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Distribution, metabolism, and localisation of cyclohexanecarboxylic acid, a naphthenic acid in Phaseolus… Padmanabhan, Usha 1972

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DISTRIBUTION, METABOLISM, AND LOCALISATION OF CYCLOHEXANECARBOXYLIC ACID, A NAPHTHENIC ACID IN PRASEOLUS VULGARIS L. by USHA PADMANABHAN B.Sc. (Hons), U n i v e r s i t y of Delhi, 1965 M.Sc. U n i v e r s i t y of Delhi, 1967 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of Botany We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the requi rements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia , I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and s tudy . I f u r t h e r agree t h a t permiss ion fo r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . It i s understood that copying o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l ga in s h a l l not be a l lowed wi thout my w r i t t e n p e r m i s s i o n . Depa rtment The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada D a t e ^ g j b f ^ x / w - 13. 1979^ ABSTRACT Naphthenic acids, obtained from crude petroleum, have been known f o r the past two decades, to act as plant growth stimulants. The present i n v e s t i g a t i o n deals with several aspects of the fate of cyclohexanecarboxylic acid (CHCA), a component of the naphthenic acid mixture, i n bush bean plants, Phaseolus v u l g a r i s L. c u l t i v a r "Top Crop" f ollowing a p p l i c a t i o n to the primary leaves. A f o l i a r spray of 0.01 M CHCA applied to the primary leaves of 14-day old bush bean plants increased the vegetative and reproductive growths. The glucose conjugate of CHCA was the f i r s t metabolite to be formed ( i n 1/8 hr) i n the leaves treated with l a b e l l e d CHCA as a K s a l t (KCHC-7- 1^C). The aspartate conjugate of CHCA and an unknown metabolite 'Y1 were detected about an hour a f t e r treatment. These three metabolites were present i n the plant u n t i l four weeks a f t e r treatment. No free CHCA was detected 8 hr a f t e r a p p l i c a t i o n . This suggests that the metabolites, rather than the free acid, were respon-s i b l e f o r the growth stimulation observed. The metabolites of CHCA hence cannot be merely detoxication products. 14 The major f r a c t i o n of the ethanol soluble C a c t i v i t y remained i n the treated primary leaves. The lowest amounts of a c t i v i t y were found i n the f r a c t i o n composed of buds, flowers and pods. This pattern of d i s t r i b u t i o n of a c t i v i t y was observed at 1, 2, 3 and 4 weeks a f t e r a p p l i c a t i o n of KCHC-7-^C. The glucose and aspartate conjugates of CHCA and the u n i d e n t i f i e d compound 'Y* were present i n the plant i i at the times mentioned above. At the end of each week, an amount of a c t i v i t y equal to 0.4% or less of the t o t a l ethanol-soluble a c t i v i t y was found i n ethanol-insoluble plant residues. 14 CHCA was decarboxylated by bean plants. The C O 2 released during a period of seven days accounted f o r 3 3 % of the a c t i v i t y absorbed. The d i s t r i b u t i o n of the a c t i v i t y i n the plant, following a p p l i c a t i o n of KCHC-7-^C to primary leaves, involved both b a s i p e t a l and acropetal movements. The basipetal movement occurred i n the phloem and the acropetal, i n the xylem and phloem. The tr a n s l o c a t i o n of CHCA was favoured by l i g h t . Evidences obtained suggest that energy i n the form of ATP was required f o r t r a n s l o c a t i o n . In the dark, the p r o v i s i o n of glucose favoured the tr a n s l o c a t i o n , perhaps by serving as a source of ATP v i a r e s p i r a t i o n . A supply of aspartate i n the dark favoured the tr a n s l o c a t i o n s l i g h t l y . The r a d i o a c t i v i t y from CHCA was l o c a l i z e d i n the chloro-p l a s t s of the c e l l . An ethanol extract of these organelles contained both glucose and aspartate conjugates of CHCA. I t i s suggested that the chloroplasts represent an important s i t e of action of CHCA. The e f f e c t s of the naphthenate mixture and the i n d i v i d u a l naphthenate, cyclohexanecarboxylate on growth show marked s i m i l a r i t i e s . I t i s pos s i b l e that the cyclohexanecarboxylic acid may be the chief growth promoting component of the naphthenic acid mixture.. TABLE OF CONTENTS . Pag ABSTRACT i TABLE OF CONTENTS i i i ABBREVIATIONS v LIST OF TABLES v i LIST OF FIGURES v i i ACKNOWLEDGEMENT x INTRODUCTION 1 LITERATURE REVIEW 6 MATERIAL AND METHODS • 22 GROWTH OF PLANTS 22 CHEMICAL 22 1. Spray experiments 22 2. Movement of acid within the plant 23 3. Time course study of the appearance of metabolites of CHCA-7-l4C 23 4. Persistence of acid and i t s metabolites i n the plant 24 5. Determination of the a c t i v i t i e s i n the plant residues a f t e r ethanol extraction 26 6. Evolution of ^ CO^ by treated plants 26 7. Translocation of CHCA out of the treated l e a f 26 8. L o c a l i s a t i o n of r a d i o a c t i v i t y from KCHC-7-^C i n the tissues 28 9. C e l l f r a c t i o n a t i o n using aqueous media 29 10. C e l l f r a c t i o n a t i o n using non-aqueous medium 30 i v Page RESULTS 33 1. Growth 33 2. Movement of acid within the plant 36 3. Time course study of the appearance of metabolites of CHCA-7- 1 4C 41 4. Persistence of the acid and i t s metabolites i n the plant. .. 46 5. A c t i v i t i e s i n the plant residues a f t e r ethanol extraction • . 55 6. Evolution of ^CC^ by treated plants 55 7. Translocation of CHCA out of the treated l e a f 5 8. L o c a l i s a t i o n of a c t i v i t y i n tissues 67 9. C e l l f r a c t i o n a t i o n using aqueous media 74 10. C e l l f r a c t i o n a t i o n using non-aqueous medium 78 DISCUSSION 83 1. Growth 83 2. Movement of acid within the plant 84 3. Appearance of metabolites of CHCA - a time course study 87 4. Persistance of the acid and i t s metabolites i n the p l a n t . . . . 89 5. A c t i v i t y i n the residues from ethanol extraction 92 6. Decarboxylation of CHCA-7- 1 4C 92 7. Translocation of CHCA out of the treated l e a f 94 8. L o c a l i s a t i o n of CHCA i n t i s s u e s . 97 9. C e l l f r a c t i o n a t i o n 97 SUMMARY 101 BIBLIOGRAPHY 103 APPENDIX 114 ABBREVIATIONS BA Benzoic a c i d BAsp Benzoyl a s p a r t i c a c i d CHCA Cyclohexane carboxylic a c i d CHC CoA Cyclohexanecarboxyl coenzyme A CHCA-aspartate or CHCA-aspartic a c i d A s p a r t i c a c i d conjugate of CHCA GA G i b b e r e l l i c a c i d Glucose-BA Glucose conjugate of BA IAA Indoleacetic a c i d IAA-glucose Glucose conjugate of IAA IAA-glutamic acid Indoleacetyl glutamate IAAsp Indoleacetylaspartate KCHC Potassium eyelohexanecarboxylate KNap Potassium naphthenate NAA Naphthalene a c e t i c acid NAA-aspartic a c i d A s p a r t i c acid conjugate of NAA NAA-d-glucose Glucose conjugate of NAA NaNap Sodium naphthenate PPO 2,5-Diphenyloxazole POPOP 1,4-Bis 2-(5-phenyloxazolyl) benz 2,4-d 2,4-Dichlorophenoxyacetic a c i d 2,4-D glutamate Glutamic acid conjugate of 2,4-D V I LIST OF TABLES Page TABLE j The percentage d i s t r i b u t i o n of carbon-14 i n plant parts, one, two, three, and four weeks a f t e r a p p l i c a t i o n of KCHC-7- 1 4C 47 I I Translocation of KCHC-y-^^C and i t s metabolies out of the l e a f under d i f f e r e n t conditions. D i s t r i b u t i o n of the carbon-14 i n leaves, stem and root, as a percentage of the t o t a l , 12 hr a f t e r a p p l i c a t i o n of KCHC-7- 1 4C 58 I I I Translocation of KCHC-7--'-4C and its metabolites out of the lea f under d i f f e r e n t conditions. D i s t r i b u t i o n of the carbon-14 i n leaves, stem, and root, as a percentage of the t o t a l , 24 hr a f t e r a p p l i c a t i o n of KCHC-7- 1 4C ,... 60 IV C e l l f r a c t i o n a t i o n . D i s t r i b u t i o n of carbon-14 i n d i f f e r e n t c e l l f r a c t i o n s , as a percentage of the t o t a l 76 v i i LIST OF FIGURES Figure Page l a Control and treated plants s i x days a f t e r a f o l i a r spray of 0.01M CHCA i n 0.3% Tween 20 34 lb Control and treated plants i n the growth room, s i x days a f t e r a f o l i a r spray of 0.01M CHCA i n 0.3% Tween 20 34 2a Control and treated plants, 18 days a f t e r a f o l i a r spray of 0.0LM CHCA i n 0.3% Tween 20 35 2b Control and treated plants, one week a f t e r a p p l i c a t i o n of 0.0005M KCHC-7- 1 4C on the adaxial surface of the primary leaves 35 3a Autoradiograph of plant parts showing the d i s t r i -bution of a c t i v i t y \ ^ r a f t e r i n j e c t i o n of KCHC-7-^ 4C into the midrib of a primary leaf 37 3b Autoradiograph of plant parts showing the d i s t r i -bution of a c t i v i t y 24 hr a f t e r i n j e c t i o n of KCHC-in t o the midrib of a primary leaf 37 4a Autoradiograph of the treated leaf and shoot apex \ hr a f t e r i n j e c t i o n of KCHC-7- 1 4C in t o the midrib of the le a f 38 4b Autoradiographs of the treated ( l e f t ) and the untreated ( r i g h t ) primary leaves, 1 hr a f t e r i n j e c t i o n of KCHC-7- 1 4C in t o the midrib 38 5 Autoradiographs of primary leaves i n j e c t e d with KCHC-7- 1 4C 39 6 Autoradiographs of ethanol extracts of treated primary leaves, 0, 1/8, 1/4, 1/2, 2/3, 1, 4, 8, 24, 48 hr a f t e r a p p l i c a t i o n of KCHC-7- 1 4C 42 7 Metabolites of CHCA i n primary leaves treated with KCHC-7- 1 4C 44 8 The percentage d i s t r i b u t i o n of carbon-14 i n plant parts, one, two, three, and four weeks a f t e r a p p l i c a t i o n of KCHC-7- 1 4C 49 v i i i F igure Page 9a Autoradiographs of ethanol extracts of plant parts, one week a f t e r a p p l i c a t i o n of KCRC-7-^C to the primary leaves 51 9b Autoradiographs of ethanol extracts of plant parts, two weeks a f t e r a p p l i c a t i o n of KCHC-7-l 4C to the primary leaves 51 10a Autoradiographs of ethanol extracts of plant parts, three weeks a f t e r a p p l i c a t i o n of KCHC-7--'-4C to the primary leaves 52 10b Autoradiographs of ethanol extracts of plant parts, four weeks a f t e r a p p l i c a t i o n of KCHC-7-14-C to the primary leaves 52 11 R e l a t i v e amounts of CHCA-glucose and CHCA-aspartic acid i n plant parts, one, two, three, and four weeks a f t e r a p p l i c a t i o n of KCHC-7 - 1 4 c 54 12 Decarboxylation of C H C A - 7 - b y bushbean plants following a p p l i c a t i o n to the primary leaves 56 13 T r a n s l o c a t i o n of CHCA and i t s metabolites out of the l e a f under d i f f e r e n t conditions. D i s t r i b u t i o n of the carbon-14 i n leaves, stem, and root, 12 and 24 hr a f t e r a p p l i c a t i o n of KCHC-7-l^C 61 14 Autoradiographs of ethanol extracts of the treated l e a f , the untreated l e a f , the stem, and the root, 24 hr a f t e r a p p l i c a t i o n of KCHC-7-l^C as a drop to the base of one primary l e a f . a. of the plant that was maintained under the growth room condition 63 b. of the plant, supplied with water from the cut end of primary l e a f i n the dark 63 15 Autoradiographs of ethanol extracts of the treated l e a f , the untreated l e a f , the stem, and the root, 24 hr a f t e r a p p l i c a t i o n of KCHC-7--'-'^ C as a drop to the base of one primary l e a f a. of the plant that was supplied with glucose from the cut end of the primary l e a f , i n £he dark 64 b. of the plant supplied with aspartate from the cut end of the primary l e a f , i n the dark , 64 i x Figure Page 16 a,b Microautoradiographs of stem of treated plant 69 17 a,b Microautoradiographs of stems of plants whose leaves were treated with KCHC-7- 1 4C 70 18 a,b Microautoradiographs of leaves treated with KCHC-7-14C 71 19 a,b Microautoradiographs of leaves treated with KCHC-7- 1 4C 72 20 a,b Microautoradiographs of leaves treated with KCHC-7- 1 4C 73 21 Schematic representation of the aqueous f r a c t i o n -a t i o n procedure 75 22 Carbon-14 a c t i v i t y p r o f i l e of a CCl^-hexane density gradient of homogenate of CHCA-7-l 4C treated leaves 79 23 Carbon-14 a c t i v i t y p r o f i l e of a CCl,-hexane density gradient of homogenate of CHCA-7-l 4C treated leaves 80 24 a Photomicrograph of the chloroplast f r a c t i on incubated with t e l l u r i t e and succinate 81 b Photomicrograph of f r a c t i o n s from the bottom of the c e n t r i f u g a t i o n tube, incubated with t e l l u r i t e and succinate 81 ACKNOWLEDGEMENTS I am deeply g r a t e f u l to Professor D. J . Wort f or his guidance and encouragement throughout the course of t h i s study and f o r his c r i t i c a l review of the manuscript. I wish to express sincere thanks to Drs. Beverley R. Green, T. B i s a l p u t r a , G. C. Hughes, and G. E. Rouse of the Department of Botany and to Professor W. J . Polglase of the De-partment of Biochemistry, f o r t h e i r advice and assistance concern-ing t h i s i n v e s t i g a t i o n and t h e i r valuable c r i t i c i s m s of the manuscript. The constructive suggestions from Dr. R. T. Wedding, Department of Biochemistry, U n i v e r s i t y of C a l i f o r n i a , Riverside, are g r a t e f u l l y acknowledged. I wish to thank the U n i v e r s i t y of B r i t i s h Columbia for the f i n a n c i a l assistance i n the form of a Graduate fellowship and the Department of Botany f o r the p r o v i s i o n of excellent f a c i l i t i e s . This thesis i s dedicated to my husband Paddy and my son Ram, whose patience and cooperation helped make th i s work p o s s i b l e . INTRODUCTION The discovery of the natural plant hormone i n d o l e a c e t i c acid i n the 1930's led to s p i r i t e d research for synthetic compounds that would have s i m i l a r e f f e c t s on plant growth. Thus, phenoxyacetic a c i d , naphthaleneacetic acid, benzoic acid, the indoles and the various d e r i v a t i v e s of the above, attained recognition as plant growth stimulators and l a t e r became well known i n a g r i c u l t u r e and h o r t i c u l t u r e f o r the various a p p l i c a t i o n s to which they could be put. I t i s i n t e r e s t i n g to note that the majority of plant growth substances, i n c l u d i n g the ones mentioned above, are aromatic compounds. To the few non-aromatic plant growth regulators can be added the naphthenic acids group. The name, naphthenic acids, was suggested by Markovnikoff and Oglobin (75) f o r the C11^20^2 a°fds °f unknown structure which had been recovered from Rumanian crude o i l . Commercial"naphthenic a c i d " contains a l l the a c i d i c components of the crude petroleum and varying amounts of non-acidic compounds, mostly hydrocarbons. J o l l y (64) described naphthenic acids as monocarboxylic acids of the naphthene s e r i e s of hydrocarbons. He suggested a general formula R(CH2) nC00H where R represented a c y c l i c nucleus of one or more r i n g s . Cyclopentane rings predominated but cyclohexane rings were also shown to be present (23). Using g a s - l i q u i d chromatography, Eider (31) showed the presence of cyclopentane-, cyclohexanecarboxylic acids, and cyclo-hexaneacetic acid along with formic, a c e t i c , i s o b u t y r i c , methyl pentanoic, COOH Cyclohexanecarboxylic acid C H A , molecular weight 128. Naphthenic acid - general formula n-hexanoic acids and some phenols i n the naphthenic acid f r a c t i o n from an Aruba crude o i l . Borisova et al (21) separated cyclopentane-, cyclohexanecarboxylic acids, and cyclohexylpropionic acid from C^_g napthenic a c i d by gas l i q u i d chromatography. The a c i d f r a c t i o n from an Austrian crude, contained about 120 d i f f e r e n t compounds, 20 of which constituted more than 507, of the sample (20)• P o s i t i v e i d e n t i f i c a t i o n was made for 3-methyl and 3-ethyl branched chain f a t t y acids, The presence, i n small amounts,of substituted cyclopentyl and cyclohexyl acids, was also detected. S e i f e r t et a l (93,94,95) found that carboxylic acids comprised 2.57o by weight of C a l i f o r n i a crude o i l . They also showed the presence of terpenoid, poly-nuclear saturated and mono- and polynuclear aromatic as well as naphtheno aromatic r i n g s t r u c t u r e s . The authors i d e n t i f i e d 20 new compound classes of carboxylic acids that represented ca 257, of the carboxylic acids. Naphthenic acids occur i n almost a l l crude petroleums i n varying amounts. The a c i d content of the American crudes varies from 0.03 to 3.07». Usually, the lower the p a r a f f i n content, higher the acid content. In most cases the f r a c t i o n s of b o i l i n g - p o i n t range up to 700°F have the highest concentration of naphthenic acids. Naphthenic acids are not present i n the gasoline f r a c t i o n . Naphthenic acids are o i l y l i q u i d s with a c h a r a c t e r i s t i c odour, which i s due to contamination with phenols and sulphur compounds. The mixture i s hig h l y soluble i n hydrocarbons. The lower molecular members are s l i g h t l y soluble i n water while the rest are i n s o l u b l e . Naphthenic acids can be extracted from the crude o i l i n several ways. One of the easier methods involves treatment with a l k a l i . D i e s e l o i l i s shaken with sodium or potassium hydroxide f o r about an hour. The amount of a l k a l i used depends on the acid content of the o i l . The a l k a l i - d i e s e l o i l emulsion i s l e f t to stand for several hours. The aqueous layer contains the naphthenic acids as t h e i r potassium or sodium s a l t s . The i n t e r e s t i n naphthenic acids as growth promoters i s f