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Studies with naphthenic acids in the bush bean, phaseolus vulgaris L. Severson, John George 1971

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STUDIES WITH NAPHTHENIC ACIDS IN THE BUSH. BEAN, Phaseolus vulgaris L. I. THE EFFECT OF POTASSIUM NAPHTHENATES ON THE UPTAKE, DISTRIBUTION, AND INCORPORATION OF PHOSPHORUS-32. I I . THE METABOLISM OF CYCLOHEXANECARBOXYLIC ACID. I I I . THE EFFECT OF POTASSIUM CYCLOHEXANECARBOXYLATE AND POTASSIUM NAPHTHENATES ON THE UPTAKE AND METABOLISM OF W GLUCOSE BY EXCISED ROOT TIPS. by John George Severson, J r . B.Sc..,. Washington State University, 196^. M.A.T., Washington State University, 1 9 6 8 . A thesis submitted i n p a r t i a l f u l f i l l m e n t 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 July 1971 In presenting this thesis in partial fulfilment of the requirements fo an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of BOTANY The University of British Columbia Vancouver 8 , Canada Date 16 July 1971 ABSTRACT The o v e r a l l objective of these experiments was to augment our understanding of how naphthenic acids stimulate metabolism and growth of bean plants. Three separate studies were carried out with bush bean plants (Phaseolus vulgaris L. c u l t i v a r Top Crop) to determinei 1) the e f f e c t of potassium naphthenates (KNap) on the uptake, d i s t r i b u t i o n , and incorporation of phosphorus-32, 2) the metabolism of the i n d i v i d u a l naphthenic acid, potassium cyclohexanecarboxylate (KCHC), i n leaves and roots, and 3) the ef f e c t of KNap and KCHC on the uptake and metabolism of glucose by excised root t i p s . 1) Fourteen-day-old plants growing i n a phosphate-free (-P) or a complete (+P) nutrient s o l u t i o n were sprayed to drip with a 0.5% s o l u t i o n of KNap. Twenty-four hours a f t e r spraying, the roots of both control and treated plants were exposed for 2 hours to a nutrient s o l u t i o n containing ^ 2P. Following the exposure to -^2P, the plants were returned to t h e i r o r i g i n a l nutrient solutions. Control and treated plants were withdrawn 4, 8 , 12, and 2k hours af t e r exposure to 3 2P, and were separated into l e a f blades, stems, and roots. Acid soluble, acid insoluble, and t o t a l 3 2P a c t i v i t y , or t o t a l phosphorus were determined at each sampling time. KNap treatment increased by 7 to 9% the intake of ^ 2P by plants grown i n the -P or +P nutrient solution. The increases, however, lacked s t a t i s t i c a l s i g n i f i c a n c e at the 0 . 0 5 l e v e l . The rate at which ^ 2P was translocated out of the roots of plants grown i n the -P nutrient only was increased s i g n i f i c a n t l y by treatment, i n s p i t e of the f a c t that at the 24 hour sampling time Qk% of the t o t a l 3 2P l a b e l remained i n root tissues. At the same sampl-ing time J2% of the t o t a l 3 2P l a b e l was found i n the roots of plants grown i n the +P nutrient. While KNap treatment s i g n i f i -cantly increased 32p a c t i v i t y i n stems of -P grown plants over the sampling period, a c t i v i t y i n stems of control and treated plants grown i n the +P nutrient was s i m i l a r . Naphthenate treatment increased the rate of incorporation of 32p into both the acid soluble (sugar phosphates, nucleotides, phospholipids) and acid insoluble (nucleic acids, phosphoproteins) fractio n s of leaves of plants grown i n the +P nutrient solution. Acid soluble 3 2P a c t i v i t y declined i n a l l root tissues over the sampling period as acid soluble 3 2p-containing compounds, priT marily orthophosphate, were translocated acropetally. The per-centage acid insoluble 32p a c t i v i t y i n the roots of KNap-treated plants was s i g n i f i c a n t l y greater than that found i n the roots of control plants at the 2k hour sampling time. Naphthenate treat-ment did not a f f e c t the amount of t o t a l P ( 3 l p + 32p) i n -the two P fractions of the three plant organs. The augmented incorporation of -^2P into the ac i d soluble and acid insoluble fractions i s further evidence of the KNap-stimulated P metabolism reported by other workers. 2) KCHC-?- 1^ administered to l e a f disks i n the l i g h t or to roots of i n t a c t seedlings i n the dark was r a p i d l y converted to a mixture of two conjugated metabolites: the glucose ester and the aspartic acid amide. The root-feeding experiment indicated that following t h e i r synthesis i n root tissues both conjugates were translocated acropetally. The r e s u l t s of amino acid analyses of the acid hydrolysates of several unidentified metabolites strongly suggest that KGHC-7-li*'C was also conjugated with a low molecular weight polypeptide. 3) Three sets of root t i p s cut from 7-day-old seedlings were incubated i n a medium containing glucose for 3 hours. Two of the three sets were pretreated i n a solution of KCHC or KNap for 6 hours. Each naphthenate treatment s i g n i f i c a n t l y increased a c t i v i t y i n the ethanol-soluble (amino acids, glucose, etc.), ethanol-insoluble (polysaccharides, protein, etc.), and respired CO2 f r a c t i o n s . The i n d i v i d u a l naphthenic acid, KCHC, had the greater e f f e c t on the uptake and metabolism of l a b e l l e d glucose. Results also indicated that not only were the uptake of glucose and C 0 2 production increased S i g n i f i c a n t l y by each treat-ment, but also amino acids containing the glucose carbon passed more quickly through soluble amino acid pools i n root tissues, and were more r a p i d l y fixed into protein. In l i g h t of the f i n d i n g that naphthenate conjugates and not the free acid were detected i n the tissue, i t may be that the conjugates were associated i n a causal way with the stimulated uptake and metabolism of l a b e l l e d glucose. TABLE GF CONTENTS page ABSTRACT . . i TABLE OF CONTENTS . . i v ABBREVIATIONS . . v i i LIST OF TABLES v i i i LIST OF FIGURES x i i ACKNOWLEDGEMENT x i v NAPHTHENIC ACIDS 1 Chapter 1. THE EFFECT OF POTASSIUM NAPHTHENATES ON THE UPTAKE, DISTRIBUTION, AND INCORPORATION OF PHOSPHORUS-32. INTRODUCTION 17 LITERATURE REVIEW 18 MATERIALS AND METHODS A. ) GROWTH OF PLANTS 2^ B. ) PREPARATION OF POTASSIUM NAPHTHENATES (KNap) AQUEOUS SOLUTION FROM NAPHTHENIC ACIDS (HNap) 25 C. ) TREATMENT WITH KNap 25 D. ) EXPOSURE TO 32p 26 E. ) HARVEST, DIGESTION, AND COUNTING 26 F. ) PHOSPHORUS FRACTIONATION 27 G. ) TOTAL INORGANIC PHOSPHATE DETERMINATIONS 28 RESULTS A. ) TOTAL UPTAKE BY THE PLANT 30 B. ) PERCENTAGE DISTRIBUTION OF THE TOTAL 3 2 P ACTIVITY AMONG LEAF BLADES, STEMS, AND ROOTS k2 C. ) PERCENTAGE DISTRIBUTION OF ACID SOLUBLE 32p ACTIVITY AND TOTAL ACID SOLUBLE P AMONG LEAF BLADES, STEMS, AND ROOTS .... 53 D. ) PERCENTAGE DISTRIBUTION OF ACID INSOL-UBLE 32p ACTIVITY AND TOTAL ACID INSOL-UBLE P AMONG LEAF BLADES, STEMS, AND ROOTS 62 E. ) DISTRIBUTION OF ACID SOLUBLE AND ACID INSOLUBLE 32p ACTIVITY OR P, EXPRESSED AS A PERCENTAGE OF THE TOTAL, AMONG LEAF BLADES, STEMS, AND ROOTS 71 DISCUSSION A. ) PHOSPHORUS UPTAKE 77 B. ) PHOSPHORUS DISTRIBUTION 78 C. ) INCORPORATION OF 32p AND P INTO ACID SOLUBLE AND ACID INSOLUBLE FRACTIONS ... 80 Chapter 2 . THE METABOLISM OF CYCLOHEXANECARBOXYLIC ACID. INTRODUCTION 84 LITERATURE REVIEW 85 MATERIAL AND METHODS A. ) LEAF DISK FEEDING EXPERIMENT 88 B. ) SYNTHESIS OF 1 -CYCLOHEX ANEC ARBO N YL-/0-D-GLUCOSE 89 C. ) SYNTHESIS OF N-CYCLOHEXANECARBONYL-L-ASPARTIC ACID 90 D. ) HYDROLYSIS PROCEDURES 90 E. ) ROOT FEEDING EXPERIMENT 91 RESULTS AND DISCUSSION 93 Chapter 3 . THE EFFECT OF POTASSIUM NAPHTHENATES AND POTASSIUM CYCLOHEXANECARBOXYLATE ON THE UPTAKE AND METABOLISM OF l^C GLUCOSE BY EXCISED BEAN ROOT TIPS INTRODUCTION 103 LITERATURE REVIEW 106 MATERIALS AND METHODS A. ) INCUBATION 112 B. <> TISSUE ANALYSIS 113 RESULTS AND DISCUSSION 116 SUMMARY 129 BIBLIOGRAPHY 133 APPENDIX 1^9 ABBREVIATIONS Naphthenic acids HNap Potassium naphthenates KNap Sodium naphthenates NaNap Cyclopentanecarboxylic acid CPCA Potassium cyclopentanecarboxylate KCPC Cyclohexanecarboxylic acid CHCA Potassium Cyclohexanecarboxylate KCHC Potassium cyclohexaneacetate KCHAc Potassium cyclohexanepropionate KCHP Potassium cyclohexanebutyrate KCHB A compound of the cyclohexylbutanol class Sh - 8 2,^-Dichlorophenoxyacetic acid 2,4-D Indole-3-acetic acid IAA Naphthaleneacetic acid NAA Benzoic acid BA Adenosine triphosphate ATP 2 , 5-Diphenyloxazole PPO 1 , 4 -bis - [ _ 2 - ( 5-Phenyloxazolyl)j -benzene POPOP Phosphoglyceric acid PGA Phosphoenolpyruvic acid PEP Uridine diphosphoglucose UDPG LIST GF TABLES TABLE PAGE Chapter 1 I. EFFECTS OF NAPHTHENATES ON GROWTH, YIELD, AND COMPOSITION OF PLANTS 9 I I . VOLUMES OF MOLAR STOCK SOLUTIONS USED FOR PREPARING ONE LITER OF A lx NUTRIENT SOLU-TION 24 I I I . EFFECT OF KNap ON TOTAL UPTAKE AND DISTRIBU-TION OF 3 2 p , ON A PER GRAM BASIS, BY BUSH BEAN PLANTS AT VARIOUS SAMPLING TIMES 32 IV. EFFECT OF KNap ON TOTAL UPTAKE AND DISTRIBU-TION OF 3 2 p , ON A PER GRAM BASIS, BY BUSH BEAN PLANTS AT VARIOUS SAMPLING TIMES 33 V. EFFECT OF KNap ON TOTAL UPTAKE OF 3 2 p t ON A PER PLANT BASIS, BY BUSH BEAN PLANTS 34 VI. EFFECT OF KNap ON TOTAL UPTAKE AND DISTRIBU-TION OF P, ON A PER GRAM BASIS, BY BUSH BEAN PLANTS AT VARIOUS SAMPLING TIMES 37 VII. EFFECT OF KNap ON TOTAL UPTAKE OF P, ON A PER PLANT BASIS, BY BUSH BEAN PLANTS AT VARIOUS SAMPLING TIMES 38 VIII. EFFECT OF KNap ON THE RATIO OF 32p ACTIVITY TO TOTAL P, ON A PER GRAM BASIS, IN BUSH BEAN PLANTS AT VARIOUS SAMPLING TIMES 39 IX. EFFECT OF KNap ON THE PERCENTAGE DISTRIBU-TION OF 3 2 p , ON A PER GRAM BASIS, AMONG LEAF BLADES, STEMS, AND ROOTS5:OF BUSH BEAN PLANTS AT VARIOUS SAMPLING TIMES 44 X. EFFECT OF KNap ON THE PERCENTAGE DISTRIBU-TION OF 3 2 p , ON A PER GRAM BASIS, AMONG LEAF BLADES, STEMS, AND ROOTS OF BUSH BEAN PLANTS AT VARIOUS SAMPLING TIMES 46 XI. EFFECT OF KNap ON THE PERCENTAGE DISTRIBU-TION OF P, ON A PER GRAM BASIS, AMONG LEAF BLADES, STEMS, AND ROOTS OF BUSH BEAN PLANTS AT VARIOUS SAMPLING TIMES 49 XII. EFFECT OF KNap ON THE PERCENTAGE DISTRIBU-TION OF ACID SOLUBLE PHOSPHORUS-32, ON A PER GRAM BASIS, AMONG LEAF BLADES, STEMS, AND ROOTS OF BUSH BEAN PLANTS AT VARIOUS SAMPLING TIMES 5^ XIV. XV. XVI. XVII. XX. XXI. EFFECT OF KNap ON THE PERCENTAGE DISTRIBUT-I O N OF ACID SOLUBLE P, ON A PER GRAM BASIS, AMONG LEAF BLADES, STEMS, AND ROOTS OF BUSH BEAN PLANTS AT VARIOUS SAMPLING TIMES .... EFFECT OF KNap ON THE PERCENTAGE DISTRIBU-TION OF ACID SOLUBLE 3 2 p , ON A PER PLANT 1 BASIS, AMONG LEAF BLADES, STEMS, AND ROOTS OF BUSH BEAN PLANTS AT VARIOUS SAMPLING TIMES , EFFECT OF KNap ON THE PERCENTAGE DISTRIBU-TION OF ACID SOLUBLE P, ON A PER PLANT BASIS, AMONG LEAF BLADES, STEMS, AND ROOTS OF BUSH BEAN PLANTS AT VARIOUS SAMPLING TIMES EFFECT OF KNap ON THE PERCENTAGE DISTRIBU-TION OF ACID INSOLUBLE 3 2 p , ON A PER GRAM BASIS, AMONG LEAF BLADES, STEMS, AND ROOTS OF BUSH BEAN PLANTS AT VARIOUS SAMPLING TIMES ... EFFECT OF KNap ON THE PERCENTAGE DISTRIBU-TION OF ACID INSOLUBLE P, ON A PER GRAM BASIS, AMONG LEAF BLADES, STEMS, AND ROOTS OF BUSH BEAN PLANTS AT VARIOUS SAMPLING TIMES EFFECT OF KNap ON THE PERCENTAGE DISTRIBU-TION OF ACID SOLUBLE, ACID INSOLUBLE, AND TOTAL 32p OR' TOTAL P, ON A PER GRAM AND A PER PLANT BASIS, IN LEAF BLADES OF BUSH BEAN PLANTS AT VARIOUS SAMPLING TIMES EFFECT OF KNap ON THE PERCENTAGE DISTRIBU-TION OF ACID SOLUBLE, ACID INSOLUBLE, AND TOTAL 32p OR TOTAL P, ON A PER GRAM AND A PER PLANT BASIS, IN STEMS OF BUSH BEAN PLANTS AT VARIOUS SAMPLING TIMES , EFFECT OF KNap ON THE PERCENTAGE DISTRIBU-TION OF ACID SOLUBLE, ACID INSOLUBLE, AND TOTAL 32p OR TOTAL P, ON A PER GRAM AND A PER PLANT BASIS, IN ROOTS OF BUSH BEAN PLANTS AT VARIOUS SAMPLING TIMES 56 58 59 63 65 XVIII. EFFECT OF KNap ON THE PERCENTAGE DISTRIBU-TION OF ACID INSOLUBLE 3 2 p , ON A PER PLANT BASIS, AMONG LEAF BLADES, STEMS, AND ROOTS OF BUSH BEAN PLANTS AT VARIOUS SAMPLING TIMES .... 68 XIX. EFFECT OF KNap ON THE PERCENTAGE DISTRIBU-TION OF ACID INSOLUBLE P, ON A PER PLANT BASIS AMONG LEAF BLADES, STEMS, AND ROOTS OF BUSH BEAN PLANTS AT VARIOUS SAMPLING TIMES 69 72 73 Chapter 2 XXIII. GLUCOSE ESTER AND ASPARTIC ACID AMIDE FOR-MATION FOLLOWING THE ADMINISTRATION OF VARIOUS ORGANIC ACIDS TO PLANT TISSUES 86 XXIV. CHROMATOGRAPHIC DATA 96 Chapter 3 XXV. TOTAL RADIOACTIVITY, AS muCi, IN THE ETHANOL-SOLUBLE, ETHANOL-INSOLUBLE, AND RESPIRED G0 2 FRACTIONS OF CONTROL AND NAPHTHENATE-TREATED BEAN ROOT TIPS AFTER SUPPLYING l^C GLUCOSE ... 117 XXVI. TOTAL RADIOACTIVITY, AS muCi, FOUND IN INDIVIDUAL ETHANOL-SOLUBLE AMINO ACIDS AND IN GLUCOSE FROM CONTROL AND NAPHTHENATE-TREATED ROOT TIPS AFTER SUPPLYING l^C GLUCOSE 119 XXVII. TOTAL RADIOACTIVITY, AS muCi, FOUND IN AMINO ACIDS FROM THE ETHANOL-INSOLUBLE HYDROLYSATE FROM CONTROL AND NAPHTHENATE-TREATED ROOT TIPS AFTER SUPPLYING l^C GLUCOSE 123 Appendix XXVIII. EFFECT OF KNap ON THE DISTRIBUTION OF ACID SOLUBLE 3 2 p , ON A PER GRAM BASIS, BY BUSH BEAN PLANTS AT VARIOUS SAMPLING TIMES 153 XXIX. EFFECT OF KNap ON THE DISTRIBUTION OF ACID SOLUBLE P, ON A PER GRAM BASIS, BY BUSH BEAN PLANTS AT VARIOUS SAMPLING TIMES 15^ XXX. EFFECT OF KNap ON THE DISTRIBUTION OF ACID SOLUBLE 3 2 p , ON A PER PLANT BASIS, BY BUSH BEAN PLANTS AT VARIOUS SAMPLING TIMES 155 XXXI. EFFECT OF KNap ON THE DISTRIBUTION OF ACID SOLUBLE P, ON A PER PLANT BASIS, BY BUSH BEAN PLANTS AT VARIOUS SAMPLING TIMES 156 XXXII. EFFECT OF KNap ON THE DISTRIBUTION OF ACID INSOLUBLE 3 2 p , ON A PER GRAM BASIS, BY BUSH BEAN PLANTS AT VARIOUS SAMPLING TIMES 157 XXXIII. EFFECT OF KNap ON THE DISTRIBUTION OF ACID INSOLUBLE P, ON A PER GRAM BASIS, BY SUSH BEAN PLANTS AT VARIOUS SAMPLING TIMES 158 XXXIV. EFFECT OF KNap ON THE DISTRIBUTION OF ACID INSOLUBLE 3 2 p , QN A PER PLANT BASIS, BY BUSH BEAN PLANTS AT VARIOUS SAMPLING TIMES 159 XXXV. EFFECT OF KNap ON THE DISTRIBUTION OF ACID INSOLUBLE P, ON A PER PLANT BASIS, BY BUSH BEAN PLANTS AT VARIOUS SAMPLING TIMES l 6 0 XXXVI. EFFECT OF KNap ON THE DISTRIBUTION OF ACID SOLUBLE, ACID INSOLUBLE, AND TOTAL 3 2 p , ON A PER GRAM BASIS, BY BUSH BEAN PLANTS AT VARIOUS SAMPLING TIMES l 6 l XXXVII." EFFECT OF KNap ON THE DISTRIBUTION OF ACID SOLUBLE, ACID INSOLUBLE, AND TOTAL P, ON A PER GRAM BASIS, BY BUSH BEAN PLANTS AT VARIOUS SAMPLING TIMES 162 XXXVIII. EFFECT OF KNap ON THE DISTRIBUTION OF ACID SOLUBLE, ACID INSOLUBLE, AND TOTAL 3 2 p , ON A PER PLANT BASIS, BY BUSH BEAN PLANTS AT VARIOUS SAMPLING TIMES 163 XXXIX. EFFECT OF KNap ON THE DISTRIBUTION OF ACID SOLUBLE, ACID INSOLUBLE, AND TOTAL P, ON A PER PLANT BASIS, BY BUSH BEAN PLANTS AT VARIOUS SAMPLING TIMES 164 XL. KEY TO THE AMINO ACIDS AND GLUCOSE SHOWN IN FIGURE 15. * 65 FIGURE LIST OF FIGURES PAGE Chapter 1 1, EFFECT OF KNap ON TOTAL 32p UPTAKE, ON A PER GRAM OR PER PLANT BASIS, WHEN BUSH BEAN PLANTS WERE GROWN IN A PHOSPHATE-FREE OR A COMPLETE NUTRIENT 35 2 . EFFECT OF KNap ON TOTAL PHOSPHORUS PRESENT, ON A PER GRAM OR PER PLANT BASIS, IN BUSH BEAN PLANTS GROWN IN COMPLETE NUTRIENT 40 3. EFFECT OF KNap ON THE RATIO OF 32p ACTIVITY TO TOTAL P, ON A PER GRAM BASIS, IN LEAF BLADES, STEMS, AND ROOTS OF BUSH BEAN PLANTS ... 4l 4. EFFECT OF KNap ON THE PERCENTAGE DISTRIBUTION OF 3 2 p , ON A PER GRAM BASIS, AMONG LEAF BLADES, STEMS, AND ROOTS OF BUSH BEAN PLANTS GROWN IN A PHOSPHATE-FREE NUTRIENT 45 5. EFFECT OF KNap ON THE PERCENTAGE DISTRIBUTION OF 3 2 p , ON A PER GRAM BASIS, AMONG LEAF BLADES, STEMS, AND ROOTS OF BUSH BEAN PLANTS GROWN IN A COMPLETE NUTRIENT 47 6. EFFECT OF KNap ON THE PERCENTAGE DISTRIBUTION OF P, ON A PER GRAM BASIS, AMONG LEAF BLADES, STEMS, AND ROOTS OF BUSH BEAN PLANTS GROWN IN A COMPLETE NUTRIENT 50 7. EFFECT OF KNap ON THE PERCENTAGE DISTRIBUTION OF ACID SOLUBLE 32p, ON A PER GRAM BASIS, AMONG LEAF BLADES, STEMS, AND ROOTS OF BUSH BEAN PLANTS GROWN IN COMPLETE NUTRIENT 55 8. EFFECT OF KNap ON THE PERCENTAGE DISTRIBUTION OF ACID SOLUBLE P, ON A PER GRAM BASIS, AMONG LEAF BLADES, STEMS, AND ROOTS OF BUSH BEAN PLANTS GROWN IN COMPLETE NUTRIENT 57 9 . EFFECT OF KNap ON THE PERCENTAGE DISTRIBUTION OF ACID INSOLUBLE 3 2 p , ON A PER GRAM BASIS, AMONG LEAF BLADES, STEMS, AND ROOTS OF BUSH BEAN PLANTS GROWN IN COMPLETE NUTRIENT 64 1 0 . EFFECT OF KNap ON THE PERCENTAGE DISTRIBUTION OF ACID INSOLUBLE P, ON A PER GRAM BASIS, AMONG LEAF BLADES, STEMS,. AND ROOTS OF BUSH BEAN PLANTS GROWN IN COMPLETE NUTRIENT 66 Chapter 3 11. RADIOACTIVITY, AS muCi, IN THE ETHANOL-SOLUBLE, ETHANOL-INSOLUBLE, AND RESPIRED CO? FRACTIONS OF CONTROL AND NAPHTHENATE-TREATED BEAN ROOT TIPS ATER SUPPLYING l^G GLUCOSE 118 12. RADIOACTIVITY, AS muCi, IN INDIVIDUAL ETHANOL-SOLUBLE AMINO ACIDS AND IN GLUCOSE FROM CONTROL AND NAPHTHENATE-TREATED BEAN ROOT TIPS AFTER SUPPLYING l^C GLUCOSE 120 13. RADIOACTIVITY,. AS muCi, FOUND IN ASPARTIC ACID, GLUTAMIC ACID, AND ALANINE FROM THE ETHANOL-INSOLUBLE HYDROLYSATE FROM CONTROL AND NAPH-THENATE-TREATED BEAN ROOT TIPS AFTER SUPPLY-ING 1*C GLUCOSE 124 Appendix 14. STANDARD CURVE FOR INORGANIC PHOSPHATE DETERMINATIONS 152 1 5 . A SCHEMATIC REPRESENTATION OF A TWO-DIMENSIONAL CHROMATOGRAM SHOWING THE POSITIONS OF THE STANDARD AMINO ACIDS AND GLUCOSE 166 ACKNOWLEDGEMENTS I f e e l a deep gratitude to the many people who hav£ helped with the various aspects of the o v e r a l l study. Most sincere appreciation i s extended to my advisor, Professor D. J . Wort, for his counsel, guidance, and support throughout the course of these studies. In addition, the author wishes to thank him for his c r i t i c a l review of t h i s manuscript. The assistance and advice given by the other members of my committee, Drs. B. A. Bohm, Department of Botany; G. W. Eaton, Department of Plant Science; A. F. Gronlund, Department of Micro-biology; and S. H. Zbarsky, Department of Biochemistry are grate-f u l l y acknowledged. Also, to Drs. B. A. Bohm and C. E. Seaforth (Division of Chemistry, University of the West Indies, Trinidad) who was a guest researcher i n our department during 1968 - 1969» I extend c o r d i a l thanks for the very i n t e r e s t i n g and informative discussions concerning a portion of the material presented i n Chapter 2. Constructive remarks concerning t h i s manuscript given by the External Examiner, Dr. R. T. Wedding (Department of Biochemistry, University of C a l i f o r n i a , Riverside) were greatly appreciated. Fin a n c i a l assistance from the University of B r i t i s h Columbia i n the form of a Graduate Fellowship, and a Graduate Research Grant from the Northwest S c i e n t i f i c Association are acknowledged with thanks. The studies were carried out i n the Department of Botany, University of B r i t i s h Columbia, and the author wishes to express his appreciation of the excellent f a c i l i t i e s made ava i l a b l e to him. F i n a l l y , I extend warm and sincere thanks to my wife, Gerrie, and to my family for t h e i r help and encouragement throughout. For t h i s , I dedicate t h i s d i s s e r t a t i o n to them. NAPHTHENIC ACIDS The name, naphthenic acid, was f i r s t suggested by Mark-ovnikoff and Ogloblin ( 101 ) for the C ^ H g ^ acids of unknown structure which H e l l and Medinger ( 73 ) had recovered from Rumanian crude o i l . Naphthenic acids extracted from petroleum are known to be a very complex mixture of chemical compounds. As noted by J o l l y ( 78 )» cyclopentyl derivatives predominate i n the naphthenic acid mixture followed by cyclohexyl compounds. Naphthenic acids are also known as petroleum acids, because branched-chain a l i p h a t i c acids and phenols are present i n some crude o i l s . Those naphthenic acids having an average molecular weight of 2 l 4 have been shown by Cason and Khodair ( 3 8 ) to consist c h i e f l y of compounds which contain 10 to 20 carbon atoms. The commercially available HNap used i n these i n v e s t i -gations has a reported molecular weight of 2 3 0 . Baivarovskaya et a l ( 21 ) investigated the properties of a HNap mixture which was obtained from crude o i l of the Perm region i n Russia. Petroleum from the southern Perm region had an acid number of 0 . 2 3 mg KOH/g HNap. The d i e s e l o i l f r a c t i o n ( 1 8 0 ° - 400° C) contained 0 . 0 3 2 ^ HNap which was extractable with k% NaOH. The NaOH was mixed with the d i e s e l o i l for 15 minutes, and the mixture was allowed to s e t t l e for 4 to 5 hours. The molecular weight of the NaNap was ca 3 0 0 . The s o l u b i l i t y of NaNap i n water was 2 0 , 3 0 , and ko g/100 ml at 2 0 , 5 0 , and 100° C, respectively. I t i s very i n t e r e s t i n g to note that under the heading of 'naphthenic acid* i n the Merck Index ( 133 ) only one compound COOH CO OH Cyclopentanecarboxylic a c i d C5H 1 0°2 ~ m o 1 w t Cyclohexanecarboxylic acid C7 H12°2 ~ m o 1 w t 1 2 8 3-dimethyl-4-ethyl-cyclop en tyl-n-butanoic acid C 1 3 H24°2 " m o 1 w t 2 1 2 2,4-dimethyl-5-dimethyl-cyclohexyl-n-pentanoic acid * C15 H28°2 " m o 1 w t COOH COOH N A P H T H E N I C A C I D S i s described, namely CHCA ( C ^ H ^ ^ ) * appears that t h i s c l a s s i f i c a t i o n system has persisted since 1910 - 1920 when the Chemical Abstracts' indices gave hexahydrobenzoic acid (CHCA) as a cross reference for naphthenic acid. However, i n the l a t e s t edition of the Merck Index ( 13^ ) HNap, as an entry, has been deleted, and CHCA i s entered separately with no cross reference to HNap. The carboxyl group of most naphthenic acids i s not attached d i r e c t l y to the a l i c y c l i c r i n g , but i s separated from the r i n g by an a l i p h a t i c side chain containing one to f i v e or more methylene groups (preceding page). According to J o l l y ( 78 ), a general formula may be written as R(CH 2 ) n C 0 0 H , where R i s an a l i c y c l i c nucleus composed of one or more rings. Other authorities i n the f i e l d of petroleum chemistry ( 58 ) have stated that the naphthenic acid mixture contains a wide v a r i e t y of substituted cyclopentane^ and cyclohexanes. The molecular weight of i n d i v i d -ual acids i n the mixture ranges from the lowest (CPCA, mol wt 114, shown on the preceding page) to compounds with molecular weights of over 1 , 0 0 0 . The authors also stated that there i s no evidence that cyclopropane, cyclobutane, cycloheptane, or higher r i n g derivatives are present i n crude petroleum. Cason and Liauw ( 37 ) and Cason and Khodair ( 38 ) have demonstrated that the a l i c y c l i c nucleus (either cyclopentane or cyclohexane) can be substituted with methyl or ethyl groups. Gas-liquid chromatography i n our laboratory has revealed that the HNap mixture i s indeed very complex. Gas chromatograms of the methyl esters (MeNap) of HNap showed that peaks of lower molecular weight components comparable to cyclopentane-, cyclo-hexane-, and cycloheptanecarboxylic acids were not evident i n the t r a c i n g of the commercial HNap preparation. A gas chromato-gram of the naphthenates derived from the unrefined d i e s e l f r a c t i o n of a Venezuelan crude o i l yielded a t r a c i n g i n which most of the components appeared to be i n the to C ^ range, and within t h i s range there were 25 major peaks. Present, though i n r e l a t i v e l y smaller amounts, were C 5 , C 7 , and C3 compounds. When compared with the cyclohexyl-n-butryrate standard, i t appeared as though a C 1 Q acid was present i n the highest concentration i n both HNap preparations. The chemical structure below might represent t h i s C 1 0 compound. COOH The range of components i n both MeNap preparations was s i m i l a r . I t should also be noted that the bases of the peaks seemed to have been obscured by what may have been a phenolic component. When the phenolic compound(s) were p a r t i a l l y removed from the naphthenate mixture on one occasion and were subsequently chromatographed, a single spot was obtained which gave a po s i t i v e phenolic reaction when sprayed with diazotized p - n i t r o a n i l i n e . Also using gas-liquid chromatography, Eider ( 46 ) confirmed the presence of formic, a c e t i c , i s o b u t r y i c , CPCA, CHCA, cyclohexaneacetie, and several other low molecular weight acids i n the naphthenic acid f r a c t i o n from an Aruba crude o i l . Some idea of the complexities involved i n t r y i n g to separate i n d i v i d u a l naphthenate compounds from the mixture i s given by Bock and Behrends ( 32 ). Using ga s - l i q u i d chromato-graphy, mass spectroscopy, and f r a c t i o n a l d i s t i l l a t i o n procedures they found the acid f r a c t i o n from an Austrian crude o i l to con-t a i n ca 120 compounds, 20 of which constituted more than 50% of the sample. 