. , . __ x“ .n .6 .o... .m H. J L...» Q‘- ..~.v d ..o a Q n. r \ . 1s \ . O r . — . q I». I y C u "u A .y .0 u u 0 ‘ . v s _. _ ____‘_._-.. THESIS 0-169 2.“O O: .14— ‘ w L. 4 Al .L-f-ll ._"‘.oll \ This is to certify that the thesis entitled Some Effects of Maleic Hydraude on Phaseolue vulgaris presented by John E. Landgraf has been accepted towards fulfillment of the requirements for ._u._s._degree infionucu-Lture Major professor Date __m 281 1-9152 _ I ’4——y 7—.“ ”\ _ _—~—‘ w.— w s J I ' . I I In - -*~"'-' I" SOME EFFECTS OF MALEIC HYDRAZIDE 0N PHASEOLUS VULGARIS By JOHN ELSEB’LORE LANDGRAF A THESIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Horticulture 1952 ACKNOWLEDGMENTS The author wishes to express his sincere appreciation to Dr. Charles Hamner for the encouragement and assistance he has given during the course of this investigation; and to those, especially Dr. Harold Sell, who have examined this manuscript during its preparation. I also wish to thank Dr. Erwin Benne and his assistants under whose keen guidance the chemical analyses were made. TABLE OF CONTENTS Page Acknowledgments I. Introduction . . . . . . . . . . . . . . 1 II. Review of literature . . . . . . . . . . h III. Experimental studies: Effects of 2,h dichlorOphenoxyacetic acid on maleic hydrazide treated red kidney beans . . 6 A. Materials and methods. . . . . . . . 6 B. Results . . . . . . . . . . . . . . 8 IV. Experimental studies: The effects of maleic hydrazide on two plant viruses, common bean mosaic and tobacco mosaic . 12 A. Materials and methods... . . . . . . 12 B. Results . . . . . . . . . . . . . . 18 V. Discussion . . . . . . . . . . . . . . . 19 VI. Summary . . . . . . . . . . . . . . . . 30 Literature . . . . . . . . . . . . . . . . . . . 31 Appendix 0 O O O I O O O O O O O O I O O O O O 0 3L;- INTRODUCTION Maleic hydrazide is one of the many chemicals whose presence alters the appearance and the physiological activ- ity of plants. The cessation of apical bud growth is the earliest and probably the most characteristic action of maleic hydrazide. This symptom may be followed by others which are not manifested in all species and vary much with the plant's metabolic state. A few of these other symptoms are: l. The fully expanded leaves upon treatment increase in size, bebome darker green, and more brittle.. 2. Narrow, strap-shaped leaves may appear close to the apical bud, and shoots may arise from buds in lower leaf axils. 3. Freq- uently the stems increase in diameter. A. Sugar exudations may appear on the leaf surface, while within the leaves anthocyanin pigments increase. Chemical analyses of a few species of maleic hydrazide treated plants reveal an accu- mulation of carbohydrates (6, 8, 35), a decrease in the activity of some of the respiratory enzymes (11), and a lower rate of oxygen uptake (19). Thus, accompanied with an inhibition of apical meristematic processes, maleic hydra- zide appears to cause a reduction in the rate of plant respiration and an accumulation of the respiratory reactants, the carbohydrates. -2- Schoene and Hoffman (25) were the first to describe the activity of maleic hydrazide, and since their report this compound has attracted the attention of many in ag- ricultural research. Attempts have been made in using maleic hydrazide to reduce the cutting of lawns (25) and hedges (12); to delay blossoming and fruit formation in blackberries (33) and strawberries (2h); to thin peach fruits (13); in preventing ginko fruit formation (18); and as a selective herbicide (9). Of more success has been the work of Wittwer and his coworkers, who found that preharvest spraying with maleic hydrazide prevented the sprouting of carrots, onions (36) and potatoes (21). The changes in the appearance of a plant caused by biologically active chemicals are undoubtedly an index to changes in chemical composition. Fy the addition of rel- atively small amounts of these