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The effect of 3-amino-1,2,4-triazole on the uptake, retention, distribution, and utilization of labelled.. LaBerge, Donald Emmanuel 1961-12-31

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THE EFFECT OF 3-AMIN0-l,2,4-TRIAZ0LE ON THE UPTAKE, RETENTION, DISTRIBUTION, AND UTILIZATION OF LABELLED PHOSPHORUS BY YOUNG- BEAN PLANTS.  by Donald E . LaBerge B.Sc.,. University  of B r i t i s h Columbia,  1959  A T h e s i s Submitted i n P a r t i a l F u l f i l m e n t the Requirements f o r the Degree of Master of Science In the Department Botany  of  of  We accept t h i s t h e s i s as conforming r e q u i r e d standard  The U n i v e r s i t y of B r i t i s h Columbia June,. 1961  to  the  In p r e s e n t i n g  t h i s thesis i n p a r t i a l f u l f i l m e n t of  the requirements f o r an advanced degree a t t h e U n i v e r s i t y British  Columbia, I agree t h a t the  a v a i l a b l e f o r reference  and  study.  of  L i b r a r y s h a l l make i t f r e e l y I f u r t h e r agree t h a t  permission  f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may g r a n t e d by  the  Head o f my  It i s understood t h a t f i n a n c i a l gain  Department o r by h i s  representatives.  copying or p u b l i c a t i o n of t h i s t h e s i s f o r  s h a l l not  be  a l l o w e d w i t h o u t my  Department The U n i v e r s i t y o f B r i t i s h Vancouver 8 , Canada. Date  be  *&k\  Columbia,  written  permission.  ABSTRACT  Bean plants were grown i n a phosphate-free nutrient solution to the early t r i f o l i a t e stage.  At this time,  they were transferred to a minus phosphate nutrient solution containing 100 p.p.m.  3~amino-l,2,4-triazole  for 48 hours, and then placed into a l a b e l l e d phosphate The plants were  nutrient solution for another hour. then returned  to a phosphate-free nutrient solution and  harvested one,  24, 48, and 96 hours a f t e r the period of  i n i t i a l phosphate uptake. AT-treatment did not a f f e c t uptake of P52 but did decrease loss of p32 to the phosphate-free nutrient solutions a f t e r i t had been absorbed by the plants. The proportion of absorbed phosphate found i n the stems and leaves of AT-treated plants was  higher than i n  these organs i n the control plants.  This phosphate  represented an increase i n both acid-30luble and acid-insoluble a c t i v i t y .  activity  The accumulation of a c i d -  soluble a c t i v i t y i n the shoots of AT-treated plants  was  an accumulation of inorganic phosphates, sugar phosphates, and nucleotides.  AT appeared to i n h i b i t downward trans-  location of acid-soluble and acid-insoluble a c t i v i t y . The incorporation of p32  into e s t e r i f i e d compounds  ( i . e . , nucleotides and sugar phosphates) was  unaffected  by AT i n d i c a t i n g that AT does not i n t e r f e r e with oxidative phosphorylation  nor with g l y c o l y s i s .  i n h i b i t transfer of P ^  2  However, AT did  from the acid-soluble f r a c t i o n  - ii to the a c i d - i n s o l u b l e effect  of AT i s  -  fraction.  to i n h i b i t the  i n t o one or more of the n u c l e i c phosphoprotein  fractions.  Therefore,  the p r i n c i p a l  i n c o r p o r a t i o n of phosphate acid, phospholipid,  or  -  iii  -  TABLE OF CONTENTS INTRODUCTION  1  EXPERIMENTAL PROCEDURES  14  The M6 l i q u i d counter  19  A n a l y s i s of r e s u l t s  20 22  RESULTS Intake, Fig.  r e t e n t i o n and d i s t r i b u t i o n of P ^ 1:  23  2  The e f f e c t of AT-treatment on the uptake and r e t e n t i o n of phosphate by whole bean p l a n t s  24  Table I : Up:take and r e t e n t i o n of P - . . ;  24  5 2  Table I I : D i s t r i b u t i o n of P?2 in leaves, stems, and r o o t s E f f e c t of AT-treatment on the d i s t r i b u t i o n of 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^2 Fig.  2 : D i s t r i b u t i o n of P ^ stems and l e a v e s  Table I I I :  2  25 26  i n roots, 27  D i s t r i b u t i o n of a c i d - s o l u b l e P32 i bean p l a n t s  29  n  Table IV: D i s t r i b u t i o n of a c i d - i n s o l u b l e P32 i bean p l a n t s  30  n  Fig.  Fig.  3s D i s t r i b u t i o n of a c i d - s o l u b l e P ^ i n l e a v e s , stems, and r o o t s of young bean p l a n t s  2  31  4 : D i s t r i b u t i o n of a c i d - i n s o l u b l e P ^ i n the l e a v e s , stems, and r o o t s of young bean p l a n t s  2  I n c o r p o r a t i o n of P32 i n t o a c i d - i n s o l u b l e fractions • Table V: D i s t r i b u t i o n of P ^ between 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 i n each organ  32 33  2  Fig.  5 : I n c o r p o r a t i o n of p32 i n t o insoluble fractions  34  acid35  - ivE f f e c t of AT-treatment on the e s t e r i f i c a t i o n of phosphate and on the d i s t r i b u t i o n of the various a c i d - s o l u b l e components  36  Table V I : E f f e c t of AT on the e s t e r i f i c a t i o n of phosphate  37  F i g . 6: D i s t r i b u t i o n of inorganic P32 w i t h i n the p l a n t  38  F i g . 7i D i s t r i b u t i o n of sugar phosphate i n young bean p l a n t s  40  F i g . 8: D i s t r i b u t i o n of nucleotide P  i n young bean plants  32 42  DISCUSSION  43  BIBLIOGRAPHY;  50  BIOGRAPHY  54  -  V  -  ACKNOWLEDGMENTS. The author wishes to express s i n c e r e Dr.  D . J . Wort, Dept.  course  of  encouragement  t h i s work and f o r h i s k i n d help  d u r i n g the p r e p a r a t i o n o f t h i s  thesis.  The author i s a l s o indebted  to Dr. P. Townsley of  the Research L a b o r a t o r y , C D . A . , and to w i t h i n the Dept.  to  of Botany, U n i v e r s i t y of B r i t i s h  Columbia, f o r h i s v a l u a b l e a d v i c e and d u r i n g the  thanks  individuals  of Botany who gave t h e i r  assistance.  These s t u d i e s were a i d e d through funds by the N a t i o n a l Research C o u n c i l of Canada.  supplied  - 1INTRODUCTION. Formerly used only i n s m a l l q u a n t i t i e s work (6), 3 - a m i n o - l , 2 , 4 — t r i a z o l e  i n photographic  underwent f i e l d t e s t s i n  1952 as a new a b s c i s s i o n - p r o m o t i n g and g r o w t h - i n h i b i t i n g chemical  (5,21).  In 1954, W.W. A l l e n ,  chief formulating  chemist of the American P a i n t Co. (now known as Amchem Products I n c . ) ,  was granted a patent f o r the use of  3-amino-l,2,4-triazole amino t r i a z o l e ,  as a h e r b i c i d e (6).  a m i t r o l , A T , or ATA, t h i s  Known a l s o as herbicide  is  formulated and s o l d by Amchem under the trade name of "Amizol."  The h e r b i c i d e i s a l s o s o l d under the  trade  names of "Weedazol" and "Amino T r i a z o l e W e e d - k i l l e r . " AT i s a h e t e r o c y c l i c ing  compound p o s s e s s i n g the  follow-  s t r u c t u r a l formula: HN  N  HC  C —NH-  I  I  2  AT has a m o l e c u l a r weight of 84.5 and a m e l t i n g p o i n t between 153 - 159°C (21). I t i s water s o l u b l e and w i l l r e a c t w i t h a c i d s and bases to form a c e t a t e ,  lactate,  h y d r o c h l o r i d e , n i t r a t e , phosphate and sodium s a l t s (29). AT can a l s o be d i a z o t l z e d i n the presence phenol to form a y e l l o w azo dye (3). appears to be s p e c i f i c  of n i t r i t e and  Because t h i s  reaction  for AT, i t affords a u s e f u l  technique f o r i d e n t i f y i n g amino t r i a z o l e on paper chromatograms.  Other chromatographic techniques  developed f o r d e t e c t i n g AT (31,33,40).  have a l s o been  Amino t r i a z o l e  - 2 forms s t a b l e complexes w i t h s e v e r a l m e t a l s , iron,  nickel,  cobalt,  copper and magnesium  importance of amino t r i a z o l e - m e t a l w i l l be d i s c u s s e d  including (40).  The  complex f o r m a t i o n  later.  Amino t r i a z o l e  came to a p o s i t i o n of n a t i o n a l  prominence i n the United States d u r i n g the Thanksgiving h o l i d a y of 1959 i n what has become known as the Crisis." harvest  "Cranberry  Amchem recommended amino t r i a z o l e as a p o s t spray f o r the c o n t r o l of r e d r o o t  tlnctoria)  (Lachnanthss  i n c r a n b e r r i e s , and In A p r i l of 1958 a p p l i e d  to the F e d e r a l Department of A g r i c u l t u r e (FDA) f o r a one p . p . m . t o l e r a n c e investigating  f o r AT on c r a n b e r r i e s  toxicity  data,  of 1959 t h a t a t o l e r a n c e be e s t a b l i s h e d .  After  (17).  FDA n o t i f i e d Amchem i n May  f o r amino t r i a z o l e  c o u l d not  The compound caused t h y r o i d adenomas  i n r a t s a t a l e v e l a;s low as 10 p . p . m . i n the Amchem t h e r e f o r e withdrew i t s  diet.  petition.  