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. Small amounts of sub-s t i t u t e d cyclopentyl and cyclohexyl acids were found. No straight-chain acids were detected. In several recent publica-tions, S e i f e r t ( 123 ,124 , 1 2 5 ) has considerably extended our knowledge of the compounds which are present i n the carboxylic acid f r a c t i o n of crude o i l . Results of these studies have shown that terpenoid polynuclear saturated and polynuclear aromatic, as well as heterocyclic carboxylic acids occur n a t u r a l l y i n petroleum. The conventional view of the naphthenic acids i n petroleum being p r i m a r i l y mononaphthenic and alkanoic acids was expanded to include 40 new classes of carboxylic acids. Naphthenic acids have a c h a r a c t e r i s t i c odor which varies with the acid source, degree of refinement and content of phenol and s u l f u r compounds. These acids are r e a d i l y soluble i n non-polar solvents; however, the lower molecular weight members, e.g. CHCA and CPCA, may be s l i g h t l y soluble i n water. The occurrence, composition, chemical properties, recovery, and non-biological uses of HNap have been given by J o l l y (78 ). As a fact i n passing, i n 1970 ca 7 0 $ of the 1 5 . 7 0 0 tons of naph-thenic acids (as metal s a l t s ) was used i n the United States p r i m a r i l y as d r i e r s i n oil-based paints, catalysts i n r e f i n e r y operations, and lubricants. The use of naphthenic acids and t h e i r s a l t s as plant growth stimulators i s rather recent, and as such, they have been u t i -l i z e d p r i m a r i l y by Bulgarian, Russian, Lithuanian, Canadian, and Albanian plant physiologists. In low concentrations naph-thenates have been found to promote vegetative and reproductive growth, and to a f f e c t other p h y s i o l o g i c a l and biochemical a c t i v i t i e s i n a large number of plants. There are also several reports that when applied at high concentrations naphthenates act as herbicides ( 9 8 , 157 ). I t has been demonstrated that naphthenic acids stimulate the growth of a number of animal species ( 5 7 , 126 ). The mechanism or mode by which naphthenates influence plant metabolism, or i t s 'modus operandi', i s imperfectly known. I t has been established that to obtain a maximum response a single application of HNap or i t s potassium s a l t should be applied to bean plants at a p a r t i c u l a r ontogenetic stage and at a s p e c i f i c concentration ( 142 ). However, as i s the case with many other chemical growth regulating compounds, the f i n a l r e s u l t of naphthenate a p p l i c a t i o n may be determined not only by the two parameters given above, but also by the pH, the method of application, and s i z e of the droplet or dust p a r t i c l e applied. The species of plant, the part of the plant to which the chemical i s applied, and the plant's vigor may also play a part i n deter-mining the f i n a l response. Moreover, environmental conditions such as temperature, l i g h t i n t e n s i t y and q u a l i t y , humidity and the a v a i l a b i l i t y of water and nutrients are also important i n determining the f i n a l outcome;. Preliminary work i n our laboratory has indicated that when i n a b i o l o g i c a l system, naphthenates e x i s t mainly i n a bound or conjugated form, and therefore^ t h e i r i d e n t i f i c a t i o n i s d i f f i c u l t . When i n the free form, the reaction of the carboxyl group with an acid-base indicator, such as bromophenol blue, together with R f values, provides the only means of i d e n t i f i c a t i o n when non-radioactive material i s used. The l o c a t i o n of a c t i v i t y on radiochromatograms of extracts of plant material may serve to i d e n t i f y the free acids and t h e i r derivatives following the a p p l i c a t i o n of l a b e l l e d naphthenic acids. Following t h e i r a p p l i c a t i o n to bean plants, naphthenates stimulate many phys i o l o g i c a l and biochemical processes. Stim-u l a t i o n of photosynthesis ( 5 2 , 53 )» dark r e s p i r a t i o n ( 5 2 , 53 ) , protein synthesis ( 1 3 0 , 1^7 ), and the s p e c i f i c a c t i v i t i e s of numerous enzymes i n crude extracts ( 40, 5 2 , 53» 1^7 ) suggests that naphthenate stimulation of plant growth i s the r e s u l t of the action of the chemical, or i t s metabolites, at both the genetic and metabolic l e v e l s ( 1^7 ). I t was also suggested that since numerous metabolic l o c i are involved, the stimulation must be a general one. Where experiments coincide, the r e s u l t s obtained i n our laboratory tend to confirm for the most part the data reported by s c i e n t i s t s i n the Iron Curtain countries. Also, during the past two years the metabolism of KCHC-?- 1^ ( 108, 127 , 131 ) , and KNap ( 122 ) have been studied i n Phaseolus vulgaris L. As f a r as the b i o l o g i c a l degradation of naphthenic acids i s concerned, Osnitskaya ( 107 ) reported that a pure culture of microorganisms capable of degrading HNap was i s o l a t e d from naphthene petroleum and waste water of a r e f i n e r y . A pure culture of these aerobic organisms (unnamed) oxidized 90?S of the naphthenic acids on which they grew within/.,!0.2days. S i g n i f i c a n t amounts of intermediate metabolic breakdown pro-ducts were not detected i n the growth medium. This was taken to indicate that oxidation of HNap had progressed to CO2 and H 2 0 . The plant growth stimulators obtained from petroleum have been named and abbreviated d i f f e r e n t l y by various researchers, v i z . , naphthenic growth substances (P.G.S.), naphthenic acids (HNap), potassium naphthenates (KNap), o i l hormone substance (O.H.S.), naphtha growth matter, o i l growth matter, petroleum growth promoters, petroleum nutrient, s i n t o v i t (an oxidized petroleum product), Sh-8 (a cyclohexylbutanol), and petroleum or naphthenic growth-helping substances (P.R.V. or N.R.V.). In the Russian language the i n i t i a l s R. V. stand for 'growth helping substance.' S c i e n t i s t s i n several Iron Curtain countries also report the use of a Bulgarian naphthenate preparation, H.T.I., which i s very s i m i l a r to N.G.S. What appears to be the f i r s t published report of the use of naphthenic acids i n b i o l o g i c a l research dates back to 1921. However, the p r a c t i c a l a p p l i c a t i o n of naphthenates to plants for purposes of stimulating plant growth appeared i n the Russian l i t e r a t u r e i n 1956. During the past f i f t e e n years the effects of naphthenic acid compounds on the p h y s i o l o g i c a l and biochemical processes i n plants have been studied p r i m a r i l y by investigators i n the Iron Curtain countries and i n Canada. The effects of naphthenates on plant growth, y i e l d , and composition are summarized i n Table I. TABLE I. E f f e c t s of naphthenates on growth, PLANT Apple, seedlings Beans Beans Beans, bush MODE OF NAPHTHENATE APPLICATION CONCENTRATION seed soak 0.00k% to cuttings f o l i a r 0.5% Beans, bush f o l i a r 0.5% Beans, bush Beans, bush Beans, bush f o l i a r 0.25-0.5% f o l i a r (dust ) 2 5 0 - 5 0 0 g/ha to s o i l 0.001-0.01 g/ 1800 g s o i l seed soak 0.001-0.01^ f o l i a r (emu-ls i o n ) f o l i a r f o l i a r 0.5% 0.5% Beans, bush Beans, bush f o l a i r f o l i a r 0.5% 0.5% Beans, bush f o l i a r 0.5% , and composition of plants EFFECT* REFERENCE Growth and development* + ( 1 2 l ) Number of and fresh weight of roots* + ( 44 ) Growth* + ( 115 ) Plant height* + ( 52 ) Number of and area of l e a f -l e t s * + Dry weight of roots, stems, and leaves* + Green pod y i e l d * + (20%) ( 1^2 ) Ripe seed production* + (8%) Dry weight of vegetative parts* + (20.7%) Yield of green pods* + ( 144 ) (23.4 - 26.5%) . Yield of green pods: + ( 145 ) Yield of green pods « + Yield of green pods* + (12%) Y i e l d of green pods* no s i g n i f i c a n t e f f e c t Vegetative and reproductive ( 5^ ) growth * + Fresh and dry weights* + ( 143 ) (21 and 22%) Weight of green pods* + (2338) Weight of r i p e seed* + (24$) Transpiration* - (12%) (128) Chlorophyll and carotenoid ( 52 ) content of leaves * + Ascorbic acid of pods* + ( 52 ) (26%) Beans, china Beans, feed Beans, f i e l d Beet Beet Cabbage Cabbage Cabbage Cabbage Cabbage Cabbage seed soak & f o l i a r seed soak to s o i l seed soak to s o i l f o l i a r to s o i l f o l i a r f o l i a r to s o i l to s o i l f o l i a r 0 . 0 0 5 & 0.01% 10-40 g/seed 100 g/ha 0.005-0.055S 0 . 2 5 $ 0 . 0 0 5 $ 5 0 - 1 0 0 g/ha 2 5 - 1 0 0 g/ha 100 g/ha 0 . 0 0 0 5 - 0 .01% Cabbage Cabbage Cabbage Carnation seed soak seed soak f o l i a r seed soak & f o l i a r 0 . 0 0 5 - 0 . 0 5 $ 0 . 0 0 0 1 - 0 . 0 0 1 $ 20 mg/g seed & 20 mg/m2 Carrot Carrot to s o i l f o l i a r 100 g/ha 0 . 0 0 0 5 - 0 . 0 1 $ Carrot Cherry Corn (maize) seed soak to cuttings seed soak 0 . 0 0 5 - 0 .05% 0.01% 0.5% Corn f o l i a r 0.005% Corn f o l i a r 0 . 0 0 5 $ seed soak 0 . 0 0 5 - 0 . 0 5 $ Corn f o l i a r 0 . 5 $ Pod y i e l d : + Germination and growths + Y i e l d i + (2 to 1 6 $ ) Y i e l d * + (20 to 2 5 $ ) Y i e l d i + (20.5$) Y i e l d j + (206$) Yield 1 + (15 to 20$) Ripening of heads: + Yield* + (13 to 32$) Yieldt + (15 to 26$) Yieldt + (12 to 28$) Yields + (20 to 2 5 $ ) Metabolism, growth, and development 1 + Yields + ( 3 0 $ ) Y i e l d i + ( 1 5 . 7 $ ) Seed moisture content* + Ascorbic acid* + Size of flowers * + Number of flowers* + (2?$) Number of seeds produced* + (54$) Yield* + (20 to 2 5 $ ) Metabolism, growth, and development* + Yield* + Yield* + (16$) Root formation* + Hay production* + (21 to 33$) Corn s i l a g e and corncobs* + (13-43 and 6-28 metric centners/ha, resp.) Growth and y i e l d * + Yie l d * + (18 to 35$) Fresh and dry weights of f o l i a g e * + (18 & 16$, resp. Corn Corn Corn Corn Com Corn Cotton Cotton Cotton Cotton Cotton f o l i a r seed soak f o l i a r 0 . 0 0 5 $ 0 . 0 0 0 1 - 0 . 0 0 1 $ 0 . 0 0 5 $ 0 . 0 0 0 1 $ to s o i l with mulch seed soak seed soak 0 . 0 1 $ seed soak 0 . 0 1 $ seed soak & 0 . 0 1 $ f e r t i l i z e r s f o l i a r d u r i n g 2 5 0 g/ha vegetative phase f o l i a r at 250 g/ha & flowering & others, the two p r i o r treatments Cotton f o l i a r 0.01$ Cotton Cotton to s o i l 100 mg/40 kg dry s o i l Cotton f o l i a r 0 . 0 0 5 $ Cranberry to cuttings — Cucumber seed soak 0 . 0 0 0 1 $ f o l i a r 0 . 0 5 $ Cucumber seed soak 0 . 0 0 5 - 0 . 0 5 $ Y i e l d : + ( 138 ) Dry weight* + ( 3 3 $ ) ( 1 1 3 ) Growth* + ( 89 ) Corn s i l a g e * + (132 metric centners/ha) Water retention* + ( 149 ) Seed moisture content* + ( 90 ) Free and bound water* + Chlorophyll content* + ( 149 ) Root growth* + ( 75 ) S o i l temperature* + ( 20 ) Germination and f r u i t i n g * + B o l l y i e l d * + ( 3 . 1 $ ) ( 65 ) Y i e l d * + ( 2 3 $ ) ( 132 ) Yield* + (5 to 10$) ( 104 ) Yi e l d * + (10 to 15$) Growth and ripening* + Yiel d * + (20$) Crude f i b e r y i e l d * + ( 2 3 ) Chlorophyll and ascorbic acid i n leaves* + Ascorbic acid i n leaves* + ( h ) Total and active absorbing ( 17 ) surface of roots* + Dry weight of roots* + Auxin transport* + ( 25 ) Auxin a c t i v i t y * + Root formation* + ( 150 ) Root growths + (75) Y i e l d * + (40$) Yi e l d * + (20.1$) ( 79 ) Currants, black to cuttings 0 . 0 1 $ Eggplant f o l i a r 0 . 0 5 $ Eggplant F o x t a i l , meadow Gooseberry Gooseberry Grape Grape Hemp Lucerne Melon M i l l e t Mulberry Muskmelon f o l i a r f o l i a r 0 . 0 0 5 $ 0 . 0 0 5 - 0 . 1 $ & NH4NO0 0 . 0 1 $ J to cuttings to cuttings - — f o l i a r ( b e f o r e 8 0 - 1 0 0 ppm and a f t e r flowering f o l i a r f o l i a r f o l i a r seed soak & ultraso n i c treatment f o l i a r seed soak f o l i a r f o l i a r 0 . 0 0 5 $ 0 . 0 0 5 $ 0 . 0 0 0 5 $ 0 . 0 0 5 $ 0 . 0 0 0 5 $ 0 . 0 0 0 5 - 0 . 0 1 $ Oat f o l i a r 0 . 1 $ Olive f o l i a r 0 . 0 0 0 5 $ Onion to s o i l 100 g/ha Onion 0 . 0 0 0 1 $ Onion f o l i a r 1 0 - 3 - 1 0 - 5 $ Onion seed soak & 0 . 0 0 0 1 - 0 . 0 0 1 ! seed i r r a d -i a t i o n Onion seed soak 0 . 0 0 5 - 0 . 0 5 $ Peas seed soak 5-^5 g/seed Root formation: + (26$) ( 114 ) Height, a e r i a l mass and ( 8 ) t o t a l weight: + ( 2 1 , 1 2 , and 6$, resp.) Y i e l d : + ( 3 2 $ ) (75) Y i e l d of green mass: + ( 3 ) ( 2 3 $ ) Root formation: + ( 4 5 $ ) ( 114 ) Root formation: + ( 91 ) Weight of c l u s t e r s : + ( 118 ) (28$) Y i e l d : + ( 1 0 $ ) ( 87 ) Growth: + ( 77 ) Y i e l d : + ( 2 0 $ ) ( 47 ) P h y s i o l o g i c a l responses: ( 2 ) + Ascorbic a c i d i n f r u i t s : + Grain y i e l d : + ( 8 $ ) ( 103 ) Growth and development: + ( 67 ) Metabolism, growth, and ( 15 ) development: + Yi e l d : + ( 3 0 $ ) Yield of green mass: + ( 3 ) (10 to 14$) Growth and development: + ( 67 ) Y i e l d : + (20 to 2 5 $ ) ( 157 ) Root growth: + ( 75 ) Rate of c e l l d i v i s i o n and ( 63 ) r o o t l e t length: + Rate of c e l l d i v i s i o n : ( 62 ) Y i e l d : + ( 3 6 $ ) ( 79 ) Y i e l d of f r u i t : + (7 to ( 118 ) 1 0 $ ) Peas Pasture (grass, legumes, U r t i c a , Stachys) Phlox Phlox Pine, Eldar (Pinus eldarica) Poplar Potato f o l i a r f o l i a r f o l i a r to cuttings seed soak to cuttings f o l i a r 0 . 0 0 5 - 0 . 0 1 $ 10$ NH4NO3 & 2 0 $ KNap (800 1/ha) 1 2 . 5 , 2 5 . 5 9 - 4 1,00 ppm 0 . 0 0 5 $ 0 . 0 2 - 0 . 0 5 $ Potato Potato Potato, early Potato, early (Lorkh) Potato, early (Warba) tuber soak 0 . 0 0 0 5 $ to s o i l f o l i a r f o l i a r f o l i a r 250 cm2/ha 63 ppm 125 g/ha 0 . 5 $ Potato tuber soak 0 . 0 0 5 $ P r i v e t Radish Rice Rice Rye, winter to cuttings f o l i a r 0 . 5 $ f o l i a r seed soak seed soak Seeds(unspecified) seed soak 0 . 0 5 $ 10 g/grain 8-16 g/100 kg of seed Growth 1 + H e r b i c i d a l e f f e c t s : ( 7 9 ) ( 98 ) Growth 1 + ( 112 ) Root formations + ( 150 ) Seedlings had a greater ( 6 ) s a l t tolerance Root formations 0 ( 150 ) Leaf areas + ( 94 ) Tuber y i e l d s + (1?.4$) Number of shoots/plants + Tuber y i e l d s + ( 9 . 9 $ ) Tuber y i e l d s + (48$) ( 92 ) Water absorptions + Tuber y i e l d s + (35 to ( 2 ) 40$) Tuber y i e l d s + (2?$) ( 118 ) Tuber y i e l d s + ( 1 ) Starch concentration of tuberss + Weight of tubers/plants ( 146 ) + (42$) Tuber numbers 0 Dry matters 0 Starch concentration of tubers s 0 Assimilative surfaces and ( 94 ) chlorophyll content of leaves s + Root formations + ( 150 ) Fresh and dry weight of ( 143 ) plants s + (11 & 11$, resp.) Yields + (28$) ( 75 ) Grain y i e l d s + (11.1$) ( 118 ) F r u i t bearing stemss + ( 118 ) Grain y i e l d s + (10$) Growths + ( 9 ) ^ Sophore, seedlings seed soak 0 .005$ (Sophora .iaponica L.) Spindle-tree (Euonymus sp.) Sugar beet Sugar beet Sugar beet Sunflower to cuttings seed soak 0 . 0 0 5 - 0 . 0 1 $ f o l i a r f o l i a r seed soak 0.5$ 0.005$ 0.005 & 0.01$ Tangerine Tea Tobacco Tobacco Tobacco,.Oriental Tomato Tomato Tomato Tomato Tomato Tomato f o l i a r f o l i a r f o l i a r to s o i l & f o l i a r to s o i l f o l i a r to s o i l f o l i a r f o l i a r to f l o s c -ules seed soak 0.05$ 0.05$ 0.005$ 0 15 mg/m2 & 2 mg/plant 15 mg/m2 0 . 0 0 0 5 - 0 . 0 1 $ 0.25$ 0 . 0 0 5 - 0 . 0 5 $ 50-100 g/ha during planting + 0.005$ l a t e r at 500 1/ha 0 . 0 5 - 0 . 0 1 $ 0 . 0 0 5 - 0 . 0 5 $ f o l i a r dur-ing flower-ing 0.01$ Greater s a l t tolerance in s a l i n i z e d s o i l s Root formation! 0 (150 ) Beet root crops + (26 ( 149 ) metric centners/ha) Fresh and dry weights of ( 143) foliages + (13 & 14$, resp.) Water content! + ( 149 ) Chlorophyll content! + Stem s i z e i + ( 88 ) Flower number* + Weight of seeds! + O i l y i e l d s + Yi e l d ! + (18.6 to 21 .2$) ( 102 ) Y i e l d ! + (39$) (75) Leaf area and growth* + ( 151 ) Growth and l e a f area* + (75) Y i e l d of leaves* + (7$) ( 116 ) Total fresh weight of 100 plants* + (23 .5$) Metabolism, growth, develop- ( 15 ) ment, and y i e l d * + F r u i t y i e l d * + (38$) ( 64) F r u i t y i e l d * + (40 to 50$) ( 7 5 ) F r u i t y i e l d ! + (30 to 37$) ( 7 ) Ovary formation and f r u i t ( 79 ) maturation * + Onset of flowering* + ( -79.) Bud formation (flower)i + F r u i t y i e l d ! + Yield of f r u i t i + (22$) Tomato Tomato Tomato Tomato Trees(unspecified) Turnsole (Chrocophora sp.) Weeds(unspecified) Weeds(unspecified) Wheat, winter Wheat Wheat, Marquis spring Wheat, winter Wheat, winter Vine Vine ( 7-year-old, var. Tamyanka) f o l i a r f o l i a r f o l i a r f o l i a r during c u l t i v a t i o n f o l i a r seed soak seed soak seed soak seed soak f o l i a r seed soak 0 . 5 $ 0 . 0 0 5 $ 0 . 0 0 5 $ 0 . 5 $ 0 . 0 0 0 1 - 0 . 0 0 0 7 $ 0 . 0 0 0 1 - 0 . 1 $ 0.4$ 8 l l 6 g/100 kg of seed 0 . 0 1 $ 0,5# 0 . 0 0 0 1 $ 1 5 . 2 g/100 kg seed to cuttings f o l i a r p r i o r 100 ppm to flowering Number of f r u i t s 1 + ( 40 ) F r u i t y i e l d : + ( 110 ) Ascorbic a c i d content of ( 7 ) f r u i t : + Ascorbic acid content of ( 40 ) f r u i t : 0 Pollen germination and p o l l e n ( 93 ) tube growth: + Sucrose content/flower, and ( 1 1 1 ) honey y i e l d / h a : + H e r b i c i a l e f f e c t s : + ( 1 5 7 ) Germination and growth* + ( 80 ) Grain y i e l d : + ( 3 0 $ ) ( 1 3 2 ) Grain y i e l d * + (10$) (118.) Weight of grain/plant: + ( 12$) ( 143 ) Fresh and dry weight of leaves* + (8 & 8$, resp.) Weight of ripe g r a i n : + (10$) Root growth: + ( 75 ) Grain y i e l d * + ( 9 . 9 $ ) ( 118 ) Root formation* + Chlorophyll a & b: Phaeophytin :""*+ Carotene: + Lutein: + Flavoxanthin: + Violaxanthin* + ( 150 ) ( 9 5 ) +* Stimulation! -: I n h i b i t i o n ! 0 : No e f f e c t ; : Followed by. Chapter 1 THE EFFECT OF POTASSIUM NAPHTHENATES ON THE UPTAKE, DISTRIBUTION, AND INCORPORA-TION OF PHOSPHORUS-32. INTRODUCTION This phase of the investigation dealt with the eff e c t which KNap had on P uptake, d i s t r i b u t i o n , and incorporation i n bush bean plants. Phosphorus was chosen for t h i s study because of the central r o l e which t h i s element plays i n c e l l u l a r metabolism, photosynthesis, and the genetics of plants. 7. Moreover, •: the amount of d e f i n i t i v e s c i e n t i f i c research describing the effects of naphthenates on P ass i m i l a t i o n i s very l i m i t e d . Both the magnitude of the P response to naphthenate treatment, and the d i s t r i b u t i o n patterns which emerged at the four sampling times during the 24 hour holding period were studied. Phosphorus absorption by plants i s a process which requires metabolic energy. Therefore, the P d i s t r i b u t i o n patterns which emerge at various sampling times following the exposure of the roots to the radioisotope, ^ 2P, would be related to the plant's metabolic a c t i v i t y , concentration of P, and i n t e r n a l requirement for t h i s p a r t i c u l a r element. The l a t t e r i s an es p e c i a l l y important point as the plants used i n several experiments were grown i n a -P nutrient solution both before and a f t e r naphthenate treatment. Actual comparisons, which have been made between l e a f blades, stems plus p e t i o l e s , or roots of control and naphthenate-treated plants, includes t o t a l , acid soluble, and acid insoluble ^ 2p activit y } and t o t a l , acid soluble, and acid insoluble P. The comparisons were made 4, 8, 12, and,24 hours a f t e r the roots of control and treated plants were removed from a nutrient solution which contained ^ 2P. LITERATURE REVIEW As far back as l 8 6 l , Hartig ( 7 1 ) suggested that soluble materials may c i r c u l a t e within plants. Later, Biddulph ( 26 ) reported that ^ 2P was very mobile i n the phloem of bean plants, and there was a p o s s i b i l i t y that some P was continually c i r c u l a t -ing i n the plant. In 1957 . Helder ( 72 ) confirmed the f a c t that a continual circulation of P did occur i n barley and bean plants. Biddulph et a l ( 2 7 ) also observed that a portion of the absorbed -^2P displayed a sustained c i r c u l a t i o n throughout the plant during a 96 hour experimental period. I t i s not inconceivable that an i n d i v i d u a l P atom, i f not metabolically captured, may make several cycles within a plant d a i l y . Numerous investigations have shown that P uptake from an external medium i s an a c t i v e process ( 1 1 9 ) . To augment t h i s f a c t i t has been recently demonstrated by Bledsoe et a l ( 31 ) that P uptake and ATP synthesis i n excised corn roots were concurrently decreased when oligomycin was present i n the eulturing medium. Edwards ( 4 5 ) reported that when the P concentration i n the external solution was less than 1 mM, P absorption was characterized by two d i f f e r e n t reactions which represented s i t e s of d i f f e r e n t a f f i n i t y . He suggested that either both s i t e s of P absorption were located at the plasmalemma, or one was at the plasmalemma with the other representing absorption by the microbial population on the root surface. However, at concentra-tions greater than 1 mM one l i n e a r absorption curve was obtained. The author suggested that at higher P concentrations the curve was consistent with the involvement of a passive P absorption across the plasmalemma. Edwards concluded that the data estab-l i s h beyond a doubt that the two low concentration mechanisms of P absorption do not correspond to system 1 and system 2 of the dual absorption mechanism for cations as described by Epstein ( 48 ) and Luttge and Laties ( 9 6 ) . In the present experiment the P concentration was 1 mM i n the complete nutrient solution. As a point of int e r e s t , Bowen and Rovira ( 3 4 ) and Barber and Loughman ( 22 ) were able to show that the presence of micro-organisms on root surfaces of barley had marked effects on P absorption. An examination of cut-stem exudate from bean plants which were root-fed -^ P^  revealed that i t was the only l a b e l l e d compound present ( 4 9 * 5 0 )« However, i t has also been shown that i n barley plants a small f r a c t i o n of the P i n the roots may ascend v i a xylem i n the form of phosphorylcholine (100 ), and glycero-phosphorylcholine ( 99 ). Depending on the concentration of P i n the external solution and on the i n t e r n a l requirement, t h i s element can move acropetally i n the phloem ( 2 9 • 3 3 , 3 9 ) . In 1969. B i e l e s k i ( 30 ) reported that inorganic phosphate was also the primary form i n which P moved in the phloem of turnip and pumpkin. When -^2P was o r i g i n a l l y moving b a s i p e t a l l y i n the phloem of l e a f traces i n the bean plant, the concentration of i n the phloem compared to that i n the xylem was ca 3:1. As the data suggest, there was an interchange of P between the phloem and xylem v i a the intervening cambium. Movement of root P to the a e r i a l portion of a bean plant may occur at the rate of 1 m/hr, i . e . 