Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

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

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1971_A1 S48.pdf [ 9.03MB ]
Metadata
JSON: 831-1.0101869.json
JSON-LD: 831-1.0101869-ld.json
RDF/XML (Pretty): 831-1.0101869-rdf.xml
RDF/JSON: 831-1.0101869-rdf.json
Turtle: 831-1.0101869-turtle.txt
N-Triples: 831-1.0101869-rdf-ntriples.txt
Original Record: 831-1.0101869-source.json
Full Text
831-1.0101869-fulltext.txt
Citation
831-1.0101869.ris

Full Text

STUDIES WITH NAPHTHENIC ACIDS IN THE BUSH. BEAN, Phaseolus v u l g a r i s L. I.  II. III.  THE EFFECT OF POTASSIUM NAPHTHENATES ON THE UPTAKE, DISTRIBUTION, AND INCORPORATION OF PHOSPHORUS-32. THE METABOLISM OF CYCLOHEXANECARBOXYLIC ACID. 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 S t a t e U n i v e r s i t y , M.A.T., Washington S t a t e U n i v e r s i t y ,  A t h e s i s submitted i n p a r t i a l  196^. 1968.  fulfillment  o f the requirements f o r the degree o f DOCTOR OF PHILOSOPHY i n the Department o f BOTANY We a c c e p t t h i s t h e s i s as conforming to the r e q u i r e d 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 o b j e c t i v e o f these experiments was to augment our understanding o f how naphthenic a c i d s s t i m u l a t e metabolism and growth o f bean p l a n t s .  Three s e p a r a t e s t u d i e s were c a r r i e d out  w i t h bush bean p l a n t s (Phaseolus v u l g a r i s L. c u l t i v a r Top Crop) to d e t e r m i n e i  1)  the e f f e c t o f potassium naphthenates  (KNap) on  the uptake, d i s t r i b u t i o n , and i n c o r p o r a t i o n o f p h o s p h o r u s - 3 2 , 2)  the metabolism o f the i n d i v i d u a l naphthenic a c i d , potassium  c y c l o h e x a n e c a r b o x y l a t e (KCHC), i n l e a v e s and r o o t s , and 3 )  the  e f f e c t o f KNap and KCHC on the uptake and metabolism o f  glucose  by e x c i s e d r o o t 1)  tips.  Fourteen-day-old p l a n t s growing i n a phosphate-free (-P)  or a complete (+P) n u t r i e n t s o l u t i o n were sprayed t o d r i p w i t h a 0.5% s o l u t i o n o f KNap.  Twenty-four hours a f t e r s p r a y i n g , the r o o t s  o f both c o n t r o l and t r e a t e d p l a n t s were exposed f o r 2 hours to a nutrient solution containing ^ P. 2  the p l a n t s were r e t u r n e d  F o l l o w i n g the exposure to -^ P, 2  to t h e i r o r i g i n a l n u t r i e n t s o l u t i o n s .  C o n t r o l and t r e a t e d p l a n t s were withdrawn 4, 8 , 12, and 2k hours a f t e r exposure to 3 P , and were s e p a r a t e d i n t o l e a f b l a d e s , 2  A c i d s o l u b l e , a c i d i n s o l u b l e , and t o t a l 3 P  and r o o t s .  2  stems,  activity,  or t o t a l phosphorus were determined a t each sampling time. KNap treatment i n c r e a s e d by 7 to 9% the i n t a k e o f ^ P by 2  p l a n t s grown i n the -P o r +P n u t r i e n t s o l u t i o n .  The i n c r e a s e s ,  however, l a c k e d s t a t i s t i c a l s i g n i f i c a n c e a t the 0 . 0 5 l e v e l .  The  r a t e a t which ^ P was t r a n s l o c a t e d out o f the r o o t s o f p l a n t s grown 2  i n the -P n u t r i e n t o n l y was i n c r e a s e d s i g n i f i c a n t l y by treatment, i n s p i t e o f the f a c t t h a t a t the 24 hour sampling time Qk% o f the t o t a l 3 P l a b e l remained i n r o o t t i s s u e s . 2  At the same sampl-  i n g time J2% o f the t o t a l 3 P l a b e l was found i n the r o o t s o f 2  p l a n t s grown i n the +P n u t r i e n t . cantly increased  While KNap treatment  signifi-  32p a c t i v i t y i n stems o f -P grown p l a n t s  over  the sampling p e r i o d , a c t i v i t y i n stems o f c o n t r o l and t r e a t e d p l a n t s grown i n the +P n u t r i e n t was s i m i l a r . Naphthenate treatment i n c r e a s e d o f 32p i n t o both the a c i d s o l u b l e phospholipids)  the r a t e o f i n c o r p o r a t i o n  (sugar phosphates,  nucleotides,  and a c i d i n s o l u b l e ( n u c l e i c a c i d s , phosphoproteins)  f r a c t i o n s o f leaves  o f p l a n t s grown i n the +P n u t r i e n t s o l u t i o n .  A c i d s o l u b l e 3 P a c t i v i t y d e c l i n e d i n a l l r o o t t i s s u e s over the 2  sampling p e r i o d as a c i d s o l u b l e 3 p - c o n t a i n i n g 2  compounds, p r i T  m a r i l y orthophosphate, were t r a n s l o c a t e d a c r o p e t a l l y .  The per-  centage a c i d i n s o l u b l e 32p a c t i v i t y i n the r o o t s o f KNap-treated p l a n t s was s i g n i f i c a n t l y g r e a t e r  than t h a t found i n the r o o t s o f  c o n t r o l p l a n t s a t the 2k hour sampling time.  Naphthenate t r e a t -  ment d i d n o t a f f e c t the amount o f t o t a l P ( 3 l p + 3 2 p ) i  n  -the two  P f r a c t i o n s o f the three p l a n t organs. The and  augmented i n c o r p o r a t i o n o f -^ P i n t o the a c i d s o l u b l e 2  a c i d i n s o l u b l e f r a c t i o n s i s f u r t h e r evidence o f the KNap-  stimulated 2)  P metabolism r e p o r t e d  by o t h e r workers.  K C H C - ? - ^ a d m i n i s t e r e d to l e a f d i s k s i n the l i g h t o r to 1  roots o f i n t a c t seedlings  i n the dark was r a p i d l y converted t o a  mixture o f two conjugated m e t a b o l i t e s : a s p a r t i c a c i d amide.  The r o o t - f e e d i n g  following t h e i r synthesis translocated acropetally. the a c i d h y d r o l y s a t e s  the g l u c o s e e s t e r and the experiment i n d i c a t e d  that  i n r o o t t i s s u e s both conjugates were The r e s u l t s o f amino a c i d a n a l y s e s o f  o f several u n i d e n t i f i e d metabolites  s t r o n g l y suggest t h a t KGHC-7- *'C was a l s o conjugated w i t h a low li  molecular 3)  weight  polypeptide.  Three s e t s o f r o o t t i p s c u t from 7-day-old  were incubated  i n a medium c o n t a i n i n g  glucose  seedlings  f o r 3 hours.  Two o f the t h r e e s e t s were p r e t r e a t e d i n a s o l u t i o n o f KCHC o r KNap f o r 6 hours. increased  Each naphthenate treatment  significantly  a c t i v i t y i n the e t h a n o l - s o l u b l e  (amino a c i d s ,  glucose,  etc.), ethanol-insoluble  ( p o l y s a c c h a r i d e s , p r o t e i n , e t c . ) , and  r e s p i r e d CO2 f r a c t i o n s .  The i n d i v i d u a l naphthenic a c i d , KCHC,  had  the g r e a t e r e f f e c t on the uptake and metabolism o f l a b e l l e d  glucose.  R e s u l t s a l s o i n d i c a t e d t h a t n o t o n l y were the uptake o f  glucose and C 0 p r o d u c t i o n 2  i n c r e a s e d S i g n i f i c a n t l y by each t r e a t -  ment, but a l s o amino a c i d s c o n t a i n i n g the glucose more q u i c k l y through s o l u b l e amino a c i d pools  carbon passed  i n root tissues,  and were more r a p i d l y f i x e d i n t o p r o t e i n . In l i g h t o f the f i n d i n g t h a t naphthenate conjugates and not the f r e e a c i d were detected  i n the t i s s u e , i t may be t h a t the  conjugates were a s s o c i a t e d i n a c a u s a l way w i t h the s t i m u l a t e d uptake and metabolism o f l a b e l l e d  glucose.  TABLE GF CONTENTS page ABSTRACT  . .  i  TABLE OF CONTENTS  . .  iv  ABBREVIATIONS  . .  v i i  LIST OF TABLES  viii  LIST OF FIGURES  x i i  ACKNOWLEDGEMENT  xiv  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 B. ) PERCENTAGE DISTRIBUTION OF THE TOTAL P ACTIVITY AMONG LEAF BLADES, STEMS, AND ROOTS  30  3 2  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 INSOLUBLE 32p ACTIVITY AND TOTAL ACID INSOLUBLE 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 Chapter 2 .  THE METABOLISM OF ACID.  ... 80  CYCLOHEXANECARBOXYLIC  INTRODUCTION  84  LITERATURE REVIEW  85  MATERIAL AND METHODS A. )  LEAF DISK FEEDING EXPERIMENT  B. ) SYNTHESIS OF 1 -CYCLOHEX ANEC ARBO N YL-/0D-GLUCOSE C. ) SYNTHESIS OF  88 89  N-CYCLOHEXANECARBONYL-L-  ASPARTIC ACID  90  D. ) HYDROLYSIS PROCEDURES  90  E. ) ROOT FEEDING EXPERIMENT  91  RESULTS AND DISCUSSION Chapter 3 .  93  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 a c i d s  HNap  Potassium  KNap  naphthenates  NaNap  Sodium naphthenates Cyclopentanecarboxylic Potassium  acid  cyclopentanecarboxylate  Cyclohexanecarboxylic  CPCA KCPC CHCA  acid  Potassium  Cyclohexanecarboxylate  KCHC  Potassium  cyclohexaneacetate  KCHAc  Potassium  cyclohexanepropionate  KCHP  Potassium  cyclohexanebutyrate  KCHB  A compound o f t h e c y c l o h e x y l b u t a n o l c l a s s  Sh-8  2,^-Dichlorophenoxyacetic  2,4-D  Indole-3-acetic  acid  Naphthaleneacetic Benzoic  acid  acid  IAA NAA BA  acid  Adenosine t r i p h o s p h a t e  ATP  2,5-Diphenyloxazole  PPO  1 , 4 - b i s - [ _ 2 - ( 5 - P h e n y l o x a z o l y l ) j -benzene  POPOP  Phosphoglyceric  PGA  acid  Phosphoenolpyruvic Uridine  acid  diphosphoglucose  PEP UDPG  LIST GF TABLES TABLE  PAGE Chapter 1  I. II.  III.  IV.  V. VI.  VII.  VIII.  IX.  X.  XI.  XII.  EFFECTS OF NAPHTHENATES ON GROWTH, YIELD, AND COMPOSITION OF PLANTS  9  VOLUMES OF MOLAR STOCK SOLUTIONS USED FOR PREPARING ONE LITER OF A l x NUTRIENT SOLUTION  24  EFFECT OF KNap ON TOTAL UPTAKE AND DISTRIBUTION OF 3 2 p , ON A PER GRAM BASIS, BY BUSH BEAN PLANTS AT VARIOUS SAMPLING TIMES  32  EFFECT OF KNap ON TOTAL UPTAKE AND DISTRIBUTION OF 3 2 p , ON A PER GRAM BASIS, BY BUSH BEAN PLANTS AT VARIOUS SAMPLING TIMES  33  EFFECT OF KNap ON TOTAL UPTAKE OF 3 2 p PER PLANT BASIS, BY BUSH BEAN PLANTS  34  t  ON A  EFFECT OF KNap ON TOTAL UPTAKE AND DISTRIBUTION OF P, ON A PER GRAM BASIS, BY BUSH BEAN PLANTS AT VARIOUS SAMPLING TIMES  37  EFFECT OF KNap ON TOTAL UPTAKE OF P, ON A PER PLANT BASIS, BY BUSH BEAN PLANTS AT VARIOUS SAMPLING TIMES  38  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  EFFECT OF KNap ON THE PERCENTAGE DISTRIBUTION OF 3 2 p , ON A PER GRAM BASIS, AMONG LEAF BLADES, STEMS, AND ROOTS5:OF BUSH BEAN PLANTS AT VARIOUS SAMPLING TIMES  44  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 AT VARIOUS SAMPLING TIMES  46  EFFECT OF KNap ON THE PERCENTAGE DISTRIBUTION OF P, ON A PER GRAM BASIS, AMONG LEAF BLADES, STEMS, AND ROOTS OF BUSH BEAN PLANTS AT VARIOUS SAMPLING TIMES  49  EFFECT OF KNap ON THE PERCENTAGE DISTRIBUTION 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.  XVIII.  XIX.  XX.  XXI.  EFFECT OF KNap ON THE PERCENTAGE DISTRIBUTI 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 ....  56  EFFECT OF KNap ON THE PERCENTAGE DISTRIBUTION 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 ,  58  EFFECT OF KNap ON THE PERCENTAGE DISTRIBUTION OF ACID SOLUBLE P, ON A PER PLANT BASIS, AMONG LEAF BLADES, STEMS, AND ROOTS OF BUSH BEAN PLANTS AT VARIOUS SAMPLING TIMES  59  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 AT VARIOUS SAMPLING TIMES ...  63  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 AT VARIOUS SAMPLING TIMES  65  EFFECT OF KNap ON THE PERCENTAGE DISTRIBUTION 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 EFFECT OF KNap ON THE PERCENTAGE DISTRIBUTION OF ACID INSOLUBLE P, ON A PER PLANT BASIS AMONG LEAF BLADES, STEMS, AND ROOTS OF BUSH BEAN PLANTS AT VARIOUS SAMPLING TIMES  69  EFFECT OF KNap ON THE PERCENTAGE DISTRIBUTION 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  72  EFFECT OF KNap ON THE PERCENTAGE DISTRIBUTION 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 ,  73  EFFECT OF KNap ON THE PERCENTAGE DISTRIBUTION 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  Chapter 2 XXIII.  XXIV.  GLUCOSE ESTER AND ASPARTIC ACID AMIDE FORMATION FOLLOWING THE ADMINISTRATION OF VARIOUS ORGANIC ACIDS TO PLANT TISSUES  86  CHROMATOGRAPHIC DATA  96  Chapter 3 XXV.  TOTAL RADIOACTIVITY, AS muCi, IN THE ETHANOLSOLUBLE, ETHANOL-INSOLUBLE, AND RESPIRED G0 FRACTIONS OF CONTROL AND NAPHTHENATE-TREATED BEAN ROOT TIPS AFTER SUPPLYING l ^ C GLUCOSE ... 117 2  XXVI.  XXVII.  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  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.  XXIX.  XXX.  XXXI.  XXXII.  XXXIII.  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  EFFECT OF KNap ON THE DISTRIBUTION OF ACID SOLUBLE P, ON A PER GRAM BASIS, BY BUSH BEAN PLANTS AT VARIOUS SAMPLING TIMES  15^  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  EFFECT OF KNap ON THE DISTRIBUTION OF ACID SOLUBLE P, ON A PER PLANT BASIS, BY BUSH BEAN PLANTS AT VARIOUS SAMPLING TIMES  156  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  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.  XXXV.  XXXVI.  XXXVII."  XXXVIII.  XXXIX.  XL.  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  EFFECT OF KNap ON THE DISTRIBUTION OF ACID INSOLUBLE P, ON A PER PLANT BASIS, BY BUSH BEAN PLANTS AT VARIOUS SAMPLING TIMES  l60  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  l6l  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  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  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  KEY TO THE AMINO ACIDS AND GLUCOSE SHOWN IN FIGURE 15.  * 5 6  LIST OF FIGURES FIGURE  PAGE Chapter 1  1,  2.  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  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 ... 4 l  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  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  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  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  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  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  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  5.  6.  7.  8.  9.  10.  Chapter 3 11.  12.  13.  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  RADIOACTIVITY, AS muCi, IN INDIVIDUAL ETHANOLSOLUBLE AMINO ACIDS AND IN GLUCOSE FROM CONTROL AND NAPHTHENATE-TREATED BEAN ROOT TIPS AFTER SUPPLYING l ^ C GLUCOSE  120  RADIOACTIVITY,. AS muCi, FOUND IN ASPARTIC ACID, GLUTAMIC ACID, AND ALANINE FROM THE ETHANOLINSOLUBLE HYDROLYSATE FROM CONTROL AND NAPHTHENATE-TREATED BEAN ROOT TIPS AFTER SUPPLYING 1*C GLUCOSE  124  Appendix 14.  STANDARD CURVE FOR INORGANIC PHOSPHATE DETERMINATIONS  152  15.  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 g r a t i t u d e t o the many people who hav£ helped w i t h t h e v a r i o u s aspects o f t h e o v e r a l l study. a p p r e c i a t i o n i s extended his  Most s i n c e r e  to my a d v i s o r , P r o f e s s o r D. J . Wort, f o r  c o u n s e l , guidance, and support throughout t h e course o f these  studies.  I n a d d i t i o n , the author wishes to thank him f o r h i s  c r i t i c a l review o f t h i s manuscript. The a s s i s t a n c e and a d v i c e g i v e n by the o t h e r members o f my committee, Drs. B. A. Bohm, Department o f Botany;  G. W. Eaton,  Department o f P l a n t S c i e n c e ; A. F. Gronlund, Department o f M i c r o b i o l o g y ; and S. H. Zbarsky, Department o f B i o c h e m i s t r y a r e g r a t e f u l l y acknowledged.  A l s o , to Drs. B. A. Bohm and C. E. S e a f o r t h  ( D i v i s i o n o f Chemistry, U n i v e r s i t y o f the West I n d i e s , T r i n i d a d ) who was a guest r e s e a r c h e r i n our department  d u r i n g 1968 - 1969»  I extend c o r d i a l thanks f o r the v e r y i n t e r e s t i n g and i n f o r m a t i v e d i s c u s s i o n s c o n c e r n i n g a p o r t i o n o f the m a t e r i a l presented i n Chapter 2. C o n s t r u c t i v e remarks c o n c e r n i n g t h i s manuscript g i v e n by the E x t e r n a l Examiner, Dr. R. T. Wedding (Department  o f Biochemistry,  U n i v e r s i t y o f C a l i f o r n i a , R i v e r s i d e ) were g r e a t l y a p p r e c i a t e d . F i n a n c i a l a s s i s t a n c e from the U n i v e r s i t y o f B r i t i s h  Columbia  i n t h e form o f a Graduate F e l l o w s h i p , and a Graduate Research from the Northwest  Grant  S c i e n t i f i c A s s o c i a t i o n a r e acknowledged w i t h  thanks. The s t u d i e s were c a r r i e d out i n the Department o f Botany, U n i v e r s i t y o f B r i t i s h Columbia, his  and the author wishes to express  a p p r e c i a t i o n o f t h e e x c e l l e n t f a c i l i t i e s made a v a i l a b l e to him.  F i n a l l y , I extend warm and s i n c e r e thanks to my w i f e , G e r r i e , and to my f a m i l y f o r t h e i r h e l p and encouragement throughout.  For t h i s , I d e d i c a t e t h i s d i s s e r t a t i o n  to them.  NAPHTHENIC ACIDS The name, naphthenic a c i d , was f i r s t suggested by Marko v n i k o f f and O g l o b l i n ( 1 0 1 ) f o r the C ^ H g ^ s t r u c t u r e which H e l l and Medinger Rumanian crude o i l .  a c i d s o f unknown  ( 73 ) had r e c o v e r e d from  Naphthenic a c i d s e x t r a c t e d from petroleum  are known to be a v e r y complex mixture o f chemical compounds. As noted by J o l l y  ( 78 )» c y c l o p e n t y l d e r i v a t i v e s  predominate  i n the naphthenic a c i d mixture f o l l o w e d by c y c l o h e x y l compounds. Naphthenic a c i d s a r e a l s o known as petroleum a c i d s ,  because  branched-chain a l i p h a t i c a c i d s and phenols a r e p r e s e n t i n some crude o i l s .  Those naphthenic a c i d s having an average m o l e c u l a r  weight o f 2 l 4 have been shown by Cason and Khodair ( 3 8  ) to  c o n s i s t c h i e f l y o f compounds which c o n t a i n 10 to 2 0 carbon atoms.  The commercially a v a i l a b l e HNap used i n these i n v e s t i -  g a t i o n s has a r e p o r t e d m o l e c u l a r weight o f 2 3 0 . B a i v a r o v s k a y a e t a l ( 21 ) i n v e s t i g a t e d the p r o p e r t i e s o f a HNap mixture which was o b t a i n e d from crude o i l o f the Perm region i n Russia.  Petroleum from the southern Perm r e g i o n had  an a c i d number o f 0 . 2 3 mg KOH/g HNap. (180°  The d i e s e l o i l f r a c t i o n  - 400° C) c o n t a i n e d 0 . 0 3 2 ^ HNap which was e x t r a c t a b l e  w i t h k% NaOH.  The NaOH was mixed w i t h the d i e s e l o i l f o r 15  minutes, and the mixture was allowed to s e t t l e f o r 4 to 5 hours. The m o l e c u l a r weight o f the NaNap was ca 3 0 0 .  The s o l u b i l i t y  o f NaNap i n water was 2 0 , 3 0 , and ko g / 1 0 0 ml a t 2 0 , 5 0 , and 100°  C, r e s p e c t i v e l y . I t i s v e r y i n t e r e s t i n g to note t h a t under the heading o f  'naphthenic a c i d *  i n the Merck Index  ( 1 3 3 ) o n l y one compound  COOH  CO OH  Cyclopentanecarboxylic C5H °2 ~  m  o  1  w  acid  Cyclohexanecarboxylic a c i d  t  C  1 0  7 12°2 ~ H  m  o  1 w  t  1  2  8  COOH  3-dimethyl-4-ethylc y c l o p en t y l - n - b u t a n o i c a c i d C  13 24°2 " H  m  o  1  w  t  2  1  2  COOH  2,4-dimethyl-5-dimethylcyclohexyl-n-pentanoic acid * 15 28°2 " C  H  m  o  N A P H T H E N I C  1  w  t  A C I D S  i s d e s c r i b e d , namely CHCA ( C ^ H ^ ^ ) *  appears  c l a s s i f i c a t i o n system has p e r s i s t e d s i n c e  1910  that -  1920 when  the Chemical A b s t r a c t s ' i n d i c e s gave hexahydrobenzoic (CHCA) as a c r o s s r e f e r e n c e f o r naphthenic a c i d .  this  acid  However,  i n the l a t e s t e d i t i o n o f the Merck Index ( 13^ ) HNap, as an entry, has been d e l e t e d , and CHCA i s entered s e p a r a t e l y w i t h no c r o s s r e f e r e n c e to HNap. The c a r b o x y l group o f most naphthenic a c i d s i s n o t a t t a c h e d d i r e c t l y to the a l i c y c l i c r i n g , b u t i s separated from the r i n g by an a l i p h a t i c s i d e c h a i n c o n t a i n i n g one to f i v e o r more methylene groups ( p r e c e d i n g page).  A c c o r d i n g to J o l l y  g e n e r a l formula may be w r i t t e n as R ( C H ) C 0 0 H , 2  where R i s an  n  a l i c y c l i c nucleus composed o f one o r more r i n g s . i n the f i e l d o f petroleum  ( 78 ), a  Other  authorities  chemistry ( 58 ) have s t a t e d t h a t the  naphthenic a c i d mixture c o n t a i n s a wide v a r i e t y o f s u b s t i t u t e d cyclopentane^ and cyclohexanes. ual  The molecular weight o f i n d i v i d -  a c i d s i n the mixture ranges from the lowest (CPCA, mol wt 1 1 4 ,  shown on the p r e c e d i n g page) to compounds w i t h m o l e c u l a r o f over 1 , 0 0 0 .  weights  The authors a l s o s t a t e d t h a t t h e r e i s no evidence  t h a t cyclopropane, cyclobutane, cycloheptane, o r h i g h e r r i n g d e r i v a t i v e s a r e p r e s e n t i n crude petroleum. and Cason and Khodair  ( 38 ) have demonstrated  nucleus ( e i t h e r cyclopentane o r cyclohexane) w i t h methyl o r e t h y l  Cason and Liauw ( 37 ) t h a t the a l i c y c l i c  can be s u b s t i t u t e d  groups.  G a s - l i q u i d chromatography i n our l a b o r a t o r y has r e v e a l e d t h a t the HNap mixture i s indeed v e r y complex. of  t h e methyl  Gas chromatograms  e s t e r s (MeNap) o f HNap showed t h a t peaks o f lower  molecular weight  components comparable to cyclopentane-,  cyclo-  hexane-, and c y c l o h e p t a n e c a r b o x y l i c a c i d s were n o t e v i d e n t i n  the t r a c i n g o f the commercial HNap p r e p a r a t i o n .  A gas chromato-  gram o f t h e naphthenates d e r i v e d from t h e u n r e f i n e d  diesel  f r a c t i o n o f a Venezuelan crude o i l y i e l d e d a t r a c i n g i n which most o f the components appeared to be i n the and w i t h i n t h i s range t h e r e were 25 major peaks.  to C ^ range, Present,  though i n r e l a t i v e l y s m a l l e r amounts, were C 5 , C 7 , and C 3 compounds.  When compared w i t h the c y c l o h e x y l - n - b u t r y r a t e  standard, i t appeared as though a C  1 Q  a c i d was p r e s e n t i n the  h i g h e s t c o n c e n t r a t i o n i n both HNap p r e p a r a t i o n s . s t r u c t u r e below might r e p r e s e n t t h i s C  1 0  The chemical  compound.  COOH  The range o f components i n both MeNap p r e p a r a t i o n s was s i m i l a r . I t should a l s o be noted t h a t the bases o f the peaks seemed to have been obscured by what may have been a p h e n o l i c component. When the p h e n o l i c compound(s) were p a r t i a l l y removed from the naphthenate mixture on one o c c a s i o n and were subsequently chromatographed,  a s i n g l e s p o t was o b t a i n e d which gave a  p o s i t i v e p h e n o l i c r e a c t i o n when sprayed w i t h d i a z o t i z e d p-nitroaniline.  Also u s i n g g a s - l i q u i d chromatography,  ( 46 ) confirmed the presence o f f o r m i c , a c e t i c ,  Eider  isobutryic,  CPCA, CHCA, c y c l o h e x a n e a c e t i e , and s e v e r a l o t h e r low m o l e c u l a r weight a c i d s i n the naphthenic a c i d f r a c t i o n from an Aruba crude  oil. Some i d e a o f the c o m p l e x i t i e s i n v o l v e d 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  graphy, mass spectroscopy, and  ).  U s i n g g a s - l i q u i d chromato-  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 a c i d f r a c t i o n from an A u s t r i a n crude o i l to cont a i n ca 120  compounds, 20 o f which c o n s t i t u t e d more than  o f the sample. and  P o s i t i v e i d e n t i f i c a t i o n was  3 - e t h y l branched-chain f a t t y a c i d s .  s t i t u t e d c y c l o p e n t y l and  (123  tions, Seifert  Small amounts o f sub-  ,125  ,124  ) has  a c i d f r a c t i o n o f crude o i l . polynuclear  No  In s e v e r a l r e c e n t considerably  knowledge o f the compounds which are present  that terpenoid  made f o r 3-methyl  c y c l o h e x y l a c i d s were found.  s t r a i g h t - c h a i n a c i d s were d e t e c t e d .  50%  publica-  extended  i n the  our  carboxylic  Results o f these s t u d i e s have shown saturated  and  polynuclear  aromatic,  as w e l l as h e t e r o c y c l i c c a r b o x y l i c a c i d s occur n a t u r a l l y i n petroleum.  The  conventional  view o f the naphthenic a c i d s i n  petroleum b e i n g p r i m a r i l y mononaphthenic and expanded to i n c l u d e 40 new  alkanoic acids  was  classes of carboxylic acids.  Naphthenic a c i d s have a c h a r a c t e r i s t i c odor which v a r i e s w i t h the a c i d source, degree o f r e f i n e m e n t and and  s u l f u r compounds.  content o f phenol  These a c i d s are r e a d i l y s o l u b l e i n  non-  p o l a r s o l v e n t s ; however, the lower m o l e c u l a r weight members, e.g.  CHCA and  CPCA, may  be  s l i g h t l y s o l u b l e i n water.  The  occurrence, composition, chemical p r o p e r t i e s , recovery, n o n - b i o l o g i c a l uses o f HNap have been given by J o l l y a f a c t i n passing, thenic acids  i n 1970  (78  and ).  As  ca 7 0 $ o f the 1 5 . 7 0 0 tons o f naph-  (as metal s a l t s ) was  used i n the U n i t e d  States  p r i m a r i l y as d r i e r s i n o i l - b a s e d p a i n t s , c a t a l y s t s i n r e f i n e r y operations, The  and l u b r i c a n t s .  use o f naphthenic a c i d s and  t h e i r s a l t s as p l a n t growth  stimulators  i s r a t h e r r e c e n t , and  as such, they have been u t i -  l i z e d p r i m a r i l y by B u l g a r i a n , Russian, L i t h u a n i a n , and  Albanian  plant physiologists.  In low  concentrations  thenates have been found to promote v e g e t a t i v e growth, and  and  to a f f e c t other p h y s i o l o g i c a l and  a c t i v i t i e s i n a l a r g e number o f p l a n t s .  ( 98,  157  naphthenic a c i d s s t i m u l a t e species  ( 57,  126  ).  naph-  reproductive  biochemical  There are a l s o  r e p o r t s t h a t when a p p l i e d a t high c o n c e n t r a t i o n s a c t as h e r b i c i d e s  Canadian,  several  naphthenates  I t has been demonstrated t h a t  the growth o f a number o f animal  ).  The mechanism or mode by which naphthenates i n f l u e n c e p l a n t metabolism, or i t s 'modus operandi',  i s i m p e r f e c t l y known.  It  has been e s t a b l i s h e d t h a t to o b t a i n a maximum response a s i n g l e a p p l i c a t i o n o f HNap o r i t s potassium s a l t should be a p p l i e d to bean p l a n t s a t a p a r t i c u l a r ontogenetic concentration  ( 142  ).  stage and  naphthenate a p p l i c a t i o n may  final result  the method  s i z e o f the d r o p l e t or dust p a r t i c l e  the p l a n t ' s v i g o r may  mining the f i n a l response.  two  of applied.  s p e c i e s o f p l a n t , the p a r t o f the p l a n t to which the  i s a p p l i e d , and  of  be determined not o n l y by the  parameters g i v e n above, but a l s o by the pH,  The  specific  However, as i s the case w i t h many other  chemical growth r e g u l a t i n g compounds, the  a p p l i c a t i o n , and  at a  chemical  also play a part i n deter-  Moreover, environmental  such as temperature, l i g h t i n t e n s i t y and  conditions  q u a l i t y , humidity  the a v a i l a b i l i t y o f water and n u t r i e n t s are a l s o  and  important  i n d e t e r m i n i n g the f i n a l outcome;. P r e l i m i n a r y work i n our l a b o r a t o r y has  i n d i c a t e d t h a t 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^  their identification i s d i f f i c u l t .  When i n the f r e e form, the r e a c t i o n o f the c a r b o x y l group w i t h an a c i d - b a s e i n d i c a t o r , such as bromophenol b l u e , together R  f  values, provides  the o n l y means o f i d e n t i f i c a t i o n when non-  r a d i o a c t i v e m a t e r i a l i s used.  The  location of a c t i v i t y  radiochromatograms o f e x t r a c t s o f p l a n t m a t e r i a l may i d e n t i f y the f r e e a c i d s and a p p l i c a t i o n of Following  with  on  serve  their derivatives following  to  the  l a b e l l e d naphthenic a c i d s . t h e i r a p p l i c a t i o n to bean p l a n t s , naphthenates  s t i m u l a t e many p h y s i o l o g i c a l and b i o c h e m i c a l ( 52,  u l a t i o n o f photosynthesis protein synthesis  (130,  1^7  53  ), and  numerous enzymes i n crude e x t r a c t s  processes.  Stim-  )» dark r e s p i r a t i o n ( 5 2 , the s p e c i f i c a c t i v i t i e s ( 40,  52,  53»  1^7  53  ),  of  ) suggests  t h a t naphthenate s t i m u l a t i o n o f p l a n t growth i s the r e s u l t o f the a c t i o n o f the chemical,  or i t s m e t a b o l i t e s ,  g e n e t i c and m e t a b o l i c l e v e l s ( 1^7  ).  I t was  a t both  a l s o suggested  t h a t s i n c e numerous m e t a b o l i c l o c i are i n v o l v e d , the must be a g e n e r a l  stimulation  one.  Where experiments c o i n c i d e , the r e s u l t s o b t a i n e d l a b o r a t o r y tend to confirm  f o r the most p a r t the data  by s c i e n t i s t s i n the I r o n C u r t a i n c o u n t r i e s . p a s t two  i n our reported  Also, during  years the metabolism o f K C H C - ? - ^ ( 108,  and KNap ( 122  the  1  127,  the  131  ),  ) have been s t u d i e d i n Phaseolus v u l g a r i s L.  As f a r as the b i o l o g i c a l d e g r a d a t i o n o f naphthenic a c i d s i s concerned, O s n i t s k a y a ( 107  ) reported  t h a t a pure c u l t u r e o f  microorganisms capable o f degrading HNap was  i s o l a t e d from  naphthene petroleum and waste water o f a r e f i n e r y .  A pure  c u l t u r e o f these a e r o b i c organisms (unnamed) o x i d i z e d 90?S o f the naphthenic a c i d s on which they grew within/.,!0.2days.  S i g n i f i c a n t amounts o f i n t e r m e d i a t e ducts were not d e t e c t e d  metabolic  breakdown pro-  i n the growth medium.  T h i s was  taken  to CO2  to i n d i c a t e t h a t o x i d a t i o n o f HNap had progressed  and  H 0. 2  The  p l a n t growth s t i m u l a t o r s obtained  been named and  abbreviated  from petroleum have  d i f f e r e n t l y by v a r i o u s  researchers,  v i z . , naphthenic growth substances (P.G.S.), naphthenic a c i d s (HNap), potassium naphthenates (KNap), o i l hormone  substance  (O.H.S.), naphtha growth matter, o i l growth matter, petroleum growth promoters, petroleum petroleum p r o d u c t ) ,  Sh-8  nutrient, sintovit  (a c y c l o h e x y l b u t a n o l ) ,  (an o x i d i z e d and  petroleum  or naphthenic growth-helping substances (P.R.V. or N.R.V.). In the R u s s i a n language the i n i t i a l s R. V. stand h e l p i n g substance.'  f o r 'growth  S c i e n t i s t s i n several Iron Curtain  countries  a l s o r e p o r t the use o f a B u l g a r i a n naphthenate p r e p a r a t i o n , H.T.I., which i s v e r y s i m i l a r to N.G.S. What appears to be the f i r s t p u b l i s h e d r e p o r t o f the use naphthenic a c i d s i n b i o l o g i c a l r e s e a r c h dates back to  of  1921.  However, the p r a c t i c a l a p p l i c a t i o n o f naphthenates to p l a n t s f o r purposes o f s t i m u l a t i n g p l a n t growth appeared i n the R u s s i a n l i t e r a t u r e i n 1956.  