a i r l y recent. Considerable research has been done i n the Iron Curtain countries and i n Canada. The naphthenic acids and t h e i r s a l t s are applied on a large scale i n Russia and Bulgaria to improve the y i e l d s of crops. In low concentrations naphthenates were shown to stimulate vegetative and reproductive growths and to a f f e c t several metabolic processes (2,12,15). At high concentrations, the chemical acted as a he r b i c i d e . There are a l s o reports on the promotion of growth of animals due to naphthenate feeding (30). The e f f e c t s of potassium naphthenate have been studied i n our laboratory with i n t e r e s t i n g r e s u l t s . A f o l i a r a p p l i c a t i o n of KNap to bush bean plants resulted i n increased y i e l d s of green pods , (39,116) higher rates of photosynthesis,and dark r e s p i r a t i o n (41), and greater s p e c i f i c a c t i v i t i e s of several enzymes (41,119). Increased p r o t e i n synthesis and higher n u c l e i c acid content were a l s o observed (86) . Wort et a l (119) have suggested that the stimulation of plant growth by KNap i s due to the action of the chemical, or i t s metabolites, at both the genetic and metabolic l e v e l s . The mechanism of a c t i o n of naphthenates may, perhaps, be explained more p r e c i s e l y 5. with the a d d i t i o n a l knowledge of the f a t e of the growth stimulants i n the p l a n t s . This must include not only the biochemistry of KNap but also i t s organo-, h i s t o - and c e l l u l a r d i s t r i b u t i o n i n the plant. A s i n g l e compound i s more s u i t a b l e for such an i n v e s t i -gation than the mixture ,naphthenates. Cyclohexanecarboxylic acid (CHCA) has been proved to be a component of naphthenic acids (2l,32) and has been shown to have growth stimulating properties comparable to KNap ( 1 1 8 ) . For these reasons CHCA was chosen i n preference to naphthenic acids. An a d d i t i o n a l f a c t o r i n favour of employing CHCA i n t h i s study 1 4 was i t s a v a i l a b i l i t y i n the unlabelled and C-labelled forms. The present i n v e s t i g a t i o n deals with the following, with reference to CHCA and i t s metabolites: t h e i r d i s t r i b u t i o n i n the bean plant at d i f f e r e n t time i n t e r v a l s following a p p l i c a t i o n ; t h e i r movement withi n the plant as influenced by methods of a p p l i c a t i o n and growth conditions; t h e i r l o c a l i z a t i o n i n various t i s s u e s ; and t h e i r associa-t i o n with c e l l organelles. -The i n v e s t i g a t i o n may reveal some important aspects of the mechanism of a c t i o n of CHCA and hence suggest ways to increase i t s e f f e c t i v e n e s s . Moreover, i t may be of further p r a c t i c a l value since the amounts of the foreign compound CHCA i n the various organs of the plant, e s p e c i a l l y the pods, w i l l be revealed. LITERATURE REVIEW In t h i s chapter, the responses of d i f f e r e n t plants to naphthenates and cyclohexanecarboxylates are discussed. The review although not exhaustive, has been based on reports that i l l u s t r a t e the wide v a r i e t y of crops that respond to naphthe-nates, and the many aspects of growth and metabolism that are affec t e d by them. The a p p l i c a t i o n of naphthenic acids to stimulate plant growth dates back to 1956, when i t appeared f i r s t i n the Russian l i t e r a t u r e . During the subsequent years, the e f f e c t s of the naphthenic acid compounds on the p h y s i o l o g i c a l and biochemical processes have been investigated by s c i e n t i s t s i n Russia, Bulgaria, Albania, and Canada. Considerable information i s a v a i l a b l e on the naphthenate stimulation of plant growth and metabolism, but only a few reports e x i s t on cyclohexanecarboxylate stimulation. There i s no published account of the metabolism and d i s t r i b u t i o n of naphthenic acids, whereas the fa t e of cyclohexanecarboxylic acid i n plants has been known f o r the past three years. Studies on the transport, d i s t r i b u t i o n and metabolism of a few other plant growth substances have also been considered i n thi s chapter, f o r comparative purposes. These w i l l be ref e r r e d to again i n the dis c u s s i o n . 7. Naphthenates Improved growth and development of apple resulted from soaking the seeds i n 0.004% naphthenate s o l u t i o n p r i o r to sowing (91). Marshaniya et a_l (76) observed increases of 18.6 to 21.2%, i n the y i e l d of tangerines, whose f o l i a g e had been sprayed with 0.05% naphthenates. A f o l i a r spray of 0.05% naphthenate s o l u t i o n invoked 40% and 28%, increases i n the y i e l d s of cucumber and r i c e r e s p e c t i v e l y (60). A t a u l l a e v (11) reported increased metabolism, growth, development, and y i e l d of cabbage, muskmelon, tomato and carrot a f t e r the f o l i a g e of these crops was sprayed with potassium naphthenate (KNap) at a con-centration of 0.0005 to 0.01%. The y i e l d of cabbage was increased by 30%. F o l i a r sprays of 0.5%, KNap on bush bean, radish, sugar beet, maize and spring wheat produced increases i n the vegetative growths of a l l these crops (118). Weights of green pods and r i p e seed i n bush bean were increased by 23 and 25%, r e s p e c t i v e l y . Soaking spring wheat i n 0.01 and 0.001% KNap produced s t a t i s t i c a l l y s i g n i f i c a n t increments i n the weight of r i p e seed per pl a n t . In oat, the y i e l d of green mass was increased by 10 to 14% by a f o l i a r a p p l i c a t i o n of naphthenates at a concentration of 0.1% (3). Wort and Hughes (117) obtained increases of 42% i n the tuber y i e l d of early potatoes a f t e r spraying the f o l i a g e with 0.5% KNap. Root formation on phlox and vine cuttings was improved by treatment with KNap (125). Seeds of sunflower, when soaked i n 0.01% and 0.005% s o l u t i o n of naphthenic s a l t s 6 to 12 hr before planting, produced plants with larger stems and greater number of flowers. The y i e l d s of seeds and o i l were also increased (69). 8. Sophore (Sophora japonica L.) seeds soaked i n 0.0057» naphthenates produced plants with greater s a l t tolerance i n s a l i n i z e d s o i l (6). Agakishiev et a l (4) reported stimulating e f f e c t s on growth of cotton plants on sulphate s a l i n i z e d s o i l . Gurvich (47) reported an i n t e r e s t i n g e f f e c t of NaNap. i r r a d i a t e d seeds of Al l i u m fistulosum (welsh onion) showed a delay i n the onset of mitosis a f t e r germination began. This u s u a l l y l a s t e d f o r 54 to 57 hours but was compensated f o r i n the next 60 to 63 hours. When -4 -5 i r r a d i a t e d seeds germinated i n NaNap sol u t i o n s , 1 x 10 and 1 x 10~J%, i n t e n s i f i c a t i o n of m i t o t i c a c t i v i t y was seen. The m i t o t i c stimulation surpassed the pr o t e c t i v e e f f e c t of other ra d i o p r o t e c t i v e compounds re-ported i n the l i t e r a t u r e . Addition of 0.0017<, naphthenate s o l u t i o n to a culture of Chaetocerus curvisetus, a diatom, stimulated the rates of c e l l d i v i s i o n and photosynthesis (129). x Goryaev et a l (45) reported that naphthenic acids used i n combination with autolyzates of wine, beer and alcohol yeasts stimulated the growth of micro-organisms. Voinova-Raikova (106) showed that the addit i o n of naphthenate s o l u t i o n to s o i l stimulated the development of ammonifying ba c t e r i a but i n h i b i t e d the number of d e n i t r i f y -ing b a c t e r i a . I n h i b i t i o n of growth can also r e s u l t from naphthenate t r e a t -ment. With 107, NH4NO3, 207o KNap applied at 800 1/ha produced h e r b i c i d a l e f f e c t s on grass, legumes, U r t i c a , Stachys etc. (74). Zhukova observed h e r b i c i d a l e f f e c t s of naphthenates on some unspecified weeds (130). Sodium naphthenate,0.057o .decreased the weight of root systems of eggplants although a concurrent increase was observed i n the weights of the a e r i a l system (48). Photosynthetic rate i s increased by naphthenates (4) Fattah (39) and Fattah and Wort (41) reported s t a t i s t i c a l l y s i g n i f i c a n t increases i n photosynthetic rates of bush bean plants grown under three d i f f e r e n t l i g h t i n t e n s i t i e s at 26°C for 7, 14, and 21 days a f t e r a f o l i a r a p p l i c a t i o n of a 0.57, s o l u t i o n of KNap to 14-day-old p l a n t s . A spray of 0.017, naphthenate s o l u t i o n on young cotton plants stimulated t h e i r r e s p i r a t o r y rates (14). Chu (24) however, reported a decrease i n r e s p i r a t o r y rate i n tomato two weeks a f t e r treatment with 0.57, KNap. Four weeks a f t e r treatment, the r e s p i r a t o r y rate increased by 9.77,. Fattah and Wort (41) observed an increase i n dark r e s p i r a t i o n i n bush bean following naphthenate treatment. Severson (99) reported that KNap stimulated the uptake and metabolism of l^C-glucose by bean root t i p s . The amount of ascorbic a c i d was increased i n cotton leaves following naphthenate treatment ( 4 ) . Vitamin C content was greater i n naphthenate treated cabbage (10) and melon ( 2 ) . Chu (24) however, found decreased vitamin C content i n tomato f r u i t s from the plants that had been sprayed with 5000 ppm KNap s o l u t i o n . Guyot (49) observed a higher concentration of sugar i n sugarcane, whose f o l i a g e was sprayed with naphthenate s o l u t i o n 15 to 30 days p r i o r to harvest. KNap treatment increased reducing sugars, sucrose, and t o t a l sugars i n the mature f r u i t s of tomato (24). The loss of sugar during storage was a l s o less i n tomato f r u i t s from KNap treated p l a n t s . Protein and starch contents of potato tubers were observed to be greater following naphthenate treatment (2 ). Wort and Hughes ( 117) however, found no increase i n the starch content of early potatoes whose f o l i a g e had been sprayed with KNap. Yur'eva (124) obtained corn grains with more protein and starch, and corn s i l a g e with a higher p r o t e i n content. S p e c i f i c a c t i v i t i e s of catalase and peroxidase were greater following 0.0 1% naphthenate treatment (15). Fattah and Wort (41) reported increases i n the a c t i v i t i e s of phosphoglyceryl kinase, phosphorylase, n i t r a t e reductase, and glutamic pyruvic transaminase i n bush bean. Phosphorylase a c t i v i t y i n tomato was reported to be increased (24). NaNap 5 x 10" "Vo promoted amylase a c t i v i t y i n A s p e r g i l l u s  usamii (22). Wort et a l (119) reported increases i n the s p e c i f i c a c t i -v i t i e s of n i t r a t e reductase, glutamic-oxaloacetic transaminase, glutamine synthetase and cytochrome oxidase. Enzyme p r o t e i n was greater by 10 to 14 20%. The incorporation of C-L-Leucine by leaves of treated plants was increased by approximately 10%. No increase i n enzyme a c t i v i t y followed i n v i t r o a d d i t i o n of 10 KNap. These authors suggested that naphthe-nate stimulation of plant growth i s the r e s u l t of the a c t i o n of the chemical, or i t s d e r i v a t i v e s , at both the genetic and metabolic l e v e l s . Metabolism of naphthenates Studies on metabolism of naphthenates i n our laboratory (9 2 ) revealed that KNap, when applied to primary leaves of bush bean plants, was conjugated with glucose and with as p a r t i c a c i d . When the naphthenate mixture was root fed, the conjugates could be detected i n the leaf blades a f t e r 24 hours. Cyclohexanecarboxylic acid Very l i t t l e information i s a v a i l a b l e on the stimulation of plant growth by cyclohexanecarboxylic a c i d . Wort and Patel (118) tested the growth stimulation pro-p e r t i e s of several cycloalkanecarboxylic acids. They sprayed the - 2 9 f o l i a g e of bush bean plants with a 1 x 10 M or 2 x 10"ZM s o l u t i o n of the potassium s a l t of the following: cyclopentane-, cyclohexane-, and cycloheptanecarboxylic acids. The conclusion was based on the y i e l d of green pods harvested 5 weeks l a t e r . The y i e l d of green pods i n each treatment was numerically greater than that from the c o n t r o l . However, only i n the case of cyclohexanecarboxylic a c i d did the increase i n y i e l d reach s t a t i s t i c a l s i g n i f i c a n c e . The percentage increases were 35 and 24 f o r the 1 x 10"' and 2 x 10 _ 2M potassium cyclohexanecarboxylate (KCHC) s o l u t i o n s . Agakishiev et a l (5) observed that d i f f e r e n t substituted cyclohexyl butanones and cyclohexyl butanols increased the rate of cotton seed germination and improved the y i e l d and q u a l i t y of cotton. The authors of the above two reports suggested that a s i x -carbon saturated r i n g was e s s e n t i a l for the growth stimulation proper-t i e s . Agakishiev et a l (4) found that cotton plants treated with Sh-8 (a cyclohexenyl butanol) and grown on sulphate s a l i n i z e d s o i l s , showed a higher peroxidase a c t i v i t y i n the leaves. Bazanova (16) reported that a spray of Sh-8 at 0.005% affected the a c t i v i t i e s , d i s t r i b u t i o n and t r a n s l o c a t i o n of natural growth regulators and i n h i b i t o r s i n cotton p l a n t s . Metabolism of CHCA The f i r s t reports of the metabolism of CHCA were with reference to animals. Friedmann i n 1911 (42) and Bernhard i n 1937 (18), observed increased excretion of hippuric acid (N-benzoylglycine) when CHCA was administered to dogs. Bernhard et a l (19) used deuterated cyclohexanecarboxylic acid and found that deuterated hippuric acid was excreted. They concluded that aromatization of CHCA occurred when the acid was fed to dogs. Babior and Bloch (13) showed that l i v e r t i s s u e and l i v e r mitochondria from guinea pigs could aromatize CHCA. A c e t y l CoA was involved. The enzyme that aromatized the CHCCoA to benzoyl CoA was p a r t i a l l y p u r i f i e d and studied. Severson e j t a l (91) reported the conjugation of cyclo-hexanecarboxylic acid to ft -6-D glucose and to L-aspartic acid i n bean le a f discs that were incubated with KCHC-7-^4C. Severson (98) also suggested the pos s i b l e conugation of CHCA to some small peptides Metabolism of other plant growth regulators  Indoleacetic acid Good e_t a_l (44) reported the formation of indoleacet-amide and indol e a c e t y l a s p a r t i c acid (IAAsp) as the der i v a t i v e s of ind o l e a c e t i c acid (IAA) i n tissues of 12 plant species. There was also an a c i d i c metabolite which they did not i d e n t i f y . Andreae and Y s s e l s t e i n (7) found that i n pea ep i c o t y l s 207« of the IAA taken up was present as IAA and IAAsp. 13. Pea e p i c o t y l s , when incubated with several indole compounds, were found to contain the respective amides and aspartates (8). Morris (80) stated that the conjugation of IAA to a s p a r t i c acid was favoured by l i g h t . Fang et al (38) and Morris et a_l (79) separately reported the occurrence of several u n i d e n t i f i e d metabolites of IAA i n corn and i n bean roots r e s p e c t i v e l y . Klambt (67) observed eight metabolites other than IAAsp i n wheat c o l e o p t i l e t i s s u e . One of them was IAA-glucose. Thurman and Street (102) reported the formation of an IAA-glutamic a c i d conjugate i n ad d i t i o n to IAAsp i n tomato roots. Very l i t t l e free IAA remained. Zenk (128) found that IAA-glucose i s the f i r s t conjugate to be formed i n Hypericum hircinum. IAAsp appeared l a t e r , two to three hours a f t e r treatment with IAA. A new conjugate between IAA and l y s i n e was discovered i n Pseudomonas savastanoi by Hutzinger et a l (61). ATP was required fo r the conjugation. The conjugate showed a s l i g h t a c t i v i t y i n the Avena s t r a i g h t growth t e s t . The occurrence of monomethyl 1-4-c h l o r i n d o l y l - 3 - a c e t y l - L - a s p a r t i c acid i n immature seeds of pea was shown by H a t t o r i (53). Winter and Thimann (113) reported that, i n Avena coleop-t i l e s , a f r a c t i o n of the applied radioactive IAA was bound to the t i s s u e and was not extractable with water, buffer s o l u t i o n , or r i b o -nuclease. However, the a c t i v i t y was r e a d i l y released by treatment with 107» urea, t r y p s i n , and chymotrypsin. Zenk (128) obtained 0.0033 ug bound IAA per milligram of p r o t e i n . Morris et a_l (79) discovered 14 the a s s o c i a t i o n of the applied C-labelled IAA with p r o t e i n . They found that the IAA molecule was unchanged. Evidences were obtained to i n d i c a t e that IAA was chemically bound to the p r o t e i n and not merely adsorbed. 