such an artificially produced carbonwas affected by narcotics in the same way as the blood charcoal.Hydrocyanic acid was found to depress the oxidative activityof the charcoal to a remarkable degree, due, in Warburg'sopinion, to its combining with the iron to form complex ferro-cyanides; the depression was, however, not of a permanentnature, since the charcoal regains its original activity aftersome time. Warburg's opinion is that the active surface ofa cell is to be regarded as a mosaic of areas, some rich andsome poor in iron; the colloidal conditions being the same,both areas equally adsorb dissolved constituents of the cellsap, but metabolic changes only take place in those regionswhere iron is present, for the addition of a small amount ofhydrocyanic acid brings the activity to an end owing to theinactivation of the iron; there is, however, no measurablereduction in the amount of material adsorbed, since the ironareas form, but a small part of the entire surface.

While this theory fits the facts fairly well as regards theoxidation of amino acids, it is less satisfactory when applied tothe oxidation of carbohydrates, since the carbon model haspractically no action upon glucose and still less upon fructose,although hexosephosphates are attacked, a fact of some interestin connection with the significance of the liexosephosp hates asan intermediate stage of carbohydrate metabolism in general;furthermore, some substances such as oxalic or formic acidswhich are oxidised with diflBculty in the living cell are fairlyeasily attacked by the carbon model.

DEHYDRASE.

The underlying idea of Warburg's theory is that for oxi-dation to take place atmospheric oxygen has to be activatedat same surface by iron, possibly by the formation of anunstable peroxide which sets free active oxygea. A theoryentirely opposed to that of Warburg is that of Wieland *

** Bar. tesvk. ckem. Ges.," 1912,45, 679, 2613 ; 1913, 46,

DEHYDRASE 139

according t o wh ich the activation of hydrogen is the all-im-portant p re l imina ry condition for oxidation. It is well knownthat the o x i d a t i o n of a substance may be effected either by theaddition of o x y g e n or the removal of hydrogen ; as an exampleof the l a t t e r t y p e may be taken the conversion of hydroquinoneinto quinone—-

OH O

OH O

or the convers ion of ethyl alcohol into acetic aldehyde—

CH3CH2OH -f O-> CHaCHO 4 H2O.

But even t h e conversion of acetic aldehyde into acetic acid.

> = CH3COOH

which might b e regarded as a direct addition of oxygen, must,according t o Wieland, be regarded as a dehydrogenation, sincehe has sho^wn tha t the reaction

CH,CHO + Ag,O = CHaCO0H -f 2Ag

cannot t ake place in the entire absence of water; this heregards as ev idence for the preliminary formatiori of a hydratewhich m a y t h e n be oxidised by dehydrogenation accordingto the fol lowing stages :—

/OKCH3CHO -f- FLO * CH3cA>H

yOH /OHCHsCf OlH + O = CHSC^ -f H20.

This convers ion of aldehyde into acid may be effected inthe entire absence of oxygen, provided some other acceptorfor the h y d r o g e n is substituted; such a substance is inethy-lene blue, ^which, provided a suitable catalyst be present, willremove t h e hydrogen, becoming itself reduced to the colourlessleuco-compound :—

DHCH s Cfo ; H : 4 MB - MBHa 4

i4o RESPIRATION

Palladium black will serve as the catalyst; the knownaffinity of this element for hydrogen enables it to eliminatehydrogen according to the equation

/OH /OHCH3Cf-OH + Pd = PdH2 -f CH3C4

\ H V>

but unless methylene blue, or some other hydrogen acceptor,is present to remove the hydrogen from the palladium, thereaction soon comes to an end. The part played by water isconvincingly shown by the fact that whereas chloral CC13CHOis unacted upon by palladium and methylene blue, chloralhydrate CC13CH(OH)2 is at once oxidised.

Even the oxidation of carbon monoxide to the dioxideCO + O = CO*

has been shown by Wieland * to involve the intermediateformation of formic acid

/OHCO + H2O = H. C4

^O

which is then further oxidized to carbon dioxide and water,

/OHH ' C \ n + ° " H*° + C°*'

The principles outlined above have been applied by Wielandto explain enzymic oxidations; in his opinion oxidizing; en-jymes are in reality activators p i ^dehydrases. Two examples will suffice to illustrate this, theSchardinger reaction of milkf and the nitrate-reducing en-zyme of the potato.

The Schardinger reaction was originally designed for dis-tinguishing boiled from unboiled milk; it depends upon thefact that the latter, when wanned with methylene blue anda drop of acetic aldehyde, decolorizes the dye, whereasboiled milk produces no such change. According to Wielandthis reaction is due to a dehydrase which activates the hydro-gen of the aldehyde hydrate, thus enabling the methylene blueto exert its action as hydrogen acceptor.

• Wieland: *' Ber. dtmt. cltem. Ges*/* 1912, 45, 679, 2613.f IMS., 1^4. 47, 2085.

DEHYDRASE 141

By slightly varying the conditions, the same enzyme isable to produce from two molecules of salicylic aldehyde onemolecule each of the corresponding acid and alcohol. Thisreaction, which is of the type kn<rwn as a Cannizarro reaction,is usually represented by the equation

2CaH4 (OH) CHO -f H2O - C«H4(OH)COOH + C,H4 (OH) CH,ORand was originally attributed by Parnas * to a special enzymeto which he gave the name of aldehyde mutase. Accordingto the view of Wieland, however, the reaction may be explainedby assuming that one of the two molecules of salicylic aldehydemerely acts as the hydrogen acceptor to the hydrate of theother molecule as follows :—

/OHCaH4 . O H . C ^ O H f C,H4 . OH. CH;6i

N/OH

= C,H4 . OH . C< -f C8H4. OH . CH2OH.

Salicylic acid Salicylic alcohol

The Nitrate-reducing Enzyme oj the Potato.—The existenceof this was first demonstrated by Bach in the following simpleexperiment. One gram of freshly pounded potato is heatedin a test tube to 6o° C. with 10 c.c. of 4 per cent, aqueoussolution of sodium nitrate together with 3 drops of lOper cent.solution of acetic aldehyde. After two minutes the solutiongives a strong reaction for nitrite by theGriess Ilosvay reagent.This reaction was originally thought to be due to a so-calledhydroiytic oxidation reduction—as expressed by the equation

NaNO, -f H4O +- CH»CHO = NaNTO2 -f HtO +- CHSCOOHaccording to which the sodium nitrate and acetic aldehydebetween them shared the hydrogen and oxygen of a moleculeof water, thereby becoming reduced and oxidized respectively.

On WielancPs hypothesis, however, this is simply explainedby the dehydrase activating the hydrogen of the aldehydehydrate whilst the sodium nitrate acts as the hydrogen ac-ceptor, f

* Paxnas : " Biochexn. Zeitsch.," 1910, 28, 274.f The ability to oxidize acetic aldehyde and effect the simultaneous

reduction of nitrate to nitrite is not uncommon in vegetable and animal

I42 RESPIRATION

/ O H1. CHsCf + HOH - CH3C^OH

\ H \ H/OH X)H

2. CH3Cf OH -* CH3C^ -f 2H

3. NaNO, + 2H = NaNO2 -f H2O.

Even the biological conversion of alcohol into acetic acid b ythe vinegar plant, Bacterium aceti, was shown by Wieland t obe a dehydrase action which could be effected in an atmosphereof nitrogen by leaving freshly washed cultures of the p l a n tin contact with alcohol and methylene blue in a flask fromwhich air had been displaced by nitrogen. In a comparativelyshort time the methylene blue was decolorized and after somedays a measurable quantity of acetic acid had been produced.*

It is evident that the theories of Warburg and Wieland a r efundamentally different, the former insisting upon the acti-vation of oxygen while the latter postulates the activation,of hydrogen.

Warburg's criticism of Wieland's hypothesis is that acti-vated hydrogen when finally oxidized should produce hydrogenperoxide and not water, and that hydrogen peroxide is n o tfound in living cells. To this Wieland replies that all aerobic

tissues. Thus milk in anaerobic conditions can reduce nitrate, the nitrateacting as the hydrogen acceptor; if, however, the reaction is allowed totake place in the presence of air a peroxide is formed (Haas and Hill," Btochem. J.," 1923, 17, 671, and Haas and Lee," Biochera. J.," 1924,18, 614) which was shown by Thurlow (id., 1925, 19, 175) to be hydrogenperoxide resulting from atmospheric oxygen acting as hydrogen acceptorin place of the nitrate; in such circumstances no nitrate is reduced.

It has since been shown by Thurlow (he. cit.), Dixon and Thurlow(" Biochem. J.," 1924, 18, 989), and Bernheim (" Biochem. J.," 1928, 22*344) that, in the nitrate-reducing system, either xantbine or hypoxanthineor adenine may replace the aldehyde as hydrogen donator and methyleneblue may replace nitrate as hydrogen acceptor.

Subsequently Bernheim, comparing the Schardinger reaction of milk:withthenitrate-reducingactionof potatoin thepresenceof aldehyde, arrivedLat Urn conclusion that the potato enzyme is able to reduce methylene bluein the presence of aldehyde as hydrogen donator in the same way as theSchardinger enzyme of milk. Bach (" Biochem. Zeit.," 1913, 53, 412)and Michlin (id,. 1927, 185, 216), who had tried unsuccessfully to do this,failed, presumably owing to their working at an unsuitable ptt range,Bernheim having shown that the methylene blue reducing range of theenzyme of the potato to be 5*6 to 7-8, while the nitrate reducing range isiKWH 5 to 8*6, with aa optimum at 5*5.

* Wieland : " Bar. dent. chem. Ges./* 1913,46, 3336.

MECHANISMS OF OXIDATION 143

cells contain catalase, an enzyme whose function it is to destroyhydrogen peroxide as it is formed.

In support of his own theory Warburg points out that whileit is true that the oxidation of succinic acid in the presence ofinethylene blue proceeds both in the absence and in the presenceof hydrocyanic acid, the latter substance inhibits the reactionwhen oxygen is employed as the hydrogen acceptor in placeof rnethylene blue ; this, according to him, shows that theoxygen is unable to function when the iron is inactivatedby hydrocyanic acid. Wieland, on the other hand, explainsthe action of hydrocyanic acid as being due to its destructiveeffect upon catalase which is thereby rendered unable toprevent the accumulation of hydrogen peroxide, which sub-stance is toxic to the cell. To this suggestion Warburg objectson the ground that such an effect would be permanent whereashe has shown the retarding effect of hydrocyanic acid to betemporary.

A difficulty in the way of accepting the idea that ironis an essential element in any oxidizing mechanism is alsopresented by the fact tha t Willstatter has shown that thepurest preparations of peroxidase are free from iron. Further-more, it has been pointed out that the action of hydrocyanicacid is not always to depress oxidation ; it may occasionallystimulate * A further obstacle in the way of interpretingthe inhibiting action of hydrocyanic acid as being due to itscombining with iron, is the observation of Moureu and Du-fraisse f tha t hydrocyanic acid, in the entire absence of iron, isable to act as a powerful depressant of oxidation; it is as theseauthors describe it, an antoxygen. According to the theoryof Duf raisse and Moureu, most oxddi2able substances remainunattacked by atmospheric oxygen owing to the presence oftraces of a negative catalyst or antoxygen.; a substance "whichis particularly active in this respect is hy^o^ujnoiie, which,in small quantity, is able to prevent entirely the autoxidationof benzaldehyde through benzaldehyde peroxide to benzoicacid, a reaction which, as is well known, takes place very

* Buchanan : " Science/' 1927, 645, 23S.f Mourem and Dniraisse: " Compt rend.," 1926, 183, 685.

144 RESPIRATION

readily in ordinary circumstances. Dufraisse and Moureuhave shown that not only hydrocyanic acid but the ferrocyan-ides, which according to Warburg should be without a c t i o nof any kind, act as depressants of oxidation. The s a m eauthors * draw attention to the fact that among the most a c t i veantoxygens are the phenols and tannins and they suggest t h a tthe wide distribution of these substances in the plant w o r l dmay be the cause of the relatively slow rates of oxidationin the plant as compared with the animal; they also suggestthat the toxic action of phenols is due to their antoxygenicproperty.

Support for Wieland's theory of dehydrogenation is fur -nished by the work of Thunberg, who has shown that freshlyminced and wTashed muscle contains an enzyme which in t h epresence of methylene blue, acting as a hydrogen acceptor,is able to convert succinic acid into fumaric acid :—

CHS. COOH CH . COOH| + MB = || -f MBH2.CHtCOOH CH • COOHSuccinic acid Fumaric acid

Thunbergf regards hydrogen as the essential fuel of t h eliving cell and considers only those substances as possibleintermediate metabolites which when left in contact with.methylene blue in the absence of oxygen decolorize this sub-stance and therefore act as hydrogen donators. His tech-nique consists in placing the material under examination i na test tube with freshly washed frog's muscle, to supply t h edehydrase, and methylene blue, filling the tube with boiledwater, and leaving the whole in a thermostat and examiningat intervals. If the methylene blue is decolorized, the sub-stance in question is a hydrogen donator and consequently a.possible intermediate metabolite.

It will be noted that the theory of Wieland envisages onlythe activation of hydrogen^atoms, thus making the substance

tidy^r^ a hydrogen donator.

and Dmfralsse : "Comp. read./' 1922, 174, 258.f TJHinberg: "Stand. Axduv. Physiof.," 1920, 40, 1; " Naturwiss./*

19a*. 1% 417.$ Quastel; " J. Hygiene," 1928, a8,139, and the literature there quoted.

RESPIRATORY PIGMENTS 145

however, from his observations on the oxidative activities oibacteria, found that furnaric acid could be activated as ahydrogen acceptor, a. state of affairs not provided for by theWieland theory. To quote the author's own words: " Theinadequacy consisted in confining the activation only to hydro-gen atoms. A more reasonable interpretation of the pheno-mena was that activation of the substrate molecule #s a wk^e

occurred, the activated molecule behaving as a hydrogendonator or acceptor, according to the nature of the moleculeand the general circumstances under which the reactionsoccurred. Further investigations with bacteria showed thata variety of substances are activated as hydrogen acceptors,e.g. nitrates and chlorates."

RESPIRATORY PIGMENTS.

Palladin * observed that a boiling water extract of manyplants contains a colourless chrornogen which by the action ofa mixture of a peroxidase and hydrogen peroxide undergoesa series of colour changes and ultimately produces a more orless deeply coloured substance known as a respiratory pigment.These pigments are concerned with^the oxidation of hydrogen,not with the oxidation of the carbon of organic compounds.The role.of respiratory pigments is therefore that of hydrogenacceptors whereby they are reduced to the chromogen

R-f 2H= RH2.

The chrornogens are then oxidized by active oxygen arisingfrom the activity of an oxidase which results in the regenera-tion of the pigment

RH3

These chrqmogens, which occur in the plant in the form jofglucosides, are supposed to belong to the group of polyhydricphenols and are therefore bodies of a tannin-like nature.During1 regular respiration an equilibrium obtains between theoxidation arid reduction; but if the oxygen absorption is

* Palladin: " Ber. dent. Bot. Ges.," 1908, 26a, 125, 378, 389; 1909,27, ir0 ; see a.lso " Jahrb. wiss. Bot," 1910, 47, 431 ; " Zeit. G&nmgs.PhysicL," 1912, i9 91,

II. IO

I 46 RESPIRATION

excessive, aerobic respiration ends, since the respiratorychromogen is transformed into the stable pigment.

Palladin thinks that most pigments take part in respiration,but the essential character of such bodies, that of reversibleoxidation and reduction with great ease, is not possessed bythem ; only by drastic chemical operations is the reversibilityeffected. But three pigments which meet with physiologicalrequirements have since been discovered, namely chlorogenicacid, cytochrome and hermidin. Oparin * isolated from sun-flower seeds the depside chlorogenic acid which acts like arespiratory pigment; by oxidation it loses four atoms ofhydrogen and is converted into a green pigment.

Oparin considers that aerobic respiration essentially con-sists of two phases: the respiratory chromogen is oxidizedto a pigment by atmospheric oxygen in the presence of aphenolic oxidase; and that the pigment is reduced to thechromogen by the hydrogen contained in the water molecule,the oxygen being taken up by the living cell. From Oparin'sexperiments it would appear that chlorogenic acid is moreparticularly active in the oxidation of natural arnino acids andother compounds (see p. 167).

Cytochrome is oxidized by a thermolabile indophenoloxidase and is reduced by a dehydrase, it appears to act asa carrier between the two activating mechanisms of oxidaseand dehydrase respectively.f

But this, together with hermidin, has already been com-mented on.J

GLUTATHIONE.

Glutathione is the name given to a substance first isolatedby Hopkins § from yeast. It is a thermostable substance,and consequently non-enzymic, whose function would appearto facilitate the oxidation of hydrogen by alternately com-bining with and yielding up this element, in' this way actingas an h^ rpge^ .transporter. The constitution of glutathione

* Oparin : " Biochem. Zeit./' 1921, 124, 90 ; 1927, 182, 155.t Keilin : " Proc. Roy. Soc./' B, 1929, 104, 206.t Vol. I.. 305, 343.§ Hopkins : " Biochem. Journ./' 1921, 15, 286.

GLUTATHIONE 147

is that of a dipeptide of cystein and glutamic acid and isrepresented by the formula

CH2 - SH

CH . NH . CO . CH2 . CH2 . CHNH2. COOH

COOH

This substance, when oxidized by the removal of thehydrogen between two molecules, yields the compound

CHa - S S - CH2

CH . NH. CO. CHs. CH2. CHNH2. COOH CH. NH. CO. CH.. CH, . CHNH*. COOHCOOH COOH

For simplicity of expression these changes may be repre-sented as follows :—

- 2 H2GSH ^ GS.SG

+ 2HIn its oxidized form, GS . SG, it serves as an acceptor

of the hydrogen set free from, say, succinic acid by the de-hydrase which occurs in minced muscle, giving rise to thereduced form, GSH, but it is not by itself able to remove thishydrogen in the absence of the enzyme succinic acid dehy-drase. The reduced form is then ready to give up its hydrogento oxygen activated by iron with the formation of G S . SGand water. The optimum pH for the reaction involving theremoval of hydrogen

is 7-4, and at pH 6 it is much retarded, and below this thereduced form GSH is stable ; on the other hand, the accept-ance of hydrogen by the oxidized form

GS .SG + 2H-* 2GSH

is much the same at pH. 6 and pH 7-4.The question whether glutathione is able to act in the

absence of metals has been much discussed, and it would ap-pear that the balance of evidence is in favour of an essential partbeing played byjironj this could be explained by assumingthat its function is to activate the oxygen, as postulated byWarburg, previous to its oxidising the hydrogen

2GSH -j- O = GS . SG -f H.O.10*

i48 RESPIRATION

Further, this view is supported by the fact that hydrocyanicacid retards the activity of glutathione as it does other oxi-dations supposed to be dependent upon iron.

The exact function of glutathione in the respiratory mechan-ism is still largely a matter of speculation ; it has been sug-gested that it acts only as a carrier in transferring hydrogenfrom the substance to be oxidised to the activating iron sur-faces where the hydrogen is to be oxidized, the iron beingsupposed to be localized upon surfaces and thus to be incapableof moving towards the oxidizable materials, a function whichis broadly comparable with that assigned to the respiratorypigments.

Glutathione is not uncommon in plant tissues althoughthe coloration by which it is recognized usually is far lessintense in vegetable than in animal tissue. Its presence inyeast may be demonstrated by grinding the cells in a mortarwith a little sand and some saturated solution of ammoniumsulphate. On pouring off and adding to the supernatantliquid a few drops of 5 per cent, solution of sodium nitro-prusside and a little strong ammonia, a pink colour is produced.This colour reaction, which also is given by a number of otherreducing substances such as aldehydes, acetone, hydrogensulphide, etc., is only given by the reduced or cysteine formof the dipeptide and not by the cystine or oxidized modification.

OXIDASES.

Oxidates * are of wide occurrence in the vegetable kingdom,as should be the case if they are primarily concerned in aerobicrespiration, but whether they are present in all living cellsis doubtful. According to Atkins j* they are absent or inactivein tissues markedly acid in reaction or containing large amountsof reducing substances. Bunzel J also has shown that theactivity of these enzymes is inhibited by acids, their greatestactivity being at or near the point of neutrality ; the limitsof the pH value corresponding to complete inhibition in the

* See Vol. I., p. 498.f Atkins : " Proc. Roy. Dublin Soc.," 1913, 14, 144.X Buazel: " Journ. Biol. Chem./' X916,28,153.