On Nov. 9, 1959, H e a l t h , E d u c a t i o n and Welfare S e c r e t a r y A r t h u r S. Fleming urged that there be no f u r t h e r s a l e s of c r a n b e r r i e s or cranberry products produced i n Washington and Oregon because of contamination w i t h amino t r i a z o l e .  possible  He s a i d that  this  h e r b i c i d e caused cancer i n the t h y r o i d of r a t s when was contained i n t h e i r d i e t s .  it  He was a c t i n g i n con-  j u n c t i o n w i t h the Delaney c l a u s e of the Food A d d i t i v e Amendment (24)  which p r o h i b i t s the a d d i t i o n to foods of  any substance that has been shown to produce cancer  in  l a b o r a t o r y animals when fed at any dosage.  all  Because  - 3 c r a n b e r r i e s became suspect i n the eyes of the p u b l i c , c r a n b e r r i e s from W i s c o n s i n , New J e r s e y ,  and Massachusetts  were i m p l i c a t e d and the bottom f e l l from the cranberry market.  In order to c l e a r s u f f i c i e n t  cranberries for  the  Thanksgiving t r a d e , a c r a s h program i n v o l v i n g 100 FDA inspectors  and 60 chemists was e s t a b l i s h e d  cranberry c r o p .  The cranberry c r i s i s  to analyze  cost the U . S .  the  Federal  Government $>10 m i l l i o n i n Indemnity to c r a n b e r r y growers (25) and i n a d d i t i o n ,  the government bought the unsold  c r a n b e r r i e s f o r government  use.  The wave of controversy which r e s u l t e d from c r i s i s has done much to s t i m u l a t e effects  interest  this  i n the  metabolic  of AT-treatment b o t h i n animal and i n p l a n t  Numerous a r t i c l e s  have appeared r e c e n t l y  of p l a n t metabolism as a f f e c t e d  on s e v e r a l  text.  have i n d i c a t e d t h a t many p l a n t s  r e a d i l y absorb amino t r i a z o l e through the r o o t s the a e r i a l p a r t s a r y l sulfonate  (7,26,30).  type  (e.g.,  oxyethelene g l y c o l type were b o t h e f f e c t i v e through the l e a v e s  aspects  by amino t r i a z o l e and these  w i l l be d i s c u s s e d b r i e f l y i n the f o l l o w i n g E x t e n s i v e experiments  tissues.  or through  Surface agents of the X - 7 7 ) and the a l k y l a r y l  (e.g.,  Multifilm  alkylpoly-  C and M u l t i f i l m L)  i n i n c r e a s i n g the amount of AT absorbed (19).  G i r d l i n g experiments were used to determine how AT i s translocated  (21).  When AT i s a p p l i e d to the s o i l or  taken up from a n u t r i e n t s o l u t i o n , upward i n the xylem.  This i s  it  is  is  translocated  i n d i c a t e d by the  chlorosis  which occurs i n the growing r e g i o n s above the b a r k -  - 4 g i r d l e d regions applications is  -  of c o t t o n p l a n t s .  o f amino t r i a z o l e  However,  indicate  that  the phloem  i n v o l v e d i n t r a n s l o c a t i o n when amino t r i a z o l e  absorbed through the l e a v e s .  i s not t r a n s l o c a t e d past  is  When c o t t o n p l a n t s  b a r k - g i r d l e d half-way up the main stem,  is  foliar  are  the amino  the g i r d l e whether  the  triazole  herbicide  a p p l i e d to the l e a v e s above the g i r d l e d bark or to  l e a v e s below the g i r d l e d bark.  the  Thus, p l a n t s appear to  translocate  AT i n a manner s i m i l a r to that used  translocate  other  to  herbicides.  Further d e t a i l s  r e g a r d i n g the t r a n s l o c a t i o n and  a c c u m u l a t i o n of AT by p l a n t s have r e s u l t e d from the use  of  amino t r i a z o l e - 5 - C ^ and from radioautography  (7,16,33,35).  When l a b e l l e d AT was a p p l i e d to the cotyledons  of  1  plants,  i t was absorbed and t r a n s l o c a t e d  w i t h i n two hours  (16).  to the  A n a l y s i s of the c u l t u r e  cotton  roots solution  showed no i n d i c a t i o n of leakage from the r o o t s .  Amino  t r i a z o l e moved I n the d i r e c t i o n of food t r a n s p o r t and a t the  same r a t e . The  ability  efficiency to p e n e t r a t e  of AT as a h e r b i c i d e depends upon and to t r a n s l o c a t e  r e a d i l y to  p a r t s of a p l a n t w h i l e r e t a i n i n g s u f f i c i e n t kill  the v i t a l ' o r g a n s .  accumulate i n the meristems, etc.  It  is  scarce  all  toxicity  to  Radioautograms i n d i c a t e t h a t AT  (or some e f f e c t i v e m e t a b o l i t e )  tips,  its  definitely  tends  to  such as a c t i v e buds,  root  or l a c k i n g i n dormant buds,  storage parenchyma and mature t i s s u e s i n g e n e r a l T h i s a c c u m u l a t i o n of AT i n young growing t i s s u e  (7 ). 1  coincides  -  5  -  w i t h t h e s i t e s where AT i s known t o a t t a i n i t s maximum toxic  effect. Further  e x p e r i m e n t s have i n d i c a t e d t h a t movement o f  AT t h r o u g h t h e p h l o e m i s d e p e n d e n t u p o n p r o d u c t i o n o f photosynthate.  Starch-depleted  n u t g r a s s p l a n t s were (7).  g i v e n one d r o p o f r a d i o a c t i v e AT o n one l e a f  t h i s l e a f was e x p o s e d t o s u n l i g h t , AT c o u l d move from t h i s  l e a f t o a l l p a r t s of t h e p l a n t s .  When readily  Hov/ever, i f t h e  l e a f was k e p t i n d a r k n e s s w h i l e t h e r e m a i n d e r o f t h e p l a n t was  exposed t o l i g h t ,  t h e AT r e m a i n e d i n t h e d a r k e n e d  This I n d i c a t e d that photosynthesis  leaf.  was e s s e n t i a l t o t h e  t r a n s l o c a t i o n o f AT by n u t g r a s s . Besides  a f f e c t i n g g r o w t h , AT i s known t o c a u s e c h l o r o s i s  i n many p l a n t s a n d i n many c a s e s , by  the. d e a t h o f t h e p l a n t .  content  chlorosis i s followed  Measurements of c h l o r o p h y l l  i n cotton regrowth-leaves  h a v e shown t h a t AT  reduces the concentrations  o f b o t h c h l o r o p h y l l a and b  (28).  i n c h l o r o p h y l l content  S i m i l a r depressions  have  been measured i n beans ( 3 0 ) , tomatoes (41), b a r l e y and potatoes  (32).  xanthophyll  AT-treatment a l s o decreases carotene and  content  of various plants  (1,28).  The  overall effect  o f AT i s a t w o - f o l d i n c r e a s e i n t h e a n t h o -  cyanin  of cotton leaves  content  of t h e c o m p o s i t i o n AT  (28).  of the anthocyanins  Spectral analysis i s u n a l t e r e d by  (28). I n t h e i r e a r l y work, M i l l e r e t a l (21) found  that  t i s s u e s formed a t t h e time o f , o r subsequent t o the a b s o r p t i o n o f amino t r i a z o l e were c h a r a c t e r i z e d by c h l o r o s i s .  - 6 T h i s suggested that c h l o r o p h y l l s y n t h e s i s was  affected-  F u r t h e r work i n d i c a t e d that AT d i d not e n t i r e l y  inhibit  the change from p r o t o c h l o r o p h y l l t o c h l o r o p h y l l but AT had i t s  that  p r i n c i p l e e f f e c t d u r i n g the b i o g e n e s i s of  chlorophyll.  Laboratory s t u d i e s have shown that AT  possesses the a b i l i t y to form s t a b l e complexes w i t h i r o n copper, and magnesium  (40).  Though M i l l e r et a l  (21)  c o u l d not demonstrate  that the r e s t r i c t i o n of c h l o r o p h y l l  s y n t h e s i s was due to an I m m o b i l i z a t i o n of Mg, Fe, Mn, N, P or K, i t has been shown r e c e n t l y p a r t i a l l y reverse  that f e r r o u s i o n can  the i n h i b i t i o n of c h l o r o p h y l l s y n t h e s i s  as w e l l as completely  reverse  the i n h i b i t i o n of  carotenoid  s y n t h e s i s and m u l t i p l i c a t i o n of s e v e r a l microorganisms  (1).  This suggests that AT a c t s as a c h e l a t i n g agent. There i s f u r t h e r evidence which suggests that amino t r i a z o l e chelates metals.  AT i n h i b i t e d the a c t i v i t y  phosphorylase p r e p a r a t i o n from the b l u e - g r e e n O s c i l l a t o r i a princeps be the r e s u l t  (20).  ferric  alga,  T h i s i n h i b i t i o n appeared  of the c h e l a t i o n by AT of the  metal r e q u i r e d by t h i s enzyme s i n c e effectively  of a  to  essential  i n h i b i t i o n could be  r e v e r s e d by the a d d i t i o n of manganese  or  ions.  I t has been suggested  (1,21,40) that the  similarity  of s t r u c t u r e of the t r i a z o l e r i n g to the p y r r o l e r i n g s of c h l o r o p h y l l together w i t h the a b i l i t y of AT to form s t a b l e complexes w i t h metals was such that amino t r i a z o l e might substitute  f o r at l e a s t one p y r r o l e r i n g thus b l o c k i n g  c h l o r o p h y l l s y n t h e s i s p r i o r to the p r o t o c h l o r o p h y l l stage.  