chemicals, the composition of plants might be altered to produce a more nutritious or palatable food. Of the three major foodstuffs carbohydrates, fats, and proteins, the latter are not only the most expen- sive but also an essential component of the diet. Thus, an increase in the protein content of plants would often be of nutritive and economic value. The plant growth substance 2,h dichlorOphenoxyacetic acid has been reported to increase the protein content (Kjeldahl nitrogen x 6.25) in the stems of red kidney bean plants (26), while in the leaves and roots -3- few changes in composition were noted (32)1. The value of this apparent protein increase is markedly offset by a drastic decrease in both the reserve and available carbohy- drate content. In contrast, carbohydrates tend to accu- mulate with.ma1eic hydrazide treatment. The investigation of this paper was an attempt to increase the protein content of plants by 2,h D treatment, without depleting the carbohy- drate content by previously applying maleic hydrazide. A second investigation resulted from an observation and preliminary experiment in which maleic hydrazide treated bean plants seemed to have a resistance to common bean mosaic infection. Because many of the respiratory enzymes and plant viruses are nucleOproteins, an inhibitor of a respiratory enzyme might conceivably destroy the self reproductivity of plant viruses. 1Expressed on a percentage basis REVIEW OF LITERATURE The observations of several investigators enabIe us to speculate as to where maleic hydrazide interferes with the metabolic processes of plants. First, the respiratory reactants, the carbohydrates, accumulate in maleic hydrazide treated plants. Girolami (8) observed that bean plants were gorged.with starch grains twenty-five days after maleic hydrazide treatment. The analyses of Currier, Day, and Crafts (6) revealed that the shoots of maleic hydrazide treated barley accumulated large amounts of polysaccharides (fructosans), while there was little change in the soluble carbohydrate content. Wittwer and Hansen (35) showed that sugar beets lost less sugar and were lower in temperature during storage if they were sprayed several weeks before harvesting with maleic hydrazide. Second, the activity of some of the respiratory enzymes is lowered upon maleic hydrazide treatment. Isenberg 3E g1 (11) determined the dehydrogenase activity in onions by measuring the rate cf hydrogen liberation with triphenyl tetrazolium chloride. For example: succinic dehydrogenase succinic acid - 2HL .‘l "fumaric acid + 2H 2H + tetrazolium chloride(colorless)-Otrimethyl formazin (red) -Lp. -5- When ethyl alcohol, succinic, fumaric, malic, and pyruvic acids were supplied with homogenate from maleic hydrazide treated onions a decrease in trimethyl formazin production was observed. This decrease in dehydrogenase activity was measured in etiolated onion plants seven days after maleic hydrazide treatment, and in the bulbs of non-etiolated plants after thirty days. Third, other evidence of respiratory inhibition is afforded by the work of Naylor and Davis (17) who observed that soon after immersing root tips of several species in a maleic hydrazide solution (pH h) the rate of oxygen uptake was lowered (at pH 6 no decrease was noted). These ob- servations-~the accumulation of respiratory reactants, the decrease in respiratory enzyme activity, and the lowering in the rate of oxygen uptake-~indicate that a most outstand- ing of perhaps several effects of maleic hydrazide is the inhibition of carbohydrate oxidation. EXPERIMENTAL STUDIES: EFFECT OF 2,L DICHLOROPHENOXYACETIC ACID ON MALEIC HYDRAZIDE TREATED RED KIDNEY DEANS Materials and Methods Red kidney beans were planted in four inch pots in a peat loam soil mixture in the greenhouse. From four seeds planted in each pot two uniform plants were selected for treatment. The treatments are illustrated in figure 1. Thirty