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The effect of 2, 4-D on certain enzymes in the bean plant Cowie, Lillian Matheson 1951

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THE EFFECT 0F2,4-D . .0N CERTAIN ENZYMES £> ^  £s m THE BEAN PLANT tf - 1 by LILLIAN MATHESON OOWIE A THESIS.SUBMITTED IN. PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF ARTS i n the Department of Biology and Botany We. accept t h i s t h e s i s as conforming to the standard.required from, candidates for the degree of MASTER OF ARTS Members of the Department of Biology and Botany THE UNIVERSITY. OF BRITISH COLUMBIA August, 1951 i i i ABSTRACT A study was undertaken of effects of 2,4-D on enzyme action i n plants ( i n vivo) and on enzyme k i n e t i c s ( i n vitDo). Bean plants were treated by spraying with a water solution of N H 4 2,4-D. Harvests were made at intervals and enzyme a c t i v i t y was estimated using standard.pro-cedures. Extracts were prepared for estimation by grinding the plant material i n water using a Waring Blendor. The enzymes amylase, perox-idase, catalase, phosphatase and phosphorylase were studied. Amylase a c t i v i t y i n stem and leaf increased.at f i r s t , then, de-creased. Peroxidase i n stem, decreased, then increased ..very greatly. In leaf, peroxidase increased steadily, Phosphorylase increased i n stem and leaf for most of the period of six day6 over which determinations were made. Phosphatase increased i n leaf, showed l i t t l e change i n stem. Cat-alase i n leaf decreased. In v i t r o studies indicate s l i g h t a ctivation of catalase.at low concentrations and up to lOOppm. Stem phosphatase shows.inhibition at concentrations of 500 and 1000 ppm. Effect on leaf phosphatase i s var-iable. Stem phosphorylase i s activated at. concentrations of 1 to 1000 ppm. ppm. Modifications of metabolism res u l t i n g from enzyme changes are discussed. A theory of action of 2,4-D i s put fo£th, which suggests that the chemical acts by i n h i b i t i o n or by activation of enzyme systems and that the plant responds by accelerated or depressed production of the enzyme in.question, i n accord with Hinshelwood 1s theory of drug adapt-ation by bacteria. AOKMOWLEDGMENTS The writer i s indebted to Dr. A.H.. Hutchinson, head of the Department of Biology and. Botany, under whose authority these invest-igations-were c a r r i e d out, and to Dr. D.J. Wort, professor, under whose d i r e c t i o n the work was planned and, executed, and. to Dr. E.C. Black, formerly of t h i s department, f o r the use of laboratory space. Thanks are also due to members.of.the. Department of Zoology, Dr. W.S. Hoar and Miss Aline Redlich, i n p a r t i c u l a r for the loan of equipment.and t e c h n i c a l assistance without which these in v e s t i g a t i o n s could not have been made. The writer also wishes to acknowledge the ai d of the Depart-ment of Ho r t i c u l t u r e i n the loan of equipment and i n the t e c h n i c a l a assistance of Mr. Robert Hughes. In addition, the cooperation of members of the Greenhouse s t a f f i s g r e a t l y appreciated. CONTENTS Introduction 1 1. Responses of Plants to 2,4-D Action 1 (a) Morphological Responses 1 (b) Biochemical Responses 2 (c) R e s p i r a t i o n 5 (4) The Death of the Plant 5 2. The Problem of Mechanism of Action 6 3. E f f e c t s on Enzymes 7 (a) Amylase (b) Peroxidase 8 (c) Enzymes of Phosphorus Metabolism 9 (d) Others 9 Methods 11 1. 1949 Experiment 12 2. 1950 Experiment.. 15 5. Preparation of Plant Extracts 14 4 . Preparation of Buffers . 14 5. Enzyme Determination Methods 15 (a) )i Amylase 15 (b) Catalase 17 (c) Peroxidase 20 (d) Phosphatase 22 (e) Phosphorylase 26 Results 51 1. Morphological 51 2. Dry Weight 52 5. Enzyme A c t i v i t y — I n Vivo 55 (a) Amylase 55 (b) Peroxidase 54 (c) Catalase 55 (d) Phosphatase 57 (e) Phosphorylase ?8 4 . Enzyme A c t i v i t y — I n V i t r o 59 Summary of Results 4 l Discussion 42 1. Amylase 42 2. Peroxidase 45 5. Catalase 44 4 . Phosphatase 44 5. Phosphorylase 45, General Considerations 45 ConciLuBion 49 Bibliography 50 1 INTRODUCTION. 1. Responses of Plants to 2,4-D Action The purpose of the. investigations reported i n t h i s t h e s i s was to elucidate the mechanism of act i o n of the herbicide 2,4-dichlorophenoxyacetic acid (abbreviated to 2,4-D). The action of the. synthetic chemical, 2,4-D, cl o s e l y ressembles that of the n a t u r a l l y occurring plant hormone or auxin, i n d o l e a c e t i c acid (abbreviated to IAA) as shown by Smith.(44). The similarity i s so close that 2,4-D and other synthetic growth regulating substances are frequently termed auxins (45). Reference w i l l be made to t h i s s i m i l a r i t y , since i t w i l l a i d i n describing the action of 2,4-D. (a) Morphological. Responses—Following treatment with 2,4-D, which may be by spray or by drops, c e r t a i n pronounced and t y p i c a l r e s u l t s are produced. As described here these w i l l be those found i n the bean plant, unless otherwise noted. Stems bend and twist(<?<). Later the nodes become enlarged, stem.elong-a t i o n i s retarded or ceases althogefeher and p r o l i f e r a t i o n s u s u a l l y appear on the internodes (l6). In one to three days, depending upon plant material and other conditions, the stems become quite t u r g i d and b r i t t l e . T u r g i d i t y i n -creases u n t i l the stems, appear.water-soaked. S t i l l l a t e r , long s p l i t s . o c c u r and these are followed f i n a l l y by necrosis. The t y p i c a l reaction of a l e a f depends upon i t s maturity. Leaves which are i n bud at time of treatment be-come lanceolate and thick, almost lacking i n laminae. Expanding leaves cease expanding,, or do so e r r a t i c a l l y , producing wavy edges and crepe-like surfaces. The response of mature leaves.is usually l i m i t e d to s l i g h t c u r l i n g and loss of color. Roots of buckwheat cease to elongate (55) and grow thickened and spongy i n the .case of bindweed (50) and develop a water-soaked appearance.. Flower buds, i f present and s u f f i c i e n t l y immature, are retarded i n development and may f a i l altogether to open (l6). The degree of e f f e c t varies i n proportion with the physiological, immaturity of the plant (5 2)» with temperature (27) and with light, conditions (51, 39, 51) p r e v a i l i n g at time, of treatment. The rate and i n t e n s i t y of response depend also upon the dose the plants receive, as shown by the work of Wort (56) using varying concentrations of 2,4-D. (b) Biochemical Responses—In the generalized picture of the biochemical ev-ents accompanying these morphological changes, stem t i s s u e of bean w i l l be con-sidered p r i n c i p a l l y , because more information i s ava i l a b l e for i t than for other t i s s u e s and the picture i s sim i l a r for l e a f and root. At six to eight days following treatment stems show considerable p r o l i f e r a t i o n but no necrosis (26, 42, 44). Treatment i n these, cases was by means of one drop of 0.1% 2,4-D ap-p l i e d to the base of the blade of one of the primary leaves. At t h i s 6tage also sugar and starch have decreased r e l a t i v e to controls and nitrogen has increased. The general trends i n buckwheat stem.(55, 5^) are i l l u s t r a t e d i n f i g . 1. These trends are also found i n bindweed (45), morning-glory (50) as well as bean (44). 140 -• 120 -100 — A — • T C 70 + 50 F i g . 1. \ I = Treated l Q Q Control \ \ \ \ """X Total Nitrogen "•Starch-Dextrine H * 1 1~ O-4 ' ' ' S Days a f t e r Bpraying Total Sugars W O R T • (94 9 In buckwheat, (55, 5^) l e a f there i s a s l i g h t increase i n total, n i t -rogen, followed by a decrease. Soluble nitrogen increases, pcobably at the ex-pense of insoluble nitrogen. Leaf.starch decreases, sugar shows a b r i e f i n -crease, l a s t i n g l e s s than one day, followed by a decrease. In root, t o t a l n i t -rogen increases, insoluble nitrogen increases and soluble nitrogen decreases. Root' starch and sugars decrease. Similar results.are obtained with bindweed (45). Because of sheer technical d i f f i c u l t i e s in.the way of discovering the f i r s t .response to 2,4-D and the immediate trends, i t i s d i f f i c u l t to say whether there is i n i t i a l l y an increase or a decrease i n a particular metabolite. As with morphological effects, the work of Wort :('56) shows that the effect upon the plant 1s metabolites varies directly i n rate and intensity with the dose, received. Since t h i s . i s the case, and since various investigators made harvests at differing intervals, i t i s not possible to compare arbitrarily the i n i t i a l observations of one experimenter with those of another. Prom four days after treatment, and on, analyses.of carbohydrates and nitrogen; show a stabilized, trend (44, 55, 56"). l * 1 the following references to the work of S e l l , Luecke and: their colleagues (26, 42) on other metabolites for which time studies are not available, i t w i l l be assumed :that' effects noted at six days after treatment are stable; At this stage stems .show considerable prolifer-ation, but no necrosis. (c) Respiration—A consideration of respiration correlates the foregoing ef-fects. Kelly and Avery (23) cultured sections of pea and of oat stems i n nut-rient solutions i n the presence and i n the absence of 2,4-D. The pea seed-lings were six to seven days old and the oats 68 to 72 hours old. The con-centrations, used were 0.01, 0.1, 1.0, 10.0, 100 and 1000 ppm. A Warburg ap-paratus was used and oxygen uptake- determined for periods of 4 to 6 hours. The authors state that "although the threshold concentration producing inhib-i t i o n was not determined, the results indicate that i n pea 2,4-D at 100 mg. per l i t e r ceases to stimulate respiration, whereas at this concentration the respiration of Avena i s s t i l l at i t s stimulatory peak." Taylor (49) performed a similar experiment, using wheat and mustard seedlings of ages from 18 to 78 hours and concentrations of 1, 2.5 aoid 5 ppm. 4 He found decreased oxygen uptake and carbon dioxide production i n a l l cases. The different results obtained by these, two investigations (25, 49) may be due to the different ages of the plant material tested. Smith (44), on the other hand, placed 2. drops .