1 4 According to Bendana et a l (17), the l a b e l from IAA- C was incorporated into the RNA f r a c t i o n . Some of i t was associated with sRNA f r a c t i o n , while the r e s t was believed by the authors to be the degraded auxin that had been reused i n the synthesis of RNA. Davies and Galston (27) incubated stem sections of pea and bean with l a b e l l e d IAA ( I A A - 1 - 1 4 C and I A A - 5 - 3 H ) . A f t e r 1 8 hr incubation 1 4 the l a b e l was found as nonindole- C i n a high molecular weight poly-saccharide. Indolealdehyde and free i n d o l e a c e t i c acid were also shown to be present i n the extract. The authors indicated that the l a b e l l e d sRNA f r a c t i o n i s o l a t e d by Bendana et al was mainly l a b e l l e d polysacc-haride. Decarboxylation of IAA was observed by Fang et a l ( 3 8 ) . Eighty to 9 2 7 c of the t o a l IAA i n peas and 3 0 to 6 5 7 » i n corn were decarboxylated. G i b b e r e l l i c acids Musgrave et a_l ( 8 1 ) observed the accumulation and r e t e n t i o n of GAj. i n the a p i c a l parts of pea stem segments. The authors suggested t h i s to be due to binding of GA to s p e c i f i c GA receptor s i t e s . Musgrave and Kende ( 8 2 ) found that H-GA^ applied to dwarf peas was 3 converted to another GA. On i n j e c t i o n into pea pods, H-GA^ was metabolized by maturing seed to more water-soluble substances and to two other a c i d i c compounds, which were not i d e n t i f i e d . In barley aleurones, polar metabolites of GA were formed ( 8 3 ) . Sembdner ( 9 6 ) screened 2 4 plant species for t h e i r endogenous g i b b e r e l l i n s and t h e i r bound forms. GA^-hexopyranoside i n elm and GA3 glucoside i n maple, i n a d d i t i o n to hydroxy compounds and (* -glucosides, were among the metabolites. Yamani jet al ( 120) i s o l a t e d a glucoside of GA35 from immature seeds of Cytisus scoparius (Scot's broom). Benzoic acid Benzoyl aspartate (BAsp) and benzamide were found i n pea e p i c o t y l t i s s u e incubated with benzoic acid (BA) ( 8 ) . Traces of BA were also present. A high amount of decarboxylation was observed. Klambt (67) reported the formation of three d i f f e r e n t glucose-BA conjugates, namely, glucose-l-benzoate, glucose-6-benzoate, and glucose-1,6-benzoate. Zenk (127) found only glucose-l-benzoate. He demonstrated that the a s p a r t i c acid i n the conjugate was of L-configuration. He also proved that benzamide was an a r t i f a c t formed during chromatography. Zenk found the decarboxylation to be less than 0.1%, although Andreae e_t a l (7) had reported i t to be rather high. Venis (105) demonstrated the presence of an enzyme system that could conjugate BA to malic a c i d . The enzyme was i n d u c i b l e by auxin and several other aromatic carboxylic acids. He a l s o found that a l l or a large part of the supposed BAsp was a c t u a l l y benzoyl-malate (104). Kaphthaleneacetic acid Wheat c o l e o p t i l e s that were incubated with l a b e l l e d naphthaleneacetic a c i d (NAA) showed 13 r a d i o a c t i v e compounds (68) Among these were the aspartate and glucose conjugates of NAA. Zenk (126) found one or both conjugates i n 18 species. Hydroxylated NAA was also present i n some. Veen (103) showed the presence of f i v e metabolites of NAA-1- C i n Coleus explants. One of these was not i d e n t i f i e d . The author suggested that the remaining four might be NAA-/3-D-glucose. NAA-aspartic acid, 8-OH-NAA, and i t s glucose ester. Kazemie and Klambt (66) tested three conjugates of NAA for auxin l i k e p r o p e r t i e s . The conjugates had only 1/25 to 1/100 of the a c t i v i t y of NAA. The uptake of NAA was 10 times as f a s t as that of NAA-aspartate- 1 4C (65). Decarboxylation of NAA-^4C i n pineapple leaves was observed by Leeper et al (71). The authors observed that sunlight was e s s e n t i a l for the process. 2,4-dichlorophenoxyacetic acid Jaworski jet a_l (63) obtained a 2,4-dichlorophenoxyacetic acid (2,4-D) complex i n bean leaves. The metabolite amounted to 33% of the applied 2,4-D, and i t s formation was independent of photo-synthesis and of influence of exogenous metabolites. Hay and Thimann reported that 2,4-D was converted by bean seedlings to some ether i n s o l u b l e compounds (54) but Andreae and Good (8 ) found that most of the 2,4-D taken up by pea e p i c o t y l s remained unchanged. However, traces of the aspartate conjugate were detected. Swets and Wedding (101),working with C h l o r e l l a , observed that a c e t y l CoA was involved i n the f i r s t r e a c t i o n that 2,4-D under-went on entering the c e l l . The next step involved a s p a r t i c acid, leading to the formation of an aspartyl-2 }4-D complex. During the early stages of c a l l u s d e d i f f e r e n t i a t i o n , i n i t i a t e d by 2,4-D, the compound was found to e x i s t as a complex with l y s i n e - r i c h histones (121) . Ring hydroxylated glycosides of 2,4-D and 2 minor aglycones have been detected i n soybean cotyledon c a l l u s (114) and also i n bean, wheat, barley, soybean and oats (34). In the soybean c a l l u s , 2,4-D-glutamate contained 50% of the 2,4-D applied. Some free 2,4-D and some u n i d e n t i f i e d metabolites were also found. Hagin et a l (51) obtained 3-(2,4-dichlorophenoxy) propionic a c i d as a major metabolite of 2,4-D from three genera of grasses that were r e s i s t a n t to the h e r b i c i d e . The compound was i d e n t i f i e d by mass spectroscopy. The authors suggested that the conversion of 2,4-D to h e r b i c i d a l l y i n a c t i v e 3-(2,4-dichlorophenoxy) propionic a c i d might be a primary mechanism of r e s i s t a n c e of grass species to 2,4-D. 14 Weintraub et a l (110) detected C0 2 from bean plants treated with methylene- and carboxyl- l a b e l l e d 2,4-D but not from those treated with r i n g l a b e l l e d 2,4-D. The ^ 4C02 accounted for ca 1.2% of the t o t a l 2,4-D applied. Fang et a l ^ however, observed that 17.5% of the applied 2,4-D was decarboxylated i n three days (36). Movement of growth regulators A large number of the i n v e s t i g a t i o n s concerned with transport of growth r e g u l a t i n g substances have involved excised t i s s u e such as c o l e o p t i l e , stem, or p e t i o l e segments. I t has been shown that i n these organs auxins move predominantly i n the b a s i p e t a l d i r e c t i o n . McCready (77), McCready et a l (78) observed polar transport of IAA and 2,4-D i n the b a s i p e t a l d i r e c t i o n i n p e t i o l a r segments of bean. Acropetal movement of the two compounds also occurred and the rate depended on the concentration of the acid and the length of segments. G i b b e r e l l i n movement i n plants i s considered to be nonpolar. However, Jacobs ^ t a_l 18. (62) demonstrated a predominantly basipetal transport of g i b b e r e l l i c acid through young Coleus p e t i o l e s . Transport of growth substances i n i n t a c t plants has been studied by a few plant p h y s i o l o g i s t s . IAA applied to cotyledon(s) of Phaseolus coccineus and of maize moved ac r o p e t a l l y i n t o t h e i r e p i c o t y l s and c o l e o p t i l e s (112). Morris et a l (79) studied the movement of I A A - l - l 4 C and lAA-2-l 4C when applied to the leaves. IAA was transported to the roots unchanged. IAAsp was not transported. Day (28) found that i n bean leaves, 2,4-D moved through c u t i c l e , epidermis, and mesophyll to the phloem with a v e l o c i t y of ca 30 p/hr. In the phloem i t s t r a n s l o c a t i o n occurred at a v e l o c i t y , ranging from 10 to 100 cm/hr. L i t t l e and Blackman (73) observed that when IAA i s applied to bean leaves, i t moves out of the leaves and through the stem v i a the phloem. The downward v e l o c i t y was about 20 to 24 cm per hour. The authors reported that 2,4-D also moved through phloem, but at much slower r a t e s . The downward v e l o c i t y was 10 to 12 cm per hour. The herbicide when applied to le a f surface reached the underlying vascular bundles i n 79 min. Another 61 min elapsed before the compound reached the f i r s t internode. Stem curvature resulted 24 min l a t e r . Times taken f o r IAA to reach the f i r s t i n t e r -node and to cause curvature was 31 and 17 min r e s p e c t i v e l y . The t r a n s l o c a t i o n of 2,4-D was reported to be favoured by the presence of sugars (88). Rohrbaugh and Rice (89) reported that t r a n s l o c a t i o n was i n e f f e c t i v e i n phosphorus d e f i c i e n t plants. Hay and Thimann (55) suggested that the increased t r a n s l o c a t i o n of 2,4-D i n presence of sucrose i s not due to increased mass flow but to the 19. f a c t that sucrose serves as the energy source for some endergonic metabolic process. Hay and Thimann (54) studied the t r a n s l o c a t i o n of 2,4-D i n bean pl a n t s . They found that only 107» of 2,4-D that entered the primary l e a f was recoverable from i t 4 to 5 days a f t e r feeding i t with the chemical. The authors also measured the amounts of extract-able 2,4-D from the root, stem, and the primary leaves at d i f f e r e n t times following 2,4-D a p p l i c a t i o n . The percentage recovery decreased from 94 a f t e r s i x hours to 27 a f t e r 120 hours. Hay and Thimann, i n another paper (55) concluded that transport of 2,4-D did not occur i n the dark. Extensive breakdown of the chemical occurred during transport. The transport was com-p l e t e l y i n h i b i t e d by r i n g i n g , although 2,4-D was shown to be d i s t r i -buted equally between xylem and cortex. No 2,4-D entered the roots. Yasumatsu et al (122) observed that a large part of the l a b e l l e d 2,4-D applied to a tobacco l e a f was translocated to the stem. Less than 27o of the t o t a l a c t i v i t y entered the roots. When 2,4-D was root fed, 797o of the t o t a l was moved to the other parts of the plant. About one h a l f of the a c t i v i t y that remained i n the roots was ethanol-insoluble. L o c a l i z a t i o n of growth regulators By means of autoradiography, Liao et a_l (72) showed nuclear and cytoplasmic l a b e l l i n g i n root t i p c e l l s of Al l i u m cernuum (w i l d onion) and V i c i a faba (broad bean), that had been treated with methy-le n e - l a b e l l e d IAA- 1 4C, or car b o x y l - l a b e l l e d 2,4-D-14C. Cytoplasmic l a b e l l i n g decreased with time a f t e r removal of the auxins, whereas 20. nuclear and chromosomal l a b e l l i n g was retained f o r at l e a s t 120 hr. A well known method of a p p l i c a t i o n of auxins involves p l a c i n g an agar block, that contains the auxin, on a stem, e p i c o t y l , or p e t i o l e segment. This method of a p p l i c a t i o n was employed by the authors of the following reports. Whitehouse and Z a l i k (111) obtained microautoradiographs of e p i c o t y l segments of Phaseolus coccineus treated with IAA- H. Greatest blackening was observed i n the epidermal and cambial regions. In sections taken near the donor block, s i l v e r grains were also observed i n the xylem t i s s u e . IAA- H was fed to Coleus internodes (90). The a c t i v i t y was l o c a l i z e d i n the secondary wall of the tracheary elements. Veen (103) supplied NAA-l-^C to Coleus explants. Micro-autoradiographs revealed a large number of s i l v e r grains i n the c o r t i c a l region. In the p i t h zone, the density of grains was much less but the grains were seen to follow the contour of plasmolysed cytoplasm. Galston and Kaur (43) f r a c t i o n a t e d pea stem c e l l s that had been treated with carboxyl l a b e l l e d 2,4-D--'"4C, by c e n t r i f ugation. The a c t i v i t y from the chemical was seen to be l o c a l i z e d i n the supernatant f r a c t i o n , that was free of a l l organelles. Homogenates of 2,4-D-^C treated leaves of bean were sub-jected to c e n t r i f u g a t i o n i n two d i f f e r e n t density gradients (52). One was aqueous, the other, non-aqueous. In the aqueous density gradient, high ^ 4C a c t i v i t i e s were associated with the c e l l f r a c t i o n that con-tained soluble components. In the non-aqueous density gradient, however, the a c t i v i t y was l a r g e l y associated with the c h l o r o p l a s t s . Leaching was 21. suggested to be the main reason for the a c t i v i t y i n the soluble f r a c t i o n when aqueous medium was employed. MATERIAL AND METHODS Growth of plants Bush bean plants, Phaseolus v u l g a r i s L., c u l t i v a r 'Top Crop 1, were used i n a l l the experiments. The seeds were purchased from B u c k e r f i e l d 1 s Ltd., New Westminster, B. C. The plants were grown i n wet vermiculite f o r the short term experiments (2 and 3). For a l l other experiments they were grown i n p l a s t i c pots, 15 cm i n diameter, containing composted s o i l . Seven seeds were sown, i n each pot. Within two weeks from sowing, the seedlings were thinned i n stages to one per pot. The room where the plants were grown was provided with a 14 hr photoperiod. The temperature regime was 26 + 1°C i n l i g h t and 21 + 1°C i n dark. The r e l a t i v e humidity was. 60 to 70% i n l i g h t and 70 to 807o i n dark. Light i n t e n s i t y at the l e v e l of the f o l i a g e was 16.1 k l x . Chemical Cyclohexanecarboxylic acid (CHCA) ( A l d r i c h Chemicals, 14 Milwaukee, Wis.) was used i n the spray experiments. CHCA-7- C, purchased from International Chemical and Nuclear Corporation, was used as the potassium s a l t (KCHC-7-^4C) i n a l l the other experiments. The s p e c i f i c a c t i v i t y of the sample was stated as 20 mc/m mole. 1. Spray experiments Fourteen days a f t e r sowing, the f o l i a g e of 30 bush bean plants was sprayed to drip with a 0.01 M aqueous s o l u t i o n of CHCA that contained 0.37o (v/v) Tween 20 (polyoxyethylene sorbitan mono-laurate (Atlas Powder Co., Wilmington, Del.) as a wetting agent. The pH of the s o l u t i o n was 5. T h i r t y unsprayed plants served as the c o n t r o l s . F i v e weeks l a t e r , the green pods were harvested from the treated and control plants and weighed. The r e s u l t s were subjected to analysis of variance. 2. Movement of acid within the plant KCHC-7-^4C was applied to one primary l e a f of each plant i n each of following three ways. Two ;ul (0.02 )ic) of KCHC-7-^C was (1) i n j e c t e d i n t o the midrib approximately i n the proximal one t h i r d of the leaf (2) applied as a drop to the base of the l e a f at the junction of the p e t i o l e (3) as a t h i n coating on the upper surface of the lamina. A f t e r selected t r a n s l o c a t i o n periods, each of the plants was separated i n t o the treated primary l e a f , the untreated primary l e a f , the stem together with the shoot apex, and the roots. The treated primary leaf was rinsed with d i s t i l l e d water to remove adhering KCHC and blotted dry. The parts were then mounted on a sheet of paper and exposed to Medical X-ray f i l m (Kodak Blue Brand) fo r 3 weeks at ca -20°C. The exposed X-ray films were developed i n the usual manner to obtain autoradiographs. 3. Time course study of the, appearance of metabolites of CHCA-7-^4C Twenty ,ul of KCHC-7-l4C (0.20 pc) was applied i n the form of small droplets to each primary l e a f of t h i r t y p l a n t s . A f t e r each of the time i n t e r v a l s of 0, 1/8, 1/4, 1/2, 2/3, 1, 4, 8, 24 and 48 hr the primary leaves of three plants were removed, rinsed with d i s t i l l e d water, b l o t t e d dry, chopped in t o small pieces and extracted i n b o i l i n g 80% ethanol. The extraction was considered complete when leaf residue was white. The bulked extracts from each sample were evaporated to dryness and the residues redissolved i n minimal amounts of methanol. The methanolic solutions were spotted on a Whatman No. 3 Chromatography paper. The chromatogram was run f o r approximately four hours i n a solvent system containing isopropanol: Tk ammonia : water:: 8:1:1 (IAW) or n butanol: a c e t i c acid : water :: 4:1:5 (top phase) (BAW) The a i r - d r i e d chromatogram was l e f t i n contact with X-ray f i l m f o r three weeks, to obtain autoradiographs. The r a d i o a c t i v e spots were eluted i n methanol and the a c t i v i t y i n each determined by l i q u i d s c i n t i l l a t i o n . The s c i n t i l l a t i o n mixture was prepared according to Durbin and Christensen (31) and contained toluene 600 ml, absolute ethanol 378 ml, 0.4% PPO (2,5 -diphenyloxazole) (Fraser Medical Supplies Ltd., Vancouver, B. C.) and 0.0015% POPOP (1,4-Bis 2-(5-phenyloxazolyl) - benzene) (Fraser Medical Supplies Ltd, Vancouver, B. C ) . The samples were counted i n a Nuclear-Chicago L i q u i d S c i n t i l l a t i o n Counter, Model 724. Counting e f f i c i e n c i e s of the samples were determined by the channels r a t i o method (108). A l l counts were corrected to 100% e f f i c i e n c y . 4. Persistence of a c i d and i t s metabolites i n the plant One, 2, 3, and 4 weeks a f t e r coating the upper surface of 14 each primary l e a f with 25 yx\ (0.25 yic) of KCHC-7- C, the plants were divided i n t o the following parts: primary leaves, t r i f o l i a t e leaves, stems, buds-flowers-pods, and roots. These were separately extracted i n b o i l i n g 80% ethanol. The extracts were processed f o r the prepara-t i o n of autoradiographs as described i n Expt. 3. The t o t a l a c t i v i t y i n each extract was also determined. The values were corrected f o r color quenching caused by ch l o r o p h y l l as follows. A non-radioactive, methanol s o l u t i o n was prepared from un-l a b e l e d leaves of bean, as described i n Expt. 3. Equal amounts of the green s o l u t i o n were added to 15 ml of s c i n t i l l a t i o n mixture (31) containing known amounts of a c t i v i t y and to 15 ml of ethanol. Paired s e r i e s of s c i n t i l l a t o r and ethanol with graded amounts of the green s o l u t i o n were prepared. The absorbance of each ethanol s o l u t i o n was measured at 660 nm with a Spectronic 20 (Bausch and Lomb). The a c t i -v i t i e s of the s c i n t i l l a t i o n mixtures containing KCHC-7-^4C and the green extract were determined. A p l o t of the absorbance versus the counting e f f i c i e n c y yielded a quench co r r e c t i o n curve. Knowing the absorbance of a green, r a d i o a c t i v e sample, i t s counting e f f i c i e n c y could be e a s i l y ascertained from the curve. The metabolites of KCHC-7- 1 4C were i d e n t i f i e d from t h e i r Rf values and by co-chromatography with standards. The r e l a t i v e amounts of the chief metabolites of KCHC, the glucose and aspartate conjugates, were determined by measuring the a c t i v i t i e s i n the respective spots. The r a d i o a c t i v e spots were cut out from the chromatograms and immersed i n s c i n t i l l a t i o n v i a l s containing 15 ml of 0.4% PPO i n toluene. These were counted and the values were corrected as i n Expt. 3. 