During the p a s t f i f t e e n years  the e f f e c t s  o f naphthenic a c i d compounds on the p h y s i o l o g i c a l and processes  i n p l a n t s have been s t u d i e d p r i m a r i l y by i n v e s t i g a t o r s  i n the I r o n C u r t a i n c o u n t r i e s and The  biochemical  e f f e c t s o f naphthenates  composition  i n Canada. on p l a n t growth,  are summarized i n Table  I.  yield,  and  TABLE I.  E f f e c t s o f naphthenates on growth,  PLANT  MODE OF APPLICATION  , and composition o f p l a n t s  NAPHTHENATE CONCENTRATION  Apple, s e e d l i n g s Beans  seed soak to c u t t i n g s  0.00k%  Beans Beans, bush  foliar  0.5%  Beans, bush  foliar  0.5%  Beans, bush  foliar  0.25-0.5%  Beans, bush  f o l i a r ( d u s t ) 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 (emulsion) foliar 0.5%  Beans, bush  foliar  0.5%  EFFECT* Growth and development* + Number o f and f r e s h weight of r o o t s * + Growth* + Plant height* + Number o f and a r e a o f l e a f lets* + Dry weight o f r o o t s , stems, and l e a v e s * + Green pod y i e l d * + (20%) Ripe seed p r o d u c t i o n * + (8%) Dry weight o f v e g e t a t i v e p a r t s * + (20.7%) Y i e l d o f green pods* + (23.4 - 26.5%) Y i e l d o f green pods: + Y i e l d o f green pods « + Y i e l d o f green pods* + (12%) Y i e l d o f green pods* no significant effect V e g e t a t i v e and r e p r o d u c t i v e growth * + Fresh and d r y weights* + (21 and 22%) Weight o f green pods* +  REFERENCE ( 12l ) ( 44 ) ( 115 ) ( 52 )  ( 1^2 )  ( 144 ) . ( 145 )  (  5^ )  ( 143 )  (2338)  Beans, bush Beans, bush  folair foliar  0.5% 0.5%  Beans, bush  foliar  0.5%  Weight of r i p e seed* + (24$) T r a n s p i r a t i o n * - (12%) C h l o r o p h y l l and c a r o t e n o i d content o f l e a v e s * + A s c o r b i c a c i d o f pods* + (26%)  (128) ( 52 ) (  52 )  Beans, c h i n a Beans, feed Beans, f i e l d Beet Beet Cabbage Cabbage Cabbage Cabbage Cabbage Cabbage Cabbage Cabbage Cabbage Carnation  seed soak & foliar seed soak to s o i l seed soak to s o i l foliar to s o i l foliar foliar to s o i l to s o i l foliar  Pod y i e l d : 0 . 0 0 5 & 0.01% 10-40 g/seed 100 g/ha 0.005-0.055S 0.25$ 0.005$ 5 0 - 1 0 0 g/ha 2 5 - 1 0 0 g/ha 100 g/ha 0.0005-0.01% 0.005-0.05$ 0.0001-0.001$  seed soak seed soak foliar seed soak & foliar  20 mg/g seed & 20 mg/m  Carrot Carrot  to s o i l foliar  100 g/ha 0.0005-0.01$  Carrot Cherry Corn (maize)  seed soak to c u t t i n g s seed soak  0.005-0.05%  0.01% 0.5%  Corn  foliar  0.005%  Corn  foliar seed soak foliar  0.005$ 0.005-0.05$ 0.5$  Corn  2  +  Germination and growths + Y i e l d i + (2 t o 1 6 $ ) Y i e l d * + (20 to 2 5 $ ) Y i e l d i + (20.5$) Y i e l d j + (206$) Y i e l d 1 + (15 to 20$) R i p e n i n g o f heads: + Y i e l d * + ( 1 3 to 3 2 $ ) Y i e l d t + ( 1 5 to 26$) Y i e l d t + (12 t o 28$) Y i e l d s + (20 to 2 5 $ ) Metabolism, growth, and development 1 + Yields + (30$) Yieldi + (15.7$) Seed moisture c o n t e n t * + Ascorbic acid* + Size of flowers * + Number o f f l o w e r s * + (2?$) Number o f seeds produced* + (54$) Y i e l d * + (20 to 2 5 $ ) Metabolism, growth, and development* + Yield* + Y i e l d * + (16$) Root f o r m a t i o n * + Hay p r o d u c t i o n * + (21 t o 33$) Corn s i l a g e and corncobs* + (13-43 and 6-28 m e t r i c centners/ha, r e s p . ) Growth and y i e l d * + Y i e l d * + (18 to 3 5 $ ) Fresh and d r y weights o f f o l i a g e * + (18 & 16$, r e s p .  Corn Corn Corn Corn Com  foliar seed soak  0.005$ 0.0001-0.001$  Corn Cotton Cotton  foliar  0.005$ 0.0001$  Cotton  to s o i l w i t h mulch seed soak seed soak 0.01$ seed soak 0.01$ seed soak & 0.01$ fertilizers 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 0.01$ foliar  Cotton Cotton  to s o i l  100 mg/40 kg dry s o i l  Cotton  foliar  0.005$  Cranberry Cucumber  to c u t t i n g s seed soak foliar seed soak  — 0.0001$ 0.05$ 0.005-0.05$  Cotton Cotton Cotton  Cucumber  Yield: + Dry weight* + ( 3 3 $ ) Growth* + Corn s i l a g e * + (132 m e t r i c centners/ha) Water r e t e n t i o n * + Seed moisture content* + Free and bound water* + Chlorophyll content* + Root growth* + S o i l temperature* + Germination and f r u i t i n g * + Boll yield* + (3.1$) Yield* + (23$) Y i e l d * + (5 to 10$) Y i e l d * + (10 to 1 5 $ ) Growth and r i p e n i n g * Yield*  +  (138 ) (113 ) ( 89 ) ( 149 ) ( 90 ) ( 149 ) ( 75 ) ( 20 ) ( 65 ) ( 132 ) ( 104 )  +  (20$)  Crude f i b e r y i e l d * + C h l o r o p h y l l and a s c o r b i c acid i n leaves* + Ascorbic acid i n leaves* + T o t a l and a c t i v e a b s o r b i n g surface of roots* + Dry weight of r o o t s * + Auxin t r a n s p o r t * + Auxin a c t i v i t y * + Root f o r m a t i o n * + Root growths + Y i e l d * + (40$) Y i e l d * + (20.1$)  ( 2 3 ) ( (  h ) 17 )  (  25 )  ( 150 ) (75) (  79 )  0.01$ 0.05$  Currants, black Eggplant  to cuttings foliar  Eggplant F o x t a i l , meadow  foliar foliar  Gooseberry Gooseberry Grape  to c u t t i n g s 0 . 0 1 $ to c u t t i n g s -— 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 0.005$ foliar foliar 0.005$ foliar seed soak & ultrasonic treatment 0.0005$ foliar  Grape Hemp Lucerne Melon  0.005$ 0.005-0.1$ & NH4NO0 J  Millet Mulberry Muskmelon  seed soak foliar foliar  0.005$ 0.0005$ 0.0005-0.01$  Oat  foliar  0.1$  Olive Onion Onion Onion  foliar to s o i l  0.0005$ 100 g/ha 0.0001$ 10-3 - 10-5$  Onion Onion Peas  foliar seed soak & seed i r r a d iation seed soak seed soak  0.0001-0.001! 0.005-0.05$ 5 - ^ 5 g/seed  Root f o r m a t i o n : + (26$) Height, a e r i a l mass and t o t a l weight: + ( 2 1 , 1 2 , and 6$, r e s p . ) Yield: + (32$) Y i e l d o f green mass: + (23$) Root f o r m a t i o n : + ( 4 5 $ ) Root f o r m a t i o n : + Weight o f c l u s t e r s : + (28$) Yield: + (10$) Growth: + Yield: + (20$) P h y s i o l o g i c a l responses:  +  Ascorbic acid i n f r u i t s : + Grain y i e l d : + ( 8 $ ) Growth and development: + Metabolism, growth, and development: + Yield: + (30$) Y i e l d o f green mass: + (10 t o 14$) Growth and development: + Y i e l d : + (20 to 2 5 $ ) Root growth: + Rate o f c e l l d i v i s i o n and rootlet length: + Rate o f c e l l d i v i s i o n : Yield: + (36$) Y i e l d o f f r u i t : + (7 to 10$)  ( 114 ) ( 8)  (  (75) 3)  ( 114 ) ( 91 ) ( 118 ) ( 87 ) ( 77 ) ( 47 ) ( 2)  ( 103 ) ( 67 ) ( 15 ) (  3)  ( 67 ) ( 157 ) ( 75 ) ( 63 ) ( 62 ) ( 79 ) ( 118 )  Peas Pasture ( g r a s s , legumes, U r t i c a , Stachys) Phlox Phlox Pine, E l d a r (Pinus e l d a r i c a ) Poplar Potato  Potato  foliar foliar foliar to c u t t i n g s seed soak to c u t t i n g s foliar tuber soak soil  0.005-0.01$ 10$  NH4NO3 &  2 0 $ KNap (800 1/ha) 12.5, 25. 59-4 1,00 ppm 0.005$ 0.02-0.05$ 0.0005$ 250  Potato  to  Potato, e a r l y Potato, e a r l y (Lorkh)  foliar foliar  63 ppm 125 g/ha  Potato, e a r l y (Warba)  foliar  0.5$  Potato  tuber soak  0.005$  Privet Radish  to c u t t i n g s foliar  0.5$  Rice Rice Rye, w i n t e r  foliar seed soak seed soak  0.05$ 10 g / g r a i n 8-16 g/100 kg of seed  Seeds(unspecified)  seed soak  cm /ha 2  Growth 1 + Herbicidal  effects:  ( (  79) 98 )  Growth 1 +  ( 112 )  Root formations + S e e d l i n g s had a g r e a t e r s a l t tolerance Root formations 0 Leaf areas + Tuber y i e l d s + (1?.4$) Number o f s h o o t s / p l a n t s + Tuber y i e l d s + ( 9 . 9 $ ) Tuber y i e l d s + (48$) Water a b s o r p t i o n s + Tuber y i e l d s + ( 3 5 to 40$) Tuber y i e l d s + (2?$) Tuber y i e l d s + Starch concentration of tuberss + Weight o f t u b e r s / p l a n t s + (42$) Tuber numbers 0 Dry matters 0 Starch concentration of tubers s 0 A s s i m i l a t i v e s u r f a c e s and c h l o r o p h y l l content o f leaves s + Root formations + Fresh and d r y weight o f p l a n t s s + (11 & 11$, r e s p . ) Y i e l d s + (28$) G r a i n y i e l d s + (11.1$) F r u i t b e a r i n g stemss + G r a i n y i e l d s + (10$) Growths +  ( 150 ) ( 6) ( 150 ) ( 94 )  ( 92 ) (  2)  ( 118 ) ( 1) ( 146 )  ( 94 ) ( 150 ) ( 143 ) ( 75 ) ( 118 ) ( 118 ) (  9)  ^  Sophore, s e e d l i n g s seed soak 0.005$ (Sophora .iaponica L.) Spindle-tree to c u t t i n g s (Euonymus sp.) seed soak 0.005-0.01$ Sugar beet Sugar beet  foliar  0.5$  Sugar beet  foliar  0.005$  Sunflower  seed soak  0.005 & 0.01$  Tangerine Tea Tobacco Tobacco Tobacco,.Oriental  foliar foliar  0.05$ 0.05$  foliar to s o i l & foliar to s o i l  15 mg/m & 2 mg/plant 15 mg/m  Tomato  foliar  0.0005-0.01$  Tomato Tomato Tomato  to s o i l foliar foliar  0.25$ 0.005-0.05$ 50-100 g/ha  Tomato Tomato  to f l o s c ules seed soak f o l i a r during flowering  0.005$  0 2  2  during planting + 0.005$ l a t e r a t 500 1/ha  0.05-0.01$  0.005-0.05$ 0.01$  Greater s a l t t o l e r a n c e in salinized s o i l s Root f o r m a t i o n ! 0 Beet r o o t crops + (26 m e t r i c centners/ha) Fresh and d r y weights o f f o l i a g e s + (13 & 14$, r e s p . ) Water c o n t e n t ! + Chlorophyll content! + Stem s i z e i + Flower number* + Weight o f s e e d s ! + Oil yields + Y i e l d ! + (18.6 to 21.2$) Y i e l d ! + (39$) Leaf a r e a and growth* + Growth and l e a f a r e a * + Y i e l d o f leaves* + (7$) T o t a l f r e s h weight o f 100 plants* + (23.5$) Metabolism, growth, development, and y i e l d * + F r u i t y i e l d * + (38$) F r u i t y i e l d * + (40 t o 50$) F r u i t y i e l d ! + (30 t o 37$)  Ovary f o r m a t i o n and f r u i t maturation * + Onset o f f l o w e r i n g * + Bud f o r m a t i o n ( f l o w e r ) i + Fruit yield! + Y i e l d o f f r u i t i + (22$)  (150 ) ( 149 ) ( 143) ( 149 ) ( 88 )  ( 102 ) (75) ( 151 ) (75) ( 116 )  ( 15 ) ( 64) ( 75) ( 7 )  ( 79 ) ( -79.)  Tomato Tomato Tomato  foliar foliar foliar  0.5$ 0.005$ 0.005$  Tomato  foliar  0.5$ 0.0001-0.0007$  Trees(unspecified) Turnsole (Chrocophora sp.) Weeds(unspecified) Weeds(unspecified) Wheat, w i n t e r Wheat  during cultivation foliar seed soak seed soak seed soak  Wheat, Marquis spring  seed soak foliar  0.0001-0.1$ 0.4$ 8 l l 6 g / 1 0 0 kg of seed 0.01$ 0,5# 0.0001$ 1 5 . 2 g / 1 0 0 kg seed  Wheat, w i n t e r Wheat, w i n t e r  seed soak  Vine Vine ( 7 - y e a r - o l d , v a r . Tamyanka)  to c u t t i n g s f o l i a r p r i o r 100 ppm to f l o w e r i n g  +*  Stimulation!  -:  Inhibition!  0:  Number o f f r u i t s 1 + Fruit yield: + Ascorbic a c i d content of fruit: + Ascorbic a c i d content o f fruit: 0 P o l l e n g e r m i n a t i o n and p o l l e n tube growth: + Sucrose c o n t e n t / f l o w e r , and honey y i e l d / h a : + Herbicial effects: + Germination and growth* + Grain y i e l d : + ( 3 0 $ ) G r a i n y i e l d * + (10$) Weight o f g r a i n / p l a n t : + ( 1 2 $ ) Fresh and d r y w e i g h t o f l e a v e s * + (8 & 8$, r e s p . ) Weight o f r i p e g r a i n : + (10$) Root growth: + Grain y i e l d * + ( 9 . 9 $ ) Root f o r m a t i o n * + C h l o r o p h y l l a & b: Phaeophytin :""*+ Carotene: + Lutein: + Flavoxanthin: + Violaxanthin* +  No e f f e c t ;  :  Followed by.  ( 40 ) ( 110 ) ( 7 ) ( 40 ) ( 93 ) (111  )  (157 ) ( 80 ) (132 ) (118.) ( 143 )  ( 75 ) ( 118 ) ( 150 ) ( 95)  Chapter 1  THE EFFECT OF POTASSIUM NAPHTHENATES ON THE UPTAKE, DISTRIBUTION, AND INCORPORATION OF PHOSPHORUS-32.  INTRODUCTION T h i s phase o f the i n v e s t i g a t i o n d e a l t with the e f f e c t which KNap had on P uptake, d i s t r i b u t i o n , and i n c o r p o r a t i o n i n bush bean p l a n t s .  Phosphorus was chosen f o r t h i s study because o f  the c e n t r a l r o l e which t h i s element p l a y s i n c e l l u l a r metabolism, photosynthesis,  and the g e n e t i c s o f p l a n t s .  7. Moreover, •: the  amount o f d e f i n i t i v e s c i e n t i f i c r e s e a r c h d e s c r i b i n g the e f f e c t s o f naphthenates on P a s s i m i l a t i o n i s v e r y l i m i t e d . magnitude  Both the  o f the P response t o naphthenate treatment, and the  d i s t r i b u t i o n p a t t e r n s which emerged a t the f o u r sampling times d u r i n g t h e 24 hour h o l d i n g p e r i o d were s t u d i e d . Phosphorus a b s o r p t i o n by p l a n t s i s a process metabolic  energy.  Therefore,  which r e q u i r e s  the P d i s t r i b u t i o n p a t t e r n s  which  emerge a t v a r i o u s sampling times f o l l o w i n g the exposure o f the r o o t s to the r a d i o i s o t o p e , ^ P , would be r e l a t e d to the p l a n t ' s 2  metabolic  activity,  c o n c e n t r a t i o n o f P, and i n t e r n a l requirement  f o r 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 e s p e c i a l l y  important p o i n t as the p l a n t s used i n s e v e r a l experiments were grown i n a -P n u t r i e n t s o l u t i o n both b e f o r e and a f t e r naphthenate treatment. A c t u a l comparisons, which have been made between l e a f  blades,  stems p l u s p e t i o l e s , o r r o o t s o f c o n t r o l and n a p h t h e n a t e - t r e a t e d plants, includes  t o t a l , a c i d s o l u b l e , and a c i d i n s o l u b l e ^2p  a c t i v i t y } and t o t a l , a c i d s o l u b l e , and a c i d i n s o l u b l e P.  The  comparisons were made 4, 8, 12, and,24 hours a f t e r t h e r o o t s o f c o n t r o l and t r e a t e d p l a n t s were removed from a n u t r i e n t s o l u t i o n which contained ^ P . 2  LITERATURE REVIEW As f a r back as l 8 6 l , m a t e r i a l s may reported and  ) suggested t h a t  circulate within plants.  that ^ P  was  2  there was  Hartig ( 7 1  L a t e r , Biddulph ( 26  In 1 9 5 7 .  Helder ( 72  continually circulat-  ) confirmed the  fact  t h a t a c o n t i n u a l c i r c u l a t i o n o f P d i d occur i n b a r l e y and plants.  B i d d u l p h et a l ( 2 7  the absorbed -^ P 2  displayed a sustained  c i r c u l a t i o n throughout I t i s not  inconceivable  t h a t an i n d i v i d u a l P atom, i f not  captured, may  make s e v e r a l c y c l e s w i t h i n a p l a n t d a i l y .  i n v e s t i g a t i o n s have shown t h a t P uptake from an i s an a c t i v e p r o c e s s ( 1 1 9 ) .  bean  ) a l s o observed t h a t a p o r t i o n o f  the p l a n t d u r i n g a 96 hour experimental p e r i o d .  metabolically Numerous  e x t e r n a l medium  To augment t h i s f a c t i t has  been  r e c e n t l y demonstrated by Bledsoe et a l ( 31 ) t h a t P uptake ATP  synthesis  i n excised  when o l i g o m y c i n  was  Edwards ( 4 5 )  corn r o o t s were c o n c u r r e n t l y  present  P absorption  t h a t when the P c o n c e n t r a t i o n  l e s s than 1 mM,  He  absorption  on the r o o t s u r f a c e .  than 1 mM  one  was  by  sites  curve was  The  author suggested t h a t a t higher P c o n c e n t r a t i o n s  was  c o n s i s t e n t w i t h the involvement o f a p a s s i v e P  at  of the  the  However, a t  l i n e a r absorption  in  was  suggested t h a t e i t h e r both s i t e s  plasmalemma w i t h the other r e p r e s e n t i n g  tions greater  P absorption  were l o c a t e d a t the plasmalemma, or one  microbial population  decreased  d i f f e r e n t r e a c t i o n s which r e p r e s e n t e d  of d i f f e r e n t a f f i n i t y .  and  i n the e u l t u r i n g medium.  reported  the e x t e r n a l s o l u t i o n was c h a r a c t e r i z e d by two  )  very mobile i n the phloem o f bean p l a n t s ,  a p o s s i b i l i t y t h a t some P was  i n g i n the p l a n t .  soluble  the  concentraobtained. curve  absorption  across  the plasmalemma.  Edwards concluded t h a t the data  l i s h beyond a doubt t h a t the two P absorption  do not  dual absorption ( 48  concentration  correspond to system 1 and  mechanisms o f  system 2 o f  mechanism f o r c a t i o n s as d e s c r i b e d by  ) and L u t t g e  Laties ( 9 6 ) .  and  the P c o n c e n t r a t i o n  was  1 mM  In the p r e s e n t  the  Epstein experiment  i n the complete n u t r i e n t s o l u t i o n .  As a p o i n t o f i n t e r e s t , Bowen and Loughman ( 22  low  estab-  (34)  Rovira  and  Barber  and  ) were a b l e to show t h a t the presence o f micro-  organisms on r o o t s u r f a c e s o f b a r l e y had  marked e f f e c t s on  P  absorption. An  examination o f cut-stem exudate from bean p l a n t s which  were r o o t - f e d -^P^ r e v e a l e d present  (4  9*  5 0 )«  t h a t i t was  However, i t has  the o n l y l a b e l l e d compound a l s o been shown t h a t i n  b a r l e y p l a n t s a s m a l l f r a c t i o n o f the P i n the r o o t s may v i a xylem i n the form o f p h o s p h o r y l c h o l i n e ( 99  phosphorylcholine  ).  (100  ), and  ( 30  of P  on the i n t e r n a l requirement,  element can move a c r o p e t a l l y i n the phloem ( 2 9 • 3 3 , Bieleski  glycero-  Depending on the c o n c e n t r a t i o n  i n the e x t e r n a l s o l u t i o n and  1969.  ascend  ) reported  this  39).  t h a t i n o r g a n i c phosphate was  the primary form i n which P moved i n the phloem o f t u r n i p pumpkin.  When -^ P 2  was  also and  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 o f l e a f t r a c e s i n the bean p l a n t , the c o n c e n t r a t i o n i n the phloem compared to t h a t i n the xylem was data suggest, there was and  an interchange  xylem v i a the i n t e r v e n i n g cambium.  the a e r i a l p o r t i o n o f a bean p l a n t may 1 m/hr,  i . e . 17 cm  In  i n 10 minutes ( 28  ca 3:1.  As  of the  o f P between the phloem Movement o f r o o t P to occur a t the r a t e o f ).  B i d d u l p h ( 70  In 1953t Hanson and  a t i o n e x i s t e d i n the uptake and the r o o t s o f bean p l a n t s .  suggested t h a t an i n c r e a s e d  reactions 1967,  t r a n s l o c a t i o n o f 32p  acropetal  Absorption,and t r a n s l o c a t i o n o f P  g r e a t e s t midway through the 12  have been a s s o c i a t e d  ) found t h a t a d i u r n a l v a r i -  hour p h o t o p e r i o d .  The  P transolocation during  ) r e l a t e d an i n c r e a s e d  3P 2  the day  may  i n the  In  uptake by the r o o t s  of  2,4-D  to  leaves. Sh-8  Akopova ( 23  showed an  rate* ( 4  i n the i n t e n s i t y o f t h e i r p h o t o s y n t h e t i c  synthesis  m  w i t h the need f o r t h i s element i n c e r t a i n  Cotton p l a n t s t r e a t e d w i t h naphthenates and  Bazanova and  o  authors  bean p l a n t s to the d i f f e r e n t i a l d i u r n a l uptake o f P, and induced r e a c t i o n s  r  was  c h a r a c t e r i s t i c o f the a e r i a l p o r t i o n o f the p l a n t .  E t t e r ( 50  increase  f  ).  ) a l s o observed s t i m u l a t i o n o f photo-  i n cotton plants treated with 0.01$  naphthenates.  Follow-  i n g a s i n g l e f o l i a r a p p l i c a t i o n o f a 0 . 0 0 5 $ naphthenate s o l u t i o n to grape p l a n t s , K o l e s n i k photosynthesis.  The  ( 87  ) observed an i n c r e a s e  author a l s o s t a t e d t h a t two  i n the r a t e  foliar  of  applications  o f a 0 . 0 5 $ naphthenate s o l u t i o n r e s u l t e d i n an i n i t i a l r e d u c t i o n  of  the p h o t o s y n t h e t i c  the  r a t e had  r a t e , but a t the end  been i n c r e a s e d .  o f the v e g e t a t i v e  A f t e r s o a k i n g the tubers o f two  period species  o f potatoes i n a 0 . 0 0 0 5 $ naphthenate s o l u t i o n f o r 1 hour b e f o r e p l a n t i n g , Ladygina ( 9^ photosynthetic  ) observed o n l y a s l i g h t change i n the  r a t e o f maturing p l a n t s .  Abolina  and  Ataullaev  ( 2 )  a l s o observed t h a t p h o t o s y n t h e s i s proceeded more ' e n e r g e t i c a l l y * i n potato p l a n t s ( 125 at *  a  f o l l o w i n g treatment w i t h a naphthenate s o l u t i o n  cm /ha ) as a f o l i a r spray. 2  concentration  of  0 . 0 0 1 $ was  A f t e r a naphthenate s o l u t i o n added to  an A l l e n -  Because o f the involvement o f P - c o n t a i n i n g compounds, data d e a l i n g w i t h p h o t o s y n t h e s i s are i n c l u d e d i n t h i s review.  Nelson's c u l t u r i n g medium c o n t a i n i n g the p h y t o p l a n k t o n i c Chaetocerus c u r v i s e t u s , Zgurovskaya (156  ) reported  the r a t e o f c e l l d i v i s i o n and p h o t o s y n t h e s i s P o p o f f et a l (11?  the p h o t o s y n t h e t i c F a t t a h and  r a t e was  Wort ( 5 3  $ naphthenate s o l u t i o n ,  i n c r e a s e d by 8 . 5 $ .  ( 52  Fattah  significant  5.38  k l x a t 26° C f o r  21 days f o l l o w i n g a f o l i a r a p p l i c a t i o n o f a 0 . 5 $  o f KNap to 14-day-old p l a n t s .  The  apparent p h o t o s y n t h e t i c  a f t e r treatment ( 4 0 i n c r e a s e o f 4.2$ after  ),  diminished  when measured two  7.  solution  o f greenhouse grown 3-week-old tomato p l a n t s t r e a t e d with KNap as a f o l i a r spray was  )  r a t e s o f bush bean p l a n t s grown under  l i g h t i n t e n s i t i e s o f 16.10, 10.76, and and  were s t i m u l a t e d .  ) reported a s t a t i s t i c a l l y  increase i n photosynthetic  14,  t h a t both  ) s t a t e d t h a t a f t e r the p e t i o l e s o f bean  l e a v e s had been immersed i n a 8 x 10"^  and  diatom,  rate 0.5$  weeks  However, the treatment r e s u l t e d i n an  i n r a t e s o f apparent p h o t o s y n t h e s i s  f o u r weeks  treatment. F o l l o w i n g the a p p l i c a t i o n o f f e r t i l i z e r and naphthenates to  the s o i l , c o n c e n t r a t i o n s the cabbage heads ( 13  ).  o f N, P, and By s o a k i n g  sugar were i n c r e a s e d i n seeds i n or  spraying  p l a n t s w i t h a 0 . 0 0 5 $ naphthenate s o l u t i o n d u r i n g the p e r i o d , Yur'eva ( 149 ) obtained  vegetative  i n c r e a s e s i n p r o t e i n and  starch  i n corncobs, and more p r o t e i n and P i n the l e a v e s o f sugar beet. A f t e r s p r a y i n g tomato p l a n t s w i t h (  7  ) observed t h a t the uptake o f N and P by t r e a t e d p l a n t s  been i n c r e a s e d . at  0 . 0 0 5 $ naphthenates, A l i e v  Asadov (  the r a t e o f 50 or 100  p l a n t e d , and  had  14 ) s t a t e d t h a t naphthenate treatments g/ha  a t the time cabbage s e e d l i n g s were  d u r i n g the v e g e t a t i v e p e r i o d as a f o l i a r  i n c r e a s e d the amount o f N and P i n the heads.  Abolina  spray, and  Ataullaev ( 2 the  ) r e p o r t e d t h a t the v e g e t a t i v e organs o f the  p o t a t o p l a n t showed i n c r e a s e s i n the l e v e l s o f N and P when  compared w i t h c o n t r o l s . increases i n t o t a l  S t i m u l a t i o n o f N and P metabolism, and  and p r o t e i n N content i n the r o o t s o f c o t t o n  were observed by Babaev ( 16 ) a f t e r the c o t t o n seeds were t r e a t e d w i t h a s o l u t i o n c o n t a i n i n g 0.0001% naphthenates p r i o r to  sowing.  The d a t a o f Guseinov and Guseinov ( 6 5 ) showed t h a t  naphthenates ( 6 5 to 280 g/ha) combined  with N-P-K  fertilizers  and a p p l i e d to s o i l i n c r e a s e d the uptake o f these elements by 78$.  The a p p l i c a t i o n o f naphthenates alone o r i n combination  with m i n e r a l f e r t i l i z e r s  (N and P) to the s o i l i n c r e a s e d the  P content i n tomatoes and cabbage  ( 64 ).  Moreover,  the sum o f  ammonium and n i t r a t e N i n the p l a n t t i s s u e was i n c r e a s e d . Ejubov and I s s a e v a ( 47 ) r e p o r t e d t h a t the c o n c e n t r a t i o n o f a s s i m i l a b l e P forms and o f m i n e r a l N compounds i n l u c e r n e , as w e l l as the t o t a l increased.  maize and  N and P c o n c e n t r a t i o n s , were  P e t e r b u r g s k y and Karamete ( 1 1 3 ) observed t h a t  naphthenates i n c r e a s e d ^ P u t i l i z a t i o n 2  when maize was grown i n  three d i f f e r e n t c u l t u r e s .  Naphthenate-treated p l a n t s grown i n  sand c u l t u r e showed the h i g h e s t r a t e o f ^ P u t i l i z a t i o n . 2  Utili-  z a t i o n o f P was lower when the p l a n t s were grown i n a hydroponic medium, o r i n s o i l .  The authors a l s o r e p o r t e d t h a t the P  content i n a l l organs o f maize was i n c r e a s e d by 5 0 $ a f t e r the seeds had been soaked i n a 0 . 0 0 5 $ naphthenate s o l u t i o n p r i o r to  sowing i n sand.  A f t e r maize had r e c e i v e d a f o l i a r  appli-  c a t i o n o f a 0 . 0 0 5 $ naphthenate s o l u t i o n , P content was i n c r e a s e d by 3 3 $ .  F o l l o w i n g a seed soak f o r 12 or 24 hours i n a s o l u t i o n  c o n t a i n i n g 200 to 1,000 u C i / 1 , as K H P 0 ^ , a l o n e , o r w i t h 0.01$ 32  2  naphthenates, Guseinov and Issaeva ( 6 6 ) r e p o r t e d  t h a t the  a l f a l f a hay crops were i n c r e a s e d by 1? and 24$, r e s p e c t i v e l y . A f t e r a l f a l f a had r e c e i v e d a f o l i a r treatment w i t h a 0 , 0 1 $ naphthenate s o l u t i o n d u r i n g the v e g e t a t i v e p e r i o d , the hay y i e l d was i n c r e a s e d by 24 ...to 3 7 $ .  Peterburgsky and  Karamete  ( 1 1 3 ) observed t h a t naphthenate treatments had an e f f e c t on 3^p  l e v e l s i n corn.  T h e i r r e s u l t s showed t h a t ^2p l e v e l s i n  plants treated with 0 . 0 0 5 , 125»  0 . 0 1 , and 0 . 0 1 5 $ naphthenates were  1 0 6 , and 1 5 2 $ o f the c o n t r o l v a l u e s , r e s p e c t i v e l y . Following  a f o l i a r a p p l i c a t i o n o f naphthenates  (0.005$)  a t the b e g i n n i n g o f the f l o w e r i n g phase, the c o n c e n t r a t i o n o f ascorbic acid  and the s p e c i f i c a c t i v i t i e s o f c a t a l a s e  and  p e r o x i d a s e i n the l e a v e s o f t r e a t e d p l a n t s were i n c r e a s e d as compared  with c o n t r o l values  ( 2 4 ),  However, when a N-P  f e r t i l i z e r was a p p l i e d to the s o i l a t the  same time the foliage  r e c e i v e d the naphthenate treatment, a s c o r b i c content and the a c t i v i t i e s o f the two enzymes were decreased i n the l e a v e s o f treated plants.  MATERIAL AND METHODS A.)  Growth o f p l a n t s . Uniform seeds o f the dwarf bush bean p l a n t , Phaseolus  v u l g a r i s L. c u l t i v a r Top Crop ( B u c k e r f i e l d ' s , L t d . , New Westm i n s t e r , B.C.), were sown i n 5 0 x 3 3 x 7 cm wooden f l a t s t a i n i n g v e r m i c u l i t e , and the f l a t s were p l a c e d While the s e e d l i n g s  i n a growth room.  were i n v e r m i c u l i t e , they were watered w i t h  a one-quarter s t r e n g t h TABLE I I .  Hoagland-Arnon's s o l u t i o n minus  -P  KH P0^  1  KNO3  5  5  5  5  2  2  1  1  2  2  • 4 H 0 2  MgSO^ • 7 H 0 2  ( 0 . 1 M)  NaCl KC1  1  Fe EDTA A-  5  ( 5 mg/ml)  b  D e i o n i z e d water b  or  a  Complete  Stock s o l u t i o n  3  P  Volumes o f molar s t o c k s o l u t i o n s used f o r p r e p a r i n g one l i t e r o f a i x n u t r i e n t s o l u t i o n . M o d i f i e d a f t e r Hoagland and Arnon ( 74 ).  inl/1  Ca(N0 )  con-  1  1  1  1  ( 0 . 2 ppm, as N a C l ) .  The A - 5 m i c r o n u t r i e n t s o l u t i o n was prepared by d i s s o l v i n g the f o l l o w i n g i n 1 1 o f d i s t i l l e d water: 2.86 g H 3 B O 3 , 1.8l g MnCl2 • 4 H2O, 2 2 0 mg ZnSO^ . 5 H 0 , 80 mg CuSOij. . 5 H 2 O , 2  and  2 0 mg Na2Mo0ij,  •  w i t h a complete s o l u t i o n . t a i n e d f o r the d u r a t i o n  H2O.  The f o l l o w i n g c o n d i t i o n s  o f the experiment!  top o f the p l a n t s , a 14 hour p h o t o p e r i o d ,  were main-  16.3$ k l x a t the a day/night  temperature o f 26-l°/2l-l° G, and a day/night p e r c e n t r e l a t i v e humidity o f 60 to 7 0 / 7 0 t o 80.  The l i g h t i n the growth room  was s u p p l i e d by cool-white f l u o r e s c e n t tubes (Westinghouse, U.S.A.) and 60-watt incandescent lamps. A f t e r e i g h t days i n v e r m i c u l i t e , 48 uniform s e e d l i n g s were transplanted? i n t o f o u r 33 x 26 x 13 cm c u l t u r e t r a y s which were covered w i t h aluminum growth.  f o i l and b l a c k p a i n t to prevent a l g a l  Each t r a y h e l d 12 p l a n t s and contained 4 l i t e r s o f a  c o n t i n u o u s l y a e r a t e d Hoagland-Arnon's (pH 5 . ^ ) n u t r i e n t (Table I I ) .  solution  A f t e r 3 days, the arrangement o f the t r a y s was  changed t o average l o c a l environmental v a r i a b i l i t y . B. ) P r e p a r a t i o n o f potassium naphthenates (KNap) aqueous from naphthenic a c i d s  solution  (HNapjT  Seventeen ml o f a 12.3$ (w/v) KOH s o l u t i o n  (2.1 g KOH i n  17 ml d i s t i l l e d water) was added to a f l a s k c o n t a i n i n g  5 g  naphthenic a c i d s ( P r a c t i c a l grade, average mol wt 2 3 0 , Eastman Organic Chemicals, Rochester, N.Y.).  The f l a s k was shaken f o r  10 t o 15 minutes and the s o l u t i o n was made to a volume o f w i t h d i s t i l l e d water.  25 ml  The s o l u t i o n thus prepared was the stock  s o l u t i o n c o n t a i n i n g 2 5 0 mg o f KNap p e r ml.  By d i l u t i n g 1.0 ml  o f the s t o c k s o l u t i o n (see next page), the f i n a l o f KNap was 2 x 10"" M ( 5 0 0 0 ppm o r 0 . 5 $ ) . 2  concentration  The pH o f the  d i l u t e d s o l u t i o n was a d j u s t e d to about 10 by the a d d i t i o n o f 0.1 N HC1. C. ) Treatment w i t h KNap. Fourteen days a f t e r sowing, bean p l a n t s i n two t r a y s were sprayed to d r i p w i t h the 2 x 10"" m aqueous s o l u t i o n 2  of  KNap  described  i n B).  Even though KNap i s a s u r f a c t a n t , the KNap  s o l u t i o n contained  0,3$ (v/v) Tween 20  (polyoxyethylenesorbitan  monolaurate) ( A t l a s Powder Co., Wilmington, D e l . ) to serve as an a d d i t i o n a l w e t t i n g agent.  The p l a n t s i n the other two t r a y s  remained as unsprayed c o n t r o l s . 32 D. )  Exposure to  P.  Twenty-four hours a f t e r s p r a y i n g , the r o o t s o f both t r e a t e d and  c o n t r o l p l a n t s were immersed i n a c o n t i n o u s l y a e r a t e d 32  plete nutrient solution containing Energy o f Canada, Ottawa, Ont.).  com-  P, as orthophosphate (Atomic I n the f i r s t s e r i e s o f e x p e r i -  ments where the p l a n t s were grown i n a phosphate-free n u t r i e n t 32 P l e v e l was 15 uCi/1. When the p l a n t s were grown 32 i n complete n u t r i e n t , the P l e v e l was 20 u C i / 1 : and f o r the phosphorus f r a c t i o n a t i o n experiment a t a l e v e l o f 25 uCi/1. 32 s o l u t i o n the  F o l l o w i n g a 2 hour exposure to the ^ P n u t r i e n t s o l u t i o n , the r o o t s o f a l l p l a n t s were w e l l r i n s e d a c c o r d i n g sequences  to the f o l l o w i n g  Four changes o f tapwater, a n o n - r a d i o a c t i v e  (210 mg/l) s o l u t i o n , and another change o f tapwater. i n g media were h e l d a t room temperature, and the t o t a l time was 1 hour. returned  phosphate A l l rinsrinsing  A f t e r the r i n s i n g p e r i o d , the p l a n t s were  to a c o n t i n u o u s l y aerated phosphate-free n u t r i e n t o r  to a complete n u t r i e n t s o l u t i o n f o r h o l d i n g times o f 4, 8, 12 and E. )  24 hours b e f o r e  harvesting.  