17 cm i n 10 minutes ( 28 ). In 1953t Hanson and Biddulph ( 70 ) found that a diurnal v a r i -ation existed i n the uptake and acropetal translocation of 32p f r o m the roots of bean plants. Absorption,and translocation of P was greatest midway through the 12 hour photoperiod. The authors suggested that an increased P transolocation during the day may have been associated with the need for t h i s element i n c e r t a i n reactions c h a r a c t e r i s t i c of the a e r i a l portion of the plant. In 1967, Etter ( 50 ) r e l a t e d an increased 3 2P uptake by the roots of bean plants to the d i f f e r e n t i a l diurnal uptake of P, and to 2,4-D induced reactions i n the leaves. Cotton plants treated with naphthenates and Sh-8 showed an increase i n the i n t e n s i t y of t h e i r photosynthetic r a t e * ( 4 ) . Bazanova and Akopova ( 23 ) also observed stimulation of photo-synthesis i n cotton plants treated with 0 . 0 1 $ naphthenates. Follow-ing a s i n g l e f o l i a r a p p l i c a t i o n of a 0 . 0 0 5 $ naphthenate solu t i o n to grape plants, Kolesnik ( 87 ) observed an increase i n the rate of photosynthesis. The author also stated that two f o l i a r applications of a 0 . 0 5 $ naphthenate solution resulted i n an i n i t i a l reduction of the photosynthetic rate, but at the end of the vegetative period the rate had been increased. After soaking the tubers of two species of potatoes i n a 0 . 0 0 0 5 $ naphthenate solution for 1 hour before planting, Ladygina ( 9^ ) observed only a s l i g h t change i n the photosynthetic rate of maturing plants. Abolina and Ataullaev ( 2 ) also observed that photosynthesis proceeded more 'energetically* i n potato plants following treatment with a naphthenate solu t i o n ( 125 cm2/ha ) as a f o l i a r spray. After a naphthenate solu t i o n at a concentration of 0 . 0 0 1 $ was added to an A l l e n -* Because of the involvement of P-containing compounds, data dealing with photosynthesis are included i n t h i s review. Nelson's c u l t u r i n g medium containing the phytoplanktonic diatom, Chaetocerus curvisetus, Zgurovskaya (156 ) reported that both the rate of c e l l d i v i s i o n and photosynthesis were stimulated. Popoff et a l (11? ) stated that a f t e r the p e t i o l e s of bean leaves had been immersed i n a 8 x 10"^ $ naphthenate solution, the photosynthetic rate was increased by 8 . 5 $ . Fattah ( 52 ) and Fattah and Wort (5 3 ) reported a s t a t i s t i c a l l y s i g n i f i c a n t increase i n photosynthetic rates of bush bean plants grown under l i g h t i n t e n s i t i e s of 16.10, 10.76, and 5 . 3 8 klx at 26° C for 7 . 14, and 21 days following a f o l i a r a p p l i c a t i o n of a 0 . 5 $ s o l u t i o n of KNap to 14-day-old plants. The apparent photosynthetic rate of greenhouse grown 3-week-old tomato plants treated with 0 . 5 $ KNap as a f o l i a r spray was diminished when measured two weeks af t e r treatment ( 4 0 ), However, the treatment resulted i n an increase of 4.2$ i n rates of apparent photosynthesis four weeks af t e r treatment. Following the a p p l i c a t i o n of f e r t i l i z e r and naphthenates to the s o i l , concentrations of N, P, and sugar were increased i n the cabbage heads ( 13 ). By soaking seeds i n or spraying plants with a 0 . 0 0 5 $ naphthenate solution during the vegetative period, Yur'eva ( 149 ) obtained increases i n protein and starch i n corncobs, and more protein and P i n the leaves of sugar beet. After spraying tomato plants with 0 . 0 0 5 $ naphthenates, A l i e v ( 7 ) observed that the uptake of N and P by treated plants had been increased. Asadov ( 14 ) stated that naphthenate treatments at the rate of 50 or 100 g/ha at the time cabbage seedlings were planted, and during the vegetative period as a f o l i a r spray, increased the amount of N and P i n the heads. Abolina and Ataullaev ( 2 ) reported that the vegetative organs of the the potato plant showed increases i n the le v e l s of N and P when compared with controls. Stimulation of N and P metabolism, and increases i n t o t a l and protein N content i n the roots of cotton were observed by Babaev ( 16 ) af t e r the cotton seeds were treated with a solution containing 0.0001% naphthenates p r i o r to sowing. The data of Guseinov and Guseinov ( 65 ) showed that naphthenates (65 to 280 g/ha) combined with N-P-K f e r t i l i z e r s and applied to s o i l increased the uptake of these elements by 78$. The app l i c a t i o n of naphthenates alone or i n combination with mineral f e r t i l i z e r s (N and P) to the s o i l increased the P content i n tomatoes and cabbage ( 64 ). Moreover, the sum of ammonium and n i t r a t e N i n the plant tissue was increased. Ejubov and Issaeva ( 47 ) reported that the concentration of assimilable P forms and of mineral N compounds i n maize and lucerne, as well as the t o t a l N and P concentrations, were increased. Peterburgsky and Karamete (113) observed that naphthenates increased ^ 2P u t i l i z a t i o n when maize was grown i n three d i f f e r e n t cultures. Naphthenate-treated plants grown i n sand culture showed the highest rate of ^ 2P u t i l i z a t i o n . U t i l i -zation of P was lower when the plants were grown i n a hydroponic medium, or i n s o i l . The authors also reported that the P content i n a l l organs of maize was increased by 50$ a f t e r the seeds had been soaked i n a 0 . 0 0 5 $ naphthenate solution p r i o r to sowing i n sand. After maize had received a f o l i a r a p p l i -cation of a 0 . 0 0 5 $ naphthenate solution, P content was increased by 3 3 $ . Following a seed soak for 12 or 24 hours i n a solution containing 200 to 1,000 uCi/1, as K 2H 3 2P0^, alone, or with 0.01$ naphthenates, Guseinov and Issaeva ( 66 ) reported that the a l f a l f a hay crops were increased by 1? and 24$, respectively. After a l f a l f a had received a f o l i a r treatment with a 0 , 0 1 $ naphthenate solution during the vegetative period, the hay y i e l d was increased by 24 ...to 3 7 $ . Peterburgsky and Karamete ( 1 1 3 ) observed that naphthenate treatments had an e f f e c t on 3^p l e v e l s i n corn. Their r e s u l t s showed that ^2p l e v e l s i n plants treated with 0 . 0 0 5 , 0 . 0 1 , and 0 . 0 1 5 $ naphthenates were 125» 1 0 6 , and 152$ of the control values, respectively. Following a f o l i a r a p p l i c a t i o n of naphthenates ( 0 . 0 0 5 $ ) at the beginning of the flowering phase, the concentration of ascorbic acid and the s p e c i f i c a c t i v i t i e s of catalase and peroxidase i n the leaves of treated plants were increased as compared with control values ( 2 4 ), However, when a N-P f e r t i l i z e r was applied to the s o i l at the same time the foliage received the naphthenate treatment, ascorbic content and the a c t i v i t i e s of the two enzymes were decreased i n the leaves of treated plants. MATERIAL AND METHODS A.) Growth of plants. Uniform seeds of the dwarf bush bean plant, Phaseolus  vul g a r i s L. c u l t i v a r Top Crop (Buckerfield's, Ltd., New West-minster, B.C.), were sown i n 50 x 33 x 7 cm wooden f l a t s con-t a i n i n g vermiculite, and the f l a t s were placed i n a growth room. While the seedlings were i n vermiculite, they were watered with a one-quarter strength Hoagland-Arnon's solut i o n minus P or TABLE I I . Volumes of molar stock solutions used for preparing one l i t e r of a ix nutrient solution. Modified a f t e r Hoagland and Arnon ( 74 ). inl/1 a Stock solu t i o n Complete -P KH 2 P 0 ^ 1 KNO3 5 5 C a ( N 0 3 ) 2 • 4 H 2 0 5 5 MgSO^ • 7 H 2 0 2 2 NaCl ( 0 . 1 M) 1 1 K C 1 1 Fe EDTA (5 mg/ml) 1 1 A - 5 b 1 1 Deionized water ( 0 . 2 ppm, as NaCl). b The A-5 micronutrient solution was prepared by d i s s o l v i n g the following i n 1 1 of d i s t i l l e d water: 2.86 g H 3 B O 3 , 1.8l g M n C l 2 • 4 H 2 O , 2 2 0 mg ZnSO^ . 5 H 2 0 , 80 mg CuSOij. . 5 H 2 O , and 2 0 mg Na2Mo0ij, • H 2 O . with a complete solution. The following conditions were main-tained for the duration of the experiment! 16.3$ klx at the top of the plants, a 14 hour photoperiod, a day/night temperature of 26-l°/2l-l° G, and a day/night percent r e l a t i v e humidity of 60 to 70/70 t o 80. The l i g h t i n the growth room was supplied by cool-white fluorescent tubes (Westinghouse, U.S.A.) and 60-watt incandescent lamps. After eight days i n vermiculite, 48 uniform seedlings were transplanted? into four 33 x 26 x 13 cm culture trays which were covered with aluminum f o i l and black paint to prevent a l g a l growth. Each tray held 12 plants and contained 4 l i t e r s of a continuously aerated Hoagland-Arnon's (pH 5 .^) nutrient solution (Table I I ) . After 3 days, the arrangement of the trays was changed to average l o c a l environmental v a r i a b i l i t y . B. ) Preparation of potassium naphthenates (KNap) aqueous solution from naphthenic acids (HNapjT Seventeen ml of a 12.3$ (w/v) KOH solution (2.1 g KOH i n 17 ml d i s t i l l e d water) was added to a flask containing 5 g naphthenic acids ( P r a c t i c a l grade, average mol wt 2 3 0 , Eastman Organic Chemicals, Rochester, N.Y.). The fl a s k was shaken for 10 to 15 minutes and the solution was made to a volume of 25 ml with d i s t i l l e d water. The solution thus prepared was the stock solut i o n containing 250 mg of KNap per ml. By d i l u t i n g 1.0 ml of the stock solu t i o n (see next page), the f i n a l concentration of KNap was 2 x 10""2 M ( 5 0 0 0 ppm or 0 . 5 $ ) . The pH of the dil u t e d solution was adjusted to about 10 by the addition of 0.1 N HC1. C. ) Treatment with KNap. Fourteen days a f t e r sowing, bean plants i n two trays were sprayed to drip with the 2 x 10""2 m aqueous solut i o n of KNap described i n B). Even though KNap i s a surfactant, the KNap solutio n contained 0,3$ (v/v) Tween 20 (polyoxyethylenesorbitan monolaurate) (Atlas Powder Co., Wilmington, Del.) to serve as an add i t i o n a l wetting agent. The plants i n the other two trays remained as unsprayed controls. 32 D. ) Exposure to P. Twenty-four hours a f t e r spraying, the roots of both treated and control plants were immersed i n a continously aerated com-32 plete nutrient solution containing P, as orthophosphate (Atomic Energy of Canada, Ottawa, Ont.). In the f i r s t s eries of experi-ments where the plants were grown i n a phosphate-free nutrient 32 solution the P l e v e l was 15 uCi/1. When the plants were grown 32 i n complete nutrient, the P l e v e l was 20 uCi/1: and for the phosphorus f r a c t i o n a t i o n experiment at a l e v e l of 25 uCi/1. 32 Following a 2 hour exposure to the ^ P nutrient solution, the roots of a l l plants were well rinsed according to the following sequences Four changes of tapwater, a non-radioactive phosphate (210 mg/l) solution, and another change of tapwater. A l l r i n s -ing media were held at room temperature, and the t o t a l r i n s i n g time was 1 hour. After the r i n s i n g period, the plants were returned to a continuously aerated phosphate-free nutrient or to a complete nutrient s o l u t i o n for holding times of 4, 8, 12 and 24 hours before harvesting. E. ) Harvest, digestion, and counting. At the end of each of the four holding times, four treated and four control plants were taken at random, immediately divided into the three f r a c t i o n s roots; stems plus p e t i o l e s : and l e a f blades, and the fresh weight of each f r a c t i o n was recorded. Each treatment had two r e p l i c a t e s for each harvest or sampling time with the same organs from two plants per r e p l i c a t e . The plant f r a c t i o n s were placed i n separate 100 ml Kjeldahl digestion f l a s k s , and the plant material was wet ashed for 30 minutes at 1 1 5 - 1 2 5 ° C using 2 ml of 60$ HCIO^ and 3 ml of 70$ HNO^*. Following digestion, the volume of each digested sample was brought to 10 ml with d i s t i l l e d water. The 32 P a c t i v i t y i n the samples was determined by using a 10 ml l i q u i d G-M counting tube type M-6 ( 2 0 ^ Century El e c t r o n i c s , New Addington, Croydon, Surrey, England) which was housed i n a lead castle connected to a decade scaler (Nuclear-Chicago, Model 8 7 0 3 ) . The mean of two one minute counts was corrected for background and decay, F.) Phosphorus f r a c t i o n a t i o n . Bean plants were grown and harvested as previously describ-ed (see Section A & E). Leaf blades, stems plus p e t i o l e s , and roots from treated and control plants c o l l e c t e d at the four d i f f e r e n t harvest times were digested as before (see Section E). P r i o r to the extraction procedure, the plant samples were stored at - 1 5 ° C. The extraction procedures outlined below were based on those described by Cole and Ross ( 41 ). Tissue samples weighing ca 3 g were placed i n an ethanol (Et0H)/dry i c e bath at ca - 4 5 ° C for 5 minutes. The frozen tissue was transferred to a cold porcelain mortar, 1 ml of 80$ •Preliminary investigations showed that the white p r e c i p i t a t e (perchlorates) formed i n the f l a s k s during digestion was not radioactive, and that during the digestion process 32p a c t i v i t y was not l o s t . formic acid was added, and the tissue was ground. An a d d i t i o n a l 2 ml of the acid was added to bring the pH of the f i n a l extract to 2 , 2 to 2 , 4 , and grinding continued u n t i l the sample formed a t h i n s l u r r y . The sample was then rinsed into a Buchner funnel with 80$ EtOH, and the s l u r r y was f i l t e r e d using Whatman No, 43 ashless, medium-fast (phosphate-free) f i l t e r paper. The residue, containing acid-insoluble phosphorus compounds, was rinsed three times with 80$ EtOH. F i n a l l y , the residue and the f i l t e r paper were placed i n a 100 ml Kjeldahl digestion f l a s k , wet ashed at 1 5 0 ° G and ^ 2P a c t i v i t y determined as described previously. The f i l t r a t e , containing acid-soluble phosphorus compounds, was brought to a volume of 70 ml with 80$ EtOH. After determin-ing the ^ 2P a c t i v i t y i n a 10 ml aliquot, 25 ml was placed i n a Kjeldahl f l a s k . The extract was heated at 50° C u n t i l a l l EtOH had been removed, and the volume had been reduced to ca 4 ml. The n i t r i c / p e r c h l o r i c acid mixture was added, and the sample was digested as before. In both cases the volume of the digested samples was brought to 10 ml, and t o t a l inorganic phosphate was determined (see Section G). G.) Total inorganic phosphate determinations. The concentration of inorganic phosphate present i n each digested f r a c t i o n was determined following the procedure described by Fiske and Subbarow ( 5 5 ). The colorimeter tube contained 0 . 2 ml of digest, 1 . 0 ml of molybdate reagent, 4 . 0 ml of 10$ t r i c h l o r o a c e t i c acid, 0 . 4 ml of aminonaphtholsulfonic acid (ANSA), and 4 . 4 m l of d i s t i l l e d water. Exactly 5 minutes a f t e r the digest had been added, the i n t e n s i t y of the blue color was measured using a Klett-Summerson colorimeter equipped with a No, 66 red f i l t e r (range 640 - 700 nm). Total inorganic phosphate analysis was not done on the organs from control and treated plants which were grown i n phosphate-free nutrient. See pages 151 - 152 i n the Appendix for reagents and the standard phosphate curve (Figure 14). RESULTS A.) Tot a l uptake by the plant. Fifteen-day-old control and naphthenate-treated bean plants growing i n a phosphate-free or a complete nutrient solution were exposed to a -^2P nutrient solution for 2 hours. The cpm per gram fresh weight i n the whole plant was taken to be a measure of the rate of phosphate uptake by the roots of treated and control plants during the exposure time. This assumption i s v a l i d provided the r i n s i n g of the roots was complete a f t e r t h e i r exposure to J P, and there was not a s i g n i f i c a n t efflux of ^  P during the holding period. In these experiments the two require-ments were met, as the J P a c t i v i t y i n the cu l t u r i n g trays was very low when determined a f t e r the plants had been removed. This observation concurs with the data of Rovira and Bowen (120). When the roots of wheat seedlings were fed 32p f o r 15 minutes at a l e v e l of 300 uCi/1, the authors concluded that the efflux of 3 2P had been washed out of the 'apparent free space' with just 5 to 7 minutes of root r i n s i n g i n tapwater. In the present experiments the roots were rinsed for 1 hour. To investigate the e f f e c t of naphthenates on the uptake of 3 2 p and on the t o t a l phosphorus content, experiments were designed to study these variables. Even though the plants were harvested at four d i f f e r e n t sampling times and divided into three d i f f e r e n t f r a c t i o n s , the sum of the ^ 2P a c t i v i t y from these fractions represents the amount of 32p l a b e l l e d phosphate which was absorbed by the roots of control and treated plants during the 2 hour exposure period. Average values from i d e n t i c a l uptake and d i s t r i b u t i o n experiments are given i n Tables III to XI. In a l l instances the v a r i a t i o n between the values from i d e n t i c a l experiments was not s t a t i s t i c a l l y s i g n i f i c a n t . Phosphorus-32 a c t i v i t y on a cpm per gram basis i n l e a f blades, stems, roots, and i n the t o t a l plant i s shown i n Table I I I . In these experiments the only phosphorus the plants received was ^ 2P l a b e l l e d orthophosphate during a 2 hour exposure or feeding period. I t was determined that naphthenate-treated plants had taken up 102$ as much a c t i v i t y as the control plants, and that t h i s increase was not s t a t i s t i c a l l y s i g n i f i c a n t . 32 When P a c t i v i t y i n the plant i s expressed on a per plant basis (Table V), the difference between treated and control values also lacked s i g n i f i c a n c e . a t the 5$ l e v e l . However, the value for the treated plants was 109$ of the control value (Figure 1). The data shown i n Table IV, VIII also represent r e s u l t s from i d e n t i c a l experiments. In these experiments control and treated plants were grown i n a complete nutrient solution 32 before and a f t e r they were exposed for 2 hours to a ^ P solution. When -^2P a c t i v i t y i n the plant i s expressed as cpm per gram, the naphthenate. treatment did not have a s t a t i s t i c a l l y s i g n i f i c a n t e f f e c t on ^ 2p uptake (Table IV). On a per plant basis (Table V), a c t i v i t y i n treated plants was 107$ of the control value, and the pattern was very s i m i l a r to the one obtained when plants were grown i n the phosphate-free nutrient solution (Figure l ) . Values for -*2p a c t i v i t y on a gram basis i n l e a f blades, stems, and roots from treated plants grown either i n the -P or i n the complete nutrient solution were not s i g n i f i c a n t l y d i f f e r e n t from the control values (Table. I l l , IV). TABLE III. E f f e c t of KNap on t o t a l uptake and d i s t r i b u t i o n of phosphorus-32, on a per gram basis, by bush bean plants at various sampling times a . Sampling Phosphorus-32 a c t i v i t y b times i n hours Leaf blades Stems Roots Total C c T C T C T C T 4 d 3.79. 1,268 1 ,202 2 , 1 6 0 35 ,724 3 1 , 1 0 7 37 ,305 34 ,536 8 873 1,626 1,648 2 , 3 9 2 31,818 34 ,273 3 5 , 0 8 9 3 8 , 2 9 1 12 863 1,751 1 ,532 2 , 5 0 3 26,799 26 ,255 2 9 , 1 9 4 3 0 , 5 U 24 928 2 , 1 3 8 1 ,899 3 ,211 28,146 2 7 , 1 5 1 3 0 , 9 7 3 3 2 , 5 0 0 Mean 761 l , 6 9 6 n s 1 ,570 2 , 5 6 7 n s 30,621 2 9 , 6 9 7 n s 33,140 3 3 , 9 6 0 n S Percent of control 222.9 1 6 3 . 4 96.9 102 .4 Plants grown i n a -P nutrient solution before and aft e r the plants were exposed f o r 2 hours to a phosphorus-32 nutrient solution. Counts/minute/gram fresh weight. C = no spray; T «= 5000 ppm KNap. Average of four values. Not s i g n i f i c a n t l y d i f f e r e n t from the control value at the 0 .05 l e v e l . This designation on a l l subsequent tables w i l l have the same meaning. TABLE IV. E f f e c t of KNap on t o t a l uptake and d i s t r i b u t i o n of phosphorus -32, on a per gram basis, by bush bean plants at various sampling times a . Sampling times i n hours Phosphorus- 32 a c t i v i t y b Leaf blades Stems Roots Total c c T c T c T C T 4 6 2 , ? l l d 6 6 , 6 6 6 2 7 , 4 1 7 2 9 , 2 2 2 64 ,582 5 7 , 6 9 1 1 5 4 , 7 1 0 1 5 3 , 5 7 8 8 66,428 7 0 , 4 0 9 2 5 , 9 5 2 28 ,768 5 7 , 0 5 6 52,421 1 4 9 , 4 3 6 1 5 1 , 5 9 8 12 68,042 71 ,923 2 5 , 0 6 9 2 9 . 0 6 3 5 1 . 2 2 9 5 0 , 5 0 6 144 ,339 1 5 1 , 4 9 2 24 6 8 , 4 4 7 6 9 , 1 1 7 24,428 2 5 . 5 7 8 47,860 4 4 , 4 9 7 140 ,734 1 3 9 , 1 9 2 Mean 6 6 , 4 0 7 6 9 , 5 2 8 n s 25,716 2 8 , l 5 8 n s 5 5 , 1 8 2 5 1 , 2 7 9 n s 147 ,305 l 4 8 , 9 6 5 n s Percent of Control 104.7 1 0 9 . 5 9 2 . 9 101 .1 a Plants grown i n a complete nutrient solution before and a f t e r the plants were exposed f o r 2 hours to a phosphorus-32 nutrient solution. b See Table III. c See Table I I I . d See Table I I I . TABLE V . E f f e c t of KNap on t o t a l uptake of phosphorus-32, on a per plant basis, by bush bean plants. Sampling times i n hours Total phosphorus- 32 a c t i v i t y a Grown i n a -P nutrient Grown i n complete nutrient T C T 4 80 ,300 ° 8 8 , 2 9 5 2 7 4 , 7 8 5 2 7 6 , 9 6 1 8 9 1 , 9 9 9 113.