OXIDASES 149

various subjects of experiment are narrow and the figure ofacid sensitiveness is almost invariable in a particular genus.

Two kinds of oxidases are generally recognized in the plantworld, namely, the direct acting oxidases which turn bluean alcoholic solution of guaiacum without the addition ofhydrogen peroxide, and the indirect acting peroxidases whichare unable to produce a change in colour until hydrogen per-oxide has been added.

The direct acting oxidases are supposed to consist of twoparts :—

(a) An autoxidisable thermostable substrate which isgenerally considered to be a derivative of catechol

OHDH

(6) The enzyme,proper or peroxidase, which is thermolabile.The function of this enzyme (b) is to liberate active oxygen

by decomposing the peroxide formed according to (a) in thedirect acting oxidase, or by decomposing hydrogen peroxidewhen added to the indirect oxidase system.

It is to be remembered that the distinction between thedirect and indirect oxidase depends upon the reagent guaiacum ;it may be that the indirect oxidases differ only in strengthfrom the direct oxidases, or, alternatively as has been suggestedby Szent Gyorgyi, that the indirect acting enzymes differfrom the direct acting in the nature of their substrate whichmay not be of a catechol nature (see Vol. L, p. 501).

The mechanism of the " direct " and " indirect " oxidaseshas been questioned by Pugh and Raper,* but the views ofthese authors are disputed by Onslow and Robinson.j*

CATALASE.

Catalase has the power of setting free gaseous oxygen fromhydrogen peroxide ; although of common occurrence, it isnot universally present in living cells and is often associatedwith oxidase. For this reason many observations have been

*Pugh and Raper: " Biochem. J.," 1927, 21, 1370.f Onslow and "Robinson : id., 1928, 22, 1327.

150 RESPIRATION

made on their distribution and relation to the oxidative aspectsof respiration.

Wieland * has drawn attention to observations by Lesser,fRywosch,} and Jorns § that anaerobes such as Bacillus tetanusand B~ botulinus are deficient in catalase whilst aerobic or-ganisms such as Pneumococcus and Sarcina, and facultativeaerobes such as yeast contain catalase in quantity. It isconsidered that the occurrence of catalase in those cells andtissues which require oxygen is necessary for the twofoldpurpose of preventing the accumulation of hydrogen peroxidewhich is toxic to the cell, and for the liberation of oxygen.Hydrogen peroxide is regarded as the first product of theoxidation of hydrogen by oxygen which acts as the hydrogenacceptor in the cell,

02 -f 2H = H2Oa.

The catalase then acts upon the resulting hydrogen peroxide,breaking it up into water and oxygen, which latter is thenavailable for further oxidation,

HjO, = H2O -f 0.

This cycle of changes does not occur in anaerobic oxidations,which may explain the absence of catalase in these conditions.

Appleman || fouad that the oxidase content of the expressedjuice of the potato is not indicative of the intensity of therespiration of the tuber, whilst the catalase activity showsa striking correlation. Similarly for sweet corn, in which in-stance the respiration in the milk stage is high when firstcollected but in storage rapidly decreases, the decrease beingaccompanied by a nearly proportional fall in catalase activity.In the pine-apple, Reed [ found that oxidase and catalaseare independent; the amount of the former remains constantduring ripening of the fruit whilst the catalase increases. Inthe wheat and certain allied plants, the embryo shows a twenty-eight to twenty-nine-fold greater catalase and oxidase activity

* Wieland : " Ber. deut. diem. Gesells.," 1921, 54, [B], 2353.f Lesser : " Zeitsch. Biot.," 1906, 48, 1.• Rywosch: " 2entr. Bact.," 1907, 44, 295.§ J t o s : " Arcbiv. d. Hyg./' 1908, 67,134.|| Appieman : " A.mcr. Jcrarn. Bot," 1916, 3, 223 ; 1918, 5, 207,^ B d : €* Bot. Ga.z./' 6 &

CATALASE 151

than the endosperm, and this also holds for the intensityof respiration. In air-dry fruits of Andropogon halepensis,catalase activity runs parallel to respiration, a correlationwhich does not obtain in the seeds of AmaranthtLS.* In Acersaccharinum, Jones f found that the intensity of respirationof seeds during the process of desiccation at 250 C. at firstdecreases, then rises to a maximum and finally graduallydeclines to zero : with regard to the catalase activity, there isa slight initial increase, then a decrease as desiccation proceeds,during which process there is also a gradual decrease in perox-idase activity. Nemec and Duchon % state that the catalaseactivity of seeds is closely correlated with their vitality asmeasured by the percentage of germinations, Gracanin,§ onthe other hand, finds that this activity is not a good indexof vitality since dead seeds may possess active catalase.Shull and Davis || find that the upper seeds of Xanihiwn,which have a delayed germination, exhibit a lower catalaseactivity than the lower seeds, which on germination showan increased catalase activity, Rhine f observed a decreasein catalase during the early stages of germination, followed byan increase in the later stages ; there is thus a wide divergencein the respiration and the catalase curves. Lautz ** also drawsattention to this initial decrease, followed by an increase, inthe catalase activity of germinating seeds; he also observedthat the rapid increase in respiration following a rise in tem-perature is not accompanied by a corresponding increase incatalase activity. This is contrary to the observations ofBurge and Burge "ff who found that the catalase of Spirogyravaries directly with temperature and illumination, temperaturehaving the greater effect. They conclude that whatever affectsrespiratory metabolism, similarly affects catalase activity,and suggest that catalase is of prime importance in respiration.

* Crocker and Harrison : " Journ. Agric. Res.," 1918, 15, 137.t Jon.es : " Bat. Gaz.," 1919, 69, 127.j Nemec and Duchon : " Compt. rend./' 1922, 174, 632.§ Gra.ca.nin : " Biochem. Zeit.,"' 1927, 180, 205.|| Shull and Davis : " Bot. Gaz.," 1923, 75, 268.[ Rhine: id., 1924, jS, 46.

** Lautz : " Ajner. Journ. Bot.," 1927, 14, 85.tf Barge and Barge : " Bot. Gaz.," 1924, 77, 220.

152 RESPIRATION

Drain * finds no clear correlation between catalase activityand respiration of apples, and the same lack was observedby Davis f in potato tubers suffering from the blackheartdisease. Rhine $ observed that catalase markedly decreaseswhen oxygen is withheld from seeds by prolonged soakingin oxygen-free water, and Morinaga § concluded that in-crease in catalase activity is a function of free oxygen fromthe facts relating to the germination of rice. Rice is grownon swampy ground and is capable of germination and sub-terranean growth in lower oxygen pressures than land plants.Rice grains contain about one-tenth the amount of catalase aswheat, oat, rye and like cereals which are grown on dry land ;but when rice is germinated in aerobic conditions, the amountof catalase rises to about seven-tenths of that of the othercereals mentioned. If germinated in an atmosphere with alow oxygen pressure there is still an increase in catalase, whichdoes not obtain if germinated in anaerobic conditions ; indeed,the increment due to aerobic cultivation rapidly decreases inthe absence of oxygen. It was further observed that seedlingswith a higher catalase content use more oxygen in their growththan those with a lesser amount of catalase.

It is hardly possible to draw definite general conclusionsfrom these facts, many of which are contradictory. Theevidence does not show that catalase is chiefly concernedin respiration, and the same applies to the oxidases, butcatalase may, as Lautz suggests, be a regulatory force pre-venting excessive oxidation by removal of superabundanthydrogen peroxide.

FERMENTATION : ZYMASE AND CARBOXYLASE.

An account of zymase and alcoholic fermentation has beengiven in Vol. I. ; it is, however, desirable again to considerthe subject in view of the place it occupies in current opinionson the mechanism of respiration.

Zymase hardly requires introduction, it is the enzyme

• Drain : " Bot. Gaz.," 1926, 82, 183.t Davis : id., 1926, 82, 323.% loc. cit.§ Morinaga: " Bot. Gaz.," 1925, 78, 73.

FERMENTATION 153

particularly associated with yeast and is responsible for thealcoholic fermentation of sugar. It has been described asoccurring in higher plants such as the beetroot, potato,lupin, and others,* and has been isolated from germinatedseeds ; in general the presence of a zymase like enzyme isconcluded from the appearance of alcohol in plant tissueskept in anaerobic conditions. A large quantity of zymase,comparable to that which occurs in yeast, is not to be ex-pected in the higher plants, since its function is apparentlyonly that of preparing material for oxygen respiration.

Carboxylase has the property of removing carbon dioxidefrom carboxyl groups, thereby converting pyruvic acid, forexample, into carbon dioxide and acetic aldehyde ; it occursin yeast, where it is associated with zymase, and, according toBodnar,f in the beetroot and potato tuber.

On an earlier page mention has been made of the formationof lactic and butyric acids in addition to alcohol from .sugar,Kostytschev considers that these fermentative mechanismsare fundamentally similar and are based on one and the sameinitial stage, the breaking of a molecule of sugar into two mole-cules of pyruvic acid with the liberation of four atoms ofhydrogen—

C6H12O, = 2CH3 . CO . COOH + 4H,Pyruvic acid

The subsequent reactions varies according to the conditionswhich determine the formation of the three different endproducts.

I. Alcoholic Fermentation.—This will result if the pyruvicacid is decomposed before the hydrogen has time to exertits reducing action—

2CH3. CO . COOH = 2CH3CH0 -f- 2CO2.Acetaldehyde

The hydrogen then reacts with the acetaldehyde to formalcohol—

2CH3CHO + 4H = 2CH3CH2OH.

*Palladin and Kostytschev: " Ber. deut. bot. Gesells./' 1906,24,273.Stoklasa and Chocensky ; id., 1907, 25, 122.

fBodnar: " Biochem. Zeitsch.," 1916,73, 193. See also Zaleski:" Ber. deut. bot. Gesells./* 1913, 31, 349.

154 RESPIRATION

2. Lactic Acid Fermentation.—Here the action of the hydro-gen is immediate, the pyruvic acid being reduced to lacticacid before either of the two other alternatives is possible—

2CH3. CO . COOH + 4H = 2CH3 . CHOH . COOH.Lactic acid

3. Butyric Acid Fermentation.—This results from the slowreducing action of the hydrogen: acetaldehyde is first formed—

2CH3 . CO . COOH + 4H = 2CH3CHO + 2CO2 + 4H.

The acetic aldehyde then undergoes an aldol condensation,

2CH3CHO = CH, . CHOH . CH2 . CHO

which, by molecular rearrangement, gives rise to butyric acid—CH3 . CHOH . CH2. CHO = CH8 . CH, . CH2 . COOH.

These three reactions can be expressed by the single equation—

C8HiaO, = CH3. CH2 . CH2 . COOH + 2CO2 + 2H2.

Reverting to the subject of alcoholic fermentation, theviews of Kostyschev are not shared by Neuberg who con-siders that pyruvic aldehyde is first formed by the removalof two molecules of water—

C6H12O6-> 2CH3. CO . CHO + 2H2O.

Assuming one molecule of pyruvic aldehyde to act in itstautomeric form, the two molecules are supposed to react inthe presence of water according to the Cannizarro reaction—

CH3CO CHO + O CH3CO COOHI ->• Pyruvic acid

CH2 = COH CHO + H2 + H2O CH2OH . CHOH CII2OH.Glycerol

The pyruvic acid thus produced is decomposed by the enzymecarboxylase into acetic aldehyde and carbon dioxide —

CH,CO COOH = CH,CHO -f HaO.

The acetic aldehyde then becomes reduced to ethyl alcoholby acting as hydrogen acceptor for the hydrogen set freefrom water when a second molecule of pyruvic aldehyde be-comes oxidised—

CH8 . CO . CHO 4- O CH3CO . COOHI ->> Pyruvic acid

CH3CHO -f H4 CHSCH,OH.Ethyl alcohol

By causing ftrmt/ntation to take j>hre i: *] *r,.. - fsodium hydrogen sulphite or of dmkih\I *1HA .r r - r : • 'either of which combine with a< *ti«* *iM»liy\li* \ i * ; ;•creased the yield «»f ^lycerol prodiuv<i in al »h >',<- f«r\. rt *•tion. Tliis, hesu^es t s , is due to tin* hydn^rr, \\L: ; \v ,r inormally reduce the acetic aldehyde to eth^l al % h* 1 I • p;^forced to find some other acceptor which he presurr*'- ;- ^naldehydic precursor of glycerol.

The main result of his experiments indicate that aceticaldehyde is an intermediate product of alcoholic fermentation,and he provided the means of establishing the presence ofsmall quantities of acetic aldehyde which has proved ot greatvalue in determining Indirectly whether, in any proa^s ofcarbohydrate degradation, a change of the nature of akobolicfermentation is operative—the significance of this will be seenlater (p. 160).

Factors influencing alcoholic fermentation by yeast.—>Qm£space must be devoted to a consideration of the influenceof oxygen upon alcoholic fermentation with a view to tracingthe relation between the oxygen respiration of the yeast plantwith its activity in alcoholic fermentation, which, accordingto the equation

C«HltO4 - 2C,H,OH + *COt,

does not involve atmospheric oxygen.Pasteur, to whom we owe much of our earlier knowledge

of alcoholic fermentation, held the opinion that alcoholicfermentation was a mode of life of the yeast plant when unableto obtain air, ihla vie sans air" but lie also knew that whenair is entirely withheld the plant sooner or later stops growingand gradually dies, but the introduction of a little oxygen,if not too late, restores the plant to activity. During fermen-tation in anaerobic conditions, although the breakdown ofsugar continues, the reproduction of the cells stops. In at-tempting to explain the role of oxygen in fermentation, sub-sequent workers came to the erroneous conclusion that oxygenstimulated fermentation since they obtained an increasedyield of alcohol when the fermenting liquors were aerated,

156 RESPIRATION

but in their observations they omitted to take account of theincrease in the number of yeast cells consequent upon thestimulation of reproduction by the oxygen. So for years thematter rested ; brewers and distillers, aware of the beneficialaction of oxygen, continued to practise a certain degree ofaeration of the fermenting liquor, a process which has theadditional advantage of stirring the liquid and removingexcess of carbon dioxide.

The difficulty encountered by Pasteur in his theory of" vie sans air " was that a plentiful supply of air should sup-press alcoholic fermentation rather than stimulate it. Anexplanation of this apparent contradiction was furnished froman entirely different source, namely from animal physiology.

Fletcher and Hopkins * in a study of the relationshipbetween muscular contraction and lactic acid production,showed that lactic acid is formed by resting muscle, and thisin increased quantity in an atmosphere of nitrogen; onexposing the muscle to air, the lactic acid disappears with theformation of carbon dioxide and water. The lactic acid isderived from a precursor, or lactacidogen, which is probablya hexosephosphate, the hexose constituent of which is formedfrom the store of glycogen contained in the muscle. A. V.Hill f by accurate measurements found that when a muscleis stimulated, heat is liberated and continues to be liberatedso long as contraction is maintained and also while the musclerelaxes. This constitutes the initial process in muscularcontraction, and it is noteworthy that the production ofheat occurs, in the absence of oxygen, the process beinganaerobic. Recovery next follows: the muscle after re-laxing ceases to give off heat, but after a short intervalheat production begins again, provided oxygen is present,reaches a maximum and then falls away to zero. This secondevolution of heat, which is about 1-5 times as great as thatoccurring in the initial stage, is accompanied by an absorptionof oxygen and a disappearance of lactic acid; the amount ofheat evolved is, however, only sufficient to account for the

* Fletcher and Hopkins ; " J. Physiol.," 1907, 35, 247.f A. V. Hill r id., 1914, 48, 10.

FERMENTATION 157

oxidation of about one-fifth of the total amount of lactic acidwhich has disappeared.

About the same time, Meyerhof * showed that when musclecontracts glycogen disappears in an amount exactly equivalentto the lactic acid produced, and that during the recoveryprocess the lactic acid disappears only in part by oxidation,the remainder being resynthesized to glycogen.

He found, moreover, tha t for every molecule of oxygenabsorbed, one and a half molecules of lactic acid disappeared ;but from the equation

C3H6O3 + 3Oa = 3CO2 + 3H2O

one molecule of oxygen could only oxidize one-third of a mole-cule of lactic acid, hence the ratio

Lactic acid disappeared I \

Lactic acid oxidized J

Hill's experiments show tha t this ratio is approximately 5 ;thus it appears that four-fifths of the lactic acid producedare reconverted into glycogen at the expense of the energysupplied by the oxidation of one-fifth, hence the anaerobicprocess is reversed by the aerobic phase introduced by theoxygen. Expressed in another way, when the oxidative pro-cess keeps pace with the anaerobic stage the products of thatstage will not accumulate. Meyerhof f subsequently foundtha t much the same phenomena occur in yeast. This he wasonly able to establish on realising the fact, ignored by earlierworkers, that some yeasts are more sensitive to the influenceof oxygen than others. Thus he established that ordinarybrewers' yeast, the so-called bottom fermentation yeast, thetype used by Pasteur in his experiments, oxidises only onemolecule of sugar for every 99 molecules of sugar which itferments ; in this type of yeast oxygen respiration is, therefore,very low, a state of affairs which Meyerhof considers to be dueto long-continued selective cultivation in special conditionswith the result that the yeast has almost lost the habit of

* See A. V. Hill and Meyerhof: " Ergeb. Physiol.," 1923, 22, 299.f Meyerhof : " Biochem. Zeit./' 1925, 162, 43 ; " J, Gen. Physiol./'

1927, 8, 531-

158 RESPIRATION

oxygen respiration. On the other hand, distillers' top fer-mentation yeast was found to ferment only 80 per cent, ofthe sugar available, the remaining 20 per cent, being oxidized.In wild yeasts, Torula so called, oxygen respiration is muchhigher, and in these plants oxygen is able to depress alcoholicfermentation to extinction. These observations provide ex-perimental proof of Pasteur's thesis " la vie sans air " whichhe could not establish owing to his use of a yeast whose oxygenrespiration was too small for detection by his experimentalprocedure.

Meyerhof also showed that for each molecule of oxygentaken up in the oxygen respiration of yeast, one moleculeof sugar less is fermented, but, since according to theequation

CeH12O6 + 6O2 = 6CO2 + 6H2O

one molecule of oxygen is only able to oxidize one-sixth of amolecule of sugar, it would appear that the oxidation of onlyone-sixth of a molecule of sugar is sufficient to protect fromfermentation one molecule of sugar; otherwise expressed,one molecule of sugar by oxidation protects six moleculesfrom fermentation, a figure approximately equivalent to thatfound by Meyerhof and by Hill to hold good for musclecontraction.

The close connection between the chemical changes inmuscle and those occurring in alcoholic fermentation is shownby the fact that yeast whose fermenting power has beendestroyed by washing out the thermostable coenzyme canbe restored to activity by the addition of an aqueous extractof muscle; similarly, the addition of the coenzyme of yeastto a washed muscle will restore its power of respiration.

Furthermore, in both there is an intermediate formationof a hexose phosphate, which in muscle gives rise to lacticacid, and is hence termed lactacidogen, but in fermentationgives rise to alcohol.

If respiratory processes are essentially enzymatic, it isnatural to suppose that the respiration intensity would increasein the presence of accelerators : various substances have been

MECHANISM OF RESPIRATION 159

so described, amongst them being phosphates * which are im-portant accelerators in zymase action.f

Palladin looks upon lipoids as being of the nature ofcoenzymes, since the more they are extracted from the planttissue, the greater is the reduction in the intensity of respira-tion although not in an exact proportion.J

Galitsky and Wassiljeff § observed that the addition ofboiled water extracts of bean seeds and wheat grains increasedthe output of carbon dioxide in living and in dead seedlings toa degree depending on the acidity of the culture medium :in neutral cultures the increase reached 117 per cent., whilstin a slightly alkaline medium an increase of 86 per cent, ob-tained as compared with an increase of 60 per cent, in a slightlyacid medium.

CURRENT IDEAS ON THE MECHANISM OFRESPIRATION.