(.05. ml. )each.) of ,Y?o 2,4-D on the base of each primary leaf of bean plants at the stage when the second internode was beginning to elongate. Determinations were made of oxy-gen uptake by slices from the stems of the treated plants and compared with that of slices from corresponding positions on untreated plants. On a dry weight basis, treated tissue took up oxygen more rapidly than untreated tissue. Considering the probable size of the plant (20 to 40 g.) and the amount of moisture (approximately 80$) the dose actually distributed would be .0002 g. diluted.by the plant material to 8.5 ppa« While care must be taken i n com-paring results of experiments performed under widely differing circumstances, i t is likely that the concentration of 2,4-D i n the bean plant f e l l within the range of concentrations which caused increased oxygen uptake in. the ex-periments of Kelly and Avery (23). In the sxper.iiments to be' cited, bean plants were treated when the f i r s t t r i f o l i a t e leaf was expanding. The-method was to apply 1 drop of 1000 ppm 2,4-D to the base of one primary leaf. Thus the dose on a basis of plant size snd maturity was roughly comparable to that of Smith1s experiment (44). The conclusion of some authors (44, 45) , that there is an increase i n the respiratory capacity of the.plant is further supported, by Luecke et al / s\ i n stem, (26) who found increases in certain B vitamins/^ namely, thiamin, niacin and riboflavin, and.Sell et al (42), who reported increases i n organic acids. The capacity of the respiratory coenzymes and of the Krebs cycle is thus aug-mented. Following the suggestion (55) that carbohydrates are converted into proteins i t can be seen that the rapid breakdown and respiration of carbo-hydrates probably supplies carbon skeletons (15) f o r the increased production of insoluble nitrogen compounds. The connecting evidence i s found i n the i n -creased production of organic acids, the keto-acids of which by amination would become.amino acids. An increase i n amino acids ,found by S e l l et a l (42) was also r e f l e c t e d i n the increase of soluble nitrogen compounds. The foregoing trends—-high r e s p i r a t i o n , p r o t e i n synthesis^ and i n -creased B vitamins are c h a r a c t e r i s t i c of the meristematic condition, which i s t y p i c a l l y augmented by 2,4-D and other growth regulators. Withner(54), studying vitamin B content.of developing*tomato and cucurbit f r u i t s , concluded that the duration of growth depended upon the vitamin B.content and that the larger f r u i t e d v a r i e t i e s were larger because the l e v e l of vitamin content was maintained for a longer period. YJhen the B vitamin l e v e l began to f a l l , growth ceased. In regard-to other growth regulators, Fraser ( l l ) found that IAA placed' i n the wood of a pine i n June caused continued production of spring wood throughout the summer. (d) The Death of the P l a n t — I f , then, 2,4-D maintains,the meristematic condi-t i o n , i t may well be asked, how does i t k i l l the plant? Smith et a l (45) point out s t r u c t u r a l damage to the plant, p a r t i c u l a r l y of phloem,,and.epidermis. This damage i s a r e s u l t of invasion by the p r o l i f e r a t i n g meristem,. Chloro-p h y l l i s depleted and food reserves reduced, but these authors consider that none of these i s enough to k i l l the plant. An additional, explanation may be' offered-. In the heavy protein synthesis which follows spraying, carbohydrates are depleted. / The fcannot be replaced because the leaves are mechanically damaged. Even without apparent damage, photosynthesis i s decreased^ (12). As a r e s u l t , the t i s s u e s r e s p i r e only protein, that of the protoplasm,, c h i e f l y , Bince there i s very l i t t l e storage of p r o t e i n i n vegetative plant parts. The r e s p i r a t i o n of p r o t e i n r e s u l t s i n the release of amino acids, which i n t u r n release organic acids and ammonia.. The former may be broken.down to water and carbon dioxide, but ammonia remains. I t i s t o x i c and cannot be deto x i f i e d by semination or amidation, since, the process of r e s p i r a t i o n .has removed the necessary organic acids. There are, therefore, two possible causes of death: destruction .of the cytoplasm and the production of a t o x i c substance, namely, ammonia. 2. The Problem, of Mechanism,.of Action There s t i l l remains .the problem of the basic e f f e c t of 2,4-D upon the plant. In studies of 2,4-D a c t i o n i t i s necessary to d i s t i n g u i s h between a d i r e c t e f f e c t of 2,4-D and the plant's response to 2,4-D. For the purposes of t h i s t h e s i s a d i r e c t e f f e c t i s an e f f e c t upon a s p e c i f i c (as yet unknown) re a c t i o n or reactions. An example of such a " d i r e c t e f f e c t " i s iodoacetate i n h i b i t i o n of malic dehydrogenase, which i s by combination with s u l f h y d r y l (28) groups of the enzyme. On the other hand, observed changes i n plants may be d i r e c t e f f e c t s of 2,4-D i n the above sense, a r e s u l t of the plant's response to 2,4-D or a combination of e f f e c t and response. In anextensive review, Audus ( l ) f i n a l l y concludes that growth regulators must act upon some funda-mental aspect of the plant's metabolism. Audus. remarks: "We therefore f i n d ourselves face to face with the old-dilemma..as to whether auxins exert a series, of discrete actions on a number of c e l l systems.or one action on a 'master system'. The former a l t e r n a t i v e has, to a large extent, f a l l e n . i n t o disrepute, and. Thimann's idea of a 'master reaction' appears, at l e a s t i n p r i n c i p l e , i n most recent theories. The explanation of the d i v e r s i t y of auxin e f f e c t s must then be sought, ei t h e r i n a series of secondary phenomena a r i s i n g as a r e s u l t of the main master reaction,, or i n the modifying e f f e c t of various c e l l con-d i t i o n s on the master r e a c t i o n i t s e l f . " Among the theories rejected by Audus was that growth regulators act as coenzymes or as enzyme..activators. This was c h i e f l y because of lack of 7 conclusive evidence furnished i n the. p r i n c i p a l studies on the subject, those of Berger and Avery (2) i n v e s t i g a t i o n e f f e c t s i n v i t r o of IAA on dehydrogen-ases of the oat c o l e o p t i l e . The dehydrogenases s t u d i e d .were those acting on alcohol and on malic.acid. Fumarase was also studied. An examination of the data, however, suggests a s l i g h t . t r e n d toward i n h i b i t i o n . Refinement of tech-nique and s t a t i s t i c a l c ontrol might e s t a b l i s h t h i s trend. The very f a c t of a trend, however small, i s of importance i n enzyme catalyzed reactions. In the h i g h l y i n t e r r e l a t e d reactions which make up c e l l metabolism, a v a r i a t i o n i n rate of one r e a c t i o n w i l l probably a f f e c t the. rate of several others coupled to i t . This e f f e c t has been..frequently observed, i n studies of the Krebs and g l y c o l y t i c cycles, a recent example of which i s found in . a review by Ratner and Racker (27)- I n t h i s case i t . i s noted that i f dephosphoryla-tion of ATP i s too slow, fermentation ceases because of lack of ADP or phosphate acceptors. In spite of Audus' conclusion, then i t remains possible that the- mechanism of a c t i o n may be v i a a s e r i e s of discrete e f f e c t s on a number of c e l l systems and the presence of a single master r e a c t i o n need not be assumed. The s i g -n i f i c a n c e of these discrete e f f e c t s l i e s i n the i n t e r r e l a t i o n of c e l l systems. 3. E f f e c t s on .Enzymes Two main types of i n v e s t i g a t i o n have been c a r r i e d out. One consists of t r e a t i n g a plant or t i s s u e with 2,4-D and. subsequently extracting.the enzyme i n question and determining i t s . a c t i v i t y , thus i n d i c a t i n g i n vivo e f f e c t s . The other determines the-effects of-2,4-D upon an enzyme rea c t i o n i n . v i t r o . (a) Amylase—The prompt loss of starch .from ti s s u e s treated with 2,4-D has led to investigations on the e f f e c t on amylase. Neely et a l (34) determined alpha- and beta-amylase a c t i v i t i e s i n bean stem, and l e a f 6 days a f t e r t r e a t -ment with 2,4-D and found a decrease.in stem alpha- and beta-amylase .activity and-no s i g n i f i c a n t change i n l e a f beta-amylase. Alpha-amylase was.absent i n both treated and untreated leaves. On the other.hand, &all (13) cultured bean stem s l i c e s on a starch substrate containing 2,4-D at a concentration of 10 ppm and compared starch degradation with that occurring i n a culture similar i n every respect except that 2,4-D was not.included. At 14 days from the s t a r t of the culture, starch degradation, as indicated by iodine test,, was greater i n the presence of 2,4-D than i n i t s absence. During t h i s time the stem s l i c e s , developed p r o l i f e r a t i o n s t y p i c a l , of 2,4-D action.. Both o f t h s e s may be consid-ered as i n vivo experiments, since the response to 2,4-D involved l i v i n g c e l l s . In v i t r o experiments provide a d i f f e r e n t p i c t u r e . In describing these experiments the words " i n h i b i t " and " a c t i v a t e ^ w i l l , be used i n the sense that a c t i v i t y i s decreased or increased, when the chemical in.question i s i n -cluded with the enzyme and suhstrate i n the incubation.procedure. In the case of 2,4-D the s p e c i f i c manner i n which i t a f f e c t s enzyme a c t i v i t y i s not known. Tekey et a l (50) found a marked i n h i b i t i o n of amylase a c t i v i t y when 2,4-D was included i n the digest at concentrations of 100 and 1000 ppm and. a s l i g h t i n h i b i t i o n at concentrations of 10 and 1 ppm. I t might be suggested that the concentrations of 100 and 1000 ppm are too great to occur i n plant c e l l s . But suppose, for example that a. bean plant weighing 20 grams, of ap-proximately 80$ moisture content, i s sprayed with 2,4-D of 2000 ppm. Suppose further that one cubic centimeter of the spray i s retained on the stem and leaves and i s absorbed. The water content of the p l a n t , i s 16 g., which d i l -utes the 2,4-D absorbed down to.125 ppm. It i s therefore possible that 2,4-D might enter the c e l l i n quantities s u f f i c i e n t to influence amylase a c t i v i t y . (b) Peroxidase—Felber (10) observed increased peroxidase a c t i v i t y i n protub-erances produced by 2,4-D action on bean leaves and noted that i n t h i s res-pect 2,4-D action ressembled that of B. tumefaciens and of f r e e z i n g . Tumors produced i n plants by the l a t t e r agencies are also r i c h i n peroxidase ( 4 7 ) . 9 (c) Enzymes, of phosphorus .metabolism.:—Nee-ly et. a l (55) observed decreased, phosphorylase a c t i v i t y i n bean stem.and leaf, six days af t e r treatment with 2,4-D. Ravazzoni (58) reported, that-"an. increased: a c t i o n .of phosphate en-zyme systems i n tomato, was-observed.when a. 0.1%.lanoline suspension of 2,4-D was applied." (Quoted from Chemical Abstracts). (d) OtherB--Pectin,methoxylase (55) i n bean stem. and. leaf.. was found.to be de-creased,when extimated.six.days .after treatment with 2,4-D. Lipase, of castor bean ( 1 7 ) and of. wheat (24). were.inhibited i n . v i t r o i n the presence of 2,4-D. However, castor, bean lipase .was. apparently i n h i b i t e d . to a- greater, extent, than. wheat l i p a s e . This difference.: was ..suggested as .a possible basis f o r the castor greater s e n s i t i v i t y of/bean .than wheat to.treatment with.2,4-D ( 2 4 ) . While the. foregoing enzyme data Sons l i t t l e t o present a correlated picture of 2,4-D action, they support the; conclusion of workers ( 5 5 , 44) that plant metabolism, i n general and carbohydrate, metabolism, i n particular,. are profoundly .modified. That the problem i s complex may be concluded from the following, miscellaneous...observations: 1 . Amylase of. p l a n t s . i s activated by dehydroascorbic a c i d (56) and i s i n a c t i v a t e d by ascorbic acid, thiamine, r i b o f l a v i n and calcium pantothen-ate ( 2 5 ) . Variations, i n ascorbic acid (55, 40) and increases in: these B v i t -amins .,follow, treatment with 2,4-D. Thus an.observed effect.on amylase must be considered, as.produced by one or more of the following means: d i r e c t l y by 2,4-D act i o n on the enzyme or its.precursor,.or by way of v a r i a t i o n s i n cer-t a i n metabolites which.in t u r n modify amylase a c t i v i t y . 