26. 5. Determination of the a c t i v i t i e s i n the plant residues a f t e r  ethanol ex t r a c t i o n . The residues from ethanol extraction were ground to a f i n e powder i n a mortar with p e s t l e . A known weight of each residue was suspended i n the s c i n t i l l a t i o n s o l u t i o n by using a t h i x o t r o p i c gel, Cab-O-Sil (108). The a c t i v i t y was determined as i n Expt. 3. 6. Evolution of -*-4C02 by treated plants Twenty u l (0.2 uc) KCHC-7-^-4C was spread on the upper sur-face of each primary leaf of two 10-day-old bush bean pl a n t s . Half an hour l a t e r , the two plants, grown i n the same pot, were placed i n a large glass desiccator. The s o i l surface and the pot were covered with polythene. A slow stream of a i r was passed over the plants. The a i r escaping from the chamber was bubbled through two successive traps of saturated barium hydroxide s o l u t i o n . Every day the Ba(0H)2 s o l u t i o n was removed and a fresh s o l u t i o n added to the traps. The milky s o l u t i o n removed from the traps was centrifuged and the p e l l e t resuspended i n acetone. The sus-pension was c a r e f u l l y spread on aluminium planchets to get a uniform d i s t r i b u t i o n , and d r i e d . A Nuclear Chicago Scaler Model 151 A was used for counting. A l t e r n a t i v e l y , the p r e c i p i t a t e d BaC03 was dried and known quan t i t i e s transferred to s c i n t i l l a t i o n v i a l s . The p r e c i p i t a t e was counted as a suspension with Cab-O-Sil i n the s c i n t i l l a t i o n mixture (108). 7. Translocation of CHCA Eleven days a f t e r sowing, s i x plants from a group of eight, were removed to a l i g h t p r o o f , v e n t i l a t e d box i n the growth room. The remaining two plants (set 1) were maintained under normal growth room conditions. Three days l a t e r , one primary l e a f of every plant i n the dark was detipped under water. The cut end was immersed i n a v i a l containing one of the following: glucose 0.25 M, sodium aspartate 0.1 M, d i s t i l l e d water. Two plants received glucose (set 2), two plants, aspar-tate (set 3), and the l a s t two, d i s t i l l e d water (set 4). The primary leaves of the plants i n l i g h t were l e f t i n t a c t . One hour l a t e r , KCHC-7-''"4C was applied to one primary leaf of each plant i n the l i g h t , and to each detipped l e a f i n the dark, as a drop (0.5 pc i n 50 jul) at base of the midrib. Twelve hours a f t e r a p p l i c a t i o n of the r a d i o a c t i v e material, each plant was divided into the treated l e a f , the untreated l e a f , the stem, and the roots. The corresponding f r a c t i o n s from both plants of a set were combined and extracted i n b o i l i n g 80% ethanol. The residues from ethanol extracts, a f t e r evaporation of the solvent, were redissolved i n small amounts of methanol. A portion of the methanolic solutions was used f o r chromatography, and another aliq u o t f o r determination of t o t a l a c t i v i t y i n each organ. Autoradiographs were made from the chromato-grams. A s i m i l a r experiment was performed simultaneously with one a l t e r a t i o n . The ethanol e x t r a c t i o n of the plant parts was c a r r i e d out 24 hr a f t e r a p p l i c a t i o n of KCHC-7-*4C instead of 12 hr as i n the above experiment. v 28. 8. L o c a l i s a t i o n o f r a d i o a c t i v i t y from KCHC-7- C i n t h e t i s s u e s . A g l a s s r i n g , a p p r o x i m a t e l y one cm i n d i a m e t e r and two mm deep, was p l a c e d on t h e a d a x i a l s u r f a c e a c r o s s t h e m i d r i b c a t h r e e cm fr o m t h e base o f b o t h p r i m a r y l e a v e s o f f o u r 1 4 - d a y - o l d bush bean p l a n t s . The r i n g was h e l d i n p l a c e by s m a l l amounts o f v a s e l i n e . F i f t y }il (0.5 juc) o f KCHC-7--'-4C was dropped on t h e l e a f s u r f a c e d e l i m i t e d by t h e g l a s s r i n g . A c o v e r s l i p was p l a c e d on t h e r i n g t o m i n i m i s e e v a p o r a t i o n o f t h e s o l u t i o n . One and f o u r days a f t e r t h e a d d i t i o n o f t h e c h e m i c a l , t h e t r e a t e d a r e a o f t h e p r i m a r y l e a f was removed w i t h a s h a r p r a z o r b l a d e ( l e a f d i s c ) and r i n s e d w i t h d i s t i l l e d w a t e r t o remove s u p e r f i c i a l KCHC. D i s c s f r o m p r i m a r y l e a v e s o f two p l a n t s were used a t each t i m e i n t e r v a l . S m a l l p i e c e s , one t o two s q u a r e mm were c u t f r o m t h e l e a f d i s c , p e t i o l e , a n d stem. These were f r o z e n i n i s o p e n t a n e c o o l e d by l i q u i d n i t r o g e n and q u i c k l y t r a n s f e r r e d t o t h e c o o l e d s t a g e of an Edwards T i s s u e F r e e z e - D r i e r M o d e l B5A. F r e e z e - d r y i n g o f t h e t i s s u e s was c a r r i e d out a t -40°C f o r 72 h o u r s . The t i s s u e s were t h e n d i r e c t l y t r a n s f e r r e d t o S p u r r ' s low v i s c o s i t y medium ( P o l y s c i e n c e s I n c . , W a r r i n g t o n , Pa.) (100) and i n f i l t r a t e d i n vacuo a t room t e m p e r a t u r e f o r s e v e r a l d a y s . The p l a s t i c was changed whenever i t s v i s c o s i t y tended t o i n c r e a s e . The t i s s u e s were embedded i n t h e same medium a t 60°C f o r 6 h r . S e c t i o n s , c a 3 p t h i c k , were c u t w i t h a g l a s s k n i f e i n a S o r v a l l MT-2 P o r t e r Blum u l t r a m i c r o t o m e . A f i n e n e e d l e was used t o t r a n s f e r t h e s e c t i o n s f r o m t h e k n i f e t o t h e m i c r o s c o p e s l i d e s . The s l i d e s w i t h t h e s e c t i o n s were b r e a t h e d upon t o e n s u r e a d h e r e n c e . The 29. s l i d e s were then coated with Kodak nuclear track emulsion, NTB^ (Rochester, N.Y. )as follows. The whole procedure i s c a r r i e d out i n a dark room provided with a safety l i g h t (Wratten s e r i e s #2). The emulsion, stored i n a r e f r i g e r a t o r , was allowed to warm up to room temperature. Using a spatula, the required amount of the emulsion (a gel below 30°C) was transferred to a glass container. The container with the emulsion was placed i n a water bath at 43°C, f or ca 15 min. The emulsion was s t i r r e d gently as i t melted. Care was taken to avoid a i r bubbles. The s l i d e s with sections were placed on a s l i d e warmer at 43°C for a minute. A few drops of the molten emulsion were placed on one end of the warmed s l i d e and spread over the s l i d e with a s t a i n l e s s s t e e l r o l l e r designed s p e c i a l l y for t h i s purpose. The thickness of the r e s u l t i n g emulsion layer was approximately 50u. The emulsion coated s l i d e s were placed h o r i z o n t a l l y i n a l i g h t t i g h t box f o r an hour or two. During t h i s time the emulsion gelled and dried on the s l i d e s . The s l i d e s were placed i n a l i g h t p r o o f s l i d e box, containing d r i e r i t e tubes, which was then stored at -20° for a week. The s l i d e s were developed and fixed i n Kodak D-19 developer and f i x e r . The microautoradiographs thus obtained were washed for 10 min and stained with s a f r a n i n . Temporary mounts using glycerine were prepared and viewed under a l i g h t microscope. Photographs were taken with phase contrast, l i g h t - and d a r k - f i e l d microscopy. 9. C e l l f r a c t i o n a t i o n using aqueous media 14 KCHC-7- C was spread on the adaxial surface of both primary leaves (0.25 juc/leaf) of four 14-day-old bush bean pl a n t s . A f t e r 24 hr the treated leaves were removed, ri n s e d with d i s t i l l e d water to remove any s u p e r f i c i a l KCHC and blotted dry. The treated primary leaves from two plants were then homogenized i n 0.35 M sodium ch l o r i d e i n a Waring blendor. The treated leaves from the other two plants were homogenized i n 307o carbowax i n 0.5 M phosphate buffer of pH 7.0 containing 0.01 M potassium c h l o r i d e (123). The b r e i of each sample was f i l t e r e d through two layers of cheese c l o t h and the f i l t r a t e s centrifuged at 20,000 g for 10 min at o 4 C i n a S o r v a l l Superspeed RC-2 r e f r i g e r a t e d centrifuge. The super-natant was drawn o f f with a p i p e t t e . The sediment was resuspended i n the grinding medium and the suspension recentrifuged at 10,000 g for 10 min. The procedure was repeated at 1000 and 600 g f o r 10 min each (see F i g . 21). The sediment and the various supernatahts were freeze-dried f o r 72 hr i n V i r T i s Freeze D r i e r . The residues were shaken with small amounts of methanol. The aliquots from these were assayed for t o t a l r a d i o a c t i v i t y . 10. C e l l f r a c t i o n a t i o n using non-aqueous medium 14 The leaves were treated with KCHC-7- C the same way as i n Expt. 9. Twenty-four hours l a t e r , the primary leaves from two plants were rinsed with d i s t i l l e d water, blotted dry, and immersed i n l i q u i d nitrogen. The b r i t t l e l e a f material was ground f i n e l y i n a mortar with p e s t l e . The powder was freeze-dried i n a V i r T i s Freeze Drier f o r 48 hr. The dried powder was stored over ^2^5 ^ n a desiccator. For f r a c t i o n a t i o n , 0.1 g of each leaf sample was ground with about 0.3-0.4 ml of carbon tetrachloride-hexane mixture of sp. gr. 1.5, 31. i n a Pyrex hand model t i s s u e grinder. The homogenate was d i l u t e d to 1 ml with the grinding medium and transferred to the bottom of a c e l l u l o s e n i t r a t e tube (0.5" x 2"). On top of t h i s homogenate 1.5 ml. of carbon tetrachloride-hexane mixture of sp. gr. 1.36 was layered care-f u l l y . Another layer of 1.5 ml of the above mixture of sp. gr. 1.34 and a fourth layer of sp. gr. 1.052 on top of the f i r s t two layers produced a discontinuous density gradient. Three such tubes were then centrifuged at 10,000 g i n a Beckman SW 39 model L preparative u l t r a c e n t r i f u g e . A d i s t i n c t green band was formed at the junction of 1.052 and 1.34 s p e c i f i c g r a v i t y l a y e r s . According to Hallam and Sargent t h i s band was comprised e n t i r e l y of chloroplasts (52). An automatic pipe t t e was used to draw consecutive samples of 0.2 ml. from the top. The samples were counted i n a Nuclear-Chicago Model Mark I l i q u i d s c i n t i l l a t i o n system, provided with an external standard. This was necessary to correct f o r the double quenching that occurred due to the presence of c h l o r o p h y l l (color quench) and carbon t e t r a c h l o r i d e (chemical quench). A f t e r every two consecutive samples for assaying a c t i v i t y , a drop was placed on a microscope s l i d e . This was examined under a l i g h t microscope for the c e l l components, i . e . n u c l e i , chloroplasts etc.present i n each sample. The presence of mitochondria i n the f r a c t i o n s was tested histochemically with t e l l u r i t e (107). The sample to be tested was incubated with 0.05 M s o l u t i o n of potassium t e l l u r i t e and a few drops of 0.1 M sodium succinate (optional) f o r 4 hours. The samples were examined under the microscope for dark granules - reduced i n s o l u b l e t e l l u r i u m - that i n d i c a t e the presence of mitochondria. 33. RESULTS 1. Growth The f o l i a g e of 14-day-old bush bean plants was sprayed with a 0.01 M aqueous s o l u t i o n of CHCA i n 0.37, Tween 20. At harvest, f i v e weeks l a t e r , the mean fresh weight of the green pods from the treated plants (36.2g) was greater than that from the controls (33.91g) by 127, (see Appendix p. 114). Analysis of variance showed that the increase i n pod y i e l d lacked s i g n i f i c a n c e at the 0.05 l e v e l . There was considerable v a r i a t i o n between pl a n t s . A s i m i l a r experiment showed that CHCA did have an e f f e c t on the growth rate. During the f i r s t week a f t e r spraying, the treated plants were seen to be d i s t i n c t l y t a l l e r than the control plants ( F i g . 1). This c h a r a c t e r i s t i c p e r s i s t e d 18 days a f t e r spraying, at which time the mean height of the treated plants(31.2 cm) was greater than that of the controls (27.8 cm) by 12.27, ( F i g . 2a). A t - t e s t showed that the two means were s i g n i f i c a n t l y d i f f e r e n t from each other at the 0.05 l e v e l . At t h i s time, 467, of the treated plants bore one or more open flowers, together with flower buds and 337, bore only buds, while 207, of the controls had open flowers and buds and 167, had buds only. The a p p l i c a t i o n of KCHC-7-'''4C at a r e l a t i v e l y low concentration of 0.0005 M to leaves of bush bean plants also produced pronounced morphological differences between the treated and control p l a n t s . Figure 2b shows that i n the treated plant the f i r s t and second t r i f o l i a t e leaves were large and more or less equal i n s i z e . In the control plant however, the second t r i f o l i a t e had not f u l l y expanded and was much smaller. Generally speaking, the plants to which cyclohexanecarboxylic acid had been applied were v i s i b l y larger than untreated plants. Figure l a Control (C) and treated (T) plants, 6 days af t e r a f o l i a r spray of O.OlM CHCA i n 0.3% Tween 20. Figure l b Control (1) and treated (2) plants i n the growth room, 6 days a f t e r a f o l i a r spray of O.OlM CHCA i n 0.3% Tween 20. Figure 2a Control (C) and treated (T) plants, 18 days a f t e r a f o l i a r spray of O.OlM CHCA i n 0.3% Tween 20. Figure 2b Control ( r i g h t ) and treated ( l e f t ) plants, one week a f t e r a p p l i c a t i o n of 0.0005M KCHC-7- 1 4C on the adaxial surface of the primary leaves. FIGURE 2 36. 2. Movement of acid within the plant The i n j e c t i o n of KCHC-y-^C resulted i n a rapid d i s t r i b u t i o n of a c t i v i t y i n the plant. Within 30 min of a p p l i c a t i o n of K C H C - 7 - ' l 4 C the a c t i v i t y had reached both the roots and the shoot apex (F i g s . 3a, 4a). The opposite primary l e a f was the l a s t plant organ to become radi o -a c t i v e . The time taken for the a c t i v i t y to reach the opposite l e a f was 1 hour or longer ( F i g . 4b). Presumably the a c t i v i t y moved out of the treated l e a f , down and up the stem, and was then transported to the opposite primary l e a f . The distance between the point of i n j e c t i o n on the le a f and the main root was approximately 16 cm. The r e s u l t s would thus suggest that the chemical moved with a v e l o c i t y of about 32 cm/hr. Figure 3b shows the d i s t r i b u t i o n of a c t i v i t y i n the plant parts, 24 hr a f t e r i n j e c t i o n . Figure 5 shows autoradiographs of treated leaves \, h, 1 and 4 hr a f t e r i n j e c t i o n of the l a b e l l e d chemical. The a c t i v i t y appears to spread from the base upward, r e s u l t i n g i n a wedge shaped d i s t r i b u t i o n ( F i g . 5. a, b, c ) . At 4 hr however, the a c t i v i t y i s seen to have receded from the marginal zones of the treated l e a f ( F i g . 5 d). Cotyledonary i n j e c t i o n s resulted i n a much slower export of KCHC-7-^4C. A c t i v i t y was detected i n the internode above the cotyledonary node 60 min a f t e r i n j e c t i o n . The primary leaves whose adaxial surfaces had been coated with KCHC-7-^4C retained a l l the detectable a c t i v i t y one hour l a t e r . The a p p l i c a t i o n of KCHC-7-^"4C as a drop to the base of the le a f also resulted i n a slow movement of the chemical. Only the basal part of the le a f around the region of a p p l i c a t i o n contained a c t i v i t y 1 hr a f t e r a p p l i c a t i o n . Twelve and 24 hr a f t e r a p p l i c a t i o n , the a c t i v i t y Figure 3a Autoradiographs of plant parts showing the d i s t r i b u t i o n 14 of a c t i v i t y % hr a f t e r i n j e c t i o n of KCHC-7- C into the midrib of a primary l e a f . T - Treated l e a f ; U - Untreated l e a f ; S - Stem; R - Roots ; a - Shoot apex. Figure 3b Autoradiograph of plant of a c t i v i t y 24 hr a f t e r the midrib of a primary parts showing the d i s t r i b u t i o n i n j e c t i o n of KCHC-7- 1 4C i n t o l e a f . T - Treated l e a f ; U - Untreated l e a f ; S - Stem ; R - Roots ; a - Shoot apex. FIGURE 3 F i g u r e 4a Autoradiograph of the treated l e a f and shoot apex \ hr a f t e r i n j e c t i o n of KCHC-y-^C into the midrib of the l e a f . T - Treated l e a f ; a - Shoot apex. Figure 4b Autoradiographs of the treated ( l e f t ) and the untreated (right) primary leaves, 1 hr a f t e r i n j e c t i o n of KCHC-7-1 4 C into the midrib. FIGURE 4 gure 5 Autoradiographs of primary leaves i n j e c t e d with KCHC-7-i^'C. Leaves removed from the p l a n t ' % , 1, and 4 hr a f t e r i n j e c t i o n (a-d re s p e c t i v e l y ) and exposed to X-ray f i l m . Note the wedge of a c t i v i t y i n the leaves, d i s t a l to the point of i n j e c t i o n . Details see page 36, 84. FIGURE 5 was detected i n the stem, roots and the opposite, untreated l e a f (Expt. 7. p. 57 Tables I I and I I I ) . The ethanol extracts of the plant parts were assayed f o r a c t i v i t y . The stems had more a c t i v i t y than the roots and untreated leaves at both times. The untreated leaves, however, had the lowest a c t i v i t y . This suggests that the opposite l e a f was the l a s t plant part to acquire a c t i v i t y . The same observation was made when the l a b e l l e d chemical was i n -jected into the midrib. 