HN  HG  N  PH  I I  C  C  H  amino t r i a z o l e Hov/ever, M i l l e r and H a l l  pyrrole (28)  have almost  completely  excluded the l i k e l i h o o d that AT e n t e r s i n t o the of p s e u d o c h l o r o p h y l l o u s p o r p h y r i n s . they recovered only extremely pigment  synthesis  Using r a d i o a c t i v e A T ,  low C ^ a c t i v i t y from t h e i r  fractions.  G y t o l o g i c a l data has i n d i c a t e d that c h l o r o s i s to a l a c k of c h l o r o p l a s t s , c h l o r o p h y l l per se  (34).  r a t h e r than some e f f e c t  is  due  on '  M i c r o s c o p i c examination of  the  c h l o r o t i c t i s s u e s of s e v e r a l p l a n t s has i n d i c a t e d that plastids  are few i n number, shrunken and misshapen.  and Loughman (44)  the  Wort  have shown that an a l t e r e d p h o s p h o l i p i d  metabolism may be r e s p o n s i b l e p l a s t i d development.  f o r the i n h i b i t i o n by AT of  Sund (41)  found that AT blocked the  s y n t h e s i s of r i b o f l a v i n , and the need f o r c e r t a i n f l a v i n coenzymes might account f o r a decrease i n p l a s t i d development.  Both groups of i n v e s t i g a t o r s  also  presented  evidence which i n d i c a t e d t h a t AT i n h i b i t e d p r o t e i n synthesis. It  is  Interesting  to note the e f f e c t  iron-*porphyrin enzymes s i n c e c o n t a i n i n g compounds. first  investigators  activity.  of AT on v a r i o u s  these enzymes a r e a l s o p y r r o l e  Pyfrom et a l  (32)  to study the e f f e c t s  were among the of AT on c a t a l a s e  Using b a r l e y and potato p l a n t s w i t h low c o n -  - 8c e n t r a t i o n s of A T , they showed that c a t a l a s e a c t i v i t y depressed, whenever AT i s present i n the t i s s u e s .  It  been shown r e c e n t l y that AT i n h i b i t s the s y n t h e s i s tryptophane p e r o x i d a s e - o x i d a s e , found i n the l i v e r of r a t s  (8).  has  of  an i r o n - p o r p h y r i n enzyme Whereas the cytochrome  oxidase enzyme i n c h l o r o t i c . corn t i s s u e was not by A T - t r e a t m e n t ( 2 7 ) ,  is  affected  the cytochrome oxidase a c t i v i t y of  e t i o l a t e d wheat s e e d l i n g s  is  i n c r e a s e d by AT-treatment  (43).  There i s no i n f o r m a t i o n a v a i l a b l e to i n d i c a t e whether or not AT i n t e r f e r e s w i t h other cytochrome•enzymes which commonly occur i n p l a n t s . I t has been suggested in vivo is  (l)  that the main a c t i o n of AT  to i n t e r f e r e w i t h the s y n t h e s i s  of  porphyrin-  c o n t a i n i n g enzymes, and only when AT i s present i n h i g h c o n c e n t r a t i o n s does i t  i n h i b i t e x i s t i n g enzymes.  a c t s p r i m a r i l y as an i r o n c h e l a t o r ,  it  I f AT  i s more l i k e l y t o  prevent s y n t h e s i s by making i r o n u n a v a i l a b l e r a t h e r than by making i t i n e f f e c t i v e  when i t i s a l r e a d y p a r t of  the  enzyme m o l e c u l e . I n h i b i t i o n of c a t a l a s e a c t i v i t y r e s u l t s i n an i n c r e a s e of H2O2 i n p l a n t t i s s u e s .  Racusen ( 3 3 )  has proposed that  an i n c r e a s e i n HgOg would lower the amount of IAA by a c t i o n of i n d o l e a c e t i c a c i d oxidase.  T h i s would r e s u l t  i n an i n d i r e c t A T - i n d u c e d growth i n h i b i t i o n . (36)  Russell  has found that AT does i n c r e a s e the peroxide l e v e l  of i n d i v i d u a l r o o t c e l l s .  He r e c o g n i z e d the  significance  of an i n c r e a s e d peroxide l e v e l w i t h r e s p e c t to IAA r e d u c t i o n sequence.  the  He t h e r e f o r e attempted to  e s t a b l i s h whether or not IAA would r e v e r s e AT-growth inhibition.  H i s experiments  such i n t e r a c t i o n .  However,  that AT does antagonize  i n d i c a t e d that there v/as no other workers  (21)  the growth e f f e c t s  l e n d i n g support to Racusen's  have shown  of IAA thus  hypothesis.  Reduction of growth i n h i b i t i o n has a l s o been by adding p u r i n e s and p y r i m i d i n e s to p l a n t s w i t h AT ( 4 , 4 1 , 4 2 ) . for  simultaneously  Because these compounds are  the p r o d u c t i o n of n u c l e i c a c i d s ,  obtained  necessary  i t - h a s been proposed  that AT i n t e r r u p t s p r o t e i n s y n t h e s i s since n u c l e i c a c i d i s necessary  f o r p r o t e i n p r o d u c t i o n (41,44).  Many workers have noted that AT s t i m u l a t e s (21,27,29,36). applications  Wort and Shrimpton (43)  found t h a t  foliar  of 4000 p . p . m . AT r e s u l t e d i n an immediate  and continued i n c r e a s e bean p l a n t s .  respiration  i n r e s p i r a t i o n of b o t h wheat and  A l l concentrations  of AT up to 840 m g / l J  i n c r e a s e d the r e s p i r a t o r y r a t e of c o t t o n l e a f d i s c s  (21,29).  A p p l i c a t i o n of AT to homogenates of wheat s e e d l i n g s grown i n the dark f o r 6 days a l s o i n c r e a s e d the o x i d a t i o n of reduced cytochrome c (43)  i n d i c a t i n g that  cytochrome  oxidase a c t i v i t y v/as i n c r e a s e d by A T - t r e a t m e n t . Thus,  i t appears that there may be s e v e r a l l o c i  which AT induces growth i n h i b i t i o n .  Briefly,  seem to i n c l u d e such t h i n g s as i n a c t i v a t i o n of enzyme systems through c h e l a t i o n of e s s e n t i a l  these would certain metal,  o x i d a t i o n of IAA by H 0 2 , decreased photosynthesis 2  at  (43)  and i n c r e a s e d r e s p i r a t i o n which lower the amount of a v a i l a b l e sugars,  and f i n a l l y ,  a decreased p h o s p h o l i p i d and  -  10 -  nucleic a c i d production i n AT-treated plants. Many p l a n t s can metabolize A T .  The use of paper  chromatography f o r the d e t e c t i o n of AT i n p l a n t  extracts  has g i v e n an i n s i g h t i n t o t h i s problem of AT metabolism. Aldrich  (4)  removed AT from p l a n t t i s s u e by g r i n d i n g  t i s s u e s i n Q0% e t h a n o l .  The e l u a t e was c h r o m a t o g r a p h i c a l l y  separated on Whatman No. 1 paper u s i n g 1-butanol  (1:1:4) as the s o l v e n t .  f i r s t w i t h 5% KN0  2  The paper was sprayed  technique,  appeared on the chromatograms.  Is,  ethanol:water:  and then w i t h phenol i n 20% H C l .  the experiments u s i n g t h i s  that the  the  two y e l l o w  In  spots  The assumption was made  spot w i t h the lower Rf v a l u e was "bound" A T , t h a t  AT bound to p r o t e i n .  The other spot was designated  as  "free" AT and appeared a t a h i g h e r Rf v a l u e . While s t u d y i n g the t r a n s l o c a t i o n and f a t e Canada t h i s t l e ,  Johnson grass and soybeans,  of AT i n  Rogers  was a b l e to demonstrate that even though soybeans very s u s c e p t i b l e all  In the case of soybeans and  he demonstrated the presence of an unknown  e n t i t y having a s m a l l Rf v a l u e .  Chromatograms prepared  from Johnson g r a s s , which was l e a s t s u s c e p t i b l e treatment,  were  to A T , the p l a n t s were able to m e t a b o l i z e  of the AT s u p p l i e d .  Canada t h i s t l e ,  (35)  d i d not have t h i s  entity,  to_ A T -  a f a c t which he  felt  might be of importance f o r e x p l a i n i n g t o l e r a n c e of p l a n t s to A T . Herrett  (22)  has found that the r a t e of AT-metabolism  w i t h i n bindweed i s more r a p i d than i n t h i s t l e . two m e t a b o l i t e s  He I s o l a t e d  which he has d e s i g n a t e d as Unknown I and  -  Unknown I I .  11 -  I n Canada t h i s t l e ,  he noted that there was a  l a g i n the t r a n s p o r t of AT from the t r e a t e d l e a f stem.  to  the  The f o r m a t i o n of a t r a n s p o r t a b l e form of AT v/as  p o s t u l a t e d as the l i m i t i n g r e a c t i o n f o r i t s through the phloem from the l e a f remainder of the p l a n t .  distribution  of a p p l i c a t i o n to  the  H e r r e t t showed that movement of  AT through the xylem d i d not r e q u i r e s y n t h e s i s of a t r a n s p o r t a b l e form of AT and he supported the claim the xylem i s not the major pathway of AT movement leaves.  The d i f f e r e n c e  and t h i s t l e  in sensitivity  that  out.of  to AT by bindweed  was concluded to be a r e s u l t  of major d i f f e r -  ences i n a b s o r p t i o n and metabolism of the h e r b i c i d e . In 1 9 5 7 j  Racusen ( 3 3 ) undertook some experiments  determine whether AT i t s e l f  or some product of AT-metabolism  was the a c t u a l m a t e r i a l which was t o x i c activities.  