plants were used in each of the four treatments, and each treatment was replicated three times. Preparattgg for analysis. Twenty-three days1 after planting all of the leaf blades were removed, and the re- maining stem tissue, consisting of the main axis and petioles, was cut from the roots at the soil level. This latter material was rapidly weighed and quickly killed by placing in a hot air oven at 100°C for three hours, and was further dried in a circulating hot air oven at 70°C for 60 hours. After cooling to room temperature, the stem tissue was weighed and then ground in a'Wiley mill to pass through a hfl mesh screen. Analyses. Total sugar. Dried, ground tissue (2.5 gms.) lApril 27, 1951 at k P.w. ~6- Fig. l The effect of M.H.1 and 2,hD2 treatments on the growth of red kidney bean plants. Plants from left to right treated with (1) control, (2) sprayed with 7.5 ml. of 1000 p.p.m. M.H. solutiOn 12 days after planting, (3) treated with 0.1 ml. of 1000 p.p.m. 2,1;1) solution3 17 days after planting, (h) sprayed with 7.5 ml. 1000 p.p.m. M.H. solution 12 days after lanting an treated with 0.1 ml. 1000 p.p.m. 2,fiD solution 17 days after planting. Photograph.was taken at time of harvest, 23 days after planting. .a.- lMaleic hydrazide (M.H.) used was formulated as the diethanolamine salt of 1,2 dihydro 3,6 pyridazinedione. Throughout these experiments 0.001 percent Aerosol was used as a wetting agent. 2The sodium.salt of 2,h dichlorophenoxyacetic acid (2,LD) solutions were adjusted to pH 3 with phosphoric acid. 3One 0.05 ml. drop of 2,hD was placed on each primary leaf blade. -8- was extracted with 80 percent ethanol. Proteins and tannins were removed from the extract by lead acetate precipitation, and the excess lead was removed with potassium oxalate. The extract was hydrolysed for 15 hours in 0.5M hydrochloric acid, and after neutralizing the acid with sodium hydroxide, reducing sugars were determined by the Munsen Walker proce- dure (2). Ether extract. One gram of dried, ground material was extracted with anhydrous ethyl ether for 16 hours in Bailey Walker extraction apparatus. The difference between the weights before and after extraction was expressed as ether extract. Protein. The standard procedure of A.0.A.C. (2) was followed to determine the Kjeldahl nitrogen content in one gram of the dried sample. The Kjeldahl nitrOgen, assumed to be completely derived from protein, was multiplied by 6.25 and expressed as protein. Results As illustrated in figure 1, the 2,h D and the M.H. treat- ments most markedly modified the external features of the younger leaves and buds, while the primary leaves, which at the time of treatment were fully expanded, seemed least affected. The 2,h.D produced striking proliferation in stem tissue on plants receiving no previous treatment, while surprisingly the proliferation was absent in the plants Table 1. -9- THE EFFECT OF M.H.; 2,kD ; AND M.H. 4- 2,1713 TREATMENTS ON THE CHEMICAL COMPOSITION OF THE STEMS OF RED KIDNEY BEAN PLANTS (expressed as milligrams per stem) ‘Repli- Treat- Free Dry ProteIn Total Etfier cation. ment was. Wgt. Water (Nx6.25) Sugar Extract 1 1730 258 1t70 32.6 23.2 6.70 2 Control 1770 258 1510 3k.0 23. 6.57 3 1800 250 1550 32.6 15. 9.32 Average 1770 255 1510 33.1 20.8 7.53 1 1170 215 95k k3.2 1i.8 fi.30 2 M.H. 1100 200 900 39. 1 .3 .37 3 1200 21k 986 i9.6 17. n.77 Average 1160 210 9N3 0.9 15.5 4.81 1 1960 2 0 1720 N9.3 18. 11.0 2 2,h-D 1930 zgu 1710 his? 19.3 6.86 3 19 0 2N2 1690 u%.0 18.8 5.72 Average 19 235 1710 a .0 19.0 7.8 1 1230 203 1030 H1.0 12.0 k.t6 2 N.H.e 1230 201 1030 N1.0 18.2 5.t7 3 2,NPD 1230 200 1030 N1.0 1k.1 5.1u Average 1230 201 .1030 N1.0 1t.8 5.02 Table 2. THE EFFECT OF M.H.; 2,hD; AND M.H. + 2,hD TREATMENTS ON THE CHEMICAL COMPOSITION OF THE STEMS 0F RED KIDNEY BEAN PLANTS (expressed as the average percentage of the control total yield) FreshIIDry Treatm°nt Wgt. Wgt. water IN:6?2§)2322: Eifiiiie Control 100 100 100 100 100 100 N. H. 65.5 83.3 62.5 123 7N.5 63.9 2,hrD 110 92.3 113 139 91.k 10k M.H. e 2,APD 69.5 78.2 68.3 12h. 71.2 66.7 -10- previously Sprayed with maleic hydrazide. In fact, few visual or chemical differences (as shown in figure 1 and tables 1 and 2) could be detected between the 1.3.1.. and the 1.1.11. 