2 . P r o t e o l y t i c a c t i v i t y ie also i n h i b i t e d by ascorbic a c i d and these B vitamins ( 2 5 ) , and the same.considerations apply.in t h i s . r e s p e c t to proteolysis-as to amylolysis. I t is. also, possible, that, the accumulation, of prot e i n following treatment with 2,4-D may be p a r t l y due to an i n h i b i t i o n 10 of proteolysis whiles synthesis s t i l l , continues. In an. attempt to elucidate the action of 2,4-D, time, studies.were made of enzyme activity in.bean.plants following treatment.. As nothing of. the kind had been previously.attempted,. i t . seemed advisable to cover.as broad a f i e l d as possible, and.me.thods were selected with that aim in view. A study of amylase activitywwe.8 undertaken because of the previously mentioned exten-sive starch .degradation. Catalase. and. peroxidase were: studied in. the expec-tation that, they might yield information on the ..general physiological, state. s u r e s t Effects,upon, respiration and protein synthesis^changes .in the energy relations. of the cell,, which suggest, i n turn effects upon phosphorus metabolism.. Accord-ingly, phosphatase.and phosphorylase.were investigated.. An attempt.was also made to determine,..in; vitro effects of.2,4-D -upon the last four of these en-zymes. Amylase had already been so investigated by Tukey et al (50). 11 METHODS The general method consisted of spraying .bean plants with 2,4-D and of e stimating . enzyme a c t i v i t y at i n t e r v a l s following treatment. Two experi-ments of t h i s kind were c a r r i e d out, one each i n the summers of 1949 and 1950-Bean plants .were selected, as the experimental material f o r t h i s i n v e s t i g a t i o n because they have been used i n a number..of 2,4-D experiments and.because of t h e i r high uniformity. V a r i a b i l i t y of enzyme/activity i n bean was not .known, however, but amylase a c t i v i t y i n tobacco leaves, f o r example,' shows.a high v a r i a b i l i t y from plant to plant as seen i n the work of B a l l s and Martin.(2)* These i n v e s t i -gators made determinations 1 of amylase a c t i v i t y in.the leaves of 5 plants and found that values ranged from.544 to 1.024 u n i t s . Thus v a r i a b i l i t y would appear to be very high. Table 1 shows r e s u l t s obtained i n an estimate, of var-i a b i l i t y i n bean plants. Plants which had emerged from, the s o i l four weeks e e a r l i e r were harvested, weighed and.ground, i n a Waring Blendor using 400- cc. of d i s t i l l e d water. The contents of the Blendor cup were f i l t e r e d through four layers of cheesecloth ( s u r g i c a l grade). The f i n a l volume of extract was measured and 25 cc. of each extract' added to starch substrates. . Digestion proceeded.for-^0 minutes following which reducing sugars produced were e s t i -mated using the Weinmann .procedure (55)* Blanks were run using b o i l e d ex-t r a c t s . Results were calculated to milligrams of reducing sugar produced per gram f r e s h weight per plant. 12 Table 1. V a r i a b i l i t y of Amylase A c t i v i t y i n Bean Plants Plant Reducing # sugar-mg.-3 62-5 7 66.5 4 . 72.4 2 74.7 9 75-7 10 76.2 11 77.1 8 88.0 5 101.7 13 105.5 6 108.8 12 109.0 Average = 84.7 Standard Deviation =» 16.9 The formula of Denny (9) was used to calculate the number of plants which should be included i n a sample i n order to obtain a 20:1 chance that the values as. estimated .would.be within ±4$ of the mean. The number so c a l -culated was 25.4 plants. The actual..number, of plants used were, i n 1949, 50 to 40 (as included i n the 100-gram sample: which was.extracted) and i n 1950, 50 i n each of. two samples, which-were harvested and estimated separately. . Var-i a b i l i t y determinations, were not made. for.enzymes other than amylase. The use of two separate harvests as i n the 1950 experiment allowed the a p p l i c a t i o n of s t a t i s t i c a l reasoning, i f not s t a t i s t i c a l c a l c u l a t i o n s , i n the studies i n which v a r i a b i l i t y was not estimated. 1. 1949 Experiment The seed used was dwar£ bean, v a r i e t y Masterpiece, registered 1948 crop, obtained from MacFarlane's Seed Store, Vancouver, B.C. The beans were sown on'Aug. 15 i n the greenhouse,.4 to a 5" P0"t, 570 pots i n a l l . The s o i l was a mixture of equal parts of old compost, new compost and sandy s o i l , to which Buckerfield' s 4-10-10 f e r t i l i z e r was added. The seedlings he.d emerged and the primary leaves loosed from'the seed coat during the two days Aug. 26 and 27. The plants grew r a p i d l y and appeared uniform. At time of spraying 1? the second pair of compound leaves waeebeginning to expand and flower buds with no white showing were v i s i b l e on a l l plants. On Sept. 15, one-rhalf the, crop was sprayed.with. a.2000 ppm.aqueous, s o l u t i o n of the ammonium.salt of. 2,4-D, using: a.Dobbins pressure sprayer. Thepppbants were thoroughly soaked from a l l . sides, special, care having, been taken to get the spray.on the under sides of the leaves. The<whole spraying process lasted, an.hour and was begun,at 8 P.M. While i t . i s advisable to spray i n the day, because plant response.is more rapid.and c e r t a i n , i t was not pos-s i b l e to. do so in. t h i s experiment-because .of length dpcsparatmons. • Delaying the schedule u n t i l the next morning would have.caused even greater d i s l o c -a t i o n of harvest schedules than did occur, as the U n i v e r s i t y session was about to begin. Harvests.were made at 0 and 17 hours and. at three, seven, nine and 16 and JO days from treatment. Eighty to ninety plants were, harvested each time. The: plant. parts were separated, i n t o the-groups: stems and,, p e t i o l e s ; leaves; roots^ Extracts were prepared and amylase:, . catalase and, per oxidase a c t i v i t i e s were determined.on stems, leaves and.roots by methods.to.be de-scribed, later.. Duplicate 100-gram: samples of each group were prepared- for dry weight determinations by drying at 70° C. to constant weight. 2. 1950 Experiment Dwarf beans,, registered. 1949 crop, of the- same va r i e t y , from the same source.were sown on July.17, 10 to a 6" pot, 200 pots i n a l l . Composted s o i l mixed with Buckerfield',s 4-10^10 f e r t i l i z e r was.used. Weather throughout germination.was. warm: and the seedlings Bad,, emerged and. were free from t h e i r seed coats i n , f i v e to s i x days. Uniformity was good* At the time o f s p r a y i n g the f i r s t compound leaves were approximately h a l f expanded and.flower buds were barely v i s i b l e on a few plants. 14 Spraying, which began at 8:55 A.M. on Aug. 7 and. occupied 20 min-utes, was c a r r i e d out i n the same way as the 1949 experiment. Harvests were made at 0, i j - , 4 and 8§- hours and at 1, 2, 4 and 6 days from spraying. Water extracts were prepared and determinations, made of the a c t i v i t i e s of amylase, catalase, peroxidase, phosphatase and phosphorylase, using the methods to be described l a t e r . 5. Preparation, of Plant Extracts A l l extracts were prepared by grinding the fresh.plant material with f i v e t i m e s . i t s weight, of water i n a Waring Blendor. This was found to be the lowest r a t i o of water to plant, material which could be s a t i s f a c t o r i l y ground. One hundred grams each of leaves, stems (including p e t i o l e s ) and roots, repre-senting i n a l l 50 to 40 plants were blended i n 500 cc. of cold demineralized water, which had been stored i n the r e f r i g e r a t o r overnight and had reached a temperature of 6 to 8° 0. In the 1950 experiments 50 plants were used i n each determination, leaves being removed from, stems and p e t i o l e s . In each case the weight of leaves and of stems (including p e t i o l e s ) was determined using a b a l -ance weighing to one-tenth of a gram and the material blended with f i v e times i t s weight of Refrigerator stored d i s t i l l e d water (^"c) Leaves and stems were blended f o r 2 minutes.and roots f o r 5 minutes. Following blending, the con-tents of. the Blendor cup were f i l t e r e d through c l o t h . In the 1949 experiment four layers of cheescloth, ( s u r g i c a l grade) were used; i n 1950 °ne layer of f i n e q u a l i t y broadcloth was used. 4. Preparation.of Buffers Buffer solutions were prepared following the p r i n c i p l e of Mcllvaine's method (18, page l405). Other f a c t o r s being equal, buffers were chosen from buffer pairs (18, page 1412) which gave maximum buf f e r i n g capacity nearest the desired pH. Molar solutions of each member of aabuffer pair were prepared and mixed to approximately the desired pH, as shown by "Accutint" t e s t paper. 15 F i n a l adjustment of pH was made using a McBeth pH meter. Enough buffer solu-t i o n was made f o r a l l determinations of the experiment. A c r y s t a l of thymol was added asaa preservative. Ohemicals used i n each buffer are l i s t e d i n the enzyme procedures concerned. Using the pH meter, a l l buffered substrates were tested f o r accur-acy of pH both before and a f t e r addition of plant extracts, and no deviation from the desired pH was found. 5. Enzyme Betermination Methods (a) Amylase—The a c t i v i t y of amylase was determined using the method of W i l l -s t a t t e r et a l as described i n Sumner and Somers (48, page 105), which consists of incubating the plant extract with a buffered starch substrate and measuring i o d i m e t r i c a l l y the quantity of maltose produced. Apparatus—Bottles, glass stoppered, 100 c c , 2 per determination Pipettes, volumetric, 1 cc. 1 20 1 Burettes, rapid d e l i v e r y , 2 a n a l y t i c a l 1 Incubator, 37° 0. Timer, with sweep seconds hand. Reagents—Buffered starch substrate Iodine-potassium iodide, 0.01 N Sodium f h i o s u l f a t e , 6.01 N Hydrochloric acid, 1.0 N Sodium hydroxide, 1.0 N The buffered starch substrate was.prepared by mixing .10 g. soluble starch (Baker 1s, prepared according to Lintner) i n t o a paste, using cold d i s -t i l l e d water. This paste was poured i n t o approximately 600 cc. b o i l i n g d i s -t i l l e d water and allowed to b o i l f o r 2 to 2 minutes. After removing from the heat, the container was covered to prevent loss of steam and allowed to cool. Retention of steam prevents formation of a skin on the surface. Y/hen cool, 80 cc. of acetate buffer, prepared by mixing molar solutions of sodium acetate and acetic acid as previously described, were added and the whole brought to 1000 cc. This substrate wull keep 'drily 2,days, and may not be stored i n a 16 r e f r i g e r a t o r . Iodine-potassium iodide was only approximately 0.01 N since i t was r e f e r r e d to accurately standardized sodium t h i o e u l f a t e . The former was prepared by d i s s o l v i n g 12 g. potassium iodide i n the smallest possible amount of d i s t i l l e d water and d i s s o l v i n g 6.5 g. iodine i n the potassium iodide s o l -u t i o n . When f u l l y dissolved the s o l u t i o n was d i l u t e d to 500 cc. This d e c i -normal.stock was stored i n a dark b o t t l e and d i l u t e d f o r use as required. The d i l u t e d s o l u t i o n was also stored i n a dark b o t t l e . Sodium t h i o e u l f a t e was prepared by accurate d i l u t i o n from standard decinormal stock. I t was found that these d i l u t e d iodine and t h i o s u l f a t e solutions would r e t a i n t h e i r t i t r e f o r approximately one-half day. Procedure—Using a burette, 25 cc. of buffered starch substrate were run i n t o each of 2 glass stoppered b o t t l e s . (A serie s of b o t t l e s may be set up f o r determination of a number of extracts.) One bo t t l e was placed i n the incubator f o r one-half hour before d i g e s t i o n was to begin. The other b o t t l e served as a blank and was not put i n the incubator. One cc. of plant extract was prepared i n a volumetric pipette and the timer started. At zero time the extract was blown into the substrate, mixed by gentle r o t a t i o n and placed i n the incubator. At 20 minutes from s t a r t , d i g e s t i o n was stopped by addi t i o n of 5 cc. of normal hydrochloric acid, run £n from a rapid d e l i v e r y burette. The maltose present as a r e s u l t of digestion, and other reducing substances were determined by means of the hypoiodite method of W i l l s t a t t e r and Schudel, as dexcribed i n Sumner and Somers (48). The digest was neutral-ized by addi t i o n of 5 cc. of normal sodium hydroxide, using a burette and 20 cc. 0.01 N iodine-potassium iodide were added using a volumetric pipette. The method d i f f e r e d from that published i n that the l a t t e r requires addition of 6.6 cc. of 0.1 N iodine f o r every milligram of maltose expected. The mod-i f i c a t i o n used here allowed greater speed—desirable when a large number of determinations were to be made and greater accuracy, since means were lacking 17 to measure out f r a c t i o n s of a cubic centimeter. Following a d d i t i o n of iodine, 4 cc. of normal sodium hydroxide were added and the b o t t l e placed i n a dark cupboard at room temperature f o r 45 minutes. Then the mixture was a c i d i f i e d by adding. 