He demonstrated  to  to c e r t a i n p l a n t  that amino t r i a z o l e was t r a n s -  formed i n t o two new products i n the leaves of young bean plants.  He designated  .and Compound Y .  these A T - m e t a b o l i t e s as Compound X  Because i t v/as not c e r t a i n t h a t X and Y  were a c t u a l l y t r a n s f o r m a t i o n products of A T , proof of o r i g i n v/as e s t a b l i s h e d  their  u s i n g C ^"-labelled A T . 1  Compound X v/as the major product of AT metabolism.  It  v/as formed a t a uniform r a t e i n young bean leaves f o r a t l e a s t 4 days.  A f t e r 5 days,  corporated i n t o Compound X.  9 3 $ of the AT had been i n Racusen e l u t e d Compound X from  h i s chromatograms and determined i t s  toxicity  u s i n g Lemna  minor.  L i k e A T , Compound X produced c h l o r o s i s and s t u n t i n g  but i t s  t o x i c i t y v/as lov/er than that of AT a l o n e .  Racusen  - 12 also isolated  Compound Y which he found to be n o n - t o x i c  Lemna c u l t u r e s .  to  He could not e s t a b l i s h whether or not X  and Y were merely d e t o x i f i c a t i o n  by-products of AT metabolism  as suggested by t h e i r lower t o x i c i t y , formed as a d i r e c t consequence  or whether they were  of some t o x i c r e a c t i o n of A T .  However, both compounds were c h a r a c t e r i z e d by the same r i n g system and f r e e amino group of A T .  Because h e a l t h y ,  turgid  bean l e a v e s were r e q u i r e d f o r t h e i r f o r m a t i o n , Racusen has suggested that the t r a n s f o r m a t i o n of AT i n t o X and Y i s probably c a r r i e d out by some enzyme system i n the  leaf.  Another approach to the problem of AT metabolism been i n v e s t i g a t e d  (12,15).  Guided by the h y p o t h e s i s  has that  AT i n t e r f e r e s w i t h the p r o d u c t i o n of p o r p h y r i n s which are essential  f o r c h l o r o p h y l l p r o d u c t i o n , C a r t e r and Naylor (15)  s t u d i e d the metabolism of c e r t a i n p o r p h y r i n p r e c u r s o r s i n AT-treated plants. exposed  When e x c i s e d  tips  of bean p l a n t s  to g l y c i n e - 1 , 2 - C ^ i n a s o l u t i o n of A T , l a r g e  quantities  1  of r a d i o a c t i v i t y appeared i n an unknown, n i n -  h y d r i n s e n s i t i v e compound which was not present controls.  The unknown was designated  Glycine-l-C *', 12  i n the  compound "1".  glycine-2-C ", s e r i n e - 1 , 2 , 3 - 0 ^ reacted li{  s i m i l a r i l y whereas g l u c o s e ,  1  s u c c i n a t e and bicarbonate  l a b e l l e d w i t h G ^ were i n e f f e c t i v e . 1  used,  were  When A T - 5 - C  1 2 f  was  compound "1_" became h e a v i l y l a b e l l e d i n d i c a t i n g that  a complex between AT and s e r i n e  or g l y c i n e was formed by  bean p l a n t s . Massinl  (26)  has r e c e n t l y  i s o l a t e d and p u r i f i e d an  AT m e t a b o l i t e which he c a l l s ATX.  ATX i s a white  powder  - 13 -  composed of f i n e n e e d l e - l i k e c r y s t a l s .  I t has a m e l t i n g  p o i n t of 230-232°C. as compared to AT a t approximately 155°C  This compound has been i d e n t i f i e d as 3-amino-  1 , 2 , 4 - t r i a z o l y l alanine.  I n d i c a t i o n s at present are that  t h i s compound i s not the same as compound "1" described by Carter and Naylor nor i s l t s i m i l a r t o Compound X described by Racusen. be i d e n t i c a l  Rather, these l a t t e r compounds are thought to (13,14).  Recent studies by Carter and Naylor (13) have shown that compound "1" ( i . e . Compound X) i s the p r i n c i p a l metabolic product formed i n beans, a l f a l f a , s i l v e r maple, and honeysuckle when treated v/ith AT.  However, they a l s o 14  demonstrated that nine other compounds derived C  from AT  thus demonstrating the complexity of AT metabolism i n plants. The present study i s an attempt t o d i s c o v e r the e f f e c t of AT on s e v e r a l aspects of phosphate metabolism i n young bean p l a n t s , the roots of which were f i r s t exposed to 100 p.p.m. AT f o r 48 hours followed by a one hour exposure to r a d i o a c t i v e phosphorus.  The i n f l u e n c e of AT on the uptake  and r e t e n t i o n of r a d i o a c t i v i t y and on the d i s t r i b u t i o n of a c t i v i t y w i t h i n the plant was determined.  Attempts were  a l s o made to study the manner i n which AT a f f e c t e d the i n c o r p o r a t i o n of inorganic phosphate i n t o organic compounds. A comprehensive study of the e f f e c t of AT on the phosphate metabolism of barley has a l r e a d y been made (44) and t h i s work w i l l be discussed l a t e r .  - 14 EXPERIMENTAL. Bean seeds (Phaseolus v u l g a r i s ,  i960)  Top Crop,  obtained from B u c k e r f i e l d Seed Co. were sown i n v e r m i c u l i t e s a t u r a t e d w i t h phosphate-free were grown i n a constant of 7 2 ° F . ,  a relative  light daily.  The p l a n t s  environment chamber at a temperature  humidity of 62%, and a l i g h t  of approximately 2500 f . c . of  nutrient solution.  intensity  The p l a n t s were g i v e n 14 hours  A f t e r s i x days, uniform s e e d l i n g s were  t r a n s p l a n t e d to b o t t l e s c o n t a i n i n g f r e s h nutrient solution.  These s o l u t i o n s  phosphate-free  were g i v e n  continuous  aeration. E l e v e n days a f t e r g e r m i n a t i o n , when the p l a n t s had grown to the e a r l y t h r e e - l e a f  stage,  p l a n t s were a g a i n  s e l e c t e d f o r u n i f o r m i t y so that only e i g h t b o t t l e s , containing four plants,  remained.  to seven hours of l i g h t ) ,  A t midday  the p l a n t s  each  (corresponding  i n f o u r b o t t l e s were  q u i c k l y t r a n s f e r r e d to b o t t l e s c o n t a i n i n g 100 p . p . m . AT i n f r e s h phosphate-free  nutrient solution.  The c o n t r o l  p l a n t s were' t r a n s f e r r e d to f r e s h phosphate-free solutions.  These s o l u t i o n s  were c o n t i n u o u s l y  A t midday, 48 hours l a t e r ,  the r o o t s  nutrient  aerated.  of a l l the  plants  were q u i c k l y r i n s e d w i t h d i s t i l l e d water and then immersed i n a f r e s h c u l t u r e s o l u t i o n to which had been added NaHgP^O^.. 3000 mis.  T h i s s o l u t i o n had an a c t i v i t y of 80 uC P of c u l t u r e s o l u t i o n .  to P ^ , the r o o t s 2  5 2  per  A f t e r a one hour exposure  of a l l the p l a n t s were q u i c k l y r i n s e d i n  d i s t i l l e d water and the p l a n t s were r e t u r n e d to b o t t l e s  - 15 c o n t a i n i n g f r e s h phosphate-free  nutrient solution.  A T - t r e a t e d and 4 c o n t r o l p l a n t s were removed one, and  96 hours a f t e r  the s t a r t  of P ^  2  Four 24,  absorption for  48,  the  e x t r a c t i o n of phosphorus compounds. EXTRACTION OF LABELLED COMPOUNDS. L a b e l l e d compounds were e x t r a c t e d from p l a n t by  the procedures of Wort and Loughman (44).  tissues  The two  groups of f o u r p l a n t s from the A T - t r e a t e d and c o n t r o l groups were q u i c k l y d i v i d e d i n t o r o o t s ,  stems and l e a v e s .  Each p l a n t p a r t was cut i n t o s m a l l e r p i e c e s and immersed in  12 m i s .  of i c e - c o l d 16% t r i c h l o r a c e t i c a c i d (TCA)  contained i n c h i l l e d m o r t a r s .  The c o l d TCA caused an  immediate c e s s a t i o n of m e t a b o l i c a c t i v i t y . for  approximately f i v e minutes,  quantitatively  the m a t e r i a l was t r a n s f e r r e d  to 25 m l . screw cap v i a l s u s i n g a minimum  amount of i c e - c o l d wash water. rifuged for  10  minutes a t  3000  The v i a l s were then r.p.m.  was decanted i n t o another v i a l . extracted with f i v e mis.  and the  cent-  supernate  The r e s i d u e was r e -  of i c e - c o l d 8% TCA and c e n t r i f u g e d  in a refrigerated centrifuge  for  10  minutes a t  T h i s supernate was poured i n t o the f i r s t and  After grinding  3750  supernate  r.p.m. solution  the combined supernate was made to 25 m i s . w i t h i c e - c o l d  water.  This s o l u t i o n comprised the a c i d - s o l u b l e  compounds  (i.e.,  i n o r g a n i c phosphates,  sugar phosphates). count,  phosphate  nucleotides,  To determine the t o t a l  and  acid-soluble  a two m l . a l i q u o t was removed and made to 10 m i s .  a volumetric.  