4- 2,11D treatments. Chemical analyses Showed that the stems of the M.H. and the M.H. + 2,hD treatments similiarly decreased in dry matter, water, ether extractable material, and total sugars; and markedly increased in protein. The total sugar analyses can hardly indicate either an increase or decrease in the total carbohydrate content, but in maleic hydrazide treated barley Currier, Crafts, and Day(6) reported an accu- mulation of polysaccharides and little change in the reducing and non-reducing sugar content. Girolami (8) also noted that maleic hydrazide treated bean plants were gorged with.starch grains, and it is probable that analyses of the maleic hydrazide treated beans of this experiment would reveal an accumulation of polysaccharides. The analyses of the stems of the 2,hD treated plants revealed an increase in water and protein content and a decrease in the total sugar and dry matter content. A decrease in the polysaccharide content of these 2,hD treated plants would be anticipated; for Sell, Luecke, Taylor, and Hamner (26) reported a very marked deple- tion in both readily available and reserve carbohydrates in the stems of 2,hD treated red kidney bean plants. The source of the protein (N x 6.25) increase in.all of the treatments remains an important point of speculation. It is the author's opinion that the apparent increase in protein (N x 6.25) -11- content of bean stems with maleic hydrazide or with 2,hD treatment is but an accumulation which in non-treated plants is used in further leaf synthesis. Without additional analyses of leaves and roots it is not possible to assuredly interpret these changes in chemical composition, yet it is clear that maleic hydrazide does markedly impede both the visual and the chemical manifestations of the otherwise very potent 2,h dichlorophenoxyacetic acid. EXPERIMENTAL STUDIES: THE EFFECTS OF MALEIC HYDRAZIDE ON TWO PLANT VIRUSES, COMMON BEAN NOSAIC AND TOBACCO MOSAIC Materials and Methods The effect of maleic hydrazide on the self-reproductivity of both common bean and tobacco mosaic viruses was studied. Inoculation with common bean.mosaic strain 151 were made on Michelite2 bean plants grown in four inch pots. Inoculations with tobacco mosaic were made on Common Havana tobacco plants3 grown in eight indh pots. In order to demonstrate the self-reproductive ability of maleic hydrazide treated viruses the following procedures were used: Procedure I. Virus inoculations were made on untreated bean and tobacco plants. After the virus symptoms were 1Beanmosaic inoculation technique. A thin.line of carborundum (grit 320) was Spread across the upper surface of one of the primary leaves. The inoculum, prepared by macerat- ing virus infected leaves in a Waring blender, was applied to the carborumdum surface. Minute punctures were made in the leaf by rubbing this surface. Immediately after, the leaves were waShed with water. 2The variety Michelite was used because it expressed distinct symptoms of common bean mosaic. 