Modern views on the mechanism of respiration includean anaerobic phase even in organisms which are obligateaerobes. It is therefore desirable to draw attention to thesalient phenomena presented in the anaerobic respiration ofhigher plants, an extravagant method of obtaining energygiving origin to carbon dioxide and ethyl alcohol which, ina sense, is wasted unless further oxidized to carbon dioxideand water. ||

It has previously been mentioned that the higher plants,although normally aerobic, may be facultatively anaerobic ;the anaerobic respiration of germinating pea seeds, for example,is a commonplace of the laboratory. To take a specificexample. Parija f has compared the respiration of the applefruit in normal air and in an atmosphere of nitrogen. Thework of Blackman and Parija on the aerobic respiration of the

•Iwanoff: " Biochem. Zeitsch.," 1910, 25, 171 ; 1911, 32, 74; Za-leski and Reinhard : id., 1910, 27, 450.

f See Vol. I., p. 494.% Palladin : " Ber. deut. bot. Gesells.," 1910, 28, 120.§ Galitsky and Wassiljeff: id., 182.|| See Kostytschev : " J. Russ. Bot. Soc," 1916, 1, 182.If Parija : " Proc. Roy. Soc./' B, 1928, 103, 446.

160 RESPIRATION

apple has been mentioned on an earlier page ; they found thatat a temperature of 22° C, the rate of respiration followed acertain definite course. Parija found that in general a longperiod in an atmosphere of nitrogen has no permanent effecton metabolism, for when the apple is replaced in air, the rateof respiration returns to that level of intensity it would haveattained in aerobic conditions in the same period of time.This recovery of position may take two days and may be as-sociated with a temporary phase of very low production ofcarbon dioxide. On the other hand, transference from aero-bic to anaerobic conditions is accompanied by a short transi-tion phase. Thus on plotting the experimental results, twotypes of anaerobic respiration are demonstrated. In the onethere is an initial outburst of carbon dioxide when the appleis placed in nitrogen, the curve then drops and coincides withthe line of normal respiration in air; after a time it graduallydescends below this line of normal respiration. When theapple is replaced in air, the respiration curve fluctuates andultimately coincides with the normal aerobic curve. Theother type shows the same early burst of carbon dioxide fol-lowed by a fall, but the curve remains above the normalaerobic curve. When the apple is replaced in air, there isa depression to below the normal line, followed by a gradualrise leading to coincidence with the curve of respiration in air.

The main conclusion drawn from this comparative studyis that the metabolic factors which determine the progress ofrespiration with time are different for the aerobic and anaerobicconditions. A consideration of these facts has led to certaingeneralizations which will be dealt with later.

Comparison of anaerobic respiration with the alcoholicfermentation of sugar is an obvious pursuit and "the twophenomena show remarkable resemblances. The occurrenceof acetic aldehyde in respiring poplar buds was observed byGodlewski and Polzeniusz.* Neuberg and Gottschalk f ger-minated seeds of pea and of bean, previously sterilized by

* Godlewski and Polzeniusz: " Zeit. physiol, Chem.," 1913, 83, 105.f Neoberg and Gottschalk : " Biochem. Zeit./' 1924, 151, 167 ; 1925,

160, 256.


washing with mercuric chloride, in sterile conditions in asugar solution in the absence of oxygen. In other experi-ments the seeds or seedlings were coarsely grown and suspendedin the nutrient solution, with all precautions against bacterialactivity, in anaerobic conditions. It was found that alcoholwas produced together with some acetaldehyde ; the additionof calcium sulphite led to a considerable increase in the amountof acetaldehyde. Klein and Pirschle * established acetic alde-hyde as an intermediate product in the respiration of cerealsand pulses, and Pirschle f states that larger quantities arepresent in germinating fatty seeds, but here some of the alde-hyde may represent a stage in the conversion of fat intosugar previous to the respiration of the sugar.

Thomas % found that in the respiration of apples in.normalaerobic conditions, neither alcohol nor acetaldehyde accumu-lated, but in the absence of oxygen the respiration is of thezymasic type, and both ethyl alcohol and acetaldehyde areformed and accumulate; even if much oxygen be present inartificial mixtures of oxygen and carbon dioxide, the zymasicform of respiration may occur owing to the increased amountof the latter gas. In a mixture containing 10 to 20 per cent, ofcarbon dioxide, the results are irregular, but when the amountof carbon dioxide exceeds 50 per cent., the zymasic type ofrespiration begins, and in such a mixture the ratio ethyl al-cohol/acetaldehyde = 2. This is called, by Thomas, carbondioxide zymasis. In anaerobic zymasis, i.e. complete absenceof oxygen, the ratio is 50.

Karlsen § studied the problem by ascertaining the effectof ether, benzene and alcohol on the aerobic respiration ofwheat seedlings. He found they yielded results similar tothose obtaining in anaerobic conditions, and concluded thatthe fundamental processes in aerobic and anaerobic respirationare essentially the same.

The amount of alcohol and carbon dioxide produced is avariable quantity depending on the amount of carbohydrate

• Klein and Pirschle : " Biochem. Zeit.," 1926, 168, 340:I Pirschle : id., 1926, 169, 482.X Thomas : " Biochem. Journ./' 1925, 19, 927.§ Karlsen : " Amer. Journ. Bot.," 1925, 12, 619.

VOL. II.—11

id2 RESPIRATION

available for reduction in the plant and on the experimentalconditions employed.* Thus the amount of carbon dioxideevolved from etiolated bean leaves, in an atmosphere freefrom oxygen, is negligible, but if kept with their petiolesimmersed in a solution of sugar for some time previousto their being placed in anaerobic conditions much carbondioxide is produced and their life is prolonged. With regardto alcohol, a similar correlation obtains ; in a specific in-stance etiolated bean leaves in anaerobic conditions gave256-8 mgs. carbon dioxide and 68*3 mgs. of alcohol in thirtyhours, a ratio of IOO : 26-5, whereas leaves previously givensugar yielded in precisely similar conditions 782-4 mgs. of car-bon dioxide and 724-6 mgs. of alcohol, a ratio of HO : 92-6-f

Boysen-Jensen, $ from his study on the relationship of thealcohol produced in anaerobic respiration and the carbondioxide evolved in aerobic respiration, divides the higherplants into two classes in which the ratio anaerobic alcohol/aerobic carbon dioxide is respectively > 0-35 and < 0*35.To the first group belong most of the classical examples ofanaerobic respiration and to the second group those plantswhich show a weak anaerobic respiration, such as potato tubers,leaves of Tropceolum, seedlings of Sinapis alba, and fungi suchas Aspergillus and Penicillium. He selected alcohol for ameasure of anaerobiosis in that it has its origin in monosac-charides, whilst the carbon dioxide produced in the absence ofoxygen may have diverse origins.

In the lower plants this parallelism is not so obvious, thusKostytschev § found that mushrooms containing no sugar ||give origin to much carbon dioxide but no alcohol whengrown in anaerobic conditions, possibly because the alcohol

* Palladin : " Rev. ge*n. Bot./' 1894, 6, 201.t PaUadin and Kostyschev : " Abderhalden's Handbuch," 1910, 3,

479.t Boysen-Jensen : " Kgl. Dansk. Videns. Selskab. Biol.," 1923, 4, 1.§ Kostytschev.: " Ber. deut. bot. Ges.," 1908, 25, 188 ; 26a, 1674 ;

" Zeit. physiol. Chem.," 191 o, 65, 350.|| Glucose has been described as occurring in the mushroom and allied

plants. The amount varies greatly, according to the conditions in whichthe plants are grown, and varies, not only in different batches, but also inindividual plants of one batch. These fungi also contain carbohydratescapable of yielding sugars.


is oxidized almost as soon as it is formed: but however thismay be, more information is necessary before an adequateattempt can be made to correlate the catabolic processes ofthese and like plants possessed of such plastic metabolicmethods.

The work of Blackman and Parija on the respiration ofripening apples has been mentioned on earlier pages. Fromthe results obtained, Blackman * has formulated a workinghypothesis of respiration. The phenomenon is a series ofphases, the ultimate products of one being the reactants ofthe next, but the fate of the reactants of the last phase, beingcontrolled by the supply of oxygen, is diverse.

A.—The first phase is essentially that of hydrolysis, thesplitting of various carbohydrate reserves to form free hexosewhich is not directly respired.

B.—The hexose is activated to a " heterohexose " pos-sessed of a less stable type of internal ring structure,

C.—The heterohexose undergoes disintegration, glycolysis,to various intermediate products, of which methyl glyoxal,lactic acid, pyruvic acid and acetaldehyde are examples,which form the raw material for the last phase D. This gly-colysis proceeds at a greater rate in air than in nitrogen andis still further accelerated by higher oxygen pressures. Thisacceleration is not due to oxidation but to the hastening ofthe rate of production of the substrate for glycolysis.

D.—The fate of the intermediate products of the phase Cis dependent on the absence or presence of oxygen. In an-aerobic conditions alcohol and carbon dioxide are produced ;in aerobic conditions water and carbon dioxide are the finalwaste products, together with an oxidative anabolite.

The early processes are reversible reactions; hexoses bycondensation can give rise to the original carbohydrate reserve,and the heterohexoses can pass back to hexoses. The otherphases are considered to be irreversible.

The oxidative anabolism in the last phase is a deduction toexplain the differences which obtain in aerobic and anaerobic

* Blackman : " Proc. Roy. Soc./' B, 1928, 103, 491.I I *

164 RESPIRATION

conditions. It "will be remembered that in the apple the for-mation of carbon dioxide in an atmosphere of nitrogen isgreater than in air, but in air the only detectable final productsare carbon dioxide and water, no other carbon derivativeaccumulates in the tissues ; it is therefore concluded that someof the intermediate products in the phase C, in the presence ofoxygen, undergo an oxidative anabolisni and are passed backinto the system.

Of this hypothesis the following are significant: (a)Emphasis is laid on the rate of glycolysis, which is regardedas the common measure of respiration in all conditions.(b) Hexoses are not the immediate substrate for glycolysis :they are first converted into the more reactive " hetero hexoses.''(c) That in the final phase there is an oxidative anabolism.

This scheme is an hypothesis based on the observed factsof respiration of isolated structures : it includes certain de-ductions which are yet to be proven, a point fully appreciatedby its authors. Exigences of space forbid a full considerationof the arguments advanced in its support, even if it weredesirable in this general survey ; it may, however, be remarkedthat Blackman. apparently accepts Meyerhof and A. V. Hill'shypothesis that there is a re-oxidation of reduced material.

The view is generally held that respiration is not a singleoperation but two groups of events controlled by enzymes,some closely associated with the protoplasm and others in-dependent of the protoplasm and therefore not concomitantwith life. The first phase is of the nature of fermentationand the last is oxidative- This is illustrated by the followingobservations:—

Palladin and Kostytschev showed that germinated peaswhich had been killed without injury to the enzymes may giveoff more carbon dioxide than during life, and so also may thebulb of an onion, killed by exposure to a temperature of —20° C.on thawing, although in this instance the amount of oxygenabsorbed is less than in the living condition. A. R. C. Haas *found that plants of Laminaria poisoned by various substancessuch as ethyl bromide, acetone, or alcohol first showed a rise

* Haas, A. R. C.: '* Bot. Gaz. / ' 191$, 67,34 7.

M EC I IAN I? M OF R E? PIR AT ION 165

in respiration followed by a smooth decline to zero; at thedeath point the evolution of carbon dioxide was not markedlysmaller, and it may be considerably greater, than the normalrate in living tissue. But this post-mortem respiration isnot shown ia all instances. Palladia observed that the evolu-tion of carbon dioxide from finely ground wheat is less thanfrom the living intact grains.

He also made a comparative study of the effects of variouspoisons on the evolution of carbon dioxide from living anddead tissues : quinine hydrochloride in a -09 per cent, solutiongave a threefold increase in the output of carbon dioxide fromliving stem apices of the broad bean, but was without effecton killed apices ; a dose of I per cent, gave the same increasedyield from the live stems and reduced the evolution of carbondioxide from the dead. Arbutin in a 1 to 2 per cent, solutiondepressed the respiration of wheat seedlings to a greater degreein dead than in live seedlings.

Quastel* remarks on the fact that if a suspension of Bacilluscolt "be shaken up with toluol or with ether, the organism isno longer capable of reproduction ; it is, in ordinary parlance,dead; but it can still activate formic, lactic and suceinieacids and this at rates not markedly divergent from thosedue to the untreated organism.

The parallelism between alcoholic fermentation and anae-robic respiration is so close that the common opinion is that inthe ordinary plant the first phase of respiration is anaerobic.As has been described elsewhere,| the precise sequence ofevents in alcoholic fermentation is a matter of opinion, but ofthe end products, alcohol and carbon dioxide, there is no doubt.The carbon dioxide evolved in respiration has its origin inthis anaerobic phase by the action of carboxylase upon thecarboxyl groups, produced in the disruption of the sugar.If oxygen be withheld, the non-gaseous products of the an-aerobic phase will accumulate and ultimately kill the plantif it be a typical aerobe. The second phase is aerobic and isconcerned with the oxidation of the products of anaerobiosis.

* Quastel: " J . Hgyiene," 1928, 28, 139.•f See Vol. I., p. 489.

166 RESPIRATION

And here opinions differ as to the starting-point of oxidation.In all probability it is not alcohol, but a precursor of alcohol;and in tracing the various stages in the oxidative degradationof these substances, the realms of speculation are entered.Of the suggestions made, the following have the merit of atleast being within the range of possibility :—

The close connection between sugar and organic acids hasbeen remarked upon on an earlier page, and also the formationof aldehyde in fermentation : the oxidation of aldehyde toacetic acid is a familiar activity of bacteria.

The dehydrogenation of two molecules of acetic acid wouldgive rise to succinic acid—

CH, . COOH CHa . COOH-* | + 2H.

CH,.COOH CHa.COOHSuccinic acid

The latter, as shown by Thunberg,* is readily converted bythe enzyme contained in muscle into furnaric acid—

CH,. COOH CH. COOH-+ II + aH.

CHa.COOH CH.COOHFumaric acid

It has further been found by Quastel and Whetham f thatresting Bacillus colt and B. pyocyantus are able to effect thesame conversion of succinic into fumaric acid. These authorsalso found evidence of the conversion of fumaric into malicacid by these bacteria, and Batelli and Stern % have shownthat muscle also contains an enzyme capable of bringing aboutthis change by the hydration of the fumaric acid—

CH . COOH CH, . COOHB -f HOH -+ |CH . COOH CHOH . COOH.

Malic acid

Malic acid, as was shown by Harm and Haarmann,§ yieldson dehydrogenation oxalacetic acid—

CH*. COOH CH* . COOHI -* 1CHOH . COOH CO . COOH

• Thanberg: '* ZentrlbL FhysiaL," 1916, 31, 91.f Quastel aad "Wlietfaam : " Biocfeenu Joura./* 1924, i&, 51:9.t Batelli and Stem: " Q^apL read. Soc. Wol.," 1921, 84, 305.{ Kabn and Hajunmun: " Zmt ^ ^ / ' 1928,87,465 ; 88,91.

MECHANISM OF RESPIRATION' 167

which Is known to be attacked by carboxylase giving rise topyruvic acid—

CH, . COOH CH3: - ^ C O •+• 1

C O . C O O H * ' CO . C O O H .

T h e r e s u l t i n g p y r u v i c a c i d c a n t h e n b e f u r t h e r c o n v e r t e d

i n t o a c e t i c a l d e h y d e w h i c h i s i t s e l f b r o k e n d o w n b y a r e p e t i t i o n

o f t h e c y c l e o f c h a n g e s o u t l i n e d a b o v e .

A l t h o u g h t h e s c h e m e a p p e a r s t o l e a d b a c k t o t h e o r i g i n a l

m a t e r i a l , i t w i l l b e n o t e d t h a t t w o m o l e c u l e s o f a c e t a l d e h y d e

w e r e i n v o l v e d a t t h e b e g i n n i n g , b u t o n l y o n e r e m a i n s a t t h e

e n d o f t h e c y c l e .

I t w i l l b e n o t i c e d t h a t m a n y o f t h e s e o x i d a t i v e p r o c e s s e s

a r e e f f e c t e d b y t h e r e m o v a l o f h y d r o g e n ; b u t s i n c e i n t h e

r e s p i r a t i o n o f t h e g r e e n p l a n t h y d r o g e n , a s s u c h , i s n e v e r s e t

f r e e , t h e h y d r o g e n m u s t i n s o m e w a y b e fixed ; t h e c u r r e n t

v i e w i s t h a t i t i s o x i d i z e d t o w a t e r w i t h t h e i n t e r v e n t i o n o f

a r e s p i r a t o r y p i g m e n t , g l u t a t h i o n e o r s o m e o t h e r m e c h a n i s m .

P a l l a d i n w a s t h e f i r s t t o v i s u a l i z e t h a t a t m o s p h e r i c o x y g e n

w a s c o n c e r n e d o n l y w i t h t h e o x i d a t i o n o f t h e h y d r o g e n o f t h e

r e s p i r a b l e m a t e r i a l t o w a t e r , a v i e w s u p p o r t e d b y T h u n b e r g

w h o r e g a r d s h y d r o g e n a s t h e e s s e n t i a l f u e l o f t h e l i v i n g c e l l .

T h e a b o v e c o n s i d e r a t i o n s i n t h e m a i n a r e c o n c e r n e d w i t h

s u g a r a s t h e r e s p i r a b l e s u b s t a n c e ; b u t , a s h a s a l r e a d y b e e n

m e n t i o n e d , f a t s a n d p r o t e i n s a r e a l s o a v a i l a b l e t o m e e t t h e

e n e r g y r e q u i r e m e n t s o f p l a n t s , c o n c e r n i n g w h i c h t h e r e i s l e s s

p r e c i s e i n f o r m a t i o n . O p a r i n * h a s e x a m i n e d j c h l o r o g e n j | c a c i d ,

w h i c h h e h a s f o u n d t o o c c u r i n o v e r a h u n d r e d

different plants, and finds it to be a substance winch readilyoxidizes in the air, losing 4 atoms of hydrogen and forminga green pigment- The latter caa act as a hydrogen acceptorand so act as an oxidizing agent. The calcium salt of thefully oxidized acid is represented by the formula CaCajH^Oj^-f- 2H2O, whilst the salt of the unoxidized acid has the for-mula CaC^H^Oj© -f 2H a 0. Chiorogertic acid is more par-ticularly active in the oxidation of natural amino acids,

p "Bioc&eaa. Zettsch.," 2921, 124,90. Garter : " Ajinalen d.Cfceoaie," 1908. 359* 217.

168 RESPIRATION

pep tides and peptones giving origin to ammonia, carbon di-oxide, and an aldehyde

K . CH . NH8 . COOH ~f O-> R . CHO -f NH3 -f CO,.

The process is accelerated by the presence of certain reagentssuch as phosphoric acid.

Pirschle * lias shown that in the respiration of manyfat-containing seeds such as Helianthus, Linum, Camiabis,Brassica, Raphanus, and Cncurbita there is produced, as inthe germination of carbohydrate and protein-containing seeds,acetic aldehyde ; it is, however, not certain whether the aceticaldehyde in this instance is a direct product of the respirationof the fat, it may in part be an intermediate product in thetransformation of fat into sugar previous to the respiratory-consumption of the resulting sugar.

The foregoing account is almost entirely confined to theactivities of higher plants. The lower plants, more especiallythe bacteria, obtain their respiratory energy in diverse waysas is indicated by their physiological classification into sul-phur, iron and nitrifying bacteria. For information on this,text-books on bacteriology must be consulted.

• Pirschle: " Biochem. Zeit.," 1926, KS9, 482.

C H A P T E R V I .

GROWTH.

T H E term growth not infrequently is used to imply mere in-crease of the plant or plant member in various directions withlittle or no attempt to correlate or to analyse these and otherrelated expressions of the activity of the organism. Thusincrease in surface is not necessarily growth : a pound pat ofbutter may be spread over a number of slices of bread wherebyits area is increased but not its mass. An etiolated seedlingmay show a much greater length of internode than its fellowof the same age grown in normal conditions: but the com-parison of the dry weights of the two will show no increase inmass in the etiolated example.

From considerations such as these, the conclusion isreached that growth is properly speaking an expression ofthe metabolism of the organism.

Metabolism has two sides, debit and credit: if the ana-bolic processes are more intense than the catabolie, growthwill result ; if the catabolic processes are in the ascendant,decretion results. From this it follows that the sure indexof growth is increase in dry weight, the credit balance of thetwo opposing activities. Thus Boysen-Jensen * found thatunder maximum illumination the carbon assimilation of Sina-pis, a sun plant, was 6 mg. of carbon dioxide per 50 sq. cm.of leaf surface per hour at 20>° C, whilst the respiration at thesame temperature and for the same units of surface and timewas 0 8 mg. of carbon dioxide. This means that for an averageplant of Sitiapis, the amount of dry matter made in one dayin July is 60 rng. whilst the loss of dry matter due to respira-tory processes is 14 mg., leaving a balance of 46 rng.f which

* Baymm-Jmsea: " Bot. Tidsskr./* 1919, 3&> 219.169

170 GROWTH

is equivalent to 16-5 per cent, of the dry weight of the plant.A similar relation is shown by Oxalis, a shade plant, but theamounts are much smaller; 0*8 nig. of carbon dioxide beingassimilated per 50 sq. crn. per hour at 20° C , whilst the lossdue to respiration is 0-15 nig. at the same temperature for thesame units.