T h i s s o l u t i o n was counted i n a  20th  in  Century  - 16 E l e c t r o n i c s M6 l i q u i d counter (see below). changes,  the remainder of  the a c i d - s o l u b l e  To reduce phosphate  s o l u t i o n was f r o z e n s o l i d l y . The screw cap v i a l  c o n t a i n i n g the p l a n t r e s i d u e was  i n v e r t e d on a paper towel to d r y .  This r e s i d u e ,  of a c i d - i n s o l u b l e phosphate compounds ( i . e . ,  comprised  phospholipids,  n u c l e i c a c i d s and phosphoproteins) was t r a n s f e r r e d to I 5 0 ml.  Kjeldahl flasks  and d i g e s t e d u s i n g f i v e m i s . of c o n -  c e n t r a t e d n i t r i c a c i d and f i v e m i s .  of 60% _perchloric  The l i q u i d was reduced to f i v e m i s . w i t h h e a t i n g . r e q u i r e d from two to three h o u r s .  The cooled  acid.  This  solutions  were then made to 10 m i s . w i t h water and counted i n the M6 l i q u i d c o u n t e r .  T h i s count r e p r e s e n t e d the t o t a l  acid-  i n s o l u b l e phosphate compounds. SEPARATION Q | ACID-SOLUBLE PHOSPHATE COMPOUNDS. The a c i d - s o l u b l e s o l u t i o n s , phosphates, to thaw.  containing inorganic  sugar phosphates and n u c l e o t i d e s ,  were allowed  The s o l u t i o n s were c e n t r i f u g e d a t 3750 r . p . m .  in  order to remove p o l y s a c c h a r i d e m a t e r i a l formed d u r i n g freezing.  A 1 0 - m l . a l i q u o t was taken from each s o l u t i o n  and p l a c e d . i n a continuous l i q u i d - l i q u i d e x t r a c t i o n  system.  A three to f o u r hour e x t r a c t i o n w i t h e t h y l ether was needed to remove the TCA. A f t e r e x t r a c t i o n ,  the s o l u t i o n s  were  poured i n t o c h i l l e d v i a l s and i f any ether l a y e r remained on these s o l u t i o n s , If any  i t was removed w i t h a c a p i l l a r y p i p e t t e .  stored overnight, chemical changes.  these s o l u t i o n s were f r o z e n to reduce When thawed,  the s o l u t i o n s  were  - 17 again centrifuged  at 3750 r . p . m .  to remove any p o l y s a c c h a r i d e  material. T v / o - m i l l i l i t e r samples of these s o l u t i o n s  were reduced  to approximately o n e - h a l f m l . u s i n g an i n f r a r e d lamp and a c u r r e n t of warm a i r from a h a i r d r i e r . solutions  were spotted  The reduced  on acid-washed 3 MM Whatman papers.  (Sheets of 3 MM Whatman paper were placed i n a t r a y ,  covered  w i t h 2N a c e t i c a c i d , and allowed to stand o v e r n i g h t . a c i d was removed next morning and t h i s wash was several  times.  The  repeated  When the a c i d s o l u t i o n no longer gave a  p r e c i p i t a t e when ammonium hydroxide and ammonium o x a l a t e were added, water.  the papers were thoroughly r i n s e d w i t h d i s t i l l e d  T h i s washing removed metal i m p u r i t i e s , p a r t i c u l a r l y  c a l c i u m , which i n t e r f e r e w i t h the chromatography of phosphate  compounds.)  certain  The chromatograms were e q u i l i b r a t e d  i n the chromatography chambers i n an atmosphere  of HgS.  T h i s converted any metals which remained on the papers sulfides  and these s u l f i d e s  remained a t the o r i g i n .  gas was generated by dropping c r y s t a l s i n t o 6N H C l .  of sodium  A f t e r one hour e q u i l i b r a t i o n , the  v/as added to the t r a y s . v/ater:picric acid  The s o l v e n t used v/as  (80 mls:20 mls:2.0 gms).  to  This  sulfide solvent  tert-butanol:  The s o l v e n t v/as  allowed to run f o r 20 hours. Ih order to f i n d the areas  of a c t i v i t y  atogram, autoradiograms were p r e p a r e d .  on each chrom-  Each chromatogram  v/as p l a c e d on a p i e c e of plywood covered w i t h f i l t e r  paper.  The chromatogram was covered v/ith a sheet of Saran Wrap and i n the darkroom, X - r a y f i l m ,  Du Pont M e d i c a l Type 508, v/as  - 18 p l a c e d over the chromatogram. a second p i e c e  A piece  of b l a c k paper and  of plywood were p l a c e d over the X - r a y  film.  The two p i e c e s of plywood, together w i t h t h e i r chromatograms, X - r a y f i l m , b l a c k paper.  etc.,  were wrapped w i t h l i g h t - p r o o f  These packages were stored i n a press  48 to 72 hours depending on the r e l a t i v e a c t i v i t y spots.  for  of  the  Before the f i l m s were removed f o r development,' a  p i n was t h r u s t through each f i l m and chromatogram i n s e v e r a l spots to i n s u r e the c o r r e c t replacement of  the  film. The chromatograms were cut i n t o segments c o n t a i n i n g i n o r g a n i c phosphates,  nucleotides  or sugar  phosphates.  Each segment was wet ashed i n a 150 m l . K j e l d a h l u s i n g two m i s . acid.  of n i t r i c a c i d and 2.2 m i s .  flask  of p e r c h l o r i c  The d i g e s t s were made to 10 mis. and counted i n the  M6 l i q u i d counter.  T h i s count represented the  a c t i v i t y of the a c i d - s o l u b l e f r a c t i o n s .  These  relative fractions  were not f u r t h e r c h a r a c t e r i z e d . RETENTION OF P ^ . 2  To determine the amount of P-^ which had been 2  from the p l a n t s to the n u t r i e n t s o l u t i o n s , which remained a f t e r  the 24,  48,  the  a l i q u o t of each of these s o l u t i o n s counter.  solutions  and 96 hour harvests  each made to one l i t e r i n a v o l u m e t e r i c f l a s k .  liquid  lost  were  A 10-ml.  was counted i n the M6  -  19 -  THE M6 LIQUID COUNTER. The r a d i o a c t i v e s o l u t i o n s were counted i n a 20th Century E l e c t r o n i c s M6 l i q u i d c o u n t e r . and uses of t h i s and M a r t i n  The c o n s t r u c t i o n  counter a r e f u l l y d e s c r i b e d by R u s s e l l  (37,38).  The counter tube was supported i n a  l e a d c a s t l e i n order to s h i e l d the tube from a l l but the: most p e n e t r a t i n g cosmic r a y s thereby r e d u c i n g the b a c k ground count to below 16 c . p . m . number of emissions  In order to r e c o r d the  p a s s i n g from the r a d i o a c t i v e  and through the counter, Nuclear-Chicago s c a l e r  solution  the tube was connected to a  (Model 1 5 1 A ) equipped with, a Model  T l timer. The 20th Century E l e c t r o n i c s M6 l i q u i d counter i s  a  t h i n - w a l l e d G e i g e r - M u l l e r counter surrounded by a g l a s s j a c k e t i n t o which a sample of l i q u i d 10 m i s . be p l a c e d .  i n volume can  The counter i s designed to r e c o r d up to 10% "52  of the emissions  from a l i q u i d sample of Br .  of counter has two d i s t i n c t advantages  This  over c o n v e n t i o n a l  end window counters designed to assay d r y samples material.  Firstly,  type  of p l a n t  the p o s i t i o n of the r a d i o a c t i v e  sample  r e l a t i v e to the p o s i t i o n of the counter i s always the  same  when a 10-ml. a l i q u o t i s added to the j a c k e t surrounding the l i q u i d c o u n t e r .  Small v a r i a t i o n s i n the p o s i t i o n of  d r y samples r e l a t i v e to the end window counter often i n l a r g e v a r i a t i o n s w i t h regard to the number of which w i l l pass from the d r y sample through the The M6 tube i s a l s o designed  results  particles counter.  so t h a t samples of P ^  2  slightly  - 20 in  e x c e s s o f 10 m i s .  counting  rate.  g i v e no  Secondly,  to prepare  than are dry  during  preparation  the  negligible small one  compared  liquid  of l i q u i d  result  i n appreciable prevented  liquid  t h a n one  p.p.m. P ? .  l o s e a s much, a s  due  to a d s o r p t i o n  83$  of P ^  of 2  on  This  errors. sample  t o pH  concentrations  their activity the w a l l s  after  of g l a s s  CORRECTION AND A N A L Y S I S OF  variation, larger all  i n t e r v a l between c o n s e c u t i v e  greater  accuracy  52  was  t o t a l number o f c o u n t s .  i s subject  Further,  obtained  by  T h i s was  done by  any  in the  order  they  c o u n t e d below  to prevent  s c a l i n g u n i t was  RESULTS. dis-  counting  a  counting depending  s o l u t i o n s which, were  more a c t i v e t h a n 6800 c o u n t s p e r m i n u t e diluted until  2  t o random  samples f o r 20 m i n u t e s o r f o r 10,000 c o u n t s  u p o n w h i c h came f i r s t .  P-^  containers  PROCEDURE AND  sample o f P-  taken,  s i x days  COUNTING-  in a  by  less  of  stored.  integrations  may  3 or  are not  solutions are  time  This  loss  when t h e  Because the  have  Adsorption  precautions  s o l u t i o n s w h i c h c o n t a i n low  may  may  s o l u t i o n s which contain  i f these  2  preparing  2  a d j u s t i n g the  adding c a r r i e r phosphate to  the  occur  s a m p l e s o f P-^ .  surfaces.  