Harvest, d i g e s t i o n , and  counting.  At the end o f each o f the f o u r h o l d i n g times, and  four treated  f o u r c o n t r o l p l a n t s were taken a t random, immediately  d i v i d e d i n t o the t h r e e f r a c t i o n s r o o t s ; stems p l u s p e t i o l e s :  and  l e a f blades,  recorded.  and  the f r e s h weight o f each f r a c t i o n  Each treatment had  two  was  r e p l i c a t e s f o r each h a r v e s t  sampling time w i t h the same organs from two  p l a n t s per  The  100 ml K j e l d a h l  p l a n t f r a c t i o n s were p l a c e d i n separate  d i g e s t i o n f l a s k s , and  the p l a n t m a t e r i a l was  minutes a t 1 1 5 - 1 2 5 ° C u s i n g 2 ml o f 60$ HNO^*. was  tube type M-6  The  32  (20^  Century E l e c t r o n i c s , New  England) which was  one minute counts was  70$ sample  activity in  P  determined by u s i n g a 10 ml l i q u i d G-M  counting  Addington, Croydon,  housed i n a l e a d c a s t l e connected to  a decade s c a l e r (Nuclear-Chicago,  F.)  3 ml o f  F o l l o w i n g d i g e s t i o n , the volume o f each d i g e s t e d  the samples was  Surrey,  replicate.  ashed f o r 30  wet  HCIO^ and  brought to 10 ml with d i s t i l l e d water.  or  Model 8 7 0 3 ) .  The  c o r r e c t e d f o r background and  mean o f  two  decay,  Phosphorus f r a c t i o n a t i o n . Bean p l a n t s were grown and  ed (see S e c t i o n A & E ) . r o o t s from t r e a t e d and d i f f e r e n t harvest  harvested  Leaf blades,  as p r e v i o u s l y d e s c r i b -  stems p l u s p e t i o l e s , and  c o n t r o l p l a n t s c o l l e c t e d a t the  times were d i g e s t e d as b e f o r e  four  (see S e c t i o n E ) .  P r i o r to the e x t r a c t i o n procedure, the p l a n t samples were s t o r e d at - 1 5 °  C.  The  e x t r a c t i o n procedures o u t l i n e d below were based  on those d e s c r i b e d by Cole and Ross ( 41  ).  T i s s u e samples weighing ca 3 g were p l a c e d C f o r 5 minutes.  i n an  ethanol  (Et0H)/dry  i c e bath a t ca - 4 5 °  t i s s u e was  t r a n s f e r r e d to a c o l d p o r c e l a i n mortar, 1 ml o f  The  frozen 80$  • P r e l i m i n a r y i n v e s t i g a t i o n s showed t h a t the white p r e c i p i t a t e ( p e r c h l o r a t e s ) formed i n the f l a s k s d u r i n g d i g e s t i o n was not r a d i o a c t i v e , and t h a t d u r i n g the d i g e s t i o n process 32p a c t i v i t y was not l o s t .  formic a c i d was  added, and  2 ml o f the a c i d was to 2 , 2  to 2 , 4 ,  thin slurry.  the t i s s u e was  sample was  w i t h 8 0 $ EtOH, and  An a d d i t i o n a l  added to b r i n g the pH o f the f i n a l  and g r i n d i n g continued The  ground.  u n t i l the sample formed a  then r i n s e d i n t o a Buchner  the s l u r r y was  c o n t a i n i n g a c i d - i n s o l u b l e phosphorus compounds, was  were p l a c e d 150°  G and The  Finally,  the r e s i d u e and  filtrate,  brought to a volume o f 70 ml w i t h 80$  ing  the ^ P  The  e x t r a c t was  EtOH.  heated a t 50°  Section G.)  and  in a  C u n t i l a l l EtOH ml.  the sample  t o t a l i n o r g a n i c phosphate was  samples was  determined  brought  (see  G).  The digested  concentration f r a c t i o n was  d e s c r i b e d by F i s k e and contained  acid  placed  before.  T o t a l i n o r g a n i c phosphate  o f 10$  A f t e r determin-  added, and  In both cases the volume o f the d i g e s t e d to 10 ml,  paper  ashed a t  the volume had been reduced to ca 4  The n i t r i c / p e r c h l o r i c a c i d mixture was d i g e s t e d as  three  previously.  a c t i v i t y i n a 10 ml a l i q u o t , 25 ml was  had been removed, and  was  rinsed  c o n t a i n i n g a c i d - s o l u b l e phosphorus compounds,  was  Kjeldahl flask.  43  residue,  the f i l t e r  a c t i v i t y determined as d e s c r i b e d  2  2  The  i n a 100 ml K j e l d a h l d i g e s t i o n f l a s k , wet ^P  funnel  f i l t e r e d u s i n g Whatman No,  a s h l e s s , medium-fast (phosphate-free) f i l t e r paper.  times w i t h 80$ EtOH.  extract  determinations.  o f i n o r g a n i c phosphate p r e s e n t  i n each  determined f o l l o w i n g the procedure Subbarow ( 5 5  0 . 2 ml o f d i g e s t , 1 . 0  ).  The  colorimeter  tube  ml o f molybdate reagent, 4 . 0  t r i c h l o r o a c e t i c a c i d , 0 . 4 ml o f a m i n o n a p h t h o l s u l f o n i c  (ANSA), and  4 . 4 m l o f d i s t i l l e d water.  E x a c t l y 5 minutes  ml  a f t e r the d i g e s t had been added, the i n t e n s i t y o f the b l u e c o l o r was  measured u s i n g a Klett-Summerson c o l o r i m e t e r  a No,  66 r e d f i l t e r  (range 640  phosphate a n a l y s i s was  - 700  nm).  Total  equipped w i t h inorganic  not done on the organs from c o n t r o l  and  t r e a t e d p l a n t s which were grown i n phosphate-free n u t r i e n t . pages 151  -  152  i n the Appendix f o r reagents and  phosphate curve ( F i g u r e  14).  the  standard  See  RESULTS A.)  T o t a l uptake by the p l a n t . F i f t e e n - d a y - o l d c o n t r o l and n a p h t h e n a t e - t r e a t e d bean p l a n t s  growing i n a phosphate-free o r a complete n u t r i e n t s o l u t i o n were exposed to a -^ P n u t r i e n t s o l u t i o n f o r 2 hours. 2  The cpm per  gram f r e s h weight i n the whole p l a n t was taken to be a measure o f the r a t e o f phosphate uptake by the r o o t s o f t r e a t e d and c o n t r o l p l a n t s d u r i n g the exposure time.  T h i s assumption i s  v a l i d p r o v i d e d the r i n s i n g o f the r o o t s was complete a f t e r exposure to  J  their  P, and t h e r e was n o t a s i g n i f i c a n t e f f l u x o f ^ P  d u r i n g the h o l d i n g p e r i o d . ments were met, as the  J  In these experiments the two r e q u i r e -  P a c t i v i t y i n the c u l t u r i n g t r a y s was  v e r y low when determined a f t e r the p l a n t s had been removed. T h i s o b s e r v a t i o n concurs w i t h the data o f R o v i r a and Bowen (120). When the r o o t s o f wheat s e e d l i n g s were f e d 32p f o r 15 minutes a t a l e v e l o f 300 u C i / 1 , the authors concluded t h a t the e f f l u x o f 3 P had been washed out o f the 'apparent f r e e space' w i t h 2  j u s t 5 to 7 minutes o f r o o t r i n s i n g i n tapwater.  In the p r e s e n t  experiments the r o o t s were r i n s e d f o r 1 hour. To i n v e s t i g a t e the e f f e c t o f naphthenates on the uptake o f 3  2 p  and on the t o t a l phosphorus  content, experiments were  designed to study these v a r i a b l e s .  Even though the p l a n t s were  harvested a t f o u r d i f f e r e n t sampling times and d i v i d e d  into  t h r e e d i f f e r e n t f r a c t i o n s , the sum o f the ^ P a c t i v i t y from 2  these f r a c t i o n s r e p r e s e n t s the amount o f 32p l a b e l l e d  phosphate  which was absorbed by the r o o t s o f c o n t r o l and t r e a t e d p l a n t s d u r i n g the 2 hour exposure p e r i o d .  Average v a l u e s from  identical  uptake and d i s t r i b u t i o n experiments a r e given XI.  In a l l i n s t a n c e s  i n Tables I I I to  the v a r i a t i o n between the v a l u e s from  i d e n t i c a l experiments was n o t 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 p e r gram b a s i s i n l e a f blades, III.  stems, r o o t s , and i n the t o t a l p l a n t i s shown i n T a b l e  In these experiments t h e o n l y phosphorus the p l a n t s  r e c e i v e d was ^ P l a b e l l e d orthophosphate d u r i n g a 2 hour 2  exposure o r f e e d i n g p e r i o d .  I t was determined t h a t naphthenate-  t r e a t e d p l a n t s had taken up 102$ as much a c t i v i t y as the c o n t r o l p l a n t s , and t h a t t h i s i n c r e a s e was n o t 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 p l a n t i s expressed on a p e r p l a n t  basis  (Table V ) , the d i f f e r e n c e between t r e a t e d and c o n t r o l v a l u e s 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 v a l u e  f o r the t r e a t e d p l a n t s was 109$ o f the c o n t r o l v a l u e ( F i g u r e 1 ) . The  data shown i n Table IV, V I I I a l s o r e p r e s e n t  from i d e n t i c a l experiments.  results  I n these experiments c o n t r o l and  t r e a t e d p l a n t s were grown i n a complete n u t r i e n t s o l u t i o n 32 b e f o r e and a f t e r they were exposed f o r 2 hours t o a ^ P s o l u t i o n . When -^ P a c t i v i t y i n the p l a n t i s expressed as cpm p e r gram, the 2  naphthenate. treatment d i d n o t 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  I V ) . On a p e r p l a n t b a s i s  (Table V ) ,  a c t i v i t y i n t r e a t e d p l a n t s was 107$ o f the c o n t r o l v a l u e , and the p a t t e r n was v e r y s i m i l a r to the one o b t a i n e d when p l a n t s were grown i n t h e phosphate-free n u t r i e n t s o l u t i o n ( F i g u r e l ) . Values f o r -*2p a c t i v i t y on a gram b a s i s i n l e a f  blades,  stems, and r o o t s from t r e a t e d p l a n t s grown e i t h e r i n t h e -P o r i n the complete n u t r i e n t s o l u t i o n were n o t s i g n i f i c a n t l y d i f f e r e n t from the c o n t r o l v a l u e s (Table.  I l l , IV).  TABLE I I I .  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 o f p h o s p h o r u s - 3 2 , gram b a s i s , by bush bean p l a n t s a t v a r i o u s sampling times .  on a p e r  a  Sampling times i n hours  Phosphorus-32 a c t i v i t y b Leaf blades C  c  Roots  Stems  T  C  T  C  Total T  C  T  4  d 3.79.  1,268  1,202  2,160  35,724  31,107  37,305  34,536  8  873  1,626  1,648  2,392  31,818  34,273  35,089  38,291  12  863  1,751  1,532  2,503  26,799  26,255  29,194  30,5U  24  928  2,138  1,899  3,211  28,146  27,151  30,973  32,500  Mean  761  l,696  1,570  2,567  30,621  29,697  33,140  33,960  Percent of c o n t r o l  n s  163.4  222.9  n s  n s  96.9  102.4  P l a n t s grown i n a -P n u t r i e n t s o l u t i o n before and a f t e r the p l a n t s were exposed f o r 2 hours to a phosphorus-32 n u t r i e n t s o l u t i o n . Counts/minute/gram C = no spray; Average o f f o u r  f r e s h weight.  T «= 5000 ppm  KNap.  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 c o n t r o l value a t the 0 . 0 5 l e v e l . This d e s i g n a t i o n on a l l subsequent t a b l e s w i l l have the same meaning.  n S  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 o f p h o s p h o r u s - 3 2 , gram b a s i s , by bush bean p l a n t s a t v a r i o u s sampling times .  on a p e r  a  Sampling times i n hours  Phosphorus- 32 a c t i v i t y Leaf blades c  Roots  Stems c  T  c  b  c  T  Total T  C  T  66,666  27,417  29,222  64,582  57,691  154,710  153,578  66,428  70,409  25,952  28,768  57,056  52,421  149,436  151,598  12  68,042  71,923  25,069  29.063  51.229  50,506  144,339  151,492  24  68,447  69,117  24,428  25.578  47,860  44,497  140,734  139,192  Mean  66,407  69,528  25,716  28,l58  55,182  51,279  147,305  l48,965  4  62,?ll  8  Percent of Control  d  104.7  n s  109.5  n s  92.9  n s  n s  101.1  a  P l a n t s grown i n a complete n u t r i e n t s o l u t i o n before and a f t e r the p l a n t s were exposed f o r 2 hours to a phosphorus-32 n u t r i e n t s o l u t i o n .  b  See Table I I I .  c  See Table I I I .  d  See Table I I I .  TABLE V .  E f f e c t o f KNap on t o t a l uptake o f phosphorus-32, on a per p l a n t b a s i s , by bush bean p l a n t s .  Sampling times i n hours  Total  phosphorus- 32 a c t i v i t y  Grown i n a -P n u t r i e n t  a  Grown i n complete nutrient T  C  T  88,295  274,785  276,961  4  80,300  8  91,999  113.^33  253.923  283,530  12  76,729  81,326  268,883  291,474  24  97.892  96,781  305.192  329.131  Mean  86,730  94,959  275.695  295.274  °  Percent of control  n s  109.5  a  Counts/minute/plant.  b  G = No spray;  c  Average o f f o u r v a l u e s .  T = 5 0 0 0 ppm KNap.  107.1  n S  FIGURE 1.  E f f e c t o f KNap on t o t a l phosphorus-32 uptake, on a per gram or p e r p l a n t "basis, when bush bean p l a n t s were grown i n a phosphate-free or a complete n u t r i e n t .  300 .  250 . o  •p  200  •H  >  •H  -P O a (Si  150  i  to  U O .C ft  to  o  100  £i  ft  rH  «J  50  0 C  T  per gram  C  T  per plant  PHOSPHATE-FREE  C  T  per gram  C  T  per plant  COMPLETE  T o t a l phosphorus on a per gram b a s i s f o r l e a f b l a d e s , and r o o t s from t r e a t e d and and  c o n t r o l p l a n t s i s shown i n T a b l e VI,  i n Table VII t o t a l phosphorus f o r t r e a t e d and  i s shown on a per p l a n t b a s i s .  No matter how  control plants  the data iare  expressed, naphthenate treatment d i d not have an e f f e c t phosphorus uptake ( F i g u r e 2 ) .  On a per gram b a s i s ,  an average o f 8 . 7 7  p l a n t s had plants 9.00  stems,  mg  on  treated  o f i n o r g a n i c phosphate; c o n t r o l  mg.  r a t i o o f average ^2p a c t i v i t y to average t o t a l phosphorus  The (32p/ ) t P  on a per gram b a s i s i n l e a f b l a d e s , c o n t r o l and  t r e a t e d p l a n t s i s shown i n Table V I I I .  between the ^ p / ^ p p l a n t s 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  greater  T h i s d i f f e r e n c e was  for  for control  between the ?§P/tP r a t i o s was value  t r e a t e d p l a n t s the  (Table V I I I , F i g u r e  J).  110.8$ of  level. the  by  For  difference  s i g n i f i c a n t a t the 2 . 5 $  f o r n a p h t h e n a t e - t r e a t e d p l a n t s was  c o n t r o l value  value  than t h a t o f c o n t r o l p l a n t s  s i g n i f i c a n t a t the 5 $ l e v e l .  l e a f blades from c o n t r o l and  The  control  the value  9 6 . 8 $ o f the v a l u e  of  difference  For the stem f r a c t i o n the d i f f e r e n c e between the  from t r e a t e d p l a n t s was 12.3$.  roots  The  r a t i o s f o r r o o t s from t r e a t e d and  naphthenate-treated p l a n t s was plants.  stems, and  TABLE V I .  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 o f phosphorus, on a p e r gram b a s i s , by bush bean p l a n t s a t v a r i o u s sampling times . a  Sampling times i n hours  T o t a l i n o r g a n i c phosphate Stems  Leaf blades C  C  T  c  b  Roots C  T  Total c  T  T  3-33  1.48  1.34  3 . 06  2.77  7.74  7.45  3.79  3.83  1.51  1.49  3.89  3.25  9.19  8.57  12  3-89  3-94  1.59  1.73  3.69  3.76  9.17  9.34  24  4.02  3.84  1.77  1.76  4.11  4.02  9.89  9.62  Mean  3.72  3.74  1.59  1.58  3.69  3.45  9.00  8.77  4  3.19  8  d  Percent of c o n t r o l  n s  100.5  a  See Table IV.  b  M i l l i g r a m s i n o r g a n i c phosphate/gram  c  See Table I I I .  d  See Table I I I .  n s  99.3  f r e s h weight.  93.5  n s  97.4  n S  TABLE V I I .  E f f e c t o f KNap on t o t a l uptake o f phosphorus, on a per p l a n t b a s i s , by bush bean p l a n t s a t the v a r i o u s sampling times . a  T o t a l i n o r g a n i c phosphate  ^  Sampling times i n hours  Control  4  13.8  8  15. G  15.6  12  17.5  18.0  24  20.9  22.0  Mean  16.8  17.3  c  d  Percent of c o n t r o l  ~  See Table IV.  b  M i l l i g r a m s i n o r g a n i c phosphate/plant.  c  See Table I I I .  d  See Table I I I .  Treated  13-5  102.6  TABLE V I I I .  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 b a s i s , i n bush bean p l a n t s a t v a r i o u s sampling times . a  Phosphorus-32 a c t i v i t y / t o t a l i n o r g a n i c phosphate ^  Sampling times i n hours C  c  Roots  Stems  Leaf blades T  C  T  C  T  24.27  18.51  21.90  20.41  18.00  4  20.41  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  Percent of c o n t r o l  d  110.8  a  See Table IV.  b  Counts/minute/gram and m i l l i g r a m s  c  See Table I I I .  d  See Table I I I .  112.3  i n o r g a n i c phosphate.  Means d i f f e r i n g s i g n i f i c a n t l y a t the 0.05 l e v e l from the r e s p e c t i v e c o n t r o l mean. This d e s i g n a t i o n on a l l subsequent t a b l e s w i l l have the same meaning.  96.8  ns  FIGURE 2.  E f f e c t of KNap on t o t a l phosphorus p r e s e n t , on a per gram or per p l a n t b a s i s , i n bush bean p l a n t s grown i n complete n u t r i e n t .  24 22  h  1  20  +>  a  18  X!  P.  in o x:  16  o  14  s u o  12  to  10  •H rH  8  •H  6 4 2 0 C per  T gram  C  T  per  plant  FIGURE 3 .  E f f e c t of KNap on the r a t i o o f phosphorus-32 a c t i v i t y to t o t a l phosphorus, on a p e r gram b a s i s , i n l e a f blades stems, and r o o t s of bush bean p l a n t s .  24 22 fciD  20  s CO  o  Xi  ft  18 16  CO  o  xi  ft  H  14  cd •p o •p \  12  1  10  ft o  CM  c-\ I 05  o  ft  m o  8 6 4  Xi  PH  2 0 C  Leaf blades  T  T  T Stems  Roots  B.)  Percentage d i s t r i b u t i o n o f the t o t a l 3 P a c t i v i t y among l e a f b l a d e s , stems, and r o o t s . 2  When bean p l a n t s grown i n a -P n u t r i e n t were exposed f o r 2 hours t o a s o l u t i o n c o n t a i n i n g ^ P a t a l e v e l o f 15 u C i / 1 2  without c a r r i e r phosphate and then h e l d f o r 24 hours, 90.7$ o f the t o t a l a c t i v i t y remained i n the r o o t s o f c o n t r o l p l a n t s , while o n l y 8 3 . 8 $ remained i n the r o o t s o f t r e a t e d p l a n t s IX).  (Table  At the same sampling time 6.2$ o f the t o t a l ^ p absorbed 2  was found i n the l e a f blades l e a f blades  o f t r e a t e d p l a n t s , and 3.0$ i n the  o f the c o n t r o l s ( F i g u r e 4 ) .  Consequently, a t the  24 hour sampling time a c t i v i t y i n t r e a t e d p l a n t s was l e s s i n the r o o t s , and g r e a t e r i n stems and l e a f b l a d e s , 155.G, and 202.9$ o f the c o n t r o l v a l u e s .  i . e . 9^.4,  T h i s same g e n e r a l  p a t t e r n was a l s o noted a t the other t h r e e sampling times.  The  32 amount o f  P present  i n the r o o t s , stems, and l e a f blades o f  t r e a t e d p l a n t s d i f f e r e d s i g n i f i c a n t l y from the c o n t r o l v a l u e a t the 2 , 5 $ l e v e l . When p l a n t s were grown i n complete n u t r i e n t both 32 and a f t e r exposure o f the r o o t s to  before  P, the percentage d i s t r i b u -  32 t i o n o f the  P l a b e l i n the l e a f b l a d e and r o o t f r a c t i o n s from  c o n t r o l and t r e a t e d p l a n t s was s i m i l a r t o the d i s t r i b u t i o n p a t t e r n which emerged a f t e r p l a n t s were grown i n a -P n u t r i e n t . However, the amount o f l a b e l p r e s e n t  i n the p l a n t organs  d i f f e r e d c o n s i d e r a b l y when compared with p l a n t s grown i n the -P n u t r i e n t ( F i g u r e 4,5). The  r e s u l t s o f an a n a l y s i s o f p l a n t s grown i n the complete  n u t r i e n t and sampled 24 hours a f t e r exposure t o -^ P r e v e a l e d 2  t h a t o n l y 34.0$ o f t h e t o t a l a c t i v i t y remained i n t h e r o o t s o f  c o n t r o l p l a n t s , w h i l e 31.8$ remained i n the r o o t s o f t r e a t e d plants.  At the same sampling time 48.7$ o f the t o t a l -^ P l a b e l 2  i n the p l a n t was found i n l e a f b l a d e s o f c o n t r o l p l a n t s , and 49.8$ i n the l e a f blades o f t r e a t e d p l a n t s .  The amount o f ^ P 2  p r e s e n t i n the r o o t s o f t r e a t e d p l a n t s d i f f e r e d s i g n i f i c a n t l y from the c o n t r o l v a l u e a t the 1$ l e v e l , and f o r the stems a t the 3$ l e v e l .  The amount o f  32  P  p r e s e n t i n the l e a f b l a d e s o f t r e a t e d p l a n t s was n o t s i g n i f i c a n t l y d i f f e r e n t from the c o n t r o l v a l u e a t the 5$ l e v e l  (Table X ) .  On a percentage o f c o n t r o l  b a s i s , the d i s t r i b u t i o n o f a c t i v i t y among the l e a f b l a d e s , stems, and  r o o t s o f t r e a t e d p l a n t s was 103.5*  t i v e l y (Figure  1 0 8 . 1 , and 91.8$, r e s p e c -  5).  I t i s i n t e r e s t i n g t o note t h a t i n c o n t r o l and t r e a t e d p l a n t s the amount o f -^ P i n the stem f r a c t i o n s remained r e l a t i v e 2  l y constant through the f o u r sampling times, i . e . 17.2 to 17.7$ and  1 8 . 3 to 19«3$» r e s p e c t i v e l y .  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 o f ^ P which d i d n o t markedly a f f e c t 2  the percentages i n the stems.  E t t e r ( 4 9 , 51  ), working w i t h  the e f f e c t o f 2,4-D on phosphorus metabolism i n bean p l a n t s , 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 o f phosphorus-32, on a p e r gram b a s i s , among l e a f blades, stems, and r o o t s o f bush bean p l a n t s a t v a r i o u s samplings times . a  % °>  Sampling times i n hours  of t o t a l  phosphorus--32 a c t i v i t y Stems  Leaf blades C  c  b  Roots  T  C  T  C  T  4  . d 1.04  3.61  3.31  6.10  95.68  90.35  8  2.62  4.23  4.83  6.41  92.55  89.36  12  2.92  5.44  5.37  8.26  91.72  8 6 . 30  24  3.02  6.25  6.28  9.90  90.71  83.84  Mean  2.40  4.8?*  4.95  7.67  92.68  87.46*  Percent of c o n t r o l  a  See Table I I I .  b  See Table I I I .  ° d  See Table I I I . See Table I I I .  202.9  155.0  94.4  rlOO  10  .96  -92  -88  -84  2 -  .80  k  8  12  2k  Stems  Leaf blades C =  FIGURE k.  8  12  2k  8  12  Roots  2k - Sampling time in hrs  E f f e c t o f KNap on the percentage d i s t r i b u t i o n o f p h o s p h o r u s - 3 2 , on a p e r gram b a s i s , among l e a f blades, stems, and r o o t s o f bush bean p l a n t s grown i n a phosphate-free n u t r i e n t .  %1 o f t o t a l phosphorus--32 a c t i v i t y  Sampling times i n hours  Stems  Leaf blades c  c  d  b  T  C  Roots T  C  T  4  41.16  44.10  17.79  18.99  41.05  36.90  8  44.11  46.47  17.47  19.02  38.43  34.52  12  46.96  49.10  17.41  19.30  35.62  33.60  24  48.78  49.75  17.29  18.37  33.94  31.88  Mean  45.25  46.85  17.49  18.92*  37.26  34.22  Percent of c o n t r o l a  See Table IV.  b  See Table I I I .  c  See Table I I I .  d  See Table I I I .  103.5  n S  108.1  #*  91.8  o o  ro o  o  o  o  I  o a* w o P H) CQ  P« H> H CQ COO a> • <+ c+ (D  P H>  O  3  P P.  3 3"  a"  H P CD P-  1 I II1 1 I1 II  I  I III I IIT  ro  CD CQ  P CD CD p O H>  OO  ro  O  CD « CQ CD  o  CQ CD c+ CD e+  3  _1  -P-  3 P CQ ati  -  CD  P P. 3 H* P. CQ 4 O O c+ CQ O H,  c+ hi H* C C c+ H* O  3  CO  c+ CD  3  CQ  ro ro  ^3  c o CQ  3"id  3*  o* o CD CQ  3 3" O  Si'  c+V*> CQ  ro  w o o  3 o o 3  3  P  3  CD  c+ CQ  ro ro  P  1  00  3 3* CQ  i w 3p  <+ H' H* 3 (w CD CQ  3 i  ro o  o  o  o  An a n a l y s i s o f t o t a l phosphorus i n the three p l a n t f r a c t i o n s from c o n t r o l and  t r e a t e d p l a n t s grown i n a complete n u t r i e n t  s o l u t i o n i s shown i n Table XI. t h i s n u t r i e n t , t h e r e was ( F i g u r e 5)»  and  When the p l a n t s were grown i n  c l o s e agreement between -* P  t o t a l phosphorus ( F i g u r e 6 )  a percentage b a s i s .  activity  2  A l s o , as was  the case w i t h -* P  times, i . e . l ? . l to 1 8 . 3 $  o f t r e a t e d and  constant through the f o u r and  1.6.4  control sampling  to 19.2$, r e s p e c t i v e l y .  the 8 hour sampling time, 4 l . 4 $ o f the t o t a l phosphorus found i n the l e a f blades o f the c o n t r o l s , and plants.  stems, and  4 hour sampling time.  T h i s p a t t e r n was  However, there was  IO3.3,  blades a t the 12 and  not a g r e a t d e a l  24 hour sampling times.  phosphorus p r e s e n t i n r o o t s  The  quite significant  was  of  amount o f sign-  As f a r as  concerned, t h i s d i f f e r e n c e was  a t the 5$ l e v e l , and  the  leaf  from t r e a t e d p l a n t s d i f f e r e d  i f i c a n t l y from the c o n t r o l v a l u e a t the 5$ l e v e l . the l e a f blade f r a c t i o n  101.6,  a l s o observed a t  d i f f e r e n c e between the percentage v a l u e s f o r r o o t s and  plants.  was  44.8$ i n t r e a t e d  r o o t s o f t r e a t e d p l a n t s was  96.0$, r e s p e c t i v e l y .  almost no  At  On a p e r c e n t o f c o n t r o l b a s i s t o t a l phosphorus i n the  l e a f blades, and  on  activity,  2  t o t a l phosphorus i n the stem f r a c t i o n p l a n t s remained r e l a t i v e l y  when compared  f o r stems there  d i f f e r e n c e between the v a l u e s from c o n t r o l and  not  was treated  % of t o t a l phosphorus  Sampling • • • times i n hours  Leaf blades C  c  b  Stems T  Roots  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  42.97  17.67  17.95  40.70  39.08*  Percent of c o n t r o l  a  See Table IV.  b  See Table VI.  c  See Table I I I .  d  See Table I I I .  103-3  n s  101.6  n S  96.0  50  r5o  40  -40  30  30  CO  u  o  X2  P. CO  o x!  Pi rH  cd o -p  20 -  -20  10  -10  o  o 4  8  12  24  4  8  12  8  24  Roots  Stems  t e a f blades C =  12  T =  "D  24 - Sampling times i n hrs  o  In t h e experiments d e s c r i b e d below, bean p l a n t s were grown h y d r o p o n i c a l l y i n a -P n u t r i e n t s o l u t i o n both b e f o r e and a f t e r the r o o t s o f c o n t r o l and t r e a t e d p l a n t s were exposed to ^ P . 2  However, i n each experiment the experimental variable was changed slightly. 1)  The r e s u l t s o f these experiments a r e b r i e f l y o u t l i n e d . When the p l a n t s were sprayed w i t h a 2500 ppm ( 0 . 2 5 $ )  KNap s o l u t i o n 24 hours p r i o r t o the ^ P f e e d i n g , the e f f e c t o f 2  32 the naphthenate treatment on  J  P uptake was n o t s i g n i f i c a n t .  The e f f e c t o f t h i s treatment on the percentage d i s t r i b u t i o n o f ^ p among the three p l a n t organs was v e r y s i m i l a r to the d i s t r i 2  b u t i o n p a t t e r n observed f o l l o w i n g the 5000 ppm naphthenate treatment. 2)  When bean p l a n t s were sprayed w i t h a 5000 ppm KNap  s o l u t i o n 8 hours p r i o r to the ^ P f e e d i n g , the uptake and 2  percentage d i s t r i b u t i o n were a g a i n v e r y s i m i l a r to the e x p e r i mental r e s u l t s p r e v i o u s l y d e s c r i b e d 3)  ( F i g u r e 1,4).  As was the case i n the experiments where bean p l a n t s  were grown i n a -P n u t r i e n t s o l u t i o n , i n t h i s experiment the p l a n t s were sprayed w i t h a 5000 ppm KNap s o l u t i o n 24 hours p r i o r to t h e -^ P f e e d i n g . 2  E i g h t hours a f t e r the r o o t s were exposed  to a ^ P n u t r i e n t , c o n t r o l and t r e a t e d p l a n t s were withdrawn 2  1  from the -P n u t r i e n t s o l u t i o n .  The a e r i a l p o r t i o n o f the  p l a n t s was removed, and cut-stem exudate was c o l l e c t e d over a p e r i o d o f 2 hours.  A comparison o f the a c t i v i t y i n the exudate  from two s e t s o f t r e a t e d p l a n t s  ( 0 . 2 ml exudate/set o f 4 p l a n t s )  was almost twice t h e l e v e l o f a c t i v i t y i n exudate from two s e t s of control plants.  4)  In a p r e l i m i n a r y  experiment i t was determined t h a t a  f o l i a r a p p l i c a t i o n o f a 0 . 3 $ Tween 20 s o l u t i o n to l4-day-old oean p l a n t s d i d n o t have an e f f e c t on phosphorus uptake and distribution. A l s o , an experiment was designed to determine the e f f e c t o f KNap and Tween 20 on the r a t e o f t r a n s p i r a t i o n . l4-day-old  Two s e t s o f  bean p l a n t s growing i n s o i l were sprayed with 0 . 5 $  KNap i n 0 . 3 $ Tween 20 or w i t h 0 . 3 $ Tween 20 alone. p l a n t s remained as unsprayed c o n t r o l s . pots w i t h two bean p l a n t s p e r pot.  One s e t o f  Each s e t contained  two  A f t e r b r i n g i n g the s o i l  water to f i e l d c a p a c i t y , the pots were c a r e f u l l y enclosed i n polyethylene  bags.  The r a t e o f t r a n s p i r a t i o n from the bean  p l a n t s i n two i d e n t i c a l experiments was determined by measuring the number o f grams o f water t r a n s p i r e d / d e c / h o u r . 2  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 r a t e o f t r a n s p i r a t i o n o f KNap t r e a t e d p l a n t s was lower than t h a t o f unsprayed p l a n t s and those which r e c e i v e d Tween 20 by 12$. A f t e r corn p l a n t s had r e c e i v e d a 0.005$ naphthenate t r e a t ment i n the form o f a f o l i a r  spray, Yur'eva (149 ) observed  t h a t t r e a t e d p l a n t s r e t a i n e d more water when compared with unsprayed c o n t r o l p l a n t s .  In a s i m i l a r experiment the author  ( l 4 9 ) a l s o observed the same r e s u l t w i t h sugar beet p l a n t s .  C.)  Percentage d i s t r i b u t i o n o f a c i d s o l u b l e 32p a c t i v i t y and t o t a l a c i d s o l u b l e P among l e a f b l a d e s , stems, and r o o t s . I d e a l l y , a procedure f o r e x t r a c t i n g metabolic products  from p l a n t t i s s u e would accomplish the f o l l o w i n g :  Stop a l l  enzymatic a c t i v i t y a t the i n s t a n t o f k i l l i n g , p r o t e c t t h e m e t a b o l i c products from chemical degradation, and permit quantitative separation  o f a c i d s o l u b l e and i n s o l u b l e components.  A c c o r d i n g to Cole and Ross ( 4 1 ), the use o f formic and  acid  ethanol to e x t r a c t a c i d s o l u b l e P compounds from p l a n t  t i s s u e 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 o f  t h i s , the procedures, as o u t l i n e d by Cole and Ross, have been employed to f r a c t i o n a t e P compounds from the organs o f c o n t r o l and  t r e a t e d bean p l a n t s  i n t o a c i d s o l u b l e and a c i d  insoluble  components. The  a c i d s o l u b l e P p r e s e n t i n the organs o f c o n t r o l and  t r e a t e d p l a n t s was e x t r a c t e d  w i t h c o l d formic  a c i d and ethanol.  U s i n g t h i s method o f e x t r a c t i o n , the f o l l o w i n g P - c o n t a i n i n g compounds o r groups o f compounds would be found i n t h i s f r a c t i o n : free nucleotides  and n u c l e o s i d e s ,  UDPG, PEP, PGA, p h o s p h o l i p i d s ,  sugar mono- and diphosphates,  and i n o r g a n i c phosphate,( 41 ).  In Table X I I the percentages o f the t o t a l a c i d s o l u b l e 3 p 2  a c t i v i t y f o r the l e a f b l a d e s , stems, and r o o t s o f c o n t r o l and t r e a t e d p l a n t s a r e given. weight b a s i s . age  The cpm were on a per gram f r e s h  Through the 24 hour h o l d i n g p e r i o d ,  o f the t o t a l a c i d s o l u b l e a c t i v i t y i n the r o o t s o f both  c o n t r o l and t r e a t e d p l a n t s decreased. and 49.3  the p e r c e n t -  treated plants  I n the r o o t s o f c o n t r o l  the v a l u e decreased from 5 1 . 