^33 2 5 3 . 9 2 3 2 8 3 , 5 3 0 12 7 6 , 7 2 9 81 ,326 2 6 8 , 8 8 3 2 9 1 , 4 7 4 24 9 7 . 8 9 2 9 6 , 7 8 1 3 0 5 . 1 9 2 3 2 9 . 1 3 1 Mean 8 6 , 7 3 0 9 4 , 9 5 9 n s 2 7 5 . 6 9 5 2 9 5 . 2 7 4 n S Percent of control 1 0 9 . 5 1 0 7 . 1 a b c Counts/minute/plant. G = No spray; T = 5000 ppm KNap. Average of four values. FIGURE 1. E f f e c t of KNap on t o t a l phosphorus-32 uptake, on a per gram or per plant "basis, when bush bean plants were grown i n a phosphate-free or a complete nutrient. o •p •H > •H -P O a (Si i to U O .C ft to o £i ft rH «J 300 . 250 . 200 150 100 50 0 C T C T per gram per plant PHOSPHATE-FREE C T C T per gram per plant COMPLETE Total phosphorus on a per gram basis for l e a f blades, stems, and roots from treated and control plants i s shown i n Table VI, and i n Table VII t o t a l phosphorus for treated and control plants i s shown on a per plant basis. No matter how the data iare expressed, naphthenate treatment did not have an e f f e c t on phosphorus uptake (Figure 2 ) . On a per gram basis, treated plants had an average of 8 . 7 7 mg of inorganic phosphate; control plants 9 . 0 0 mg. The r a t i o of average ^2p a c t i v i t y to average t o t a l phosphorus ( 3 2 p / t P ) on a per gram basis i n l e a f blades, stems, and roots of control and treated plants i s shown i n Table VIII. The difference between the ^p/^p r a t i o s for roots from treated and control plants was not s t a t i s t i c a l l y s i g n i f i c a n t , and the value for naphthenate-treated plants was 9 6 . 8 $ of the value for control plants. For the stem f r a c t i o n the difference between the value from treated plants was greater than that of control plants by 1 2 . 3 $ . This difference was s i g n i f i c a n t at the 5 $ l e v e l . For l e a f blades from control and treated plants the difference between the ?§P/tP r a t i o s was s i g n i f i c a n t at the 2 . 5 $ l e v e l . The value for naphthenate-treated plants was 1 1 0 . 8 $ of the control value (Table VIII, Figure J). TABLE VI. E f f e c t of KNap on t o t a l uptake and d i s t r i b u t i o n of phosphorus, on a per gram basis, by bush bean plants at various sampling times a . Sampling Total inorganic phosphate b times i n hours Leaf blades Stems Roots Total C c T C T C T c T 4 3 .19 d 3-33 1.48 1 . 3 4 3 . 06 2 . 7 7 7 . 7 4 7 . 4 5 8 3 . 7 9 3 . 8 3 1.51 1.49 3 . 8 9 3 . 2 5 9 . 1 9 8 . 5 7 12 3 - 8 9 3-94 1 .59 1 .73 3 . 6 9 3 . 7 6 9 . 1 7 9 . 3 4 24 4 . 0 2 3.84 1 .77 1 .76 4 . 1 1 4 . 0 2 9 . 8 9 9.62 Mean 3 . 7 2 3 . 7 4 n s 1 .59 1 . 5 8 n s 3 . 6 9 3 . 4 5 n s 9 . 0 0 8 . 7 7 n S Percent of control 1 0 0 . 5 9 9 . 3 9 3 . 5 9 7 . 4 a See Table IV. b Milligrams inorganic phosphate/gram fresh weight. c See Table III. d See Table III. TABLE VII. E f f e c t of KNap on t o t a l uptake of phosphorus, on a per plant basis, by bush bean plants at the various sampling times a . Sampling Total inorganic phosphate ^ times i n hours c Control Treated d 4 13.8 13-5 8 15. G 15.6 12 17 .5 18.0 24 20.9 22.0 Mean 16.8 17.3 Percent of control 102.6 ~ See Table IV. b Milligrams inorganic phosphate/plant. c See Table I I I . d See Table I I I . TABLE VIII. E f f e c t of KNap on the r a t i o of phosphorus-32 a c t i v i t y to t o t a l phosphorus, on a per gram basis, i n bush bean plants at various sampling times a . Sampling Phosphorus-32 a c t i v i t y / t o t a l inorganic phosphate ^ times i n hours Leaf blades Stems Roots C c T C T C T 4 20.41 d 24.27 18.51 21 .90 20.41 18.00 8 19.27 20.82 17.44 20.17 14.07 14.62 12 17.93 19.58 15-81 16.99 12.65 13.38 24 19.24 20.45 13.81 14.62 10.57 9.89 Mean 19.21 21.28* 16.39 18.42* 14.43 13.97 n s Percent of control 110.8 112.3 96.8 a See Table IV. b Counts/minute/gram and milligrams inorganic phosphate. c See Table I I I . d See Table I I I . Means d i f f e r i n g s i g n i f i c a n t l y at the 0.05 l e v e l from the respective control mean. This designation on a l l subsequent tables w i l l have the same meaning. FIGURE 2. E f f e c t of KNap on t o t a l phosphorus present, on a per gram or per plant basis, i n bush bean plants grown i n complete nutrient. +> a X! P. in o x: o s u o to •H r H •H 24 22 h1 20 18 16 14 12 10 8 6 4 2 0 C T per gram C T per plant FIGURE 3 . E f f e c t of KNap on the r a t i o of phosphorus-32 a c t i v i t y to t o t a l phosphorus, on a per gram basis, i n leaf blades stems, and roots of bush bean plants. fciD s CO o Xi ft CO o xi ft H cd •p o •p \ 1 ft o CM c-\ I 05 o ft m o Xi PH 24 22 20 18 16 14 12 10 8 6 4 2 0 C T Leaf blades T T Stems Roots B.) Percentage d i s t r i b u t i o n of the t o t a l 3 2P a c t i v i t y among  l e a f blades, stems, and roots. When bean plants grown i n a -P nutrient were exposed for 2 hours to a solution containing ^ 2P at a l e v e l of 15 uCi / 1 without c a r r i e r phosphate and then held for 24 hours, 90.7$ of the t o t a l a c t i v i t y remained i n the roots of control plants, while only 8 3 . 8 $ remained i n the roots of treated plants (Table IX). At the same sampling time 6.2$ of the t o t a l ^ 2p absorbed was found i n the l e a f blades of treated plants, and 3.0$ i n the le a f blades of the controls (Figure 4). Consequently, at the 24 hour sampling time a c t i v i t y i n treated plants was less i n the roots, and greater i n stems and l e a f blades, i . e . 9^.4, 155.G, and 202.9$ of the control values. This same general pattern was also noted at the other three sampling times. The 32 amount of P present i n the roots, stems, and l e a f blades of treated plants d i f f e r e d s i g n i f i c a n t l y from the control value at the 2 , 5 $ l e v e l . When plants were grown i n complete nutrient both before 32 and a f t e r exposure of the roots to P, the percentage d i s t r i b u -32 tio n of the P l a b e l i n the l e a f blade and root fractions from control and treated plants was s i m i l a r to the d i s t r i b u t i o n pattern which emerged a f t e r plants were grown i n a -P nutrient. However, the amount of l a b e l present i n the plant organs d i f f e r e d considerably when compared with plants grown i n the -P nutrient (Figure 4,5). The r e s u l t s of an analysis of plants grown i n the complete nutrient and sampled 24 hours a f t e r exposure to -^2P revealed that only 34.0$ of the t o t a l a c t i v i t y remained i n the roots of control plants, while 31.8$ remained i n the roots of treated plants. At the same sampling time 48.7$ of the t o t a l -^2P l a b e l i n the plant was found i n l e a f blades of control plants, and 49.8$ i n the l e a f blades of treated plants. The amount of ^ 2P present i n the roots of treated plants d i f f e r e d s i g n i f i c a n t l y from the control value at the 1$ l e v e l , and for the stems at the 3$ l e v e l . The amount of 3 2 P present i n the l e a f blades of treated plants was not s i g n i f i c a n t l y d i f f e r e n t from the control value at the 5$ l e v e l (Table X). On a percentage of control basis, the d i s t r i b u t i o n of a c t i v i t y among the l e a f blades, stems, and roots of treated plants was 103.5* 108 . 1 , and 91.8$, respec-t i v e l y (Figure 5 ) . I t i s i n t e r e s t i n g to note that i n control and treated plants the amount of -^2P i n the stem fracti o n s remained r e l a t i v e -l y constant through the four sampling times, i . e . 17 .2 to 17.7$ and 18 .3 to 19«3$» respectively. Consequently, changes occurred i n l e a f - r o o t d i s t r i b u t i o n s of ^ 2P which did not markedly a f f e c t the percentages i n the stems. Etter ( 4 9 , 51 ), working with the e f f e c t of 2,4-D on phosphorus metabolism i n bean plants, made a s i m i l a r observation. TABLE IX. E f f e c t of KNap on the percentage d i s t r i b u t i o n of phosphorus-32, on a per gram basis, among leaf blades, stems, and roots of bush bean plants at various samplings times a . Sampling times i n °> % of t o t a l phosphorus--32 a c t i v i t y b hours Leaf blades Stems Roots C c T C T C T 4 . d 1.04 3 . 6 1 3 .31 6 . 1 0 9 5 . 6 8 9 0 . 3 5 8 2 . 6 2 4 . 2 3 4 . 8 3 6.41 9 2 . 5 5 8 9 . 3 6 12 2 . 9 2 5.44 5 . 3 7 8.26 9 1 . 7 2 8 6 . 30 24 3 . 0 2 6 . 2 5 6.28 9 . 9 0 90.71 8 3 . 8 4 Mean 2.40 4 . 8 ? * 4 . 9 5 7 . 6 7 9 2 . 6 8 8 7 . 4 6 * Percent of control 2 0 2 . 9 1 5 5 . 0 9 4 . 4 a See Table III. b See Table I I I . ° See Table I I I . d See Table I I I . 10 rlOO . 9 6 -92 -88 2 - -84 k 8 12 Leaf blades 2k C = 8 12 2k Stems 8 12 Roots .80 2k - Sampl-ing time i n hrs FIGURE k. E f f e c t of KNap on the percentage d i s t r i b u t i o n of phosphorus-32, on a per gram basis, among le a f blades, stems, and roots of bush bean plants grown i n a phosphate-free nutrient. Sampling % 1 of t o t a l phosphorus--32 a c t i v i t y b times in hours Leaf blades Stems Roots c c T C T C T 4 d 41.16 44 .10 17 .79 1 8 . 9 9 4 1 . 0 5 3 6 . 9 0 8 44 .11 46 .47 1 7 . 4 7 1 9 . 0 2 3 8 . 4 3 3 4 . 5 2 12 46.96 4 9 . 1 0 17.41 1 9 . 3 0 3 5 . 6 2 3 3 . 6 0 24 4 8 . 7 8 4 9 . 7 5 1 7 . 2 9 1 8 . 3 7 3 3 . 9 4 3 1 . 8 8 Mean 4 5 . 2 5 4 6 . 8 5 n S 1 7 . 4 9 1 8 . 9 2 * 37.26 #* 3 4 . 2 2 Percent of control 1 0 3 . 5 1 0 8 . 1 9 1 . 8 a See Table IV. b See Table I I I . c See Table I I I . d See Table I I I . o o o H a> • c+ (D 3 a* P CQ P« CQ w H) H> CO O <+ O CD P CD p O H> 3 a" H P P-CD « CQ CD 3" CD CQ c+ CD 3 CQ ati - CD o CD 3 e+ P P P. 3 H* P. CQ c+ 4 hi O H* O C c+ C CQ c+ H* O O H, 3 c o CQ 3"id 3* o* o CD CQ 3" 3 O S i ' c+V*> CQ ro 3 o o 3 3 P 3 P CD 1 P H> OO P P. CD CQ O ro ro _1 -P-c+ CD 3 CQ 3^ CO ro ro o I ro o o o o 1 I I I 1 1 I 1 I I I I I I I I I I T w o o c+ CQ 3 3* CQ <+ H' H* 3 3 (w CD CQ 00 ro ro i w p 3 i ro o o o o An analysis of t o t a l phosphorus i n the three plant f r a c t i o n s from control and treated plants grown i n a complete nutrient solution i s shown i n Table XI. When the plants were grown i n t h i s nutrient, there was close agreement between -*2P a c t i v i t y (Figure 5)» and t o t a l phosphorus (Figure 6 ) when compared on a percentage basis. Also, as was the case with -*2P a c t i v i t y , t o t a l phosphorus i n the stem f r a c t i o n of treated and control plants remained r e l a t i v e l y constant through the four sampling times, i . e . l ? . l to 18 . 3 $ and 1.6.4 to 19.2$, respectively. At the 8 hour sampling time, 4l.4$ of the t o t a l phosphorus was found i n the l e a f blades of the controls, and 44.8$ i n treated plants. On a percent of control basis t o t a l phosphorus i n the l e a f blades, stems, and roots of treated plants was I O 3 . 3 , 101 .6 , and 96.0$, respectively. This pattern was also observed at the 4 hour sampling time. However, there was not a great deal of difference between the percentage values for roots and l e a f blades at the 12 and 24 hour sampling times. The amount of phosphorus present i n roots from treated plants d i f f e r e d sign-i f i c a n t l y from the control value at the 5$ l e v e l . As f a r as the l e a f blade f r a c t i o n was concerned, t h i s difference was not quite s i g n i f i c a n t at the 5$ l e v e l , and for stems there was almost no difference between the values from control and treated plants. Sampling • • • % of t o t a l phosphorus b times i n hours Leaf blades Stems Roots c C T C T C T 4 41.24 45.11 19.20 18.04 39-56 36.85 8 41 .47 44.89 16 .45 17.16 42 .07 37.95 12 42 .76 41 .78 17.28 18.34 39.96 39.88 24 41 .05 40.10 17 .73 18.26 41 .22 41.64 Mean 41 .63 42 . 9 7 n s 17.67 1 7 . 9 5 n S 40 .70 3 9 . 0 8 * Percent of control 103-3 101.6 9 6 . 0 a See Table IV. b See Table VI. c See Table III. d See Table I I I . 50 r5o 40 -40 CO u o X2 P . CO o x! P i rH cd o -p o 30 20 -30 -20 10 -10 4 8 12 teaf blades 24 4 8 12 Stems 24 8 12 Roots C = T = "D o 24 - Sampl-ing times i n hrs o In the experiments described below, bean plants were grown hydroponically i n a -P nutrient solution both before and a f t e r the roots of control and treated plants were exposed to ^ 2P. However, i n each experiment the experimental variable was changed s l i g h t l y . The r e s u l t s of these experiments are b r i e f l y outlined. 1) When the plants were sprayed with a 2500 ppm (0 .25$) KNap solution 24 hours p r i o r to the ^ 2P feeding, the e f f e c t of 32 the naphthenate treatment on J P uptake was not s i g n i f i c a n t . The e f f e c t of t h i s treatment on the percentage d i s t r i b u t i o n of ^ 2p among the three plant organs was very s i m i l a r to the d i s t r i -bution pattern observed following the 5000 ppm naphthenate treatment. 2) When bean plants were sprayed with a 5000 ppm KNap solution 8 hours p r i o r to the ^ 2P feeding, the uptake and percentage d i s t r i b u t i o n were again very s i m i l a r to the experi-mental r e s u l t s previously described (Figure 1,4). 3) As was the case i n the experiments where bean plants were grown i n a -P nutrient solution, i n t h i s experiment the plants were sprayed with a 5000 ppm KNap solut i o n 24 hours p r i o r to the -^2P feeding. Eight hours a f t e r the roots were exposed to a ^ 2P nutrient, control and treated plants were withdrawn 1 from the -P nutrient solution. The a e r i a l portion of the plants was removed, and cut-stem exudate was coll e c t e d over a period of 2 hours. A comparison of the a c t i v i t y i n the exudate from two sets of treated plants ( 0 . 2 ml exudate/set of 4 plants) was almost twice the l e v e l of a c t i v i t y i n exudate from two sets of control plants. 4) In a preliminary experiment i t was determined that a f o l i a r a p p l i c a t i o n of a 0 .3$ Tween 20 solution to l4-day-old oean plants did not have an e f f e c t on phosphorus uptake and d i s t r i b u t i o n . Also, an experiment was designed to determine the e f f e c t of KNap and Tween 20 on the rate of transpiration. Two sets of l4-day-old bean plants growing i n s o i l were sprayed with 0 .5$ KNap i n 0 .3$ Tween 20 or with 0 .3$ Tween 20 alone. One set of plants remained as unsprayed controls. Each set contained two pots with two bean plants per pot. After bringing the s o i l water to f i e l d capacity, the pots were c a r e f u l l y enclosed i n polyethylene bags. The rate of tran s p i r a t i o n from the bean plants i n two i d e n t i c a l experiments was determined by measuring the number of grams of water transpired/dec 2/hour. Even though not s t a t i s t i c a l l y s i g n i f i c a n t , the rate of tr a n s p i r a t i o n of KNap treated plants was lower than that of unsprayed plants and those which received Tween 20 by 12$. After corn plants had received a 0.005$ naphthenate t r e a t -ment i n the form of a f o l i a r spray, Yur'eva (149 ) observed that treated plants retained more water when compared with unsprayed control plants. In a s i m i l a r experiment the author (l49 ) also observed the same r e s u l t with sugar beet plants. C.) Percentage d i s t r i b u t i o n of acid soluble 32p a c t i v i t y and  t o t a l acid soluble P among l e a f blades, stems, and roots. Ideally, a procedure for extracting metabolic products from plant tissue would accomplish the following: Stop a l l enzymatic a c t i v i t y at the instant of k i l l i n g , protect the metabolic products from chemical degradation, and permit quantitative separation of acid soluble and insoluble components. According to Cole and Ross ( 4 1 ), the use of formic acid and ethanol to extract acid soluble P compounds from plant tissue seemed to s a t i s f y the c r i t e r i a l i s t e d above. Because of t h i s , the procedures, as outlined by Cole and Ross, have been employed to fractionate P compounds from the organs of control and treated bean plants into acid soluble and acid insoluble components. The acid soluble P present i n the organs of control and treated plants was extracted with cold formic acid and ethanol. Using t h i s method of extraction, the following P-containing compounds or groups of compounds would be found i n t h i s f r a c t i o n : free nucleotides and nucleosides, sugar mono- and diphosphates, UDPG, PEP, PGA, phospholipids, and inorganic phosphate,( 41 ). In Table XII the percentages of the t o t a l acid soluble 3 2p a c t i v i t y for the l e a f blades, stems, and roots of control and treated plants are given. The cpm were on a per gram fresh weight basis. Through the 24 hour holding period, the percent-age of the t o t a l acid soluble a c t i v i t y i n the roots of both control and treated plants decreased. In the roots of control and treated plants the value decreased from 51.4 to 37.5$» and 49 .3 to 40 . 9 $ , respectively. The differences between the values Sampling times i n hours Phosphorus-32 a c t i v i t y Leaf blades Stems Roots T 4 8 12 24 34.09 37.21 40 .89 43.36 32.42 37.80 42.61 48.78 16.56 13.65 16.82 15.73 16.09 14.97 15.61 13.67 49 .35 49 .13 42 .30 40.91 51.49 47 .23 41.78 37.55 Mean 38.89 40.40 n S 15.69 1 5 . 0 8 n s 45.42 4 4 . 5 1 n s Percent of control 103.9 96.1 97.0 a See Table IV. b See Table III. c See Table III. d See Table I I I . o * ' • ' ' 0 4 8 12 24 Sampling time (hours) C = T = Sampling Total inorganic phosphate b times i n hours Leaf blades Stems Roots C c T C T C T 4 d 44.49 46 .44 14.11 14.08 41 .39 39^48 8 42 .99 42 .88 12.99 15 .02* 44.02 42.11 12 46 .29 43.43 l 4 . ? 2 17 .99 38.99 38.58 24 44.28 44.98 15.59 i 4 . 4 5 40.14 40 .57 Mean 44.51 4 4 . 4 3 n s 14 .35 I 5 . 3 8 n s 41.14 4 0 . l 8 n s Percent of control 99.8 107.2 97.7 a See Table IV. b See Table VI. c See Table I I I . d See Table I I I . CO U O Xi ft ra o Xi ft CD r-l X> d H o ra n •H o ct$ •P o -p o 40 30 • 20 10 Leaf — — ft Root o . •« Stem ° Stem —T" 8 — i — 12 24 Sampling time (hours) C = T = -o Sampling times i n hours Phosphorus-32 a c t i v i t y Leaf' blades Stems Roots C c T c T C T 4 35i9? d 32.67 16.55 16.55 4 7 . 4 8 50.78 8 37.79 38.43 12.83 1 3 . 9 0 4 9 . 3 8 47.67 12 ki.kk 44.72 15.98 1 4 . 8 9 4 2 . 5 8 4 0 . 3 9 2 4 44.35 4 8 . 5 7 1 5 . 2 4 1 4 . 0 7 4 0 . 4 1 37.37 Mean 3 9 . 8 9 4 i . l 0 n s 15.15 l 4 . 8 6 n s 4 4 . 9 6 4 4 . 0 5 n s Percent of control 103.1 9 8 . 0 97.8 a See Table IV. b See Table I I I . c See Table I I I . d See Table I I I . Sampling Total inorganic phosphate b times i n hours Leaf blades Stems Roots C c T C T G: T 4 45.6? d 46.24 11.85 14.46* 42.48 39.30 8 43.41 43.77 12.14 * 13.81 44.45 42.41 12 46 .83 44.15 13.95 16.61* 39.23 39.24 24 44.62 44.83 14 .93 14.70 40.46 40.46 Mean 45.13 4 4 . 7 5 n S 13.22 14 .89* 41 .65 4 0 . 3 6 n s Percent of control 99.2 112.6 96.9 a See Table IV. b See Table VI. c See Table I I I . d See Table I I I . for control and treated plants were not s t a t i s t i c a l l y s i g n i f i c a n t . Corresponding to t h i s decrease, the percentage of the t o t a l soluble a c t i v i t y i n the l e a f blades of control and treated plants increased. Even though the difference was not s t a t i s -t i c a l l y s i g n i f i c a n t , acid soluble a c t i v i t y i n the l e a f blades of treated plants increased from 32.4 to 48.?$; controls from 34 .0 to 4 3 . 3 $ . The percentage of the t o t a l acid soluble a c t i v i t y i n stem tissue from control and treated plants varied s l i g h t l y from one sampling time to another, but the percentage acid soluble a c t i v i t y remained r e l a t i v e l y constant through the 24 hour holding period (Figure 7 ) . Compared with the acid soluble ^2p pattern described above, the t o t a l soluble P pattern expressed on the same bases was s l i g h t l y d i f f e r e n t . The percentages of t o t a l acid soluble P i n the l e a f blades and roots of both control and treated plants remained r e l a t i v e l y constant over the 24 hour period. However, i n the stem f r a c t i o n of treated plants the percentage soluble P, on a per gram basis, was s i g n i f i c a n t l y greater (0 .05 l e v e l ) than the respective control values at the 8 and 12 hour sampling times (Table XIII* and Figure 8). Comparison of the percentages of the t o t a l acid soluble 3 2p a c t i v i t y , on a per plant basis, i n the organs of control and treated plants (Table XIV) with the percentages, on a per gram basis (Table XII), revealed very s i m i l a r d i s t r i b u t i o n patterns. This was also true for the acid soluble P data (Table XIII, XV). Table XXVIII . XXIX , XXX , XXXI i n the Appendix gives the actual values from which the percentage d i s t r i b u t i o n s were determined. The data indicate that a mobile ^ 2P-containing compound, probably l a b e l l e d orthophosphate ( 5 0 )» i n the acid soluble f r a c t i o n was being acropetally translocated from root tissues to the a e r i a l portion of the plant. Naphthenate treatment seemed to augment t h i s translocation process. However, with the exception of stem tissue from treated plants, the percentage of the t o t a l soluble P remained r e l a t i v e l y constant i n both control and treated plants through the holding period. D.) Percentage d i s t i b u t i o n of acid insoluble phosphorus-3 2p  a c t i v i t y and t o t a l acid insoluble P among l e a f blades,  stems, and roots. Using the formic/ethanol method of extraction, the acid insoluble f r a c t i o n Would include P-containing proteins and nucleic acids. As before, the cpm and t o t a l P data i n t h i s section are expressed on both a per gram and per plant basis. When 3 2p a c t i v i t y of the acid insoluble f r a c t i o n i n each plant organ i s expressed a percentage of the t o t a l count on either a per gram or a per plant basis, the values i n Table XVI, XVIII are obtained. Whether on a per gram or a per plant basis, the percentage d i s t r i b u t i o n s of acid insoluble 32p a c t i v i t y among the three organs from control and treated plants at the four sampling times were very s i m i l a r . The percentage of the t o t a l acid insoluble -^P a c t i v i t y on a per gram basis i n root tissue of treated plants increased from 69 .5 to 75.1$ over the 24 hour sampling period, while the increase i n root tissue of control plants was from 71.1 to 72.7$ (Figure 9 ) . When on a gram or plant basis, the difference between the values for control and treated plants at the 24 hour sampling time was s i g n i f i c a n t at the 0.05 l e v e l . In contrast to the d i s t r i b u t i o n patterns obtained for root tissues, on a per gram or a per plant basis the percentage of the t o t a l acid insoluble a c t i v i t y i n the stems of control and treated plants decreased. The values decreased from 10.0 to 7 . 9 $ , and from 10.0 to 6.8$ i n control and treated plants, respectively. The differences between the percentage values for stems from control and treated plants were not s t a t i s t i c a l l y Phosphorus-32 a c t i v i t y Sampling times i n hours Leaf blades Stems Roots C c T C T C T 4 18.69 d 20.37 10.14 10.08 71.17 69.54 8 19.14 21 .65 9.26 9.50 71.61 68.85 12 19.09 18.41 8.80 8 .70 72.11 72.89 24 19.40 18 .05 7.89 6.79 72.71 75-16* Mean 19.08 1 9 . 