4 to 5 cc. of normal hydrochloric acid and t i t r a t e d at once with 0.01 N sodium t h i o e u l f a t e . For the blank determination, plant extract waH added after the f i r s t a d d i t i o n of hydrochloric a c i d and the sugar-determining procedure c a r r i e d through from there. This was.a.change, from the published method i n that the l a t t e r i n s t r u c t s a d d i t i o n of the enzyme just before t i t -r a t i o n . This m o d i f i c a t i o n was necessary because i n these experiments the plant extracts contained a considerable and var i a b l e quantity of sugar. I f t h i s were estimated i t would be interpreted as an increase or decrease i n amylase a c t i v i t y where such did not necessa r i l y e x i s t . Accordingly the amount of sugar present before d i g e s t i o n was subtracted from that present a f t e r d i -gestion and the net used to calculate amylase a c t i v i t y . The t i t r a t i o n value obtained f o r the active digest was subtracted from that f o r the blank run t o obtain the net t i t r a t i o n value T n . Milligrams maltose = 1.715 x T n The maltose produced was taken to indiaate-amylase a c t i v i t y . $b) Oatalase—The a c t i v i t y of catalase was measured by using Sumner and Somers modif i c a t i o n (48, page 209) of a procedure published by von Euler and Joseph-son i n which' the decomposition of Hydrogen peroxide was measured by t i t r a t i o n . As given i n the l i t e r a t u r e , t h i s method involves three determinations of oxy-gen produced over a period of 12 minutes and with t h i s information the mono-molecular K i s calcu l a t e d . I t was abbreviated i n order to gain time f o r other enzyme determinations and a determination was made of oxygen production during i.. 5 minutes only. 18 Apparatus—Bottles, glass stoppered, wide mouth, of approximate capacity JOO c c , having sufficient weight to standin the ice bath,--1 per determination Flasks, Erlenmeyer, 125 c c , 2 per determination Pipettes, volumetric, 1 c c 1 ..5 1 (rapid delivery) 10 1 50 1 Burettes—2 (l analytical) Timer with sweep seconds hand. Ice-water bath Reagents—Buffered suhstrate Sulfuric acid, 2N Potassium iodide, 5$; Ammonium molybdate, 1% (in dropping bottle) Sodium thiosulfate, 0.005 N Starch indicator. A phosphate buffer, pH 6.8 was used, mixed from molar solutions of potassium acid phosphate and disodium phosphate, by the method previously de-scribed. Substrate consisted of 6.7 cc. of phosphate buffer and 11.2 cc. of hydrogen peroxide mixed in water to make 1000 . c c Enough substrate was pre-pared for a l l determinations of an experiment, in order that comparisons could readily be made between determinations made on different days. Substrate was stored in the refrigerator. Procedure—Fifty cu. of cold buffered substrate were pipetted into a.dry glass bottle and placed in the ice-water bath an hour before start of a determination. For each determination, 2 Erlenmeyer flasks, 125 cc. were set out and 5 c c « 2 N sulfuric acid run into each.flask. When the bottles had thoroughly cooled the timer was started and at zero time one c c of plant ex-tract was blown into the substrate and mixed.. Immediately 5 c c were with-drawn and blown into one of the flasks containing sulfuric acid. This stopped the reaction. From addition of the enzyme extract until the whole of the 5 cc. aliquot of substrate-enzyme mixture.was in the acid took 15 seconds. If more one determination is to be carried out, the above steps, may be performed for two more extracts. The reaction goes for only 5 minutes, therefore only 2 determinations can be made in a ba^bh, since i t takeH about 45 seconds to . . - 1 9 carry out the above step6. At 3 minutes 0 seconds from s tar t of blowing i n the £ i r s t ex t r ac t , a second 5 c c « a l i q u o t was withdrawn and blown in to the second f l a s k containing ac id so that the. p ipet te contents were i n the a c i d at 3 minutes 15 seconds. Thus the 5 minute i n t e r v a l was maintained. In the determination of oxygen,. 10 cc . of 5% potassium iodide were added, us ing a volumetr ic p i p e t t e , aA volumetr ic p ipet te was necessapy because the potassium iodide used contained iodates , which react with ac id and iodides t o produce f ree i o d i n e . I f the quantity of iodates so added.were not con-t r o l l e d , r e p r o d u c i b i l i t y of r e s u l t s would be a f fec ted . An a l t e r n a t i v e method would be to use potass ium'iodide which i s f ree of iodates , but t h i s chemical i s much more expensive than the other . Fo l lowing a d d i t i o n of potassium iodide one drop of \% ammonium.molybdate was added to cata lyze the re lease of i o d i n e . Af ter wai t ing 5 minutes the contents of the f l a s k were t i t r a t e d with sodium thiosulfate- , 0 . 0 0 5 N , us ing a s tarch i n d i c a t o r near the end of the t i t r a t i o n . To f i n d the amount of oxygen produced, the t i t r a t i o n value of the 3-minute a l iquot was subtracted from that, of the 0-time a l iquot to get the net t i t r a t i o n , T n . ' -M i l l i g r a m s oxygen = T n x 8 x normal i ty of t h i o e u l f a t e / L O T r i a l runs were made to a s c e r t a i n whether a comparison of amounts of oxygen re leased over a.J-minute'.-.period .would give r e l i a b l e comparisons of ac tua l a c t i v i t y . Thi s was done using, an .undi luted extract and the same ex-t r a c t which had been d i l u t e d with an equal quanti ty of water.. In each case, one c c . of enzyme mater i a l was used i n the determinat ion. A r e l i a b l e method would be expected to show the a c t i v i t y of the d i l u t e d extract to be one-half tha t of the und i lu ted e x t r a c t . T r i a l s on d i f f e r e n t occasions with d i f f e ren t extract s are entered i n - t a b l e 2. The values are cubic centimeters of 0.005 N sodium t h i o s u l f a t e . 20 Table 2. Catalase Activity in Diluted and Undiluted Extracts Trial #1 Trial #2 Extracts 0-time 3 min. Net 0-time 2 min. Net Dil'd. 10.15 8.35 1.80 12.65 11.15 1.50 Undil. 10.25 6.70 2-55 12.50 9.70 2.80 These-results indicate that relative differences in activity of 6$ or greater can be detected by this .method. (c) Peroxidase—Activity was measured by the method of Davis (8), which in-volves delayed oxidation of an iodide, liberating iodine. As the iodine is liberated i t is reduced by .sodium thiosulfate. The. quantity of thiosulfate is fixed and when i t has been completely oxidized the iodine which is s t i l l being liberated by peroxidase reacts with starch to produce the familiar starch-iodine blue. The time taken for the blue color to appear is inversely pro-portional to the peroxidase activity of the sample, thus a reciprocal of the time gives a relative indication of activity.(8). Apparatus—Flasks, Erlenmeyer., 125 cc. 1 per determination and & for the blank Timer, with sweep seconds hand. Pipettes, volumetric, 5 c c ) one 50 ) of Mohr, 10 ) each. Reagents-—Buffered, substrate Hydrogen peroxide, 0.9$ The buffered substrate consisted.of sodium thiosulfate, 0.001 N, in which was incorporated starch 0.1$, potassium,iodide, 0.027 M, and acetate buffer (mixed by general method, using sodium acetate and acetic acid) pH 4.7 0.02 M (Schwimmer, 4 l ) . It was mixed, by first making a starch paste--1 g. soluble starch mixed with 5 to 10 c c cold distilled water and poured into 100 c c boiling distilled water. Addition of 500 cc. coid distilled water cooled the mixture. Ten ec. 0.1 N sodium thiosulfate, 4.5 g« potassium iodide and 40 c c acetate buffer were added and the solution made up to 1000 cc. 21 • Enough substrate was prepared f o r the determinations on one harvest. For com-parisons of r e s u l t s obtained on d i f f e r e n t substrates a c a l c u l a t i o n as, given below was applied. P r o c e d u r e — F i f t y cc. of buffered substrate were pipetted into 125 cc. Erlenmeyer f l a s k s , one f o r each determination and one f o r a blank. Five cc. of enzyme s o l u t i o n ( i n t h i s case, plant extract)we"r-e added to each .flask, exclusive of blank, and mixed by gentle rotations. Five cc. of d i s t i l l e d water were added to the blank. .The timer v/as. started .and at zero time. 1 cc. of 0.9f° hydrogen, peroxide was. added to each f l a s k i n order, and the time of a d d i t i o n noted. Flasks were-gently rotated two or three: times and.then ob-served f o r time. of. color change. When, judging the end, .point of. the reaction, the f i r s t appearance. of. permanent- blue ..in, the body of the l i q u i d , not at. the walls of the,flask, was taken as the i n d i c a t i o n that-the r e a c t i o n was.: complete. As indicated the a c t i v i t y of-an. extract i s , proportional to the r e -c i p r o c a l of the. time taken f o r the: blue color, to appear.. However, even i n the absence of peroxidase, oxidation fcakes place eventually,, and, t h i s . e f f e c t , determined by making a blank run without.plant, extract, contributes to the. t o j a l apparent, a c t i v i t y . -The. r e c i p r o c a l of the time .taken.for. the..blank, to change color was there fore subtracted from that of. the.active determinations. Reciprocals were m u l t i p l i e d by 1000 i n order, to avoid inconvenient decimal., f r a c t i o n s . Table 3 shows r e s u l t s of a t r i a l run.designed to t e s t the above c a l c u l a t i o n s . 22 Table 2 « Peroxidase.Activity T r i a l s E x t r a c t Time 1000 Perox. (cc.) (sec) time' u n i t s * 0. (blank) 360 2.78 • • • 1. (active 345 2.90 .12. 2 . 330 3 . O 2 .25 4 300 . 2 . 