experimental by  in  samples a r e u s u a l l y  L i q u i d counting  tends t o adsorb to g l a s s  l o s s e s are  losses which  t o l o s s e s e n c o u n t e r e d when  samples of d r y m a t e r i a l .  isotope  alteration  s a m p l e s a r e much e a s i e r  s a m p l e s and  d i s a d v a n t a g e when u s i n g  then  appreciable  (c.p.m.) were  this rate.  T h i s was  l o s s e s of counts which occured r e c o r d i n g a b o v e t h i s maximum  done  when counting  - 21 -  rate. All  samples were c o r r e c t e d f o r background and r a d i o -  a c t i v e decay.  The a c t i v i t y of each sample was c o r r e c t e d  to a chosen -reference time, namely,  to the a c t i v i t y  of the  sample one hour a f t e r i n i t i a l phosphate uptake by the p l a n t . In order t o determine whether or not the d i f f e r e n c e between  sample means was due to treatment or t o chance,  the means of the small samples were compared u s i n g the 'Student's' t - t e s t  (9).  Xi S.D. / y  1  n  i  x ,  2  1 *2  To determine the' s i g n i f i c a n c e of the t-value g i v e n by the above equation, the one-sided t a b l e f o r 'Student's' t - d i s t r i b u t i o n was used,..  - 22 -  RESULTS.  1  The response of bean p l a n t s striking.  For the f i r s t  to AT-treatment i s  24 hours a f t e r  treatment w i t h 100  p . p . m . A T , t r e a t e d p l a n t s l o o k very s i m i l a r to p l a n t s w i t h the e x c e p t i o n p e t i o l e s of the f i r s t found.  that  quite  some c h l o r o s i s  control of  the  trifoliate  l e a v e s can u s u a l l y be  However, a f t e r 48 h o u r s ,  the b a s a l p o r t i o n s of  expanding f i r s t concentration  trifoliates  become p a r t i a l l y c h l o r o t i c .  of 100 p . p . m . AT does not r e s u l t  f o l i a t e s which appear a t t h i s time are i n d i c a t i n g a complete  carotenoids.  tri-  characteristically  l a c k of c h l o r o p h y l l and  Areas w i t h i n the laminae of primary l e a v e s  a l s o b e g i n to l o s e t h e i r normal green c o l o r 48 hours treatment.  These l e a v e s are u s u a l l y q u i t e f l a c c i d  after  as  compared to the primary l e a v e s of c o n t r o l p l a n t s ,  with  result  curl  upward.  that the margins of t r e a t e d leaves tend to  the  Some of the primary leaves may a b s c i z e d u r i n g  this period.  A f t e r 96 h o u r s , many of the  have a b s c i z e d both of  treated  jarred,  trifoliate  plants  t h e i r primary leaves or i f n o t ,  l e a v e s a r e very n e a r l y ready f o r a b s c i s s i o n . is  A  i n the  immediate c e s s a t i o n of growth so that any secondary  white,  the  these l e a v e s w i l l u s u a l l y f a l l .  If  the  are w h i t e .  Growth appears  terminated 96 hours a f t e r A T - t r e a t m e n t .  plant  The o l d e r  l e a v e s are c h a r a c t e r i s t i c a l l y y e l l o w while  young t r i f o l i a t e s  these  to have  the  - 23 -  INTAKE, RETENTION AND DISTRIBUTION OF Y  .  J  The 48 hour exposure  of the r o o t s  to 100 p . p . m . AT had very l i t t l e of P - .  This i s  5 2  after  of young bean p l a n t s  effect  on the t o t a l uptake  c l e a r l y i l l u s t r a t e d i n F i g . 1.  the b e g i n n i n g of phosphate uptake,  "52 the P^ content significantly  One hour  the t - v a l u e  of A T - t r e a t e d p l a n t s d i d not  for  differ  from t h a t of the c o n t r o l p l a n t s .  Thereafter,  however,  the r o o t s  of c o n t r o l p l a n t s  lost  ~ >2 z  •more P-'  to the f i n a l phosphate-free  than d i d the r o o t s  of A T - t r e a t e d p l a n t s  treatment by which time the r o o t s of t h e i r o r i g i n a l a c t i v i t y  of c o n t r o l p l a n t s had l o s t to the n u t r i e n t  solution  A l l plants  to the n u t r i e n t s o l u t i o n s  d u r a t i o n of the experiment.  for  the  This l o s s appeared to r e a c h a  steady s t a t e between the 48 hour and 96 hour N i n e t y - s i x hours a f t e r  This  the end of A T -  whereas the A T - t r e a t e d p l a n t s l o s t only 1.7$. continued l o s i n g a c t i v i t y  solutions  (Table I ) .  24 hours a f t e r  e f f e c t was most n o t i c e a b l e  31.8$  nutrient  harvests.  the b e g i n n i n g of phosphate  uptake,  A T - t r e a t e d p l a n t s had r e t a i n e d 6.9$ more of t h e i r o r i g i n a l activity  than had the  controls. 32  There v/as a marked i n c r e a s e  i n the amount of P  t r a n s l o c a t e d to the l e a v e s of A T - t r e a t e d bean p l a n t s (Table I I ) .  T-values d i f f e r e d s i g n i f i c a n t l y  the b e g i n n i n g of phosphate u p t a k e . the b e g i n n i n g of phosphate uptake,  one hour a f t e r  N i n e t y - s i x hours  the l e a v e s of A T - t r e a t e d  p l a n t s had approximately 40,000 c . p . m . more a c t i v i t y the l e a v e s of c o n t r o l p l a n t s .  after  than  24 300,000  AT-treated  250,000 Average c.p.m. per  200,000  plant  150,000  100,000 0  24  48  Hours a f t e r the b e g i n n i n g Fig.  1.  96  o f p32 u p t a k e  The e f f e c t o f A T - t r e a t m e n t on t h e u p t a k e a n d r e t e n t i o n o f p h o s p h a t e by whole b e a n p l a n t s .  TABLE I UPTAKE AND Hours a f t e r initial contact w i t h P32  1 24  Plant  Percentage of t o t a l initial activity lost  268,880 266,675  AT  264,325  182,255  4,555 84,420  1.7 31.8  AT  158,418 125,154  110,462 141,521  41.0 53.7  141,006 122,886  127,874 143,789  47.5 53.9  Control 96  Activity l o s t to the nutrient solution (cpm)  AT Control  Control  48  Total activity per plant (cpm)  RETENTION OF p32  AT Control  - 25 -  TABLE I I DISTRIBUTION Hours a f t e r initial contact with P 3  OF P  Plant  5 2  IN LEAVES,  Organ  Total activity (cpm/organ)  2  48  96  2  1,932 13,637 253,311  0.71 5.06 94.22  Control  Leaves Stem Roots  281 4,564 261,830  0.10 1.67 98.22  Treated  Leaves Stem Roots  7,265 41,905 15,154  -l5 20.10 77.74  2  Leaves Stem Roots  ,950 13,549 165,756  1.64 7.49 90.87  Treated  Leaves Stem Roots  33,102 35,827 89,489  20.85 22.57 56.57  Control  Leaves Stem Roots-  5,518 12,718 106,918  4.11 10.34 85.54  Treated  Leaves Stem Roots  44,721 22,966 73,319  31.96 16.27 51.76  Leaves Stem Roots  3,940 15, 5 103,721  .77 12.12 85.10  significant significant *** s i g n i f i c a n t  2  a t 0.05 l e v e l a t 0.01 l e v e l a t 0.001 l e v e l  2 2  t-value  32.96 15.38 16.87  2  Control  Control  *  Percentage of t o t a l P3 found i n each organ  Leave s Stem Roots  Treated  24  STEMS AND ROOTS.  2  0.49 4.21 3.27 «  37.61 41.73 93.28  4 . 4 1 it 7.70 # 5.48 •i'c  - 26 When the a c t i v i t y  i n the l e a v e s ,  expressed as a percentage plant,  stems and r o o t s  of the t o t a l a c t i v i t y  is  i n the  a much c l e a r e r p i c t u r e of the d i s t r i b u t i o n of P ^  w i t h i n the p l a n t i s  given  (Fig. 2).  treated plants l o s t a c t i v i t y  to the  The r o o t s  2  of A T -  shoots s t e a d i l y  for  96 h o u r s , a t which, time only 51-8$ of the a c t i v i t y remained in  the r o o t s .  of  the a c t i v i t y  For the f i r s t 24 hours, the l a r g e s t  amount  t r a n s l o c a t e d from the r o o t s appeared  p r i m a r i l y i n the stems.  Thereafter,  the l e a v e s of A T -  t r e a t e d p l a n t s r a p i d l y accumulated i n the stems decreased  slightly.  w h i l e the Thus,  the  activity  over-all  p i c t u r e i s a steady l o s s of a c t i v i t y from the r o o t s m i r r o r e d by a steady accumulation of P32 i n the  leaves.  The r o o t s of c o n t r o l p l a n t s l o s t only 14.4$ i n i t i a l activity after  to the  Of t h i s  10.3$ was l o c a t e d i n the stems and only 4.1$  P^  2  14.4$,  i n the  leaves.  the r o o t s d i d not change i n t h e i r  content whereas the l e a v e s l o s t some of t h e i r  which reappeared i n the  their  shoots d u r i n g the f i r s t 48 hours  the b e g i n n i n g of phosphate .uptake.  However, a f t e r 48 h o u r s ,  of  stems.  . This f l u c t u a t i o n  t h a t the l e a v e s of c o n t r o l p l a n t s  activity indicates  accumulate P  32 for  the  f i r s t 48 hours a f t e r which time there i s a downward t r a n s l o c a t i o n of  activity.  EFFECT OF AT-TREATMENT ON THE DISTRIBUTION  OF ACID-SOLUBLE  AND ACID-INSOLUBLE ACTIVITY. G r i n d i n g p l a n t m a t e r i a l w i t h c o l d 16$ TCA separates the a c i d - s o l u b l e phosphate  compounds from the  acid-insoluble  - 27 4o.o r  Percentage of a c t i v i t y i n the leaves  Hours a f t e r  the b e g i n n i n g of phosphate uptake  25.0  Percentage of a c t i v i t y i n the stems  o L  i  24  0  i  48  96  Hours a f t e r  the b e g i n n i n g of phosphate uptake  Hours a f t e r  the b e g i n n i n g of phosphate uptake  Percentage of a c t i v i t y i n the roots  Fig.  2:  D i s t r i b u t i o n of P  32  in roots,  stems, and l e a v e s .  - 28 phosphates. phosphates,  The a c i d - s o l u b l e sugar phosphates  nucleic acids,  phosphates  include inorganic  and n u c l e o t i d e s  while  p h o s p h o l i p i d s , and phosphoproteins  i n c l u d e d i n the a c i d - i n s o l u b l e f r a c t i o n .  the are  The r e l a t i v e  a c t i v i t y and the d i s t r i b u t i o n of 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 w i t h i n bean p l a n t s i s presented  in  Tables I I I and IV. I t was noted i n the p r e v i o u s s e c t i o n  that  treatment  of bean p l a n t s w i t h 100 p . p . m . AT f o r 48 hours  increased  the amount of P-^ that moved from the r o o t s 2  and l e a v e s . represents P  Figs.  3 and 4 i n d i c a t e  an i n c r e a s e  i n t o the stems  that t h i s  i n b o t h the amount of  increase acid-soluble  32  and the amount of a c i d - i n s o l u b l e P ^ the r o o t s  i n t o the upper p o r t i o n s of the t r e a t e d  The r o o t s steadily  that moves from  2  plants.  of A T - t r e a t e d p l a n t s l o s t a c i d - s o l u b l e P ^  f o r 96 hours u n t i l only 35.4% of the t o t a l  i n the p l a n t remained i n the r o o t s .  The leaves of  i n the p l a n t .  activity  The leaves of c o n t r o l p l a n t s d i d  not accumulate a c i d - s o l u b l e P^2 ±  n  a s i m i l a r manner.  Though 29% of 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 had been t r a n s l o c a t e d  activity treated  p l a n t s accumulated 43.9% of the t o t a l a c i d - s o l u b l e present  2  to the shoots a f t e r  of the  96 hours,  k% of t h i s a c t i v i t y appeared i n the l e a v e s .  plant only  The f l u c t u a t i o n  of a c i d - s o l u b l e P-^ w i t h i n the leaves between the 24 hour 2  and 96 hour harvests activity  i n the  soluble P32 i  s  together w i t h the steady i n c r e a s e  stems of c o n t r o l p l a n t s being freely  suggests that  t r a n s l o c a t e d from the  to the r o o t s as w e l l as i n the r e v e r s e  direction.  of acid-  leaves  - 29 -  TABLE I I I DISTRIBUTION OF AGID-SOLUBLE P Hours a f t e r initial contact with p 3  Plant  Organ  Treated  Leaves Stem Roots  24  Treated  Control  Leaves Stem Roots Leaves Stem Roots Leaves Stem Roots  Percentage of t o t a l acid-sol. activity i n organ  1,630 11,765 167,281 180,676  0.9 6.5 92.6  155  0.1 2.2 97.6  3,631  153,525 157,311 3,375 27,840 103,500 134,715  2.1 26.0 71.9  1,043 6,547 44,784  2.2  52,374 48  96  Treated  IN BEAN PLANTS.  Acid-soluble activity (cpm/organ)  2  Control  3 2  26.7 49.5  Control  Leaves Stem Roots  1,884 6,298 24,785 32,967  5.318.9 75.8  Treated  Leaves Stem Roots  19,434  43.9 20.7 ' 35.4  15,859  Control  Leaves Stem Roots  1,003 6,259 18.256  25,518 * s i g n i f i c a n t a t 0.05 l e v e l * * s i g n i f i c a n t a t 0.01 l e v e l  1.11 2.68 2.07  13.7  16,865 18,890 34,684 70,439  44,543  4.02 8.90 12.74  84.1  Leaves Stem Roots  9,250  t-test  23.8  5.71 tt  13.52  3.9 24.5  71.5  16.85  9.50 tt  1.99 17.54  - 30 -  TABLE IV DISTRIBUTION OF ACID-INSOLUBLE P Hours a f t e r initial contact w i t h P32  1  24  Plant  Organ  Treated  Leaves Stem Roots  301 . 1,871 86,030 88,202  Percentage of t o t a l acid-insol. activity i n organ 0.2 2.1 97.5  Leaves Stem Roots  126 933 108,305 109,364  0.1 0.8 99.0  Treated  Leaves Stem Roots  3,890 14,065 111,655 129,610  2.2 13.7 84.0  Leaves Stem Roots  1,906 7,002 120,972  1.5 5.4 91.1  Leaves Stem Roots  16,236 16,936 54,805 87,977  18.5 19.3 62.2  Leaves Stem Roots  3,634 6,421  3.7 7.2 89.1  Treated  Control  96  Acid-insoluble activity (cpm/organ)  IN BEAN PLANTS.  Control  Control  48  3 2  Treated  Control  Leaves Stem Roots Leaves Stem Roots  129,880  82,133  92,188 25,287  13,716 57,460 98,963 2,937 8,966 85,465 97,368  * s i g n i f i c a n t a t 0.05 l e v e l ** s i g n i f i c a n t a t 0.01 l e v e l s i g n i f i c a n t a t 0.001 l e v e l  26.2 14.2 59.6  t-test  9.18 12.16 4.10  0.96 3.61 2.85  39.83 35.22 60.49  6.50  1.89 2.4 8.7 88.9  15.90  -  31 -  50.0  Percentage of a c i d soluble activity i n the leaves  Hours a f t e r the b e g i n n i n g of phosphate uptake 30.0 p  ,  •  i  1  24  48  96  Percentage of a c i d soluble activity i n the stems  nl •  0  Hours a f t e r the b e g i n n i n g of phosphate uptake 100.0  Percentage of a c i d soluble activity i n the roots  r  '  1  62.5 -  25.0 I  0  1  1  — '  24  48  96  Hours a f t e r the b e g i n n i n g of phosphate uptake Fig.  3:  D i s t r i b u t i o n of a c i d - s o l u b l e P  32  and r o o t s of young bean p l a n t s .  i n leaves,  stems,  - 32 50.0  Percentage of a c i d insoluble activity i n the leaves  Hours a f t e r  30.0 r  the beginning of phosphate 1  uptake  "  Percentage of a c i d insoluble activity i n the stems  Fig.  4:  Hours a f t e r  the b e g i n n i n g of phosphate  uptake  Hours a f t e r  the b e g i n n i n g of phosphate  uptake  D i s t r i b u t i o n of a c i d - i n s o l u b l e P 3 2 i n the l e a v e s , and r o o t s of young bean p l a n t s .  stems,  -  33 -  The d i s t r i b u t i o n of  acid-insoluble P  32  and u n t r e a t e d bean p l a n t s a l s o d i f f e r e d . percentage  In wai tl hl i ncases, t r e a t ethe d  of 32  P  i n the l e a v e s of t r e a t e d p l a n t s , to the a c t i v i t y  i n the e n t i r e p l a n t , A f t e r 9 6 hours,  control plants.  i n c r e a s e d over that  of A T - t r e a t e d  During t h i s p e r i o d , 2 7 $ of the a c t i v i t y  p l a n t had accumulated i n the l e a v e s . roots  of  only 59.5% of the a c i d -  i n s o l u b l e a c t i v i t y remained i n the r o o t s plants.  relative  i n the  A f t e r 9 6 hours,  the  of c o n t r o l p l a n t s had 88.9% of 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 whereas the l e a v e s had only 2 . 4 $ of a c i d - i n s o l u b l e P-  3  .  The r e s u l t s  the  i n d i c a t e that r a t h e r than  accumulating P  32  in leaves,  the a c i d - i n s o l u b l e form w i t h i n  control plants  tend to t r a n s l o c a t e  activity  the in  either direction. INCORPORATION OF P 3 £ INTO ACID-INSOLUBLE FRACTIONS. When the P32 content fractions  i n the r o o t s ,  percentage the r e s u l t s  of 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  stems and leaves was expressed as a  of the t o t a l P-^ content  i n these organs  2  i n d i c a t e d t h a t AT-treatment a f f e c t s  b u t i o n of P32  the  (Table V ) , distri-  b  etween these two f r a c t i o n s . p l a n t s continued to i n c o r p o r a t e P-^ i n t o 2  Though a l l  acid-insoluble  f r a c t i o n s r e s u l t i n g i n a corresponding decrease i n a c i d soluble a c t i v i t y , AT-treated plants  i n c o r p o r a t e d l e s s of  the  t o t a l P ^ i n t o t h i s f r a c t i o n . A f t e r 9 6 hours, 5 6 . 6 $ of the whereas P the 32 li ena vthe 7e 3s . 9of le $av of the e s the was c o natpresent cr toilv iptlya n appeared ti sn the ( F i ga. c 5 in d)-.itnhsThough i so l uf rbal ec tthe iforna c itn ion 2  - 4 3  TABLE V DISTRIBUTION OF P ^ BETWEEN ACID-SOLUBLE AND ACID-INSOLUBLE FRACTIONS IN EACH PLANT ORGAN 2  Hours a f t e r initial Plant contact with P 3  Organ  2  1  T  C  2 4  T  C  4 8  T  C  9 6  T  C  Leaves Stem Roots Leaves Stem Roots Leaves Stem Roots Leaves Stem Roots ' Leaves Stem Roots Leaves Stem Roots Leaves Stem Roots Leaves Stem Roots  Acid-soluble as percent total activity i n organ  8 4 . 7 8  Acid-insoluble as percent total activity i n organ 15.22  8 6 . 4 9  13.51  6 6 . 3 7  3 3 . 6 2  5 -27  4 7 . 7 3  2  t-test  7 9 . 0 7  2 1 . 3 8  5 8 . 6 2  4 1 . 3 7  4 7 . 1 0  52.90  66.05  3 3 . 9 4  4 8 . 1 0  51.89  7 . 7 9 6 . 1 8  *  3.51  *  6 . 2 8  *  1 8 . 5 1 3 4 . 8 4  6 5 . 1 6  4 8 . 6 4  5 1 . 3 6  2 6 . 9 4  73.05  49.91  50.09  51.77  4 8 . 2 2  3 8 . 2 4  6 1 . 7 5  1 0 . 7 4  *  5 . 6 3  *  *  0 . 7 3 3 3 . 9 1  6 6 . 0 9  4 9 . 9 2  50.08  2 4 . 6 1  7 5 . 3 9  4 3 . 4 2  5 6 . 5 8  5 . 2 0  *  4 0 . 1 9  5 9 . 8 1  2 2 . 5 2  7 7 . 4 7  7 . 1 6  *  3 . 2 0  *  26.13  7 3 . 8 7  4 1 . 1 7  5 8 . 8 2  17.60  8 2 . 