6 l n s 9.01 8 . 7 7 n s 71.90 7 1 . 6 l n s Percent of control 102.7 97 .3 99.5 a See Table IV. b See Table I I I . c See Table I I I . d See Table I I I . <M I CO U o si Q, CO O Xi p, £> H O W C •a •H O a +> o -p « H o FIGURE 9 70 60 -_ A -_________ — * Root —— A Root 20 10 -— X Leaf Stem i 4 12 Sampling time (hours) 24 C = T = ON Total inorganic phosphate b Sampling times i n Leaf blades Stems Roots C c T C T C T 4 42.12 d 43.17 23.44 21.13 34.43 35.70 8 44.61 42.70 20.44 22.17 34.95 35.14 12 42.64 42.98 21.28 21.37 36.08 35.65 24 40.14 43.41 23.86 21.28 35.99 35.30 Mean 42.38 4 3 . 4 l n s 22.26 21.49 n S 35.36 35.45 n s Percent of control 101.6 96.5 100.2 a See Table IV. b See Table VI. c See Table I I I . d See Table I I I . Sampling time (hours) C = T = s i g n i f i c a n t . The percentage of the t o t a l acid insoluble a c t i v i t y i n l e a f blade tissue from control and treated plants varied s l i g h t l y from one sampling time to another, but the percentage acid insoluble a c t i v i t y remained r e l a t i v e l y constant through most of the 24 hour holding period. As was the case when the percentages of the t o t a l acid insoluble -^2P a c t i v i t y were compared, the percentage d i s t r i b u t i o n s of acid insoluble phosphorus among the three organs were also very s i m i l a r . Over the 24 hour holding period the percentages of the t o t a l acid insoluble phosphorus on a per gram or per plant basis ranged between 40 and 45$, 19 and 23$, and 34 and 36$ f o r l e a f blades, stems, and roots of control and treated plants, r e s p e c t i v e l y (Table XVII, XIX). Only the difference between values for l e a f blades of control and treated on a per gram basis at the 24 hour sampling time was s i g n i f i c a n t at the 0.05 l e v e l . The percentage of the t o t a l acid insoluble phosphorus on a per gram basis over the 24 hour holding time i s shown graphically i n Figure 10. Table XXXII ,XXXIII , XXXIV, XXXV i n the Appendix gives the actual values from which the percentage d i s t r i b u t i o n s were determined. Data shown i n Table XVI reveal that with exception of the 4 and 8 hour sampling times, the rate of 3 2p incorporation into acid insoluble compounds was stimulated i n roots of naphthenate-treated plants. While the percentage acid insoluble 3 2p i n control and treated plants showed an o v e r a l l decrease i n stem Phosphorus-32 a c t i v i t y b Sampling times i n ^ j s a^ blades Stems Roots hours C c T C T C T 4 I 8 . 3 6 d 20.65 8 . 2 2 8 . 5 7 71.28 6 9 . 5 9 8 19.42 21.93 8 . 6 7 8.77 7 1 . 9 2 6 2 . 2 9 12 19.29 19 .69 8.32 8 . 4 8 7 2 . 3 8 71.82 24 2 0 . 0 5 1 8 . 0 7 7 .69 6.82 72.26 7 5 . 1 1 * Mean 19.28 1 9 . 9 8 n s 8 . 2 3 8 . l 6 n s 7 1 . 9 6 7 1 . 4 5 n S Percent of control I O 3 . 6 99.1 99.2 See Table IV. b See Table I I I . c See Table III. d See Table III. Total inorganic phosphate b Sampling times i n hours Leaf blades Stems Roots G c T C T C T 4 8 12 24 43.89 d 45.35 43.17 40.59 43.29 43.50 43.75 43.36 20.08 19.16 20.26 22.95 20.95 20.78 19.87 21.31 36 .03 35.49 36.56 36.46 35.76 35.73 36.38 35.33 Mean 43.25 4 3.47 n s 20.61 20.73 n S 36.14 35.80 n s Percent of control • 100.5 100.6 99.0 a See Table IV. b See Table VI. c See Table I I I . d See Table I I I . tissues over the holding period, the decrement was not s t a t i s -t i c a l l y s i g n i f i c a n t . The percentage of the t o t a l acid insoluble P i n the three plant organs from control and treated plants remained r e l a t i v e l y constant through the f i r s t 12 hours of the 24 hour sampling period. However, at the 24 hour sampling time the percentage acid insoluble P on a per gram basis i n l e a f blades of treated plants was s i g n i f i c a n t l y greater (0 .05 l e v e l ) than the respective control value. This increase was not r e f l e c t e d i n the ^ 2P data. E.) D i s t r i b u t i o n of acid soluble and acid insoluble 32p  a c t i v i t y or phosphorus, expressed as a percentage "of  the t o t a l , among l e a f blades, stems, and roots. Values for acid soluble and acid insoluble 32p a c t i v i t y or phosphorus (as P^)» expressed as a percentage of the t o t a l and di s t r i b u t e d among l e a f blades, stems, and roots of control and naphthenate-treated plants, are given i n Table XX, XXI, XXII. Since there was very l i t t l e difference between the percentage d i s t r i b u t i o n s of -^2p and t o t a l phosphorus when expressed on a per gram or on a per plant basis, subsequent discussion w i l l be confined to the per gram data. As f a r as the per gram data are concerned, the percentage d i s t r i b u t i o n over the 24 hour sampling period was much more informative than the r e s u l t i n g mean. Over the sampling period the percentage a c i d soluble -^2P a c t i v i t y i n the l e a f blade f r a c t i o n of control and treated plants increased (Table XX). With an exception at the 4 hour -sampling time, the percentages of the t o t a l 3 2 P a c t i v i t y i n the acid soluble and acid insoluble fractions of l e a f blades from treated plants were greater than the respective control values. 32 Of the P a c t i v i t y reaching the l e a f blades over the sampling period a greater proportion remained i n the aci d soluble f r a c t i o n . However, i t also appears that acid soluble -^2P a c t i v i t y was incorporated into the acid insoluble f r a c t i o n . Compared with the 3 2P data, the d i s t r i b u t i o n patterns for the percentages of the t o t a l inorganic phosphate i n acid soluble and acid insoluble fractions of l e a f blades from control and treated plants were d i f f e r e n t . Generally, the percentages of the t o t a l P i n the two P fractio n s remained r e l a t i v e l y constant. TABLE XX. Ef f e c t of KNap on the percentage d i s t r i b u t i o n of acid soluble, acid insoluble, and t o t a l phosphorus-32 or t o t a l phosphorus, on a per gram and a per plant basis, i n leaf blades of bush bean plants at various sampling times a . Phosphorus-32 a c t i v i t y b Sampling times in hours P E R G R A M P E R P L A N T Acid soluble Acid insoluble Total Acid soluble Acid insoluble Total C c T C T C T C T C T C T 4 8 12 24 2 7 . 7 3 d 28.71 30.34 30.19 25.87 29.29 31.02 32.61 3-49 4.36 4.90 5.85 4.11 4.87 5.00 5.98 31.22 33.07 35.24 36.04 29.98 34.16 36.02 38.59 28. 33 29.16 30.72 30.97 25.80 29.75 32.71 32.52 ; 3 . 9 0 4.42 4.97 6.00 4.25 4 .95 5.28 5.96 32.23 33.58 35.69 36.97 30.05 34.70 37.99 38.49 Mean % of control 29.24 2 9 . 6 9 n s 4.65 4 . 9 9 r i B 3 3 . 8 9 3 4 . 6 8 n s 29.79 3 0 . 2 1 n s 4.82 5 . 1 1 i i 0 3 4 . 6 l 3 5 - 3 l J 101.5 107.0 102.3 101.4 106 .0 102.0 Total inorganic phosphate e P E R G R A M P E R P L A N T C c T C T C T c T C T C T 4 8 12 24 41 .13 39.49 42.39 40.82 42 .85 39.40 39.64 41 .74 3.18 3.63 3.51 3.13 3.3^ 3.46 3.75 3.13 44.31 43.12 45.90 43.95 46.19 42 .86 43.39 44.87 42.26 39.89 42 .97 40 .89 42 .68 40.26 40.31 41 .63 3.27 3.68 3.56 3.14 3.34 3.49 3.81 3.11 45.53 43.57 46 .53 44. 03 46 .02 43.75 44.12 44.74 Mean fo O f control 40 .98 40.91 n s 3.36 3.42 n s 4 4 . 3 4 4 4 . 3 3 n s 41 .57 41.2 2 n s 3.41 3 . 2 j 4 n s ^ > 9 8 44.66 : 99.8 .101.7 99.9 99.1 100.8 99.3 a „ See Table IV. C See Table III. e See Table IV. ns TABLE XXI. E f f e c t of KNap on the percentage d i s t r i b u t i o n of acid soluble, acid insoluble, and t o t a l phosphorus-32 or t o t a l phosphorus, on a per gram and a per plant basis, i n stems of bush bean plants at various samplings times a . Phosphorus-32 a c t i v i t y Sampling times i n P E R G R A M P E R P L A N T hours Acid soluble Acid insoluble Total Acid soluble Acid insoluble Total C c T C T C T C T C T C T 4 8 12 24 13.46 d 10.52 12.50 10.97 12.85 11.59 11.36 9.15 1.89 2.11 2.26 2.38 2.03 2.14 2. 36 2.24 15.35 12.63 14,76 13.35 14.88 13.73 13.72 11.39 13.04 9-89 11.87 10.64 13 .07 10.75 10.90 9.42 1.74 1.97 2.14 2,32 1.80 1.99 2.27 2.25 14.78 11.86 14,01 12.96 14.87 12.74 13.17 11.67 Mean 11.86 11.24 n s 2.16 2.19 n sl4 . 0 2 13.43 n s 11.36 1 1 . 0 3 n s 1 2.04 2 . 0 8 n s 1 3.40 I 3 . l l n s % of control 94.7 101.3 95.7 97 .0 101.9 97 .8 Total inorganic phosphate e P E R G R A M P E R P L A N T C c T C T C T C T C T C T 4 8 12 24 11.91 10.42 12.11 13.33 11.62 ? 12.47* 14.89 12.11 >2v9l 3.18 3.15 2.91 3 .00 3.13 3.39 2.83 14.82 13.60 15.26 16.24 14 .62^ 15.60* 18.28* 14 .94 10.98 11.16 12.80 13.76 13.34„ 12.70* 15.16* 13.64 1.48 1.55 1.67 1.79 1.62 1.67 1.73 1.53 14.46 12.71 14.47 15.55 14 .96* 14 .37* 16 .89* 15.17 Mean 11.94 I 2.77 n s 3.04 3 . 0 9 n s l 4 . 9 8 15.86* 12.18 13.71* 1.62 1.64 n s 1 3 . 8 0 15.35* % of control 106.9 101.6 105.9 112.6 101.2 111.2 TABLE XXII. E f f e c t of KNap on the percentage d i s t r i b u t i o n of acid soluble, acid insoluble, and t o t a l phosphorus-32 or t o t a l phosphorus, on a per gram and a per plant basis, i n roots of bush bean plants at various sampling times a . Sampling times i n hours Phosphorus-32 a c t i v i t y D P E R G R A Acid soluble T Acid insoluble Total P E R P L A N T Acid soluble Acid insoluble _ Total T 4 4 o . i 5 d 41 .15 13.27 8 37.95 36.58 16.35 12 32.41 30.41 19.09 24 28.49 25.04 22.11 Mean % of control 13-99 15.52 19.85 f t 24 .98* 53.42 54.30 51.50 50.60 55.14 52.10 50.26 50.02 37.39 38.14 31.65 28.17 40 .15 36.90 30.19 24 .50 15.59 16.43 18.65 21.91 14 .92 15.66 19 .65* 24.34* 52.98 54.57 50.30 50.08 55.07 52.56 49.84 48.84 34.47 33 .07ns17 .6 l l 8 . 8 l n s 5 2 . 0 8 5 1 . 8 8 n s 33.84 3 2 . 9 4 n s l 8 . l 4 l 8 . 6 4 n s 5 l . 9 8 5 1 . 5 8 n s 95.9 106.8 99.6 97 .3 102.7 99.2 Total inorganic phosphate P E R G R A M P E R P L A N T T T T 4 8 12 24 Mean foOf control 38.28 36.42 40.42 38.69 35.77 35.21 37.00 37.65 2.59 2.86 2.98 2.81 2.77 2.85 3.12 2.54 40 .87 43.28 38.75 39.81 39.19 41.54 38.33 40.19 39.33 40.84 35.98 37.31 36.27 38.99 35.82 37.55 2.67 2.88 3.03 2.84 2.76 2.88 3.17 2.54 42.00 43 .72 39.00 40.15 97.6 100.3 97.8 96.8 9 9 . 6 39.03 41.87 38.99 40 .09 37.87 36.99 n s 2.81 2.82 n s40.68 3 9 . 8 l n s 38.37 37 . 1 5 n s 2.85 2.84 n s4l.22 39.99 n s 97.0 In stem tissue the percentage d i s t r i b u t i o n s of acid soluble and acid insoluble ^ 2P a c t i v i t y were si m i l a r to the d i s t r i b u t i o n s of acid soluble and acid insoluble P. As f a r as the ^ 2P data are concerned, differences between the acid soluble and acid insoluble percentage values of eontrol and treated plants were not s t a t i s -t i c a l l y s i g n i f i c a n t . However, at the 8 and 12 hour sampling times the percentage acid soluble P i n treated plants was s i g n i f i c a n t l y greater (0.05 l e v e l ) than the control values. While increases i n the percentage acid insoluble 3 2p a c t i v i t y occurred i n l e a f blade and stem tissues over the 24 hour holding time, incorporation of 32p a c t i v i t y into the a c i d insoluble f r a c t i o n of roots from control and treated plants was greatly increased when compared with percentages of acid soluble 3 2p a c t i v i t y . At the 24 hour sampling time, the percentage acid insoluble -^2P a c t i v i t y i n roots of treated plants was s i g n i f i c a n t l y greater (0.05 l e v e l ) than the control value. The values shown on the next page represent the r a t i o of acid soluble to acid insoluble 32p a c t i v i t y or inorganic phos-phorus i n control and naphthenate-treated plants. At the 24 hour sampling time percentage values from Table XX, XXI, XXII were used to calculate the r a t i o . Ratio of acid soluble/acid insoluble Phosphorus-32 a c t i v i t y Control Treated Leaf blades 5.1 Stems 4.6 4.0 Roots 1.2 1.0 Inorganic phosphate • Leaf blades 13.0 13.3 Stems 4.5 Roots 13.1 14.8 Depending upon the actual percentage values, an increase i n the r a t i o may indicate either a greater l e v e l of ^ 2P or P i n the acid soluble f r a c t i o n i n treated plants, or less incorporation of ^ 2P or P into the acid insoluble f r a c t i o n . Tables XXXVI through XXXIX i n the Appendix contain the data from which the percentage values i n Table XX, XXI, XXII were determined. Since the bean plants used i n t h i s p a r t i c u l a r 'pulse and chase' experiment were grown i n a complete nutrient, t o t a l inorganic phosphate concentrations (31p + ^ 2P) i n l e a f blades, stems, and roots of control plants represent the 'normal' d i s t r i b u t i o n of acid soluble and acid insoluble P. The 3 2 P data alone r e f l e c t the fate of a small pulse of t h i s p a r t i c u l a r radioisotope at a p a r t i c u l a r point during the vegetative growth of control and treated bean plants. Even though the r a t i o of to 3 2P i n the t o t a l was not determined, one could hypothesize that t h i s r a t i o would have been very small indeed. DISCUSSION A.) Phosphorus uptake. Uptake of 3 2p t>y "bean plants which received a f o l i a r naph-thenate treatment was greater than that which occurred i n control plants (Figure 1). When expressed on a per plant basis, naph-thenate treatment increased the ^ 2p content of plants grown i n the -P nutrient by 9.5$« The increase observed i n plants grown i n complete nutrient was 7.1$. Results of several experiments performed i n our laboratory have indicated that naphthenates stimulated both vegetative and reproductive growth, and various metabolic reactions c h a r a c t e r i s t i c of the a e r i a l portion of the plant. In 1970, Fattah and Wort ( 53 ) reported that the rates of photosynthesis and dark r e s p i r a -t i o n were s i g n i f i c a n t l y increased by naphthenate treatment. An increase i n the rate of photosynthesis i n treated plants would have provided greater amounts of reduced nucleotides, ATP, and sugar phosphates. U t i l i z a t i o n of larger quantities of photo-synthate by res p i r a t o r y processes would have supplied treated plants with a greater amount of ATP for use i n the pathways of active and maintenance metabolism. Because P uptake i s an energy requ i r i n g process, one might have expected a greater difference between the amount of P absorbed by control and treated plants. In a d i s t r i b u t i o n experiment, Padmanabhan ( 109 ) observed that a f t e r 1 week 72$ of the t o t a l ^ C a c t i v i t y remained i n the primary leaves of bean plants which had received a f o l i a r applica-t i o n of KCHC-7-1^C. Other r e s u l t s by the same author revealed that 24 hours a f t e r KCHG-7-l2fC was spotted on the midrib of a primary bean l e a f less that 1$ of the t o t a l ^ C a c t i v i t y was detected i n the roots. In 1969. Fattah ( 52 ) reported the following* fresh and dry weights of l e a f blades of naphthenate-treated bean plants were s i g n i f i c a n t l y increased, when fresh and dry weights of root tissues were not increased. S i m i l a r l y , the increase i n the a c t i v i t y of n i t r a t e reductase i n the l e a f blades of treated plants was s i g n i f -icant at the 0.05 l e v e l , while the increase i n the a c t i v i t y of the enzyme i n root tissue lacked s t a t i s t i c a l s i g n i f i c a n c e . Results obtained by these two investigators and the suggest-ion that the metabolites of CHGA ( 130 ) or KNap ( 14? ), rather than the free a c i d ( s ) , stimulate metabolic reactions may be correlated. Since the majority of the naphthenate metabolites remain i n the f o l i a g e following treatment ( 109 ) compared with the roots, r e s p i r a t o r y processes i n root tissue may not have been affected appreciably. Therefore, the amount of ATP a v a i l -able for the process of P uptake i n control and treated plants would have been s i m i l a r . Further, the r e s u l t s obtained are contrary to those reported by Russian plant physiologists (47, 64, 65, 113 ). B.) Phosphorus d i s t r i b u t i o n . When plants were grown i n a -P nutrient, naphthenate t r e a t -32 ment stimulated the acropetal translocation of absorbed J P. Plants grown i n complete nutrient had a s i m i l a r d i s t r i b u t i o n pattern, but compared with minus P-grown plants the percentages of the t o t a l 3 2P i n l e a f blades, stems, and roots were quite d i f f e r e n t . When grown i n the - P nutrient, at the 2k hour sampling time f o u r - f i f t h s of the t o t a l ^ 2 P l a b e l remained i n the roots of treated plants. However, roots of treated plants grown i n complete nutrient contained s l i g h t l y l e s s than one-third of the t o t a l 32p l a b e l . Root c e l l s of plants grown i n the complete nutrient would have had a "normal* complement of P , and there would not have been a great demand for the 3 2 P which entered the roots during the feeding period. Consequently, the majority of the 3 2 P absorbed would have entered the tran s p i r a t i o n stream, probably as acid soluble h3 2 P04~ 2. The r e l a t i v e l y constant l e v e l of ^ 2 P and t o t a l P i n stem tissue over the 2k hour sampling period indicates that the majority of the absorbed 3 2 P was translocated to the leaves and meristematic areas (Figure 5). This d i s t i b u t i o n pattern was not evident when plants were grown i n the - P nutrient. The only P a v a i l a b l e to these plants p r i o r to the 3 2 P feeding was seed P . Even though the plants did not exhibit symptoms of P deficiency, some degree of P d e f i c i t probably existed. When exposed to ^ 2 P , root c e l l s , e s p e c i a l l y the vacuoles, would have absorbed and subsequently retained a great deal of the - ^ 2 P to s a t i s f y t h e i r need for P . However, naphthenate treatment had a s i g n i f i c a n t e f f e c t on the amount of 3 2p which was translocated from root tissues to the shoot (Figure k). The more rapid rate of P translocation probably occurred as a r e s u l t of an accentuated need for P i n the a e r i a l portion of treated plants, i . e . a sink e f f e c t . Results of an e a r l i e r experiment revealed that the rate of transp i r a t i o n i n treated plants was 88$ of that i n control plants. Other investigators have reported that naphthenate treatment increased the water content of corn ( 149, 90 )» and sugar "beets ( 149 ), suggesting a decrease i n the rate of tr a n s p i r a t i o n . On the assumption that t r a n s p i r a t i o n was s i m i l a r l y reduced i n the present experiment, the r e s u l t s suggest that P translocation occurred i n the symplast. This mode of P translocation would require the augmented energy production which i s believed to occur i n the a e r i a l portion of the plant following naphthenate treatment. An increase i n metabolic rates would have also occurred i n treated plants grown i n the complete nutrient medium. However, with a normal complement of P, the sink e f f e c t i n treated plants would have been greatly reduced when compared with plants i n which P was l i m i t i n g . C.) Incorporation of phosporus-32 and phosphorus into a c i d  soluble and acid insoluble f r a c t i o n s . The incorporation of -^2P into the acid soluble and acid insoluble fractions of l e a f blades was enhanced by naphthenate treatment. In root tissues of control and treated plants acid soluble 3 2P a c t i v i t y decreased, however, -^2P a c t i v i t y i n the a c i d insoluble f r a c t i o n of roots from treated plants increased s i g n i f i c a n t l y . With few exceptions, the amount of t o t a l P i n the two P fractions of control and treated plants did not change over the 24 hour holding period. An increase i n the photosynthetic and r e s p i r a t o r y rates of treated plants would r e s u l t i n a greater incorporation of -*2P among components of the acid soluble f r a c t i o n , e.g. free nucleotides and sugar phosphates. Related to t h i s , Wort et a l ( 1^7 ) observed that the s p e c i f i c a c t i v i t y of cytochrome oxidase i n the leaves of naphthenate-treated plants was 1 7 2 $ of the control value. This suggests that the u t i l i z a t i o n of reduced nucleotides and the production of ATP by the electron transport system were increased. The fact that the a c t i v i t y of starch phosphorylase was 1 3 7 $ of the control value indicates that sugar phosphate production was also augmented i n treated bean plants ( 52 ). Further, an increase i n the production of acid soluble 3 2 P compounds would play an important r o l e i n the synthesis of components found i n the acid insoluble f r a c t i o n . For example, ATP i s required f o r the synthesis of i n o s i n i c acid, a nucleotide from which other purine nucleotides are derived, and for o r o t i c acid, the parent pyrimidine. An increase of acid insoluble 3 2P a c t i v i t y i n root tissues suggests that the synthesis of n u c l e i c acids may have been among the components of t h i s f r a c t i o n which were affected by naphthenate treatment. In 1 9 6 5 t Pakhomova ( 110 ) reported that naphthenate treatment increased the rate of nucleic acid synthesis.in the leaves of tomato. Wort et a l ( 1^7 ) suggested that naphthenate stimulation of plant growth was the r e s u l t of the action of naphthenate, or i t s derivatives, at the genetic l e v e l , i . e . at the t r a n s c r i p t i o n l e v e l . An enhanced incorporation of -^2P into the acid insoluble f r a c t i o n of root tissue from treated plants may have also been due to an increase i n the rate of phosphoprotein formation. The hydroxyl group of serine or threonine may serve as l o c i i n the primary structure of a protein where phosphorylation could occur. Phosphoproteins exi s t i n plant c e l l s and i t has been reported that naphthenate treatment increased both the amount of protein and the l e v e l of incorporation of 1^C-L-leucine into protein of l e a f blades ( 147 ), perhaps the phosphoprotein content of the acid insoluble f r a c t i o n was correspondingly increased by treatment. In conclusion, the rate at which the l e v e l of acid soluble 3 2P a c t i v i t y was reduced i n roots of control and treated plants suggests that acid soluble ^ 2P a c t i v i t y was translocated acropetally to the a e r i a l portion of the plant. An increase i n the acid insoluble 32p f r a c t i o n of roots also indicates that one or more J P-containing compounds were incorporated into t h i s f r a c t i o n , and the process of incorporation was enhanced by naphthenate treatment. In leaves, naphthenate treatment increased the 32p l e v e l i n the acid soluble f r a c t i o n . This increase was probably c l o s e l y related to the enhanced acropetal movement of J P, and to a stimulation of metabolic reactions i n the a e r i a l portion of the plant. Chapter 2 THE METABOLISM OF CYCLOHEXANECARBOXYLIC ACID. INTRODUCTION The metabolism of plant growth regulators and rela t e d compounds has received attention from several