The following table gives Boysen-Jensen's results forvarious plants. The figures for respiration are milligrams ofcarbon dioxide given out per 50 sq. cm. of leaf surface perhour at 20° C.? and those for assimilation are milligrams ofcarbon dioxide synthesized by the same area, in the sametime and at the same temperature. All the figures aremaximal values:—

iSambtuus nigra} (Shade leaves)

Shade plants iAjuga fept4ins

^OxaJis acetcsella1 Sin&pis alba

Sun plants J Rumf* ***>*** •Sambucus mgra

( (Sun leaves)

Respiration.O*2O

O-150-100-8

o-3

Assimilation.0*85

O-70o-8o6-04*52*3

In all these plants the carbon assimilation is much greaterthan respiration, and since the amount of synthesized materialis more than eaough to provide for respiration during thehours of darkness, growth takes place. This is illustrated inthe fallowing table which, represents the percentage of in-crease in the dry matter of Sinapis alba.

Peiiod.Dec. 1—20Dec. 22—Jan. 15Jan. 21—Feb. 14Feb. 19—March 14March. 21—April 20April 24—May 22June 5 .

Percentage-Increasein Dry Weight.

105130

7\5»16-70i8-8o

The flowerfag period occurred early in June which resultedin a fall (Fig. 7}. When these figures are plotted, an S-shapedetirve is formed - which, as will be seen later, is typical ofgrowth curves.

In fartlier ilhistratba a saprophyte may be takea: Ter-

RATE OF GROWTH 171

roine and Wurmser * in their work on Aspergillus niger usedthe dry-weight method which they considered in relation tothe amount of organic matter used up in growth ; the ratioof the dry weight of the organism to the organic matter con-sumed, giving the coefficient of utilization. For Aspergillusgrown in a culture medium of salts together with 3 per cent,glucose, the sole source of carbon, and 0*05 per cent, ammoniumsulphate, the only source of nitrogen, the coefficient of utiliza-tion was found to be 0-44. This value is unaltered when

18

16

i »

r 10

Dec. Jan. Feb. Mclv, Apr. MCLV JuneFIG. 7.

g lucose is rep laced b y o ther sugars and w h e n t h e c o n c e n t r a t i o n

of t h e a m m o n i u m s u l p h a t e is m u c h increased or is rep laced

b y guanid ine , urea or nitr ic ac id ; n i tra tes , o n t h e o t h e r

h a n d , br ing a b o u t a r e d u c t i o n in t h e coefficient. F o r t h e

p r o d u c t i o n of I g r a m of m y c e l i u m in ordinary c o n d i t i o n s of

g r o w t h , 2*3 g r a m s of g lucose are u s e d ; 2-2 g r a m s are required

for a c t u a l g r o w t h , t h e rest be ing c o n s u m e d in resp iratory

processes .

T h e rate of g r o w t h of Aspergillus niger is inf luenced b y t h e

h y d r o g e n ion c o n c e n t r a t i o n of t h e cul ture m e d i u m ; a range

•Terroine and Wurmser: " Bull. Soc. Chim. Biol.," 1922, 4, 519.

i;2 GROWTH

in pi I from 77 to 17 is without effect, but at pK 1-2 thegrowth is much slower and the coefficient of utilization fallsto O-2Q. The influence of temperature will be consideredlater.

A disposal to go further may be evinced and to select thenumber or weight of offspring as being the true measure of anaturally growing organism's growth, since the selfish needsof the parent are thereby eliminated. For obvious reasonssuch a measure is impracticable except in special cases wherereproduction takes place with extreme rapidity, as for instancein bacteria, or where crop yield in response to methods ofcultivation is required.

The employment of the dry weight method has a disad-vantage in that it forbids the study of progressive change inone and the same member, since to find the dry weight, theplant or plant member must be killed. For this reason otherindices sometimes must be employed. Thus change in thesize and area of leaves in certain investigations serve as areliable measure, a fact which becomes evident when it isrealized that an increase in the size of a leaf, or of the entirechlorenchyma system of the plant, means an increase in theplant's factory and all that this connotes. Thus Johnston*found that the total dry weight and total leaf-area of thebuckwheat ran on parallel lines during the season Februaryto October, the greatest rapidity of growth occurring in thesummer months.

In further illustration the observations of Briggs, Kidd andWest,"f based on the investigations of Kreusler and otherson the maize, may be considered. Briggs, Kidd and Westanalysed the growth of the maize in terms of dry weight, leafarea and time, and employed the relative growth rate and theleaf area ratio*. The relative growth rate curve is the weeklypercentage increase in dry weight platted against time, andthe leaf area ratio curve is the leaf area in square centimetresper gram of dry weight plotted against time.

The growth rate of the maize varies much in magnitude at

"•Johnston: " Jotas Hopkins Univ. Cure./' 1917, 211.iri^»# Kfcld and West: " Ana. AppL Biol./' 1920, 7,103, 202.

RATE OF GROWTH

different periods of its life. This is expressed in a generalizedform in Fig. 8.

The early seedling stage is shown by the portion ab, andis characterized bv a decretion owing to the young leaves beingin a low phase of carbon assimilation activity, and providingless material than is consumed In respiratory processes, anobservation which confirms the results of Irving and of Briggs.The phase bt corresponds to the morphological developmentof the plant during which the leaf area per unit dry weightincreases to a maximum. Finally the phase cd is the latterpart of the plant's life and includes the formation of the

FIG. 8.—The continuous line represents growth rate and the broken lineleaf area per unit dry weight.

flower and the seed. This portion shows two secondarymaxima at r a n d / which are respectively coincident with theappearance of the male and female inflorescences and each ispreceded by a minimum, g and A, whicli corresponds to theearly stages of flower development at which epoch there is amarked increase in respiration activity.

The incidence of the maxima is dependent on environ-mental conditions operating not at the time but at a previousstage in the life of the plant.

The fact that the curve for leaf area per unit dry weightcorresponds with the growth rate curve, indicates the closephysiological connection betweea the relative growth rate and

I 7 4 GROWTH

carbon assimilating area per unit of dry weight (Fig. 8). Thecorrespondence, however, is not precise ; instead of shoeinga definite type of variation, as does the relative growth rate,the leaf area per unit dry weight curve fluctuates about amean value. These fluctuations are due to the conditioningfactors of growth of which factors temperature is amongst themore significant. The importance of leaf area in the economyof the higher plant is so obvious that no elaboration of thestatement is required. The growth in area of the leaves ofthe cucumber has been closely studied by Gregory * and maybe alluded to at this stage in that it introduces some aspectsof growth which properly belong to a general consideration.But before this is done, the " grand period of growth " mustbe recalled to memory. As is well known, Sachs used thisexpression to designate the period through which the plant, orplant member, exhibits its sequence of growth rates. Thusthe growth rate of the first internode of Pkaseolus multiflorusis at first slow, then quickens to a maximum, after which adecrease in the rate to zero obtains, which point is coincidentwith the attainment of maturity. The sequence is illustratedin Fig. 9 which is based on Sachs's measurements.

The grand period of growth does not include an analysisof the fluctuations in the rate during the periods between themeasurements; thus the growth rate between the third andfourth day is the summation of the growth during that period.Priestley and Evershed "j* in their study on the root growth ofTradescaftiia and tomato, based on the increase in dry and wetweights of the roots produced on cuttings, find the curvesobtained are a sequence of these grand period curves whichare a series of S curves, a type which is very common in graphsillustrating growth phenomena (see Figs. 7 and 10). The timeof transition from one S curve to the next coincides with thetistt ©i appearance of a crop of roots of a subordinate branchorder.

Gregory found that the growth ia length, ia breadth, andin mtm <£ the leaYes qf Cucumis satknis show a grand period

: " Aaa. Bpi/ ' 1921, 55, 93.& E i 6 , 225.

RATE OF GROWTH 175

of growth when grown in natural conditions; but, whencontinuously illuminated by electric light, a method some-times adopted by market gardeners to secure early crops, therate of increase falls from the first measurement of area. Thecurves of increase in linear dimensions and in area can be

14

109

1 5

CO

?

Temp. 12-75°-1375'C

1 2 3 4 5 6 7Time in days

FIG. 9.

8 9 10

represented by the formula of an autocatalytic reaction.*The increase in area of the total leaf surface varies with theseason of the year : in March and June in daylight, the rate ofincrease is proportional to the area existing at that time, thatis, the growth follows a compound interest law ; but duringDecember a detrimental factor intervenes so that the rate of

* This term is explained on p. 178.

176 GROWTH

increase, although still proportional to the extant leaf area,is n o t ma in -

5 4 h , * * • tained but falls

CO12

fiO= 8•£ 6

52504846444240383634323028262422

20. - _ » tion was intensified whilst carbon as-.- similation was depressed.

x . In this consideration mention hasbeen made of the law of compound

; • away in time.

t • W h e n g r o w n, " under artificial light, re-

sults comparable to those* grown in daylight during

December obtain. Thedetrimental factor is hard toformulate: in December the lowlight intensity and the low tem-perature militate against photo -synthetic activity and thus the

amount of available food may be ad-versely affected. When grown underartificial light, the energy value of theilluminant was about equal to wintersunshine, but the temperature of thegreenhouse was that of the summer,conditions which indicate that respira-

„ interest and of the law of auto catalyticreactions.

V. H. Blackman * has drawn at-tention to the fact that in many natural

phenomena the, , , , , , , , , , rate of change

5 7 9 11 13 15 17 19 21 23 25 of some quan-in days tity is propor-

FIG. io.—Growth of the fruit of Cucurbita pefio; . . . observed value,xxx calculated value.

* Blackman : " Aim. Bot.," 1919, 33, 353 ; " New Phytol.," 1920,1% 97. See also Kidd, West and Briggs: id.t 88 ; Schmidt: " New JerseyAgrlc. Exp. Sta./' BulL 404,1924.

COMPOUND INTEREST LAW 177

tional to the quantity itself. This is the law of compoundinterest; money left to accumulate at compound interestincreases to an amount the magnitude of which depends onthe initial capital, the rate of interest and the time themoney is accumulating ; the dry weight of an annual plantdepends upon the dry weight of the food reserves in the seed,the percentage increase in the dry weight over the selectedperiod, and the time during which the plant is increasing inweight. This may be represented by the equation

where Wx is the final weight, Wo the initial weight, r theaverage rate of interest, t time, and e the base of naturallogarithms.*

The rate of interest is obviously of first-rate importanceand if constant the final weight will vary directly as theinitial weight, wherefore a large seed, with more initial capital,will give a much larger plant than a small seed with a rela-tively smaller initial capital.

From considerations such as these Blackman arrives atthe conception that the rate of increase observed in the plantis the index of efficiency of the plant, a conception which givesa useful basis for comparison but which is not a constantsince it is the average of a number of rates which may show-variations through a wide range, for it is affected by the ex-ternal conditions. The rate of increase is highest in the earlystages of growth and falls with the inception of the repro-ductive period.

The laws governing autocatalytic reactions are the logicaloutcome of the laws of monomolecular reactions.")* An auto-

* This equation is perhaps the simplest suggested for a mathematicalexpression of growth ; but simple or complex, the objection may be justlyraised that in view of the impossibility of evaluating many of the factorsgoverning the rate of growth, more particularly environmental and internalfactors, all formulae, even at their best, are mere approximations and there-fore of but little scientific value. However this may be, many authorsgive a mathematical consideration of their measurements, which, withone exception, are excluded from the following pages. In some instances,such expressions have become polemical (see, for example, Briggs: " Ann.Bot./' 1925, 39, 475 ; " Proc. Roy. Soc.," B, 1928, 102, 280; Gregory:" Ann. Bot./' 1926, 40,1 ; 1928, 42, 531).

t Vol. I., p. 469.VOL. II.—12

178 GROWTH

catalytic reaction is one in which one of the products of dis-sociation of the original material acts as a catalyst on thematerial which is obviously decreased in amount as the reactionproceeds. Thus a solution of methyl acetate undergoes auto-dissociation into methyl alcohol and acetic acid ; of theseproducts the acetic acid catalyses the methyl acetate so thatthe rapidity of the reaction is continually accelerated, owingto the diminishing amount of methyl acetate and the increas-ing amount of acetic acid, until the whole of the substrate isdissociated.

From his study of the growth of various organisms, Robert-son * concludes that in any particular growth cycle, either ofan organism or of a member of an organism, the maximumincrease in volume or in weight in a unit of time occurs whenthe total growth due to the cycle is half completed. Such acycle of growth conforms to the formula

where x is the amount of growth in weight or volume whichhas been attained in time t. A is the total amount of growthattained during the cycle, K is a constant and t is the time atwhich growth is half completed. These relations are such aswould be expected to hold good were growth the expressionof an autocatalytic chemical reaction, and the growth ofthe organism should, from the hypothesis, remain constant,having once attained its maximum. But the contrary obtains,in senescence a loss of weight occurs ; this is supposed to bedue to the action of secondary factors which are imposed onthe phenomena of growth itself.

An inspection of Robertson's figures, for example thosegiven for the oat,f or for Cucurbita, shows differences betweenthe observed and calculated growth values of varying magni-tude, and these differences when expressed in percentagessometimes appear too large to support the thesis ; but whenthe observed and the calculated values are expressed in the

• Robertson: " Arch. Entwicklungsmech. Org./' 1908, 25, 581.t Jd.t 26,108.

AUTOCATALYTIC REACTION

form of a curve (Fig. io) their incidence is sufficiently closeto give support to the thesis, especially when allowance ismade for the experimental errors.

To what extent and in what degree growth processes may becorrelated with this law is uncertain. The observations ofGregory show that the curves of increase in the area of theleaf surface of cucumber plants are of the S form and can berepresented by the formula of such a reaction. In the earlierstages of growth this increase also is in accordance with the lawof compound interest. There is thus a choice between thetwo expressions : the compound interest law is a conceptionrather than a physiological constant, but even so Gregoryconsiders it to have the advantage over the law of autocata-lytic reactions in that it is independent of hypothetical mech-anisms of growth. For it is obvious that if growth is anexpression of the activity of some catalytic agent, that agentmust be sought out ; according to Robertson * the lipoidsmay subserve the required function.

Robertson's thesis, in essentials, is supported by the ob-servations of many investigators : Rippel f finds it moresatisfactory than others, and has applied it to his andOskar's J observations on the increase of growth in responseto nitrate manuring. Prescott § is satisfied that the floweringcurve of the cotton plant can be accurately expressed by anequation which is essentially that of an autocatalytic chemicalreaction. Reed and Holland || found that the rate of growthof the sunflower closely approximates the course of such areaction. Reed f also observed that the rate of increasein the height of walnut and pear trees showed growth cyclesthroughout the growing season; in each cycle the growthrate corresponded to an autocatalytic reaction. Similar re-sults have been obtained by Gustafson ** for Cucurbita Pepo

* Robertson : " Arch. Entwicklungsmech. Org./' 1913, 37, 497.f Rippel: " Ber. deut. bot. Ges./' 1919, 37, 169.t Rippel and Oskar : " Biochem. Zeit./* 1925, 155, 133.§ Prescott: " Ann. Bot./' 1921, 36, 121.|| Reed and Holland : " Proc. Nat. Acad. Sci./' 1919, 5> 135-*§ Reed : " Journ. Gen. Physiol./* 1920, 2, 545 ; " Amer. Naturalist/*

1924* 58> 337-** Gustafson: " Plant Physiol./' 1926,1,265; see also id., 1927, 2, 153.

i8o GROWTH

var. condensa, Cucumis Melo, C. Sativus, and Lyopersicumesculentum.

On the other hand, van de Sande-Bakhuyzen * considersthat the formula is but an approximation, and that it is in-correct to regard growth as a simple autocatalytic process,whilst Hooker,f considers that growth is best considered as aconsecutive reversible monomolecular reaction.

Returning to the terms in which increase in growth maybe expressed, allusion has been made to offspring especiallyof unicellular organisms in which the unit grows to a certainsize and then divides. The yeast plant may be selected forillustration, more especially as a consideration of its repro-duction rate will introduce other aspects of the subject ofgrowth.

A young yeast cell on being placed in a suitable mediumincreases to a certain size, the magnitude of which depends tono small extent upon the osmotic strength of the medium, $and then reproduces itself by gemmation. The phenomenonmay be illustrated by the accompanying Fig. II which isbased upon observations made by Slator.§ This figure repre-sents the offspring of a single cell up to and including the fourthgeneration. The cycle was completed in 232 minutes fromthe second generation, the average time for the interval betweenone generation and the next, that is the generation time, beingseventy-six minutes.

Elaborating this general statement, the growth of the yeastexhibits a sequence of phases the conspicuousness and durationof which varies with the conditions. The " seed " on beingsown in the wort may remain inactive for a time ; this is thelag phase, the duration of which depends in the main on theage of the seed, old plants showing a longer quiescent periodthan young plants grown from spores which may show no lagphase. When once growth has started, it is unrestricted andfollows the logarithmic law, for which reason this phase often

•Van de Sande-Bakhuyzen : " Science," 1926, 64, 653.t Hooker : " Proc. Nat. Acad. Sci. Wash.," 1925, 11, 710.% See Drabble, E. and H., and Scott: " Biochem. Journ./' 1907, 2,

221.§ Siator : id., 1918, 12, 248.

GROWTH OF YEAST 181

is termed the logarithmic phase. This is followed by a periodof retardation due to the operation of factors such as the ac-cumulation of carbon dioxide and the lack of oxygen. This

in

n 10520330

4050607080

120

153

180

218

232240

FIG. I I .

is a normal sequence: but departures from the normal mayresult on varying the conditions of growth; thus if theseeding be heavy, but not excessive, the logarithmic phasemay disappear and retardation set in immediately after the

182 GROWTH

lag phase. If the seeding be excessive, the retarding factorsmay prevent any measurable growth.* Priestley and Pearsall flook upon the logarithmic phase as the natural rate of increasewhere increase in mass is an exponential function of time ;the retardation phase, in which the growth rate is directlyproportional to time, is due to adverse operatnig factors,such as the accumulation of end products, which are theoutcome of the earlier unrestricted growth. In addition tothe obvious products, carbon dioxide and alcohol, of theactivity of yeast, oxygen and sugar may also influence itsgrowth.

The influence of oxygen is important, and this notwith-standing the fact that yeast is capable of action in anaerobic,as well as in aerobic conditions. The growth exhibited inthese different circumstances is not the same : when grownanaerobically, yeast cells quickly acquire a static condition ofequilibrium with regard to the medium in which they aresuspended ; J the lack of oxygen, especially prior to the be-ginning of gemmation, arresting the reproduction function.It is the small amount of oxygen initially present in the wortwhich is considered to explain the fact that in fixed condi-tions the maximum cell increase is independent of the num-ber of cells of seed yeast per unit volume of yeast. ThusHorace Brown § found that up to 65 or 70 per cent, of completeaeration, the cell increase is a linear function of the availablefree oxygen at the beginning of growth. In other words,during the period of active reproduction in a suitable mediumin which access to oxygen is limited to that initially presentin solution in the liquid and in conditions of culture whicheliminate the competition factor, the number of yeast cellspresent at any moment is directly proportional to the time.Reproduction can, however, take place to a small extentin anaerobic conditions : Horace Brown found the limit tobe 6*5 cells for each initial cell of seed yeast. This is ex-

• Slator: •' Biochem. Journ./* 1918, 12, 248.f Priestley and Pearsall; " Ann. Bot.," 1922, 36, 239.% Adrian Brown : " Journ. Chem. Soc.," 1905, 87, 1395.§ Horace Brown: " Ann. Bot./' 1914, 28,197.

GROWTH OF YEAST 183

plained by the fact that before reproduction takes place, thecells of the seed yeast absorb and fix oxygen which renderspossible this limited reproduction in anaerobic conditions.This absorption of oxygen, which is a linear function of time,takes place with great rapidity; thus in one instance it wasfound that -3 gram of pressed yeast per 100 c.c. of liquid com-pletely absorbed the oxygen in two and a half hours. HoraceBrown concludes that the power of reproduction is impressedin the cell at the very outset by the absorbed oxygen and that aquantitative relation exists between this absorbed oxygen andthe number of units which the initial yeast cell can finallygemmate. The action of the oxygen is one of induction and,according to Horace Brown, all the known facts can beexplained on the assumption that the available oxygen, isequally divided between the initial cells and the consequentvariation in the oxygen charge which these cells must receivewhen the ratio of the seed yeast to the available oxygenvaries.