41. 3. Time course study of the appearance of metabolites of CHCA-7- C Severson et a l (97) reported that when bean leaf discs were incubated with KCHC-7-^4C for 24 hours, the chemical was con-verted by the le a f c e l l s to CHCA-0 -D-glucose and CHCA-L-aspartic a c i d . The study did not reveal any other metabolites and i t did not show the sequence i n which the conjugates were formed. In the present experiment, a time course study of the appearance of metabolites of CHCA was made. A radiochromatographic analysis of the le a f extracts showed f i v e r a d i o a c t i v e spots ( F i g . 6; a and b). Both TAW and BAW solvent systems were used to develop the chromatograms. Although a c e r t a i n amount of hydrolysis of the CHCA-glucose occurred i n the IAW ystem, the separation of the spots was better than that i n the BAW system. The BAW system was hence used mainly to confirm the presence or absence of free CHCA. A r a d i o a c t i v e spot with an Rf of 0.75 (C i n F i g . 6; a and b) was evident i n the extracts at zero time to ca 4 hr. A f t e r 4 hr, the spot was less conspicuous. KCHC-7--'-4C and CHCA have also an Rf value of 0.75. Thus spot 1 was i d e n t i f i e d as CHCA-7- 1 4C. The i n t e n s i t y of the CHCA-7--'-4C spots decreased with time. The glucose conjugate of CHCA (Rf 0.85),G i n F i g . 6; a and b , was apparent i n the le a f 'one eighth hour a f t e r a p p l i c a t i o n of KCHC-7-^4C. The CHCA-glucose was observed i n c e r t a i n other experiments as ear l y as one minute a f t e r a p p l i c a t i o n of KCHC. The i n t e n s i t y of CHCA-glucose spot increased u n t i l ca 4 hr. The spot was less conspicuous at 8, 24, and 48 hr. The a s p a r t i c a c i d conjugate of CHCA (Rf 0.17) (A i n F i g . 6; Figure 6 Autoradiographs of extracts of treated primary leaves, 0, 1/8, 1/4, 1/2, 2/3, 1, 4, 8, 24, 48 hr a f t e r a p p l i c a t i o n of KCHC-7-'''^ C. (Isopropanol : 1°L ammonia : water G - CHCA-glucose; C - CHCA; X - a r t i f a c t 'Y' - unknown metabolite; A - CHCA-aspartic acid G FIGURE 6 43. a and b) did not appear u n t i l an hour a f t e r a p p l i c a t i o n of the chemical to the leaves. The spot with an Rf of 0.56 (Y i n F i g . 6 b) has not been reported previously. I t apparently was formed i n the leaves approximately one hour a f t e r the KCHC had been applied. This spot i s termed ' Y 1 i n the present work f or the sake of convenience. The compound 'Y' appeared to increase with time. The spot termed X i n Figure 6; a and b was seen to cor-respond to the impurity i n the sample of K C H C - 7 - . This was not i d e n t i f i e d f u r t h e r . The o u t l i n e s of the spots G, C, A and Y were traced out on the chromatogram. Even though there was a c e r t a i n amount of overlapping of spots, they could be distinguished. The respective areas were cut out, and eluted i n methanol. The a c t i v i t y i n each eluate was determined. A plo t of the a c t i v i t y of each metabolite, as a percentage of the t o t a l a c t i v i t i e s of the four metabolites i n each extract, versus time i s shown i n Figure 7. The percentage of CHCA increased from ca 68% at zero time to ca 83% at one fourth hour a f t e r a p p l i c a t i o n of KCHC. Thereafter i t declined to about 10%, i n four hours. During the next 44 hours there was only a s l i g h t v a r i a t i o n i n the le v e l s of CHCA. The CHCA-glucose, apparent at one eighth hour increased gradually to con s t i t u t e ca 73%, of the t o t a l a c t i v i t y at 4 hr. There was a decline i n the percentage value i n the next four hours followed by a more gradual decrease during the next 44 hr. The CHCA-aspartic acid, was not evident on the autoradio-100 graph u n t i l one hour a f t e r a p p l i c a t i o n . However the eluates of t h i s region on the chromatogram contained low l e v e l s of a c t i v i t y . During the i n t e r v a l between four and eight hours, the l e v e l of this metabolite increased r a p i d l y . In the next 40 hours, a slow build-up of the aspartate conjugate occurred. The metabolite 'Y1, observed one hour a f t e r a p p l i c a t i o n of KCHC, increased to almost double the amount at one hour i n the i n t e r v a l four to eight hours. The amount of 'Y' continued to increase during the following 40 hr. 4 6 . 4. Persistence of acid and i t s metabolites i n the plant In t h i s experiment the d i s t r i b u t i o n of the applied chemical i n the d i f f e r e n t parts of the plant at d i f f e r e n t times a f t e r a p p l i c a -t i o n was studied. Only a few reports of s i m i l a r experiments e x i s t . Even i n these, the time i n t e r v a l s used were rather short or the authors did not in v e s t i g a t e the nature of metabolites present at each time. In the present i n v e s t i g a t i o n the plants were divided into the treated primary leaves, t r i f o l i a t e leaves, stem, buds-flowers-pods, and roots. A c t i v i t y i n an ethanol extract of each part was determined 1 , 2 , 3 , and 4 weeks a f t e r a p p l i c a t i o n of the chemical and expressed as a percentage of the t o t a l ethanol-soluble a c t i v i t y ( T a b l e l ). The radiochromatographic analyses of the extracts indicated the compounds i n which the a c t i v i t y was present i n each plant part (Figs. 9 and 1 0 ) . Table I and Figure 8 show the d i s t r i b u t i o n of a c t i v i t y i n / the various parts of the plants at d i f f e r e n t times. The v a r i a b i l i t y i n the values between the r e p l i c a t e s i s very obvious (Table I ) . However, the trends i n the d i s t r i b u t i o n pattern are s i m i l a r for both r e p l i c a t e s . Hence the means were ca l c u l a t e d ( i n every case) and used to show the general pattern of d i s t r i b u t i o n ( F i g . 8 ) The treated primary leaves were seen to have the highest per-centage of t o t a l a c t i v i t y at a l l times of i n v e s t i g a t i o n . The a c t i v i t y decreased from 7 5 . 0 7 ° at the end of the f i r s t week, to 6 1 . 8 7 o at the end of four weeks. The a c t i v i t y i n the untreated t r i f o l i a t e s at the end of the f i r s t week was less than that at the end of four weeks. The percent a c t i v i t y increased from 5 . 1 at the end of f i r s t week to 8 . 5 at the end \ TABLE I . The percentage d i s t r i b u t i o n of carbon-14, i n plant p a r t s , one, two, three, and four weeks a f t e r a p p l i c a t i o n of KCHC-7-*4C. Weeks a f t e r a p p l i c a t i o n of KCHC-7-Part(s) of 1 2 3 4 the plant a R l R 2 Mean R l R 2 Mean R l R 2 Mean R l R 2 Mean Primary 1 eaves 77.8 72.2 75.0 71.3 60.6 66.0 59.7 56.6 58.2 60.4 63.2 61.8 T r i f o l i a t e leaves 3.8 6.3 5.1 5.4 1.0 3.2 9.6 3.6 6.6 8.8 8.2 8.5 Stem 8.8 4.2 6.5 9.6 3.2 6.4 7.3 10.1 8.7 7.3 11.3 9.3 Buds, flowers and pods 1.2 4.1 2.7 1.8 0.9 1.4 2.8 4.4 3.6 2.1 1.0 1.6 Roots 8.2 13.1 10.7 11.8 34.2 23.0 20.5 25.0 22.8 21.2 16.3 18.8 Tot a l a c t i v i t y per plant as 25.2 10.3 17.8 14.1 10.4 12.3 9.0 18.4 13.7 8.4 33.3 20.9 muc aR 1 =; Replicate 1 R 2 = Replicate 2 of four weeks. A s l i g h t decrease to 3.2 was observed at the end of second week. The stems contained at a l l t i m e s . a c t i v i t i e s higher than those i n the t r i f o l i a t e s . The range was from 6.5% at the end of one week to 9.3% at the end of four weeks. The extract of buds-flowers-pods contained the lowest a c t i v i t y among the organs at a l l times. At the end of the f i r s t week, the percent a c t i v i t y was 2.7 and i t decreased to 1.6 at the end of four weeks. A somewhat higher a c t i v i t y , 3.6%, was observed at the end of the t h i r d week. The roots contained a c t i v i t i e s that ranked next to those i n the primary leaves. One week a f t e r a p p l i c a t i o n , the a c t i v i t y was 10.7%, i of the t o t a l . I t increased to 23.0%, at the end of second week but gradually decreased to 18.8%, i n the next two weeks. The t o t a l a c t i v i t y contained i n each plant w i l l depend d i r e c t l y on the amount of a c t i v i t y absorbed by the leaves. I t w i l l also be a f f e c t e d by metabolic l o s s , i f any occurred. In the present experi-14 ment the t o t a l C a c t i v i t i e s i n the plants were found to be v a r i a b l e . In r e p l i c a t e 1, 25.2, 14.1, 9.0 and 8.4 muc per plant were found 1, 2, 3, and 4 weeks a f t e r KCHC a p p l i c a t i o n , r e s p e c t i v e l y . If the uptakes by the plants were equal, the above observation would i n d i -cate a loss of with time, due perhaps to decarboxylation. In r e p l i c a t e 2, on the other hand, 10.3, 10.4, .18.4, and 33.3 muc per plant were found at the end of 1, 2, 3, and 4 weeks a f t e r a p p l i c a t i o n of the chemical. This suggested a continued uptake of the chemical, with or without a simultaneous loss of ^ 4C. Weeks a f t e r a p p l i c a t i o n Primary leaves T r i f o l i a t e -leaves Stem Buds-flowers- 1 pods Roots 7o of t o t a l a c t i v i t y • i 0 25 50 75 100 0 25 50 75 100 0 25 50 75 100 0 25 50 75 100 F I G U R E 8 . The percentage d i s t r i b u t i o n of carbon-14 i n plant parts, one, two, three, and four weeks a f t e r a p p l i c a t i o n of KCHC-7- 1 4C. 4> 50. The amount of a c t i v i t y applied as KCHC-7- 1 4C to each 14-day-old plant was 0.5 p.c. The mean t o t a l a c t i v i t i e s i n the plants detected 1, 2, 3 and 4 weeks a f t e r a p p l i c a t i o n were 17.8, 12.3, 13.7 and 20.9 mp.c/plant. These values represented 3.7, 2.5, 2.7 and 4.27» of the o r i g i n a l 0.5 pc KCHC-7-- l 4C applied, r e s p e c t i v e l y . The autoradiographs of the various extracts showed that the CHCA-glucose was the most prominent metabolite i n a l l the organs at 1, 2, 3 and 4 weeks (Figs. 9; a,b and 10; a,b). In chromatograms run i n the LAW system, a hydrolysis of the CHCA-glucose to CHCA occurred. Consequently a spot corresponding to the Rf of CHCA was observed. This however, did not occur i n the chromatograms that were run i n the BAW system. Hence i t was concluded that no free CHCA was present i n the plant 1, 2, 3, and 4 weeks a f t e r a p p l i c a t i o n of CHCA. The presence of the as p a r t i c acid conjugate was indicated i n most of the extracts but the spot was not always prominent. I t was observed i n the extracts of treated primary leaves and of roots at a l l times. The metabolite ' Y 1 was not detected i n the extracts of buds-flowers-pods at any time. The stem extracts at the end of 1 and 3 weeks and the extracts of untreated l e a f at 3 weeks also did not contain ' Y 1 . In a l l the other extracts 'Y' was present. The a c t i v i t i e s i n the glucose and aspartate conjugates of CHCA on the chromatograms of the extract were compared. As mentioned e a r l i e r , the free CHCA. observed on the chromatograms developed i n IAW system was i n f e r r e d to be a r e s u l t of hydrolysis of CHCA-glucose. Hence, f o r determining the a c t i v i t y i n the CHCA-glucose, both CHCA and CHCA-glucose spots were, eluted together and counted as CHCA-glucose. Figure 9a. Autoradiographs of ethanol extracts of plant parts, one week a f t e r a p p l i c a t i o n of KCHC-7-^"4C to the primary leaves. (Isopropanol : 1% ammonia : water) Figure 9b. Autoradiographs of ethanol extracts of plant parts, two weeks a f t e r a p p l i c a t i o n of KCHC-y-^^C to the primary leaves. (Isopropanol : TL ammonia : water) T - Treated leaves; B - Buds-flowers-pods; U - Untreated leaves; S - stem; R - Roots G - CHCA-glucose;- C - CHCA; Y - Unknown metabolite 'Y'; A - CHCA - a s p a r t i c a c i d . 0 Q " 0 0 0 " 03 R O n O O O C? n • • 1 i ' j 1— — 1 1 . • I T U B S R CHCA FIGURE 9 Figure 10a. Autoradiographs of ethanol extracts of plant parts, three weeks a f t e r a p p l i c a t i o n of KCHC-7-^4C to the primary leaves. (Isopropanol : 1% ammonia : water) Figure 10b. Autoradiographs of ethanol extracts of plant parts, four weeks a f t e r a p p l i c a t i o n of KCHC-7--^C to the primary leaves. (Isopropanol : 1% ammonia : water) T - Treated leaves; B - Buds-flowers-pods; U - Untreated leaves; S - Stem; R - Roots. G - CHCA - glucose; C - CHCA; Y - Unknown metabolite *Y'; A - CHCA - a s p a r t i c a c i d . c Y 0 o 0 0 0 o 0 —I— u Q 0 Q 0 0 0 0 U B S R CHCA FIGURE 10 F i g . 11 shows a plo t of time versus the r a t i o G/A, where G represents the a c t i v i t y i n CHCA-glucose and A, the a c t i v i t y i n CHCA-aspartate spots. Since a c t i v i t i e s are i n d i c a t i v e of amounts, i t can be said that G/A was greater than 1, when the amount of CHCA-glucose i n an extract was greater than the aspartate and was less than one when the amount of CHCA-aspartate i n the extract was greater than the CHCA-glucose. No consistent r e l a t i o n s h i p between G and A were observed. In the extracts of plant parts, 1, 2, and 4 weeks a f t e r treatment, the amounts of CHCA-glucose were greater than or equal to those of CHCA-aspartate. In the extracts of plant parts 3 weeks a f t e r a p p l i c a t i o n of KCHC, the CHCA-aspartate was i n greater amount than CHCA-glucose. 54. 1 2 3 4 WEEKS T - Treated leaves U - Untreated leaves B - buds,flowers,pods S - Stem R - Roots G - A c t i v i t y i n CHCA-glucose A - A c t i v i t y i n CHCA-aspartic acid FIGURE 11. Relative amounts of CHCA-glucose and CHCA-aspartic acid i n plant parts, one, two, three and four weeks a f t e r a p p l i c a t i o n of KCHC-7- 1 4C 5 5 . 5 . A c t i v i t i e s i n the plant residues a f t e r ethanol e x t r a c t i o n . The ethanol-insoluble plant residues were counted i n a powder form. The t o t a l a c t i v i t y per plant ranged from 0 . 0 5 muc to 0 . 0 8 nyjc. This represented less than 0 . 0 0 0 2 7 „ of the applied K C H C - 7 - - ' - 4 C and ca 0 . 4 7 ° or less of the ethanol-soluble a c t i v i t i e s . A major part of the ethanol-insoluble a c t i v i t y i n each case was contributed by the residues of the treated primary leaves. 6 . Evolution of - ^ C ^ by treated plants ^ 4 C 0 2 was released by a e r i a l portions of the plants whose primary leaves were treated with K C H C - 7 - ^ 4 C . Figure 1 2 shows the amount of ^ ( X ^ released by a plant during a 7-day period. A peak i n the evolution of ^ 4 C 0 2 was seen 5 days a f t e r treatment of leaves with K C H C - . The r a d i o a c t i v i t y l o s t as • ' " 4 C 0 2 during the 7-day period amounted to ca 1 . 7 7 o of the t o t a l a c t i v i t y of the applied K C H C . Although t h i s f i g u r e does not appear to be very high, i t i s approximately h a l f of the t o t a l ethanol sdluble a c t i v i t y (as percentage of the t o t a l a c t i v i t y applied to the leaves) i n the plants at the end of one week. This indicated that approximately a t h i r d of the chemical which had been absorbed by the leaves was decarboxylated at the end of one week. FIGURE 12. Decarboxylation of CHCA-7- C by bush bean plants following a p p l i c a t i o n to the primary leaves. 7. Translocation of KCHC-7-1^C out of the treated l e a f . The t r a n s l o c a t i o n of 2 , 4 - D has been shown to be dependent on l i g h t ( 5 5 ) . According to some authors ( 5 5 , 8 8 , 1 0 9 ) the presence of sugars favours i t s transport out of the treated leaves. In the following experiments the aim was to f i n d out i f l i g h t , sugar, or substrates for conjugation were necessary f o r the t r a n s l o c a t i o n of CHCA. The r e s u l t s are given i n Tables H a n d III,and F i g . 1 3 . As in Expt. 4 . , a c e r t a i n amount of v a r i a t i o n among the values of the r e p l i c a t e s was seen. Nevertheless, the conclusions reached from the r e s u l t s of one r e p l i c a t e were s i m i l a r to those from the other r e p l i c a t e . Hence the r e s u l t s are discussed using the mean values of r e p l i c a t e s 1 and 2 . In the plant that had been i n l i g h t f o r 1 2 hr, 76.77, of the t o t a l a c t i v i t y was i n the treated primary l e a f , whereas i n the plant supplied with glucose i n the dark, 9 6 . 1 7 , of the t o t a l a c t i v i t y remained i n the treated l e a f . When aspartate was provided for 1 2 hr to the plant i n the dark, the a c t i v i t y i n the treated leaf was 9 6 . 2 7 , of the t o t a l . There w a s ^ 9 8 . 5 7 o of the t o t a l a c t i v i t y i n the treated primary l e a f of the plant that had received d i s t i l l e d water i n the dark. From these values one can conclude that l i g h t favoured the movement of the chemical out of the l e a f . Glucose and aspartate promoted the movement i n the dark only s l i g h t l y , i . e . ca 2 . 3 7 , . When the a c t i v i t i e s i n the stems 1 2 hr a f t e r a p p l i c a t i o n of KCHC were compared, i t was seen that 2 0 . 2 7 , of the a c t i v i t y had moved into the stem i n the l i g h t compared to 0 . 