laboratories. Interest i n the metabolism of a l i c y c l i c carboxylic acids arose from current work being conducted i n our laboratory on the ef f e c t of KNap, HNap, and of model naphthenic acids, e.g. KCHC, KCPC, KCHAc, KCPA, and KCHB, on the growth, y i e l d , and metabolic processes of various crop plants. The i n d i v i d u a l naphthenic acid, CHCA, was used i n t h i s phase of the present investigation for the following reasons, l ) i t i s a component of the complex naphthenic acid mixture ( 46 ), 2) A i t stimulates both vegetative and reproductive growth of bean plants ( 108, 143, 145 ), and 3) i t i s the only naph-thenic acid which can be purchased i n the radioactive form. The purpose of these studies was to investigate the meta-bolism of the b i o l o g i c a l l y foreign petrochemical, CHCA, i n l e a f blades and roots of bush bean plants. After a careful review of pertinent l i t e r a t u r e , i t appears that t h i s i s the f i r s t metabolic study conducted with cyclohexanecarboxylic acid i n a higher plant. LITERATURE REVIEW Even though the compilation i s by no means exhaustive, Table XXIII l i s t s the i n i t i a l products of the metabolism of several naturally-occurring and synthetic organic acids i n plant tissues. While in v e s t i g a t i n g the metabolism of IAA i n tomato roots, Thurman and Street ( 1 3 9 ) suggested that i n addition to the endogenous metabolite, indoleacetylaspartate, indoleacetylglutamate was also formed. However, the presence of the glutamate conjugate was not confirmed. In several publications Wort and Patel ( 143, 144) have shown that KCHC s i g n i f i c a n t l y stimulated the growth of bean plants. In one of t h e i r experiments a f o l i a r a p p l i c a t i o n of an aqueous 2 5 0 0 ppm (2 x 10"" 2 M) KCHC soluti o n invoked a 3 5 $ increase i n the weight of green pods per plant. Working i n the Soviet Union, Agakishiev et a l ( 5 ) observed that the a p p l i -cation of several d i f f e r e n t substituted cyclohexylbutanones and cyclohexylbutanols increased the rate of cotton seed germination, and the y i e l d and q u a l i t y of cotton during a three year study. Wort and Patel and also Agakishiev et ;al have both suggested that the presence of the six-carbon saturated r i n g i n these molecules was required for the most e f f e c t i v e stimulation of plant growth. I t has also been suggested ( 1 2 9 , 1 3 0 , 14?) that the presence of the conjugated or bound forms of naphthenate may be responsible for the stimulation of metabolic reactions which lead to increases i n vegetative and reproductive growth i n vivo. TABLE XXIII. Glucose ester and aspartic acid amide formation following the administration of various organic acids to plant tissues. PLANT COMPOUND GLUCOSE ASPARTIC REFERENCE ESTER ACID AMIDE Pea seedlings IAA X ( 1 0 ) Pea epicotyls IAA X X ( 1 5 2 ) Pea epicotyls NAA X X ( 1 5 3 ) Wheat coleoptiles NAA X X ( 86) Wheat cole o p t i l e s BA X X ( 84) Pea epicotyls BA X X ( 1 5 4 ) Wheat cole o p t i l e s 2,4-D X X ( 8 5 ) Pea epicotyls 2,4-D X ( U ) Ch l o r e l l a 2,4-D X ( 1 3 7 ) Bean l e a f disks KCHC X X ( 1 2 7 ) Bean seedlings KCHC KCPC KNap X X X X X X ( 1 2 2 ) Bean roots and cut-stem exudate KCHC X X ( 1 3 1 ) x denotes the presence of the metabolite. Other metabolic studies i n our laboratory have revealed that when the complex naphthenate mixture, KNap, was applied to the primary leaves of young bush bean plants these acids were r e a d i l y converted to a mixture of conjugates with glucose and aspartic a c i d . Twenty-four hours a f t e r a pplication, free acids were not detected chromatographically i n extracts of l e a f blades. When KNap, KGHG, and KCPC were fed to the roots of bean plants, both conjugates were detected i n the l e a f blades a f t e r 2k hours ( 122 ) . Results of investigations performed i n our laboratory dealing with the metabolism of naphthenic acids i n bean plants were communicated by the author and Dr. G. E. Seaforth at the Annual Meeting of the Northwest S c i e n t i f i c Association, 19^9. This information was also published i n Phytochemistry ( 127 ) . Additional information on CHCA metabolism i n bean plants was communicated at the Western Section meeting of the Canadian Society of Plant Physiologists, 1971 ( 1 0 9 , 1 3 1 ) . MATERIALS AND METHODS A.) Leaf disk feeding experiment. Leaf disks were cut from the primary leaves of a 14-day-old bush bean plant using a cork borer (l/2 inch i n diameter) and were floated on 6 ml of d i s t i l l e d water i n a p e t r i dish. To t h i s was added 2.5 JiCi of cyclohexanecarboxylie acid-7-14 c (Inter-national Chemical and Nuclear) as the K s a l t . This represented a concentration of 20 jaM, The dish was wrapped with Saran wrap to minimize water loss by evaporation, and then placed i n the l i g h t (5400 lux) for 24 hours at 24° C. Control dishes contained the l e a f disks, water, but no acid. At the end of the metabolism period the l e a f disks were washed several times with water to remove adhering l a b e l l e d compound, then extracted with several changes of 80$ ethanol. Extraction was considered complete when the l e a f disks were white. The combined ethanol extracts were evaporated to dryness, extracted with b o i l i n g water, and f i l t e r e d through C e l i t e . An 8 hour ethyl acetate extraction of the aqueous f i l t r a t e yielded only a p a r t i a l separation of the major compounds, the glucose ester (compound number 1) being more soluble i n ethyl acetate, while the aspartic acid amide (compound number 4) remained p r i -marily i n the aqueous phase. A l l chromatography was done using thin-layer plates of c e l l u l o s e MN 3 0 0 G (Macherey and Nagel, Co.). Radioactive compounds were detected with Kodak Medical X-ray f i l m . B.) Synthesis of l-Cyclohexaneearbonvl-yg-D-glucose. Cyclohexanecarboxylic acid was converted into the a c i d chloride by r e f l u x i n g the acid with a s l i g h t excess of thio n y l chloride i n dry benzene for 4 hours. After removal of benzene and excess thio n y l chloride under reduced pressure, the r e s i d u a l acid chloride was dissolved i n a small volume of dry pyridine and treated with an equimolar amount of /l-D-glucose-2,3,4 ,6-tetraacetate,, ( 9 7 ). The stoppered reaction f l a s k was shaken to f a c i l i t a t e s o l u t i o n and allowed to stand f o r 3 days at room temperature. The reaction mixture was poured into a mixture of 12 N HC1 and crushed i c e . The p r e c i p i t a t e was c o l l e c t e d , washed with water, then with 10$ sodium bicarbonate several times, f i n a l l y with water, and then dried at room temperature. Several r e c r y s t a l l i z a t i o n s from anhydrous methanol yielded colourless c r y s t a l s of l-cyclohexanecarbonyl-2,3,4 ,6-tetraacetyl -/3-D-glucose, m.p. 114.5 - 1 1 5 . 5 ° C. The y i e l d was 6 8 $ . Deacetylation was accomplished by passing a stream of anhydrous NH^ through a methanol solution of the product from above. Excess NH^ and solvent were removed under reduced pressure, the residue was dissolved i n water, and t h i s s o l u t i o n was extracted continuously with ethyl acetate for 12 hours. The ethyl acetate soluble material was p u r i f i e d by chromatography i n BAW to y i e l d a white product with m.p. l 4 l - 143° 0. A c r y s t a l l i n e product was not obtained. This product was chromato-graphically homogeneous and yielded only cyclohexanecarboxylic acid and glucose on hydrolysis. Further p u r i f i c a t i o n was not attempted. C. ) Synthesis of N-Cyclohexanecarbonyl-L-aspartic Acid. L-Aspartic a c i d ( O . 6 7 grams, 0 . 0 0 5 M) and sodium bicarbonate ( I . 4 3 grams, 0 . 0 1 ? M) were s t i r r e d i n 6 ml of water at room temperature. Cyclohexanecarboxylic a c i d chloride (0.64 grams, 0 . 0 0 5 M) was added i n 5 portions over a period of 3 0 minutes. The mixture was a c i d i f i e d to pH 2 and continuously extracted with ether for several hours. Chromatography of the ether extract showed i t to be a mixture of cyclohexanecarboxylic acid and the desired amide. Separation was done chromatographically. E l u t i o n of the desired band with methanol was followed by c r y s t a l l i z a t i o n of the material from ethanol off ethyl acetate. This procedure yielded a white, waxy s o l i d whose m.p.was s l i g h t l y above room temperature. D. ) Hydrolysis Procedures. Bands of unknown conjugates were scraped from the TLC^plates and eluted with methanol:water ( l : l ) . The c e l l u l o s e was removed by f i l t r a t i o n and the f i l t r a t e s evaporated to dryness. Band 1 (banded material corresponding to spot l ) material was hydrolyzed into i t s component parts by 2 N NaOH for 3 0 minutes at 7 5 ° C. A c i d i f i c a t i o n and extraction with ether separated the acid and sugar portions of t h i s compound. Band 4 material was e f f e c t i v e l y hydrolyzed with 6 N H C 1 f o r 3 0 minutes at 7 0 ° C . The mixture was neutralized with sodium bicarbonate, evaporated to dryness, and the residue extracted with small portions of anhydrous methanol. This methanolic solu t i o n was used for chromatography. Band 2 material was also hydrolyzed t h i s way. Band numbers correspond to compound numbers. E.) Root feeding experiment. Uniform seeds of the hush bean plant, Phaseolus vulgaris L. c u l t i v a r Top Crop were sown i n vermiculite, and were placed i n a growth room as described i n the preceding section. After s i x days, uniform seedlings were transplanted into beakers containing a one-quarter strength Hoagland-Arnon*s complete nutrient s o l u t i o n ( 74 ) . The roots of two sets of uniform bean seedlings, with four 2-week-old plants i n each set, were immersed i n 2 5 ml of d i s t i l l e d water containing only 5 " C i (1 x 1 0 - 5 m) of KCHC^-^C, or 5 juGi ( 3 x 1 0 * 5 M) of D-glucose-UL- l Z*C (Amersham/Searle, Toronto) with 1Q~5 M unlabelled KCHC at pH 8 . 3 . After s i x hours of incubation, the a e r i a l portions of the plants i n each set were excised, and cut-stem exudate was coll e c t e d for ca 1 hour. After c o l l e c t i o n , the exudate was applied d i r e c t l y to separate TLC plates. The a e r i a l portions of the plants i n each set were divided into stems plus p e t i o l e s and l e a f blades. Upon completion of exudate c o l l e c t i o n , the roots of the plants from each set were well rinsed, and the three organs from the plants i n each set were extracted separately with b o i l i n g 80$ EtOH f o r 1 hour. Each extract was evaporated to a volume of ca 1 ml under reduced pressure at 40° C. The concentrated extracts were used d i r e c t l y f o r chromatographic purposes. A l l chromatography was done using TLC plates of c e l l u l o s e MN 3 0 0 G (wet thickness O .50 mm), and the plates were developed i n an isopropanol1 7 $ NH^0H;water ( 8 1 I 1 I ) solvent system. In certai n instances, extracts spotted on TLC plates were developed i n n-butanolj g l a c i a l a c e tic aeidj water ( 4 : 1 : 5 , top phase). Radioactive areas on the chromatograms were detected by exposing the chromatograms to Kodak Blue Brand Medical X-ray film,. RESULTS AND DISCUSSION After 24 hours of metabolism i n the presence o f KCHC-7-^C, l e a f disk tissue was extracted and analyzed by TLC using a basic solvent system, isopropanol» 7 $ NHjjOH.water (IAW), and an a c i d i c one, n-butanol:glacial a c e t i c acid.water (BAW). Chromatography i n the IAW system yielded four major l a b e l l e d compounds, while chromatography i n BAW gave only two. An analysis of the l a b e l l e d compounds of extracts ehromatographed i n the IAW system follows. Compound No. 1 This was the l a r g e s t spot observed on the radiochromatogram. The R f was 0 . 8 5 which agreed c l o s e l y with that for the synthetic ester, l-cyclohexanecarbonyl-/3-D-glucose (Table XXIV). This area on the chromatogram gave no color when sprayed with a bromophenol blue reagent, but the area quickly darkened when sprayed with ammoniacal s i l v e r n i t r a t e . Removal of t h i s region of the chromatogram, followed by hydrolysis of the eluted material yielded only two compounds, glucose and CHCA. Hydrolysis of the synthetic glucose ester under i d e n t i c a l conditions yielded the same r e s u l t s . Synthetic ester and the i s o l a t e d metabolite also had i d e n t i c a l R^a i n three a d d i t i o n a l solvent systems: BAW, isopropanol*benzene:water ( 5 5 * 3 0 * 1 1 ) , and chloroform:ethyl acetate:formic acid ( 3 5 * 5 5 * 1 0 ) . Compound number 1 was therefore the glucose ester of cyclohexanecarboxylic acid (CHCGluc). The color reaction with ammoniacal s i l v e r n i t r a t e evidently occurred as a r e s u l t of some hydrolysis due to the a l k a l i n e nature of the spray reagent. Compound No. 2 This compound was observed only i n the basic solvent system where i t ran just behind the glucose ester. The com-pound gave no color reaction with either bromophenol blue or ammoniacal s i l v e r n i t r a t e , but did give a p o s i t i v e reaction with iodine vapor. Acid hydrolysis of eluted material yielded CHCA as the sole organic product. Appearance of t h i s compound only i n the ammoniacal solvent system, and the r e s u l t s of the acid hydrolysis suggest that i t might be cyclohexanecarboxylic acid amide (CHCAm). Comparison of eluted material with authentic amide,by means of chromatography and hydrolytie behavior supported t h i s view. I t was then found that chromatography of the synthetic glucose ester i n the IAW system led to the formation of t h i s compound i n a proportion roughly equivalent to that which was observed by chromatography of l e a f extracts. Since t h i s com-pound was not detected when l e a f extracts were chromatographed i n the a c i d i c BAW solvent system, the amide was i n a l l proba-b i l i t y an a r t i f a c t . Zenk reported s i m i l a r a r t i f a c t formation with IAA ( 1 5 2 , 1 5 5 ) . Compound No. 3 This was the smallest spot observed on the radiochromato-gram, and again was seen only when extracts were chromatographed i n the IAW system. I t gave an a c i d i c reaction with bromophenol blue, and had a R f i d e n t i c a l to that of free CHCA. Formation of t h i s a r t i f a c t by a l k a l i n e hydrolysis of the glucose ester was demonstrated using synthetic material. Since synthetic N-eyclohexaneearbonyl-L-glycine ( 60 ) hasr; almost the same as CHCA i n IAW and BAW, eluted material was hydrolyzed with acid and the hydrolysate examined for amino acids but none was found. Compound No. 4 This spot on the chromatogram represented a compound which was present i n a r e l a t i v e l y high concentration with low mobility i n the basic solvent system (H^ 0.l6). The compound gave a p o s i t i v e test with bromophenol blue, and upon hydrolysis, yielded CHCA and aspartic acid. Comparison with synthetic N-cyclohexane-carbonyl-L-aspartic acid (CHCAsp) with regard to chromatography, color reaction with the acid-base indicator, and products of hydrolysis showed t h i s compound to be present. The two radioactive compounds observed on radiochromato-grams developed i n BAW had R fs of O.78 and 0.90. The slower moving compound was i d e n t i c a l i n a l l respects to authentic glucose ester, and the faster moving to the aspartic acid amide. The r e s u l t s of radiochromatography analyses of ethanol extracts of roots, stems plus p e t i o l e s , l e a f blades, and cut-stem exudate of bean seedlings root-fed KCHC-7-"^C revealed that CHCGluc and CHCAsp were present. When developed i n the IAW solvent system, t h e i r R fs were 0.88 and 0.17, respectively. The i d e n t i f i c a t i o n of CHCGluc and CHCAsp was based on the c r i t e r i a outlined previously. The concentration of the two conjugated metabolites was highest i n the extract of root tissue, and lowest i n the l e a f blade and i n cut-stem exudate fr a c t i o n s . When root extracts were chromatographed i n IAW, a p a r t i a l ammonolysis of CHCGluc occurred producing the chromato-graphic a r t i f a c t s , i . e . CHCA and CHCAm. As before, when TABLE XXIV. Chromatographic data a . -Compound R f IAW BAW Cyclohexanecarboxylic acid 0.73 0.92 Cyclohexanecarboxylic acid (K s a l t ) 0.75 0.92 Benzoic acid 0.66 0.90 Glucose 0.40 0.27 Aspartic acid 0.08 0.25 Cyclohexanecarboxylic acid amide 0.80 0.85 1 - Cy clo h exane carbonyl-/3- D- glu co s e 0.85 0.78 N-Cyclohexaneearbonyl-L-aspartic acid 0.16 0.90 N-Cyclohexanecarbonyl-L-glycine 0.71 0.91 A l l analyses were done using 0.50 mm (wet thickness) Cellulose MN 300 G plates. Solvent abbreviations! IAW, isopropanoli7$ NHirOHtwater (8il*l)« BAW, n-butanolt g l a c i a l a c e t i c acidiwaxer (4:ls5, top phase). extracts were chromatographed i n BAW, free acid and the amide were not detected. A radiochromatographic analysis of extracts the three plant organs, and of cut-stem exudate from plants root-fed uniformly l a b e l l e d glucose and unlabelled KGHC revealed the 14 presence of CHCGluc and CHCAsp i n roots and stems. Free C glucose was detected i n roots, stems, and cut-stem exudate fr a c t i o n s . The l e a f blade f r a c t i o n was void of l a b e l l e d metabolites. The appearance of GHCAsp i n the extracts indicates that the aspartate moiety of the conjugate was l a b e l l e d . This i s not an unusual r e s u l t as the metabolism of ^ C glucose by bean roots would have produced a v a r i e t y of l a b e l l e d compounds, including l a b e l l e d aspartate, v i a pathways of dark r e s p i r a t i o n . Since several of the l a b e l l e d amino acids would have been present i n the same R_. region as CHCAsp i n the IAW system, the extracts of stems and roots were also chromatographed i n BAW. In t h i s solvent system most amino acids are r e l a t i v e l y immobile, while the glucose ester and the aspartic acid amide were present at high R f positions (Table XXIV). The absence of l a b e l l e d metabolites i n the l e a f blades, and generally, the lower concentrations of l a b e l l e d compounds i n stems, roots, and i n cut-stem exudate fracti o n s of plants root-fed l a b e l l e d glucose and unlabelled KCHC was probably related to the reduced a v a i l a b i l i t y of l a b e l l e d glucose and aspartate for purposes of conjugation i n root tissues. Once inside root tissues the ^ G glucose concentration would have been d i l u t e d as t h i s compound entered several d i f f e r e n t metabolic pathways, e.g. the incorporation of glucose carbon into c e l l walls v i a the metabolism of myo-inositol (the Loewus pathway), and the g l y c o l y t i c , pentose phosphate, and shikimic pathways. The presence of CHCGluc and CHGAsp i n cut-stem exudate also indicates that these two conjugates were translocated acropetally through the plant. Cyclohexanecarboxylic acid a c t i v i t y was uniformly d i s t r i b u t e d i n a l l organs of the bean plant s i x hours a f t e r the root feeding had begun. This observation plus the fact that free acid was not detected i n l e a f blades, stems, and cut-stem exudate a f t e r 6 hours tends to support the idea that the metabolites, and not the free acid, were mobile. Also, i n a time course study of metabolite formation i n l e a f blades of bean plants, Padmanabhan ( 108) reported that the glucose ester was the f i r s t metabolite produced, and was present one-eighth hour a f t e r a p p l i c a t i o n of KCHC-7-1^C. The aspartate derivative appeared a f t e r one-hour. Free acid i n extracts of l e a f blades was not detected a f t e r 6 hours. In a l l plant fractions and i n cut-stem exudate from the KCHG-7-14G and glucose-^C/KCHC feeding experiments a metabolite was present at Rf 0.68 on chromatograms developed i n IAW. In the early experiments i t was thought that t h i s compound was not a metabolite, but rather a l a b e l l e d contaminant present i n the 11s KCHC-7- C stock solution. However, since t h i s compound was detected i n extracts of plants which received l a b e l l e d glucose and unlabelled KCHC, i t appears that future investigations should include an analysis of t h i s unknown metabolite. In addition to the metabolites and a r t i f a c t s already-discussed several other unknown metabolites were present i n extracts of both l e a f blade^and root tissues following KCHC treatment. In IAW, these metabolites were detected i n the R f range G .20 to 0.40. In a preliminary inves t i g a t i o n three unknown metabolites were removed i n d i v i d u a l l y from a ehromatogram which had been developed i n IAW. Aqueous solutions containing the unknown 'conjugated' metabolites were run through a Dow.exi 5QW-X8(Hv) cation-exchange column to remove free amino acids, and the eluates were subjected to an acid hydrolysis at 1 0 5 ° C for 8 hours i n vacuo. Individual hydrolysates were then analyzed using a Beckman 120 C automatic amino acid analyzer. Results indicated that from 10 to 13 d i f f e r e n t amino acids were present i n each hydrolysate. Even though the amino acids present i n each hydrolysate were very s i m i l a r , the con-centration of i n d i v i d u a l amino acids varied from one sample to the other. Further, i n a separate procedure the aqueous solutions containing i n d i v i d u a l unhydrolyzed 'conjugated' metabolites were run through the cation-exchange column, and the eluates were subjected to an amino acid analysis. The r e s u l t s of these analyses revealed that free amino acids were absent. I t i s very tempting to speculate that i n addition to the conjugated metabolites already discussed CHCA may have also been a component of a small peptide. I t seems very u n l i k e l y that CHCA would have formed i n d i v i d u a l conjugates with ten or more amino acids. Also, a host of i n d i v i d u a l conjugates would not have been located at one R f p o s i t i o n . According to Towers ( 140 ), tissues would be expected to contain pools of free amino acids, and hence i t would seem reasonable that conjugation of introduced acids would occur with amino acids present i n these pools. However, the employment of several d i f f e r e n t amino acids i n the conjugation process may be l i m i t e d by the lack of the appropriate N-acyl synthetase enzymes. This conjecture may be relevant to what i s written i n the 'Discussion Section' of the next chapter. I t has been established that i n some animal tissue CHCA i s r e a d i l y dehydrogenated ( 18, 56 ), and then conjugated to form hippuric a c i d (N-benzoyl-L-glycine) ( 18 ). Hydrolysis of the metabolites from the present experiments f a i l e d to y i e l d any trace of benzoic acid. I t has been shown that the tissues of certain higher plants can hydroxylate administered benzoic acid ( 76 ). Either o- or p-hydroxybenzoic acid was formed, depending on the plant used, and further hydroxylation was also shown to occur. This route of metabolism of CHCA was discounted i n the bush bean by the observations that benzoyl metabolites were not detected and no radioactive compound gave a p o s i t i v e phenolic reaction with diazotized p - n i t r o a n i l i n e . Cyclohexanecarboxylic acid, when administered to l e a f disks or to bean seedlings v i a a root feeding, i s converted into a mixture of the glucose ester, the amide of aspartic acid, and several unknown 'conjugates.' Because CHCA i s not toxic to bean plants even when applied at concentrations as high as 2500 ppm (2 x 10"" 2 M), i t may be that the conjugates described i n t h i s chapter may not be mere 'detoxication products', but rather metabolites which may play a r o l e i n stimulating growth of CHCA-treated plants. The fac t that the metabolites, and not the free acid, were detected i n treated plantsalends support to t h i s hypothesis. In conclusion, the r e s u l t s of these experiments have shown that bean plants contain enzymes which can chemically conjugate t h i s b i o l o g i c a l l y foreign petrochemical with glucose, aspartic acid, and perhaps with other endogenous compounds. The occur-rence of the glucose and aspartic acid conjugates, and the concurrent absence of free KCHC-?