The amount of oxygen in aerated wort may be very small,but its effect may be very great: thus 1 c.c. of oxygen inaerated wort brings about a growth sixty times greater thanthe same amount of oxygen in non-aerated wort.* Slatoris impressed by the importance of carbon dioxide as a con-ditioning factor in the growth of yeast, and he considers thatthe influence of this gas is much greater than is generallysupposed, and that some of the observed effects generallyascribed to the direct influence of oxygen may be due to itsindirect action in lessening the supersaturation of the wortwith carbon dioxide. For measurements of the rate of growthin wort and in wort saturated with carbon dioxide show muchretardation, possibly due to the carbon dioxide rather thanto the lack of oxygen. These measurements are confirmedby controlled experiments in which the carbon dioxide wasthe limiting factor. In fact, a correlation can be made outbetween the crop of cells and the concentration of carbondioxide in the medium :—

* Slator : " Journ. Chem. Soc," 1921, H9» 115-

184 G R O W T H

Proportionate Concentration Crop ofof Carbon Dioxide = a. Cells.*

I ' o 8 3*°i -o8 3 ' °1-46 4-11*48 4-52-13 6-12-33 6-5

Cropa

2-82-82-83-12-92-8

W i t h r e g a r d t o t h e i n f l u e n c e o f s u g a r , t h e r e i s , a c c o r d i n g

t o S l a t o r , a p r o p o r t i o n b e t w e e n t h e s i z e o f t h e c r o p a n d t h e

i n i t i a l c o n c e n t r a t i o n o f g l u c o s e u p t o a b o u t 1 p e r c e n t . T h e

- 5 1 0 1-5 2 - 0 2 5

G m s . G l u c o s e p e r 1 0 0 c c .

F I G . 12 .

a c c o m p a n y i n g c u r v e ( F i g . 1 2 ) s h o w s t h e r e t a r d i n g i n f l u e n c e o f

s u g a r i n i n c r e a s i n g c o n c e n t r a t i o n , w h e n s u g a r i s t h e l i m i t i n g

f a c t o r . I t s s l o p e c o r r e s p o n d s t o 3 9 0 0 X I O 1 6 c e l l s p e r g m . ,

a figure i n f a i r a g r e e m e n t w i t h t h e c a l c u l a t e d n u m b e r .

I n o r d i n a r y c o n d i t i o n s o f b r e w i n g t h e a c c u m u l a t i o n o f

a l c o h o l i s n e v e r s o g r e a t a s t o b e a s i g n i f i c a n t f a c t o r i n t h e

g r o w t h of t h e y e a s t ; if, h o w e v e r , a l c o h o l b e p r e s e n t i n e x -

c e s s i v e a m o u n t , i t h a s a l i m i t i n g a c t i o n ; t h u s t h e p r e s e n c e

o f 8 p e r c e n t , o f a l c o h o l i n t h e f e r m e n t i n g l i q u o r m a y i n h i b i t

r e p r o d u c t i o n , e s p e c i a l l y if t h e s u p p l y o f o x y g e n i s l i m i t e d .

• U n i t of c r o p of cel ls ~ 7*65 x i o 6 cel ls p e r c c .

GROWTH OF LEMNA 185

Richards * also has studied the growth of yeast by ascer-taining the increase in the number of cells and also by findingthe volume of the population per unit volume of 1/250 cmm.,the separation of the cells from the nutrient medium beingeffected by the centrifuge with appropriate precautions anda standardized routine. By both methods it was found thatthe growth curve was asymmetrically S-shaped, attaining amaximum yield within IOO hours at a temperature of 300 C.At first the increase in volume is greater than the increasein cell number, which is presumably due to the increase insize of the individual cells before gemmation begins. After15 hours the increase in the cell volume becomes slower untilit equals the increase in cell number at about 90 hours. Thecomplexity of the growth curve is due to a limiting factor,possibly the accumulation of the products of the plant's me-tabolism in a limited environment.

This survey, incomplete though it be, shows the usefulnessof offspring as an index of growth f and emphasizes the im-portance of various conditioning factors in the governance oflife processes. It must, however, be pointed out that thenumber of offspring is not always a true index of growth.This is shown in the accompanying table which relates to the

Solution.

Normal .— Calcium .— Nitrogen— Phosphorus— Potassium

Per cent. Increasein Numbers.

3OO111-25287.52IO-O25O*O

Per cent. Increase or Decrease inDry Weight of Crop.

4- 150- 37-5+ 25+ 25-i-87-5

growth of Lemna in a normal culture solution and in solutionslacking various essential elements.% It will be seen that thepercentage increase in the number of plants is ever greater

* Richards : " Ann. Bot.," 1928, 42, 271.f A little thought will show that measurements of increase in length

and girth of those parts of plants exhibiting merismatic activity are inreality measurements of offspring of the dividing elements.

X Based on. observations made by Messrs. Williams and Cole in thelaboratory of the authors. See also Ashby, Bolas and Henderson : " Ann.Bot.," 1928, 43, 771,

186 GROWTH

than the percentage increase in the dry weight of the offspring.An extreme case is presented when calcium is lacking, foralthough the number of offspring is more than double thenumber of the parent plants, the dry weight of the offspring is37*5 per cent, less than the original dry weight of the parentplants.

The growth rate of a plant varies with the age of the or-ganism and also may show periodic and seasonal variation.

Eucalyptus regnans shows the greatest rate of increase ofarea between the age of forty and fifty years.* The measure-ment of the diameter of the annual rings indicates that thegrowth rate falls off with time. In many examples growth inthickness is periodic through the seasons on account of climaticfactors, thus at Peradeniya Hevea brasiliensis shows no growthin thickness during the dry season January to March. Fromthe end of March to the beginning of October, the wet season,growth is uniform; whilst during the dry season, October toDecember, growth is considerably less and sometimes maycease altogether.-)* In instances such as this it would appearthat climate is all-important, especially as regards the pro-vision of adequate supplies of soil water. Thus the erraticgrowth of Hevea observed during the second dry season maybe pre-determined by the amount of rainfall during the previouswet season.

In more temperate climates, soil temperature is a significantfactor; the observations of McDougall $ show that the rootgrowth of forest trees begins in the early part of the year whenthe soil temperature reaches a degree sufficiently high forabsorption of water to take place, and stops in the autumnwhen the soil becomes too cold. There is no inherent tendencyto periodicity, and when a resting period obtains during thesummer months, its cause may be found in the decreasingwater supply. There is, however, as Petch has shown forHewa, a personal as well as a specific physiology of plants,

* Patton : *' Proa Roy. Soc. Victoria/' 1917, 30, 1.f Pfetek: "Ana. Roy. Bot. Card. Peradeniya," 1916, 6, 77.t McDougall: " Aiaer, Jowra. Bot/* 1916,3, 384.

PREDETERMINATION 187

an almost untrodden field, and some of the results observedmay be ascribed to individual peculiarity rather than to thisor to that factor.

Gregory,* in his study of the effect of climatic conditionson the growth of barley, recognizes eight environmentalfactors : maximum day temperature, average day temperature,minimum night temperature, average night temperature, totalradiation in calories per sq. cm. per week, hours of brightsunshine, evaporating power of the air and the nitrogen factor,which is important in the rate of growth of the leaf.

Allusion has been made to the importance of the previoushistory of the conditions and of the plant in determiningthe activity of the plant at any moment- In the presentconnection, the important and extensive work of Balls on thecotton plant must be mentioned, f From a long series ofobservations, Balls concludes that the different behaviour ofthe plants, as indicated by the crop of cotton, are the inevitableconsequences of the known environmental conditions, pro-vided that proper regard is paid to the distinction in timebetween the incidence of the conditioning factor and its mani-festation in the crop. Thus the daily fluctuations in theflowering curve are predetermined and controlled by theweather conditions which obtained a month before the floweropens. This is the principle of predetermination: its im-portance is obvious ; an accurate knowledge of predeterminingfactors will amongst other things give to certain aspects ofphysiology, ecology, and agriculture a precision which now issometimes lacking.^

THE CONDITIONING FACTORS.

TEMPERATURE.

The factors which condition growth and its rate are pre-cisely those which influence anabolic and catabolic activities,

• Gregory : " Ann. Bot.," 1926, 40, 1.t Balls: " Phil. Trans. Roy. Soc," B, 1917, 208, 157.% For an application of this principle, see Appleman's paper on " Fore-

casting the Date and Duration of the Best Canning Stage for Sweet Corn "(" Univ. Maryland Agric. Exp. Sta.," 1923, Bull, 254).

188 GROWTH

and the effect of any one such factor on growth is the result-ant of its action on the opposing components of growth.Thus temperature accelerates respiration and also car-bon assimilation, but since in a vigorous green plant theproducts of one hour's carbon assimilation is a sufficientprovision for many hours' respiration,* there will be a balanceon the credit side; wherefore an increased temperature willresult in an increased rate of growth, other things being equal.But there is a limit to the height of the temperature ; if acertain degree be exceeded, growth will be retarded, come toa halt, and ultimately a decrement will result. In illustration,it is a common laboratory experience that the increase inlength of the radicle of a seedling is a linear function of timeand that there is a gradual increase in the rate of growth upto about 28° C. Talma f observed the growth of the radicleof Lepidium sativum from o° to 400 C. In conditions ofconstancy of temperature for at least three and a half hours,it was found that measurable growth occurred at 0° C. andthat the greatest rate was at 280 C. Van't Hoff's law isapplicable only for small ranges of temperature a^d, ingeneral terms, the temperature coefficient decreases with arising temperature.

The same general observations have been recorded forlower plants. Thus Fawcett J found that the radial growthrate of cultures of the fungus Pythiacystes atrophthora remainedconstant as long as the environmental conditions did notchange. With increasing temperature a greater rate of growthis exhibited up to a certain degree; when this is passed, therate of growth falls off. Thus—

At io° C, radial growth rate == 2-5 mm. per 24 hours.„ 200 C, „ ,, ,, = 6-o „ „ „„ 280 C, „ „ „ - 7'5 .. .. .,,. 33° C, „ „ „ =2-6 „ „ „

In Aspergillus niger Terroine and Wurmser § found thatthe influence of temperature on the rate of growth varies with

• See Boysen-Jensen: " Bot. Tidsskr.," 1918, 36, 219.t Talma : " KoninkL Akad. Wetensch.," 1916, 24, 1840.X Fawcett: " Johns Hopkins Univ. Circ./' 1917, 193.§ Terroine and Wurmser: " Bull. Soc. Chim. Biol.," 1922, 4, 519.


the stage of development; in the earliest phase, when thespores are germinating, Q10 = 2*8 i when the mycelium isestablished and growing well, Q10 = 1*6.

Balls * measured the growth in the length of the hyphaeof the fungus causing " sore skin " on the cotton plant. Thegrowth rate at different temperatures is what might be ex-pected from -van't Hoff's law; but at higher temperatures,38°C , there is a decrease in growth followed by completecessation probably due to the accumulation within the cell ofcertain products of catabolism; these deleterious substancespresumably are formed at lower temperatures but at a muchslower rate ; they diffuse out into the surrounding medium,especially at higher temperatures, possibly on account ofalterations in the permeability of the protoplasm at thesehigher ternperatures.f In higher plants, the outward dif-fusion of these harmful bodies, provided they be formed,must be slower on account of the more massive nature of thestructures, or they are oxidized within the tissues themselves.

As Balls points out, since the conditions in which thisdecomposition takes place must "be fairly uniform in a higherplant, growth optima are shown which are the expressionsaf the internal struggle between the increasing rapidity ofchemical change with the rise in temperature and the inhibitoryaction of the accumulating products of catabolism.

Brown f ia his study of the growth of various fungi, asindicated by the increasein the diameter of the colonies growingin the culture media and by the estimation of the amount ofrotted portions of fruits artificially infected, paid particularattention to the relation between temperature and carbondioxide concentration, which factors are of great economicsignificance in the storage of fruit. His main conclusion isthat the relative retarding effect of a given concentration ofcarbon dioxide on the growth of fungi is greater at a low thanat a Mgh temperature, which can only partly be explained bythe fact that this gas is more soluble in water at a low than

* Balls : " Ann. Bot.," 1908, 23, 557.t See Eckerson.: " Bot. Gaz.," 19x4, 58, 254.} Brcyvm : *' Ann. Bot.," 1922, 36, 257.

i 9 o GROWTH

at a higher temperature. The following figurrs, illustratingthe phenomenon in a culture of Botrytis, are relative t«» thegrowth in air which is taken a.s ioo:

i5 ' C.3" C

Air.

urn

Jc» {Iff (.1*1!50n

It is hardly necessary to remark that temperature anil con-centration of carbon dioxide are but two *>f the several factorswhich influence the growth of these plants ; in addition, thoseof nutrition, moisture and illumination arc of importance,*

To return to the higher plants, the investigationn *>f hriteh fon the; influence of temperature on the rate of growth <*f I lit*roots of Pimm sativum, show that the relationship ran beexpressed as a uniform curve for the range of tempera tun*— 2° C. to 29° C. and resembles those; of Kuijpcr for respiration,Above 290 C. there is m> much fluctuation that relationshipcannot be expressed in a single curve, wherefore for each highertemperature a different curve must be made to express therate of growth in successive periods of time. Thin in owing tothe operation of one or more of those imperfectly knownfactors termed by F. F. Hiarkman the time factor. For in-stance, at 30* and 35* (!. the rate of growth in the fir Hi Urnminutes is the highest attained, in the first half hour there ba rapid fall followed by a recovery marked by a rbe to 11 secondmaximum, after which there in a gradual fall, At 40' C. thedecrease in growth rate* is uniform and rapid, then? bring norecovery.! As in the cress, observed by Talma, so in the pern ;the coefficient for a rise in the temperature of ten degreesshows a distinct falling off as the temperature rises, and, ac-cording to the observations of Ldtch, it is only b*:twetm ttfand 22° that the coefficient value 1km between 2 and 3, forwhich reason the complete curve m not regarded a* a van'tHoff curve. The extremes of measurable growth wan observedat — 2° and 44*5° C , the highest rate being at 30*3** C Withreference to these observations, I*dich di»tingui»hi*ft four

* See, for example, Robinson ; " Ann, SicriL/' x afi, 40, 343,f Untch : id,t 1916, 30, 25,

S S M Bw. drat, tot* titt.," 191a, 3ff %\ '* Hot* Ztntitbl./'


cardinal points : the minimum temperature, the maximumtemperature, the optimum temperature and the maximum ratetemperature. The minimum temperature for any physio-logical process is the lowest temperature at which the pro-cess takes place ; the maximum temperature is the highesttemperature at which the process takes place; the optimumtemperature is the highest temperature at which there is notime factor operating ; and the maximum rate temperatureis that temperature at which the process attains its highestintensity.

The fluctuations observed at higher temperatures may be dueto several factors ; in such conditions some biochemical re-actions may be intensified but not necessarily at the same rate ;and some may be inhibited in varying degrees. The operationsof enzymes, for example, are not affected in precisely the sameway by the same high degree of temperature. Such a contin-gency may interfere with the supply of soluble food, and, con-currently, the rapid accumulation of the products of respiratoryprocesses, which also are accelerated by an increased tempera-ture, may have a toxic action. In a few words, up to a certaindegree a rise in temperature accelerates physiological actionsand here a van't Hoff curve may be expected : beyondthis degree, co-ordination becomes less and less, owing tometabolic derangement. Priestley and Pearsall,* in theirexamination of Leitch's results, point out that the growthrate of radicles is dependent on the chemical reactions involvedin the merismatic activity of the growing point and on thehydrolysis of the reserve food in the seed and the translo-cation of the products to the growing parts. The situationtherefore is this : the increase in temperature acceleratesboth growth and hydrolysis, but the merismatic tissue quicklyuses up the food immediately available, wherefore a decreasein the rate of growth must ensue for that period of time takenby the food materials in their translocation from the seed tothe apical regions of the root. The arrival of this food ac-counts for the second maximum in Leitch's curves. Finally,

* Priestley and Pearsall: " Ann. Bot./' 1922, 36, 239.

I 9 2 GROWTH

the growth rate is diminished by the dislocation of the meta-bolic processes. If this contention be correct, a close corre-lation between the length of the root and the time of theincidence of the second maximum should obtain.

In further illustration of the interaction of temperaturewith other factors, the work of Bushnell, Tottingham andGregory may be referred to. Bushnell * examined the growthof the potato in controlled conditions at temperatures vary-ing between 20° to 290 C. Both the leaves and the tubersdecreased in size with increasing temperature, no tubers beingformed at 290 C. Respiration in the sub-aerial parts also in-creased with rising temperature, which means a greater con-sumption of carbohydrate and thus a reduction in the amountof material available for translocation which roughly corre-sponded to the reduction in tuber growth in the experimentalconditions employed; for although the carbon dioxide ofrespiration during the day is immediately available for re-synthesis, that emitted during the night is not. It thus ap-pears that the excessive respiration at higher temperatures isan important factor, if not the limiting factor, in the growthof the potato.

Tottingham f studied the effect of temperature and lighton the growth of the wheat plant. When the light intensityis low a lower temperature is favourable to the production ofdry matter, and in these conditions the tissues show a higherpercentage and yield of protein. When the light intensityis high and the temperature low, young plants contain moresucrose and protein and mature plants contain more carbo-hydrate and give a higher yield of grain : at higher tempera-tures the percentage of protein is increased, although the totalamount may be less owing to the increased loss of carbohy-drate due to the more intense respiration.

Gregory,^ in continuance of his study of the energy re-lations of plants, has investigated the effect of temperatureon the increase in the area of the leaf surface of Cucumis sativus.

* BushneU: " Univ. Minn. Agric. Exp. Sta./' Technical Bull. 34, 1925.t Tottingham : " Plant PhysioL," 1926, I, 307.% Gregory: u Ann. Bat," 192a, 42, 469.


He finds that at sub-optimal temperatures, 17-3° to 21-6° C,the relative growth rate of the leaf is controlled by lightintensity not by temperature. At this sub-optimal range,temperature affects leaf growth by its action on the rate ofdevelopment in the apical bud, thus controlling the rate ofunfolding of the first foliage leaf. In other words, the delayin the development of the first foliage leaf is determined bythe temperature coefficient of developmental processes; withhigher temperatures the development of the first foliage leafis progressively delayed owing to the derangement of develop-mental processes leading to a fall in the temperature coefficient.This is illustrated by the following figures :—

At sub-optimal temperatures (17-3° to 21-6° C.)At supra-optimal temperatures (29-1° to 32*4° C.)

This retardation in the apical development at supra-normaltemperatures is partly responsible for the reduction in therelative growth rate of the leaf, which also is retarded by aredistribution of material, more growth being made by thestem. These higher temperatures thus introduce a time-factor.At all temperatures, with the light intensity used, a fall inthe relative growth rate of the leaf occurs. A second time-factor is thus operating due to the low light intensity, for itdisappears on increasing the amount of light. There wouldthus seem to be a minimum light intensity below which normalgrowth cannot proceed, and unless this degree of light intensitybe maintained, a time-factor appears which continually re-duces the relative rate of growth of the leaf surface. Thisdetrimental effect is not due to a derangement of the corre-lation between stem and leaf growth, nor does it appear to bedue to incipient starvation following the low rate of carbonassimilation. The detrimental effect of a low light intensitypossibly indicates-an effect on the root system, for, in the barley,there is a close relation between leaf growth and root growthwhich, possibly, is reciprocal, the leaf supplying carbohydrates,which are of great importance in determining root growth, andthe roots supplying the nitrogen requisite for the growth and

VOL. 11.—13

<* * • * i* 1* ', r* !„ « ! .iri5 1 \dra!e loads to, « v+* r <* : t *- * a tall 5 21 iK relative gr*»\vth

\ t it ». ' ! ! ** >^ir^ t<» tin n. i> a-nii^ di>propor-

kr • * «. M / • * . ».ji: Ivuth ! l" *e*»f Priestley *J f * ' ». „, * I 4\ IT*» *J ' >n» rptn n nf a growth

r . 5 * ' ft • i r* a ti n, amk j itidi nt of carb«">n

if * i *.'^ • " a )*fr f n su- > u b t

LIGHT,

The mliutTuv of ligfit. mi gn.>wth is a subject of consider-able magnitude, optTiaHy wlitii the term growth is used inits general sense : the directive action of light in tropistieand kindrtd phenomena, its influence in the determinationof growth form and the lacks of a flora are aspects of thesubject outside the scope of the present consideration.