6 7 , i n the stem of the water-fed 14 TABLE I I . Translocation of KCHC-7- C and i t s metabolites out of the l e a f under d i f f e r e n t conditions. D i s t r i b u t i o n of the carbon-14 i n leaves, stem, and root, as a percentage of the t o t a l , 12 hr a f t e r a p p l i c a t i o n of KCHC-7- 1 4C. Part of the plant In l i g h t In dark, with glucose In dark, with aspartate In dark, with water Replicates Replicates Replicates Replicates 1 2 Mean 1 2 Mean 1 2 Mean 1 2 Mean Treated primary l e a f 78.2 75.2 76.7 Untreated primary l e a f 1.3 1.1 1.2 94.2 97.9 96.1 0.4 0.7 0.6 96.8 95.6 96.2 0.4 1.4 0.9 98.2 98.7 98.5 0.9 0.9 0.9 Stem 19.5 20.8 20.2 5.1 1.1 3.1 2.7 2.9 2.8 0.8 0.3 0.6 Root 1.0 2.8 1.9 0.2 0.2 0.2 0.1 0.0 0.1 T o t a l C per plant 57.5 49.0 53.3 36.6 29.1 32.9 as m u^. c 0.0 0.0 0.0 60.0 40.0 50.0 55.8 30.8 43.3 oo plants i n the dark. Of the t o t a l r a d i o a c t i v i t y , 3.17„ was present i n the stem of the plant that had received glucose i n the dark. The stem of the plant that had been fed aspartate i n the dark contained 2.87, of the t o t a l a c t i v i t y . Thus the stem of the plant provided with glucose or aspartate for 12 hr i n the dark had f i v e times, and that of the plant i n l i g h t , 35 times, as much a c t i v i t y as the stem of the plant supplied with d i s t i l l e d water i n the dark. The untreated l e a f of the plant i n l i g h t contained 1.2% while that of the plant fed with glucose i n the dark had 0.67, of the t o t a l a c t i v i t y . The untreated leaves of the plants supplied with aspartate or water contained 0.97, of the t o t a l a c t i v i t y . The roots of the plant i n l i g h t had 1.9% of the t o t a l a c t i v i t y . The roots of the plant supplied with glucose or aspartate i n the dark had 0.17, of the t o t a l a c t i v i t y . There was no detectable a c t i v i t y i n the roots of the plants that received water i n the dark. At 24 hr, a s i m i l a r trend as at 12 hr was observed. The treated primary l e a f of the plant i n l i g h t contained 76.37, of the t o t a l a c t i v i t y . Of the r e s t , 20.17, was i n the stem, 1.07. i n the untreated l e a f and 2.67, i n the roots. In the plant supplied with d i s t i l l e d water i n the dark, about 98.2% a c t i v i t y stayed i n the treated l e a f , only 1.4% i n the stem. There was only 0.4% i n the untreated leaves, while the roots had no a c t i v i t y at a l l . The glucose fed to the l e a f i n dark appeared to have 14 favoured the t r a n s l o c a t i o n of C from the leaf to the stem. The stem had 9.4% of the t o t a l a c t i v i t y , the roots 0.4% and the untreated le a f , 0.5%. The treated leaf retained the remaining 89.8%. In the plant TABLE I I I . Translocation of KCHC-7- C and i t s metabolites out of the le a f under d i f f e r e n t c o n d i t i o n s . D i s t r i b u t i o n of the carbon-14 i n leaves, stem, and root, as a percentage of the t o t a l , 24 hr a f t e r a p p l i c a t i o n of KCHC-7- 1 4C In l i g h t In dark, with glucose In dark, with aspartate In dark, with water Part of Replicates Replicates Replicates Replicates the plant ^ 2 Mean \ 2 Mean 1 2 Mean i 2 Mean Treated primary l e a f 71.0 81.6 76.3 85.7 93.8 89.8 95.9 95.9 95.9 98.9 97.5 98.2 Untreated primary l e a f 1.0 0.9 1.0 0.4 0.5 0.5 0.5 0.8 0.7 0.3 0.5 0.4 Stem 26.0 14.3 20.1 13.8 5.0 9.4 3.6 3.3 3.5 0.8 1.9 1.4 Root 2.0 3.2 2.6 0.1 0.6 0.4 0.0 0.0 • 0.0 0.0 0.0 0.0 To t a l C per plant 123.3 123.1 123.2 180.0 165.0 172.5 191.6 93.3 142.5 259.2 81.6 170.4 as m^u c Replicate 1 Replicate 2 Light 12 hr msmam u s R 1 T mmmimmmmmm Dark U 1 & glucose S • R 1 T Dark U 1 & S 1 aspartate 1 R T Dark U 1 & 1 water S R 24 hr 8 I I 12 hr 24 hr I I 1 B I 1 I I a i I 1 i % of t o t a l 14„ FIGURE 13. 0 25 50 75 100 0 25 50 75 100 0 25 50 75 100 0 25 50 75 100 T - Treated primary leaf U - Untreated primary leaf S - Stem R - Root Translocation of CHCA and i t s metabolites out of the leaf under d i f f e r e n t conditions. D i s t r i b u t i o n of the carbon-14 i n leaves, stem and root, 12 and 24 hr a f t e r a p p l i c a t i o n of KCHC-7-^4C. which was supplied with aspartate, the a c t i v i t y i n the treated leaf was 9 5 . 9 7 o and i n the stem 3 . 5 7 » . The untreated l e a f had 0 . 7 7 „ and the roots, n e g l i g i b l e amounts. The t o t a l a c t i v i t y per plant was hig h l y v a r i a b l e (Tables I I and III) e.g. the a c t i v i t i e s i n the r e p l i c a t e s of water-fed plants d i f f e r e d by a f a c t o r of two, 1 2 hr a f t e r a p p l i c a t i o n of KCHC, and by a fa c t o r of three, 2 4 hr a f t e r a p p l i c a t i o n of KCHC. The t o t a l a c t i v i t i e s i n the r e p l i c a t e s of aspartate-fed plants d i f f e r e d by a fa c t o r of two 2 4 hr a f t e r a p p l i c a t i o n of KCHC. Compared to the above, the differences between r e p l i c a t e s of plants i n l i g h t were much l e s s . The v a r i a t i o n found i n these experiments i s reminiscent of that i n Expt. 4 . Twelve hours a f t e r a p p l i c a t i o n of KCHC - 7- 1 4C, the t o t a l a c t i v i t y i n the plant i n l i g h t was higher than that i n the plants i n dark given glucose, aspartate or water. This trend was however not observed 2 4 hr a f t e r a p p l i c a t i o n of the chemical. Considering only the mean values, i t i s seen that 2 4 hr a f t e r a p p l i c a t i o n the plants that had been i n . l i g h t contained about two and a h a l f times as much a c t i v i t y as at 1 2 hr. S i m i l a r l y the glucose-fed plants contained f i v e times as much and the water-fed plants four times as much a c t i v i t y at 2 4 hr as at 1 2 hr. The radiochromatographic analyses of the extracts (Figs. 1 4 ; a , b and 1 5 ; a , b ) revealed the following: T h e treated leaves from the plants i n l i g h t contained glucose and aspartate conjugates, and also the metabolite 'Y1. The presence of CHCA was also i n d i c a t e d . However, th i s spot was not present i n the autoradiograph of the chromatogram run i n a BAW system. Hence i t Figure 14. The autoradiographs of-extracts of the treated l e a f , the untreated l e a f , the stem, and the root, 24 hr a f t e r a p p l i c a t i o n of KCHC-7-^4C as a drop to the hase of one primary l e a f , a. of the plant that was maintained under the growth room conditions b. of the plant that was removed to dark 72 hr p r i o r to the a p p l i c a t i o n of KCHCW-^C. The treated l e a f was supplied with d i s t i l l e d water through the cut end. T - Treated l e a f ; U - Untreated l e a f ; S - Stem; R - Root. G - CHCA-glucose; C- CHCA; Y - 'Y1; A - CHCA-aspartic a c i d . Solvent system used for the chromatography: Isopropanol : 1% ammonia : water FIGURE 14 Autoradiographs of extracts of the treated l e a f , the untreated l e a f , the stem and the'root, 24 hr a f t e r a p p l i c a t i o n of KCHC-7- C as a drop to the base of one primary l e a f , of the plant that was removed to the dark 72 hr p r i o r to the a p p l i c a t i o n of KCHC-7- 1 4C. The treated l e a f was supplied with a glucose s o l u t i o n through i t s cut end. of the plant that was removed to the dark 72 hr p r i o r to the a p p l i c a t i o n of KCHC-7- l 4C. The treated leaf was supplied with a s o l u t i o n of sodium aspartate through i t s cut end. T - Treated l e a f ; U - Untreated l e a f ; S - stem; R - Root. G- CHCA-glucose; C - CHCA; Y - ' Y ' ; A - CHCA-aspartic a c i d . Solvent system used for the chromatography: Isopropanol 7% Ammonia : water FIGURE 15 6 5 . was concluded that no free acid was present i n the treated l e a f . The stem extracts contained the glucose and aspartate conjugates, and ' Y 1 , but i n much smaller amounts than the leaf extracts. Traces of spots corresponding to glucose and aspartate conjugates were observed i n the root extracts, e s p e c i a l l y 24 hr a f t e r a p p l i c a t i o n of KCHC. A prominent spot corresponding to CHCA-glucose was observed on the autoradiograph of the extract from the treated l e a f of plants supplied with glucose i n the dark CHCA-aspartate and ' Y 1 were also present. Small amounts of free acid were d i s c e r n i b l e i n the chromato-gram run i n BAW system. In the stem extract only traces of glucose conjugate were detected. At 24 hr, the glucose conjugate was very prominent i n the l e a f . ' Y ' and CHCA-aspartate were also quite d i s t i n c t . The stem extract also contained the above three metabolites. In the plants provided with aspartate i n the dark, the treated l e a f showed a d i s t i n c t spot of CHCA-aspartate on the auto-radiograph. At 24 hr t h i s spot was extremely'intense. The presence of free CHCA was indicated at both times. The glucose conjugate was present f n le a f extracts at 12 and 24 hr. This indicated that the plants were not t o t a l l y f r ee of sugars a f t e r they had been deprived of l i g h t for more than 72 hours. Spot ' Y * was also d i s c e r n i b l e . In the stem extracts at 12 hr, only the CHCA-aspartate was c l e a r . At 24 hr the CHCA-aspartate was more prominent and traces of CHCA-glucose and ' Y ' were also seen. In the plants given d i s t i l l e d water i n the dark, only the extracts of treated leaf showed the rad i o a c t i v e metabolites. These i n -cluded the glucose conjugate, ' Y ' , CHCA-aspartate and some free CHCA. In a l l the autoradiographs, spots with Rf values between those of 'Y' (0.56) and of CHCA-aspartate (0.17) were observed. These were not seen r e g u l a r l y i n the autoradiographs of extracts. The Rf values suggest that these r a d i o a c t i v e metabolites may correspond to the peptide conjugate of CHCA, obtained by Severson (98). 67. 8. L o c a l i z a t i o n of a c t i v i t y i n tissues This part of the i n v e s t i g a t i o n presented several d i f f i c u l t i e s . Freeze-drying of the tissues was necessary since CHCA and i t s metabolites are water soluble. An automatic f r e e z e - d r i e r was not a v a i l a b l e at the time and a freeze-drying apparatus was set up i n the laboratory. Considerable time and e f f o r t went into the improve-ment of t h i s . The r e s u l t s however were not s a t i s f a c t o r y . The apparatus was not consistent i n i t s operation. A f r e e z e - d r i e r was procured l a t e r and was employed i n getting the r e s u l t s described. The freeze-dried tissues were i n f i l -trated i n a p l a s t i c medium for several days. The i n f i l t r a t i o n was the most d i f f i c u l t part of the procedure. Only a few pieces became p a r t l y i n f i l t r a t e d even a f t e r a week. This p a r t l y affected the next step, the c u t t i n g of sections. Many sections crumbled. The sections that were r e l a t i v e l y e n t i r e , were placed on a glass s l i d e . Normally each s e c t i o n i s placed on a drop of water. The sec t i o n spreads out, and as the water drop dries the section becomes a f f i x e d to the s l i d e . However, i f water i s used to spread poorly i n -f i l t r a t e d t i s s u e s , leaching can occur. Hence, i n th i s experiment, the normal procedure was not followed. A mixture of CCl^-hexane was t r i e d as a su b s t i t u t e f o r water but was found to be i n e f f e c t i v e i n spreading the sections. Eventually the dry sections had to be placed on a glass s l i d e and breathed upon to ensure adherence. The e n t i r e s e c t i o n did not l i e i n one plane, with the r e s u l t that the microautoradiographs showed only a few c e l l s at focus. In the microautoradiographs of leaves the s i l v e r grains 68. that i n d i c a t e the l o c a t i o n of r a d i o a c t i v i t y , are d i s t r i b u t e d through-out the mesophyll (Fig.18a). Presence of a c t i v i t y i n the vein endings i s also evident. In the mesophyll c e l l s , marked as s o c i a t i o n of the grains with the chloroplasts was observed (Figs. 18b, 19, 20). In the stem, s i l v e r grains were observed i n the elements of the conducting t i s s u e s . The lumina of the xylem elements showed s i l v e r grains (Fig.16a). Figure 16b shows the presence of s i l v e r grains on the thickenings of two xylem elements. The adjacent par-enchymatous c e l l s also contained a few grains. In the phloem, grains were observed i n the sieve tubes ( F i g . 17a). Figure 17b shows s i l v e r grains i n ass o c i a t i o n with what appear to be slime bodies. Figure 16. Microautoradiographs of stem of treated p l a n t . a. Longitudinal s e c t i o n of stem. S i l v e r grains i n the lumina of the xylem elements. Phase contrast. X 900 b. Longitudinal section of stem. S i l v e r grains i n the xylem elements, and i n close a s s o c i a t i o n with the thicken-ings. The adjacent parenchyma c e l l s also contain a few grains. X 800 FIGURE 16 Microautoradiographs of stems of plants whose leaves were treated with KCEC-l-^C L.S. stem, showing prominent s i l v e r grains i n the sieve tube elements. X 900 L.S. stem, S i l v e r grains associated with structures resembling slime bodies. Phase contrast. X 1050 Figure 18. Microautoradiographs of leaves treated with KCHC-7- C. a. T.S. Leaf. The white grains representing a c t i v i t y are d i s t r i b u t e d throughout the mesophyll. A part of vascular t i s s u e i s seen as a white patch i n the centre. Dark f i e l d . X 500 b. T.S. Leaf. A row of black s i l v e r grains are seen.across chloro p l a s t s (arrow). A few other c h l o r o p l a s t s also show grains. The grains on the other chloroplasts are not i n focus. Phase contrast. X 1350 FIGURE 18 Microautoradiography of leaves treated with KCHC-7-T.S. Leaf. S i l v e r grains associated with c h l o r o p l a s t s . Phase contrast. X 900. The grey d i s c o i d bodies are the c h l o r o p l a s t s . The s i l v e r grains on some are i n focus. X 990. Groups of grains are associated with c h l o r o p l a s t s . The black d i s c o i d bodies are the chloroplasts that are over focussed. FIGURE 19 Microautoradiographs of leaves treated with KCHC-7 T . S . of leaf, focussed on the chloroplasts Phase contrast. X 900/ Same section as (a), focussed on the silve r grains Groups of grains can be seen to correspond to the location of chloroplasts in (a). FIGURE 20 74. 9. C e l l f r a c t i o n a t i o n using aqueous media C e l l f r a c t i o n a t i o n i s the simplest and commonest method of separation of c e l l organelles. Reasonably pure, homogenous f r a c t i o n s of c e l l s can be obtained by th i s method. F r a c t i o n a t i o n of KCHC-7--Re-treated t i s s u e and the subsequent counting of the d i f f e r e n t f r a c t i o n s would i n d i c a t e i f an as s o c i a t i o n of the chemical and a p a r t i c u l a r group of organelles existed. The procedure followed i s shown schematically i n F i g . 21. A descending order of c e n t r i f u g a t i o n was employed to minimize leaching from the organelles. A s o l u t i o n of 0.35 M NaCl has been i n use for several years as a c e n t r i f u g a t i o n medium ( 9 )• The other medium, phosphate buffer with carbowax and KCl, has also been used s u c c e s s f u l l y to i s o l a t e chloroplasts ( 123). The r e s u l t s of c e l l f r a c t i o n a t i o n using aqueous media are shown i n Table IV. The values show thatv, i n both c e n t r i f u g a t i o n media, more than 907» of the t o t a l a c t i v i t y i s present i n the 20,000 super-natant. This supernatant f r a c t i o n would be expected to contain the microsomes and the soluble materials of the c e l l . The supernatant of 20,000 was not centrifuged at higher speeds. Hence no conclusion regarding the d i s t r i b u t i o n of a c t i v i t y i n the ribosomes, microsomes, or the soluble phase of the c e l l per se was p o s s i b l e . The a c t i v i t i e s i n the nuclear and chloroplast f r a c t i o n s (Fractions 5 and 4 i n Table IV) are very low as compared to the a c t i v i t y i n the soluble f r a c t i o n . The differences i n the a c t i v i t i e s of f r a c t i o n s 4 and 5 i n 75. Leaf homogenate F i l t e r e d through 2 layers of cheesecloth F i l t r a t e x 20,000 £ Sediment Resuspended i n grinding medium x 10,000 g Sediment Resuspended i n grinding medium x 1000 £ Sediment Resuspended i n grinding medium x 600 £ Sediment F r a c t i o n 5 Supernatant F r a c t i o n 1 Supernatant F r a c t i o n 2 Supernatant F r a c t i o n 3 Supernatant F r a c t i o n 4 FIGURE 21. Schematic representation of the aqueous f r a c t i o n a t i o n procedure. TABLE IV. C e l l f r a c t i o n a t i o n . D i s t r i b u t i o n of carbon-14 i n d i f f e r e n t c e l l f r a c t i o n s , as a percentage of the t o t a l a c t i v i t y . Organelles F r a c t i o n # %, of t o t a l carbon-14 present b c Medium 1 Medium 2 1 Microsomes, soluble material of homogenate 96.45 91.57 2 Smaller mitochondria 1.06 3 Larger mitochondria 1.71 1.87 4 Chloroplasts 0.09 2.98 5 * Intact c e l l s , n u c l e i , few chloroplasts 0.66 ' 3.57 a See Figure 21 b Medium 1 for homogenisation and c e n t r i f u g a t i o n : 30% carbowax 4000 i n 0.5M phosphate buffer, pH 7.0, containing 0.01 M potassium chl o r i d e (123) c Medium 2 f o r homogenisation and c e n t r i f u g a t i o n : 0.35 M sodium c h l o r i d e (9). the two aqueous c e n t r i f u g a t i o n media are conspicuous. Differences i n degree of homogenization could r e s u l t i n greater a c t i v i t i e s i n c e r t a i n f r a c t i o n s i n one medium as compared to those i n the other. However, i t i s believed that t h i s could not have been a major source of d i f f -erence since the blendings of l e a f t i s s u e were c a r r i e d out for the same lengths of time. Another possible reason might be the leaching of a c t i v i t y from the organelles. Medium 1 perhaps leached out more a c t i v i t y from the components of f r a c t i o n s 4 and 5 than did medium 2. 