4 to 37.5$» and  to 4 0 . 9 $ , r e s p e c t i v e l y .  The differences between t h e values  Phosphorus-32 a c t i v i t y  Sampling times i n hours  Stems  Leaf blades  Roots  T 4  34.09  32.42  16.56  16.09  49.35  51.49  8  37.21  37.80  13.65  14.97  49.13  47.23  12  40.89  42.61  16.82  15.61  42.30  41.78  24  43.36  48.78  15.73  13.67  40.91  37.55  Mean  38.89  40.40  15.69  15.08  45.42  44.51  Percent of c o n t r o l  a  See Table IV.  b  See Table I I I .  c  See Table I I I .  d  See Table I I I .  103.9  nS  96.1  n s  n s  97.0  o  *  '  •  '  0  4  8  12 Sampling time  C =  ' 24 (hours) T =  Total inorganic  Sampling times i n hours  T  c  d  b  Stems  Leaf blades C  phosphate  C  Roots T  C  T  4  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  l4.?2  17.99  38.99  38.58  24  44.28  44.98  15.59  i4.45  40.14  40.57  Mean  44.51  44.43  14.35  I5.38  41.14  40.l8  Percent of c o n t r o l  a  See Table IV.  b  See Table VI.  c  See Table I I I .  d  See Table I I I .  n s  99.8  107.2  n s  97.7  n s  Leaf CO  U O Xi  40  — — ft Root  ft ra o  Xi ft  CD r-l  X>  30 •  d H  o ra  n  •H  o  20 o  ct$ •P o -p  o  .  •« Stem ° Stem 10  —T"  8 C =  —i—  12 Sampling time  24 (hours) T = -o  Phosphorus-32 a c t i v i t y  Sampling times i n hours  Stems  Leaf' blades C  c  Roots  T  c  T  C  T  32.67  16.55  16.55  47.48  50.78  4  35i9?  8  37.79  38.43  12.83  13.90  49.38  47.67  12  ki.kk  44.72  15.98  14.89  42.58  40.39  24  44.35  48.57  15.24  14.07  40.41  37.37  Mean  39.89  4i.l0  15.15  l4.86  44.96  44.05  Percent of c o n t r o l  a  See Table IV.  b  See Table I I I .  c  See Table I I I .  d  See Table I I I .  d  n s  103.1  n s  98.0  n s  97.8  T o t a l i n o r g a n i c phosphate  Sampling times i n hours  Stems  Leaf blades C  T  c  4  45.6?  8  b  C  Roots T  G  T  :  46.24  11.85  14.46*  42.48  39.30  43.41  43.77  12.14  *  44.45  42.41  12  46.83  44.15  13.95  13.81 16.61*  39.23  39.24  24  44.62  44.83  14.93  14.70  40.46  40.46  Mean  45.13  44.75  13.22  14.89*  41.65  40.36  Percent of c o n t r o l  a  See Table IV.  b  See Table V I .  c  See Table I I I .  d  See Table I I I .  d  n S  99.2  112.6  n s  96.9  f o r c o n t r o l and  t r e a t e d p l a n t s were not s t a t i s t i c a l l y  Corresponding to t h i s decrease, the percentage o f the s o l u b l e a c t i v i t y i n the l e a f blades o f c o n t r o l and plants increased.  Even though the d i f f e r e n c e was  significant. total  treated not  statis-  t i c a l l y s i g n i f i c a n t , a c i d s o l u b l e a c t i v i t y i n the l e a f blades o f treated plants increased 34.0  to 4 3 . 3 $ .  The  from 32.4  percentage o f the t o t a l a c i d s o l u b l e  i n stem t i s s u e from c o n t r o l and from one  to 48.?$; c o n t r o l s from  treated plants varied  sampling time to another, but  slightly  the percentage a c i d  s o l u b l e a c t i v i t y remained r e l a t i v e l y constant hour h o l d i n g p e r i o d  through the  Compared w i t h the a c i d s o l u b l e ^2p  pattern described  the t o t a l s o l u b l e P p a t t e r n expressed on the same bases  the l e a f blades and  The  24  7).  (Figure  slightly different.  activity  above, was  percentages o f t o t a l a c i d s o l u b l e P i n  r o o t s o f both c o n t r o l and  remained r e l a t i v e l y constant  treated  over the 24 hour p e r i o d .  plants However,  i n the stem f r a c t i o n o f t r e a t e d p l a n t s the percentage s o l u b l e P, on a per gram b a s i s , was  s i g n i f i c a n t l y greater  than the r e s p e c t i v e c o n t r o l values times (Table X I I I * and  Figure  a t the 8 and  (0.05  level)  12 hour sampling  8).  Comparison o f the percentages o f the t o t a l a c i d s o l u b l e 3p  a c t i v i t y , on a per p l a n t b a s i s , i n the organs o f c o n t r o l  and  treated plants  2  (Table XIV)  with the percentages, on a  gram b a s i s  (Table X I I ) , r e v e a l e d v e r y s i m i l a r d i s t r i b u t i o n  patterns.  T h i s was  (Table X I I I ,  per  a l s o t r u e f o r the a c i d s o l u b l e P data  XV).  Table XXVIII .  XXIX  g i v e s the a c t u a l values  ,  XXX  , XXXI  i n the  Appendix  from which the percentage d i s t r i b u t i o n s  were determined. The  data i n d i c a t e t h a t a mobile ^ P - c o n t a i n i n g 2  p r o b a b l y l a b e l l e d orthophosphate ( 5 0  )» i n the a c i d  f r a c t i o n was b e i n g a c r o p e t a l l y t r a n s l o c a t e d to the a e r i a l p o r t i o n o f the p l a n t .  from r o o t  soluble tissues  Naphthenate treatment  seemed to augment t h i s t r a n s l o c a t i o n p r o c e s s . the exception  compound,  However, w i t h  o f stem t i s s u e from t r e a t e d p l a n t s , the percentage  o f the t o t a l s o l u b l e P remained r e l a t i v e l y constant i n both c o n t r o l and t r e a t e d p l a n t s through the h o l d i n g  period.  D.)  Percentage d i s t i b u t i o n o f a c i d i n s o l u b l e phosphorus-3 p a c t i v i t y and t o t a l a c i d i n s o l u b l e P among l e a f blades, stems, and r o o t s . 2  U s i n g the f o r m i c / e t h a n o l  method o f e x t r a c t i o n , the a c i d  i n s o l u b l e f r a c t i o n Would i n c l u d e P - c o n t a i n i n g nucleic acids.  As b e f o r e ,  p r o t e i n s and  the cpm and t o t a l P data i n t h i s  s e c t i o n a r e expressed on both a p e r gram and p e r p l a n t  basis.  When 3 p a c t i v i t y o f the a c i d i n s o l u b l e f r a c t i o n i n each 2  p l a n t organ i s expressed a percentage o f the t o t a l count on e i t h e r a p e r gram o r a p e r p l a n t b a s i s , the v a l u e s i n Table XVI,  XVIII a r e o b t a i n e d .  Whether on a p e r gram o r a p e r p l a n t  b a s i s , the percentage d i s t r i b u t i o n s o f a c i d i n s o l u b l e 32p a c t i v i t y among the three organs from c o n t r o l and t r e a t e d a t the f o u r sampling times were v e r y s i m i l a r .  plants  The percentage  o f the t o t a l a c i d i n s o l u b l e -^P a c t i v i t y on a p e r gram b a s i s i n root tissue o f treated plants  increased  from 6 9 . 5 t o 7 5 . 1 $  over the 24 hour sampling p e r i o d , w h i l e the i n c r e a s e  i n root  t i s s u e o f c o n t r o l p l a n t s was from 71.1 to 7 2 . 7 $ ( F i g u r e 9 ) . When on a gram o r p l a n t b a s i s , the d i f f e r e n c e between the v a l u e s f o r c o n t r o l and t r e a t e d p l a n t s a t the 24 hour sampling time was s i g n i f i c a n t a t the 0 . 0 5 l e v e l . In c o n t r a s t t o the d i s t r i b u t i o n p a t t e r n s  obtained f o r root  t i s s u e s , on a p e r gram o r a p e r p l a n t b a s i s the percentage o f the t o t a l a c i d i n s o l u b l e a c t i v i t y i n the stems o f c o n t r o l and t r e a t e d p l a n t s decreased. 7.9$,  The v a l u e s decreased from 1 0 . 0 to  and from 1 0 . 0 to 6.8$ i n c o n t r o l and t r e a t e d  respectively.  plants,  The d i f f e r e n c e s between the percentage v a l u e s f o r  stems from c o n t r o l and t r e a t e d p l a n t s were n o t 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  Stems  Leaf blades C  c  Roots  T  C  T  C  T  20.37  10.14  10.08  71.17  69.54  4  18.69  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  19.6l  9.01  8.77  71.90  71.6l  Percent of control  a  See Table IV.  b  See Table I I I .  c  See Table I I I .  d  See Table I I I .  d  102.7  n s  97.3  n s  n s  99.5  _________  <M I CO  _A-  70  U o si  — * Root  ——  A Root  Q, CO O Xi  p,  60 £> H O W  C  — X  •a  20  •H O  Leaf  a  +> o -p  10 -  «H  o  Stem  i  12  4  24  Sampling time (hours) C =  FIGURE  9  T = ON  T o t a l i n o r g a n i c phosphate Sampling times i n  Stems  Leaf blades C  Roots  C  T  C  T  43.17  23.44  21.13  34.43  35.70  T  c  b  4  42.12  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  43.4l  22.26  21.49  35.36  35.45  Percent of control  a  See Table IV.  b  See Table VI.  c  See Table I I I .  d  See Table I I I .  d  n s  101.6  nS  96.5  ns  100.2  Sampling time C =  (hours) T =  significant. The percentage o f the t o t a l a c i d i n s o l u b l e a c t i v i t y i n l e a f b l a d e t i s s u e from c o n t r o l and t r e a t e d p l a n t s  varied  s l i g h t l y from one sampling time to another, but the percentage a c i d i n s o l u b l e a c t i v i t y remained r e l a t i v e l y constant through most o f the 24 hour h o l d i n g As was  period.  the case when the percentages o f the t o t a l a c i d  i n s o l u b l e -^ P 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 2  o f a c i d i n s o l u b l e phosphorus very s i m i l a r .  among the t h r e e organs were a l s o  Over the 24 hour h o l d i n g p e r i o d  the percentages  o f the t o t a l a c i d i n s o l u b l e phosphorus  on a per gram o r per  p l a n t b a s i s ranged between 40 and 4 5 $ ,  19 and 23$,  and 34 and  36$ f o r l e a f b l a d e s , stems, and r o o t s o f c o n t r o l and plants, respectively  (Table  XVII, XIX).  treated  Only the d i f f e r e n c e  between v a l u e s f o r l e a f blades o f c o n t r o l and t r e a t e d on a per gram b a s i s a t the 24 hour sampling time was 0.05  level.  phosphorus  s i g n i f i c a n t a t the  The percentage o f the t o t a l a c i d  insoluble  on a per gram b a s i s over the 24 hour h o l d i n g  i s shown g r a p h i c a l l y i n F i g u r e Table XXXII ,XXXIII  time  10.  , XXXIV, XXXV  i n the Appendix  gives  the a c t u a l v a l u e s 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 r e v e a l t h a t with e x c e p t i o n o f the 4 and 8 hour sampling times, the r a t e o f 3 p 2  a c i d i n s o l u b l e compounds was treated plants.  stimulated  incorporation  i n roots  o f naphthenate-  While the percentage a c i d i n s o l u b l e 3 p i  c o n t r o l and t r e a t e d p l a n t s  into  2  n  showed an o v e r a l l decrease i n stem  Phosphorus-32 a c t i v i t y Sampling times i n hours  ^ C  j s a  ^ blades  Stems  T  c  b  C  Roots T  C  T  20.65  8.22  8.57  71.28  69.59  19.42  21.93  8.67  8.77  71.92  62.29  12  19.29  19.69  8.32  8.48  72.38  71.82  24  20.05  18.07  7.69  6.82  72.26  75.11*  Mean  19.28  19.98  8.23  8.l6  71.96  71.45  4  I8.36  8  Percent of control  See Table IV. b  See Table I I I .  c  See Table I I I .  d  See Table I I I .  d  n s  IO3.6  n s  99.1  99.2  n S  Total i n o r g a n i c Sampling times i n hours  T  c  b  Stems  Leaf blades G  phosphate  C  Roots T  C  T  43.29  20.08  20.95  36.03  35.76  45.35  43.50  19.16  20.78  35.49  35.73  12  43.17  43.75  20.26  19.87  36.56  36.38  24  40.59  43.36  22.95  21.31  36.46  35.33  Mean  43.25  43.47  20.61  20.73  36.14  35.80  4  43.89  8  Percent of c o n t r o l  a  See Table IV.  b  See Table VI.  c  See Table I I I .  d  See Table I I I .  d  •  100.5  ns  nS  100.6  99.0  ns  t i s s u e s over the h o l d i n g p e r i o d , the decrement was n o t s t a t i s tically  significant.  The percentage o f the t o t a l a c i d i n s o l u b l e P i n the t h r e e p l a n t organs from c o n t r o l and t r e a t e d p l a n t s remained  relatively  constant  through the f i r s t 12 hours o f the 24 hour  sampling  period.  However, a t the 24 hour sampling time the percentage  a c i d i n s o l u b l e P on a per gram b a s i s i n l e a f b l a d e s o f t r e a t e d p l a n t s was s i g n i f i c a n t l y g r e a t e r control value.  ( 0 . 0 5 l e v e l ) than the r e s p e c t i v e  T h i s i n c r e a s e was n o t r e f l e c t e d i n the ^ P 2  data.  E.)  D i s t r i b u t i o n o f a c i d s o l u b l e and a c i d i n s o l u b l e 32p a c t i v i t y o r phosphorus, expressed as a percentage "of the t o t a l , among l e a f b l a d e s , stems, and r o o t s . Values f o r a c i d s o l u b l e and a c i d i n s o l u b l e 32p a c t i v i t y o r  phosphorus (as P^)» expressed as a percentage o f the t o t a l and d i s t r i b u t e d among l e a f b l a d e s ,  stems, and r o o t s o f c o n t r o l and  naphthenate-treated p l a n t s , a r e g i v e n i n Table XX, XXI, XXII. Since  t h e r e was v e r y l i t t l e d i f f e r e n c e between the percentage  d i s t r i b u t i o n s o f -^ p and t o t a l phosphorus when expressed on a 2  per gram o r on a p e r p l a n t b a s i s , subsequent d i s c u s s i o n w i l l be confined  t o the p e r gram data.  As f a r as the p e r gram data a r e  concerned, the percentage d i s t r i b u t i o n over the 24 hour sampling p e r i o d was much more i n f o r m a t i v e  than the r e s u l t i n g mean.  Over the sampling p e r i o d the percentage a c i d s o l u b l e -^ P 2  a c t i v i t y i n the l e a f blade f r a c t i o n o f c o n t r o l and t r e a t e d plants increased  (Table XX).  With an exception  -sampling time, the percentages o f the t o t a l 3 2  a t the 4 hour P  a c t i v i t y i n the  a c i d s o l u b l e and a c i d i n s o l u b l e f r a c t i o n s o f l e a f b l a d e s from t r e a t e d p l a n t s were g r e a t e r than the r e s p e c t i v e c o n t r o l v a l u e s . 32 Of the  P a c t i v i t y reaching  the l e a f blades over the sampling  p e r i o d a g r e a t e r p r o p o r t i o n remained i n the a c i d s o l u b l e  fraction.  However, i t a l s o appears t h a t a c i d s o l u b l e -^ P a c t i v i t y was 2  incorporated  i n t o the a c i d i n s o l u b l e f r a c t i o n .  the 3 P data, 2  the d i s t r i b u t i o n p a t t e r n s  Compared w i t h  f o r the percentages o f  the t o t a l i n o r g a n i c phosphate i n a c i d s o l u b l e and a c i d i n s o l u b l e f r a c t i o n s o f l e a f b l a d e s from c o n t r o l and t r e a t e d p l a n t s were different.  Generally,  t h e percentages o f the t o t a l P i n the  two P f r a c t i o n s remained r e l a t i v e l y  constant.  TABLE  E f f e c t o f KNap on the percentage d i s t r i b u t i o n o f a c i d s o l u b l e , a c i d i n s o l u b l e , and t o t a l phosphorus-32 or t o t a l phosphorus, on a p e r gram and a p e r p l a n t b a s i s , i n l e a f blades o f bush bean p l a n t s a t v a r i o u s sampling times .  XX.  a  Phosphorus-32 a c t i v i t y Sampling times i n hours  PER  Acid insoluble T C  c  4 8 12 24 Mean % of control  27.73 28.71 30.34 30.19 29.24  d  PER  GRAM  Acid soluble T C 25.87 29.29 31.02 32.61 29.69  n s  4.11 4.87 5.00 5.98 4.99  3-49 4.36 4.90 5.85 4.65  r i B  C  T  31.22 33.07 35.24 36.04 33.89  29.98 34.16 36.02 38.59 34.68  n s  28. 33 29.16 30.72 30.97 29.79  C  c  T  4 8 12 24  41.13 39.49 42.39 40.82  42.85 39.40 39.64 41.74  Mean  40.98  40.91  fo  3.18 3.63 3.51 3.13 ns  3.3^ 3.46 3.75 3.13 3.42  3.36  ns  25.80 29.75 32.71 32.52 30.21  n s  ;3.90 4.42 4.97 6.00 4.82  phosphate  99.8  PER T  T  c  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  44.34  44.33  41.57  41.22  „ See  Table IV.  .101.7  P L A N T C T 3.27 3.68 3.56 3.14 n s  3.41  C  See Table I I I .  99.1  99.9 e  i i 0  C  T  32.23 33.58 35.69 36.97 34.6l  30.05 34.70 37.99 38.49 35-3l  J  102.0  e  C  n s  4.25 4.95 5.28 5.96 5.11  Total  106.0  Of  control a  C  GRAM T  Acid insoluble C T  101.4  102.3 Total inorganic  P E R  P L A N T  Acid soluble C T  Total  107.0  101.5  b  See Table IV.  C  3.34 45.53 3.49 4 3 . 5 7 3.81 46.53 44. 03 3.11 3.2j4ns^ > 9 8  100.8  T 46.02 43.75 44.12 44.74 4 4 . 6 6 ns :  99.3  TABLE  XXI. E f f e c t o f KNap on the percentage d i s t r i b u t i o n o f a c i d s o l u b l e , a c i d i n s o l u b l e , and t o t a l phosphorus-32 or t o t a l phosphorus, on a p e r gram and a p e r p l a n t b a s i s , i n stems o f bush bean p l a n t s a t v a r i o u s samplings times . a  Phosphorus-32 a c t i v i t y Sampling times i n hours  P E R  4 8 12 24 Mean % of control  Acid insoluble  Acid soluble C  T  c  13.46 10.52 12.50 10.97 11.86  d  P E R  GRAM  12.85 11.59 11.36 9.15 11.24  C  ns  1.89 2.11 2.26 2.38 2.16  T  C  2.03 15.35 2.14 12.63 2. 36 14,76 2 . 2 4 13.35 2.19 l4.02 n s  101.3  94.7  Acid soluble  Total T 14.88 13.73 13.72 11.39 13.43  C  ns  13.04 9-89 11.87 10.64 11.36  T  C  13.07 10.75 10.90 9.42 11.03  1.74 1.97 2.14 2,32 2.04  T o t a l i n o r g a n i c phosphate P E R C 4 8 12 24 Mean % of control  c  11.91 10.42 12.11 13.33 11.94  11.62 ?>2v9l 12.47* 3.18 14.89 3.15 12.11 2.91 3.04 I2.77 n s  106.9  C  3.00 14.82 3.13 13.60 3.39 15.26 2.83 16.24 3.09 l4.98 n s  T 14.62^ 15.60* 18.28* 14.94 15.86* 105.9  C 10.98 11.16 12.80 13.76 12.18  T  C  1.80 14.78 1.99 11.86 2.27 14,01 2.25 1 2 . 9 6 2.08 13.40 ns  101.9  P E R  T  101.6  n s 1  Total T 14.87 12.74 13.17 11.67 I3.ll  n s  97.8  e  GRAM C  T  Acid insoluble  97.0  95.7  P LA N T  T 13.34„ 12.70* 15.16* 13.64 13.71* 112.6  C 1.48 1.55 1.67 1.79 1.62  P LA N T T  C  T  1.62 14.46 1.67 12.71 1.73 14.47 1.53 15.55 1.64 13.80  14.96* 14.37* 16.89* 15.17  101.2  111.2  ns  15.35*  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 o f a c i d s o l u b l e , a c i d i n s o l u b l e , and t o t a l phosphorus-32 or t o t a l phosphorus, on a p e r gram and a p e r p l a n t b a s i s , i n roots of bush bean p l a n t s a t v a r i o u s sampling times . a  Phosphorus-32 a c t i v i t y Sampling times i n hours  PER  PER  G R A Acid insoluble  Acid soluble  D  Total  P L A N T Acid insoluble _  Acid soluble  Total T  T 4 8 12 24 Mean % of control  4o.i5 37.95 32.41 28.49 34.47  d  41.15 13.27 36.58 16.35 30.41 19.09 25.04 22.11 33.07ns17.6l  53.42 13-99 54.30 15.52 1 9 . 8 5 51.50 2 4 . 9 8 * 50.60 l8.8l 52.08 ft  n s  n s  foOf  control  38.28 40.42 35.77 37.00 37.87  36.42 38.69 35.21 37.65 36.99 97.6  ns  2.59 2.86 2.98 2.81 2.81  GRAM  14.92 15.66 19.65* 24.34* l8.64  n s  52.98 54.57 50.30 50.08 5l.98  ns  97.8  n s  n s  P L A N T T  T 39.19 41.54 38.33 40.19 39.8l  55.07 52.56 49.84 48.84 51.58 99.2  102.7  PER  40.87 2.77 2.85 43.28 3.12 38.75 2.54 39.81 2.82 40.68 100.3  n s  15.59 16.43 18.65 21.91 l8.l4  phosphate  T 4 8 12 24 Mean  40.15 36.90 30.19 24.50 32.94 97.3  Total inorganic PER  37.39 38.14 31.65 28.17 33.84  99.6  106.8  95.9  55.14 52.10 50.26 50.02 51.88  39.33 40.84 35.98 37.31 38.37  36.27 38.99 35.82 37.55 37.15 96.8  n s  2.67 2.88 3.03 2.84 2.85  2.76 2.88 3.17 2.54 2.84 99.6  ns  42.00 43.72 39.00 40.15 4l.22  39.03 41.87 38.99 40.09 39.99 97.0  ns  In stem t i s s u e the percentage d i s t r i b u t i o n s o f a c i d and  acid insoluble  of acid soluble  ^P 2  and  a c t i v i t y were s i m i l a r to the  a c i d i n s o l u b l e P.  concerned, d i f f e r e n c e s  f a r as  between the a c i d s o l u b l e  percentage v a l u e s o f e o n t r o l tically significant.  As  and  treated  However, a t the  times the percentage a c i d s o l u b l e s i g n i f i c a n t l y g r e a t e r (0.05  8 and  the  ^P acid  hour h o l d i n g time, i n c o r p o r a t i o n insoluble  statis-  plants  control  was  values. 3p 2  stem t i s s u e s over the  o f 32p  24  a c t i v i t y i n t o the  f r a c t i o n o f r o o t s from c o n t r o l and  treated  acid  plants  g r e a t l y i n c r e a s e d when compared w i t h percentages o f a c i d 3p 2  activity.  At the 24 hour sampling time, the  acid insoluble  -^ P 2  a c t i v i t y i n roots of treated  s i g n i f i c a n t l y g r e a t e r (0.05 The  l e v e l ) than the  to a c i d i n s o l u b l e  phorus i n c o n t r o l and  32p  percentage plants  control  was  value.  a c t i v i t y or i n o r g a n i c  naphthenate-treated p l a n t s .  hour sampling time percentage v a l u e s from Table XX, were used to c a l c u l a t e the  ratio.  was  soluble  v a l u e s shown on the next page r e p r e s e n t the r a t i o  acid soluble  are  insoluble  were not  While i n c r e a s e s i n the percentage a c i d i n s o l u b l e a c t i v i t y o c c u r r e d i n l e a f b l a d e and  data  2  12 hour sampling  P i n treated  l e v e l ) than the  distributions  and  plants  soluble  of  phos-  At the  24  XXI,  XXII  Ratio of a c i d soluble/acid insoluble Control  Phosphorus-32 a c t i v i t y  Treated  L e a f blades  5.1  Stems  4.6  4.0  Roots  1.2  1.0  Inorganic  •  phosphate  Leaf blades  13.0  Stems  4.5  Roots  13.1  13.3  14.8  Depending upon the a c t u a l percentage v a l u e s , i n the r a t i o may  an  increase  i n d i c a t e either a greater l e v e l of ^ P 2  or P  i n the a c i d s o l u b l e f r a c t i o n i n t r e a t e d p l a n t s , or l e s s incorporation of ^ P 2  Tables XXXVI  or P i n t o the a c i d i n s o l u b l e f r a c t i o n .  through XXXIX  i n the Appendix c o n t a i n  data from which the percentage v a l u e s  i n T a b l e XX,  XXI,  the XXII  were determined. Since  the bean p l a n t s used i n t h i s p a r t i c u l a r 'pulse  chase' experiment were grown i n a complete n u t r i e n t , i n o r g a n i c phosphate c o n c e n t r a t i o n s  (31p  + ^ P) 2  stems, and r o o t s o f c o n t r o l p l a n t s r e p r e s e n t d i s t r i b u t i o n o f a c i d s o l u b l e and data alone r e f l e c t  total  i n l e a f blades,  the  'normal'  a c i d i n s o l u b l e P.  The  to 3 P 2  3 2  P  the f a t e o f a s m a l l p u l s e o f t h i s p a r t i c u l a r  r a d i o i s o t o p e a t a p a r t i c u l a r p o i n t d u r i n g the v e g e t a t i v e o f c o n t r o l and  and  t r e a t e d bean p l a n t s .  i n the t o t a l was  growth  Even though the r a t i o  not determined, one  t h a t t h i s r a t i o would have been v e r y s m a l l  could  indeed.  of  hypothesize  DISCUSSION A.)  Phosphorus uptake. Uptake o f 3 p t>y "bean p l a n t s which r e c e i v e d a f o l i a r naph2  thenate treatment was g r e a t e r than t h a t which o c c u r r e d plants  ( F i g u r e 1).  i n control  When expressed on a p e r p l a n t b a s i s , naph-  thenate treatment i n c r e a s e d the ^2p content the -P n u t r i e n t by 9.5$«  of  p l a n t s grown i n  The i n c r e a s e observed i n p l a n t s grown  i n complete n u t r i e n t was 7.1$. R e s u l t s o f s e v e r a l experiments performed i n our l a b o r a t o r y have i n d i c a t e d t h a t naphthenates s t i m u l a t e d both v e g e t a t i v e and reproductive  growth, and v a r i o u s metabolic  o f the a e r i a l p o r t i o n o f the p l a n t . ( 53 ) r e p o r t e d  reactions characteristic  In 1970, F a t t a h and Wort  t h a t the r a t e s o f 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 i n c r e a s e d by naphthenate treatment. i n c r e a s e i n the r a t e o f photosynthesis have p r o v i d e d  i n t r e a t e d p l a n t s would  g r e a t e r amounts o f reduced n u c l e o t i d e s , ATP, and  sugar phosphates. synthate  An  U t i l i z a t i o n o f l a r g e r q u a n t i t i e s o f photo-  by r e s p i r a t o r y processes would have s u p p l i e d t r e a t e d  p l a n t s w i t h a g r e a t e r amount o f ATP f o r use i n the pathways o f a c t i v e and maintenance metabolism. r e q u i r i n g process,  Because P uptake i s an energy  one might have expected a g r e a t e r d i f f e r e n c e  between the amount o f P absorbed by c o n t r o l and t r e a t e d p l a n t s . In a d i s t r i b u t i o n experiment, Padmanabhan ( 109 ) observed t h a t a f t e r 1 week 72$ o f the t o t a l ^ C a c t i v i t y remained i n the primary l e a v e s o f bean p l a n t s which had r e c e i v e d a f o l i a r a p p l i c a t i o n o f KCHC-7- ^C. 1  Other r e s u l t s by the same author r e v e a l e d  t h a t 24 hours a f t e r KCHG-7- C was s p o t t e d on the m i d r i b o f a l2f  primary bean l e a f l e s s t h a t 1$ o f the t o t a l ^ C a c t i v i t y was  detected  i n the r o o t s .  In 1969.  Fattah  ( 52  ) reported  dry weights o f l e a f blades  the f o l l o w i n g *  and  o f naphthenate-treated bean p l a n t s were  s i g n i f i c a n t l y i n c r e a s e d , when f r e s h and were not i n c r e a s e d .  fresh  dry weights o f r o o t t i s s u e s  S i m i l a r l y , the i n c r e a s e i n the a c t i v i t y o f t r e a t e d p l a n t s was  of  n i t r a t e reductase  i n the l e a f blades  signif-  i c a n t a t the 0.05  l e v e l , w h i l e the i n c r e a s e i n the a c t i v i t y o f  the  enzyme i n r o o t t i s s u e l a c k e d s t a t i s t i c a l s i g n i f i c a n c e . R e s u l t s obtained by these two i o n t h a t the m e t a b o l i t e s  i n v e s t i g a t o r s and  o f CHGA ( 130  ) o r KNap ( 14?  than the f r e e a c i d ( s ) , s t i m u l a t e metabolic  remain i n the f o l i a g e f o l l o w i n g treatment ( 109  been a f f e c t e d a p p r e c i a b l y . a b l e f o r the process  Further,  metabolites  not have  the amount o f ATP  o f P uptake i n c o n t r o l and  would have been s i m i l a r .  be  ) compared w i t h  i n r o o t t i s s u e may  Therefore,  ), r a t h e r  r e a c t i o n s may  c o r r e l a t e d . S i n c e the m a j o r i t y o f the naphthenate  the r o o t s , r e s p i r a t o r y processes  the suggest-  avail-  treated plants  the r e s u l t s o b t a i n e d  are  c o n t r a r y to those r e p o r t e d by Russian p l a n t p h y s i o l o g i s t s  64, 65, 113 B.)  (47,  ).  Phosphorus d i s t r i b u t i o n . When p l a n t s were grown i n a -P n u t r i e n t , naphthenate t r e a t 32  ment s t i m u l a t e d the a c r o p e t a l t r a n s l o c a t i o n o f absorbed  J  P.  Plants  grown i n complete n u t r i e n t had a s i m i l a r d i s t r i b u t i o n p a t t e r n , but compared with minus P-grown p l a n t s the percentages o f total 3 P 2  i  n  l e a f blades,  the  stems, and r o o t s were q u i t e d i f f e r e n t .  When grown i n the - P n u t r i e n t , a t the 2k hour sampling time f o u r - f i f t h s o f t h e t o t a l ^ P l a b e l remained i n the r o o t s 2  of treated plants.  However, r o o t s o f t r e a t e d p l a n t s grown i n  complete n u t r i e n t contained t o t a l 32p l a b e l .  s l i g h t l y l e s s than o n e - t h i r d o f t h e  Root c e l l s o f p l a n t s grown i n the complete  n u t r i e n t would have had a "normal* complement o f P , and there would n o t have been a g r e a t demand f o r the 3 P which  entered  2  the r o o t s d u r i n g the f e e d i n g p e r i o d .  Consequently, the m a j o r i t y  o f the 3 P absorbed would have entered 2  p r o b a b l y as a c i d s o l u b l e h 3 P 0 4 ~ . 2  2  the t r a n s p i r a t i o n stream,  The r e l a t i v e l y constant  level  o f ^ P and t o t a l P i n stem t i s s u e over the 2k hour sampling 2  p e r i o d i n d i c a t e s t h a t the m a j o r i t y o f the absorbed 3 P was 2  t r a n s l o c a t e d to the l e a v e s and m e r i s t e m a t i c areas ( F i g u r e 5). T h i s d i s t i b u t i o n p a t t e r n was n o t evident when p l a n t s were grown i n the - P n u t r i e n t .  The o n l y P a v a i l a b l e to these p l a n t s  p r i o r to t h e 3 P f e e d i n g was seed P .  Even though t h e p l a n t s d i d  2  not e x h i b i t symptoms o f P d e f i c i e n c y , some degree o f P d e f i c i t probably existed. the v a c u o l e s ,  When exposed to ^ P , r o o t c e l l s , e s p e c i a l l y 2  would have absorbed and subsequently r e t a i n e d a  great d e a l o f the - ^ P to s a t i s f y t h e i r need f o r P . 2  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 o f 3p 2  which was t r a n s l o c a t e d from r o o t t i s s u e s to the shoot  ( F i g u r e k). occurred  The more r a p i d r a t e o f P t r a n s l o c a t i o n p r o b a b l y  as a r e s u l t o f an accentuated need f o r P i n the a e r i a l  portion o f treated plants, i . e . a sink e f f e c t . R e s u l t s o f an e a r l i e r experiment r e v e a l e d  t h a t the r a t e o f  t r a n s p i r a t i o n i n t r e a t e d p l a n t s was 88$ o f t h a t i n c o n t r o l  plants.  Other i n v e s t i g a t o r s have r e p o r t e d  treatment i n c r e a s e d sugar "beets ( 149 transpiration.  On  t h a t naphthenate  the water content o f corn  ), s u g g e s t i n g  90  ( 149,  and  a decrease i n the r a t e o f  the assumption t h a t t r a n s p i r a t i o n  s i m i l a r l y reduced i n the p r e s e n t t h a t P t r a n s l o c a t i o n occurred  )»  was  experiment, the r e s u l t s suggest  i n the symplast.  t r a n s l o c a t i o n would r e q u i r e the augmented energy  T h i s mode o f P production  which i s b e l i e v e d to occur i n the a e r i a l p o r t i o n o f the  plant  f o l l o w i n g naphthenate treatment. An i n c r e a s e i n m e t a b o l i c r a t e s would have a l s o o c c u r r e d t r e a t e d p l a n t s grown i n the complete n u t r i e n t medium. w i t h a normal complement o f P,  in  However,  the s i n k e f f e c t i n t r e a t e d  p l a n t s would have been g r e a t l y reduced when compared w i t h p l a n t s i n which P was C.)  limiting.  I n c o r p o r a t i o n o f phosporus-32 and phosphorus i n t o a c i d s o l u b l e and a c i d i n s o l u b l e f r a c t i o n s . The  i n c o r p o r a t i o n o f -^ P 2  i n t o the a c i d s o l u b l e and  i n s o l u b l e f r a c t i o n s o f l e a f b l a d e s was  enhanced by naphthenate  treatment.  In r o o t t i s s u e s o f c o n t r o l and  soluble 3 P  a c t i v i t y decreased, however, -^ P  2  treated plants acid 2  a c t i v i t y i n the  a c i d i n s o l u b l e f r a c t i o n o f r o o t s from t r e a t e d p l a n t s significantly. the two  With few  exceptions,  P f r a c t i o n s o f c o n t r o l and  over the 24 hour h o l d i n g  acid  increased  the amount o f t o t a l P i n  t r e a t e d p l a n t s d i d not  change  period.  An i n c r e a s e i n the p h o t o s y n t h e t i c  and r e s p i r a t o r y r a t e s  t r e a t e d p l a n t s would r e s u l t i n a g r e a t e r  incorporation of  among components o f the a c i d s o l u b l e f r a c t i o n , e.g.  free  of  -* P 2  nucleotides ( 1^7  and  sugar phosphates.  Related  to t h i s , Wort et a l  ) observed t h a t the s p e c i f i c a c t i v i t y o f cytochrome 172$  o x i d a s e i n the l e a v e s o f naphthenate-treated p l a n t s was o f the  control value.  reduced n u c l e o t i d e s  T h i s suggests t h a t the u t i l i z a t i o n  and  the p r o d u c t i o n  t r a n s p o r t system were i n c r e a s e d . o f s t a r c h phosphorylase was  ( 52 ).  