3 3 •55 •Peroxidase, u n i t s were derived by sub-t r a c t i n g r e c i p r o c a l of blank time from that of a c t i v e . Such a c a l c u l a t i o n , y i e l d s peroxidase u n i t s which are approximately p r o p o r t i o n a l to the. amount of enzyme present, a r e s u l t which could not be obtained without considering the, size of the blank. The substrate would not keep.for more than a day and the i n d i v i d u a l substrates,used. from.day to. day probably did, not contain exactly the.same . quantity.of, t h i o s u l f a t e . This would be r e f l e c t e d i n the-time taken f o r the blank to, change color, a low blank, i n d i c a t i n g a low.amount o f ; t h i o s u l f a t e , and a peroxidase determination, made, on such a substrate would be too high. Readings.taken at d i f f e r e n t times were a l l re c a l c u l a t e d to a standard blank time of t e n minutes thus: . Recalculated reading on basis of 10-minute = Perox. units x o r i g i n a l blank i n seconds blank 600 (d) Phosphatase:—Activity was-determined by a method adapted from Gottschalk's (l4) micromethod, which, consists of.incubating the enzyme extract with d i -sodium, phenyl phosphate and measuring colorimetr.ically the phenol produced. Apparatus—Erlenmeyer f l a s k s , 125 c c with.stoppers Pipettes, volumetric, 1 c c — 1 . 2 1. 5 2 100 1 Colorimeter, Klett^Summerson,, green f i l t e r Colorimeter, tubes Timer, with sweep seconds hand Constant temperature chamber, 57° C. 23 Reagents—Buffered substrate Folin-Giocalteu reagent Sodium carbonate, 10$ Buffered substrate, as described by Hawk, Oser and Summerson (20) page 208, consisted of disodium phenyl phosphate, 1.09 g-, citrate buffer, pH 6,0, prepared from secondary sodium citrate and citric acid as previously described, 200 c c , mixed and distilled water added to make one lit e r . Cit-rate buffer was used in the absence of sodium veronal, the chemical required by the procedure of Hawk et al (20). The Folin-Ciocalteu reagent was pre-pared as in Hawk et al, page 879. It was diluted for use by adding 2 parts of water to one part of reagent. If the reagent went greenish at any time i t was restored by addition of bromine and boiled to remove any excess bre-mine. The greenish color was due to the presence of reduced phosphomolybdic acid and would have interfered with accurate color determinations. Bromine oxidized the reduced acid back to unreduced phosphomolybdic acid. Procedure—Erlenmeyer flaBks, 125 c c , in which the enzyme reaction was to take place were set in a 57° C. incubator for 30 minutes before the reaction was to begin. The timer was started and one cc. of enzyme blown into each of two flasks, one of which, for the blank run, need not have been warmed. For the active run, 5 cc. of buffered substrate was added and the reaction, whichwas timed to the second, allowed to proceed for one hour. The reaction was stopped by addition of the phenol reagent, 5 cc. Following the addition of 5 c c 10% sodium carbonate, color was allowed to develop for ex-actly 10 minutes. At the end of this time color development was complete and 100 cc. of tap water were added, to reduce the density to a readable range and to minimize further changes of density. Intensity of color was read in photoelectric colorimeter in from 10 to 20 minutes after dilution. The blank run was treated in exactly the same way, except that the phenol reagent was 24 added to the enzyme extract before the substrate was added and the color dev-elopment procedure carried on from there. The reading for the blank run was subtratfeed from that of the active run, (Both readings were taken using the same colorimeter tube) giving net colorimeter reading and the amount of phenol released was calculated: Micrograms phenol released = 2.60 x net colorimeter reading. Using the method of least squares, the figure 2.60 was calculated from readings obtained when the above color development method was applied to standard solutions of phenol. The graph oil the page following shows the standard curve obtained from these readings. In order to develop this procedure, thrial runs were made from which were selected first the color development method and next the desirable reac-tion time. In the determination of phenol, the Folin-Oiocalteu phenol reagent, which contains unreduced phosphomolybdic acid, is reduced by phenol to a mix-ture of the lower oxides of molybdenum, yielding the turbid blue known as molybdenum blue. Stability of color is best obtained by rapid color devel-opment (this requires concentrated reactants) followed by rapid dilution to a low density at which further changes of intensity occur at a reduced rate. Dilution should take place at or near the maximum color development. Trial runs were mMe^  in the presence of leaf and of stem extracts, to determine the optimum time for allowing color to stabilize before and after dilution. Table 4 shows the results of these t r i a l B . The column "before dilution" re-fers to the time allowed between addition of sodium carbonate, at which stage color development began, and dilution by addition of 100 cc. of water. The colorimeter readings were taken after dilution, at the intervals indicated. Figs. 2 and 5 show these results when graphed. Stem and leaf extracts were included in a l l runs, because i t had been observed that color development in the presence of plant extracts was more rapid than without them. 25 Table 4. Phenol Color Development T r i a l s . (Figs. 2 and 3) Time of color development (minutes) Source Before A f t e r d i l u t i o n of enz. Stem Leaf D i l ' n. 0 0 5 306 10 20 . Colorimeter readings 248 247 313 305 30 247 299 Stem Leaf 10 10 259 347 257 348 257 348 255 346 Stem Leaf. 20 20 241 .294 243 294 243 292 241 289 Stem Leaf 40 40 266 280 262 280 262 274 258 272 Times were, selected on a basis of maximum..color and s t a b i l i t y . T M s the procedure allowed, ten minutes f o r color development.before d i l u t i o n and ten t o twenty minutes f o r s t a b i l i z i n g of. color following, d i l u t i o n . Gottschalk's (l4) method c a l l s - f o r d i g e s t i o n periods of one to three hours. The following r e s u l t s were obtained i n rune made to a s c e r t a i n the op-timum incubation-time.for bean extracts. Incubation and color development procedure were as. given except for time of incubation. Table 5 shows the re-s u l t s of these t r i a l s . . The.net colorimeter reading i s that obtained after subtraction, of the blank, reading from-that given by active samples. These r e s u l t s are also graphed i n f i g s . 4 and 5' Table 5' Phosphatase Incubation Period T r i a l s (Figs. 4 and 5) Colorimeter Colorimeter readings Incubation readings Net period Net 63 ... 2 hrs. 200 137 215 ... 2 257 42 184 121 3 201 138 261 46 3 256 41 195 132 4 196 133 271 56 4 259 44 Incubation period Stem 0 (blank) Leaf 0 . Stem 30 min. Leaf 30 Stem 1 hr. Leaf 1 1'inutes Minutes Figr. 2 and J. E f f e c t of t i k e of color development on i n t e n s i t y and r t e b l l i t y of color f o l l o w i n g d i l u t i o n i n , presence of plp.nt e x t r a c t s : stem, fir;.2 and l e e f , fi£.J. Curve 1 — C o l o r was d i l u t e d by adding IOC cc. water immediately a f t e r a d d i t i o n of sodium carbonate. Curves 2, J and 4— Color was allowed to develop for 10, 2C and 40 x i n u t e e , r e s p e c t i v e l y , be-fore reading. Readinje were mode at 5, 10, 20 9nd JO minute? a f t e r d i l ' n . (To follow page 25) 26 The d i g e s t i o n period of one hour was.selected, f o r stem extracts because at t h i s point the amount of phenol produced i s approximately prop-o t t i o n a l to time. For le a f extracts, the choice of one hour was a r b i t r a r y , being made because was an appreciable production of phenol ..during, t h i s time. Other attempts to get a good time study were equally unsatisfactory, an app-re c i a b l e loss of phenol apparantly occurring i n some instances. (e) Phosphorylase—Determination was by use of the method of Sumner et al. (20) consisting of incubating,the enzyme extract, with glucose-l-phosphate and measuring the amount .of phosphorus released. . Apparatus—Test tubes, JO c c ' 2 per determination Pipettes, volumetric, 1 c c . — 1 5 5 (one rapid delivery) 10 1 Colorimeter, KlettT-Summer.son, green f i l t e r Timer, with sweep seconds hand Constant, temperature chamber, or bath, with supports fo r test-tubes, 24° C. Colorimeter tubes Reagents—Buffered substrate Starch solution,. 1$ (potato) Ammonium.molyBdate, 6.66% S u l f u r i c acid, 7«5 N EfemaiiS sulfate., 4$, f r e s h l y prepared. Glucose-l-rphosphate was prepared enzymatically by the procedure of Sumner and,Somers (48). C i t r a t e buffer, pH 6.0, molar, mixed according to procedure, using secondary sodium c i t r a t e and c i t r i c acid, was used i n the absence of the chemicals used by Sumner et a l (4^ 7) and,was found to be s a t i -sfactory, ^otato starch, 1% s o l u t i o n was prepared as i n Sumner et a l (•%?), except that f i l t r a t i o n was. found, to be impossible, since the starch solution was extremely viscous. The' solu t i o n ..was decanted, instead. The substrate so l u t i o n was made by d i s s o l v i n g 1. g. glucose-l^phos-phate. i n 50 cc. d i s t i l l e d water, shaking, with dry calcium, oxide to remove i n -organic phosphates and f i l t e r i n g . The f i l t r a t e was neutralized with drops of 27 hydrochloric acid aa i n Sumner and- Somers; The quantity of f i l t r a t e was measured and an.equal volume of mixed pH.6.0 c i t r a t e . b u f f e r added. Usually t h i s gave a t o t a l substrate volume of 90 cc. This substrate-will keep i n -d e f i n i t e l y i n the r e f r i g e r a t o r . Buffered substrate was.mixed, before use, with an equal volume; of %% potato starch and.a c r y s t a l of thymol added. This s o l u t i o n w i l l keep at room temperature f o r one to two weeks. I t cannot be stored in.the r e f r i g e r a t o r or reversion of the.starch w i l l . t a k e place. Mix-ing buffered, substrate.and.starch was a.technical, departure from the'procedure of Sumner et a l (20), but involved no change, i n p r i n c i p l e . Time was saved during actual determinations by t h i s v a r i a t i o n . Ammonium molybdate, 6.66%, was' prepared from "Acid Molybdic" 8fy%, Nichols' brand. It. i s l a r g e l y ammonium, molybdate•,. Sixty grams were dissolved, i n approximately 600 cc. of water,, and ammonium.hydroxide was.added u n t i l . t h e solution.was neutral to. "Accutint". t e s t paper. While the concentration of t h i s reagent was not exactly 6.66%, since.the assay of ammonium molybdate was unknown, i t was s u f f i c i e n t l y close, since a l l determinations and-.the construc-t i o n of the standard ..curve were .made with the. same batch, of reagent. Ferrous s u l f a t e , used i n color development.instead of the more usual stannous: chl o r i d e , was recommended by Sumner.et.al. I t could readily.be pre^ pared just before use and,.had.,the. advantage of showing when.it no longer pos-sessed reducing power, since a p r e c i p i t a t e . o f f e r r i c s ulfate formed. The incubator consisted, of a large tub of water. maintained at 24° 0. by means of a thermoregulator and a low lag immersion heater. The tolerance of t h i s apparatus was le s s than 1° 0. above and.below the desired .temperature. Long blocks, or wood d r i l l e d , with, holes into which t e s t tubes just f i t t e d served as supports to hold the di g e s t i o n tubes i n the water bath. 28 Procedure—Using a volumetric pipette, one cc. of enzyme, extract was run into each of. two J O c c . t e s t tubes and one of. them, placed i n the i n -cubator. After about.15 minutes, .2 cc.•of buffered-substrate-starch solution were, added .to.the tube i n the.incubator and.the, contents allowed to incubate f o r one hour. Digestion.was arrested by adding 5 60. ammonium:molybdate, 6.66% 6.66%. At t h i s time, 5 cc.'