- 1^ i n stems, l e a f blades, and cut-stem exudate suggest that following t h e i r synthesis i n root tissues the conjugates were translocated acropetally to the a e r i a l portion of the plant. The presence of the conjugated metabolites may play a r o l e i n enhancing the growth of CHCA-treated plants. Chapter 3 THE EFFECT OF POTASSIUM NAPHTHENATES AND POTASSIUM CYCLOHEXANECARBOXYLATE ON THE UPTAKE AND METABOLISM OF l^C GLUCOSE BY EXCISED ROOT TIPS. Although the uptake and metabolism of carbohydrate com-pounds by excised root tissue has been extensively reviewed by Butcher and Street ( 36 ), and studied by Goldsworthy ( 59 ) , there i s l i t t l e information available regarding the e f f e c t of auxins and growth promoting compounds on the uptake and sub-sequent metabolism of carbohydrate compounds by roots. There i s a wealth of information which demonstrates that naphthenates stimulate growth and increase the y i e l d s of a wide v a r i e t y of plants (Table I ) . Evidence i s also accumulating which supports the hypotheses that naphthenic acid compounds favorably influence protein synthesis ( 110, 147), nucleic acid synthesis ( 110 ), mitochondrial a c t i v i t y ( 147 ), photosynthesis ( 5 3 )» enzyme a c t i v i t y ; ( l47 ), dark r e s p i r a t i o n ( 5 3 ), and IAA synthesis ( 2 5 ). The data suggest that naphthenate stimu-l a t i o n of plant growth i s the r e s u l t of the action of the chemical, or i t s metabolites, at both the genetic and metabolic l e v e l s . Of p a r t i c u l a r importance to t h i s experiment was the fac t that dark r e s p i r a t i o n of treated plants was increased over the rates i n the corresponding untreated plants. Measured 21 days following treatment with a 0 . 5 $ KNap solution, dark r e s p i r a t i o n i n treated plants growing i n a high l i g h t i n t e n s i t y ( l 6 . 1 klx ) was increased by 4 3 $ when compared with the control value ( 5 3 ) . An experiment by Kim and Bidwell ( 83 ) demonstrated that IAA and 2,4-D had a pronounced i n v i t r o e f f e c t on the metabolism of 1^C glucose i n pea root t i p s . Their work revealed that the amount of ^COp produced, and biosynthetic pathways leading added to a culture solution containing pea root t i p s , the investigators also found that the main e f f e c t was not on the uptake of glucose from the medium, but was on i t s metabolism. They also noted that the production of certain amino acids o r i g i n a t i n g from glucose was thwarted, while the production of A^C asparagine from l a b e l l e d glucose was increased. In view of these findings control, cyclohexanecarboxylic acid-, and naphthenic acid-treated sets of bean root t i p s were incubated for 6 hours i n a 1 2 C glucose culture medium. After t h i s period of incubation, the root t i p s i n each set were incubated for 3 hours i n separate medium containing glucose. Following these incubation procedures, the l e v e l of r a d i o a c t i v i t y i n each set's ethanol-soluble, ethanol-insoluble, and respired 14 CO2 f r a c t i o n s was measured. After determining t o t a l C a c t i v i t y i n these fractions from each set, representative compounds i n the ethanol-soluble and the hydrolyzed ethanol-insoluble fractions were separated by thin-layer chromatographic methods. Following i d e n t i f i c a t i o n of the compounds on radiochromatograms, the compounds were removed, and t h e i r *^C a c t i v i t y determined i n d i v i d u a l l y . Compounds i n the ethanol-soluble f r a c t i o n whose l^C a c t i v i t y was determined were: Aspartic acid, serine, glutamic acid, glutamine, threonine, alanine, If-aminobutryic acid, v a l i n e , leucine/isoleucine, and glucose. In addition, 14 the A C a c t i v i t y i n aspartic acid, glutamic acid, and alanine from the hydrolysate of the ethanol-insoluble f r a c t i o n was determined. The basic objective of t h i s phase of the invest i g a t i o n was to augment our understanding of how naphthenate compounds stimulate the growth of bean plants. More s p e c i f i c a l l y , the purpose was to determine i f the complex naphthenic acid mixture, or an i n d i v i d u a l naphthenic acid, cyclohexanecarboxylic acid, a f f e c t s the uptake and subsequent metabolism of l a b e l l e d glucose i n bean root t i p s . Because of the close connection between enzymes and a l l aspects of metabolism, the e f f e c t s of naphthenic acid compounds on enzyme a c t i v i t y have been included i n the l i t e r a t u r e review. Of the l i t e r a t u r e c i t e d , several a r t i c l e s ( 40, 5 2 , 5 3 , 1^7 ) have considered enzymes involved i n amino acid or nitrogen metabolism, and are, therefore, relevant to the re s u l t s reported i n this chapter. LITERATURE REVIEW In 1 9 6 6 . Bazanova and Akopova ( 2 3 ) reported that a 0 . 0 1 $ naphthenate solution applied as a f o l i a r spray to young cotton plants stimulated t h e i r r e s p i r a t o r y rates. An increase i n r e s p i r a t i o n i n potato leaves following naphthenate treatment was observed by Abolina and Ataullaev ( 2 ) and Ladygina ( 94 ). Chu--i( 40 ) observed that the f o l i a r a p p l i c a t i o n of 0 . 5 $ KNap produced a decrease i n the rate of r e s p i r a t i o n i n the above ground parts of tomato plants two weeks af t e r treatment. This was followed by a 9 . 7 $ increase i n the rate of r e s p i r a t i o n four weeks a f t e r the growth stimulant was applied. After two f o l i a r applications of a 0 . 0 5 $ naphthenate solution to the leaves of eggplants and tomatoes, Guseinov ( 6 8 ) obtained an increase i n the rate of r e s p i r a t i o n . Kolesnik ( 87 ) observed that catalase a c t i v i t y i n grape plants was increased following a f o l i a r a p p l i c a t i o n of a 0 . 0 0 5 $ naphthenate solution. An i n i t i a l f o l i a r a p p l i c a t i o n of a 0 , 0 5 $ naphthenate solution , followed by spraying the plants twice during the vegetative period with a lower concentration of naphthenates ( 0 , 0 0 5 $ ) . i n i t i a l l y decreased catalase a c t i v i t y , but at the end of the vegetative period the a c t i v i t y of t h i s enzyme was higher than i n the controls. Bazanova and Akopova ( 23 ) reported that a f o l i a r 0 . 0 1 $ naphthenate application had only a s l i g h t l y stimulatory e f f e c t on catalase a c t i v i t y i n cotton. Agakishiev and Bazanova ( 4 ) found that cotton plants treated with naphthenates or Sh - 8 , and grown on s u l f a t e s a l i n i z e d s o i l s , showed a higher rate of peroxidase a c t i v i t y i n the leaves. These two treatments also increased the a c t i v i t y of peroxidase i n the roots of cotton ( l 6 ). After the seeds had been soaked i n a 10 ppm naphthenate or a 1 ppm Sh-8 solution, the author also reported that peroxidase a c t i v i t y i n cotton plants was greatly increased, but the increase i n a c t i v i t y was less when the chemicals were applied to the s o i l at the rate of 20 mg/4 ha and 10 mg/kg of dry s o i l , respectively. Alekperov and Chrjanovskaya ( 6 ) reported that young seedlings of Eldar pine, Pinus eldarica var. Eldarska, and Japanese sophore (Sophora japonia L.) which had received a 0,00$% naphthenate treatment i n the form of a 12 hour seed soak had higher res p i r a t o r y rates and an increased peroxidase a c t i v i t y , but t h e i r catalase a c t i v i t y was concurrently decreased. Treated seedlings also had a greater s a l t tolerance than did the controls. Following a seed soak i n 0 . 0 0 5 $ naphthenates, Matzjuk and Grinberg ( 1 0 3 ) observed that catalase a c t i v i t y i n the leaves of m i l l e t was 9% above the control value. The a c t i v i t y of ascorbic acid oxidase i n cotton plants was increased following naphthenate treatment when the plants were grown i n chloride s a l i n i z e d s o i l , but the a c t i v i t y of t h i s enzyme did not increase when the plants were grown i n s u l f a t e s a l i n i z e d s o i l ( 4 ). Both the formation of amylase and growth were promoted i n Aspergillus usamii a f t e r the mold had been treated with NaNap (avg. M.W, 2 0 8 , C ^ H ^ ^ ) at a concentration of 5 x 1 0 " H ( 3 5 ) . After tomato plants had received a f o l i a r treatment of a 0 . 5 $ naphthenate solution, Chu ( 4 0 ) observed that the a c t i v i t y of phosphorylase i n the leaves was increased. I t was also reported that the a c t i v i t i e s of n i t r a t e reductase, phosphoglyceryl kinase, and succinic dehydrogenase were decreased at a l l observation times. Fattah ( 5 2 ) and Fattah and Wort ( 5 3 ) obtained a s i g n i f i c a n t increase ( 0 . 0 5 l e v e l ) i n the a c t i v i t y of phosphorylase measured 7 , 14, and 21 days a f t e r the naphthenate treatment when bean plants were grown at a high l i g h t i n t e n s i t y . The stimulation of n i t r a t e reductase and glutamic-pyruvic transaminase i n treated plants reached a s i g n i f i c a n t l e v e l ( 0 . 0 5 ) between 7 and 14 days a f t e r treatment. S i g n i f i c a n t stimulation of phosphoglycerate kinase developed more slowly under lower l i g h t i n t e n s i t i e s . Wort et a l ( 147 ) reported that the a p p l i c a t i o n of 2 x 1 0 " 2 M KNap to 14-day-old bush bean plants resulted i n the following, measured i n the leaves 7 days a f t e r treatment: l ) Percentage increases i n s p e c i f i c a c t i v i t y per mg enzyme protein, compared with control plant values, were: n i t r a t e reductase, 1 5 2 ; glutamic-oxaloacetic transaminase, 1 0 ; glutamine synthetase, 17; glutamate dehydrogenase, 0 ; and cytochrome oxidase, 7 2 . The increase i n a c t i v i t y of glutamate dehydrogenase on an extract basis was 16%. 2 ) The content of enzyme protein was greater by 10 to 2 0 $ . 3) The incorporation of ^ C - L - l e u c i n e was increased i n treated plants by ca, 1 0 $ . 4) No increase i n enzyme a c t i v i t y followed i n v i t r o addition of 1 x 1 0 " ° M KNap. The authors stated that the increase i n enzyme protein, the stimulated incorporation of l a b e l l e d leucine, and the greater s p e c i f i c a c t i v i t y of the numerous enzymes suggested that naphthenate stimulation of plant growth i s the r e s u l t of the action of the chemical, or i t s derivatives, at both genetic and metabolic l e v e l s . Spraying tomato plants with a 0.05$ aqueous naphthenate solution caused changes i n the protein and nucleic content of leaves and vegetative apices. The content of the °(-nucleo-protein complex increased, while that of the ^-nucleoprotein complex decreased. Protein N i n the vegetative apices increased, but decreased i n older leaves ( 110). When potato tubers were soaked i n a 0.005$ solution of naphthenates p r i o r to sowing, the plants showed an increase i n the content of nitrogenous substances i n the leaves, stems, and roots ( 6 l ). Abolina and Ataullaev ( 2 ) observed that the application of naphthenates at the rate of 500 cm^/ha increased the starch concentration i n potato tubers by 20$. Later experiments by the same author showed that a f t e r naphthenate treatment the protein content of potato tubers was also increased. Even though the weight of tubers/plant increased following a f o l i a r treatment with 0 .5$ naphthenates to early potatoes var. Warba, Wort and Hughes ( l 46 ) reported that there was no difference between the starch content i n tubers from control and treated plants. Following a f o l i a r a p p l i c a t i o n of a 0,005$ naphthenate solution to corn plants, Yur'eva ( 149) observed that corn si l a g e contained more protein, and the corn grain contained more protein and starch when compared with the controls. Subbotina ( 136) reported that the f i e l d a p p l i c a t i o n of a 0.1$ naphthenate solution i n conjunction with a f e r t i l i z e r ( N 5 o E 6 0 K 6 o ^ led to an increased protein l e v e l i n apple tree leaves, deter-mined one month af t e r a p p l i c a t i o n . Guyot ( 69 ) observed that sugarcane which had received a f o l i a r naphthenate treatment 15 to 30 days p r i o r to harvest had a higher sugar concentration than controls. By applying a 0.05$ naphthenate solution to tangerine leaves during the flowering period, Marshaniya et a l ( 102 ) observed an increase i n the sugar content i n the f r u i t s . The f o l i a r a p p l i c a t i o n of 0.005$ naphthenates to grape plants also increased the sugar content i n the f r u i t s ( 8? ), This author also reported that using a higher concentration of naphthenate (0.05$) i n i t i a l l y , and then spraying twice with a 0.05$ solution of the chemical decreased the concentration of disaccharides. At the end of the vegetative period sucrose i n the leaves disappeared, and the sugar content of f r u i t s from treated plants was greater than i n the controls, Chu ( 40 ) reported that a f o l i a r 0.5$ KNap treatment increased the l e v e l of reducing sugar, sucrose, and t o t a l sugars i n mature tomato f r u i t . A reduction i n the concentration of sugar during the course of storage was less i n tomato f r u i t from KNap treated plants. T i t r a t a b l e acid from the mature 1 tomato f r u i t which was under the influence of KNap was higher at the end of 4 days storage, but was lower as the duration of storage was increased, Peterburgsky and Karamete ( 113) observed that a f o l i a r 0.007$ naphthenate treatment increased the synthesis of 10 important amino acids at the beginning of and at the end of the vegetative period i n maize, and increased the synthesis of protein and non-protein N substances i n the leaves and roots. Concentrations of naphthenates ranging from 0.005 "to 0.01$ considerably increased the content of t o t a l and protein N i n the leaves of tomato, carrot and beet following treatment The data of Voinova-Raikova ( 141 ) showed that the addition of a naphthenate solution to the s o i l stimulated the development of ammonifying ba c t e r i a which i n turn promoted an increase i n the amount of n i t r a t e entering the s o i l from organic nitrogenous compounds. In t h i s investigation i t was also demonstrated that the naphthenate treatment caused a considerable decrease i n the number of d e n i t r i f y i n g bacteria. As well, i t was shown that naphthenate treatment increased the quantity of N u t i l i z e d by 80$ of the Azotobacter s t r a i n s which were tested. The author concluded that naphthenate treatment was responsible for increasing the l e v e l of inorganic nitrogen compounds i n the s o i l , improving the N 2 f i x a t i o n process, and decreasing the amount of n i t r a t e l o s t by the process of d e n i t r i f i c a t i o n . What appears to be the f i r s t report of the use of naph-thenate i n a b i o l o g i c a l experiment dates back to 1921. In th i s experiment ( 105 ) one hour a f t e r 5 0 mg of NaNap was added to 10 ml of a 2 . 5 $ glucose solution which contained 2 5 0 mg of yeast, C0 2 production by treated 'Munchner' and 'Patzenhofer• yeast suspensions was 143 and 140$ of the control values, respectively. As a note i n passing, a very innovative use involving naphthenates was reported by Nussbaum ( 106 ). He stated that manual root pinching was e a s i l y skipped by pruning the roots chemically using a 5 5 $ copper naphthenates solution. A more branched, fibrous root system resulted when seedlings were grown i n a wooden f l a t the bottom of which had been pretreated with the naphthenate solution. MATERIALS AND METHODS The procedures involved i n t h i s investigation, modified somewhat, were based on those described by Kim and Bidwell ( 8 3 ). Uniform seeds of the dwarf bush been (Phaseolus  vulgaris L. c u l t i v a r Top Crop) were sown i n wooden f l a t s containing vermiculite, and placed i n a growth room. Seven-day-old seedlings with root systems of uniform s i z e were selected, the roots quickly rinsed with d i s t i l l e d water, blotted, and 5 mm terminal root sections were cut. A.) Incubation. The incubation medium used was that outlined by Andreae et a l ( 12 ). The medium consisted of 20 umoles CaCNO^^ • 4 H20, 100 umoles KHgPO^, and 280 umoles sucrose i n a t o t a l volume of 4,4 ml. To serve as a control, the complete medium was added to the main or lower compartment of a glass b o t t l e (capacity 55 ml), which contained an upper compartment, capacity 6 . 7 ml (a Skrip ink b o t t l e ) . Two i d e n t i c a l bottles also with the complete medium contained i n addition KCHC (1 x 10"-5 M) or KNap (1 x 10" 5 M). The pH of the medium i n each b o t t l e was adjusted to 6 with 1 N NaOH, autoclaved for 20 minutes at 15 l b s / i n 2 , and the K s a l t of p e n i c i l l i n G (15 ug/ml) and strepto-mycin sul f a t e ( 3 0 ug/ml) were added a f t e r cooling. Three sets of root t i p s each containing 40 t i p s , from the root systems of eight d i f f e r e n t plants, weighing from 42 to 47 mg fresh weight per set were placed i n the main compartment of three b o t t l e s , 1 . 5 ml of 1 N NaOH was added to the smaller or upper compartment, and the bottles were t i g h t l y capped. For s 1 1 3 . the f i r s t phase of incubation the media also contained glucose ( 0 . 5 % ) . After 6 hours of incubation i n the dark at 2 5 ° with constant shaking, each set of root t i p s was removed, rinsed with d i s t i l l e d water, and blotted. Each set was then placed i n a separate b o t t l e , containing the complete medium i n which glucose (Amersham/Searle: Toronto, Ontario), 2 . 5 umoles and 12 7 . 5 uCi per set of root t i p s , replaced the C glucose of the f i r s t phase of incubation. A fresh solution of NaOH was added to the upper compartment of each b o t t l e . The bottle s were again t i g h t l y capped, and incubation was continued for an additional 3 hours. B.) Tissue analysis. At the end of the second incubation period each set of root t i p s was removed, rinsed with d i s t i l l e d water, and blotted. The root t i p s inl.each set were divided into two groups of 2 0 , and each group was transferred to a beaker containing b o i l i n g 75% ethanol (EtOH). This procedure served to inactivate enzymes, and to extract the EtOH-soluble compounds. The tissue was extracted for 2 hours a f t e r which the EtOH-soluble fractions were placed i n separate v i a l s . The root t i p s i n each set were rinsed twice with b o i l i n g 75% EtOH, and the rinsi n g s were added to the EtOH-soluble fr a c t i o n s . The EtOH-soluble fractions were evaporated to dryness under streams of a i r , and 1 ml of 25% methanol (MeOH) was added to each f r a c t i o n . A 25 u l aliquot was withdrawn from each 1 ml sample and was applied to a 20 x 20 cm TLC plate coated with c e l l u l o s e MN 3 0 0 HR ( 0 . 5 mm wet thick-ness) (Macherey and Nagel, Co.). Chromatograms were developed i n n-butanol: g l a c i a l a c e tic acids water ( 4 : 1 : 5 , top phase) (BAW) and 80$ phenol. For determining t o t a l r a d i o a c t i v i t y of the EtOHr-soluble f r a c t i o n , another 25 u l aliquot was withdrawn and added to 15 ml of s c i n t i l l a t i o n solution. The s c i n t i l l a t i o n solution consisted of 126 ml EtOH, 2 0 0 ml toluene, 1 . 3 3 g PPO, and 5 mg POPOP. The ^ C a c t i v i t y was determined using a l i q u i d s c i n t i l l a t i o n counting system (Nuclear-Chicago, Model 7 2 4 ) . Root t i p s which had been EtOH extracted were divided into two groups of 1 0 . One group was transferred to a test tube which contained 1 ml of formamide, and the tissues were s o l u b i l -ized by heating the mixture at 2 0 0 ° for 5 hours. The r e s u l t i n g solution was well mixed, di l u t e d to 3 m l with d i s t i l l e d water, and a 0 . 2 ml aliquot was added to 15 ml of s c i n t i l l a t i o n solution. The other group together with 2 ml of 6 N HC1 was transferred to a 25 ml f l a s k equipped with a condenser, and was subjected to acid hydrolysis for 10 hours at 1 0 5 ° . The solution containing the hydrolyzed EtOH-insoluble compounds was evaporated to dryness, and 0 . 5 ml of 2 5 $ MeOH was added. T h i r t y - f i v e u l from each f r a c t i o n was applied to TLC plates of c e l l u l o s e MN 3 0 0 HR, and developed as before. Radioactive compounds on the TLC plates were detected by the use of Kodak Blue Brand Medical X-ray f i l m . Following the i d e n t i f i c a t i o n of compounds using amino acid standards (see pages 165 - 166 i n the Appendix), the c e l l u l o s e adsorbant with the appropriate compound was removed from the plate, and was placed i n a counting v i a l containing the s c i n t i l l a t i o n s o l u t i o n plus 0 . 