78. 10. Cell fractionation using non-aqueous medium This method of fractionation was used by Hallam and Sargent (52) for localizing 2,4-D in leaf tissue. The method has an advantage over that using an aqueous medium in that leaching of water soluble substances from the c e l l parts cannot occur. Fractions obtained after ultracentrifugation were assayed for radioactivity and the counts per minute values were corrected for background and quenching. The corrected values, disintegrations per minute, were plotted against the fraction number (Figs. 22 and 23). Each figure represents the activity p r o f i l e in one tube. In Figure 22 i t is seen that the peak of activity is found in fraction 9 and in fraction 27. In Figure 23 the peaks are in fractions 9, 20, and 24. The fraction 9 in each tube was from the green band observed at the junction of the specific gravity layers 1.34 and 1.052. An examination "of a comparable fraction under the microscope showed that i t was comprised largely of chloroplasts. A few pieces of broken c e l l wall were also seen. The other peaks in Figures 22 and 23 are from the bottom of the tube. A microscopic observation of these fractions revealed the presence of intact c e l l s , pieces of vascular tissue, guard cells, c e l l walls, etc. Incubation of chloroplast fractions with t e l l u r i t e did not produce dark granules of reduced tellurium (Fig.24a). This fact indicated the absence of mitochondria. The presence of mitochondria was however evident in the samples from the bottom of the tube, since FIGURE 22. Carbon-14 a c t i v i t y p r o f i l e of a CCl^-hexane density gradient of homogenat CHCA-7- 14C treated leaves. FIGURE 23. Carbon-14 a c t i v i t y p r o f i l e of a CCl^-hexane density gradient of homogenate of CHCA-T-^^C treated leaves. Figure 24 a. The chloroplast f r a c t i o n , incubated with 0.05M t e l l u r i t e and succinate for 4 hr. . No prominent dark granules of-reduced t e l l u r i u m are seen. X 600. b. The f r a c t i o n s from the bottom of the c e n t r i f u g a t i o n tube, incubated with 0.05 M t e l l u r i t e and succinate f o r 4 hr. Prominent dark granules of reduced t e l l u r i u m are seen. X 600. FIGURE 24 large, dark granules of reduced t e l l u r i u m were formed from t e l l u r i t e ( F i g . 24b) .The mitochondria i n the i n t a c t c e l l s would have been responsible f o r the reduction of t e l l u r i t e . The a c t i v i t y associated with the chloroplasts could have been due to the ra d i o a c t i v e CHCA and i t s metabolites. On the other 14 hand the CO2 r e s u l t i n g from decarboxylation of the a c i d might have been photosynthetically f i x e d i n the ch l o r o p l a s t s . Both of the above could have been involved. In order to determine i f the a c t i v i t y associated with the chloroplasts was due to CHCA and i t s metabolites, the green band c o n s i s t i n g of chloroplasts was extracted i n ethanol and the extracts chromatographed. The autoradiograph of the chromatogram showed that CHCA-glucose and CHCA-aspartic acid were present i n the chloroplast f r a c t i o n . The f r a c t i o n s containing the i n t a c t c e l l s etc. also showed the presence of the conjugates of CHCA. The metabolite Y which was not conspicuous i n the chlor o p l a s t f r a c t i o n was evident i n the c e l l f r a c t i o n . No other r a d i o a c t i v e spot was detected on the autoradiograph 14 suggesting that no C02 had been f i x e d i n photosynthesis during the 24 hr period. 83. DISCUSSION 1. Growth The stimulation of vegetative growth observed i n bushbean plants treated with CHCA i n the present i n v e s t i g a t i o n i s s i m i l a r to that reported by Fattah (39) and Fattah and Wort (40) with KNap-treated p l a n t s . The concentration of KNap used by the above authors (39, 40) was about four times greater than that of CHCA used i n the present study. The 127o increase i n y i e l d of pods obtained by using CHCA lacked s t a t i s t i c a l s i g n i f i c a n c e (See p. 114). However, Wort et a l (118) reported s t a t i s t i c a l l y s i g n i f i c a n t increases i n y i e l d of pods i n bushbean plants treated with 1 x 10"2M and 2 x 10"2M CHCA (35 and 247o r e s p e c t i v e l y ) . The cyclohexene-, cyclopentane-, and cyclo-heptane carboxylic acids did not invoke s i m i l a r responses. The authors suggested the presence of a 6-carbon saturated r i n g to be e s s e n t i a l for the stimulation of growth. A s i m i l a r conclusion was arr i v e d at by Agakishiev ej: a l (15) following t h e i r work with sub-s t i t u t e d cyclohexyl butanols and cyclohexyl butanones on cotton. The increased growths observed by the above authors are comparable to the naphthenate induced stimulation observed i n a number of crops (1, 2, 10, 14, 24, 39, 69, 70, 116, 117, 124). I t i s thus seen that the vegetative and reproductive growths were stimulated by KNap, and CHCA, a constituent of naphthenic ac i d s . S i m i l a r responses were not observed with the 5 or 7 carbon saturated r i n g - and cyclohexene carboxylic acids (118). I t i s p o s s i -ble that i t i s the CHCA i n the naphthenic acid mixture that i s l a r g e l y responsible for the growth stimulation observed i n the treated plants. 84. 2. Movement of acid within the plant The i n j e c t i o n of KCHC into the midrib of a primary l e a f , introduced the chemical d i r e c t l y i n t o the conducting t i s s u e s . The movement of the chemical then occurred v i a the phloem or the xylem, Consequently the a c t i v i t y spread r a p i d l y within the plant. A wedge of a c t i v i t y was seen i n the a p i c a l halves of the inj e c t e d leaves, d i s t a l to the point of i n j e c t i o n ( F i g . 5). This has been c a l l e d the apoplastic wedge (26) and i t represents the acropetal transport of the rad i o a c t i v e substance i n the xylem of the l e a f . In other words, the i n j e c t i o n introduced the CHCA in t o the xylem from where the a c t i v i t y moved a c r o p e t a l l y i n the form of a wedge. Since the a c t i v i t y could be detected i n the stem and i n the roots one h a l f hour a f t e r i n j e c t i o n , i t i s l o g i c a l to assume that the a c t i v i t y d i s t r i b u t e d i n the leaf had been trans-ferred v i a the phloem to the stem and the roots. The transfer of a c t i v i t y i n t o the phloem may have occurred at the vein endings. CHCA, when applied to the surface of the l e a f , has to pass through c u t i c l e , epidermis, and mesophyll, one or more of which perhaps provides resistance to the passage of the material toward the vascular t i s s u e . I t i s l i k e l y that t h i s slow i n i t i a l movement to the vascular tissues i s responsible f o r the longer time i n t e r v a l s taken by the chemical to reach the other organs of the plant. 85, The actual values f o r the rate of movement of 2,4-D through the leaf t i s s u e have been ca l c u l a t e d . L i t t l e and Blackman reported that 79 min elapsed before 2,4-D from the surface of a bean l e a f reached the underlying vascular bundles (73). Day determined the rate of movement of 2,4-D through the mesophyll t i s s u e to be 30 js/hr (28). The presence of a c t i v i t y i n the roots h a l f an hour a f t e r i n j e c t i o n indicated that a ba s i p e t a l transport of the compound had occurred i n the stem. At the same time the presence of a c t i v i t y i n the shoot apex i n h a l f an hour suggested that acropetal move-ment from the leaf to the shoot apex a l s o took place. Thus the movement of the chemical i n the phloem appears to be b i d i r e c t i o n a l . R a d i o a c t i v i t y i n the primary l e a f could have been acquired by acropetal transport from the .root, by basipetal transport from the shoot apex, or i n both ways. Twelve hours a f t e r a p p l i c a t i o n of CHCA to the leaf surface the stem, the roots, and the opposite leaves had become r a d i o a c t i v e . The a c t i v i t i e s i n the three organs increased i n 24 hr. The a c t i v i t y i n the opposite l e a f was less than that i n the roots at each time, suggesting that the roots acquired the a c t i v i t y p r i o r to the opposite l e a f . Thus a basipetal transport from the leaves to the roots followed by an acropetal one from the roots to the opposite l e a f probably occurred. In the CHCA-treated plant, i n one hour a f t e r i n j e c t i o n of KCHC-7- 1 4C and within 12 hr a f t e r a p p l i c a t i o n of KCHC-7- 1 4C to the base of a primary l e a f , the r a d i o a c t i v i t y had reached the opposite 86. l e a f . This suggests that a t r a n s f e r of a c t i v i t y occurred from phloem to xylem. The region where t h i s transfer occurred i s not known. I t i s i n t e r e s t i n g that L i t t l e and Blackman reported that the a c t i v i t y from the l e a f to which l a b e l l e d 2,4-D had been applied, did not reach the opposite l e a f even a f t e r 27 hr following a p p l i -cation (73). They suggested that there was no t r a n s f e r of 2,4-D from the phloem to the xylem. This transfer, however, was observed i n aspen by E l i a s s o n (33). Huseinov (60) observed that naphthenates caused bending of Avena c o l e o p t i l e s . This suggested that the movement of naph-thenates was polar, l i k e an auxin. This f i n d i n g i s not supported by the r e s u l t s obtained with CHCA, which i n d i c a t e a nonpolar move-ment. However, i t i s p o s s i b l e that the movement i n c o l e o p t i l e s d i f f e r s from that i n the i n t a c t plants. This i s supported by the f a c t that IAA, whose movement i n c o l e o p t i l e s and p e t i o l e segments i s known to be predominantly b a s i p e t a l , was shown to move upward and downward from the treated leaves of V i c i a faba (35). 87. 3. Appearance of metabolites of CHCA - a time course study The formation of conjugates of aspa r t i c acid with IAA (7, 8, 38, 44, 67, 79); BA (67, 127), NAA (68, 126), and 2,4-D (8, 101) has. been known for some years. KlHmbt (67, 68) and Zenk (126,127,128) reported the formation of glucose conjugates of IAA, BA, and NAA. Both KlSmbt and Zenk r e f e r r e d to the glucose conjugates as temporary detoxication products. The formation of glucose and aspa r t i c acid conjugates of CHCA was reported by Severson et al (97). In the present i n v e s t i g a t i o n , the sequence i n which the metabolites appeared was determined. The CHCA-glucose appeared f i r s t , followed by the aspartate conjugate approximately an hour a f t e r treatment of l e a f t i s s u e with CHCA. The same sequence was observed with the conjugates of IAA by Zenk (128). An unknown metabolite of CHCA appeared about an hour a f t e r treatment and was seen to p e r s i s t through the experiment duration of 48 hr. The CHCA-glucose was present during the 48 hr although the amount of a c t i v i t y as CHCA-glucose decreased a f t e r 4 hr. The a c t i -v i t y i n spot ' Y ' was seen to increase a f t e r 4 hr. The increase i n 'y' between 4 and 48 hr coincided with the decrease i n CHCA-glucose during that i n t e r v a l of time. This suggested that ' Y * may be another metabolite of CHCA, perhaps derived from CHCA-glucose or that i t was another isomeric form of CHCA-glucose. On the other hand i t could have been a compound e n t i r e l y d i f f e r e n t from CHCA, which had probably 1 / become rad i o a c t i v e by incorporation of C0 2, a product of decarboxy-l a t i o n of KCHC-7- 1 4C. The acid and a l k a l i n e hydrolyses of ' Y ' did not produce any conclusive r e s u l t s . 88. L i t t l e or no CHCA was present i n the treated leaves 8 hr af t e r a p p l i c a t i o n to the le a f t i s s u e . This i s not true of some other growth hormones. A part of exogenously applied IAA was found i n pea seedlings at a l l times (79) . The authors stated that the con-jugation and decarboxylation of IAA helped to maintain a p h y s i o l o g i c a l concentration of the auxin i n the plants. Of the supplied 2,4-D, 27% was recoverable as such from bean plants 5 days a f t e r a p p l i c a t i o n of the herbicide (54). In maple, 94.1% of the absorbed 2,4-D remained as the free ac i d i n the treated leaves a week a f t e r treatment while i n roots 44.7% of the 2,4-D remained unmetabolized (85). t 4• Persistence of the acid The r e s u l t s obtained with CHCA indicate- that a major part of the absorbed ^ 4C a c t i v i t y stayed i n the treated primary leaves. The roots contained the next highest amounts. The t r i -f o l i a t e s and the stem had approximately equal amounts. The f r a c t i o n from the buds-flowers-pods contained the lowest amounts of a c t i v i t y . This pattern of d i s t r i b u t i o n was observed at 1,2, 3, and 4 weeks a f t e r treatment with KCHC. There was no free CHCA present i n any of the organs at any time. The CHCA-glucose and the CHCA-aspartate were detected i n a l l the organs. The autoradiographs of the extracts of treated leaves and roots were the only ones which showed prominent spots of CHCA-aspartate. The above r e s u l t s show some s i m i l a r i t i e s and d i s s i m i l a r i -t i e s with those obtained by other authors, with other growth substances. Following a p p l i c a t i o n of IAA to the primary leaves of beans, only 10-14% of IAA taken up was translocated to other parts of the plant (37). The re s t of the a c t i v i t y remained i n the treated leaves. The findings of Morris et al_ (79) indicated that 12 hr a f t e r a p p l i c a t i o n of l a b e l l e d IAA to the shoot apex of pea plants, the high-est a c t i v i t y was present i n the apex. The roots contained the second highest amounts. Norris and Freed found that 77% of the absorbed 2,4-D stayed i n the treated leaves of maple (84). The roots con-tained a c t i v i t i e s ranging between 4.9 and 77%,. A l l these findings reveal that the major part of these growth substance remain i n the organs to which they were applied. The same i s true f o r CHCA. Good e_t al (44) reported that the metabolites of IAA could not account f o r the t o t a l IAA supplied. Norris and Freed (84) found that only 2.9% of the 2,4-D, applied to the primary leaves, was absorbed by maple plants i n three days. One week a f t e r spreading KCHC-7--'-4C on the primary leaves of bush bean, only 3.7% of the t o t a l a c t i v i t y applied was recoverable from the plant (Table I ) . However, when the chemical was applied as a drop to the base of the l e a f , the uptake was greater. Of the t o t a l a c t i v i t y supplied 10.7%, and 24.6% were recoverable from the plants, 12 and 24 hr a f t e r a p p l i c a t i o n , r e s p e c t i v e l y (Tables II and I I I ) . The persistence of CHCA-glucose for four weeks a f t e r a p p l i -c ation of CHCA i s of i n t e r e s t . An observation of t h i s type has not been made with regard to any other growth substance. KlHmbt (67,68) and Zenk (126, 127, 128) reported the glucose conjugates of IAA, NAA and BA to be temporary detoxication products that are eventually con-verted to the aspartate conjugate. The persistence of CHCA-glucose u n t i l four weeks a f t e r a p p l i c a t i o n shows that i t i s not a temporary metabolite. I t al s o occurs i n a l l parts of the plant. Perhaps, the CHCA-glucose i s responsible i n some way jfor the production of growth stimulation. The aspartate conjugates of IAA, BA, and NAA have also been re f e r r e d to as detoxication products (67, 68). Davies and Galston (27) suggested that a complex of IAA-aspartate-tRNA may have a function, s i m i l a r to that of formylmethionine, as a p r o t e i n chain i n i t i a t o r . In t h i s connection, the peptide conjugate of CHCA, suggested by Severson (98) i s of considerable i n t e r e s t . Perhaps t h i s peptide represents a formative stage of a protein chain. It i s not known i f the amounts of CHCA i n the pods are tox i c to man. CHCA i s known to be metabolized by dogs, guinea pigs, r a t s and rabbits and excreted as hippuric acid (42). It i s possi b l e that t h i s detoxication mechanism i s present also i n man. The low le v e l s of CHCA and i t s metabolites present i n the pods may well prove to be nontoxic. 92. 5. A c t i v i t y i n t h e r e s i d u e s f r o m e t h a n o l e x t r a c t i o n The e t h a n o l - i n s o l u b l e a c t i v i t y amounted t o 0.47, of t h e 14 a b s o r b e d C and 0.0027, o f t h e t o t a l a c t i v i t y a p p l i e d . I t i s p o s s i b l e t h a t t h i s a c t i v i t y may be due t o C H C A - p r o t e i n complexes. The CHCA-peptide c o n j u g a t e s , s u g g e s t e d by S e v e r s o n ( 9 8 ) , p e r h a p s r e p r e s e n t e d t h e f o r m a t i v e s t a g e s o f C H C A - p r o t e i n complexes. P r o t e i n complexes o f IAA have been r e p o r t e d (79, 1 2 8 ) . Zenk (128) f o u n d t h a t IAA was complexed w i t h p r o t e i n a t t h e r a t e o f 0. 