. ammonium molybdate. were also added to the tube con-taining, only the enzyme, followed by 2 cc. substratew This tube served as a blank. A rapid d e l i v e r y pipette should,be used when adding ammonium molybdate. Color development took place following a d d i t i o n of 5 cc. o f - s u l f u r i c acid, 7«5 N and 5 cc. ferrous s u l f a t e , h%.. Ten cc. d i s t i l l e d water were added to d i l u t e the s o l u t i o n for.reading, i n the colorimeter. The contents of each tube.were mixed by r o t a t i o n . a f t e r a d d i t i o n of each reagent. Following the a d d i t i o n of. water, the tubes were stoppered and mixed, by inverting.three or four times. Readings were taken a f t e r tubes, had .been allowed to stand f o r at, l e a s t ten.minutes and no more than 25 minutes after, addition.of water. Correction.for the colorimeter tube was eliminated by reading both the blank and the active.runs i n the same colorimeter tube. Considerations of color development made i n the phosphatase pro-cedure .apply, here also. Table 6... shows .results of t r i a l runs made to deter-mine the optimum color development periods. Table 6. Phosphorus Color Development T r i a l s Time after,adding water (minutes) 5 10 15 20 25 50 Colorimeter readings Stem 326 340 340 340 342 250 Leaf 178 179 180 181 181 181 Accordingly, readings were taken not less than ten and not more than 25 minutes a f t e r a d d i t i o n of water. 29 The procedure of Sumner et. a l (•$?) re quires an incubation period of f i v e minutes. This was found to be too short f o r the extracts i n use. T r i a l runs were made using various.incubation periods and.following i n a l l other respects the procedure- as given above. The r e s u l t s of these t r i a l s may be seen i n table 7 and f i g s . 6 to 9. Table 7« Phosphorylase Incubation Period T r i a l s . (Figs. 6 to 9) T r i a l #1 Incubation Colorimeter period readings Net Stem 0 (blank) 104 • • • Leaf 0 . " 301 • • Stem 1 hr. 10' 154 50 Leaf ti 3l4 13 Stem 2 hr. 161 57 Leaf 2 322 21 Stem 4 174 70 Leaf 4 330 29 Stem 5 179 75 Leaf 5 350 30 T r i a l #2 Stem 15 min. 137 16 Leaf 15 317 0 Stem 30 175 31 Leaf .30 324 3 Stem 1 hr. 175 55 Leaf 1 340 19 Stem 2 230 110 Leaf 2 359 38 The amount of phosphorus released was calculated from the net c o l -orimeter readings as follows: Micrograms of phosphorus = 8.42 x net colorimeter reading. The f i g u r e 8.42 was calculated, using the.method.of l e a s t squates, f r om readings obtained when the phosphorus-determining procedure was applied to standard solutions of phosphorus made up from potassium dihydrogen phos-phate . I t i s l i k e l y that the l i b e r a t e d phosphate comes from phosphorylase action, since Neely et a l (55) a n ( i Sumner et a l (47) found no evidence of a phosphatase attacking glucose-l-phosphate. 75 + J? 70-L -O o B £ 60 o o o 55 + 50 STEM X 5 Hours Piga. 6 and 7. Phosphorylase incubat ion, Btem extract . 50 t a5 V 1 O O o 20 4 10 4- + 40 + 20 + 10 + 1 2 3 4 5 .5 l 2 Hours Pig*. $ and 9. Phosphorylase incubation, l ea f ex t rac t . (To fo l low page 50) 31 RESULTS 1. Morphological In both experiments the morphological responses followed the typin-eal pattern described, ihethe introduction. . In the 19^9 experiment, c u r l i n g of the youngest leaves and bending of t h e i r p e t i o l e s were evident i n le s s than 30 minutes after spraying. By three days the stems were becoming t u r g i d and b r i t t l e and a s l i g h t touch would knock.off the leaves. At seven days the plants.were generally l i g h t e r i n color than controls, and. chlorosis, was begin-Mng to be evident i n the primary leaves. The. nodes and stems f o r 1 to. li?" down from the nodes had become greatly, enlarged, and protuberances had.dev-eloped just above s o i l l e v e l . L e a f l e t s which were expanding when the.plants were treated.had curled and f a i l e d to complete expansion. By 16 days the majority of the primary leaves were r e l a t i v e l y unchanged although they were less green than those on control plants. At t h i s stage protuberances on the stems were very numerous and some stems appeared quite.succulent,.others had extensive n e c r o t i c areas. I t was noticed that the "sleep movements" common among Leguminosae had ceased to take place. No flowers formed on treated.plants. A few plants, whose primary leaves.had not been knocked o f f , continued to l i v e forabout ten weeks i n a l l , but no apparent growth took place. . In the 1950 experiment, the plants responded more r a p i d l y , prob-ably because'these, plants, were younger, theweather.warmer and treatment was c a r r i e d out i n the morning, while i n the.19^9 experiment-spraying was done i n the evening. The secondary leaves began to c u r l and t h e i r p e t i o l e s to bend within .20 minutes.after treatment, and t h i s c u r l i n g and bending increased throughout the day. During the f i r s t day the nodes enlarged somewhat and by the second day l e a f color was f a i n t e r and.the stems thickened. At four days. 32 stem p r o l i f e r a t i o n was quite noticeable, and some necrosis was evident i n the hypocotyl. Six days after treatment p r o l i f e r a t i o n and. necrosis had increased and the secondary leaves were-wilted, and on some plants.these had died. A harvest was made at t h i s time instead.of on the eighth day.because i t seemed possible that.the plants would be too f a r gone to "fee.of any use at eight days, the stage o r i g i n a l l y planned for the l a s t harvest. Eight days a f t e r t r e a t s ment, proliferation.was.heavy, even on the p e t i o l e s , the stems appeared very water-soaked, the epidermis was peeling and necrosis was increasing to r o t t i n g . 2. Dry Weight Results, of dry weight determinations, for the 1949 experiment are given, i n table 8. These indicate, a l o s s i n percent dry matter and are i n agreement with, the r e s u l t s .of other workers (7, 55, ^>6). In t h i s and sub-sequent t a b l e s , "C" stands for control, "T" f o r treated, with respect to.the c h a r a c t e r i s t i c being measured, and T/0 s i g n i f i e s the r a t i o of treated to c o n t r o l . Table 8. Percent.Dry Weight* (1949 Experiment). Time a f t e r Stem Leaf Root treatment C T T/0 C T T/0 0 T T/0 0 14.6 • • • • • • • • 14.3 • • • • * • • • 8.2 • * • • * • 17 hrs. 14.3 14.3 1. 14.4 13.8 .96 9.4 8.0 .85 5 days 14.8 14.6 .99 14.7 14.0 •95 8.5 8.4 .99 7 16.3 12.2 •75 17.4 14.3 .82 10.8 9.0 • 83 9 18.4 11.8 .64 18.6 15.9 .85 8.9 11.6 1.30 16 22.9 10.9 .48 18.4 16.1 188 8.8 8.1 .92 30 28.8 14.2 .50 18.9 18.2 .96 8.5 12.8 1.51 * Vaili-ies are the average of two determinations. The i r r e g u l a r i t y of the r a t i o T/C for root i s probably due to the t e c h n i c a l d i f f i c u l t y of obtaining fresh, root samples, of uniform moisture content. This d i f f i c u l t y contributed to the i r r e g u l a r i t y of enzyme deter-minations on root samples. 5« Enzyme A c t i v i t y — I n vivo (a) Amylase Table 9* Amylase Activity i n Milligrams Maltose Produced 1949 Experiment (Fig. 10) Time after Stem Leaf Root treatment 0 T T/0 0 T T/0 C T T/C 0 12-64 • * • • • • I6CO3 a • * • • • 5 . 8 3 • • • • • • 17 hrs 9 . 9 5 1 0 . 1 2 . 1 . 0 2 . 1 3 . 5 6 14.58 1.08 6 . 1 5 6 .35 1 . 0 3 3 days 6 . 7 7 7-80 1.15 9 . 5 2 10.46 1.1 3.18 1 .71 . 5 4 7 1 1 . 8 3 5 . 4 9 .46 1 0 . 9 6 9 . 2 6 •85 1 . 2 0 1 . 0 3 . 8 6 9 1 0 . 8 9 4 . 3 7 . 4 0 9 . 4 3 7 . 0 3 . 7 5 1 . 2 7 1 . 7 5 I . 3 8 16 1 7 . 6 9 3 . 8 6 . 2 2 1 0 . 3 8 4.46 . 4 3 1 . 9 7 1 .11 • 56 30 6 . 8 6 2 0 . 8 3 . 0 2 7 . 0 4 5 . 4 9 . 7 8 1 .57 6 . 5 2 4 . 7 Table 10. Amylase Activity i n Milligrams Maltose Produced* 1950 Experiment (Fig. 11) Time after Stem Leaf reatment 0 1| 0 1.68 1.92 T T/C • • • C 4.68 3.48 T • • • T/C • • • if? hrs. I.56 1.68 2.04 2.16 1.3 4.08 4.56 1.92 3.56 .61 4 2.40 2.64 1.20 .48 4.08 1.08 1.20 .28 2.16 4.80 3.24 1.85 8.64 8.40 7.20 7.44 .86 1 day 1.92 3-72 .84 .48 .23 9.24 8.88 7.92 6.96 .82 2 days 2.88 3.00 •56 .12 6.36 5.64 5.40 5.52 • 91 4 2.76 5.12.' 1.92 2.04 .67 7.08 8.40 5.64 7.20 .85 6 5.c54 6.24 2.04 1.80 .32 6.36 6.24 5.84 5.24 .56 *C and T are given for each of two harvests. T/C vjas calculated from average of values of two harvests. The results of^both experiments are shown i n tables 9 and.10. The essential trends.seem to. be.an increase followed by a decrease. If har-vests are made at sufficiently frequent intervals, a decrease occurs after the f i r s t increase, which is i n turn followed by a second increase, as seen 4 AMYIASI Pig. .C. Aaylaae a c t i v i t y i n treated Dlantr compared to that i n controls, T/C, 1949. 5 -T C 2 --• • —I »-7 9 l 3 16 Days a f t e r epreying if 30 L.M.C. l&O Tig. 11. Anylaee a c t i v i t y i n treated plant* compared to that i n controlp, T/0, 1950 2 - -t 0 Daya after apraylnf 4 6 L.M.C. 1950 (To follow page 35) 34 in the 1950 results. The increases as.shown in 1949, though, slight, are con-sidered significant at the odds of 20:1, since treated plants exceed controls by more than 8$ , and the number of plants analyzed provides for significant, results i f these results deviate by more than ±4% from the mean. Thus.adif-ference of 8$ between treated,and control would just reach significance at this level and. the differences of 15% and 10$ observed for stem,and. leaf re-spectively on the third day after treatment.would be significant. In the 1950 results, the changes in stem amylase activity are pronounced and.eon-si stent over the.two replications of treated and controls. Leaf 1950 failed to show the increase.shown by leaf 1949* The possibility may be considered that an increase, i f present, did, not occur.at times, of harvest, but took place between 8g hours and 24 hours after spraying. (b) Peroxidase Table 11. Peroxidase Activity Expressed in Peroxidase Units. 1949 Experiment (Figs. 12 and 14) Time after Stem Leaf Root treatment 0 T T/C C T T/C C T T/C .0 .85 ••• • • • .0 « • • • * • 6.06 • • • • • • 17 hrs. 2.09 3.20 i.o4 2.09 2.52 1.27 20.9 25.5 1.21 5 days 11.2 8.05 • 72 2.52 2,69 1.07 25.6 2^3 1.26 7 4.02 25.5 6.35 3.64 5.22 1.44 27.9 22.8 1.17 9 2.78 30.7 11.1 2.53 4.92 -1.95 28.9 49.9 1-72 16 1.21 71.2 58.7 1.08 .82 .77 41.9 41.2 .98 30 10.73 196.6 18.4 13.6 16.4 1.2 97.4 196.6 2.02 In a l l tissues observed, the general trend of peroxidase activity is towards, an increase. The initial observations for stem tissue differ in the 1949 and 1959 experiments, so that the ini t i a l response is in doubt, although there appears to be a decrease, in activity preceding the final pror-nounced rise. Leaf tissue.Bhows a steady rkte. Table 12, showing the re-sults of the 1950 experiment is to be seen on page 55. PEROXIDASE 2 T ROOT > i — * *w - - — ^ i — i 1 — i 1 r 1 5 7 9 16 50 Days a f t e r spraying L.M.C. 1949 Pig. 12. Peroxidase a c t i v i t y i n treated plants compared to that In controls, T/C,. 1949. H  T 0 At ^ days At 6 dcys 54.6 A 1 1 1 1 1 1 8.: ' " " • " ^ •* — » — — 1 1 / \ • 1. i • 111 1 — 1 1 \ 1 2 4 6 Daya after spray ing L.M.C. 1950 Fig. l ^ . Peroxidase activity in treated l e a f compared to that in control, T/C, 1950 (To follow page 34-55 Table 12. Peroxidase A c t i v i t y Expressed i n Peroxidase U n i t s 1950 Experiment ( F i g s . 13 and 14) Time a f ter Stem Leaf T/C treatment 0 T T/0 0 T 0 1.28 >{ • • • • .82 • • • • • • 1.03 .85 lib- h r s . 