acid  by the  f a c t t h a t the  electron activity  1 3 7 $ o f the c o n t r o l v a l u e  t h a t sugar phosphate p r o d u c t i o n bean p l a n t s  The  o f ATP  Further,  was  of  indicates  a l s o augmented i n t r e a t e d  an i n c r e a s e i n the p r o d u c t i o n  of  soluble 32  P  compounds would p l a y an important r o l e i n the s y n t h e s i s o f components found i n the a c i d i n s o l u b l e f r a c t i o n . For example, ATP  i s r e q u i r e d f o r the s y n t h e s i s o f i n o s i n i c a c i d ,  a n u c l e o t i d e from which other p u r i n e n u c l e o t i d e s and  f o r o r o t i c a c i d , the parent p y r i m i d i n e .  insoluble 3 P 2  are  An i n c r e a s e o f a c i d  a c t i v i t y i n r o o t t i s s u e s suggests t h a t  s y n t h e s i s o f n u c l e i c a c i d s may  derived,  have been among the  the  components  o f t h i s f r a c t i o n which were a f f e c t e d by naphthenate treatment. In 1 9 6 5 t Pakhomova ( 110 increased tomato.  ) reported  t h a t naphthenate treatment  the r a t e o f n u c l e i c a c i d s y n t h e s i s . i n the l e a v e s Wort et a l ( 1^7  o f p l a n t growth was  of  ) suggested t h a t naphthenate s t i m u l a t i o n  the r e s u l t o f the a c t i o n o f naphthenate, or  i t s d e r i v a t i v e s , a t the g e n e t i c l e v e l , i . e . a t the t r a n s c r i p t i o n level. An  enhanced i n c o r p o r a t i o n o f -^ P 2  i n t o the a c i d i n s o l u b l e  f r a c t i o n o f r o o t t i s s u e from t r e a t e d p l a n t s may due  to an i n c r e a s e i n the r a t e o f phosphoprotein  The  hydroxyl  group o f s e r i n e or t h r e o n i n e  may  have a l s o been formation.  serve as  loci  i n the primary s t r u c t u r e o f a p r o t e i n where  phosphorylation  could occur.  Phosphoproteins e x i s t i n p l a n t c e l l s and i t has  been r e p o r t e d  t h a t naphthenate treatment i n c r e a s e d both the  amount o f p r o t e i n and the l e v e l o f i n c o r p o r a t i o n o f  1  ^C-L-leucine  i n t o p r o t e i n o f l e a f b l a d e s ( 147 ), perhaps the phosphoprotein content o f the a c i d i n s o l u b l e f r a c t i o n was  correspondingly  i n c r e a s e d by treatment. In c o n c l u s i o n ,  the r a t e a t which the l e v e l o f a c i d s o l u b l e  3 P a c t i v i t y was reduced i n r o o t s o f c o n t r o l and t r e a t e d p l a n t s 2  suggests t h a t a c i d s o l u b l e ^ P a c t i v i t y was t r a n s l o c a t e d 2  a c r o p e t a l l y to the a e r i a l p o r t i o n o f the p l a n t .  An i n c r e a s e i n  the a c i d i n s o l u b l e 32p f r a c t i o n o f r o o t s a l s o i n d i c a t e s t h a t one or more  J  P-containing  compounds were i n c o r p o r a t e d  into this  f r a c t i o n , and the p r o c e s s o f i n c o r p o r a t i o n was enhanced by naphthenate treatment.  I n l e a v e s , naphthenate treatment  the 32p l e v e l i n the a c i d s o l u b l e f r a c t i o n .  increased  This increase  was  p r o b a b l y c l o s e l y r e l a t e d to the enhanced a c r o p e t a l movement o f J  P, and to a s t i m u l a t i o n o f m e t a b o l i c r e a c t i o n s i n the a e r i a l  p o r t i o n o f the p l a n t .  Chapter 2  THE METABOLISM OF CYCLOHEXANECARBOXYLIC ACID.  INTRODUCTION The metabolism o f p l a n t growth r e g u l a t o r s and r e l a t e d compounds has r e c e i v e d a t t e n t i o n from s e v e r a l l a b o r a t o r i e s . I n t e r e s t i n t h e metabolism o f a l i c y c l i c c a r b o x y l i c a c i d s arose from c u r r e n t work b e i n g  conducted i n our l a b o r a t o r y on the  e f f e c t o f KNap, HNap, and o f model naphthenic a c i d s , e.g. KCHC, KCPC, KCHAc, KCPA, and KCHB, on the growth, y i e l d , and m e t a b o l i c processes o f various The  i n d i v i d u a l naphthenic a c i d , CHCA, was used i n t h i s  phase o f the p r e s e n t l)  crop p l a n t s .  i n v e s t i g a t i o n f o r the f o l l o w i n g reasons,  i t i s a component o f the complex naphthenic a c i d mixture  ( 46 ), 2) A i t s t i m u l a t e s both v e g e t a t i v e o f bean p l a n t s  and r e p r o d u c t i v e  growth  ( 108, 143, 145 ), and 3) i t i s the o n l y naph-  t h e n i c a c i d which can be purchased i n the r a d i o a c t i v e form. The purpose o f these s t u d i e s was to i n v e s t i g a t e the metab o l i s m o f the b i o l o g i c a l l y f o r e i g n p e t r o c h e m i c a l , l e a f b l a d e s and r o o t s o f bush bean p l a n t s .  CHCA, i n  After a careful  review o f p e r t i n e n t l i t e r a t u r e , i t appears t h a t t h i s i s the f i r s t m e t a b o l i c study conducted w i t h a c i d i n a higher  plant.  cyclohexanecarboxylic  LITERATURE REVIEW Even though the c o m p i l a t i o n Table XXIII l i s t s  i s by no means exhaustive,  the i n i t i a l products o f t h e metabolism o f  s e v e r a l n a t u r a l l y - o c c u r r i n g and s y n t h e t i c o r g a n i c plant tissues.  acids i n  While i n v e s t i g a t i n g the metabolism o f IAA i n  tomato r o o t s , Thurman and S t r e e t  ( 139)  suggested t h a t i n  a d d i t i o n to the endogenous m e t a b o l i t e ,  indoleacetylaspartate,  indoleacetylglutamate  However, t h e presence  was a l s o formed.  o f the glutamate conjugate was n o t confirmed. In s e v e r a l p u b l i c a t i o n s Wort and P a t e l ( 143, 144) have shown t h a t KCHC s i g n i f i c a n t l y s t i m u l a t e d plants.  the growth o f bean  I n one o f t h e i r experiments a f o l i a r a p p l i c a t i o n o f  an aqueous 2 5 0 0 ppm ( 2 x 1 0 " " M) KCHC s o l u t i o n invoked a 3 5 $ 2  increase  i n the weight o f green pods p e r p l a n t .  S o v i e t Union, A g a k i s h i e v e t a l ( 5  Working i n the  ) observed t h a t the a p p l i -  c a t i o n o f s e v e r a l d i f f e r e n t s u b s t i t u t e d cyclohexylbutanones and cyclohexylbutanols and  increased  t h e r a t e o f c o t t o n seed germination,  t h e y i e l d and q u a l i t y o f c o t t o n d u r i n g a t h r e e year study.  Wort and P a t e l and a l s o A g a k i s h i e v et ;al have both suggested t h a t the presence o f the s i x - c a r b o n molecules was r e q u i r e d  saturated  r i n g i n these  f o r t h e most e f f e c t i v e s t i m u l a t i o n o f  p l a n t growth. It  has a l s o been suggested ( 1 2 9 , 1 3 0 , 14?) t h a t t h e  presence o f the conjugated o r bound forms o f naphthenate may be r e s p o n s i b l e  f o r the s t i m u l a t i o n o f m e t a b o l i c  which l e a d to i n c r e a s e s in  vivo.  i n vegetative  reactions  and r e p r o d u c t i v e  growth  TABLE  XXIII.  Glucose e s t e r and a s p a r t i c a c i d amide f o r m a t i o n f o l l o w i n g the a d m i n i s t r a t i o n o f v a r i o u s o r g a n i c acids to plant t i s s u e s .  PLANT  COMPOUND  GLUCOSE ESTER  ASPARTIC ACID AMIDE  REFERENCE  X  (  X  X  (152)  NAA  X  X  (153)  Wheat c o l e o p t i l e s  NAA  X  X  ( 86)  Wheat c o l e o p t i l e s  BA  X  X  ( 84)  Pea  BA  X  X  (154)  X  X  (  Pea  seedlings  IAA  Pea  epicotyls  IAA  Pea  epicotyls  epicotyls  10)  Wheat c o l e o p t i l e s  2,4-D  Pea  2,4-D  X  (  2,4-D  X  (137)  epicotyls  Chlorella Bean l e a f Bean  disks  seedlings  Bean r o o t s and c u t stem exudate  85)  U)  KCHC  X  X  (127)  KCHC KCPC KNap  X X X  X X X  (122)  KCHC  X  X  (131)  x denotes the presence o f the m e t a b o l i t e .  Other m e t a b o l i c s t u d i e s i n our l a b o r a t o r y have r e v e a l e d t h a t when the complex naphthenate mixture, KNap, was  a p p l i e d to  the primary l e a v e s o f young bush bean p l a n t s these a c i d s were r e a d i l y converted to a mixture o f conjugates w i t h g l u c o s e aspartic acid.  and  Twenty-four hours a f t e r a p p l i c a t i o n , f r e e a c i d s  were not d e t e c t e d  chromatographically  i n extracts of l e a f  blades.  When KNap, KGHG, and KCPC were f e d to the r o o t s o f bean p l a n t s , both conjugates were detected  i n the l e a f b l a d e s a f t e r  2k  hours ( 122 ) . Results  o f i n v e s t i g a t i o n s performed i n our l a b o r a t o r y  dealing  w i t h the metabolism o f naphthenic a c i d s i n bean p l a n t s were communicated by the author and  Dr.  G. E. S e a f o r t h a t the Annual  Meeting o f the Northwest S c i e n t i f i c A s s o c i a t i o n , 19^9. i n f o r m a t i o n was  also published  Additional information  i n Phytochemistry ( 127  This ).  on CHCA metabolism i n bean p l a n t s  communicated a t the Western S e c t i o n meeting o f the S o c i e t y o f P l a n t P h y s i o l o g i s t s , 1971  (109,  131).  was  Canadian  MATERIALS AND A.)  Leaf disk feeding  METHODS  experiment.  L e a f d i s k s were cut from the primary l e a v e s o f a  14-day-old  bush bean p l a n t u s i n g a cork b o r e r ( l / 2 i n c h i n diameter)  and  were f l o a t e d on 6 ml o f d i s t i l l e d water i n a p e t r i d i s h .  To  t h i s was  added 2.5 JiCi o f c y c l o h e x a n e c a r b o x y l i e a c i d - 7 14  c  (Intern a t i o n a l Chemical  and Nuclear) as the K s a l t .  a c o n c e n t r a t i o n o f 20 jaM, to minimize light  The d i s h was  This represented  wrapped w i t h Saran wrap  water l o s s by e v a p o r a t i o n , and then p l a c e d i n the  (5400 l u x ) f o r 24 hours a t 24° C.  the l e a f d i s k s , water, but no  C o n t r o l d i s h e s contained  acid.  At the end o f the metabolism p e r i o d the l e a f d i s k s were washed s e v e r a l times w i t h water to remove a d h e r i n g l a b e l l e d compound, then e x t r a c t e d w i t h s e v e r a l changes o f 80$ e t h a n o l . E x t r a c t i o n was white.  The  c o n s i d e r e d complete when the l e a f d i s k s were  combined ethanol e x t r a c t s were evaporated  to dryness,  e x t r a c t e d 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 . 8 hour e t h y l a c e t a t e e x t r a c t i o n o f the aqueous f i l t r a t e  An  yielded  o n l y a p a r t i a l s e p a r a t i o n o f the major compounds, the g l u c o s e e s t e r (compound number 1) b e i n g more s o l u b l e i n e t h y l a c e t a t e , w h i l e the a s p a r t i c a c i d amide (compound number 4) remained p r i m a r i l y i n the aqueous phase.  A l l chromatography was  t h i n - l a y e r p l a t e s o f c e l l u l o s e MN  done u s i n g  3 0 0 G (Macherey and Nagel,  R a d i o a c t i v e compounds were d e t e c t e d w i t h Kodak M e d i c a l film.  X-ray  Co.).  B.)  S y n t h e s i s o f l-Cyclohexaneearbonvl-yg-D-glucose. C y c l o h e x a n e c a r b o x y l i c a c i d was  converted i n t o the a c i d  c h l o r i d e by r e f l u x i n g the a c i d w i t h a s l i g h t excess o f t h i o n y l c h l o r i d e i n dry benzene f o r 4 hours.  A f t e r removal o f benzene  and excess t h i o n y l c h l o r i d e under reduced p r e s s u r e , the r e s i d u a l a c i d c h l o r i d e was  d i s s o l v e d i n a s m a l l volume o f d r y p y r i d i n e  and t r e a t e d w i t h an equimolar amount tetraacetate,, ( 9 7  ).  o f /l-D-glucose-2,3,4,6-  The stoppered r e a c t i o n 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 a t room temperature. 12 N HC1  The r e a c t i o n mixture was  and crushed i c e .  w i t h water,  poured i n t o a mixture o f  The p r e c i p i t a t e was  c o l l e c t e d , washed  then w i t h 10$ sodium b i c a r b o n a t e s e v e r a l times,  f i n a l l y w i t h water, and then d r i e d a t room temperature. r e c r y s t a l l i z a t i o n s from anhydrous methanol y i e l d e d  Several  colourless  c r y s t a l s of l-cyclohexanecarbonyl-2,3,4,6-tetraacetyl-/3-D-glucose, m.p.  114.5  -  115.5°  C.  D e a c e t y l a t i o n was anhydrous NH^ above.  The y i e l d was  accomplished by p a s s i n g a stream o f  through a methanol s o l u t i o n o f the product from  Excess NH^  and s o l v e n t were removed under reduced  p r e s s u r e , the r e s i d u e was was  68$.  d i s s o l v e d i n water, and t h i s  solution  e x t r a c t e d c o n t i n u o u s l y w i t h e t h y l a c e t a t e f o r 12 hours.  The e t h y l a c e t a t e s o l u b l e m a t e r i a l was i n BAW  p u r i f i e d by chromatography  to y i e l d a white p r o d u c t w i t h m.p.  c r y s t a l l i n e p r o d u c t was  not obtained.  l 4 l - 143° 0.  T h i s p r o d u c t was  A chromato-  g r a p h i c a l l y homogeneous and y i e l d e d o n l y c y c l o h e x a n e c a r b o x y l i c a c i d and g l u c o s e on h y d r o l y s i s . attempted.  F u r t h e r p u r i f i c a t i o n was  not  C. ) S y n t h e s i s o f N-Cyclohexanecarbonyl-L-aspartic L-Aspartic acid (I.43  grams, 0 . 0 1 ?  .  0  0  5  grams,  0  .  0  0  5  M) and sodium b i c a r b o n a t e  M) were s t i r r e d i n 6 ml o f water a t room  C y c l o h e x a n e c a r b o x y l i c a c i d c h l o r i d e ( 0 . 6 4 grams,  temperature. 0  (O.67  Acid.  M) was added i n  5  p o r t i o n s over a p e r i o d o f  3  0  minutes.  The mixture was a c i d i f i e d t o pH 2 and c o n t i n u o u s l y e x t r a c t e d w i t h ether f o r s e v e r a l hours.  Chromatography o f t h e ether  e x t r a c t showed i t t o be a mixture o f c y c l o h e x a n e c a r b o x y l i c a c i d and t h e d e s i r e d amide.  S e p a r a t i o n was done c h r o m a t o g r a p h i c a l l y .  E l u t i o n o f t h e d e s i r e d band w i t h methanol was f o l l o w e d by c r y s t a l l i z a t i o n o f t h e m a t e r i a l from e t h a n o l off e t h y l a c e t a t e . T h i s procedure above room  y i e l d e d a white, waxy s o l i d whose m.p.was s l i g h t l y  temperature.  D. ) H y d r o l y s i s Procedures. Bands o f unknown conjugates were scraped from the TLC^plates and e l u t e d w i t h 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  t o dryness.  Band 1  (banded m a t e r i a l c o r r e s p o n d i n g t o spot l ) m a t e r i a l was h y d r o l y z e d i n t o i t s component p a r t s by 2 N  NaOH f o r 3 0 minutes a t 7 5 °  C.  A c i d i f i c a t i o n and e x t r a c t i o n w i t h ether separated the a c i d and sugar p o r t i o n s o f t h i s compound. Band 4 m a t e r i a l was e f f e c t i v e l y h y d r o l y z e d w i t h 6 N 3 0  minutes a t 7 0 ° C .  for  The mixture was n e u t r a l i z e d w i t h sodium  b i c a r b o n a t e , evaporated  t o dryness, and the r e s i d u e e x t r a c t e d with  s m a l l p o r t i o n s o f anhydrous methanol. was  HC1  used f o r chromatography.  T h i s methanolic  solution  Band 2 m a t e r i a l was a l s o h y d r o l y z e d  t h i s way. Band numbers correspond t o compound numbers.  E.)  Root f e e d i n g experiment. Uniform seeds o f the hush bean p l a n t , Phaseolus v u l g a r i s L.  c u l t i v a r Top Crop were sown i n v e r m i c u l i t e , and were p l a c e d i n a growth room as d e s c r i b e d  i n the p r e c e d i n g s e c t i o n .  After  s i x days, u n i f o r m s e e d l i n g s were t r a n s p l a n t e d i n t o beakers c o n t a i n i n g a one-quarter s t r e n g t h Hoagland-Arnon*s  complete  nutrient s o l u t i o n ( 74 ) . The r o o t s o f two s e t s o f u n i f o r m bean s e e d l i n g s , w i t h f o u r 2-week-old p l a n t s i n each s e t , were immersed i n 2 5 ml o f d i s t i l l e d water c o n t a i n i n g o n l y 5 " C i (1 x 1 0 - 5 m) o f K C H C ^ - ^ C , o r 5 juGi ( 3 x 1 0 * 5 M) o f D-glucose-UL- *C lZ  (Amersham/Searle,  1Q~5 M u n l a b e l l e d KCHC a t pH 8 . 3 .  Toronto) w i t h  A f t e r s i x hours o f i n c u b a t i o n ,  the a e r i a l portions o f the p l a n t s i n each s e t were e x c i s e d , and cut-stem exudate was c o l l e c t e d f o r ca 1 hour.  After collection,  the exudate was a p p l i e d d i r e c t l y to s e p a r a t e TLC p l a t e s . The a e r i a l p o r t i o n s o f the p l a n t s i n each s e t were d i v i d e d i n t o stems p l u s p e t i o l e s and l e a f b l a d e s .  Upon completion o f  exudate c o l l e c t i o n , the r o o t s o f the p l a n t s from each s e t were w e l l r i n s e d , and the t h r e e organs from the p l a n t s i n each s e t were e x t r a c t e d s e p a r a t e l y w i t h b o i l i n g 80$ EtOH f o r 1 hour. Each e x t r a c t was evaporated t o a volume o f ca 1 ml under reduced p r e s s u r e a t 40° C.  The c o n c e n t r a t e d e x t r a c t s were used  d i r e c t l y f o r chromatographic purposes. All  chromatography was done u s i n g TLC p l a t e s o f c e l l u l o s e  MN 3 0 0 G (wet t h i c k n e s s O . 5 0 mm), i n an i s o p r o p a n o l 1 7 $ NH^0H;water  and the p l a t e s were developed (81I1I)  s o l v e n t system.  In  c e r t a i n i n s t a n c e s , e x t r a c t s s p o t t e d on TLC p l a t e s were developed  i n n - b u t a n o l j g l a c i a l a c e t i c a e i d j water ( 4 : 1 : 5 , top phase). R a d i o a c t i v e areas on the chromatograms were d e t e c t e d by the chromatograms to Kodak Blue Brand M e d i c a l X-ray  exposing  film,.  RESULTS AND  DISCUSSION  A f t e r 24 hours o f metabolism i n the presence o f KCHC-7-^C, l e a f d i s k t i s s u e was  e x t r a c t e d and  analyzed  by TLC  using a basic  s o l v e n t system, i s o p r o p a n o l » 7 $ NHjjOH.water (IAW), and one,  n - b u t a n o l : g l a c i a l a c e t i c acid.water (BAW).  i n the IAW  gave o n l y two.  Compound No.  R  f  was  Chromatography while  An a n a l y s i s o f the  compounds o f e x t r a c t s ehromatographed i n the IAW  The  acidic  system y i e l d e d f o u r major l a b e l l e d compounds,  chromatography i n BAW  T h i s was  an  labelled  system f o l l o w s .  1  the l a r g e s t spot observed on the radiochromatogram. 0 . 8 5 which agreed c l o s e l y w i t h t h a t f o r the  synthetic  e s t e r , l-cyclohexanecarbonyl-/3-D-glucose (Table XXIV). area on the chromatogram gave no bromophenol b l u e reagent, but  c o l o r when sprayed w i t h  Removal o f t h i s  o f the chromatogram, f o l l o w e d by h y d r o l y s i s o f the  o f the s y n t h e t i c glucose the same r e s u l t s . a l s o had  compounds, glucose  e s t e r and  the glucose  eluted  CHCA.  the i s o l a t e d  acid (35*55*10).  and  metabolite  therefore  a c i d (CHCGluc).  c o l o r r e a c t i o n w i t h ammoniacal s i l v e r n i t r a t e e v i d e n t l y as a r e s u l t o f some h y d r o l y s i s due  BAW,  chloroform:ethyl  Compound number 1 was  ester of cyclohexanecarboxylic  spray r e a g e n t .  Hydrolysis  i n t h r e e a d d i t i o n a l s o l v e n t systems:  isopropanol*benzene:water ( 5 5 * 3 0 * 1 1 ) , acetate:formic  and  region  e s t e r under i d e n t i c a l c o n d i t i o n s y i e l d e d  Synthetic  i d e n t i c a l R^a  a  the area q u i c k l y darkened when  sprayed w i t h ammoniacal s i l v e r n i t r a t e .  m a t e r i a l y i e l d e d o n l y two  This  The  occurred  to the a l k a l i n e n a t u r e o f  the  2  Compound No.  T h i s compound was system where i t r a n pound gave no  observed o n l y i n the b a s i c  solvent  j u s t behind the glucose e s t e r .  The  com-  c o l o r r e a c t i o n w i t h e i t h e r bromophenol b l u e  or  ammoniacal s i l v e r n i t r a t e , but d i d g i v e a p o s i t i v e r e a c t i o n with i o d i n e vapor.  Acid h y d r o l y s i s of eluted material y i e l d e d  CHCA as the s o l e o r g a n i c product.  Appearance o f t h i s compound  o n l y i n the ammoniacal s o l v e n t system, and a c i d h y d r o l y s i s suggest t h a t i t might be a c i d amide (CHCAm).  I t was  pound was  equivalent  chromatography o f l e a f e x t r a c t s .  not d e t e c t e d  i n the a c i d i c BAW  ( 152 ,  this  Since  was  t h i s com-  when l e a f e x t r a c t s were chromatographed  Zenk r e p o r t e d  i n a l l proba-  similar artifact  formation  155).  Compound No. T h i s was  of  synthetic  to t h a t which  s o l v e n t system, the amide was  b i l i t y an a r t i f a c t . w i t h IAA  h y d r o l y t i e b e h a v i o r supported  system l e d to the formation  compound i n a p r o p o r t i o n r o u g h l y observed by  authentic  then found t h a t chromatography o f the  glucose e s t e r i n the IAW  the  cyclohexanecarboxylic  Comparison o f e l u t e d m a t e r i a l w i t h  amide,by means o f chromatography and t h i s view.  the r e s u l t s o f  3  the s m a l l e s t spot observed on the radiochromato-  gram, and  a g a i n was  seen o n l y when e x t r a c t s were chromatographed  i n the IAW  system.  I t gave an a c i d i c r e a c t i o n w i t h bromophenol  b l u e , and  had  a R  f  i d e n t i c a l to t h a t o f f r e e CHCA.  Formation  o f t h i s a r t i f a c t by a l k a l i n e h y d r o l y s i s o f the g l u c o s e e s t e r was  demonstrated u s i n g s y n t h e t i c m a t e r i a l .  N-eyclohexaneearbonyl-L-glycine ( 60 ) hasr  Since ;  synthetic  almost the same  as CHCA i n IAW and BAW, e l u t e d m a t e r i a l was h y d r o l y z e d w i t h a c i d and t h e h y d r o l y s a t e examined f o r amino a c i d s b u t none was found. Compound No. 4 T h i s spot on the chromatogram r e p r e s e n t e d a compound which was p r e s e n t i n a r e l a t i v e l y h i g h c o n c e n t r a t i o n w i t h low m o b i l i t y i n the b a s i c s o l v e n t system (H^ 0.l6).  The compound gave a  p o s i t i v e t e s t w i t h bromophenol b l u e , and upon h y d r o l y s i s ,  yielded  CHCA and a s p a r t i c a c i d .  Comparison w i t h s y n t h e t i c N-cyclohexane-  carbonyl-L-aspartic acid  (CHCAsp) w i t h r e g a r d to chromatography,  c o l o r r e a c t i o n w i t h the a c i d - b a s e i n d i c a t o r , and products o f h y d r o l y s i s showed t h i s compound to be p r e s e n t . The two r a d i o a c t i v e compounds observed on radiochromatograms developed  i n BAW had R s o f O.78 and 0.90. f  The slower  moving compound was i d e n t i c a l i n a l l r e s p e c t s to a u t h e n t i c g l u c o s e e s t e r , and the f a s t e r moving t o t h e a s p a r t i c a c i d amide. The r e s u l t s o f  radiochromatography  analyses o f ethanol  e x t r a c t s o f r o o t s , stems p l u s p e t i o l e s , l e a f b l a d e s , and c u t stem exudate o f bean s e e d l i n g s r o o t - f e d KCHC-7-"^C r e v e a l e d t h a t CHCGluc and CHCAsp were p r e s e n t .  When developed  i n the  IAW s o l v e n t system, t h e i r R s were 0.88 and 0.17, r e s p e c t i v e l y . f  The  i d e n t i f i c a t i o n o f CHCGluc and CHCAsp was based on the  c r i t e r i a outlined previously.  The c o n c e n t r a t i o n  o f the two  conjugated m e t a b o l i t e s was h i g h e s t i n the e x t r a c t o f r o o t t i s s u e , and lowest i n the l e a f blade and i n cut-stem fractions.  exudate  When r o o t e x t r a c t s were chromatographed i n IAW, a  p a r t i a l ammonolysis o f CHCGluc o c c u r r e d p r o d u c i n g the chromatographic a r t i f a c t s ,  i . e . CHCA and CHCAm.  As b e f o r e , when  TABLE  XXIV.  Chromatographic data  a  .  -  R  Compound  f  IAW  BAW  0.73  0.92  0.75  0.92  Benzoic a c i d  0.66  0.90  Glucose  0.40  0.27  0.08  0.25  C y c l o h e x a n e c a r b o x y l i c a c i d amide  0.80  0.85  1 - Cy c l o h exane carbonyl-/3- D- g l u co s e  0.85  0.78  N-Cyclohexaneearbonyl-L-aspartic a c i d  0.16  0.90  N-Cyclohexanecarbonyl-L-glycine  0.71  0.91  Cyclohexanecarboxylic  acid  Cyclohexanecarboxylic a c i d  Aspartic  (K s a l t )  acid  A l l a n a l y s e s were done u s i n g 0.50 mm (wet t h i c k n e s s ) C e l l u l o s e MN 300 G p l a t e s . 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, t o p phase).  e x t r a c t s were chromatographed i n BAW, were not  f r e e a c i d and  the amide  detected.  A radiochromatographic a n a l y s i s o f e x t r a c t s the p l a n t organs, and uniformly  o f cut-stem exudate from p l a n t s  l a b e l l e d glucose and  three  root-fed  u n l a b e l l e d KGHC r e v e a l e d  the  14 presence o f CHCGluc and glucose was  detected  fractions.  The  CHCAsp i n r o o t s and  i n r o o t s , stems, and  l e a f blade f r a c t i o n was  stems.  Free  C  cut-stem exudate  void of l a b e l l e d  metabolites. The  appearance o f GHCAsp i n the e x t r a c t s i n d i c a t e s t h a t  the a s p a r t a t e moiety o f the conjugate was  labelled.  not an unusual r e s u l t as the metabolism o f ^ C  This i s  glucose by bean  r o o t s would have produced a v a r i e t y o f l a b e l l e d compounds, i n c l u d i n g l a b e l l e d a s p a r t a t e , v i a pathways o f dark r e s p i r a t i o n . S i n c e s e v e r a l o f the l a b e l l e d amino a c i d s would have been i n the same R_. r e g i o n as CHCAsp i n the IAW o f stems and  system, the  r o o t s were a l s o chromatographed i n BAW.  present  extracts In  this  s o l v e n t system most amino a c i d s are r e l a t i v e l y immobile, w h i l e the g l u c o s e e s t e r and at high R The and  f  the a s p a r t i c a c i d amide were  present  p o s i t i o n s (Table XXIV).  absence o f l a b e l l e d m e t a b o l i t e s  g e n e r a l l y , the lower c o n c e n t r a t i o n s  i n stems, r o o t s , and  i n the l e a f b l a d e s , o f l a b e l l e d compounds  i n cut-stem exudate f r a c t i o n s o f p l a n t s  r o o t - f e d l a b e l l e d glucose and  u n l a b e l l e d KCHC was  probably  r e l a t e d to the reduced a v a i l a b i l i t y o f l a b e l l e d g l u c o s e  and  aspartate  Once  f o r purposes o f c o n j u g a t i o n  i n s i d e r o o t t i s s u e s the ^ G  i n root tissues.  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 i n c o r p o r a t i o n o f g l u c o s e carbon i n t o w a l l s v i a the metabolism o f m y o - i n o s i t o l and  cell  ( t h e Loewus pathway),  t h e g l y c o l y t i c , pentose phosphate, and s h i k i m i c pathways. The  presence o f CHCGluc and CHGAsp i n cut-stem exudate a l s o  i n d i c a t e s t h a t these two conjugates were t r a n s l o c a t e d a c r o p e t a l l y through the p l a n t . uniformly  Cyclohexanecarboxylic  a c i d a c t i v i t y was  d i s t r i b u t e d i n a l l organs o f the bean p l a n t s i x hours  a f t e r the r o o t f e e d i n g had begun.  This observation  f a c t t h a t f r e e a c i d was n o t detected  p l u s the  i n l e a f blades,  stems, and  cut-stem exudate a f t e r 6 hours tends to support the i d e a t h a t the m e t a b o l i t e s ,  and not the f r e e a c i d , were mobile.  a time course study o f m e t a b o l i t e  formation  bean p l a n t s , Padmanabhan ( 108) r e p o r t e d was the f i r s t m e t a b o l i t e  1  appeared a f t e r one-hour. was n o t d e t e c t e d  i n l e a f blades o f  t h a t the glucose e s t e r  produced, and was p r e s e n t  hour a f t e r a p p l i c a t i o n o f KCHC-7- ^C.  Also, i n  one-eighth  The a s p a r t a t e  derivative  Free a c i d i n e x t r a c t s o f l e a f b l a d e s  a f t e r 6 hours.  In a l l p l a n t f r a c t i o n s and i n cut-stem exudate from t h e KCHG-7- G and glucose-^C/KCHC f e e d i n g experiments a 14  was p r e s e n t  metabolite  a t Rf 0.68 on chromatograms developed i n IAW.  In the  e a r l y experiments i t was thought t h a t t h i s compound was n o t a metabolite,  but r a t h e r a l a b e l l e d contaminant p r e s e n t  i n the  11s KCHC-7-  C stock s o l u t i o n .  detected  i n e x t r a c t s o f p l a n t s which r e c e i v e d l a b e l l e d g l u c o s e  and  However, s i n c e t h i s compound was  u n l a b e l l e d KCHC, i t appears t h a t f u t u r e i n v e s t i g a t i o n s  should  i n c l u d e an a n a l y s i s o f t h i s unknown  metabolite.  I n a d d i t i o n to the m e t a b o l i t e s and a r t i f a c t s alreadyd i s c u s s e d s e v e r a l o t h e r unknown m e t a b o l i t e s were p r e s e n t i n e x t r a c t s o f both l e a f blade^and KCHC treatment. the R  In IAW,  these m e t a b o l i t e s were d e t e c t e d i n  range G.20 to 0.40.  f  root tissues following  In a p r e l i m i n a r y i n v e s t i g a t i o n  t h r e e unknown m e t a b o l i t e s were removed i n d i v i d u a l l y from a ehromatogram which had been developed c o n t a i n i n g the unknown 'conjugated' Dow.ex 5QW-X8(Hv) cation-exchange  i n IAW.  Aqueous s o l u t i o n s  m e t a b o l i t e s were r u n through  column to remove f r e e amino  i  a c i d s , and the e l u a t e s were s u b j e c t e d to an a c i d h y d r o l y s i s a t 1 0 5 ° C f o r 8 hours i n vacuo.  I n d i v i d u a l h y d r o l y s a t e s were  then a n a l y z e d u s i n g a Beckman 1 2 0 C automatic analyzer.  amino a c i d  R e s u l t s i n d i c a t e d t h a t from 10 to 13 d i f f e r e n t amino  a c i d s were p r e s e n t i n each h y d r o l y s a t e .  Even though the amino  a c i d s p r e s e n t i n each h y d r o l y s a t e were v e r y s i m i l a r , the conc e n t r a t i o n o f i n d i v i d u a l amino a c i d s v a r i e d from one to the o t h e r .  F u r t h e r , i n a separate procedure  s o l u t i o n s c o n t a i n i n g i n d i v i d u a l unhydrolyzed m e t a b o l i t e s were run through  sample  the aqueous  'conjugated'  the cation-exchange  column, and  the e l u a t e s were s u b j e c t e d to an amino a c i d a n a l y s i s .  The  r e s u l t s o f these analyses r e v e a l e d t h a t f r e e amino acids were absent.  I t i s v e r y tempting  to s p e c u l a t e t h a t i n a d d i t i o n to  the conjugated m e t a b o l i t e s a l r e a d y d i s c u s s e d CHCA may a l s o been a component o f a s m a l l p e p t i d e .  have  I t seems v e r y u n l i k e l y  t h a t CHCA would have formed i n d i v i d u a l conjugates w i t h ten or more amino a c i d s .  A l s o , a host o f i n d i v i d u a l conjugates  not have been l o c a t e d a t one R  f  position.  would  A c c o r d i n g to Towers  a  ( 140  ), t i s s u e s would be expected  to c o n t a i n p o o l s o f f r e e  amino a c i d s , and hence i t would seem reasonable t h a t c o n j u g a t i o n o f i n t r o d u c e d a c i d s would occur w i t h amino a c i d s p r e s e n t i n these pools.  However, the employment o f s e v e r a l d i f f e r e n t amino a c i d s  i n the c o n j u g a t i o n process may  be l i m i t e d by the l a c k o f the  a p p r o p r i a t e N-acyl synthetase enzymes.  This conjecture  may  be r e l e v a n t to what i s w r i t t e n i n the ' D i s c u s s i o n S e c t i o n ' o f the next  chapter.  