3Common Havana tobacco plants were inoculated with tobacco mosaic by rubbing both the upper and lower surfaces of one of the leaves with the juice from virus infected tobacco leaves. The leaf was then.washed with water. -12- -13- evident maleic hydrazide was applied. Inoculum.was prepared from each of these treated plants, and was used to inoculate untreated bean and tobacco plants. Twenty-five days later observations were made to detennine the number of these plants which had become virus infected. Procedure II. Maleic hydrazide was applied to untreated bean and tobacco plants. Later virus inoculations were made on the maleic hydrazide treated plants. Inoculum was pre- pared from eaCh of these treated plants, and was used to inoculate untreated bean and tobacco plants. Twenty-five days later observations were made to determine the number of these plants which had become virus infected. Details of these procedures are described in figures 2, 3, h, and 5. -1A- Procedure I Fig. 2 The effect of maleic hydrazide and common bean mosaic on the growth of Michelite bean plants. Plants from left to right treated with (1) control, (2) inoculated with virus lE’days after planting, (3) inoculated with virus 12 days after planting and sprayed with 10 ml. 250 p.p.m. M.H. 32 days after planting, (it) sprayed.with 10 ml of 250 p.p.m. M.H. 32 days after planting. The photograph was taken AZ days after planting. Forty-two days after planting inoculum from each of the above virus inoculated plants was prepared and used to inoculate 12 day old untreated bean plants. oinaom need natures gaowi airy Hi. .Bfir bedaiuooai (S) .Icm‘ I39 33.01905: ( 5 3 ddiw tevaxqs be u (11,: ,gx:1.3ra§ft; rr .. .o - ,.; t“ " - -:v+}£ 3". 0. .‘f o... I I s -15- Fig. 3 The effect of maleic hydrazide and tobacco mosaic on the growth of Common Havana tobacco plants. Plant on the left inoculated with tobacco mosaic when two months old. Plant on the right was inoculated with tobacco mosaic when two months old, and sprayed with 100 ml of 1000 p.p.m. M.H. twenty days after the virus inoculation. Photograph taken of h month old plants. When these virus inoculated plants were three months old an upper leaf was removed and used to inoculate one eight inch high untreated tobacco plant. -16- Procedure II K5 ' it Fig. h. The effect of maleic hydrazide and virus inoculations on the growth of Mishelite bean plants. Plants from left to right treated with (1) control, (2) inoculated with virus 22 days after planting, (3) sprayed with 5 m1 250 p.p.m. M.H. 12 days after planting and inoculated with virus 22 days after planting, (h) sprayed with 250 p.p.m. maleic hydrazide 12 days after planting. Photograph taken E2 days after planting. Forty-two days after planting inoculum from each of the above virus inoculated plants was prepared and used to inoculate 12 day old, untreated bean plants. -17- Procedure II P” l l Fig. 5 The effect of maleic hydrazide and tobacco mosaic on.the growth of Common Havana tobacco plants. Plant on the left received no treatment. Plant on the right was sprayed. with 30 ml of 250 p.p.m. maleic hydrazide when two months old. Photograph was taken of four:month old tobacco plants. When the virus inoculated plants were three months old one of the terminal narrow, strap Shaped leaves was removed and used to inoculate one eight-inch high, normal tobacco plant. -18- Results Very distinct mosaic symptoms were evident twenty-five days after the untreated tobacco and bean plants had been inoculated with samples from the maleic hydrazide, virus treated plants. Apparently maleic hydrazide does not destroy or directly influence the self-reproductive ability of these viruses. Anson and Stanley (1) report that oxidation of sulfhydryl groups of tobacco mosaic virus does not impair the virus activity. It has been suggested that maleic hydrazide inhibits plant growth by reacting with essential sulfhydryl groups. If this were true maleic hydrazide would not be expected to destroy tobacco mosaic activity. These investigators also report that iodination of the tyrosine residues 1g‘11333 destroys tobacco mosaic activity. Substances which.might be predicted to react with tyrosine groups would seem worthy of investigating as selective virus inhibitors. DISCUSSION In.this experiment maleic hydrazide prevented.the action of 2,h dichlorophenoxyacetic acid. The latter is one of a large number of plant growth substances. This group is characterized by the ability to promote cell elongation. If the