1.55 .98 .71 1.29 1.91 1.52 1.42 1.11 I.34 2.08 4 1.06 .96 .95 .83 1.17 1.54 .96 .96 .66 1.15 8t 1.42 I.05 .82 1.22 1.50 1.24 1.15 1.05 I.30 1.60 1 day 2.73 1.72 •73 I.58 1.62 1.21 2.03 1.72 1.47 2.12 2 days 1.84 5.23 2.69 1.15 1.52 1.25 1.90 4.78 1.61 4 .24 8.33 58. .05 1.86 54.6 .07 9.25 .05 1.49 6 158 32.6 43.8 .84 4.25 8.07 .j94 34.5 • 79 5-99 (c) Catalase The trends i n both.experiments ind ica te a r a p i d f a l l i n a c t i v i t y , fol lowed poss ib ly by a recovery, which i s not maintained, t h e . f i n a l obser-vat ions suggesting a continued depression of a c t i v i t y . Thought s t a t i s t i c a l ana lys i s of catalase v a r i a b i l i t y was n o t - c a r r i e d out, the reasoning of. s t a t i s t i c s may be a p p l i e d . Where each r e p l i c a t i o n . o f t reated and cont ro l i n the 1950 experiment y i e l d s cons i s tent r e s u l t s , the t rend of t rea ted i n comparison to control .may be considered reasonably s i g n i f i c a n t . . Where they are not cons i s tent , as for example, i n . the values for.8|- hours and 1 day, i t may be concluded that any di f ference between t rea ted and c o n t r o l . f a l l s w i t h i n the range of the plant mater i a l v a r i a b i l i t y , and i s probably not r e a l . Catalase values obtained i n these experiments are shown on page 36, t ab les 15 and 14. 60-• 50-. Ac--T C 30-1-10--PEROXIDASE / ° ~ ^ ^ STEM / / / / / / \ \ 1 9 5 0 / x« X / / / / ^ / / / /  / / / / / / / / \ / / \ \ \ \ \ I I \ \ \ \ 1 / 20+ / / ^ / / / r^-^^m—9-—' — \ 1 k — \ 1 h 1 3 7 9 16 50 -M—1 1 -1 1 \-2 k Days after spraying Fig. l A . Peroxidase activity in treated stem compared to that in control. Upper scale— I 9 h 9 experiment. Lower ecale—1930 experiment. (To follow p a g e 3-£? 36 Catalase A c t i v i t y i n Leaf: Milligrams of Oxygen 1949 Experiment (Pig. 15) Time af t e r -T/C treatment a T 0 2.12 • • • » • • 17 hrs. 2.40 1.84. • 77 3 days 1.48 1.52 1.03 7 1.64 .96 •58 9 2.10 .70 •33 16 1.22 1.0 .82 30 1. 69 1.16 .68 Oatalase A c t i v i t y i n Leaf: Milligrams of Oxygen 1950 Experiment ( F i g . 16) Time a f t e r treatment 0 T T/C 0 1.46 • • • • • • 1.32 !§• hrs. 2.18 1.0 .44 2.. 24 .95 4 1.20 1.06 .90 • 1.23 1.15 2.63 1.74 .85 1.23 1.57 1 day •95 1.06 •95 1.12- .90 2 l a y s • 1.29 .95 •73 1.32 • 95 4 .84 .73 .76 1.01 .67 6 .70 .45 .60 .84 .48 In the 1949 experiments catalase- a c t i v i t y was estimated f o r stem and toot, but on the majority of estimations a c t i v i t y was too low to be measured by the method used i n t h i s i n v e s t i g a t i o n . CATALASE Dave nf* T p p r a y i n g r-6 1950 F i g . 16. :«talaee a c t i v i t y o f t r e a t e d l e a f compared t c t h a t o f c e n t r e 1. T/C, 1950. (To follow page 37 (d) Phosphatase The r e s u l t s of. phosphatase determinations may be seen i n table 15. Phosphatase was not. determined i n the. 1949 experiment. In stem r e s u l t s , treated.and control.follow the same trends throughout the experiment, sug-gesting that there i s no s i g n i f i c a n t difference produced by treatment. In l e a f , though the i n i t i a l values are very s i m i l a r , a f t e r two days there i s an i r r e g u l a r increase i n a c t i v i t y . Table 15- Phosphatase A c t i v i t y Expressed as Micrograms of Phenol Released. (Figs. If and 1&) Time af t e r Stem Leaf . treatment 0 T 0 T 0 90.5 92.0 2500 • • • 92.0 27.0 I f hrs 84.3 81.6 20.8 24.4 86.3 23.9 4 86.3 90.5 28.6 23.4 87-9 86.3 24.4 21.8 79.5 81.0 16.6 17.7 83.6 82.0 17.7 18.7 1 day 76.4 78.0 33.8 34.8 77.5 77.5 36.9 54.3 2- days 96.7 98.3 21.3 42.1 96.2 98.8 23.9 46.8 4 82.6 80.0 5443 37.0 82.2 81.6 32.2. 38.0 88.8 85-3 26.5 37.5 86.9 85-3 25.5 34.3 The r a t i o s T/0 were not computed f o r phosphatase determinations because treated values varvysd Earlier g r e a t l y j as also.do controls. There, appears to be no c o n s i s t e n t . r e l a t i o n between treated and controls, espec-i a l l y i n the i n i t i a l stages. Thus, to average the r e p l i c a t i o n s and com-pute T/0 trends where such have not been demonstrated would give a f a l s e PHOSPHATASE 70 1 1 1 1 h 1 2 4 6 Days after spraying L.M.C. 1950 O 10 1 1 —I 1 \ 1 2 A 6 Days after spraying L.M.C. 1950 Figs. 17 and 18. Phoephatase activity in stem ( f i g . 17, upper) and in leaf ( f i g . 18, lower), treated and control, Ordinates are micrograms phenol releaeed. (To follow page J J 58 impression. Therefore the values, for treated and control are graphed d i r -e c t l y , without c a l c u l a t i o n of treated as a f r a c t i o n of c o n t r o l . The above c r i t i c i s m could be applied-to the c a l c u l a t i o n of catalase r e s u l t s as T/0 r a t i o s . However, the inconsistency appears i n two readings, the.remainder presenting a regular trend. The a c t i v i t y of each separate har^ vest (1950) with reference to a given enzyme, has been tabulated i n order that the v a l i d i t y of T/0 trends, where calculated, may be judged. (e) Phosphorylase. Table 16. Phosphorylase A c t i v i t y Expressed as Micrograms of Phosphorus Released. (Figs. 19 and 20). Time a f t e r treatment 0 Stem T 0 Leaf T 0 80.0 88.4 • • • 164. 175. • • e !§• hrs. 122. 118. 155. 75.7 168. 147. 147. 4 89.2 65.2 80.0 89.2 122. 96.8 126. 89.2 8* 92.6 65 . I 126. 96.8 109-96.8 126. 122. 1 day 65,7 92.6 50.5 71.6 67.5 71.6. 71.6 88.4 2 days 46.5 46.5 58.9 58.9 55-7 29-5 50.5 42.1 4 65 . I 54.7 114. 101. 4.21 4.21 25.5 29.5 6 84.2 84.2 105.? 84.2 96.& 71.6 84.2 Stem phosphorylase r e s u l t s . a r e v a r i a b l e up to one day, but from there i s an increase i n a c t i v i t y . Leaf r e s u l t s are also v a r i a b l e during the i n i t i a l stages,-but by eight hours show a s l i g h t increase which l a s t s u n t i l approximately the f i f t h day. lAO-lo PHOfFHCHYLASE STEM • • Fife. 19 «»nd 20. Phopphorylftp* a c t i v i t y i n stem ( f i s t . 19, upper) and i n l e a f ( f i e . 2C, lower), treated and control. Ordinate:? are micrograms DhoF.phorus r^leaped. (Tc f o l l o w page 3 a 39 4. Enzyme A c t i v i t y — I n V i t r o The r e s u l t s of b r i e f experiments designed to.inv e s t i g a t e some of the e f f e c t s of 2,4-D i n v i t r o may be seen i n tables 17, 18 and 19. The prep-a r a t i o n of bean plant extracts and estimation.of enzyme a c t i v i t y were ca r r i e d out using the methods already described i n t h i s t h e s i s l Table 17- E f f e c t s of 2,4-D upon Catalase A c t i v i t y i n V i t r o T h i o s u l f a t e , net t i t r a t i o n 2,4-D—ppm. (cubic centimeters) 0 2.45 1 2.7 5 2..65 10 2.|5 25 2.45 50 2.45 100 2.2 500 2.4 1000 2.2' Table 18. E f f e c t s of 2,4-D upon Phosphatase A c t i v i t y In V i t r o . Colorimeter readings Source of enzyme -D—ppm Stem Leaf 0 122, 127* 197, 199 1 122, 128 5 125, 125 10 121, 119 202, 204 25 120, 121 50 120, 120 * 100 116, 120 201, 209 500 120, 117 1000 111, 109 199, 199 .one were c a r r i e d out i n duplicate. 4o Table 19. E f f e c t s of 2,4-D upon Phosphorylase A c t i v i t y In V i t r o . Colorimeter readings Source of enzyme '2,4-D--ppm Stem Leaf 0 72, 69* 28, 28* 1 68, 67 35, 55 10 78, 79 55, 55 25, 62, 65 55, 55 50 76, 77 54, 25 100, 70, 74 76, 78 31, 31 500 32, 28 1000 78, 79 36, 57 •Determinations were c a r r i e d put i n duplicate. Catalase i s apparently s l i g h t l y activated at 1 and 5 PP m» i s un-affec t e d at 10, 25 and.50. ppm, and.may. be s l i g h t l y i n h i b i t e d at high .(1000 ppn$ concentrations... In the. l i m i t e d t r i a l s made using phosphatase of l e a f , not e f f e c t could be detected. In the case- of stem phosphatase, there may be a s l i g h t . i n h i b i t i o n , increasing with.concentration. Stem phosphorylase shows i n h i b i t i o n at. 500 and 1000 ppm. Leaf phosphorylase.: shows a s l i g h t l y , i r r e g -u l a r a c t i v a t i o n . Peroxidase t r i a l s , which could not be repeated,because of lack of time, were highly i r r e g u l a r and no trend could be seen i n them. The following table presents a summary of the e f f e c t of 2,4-D on enzymes as discussed i n t h i s t h e s i s . The l e v e l s of concentration referred to as "low" and "high" are divided according to the enzyme concerned. For cat-alase and phosphorylase the d i v i d i n g l i n e f a l l s between 10 and lOOOppm and at 5 PP m f o r stem phosphatase. Al Table 20. Summary of E f f e c t s of 2,A-D on Enzyme A c t i v i t y i n Bean Plants. Source of Enzyme In Vivo Stem 1 Leaf t In V i t r o Stem : Leaf Oonc. 2,4-D Enzynw: Amylase Peroxidase Oatalase Phosphatase Phos' ylase 2000 ppm spray Low High Low High 4- then — —- tfe'en 4- 4-f i n a l l y -4-f i n a l l y -4^ -(Taka-d L a s t a s e — -decrease (50) 4" .unknown 4-0 4-0 f i n a l l y -L_ 0 unknown 4-Kfy.— -f" s l i g h t increase ~f—t" great increase — decrease Q-no e f f e c t 42 DISCUSSION What has . a c t u a l l y b e e n done, i s t o measure t h e a c t i v i t y o f a n e x -t r a c t w i t h r e s p e c t - t o a. g i v e n . e n z y m e of. t r e a t e d t i s s u e compared t o t h a t o f u n t r e a t e d t i s s u e . F rom t h e r e t h e a s s u m p t i o n has. b e e n made t h a t enzyme a c t -i v i t y i n t h e c e l l s c o n c e r n e d i s r e f l e c t e d i n t h i s . a c t i v i t y o f e x t r a c t s . Enzyme a c t i v i t y i r l p x t r a c t s f r o m t r e a t e d and u n t r e a t e d t i s s u e s m i g h t , d i f f e r i n a c t u a l , a c t i v i t y , b u t t h i s m i g h t mean a c t u a l l y t h a t t h e enzyme c a n be e x -t r a c t e d more r e a d i l y f r o m one t h a n f r o m t h e o t h e r . Thus r e s u l t s may d i f f e r f r o m one e x p e r i m e n t t o a n o t h e r . a c c o r d i n g t o t h e method u s e d . I t m i g h t be s u g g e s t e d , t h a t i n t e r p r e t a t i o n o f r e s u l t s i s c o m p l i c a t e d by t h e . p r e s e n c e o f r e s i d u a l 2 , 4 - D i n t h e enzyme e x t r a c t . T h i s was one r e a s o n f o r m a k i n g i n . v i t r o s t u d i e s . I t . i s c o n s i d e r e d , h o w e v e r , t h a t t h e s e s t u d i e s show t h a t s u c h e f f e c t i s l i k e l y - to .be n e g l i b l e , on t h e g r o u n d s t h a t t h e c o n -c e n t r a t i o n o f 2 , 4 - D i s g r e a t l y d e c r e a s e d by d i l u t i o n i n e x t r a c t i n g and i n m i x -i n g w i t h t h e s u b s t r a t e , 2,.4-D e f f e c t s , i n v i t r o a r e l e s s t h a n t h o s e o b s e r v e d o n enzymes e x t r a c t e d f r o m t r e a t e d p l a n t s , and a r e f r e q u e n t l y i n t h e o p p o s i t e d i r e c t i o n t o t h e e f f e c t o b s e r v e d i n v i v o . As w o u l d be e x p e c t e d f r o m t h e f a c t t h a t t h e p l a n t s i n t h e 1950 e x p e r i m e n t r e s p o n d e d f a s t e r t h a n t h o s e i n 1949, t h e enzyme changes a p p e a r e d more r a p i d l y a l s o . N o t i n g t h e i r r e g u l a r i t y o f d r y w e i g h t s i n r o o t s , i t w o u l d be e x -p e c t e d t h a t enzyme a c t i v i t y as d e t e r m i n e d w o u l d . a l s o be i r r e g u l a r . F o r t h i s r e a s o n and b e c a u s e o f . t h e t i m e - c o n s u m i n g n a t u r e o f t h e t a f e k . o f p r e p a r i n g t o o t e x t r a c t s , enzyme d e t e r m i n a t i o n s o n r o o t t i s s u e were n o t a t t e m p t e d i n 1950. 