The action of light on growth is both direct and indirect,and its action i*. rn^t marked in those members which uselight as a soim i r-f vruTgy, Thus f*»r the ordinary green plant,increase without h^ht is an nnpossibrlity since light is the<M»uree >-«f energy for the making of food ; in non-green mem-bers of plant*, m total parasites, and saprophytes, on thenthtr hand, light, for obvious reasons, is not a factor of con-sequence, It is a laboratory commonplace to find that forthe subjects generally used for experimental purposes, lightinfluences growth, as indicated by its external expression ofincrease in length and area, in various ways. Thus internodesin darkness attain a ranch greater length than in light; leavesmay develop hardly at all in darkness, as in the pea, whilst inother plants, the wheat for example, the leaves attain a sizemore or less equal to those grown in light. This difference inbehaviour is possibly due to the amount of carbohydrate, rela-tively larfe in the wheat and relatively small in the pea, avail-able (of structural purposes.f To take a specific instance,

** Mm y 5 4 ?f thm wttri #1 PtkHAtof f' M#w Pfcyt.,'* 1 25, 24* 271} indicates that

v*ry ftpecial pwaatx*i» most be taken to txcted* liglit and thus to me thet*w tt*t>lAt#a ccHaditHTo. Mm will be mm Uter, a v*ty %m&l exposure toitCht wmf Mm * mm^h^mk t*Kt.

INFLUENCE OF LIGHT 195

Popp * grew soy beans in light of intensities varying from 4285to 26 foot candles ; measurements of stem lengths showed thatthe lower the light intensity the faster was the rate of elonga-tion of the internodes during the initial period of growth.The greatest general height was attained by plants grown in alight intensity of about 560 f.c. and the lowest in an averageillumination of 26 f.c. The thickness of the stem was directlyproportional to the light intensity, being greatest in an illu-mination of 4285 f.c. and least in an illumination of 26 f.c.

In general terms the plants grown in the strongest lightwere the most vigorous, showing the best vegetative develop-ment and producing the best fruit. A decrease in vigouroccurred with decreasing light, and plants grown in light of26 f.c. were completely etiolated and died in the course of amonth. In all illuminations except the lowest, the lengthgrowth curves followed the general course of a monomolecularautocatalytic reaction ; the first phase, associated with thebeginning and development of independence of the seedling,is effected by the photosynthetic ascendancy, whilst the fallingoff in the last phase is associated with the reproductive pro-cesses. In passing, attention may be recalled to the fact thatthe humidity of the atmosphere is an important factor in theconfiguration of a plant, wherefore in experiments on livingplants in closed chambers, allowance must be made for thehumidity conditions.

It is commonly accepted that of the different qualitiesof light, growth is promoted by the blue and violet rays,whilst those less refrangible retard. Here again care must betaken to ensure a just comparison : to judge the effect of, say,blue light and red light, the intensities of each must be thesame or, at any rate, known ; further, in such an experimentdue allowance for the different heating effects of light ofdifferent wave-lengths must be made, and in some cases, ifnot in all, the internal temperature must be observed ratherthan the temperature of the surrounding medium.

The problem presents three aspects: intensity, duration

* Popp : " Bot. Gaz./' 1926, 82,154.T<7 *

'H

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-* < i!» i? flit" timr within-ij iLa flu incidt;ner of.',*!'•-/\ i M'l.lHlS. Hit'i , «M 1 .»- ihf nittnsity

.* j Iant growth, Garner• tnat ios^ vi\ the tobacco,Lint *4 vc^ftativt* growthq^'Mirr to davlight, shortpLint* exhibiting a slower

^ al-o I'm! tTat the duration of light isin instituting the reproductive phase ;mis of exposure, bitnnials may be madehi^ti*nes m a

4\ hr « oiu* rtt1

- ', L* an i)!u* *\i\s 1 .it mil

few months, and, on the\\ into biennials. To take"v.vi ited tor twelve hoursa a u\r hour illumination

* kir<;^ it^an m 27 diiV^, a variety of tobaccouf jrM-d !M tht1 >anir periods of daily illumination

alter the fapn* of 1 2 tu IC>2 days and 55 to 61 days*L\ Urn** it i« <evn that shortened periods of dailyrr*ult5 in earlier flowering in these plants. But this

H n»it m^rssanlv SM for dll plant*: Lespedesa, a leguminousf Lint utri'ul a fodder, does not thrive with shortened periodsof light; red clo\rf» many grassrs and the radish when given&hurte»rd days «>huw A retardation in flowering, and thegroundsel, foxglove and tomato are nit>re ur less indifferentto rithtr l'*ng vr to $Lorf da>s,*

Results iontrary to some of the*>e are recorded; thusAdams | found that mu&tard, soy, tlax? tomato, sunflower

. bot €m,t" 1918, » « , "' Hot. Zentrlbl.," 1920,

; *' Joum Afik. RM 9 *553; ,, A4, i4# Qmmm* Bacoe aud Allan!: " Joam. Agric. Res./' 1924,l

Hi IJ». Tiadoa-: "Ana,

PHOTOPERIODISM 197

and other plants showed a greater average weight, a greateraverage height, and earlier flowering when exposed to longperiods of illumination. In fact, the phenomenon of photo-periodism * is bound up with and is influenced by other factors,especially nutrition, the carbohydrate/protein ratio, tempera-ture and humidity.f

The photoperiodicity of plants taken in conjunction withtemperature and humidity is clearly of importance in relationto the geographical distribution of plants, and a knowledgeof these factors is of economic significance in that it will in-dicate the varieties of crop plants suited for cultivation indifferent latitudes.

The results obtained by Blaauw J and of Vogt § fromtheir studies on the growth of the sporangiophore of Phy-comyces and on the coleoptile of Avena respectively areimportant. According to Blaauw, the effect of light ongrowth is an acceleration followed by a retardation to arate lesser than the normal, followed by a gradual increaseto the normal rate. The time of incidence of the initial ac-celeration varies with the intensity of the illumination; thuson exposure to a light intensity of one unit, the accelerationbegins in about eight minutes, but in an intensity of fifteenunits, the acceleration begins in about three and a half minutes.The amount and duration of the reactions vary with thedegree of illumination: for the lower light intensities the totalacceleration of growth exceeds the total retardation ; and forthe higher light intensities the total retardation exceeds thetotal acceleration. For the former, Blaauw finds that theincreased growth is proportional to the cube root of the amountof light. These results are extended by Vogt, who not only

* In passing, it may be mentioned that this duration of illumination isreflected in the reaction of the plant j uices, thus a short-day plant in responseto lengthened periods of illumination, which produce excessive vegetativegrowth, shows an increase in acidity; on the other hand, a short-day plantinduced to early flowering by shortened times of illumination has an aciditymuch below that of the vegetative plant; the same applies to long-dayplants.

t See Gilbert: " Ann. Bot.," 1926, 40, 315.X Blaauw : " Zeitsch. Bot.," T914, 6, 641.§ Vogt: id., 1915, j , 193.

111 I T* tar * iti* n in t}^ ^ri»vf! *7*i hut aJ- » *i * *»twderabR initial

t> •* | ^r»jr\ txp»*Mirr t« a Mjt-^ '] i( r^ i> * r^t a tap! ! ilr• s\ j«] a < H* r 4M M whit h is

r i~ \ ^ *\\ r } r

^ ' ,* :i *i p/ .* i \il«!t h i v t * iK t!if/ *" t!t t tal tilt't t mav be a fon»

* * * *' rat J1.*1 int1* .in\«>unt »>f growth. F« r a' r \\t' :;.'* ]t ilu't »»f tlu* hîit inti'nîtv

jv. , .f »* -, ,* ^ i • » t*tnt iing»rv* aNi fuimlthat in* ,• ' » ^ L* *i> i}r a \ i u ^ leaf area nf the rucumhtr

' t * \ ti r | r«uluct of thv infinity and the durationt ft " </t n l ' i ! ' . n

If- ^'iîal d< *Tt a<r in the growth rate is considered bv\ *v* t i ** !>4< IM t! t- * ti*m of light *il* ne, not to the combined**•'* t f 4i*'fpa!'n^ li^t't and ddrknes* nor to increased trans-

r • . , ^ i->rwi' i^ f Vi%ft illn-fraft t'u j ht totonic' . * ^ !

t% *'• i t| i.ptik' if the nat was alternately, \ s I £<v ?i^ht »'f tht -anic inten>ity, and darkened for

7 ?» i- f f rteen thirt\ and îxtv minutes, lesser growth^* - * . +.I i i<H in *he tw > latter periods of illumination. Int\ > «H "^rur ^ tht greater growth in darkness is considered• * *•*! it f t *he stimulation of the previous exjH>sures» to light.1 * t -l..%t i gr<»%th in periods of illumination is merely a part ofth* iHftipfcfe nar tun, an acctkration being followed by adepression.

Mmtion ha> beui made of the morphological appearanceoi vrdhnt;^ «-u< h a tin* pea, when grown in the dark. Themrnrk «»f Trumpt' + dnd Priestley % shows that if such etiolatedpUnU are exposed to light for a short period daily, § normal

f MAna. Bat.," 4 ^ 97t Tamp! : *' Bntv Archiv./' 1 24, $, 381, 410.J Ptfettfof • '* Ntw I%yt / ' 1925, a4, 271 ; 1926, as*. 213. See alsok M E « ^ : #gf., 1933 23, 30.

| Tli® penod vamt «awrdi^ to tt» seM tivity of t&e plant to light;t k p#i iiKi fewi^ bc in Ftwtky kmnd a r«s|x»»e after a period of

t

QUALITY OF RAD|A;f ION 199

leaves will be developed, although t h \ p^rk^i of exposure ismuch too short for the development oF*^jfior9^i^llr.a,ndJ:he-internodes will become shorter. The quahfci|y < &£ Jjgftt: >equired is very small and the change effected is independentof the products of photosynthesis. Further, if the plant beexposed to light after growth has been stopped by low tem-perature or by the action of anaesthetics, the morphologicalchange still takes place when growth is resumed, which indi-cates that the action is not exerted on the " living metabolicmachinery." Priestley considers that the response is due toa photocatajytic action on some metabolic product, a viewwhich is supported by Gregory.*

This work introduces questions relating to the irritabilityof the plant in response to the action of light, on which thereis a voluminous literature, a problem which is outside ourpresent survey.f

From these investigations it is clear that the reaction of theplant to light is very complex ; that the total effect observedis the balance of the measures of the accelerating and depres-sing influences of light; that the magnitude of the effectdepends upon the intensity and the duration of light.; andthe possibility of a photocatalytic reaction.

The above observations relate to ordinary white light;the quality of radiation affects the growth of plants accordingto the wave-length and to the physiological peculiarity of thespecies.

Schanz, J in continuance of his earlier work,§ grew Begonia,Cucumis, Lobelia, Petunia, and other plants in frames arrangedin a series of eight; by means of glass of different opacities,all but the first, which was uncovered, were illuminated bylight from which certain rays had been abstracted :—

1. Unaltered daylight. 5. Rays longer than 560 /x/x.2. Rays longer than 320 fxfx. 6. Yellow light.3. Rays longer than 380 /JL/X. 7. Green light.4. Rays longer than 420 fxfx. 8. Blue-violet light.

* Gregory : " Ann. Bot.," 1928, 42, 469.t See Priestley : " New Phyt./* 1926, 25, 227, and the literature there

cited. t Schanz : " Ber. deut. bot. Ges.," 1919, 37, 43°,§ Id., 1918, 36, 619..

**> GROWTH

; r i f |«**- r" **1 r.t ! *!j. ut 1 a regular inrrra«;r in Itnqlh

V u »,4\;,.» • illi'ti;**, h>»w AtT, m ^ not tht same; thek«rtr*.i! i" ! *: i ! it • I'Vr'I'jvi bvfttr in tb« Mue-violrtt« k« u n _» . ^ ! 1 **\r .-i tl ' i r* *n flan m th-/ vrll^wI" . * i r * i' 1 N-t+ r.4\-, * -j . urilH thf ultra-

4 * - ' ^ , i • ^ M i! r **x ^r->uth up t > a rtTtain;• ./ -A * ' -M\r4 *V it tarVer ilH^t-irt^ was pro-

!», • f \ % i* in. i* r ; m«1 4 »-f the serifs,^. - *i\s V \\ * ,;T ^ fLtTit™ in precisely the same con-

*- tt i. *. • \ ; f f, / ijiiahtv it I IT illumination. In light from*••' K ,* " r *\s «•! - ft* r than *>2>) pp had been abstracted, thej ! *rt^ 4*, . *t;p.irMl mifti those grown in normal conditions,%*n.u*.! thntnrr s t tm\ fewer branches, curled leaves, but with*> ««»il ri< vt'iopfumt uf rhlorophyll, a reduction in the amountui 4n!l t>r\afi» les* diMcrrntiati«»n in the tissue of the leaf and%t€m, U'h* i* njpact .and thinner walled cells, and a reductionin me fxant^f ttûf Flierc â^ also a delay in flowering;thf rl-m-trv u».T t^\itT the fruit weak and the seed poor.Ih it n^s a,\ i a i<vrea.v in the fresh and dry weight, a de-1, "irv\ \ ;n i f/a! • arbuJivdrate and an increase in total nitrogen.It *mi!d. thfref^re, appear that the blue-violet part of thetpeîruin u requisite for normal development but not theultra M»Ut fi»r %hen these rays were excluded the plantgrowth ma«i nnrmal.

In addition to the above-mentioned observations, theeffect 14 ultra violet radiation on the growth of the plant hasbttn tht subject of many investigations. In general, thert!ret nf these rays i* to promote a dwarf habit which isaccompanied h\ various structural changes. Since plantsflourish in the absenn of ultra-violet light, the matter is asubject of special rather than of general interest, for which

it it not necessary to pursue it further on the present

01 «®ergy other than light, field experiments with wheat

f*i tm, mmm md W«tbnx>k ; •• Mt. 3mm. Exp. BW.," 1927,X mA mi mmmm mem quoted. Also Sidson and ITiilippov:

ELECTRO CULTURE 201

and other crops show the beneficial action of overhead electricdischarge.* In the year 1915 the increase in grain and instraw was 30 and 50 per cent, respectively greater than thecontrol, and in 1916 the corresponding figures were 49 and 88per cent. The effect of the discharge also showed itself inthe year subsequent to its application ; thus in the clover andgrass crop there was a marked increase in 1916, the yearfollowing the treatment. Continued observation confirms thisearlier work: of eighteen field experiments f with variouscrops, fourteen gave increased yields ; of these three onlygave increases of less than 10 per cent, and the rest gaveincreases of over 30 per cent., sometimes of over 50 per cent.Four experimental plots showed a decreased yield of less than10 per cent., compared with the controls.

Like results were obtained with pot cultures,]: thus thepercentage increase in the dry weight of maize grown underglass was 27 ± 5-8, whilst barley under the same conditionsgave a percentage increase of 18 ± 2-4.

The effect of these minute electric currents is not alwaysthe same ; in one experiment with barley the increase in theyield of grain was greater than the increase in the total dryweight. This was due not to an increase in the number ofears but to an increase in the percentage of fertile flowers perear.§ The effect of electro-culture is stimulative, for theenergy supplied is out of all proportion to the energy value ofthe additional plant material produced ; to take one example,the additional energy supplied by the electric discharge to aculture of maize at most was 15 calories; the calorific value ofthe extra material produced, calculated on dry weight, wasof the order of 9000 calories. The results obtained from fieldexperiments, pot cultures and laboratory experiment,|| leaveno doubt about the beneficial action of electric discharge;but the reasons for this are still unknown.

* Blackmail and Jdrgensen : " Journ. Board Agric.," 1916, 23, 671 ;1917, *4> 45*

f Blackmail: " Journ. Agric. Sci.," 1924, 14, 240.% Blackmail and Legg: id., 1924, 14, 268.§ Private communication from the authors.|| Blackmail, Legg and Gregory: " Proc. Roy. Soc./' B, 1923, 95,

214. Gregory and Batten*: id., 1926, 99, 122.

j ' ] * i* i* o* . •* - r i *i iti »n>,• * ! » M( ,] f j • T 'l •:,. | lar**- hahiMl,

• ; i' ^ i*- i il ! radi » a TIN itv,*

W.VTLR

-1 .A- i} it growth i** uilv possible' j / * . «},, ^ it!]-, are in a turbid ronditinn,

•A * * • * , ^. .? r . vviiitli pr»»m»'tt% tin condition will^ * * v j? w*» hit n. t nett warily reproduction

1 ,*• .f i ^?ti" >- t w!1*)] aiiu-i'»n i *nade are humidity,1 ^ -<*•*• ' u i* r, al>-'tpti *n cf water ami transpiration.

\ ; 14 • i 1 i"r -| i'«re pr^tr* te^ vtt;itAti\e growth in that it

tran | irs*u>n he laÛ . the absorption «»f water and its con-\^\4ii<f i*» t! < frarî'Jin^ Mirt.i'»^ i in part a question of.*».,,< i»'.I t ' s ,-, '..''tr 1 u; with ] f r M* abihtv. In lllustra-

n * * ?M- . ' ^ *k4|j <* *. h\ rtar-.tll anJ Hanby J may be1 ' ti 1 t'.ir. 1 l]a: in r . , K l ^rowtli took place

** ' V > . ?«» ! ,4 \,trutv »<f plants* wtre grown in a culture--1 'ti >n 'in i-r SH r.a-ed f rewm\ 15 to 25 cm. of mercury ;ti r,tr^fsuiî, Hmail nâti \e j reûre* are sufficient to reducet r • * n - ;> ^T.^th, The available evidence indicates thatt1 t p >> \ * pr> s-urt\ uhirh obviously kads to a greater tur-1 litv pr« Tr. tis tS< rat< of cell division and the formation of

A lar fer number of veins. Incidentally it may be mentioned,for < au*al morphology does not form part of the present con-^deration, that after treatment with negative or reducedprevMjn *, pahuu Ka\e> become smaller, more dissected, andhave rediiiH-i! 1M-.I1 l^bts ^hen cunipared with similar leavesaftt i trrj*tnunt îth a p*îtive prtisure.

Although bK^phvMcal problems are outside the presentprovince, it may be remarked that any factors which inter-

$m )®kmm : " lot, Cm ." 1926, Sa, $y$ ; i# Amor. Jmira. Bot./*ift H V&» m*&imm$m< Bljuiaw and Haitsoina : " Koa. Akad.

msterdam/' if jS# **, 1.f hm MmMmm " Ann Bot., * 19*0 34* 299.


rupt the normal osmotic adjustments of a plant must adverselyaffect its vegetative growth. Thus Dufre^noy has pointed out *that growth is a function of at least six factors and a variationin any one of these will influence the velocity of growth. Heconsiders that growth rate depends upon the balance betweenthe sum of imbibition, the osmotic pressure of colloids and ofthe salts of the cell sap, and the sum of the tension of mem-branes, the osmotic pressure of the salts of the surroundingmedium and the mechanical resistance of the medium.

It is obvious that water content and sap concentration areinterlocked factors. Reed f investigated the growth of shootsof the apricot, orange and walnut throughout the growingseason and at the same time followed the concentration of theexpressed sap. In general terms, he found that rapid growthwas associated with a low concentration of sap, higher con-centrations are associated with the formation of fruit buds aswell as with slow vegetative growth. In fact it is a matter ofalmost common observation that the vegetative and repro-ductive phases of a plant are antagonistic, for which reasonconditions which promote the one will degrade the other. t

NUTRITION.

The importance of food is so obvious a factor governinggrowth that its consideration would appear to be unnecessary:but the significance of certain raw materials, more especiallyinorganic substances, is so marked as to warrant some mention.

The importance of various salts, especially phosphate andnitrate in combination with such essential bases as potassium,calcium and magnesium, on the growth of plants is a common-place of the physiological laboratory and is easily demon-strated by the use of sand and water cultures in controlledconditions. By using different combinations of salts in suchexperiments, the effect of single nutrient ions on plant yieldcan be ascertained, a subject of some complexity.§

* Dufr6noy : " Rev. gen. Sci.," 1918, 29, 323.t Reed: " Journ. Agric. Res./' 1921, 21, 81.} See Klebs : " Flora," 1918, 11, 128.§ See Johnston : " Univ. Maryland Agric, Exp. Sta./' 1924. Gregory:

" Proc. Roy. Soc./' B, 1928, 102, 311.