symptoms of maleic hydrazide inhibition are compared with the responses promoted by plant growth substances we see that maleic hydrazide often prevents the action of the plant growth substances. Bonner (4') has listed twenty responses to plant growth substances, and this list is compared with responses to maleic hydrazide in table 3. Of twenty responses to plant growth substances ten are prevented by maleic hydrazide, and with closer observation other opposite effects might be noted. Thus, it seems that maleic hydrazide not only prevents the action of 2,h dichlorOphenoxyacetic acid but also prevents the action of the naturally occurring plant growth substance, indoleacetic acid.' The question which presents itself is by what mechanism does maleic hydrazide interfere with the plant's metabolism. The chemical reactions within plants and animals are unique for they are dependent upon proteinaceous catalysts, the -19- -20- Table 3. A COMPARISON OF THE RESPONSES PROMOTED BY PLANT GROWTH SUBSTANCES TO THOSE OF MALZIC HY“RAZIDE Physiological Responses to Plant Physiological Responses to Growth Substances (4) _ Maleic Hydrazide Cell elongation Inhibition of cell elongation ! Avena curvature test I X LeOpold and Klein (1h) Avena section growth 9 X Leopold and Klein (Di) Split pea stem curvature I X Leopold and Klein (1h) Flower peduncles X numerous eg. Schone and Hoffman (25) Epinasty X in results of this paper Leaf veins Algae Cell division Inhibition of cell division New root formation Callus growth Cambial activity X numerous eg. Schone and Hoffman (25) Parflhenocarpy Inhibition Promotion Axillary buds X numerous eg. Schone and Hoffman (25) Root growth ' Flower formation Photoperiodic induction Abscission of petioles X Watson (30) Miscellaneous Promotions Miscellaneyus Inhibitions Promotion of flower initiation . Ripening of fruit X White (33) Closing of stomata . Nonosmotic water uptake X Tso (29) -21- enzymes. Because of the essentiality, the minute concentrations, and the highly reactive nature of certain functional groups of the enzymes, other biological antag- onists often interfere by attacking the enzymes. Two ways that maleic hydrazide might inhibit enzyme action are: First, maleic hydrazide might act as a competitive enzyme inhibitor, as does malonic acid. Malonic acid inhibition is represented where M is malonic acid, E the enzyme succinic dehydrogenase, and MB is the malonic acid enzyme complex (22). M & E :::2:ME The normal reaction is represented where S is succinic acid, E the enzyme succinic dehydrogenase, SE use enzyme substrate complex, and F the endproduct, fumaric acid. S 9 E :;==: SE ::=£= F + E Because of the structural similiarity between malonic and succinic acids, succinic dehydrogenase can combine with either of these two acids, yet the enzyme can dehydrOgenate only succinic acid, the natural substrate. H2 Hz H2 HOOC - C -C -COOH HOOC -C -COOH Succinic acid Malonic acid The ME complex formation ties up the enzyme and prevents it from reacting with succinic acid. Structurally fumaric acid and maleic hydrazide are similar, and a competitive type inhibition between these two seems possible. -22- 0 II /C\ HC -COOH HC NH HO C 3H Pg RH O -" .L Fumaric acid Maleic hydrazide Competitive inhibitors could be overcome by the addition of an excess of the particular normal substrate, for the interaction of an enzyme with a substrate is a reversflale reaction. Competitive inhibition is usually reported from work on isolated enzyme systems. It is doubtful that this type of reversible inhibition would occur in the intact plant; for it produces a continuous supply of sdbstrates, which might readily overcome the inhibitor. Second, maleic hydrazide might irreversibly combine with an enzyme and destroy an essential group or config- uration. Maleic hydrazide might be expected to react like the quinones, for the two are structurally similiar. The quinones, lactones, and