1. A m y l a s e R e s u l t s a r e i n agreement w i t h t h o s e o f N e e l y e t a l (j4) i n t h a t . b y t h e t i m e t h e p l a n t 1 s r e s p o n s e h a s become s t a b i l i z e d t h e r e i s a m a r k e d d e c r e a s e i n amylase a c t i v i t y . The increased digestion of starch i n culture as found by G a l l $23) i s probably due to the i n i t i a l increase i n a c t i v i t y , which i s indicated i n these experiments, but not detected i n that of Neely et a l , since determinations were made only at 6 days from treatment. The increase i n amylase a c t i v i t y probably causes the familiar loss of starch from stem tissues. The subsequent decrease of amylase a c t i v i t y could be due to the increased production of r i b o f l a v i n , thiamine and pantothenic acid, which are known to i n h i b i t amylase a c t i v i t y ( 3 2 ) . I t i s not known how soon the pro-duction of these vitamins begins to increase, so that a definite correlation with amylase a c t i v i t y throughout a time study cannot be made. In the case of leaf...amylase, Neely et a l observed.no changes. In view of the investigation reported here, i t i s possible that the former ob-servation was made, at a time when amylase intreated plants was on the decrease and was almost equal to that of controls.. I t i s more l i k e l y that the decrease of starch, i n leaf i s due to res p i r a t i o n , with f a i l u r e to replace carbohydrates (18) than to amylase action. 21 Peroxidase The very great increase i n peroxidase a c t i v i t y suggested that the production of peroxidase may be autocatalybic or related to an autocatalytic process. The rate of such processes when plotted on semi-log graph.paper, would give a straight l i n e , since when plotted on ordinary graph paper the curve i s exponential.(5)• Peroxidase a c t i v i t y i n treated stem was.plotted on the logarithmic scale and time on the. linear, scale of a sheet of semi-log graph paper. The results are.shown i n f i g . 21. and do.indicate that the production of peroxidase may be autocatalytic. Felber (10) noted an increase i n peroxidase a c t i v i t y i n protuberances i n bean leaves. In the experiments of t h i s t h esis, p r o l i f e r a t i o n s of the stem were observed, at approximately the time peroxidase began, to.increase. I t i s not known whether they began to develop prior to or following the increase i n peroxidase a c t i v i t y . A T o -fellow pa£e A~3 parallel-may be noted with growth curves generally, which are also expon-ential during the grand period. Thus the rapid production of.peroxidase may be definitely related to the production-of stem proliferations. Tumor-like tissue produced with B. tumefaciens or by freezing also posseB greater per-oxidase activity than normal tissue (l-9)« There the.similarity ends, how-ever, since these tissues also posess greater catalase activity and leaf results show that this was not observed i n these experiments. 5« Catalase An indication of the rapidity of the plant* s response may be seen i n the marked decrease i n catalase activity hours after treatment. De-creased, catalase activity was noted by Heinicke (2l) to accompany wilting and a similar correlation may be suggested here, for wilting was noticeable after treatment. Catalase activity.is also considered to parallel respir-atory activity (29), but this parallel is. doubtful i n these experiments. The slight i n vitro stimulation of catalase activity i s d i f f i c u l t to inter-pret. Further experiments are necessary to verify this result and i f poss-ible to determine the nature of stimulation. It i s evident, however, that the plant's decreased catalase activity i s not due to a direct effect upon catalase, since no inhibition was.detected with cettainty at concentrations less than 1000 ppm. Thus the decrease in catalase activity may be classed as a plant response rather than a direct effect of 2,4-D. 4. Phosphatase There appears to be no effect upon stem.phosphatase. That is not without meaning, when one considers that the other enzymes studied are aff-ected. Probably this phosphatase attacks hexose-6-phosphates and other phosphate esters of the glycolytic cycle, and possibly hastens the dephos-phorylation of ATP as mentioned Bonner's text (6). This would suggest that the amounts of these substances are not affected by phosphatase a c t i o n i n treated stem any more than i n . c o n t r o l stem, and .that interference, with.resp-i r a t i o n i s probably not accelerated or retarded by changes i n dephosphory-l a t i o n by phosphatase.. On the other hand, ..leaf phosphatase increases from 2 days on. By t h i s time leaves were: severely curled and twisted. I t i s possible that the increased a c t i v i t y of phosphorylase would remove, phos^ phorylated intermediates, thus i n t e r f e r i n g with, e f f i c i e n c y of r e s p i r a t i o n and p a r t i a l l y accounting f o r the decrease i n photosynthesis.. In v i t r o ex-periments indicate, a s l i g h t a c t i v a t i o n . o f leaf, phosphatase! f u r t h e r ex-periments to characterize the type of a c t i v a t i o n would be valuable. !». PhosphoryM.se I t i s . ' d i f f i c u l t to correlate these r e s u l t s with those of Neely et a l (27) since t h e i r work made observations at one harvest only. Moreover the method of preparing t i s s u e f o r enzyme determination may have an e f f e c t upon the indicated a c t i v i t y . The slow drying at 30° 0«- and.the water ex-t r a c t i o n of fresh material are l i k e l y to a f f e c t d i f f e r e n t enzymes i n . d i f f -erent ways, and there i s no guarantee that the enzyme a c t i v i t i e s of treated, and untreated plants are equally sensitive to any one method of extraction, much l e s s to more than one. General Considerations From stem results, i t may be suggested, that the increased amylase a c t i v i t y contributes to the modified metabolism c h a r a c t e r i s t i c of 2,4-D action, by working In the following manner: starch degradation by amyl-o l y s i s would, produce sugars, which, before being respired must be phos-r phorylated. For each molecule of hexose phosphorylated, one high energy bond of ATP i s required. The phosphate bond.so produced, has an energy of only, about.3000 c a l o r i e s , as compared with about 12,000 c a l o r i e s (per.mole) expended to produce i t . On the other hand, i f starch i s phosphorylated 46 d i r e c t l y , . b y phosphorylase. and inorganic phosphate, ATP. i s not required, and a saving of energy i s effected. A comparison of the curves of amylase and of phosphorylase. a c t i v i t y w i l l show that, there i s no c l e a r l y , indicated increase i n phosphorylase a c t i v i t y accompanying that of amylase a c t i v i t y , thus amylase would be c h i e f l y responsible for the. starch breakdown that takes place.. Under the conditions indicated, with, starch, degradation taking place mainly by amylolysis, a considerable supply of energy i s wasted. The problem of 2,4-D a c t i o n , s t i l l remains. The following consid-erations are offered as. a d e s c r i p t i o n of possible action. Amylase.activity probably. increases in. the plant, for. a short period after treatment, whereas catalase . a c t i v i t y decreases. Since these trends are... in. opposition to those observed i n v i t r o , i t i s l i k e l y that.they are an active response of the plant to 2,4-D and not a r e s u l t o f . r e s i d u a l 2,4-D i n the extract. This opposition of trends, i s not observed i n phosphatase and phoByhorylase r e s u l t s , c h i e f l y because there i.s. either no change in. the early stages, or the change cannot be distinguished from v a r i a b i l i t y ojlmaterial,... A s i m i l a r s i t u a t i o n may be seen in.experiments on alcohol and malic dehydrogenases, from oat c o l e o p t i l e s , using IAA. As previously,mentioned (20), IAA i n v i t r o s l i g h t l y i n h i b i t s the a c t i o n of malic dehydrogenase and possibly also that of alcohol dehydrogenase. In addition, oat c o l e o p t i l e s showed.a marked.increase i n a c t i v i t y of malic and alcohol dehydrogenases after having been soaked f o r 15 hours i n IAA $4). While t h i s opposition of trends may be only i l l u s o r y , the i n d i c -ations are strong enough to.suggest that an adaptation to i n h i b i t i o n or to a c t i v a t i o n may.take place.. This adaptation might occur i n a manner similar to drug adaptation i n b a c t e r i a . Hinshelwood (22) suggested an explanation f o r t h i s phenomenon as follows! When b a c t e r i a are treated with a drug 47 which impedes reaction "y", for example, of a chain of reactions A > B ^ 0 > D x y. •< . z the metabolite B accumulates. Enzyme production has been shown (46) to depend at least i n certain cases, notably fermentation, on the presence of. the sub-strate. Thus with an accumulation of B, the c e l l produces more of the enzyme which catalyzes reaction."y". The. c e l l i s now able, to carry on metabolism in the presence of the inhibiting dr^tg. Strains derived from this adapted c e l l are said to be "drug adapted". A similar mechanism may operate at certain enzyme sites, affected by 2,4-D. Thus.the opposition of tendencies of i n vivo and in vitro experi-ments would be explained. On the other hand, i f an enzyme i s slightly act-ivated by a chemical, in.this.case, the 2,4-D effect of catalase i n vitro, the substrate, namely hydrogen peroxide would be reduced.in. quantity and loss of enzyme would occur. This deadaptation also takes place (46). It i s thusysuggested that a possible mechanism, of operation of 2,4-D i s by way of a slight inhibition or activation of certain, enzymes, to which the c e l l responds by increased or decreased enzyme production, respectively. To test out this hypothesis i t . would be necessary f i r s t to estab-l i s h the extent.of inhibition.or activation and the enzyme systems affected. It i s not necessary that a l l be affected. Other enzymes, may be modified by interactions with, the system or systems i n i t i a l l y reacting.to 2,4-rD. It. would be valuable to compare 2,4-D action with that of well known specific metabolic inhibitors. Once the systems susceptible to 2,4-D had been noted with certainty, their activation and inactivation. character-i s t i c s with other chemicals might give clues as to. how.2,4-D affects them. Other growth regulating action should also be studied, with ref^-erence to enzyme, effects. It is tempting to suggest, that naturally occurring auxin operates in a manner similar to that just described for 2,4-D. 48 It may be aeked-r-if the plant, produces amylase i n response to 2,4-D treatment,' why does the activity of amylase eventually decrease? The answer would be that other factors, such as production by the plant of inhibiting substances and loss of proenzyme might operate here. CONCLUSION 49 On the basis of. a study of enzyme a c t i v i t i e s as modified by 2,4-D i n vivo and i n v i t r o , i t . i s suggested that the action of the herb-i c i d e and growth reguJkiLtor, 2,4-D may.be 1. By a c t i v a t i o n i n some cases and i n h i b i t i o n i n others of one or more enzyme systems. 2. The plant then reacts to t h i s treatment by accelerated or.depressed enzyme production. The mechanism for t h i s response may be that suggested by Hinshelwood f o r brug adaptation by b a c t e r i a . 50 LITERATURE CITED . 1. Audus, L. J . The mechanism of auxin a c t i o n . B i o l . Revs. 24:51-95- 1949. 2. B a l l s , A.K. and M a r t i n , L.F. Amylase a c t i v i t y of mosaic tobacco. Enzymologia 5:233-238. 1938. 3- Berger, J . and Avery, G.S. j r . A c t i o n of sy n t h e t i c auxins on dehydrogenases of Avena c o l e o p t i l e . Amer. Jour. Bot. 30:297-302. 1943. 4 . Berger, J . and Avery, G.S. j r . The mechanism of auxin a c t i o n . . 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