2*>4 GROWTH

In addition to the normal ingredients of the usual culturesolutions, traces of other substances may be requisite for thenormal development of many plants. The amount of thesesubstances necessary for the exercise of their beneficent actiont% m small, that the chemicals used in culture experimentsmust be of the highest order of purity before the effect of theirabsence can be ascertained.

The wnrk of Brenchley, Thornton and Warington * hasshown the importance of boron for the well-being of manyplants : for Glycine hispida, Phaseolus multiflorus, Trifoliumspp. and Cucuntis melo% boron is a necessity ; without it theshoot apex dies, the root system is ill developed and flowersAW not formed. In other plants, Iberis umbellaia, Pisutn,and various cereals, boron has a beneficial effect but is notrequisite for healthy growth.

The effect of the absence of boron on the structure of thestem and root of Viciafaba'f may be summarized : the apicalmeristems die, the cells of the cambium hypertrophy anddegenerate, or disintegration takes place without previousenlargement; the phloem and ground parenchyma frequentlydisintegrate; the xylem is poorly developed and, in extremecases, breaks down.

The quantity of boron required for normal developmentis very small, I part of boric acid in 2,500,000 of water wasfound enough for the normal growth of the broad bean ; butthe concentration of the boric acid does not much matter, pro-vided that an adequate total amount is supplied over a givenperiod; this quantity, however, must not be excessive. Thephysiological role of boron is not yet decided ; it is not ableto replace any one of the essential elements of nutrition, butit would appear to be associated in some way with calcium.From the facts that a constant supply of boron is necessaryand that in the plant it becomes fixed, it is concluded that itsaction is not that of an ordinary catalyst.

• BraiebJey and Tfaoratoa: " Proc. Roy. Sac./' B, 1925, 98, 373.kk & W i t a " AJ*H. BO*./' 1927, 41, 167. Warington:

m mm 4» 7f Wafwftcwi; •• Ana, Bofc./' 19*,

EFFECT OF BORON 205

Similarly Johnston and Dore * found that for tomatoplants grown in water culture, normal growth is impossibleif boron, in the form of boric acid, is absent. In boron-starved plants there is twice as much sugar in the leaves ascompared with plants grown in a boron-containing medium,the breakdown in translocation being due presumably to thedisintegration of the conducting tissues.

McHargue f found comparable effects in Glycine hispida,the soy bean, when manganese was withheld from the culturemedium. But Brenchley and Warington found that man-ganese was entirely without effect on this plant when deprivedof boron; it, however, had a stimulating action on thoseplants supplied with boron. There may be some interrelationbetween boron and manganese such as occurs between othernutrient elements.J

Sommer and Lipman § find that boron is essential for thegrowth of the Windsor bean, buckwheat, flax, mustard and otherplants, and zinc is indispensable for the dwarf sunflower andbarley.

In view of the observation || that the administration ofpure borax has harmful effects varying with the plant, thepotato for example being more resistant than maize and bean,it would appear that the form in which the boron is adminis-tered may be of importance.

Mention has above been made of manganese; this metalhas a wide occurrence in the plant and is possibly of universaldistribution; f it accumulates in those parts where metabolismis most active and in those regions which are known to berich in vitamins such as the pericarp of cereal fruits.*1*1 Itmay possibly play the part of a catalyst, an idea which issupported by* its association with certain oxidases.ff The

* Johnston and Dore: " Science/' 1928, 67, 324.t McHargue: " Journ. Agric. Res.," 1923, 37> 629.} See, for example, Brenchley, Maskell and Warington: " Ann.

Appl. Bid.," 1927/ I4> 45-§ Sommer and Lipman : " Plant Physiol.," 1926, I, 231.|| Skinner, Brown and Reed: " U.S. Dept. Agric," Bull. 1126, 1923.If Bertrand and Rosenblatt: " Compt. rend.," 1921, 173, 333 ; " Ann.

Inst. Pasteur/' 1921, 35, 815.•• McHargue: " Journ. Agric. Res./' 1924, 27, 417.t t See Vol.. I., p. 502.

r t* GROWTH

& ,' * i - i? ^ ii i sr rnjurt.i * r ^ ^ d growth is extremely- t< A t - p i d » i.*r ir, i . lit 'mng i ut 25-5 parts of man-

tiT, . >l ] t#i - p r ?", ;it n a*3<> 1 t» tlie M»I1 bring sufficient

* - 1 r •,! 1 •> it MI > ^\d\.\i 41 Atii anil truit in the tomato.**M s, ,T - / 11 $ * ki!» i mi an important in the

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M i n e r a h w i t h o u t } X 3 t & » f i - + • j t m m o n i u m s a l t s

I n t h i s c o n n e c t i o n t h e r e l a t i v e a b u n d a n c e o f c o l l o i d s i n t h e

f o i l w o u l d a p p e a r t o b e o f i m p o r t a n c e ; t h u s J e n n i n g s § finds

t h a t c o l l o i d s a d d e d t o t h e c u l t u r e m e d i u m p r o m o t e d o r d e -

p r e s s e d t h e g r o w t h o f w h e a t s e e d l i n g s a c c o r d i n g t o t h e s p e c i f i c

c o l l o i d e m p l o y e d a n d t h e c o n c e n t r a t i o n o f t h e c u l t u r e m e d i u m .

A g a r t f o r i n s t a n c e , i n t h e p r e s e n c e o f l o w c o n c e n t r a t i o n s o f

n u t r i e n t s a l t s i n c r e a s e s g r o w t h , b u t i n h i g h c o n c e n t r a t i o n s

a d e p r e s s i o n o b t a i n s . O f i n o r g a n i c c o l l o i d s , f e r r i c a n d a l u -

m i n i u m h y d r o x i d e s d e p r e s s , w h i l s t c o l l o i d a l s i l i c a i n c r e a s e s

g r o w t h , d o u b t l e s s b e c a u s e s i l i c a i s m u c h a b s o r b e d b y t h e

c r t t a l g t m m a m i p o s s i b l y i n t h i s f o r m . I n v i e w o f t h e

a m ! D u m a : M J o u r s . I n d . E n g > C f a e m . . " 1 9 2 7 , 1 9 , 4 0 0 .

f X f t i f W W J t * m & € # r i f t » t M : " C o m p t . r e n d . . " 1 9 2 2 , 1 7 4 , 1 2 6 9 .

t F a r t h e s e w e m m i n d e b t e d t o D r . W . R r e n c h l e y .

C/N RATIO 207

adsorptive properties of colloids, the depressing action ofsome examples may be due to their adsorbing mineral saltsand thus rendering them unavailable for the use of the plant.

Further, in considering the action of inorganic compoundson growth, certain important aspects are to be remembered :the plant has a specific and a varietal physiology; a good cropof nettles indicates a high nitrogen content in the soil andvarieties of barley differently respond to various types ofmanuring ; * the inter-relationship of the various compoundsconcerned, carbohydrate and nitrogen for instance ; and thefact that conditions which favour vegetative growth are notnecessarily those for reproductive activity. An abundanceof inorganic salts in the soil promotes vegetative activity,whilst a relatively small salt supply induces reproduction;but this only results provided that other conditions aresatisfactory. Thus increase in growth is impossible for thegreen plant if carbon assimilation be of a low order of in-tensity, since carbohydrate is required for many purposes,structural, respirative, and as raw material for the elaborationof other compounds such as proteins. And for this last purposenitrogen also is necessary ; wherefore intense carbon assimi-lation in the absence of nitrogen-containing substances cannotlead to a growth commensurable with the intensity of carbo-hydrate formation. In fact, there is between nitrogen andcarbohydrate a correlation, and growth is affected accordingto their ratio. Thus if carbon assimilation be increased bygrowing plants in an atmosphere enriched by the additionof carbon dioxide, whilst the nitrogen-containing salts of thesoil are not increased, the ratio C/N is high and the reproductivephase is induced ; if, on the other hand, the ratio C/N is low,the vegetative activity is intensified.f These results may,however, not occur if the ratio mentioned runs to extremes.Kraus and Kraybill J in their experimental work on the tomatofound that a very high C/N ratio results in but little vegetativegrowth and but poor reproduction ; a medium ratio gives

• Gregory and Crowther : " Ann. Bot./' 1928, 42, 757.f Fischer : " Gartenflora," 1916, 65, 232.J Kraus and Kraybill: " Oregon Agric. Exp. Sta./' 1918, Bull. 149.

2O8 GROWTH

05 >»!»•? iiv \rgetatiw ^rnuth and ôod reproduction; and whentl t ( N fîbt H \ t rv l««w, a vignnni:* vegetative growth andp»*.r f? f'f^diii ti^n • bi.uns. Ihn^ the best results occur whenxhtw t%o f*ii t>«r- art reasonably balanced.

'Ifm jp'j« rlaii? m^rk, wlhL is u«ll substantiated, stimu-\M*i\ rtM^ti^^- \ ,ii l i i - ^Mvt nf nutrition in relation totjT f»t'-. .in4 "..tin ]*i]tr* have hern published on this and* «' *i t*"- * i .v t- A nummary of these will be found in themem* ir< <! Hi* k and nf linektr. 1 fie former * is concernedrnuh t}i»x . .trb»>n lutroî a ratio m the wheat plant in which istru*t«l ll r , hanê in the ratio from the seedling to the grain.A tar^r irifrNr *»i anal\M'> ^how that the early stages of ger-nun.iti- n ^rc «. hara» tenzed by a low C/N ratio which steadilymrs t!ifHU|;hût the vegetative period ; when sufficientlybig!), flowenng occurs. Fruit development is characterizedh\ a hm C,S, the amount of nitrogen being high. Activen:rnstenutic tissues have a low ratio, which increases withagr ; and senescent tiûe^ have a high C N ratio. For theserta<*»n* tin* romiusi^n ^ reached that the younger the tissuetlit Wt.r I* the C N ratio. From a consideration of the avail-

AUXIMONES 209

but in lengthened illumination some long-day plants, e.g. theradish, have more soluble carbohydrate in the shape of sugarand produce flowers.

From the present aspect the value of these observationslies in the fact that they provide another instance in the in-teraction of the various factors involved in growth : there isno virtue in increasing the one without duly considering theothers ; heavy manuring with nitrates, for instance, is merewaste if there be not adequate supplies of water, and adequateand proportionate supplies of both will not promote fruitformation in a greenhouse so dimly illuminated as to depresscarbon assimilation.

AUXIMONES.

Bottomley * concluded from a large number of experi-ments that something more was requisite for the ^vigorousgrowth of a plant than is contained in the ordinary culturesolution made up with mineral substances. These promotersof growth, the nature and composition of which are unknown,he termed auximones. Bottomley selected such pJants asLemnay Salvinia and Azolla which normally lead an aquaticexistence and thus avoided the rather artificial conditioninseparable from the cultivation of a terrestrial plant in anaqueous medium. For healthy growth he found that smallamounts of organic matter are necessary: amongst the bestresults obtained were those in which an aqueous extract ofbacterized peat had been added, but other organic substances,such as autoclaved Azotobacter and crude nucleic acid deriva-tives from raw peat, will also serve.

Bacterized peat is sterilized raw sphagnum peat decomposedby nitrogen fixing bacteria of the soil; such treated peat isconsidered to act either as a food substance or indirectly as anaccessory food substance. The amount necessary for a posi-tive result is so small that a body comparable to a vitamin issuggested. Thus Rosenheim f found that plants of Primula

• Bottomley : " Proc. Roy. Soc./' B, 1917, 89, 481; " Ann. Bot.,"1920,34,345, 353. See also article in " The Exploitation of Plants/' ed. byF. W. Oliver, London, 1917. Mockeridge: " Proc. Roy. Soc.," B, 1917,89, 508. f Rosenheim : " Biochem. Journ.," 1917. 11, 7.

VOL. II.—14

21 o GROWTH

mj!a*outes treated with an aqueous extract of 018 gram ofbaetenzed prat grew taller than untreated plants. Th isaqueous extract contained 20 mg. of organic matter, of whichonly l*9 nig- represented nitrogen.

Bottomk'v's suggestion that nucleic acid derivatives a r eresponsible for the auximonic action has been further examinedby Mockeridge* who fount! that, such bodies occur in manymanurial composts* dnd her cultural experiments indicated t h a tthe greater the decomposition of such manures, the more effec-tive is a water extract of them in promoting growth. The conclu-sion is therefore reached that the free bases of nucleic acid a r emore effective than undecomposed nucleic acid. In a furtherinve&tigatinn.t it was found that the increase in the growth ofLemna miner was approximately proportional to the amountof nucleic acid derivatives added to the culture solution.

Asowb^'Ur and Bacillus radicicola, nitrogen-fixing organ-isms, werevthe subjects in this investigation: it was foundthat sterilized cultures of Azotobacter increased the growth ofLemna, although the increase was not proportional to t h eamoun;. of Azotobacier added. Autoclaved and autolyzed yeastsimilarly have growth-promoting powers, the autoclaved beingmore potent than the autolyzed. Yeast contains nucleic acidradicles and the bacteria mentioned also have the necessaryradicles for the formation of nucleic acid.

Possibly auximones are connected with the synthesis ofcomplex nitrogenous molecules, for their action on the ni t-rogen cycle organisms is to increase the rate of nitrogenfixation and nitrification and to depress the rate of denitri-fication. There is no doubt that the use of bacterized pea tmay give marked positive results in pot cultures, but to whatextent the treatment is advantageous to field crops is doubtful, j :

It must, however, be mentioned that evidence is not wantingto indicate that such auximones are not essential for plantgrowth, although they may have an accelerating action.§

• Motktndge : " BiodMsn. Jonra.," 1920, 14, 452.f Mockwklge: " Aaa. Bo*.,'* 1924, 38, 723.I Sm Russell: " Jomra. Board A$ric.," 1917, 24* **•fit* €i«rfc: " joufn. lad. Eng. Chem.," 1924, 16, 249; "Planti l " 6 I, 27$. Sacfer: "Jomm. Gm. ttjvtiL," 1925, 7* $1?.

HORMONES 211

HORMONES.

The study of regeneration, correlation, polarity andcognate subjects * leads to conclusions in some respectsindefinite in that no tangible factor is discoverable thatwill account for the beginning or for the control of certainphenomena. A cambium cell divides; the daughter celldestined to become a permanent tissue element will developinto a phloem element if cut off on one side and into a xylemelement if cut off on the opposite side ; what is it that deter-mines the fate of the cell ? The leader of a spruce is negativelygeotropic, the lateral branches are diageotropic ; if the leaderis removed, a lateral branch from the topmost whorl willchange its habit, become negatively geotropic and carryon the functions of the leader. Why must the leader beremoved before a change in tone of a plagiotropic shoot canbe effected ? The primordium of a lateral bud is laid down,and, apparently, all conditions are favourable for development,yet the bud remains dormant until the apex of the mainshoot is removed, then the bud will immediately start itsdevelopment. It is true that often the diversion of foodwill account for the subsequent phenomena, but in otherinstances such an explanation is inadequate and in suchexamples the question is : What is it that presses the trigger ?

The subject properly is beyond the scope of an introductionto the physiology of metabolism of plants, but the introductionis desirable even though the acquaintance be not cultivated.

For long it has been known that very small quantities ofvarious materials act as powerful stimulants ; the extra-ordinary effect of minute traces of zinc on the growth ofmoulds may be instanced. Animal physiologists recognizethe effect of traces of substances in stimulating variousactivities, especially those associated with secretion. Thesesubstances are produced in one organ and stimulate anotherorgan to which they are conveyed by the blood. Hence the

*See Bolin: " Compt. rend. Soc. biol./' 1918, 81, 220. Farmer:" New Phyt./' 1903, 2, 193, 217. Goebel: " Biol. ZentrlbL," 1916, 36,193. Lang : " Brit. Ass. Rep./' I9*5» 701. JLoeb : " Bot. Gaz./' 1918,65,150 ; " Journ. Gen. Physiol.," 1919, 1, 337.

XA •

212 GROWTH

i--.b:.\ ot d cht'iiiioa! messenger, or, to use the current te rm,

i rr tj. * u,t- n", *: **t tl'c rr^t of bnUuii^ tu suggest t h a tI ?i rit- | U\ a : tir* m tin1 * in»ny «»f tlu* plan! ; his eX"I !,4!,a ' i 11 * L i ? v in tf t -pru« r n>tan» id above is t h a tt1 •' a : .,! t * : M., i \ :*• -» nt.iiL; an mvi itinj sub --!a- .vi < i.^T/ ./ U " t l . r ] ar!N »f the pUn*, keeping* *" *: * j - r'i a. ! nr I lit* rx nil wil < i the k\iJtT of t h e*«|f- { nn, vt^ t1 4 - urn < t tLe inhibiting hornnne, where-! re .* , IT ia!k ral ^.J^ t a^um^ the qualities and functions of

1!^ : Ivd ua* aduptrd by Loch t who concluded fromv AVA '>I«>ir\ j!i"]> nn tin* (it^clopment of buds and roots o ntlf K i \ ^ ,1 Bryjphxllum calycinum that the apical b u d*iYntf- d Lnnr*<>ne wlurh inhibits the development of b u d sm^xt hd^il m poMtion. It is not until the apex is removedthat the bud? below will develop.

Ihv decree i»f inhibition depends on the amount of hor-ni^nv APA !;•*. iha>- >4 the lateral bud; it is presumably fortfa> ria«'«n tluit the inhibition may only extend to these p r i -T J rdia situated more immediately below the apex.

This acci»unt> brief though it be, will give some idea of t h eaction of hormones ; the hypothesis, in so far as it affectsplants, however, is not universally accepted. Thus Fysonand Venkataraman $ can find no evidence in favour of t h eexistence of such bodies, and Child and Bellamy § consider t h a tinhibition is a question of the conduction of stimuli ra therthan the movement of tangible substances. Indeed, t h eevidence regarding the occurrence of hormones in plants i s ,perhaps, less conclusive than the proof of their existence i nthe animal; actually, hormones have never been demonstratedin the plant; their presence is inferred in order to explain

• Eima: " British Ass. Rep.," 1904, 814. See also Armstrong :"JUuft, Hot,/' i$u, ig, 507.

f toafe: " B©4. Gw.," 1915, 60, 249; 1916, 6a, 295; "Science,"«f * 7» #» 547* §*• a *o &«*«* aad Halma : " Plant World/' 1919, 23, 239.

f Vymm mwJ Vcnkafarau&an: ** Joarn. Indian Bot.,'* 1920, 1, 337.vHd tad MaBsmy : " Science/' 1919, gfa 362 ; ° Bot. Gaz./' 1920,

HORMONES 213

certain very complicated physiological processes otherwiseinexplicable.

Of recent years the study of the transmission of stimuli,in relation to correlation of growth and development,* geo-tropism and heliotropism, by the movements of hormones,has resulted in the publication of a large number of obser-vations a consideration of which is hardly possible without anaccount of the phenomena associated with irritability, a bigsubject outside the scope of a treatise on the metabolic pro-cesses of plants.

•See Haberlandt: " Sitz. Preuss. Akad. Wiss.," 1921, 695, 861;" Beitr. Allegm. Bot.," 1923, 2, 1. Gilbert: " Bot. Gaz.," 1926, 81, 1.Reed : " Journ. Agric. Res./' 1921, 21, 849. Snow : " Ann. Bot.," T923,37> 43; 1924, 38, 163, 841. Soding: " Jahrb. Wiss. Bot.," 1925, 64, 587.Suebert: " Zeit. Bot.," 1925, 17, 49. Tschirch: " Vierteljahr. Natur. Ges.Zurich/' 1921, 66, 201. Went: " Kon. Akad. Wetens. Amsterdam,"1926, 30, 1.

I N D E X .

A butilon Darwini, influence of wateron carbon assimilation, 15, 16.

A cer, development of photosyntheticpower, 44.