I t has been e s t a b l i s h e d t h a t i n some animal t i s s u e CHCA i s r e a d i l y dehydrogenated ( 18, 56 ), and then conjugated to form h i p p u r i c a c i d  ( N - b e n z o y l - L - g l y c i n e ) ( 18  ).  o f the m e t a b o l i t e s from the p r e s e n t experiments any t r a c e o f b e n z o i c a c i d .  Hydrolysis f a i l e d to y i e l d  I t has been shown t h a t the t i s s u e s o f  c e r t a i n h i g h e r p l a n t s can h y d r o x y l a t e a d m i n i s t e r e d b e n z o i c ( 76 ).  E i t h e r o- or p-hydroxybenzoic  a c i d was  on the p l a n t used, and f u r t h e r h y d r o x y l a t i o n was occur.  T h i s r o u t e o f metabolism o f CHCA was  acid  formed, depending a l s o shown to  d i s c o u n t e d i n the  bush bean by the o b s e r v a t i o n s t h a t b e n z o y l m e t a b o l i t e s were not d e t e c t e d and no r a d i o a c t i v e compound gave a p o s i t i v e p h e n o l i c r e a c t i o n with d i a z o t i z e d p - n i t r o a n i l i n e . C y c l o h e x a n e c a r b o x y l i c a c i d , when a d m i n i s t e r e d to l e a f d i s k s o r to bean s e e d l i n g s v i a a r o o t f e e d i n g , i s converted i n t o a mixture o f the g l u c o s e e s t e r , the amide o f a s p a r t i c a c i d , s e v e r a l unknown 'conjugates.'  and  Because CHCA i s not t o x i c to  bean p l a n t s even when a p p l i e d a t c o n c e n t r a t i o n s as h i g h as ppm  (2 x 10""  chapter may  2  M),  i t may  2500  be t h a t the conjugates d e s c r i b e d i n t h i s  not be mere ' d e t o x i c a t i o n p r o d u c t s ' , but r a t h e r  m e t a b o l i t e s which may p l a y a r o l e i n s t i m u l a t i n g growth o f CHCAtreated plants.  The f a c t t h a t the m e t a b o l i t e s ,  a c i d , were detected  i n treated plantsalends  and n o t the f r e e  support to t h i s  hypothesis. In c o n c l u s i o n ,  the r e s u l t s o f these experiments have shown  t h a t bean p l a n t s c o n t a i n  enzymes which can c h e m i c a l l y  conjugate  t h i s b i o l o g i c a l l y f o r e i g n petrochemical with glucose, a s p a r t i c a c i d , and perhaps w i t h o t h e r endogenous compounds.  The o c c u r -  rence o f t h e g l u c o s e and a s p a r t i c a c i d conjugates, and t h e concurrent absence o f f r e e K C H C - ? - ^ i n stems, l e a f 1  and  blades,  cut-stem exudate suggest t h a t f o l l o w i n g t h e i r s y n t h e s i s i n  r o o t t i s s u e s t h e conjugates were t r a n s l o c a t e d a c r o p e t a l l y to the a e r i a l p o r t i o n o f the p l a n t .  The presence o f the conjugated  m e t a b o l i t e s may p l a y a r o l e i n enhancing the growth o f CHCAtreated  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 o f carbohydrate compounds by e x c i s e d r o o t t i s s u e has been e x t e n s i v e l y reviewed by Butcher and S t r e e t ( 36 ), and s t u d i e d by Goldsworthy ( 5 9 ) , there i s l i t t l e  information  a v a i l a b l e regarding  the e f f e c t o f  auxins and growth promoting compounds on the uptake and subsequent metabolism o f carbohydrate compounds by r o o t s . There i s a wealth o f i n f o r m a t i o n which demonstrates t h a t naphthenates s t i m u l a t e growth and i n c r e a s e the y i e l d s o f wide v a r i e t y o f p l a n t s  (Table I ) .  a  Evidence i s a l s o accumulating  which supports the hypotheses t h a t naphthenic a c i d compounds favorably influence protein synthesis synthesis (  ( 110 ), m i t o c h o n d r i a l  ( 110, 147), n u c l e i c a c i d  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 t h a t naphthenate stimu-  l a t i o n o f p l a n t growth i s the r e s u l t o f the a c t i o n o f the chemical, or i t s m e t a b o l i t e s ,  a t both the g e n e t i c and m e t a b o l i c  levels.  Of p a r t i c u l a r importance to t h i s experiment was the f a c t t h a t dark r e s p i r a t i o n o f t r e a t e d p l a n t s was i n c r e a s e d over the r a t e s i n the c o r r e s p o n d i n g untreated  plants.  Measured 21 days  f o l l o w i n g treatment w i t h a 0 . 5 $ KNap s o l u t i o n , dark r e s p i r a t i o n i n t r e a t e d p l a n t s growing i n a high l i g h t i n t e n s i t y ( l 6 . 1 k l x ) was i n c r e a s e d by 4 3 $ when compared with the c o n t r o l value An experiment by Kim and B i d w e l l  ( 53 ) .  ( 8 3 ) demonstrated t h a t  and 2,4-D had a pronounced i n v i t r o e f f e c t on the metabolism  IAA  o f ^ C glucose i n pea r o o t t i p s . 1  amount o f ^COp  T h e i r work r e v e a l e d  t h a t the  produced, and b i o s y n t h e t i c pathways l e a d i n g  added to a c u l t u r e s o l u t i o n c o n t a i n i n g pea r o o t t i p s , i n v e s t i g a t o r s a l s o found t h a t the main e f f e c t was uptake o f glucose  from the medium, but was  They a l s o noted t h a t the p r o d u c t i o n o r i g i n a t i n g from o f ^C  glucose was  not on  the  on i t s metabolism.  o f c e r t a i n amino a c i d s  thwarted, w h i l e  asparagine from l a b e l l e d glucose was  A  the  In view o f these f i n d i n g s c o n t r o l ,  the  production  increased.  cyclohexanecarboxylic  a c i d - , and naphthenic a c i d - t r e a t e d s e t s o f bean r o o t t i p s were incubated  f o r 6 hours i n a  1 2  C  glucose  c u l t u r e medium. A f t e r  t h i s p e r i o d o f i n c u b a t i o n , the r o o t t i p s i n each s e t were incubated  f o r 3 hours i n separate  Following  these i n c u b a t i o n procedures, the l e v e l o f r a d i o a c t i v i t y  i n each s e t ' s e t h a n o l - s o l u b l e ,  medium c o n t a i n i n g  glucose.  e t h a n o l - i n s o l u b l e , and  respired 14  CO2  f r a c t i o n s was  measured.  A f t e r determining  total  C activity  i n these f r a c t i o n s from each s e t , r e p r e s e n t a t i v e compounds i n the e t h a n o l - s o l u b l e  and  f r a c t i o n s were separated Following  the hydrolyzed  by t h i n - l a y e r chromatographic methods.  i d e n t i f i c a t i o n o f the compounds on radiochromatograms,  the compounds were removed, and individually.  t h e i r *^C  a c t i v i t y determined  Compounds i n the e t h a n o l - s o l u b l e  l ^ C a c t i v i t y was glutamic  ethanol-insoluble  determined were:  f r a c t i o n whose  Aspartic acid, serine,  a c i d , glutamine, t h r e o n i n e ,  a c i d , v a l i n e , l e u c i n e / i s o l e u c i n e , and  a l a n i n e , If-aminobutryic glucose.  In a d d i t i o n ,  14  the  A  C a c t i v i t y i n a s p a r t i c a c i d , glutamic  from the h y d r o l y s a t e determined.  a c i d , and  o f the e t h a n o l - i n s o l u b l e f r a c t i o n  alanine was  The b a s i c o b j e c t i v e of t h i s phase of the i n v e s t i g a t i o n to augment our u n d e r s t a n d i n g of how s t i m u l a t e the growth of bean p l a n t s . purpose was  was  naphthenate compounds More s p e c i f i c a l l y ,  to determine i f the complex  the  naphthenic a c i d mixture,  o r an i n d i v i d u a l naphthenic a c i d , c y c l o h e x a n e c a r b o x y l i c  acid,  a f f e c t s the uptake and subsequent metabolism o f l a b e l l e d glucose i n bean r o o t  tips.  Because o f the c l o s e c o n n e c t i o n between enzymes and a l l aspects o f metabolism, the e f f e c t s o f naphthenic a c i d compounds on enzyme a c t i v i t y have been i n c l u d e d 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 , s e v e r a l a r t i c l e s  ( 40, 5 2 , 5 3 , 1^7 )  have c o n s i d e r e d enzymes i n v o l v e d i n amino a c i d o r n i t r o g e n metabolism, and a r e , t h e r e f o r e , r e l e v a n t to the r e s u l t s i n t h i s chapter.  reported  LITERATURE REVIEW In 1 9 6 6 . Bazanova and  Akopova ( 2 3 ) r e p o r t e d t h a t a 0 . 0 1 $  naphthenate s o l u t i o n a p p l i e d as a f o l i a r spray to young c o t t o n plants stimulated t h e i r r e s p i r a t o r y rates. r e s p i r a t i o n i n potato  An i n c r e a s e i n  leaves f o l l o w i n g naphthenate treatment  observed by A b o l i n a and A t a u l l a e v ( 2  was  ) and Ladygina ( 94 ).  ) observed t h a t the f o l i a r a p p l i c a t i o n o f 0 . 5 $ KNap  Chu--i( 40  produced a decrease i n the r a t e o f r e s p i r a t i o n i n the above ground p a r t s o f tomato p l a n t s two was  f o l l o w e d by a 9 . 7 $  weeks a f t e r treatment.  This  i n c r e a s e i n the r a t e o f r e s p i r a t i o n  weeks a f t e r the growth s t i m u l a n t was  applied.  A f t e r two  four  foliar  a p p l i c a t i o n s o f a 0 . 0 5 $ naphthenate s o l u t i o n to the leaves eggplants  and  tomatoes, Guseinov ( 6 8 ) obtained  of  an i n c r e a s e i n  the r a t e o f r e s p i r a t i o n . Kolesnik p l a n t s was  ( 87  ) observed t h a t c a t a l a s e a c t i v i t y i n grape  increased f o l l o w i n g a f o l i a r a p p l i c a t i o n of a  naphthenate s o l u t i o n .  An i n i t i a l  f o l i a r application of a  naphthenate s o l u t i o n , f o l l o w e d by s p r a y i n g the p l a n t s  initially  decreased  0,05$  twice  d u r i n g the v e g e t a t i v e p e r i o d w i t h a lower c o n c e n t r a t i o n naphthenates ( 0 , 0 0 5 $ ) .  0.005$  of  catalase a c t i v i t y ,  but a t the end o f the v e g e t a t i v e p e r i o d the a c t i v i t y o f t h i s enzyme was ( 23 had  higher than i n the c o n t r o l s .  ) reported that a f o l i a r  Bazanova and  Akopova  0 . 0 1 $ naphthenate a p p l i c a t i o n  o n l y a s l i g h t l y s t i m u l a t o r y e f f e c t on c a t a l a s e a c t i v i t y i n  cotton.  Agakishiev  and Bazanova ( 4  t r e a t e d with naphthenates or S h - 8 , soils,  and  ) found t h a t c o t t o n p l a n t s grown on s u l f a t e s a l i n i z e d  showed a higher r a t e o f peroxidase  These two  a c t i v i t y i n the l e a v e s .  treatments a l s o i n c r e a s e d the a c t i v i t y o f  peroxidase  ( l 6 ).  i n the r o o t s o f c o t t o n i n a 10 ppm reported  A f t e r the seeds had been soaked  naphthenate or a 1 ppm  t h a t peroxidase  Sh-8  s o l u t i o n , the author a l s o  a c t i v i t y i n c o t t o n p l a n t s was  i n c r e a s e d , but the i n c r e a s e i n a c t i v i t y was chemicals were a p p l i e d to the s o i l and  6  ) reported  l e s s when the  a t the r a t e o f 2 0 mg/4  10 mg/kg o f dry s o i l , r e s p e c t i v e l y .  Chrjanovskaya (  greatly  Alekperov  ha  and  t h a t young s e e d l i n g s o f E l d a r  p i n e , Pinus e l d a r i c a v a r . E l d a r s k a , and  Japanese sophore  (Sophora j a p o n i a L.) which had r e c e i v e d a 0,00$% naphthenate treatment i n the form o f a 12 hour seed soak had r e s p i r a t o r y r a t e s and  an i n c r e a s e d peroxidase  t h e i r c a t a l a s e a c t i v i t y was s e e d l i n g s a l s o had controls.  higher  activity,  c o n c u r r e n t l y decreased.  but  Treated  a g r e a t e r s a l t t o l e r a n c e than d i d  the  F o l l o w i n g a seed soak i n 0 . 0 0 5 $ naphthenates,  Matzjuk and  Grinberg  ( 1 0 3 ) observed t h a t c a t a l a s e a c t i v i t y i n  the l e a v e s o f m i l l e t was  9% above the c o n t r o l v a l u e .  a c t i v i t y o f a s c o r b i c a c i d oxidase  The  i n c o t t o n p l a n t s was  increased  f o l l o w i n g naphthenate treatment when the p l a n t s were grown i n c h l o r i d e s a l i n i z e d s o i l , but the a c t i v i t y o f t h i s enzyme d i d not i n c r e a s e when the p l a n t s were grown i n s u l f a t e s a l i n i z e d soil  (  4  ).  Both the formation  promoted i n A s p e r g i l l u s usamii w i t h NaNap (avg. M.W, 5  x 10"  H  ( 35  o f amylase and  growth were  a f t e r the mold had been t r e a t e d  2 0 8 , C ^ H ^ ^ ) at a concentration  of  ).  A f t e r tomato p l a n t s had r e c e i v e d a f o l i a r treatment o f a 0.5$  naphthenate s o l u t i o n , Chu  ( 4 0 ) observed t h a t  a c t i v i t y o f phosphorylase i n the leaves was also reported  t h a t the a c t i v i t i e s o f n i t r a t e  the  increased. reductase,  It  was  phosphoglyceryl  kinase,  at a l l observation (53  ) obtained  and  times.  s u c c i n i c dehydrogenase were decreased Fattah  (5  2  a s i g n i f i c a n t increase  a c t i v i t y o f phosphorylase measured 7 ,  ) and  F a t t a h and  Wort  ( 0 . 0 5 l e v e l ) i n the 14,  21 days a f t e r the  and  naphthenate treatment when bean p l a n t s were grown a t a  high  light intensity.  and  glutamic-pyruvic significant level  The  s t i m u l a t i o n of n i t r a t e reductase  transaminase i n t r e a t e d p l a n t s reached a between 7 and  (0.05)  14 days a f t e r treatment.  S i g n i f i c a n t s t i m u l a t i o n o f phosphoglycerate kinase more s l o w l y under lower l i g h t  developed  intensities. t h a t the a p p l i c a t i o n o f 2 x  Wort et a l ( 147 ) r e p o r t e d  M KNap to 14-day-old bush bean p l a n t s r e s u l t e d i n the measured i n the l e a v e s increases  7 days a f t e r treatment:  i n s p e c i f i c a c t i v i t y per mg  w i t h c o n t r o l p l a n t v a l u e s , were: glutamic-oxaloacetic  l ) Percentage  enzyme p r o t e i n , compared 152;  n i t r a t e reductase,  transaminase, 1 0 ; glutamine  synthetase,  glutamate dehydrogenase, 0 ; and  The  i n c r e a s e i n a c t i v i t y o f glutamate dehydrogenase on  g r e a t e r by 10 to 2 0 $ . was  increased  2 ) The  16%.  3)  72.  cytochrome oxidase,  an  content o f enzyme p r o t e i n incorporation of  i n t r e a t e d p l a n t s by ca, 1 0 $ .  enzyme a c t i v i t y f o l l o w e d The  The  increase i n  i n v i t r o a d d i t i o n o f 1 x 1 0 " ° M KNap.  authors s t a t e d t h a t the i n c r e a s e i n enzyme p r o t e i n ,  stimulated  i n c o r p o r a t i o n o f l a b e l l e d l e u c i n e , and  the  s p e c i f i c a c t i v i t y o f the numerous enzymes suggested  metabolic  levels.  the  greater  that  naphthenate s t i m u l a t i o n o f p l a n t growth i s the r e s u l t o f a c t i o n o f the chemical,  was  ^C-L-leucine  4) No  2  following,  17;  e x t r a c t b a s i s was  10"  the  or i t s d e r i v a t i v e s , a t both g e n e t i c  and  tomato p l a n t s with a 0 . 0 5 $ aqueous naphthenate  Spraying  s o l u t i o n caused changes i n the p r o t e i n and n u c l e i c content l e a v e s and v e g e t a t i v e a p i c e s .  The  of  content o f the °(-nucleo-  p r o t e i n complex i n c r e a s e d , w h i l e t h a t o f the ^ - n u c l e o p r o t e i n complex decreased. but decreased  P r o t e i n N i n the v e g e t a t i v e a p i c e s  i n older leaves  ( 110).  When potato  increased,  tubers were  soaked i n a 0 . 0 0 5 $ s o l u t i o n o f naphthenates p r i o r to sowing, the p l a n t s showed an i n c r e a s e i n the content  of  nitrogenous  substances i n the l e a v e s , stems, and r o o t s ( 6 l Ataullaev ( at  2  Abolina  and  ) observed t h a t the a p p l i c a t i o n o f naphthenates  the r a t e o f 500  potato  ).  cm^/ha i n c r e a s e d the s t a r c h c o n c e n t r a t i o n i n  tubers by 20$.  L a t e r experiments by the same author  showed t h a t a f t e r naphthenate treatment the p r o t e i n content potato  tubers was  also increased.  of  Even though the weight o f  tubers/plant increased following a f o l i a r  0.5$  treatment with  naphthenates to e a r l y potatoes  var. Warba, Wort and  ( l 46 ) r e p o r t e d t h a t there was  no d i f f e r e n c e between the s t a r c h  content  i n tubers from c o n t r o l and  Hughes  treated plants.  F o l l o w i n g a f o l i a r a p p l i c a t i o n o f a 0 , 0 0 5 $ naphthenate s o l u t i o n to corn p l a n t s , Yur'eva ( 149) s i l a g e contained more p r o t e i n , and  observed t h a t  the corn g r a i n  corn  contained  more p r o t e i n and  s t a r c h when compared with the c o n t r o l s .  Subbotina ( 136)  r e p o r t e d t h a t the f i e l d a p p l i c a t i o n o f a  naphthenate s o l u t i o n i n c o n j u n c t i o n with a f e r t i l i z e r led  ( 5o 60 6o^  to an i n c r e a s e d p r o t e i n l e v e l i n apple t r e e l e a v e s ,  mined one month a f t e r a p p l i c a t i o n .  0.1$  N  E  deter-  Guyot ( 69 ) observed t h a t  sugarcane which had r e c e i v e d a f o l i a r naphthenate treatment to  K  30 days p r i o r to h a r v e s t had a higher sugar  concentration  15  than c o n t r o l s .  By a p p l y i n g a 0.05$ naphthenate s o l u t i o n to  t a n g e r i n e l e a v e s d u r i n g the f l o w e r i n g p e r i o d , Marshaniya e t a l ( 102 ) observed an i n c r e a s e i n the sugar content The  i n the f r u i t s .  f o l i a r a p p l i c a t i o n o f 0.005$ naphthenates to grape p l a n t s  a l s o i n c r e a s e d the sugar content  i n the f r u i t s  ( 8? ),  This  author a l s o r e p o r t e d t h a t u s i n g a higher c o n c e n t r a t i o n o f naphthenate (0.05$) i n i t i a l l y ,  and then s p r a y i n g twice with a  0.05$ s o l u t i o n o f the chemical  decreased  disaccharides.  the c o n c e n t r a t i o n o f  At the end o f the v e g e t a t i v e p e r i o d sucrose i n  the l e a v e s disappeared,  and the sugar content  of fruits  from  t r e a t e d p l a n t s was g r e a t e r than i n the c o n t r o l s , Chu  ( 40 ) r e p o r t e d t h a t a f o l i a r 0.5$ KNap treatment  i n c r e a s e d the l e v e l  o f r e d u c i n g sugar, sucrose,  i n mature tomato f r u i t .  and t o t a l  sugars  A reduction i n the concentration o f  sugar d u r i n g the course o f storage was l e s s i n tomato f r u i t from KNap t r e a t e d p l a n t s .  T i t r a t a b l e a c i d from the mature  1  tomato f r u i t which was under the i n f l u e n c e o f KNap was higher at  the end o f 4 days storage, b u t was lower as the d u r a t i o n o f  storage was i n c r e a s e d ,  Peterburgsky  and Karamete ( 113) observed  t h a t a f o l i a r 0.007$ naphthenate treatment i n c r e a s e d the s y n t h e s i s o f 10 important at  amino a c i d s a t the b e g i n n i n g  o f and  the end o f the v e g e t a t i v e p e r i o d i n maize, and i n c r e a s e d the  s y n t h e s i s o f p r o t e i n and n o n - p r o t e i n and r o o t s .  Concentrations  N substances i n the l e a v e s  o f naphthenates r a n g i n g from 0.005 "to  0.01$ c o n s i d e r a b l y i n c r e a s e d the content o f t o t a l  and p r o t e i n N  i n the leaves o f tomato, c a r r o t and beet f o l l o w i n g treatment  The  data o f Voinova-Raikova ( 141 ) showed t h a t the a d d i t i o n  o f a naphthenate s o l u t i o n to the s o i l s t i m u l a t e d the development o f ammonifying b a c t e r i a which i n t u r n promoted an i n c r e a s e i n the amount o f n i t r a t e e n t e r i n g the s o i l from o r g a n i c compounds.  nitrogenous  In t h i s i n v e s t i g a t i o n i t was a l s o demonstrated t h a t  the naphthenate treatment caused a c o n s i d e r a b l e the number o f d e n i t r i f y i n g b a c t e r i a .  decrease i n  As w e l l , i t was shown  t h a t naphthenate treatment i n c r e a s e d the q u a n t i t y o f N u t i l i z e d by 80$ o f the Azotobacter  s t r a i n s which were t e s t e d .  The  author concluded t h a t naphthenate treatment was r e s p o n s i b l e for  i n c r e a s i n g the l e v e l o f i n o r g a n i c n i t r o g e n compounds i n the  soil,  improving the N  2  f i x a t i o n process,  amount o f n i t r a t e l o s t by the process  and d e c r e a s i n g the  of denitrification.  What appears to be the f i r s t r e p o r t o f the use o f naphthenate i n a b i o l o g i c a l experiment dates back to 1921.  In  t h i s experiment ( 1 0 5 ) one hour a f t e r 5 0 mg o f NaNap was added to 10 ml o f a 2 . 5 $ glucose yeast,  C0  2  s o l u t i o n which contained  p r o d u c t i o n by t r e a t e d 'Munchner' and  2 5 0 mg o f  'Patzenhofer•  yeast suspensions was 143 and 140$ o f the c o n t r o l v a l u e s , respectively. As a note i n p a s s i n g ,  a v e r y i n n o v a t i v e use i n v o l v i n g  naphthenates was r e p o r t e d by Nussbaum ( 106 ).  He s t a t e d t h a t  manual r o o t p i n c h i n g was e a s i l y skipped by p r u n i n g  the r o o t s  c h e m i c a l l y u s i n g a 5 5 $ copper naphthenates s o l u t i o n .  A more  branched, f i b r o u s r o o t system r e s u l t e d when s e e d l i n g s were grown i n a wooden f l a t the bottom o f which had been p r e t r e a t e d w i t h the naphthenate s o l u t i o n .  MATERIALS AND The  METHODS  procedures i n v o l v e d i n t h i s i n v e s t i g a t i o n , m o d i f i e d  somewhat, were based on those d e s c r i b e d by Kim (  8 3 ).  and  Bidwell  Uniform seeds o f the dwarf bush been (Phaseolus  v u l g a r i s L. c u l t i v a r Top  Crop) were sown i n wooden f l a t s  c o n t a i n i n g v e r m i c u l i t e , and p l a c e d day-old  i n a growth room.  Seven-  s e e d l i n g s with r o o t systems o f uniform s i z e were  s e l e c t e d , the r o o t s q u i c k l y r i n s e d with d i s t i l l e d water, b l o t t e d , and A.)  i n c u b a t i o n medium used was  et a l ( 12 ). 2  100  The  ml.  t h a t o u t l i n e d by  Andreae  medium c o n s i s t e d o f 20 umoles CaCNO^^ • 4  umoles KHgPO^, and  volume o f 4,4 was  t e r m i n a l r o o t s e c t i o n s were cut.  Incubation. The  H 0,  5 mm  280  umoles sucrose i n a  total  To serve as a c o n t r o l , the complete medium  added to the main or lower compartment o f a g l a s s b o t t l e  ( c a p a c i t y 55 ml), which contained  an upper compartment,  6 . 7 ml  i d e n t i c a l b o t t l e s a l s o with  (a S k r i p ink b o t t l e ) .  the complete medium contained KNap (1 x 10" 5 M). adjusted  i n a d d i t i o n KCHC (1 x 10"-5  The pH o f the medium i n each b o t t l e  to 6 w i t h 1 N NaOH, a u t o c l a v e d  l b s / i n , and 2  Two  capacity  M) was 15  f o r 20 minutes a t  the K s a l t o f p e n i c i l l i n G ( 1 5 ug/ml) and  or  strepto-  mycin s u l f a t e ( 3 0 ug/ml) were added a f t e r c o o l i n g . Three s e t s o f r o o t t i p s each c o n t a i n i n g 40  t i p s , from  r o o t systems o f e i g h t d i f f e r e n t p l a n t s , weighing from 42 mg  f r e s h weight per s e t were p l a c e d  three b o t t l e s , 1 . 5  to  47  i n the main compartment o f  ml o f 1 N NaOH was  upper compartment, and  the  added to the s m a l l e r  the b o t t l e s were t i g h t l y capped.  or  For  113.  s  the  f i r s t phase o f i n c u b a t i o n the media a l s o  glucose ( 0 . 5 % ) .  contained  A f t e r 6 hours o f i n c u b a t i o n i n the dark a t 2 5 °  with constant shaking, each s e t o f r o o t t i p s was removed, r i n s e d w i t h d i s t i l l e d water, and b l o t t e d .  Each s e t was then p l a c e d i n  a s e p a r a t e b o t t l e , c o n t a i n i n g the complete medium i n which glucose (Amersham/Searle:  Toronto, O n t a r i o ) , 2 . 5 umoles and 12  7.5  u C i p e r s e t o f r o o t t i p s , r e p l a c e d the  f i r s t phase o f i n c u b a t i o n . to  C g l u c o s e o f the  A f r e s h s o l u t i o n o f NaOH was added  the upper compartment o f each b o t t l e .  The b o t t l e s were  again t i g h t l y capped, and i n c u b a t i o n was continued f o r an a d d i t i o n a l 3 hours. B.)  Tissue analysis. At  the end o f the second i n c u b a t i o n p e r i o d each s e t o f  r o o t t i p s was removed, r i n s e d w i t h d i s t i l l e d water, and b l o t t e d . The r o o t t i p s inl.each s e t were d i v i d e d i n t o two groups o f 2 0 , and each group was t r a n s f e r r e d to a beaker c o n t a i n i n g b o i l i n g 75%  ethanol (EtOH).  T h i s procedure served to i n a c t i v a t e  enzymes, and to e x t r a c t the EtOH-soluble compounds. was  The t i s s u e  e x t r a c t e d f o r 2 hours a f t e r which the EtOH-soluble f r a c t i o n s  were p l a c e d i n s e p a r a t e v i a l s .  The r o o t t i p s i n each s e t were  r i n s e d twice w i t h b o i l i n g 75% EtOH, and the r i n s i n g s were added to  the EtOH-soluble f r a c t i o n s .  The EtOH-soluble f r a c t i o n s were  evaporated to dryness under streams o f a i r , methanol  (MeOH) was added to each f r a c t i o n .  and 1 ml o f 25% A 25 u l aliquot  was withdrawn from each 1 ml sample and was a p p l i e d to a 2 0 x 2 0 cm TLC p l a t e coated w i t h c e l l u l o s e MN 3 0 0 HR ( 0 . 5 mm wet t h i c k ness) (Macherey and Nagel, Co.).  Chromatograms were developed  i n n-butanol: g l a c i a l a c e t i c acids water ( 4 : 1 : 5 , (BAW)  and 80$ phenol.  top phase)  For determining t o t a l r a d i o a c t i v i t y o f  the EtOHr-soluble f r a c t i o n , another 2 5 u l a l i q u o t was and added to 15 ml o f s c i n t i l l a t i o n s o l u t i o n .  The  withdrawn scintillation  s o l u t i o n c o n s i s t e d o f 126 ml EtOH, 2 0 0 ml t o l u e n e , 1 . 3 3 g and 5 mg POPOP.  The  ^C  a c t i v i t y was  determined  PPO,  using a l i q u i d  s c i n t i l l a t i o n c o u n t i n g system (Nuclear-Chicago, Model 7 2 4 ) . Root t i p s which had been EtOH e x t r a c t e d were d i v i d e d two  groups o f 1 0 .  One  group was  t r a n s f e r r e d to a t e s t  into  tube  which contained 1 ml o f formamide, and the t i s s u e s were s o l u b i l i z e d by h e a t i n g the mixture a t 2 0 0 ° f o r 5 hours. w e l l mixed, d i l u t e d to 3  s o l u t i o n was  and a 0 . 2 ml a l i q u o t was  m  The  resulting  l w i t h d i s t i l l e d water,  added to 15 ml o f s c i n t i l l a t i o n  The other group together w i t h 2 ml o f 6 N HC1  solution.  t r a n s f e r r e d to a 25 ml f l a s k equipped  was  w i t h a condenser,  and  was  s u b j e c t e d to a c i d h y d r o l y s i s f o r 10 hours a t 1 0 5 ° . The  s o l u t i o n c o n t a i n i n g the h y d r o l y z e d  compounds was was  added.  evaporated  to dryness, and  EtOH-insoluble  0 . 5 ml o f 2 5 $  T h i r t y - f i v e u l from each f r a c t i o n was  TLC p l a t e s o f c e l l u l o s e MN  3 0 0 HR,  and developed  MeOH  a p p l i e d to as b e f o r e .  R a d i o a c t i v e compounds on the TLC p l a t e s were d e t e c t e d by use o f Kodak Blue Brand M e d i c a l X-ray  the  film.  F o l l o w i n g the i d e n t i f i c a t i o n o f compounds u s i n g amino a c i d standards  (see pages 165 - 166 i n the Appendix),  adsorbant w i t h the a p p r o p r i a t e compound was p l a t e , and was  the  cellulose  removed from  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 p l u s 0 . 2 ml o f d i s t i l l e d water. lZ  *C a c t i v i t y was  determined  as b e f o r e .  the  The  Because the l e u c i n e and  i s o l e u c i n e standards  d i d n o t 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 a c i d s were c o n s i d e r e d as one. One-tenth ml o f the NaOH which contained the r e s p i r e d A  ^C02 was t r a n s f e r r e d to 15 ml o f 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 b e f o r e .  In a l l i n s t a n c e s ,  samples were c o r r e c t e d f o r quenching. The  data were s u b j e c t e d t o an a n a l y s i s o f v a r i a n c e o f a  n e s t e d d e s i g n , and a comparison o f means by Duncan's New M u l t i p l e Range T e s t ( 1 3 5 ).  The v a r i a b i l i t y which e x i s t e d  between the c o n t a i n e r s used f o r i n c u b a t i o n purposes was n o t tested.  As seen i n Table  XXV  i c a n t l y i n c r e a s e d the  A  , each naphthenate treatment  signif-  C a c t i v i t y i n the e t h a n o l - s o l u b l e ,  e t h a n o l - i n s o l u b l e , and the r e s p i r e d ^ C 0 1  2  fractions.  Using  14 t o t a l r a d i o a c t i v i t y as a b a s i s f o r comparison,  C activity  i n r o o t t i p s t r e a t e d with KCHC was g r e a t e r than the c o n t r o l v a l u e by 2 0 $ ; the i n c r e a s e observed i n KNap-treated r o o t was 1 3 $ ( F i g u r e 11).  The data o f Table  XXV  tips  i n d i c a t e t h a t 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 s t i m u l a t i v e e f f e c t on the uptake o f l a b e l l e d glucose The  from the i n c u b a t i o n media.  g r e a t e r e f f e c t was obtained by the use o f KCHC. 14  Each naphthenate treatment i n c r e a s e d the all  e t h a n o l - s o l u b l e amino a c i d s , except one.  C activity i n Carbon-l4 a c t i v i t y  i n s e r i n e was s i g n i f i c a n t l y i n c r e a s e d f o l l o w i n g KCHC and KNap treatments, while  the i n c r e a s e observed i n the case o f v a l i n e  was h i g h l y s i g n i f i c a n t .  Only the KNap treatment  significantly  14 i n c r e a s e d the Figure 12),  C a c t i v i t y i n i s o l e u c i n e / l e u c i n e (Table XXvi ,  Because o f v a r i a b i l i t y i n i n d i v i d u a l v a l u e s ,  o f 51 and 40$ i n the l e v e l o f  > C glucose  increases  i n the e t h a n o l - s o l u b l e  f r a c t i o n from KCHC- and KNap-treated t i s s u e s , r e s p e c t i v e l y , were n o t q u i t e s t a t i s t i c a l l y s i g n i f i c a n t a t the 0 . 0 5 l e v e l . 14 The  amount o f  C l e v e l i n a s p a r t i c a c i d , glutamic  and a l a n i n e from the e t h a n o l - i n s o l u b l e h y d r o l y s a t e  acid,  from KCHC-  t r e a t e d r o o t t i p s was s i g n i f i c a n t l y i n c r e a s e d , and were 1 2 3 , 1 2 6 , . a n d 1 6 2 $ o f the c o n t r o l v a l u e , r e s p e c t i v e l y . the h y d r o l y s a t e  However, i n  from KNap-treated r o o t t i p s o n l y ^C a c t i v i t y X  i n a l a n i n e was s i g n i f i c a n t l y g r e a t e r than the c o n t r o l v a l u e  TABLE XXV  .  T o t a l r a d i o a c t i v i t y , as muCi, i n the e t h a n o l s q l u b l e , e t h a n o l - i n s o l u b l e , and r e s p i r e d CO? f r a c t i o n s o f c o n t r o l and naphthenate-treated bean r o o t t i p s a f t e r s u p p l y i n g glucose . a  Treatments Control  KNap  KCHC  *  Ethanol-soluble  67.2  Ethanol-insoluble  61.1  63.1  68.5  R e s p i r e d COg  53.9  63.O  68.3  209.0*  220.4*  Total r a d i o a c t i v i t y  182.2  Percent o f c o n t r o l  b  81.2  #  **  120  113  12  83.6  Incubation sequence : S i x hours i n a C glucose medium, or i n one c o n t a i n i n g 1 0 5 M KNap or 10~5 M KCHC; then 3 hours i n a 14Q glucose medium. n  n  _  Each v a l u e r e p r e s e n t s the mean of f o u r measurements.  230  90  220  80  . 210  70  200  60  50  • 190  -  180  40 C  KNap KCHC  Ethanolsoluble  C  KNap  Ethanolinsoluble  KCHC  C  KNap  Respired  KCHC  C  KNap KCHC Total activity  KNap = I I I I C =• KCHC = I R a d i o a c t i v i t y , as muCi, i n the e t h a n o l - s o l u b l e , e t h a n o l - i n s o l u b l e , and r e s p i r e d C0 f r a c t i o n s o f c o n t r o l and naphthenate-treated bean r o o t t i p s a f t e r s u p p l y i n g 14Q glucose. 