2 ml of d i s t i l l e d water. The lZ*C a c t i v i t y was determined as before. Because the leucine and isoleucine standards did not separate adequately from each other when developed i n the BAW/phenol systems, l^ c a c t i v i t i e s from these two amino acids were considered as one. One-tenth ml of the NaOH which contained the respired A^C02 was transferred to 15 ml of the s c i n t i l l a t i o n s o l u t i o n . Carbon-14 a c t i v i t y was determined as before. In a l l instances, samples were corrected for quenching. The data were subjected to an analysis of variance of a nested design, and a comparison of means by Duncan's New Multiple Range Test ( 135 ). The v a r i a b i l i t y which existed between the containers used f o r incubation purposes was not tested. As seen i n Table XXV , each naphthenate treatment s i g n i f -i c a n t l y increased the A C a c t i v i t y i n the ethanol-soluble, ethanol-insoluble, and the respired 1^C0 2 f r a c t i o n s . Using 14 t o t a l r a d i o a c t i v i t y as a basis for comparison, C a c t i v i t y i n root t i p s treated with KCHC was greater than the control value by 2 0 $ ; the increase observed i n KNap-treated root t i p s was 1 3 $ (Figure 11). The data of Table XXV indicate that both compounds had a s t a t i s t i c a l l y s i g n i f i c a n t stimulative e f f e c t on the uptake of l a b e l l e d glucose from the incubation media. The greater e f f e c t was obtained by the use of KCHC. 14 Each naphthenate treatment increased the C a c t i v i t y i n a l l ethanol-soluble amino acids, except one. Carbon-l4 a c t i v i t y i n serine was s i g n i f i c a n t l y increased following KCHC and KNap treatments, while the increase observed i n the case of valine was highly s i g n i f i c a n t . Only the KNap treatment s i g n i f i c a n t l y 14 increased the C a c t i v i t y i n isoleucine/leucine (Table XXvi , Figure 1 2 ) , Because of var i a b i l i t y i n i n d i v i d u a l values, increases of 51 and 40$ i n the l e v e l of > C glucose i n the ethanol-soluble f r a c t i o n from KCHC- and KNap-treated tissues, respectively, were not quite s t a t i s t i c a l l y s i g n i f i c a n t at the 0 . 0 5 l e v e l . 14 The amount of C l e v e l i n aspartic acid, glutamic acid, and alanine from the ethanol-insoluble hydrolysate from KCHC-treated root t i p s was s i g n i f i c a n t l y increased, and were 1 2 3 , 126,.and 1 6 2 $ of the control value, respectively. However, i n the hydrolysate from KNap-treated root t i p s only X^C a c t i v i t y i n alanine was s i g n i f i c a n t l y greater than the control value TABLE XXV . Total r a d i o a c t i v i t y , as muCi, i n the ethanol-sqluble, ethanol-insoluble, and respired CO? fractions of control and naphthenate-treated bean root tip s a f t e r supplying glucose a . Treatments Control KNap KCHC Ethanol-soluble 6 7 . 2 b * 8 1 . 2 8 3 . 6 Ethanol-insoluble 6 1 . 1 6 3 . 1 # 6 8 . 5 Respired COg 5 3 . 9 63.O ** 6 8 . 3 Total r a d i o a c t i v i t y 1 8 2 . 2 2 0 9 . 0 * 2 2 0.4* Percent of control 113 120 Incubation sequence one containing 1 0 _ 5 14Q glucose medium. : Six hours i n M KNap or 10~5 1 2 n n a C glucose M KCHC; then medium, or i n 3 hours i n a Each value represents the mean of four measurements. 90 230 80 220 70 . 210 60 2 0 0 50 - • 190 40 C KNap KCHC Ethanol-soluble C =• Ethanol-insoluble C KNap KCHC C KNap KCHC Respired KNap = I I I I C KNap KCHC Total a c t i v i t y KCHC = I 1 8 0 Radioactivity, as muCi, i n the ethanol-soluble, ethanol-insoluble, and respired C0 2 fractions of control and naphthenate-treated bean root t i p s a f t e r supplying 14Q glucose. TABLE XXVI . Total r a d i o a c t i v i t y , as muCi, found i n i n d i v i d u a l ethanol-soluble amino acids and in glucose from control and naphthenate-treated root tips a f t e r supplying 14c glucose a . Treatments Control KNap KCHC Aspartic acid 2.22 b 2 . 2 5 n s 2 . 6 8 n s Serine 5.12 9.08 *• 7.42 Glutamic acid 1.4? 1 . 6 8 n s 1 . 7 6 n s Threonine 10.21 11.40 n S 1 3 . 0 3 n S Alanine 10.10 i o . 2 5 n s 1 2 . 5 2 n s Glutamine 1.45 1 . 4 7 n s 1 . 6 l n s IT-Aminobutyric acid 2.10 1 . 6 5 n s 2 . l 4 n s Valine 4.91 5-75 ** 7.32 Isoleucine/leucine 3-54 4 . 3 8 * 3 . 3 5 n s Glucose 17.48 2 5 . 3 4 n s 2 3 . l 7 n s Total a c t i v i t y 58.68 72.79 75.54 * See Table XXV. b See Table XXV. 12 3 " Aspartic acid Serine Glutamic acid Threonine Alanine Glutamine c = C KNap = |I I I I KCHC = ro o 24 21 18 15 a—c 12 )T-Amino-butyric acid Valine Isoleucine/ leucine Glucose 3-Phospho-hydroxypyruvate i Serine I Glycine Methionine Leucine Alanine Asparate Homoserme Threonine Isoleucine I 3-Phospho-glycerate 1 Phosphoenol-pyruvate Pyruvate I Acetyl Co A Oxaloacetate i t-keto-glutarate Glutamate - Glutamine y-Aminobutyrate TABLE XXVII Total r a d i o a c t i v i t y , as muCi, found i n amino acids from the ethanol-insoluble hydrolysate from con-t r o l and naphthenate-treated bean root t i p s a f t e r supplying glucose a . Control Treatments KNap KCHC Aspartic acid Glutamic acid Alanine 3 . 7 6 3 - 3 5 2 . 1 3 3 . 5 8 ns 3.54 3 . 0 1 ns 4 . 7 2 4 . 2 7 3 . 5 8 a See Table XXV. b See Table XXV. C KNap KCHC C KNap KCHC C KNap KCHC Aspartic Glutamic Alanine acid acid FIGURE 1 3 . Radioactivity, as muCi, found in aspartic acid, glutamic acid, and alanine from the ethanol-insoluble hydrolysate from control and naphthenate-treated bean root ti p s after supplying l 4 C glucose. £ (Table XXVII, Figure 1 3 ) . Although the uptake and metabolism of carbohydrate and the effects of growth regulators on the growth and aging of excised root t i s s u e have been extensively reviewed by Butcher and Street ( 36 ), the amount of information related to the e f f e c t of growth-promoting compounds on the uptake and metabolism of sugars i s very l i m i t e d . Results of Kim and Bidwell ( 83 ) revealed that both i n d o l e - 3 - a c e t i c acid (IAA) and 2 , 4 - d i c h l o r o -phenoxyacetie a c i d generally reduced the uptake and retarded the metabolism of l a b e l l e d glucose by excised pea root t i p s . Goldsworthy ( 59 ) demonstrated that mannose competitively i n h i b i t e d the phosphorylation of glucose by hexokinase i n root tissues, while higher concentrations of glucose i n h i b i t e d the uptake of mannose by competing for the hexokinase enzyme. Even though the e f f e c t of naphthenate compounds on t h i s p a r t i c u l a r enzyme has not been studied, the s i g n i f i c a n t increase i n glucose uptake observed i n naphthenate-treated root tissues suggests that hexokinase a c t i v i t y was increased. Fattah and Wort ( 5 3 ) and Wort et a l ( 1^7 ) have shown that the a c t i v i t i e s of nine d i f f e r e n t enzymes i n bean plants were increased following naphthenate treatment. I t was suggested ( 1^7 ) that because of these increases the stimulation of plant metabolism by naph-thenates must be a general one. Even though the naphthenate treatments increased the a c t i v i t y i n almost a l l ethanol-soluble amino acids, s i g n i f i c a n t increases of glucose carbon i n serine and v a l i n e suggest that certain metabolic pathways may be affected to a greater extent. The increased l e v e l of i n serine indicates that f o l a t e metabolism may be affected ( 42 ), while i n the case of valine the regulation of the transamination of «(-ketoisovaleric acid may be implicated. Glycine was not detected on the radio-chroma to grams, and t h i s suggests that serine was not r a p i d l y converted to glycine. Carbon-14 a c t i v i t y i n aspartic acid, glutamic acid, and alanine i n the hydrolysate of the ethanol-insoluble f r a c t i o n from root t i p s treated with KCHC was s i g n i f i c a n t l y increased. The stimulated incorporation of these amino acids into protein coupled with the lack of s i g n i f i c a n c e observed i n the a c t i v i t y i n the same three amino acids i n the treated root t i p s from the ethanol-soluble f r a c t i o n suggests that protein synthesis was augmented by naphthenate-treatment. This i s supported by r e s u l t s obtained i n our laboratory ( 147 ) which have shown that both the amount of protein, and the l e v e l of i4 incorporation of C-L-leucine into protein of l e a f blades from bean plants was increased by KCHC and KNap treatments. Zenk reported that i n various plant tissues IAA ( 152 ) and o('-naphthaleneacetic acid ( 153 ) were converted to a mixture of the glucose ester and the aspartic acid amide, and that these conjugated compounds were referred to as being 'true detoxication' products, i . e . not essential i n the growth induction process. Kim and Bidwell ( 8 3 ) also suggested that the aspartic acid conjugate of IAA was not responsible for the reduction of glucose uptake and the impairment of glucose metabolism i n pea root t i p s . Recently, i t has been shown ( 148) that IAA can form a macromolecular conjugate with tRNA. Even though the presence of t h i s macromolecular conjugate could not be confirmed ( 4 3 ) , the existence of such a conjugate has i n t e r e s t i n g implications. Davis and Galston ( 4 3 ) also suggested the p o s s i b i l i t y of indoleacetyl aspartate (IAA-Asp) becoming attached to tRNA y i e l d i n g a compound, t R N A a s p " ^ I A A A s p \ s i m i l a r to formyl-methionine-tRNA which might serve as a protein chain i n i t i a t o r . However, using l a b e l l e d aspartate the authors could not demonstrate the presence of IAA-Asp-tRNA a sP i n pea stem sections. Studies i n our laboratory ( 108, 1Q9, 127 , 131) have shown that i n the roots and leaves of bean plants KCHC-7-^C was r a p i d l y converted to a mixture of the glucose ester, the aspartic acid amide, and several unknown metabolites. In these experiments KCHC, and i n the present experiment, KCHC and KNap were not detected on the chromatograms. As stated previously, Wort et a l ( l4?) reported that the a c t i v i t i e s of several important enzymes were increased following naphthenate treat-ment. However, no increase i n enzyme a c t i v i t y was obtained following an i n v i t r o addition of the free acid. I t would appear that naphthenate stimulation of glucose uptake and metabolism observed i n t h i s experiment may have been i n some way associated with the presence of the conjugated or a bound form of naphthenate, rather than with the free a c i d f s ) . In conclusion, the data suggest that the naphthenate compounds or t h e i r metabolites stimulated glucose a s s i m i l a t i o n i n excised root t i p s from bush bean plants used i n t h i s experiment. Not only was CC»2 production increased, but also amino acids containing glucose carbon passed through soluble pools i n root tissues and were more r a p i d l y fixed into protein. Three separate studies were carried out with bean plants to determine! l ) the eff e c t of the complex naphthenic acid mixture, KNap, on the uptake, d i s t r i b u t i o n , and incorporation of phosphorus-3 2 , 2) the metabolism of KCHC-?- 1^ i n leaves and roots, and 3 ) the e f f e c t of KNap and KCHC on the uptake and metabolism of A^C glucose by root t i p s . On the basis of r e s u l t s obtained i n these experiments i t may be concluded that: 1. ) When grown i n either a minus P or, a complete nutrient solution, KNap did not have a s i g n i f i c a n t e f f e c t on P uptake. 2. ) The acropetal movement of P from the roots of plants grown i n a minus P nutrient was greatly enhanced by naphthenate treatment. The d i s t r i b u t i o n pattern i n these plants showed that the balance i n ^ 2p d i s t r i -bution between the leaves and roots of treated plants , was greatly upset i n favor of the roots. When plants were grown i n a complete nutrient, the ef f e c t of KNap on the d i s t r i b u t i o n of 3 2p w a s not as dramatic, and the 3 2P d i s t r i b u t i o n balance was upset i n favor of the leaves. 3 . ) Naphthenate treatment increased the rate of incorpora-t i o n of 3 2p into the acid soluble (sugar phosphates, free nucleotides, phospholipids) and acid insoluble (nucleic acids, phosphoproteins) fract i o n s of leaves. However, as 3 2P a c t i v i t y i n root tissues declined, due to the f a c t that the acid soluble 32 P a c t i v i t y was translocated acropetally, the amount of 32p a c t i v i t y i n the acid insoluble f r a c t i o n s i g n i f i -cantly increased. Naphthenate treatment did not a f f e c t the amount of P i n the two P f r a c t i o n s . 4 . ) When administered to l e a f disks i n the l i g h t or to roots of i n t a c t seedlings i n the dark, KCHC-7-1^C was r a p i d l y converted to a mixture of two conjugated metabolites: the glucose ester and the aspartic acid amide. I t appears that both conjugates are mobile within the plant. Also, i t i s possible that KGHC may have been conjugated with a low molecular weight peptide. 5. ) Both KNap and KCHG treatments s i g n i f i c a n t l y increased the l e v e l of i^C a c t i v i t y i n the ethanol-soluble, ethanol-insoluble, and the respired C0 2 fractions a f t e r •^C glucose was administered to bean root t i p s . The amount of ^ C a c t i v i t y i n soluble amino acids and i n protein was increased by the treatments. The i n d i v i d -ual naphthenic acid, KCHC, had the greater e f f e c t on the uptake and metabolism of l a b e l l e d glucose. One of the o v e r a l l objectives of these experiments was to augment our understanding of how naphthenates stimulate meta-bolism and growth of bean plants, or to understand more f u l l y t h e i r 'modus operandi.' I t appears that the effects of naph-thenate on phosphorus assim i l a t i o n are not only concerned with oxidative phosphorylation ( 1^7 ) and the normal g l y c o l y t i c pathway ( 5 2 , Chapter 3 ) , but also with the incorporation of P into components of the acid insoluble f r a c t i o n . A close connection between increases i n enzyme a c t i v i t y ( 53» 147 )» nucleic acid biosynthesis ( 110 ), and protein formation ( 147 ) suggests that a major e f f e c t of naphthenates may be exerted at both the metabolic and genetic l e v e l s . Results of the KCHC-7-l^C root-feeding (Chapter 2) and the l^C glucose metabolism (Chapter 3) experiments also suggest that the metabolites of the naphthenic acids may be responsible for stimulating metabolic reactions. Data obtained i n other experiments performed i n our laboratory indicate t h i s to be the case ( 147 ). Actual experimental evidence supporting t h i s suggestion follows s 1. ) Free acids did not increase the s p e c i f i c a c t i v i t i e s of f i v e d i f f e r e n t enzymes i n v i t r o ( 147 ). 2. ) Free acid i s not detected i n concentrated extracts of plant tissue six hours a f t e r a p p l i c a t i o n ( 108, Chapter 2 ). 3. ) L o c a l i z a t i o n of the metabolites i n the a e r i a l portion a f t e r the plant had received a f o l i a r naphthenate treatment seems to be associated with a corresponding increase i n metabolic a c t i v i t y ( Chapter 1 ). 4. ) The suggestion was made recently that the aspartic acid amide of IAA may not be a mere detoxication product. Davis and Galston ( 43 ) speculated that t h i s IAA metabolite may function as a chain i n i t i a t o r i n protein synthesis. Zenk ( 155 ) has also reported that IAA formed a IAA-protein complex i n peas. Perhaps, the aspartic acid amide of CHCA may play a s i m i l a r r o l e . 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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 Vesh-chestva S e l . Khoz., 2nd, Baku ( 1 9 6 3 ) , 118 - 1 2 9 : Chem. Abstr. 67_, 20832j, 1 9 6 7 . APPENDIX Detection of amino acids,(83). 0 . 2 $ ninhydrin (1,2,3»-triketohydrindene) i n acetone. Detection of mono- and disaccharides, (83). Solution A. 0 . 5 ml a n i l i n e , 20 ml g l a c i a l acetic acid, and 10 ml d i s t i l l e d water. Solution B. 5 g t r i c h l o r o a c e t i c acid i n 20 ml d i s -t i l l e d water. Mix solutions A and B at the r a t i o of 1 : 1. The acid-base indicator, bromophenol blue c (127). 50 mg bromophenol blue, 200 mg c i t r i c acid in 100 ml d i s t i l l e d waters Ammoniacal s i l v e r n i t r a t e 0 (127)0 Add concentrated NH^OH to a 5 $ s i l v e r n i t r a t e solution u n t i l the amber p r e c i p i t a t e disappears. Detection of phenolic compounds, diazotized p - n i t r o a n i l i n e i l 2 7 ) . 5 ml p - n i t r o a n i l i n e , 1 ml 5 $ sodium n i t r i t e , and 25 ml 2 5 $ sodium acetate. Over spray chromatogram with 5 $ sodium hydroxide. Determination of inorganic phosphate  i n acid digests of plant tissue,;. ( 5 5 ) . Reagents ANSA (Aminonaphtholsulfonic acid) - Dissolve 1 . 2 5 g of ANSA i n 2 2 5 ml of d i s t i l l e d water, and then add 71 .25 g of sodium b i s u l f i t e . Shake for 15 minutes. - Pour i n 75 ml of 10$ sodium s u l f i t e , and shake u n t i l a l l material dissolves. Make to $00 ml with d i s t i l l e d water, and store i n the dark. Ammonium molybdate Add 5 5 * 6 ml of concentrated s u l f u r i c acid to ca 200 ml of d i s t i l l e d water. In this volume dissolve 10 g of ammonium molybdate. Make to 400 ml with d i s t i l l e d water. Standard curve for inorganic phosphate (see next page). Phosphorus-32 a c t i v i t y Sampling times i n hours Leaf blades Stems Roots C c T C T C T 4 d 62 ,799 5 7 , 1 6 3 3 0 , 4 2 9 28 , 3 9 6 9 0 , 9 3 1 91,144 8 6 3 , 7 1 4 6 5 , 8 1 9 2 3 , 3 6 1 2 5 , 9 9 8 84 ,221 82 , 2 3 2 12 6 7 , 7 3 7 6 9 , 4 7 4 2 7 , 9 3 7 25,440 7 0 , 3 4 7 6 8 , 1 3 7 24 7 0 , 5 3 8 82 ,460 25,640 2 3 , 0 9 7 66,410 63,247 Mean 6 6 , 1 9 7 6 8 , 7 2 9 n s 26,842 ns 2 5 , 7 3 2 7 7 , 9 7 7 ns 7 6 , 1 9 0 Percent of control 1 0 3 . 9 9 5 . 9 9 7 . 7 a- See Table IV. b See Table I I I . C See Table I I I . Total inorganic phosphate Sampling — times i n Leaf blades Stems Roots hours C c T C T C T d 0.64 4 2.12 2.11 0.67 1.98 1.79 8 2.02 2.06 0.61 * 0.72 2.06 2.03 12 2.47 2.26 0.78 0.94 2.08 2.01 24 2.89 3.10 1.02 1.00 2.63 2.80 Mean 2.38 2.38 n s 0.77 0.82 n s 2.19 2 . l 6 n S Percent of control 100.0 106.5 98.6 a See Table IV. b See Table VI. c See Table III. d See Table I I I . Sampling Phosphorus-32 a c t i v i t y b times i n hours Leaf blades Stems Roots C c T C T C T 4 , 94 ,198 d 8 5 , 7 4 4 4 3 , 3 5 9 4 3 , 3 8 4 124 ,376 1 3 3 , 5 0 3 8 9 5 , 6 5 1 9 8 , 7 ^ 8 3 2 , 4 5 2 3 5 , 6 3 0 1 2 5 , 0 9 6 1 2 2 , 4 8 9 12 1 0 1 , 6 0 5 104,211 3 9 , 2 7 9 3 4 , 7 4 5 104 ,756 9 4 , 2 7 8 24 105,844 1 2 3 , 6 9 2 3 6 , 3 5 7 3 5 , 7 9 4 9 6 , 2 1 1 9 4 , 8 7 0 Mean 9 9 , 3 2 4 1 0 3 , 0 9 8 n s 37,862 3 7 . 3 8 8 n S 112,610 l l l , 2 8 5 n s Percent of control 1 0 3 . 8 9 8 . 7 9 8 . 8 a See Table IV. b See Table I I I . c See Table I I I . Total inorganic phosphorus times i n hours Leaf blades Stems Roots C c T C T C T 4 3 , 1 8 d 3 . . 1 6 0 . 8 3 0 . 9 9 2 . 9 7 2 . 6 9 8 3 . 0 3 3 . 1 3 0 . 8 5 0 . 9 9 3 . 0 9 3.04 12 3 - 7 0 3.40 1 . 1 0 1 . 2 8 3 . 1 0 . 3 . 0 2 24 4 . 3 5 4 . 6 6 1 . 4 5 1 . 5 3 3 . 9 5 4 . 2 0 Mean 3 . 5 6 3 . 5 9 n s 1 . 0 6 1 . 1 9 n s 3.28 3 . 2 3 n S Percent of control 1 0 0 . 8 1 1 2 . 3 9 8 . 5 a See Table IV. b See Table VI. c See Table III. d See Table I I I . Sampling Phosphorus-32 a c t i v i t y b times in hours Leaf blades Stems Roots C c T C T C T 4 d 7,915 9,083 4,287 4,496 30,080 31,008 8 9,677 10,965 4,660 4,807 36,298 34,892 12 10,958 11,208 5,055 5,296 41,444 44,467 24 13,686 15,112 5,575 5,664 51,544 62,931 Percent of control 109.7 103.5 108.7 Mean 10,559 U,592 n s 4,894 5,066 n s 39,842 43,324 * a See Table IV. b See Table III. c See Table I I I . d See Table I II. Sampling Total inorganic phosphate b times i n hours Leaf blades Stems Roots C c T C T C T 4 0.17 d 0.17 0.09 0.08 0.13 0.14 8 0.18 0.18 0.09 0.09 0.15 0.15 12 0.20 0.21 0.10 0.11 0.17 0.18 24 0.22 0.23 0.13 0.11 0.20 0.19 Mean 0.20 0.20ns 0.10 0.10ns 0.16 0.l6 n s Percent of control 100.0 100.0 100.0 a See Table IV. b See Table VI. c See Table I I I . d See Table I II. Sampling Phosphorus-32 a c t i v i t y b times i n hours Leaf blades Stems Roots C c T C T C T 4 12,974 d 14,118 5,805 5,982 51,828 49,620 8 14,516 16,447 6,463 6,577 53,919 51,978 12 16,438 16,813 7,099 7,232 61,766 61,347 24 20,529 22,666 7,933 8,526 74,873 94,273 Mean 16,114 17,511ns 6,825 7,079ns 60,596 64,304* Percent of control 108.6 103.7 106.1 a See Table IV. b See Table I I I . c See Table I I I . d See Table I I I . Sampling Total inorganic phosphate b times in hours Leaf blades Stems Roots C c T C T c T 4 0 . 2 5 d 0 . 2 5 0.11 0 . 1 2 0 . 2 0 0 . 2 0 8 0.28 0 . 2 7 0 . 1 2 0 . 1 3 0.22 0.22 12 0 . 3 1 0 . 3 2 0.14 0 . 1 5 0.26 0 . 2 7 24 0 - 3 3 0 . 3 5 0 . 1 9 0 . 3 0 0.28 Mean 0 . 2 9 0 . 3 0 n s 0.14 0 . l 4 n s 0 . 2 5 0.24 n s Percent of control 1 0 3 . 4 1 0 0 . 0 9 6 . 0 a See Table IV. b See Table VI. c See Table III. d See Table III. Sampling Phosphorus-32 a c t i v i t y b times i n hours Acid soluble 3 2 P Acid insoluble 3 2 p Total 3 2 p C .9 ..T C T C T 4 „ d 184 ,158 1 7 6 , 7 0 2 42,282 4 4 , 5 8 8 226,440 : : 2 2 1 , 2 9 0 8 171 ,296 174,048 5 0 , 6 3 4 5 0 , 6 6 3 2 2 1 , 9 3 1 224,711 12 1 6 6 , 0 2 0 1 6 3 , 0 5 1 5 7 , 4 5 7 6 0 , 9 7 0 223,471 224 ,021 24 1 6 2 , 5 8 8 1 6 8 , 8 0 3 70,804 8 3 , 7 0 6 2 3 3 , 3 9 2 2 5 2 , 5 0 9 Mean 1 7 1 . 0 1 5 l 7 0 , - 6 5 1 n s 55,294 5 9 , - 9 8 2 * 2 2 6 , 3 1 0 2 3 0 , 6 3 3 n s Percent of control 9 9 . 7 IO8.5 1 0 1 . 9 a See Table IV. b See Table III . values represent the sum, leaf blades + stems + roots. c See Table I I I . d See Table I I I . Samolinff'" Total inorganic phosphate b times i n hours Acid soluble P Acid insoluble P Total P C c T C T C T 4 4 . 7 7 d 4 . 5 4 0 . 3 9 O.38 5 . 1 6 4 . 9 2 8 4 . 6 9 4.81 0.42 0.42 5 . 1 1 5 . 2 3 12 5 . 3 3 5 . 2 1 0.48 0 . 5 0 5 . 8 1 5 . 7 1 24 . 6 - 5 5 6 . 9 0 O.56 0 . 5 4 7.11 7 . 4 3 Mean 5 - 3 3 5 . 3 6 n s 0.46 0 . 4 6 n s 5.80 5.82 N S Percent of control 1 0 0 . 6 1 0 0 . 0 1 0 0 . 3 a See Table IV. b See Table VI. values represent a sum, leaf blades + stems + roots. c See Table I I I . d Sampling Phosphorus-32 a c t i v i t y b times i n hours Acid soluble 3 2 P Acid Insoluble 3 2 P Total 32p c c T C C T 4 2 6 1 , 9 3 3 d 2 6 2 , 6 3 1 7 0 , 6 0 6 6 9 . 7 2 0 3 3 2 , 5 3 9 3 3 2 , 3 5 1 8 2 5 3 , 1 9 9 2 5 6 , 8 6 7 7 4 , 8 9 6 7 5 , 0 0 1 3 2 8 , 0 9 5 3 3 1 , 8 6 7 12 245,640 2 3 3 , 2 3 4 8 5 , 3 0 3 8 5 . 3 9 0 330,942 3 1 8 . 6 2 3 24 238,412 2 5 4 , 3 5 5 1 0 3 , 3 3 4 1 2 5 , 4 6 3 341,745 379,818 Mean 249,796 251 , 771 n s 8 3 , 5 3 4 8 8 , 8 9 3 * 3 3 3 . 3 3 0 340 ,665 n s Percent of control 1 0 0 . 8 1 0 6 . 4 1 0 2 . 2 a See Table IV. See Table I I I . Values represent the sum, leaf blades + stems ' + roots. C See Table I I I . d See Table I I I . Sampling Total inorganic phosphate b times i n hours Acid soluble P • Acid insoluble P Total P C c T C T C T # d 6 . 9 7 6 . 8 3 O.56 0 . 5 7 7 . 5 3 7.40 8 6 . 9 7 7.16 0 . 6 2 0 . 6 3 7 . 5 9 7 . 7 8 12 7 . 9 0 7 . 6 9 0 . 7 1 # 0 . 7 3 8 . 6 1 8.42 24 9 - 7 5 1 0 . 3 8 0 . 7 8 0 . 8 0 * 1 0 . 5 7 1 1 . 1 8 Mean 7 . 9 0 8 . 0 l n s 0 . 6 7 0 . 6 8 n s 8 . 5 8 8 . 7 0 n s Percent of control 101.4 1 0 1 . 5 101.4 a See Table IV. See Table VI. Values represent the sum, leaf blades + stems + roots. c See Table I I I . d See Table I I I . Number Amino acid, amide, or sugar 0 O r i g i n 1 Aspartic acid 2 Serine 3 Glutamic acid Glucose 5 Threonine 6 Glutamine 7 Alanine 8 )f-Aminobutyric acid 9 Valine 10 Isoleucine 11 Leucine CD CO W X J P « P . o -p Dimension 2 - 8.0$ phenol FIGURE 15. A schematic representation of a two-dimensional chromatogram snowing the standard positions of known amino acids and glucose. A l l analyses were done using 0 . 5 0 mm (wet thickness) cellulose MN 3 0 0 HR thin-layer plates. 

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