0033 ug IAA/mg p r o t e i n o f p e a e p i c o t y l s . M o r r i s e t al (79) d i s c o v e r e d t h a t t h e a c t i v i t y i n t h e e t h a n o l - i n s o l u b l e r e s i d u e s o f pea s e e d l i n g s was due t o I A A - p r o t e i n complexes. The p h y s i o l o g i c a l r o l e o f t h e s e complexes i s n o t known. The i s o l a t i o n o f t h e p r o t e i n f r o m t h e r e s i d u e s and d e t e r -m i n a t i o n o f t h e a c t i v i t y a s s o c i a t e d w i t h t h e p r o t e i n w i l l show whether CHCA i s complexed w i t h p r o t e i n s . 6. D e c a r b o x y l a t i o n o f CHCA-7- 1 4C A p p r o x i m a t e l y a t h i r d o f t h e c h e m i c a l w h i c h had been ab-s o r b e d by t h e l e a v e s was d e c a r b o x y l a t e d by t h e end o f one week. 14 The C0 2 r e s u l t i n g f r o m t h e d e c a r b o x y l a t i o n o f CHCA c o u l d be r e f i x e d i n p h o t o s y n t h e s i s and dar k f i x a t i o n and th u s f o r m a new s o u r c e o f l a b e l . However, t h e absence o f any a d d i t i o n a l s p o t o t h e r t h a n t h e c o n j u g a t e s and 'Y' on the chromatograms o f t h e . p l a n t e x t r a c t s 1, 2, 3 and 4 weeks a f t e r t r e a t m e n t s u g g e s t s t h a t perhaps t h e amounts 14 o f CO2 r e f i x e d were not v e r y s i g n i f i c a n t . The f a t e o f t h e c y c l o h e x y l m o i e t y r e s u l t i n g f r o m t h e de-c a r b o x y l a t i o n o f CHCA s h o u l d be i n t e r e s t i n g t o s t u d y . The p o s s i b i l i t y o f t h i s c y c l o h e x y l m o i e t y o r i t s d e r i v a t i v e ( i f i t i s m e t a b o l i s e d i n the plant) having some growth stimulating property cannot be ruled out. The phenomenon of decarboxylation has been observed i n the case of other growth substances a l s o . The reports are however very contradictory with respect to the amounts decarboxylated. According to some authors the rates of decarboxylation of IAA (7, 38, 79, 80), BA (8), NAA (71), and 2,4-D (36) were high. However, low amounts of decarboxylation have been observed by others following the a p p l i c a t i o n of BA (126) and 2,4-D (8, 110). Leeper e± a l (71) showed that decarboxylation of NAA was increased by l i g h t whereas Morris (80) found that loss of ^C02 from IAA was greater i n the dark than i n l i g h t . 7. Translocation of CHCA The r e s u l t s obtained (Tables II and I I I , F i g s . 13,14 and 15) suggest that the t r a n s l o c a t i o n of CHCA out of the treated leaf i s favoured by l i g h t . The p r o v i s i o n of glucose or aspartate i n -creased the export from the leaf i n the dark. The glucose was, however, more e f f e c t i v e than the aspartate. The t r a n s l o c a t i o n of 2,4-D has been shown to be dependent on l i g h t (55). Several authors have reported that a supply of sugars f a c i l i t a t e d the t r a n s l o c a t i o n of 2,4-D out of the treated l e a f (55, 63, 88, 109). Hay and Thimann found that the transport of 2,4-D out of the leaf was activated by sucrose, applied to the surface of the treated leaf (55). Mannitol, urea, and arabinose could not substitute f o r sucrose. The authors suggested that the transport of 2,4-D or, at le a s t a step l i m i t i n g i t s transport, may be a metabolic process. The carbohydrates that can f u r n i s h energy w i l l favour the transport while compounds that cannot provide energy w i l l not be e f f e c t i v e i n promoting the transport of the herbicide. Phosphorus d e f i c i e n c y has been found to e f f e c t the trans-l o c a t i o n of 2,4-D adversely (89). Greenham discovered that the tr a n s l o c a t i o n of 2,4-D was accelerated by 0.25 M phosphate buffer fed to the treated leaf (46). This was not observed when glycine or c i t r a t e was supplied. These authors pointed out that phosphory-l a t i o n and ATP involvement played a r o l e i n the t r a n s l o c a t i o n of the herbicide. I t i s possible that the t r a n s l o c a t i o n of CHCA i s dependent on the a v a i l a b i l i t y of ATP. In l i g h t , the plants would have s u f f i -c i e n t ATP, r e s u l t i n g from photosynthesis and r e s p i r a t i o n . ATP pro-duction i s l i m i t e d by the low amounts of carbohydrates i n the plants i n the dark. The p r o v i s i o n of glucose to such plants, would increase the ATP production and thus f a c i l i t a t e the tr a n s l o c a t i o n of CHCA out of the l e a f . The addi t i o n of aspartate could not stimulate ATP production unless a c e t y l CoA were a v a i l a b l e (TCA c y c l e ) . The amounts of a c e t y l CoA i n the plants i n the dark would also be l i m i t e d by low rates of g l y c o l y s i s . This suggests why the t r a n s l o c a t i o n of CHCA i n the dark was more e f f e c t i v e with glucose than with aspartate. The presence of free CHCA i n the leaves of the plants i n the dark i s of i n t e r e s t . In the glucose-fed leaves, only traces of ' acid were observed, whereas much larger amounts were present i n the leaves that were fed aspartate or water. Some f a c t o r ( s ) necessary f o r conjugation was probably not"available i n these plants. Swets and Wedding (101) reported that a c e t y l CoA was i n -volved i n the formation of an aspar t y l 2,4-D complex. The acetyl CoA was involved i n the f i r s t r e action undergone by 2,4-D i n the c e l l . The p a r t i c i p a t i o n of asp a r t i c acid followed leading to the formation of the as p a r t i c acid conjugate of 2,4-D. The factors pro-moting g l y c o l y s i s augmented the production of ac e t y l CoA and thus favoured the formation of the conjugate. The addi t i o n of exogenous acetate also favoured the re a c t i o n . The formation of CHCA-aspartate may also involve a c e t y l CoA. In dark, owing to the depletion of carbohydrates, g l y c o l y s i s would be decreased, leading to a shortage of acetyl CoA in the plants. Consequently, i n the aspartate-fed plants, conjugation could not have occurred i n s p i t e of the a v a i l a b l e a s p a r t i c a c i d . In the water-fed plants, l i m i t e d amounts of glucose, a s p a r t i c acid and a c e t y l CoA would have been a v a i l a b l e . Hence free a c i d was present i n the treated leaves of these plants u n t i l 24 hr a f t e r treatment. Morris et a l (79) stated that IAA-aspartate was immobile i n pea seedlings. In the present experiment, CHCA-glucose was present i n the stem of the glucose-fed plant 12 hr a f t e r a p p l i c a t i o n of KCHC-7--^C. In the aspartate-fed plant, the stem contained only CHCA-aspartate at 12 hr but both conjugates at 24 hr. This suggests that the large amounts of CHCA-glucose and CHCA-aspartate formed i n the treated leaves of glucose- and aspartate-fed plants, r e s p e c t i v e l y , moved out of the l e a f i n t o the stem. There i s , however, the p o s s i b i l i t y that the a c i d moved out of the l e a f into the stem and was conjugated i n the stem t i s s u e s . Radiochromatographic analyses of the metabolites of CHCA i n the internodes immediately below and above the l e a f node at shorter i n t e r v a l s of time following a p p l i c a t i o n would show con-c l u s i v e l y i f CHCA i s transported as a conjugate or as a free a c i d . From the present i n v e s t i g a t i o n no conclusive evidence can be drawn about the m o b i l i t y of these conjugates. 8. L o c a l i z a t i o n of CHCA The microautoradiographs showed the presence of radio-a c t i v i t y i n the xylem and the phloem of the stem. This suggests that the chemical i s transported i n both tissues as discussed e a r l i e r (p.84 ) . In the l e a f t i s s u e the a c t i v i t y was c l o s e l y associated with the c h l o r o p l a s t s . The a s s o c i a t i o n of a c t i v i t y from l a b e l l e d IAA with the p l a s t i d s i n the parenchyma of Coleus internode segments was reported by Sabnis est a_l (90) . The authors however did not discuss the s i g n i f i c a n c e of t h i s a s s o c i a t i o n . The l o c a l i z a t i o n of CHCA i n the chloroplasts i s d i s -cussed with the r e s u l t s from the c e l l f r a c t i o n a t i o n experiments (P- 98). 9. C e l l f r a c t i o n a t i o n s The aqueous f r a c t i o n a t i o n of CHCA-treated t i s s u e reveal-ed that the major part of the a c t i v i t y was associated with the 20,000 j> supernatant. This f r a c t i o n contained the microsomes and soluble components of the c e l l . Galston et a l reported that IAA was associated with the soluble phase of the c e l l (43). The a c t i v i t y from l a b e l l e d 2,4-D was associated with the soluble f r a c t i o n of the c e l l when the treated l e a f t i s s u e was f r a c t i o n -ated, i n an aqueous medium (52). A non-aqueous f r a c t i o n a t i o n , however, revealed that the a c t i v i t y was concentrated i n the c h l o r o p l a s t f r a c t i o n . The r e s u l t s obtained from f r a c t i o n a t i o n of CHCA-treated ti s s u e i n a non-aqueous medium did not support the conclusions obtained from f r a c t i o n a t i o n i n the aqueous media. The a c t i v i t y was found i n the chloroplasts and not i n the soluble phase. Leaching of water soluble a c t i v i t y could have occurred i n the aqueous media but not i n the non-aqueous medium. Moreover, the evidence from the microautoradiographs suggests that the a c t i v i t y was l o c a l i z e d i n the c h l o r o p l a s t s . The ethanol extract of the chloroplasts (obtained by non-aqueous f r a c t i o n -ation) contained the conjugates of CHCA. From a l l the fact s mentioned above i t would appear that the a c t i v i t y found i n the soluble phase i n the aqueous medium had been leached out of the ch l o r o p l a s t s . The l o c a l i z a t i o n of CHCA i n the chloroplasts suggests at l e a s t one s i t e of act i o n of the naphthenic a c i d . Increases i n photosynthetic pigments i n naphthenate treated plants were observed by several authors (2, 14, 24, 40, 70). Photo-synthesis was reported to be increased following a p p l i c a t i o n of naph-thenates to plants (2, 4, 14, 24, 41, 70). Electronmicrographs of le a f t i s s u e of the treated plants showed abundant starch grains i n the chloro-p l a s t s (D. J. Wort, unpublished data). The chloroplasts of the controls had much fewer starch grains. This indicated an increased production of photosynthate i n the treated plants. These increases i n the metabolism of the chloroplasts are perhaps d i r e c t l y due to the CHCA or i t s meta-b o l i t e s . According to Fattah and Wort (41) an increase i n photo-synthesis r e s u l t s i n the production of increased photosynthate, reduced nucleotides, and ATP. The increased photosynthateis a v a i l a b l e f o r the biosynthetic processes. Respiration i s also stimulated, r e s u l t i n g i n an augmented supply of reduced nucleotides, ATP, and keto acids, necessary f o r the synthesis of amino acids. Thus, a chain of events s t a r t i n g from 99. an increased photosynthesis could lead to an o v e r a l l increase i n metabolism, and f i n a l l y , i n growth. S i g n i f i c a n t increases i n the amounts of RNA and DNA ( 2 2 . 6 7 o and 1 7 . 5 % r e s p e c t i v e l y ) were observed i n the leaves of KNap-treated plants ( 8 6 ) . An increase i n RNA i s i n d i c a t i v e of an increased metabolism but an increase i n DNA suggests a greater c e l l number. I t i s not known whether the number of c e l l s per gram of the treated leaf i s greater than that of the c o n t r o l . The DNA obtained by the methods employed by the above authors would have included both the nuclear and the non-nuclear f r a c t i o n s . The non-nuclear or cytoplasmic DNA forms only a small per-centage of the t o t a l DNA. However, the increase observed could have resul t e d from an increase i n both kinds of DNA. Degani et a l reported that the increase i n the DNA of the cucumber hypcotyl treated with IAA or GA was mainly i n the non-nuclear f r a c t i o n ( 2 9 ) . It i s p o s s i b l e that a s i m i l a r increase i n the non-nuclear DNA could have occurred i n the K-Nap-treated t i s s u e . The l o c a l i s a t i o n of CHCA i n the chloroplasts suggests that the DNA i n these organelles might have been a f f e c t e d . An increase i n the chloroplast DNA need not i n d i c a t e an increase i n the number of c h l o r o p l a s t s . Herrmann ( 5 6 , 5 8 ) , Herrmann et a l ( 5 7 , 5 9 ) have shown that i n beet, the number of DNA centres per chl o r o p l a s t varied with the s i z e of the organelle. The larger ones contained a larger number of DNA areas and the t o t a l lengths of the DNA were greater. Differences i n the DNA content could be seen among chloroplasts from the same piece of t i s s u e . Woodcock et a_l ( 1 1 5 ) a l s o found that i n Acetabularia the quantity of DNA per chloroplast was v a r i a b l e . Increases i n photosynthesis have been observed i n KNap-treated plants by several authors (2, 4, 14, 24, 41, 70). Some authors have also reported increases i n the amount of photo-synthetic pigments i n naphthenate-treated plants (2, 14). An increase i n chloroplast DNA may be i n d i r e c t l y responsible for the increased a c t i v i t y of the organelle. It must be remembered, however, that no d i r e c t evidence has yet been obtained to show the increase i n the chloroplast DNA of naphthenate-treated plants. Microautoradiography of KNap-, or CHCA-treated tissues incubated with ?H-thymidine w i l l i n d i c a t e whether an increase i n the DNA of the chloroplasts occurs as a r e s u l t of the treatment. 101. SUMMARY A study of the transport, metabolism and the organo-, histo-, and cellular localization of cyclohexanecarboxylic acid in bush bean plants was carried out. The results obtained were as follows. 1. A f o l i a r spray of 0.01 M CHCA applied to the primary leaves of 14-day old bush bean plants increased the vegetative and reproductive growths. 2. The distribution of the acid in the plant, following application to primary leaves, involved both basipetal and acropetal movements. The basipetal movement occurred in the phloem and the acropetal, in the xylem and phloem. The velocity in the phloem was approximately 32 cm/hr. 3. The CHCA-glucose was the f i r s t conjugate to be formed i n the leaves treated with CHCA as a K salt. The CHCA-aspartate and an unknown metabolite 'Y' were detected about an hour after treatment. These three metabolites were present in the plant u n t i l four weeks after treatment. No free CHCA was detected 8 hr after application. This suggests that the metabolites, rather than the free acid, were respon-sible for the growth stimulation observed. The metabolites of CHCA hence cannot be merely detoxication products. 4. The major fraction of the ethanol soluble "4C activity remained in the treated primary leaves. The roots contained the next highest amounts. The lowest amounts of activity were found in the buds-flower-pods. This pattern of distribution of activity was observed at 1, 2, 3 and 4 weeks after application of KCHC-7-^ 4C. The glucose and aspartate conjugates of CHCA and the unidentified compound 'Y' were 1 0 2 . present i n the plant at the times mentioned above. 5 . At the end of each week, an amount of a c t i v i t y equal to 0 . 4 7 o or less of the t o t a l ethanol-soluble a c t i v i t y was found i n ethanol-insoluble plant residues. This perhaps represented a CHCA-protein complex. 14 6. CHCA was decarboxylated by bean pl a n t s . The CO^ released during a period of seven days accounted f o r 1.77„ of the a c t i v i t y applied. 7. The t r a n s l o c a t i o n of CHCA was favoured by l i g h t . E v i -dences obtained suggest that energy i n the form of ATP was required f o r t r a n s l o c a t i o n . In the dark, the p r o v i s i o n of glucose favoured the t r a n s l o c a t i o n , perhaps by serving as a source of ATP v i a r e s p i r -a t i o n . A supply of aspartate i n the dark favoured the tr a n s l o c a t i o n s l i g h t l y . 8. The r a d i o a c t i v i t y from CHCA was l o c a l i z e d i n the chloro-p l a s t s of the c e l l . An ethanol extract of these organelles contained both glucose and aspartate conjugates of CHCA. I t i s suggested that the chloroplasts represent one s i t e of ac t i o n of CHCA. The e f f e c t s of the naphthenate mixture and the i n d i v i d u a l naphthenate, cyclohexanecarboxylate on growth (87, 98, 118) show marked s i m i l a r i t i e s . 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Use of petroleum growth promoting substance i n the Belorussian vegetable c u l t i v a t i o n . Dokl., Vses. Soveshch. Primen. Neft. Rostovogo Veshchestva S e l . Khoz., 2nd, Baku (1963) Pub 1965, 118-129: Chem Abstr. 67, 20832 j , 1967. 114 APPENDIX E f f e c t of O.OLM CHCA on the y i e l d of bush bean pods (see p. 33) Weights of green pods i n grams Treatment Control CHCA Plant No. 1 34.43 49.52 2 15.74 6.90 3 38.35 35.22 4 33.91 30.28 5 29.04 50.16 - 6 24.65 33.41 7 47.63 47.92 8 38.19 33.31 9 17.91 33.08 10 23.94 14.67 11 31.39 37.48 12 33.30 35.08 13 34.44 36.26 14 41.10 33.26 15 14.97 27.20 16 45.59 33.30 17 36.44 29.00 18 33.78 46.32 19 20.38 37.02 20 26.09 38.10 21 30.50 46.40 22 41.69 38.12 23 42.10 44.02 24 36.20 40.97 25 39.31 48.92 26 48.22 32.91 27 43.72 46.35 28 40.55 45.76 29 36.92 37.68 30 36.93 30.12 Mean 33.91 36.62 ns - s t a t i s t i c a l l y not s i g n i f i c a n t at 0.05 l e v e l . 

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