— saccharinum, respiration, 151.Acetaldehyde in apple, 161 ; in

seeds, 161.Acidity, effect on respiration, 134.Acrose, 61.Adaptation, complementary chro-

matic, 35.Aerobic respiration, 108.Ajuga reptans, assimilation, 170;

respiration, 170.Alcohol production in apple, 161 ;

in seeds, 161-Alcoholic fermentation, 107, 153,

*55-Aldehydase, 90.Aldehyde mutase, 141.Algse, assimilation of amides, 83;

of formaldehyde, 59.Almond, fat formation, 72Alpine plants, carbon assimilation,

29.Amaranthus, respiration, 151.Amides, production, 94.Amino acids, oxidation, 137.Anaerobic respiration, 108.Anaesthetics, action on respiration,

116.Andropogon halepensis, respiration,

151.Angiosperms, occurrence of nitrite,

88 ; reduction of nitrate, 90.Anthocyanin and respiration, 113,

134.Antoxygen, 143.Aponogeton, carbon assimilation, 32.Apple, alcohol and acetaldehyde in,

161; catalase activity, 152 ; res-piration, 112, 161, 163.

Aquatic plants, assimilation of for-maldehyde, 59; carbon assimila-tion, 22, 32, 50.

Arachis, formation of carbohydratesfrom, fats, 74 ; germination, 72.

Arctium, range in carbon assimila-tion, 50.

Asparagine, origin, 94.Asparagus, respiration, 130.

Aspergillus, respiration, 162.— niger, action of anaesthetics on

growth, 118 ; growth, 171,188 ; nitrogen supply, 85 ; or-ganic acids formed by, 104;respiration, 106, 134.

Assimilation numbers, 43.Assimilatory quotient, 6.— ratio, 60, 64.A triplex, respiration, 136.Autocatalytic reaction, 175, 178.Auximones, 209.Avena sativa, growth, 196, 197, 198.Azolla, growth, 209.Azotobacter, growth, 210.

BACILLIARIALES, chromatic adapta-tion, 36.

Bacillus, respiration of Timothygrass, 105.

— botulinus, catalase, 150.— butylicus Fitzianus, fermentation.

79-— coli, activity, 166; nitrogen

supply, 84 ; oxidative activ-ity, 100; respiration, 165.

— pyocyaneus, activity, 166; oxi-dative activity, 100.

— radicicola, growth, 210.— subtilis, respiration, 118, 133.— tetanus, catalase, 150.Bacteria, chromatic adaptation, 36.Bacterium aceti, oxidation, 142.Baeyer's theory of carbon assimila-

tion, 56.Barley, electro-culture, 201; growth,

187; origin of protein, 96; res-piration, 116, 119.

Bean, respiration, 126,129, 131, 162.Beet, diurnal range in carbon as-

similation, 49 ; sugars of leaf, 53 ;zymase, 153.

Begonia, growth, 199.Blackheart disease, 125.Blackman reaction, 29.Bomarea, carbon assimilation, 32.Boron and growth, 204.Botrytis, growth, 190.Brassica, protein formation, 82 ;

respiration, 160.— alba, respiratory quotient, 103.

215

INDEX

i . nibu-t >r ^ *i , r^jH r -sitj n f> m t , 71 i IM} It iv» n t* i r \ c h r ' V . i t i c a d a p t a -

t< n î . n i p a n i i intt*ie*»t K m , X76.( 1 n i i dtahi***' of ^w<*t>^ 1 5 0 .i r a -HiiLictM1 r e N p i r a t J o n 1 0 4 ,

h S

4 < * n *a^ i ' i "> i i** - tie *\ t . i l lu -' •'rt.i "i 4, -i> I ' i i u t T i t c t f ( v t o t h r o m e ,

t \ *{ •» 4 i 5 r lut~n»eni s a l t s ,4 " r t» • • i i a i a< t c : s 4 ^ t<r~

ttv i \ \ ^ t v **> , ' a t e , 7 * , P t l n d r j

r j * ° i l t i . a ^ 1 4 , t t n a t u r a i I > t h \ t l n

T<IT i c i a t u r t * i * t r f c i m t * j " : 5 4

t i n * * i - i v* r * ^ i i i i k i i u u n

Uitî, 4s* w i t e r 1 4

••* i t a ' h . ii . t s - ^n - i l a t i o ? : . 2 5 ;

^ T ) \ \ t h , ] « » J

i , ' j f k'< d t v t V ' p n a ' i i t o f p h o t o -

s \ i i t h t j t i t p o w t T , 4 4 ; g r o u t ! ] ,J 7 S ' ! 7 ^ p r t i t t ' m f o r m a t i o n , 8 2 •

o f u i r b t n a s s i m i l a t i o n ,

3

a n d s u g a r p r o d u c t i o n ,

5 4

I h a i n t t r r s , l a w o f , i S .

D i t t u ^ o n And t\d\xiraction, 21.

l > r o u g b t a n d r e s p i r a t i o n , 1 3 2 .10

t !

I 4^

K<Tl Of

\ e, r

{ aft aft/* num, j n t f i n h\«Jrol\si3f

^4t ** ?##.*# tdib* n Hss'tnilatioii, 2b, 28,

%S ^^ piutf in foinuition, S3 ,TcînrdtK'B m , t r m i ^ r a t u r e co-r*tu » n t 1 f c«4ilH>n jwsinaiLition, 28.

C hi* »n>h>rm, t tstxt on respiration, 117.i 111 Tl^l .K Icivt I4»> P>7i ^K;»^l-h\'i and carbon Assimilation,

en 30

C hromatH. AdaptatMJB, 35,< i«#t«r*# MISIM* ourboti assimila-

tWR, 16i kdi^ph&9», c u b o a a n l i n i l a t u m , 5 0 .(Umrtc awl growtli, 1*6.C mm, tMbcm «wuailAtioii» 34,C H k ami growth, 200.

F htn\aftu>t r e s p i r a t i o n , 1 3 0 .

E f f i u e n t \ m d f x o f c a r b o n a s s i m i l a -

t u n 3 s

I i $ \ t r i t j j r n e r a : \ a,n<I c a r b o n a s -

'- n < ! . i t 1 - n 4 0

1 I t x t r o c u l t u r e 201

i 1111 a - s i m i l a t i o n n u m b e r , 4 3 .

t • "Jea, a s s i m i l a t i o n o f f o r m a l d e h y d e ,

5 a , f>o ; c a r b o n a s s i m i l a t i o n , 22,

] 3 2 ; r e d u c t i o n o f n i t r a t e , 8 9 ;

r e s p i r a t i o n , 1 3 3 ; t e m p e r a t u r e c o -

i e f f i c i e n t o f c a r b o n a s * & i r m l a t i o n > 2 7 .

Fndi'tnnesternahs, f a t f o r m a t i o n , 7 6 .

E n e r g y a b s o r b e d a n d e n e r g y u t i l i z e d

r a t i o , 3 5 ,

, —- o f f e r m e n t a t i o n , 1 0 7 .

F n z v m e o i c a r b o n a s s i m i l a t i o n , 4 6 .

E n z \ m e s o f o x i d a t i o n , 1 3 6 .

JErytkrina, n i t r i t e i n , 8B.s E t h e r , e f f e c t o n r e s p i r a t i o n , 1 1 8 .! Eucalyptus regnans, g r o w t h , 1 8 b .

Eurotiufn, a s s i m i l a t i o n o f a m i d e s ,

S 3 -E v a p o r a t i o n a n d d i f f u s i o n , 2 1 .

F A C T O R S , i n t e r a c t i o n o f , 1 3 .

Fagus, t r a u m a t i c r e a c t i o n , 1 1 5 .

F a t - c o n t a i n i n g s e e d s , g e r m i n a t i o n ,

7 2 .

F a t c o n v e r s i o n o f c a r b o h y d r a t e i n t o ,

7 4 ; f o r m a t i o n in $&cckmrotmvM$,

J5 ; h y d r o l y s i s , 7 3 ; o r i g i n f r o m

c a r b o h y d r a t e , 7 1 ; o r i g i n f r o m

p r o t e i n , 7 0 ; r e s p i r a t i o n o f , 1 0 5 ,

1 2 8 , 1 6 8 ; s y n t h e s i s , 6 9 ; t r a n s -

i o c a t i o a , 7 2 .

INDEX 217

Fatty acids, origin, 77.Feeding experiments with formalde-

hyde, 63.Fermentative processes, 106, 152.Flax, respiratory quotient, 103.Fontinalis, carbon assimilation, 10,

22, 23, 32.Food and respiration, 116, 125.— calorific values, 69.Formaldehyde and carbon assimila-

tion, 59.— hypothesis, 62.— in leaves, 64.— photochemical production, 37.— polymerization to sugars, 60, 63.— theory of carbon assimilation, 56.Formic acid hypothesis of carbon

assimilation, 66.Formose, 61.Fraxinus, traumatic reaction, 115.Fungi, assimilation of amides, 83 ;

growth, 188 ; nitrogen supply, 84 ;respiratory quotient, 106.

Galanthus nivalis, sugars of leaf, 51 ;respiration, 129.

Gentiana brevidens, sugars of leaf, 52.Germination of fat-containing seeds,

72.Glutathione, 146.Glycerol, origin, 75.Glycine hispida, growth, 204, 205.Glycolysis, 163.Gossypium, growth, 179.Grain, respiration of damp, 131.Grass, effect of fertilizers, 206.Growth, 169 ; conditioning factors,

187.— and climate, 186.

colloids, 206.nuclei acid derivatives, 210.

— grand period of, 174.— in ultra-violet radiation, 200.— influence of boron, 204.

light, 194.manganese, 205.

— nutrition, 203.temperature, 187.water, 202.zinc, 205.

— inhibition by hormones, 212.

HEAT of combustion, 100.respiration, 114.

Helianthus, absorption of carbondioxide, 20 ; carbon assimila-tion, 32 ; development ofphoto-synthetic power, 44 ;growth, 179; protein forma-tion, 183; respiration, 116,168.

— annutts, rate of carbon assimila-tion, 8; respiration, i n .

Helianthus tuberosus, carbon as-similation, 31; effect of chloroformon respiration, 117; temperaturecoefficient of carbon assimilation,27.

Hermidin, 146.Heterohexose, 163.Hevea brasilienses, growth, 186.History of material, importance of,

26.Hormones, 211.Hydrocyanic acid and oxidation,

138.protein synthesis, 98.respiration, 143, 144.

Hydrogen, an essential fuel, 144.— ion concentration and ~respira-

tion, 134.Hydrolysis of fat, 73 ; of protein,

93-Hypnum, respiration, 133.

ILLUMINATION and carbon assimila-tion, 9, 30.

Ionized air and carbon assimilation,42.

respiration, 119.Iron, r61e in respiration, 137.

LACTACTDOGEN, 156.Lactic acid fermentation, 152.Lactuca, protein formation, 82.Laminana, effect of anaesthetics on

respiration, 119; respiration, 133,164.

Larmour's Law of Diameters, 18.Law of Diameters, 18.Leaf, protein formation in, 81 ;

temperature of, 30.Lemna, growth, 185, 209, 210.Lepidium sativum, growth of radicle,

188.Lespedeza, growth, 196.Lichens, carbon assimilation, 29;

respiration, 132.Light and respiration, 135.

shade plants, carbon as-similation, 10.

effect of quality of, on growth,199.

— influence on growth, 194.— range in wave-length used in

carbon assimilation, 33.Lilac, carbon assimilation, 26.Limiting factors, doctrine of, 8 ; in-

teraction of, 9.Linum, respiration, 168.Lipase, 75.Lobelia, growth, 199.Lupin, asparagine in, 94 ; origin of

protein, 96 ; respiration, 122, 128 ;zymase in, 153.

Lycopersicum, growth, 180.

« * 11 * t n n h v i ? o t \ *

1 f«t« n H I v Aa

•

iium, a&sinulatinn of amides ,3 , respi ra t ion , 102.u *emtm respi ra t ion , 135,

a. g rowth , u&h\tcê, ch r oma t i c a d a p t a t i o n ,

11

M ^V *,l f i» a

"X - si

i f ft**. 5 %J

] ^*"

* • a J ^ * r»-t I f * * SJt

t ] 14 ^

dl it! n

l L lrf»i J ti "'•n to nitrite, 8o.f mt liani*m, 141.

Nitntf, iRfanrtke in higher rlants,

in }>rot«n s>nthe5i»,

itunJttnuifii ca rbon as-M' lil it 'H,

/ 43 f #'u . 1 \ T N 11 a3">iiiulation, 34,47 dcci m(H>sition p r o d u c t s of1 u it t i n s , 1)5 - deve lopmen t ofphito&vntheM*, 44 ; p ro te inm n t t n t of leaves, 81 ; p ro te informat ion, 82

multtHotWi, gr»">wth, 174, 204.Phormtdium fcialarum c a rbon as -

simulation, zt , ch roma t i c a d a p t a -ti* n, 3*»

1 lniHphatidic a t id. 70Photoelet trie re^ponê, 4 1 .

d 8

A I

, 134h

v quotient, 103t talon re%piration 1 *4, tr^vnutic

x 1.* J *nv itives

i h »rflMcnce 1 n

04T growth,

, aywmi.lator>T quotient,5.

Oryantc acid* and respiration, 104,Q&etUmnM, chromatic adaptation, 36.O*Mi%, carb:m assimilation, 11, 170 ;

conprasafioa point, 7 ; growtla,170; rMfxnitJofi, 136, 170,

Oxidation, enzymes, 136.Oxygea and pfaotosynthesis, 6.

odfia in cafhoa"assimilatioQ> 58.

, fMniratiosi, 130.Mi p$$?$i)dg$, rmpkttikm, 132.

, growth 190; heat of rwpira-ttm* 114; ottgin ot protan, <>5 ;

t 6122,i

nin»ni*«i Air. 42; mtrat in leaf. $3 ;respiration to ioaiieU akf 120.

, 7, artificial, 37. See

Carbon ass imila t ion.Phmgmtiis, c a rbon ass imila t ion, 50.I t i y c o e r y t h n a , 36PhwmyciSt g rowth , 197 ; respira-

t ion, 1 2 6 ; resp i ra to ry q u o t i e n t ,10*)

ikh\&it logical CDinbuhtion, 100.Fh£j t r a u m a t i c rcactit>n, 115.- exiehx, c a rbon aseimilat ion, 12.

Pigment , res j i r a to ry , 145.Iîneuppk, ca ta lase a n d ox idase , 150.Ihnus A lie tris, c a rbon ass imi la t ion ,

12.Pisum saiivum, g r o w t h , 190. See

Pea.1 leufococtui,, a ss imi la t ion of a m i d e s ,

83 .Pnrnmacmcust ca t a l a se , 150.Poll ination a n d resp i ra t ion , 115.Poiygtmum sacchahntnse, r a d i a n t

energy used b y , 3 1 .— Weyruhii, r a d i a n t ene rgy a b -

sorbed, 3 1 .Pofmlus, ass imi la t ion n u m b e r , 4 3 .— ctmedensis, p r o t e in c o n t e n t of

leaf, 8 1 .P&tattoogeton, c a r b o n ass imi la t ion , 22.Potass ium, a c t i o n in p r o t e i n forma-

t ion , 86 .P o t a t o , ca t a l a se , 150, 152 ; g r o w t h ,

1 9 2 ; n i t r a t e r educ ing e n z y m e ,141 ; r e d u c t i o n of n i t ra te* % ;respi ra t ion , 113 , 121 , 126, 130,162, 1 9 2 ; s u g a r s of leaf* 51 ;t e m p e r a t u r e effect o n resp i ra t ion ,121 ; t r a u m a t i c r eac t ion , 1 1 5 ;lymasfe, 153 .

Pmstola crispa, r esp i ra t ion , 133.Prede te rmina t ion , 186, 187.Primula, ass imi la t ion n u m b e r , 4 3 .

INDEX 2J9

Primula malacoides, growth, 209.Protein and respiration, 127.— fats formed from, 70.— formation and radiant energy, 83.— hydrolysis, 93.— origin in seed, 95.— respiration, of, 167.— synthesis, 81 ; nitrogen supply,

84.and hydrocyanic acid 98.

Prunus, carbon assimilation, 32, 33.— laurocerasus, carbon assimila-

tion, 9, 27, 29; respiration,117, 120.

Pythiacystes atrophthora, growth,188.

Pyrus malus, reduction of nitrate, 89.

RADIANT energy and carbon assimila-tion, 30.

— protein formation, 83.Radio activity and carbon assimila-

tion, 41.Raphanus, respiration, 168.Raphidium, assimilation of amides,

Reactive chlorophyll surface, 47.Reductase, 89.Respiration, 99; action of anaes-

thetics, 116; conditioning fac-tors, 120; definition, 101; effectof acidity, 134; effect of car-bon dioxide, 109; effect ofionized air, 119; effect ofpoisons, 164; enzymes in-volved, 136; heat evolved,114; influence of food, 125;influence of light, 135; in-fluence of salts, 133 ; influenceof water, 129; intensity, n o ;mechanism, 159 ; rdle of iron,137; stimulation, 114; van'tHoff's Law, 124; zymasictype, 161.

— and age, no.anthocyanins, 113, 134.catalase, 150.enzyme action, 131.fats, 128.glutathione, 148.organic acids, 103.proteins, 127.surface, 113.

— of fats, 105.Respiratory index, i n .— pigments, 145.— quotient, 102.Rhizoclonium, carbon assimilation,

5O.Rice, catalase activity, 152.Ricinus, development of photo-

synthetic j>ower, 44.Rubus, assimilation number, 43.

Rumex acetosella, assimilation, 170 ;respiration, 170.

— conglomerates, nitrate content,87.

Saccharomyces, alcoholic fermenta-tion, 107; fat formation, 75 ;nitrogen supply, 84. See Yeast.

Salts and carbon assimilation, 47.— influence on respiration, 133.Salvia, effect of ether on respiration,

118.Salvinia, growth, 209.Sambucus, nitrate in leaf, 82.— nigra, carbon assimilation, 170 ;

compensation point, 7; res-piration, 170

Sarcina, oxidase, 150.Schardinger reaction, 140.Seed, catalase activity, 151; matura-

tion, 71 ; respiration, 131.Shade and sun leaves, carbon as-

similation, 12.— plants, carbon assimilation, 10,

170 ; respiration, 136, 170.Sinapis, assimilation of formalde-

hyde, 59 ; growth, 169.— alba, carbon assimilation, 170;

compensation point, 7; res-piration, 162, 170.

Snowdrop, respiration, 129 ; sugarsof leaf, 51.

Solatium dulcamara, nitrate content,87 ; reduction of nitrate, 90.

" Sore-skin " fungus, growth, 189.Soy, fat formation, 71 ; growth, 195,

196, 205; respiration, 116.Sparmannia africana, carbon as-

similation, 16.Spirogyra, carbon assimilation, 50,

59; catalase activity, 151.Starch production in potato, 48.Stimulation of respiration, 114.Stomatal opening, rhythm, 21.Structural factor in carbon assimila-

tion, 12.Suaeda fruticosa, nitrate content, 86.Succulents, assimilatory quotient,

6; respiration, 104, 113.Sugar, fermentation of, 107.— and respiration, 126.— of carbon assimilation, the first,

50.— production and desiccation, 54.Sugars of assimilating leaves, 50.— polymerized from formaldehyde,

60, 63.S u n a n d s h a d e l e a v e s , carbon a s -

s imi la t ion , 12 .— p l a n t s , carbon ass imi lat ion, 170 ;

respirat ion, 136, 170.Sunflower, growth , 1 7 9 ; prote in

format ion , 8 3 . See Helianthus.

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\ \ tin 1 11 t l i i r r u on c a r b o n a s s r i n u l a -

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r e v f i r i t i o n 1 2 P

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1 1 ^ 1 2 2 1 2 7 , i*»* 1 3 1 .

W i f l m » i *» t l i t ' o r \ o f o x i d a t i o n , 1 3 8 ,

W i l l s t i t t r r a n d S t o l l ' s a s s i m i l a t i o n

n u m b e r s . 4 3 .

~ — — t h e o r y t f c a r b o n a s s i m i l a -

t i o n , f>3

\ inthn m c a t a l d . b e a c t i v i t y , 1 5 1 .

X * T ] h \ t e s r e s p i r a t i o n , 1 3 0 .

\ h \ ^ i a l e c h l i t f e r m e n t a t i o n , 1 5 5 ;

SJT w t h 1 .So s o u r c e o f g l u t a -

t h 1 n p 1 4 1 Ste Saechmromyces.

7e% d e v e l o p m e n t o f p h o t o s y n t h e t i c

p o w e r , 4 4 ; p r o t e i n f o r m a t i o n ,

hi See M a i z e .

Z i n c a n d g r o w t h , 2 0 5 .

Z v n i a s e . 1 5 2

Z v m a s i c t y p e o f r e s p i r a t i o n , 1 6 1 .

Z v m a i s , i b i .

IN CiftlAT WkltAl» Wt THE UNIVERSITY IRESS, ABEKPEKN

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