2  TABLE XXVI  .  T o t a l 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 e t h a n o l - s o l u b l e amino a c i d s and i n glucose from c o n t r o l and naphthenate-treated r o o t t i p s a f t e r s u p p l y i n g 14c glucose . a  Treatments Control  Aspartic  2.22  acid  b  KNap  KCHC  2.25  n s  2.68  n s  *•  5.12  9.08  1.4?  1.68  Threonine  10.21  11.40  Alanine  10.10  io.25  1.45  1.47  n s  1.6l  n s  2.10  1.65  n s  2.l4  n s  Valine  4.91  5-75  Isoleucine/leucine  3-54  4.38*  Serine Glutamic  acid  Glutamine IT-Aminobutyric  acid  Glucose  17.48  25.34  Total  58.68  72.79  activity  *  See Table XXV.  b  See Table XXV.  7.42 n s  nS  n s  1.76 13.0  n s  n S  3  12.52  n s  ** 7.32  n s  3.35 2 .l7 3  75.54  n s  n s  12  3 "  Serine  Aspartic acid  c =  C  Glutamic acid  Threonine KNap =  |I I I I  Alanine  Glutamine KCHC =  ro o  24  21  18  15  a—c )T-Aminobutyric acid  12 Valine  Isoleucine/ leucine  Glucose  I  3-Phosphohydroxypyruvate  i  Leucine  Glycine  1  Phosphoenolpyruvate  Serine  I  3-Phosphoglycerate  Alanine  Pyruvate  I  Acetyl Co A Asparate Methionine  Homoserme Threonine  Isoleucine  Oxaloacetate it-ketoglutarate  Glutamate y-Aminobutyrate  Glutamine  TABLE XXVII  T o t a l r a d i o a c t i v i t y , as muCi, found i n amino a c i d s from the e t h a n o l - i n s o l u b l e h y d r o l y s a t e from cont r o l and naphthenate-treated bean r o o t t i p s after supplying glucose . a  Treatments  Control  Aspartic  acid  3.76  3.58  Glutamic  acid  3-35  3.54  2.13  3.01  Alanine  a  See Table  XXV.  b  See Table  XXV.  KCHC  KNap  ns ns  4.72  4.27  3.58  C  KNap KCHC Aspartic acid  FIGURE 1 3 .  C  KNap KCHC Glutamic acid  C  KNap KCHC Alanine  R a d i o a c t i v i t y , as muCi, found i n a s p a r t i c a c i d , glutamic a c i d , and a l a n i n e from the e t h a n o l - i n s o l u b l e h y d r o l y s a t e from c o n t r o l and naphthenate-treated bean r o o t t i p s a f t e r s u p p l y i n g C glucose. l 4  £  (Table XXVII, F i g u r e  13).  Although the uptake and metabolism o f carbohydrate and e f f e c t s o f growth r e g u l a t o r s on the growth and  aging of  the  excised  r o o t t i s s u e have been e x t e n s i v e l y reviewed by Butcher and ( 36 ), the amount o f i n f o r m a t i o n r e l a t e d to the e f f e c t  Street  of  growth-promoting compounds on the uptake and metabolism o f sugars i s v e r y l i m i t e d .  R e s u l t s o f Kim  and  Bidwell  r e v e a l e d t h a t both i n d o l e - 3 - a c e t i c a c i d (IAA)  83  (  )  and 2 , 4 - d i c h l o r o -  phenoxyacetie a c i d g e n e r a l l y reduced the uptake and  retarded  the metabolism o f l a b e l l e d glucose by e x c i s e d pea r o o t  tips.  Goldsworthy ( 59 ) demonstrated t h a t mannose c o m p e t i t i v e l y i n h i b i t e d the p h o s p h o r y l a t i o n t i s s u e s , while higher  o f g l u c o s e by hexokinase i n r o o t  concentrations  o f glucose  inhibited  uptake o f mannose by competing f o r the hexokinase enzyme. though the e f f e c t o f naphthenate compounds on t h i s  t h a t hexokinase a c t i v i t y was and Wort e t a l ( 1^7  Even  particular  enzyme has not been s t u d i e d , the s i g n i f i c a n t i n c r e a s e i n uptake observed i n naphthenate-treated  the  glucose  r o o t t i s s u e s suggests  increased.  F a t t a h and  Wort  ) have shown t h a t the a c t i v i t i e s o f  ( 53 ) nine  d i f f e r e n t enzymes i n bean p l a n t s were i n c r e a s e d f o l l o w i n g naphthenate treatment. these  I t was  suggested ( 1^7  ) t h a t because o f  i n c r e a s e s the s t i m u l a t i o n o f p l a n t metabolism by naph-  thenates must be a g e n e r a l  one.  Even though the naphthenate treatments i n c r e a s e d  the  a c t i v i t y i n almost a l l e t h a n o l - s o l u b l e amino a c i d s , s i g n i f i c a n t increases of glucose  carbon i n s e r i n e and  v a l i n e suggest t h a t  c e r t a i n m e t a b o l i c pathways may The  increased  l e v e l of  metabolism may  be a f f e c t e d to a g r e a t e r  i n serine indicates that  be a f f e c t e d ( 42  be  implicated.  G l y c i n e was  chroma to grams, and  folate  ), w h i l e i n the case o f v a l i n e  the r e g u l a t i o n o f the t r a n s a m i n a t i o n may  extent.  o f «(-ketoisovaleric a c i d  not detected  on the r a d i o -  t h i s suggests t h a t s e r i n e was  not r a p i d l y  converted to g l y c i n e . Carbon-14 a c t i v i t y i n a s p a r t i c a c i d , glutamic a l a n i n e i n the h y d r o l y s a t e  o f the e t h a n o l - i n s o l u b l e  from r o o t t i p s t r e a t e d with KCHC was The  stimulated  acid,  and  fraction  significantly  increased.  i n c o r p o r a t i o n o f these amino a c i d s i n t o p r o t e i n  coupled w i t h the l a c k o f 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 a c i d s i n the t r e a t e d r o o t from the e t h a n o l - s o l u b l e s y n t h e s i s was  tips  f r a c t i o n suggests t h a t p r o t e i n  augmented by naphthenate-treatment.  supported by r e s u l t s obtained  i n our l a b o r a t o r y  have shown t h a t both the amount o f p r o t e i n , and  This i s  ( 147 ) which the l e v e l  of  i4 incorporation of bean p l a n t s was  C-L-leucine  i n t o p r o t e i n of l e a f blades from  i n c r e a s e d by KCHC and KNap treatments.  Zenk r e p o r t e d  t h a t i n v a r i o u s p l a n t t i s s u e s IAA  o('-naphthaleneacetic  a c i d ( 153 ) were converted to a mixture o f  the g l u c o s e e s t e r and  the a s p a r t i c a c i d amide, and  conjugated compounds were r e f e r r e d to as b e i n g products, Kim  and  i . e . not  Bidwell  conjugate o f IAA uptake and tips.  ( 152 ) and  t h a t these  'true d e t o x i c a t i o n '  e s s e n t i a l i n the growth i n d u c t i o n  ( 8 3 ) a l s o suggested t h a t the a s p a r t i c a c i d was  not r e s p o n s i b l e  f o r the r e d u c t i o n o f glucose  the impairment o f glucose metabolism i n pea  Recently,  process.  i t has been shown ( 148)  t h a t IAA  root  can form a  macromolecular conjugate with  tRNA.  Even though the presence  o f t h i s macromolecular conjugate c o u l d not be confirmed ( 4 3 the e x i s t e n c e o f such a conjugate has Davis and  Galston  interesting implications.  ( 4 3 ) a l s o suggested the p o s s i b i l i t y  indoleacetyl aspartate  (IAA-Asp) becoming a t t a c h e d  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  l a b e l l e d aspartate  of  to tRNA formyl-  methionine-tRNA which might serve as a p r o t e i n c h a i n However, u s i n g  ),  initiator.  the authors could  demonstrate the presence o f IAA-Asp-tRNA P i n pea as  not  stem  sections. Studies  i n our l a b o r a t o r y ( 108,  t h a t i n the r o o t s and r a p i d l y converted  127,  to a mixture o f the glucose  experiments KCHC, and  131)  have shown  leaves o f bean p l a n t s KCHC-7-^C  a s p a r t i c a c i d amide, and  were not d e t e c t e d  1Q9,  ester,  was  the  s e v e r a l unknown m e t a b o l i t e s .  i n the p r e s e n t  experiment, KCHC and KNap  on the chromatograms.  Wort et a l ( l 4 ? ) r e p o r t e d  In these  As s t a t e d p r e v i o u s l y ,  t h a t the a c t i v i t i e s o f s e v e r a l  important enzymes were i n c r e a s e d f o l l o w i n g naphthenate t r e a t ment.  However, no  i n c r e a s e i n enzyme a c t i v i t y was  f o l l o w i n g an i n v i t r o a d d i t i o n o f the f r e e a c i d . appear t h a t naphthenate s t i m u l a t i o n o f glucose metabolism observed i n t h i s experiment may way  associated with  obtained I t would  uptake  and  have been i n some  the presence o f the conjugated or a bound  form o f naphthenate, r a t h e r than with  the f r e e a c i d f s ) .  In c o n c l u s i o n , the data suggest t h a t the naphthenate compounds or t h e i r m e t a b o l i t e s  s t i m u l a t e d glucose a s s i m i l a t i o n  i n e x c i s e d r o o t t i p s from bush bean p l a n t s used i n t h i s  experiment.  Not o n l y was CC»2 p r o d u c t i o n  i n c r e a s e d , but a l s o  amino a c i d s c o n t a i n i n g glucose carbon passed through s o l u b l e pools  i n r o o t t i s s u e s and were more r a p i d l y f i x e d i n t o p r o t e i n .  Three separate determine!  s t u d i e s were c a r r i e d out with bean p l a n t s to  l ) the e f f e c t o f the complex naphthenic a c i d mixture,  KNap, on the uptake, d i s t r i b u t i o n , and i n c o r p o r a t i o n o f phosphorus32,  2) the metabolism o f K C H C - ? - ^ i n leaves and r o o t s , and 1  3)  the e f f e c t o f KNap and KCHC on the uptake and metabolism o f A  ^C glucose by r o o t  tips.  On the b a s i s o f r e s u l t s obtained  i n these experiments i t  may be concluded t h a t : 1. )  When grown i n e i t h e r a minus P or, a complete n u t r i e n t s o l u t i o n , KNap d i d n o t have a s i g n i f i c a n t e f f e c t  on  P uptake. 2. )  The a c r o p e t a l movement o f P from the r o o t s o f p l a n t s grown i n a minus P n u t r i e n t was g r e a t l y enhanced by naphthenate treatment.  The d i s t r i b u t i o n p a t t e r n  in  these p l a n t s showed t h a t the balance i n ^ p d i s t r i 2  b u t i o n between the l e a v e s and r o o t s o f t r e a t e d p l a n t s , was g r e a t l y upset i n f a v o r o f the r o o t s .  When p l a n t s  were grown i n a complete n u t r i e n t , the e f f e c t o f KNap on the d i s t r i b u t i o n o f 3 p 2  3 P 2  w  a  s not as dramatic, and the  d i s t r i b u t i o n balance was upset i n f a v o r o f the  leaves. 3. )  Naphthenate treatment i n c r e a s e d the r a t e o f i n c o r p o r a t i o n o f 3 p i n t o the a c i d s o l u b l e (sugar 2  free nucleotides, phospholipids) (nucleic acids, However, as due  to  the  phosphoproteins)  3 P activity 2  f a c t that  phosphates,  and a c i d i n s o l u b l e f r a c t i o n s o f leaves.  i n root tissues declined, the a c i d  soluble 32  P  a c t i v i t y was t r a n s l o c a t e d a c r o p e t a l l y , the amount o f 32p a c t i v i t y i n the a c i d i n s o l u b l e f r a c t i o n s i g n i f i cantly increased.  Naphthenate treatment d i d n o t a f f e c t  the amount o f P i n the two P f r a c t i o n s . 4. )  When administered  to l e a f d i s k s i n the l i g h t o r to  roots o f i n t a c t seedlings  i n the dark, KCHC-7- ^C 1  was r a p i d l y converted to a mixture o f two conjugated metabolites: amide.  the g l u c o s e e s t e r and the a s p a r t i c a c i d  I t appears t h a t both conjugates a r e mobile  w i t h i n the p l a n t .  A l s o , i t i s p o s s i b l e t h a t KGHC  may have been conjugated w i t h a low m o l e c u l a r 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 o f i ^ C a c t i v i t y i n the e t h a n o l - s o l u b l e , e t h a n o l - i n s o l u b l e , and the r e s p i r e d C 0 •^C g l u c o s e was administered amount o f ^ C  2  fractions after  t o bean r o o t t i p s .  The  a c t i v i t y i n s o l u b l e amino a c i d s and i n  p r o t e i n was i n c r e a s e d by the treatments. u a l naphthenic a c i d , KCHC, had the g r e a t e r  The i n d i v i d e f f e c t on  the uptake and metabolism o f l a b e l l e d g l u c o s e . One o f the o v e r a l l o b j e c t i v e s o f these experiments was to augment our understanding o f how naphthenates s t i m u l a t e metab o l i s m and growth o f bean p l a n t s , o r to understand more f u l l y their  'modus operandi.'  I t appears t h a t the e f f e c t s o f naph-  thenate on phosphorus a s s i m i l a t i o n a r e n o t o n l y concerned with oxidative phosphorylation  ( 1^7 ) and the normal g l y c o l y t i c  pathway ( 5 2 , Chapter 3 ) , b u t a l s o w i t h the i n c o r p o r a t i o n o f  P i n t o components o f the a c i d i n s o l u b l e f r a c t i o n . connection  between i n c r e a s e s  nucleic acid biosynthesis  i n enzyme a c t i v i t y  ( 110 ), and p r o t e i n  A close  ( 5 3 » 147 )» formation  ( 147 ) suggests t h a t a major e f f e c t o f naphthenates may be exerted  a t both the m e t a b o l i c and g e n e t i c  levels.  R e s u l t s o f t h e KCHC-7-l^C r o o t - f e e d i n g  (Chapter 2) and t h e  l ^ C g l u c o s e metabolism (Chapter 3) experiments a l s o suggest t h a t the m e t a b o l i t e s  o f the naphthenic a c i d s may be r e s p o n s i b l e  f o r s t i m u l a t i n g metabolic r e a c t i o n s .  Data obtained  i n other  experiments performed i n our l a b o r a t o r y i n d i c a t e t h i s to be t h e case ( 147 ). suggestion 1. )  A c t u a l experimental evidence s u p p o r t i n g  follows s Free a c i d s d i d n o t i n c r e a s e the s p e c i f i c a c t i v i t i e s o f f i v e d i f f e r e n t enzymes i n v i t r o  2. )  this  Free a c i d i s n o t detected  ( 147 ).  i n concentrated  extracts of  p l a n t t i s s u e s i x 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 o f the metabolites  i n the a e r i a l p o r t i o n  a f t e r the p l a n t had r e c e i v e d a f o l i a r naphthenate treatment seems to be a s s o c i a t e d w i t h a c o r r e s p o n d i n g increase i n metabolic a c t i v i t y 4. )  The s u g g e s t i o n  ( Chapter 1 ).  was made r e c e n t l y t h a t the a s p a r t i c a c i d  amide o f IAA may n o t be a mere d e t o x i c a t i o n product. Davis and Galston metabolite synthesis.  ( 43 ) s p e c u l a t e d  t h a t t h i s IAA  may f u n c t i o n as a c h a i n i n i t i a t o r i n p r o t e i n Zenk ( 1 5 5 ) has a l s o r e p o r t e d  formed a IAA-protein  complex i n peas.  t h a t IAA  Perhaps, the  a s p a r t i c a c i d amide o f CHCA may p l a y a s i m i l a r r o l e .  In Chapter 2 r e s u l t s i n d i c a t e d t h a t CHCA may a l s o be bound to a s m a l l  peptide.  Kazemie and Klambt ( 81, 82 ), however, r e p o r t e d  t h a t the ,  a s p a r t i c a c i d amide o f the s y n t h e t i c a u x i n , NAA, was h y d r o l y z e d when a d m i n i s t e r e d to wheat c o l e o p t i l e t i s s u e .  The authors con-  cluded t h a t t h a t the r e l e a s e o f f r e e NAA upon h y d r o l y s i s was responsible  f o r t h e observed i n c r e a s e  i n c o l e o p t i l e growth.  Naphthenic a c i d s , though b i o l o g i c a l l y f o r e i g n p e t r o c h e m i c a l s , p e n e t r a t e p l a n t t i s s u e s r e a d i l y upon a p p l i c a t i o n , and a r e r a p i d l y metabolized. and  A f t e r a l l naphthenate m e t a b o l i t e s a r e i d e n t i f i e d  t h e i r e f f e c t s on animals have been a s c e r t a i n e d ,  the a p p l i c a -  t i o n o f the naphthenic a c i d mixture o r the i n d i v i d u a l naphthenic a c i d , CHCA, to crop p l a n t s may help to make the 'green r e v o l u t i o n ' i n North America?,even more s u c c e s s f u l .  1. )  A b o l i n a , G. I . and V. V. Berezhnova. 1963. Effect of petroleum growth promoting substance on growth, development, and y i e l d o f potatoes and on p h y s i o l o g i c a l and s o i l processes under U z b e k i s t a n c o n d i tions . Dokl., Vses. Soveshch. Primen. Neft. Rostovogo. V e s h c h e s t r a S e l . Khoz., 2nd, Baku, 73 - 91; Chem. A b s t r . 62, 20830g, 1967.  2. )  A b o l i n a , G. I . and N. A t a u l l a e v . 1969. Influence of naphthenic growth substance (NGS) upon p h y s i o l o g i c a l and b i o c h e m i c a l processes and the p r o d u c t i v i t y o f p o t a t o e s , melons, and v e g e t a b l e s i n the c o n d i t i o n s o f Uzbekistan. P l a n t S t i m u l a t i o n i A Symposium. K. L. Popoff, Ed., Bulg. Acad, o f S c i e n c e s P r e s s , S o f i a , Bulg. p? 904. c  3. )  Afanas'eva, T. A. 1969. E f f e c t of t r e a t i n g p l a n t s with a s o l u t i o n o f petroleum growth substance (NaNap) and a l s o a mixture o f t h i s s o l u t i o n and ammonium n i t r a t e on the y i e l d o f the green mass o f oats and meadow f o x t a i l under T r a n s p o l a r c o n d i t i o n s . Tr. Khar'kov. Sel'skekhoz. I n s t . 28_« 3 - 261 Chem. A b s t r . 2 i . 110078a, 1970. 2  4. )  A g a k i s h i e v , D. and T. B. Bazanova. 1965. E f f e c t o f some growth s t i m u l a n t s on c o t t o n p l a n t s a t d i f f e r e n t degrees o f s o i l s a l i n i t y . Izv. Akad. Nauk. Turkm. SSR, Ser. B i o l . Nauk j£» 22 28} Chem. A b s t r . 64, 20538h, 1966.  5. )  A g a k i s h i e v , D., G. P. Kugatova-Shemyakina, T. B. Bazanova, V. N. Gramenitskaya, and L. I . Rozhkova. 1970. Growth a c t i v i t y and chemical s t r u c t u r e . IV. Effect of 4-(2*,6*-dimethyl-3'-cyclohexenyl)-4-hydroxybutan-2-one and compounds r e l a t e d to i t on the germi n a t i o n and p r o d u c t i v i t y o f c o t t o n p l a n t s . Agrokhimiya 2* 122 - 125t  6. )  Alekperov, S. and G. Chrjanovskaya. 1969. A physiological study on the a c t i o n o f growth s t i m u l a t o r s on woody plants i n s a l i n i z a t i o n conditions. P l a n t S t i m u l a t i o n i A Symposium. K. L. Popoff, Ed., Bulg. Acad, o f S c i e n c e s P r e s s , S o f i a , Bulg. p.. 1050.  7. )  A l i e v , A. Yu. 1965. E f f e c t o f NRV (sodium on growth, development, and composition F i z i o l . A k t i v . Veshchestva Ikh Primen. Dokl. Nauch. Konf., V i l n y u s (1963), 13 A b s t r . 66, 45849n, 1967.  naphthenates) o f tomatoes. Rastenievod., - 17» Chem.  8. )  A l i - Z a d e , M. A. and Z. B. Guseinov. 1965. E f f e c t s of petroleum growth promoting substances, g i b b e r e l l i n , and heteroauxin on the growth and development o f eggplants. Dokl., Vses. Soveshch. Priraen. N e f t . Rostovogo Veshchestva S e l . Khoz., 2nd, Baku (1963), 3^3 - 347; Chem. A b s t r . 67J 20847t, 1967.  9. )  Anbrokh, R. V. , L. D. Z a g r i i c h u k , 0. L. Solov'eva, I . A. Novitskaya, and A. V. B o g a t s k i i . 1969. Composition and p h y s i o l o g i c a l a c t i v i t y o f naphthenic a c i d s o f d i e s e l f u e l and Otduv (by-product formed d u r i n g bitumen p r o d u c t i o n ) from E a s t e r n USSR petroleums, P i z i o l . A k t i v . Veshchestva 2» 176 - 186; Chem. A b s t r .  21* 13325s, 1970.  1G.)  Andreae, W. A. and N. E. Good. 1955. The formation i n d o l e a c e t y l a s p a r t i c a c i d i n pea s e e d l i n g s . P l a n t P h y s i o l . J O i 38G - 382.  of  11. )  Andreae, W. A. and N. E. Good. 1957. S t u d i e s on 3 - i n d o l e a c e t i c a c i d metabolism. IV. Conjugation w i t h a s p a r t i c a c i d and ammonia as processes i n the metabolism o f carboxylic acids. P l a n t P h y s i o l . J 2 : 566 - 572.  12. )  Andreae, W. A., J . R. Robinson, and M. W. H. van Y s s e l s t e i n . 1961. S t u d i e s on 3 - i n d o l e a c e t i c a c i d metabolism; VII. Metabolism o f r a d i o a c t i v e a c i d by pea r o o t s . P l a n t P h y s i o l . _36: 783 - 787.  13. )  Asadov, Sh. D. 1965. E f f e c t o f new types o f f e r t i l i z e r s on growth, development, and y i e l d o f v e g e t a b l e s . F i z i o l . A k t i v , Veschestva Ikh. Primen. Rastenievod., Dokl. Nauk. Konf., V i l n y u s (19^3) 19 - 2 3 ; Chem. A b s t r . 66, 4585©f, 1967.  14. )  Asadov, Sh. D. 1965. E f f e c t o f petroleum growth promoting substance on the cabbage crop. Dokl., Vses. Soveshch. Primen. N e f t . Rostovogo Veshchestva S e l . Khoz., 2nd, Baku (1963)1 230 - 236; Chem. A b s t r . 6£, 20835n, 1967.  15. )  A t a u l l a e v , N. A. 1965. E f f e c t o f growth s t i m u l a n t s o f petroleum o r i g i n and complex t r a c e element f e r t i l i z e r s on the y i e l d s o f melon and v e g e t a b l e s . Tr. Uzbek. Nauch.-Issled. I n s t . Ovoshehebakhch. K u l ' t . K a r t o f e l y a 4s 18G - 202; Chem. A b s t r . 6 6 , 8 4 9 6 m , 1967.  16. )  Babaev, D. 1966. E f f e c t o f growth s t i m u l a t o r s on some p h y s i o l o g i c a l b i o c h e m i c a l processes i n the r o o t system of c o t t o n . Izv. Akad. Nauk. Turkm. SSR $i 2 4 - 3 2 ; Chem. A b s t r . 48, 82370, 1967.  17. )  Babaev, D. 1970. E f f e c t o f some growth r e g u l a t o r s on the r o o t system o f f i n e - f i b e r e d c o t t o n a t v a r i o u s phases o f p l a n t development. I z v . Akad. Nauk. Turkm. SSR, Ser. B i o l . Nauk Ji 3-8; Chem. A b s t r . 2h.* 12053k, 1971.  18. )  B a b i o r , B. M. and K. Bloeh. 1966. Aromatization hexanecarboxylic a c i d . J . B i o l . Chem. 24ls 3643 - 3651.  19. )  Bachramov, A. B. 1957. The i n f l u e n c e o f growth h e l p i n g substance o f petroleum o r i g i n upon the crop c a p a c i t y o f China beans. Dan. Azerb. SSR 13s 221. Quoted i n Huseinov, i960.  20. )  Bahram, H. M. 1970. E f f e c t o f petroleum mulch on c o t t o n growth. Acta Agron. Acad. S c i . 12* 117 - 122; B i o l . A b s t r .  of cyclo-  I I . 92157. 1970.  21. )  Baivarovskaya, Yu. V., A. I , Preobrazhenskaya, L. M. Starkova, and E. S. Sevast'yanova. 19&3. Growth f a c t o r p r o d u c t i o n from petroleum o f the Perm r e g i o n . Neftepererabotka i Neftekhum., Nauchn.-Tekhn. Sb. 2« 8 - 9s Chem. A b s t r . 60, 6l47d, 1964.  22. )  Barber, D. A. and B. C. Loughman. 1967. The e f f e c t o f microorganisms on the a b s o r p t i o n o f i n o r g a n i c n u t r i e n t s by i n t a c t p l a n t s . I I . Uptake and u t i l i z a t i o n o f phosphate by b a r l e y p l a n t s grown under s t e r i l e and non-sterile conditions. J . E x p ' t l Bot. 18s 170 - 176.  23. )  Bazanova, T. B. and K. M. Akopova. 1966. E f f e c t of naphthenic a c i d s on s e v e r a l aspects o f metabolism and y i e l d o f f i n e - f i b e r c o t t o n p l a n t s under v a r i o u s n u t r i t i v e conditions. I z v . Akad. Nauk, Turkm. SSR, Ser. B i o l . Nauk. £s 53 58; B i o l . A b s t r . 4£, 4685, 1968.  24. )  Bazanova, T. B. 1969. E f f e c t o f growth s t i m u l a n t s on enzyme a c t i v i t i e s i n c o t t o n l e a v e s i n r e l a t i o n to n u t r i t i v e conditions. I z v . Akad. Nauk. Turkm. SSR, Ser. B i o l . Nauk. l i 9 14; Chem. Abstr. 2I» 59868h, 1969.  25. )  Bazanova, T. B. 1970. E f f e c t o f the g r o w t h - r e g u l a t i n g p r e p a r a t i o n Sh-8 on the content o f f r e e a u x i n and i n h i b i t o r s i n v a r i o u s organs o f f i n e - f i b e r e d c o t t o n . I z v . Akad. Nauk. Turkm. SSR, Ser. B i o l . Nauk. 2. 9 16; Chem. Abstr. 2i*» 12054m, 1971. 1  B i d d u l p h , 0. 19^1. D i u r n a l m i g r a t i o n o f i n j e c t e d r a d i o phosphorus from bean l e a v e s . Amer. J . Bot. 28: 348 -  352.  B i d d u l p h , 0., S. B i d d u l p h , R. Cory, and H. Koontz. 1958. C i r c u l a t i o n p a t t e r n s f o r phosphorus, s u l f u r , and calcium i n the bean p l a n t . P l a n t P h y s i o l . 2 2 293 - 300. s  B i d d u l p h , 0. 1959. T r a n s l o c a t i o n o f I n o r g a n i c S o l u t e s . In: P l a n t P h y s i o l o g y , A T r e a t i s e . V o l . I I . F. C. Steward, Ed. Academic P r e s s , New York, pp. 553 - 598. B i d d u l p h , 0. and R. Gory. i960. Demonstration o f two t r a n s l o c a t i o n mechanisms i n s t u d i e s o f b i d i r e c t i o n a l movement. P l a n t P h y s i o l . 25« 689 - 695. B i e l e s k i , R. L. 1969. Phosphorus compounds i n t r a n s l o c a t i n g phloem. P l a n t P h y s i o l . 44: 497 - 502. Bledsoe, C., C. V. Cole, and C. Ross. 1969. Oligomycin i n h i b i t i o n o f phosphate uptake and ATP l a b e l l i n g i n e x c i s e d maize r o o t s . P l a n t P h y s i o l . 44: 1040 - 1044. Bock, R. and K. Behrends. 1965. I n v e s t i g a t i o n o f a mixture o f petroleum a c i d s . The naphthenic a c i d problem. Z. Anal. Chem. 208: 338 - 352; Chem. A b s t r . 62, 10267b,  1965.  B o l l a r d , E. G. i960. T r a n s p o r t i n the xylem. Ann. Rev. P l a n t P h y s i o l . 11: l 4 l - 166. Bowen, G. D. and A. D. R o v i r a . 1966. M i c r o b i a l f a c t o r s i n short-term phosphate uptake s t u d i e s w i t h p l a n t r o o t s . Nature 211: 665 - 666. Burachevskii, I . I . 1965. A c t i v a t i o n o f t h e s y n t h e s i s o f amylase by A s p e r g i l l u s usamii. Fermentnaya i S p i r i t . Prom. 2 1 : 6 - 8; Chem. A b s t r . 62,  l6920h, 1965.  Butcher, D. N. and A. E. S t r e e t . culture. Botan. Revs. 2 2 * 513 - 586,  1964.  Excised  root  Cason, J . and K-L. Liauw. 1965. C h a r a c t e r i z a t i o n and s y n t h e s i s o f a monocyclic eleven-carbon a c i d i s o l a t e d from a C a l i f o r n i a petroleum. J . Org. Chem. 22» 1763 - 1769.  38. )  Cason, J . and A. I . A. Khodair. 1966. S e p a r a t i o n from a C a l i f o r n i a petroleum and c h a r a c t e r i z a t i o n o f geometric isomers o f 3 - e t h y l - 4 - m e t h y l c y c l o p e n t y l a e e t i c acid, J . Org. Chem. 21: 3618 - 3625.  39. )  Chen, S. L. 1951. Stimultaneous movement o f 3 P i n o p p o s i t e d i r e c t i o n s i n phloem t i s s u e . Amer. J . Bot. J_3. 203 ~ 211.  40. )  Chu,  41. )  Cole, C. V. and G. Ross. 19*>6. E x t r a c t i o n , s e p a r a t i o n , q u a n t i t a t i v e e s t i m a t i o n o f s o l u b l e n u c l e o t i d e s and sugar phosphates i n p l a n t t i s s u e s . Anal. Biochem. 12. 526 - 539.  42. )  C o s s i n s , E. A. and S. K. Sinka. 1966. The i n t e r c o n v e r s i o n o f g l y c i n e and s e r i n e by p l a n t t i s s u e e x t r a c t s . Biochem. J . 101. 542 - 549.  43. )  Davis, P. J . and A. W. Galston. 1971. Labelled indolemacromolecular conjugates from growing stems s u p p l i e d with l a b e l l e d i n d o l e a c e t i c a c i d . I . F r a c t i o n a t i o n . P l a n t P h y s i o l . ij£: 435 - 441.  44. )  D i m i t r o v , D., A. E. Stefanova, L. K . Khalacheva, M. Arnaudov, and M. Popov. 1969. S e p a r a t i o n o f s y n t h e t i c napht h e n i c a c i d s obtained by the l i q u i d - p h a s e o x i d a t i o n o f a dearomatized g a s - o i l f r a c t i o n o f Tyulenovo crude o i l and t e s t i n g the s t i m u l a t i o n a c t i v i t y o f i n d i v i d u a l narrow f r a c t i o n s . Khim. Ind. ( S o f i a , Bulg.) 2» 389 - 393: Chem. Abstr.  2  and  1 4  C  S-M. 1969. Growth and b i o c h e m i c a l responses o f the tomato (Lycopersicum esculentum L. v a r . Bonny Best) to K naphthenates. M. Sc. T h e s i s , U n i v e r s i t y o f B r i t i s h Columbia.  22,  27259k,  and  1970.  45. )  Edwards, D. G. 1970. Phosphate a b s o r p t i o n and d i s t a n c e t r a n s p o r t i n wheat s e e d l i n g s . Aust. J . B i o l . S c i . 2^: 255 - 264.  long-  46.)  E i d e r , N. G. 1970. A n a l y s i s o f naphthenic a c i d s by l i q u i d chromatography. J . P a i n t Technol. 42: 504 - 509.  47. )  Ejubov, R. and F. Issaeva, 1969. I n f l u e n c e o f the napht h e n i c growth substance on the growth, development, and y i e l d o f maize and lucerne., P l a n t S t i m u l a t i o n : A Symposium. K . L. Popoff, Ed., Bulg. Acad, o f Sciences P r e s s , S o f i a , Bulg. p. 929.  48. )  E p s t e i n , E. 1966. Dual p a t t e r n o f i o n a b s o r p t i o n by p l a n t c e l l s and by p l a n t s . Nature 212: 1324 - 1327.  gas-  E t t e r , H . M. 1966. E f f e c t o f low l e v e l s o f 2,4-D on the uptake, t r a n s l o c a t i o n , and i n c o r p o r a t i o n o f 32p by the bean p l a n t , Phaseolus v u l g a r i s L. Ph.D. T h e s i s , U n i v e r s i t y o f B r i t i s h Columbia. E t t e r , H. M. 1967. S t i m u l a t i o n by 2,4-D and 2,4-D p l u s FeEDTA o f 32P uptake by bean s e e d l i n g s , Phaseolus vulgaris. Can. J . Bot. k$ 1 535 - 537. E t t e r , H. M. 1967. The uptake and d i s t r i b u t i o n o f 32p t> bean s e e d l i n g s (Phaseolus v u l g a r i s ) growing a t two phosphate l e v e l s , and some e f f e c t s o f 2,4-D. Can. J . Bot. 4j>{ 1011 - 1017.  y  F a t t a h , Q. A. 1969. Growth and m e t a b o l i c responses o f the bush bean to potassium naphthenates. Ph.D. T h e s i s , U n i v e r s i t y o f B r i t i s h Columbia. F a t t a h , Q. A. and D. J . Wort. 1970. M e t a b o l i c responses o f bush bean p l a n t s to naphthenate a p p l i c a t i o n . Can. J . Bot. 48* 861 - 866. F a t t a h , Q. A. and D. J . Wort. 1970. E f f e c t o f l i g h t and temperature on s t i m u l a t i o n o f growth by naphthenates. Agron. J . 62. 576 - 577. F i s k e , C. H. and Y. Subbarow. 1925. d e t e r m i n a t i o n o f phosphorus. J . B i o l . Chem. 6 6 1 375 - 400.  The c o l o r i m e t r i c  Galper, J , B. and B. M. B a b i o r . 1968. Hydrogen t r a n s f e r i n t h e enzymatic d e s a t u r a t i o n o f c y c l o h e x a n e c a r b o x y l CoA. Biochim. e t Biophys. A c t a lj_8i 289 - 292. Glushkov, N. M. and A. S. Yakovlev, 1963. Bee f e e d i n g w i t h growth substances. P c h e l o r o d s t r o 6 1 25 - 27« Chem. Abstr. 6_2, 6080b, 1965. G o l d s t e i n , R. F. and A. L. Waddams. 1967. Petroleum Chemicals I n d u s t r y , 3rd E d i t i o n . E. and F. N. Spon, L t d . , London, pp. 439 - 442. Goldsworthy, A. 1964. S t u d i e s i n carbohydrate metabolism o f excised roots. Ph.D. T h e s i s , U n i v e r s i t y o f Wales. G r e e n s t e i n , J . P. and M. W i n i t z . 1961. Chemistry o f the Amino A c i d s . V o l . I I . John Wiley and Sons, New York. P. 892.  61. ) Gruodiene, J . and S. Buciene. 1967. E f f e c t o f growth s t i m u l a n t s on n i t r o g e n metabolism o f p o t a t o e s . L i e t u v o s TSR Aukst. Mokyklu Mokslo Darb. B i o l . 2« 51 - 59f Chem. Abstr. 6j>i 75779u, 1968. 62. ) Gurvich, M.L. 1968. E f f e c t o f sodium naphthenate on t h e m i t o t i c a c t i v i t y o f c e l l s under % - i r r a d i a t i o n . T s i t o l . Genet. 2: 400 - 407i Chem. A b s t r . 70.  17398d, 1969.  63. ) Gurvich, M. L. and V. K. Shcherbakov. 1968. M i t o t i c a c t i v i t y s t i m u l a t i o n and appearance o f b i n u c l e a t e d c e l l s under the a c t i o n o f s a l t s o f naphthenic a c i d s . T s i t o l o g i y a 10: 1058 - IO63: Chem. A b s t r . 6£,  85616c, 1968.  64. ) Guseinov, D. M., Sh. D. Asadov, and A. Yu. A l i e v . 1956. E f f e c t o f t h e growth substance o f petroleum o r i g i n on crops o f cabbage and tomatoes. Dokl. Acad. Nauk Azerbaidzhan. SSR, 12: 123 - 128? Chem. Abstr. ^0, 11585e, 1956. 65. ) Guseinov, D. M. and A. A. Guseinov. 1958. E f f e c t o f petroleum s t i m u l a n t onnthe y i e l d o f c o t t o n . Dokl. Acad. Nauk Azerbaidzhan. SSR. l 4 : 391 - 393: Chem. Abstr. ^2, l4952d, 1958. 66. ) Guseinov, D. M. and F. G. Issaeva. 