other unsaturated ketones often interact with the sllfhydryl groups of pnateins (7) 0 OH II I C\ C\ HC CH Ht/ 0 - SR II + RSH I ' HC CH RS -c on -23- Again maleic acid and maleic hydrazide are closely related. Llorgan and Friedmann (17) isolated the addition products of maleic acid with several sllfhydryl compounds. Cysteine formed a simple addition product with maleic acid. “Hz HOOC - OH I ll {.Hs-CHZ-CH-Coos ,- HOOC -CH NH2 HOOC - CH - S - CH2 - CH - COOH HOOC - CH2 Hopkins, Morgan, and Lutwak-Mann (10) demonstrated that the activity of succinic dehydrogenase was lowered in the presence of maleic acid, and interpreted this inhibition as an interaction with essential sulfhydryl groups of the enzyme. Other dehydrogenases were not inhibited by maleic acid or different SH group inhibitors. Apparently, the H group is not exposed or not essential for the action of these enzymes. The ions of the heavy metals,for example, mercury and (c0pper interfere by combining with the sulfhydryl group. Cysteine, hydrogen sulfide, glutathione or other compounds containing the SH group can'be used as antidotes for mercury and.copper poisoning in.animals (27). The.metal ions react with the sulfhydryl group of the antidote instead of the enzymes. If maleic hydrazide is a sulfhydryl inhibitor, per- haps, its action could be prevented with compounds containing -2u- the sulfhydryl group. Although the inhibition of carbohydrate oxidation is an outstanding effect of maleic hydrazide, if it is a sulfhydryl inhibitor its attack would not be limited to the respiratory enzymes, but would inhibit any enzyme whose SH groups were exposed and essential for'activity. The question remains as to how maleic hydrazide is able to prevent the action of both 2,4 dichlorOphenoxyacetic and indoleacetic acid. Bonner (5) has shown that inhibitors of the respiratory enzymes (the soluble salts of heavy metals, iodoacetic acid, HCN, H23, CO, and sodium azide) reduce the elongation of Azggg coleOptiles in indoleacetic acid. Leopold and Klein (1h) report that maleic hydrazide also demonstrates this action. It would appear that indoleacetic acid action is dependent upon.the rate of plant respiration, yet certain substances have been found which inhibit the action of plant growth substances but do not reduce respiratory rates. Arsenic acid, when present in low concentrations is one such substance. Heedham Pillai (20) have found that low concentrations of arsenic acid replace phOSphoric acid in triose phosphate oxidation. -25- 3 phosphoglyceraldehyde + H ASOQ l arseno, 3 phosp o g yceraldehyde (normal reaction) 3 phosphOglyceraldehyde + H3POLI- :,, 1,3 diphosphoglyceraldehyde l arseno, 3 phosphoglyceraldehyde f oxid. DPN l arseno, 3 phosphoglyceric acid + red. DPN (normal reaction) 1,3 diphospho glyceraldehyde 4 oxid. DPN au.l,3 diphospho glyceric acid 4 red. DPN l arseno, 3 phospho glyceric acid + HE ,, O 3 phosphOglyceric acid ' H3 sOu (normal reaction) 1,3 diphospho glyceric acid f H O ADP _;,,3 phosphoglyceric acid + ATP Low concentrations of arsenic acid do not reduce the rate of plant respiration, but do prevent the production of energy rich phosphate (ATP). Another substance, 2,h dinitro phenol, is a powerful inhibitor of indoleacetic acid, yet Bonner (3) has found it to increase respiratory rates. Loomis and Lipmann (16) have found 2,4 dinitro phenol to uncouple from respiration the production of ATP. The action of indoleacetic acid appears then not to be directly dependent upon the rate of plant respiration but directly upon the production.of ATP. Adenosine triphosphate is a most important product of the respiratory process; and much of the energy released in respiration is used to synthesize the high energy anhydride linkage formed between phosphoric acid and adenosine mono or di phosphate. -25- NH2 I C I \ Ester linka/ge N C"'N' 0 3,000 cal. mole. | I T::CH )’ H H \ H HC c-—-N c - c - c - c - c -