1965. E f f e c t o f combined d e l i v e r y o f petroleum growth promoting substances and r a d i o a c t i v e phosphorus on the a l f a l f a crop. Dokl., Vses. Soveshch. Primen. N e f t . Rostovogo Veshchestva S e l . Khoz., 2nd, Baku (1963), 257 - 2621 Chem. A b s t r . 6£: 20836p, 1967, 67. ) Guseinov, Z. B. and A. M. Masiev. 1965. E f f e c t o f p e t r o chemical growth r e g u l a t o r substance on the growth and development o f r o o t systems o f p l a n t s . I z v . Akad. Nauk. Azerbaidzh. SSR, S e r . B i o l . Nauk. 6: 26 - 31j B i o l . Abstr. 48, 24867, 196?. 68. ) Guseinov, Z. B. 1966. R e s p i r a t i o n i n t e n s i t y i n t h e l e a v e s o f v e g e t a b l e crops under t h e e f f e c t o f g i b b e r e l l i n and petroleum growth substance (NaNap). T r . Azerb. Naueh.-Issled. I n s t . Zemled. 1^: 305 - 310: Chem. A b s t r . 21* 42032d, 1970. 69. ) Guyot, H. M. 1964. Treatment o f sugarcane to i n c r e a s e sugar content, Esso Research and E n g i n e e r i n g Co., F r . Patent 1,433,1211 Chem. A b s t r . 65, 17655a, 1966.  70. )  Hanson, J . B. and 0. Biddulph. 1953. The d i u r n a l v a r i a t i o n i n the t r a n s l o c a t i o n o f m i n e r a l s a c r o s s bean r o o t s . P l a n t P h y s i o l . 28i 356 - 370.  71. )  H a r t i g , Th. l 8 6 l . Ueber d i e Bewegung des S a f t e s i n Holzpflanzen. Bot. Z e i t . __9_s 17 - 23.  72. )  Helder, R. J . 1957. Exchange and c i r c u l a t i o n o f l a b e l l e d i o n s i n young i n t a c t p l a n t s . I n t e r n a t i o n a l Corif, on R a d i o i s o t o p e s i n S c i . Research, R. C. Extermann, Ed., P a r i s , Sept. 1957. Quoted i n B i d d u l p h e t a l , 1958.  73. )  H e l l , C. and E. Medinger. 1874. Ber. 2' 1216. Quoted i n J o l l y , 1967.  74. )  Hoagland, D. R. and D. I . Arnon. 1938. The water c u l t u r e method f o r growing p l a n t s without s o i l . C a l i f . A g r i c u l . Exp't. S t a t i o n C i r c . No. 347. pp. 1 - 32.  75. )  Huseinov, D. M. i960. The i n f l u e n c e o f o r g a n i c compounds o f petroleum o r i g i n upon the growth o f r o o t s and crop capacity o f a g r i c u l t u r a l plants. Trans. 7th I n t . Congress S o i l S c i . (Madison, Wis.) 3.  253 - 259. 76. )  Ibrahim, R. K. and G. H. N. Towers. 1959. C o n v e r s i o n o f s a l i c y l i c a c i d to g e n t i s i c and o - p y r o c a t e c h u i e a c i d , a l l l a b e l l e d w i t h carbon-14, i n p l a n t s . Nature 1 8 4 : I 8 O 3 .  77. )  Ivanova, I . A. 1970. E f f e c t o f g i b b e r e l l i c a c i d , p e n i c i l l i n , and naphthenic a c i d on growth and p r o d u c t i v i t y o f hemp. Izv. I n s t . F i z i o l . Rast., Bulg. Akad. Nauk. 16s 191 201; Chem. A b s t r . __4. 30919b, 1971.  78. )  J o l l y , S. E. 1967. Naphthenic A c i d s . Ins Kirk-Othmer's E n c y c l o p e d i a o f Chemical Technology, H. Mark e t a l , Eds., 2nd E d i t i o n , r _ , John Wiley and Sons, New York, N. Y. pp. 727 - 73^7  79. )  Jukova, P. S. 1969. I n f l u e n c e o f p h y s i o l o g i c a l a c t i v e substances on the growth, development, and p r o d u c t i v i t y of vegetables. P l a n t S t i m u l a t i o n s A Symposium. K. L. P o p o f f , Ed., B u l g . Acad, o f S c i e n c e s P r e s s , S o f i a , Bulg. pp. 1025 -  1026.  80. )  Kazakova, I . I . 19^5. E f f e c t o f growth s t i m u l a n t s on the germination and growth o f weed seeds. Dokl., Vses. Soveshch. Primen. N e f t . Rostovogo Veshchestva S e l . Khoz. 2nd, Baku, (1963), 391 - 394; Chem. A b s t r . 6_2, 20837q. 1967.  81. ) Kazemie, M. and D. Klambt. 1969. Untersuchungen zur Wirkungsweise der Aminosaurekonjugate der Auxine i n wachsenden Geweben. P h y s i o l . P l a n t . 22s 489 - 496. 82. ) Kazemie, M. and D. Klambt. 1969. Untersuchungen zur Aufnahme von N a p h t h a l i n - l - e s s i g s a u r e und i h r e s Asparaginsaure-Konjugates i n Weizenkoleoptilgewebe. P l a n t a 89.: 76 - 81. 83. ) Kim, W. K. and R. G. S. B i d w e l l . 1967. The e f f e c t s o f i n d o l e a c e t i c a c i d and 2,4-dichlorpphenoxyacetie a c i d on the uptake and metabolism o f l ^ C g l u c o s e and on the i n c o r p o r a t i o n o f 14c l a b e l l e d amino a c i d s i n t o p r o t e i n i n pea r o o t t i p s . Can. J . Bot. 4$s 1751 - 1760. 84. ) Klambt, H. D. 196l. Wachsturnsinduktion und Wuchsstoffmetabolismus im W e i z e n k o l e o p t i l z y l i n d e r . I I . S t o f f wechsel^roduckte xle I n d o l e - 3 - e s s i g s a u r e und der Benzoesaure. P l a n t a ^6* 618 - 631. 85. )  Klambt, H. D. 1961. Wachsturnsinduktion und Wuchsstoffmetabolismus im W e i z e n k o l e o p t i l z y l i n d e r . I I I . S t o f f wechselprodukte der N a p h t h y l - l - e s s i g s a u r e und 2,4d i c h l o r o p h e n o x y e s s i g s a u r e u n d der v e r g l e i c h m i t genen der I n d o l e - 3 - e s s i g s a u r e and Benzoesaure. P l a n t a 1Z« 339 - 353. i(  86. ) Klambt, H. D. 1961. Wachsturnsinduktion und Wuchsstoffmetabolismus im W e i z e n k o l e o p t i l z y l i n d e r . IV. D i e I d e n t i f i z i e r u n g eines S t o f f w e c h s e l p r o d u k t e s der N a p h t h y l - l - e s s i g s a u r e , das mSglieherweise beziehung zur Waehstumsforderung b e s i t z t . P l a n t a j&t 391 - 401. 87. ) K o l e s n i k , Z. V. 1965. E f f e c t o f petroleum growth-promoti n g substance on t h e b i o c h e m i s t r y o f t h e grape p l a n t . Dokl., Vses. Soveshch. Primen. N e f t . Rostovogo Veshchestva S e l . Khoz., 2nd, Baku (1963), 395 - 400s Chem. A b s t r . 6£, 20838r, 1967. 88. ) K o n g j i k a , S. and Dh. Shuqja. 1970. E f f e c t o f naphthenic a c i d s a l t s , s o l a r and g a z o i l on t h e growth and y i e l d o f sunflowers. Ser. Shkencat Natyr. 24t i l l - 119s Chem. A b s t r . 24,  309l4w, 1971.  89. ) Kosobokov, V. I . 1965. E f f e c t o f petroleum growth-promoti n g substance on t h e growth and development o f feed cultures. Dokl., Vses. Soveshch. Primen. N e f t . Rostovogo Veshchestva S e l . Khoz., 2nd, Baku (1963), 401 - 404j Chem. A b s t r . 6£, 20839s, 1967.  90. ) Kotyashkina, V. F. 1968. The e f f e c t o f p l a n t growth r e g u l a t o r s on seeds and s p r o u t s . T r . Kostrom Sel'skokhoz Inst.. Karavaeo 2» 196 - 206; B i o l . A b s t r . _51, 45717. 1970. 91. ) K o z y r k i n a , V. V. and E. N. K o t l y a r o v a , 1969. Reproduction o f g o o s e b e r r i e s by green c u t t i n g s u s i n g growth stimulants , Zap. Leningrad. Sel'skokhoz, I n s t . 130. 49 - 53; Chem. A b s t r . 21* l2P3l4p, 1970. 92. )  Krasonova, E. M., V. F. Kotyashkina, and G. S. I l ' i c h e v . 1965. B i o l o g i c a l c h a r a c t e r i s t i c s o f petroleum growthpromoting substances as p l a n t a c t i v a t o r s . Dokl., Vses. Soveshch. Primen. N e f t . Rostovogo Veshchestva S e l . Khoz., 2nd, Baku (1963), 405 - 4101 Chem. A b s t r . §J_* 20840k, 1967.  93. )  Krawehanka, L. U. 1966. E f f e c t o f p e t r o c h e m i c a l growth substance on p o l l e n germination i n i n t r o d u c e d t r e e s . V y e s t s i . Akad. Navuk. B y e l a r u s k a i SSR,Syeryya B i y a l a n i c h n y k h Navuk 1: 28 - 341 B i o l . A b s t r . 48, 40382, 1967.  94. ) Ladygina, E. A. 1965. E f f e c t o f petroleum growth-promoti n g substance on growth, development, and y i e l d o f potatoes. Dokl., Vses. Soveshch. Primen. N e f t . Rostovogo Veshchestva S e l . Khoz., 2nd, Baku (1963), 162 - 167» Chem. A b s t r . 6_7_, 20833k, 1967. 95. ) L i l o v , D. 1970. E f f e c t o f NGS (naphthenic growth substance) on the content o f some p l a s t i d pigments i n v i n e l e a v e s . Dokl. Bulg. Akad. Nauk. 23.. 105 - 108$ Chem. A b s t r .  22, 110054q, 1970.  96. )  L u t t g e , U. and G. G. L a t i e s . 1966. Dual mechanisms o f i o n a b s o r p t i o n i n r e l a t i o n to l o n g d i s t a n c e .transport in plants. P l a n t P h y s i o l . 4l» 1531 -1539.  97. )  M Closkey, C. M. and G. H. Coleman. V o l 3, John Wiley and Sons, New c  98. ) M a i l o v , A. I . 1968. growth substance and p a s t u r e s . I z v . Akad. Nauk. 42} Chem. A b s t r . 99. )  1955. Organic Syntheses, York. pp. 434 - 435.  Use o f ammonium n i t r a t e w i t h (NaNap) a g a i n s t weeds i n hay  petroleum crops  Azerb. SSR, Ser. B i o l . Nauk. _>i 37 21* 37672n, 1969.  M a i z e l , J . V., A. A. Benson, and N. E. T o l b e r t . 1956. I d e n t i f i c a t i o n o f p h o s p h o r y l c h o l i n e as an important c o n s t i t u e n t o f p l a n t sap. P l a n t P h y s i o l . J i t 407 - 408.  100. )  M a i z e l , J . V. and A. A. Benson. .1956. phosphorylchoiine i n plants. P l a n t P h y s i o l . _31: (Suppl.) x x i v .  Function o f  101. ) Markovnikoff, W. and W. O g l o b l i n . 1883. J . Russ. Phys. Chem. Soc. 1_J: 34. Quoted i n J o l l y ,  1967. 102. ) Marshaniya, I . I . , M. G. Sharashenidze, and A. I . Dumbadze. 1965. E f f e c t o f petroleum growth-promoting substance on t h e t a n g e r i n e crop and q u a l i t y . Dokl., Vses. Soveshch. Primen. N e f t . Rostovogo Veshchestva S e l . Khoz., 2nd, Baku (1963), 168 - 170; Chem. A b s t r . 67., 20834m, 1967. 103. ) Matzjuk, L. S. and I . P. G r i n b e r g . 1969. E f f e c t s o f growth s t i m u l a t o r s and t r a c e elements on the growth, development, and y i e l d o f c e r e a l s and leguminous p l a n t s i n Moldavian S o v i e t S o c i a l i s t R e p u b l i c . P l a n t S t i m u l a t i o n 1 A Symposium. K. L. Popoff, Ed., Bulg. Acad, o f S c i e n c e s P r e s s , S o f i a , Bulg. pp. 1005 - 1011. 104. )  Naghibin, L a . D. 1969. I n f l u e n c e o f naphthenic growth substances (NGS) upon the development and p r o d u c t i v i t y o f t h e c o t t o n p l a n t i n the c o n d i t i o n s o f T a d j i k i s t a n . P l a n t S t i m u l a t i o n s A Symposium. K. L. Popoff, Ed.. Bulg. Acad, o f S c i e n c e s P r e s s , S o f i a , Bulg. p. 904.  105. )  Neuberg, C. and M. Sandberg. 1921. The s t i m u l a n t s o f a l c o h o l i c sugar s p l i t t i n g . IX. C h e m i c a l l y d e f i n e d catalyzers o f fermentation. Biochem. Z. 126s 153 - 178s Chem. A b s t r . l6» 159^, 1922.  106. )  Nussbaum, J . J . 1969. Chemical container p l a n t s . C a l i f . Agr. 23j 16 - 18.  107. )  O s n i t s k a y a , L. K. 1947. Decomposition o f naphthenic a c i d s and naphthenic hydrocarbons by microorganisms. Azerbaidzhanskoe Neftyanoe Khoz. 26s 23 - 26; Chem. A b s t r . 4 j , 3 l 8 l g , 1949.  pinching for roots o f  108. ) Padmanabhan, U. 1970. T r a n s p o r t and metabolism o f c y c l o h e x a n e c a r b o x y l i c a c i d i n bean p l a n t s . Northwest S c i . 44s 67 ( A b s t r . ) . 109. )  Padmanabhan, U. 1971. Metabolism o f c y c l o h e x a n e c a r b o x y l i c a c i d and the d i s t r i b u t i o n o f i t s d e r i v a t i v e s i n Phaseolus v u l g a r i s L. Can, Soc. o f P l a n t P h y s i o l o g i s t s , Western S e c t i o n meeting, Vancouver, B.C., Feb. 18-19. p., 13 ( A b s t r . )  110. )  Pakhomova, G. I . 1965. E f f e c t o f treatment w i t h p e t r o leum growth-promoting substance on b i o c h e m i c a l p r o cesses i n tomato l e a v e s . Dokl,, Vses. Soveshch. Primen. N e f t . Rostovogo Veshchestva S e l . Khoz., 2nd, Baku (1963), 4 n - 4 l 5 ; Chem. A b s t r . 6£, 2084lm, 1967.  111. )  P a p a r i s t o , K. 1968. M e l l i f e r o u s v a l u e o f t u r n s o l e and the e f f e c t o f KAN (naphthenic a c i d s a l t ) on the amount o f sugar produced i n f l o w e r n e c t a r . Bulg. Univ. S h t e t e r o r T i r a n e s , Ser. Shkencat Natyres. 22» 75- 79; Chem. A b s t r . 21* 21079P, 1969.  112. )  P a v l o v a , Z l . and L. K h r i s t o v a . 1970. E f f e c t o f some chemical substances d u r i n g r e p r o d u c t i o n o f Phlox p a n i e u l a t a w i t h green s e e d l i n g s . Izv. I n s t . F i z i o l . Rast., Bulg. Akad. Nauk. 16: 311 316j Chem. A b s t r . 21* I29856v, 1970.  113. )  Peterburgsky, A. V. and K. I . Karamete. 19&9. I n f l u e n c e o f naphthenic growth substance on growth, y i e l d , and q u a l i t y o f maize. P l a n t S t i m u l a t i o n : A Symposium. K. L. Popoff, Ed., Bulg. Acad, o f S c i e n c e s P r e s s , S o f i a , Bulg. pp. 978 -  979.  114. )  P o l i k a r p o v a , F. Ya. 19^5. Petroleum growth-promoting substance as a growth s t i m u l a t o r d u r i n g r o o t f o r m a t i o n i n green c u t t i n g s o f f r u i t c u l t u r e s . Dokl., Vses. Soveshch. Primen. N e f t . Rostovogo Veshchestva S e l . Khoz., 2nd, Baku (1963), 4l6 - 4 l 8 : Chem. A b s t r . 6£, 20842n, 1967.  115. )  Popoff, Mih. D., D. D i m i t r o v , and A. Stefanova. 1966. T e s t i n g o f the s t i m u l a t o r y e f f e c t o f naphthenates o b t a i n e d by o x i d a t i o n o f B u l g a r i a n naphtha. Izv. I n s t . F i z i o l . Rast., Bulg. Acad. Nauk. l*[j 109 115; Chem. A b s t r . 66, 10095r, 1967.  116. )  Popoff, Mih. D. and D. K. H r i s t o z o v . 196?. E f f e c t o f naphthenic growth substance on the y i e l d and q u a l i t y o f tobacco. P l a n t S t i m u l a t i o n : A Symposium. K. L. Popoff, Ed., Bulg. Acad, o f S c i e n c e s P r e s s , S o f i a , Bulg. p . 9 4 § .  117. )  P o p o f f , K. L., I v . Jordanov, and N. Usheva. 1969. E f f e c t of p h y s i o l o g i c a l l y a c t i v e substances on the i n t e n s i t y of photosynthesis. P l a n t S t i m u l a t i o n : A Symposium. K. L. Popoff, Ed., Bulg. Acad, o f S c i e n c e s P r e s s , S o f i a , B u l g . p.. 719.  118. )  Popov, Mih. D. and A l . D. Boikov. 1969. T e s t i n g the s t i m u l a t i o n e f f e c t o f naphthenic growth substance (NGS) on p l a n t s . P l a n t S t i m u l a t i o n : A Symposium. K. L. Popoff, Ed., Bulg. Acad, o f S c i e n c e s P r e s s , S o f i a , Bulg. p.. 963.  119. )  Rathore, V. S., S. H. Wittwer, W. H. Jyung, Y. P. S. B a j a j , and M. W. Adams. 1970. Mechanism o f z i n c uptake i n (Phaseolus v u l g a r i s L.) t i s s u e s . P h y s i o l . P l a n t . 23.: 908 - 919.  120. )  R o v i r a , A. D. and G. D. Bowen. 1970. T r a n s l o c a t i o n and l o s s o f phosphate a l o n g r o o t s o f wheat s e e d l i n g s . P l a n t a £ 2 : 15 - 25.  121. )  Sardarova, G. G., Z. A. A l i e v a , and A. G. G i l a n i . 1958. E f f e c t o f s t i m u l a t o r substance o f petroleum o r i g i n on the growth and development o f apple s e e d l i n g s . Dokl. Acad. Nauk Azerbaidzhan SSR. 14: 527 - 53O: Chem. A b s t r . £3., 637f, 1959.  122. )  S e a f o r t h , G. E., J . G. Severson, J r . , and D. J . Wort. 1971. Metabolic studies with cyclohexanecarboxylic and .naphthenic a c i d s i n the bean p l a n t , Phaseolus v u l g a r i s L. (In manuscript.)  123. )  S e i f e r t , W. K. and W. G. Howells. 1969. Interfacially a c t i v e a c i d s i n a C a l i f o r n i a crude o i l . Isolation o f c a r b o x y l i c a c i d s and phenols. Anal. Chem. 4 l : 554 - 562.  124. )  S e i f e r t , W. K. and R. M. T e e t e r . 1969. Preparative thinl a y e r chromatography and high r e s o l u t i o n mass s p e c t r o metry o f crude o i l c a r b o x y l i c a c i d s . A n a l . Chem. 4 l : 786 - 795.  125. )  S e i f e r t , W. K. and R. M. T e e t e r . 1970. Identification o f p o l y c y c l i c naphthenic, mono-, and d i a r o m a t i e crude o i l carboxylic acids. Anal. Chem. 42: 180 - 189.  126. )  Semenyuk, L. A. and E. N. Savchenko. 1969. Absorbing a c t i v i t y o f the g a s t r o - i n t e s t i n a l t r a c t and the growth o f dogs under the i n f l u e n c e o f petroleum growth substances (NaNap) obtained from r e f i n e r y by-products, Vop. F i z i o l . P i s h c h . 6 9 - 7 1 ; Chem. A b s t r . J2  119152c,  127. )  1970.  %  Severson, J . G., J r . , B. A. Bohm, and C. E. S e a f o r t h . The metabolism o f c y c l o h e x a n e c a r b o x y l i c a c i d i n Phaseolus v u l g a r i s . Phytochem." ±i 107 - 110.  1970.  128. ) Severson, J . G., J r . and D. J . Wort. 197©. The e f f e c t o f naphthenates on the uptake and d i s t r i b u t i o n o f phosphorus-32 i n Phaseolus v u l g a r i s L. Northwest S c i . 4 4 . 6 9 - 7 0 ( A b s t r . ) . 1 2 9 . ) Severson, J . G., J r . 1971. The e f f e c t o f potassium c y c l o hexanecarboxylate and potassium naphthenates on the uptake and metabolism o f l4c g l u c o s e i n bean r o o t t i p s . Northwest S c i e n t i f i c A s s o c i a t i o n , Annual meeting, Moscow, Idaho, Apr. 1 6 - 1 7 ( A b s t r . ) . 1 3 0 . ) Severson, J . G., J r . 1 9 7 1 . S t i m u l a t i o n o f l^C g l u c o s e uptake and metabolism i n bean r o o t t i p s by naphthenates. Phytochem. ( I n p r e s s ) . 1 3 1 . ) Severson, J . G., J r . 1 9 7 1 . S t u d i e s w i t h cyclohexanec a r b o x y l i c a c i d i n bean p l a n t s . Conjugate f o r m a t i o n i n and t r a n s p o r t from r o o t t i s s u e s . Can. Soc. o f P l a n t P h y s i o l o g i s t s , Western S e c t i o n meeting, Vancouver, B.C., Feb. 18-19. p . 12 ( A b s t r . ) . :  1 3 2 . ) Shakhi-Zade, M. G. 1 9 6 3 . I n d u s t r i a l p r o d u c t i o n o f naphthenic growth substance. N e f t e p e r e r a b o t k a i Neftekhim., Nauchn.-Tekhn. Sb. 11t 24 - 2 7 s Chem. A b s t r . 6 1 , 543le, 1964. 1 3 3 . ) S t e c h e r , P. G. e t a l , Eds. i960. The Merck Index o f Chemicals and Drugs. 7 t h E d i t i o n , Merck and Co., I n c . , Rahway, N.J. p' . 7 0 3 . 1 3 4 . ) S t e c h e r , P. G. e t a l , Eds. 1968. The Merck Index o f Chemicals and Drugs. 8th E d i t i o n , Merck and Co., I n c . , Rahway, N.J. p.;. 3 1 0 . 1 3 5 . ) S t e e l , R. G. D. and J . H. T o r r i e . i960. P r i n c i p l e s and procedures o f s t a t i s t i c s . M G r a w - H i l l P u b l i s h i n g Co., New York, N.Y. G  1 3 6 . ) Subbotina, N. V. 1965. P h y s i o l o g i c a l r e a c t i o n o f f r u i t t r e e t o petroleum growth-promoting substance. Dokl., Vses. Soveshch. Primen. N e f t . Rostovogo Veshchestva S e l . Khoz., 2nd, Baku (1963), 4 3 7 - 4 3 9 ; Chem. A b s t r . 67_, 20845r, 1967. 1 3 7 . ) Swets, W. A. and R. T. Wedding. 1964. The e f f e c t o f d i n i t r o p h e n o l and o t h e r i n h i b i t o r s on the uptake and metabolism o f 2 , 4 - d i c h l o r o p h e n o x y a c e t i c a c i d by Chlorella. New P h y t o l o g i s t __3.« 5 5 - 7 2 . 1 3 8 . ) S z e ' k e l y , A. and J . d i G l e r i a . 1 9 6 6 . The importance o f naphthenic a c i d s i n a g r i c u l t u r a l c h e m i s t r y . Agrokem. T a l a j t a n 1£. 1 2 5 - 130} B i o l . A b s t r . 48,  307G3b, 1967.  139. )  Thurman, D. A. and H. E. s t r e e t . 1962. Metabolism o f some i n d o l e auxins i n e x c i s e d tomato r o o t s . J . E x p ' t a l Bot. 13_: 369 - 377.  140. )  Towers, G. H. N. 1964. Metabolism o f p h e n o l i c compounds in plants. I n : B i o c h e m i s t r y o f P h e n o l i c Compounds. J . B. Harborne, Ed., Academic P r e s s , New York, p.. 259.  141. )  Voinova-Raikova, J . 1969. E f f e c t o f some s t i m u l a t o r s on s o i l m i c r o f l o r a . P l a n t S t i m u l a t i o n s A Symposium. K. L. P o p o f f , Ed., Bulg. Acad, o f S c i e n c e s P r e s s , S o f i a , Bulg. pp. 995 996.  142. )  Wort, D. J . 1969. S t i m u l a t i o n o f v e g e t a t i v e and r e p r o d u c t i v e growth o f bush bean p l a n t s by naphthenates. Can. J . P l a n t S c i . 4£i 791 - 796.  143. )  Wort, D. J . and K. M. P a t e l . 1970. The response o f p l a n t s to naphthenic and c y e l o a l k a n e e a r b o x y l i c a c i d s . Agron. J . 62s 644 - 646.  144. )  Wort, D. J . and K. M. P a t e l . 1970. Erhohung des Buschbohnenertrags durch Naphthenate und d i e Auswirkungen der Anwendungsmethode. Angew. Botanik 44: 179 - 185.  145. )  Wort, D. J . and K. M. P a t e l . 1970. S t i m u l a t i o n o f bush bean p l a n t s by naphthenates as r e l a t e d to s t r u c t u r e o f the a c i d , method o f a p p l i c a t i o n , , and growth conditions, Northwest S c i e n c e 4 4 : 71 ( A b s t r , ) .  146. )  Wort, D. J . and E. C. Hughes. 1970. S t i m u l a t i o n o f tuber p r o d u c t i o n o f e a r l y p o t a t o e s by naphthenates. Amer. Potato J . 4_2: 394 - 396.  147. )  Wort, D. J . , J . G. Severson, J r . , and D. R. P e i r s o n . 1971. Mechanism o f p l a n t growth s t i m u l a t i o n by naphthenates: I n c r e a s e i n the a c t i v i t y o f enzymes o f n i t r o g e n metabolism. Northwest S c i e n t i f i c A s s o c i a t i o n , Annual meeting, Moscow, Idaho, Apr. 16-17 ( A b s t r . ) .  148. )  Yamaki, T. and K. Kobayashi. 1970. 7th I n t e r n a t i o n a l Conf, on P l a n t Growth Substances, Canberra, Aust. ( A b s t r . ) .  149. )  Yur'eva, K. V. 1965. R e s u l t s o f u s i n g petroleum growthpromoting substances i n p l a n t growing. Dokl., Vses. Soveshch. Primen. N e f t . Rostovogo Veshchestva S e l . Khoz., 2nd, Baku (1963), 443 - 447; Chem. A b s t r . 67, 20846s, 1967.  1 5 0 . ) Yusufov, A.G. 1 9 6 5 . E f f e c t o f petroleum growth-promoti n g substance on r o o t f o r m a t i o n i n c u t t i n g s . Dokl., Vses. Soveshch. Primen. N e f t . Rostovogo Veshchestva S e l . Khoz., 2nd, Baku ( 1 9 ^ 3 ) , 448 - 4 5 2 s Chem. A b s t r . 6 2 , 20848u, 1 9 6 7 . 1 5 1 . ) Zamanov, P. 1 9 6 6 . I n f l u e n c e o f naphthenic growth substances upon the growth and p r o d u c t i v i t y development o f tobacco p l a n t s . P l a n t S t i m u l a t i o n s A Symposium. K. L. P o p o f f , Ed., Bulg. Acad, o f S c i e n c e s P r e s s , S o f i a , Bulg, pp. 118 119. 1 5 2 . ) Zenk, M. H. 1 9 6 1 . l - ( I n d o l e - 3 - a c e t y l ) - y 3 - D - g l u c o s e , a n e w compound i n the metabolism o f i n d o l e - 3 - a c e t i c a c i d in plants. Nature l£ls 493 W. 153. ) Zenk, M. H. 1 9 6 2 . Aufnahme und s t o f f w e c h s e l von «t-Naphthylessigsaure durch E r b s e n e p i c o t y l e . P l a n t a .58 $ 75 - 94. 154. ) Zenk, M. H. ,,1962. Z i p Frage de S t o f f w e c h s e l p r o d u k t e d e r Benzoesaure i n hoheren P f l a n z e n . P l a n t a j>8s 668 - 6 7 2 . 1 5 5 . ) Zenk, M. H. 1964. I s o l a t i o n , b i o s y n t h e t i e , and f u n c t i o n o f i n d o l e a c e t i c a c i d conjugates. R e g u l a t e u r s N a t u r e l s de l a C r o i s s a n c e Vege'tale. No. 1 2 3 , P a r i s , France, pp. 2 4 l - 249. 1 5 6 . ) Zgurovskaya, L. N. 1 9 6 9 . E f f e c t o f petroleum growth agent on the p h o t o s y n t h e t i c i n t e n s i t y and r a t e o f c e l l d i v i s i o n o f Chaetocerus c u r v i s e t u s . G i d v o b i o l . Zh. Akad. Nauk. Ukr. SSR j^s 5 5 - 5 9 ? Chem. A b s t r . 21* 48507p, 1 9 6 9 . 157. ) Zhukova, P. S. 1 9 6 5 . Use o f petroleum growth-promoting substance i n the B e l o r u s s i a n v e g e t a b l e c u l t i v a t i o n , Dokl., Vses. Soveshch, Primen. N e f t . Rostovogo Veshchestva S e l . Khoz., 2nd, Baku ( 1 9 6 3 ) , 1 1 8 - 1 2 9 : Chem. A b s t r . 67_, 20832j, 1 9 6 7 .  APPENDIX  D e t e c t i o n of amino  acids,(83).  0 . 2 $ n i n h y d r i n (1,2,3»-triketohydrindene) i n acetone. D e t e c t i o n o f mono- and d i s a c c h a r i d e s , (83). S o l u t i o n A.  0 . 5 ml a n i l i n e , 20 ml g l a c i a l a c e t i c a c i d , and 1 0 ml d i s t i l l e d water.  S o l u t i o n B.  5 g t r i c h l o r o a c e t i c a c i d i n 2 0 ml d i s t i l l e d water.  Mix s o l u t i o n s A and B a t the r a t i o o f 1 The acid-base  i n d i c a t o r , bromophenol b l u e  c  : 1.  (127).  50 mg bromophenol blue, 2 0 0 mg c i t r i c a c i d i n 1 0 0 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 s o l u t i o n u n t i l the amber p r e c i p i t a t e d i s a p p e a r s . D e t e c t i o n of p h e n o l i c compounds, d i a z o t i z e d 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 2 5 ml 2 5 $ sodium a c e t a t e . Over spray chromatogram w i t h 5 $ sodium hydroxide.  Determination o f i n o r g a n i c phosphate i n a c i d d i g e s t s o f p l a n t tissue,;. ( 5 5 ) .  Reagents ANSA  (Aminonaphtholsulfonic a c i d ) -  D i s s o l v e 1 . 2 5 g o f ANSA i n 2 2 5 ml o f d i s t i l l e d water, and then add 7 1 . 2 5 g o f sodium b i s u l f i t e . Shake f o r 1 5 minutes.  -  Pour i n 75 ml o f 10$ sodium s u l f i t e , u n t i l a l l material dissolves.  and shake  Make to $00 ml w i t h d i s t i l l e d water, and s t o r e i n the dark. Ammonium molybdate Add 5 5 * 6 ml o f concentrated s u l f u r i c a c i d to ca 200 ml o f d i s t i l l e d water. In t h i s volume d i s s o l v e 10 g o f ammonium molybdate. Make to 400 ml w i t h d i s t i l l e d water. Standard curve f o r i n o r g a n i c phosphate (see next page).  Phosphorus-32 Sampling times i n hours  Leaf blades C  c  d  activity  Stems  Roots  T  C  T  C  T  4  62,799  57,163  30,429  28,396  90,931  91,144  8  63,714  65,819  23,361  25,998  84,221  82,232  12  67,737  69,474  27,937  25,440  70,347  68,137  24  70,538  82,460  25,640  23,097  66,410  63,247  Mean  66,197  68,729  26,842  25,732  77,977  76,190  Percent of c o n t r o l  a  -  103.9  See  Table  IV.  See  Table I I I .  See  Table I I I .  b  C  n s  ns  95.9  ns  97.7  T o t a l i n o r g a n i c phosphate Sampling times i n hours  — Leaf blades C  c  d  Stems  Roots  T  C  T  C  T  2.11  0.67  0.64  1.98  1.79  2.06  2.03  2.08  2.01  2.63  2.80  2.19  2.l6  4  2.12  8  2.02  2.06  0.61  12  2.47  2.26  0.78  0.72 0.94  24  2.89  3.10  1.02  1.00  Mean  2.38  2.38  0.77  0.82  Percent of c o n t r o l  a  See Table IV.  b  See Table V I .  c  See Table I I I .  d  See Table I I I .  ns  100.0  *  ns  106.5  n S  98.6  Phosphorus-32 a c t i v i t y  Sampling times i n hours  Leaf blades C  c  b  Stems  Roots  T  C  T  C  85,744  43,359  43,384  124,376  133,503  T  4,  94,198  8  95,651  98,7^8  32,452  35,630  125,096  122,489  12  101,605  104,211  39,279  34,745  104,756  94,278  24  105,844  123,692  36,357  35,794  96,211  94,870  Mean  99,324  37,862  37.388  112,610  lll,285  Percent of c o n t r o l  a  See Table IV.  b  See Table I I I .  c  See Table I I I .  d  103,098  103.8  n s  98.7  n S  98.8  n  s  Total inorganic times i n hours  Roots  Stems  Leaf blades C  phosphorus  C  T  C  T  3..16  0.83  0.99  2.97  2.69  3.04  T  c  4  3,18  8  3.03  3.13  0.85  0.99  3.09  12  3-70  3.40  1.10  1.28  3.10  24  4.35  4.66  1.45  1.53  3.95  4.20  Mean  3.56  1.06  1.19  3.28  3.23  Percent of c o n t r o l  a  See Table IV.  b  See Table VI.  c  See Table I I I .  d  See Table I I I .  d  3  .  5 9  ns  100.8  112.3  n s  .  3.02  98.5  n S  Phosphorus-32 a c t i v i t y  Sampling times i n hours  d  C  Roots T  C  T  4  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  Mean  b  T  c  Percent of c o n t r o l  a  Stems  Leaf blades C  b  109.7 10,559  See Table IV. See Table I I I .  c  See Table I I I .  d  See Table I I I .  U,592  108.7  103.5 n s  4,894  5,066  ns  39,842  43,324 *  T o t a l i n o r g a n i c phosphate  Sampling times i n hours  Stems  Leaf blades C  c  b  Roots  T  C  T  C  T  0.17  0.09  0.08  0.13  0.14  4  0.17  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  0.20  0.10  0.10  0.16  0.l6  Percent of c o n t r o l  a  See Table IV.  b  See Table VI.  c  See Table I I I .  d  See Table I I I .  d  ns  100.0  ns  100.0  n s  100.0  Phosphorus-32 a c t i v i t y  Sampling times i n hours  Leaf blades C c  b  Roots  Stems T  C  T  C  T  14,118  5,805  5,982  51,828  49,620  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  17,511  6,825  7,079  60,596  64,304*  4  12,974  8  Percent of control  a  See Table IV.  b  See Table I I I .  c  See Table I I I .  d  See Table I I I .  d  108.6  ns  103.7  ns  106.1  Total inorganic phosphate Sampling times i n hours  Leaf blades C  Stems T  c  b  C  Roots T  c  T  0.25  0.11  0.12  0.20  0.20  0.28  0.27  0.12  0.13  0.22  0.22  12  0.31  0.32  0.14  0.15  0.26  0.27  24  0-33  0.35  0.19  0.30  0.28  Mean  0.29  0.30  0.25  0.24  4  0.25  8  Percent of control  a  See Table IV.  b  See Table VI.  c  See Table I I I .  d  See Table I I I .  d  n s  103.4  0.14  0.l4  100.0  n s  96.0  ns  Phosphorus-32 a c t i v i t y  Sampling times i n hours  Acid soluble 3 2 C .9  Acid insoluble 3 2  P  C  ..T  b  Total  p  T  C  32  p  T  4  „ d 184,158  176,702  42,282  44,588  226,440  8  171,296  174,048  50,634  50,663  221,931  224,711  12  166,020  163,051  57,457  60,970  223,471  224,021  24  162,588  168,803  70,804  83,706  233,392  252,509  Mean  171.015  l70,-651  55,294  59,-982*  226,310  230,633  Percent of c o n t r o l  a  See Table IV.  b  See Table I I I .  c  See Table I I I .  d  See Table I I I .  99.7  n s  IO8.5  values r e p r e s e n t the sum, l e a f blades + stems + r o o t s .  ::221,290  101.9  n s  T o t a l i n o r g a n i c phosphate Samolinff'" times i n hours  Acid soluble C  T  C  T  O.38  5.16  4.92  4.69  4.81  0.42  0.42  5.11  5.23  5.33  5.21  0.48  0.50  5.81  5.71  -55  6.90  O.56  0.54  7.11  7.43  0.46  0.46  5.80  5.82  12  6  5-33  Percent of c o n t r o l  d  5.36  n s  100.6  a  See  Table IV.  b  See  Table VI. values r e p r e s e n t a sum,  c  See Table I I I .  d  Total P  0.39  8  Mean  C  P  4.54  4.77  .  P T  c  4  24  Acid insoluble  b  n s  100.0  l e a f blades + stems + r o o t s .  100.3  N S  Phosphorus-32 a c t i v i t y  Sampling times i n hours  Acid soluble 3 2  c  c  Acid Insoluble 3 2  P  T  b  Total  P  C  C  32p T  262,631  70,606  69.720  332,539  332,351  253,199  256,867  74,896  75,001  328,095  331,867  12  245,640  233,234  85,303  85.390  330,942  318.623  24  238,412  254,355  103,334  125,463  341,745  379,818  Mean  249,796  251,771ns  333.330  340,665  4  261,933  8  Percent of c o n t r o l  a  d  100.8  83,534  88,893*  106.4  See Table IV. See  Table I I I .  C  See Table I I I .  d  See Table I I I .  Values r e p r e s e n t the sum, l e a f blades + stems ' +r o o t s .  102.2  ns  T o t a l i n o r g a n i c phosphate  Sampling times i n hours  Acid soluble C  c  d  Acid insoluble  P  T  C  T  C  T  6.83  O.56  0.57  7.53  7.40  7.59  7.78  8.61  8.42  P •  Total P  #  6.97  8  6.97  7.16  0.62  0.63  12  7.90  7.69  0.71  0.73  24  9-75  10.38  0.78  0.80*  Mean  7.90  0.67  0.68  Percent of c o n t r o l  See  Table  See  Table VI.  c  See  Table I I I .  d  See  Table I I I .  a  b  8.0l  n  s  #  n s  101.4  101.5  Values r e p r e s e n t the sum,  l e a f blades + stems + r o o t s .  IV.  10.57  8.58  11.18  8.70  101.4  n s  Number  Amino a c i d , amide, or sugar  0  Origin  1  Aspartic  2  Serine  3  Glutamic a c i d  acid  Glucose 5  Threonine  6  Glutamine  7  Alanine  8  )f-Aminobutyric a c i d  9  Valine  10  Isoleucine  11  Leucine  CD CO W XJ 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) c e l l u l o s e MN 3 0 0 HR t h i n - l a y e r p l a t e s .  

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0101869/manifest

Comment

Related Items