4 0  T " Treated C = Control * s i g n i f i c a n t a t 0.05 l e v e l * * s i g n i f i c a n t a t 0.01 l e v e l  *  - 35 80.0  r  Percentage of a c i d i n s o l u b l e 3 ,32 40.0 i n leaves based on total activity i n leaves  0  24  Hours a f t e r  48  the b e g i n n i n g of phosphate  96 uptake  60.0  Percentage of a c i d -, i n s o l u b l e P-^ i n stems 30.0 based on total activity i n stems 0  0  0  24  Hours a f t e r  100. O r  48  96  the b e g i n n i n g of phosphate uptake 1  Percentage of a c i d insoluble P-^ i n roots 62.5" based on total a ctivity i n roots  r  AT-treated  25.0 0  24  Hours a f t e r Fig.  5:  I n c o r p o r a t i o n of  P  48  96  the b e g i n n i n g of phosphate uptake  32 into Acid-Insoluble Fractions.  - 36 percentage of P  32 in  the acid-insoluble f r a c t i o n i n the  stems o f A T - 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 that  i n the controls f o r the f i r s t  from  24- h o u r s , t h i s d i f f e r -  e n c e was n o t s i g n i f i c a n t af-ter-. 4-8 h o u r s .  AT-treatment  a l s o i n h i b i t e d the i n c o r p o r a t i o n of P  32  into acid-insoluble fractions i n the root. EFFECT OF AT-TREATMENT ON THE E S T E R I F I C A T I O N OF PHOSPHATE AND ON THE DISTRIBUTION OF THE VARIOUS ACID-SOLUBLE COMPONENTS. When t h e 32 P  content of inorganic phosphates and n u c l e o t i d e s was  phosphates,  i n the r o o t s ,  sugar  stems, and l e a v e s  e x p r e s s e d a s a p e r c e n t a g e o f t h e t o t a l a c i d - s o l u b l e P^2  content of these organs, the r e s u l t s Indicated ment r e s u l t e d 96 h o u r s plants  that  treat-  i n n o c h a n g e i n e s t e r i f i c a t i o n o f P^2 f o r  (Table V I ) .  Thus, A T - t r e a t e d p l a n t s  and c o n t r o l  e s t e r l f i e d a p p r o x i m a t e l y t h e same p e r c e n t a g e o f t h e  inorganic  phosphates present i n each organ f o r the f o r m a t i o n  of n u c l e o t i d e s  and sugar  When t h e i n o r g a n i c  phosphates. P-^2 c o n t e n t o f e a c h o r g a n was  expressed as a percentage of the t o t a l inorganic  P-^  content of the p l a n t ,  AT-treatment  the r e s u l t s indicated that  does a f f e c t t h e d i s t r i b u t i o n o f i n o r g a n i c plant  (Fig. 6).  P^  2  2  within the  The l e a v e s o f A T - t r e a t e d a n d c o n t r o l  p l a n t s h a v e a b o u t t h e same p e r c e n t a g e o f t h e i n o r g a n i c a c t i v i t y p r e s e n t i n each p l a n t f o r the f i r s t AT-treatment. plants  Thereafter,  r a p i d l y accumulate  24- h o u r s a f t e r  however, t h e l e a v e s of t r e a t e d inorganic  P-^  f o r the duration  of t h e experiment whereas t h e l e a v e s of c o n t r o l  plants  - 37 TABLE V I EFFECT OF AT ON THE E S T E R I F I C A T I O N OF PHOSPHATE. Hours a f t e r initial Plant contact w i t h P32  24  T  48  96  T = C »  Organ  Percent acid-soluble a c t i v i t y i n the form o f : NucleoSugar Inorganic tides phosphosphates phates  Percent esterification  Leaves Stem Roots  27.4 18.6  22.4  13.2 25.0 21.2  59.4 56.4 56.4  40.6 43.6 43.6  Leaves Stem Roots  26.1 31.4 29.6  16.0 16.6 24.9  57.8 51.9 45.4  42.1 48.0 54.5  Leaves Stem Roots  26.6 21.0 28.6  19.5 22.2 18.7  53.9 56.8 52.6  46.1 43.2 47.3  Leaves Stem Roots  20.3 27.4 30.8  22.5 18.2 25.5  57.2 54.4 43.7  42.8 45.6 56.3  Leaves Stem Roots  25.5 17.3  24.0 23.0 34.6  50.4 59.7 40.5  49.5 40.3 59.4  Leaves Stem Roots  32.3  27.9 25.3  17.1 23.9  54.9 50.8  45.0 49.2  Leaves Stem Roots  26.9 24.1 32.4  18.9 20.8 14.6  54.2 55.0 53.0  45.8 44.9 47.0  Leaves Stem Roots  19.8 18.9 20.7  22.9 25.5 22.6  57.3 55.6 56.7  42.7 44.4 43.3  AT-treated Controls  24.8  - 38 50.0 r  r  Percentage of the t o t a l plant inorganic activity i n the leaves  Hours a f t e r 40.0 r  Percentage the t o t a l plant inorganic activity i n the stems  the b e g i n n i n g of phosphate uptake 1  1  24  48  of 20.0 •  0 Hours a f t e r  96  the b e g i n n i n g of phosphate  uptake  the b e g i n n i n g of phosphate  uptake  100.0 r-r  Percentage of the t o t a l plant inorganic activity i n the roots  Hours a f t e r Fig.  6:  D i s t r i b u t i o n of  Inorganic  P  32 w i t h i n the P l a n t .  - 39 a c t u a l l y decrease s l i g h t l y  i n their inorganic  activity.  N i n e t y - s i x hours a f t e r AT-treatment, the leaves  4-7.3$  treated plants contain while  the leaves  o f AT-  of the t o t a l inorganic  of the c o n t r o l p l a n t s c o n t a i n only  P-  52  1.5$  "32  o f t h e t o t a l i n o r g a n i c P^  present  i n the plant.  The  steady decrease of i n o r g a n i c P  32 in  plants reflects  the roots of AT-treated  theaccumulation of a c t i v i t y  i nthe leaves.  N i n e t y - s i x hours a f t e r AT-treatment, the AT-treated  75.6$  had  of the inorganic a c t i v i t y .  inorganic a c t i v i t y The  2  p l a n t s h a v e much more o f t h e t o t a l  t h a n do t h e stems o f c o n t r o l p l a n t s  the f i r s t 48 h o u r s a f t e r i n i t i a l 96 h o u r s a f t e r i n i t i a l the  the leaves  much i n o r g a n i c P-  P--  2  during  phosphate uptake.  However,  phosphate uptake, the a c t i v i t y i n  s t e m s o f e a c h s e t o f p l a n t s i s t h e same.  this,  This  The d i s t r i b u t i o n o f  i n t h e stems i s a l s o w o r t h y o f n o t e .  stems o f A T - t r e a t e d  i n o r g a n i c P^  roots  I n s p i t e of  o f c o n t r o l p l a n t s d o not' a c c u m u l a t e a s 52  a s do t h e l e a v e s  of AT-treated  plants.  suggests that c o n t r o l p l a n t s r e d i s t r i b u t e inorganic i n b o t h d i r e c t i o n s whereas t h e i n o r g a n i c P^  2  i n AT-  treated plants i saccumulated i n the leaves. Sugar phosphates a l s o accumulated i n t h e l e a v e s treated plants  ( F i g . 7)  whereas t h e leaves  p l a n t s f l u c t u a t e d markedly i n t h e i r Again,  c o n t r o l p l a n t s seem t o f r e e l y  phate from reverse  the leaves  o f AT-  of c o n t r o l  sugar phosphate  activity.  t r a n s p o r t sugar phos-  t o the roots as w e l l as i n the  d i r e c t i o n whereas AT-treatment r e s u l t s i n only  u p w a r d movement o f a c t i v i t y .  - 40  -  6o.o r Percentage of the t o t a l p l a n t sugar phosphate i n the leaves  Hours a f t e r the b e g i n n i n g of phosphate uptake 40. o r  i  •  — —  Percentage of the t o t a l p l a n t sugar phosphate i n the stems  Hours a f t e r  the b e g i n n i n g of phosphate uptake  Percentage of the t o t a l p l a n t sugar phosphate i n the roots  Hours a f t e r the b e g i n n i n g of phosphate uptake Fig.  7:  D i s t r i b u t i o n of sugar phosphate i n young bean p l a n t s  - 41 The d i s t r i b u t i o n of n u c l e o t i d e a c t i v i t y w i t h i n each set  of p l a n t s i s g i v e n i n F i g . 8.  As i n the case of  i n o r g a n i c phosphate a c t i v i t y and sugar phosphate a c t i v i t y , the n u c l e o t i d e a c t i v i t y i s a l s o accumulated i n the of A T - t r e a t e d p l a n t s . reaches a steady  activity  s t a t e i n these l e a v e s 48 hours a f t e r A T -  (38.7$).  treatment  The l e v e l of n u c l e o t i d e  leaves  On the other hand,  the n u c l e o t i d e  a c t i v i t y of the l e a v e s of c o n t r o l p l a n t s f l u c t u a t e s w i t h only  22.4$  of t h i s f r a c t i o n oc'curing i n the l e a v e s  AT-treatment.  hours a f t e r  The r o o t s of A T - t r e a t e d p l a n t s l o s t  a c t i v i t y s t e a d i l y f o r 48 hours and t h e r e a f t e r slightly  96  in their nucleotide a c t i v i t y .  nucleotide  increased  Though t h i s  suggests  t h a t some of the n u c l e o t i d e s may have been t r a n s p o r t e d from the stem to the r o o t s d u r i n g t h i s p e r i o d , n u c l e o t i d e s may have been manufactured w i t h i n the r o o t s d u r i n g t h i s p e r i o d s i n c e there was a c o r r e s p o n d i n g decrease and sugar P^ Thus,  2  during this  i n inorganic  same p e r i o d .  the accumulation of 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 v e s of A T - t r e a t e d p l a n t s does not appear to be confined to any of the f r a c t i o n s which comprise the a c i d - s o l u b l e activity,  but r a t h e r , the accumulation r e p r e s e n t s an accumula-  t i o n of i n o r g a n i c phosphates, tides.  sugar phosphates and n u c l e o -  The l e a v e s of c o n t r o l p l a n t s do not accumulate any of  these f r a c t i o n s . esterlfied  Furthermore,  compounds ( i . e . ,  the i n c o r p o r a t i o n of p32 i n t o  n u c l e o t i d e s and sugar phosphates),  as r e v e a l e d by the percentage e s t e r i f i c a t i o n i n each p l a n t organ,  i s u n a f f e c t e d by A T - t r e a t m e n t .  - 42 40.0  Percentage of the t o t a l plant nucleotide activity i n the leaves  Hours a f t e r the b e g i n n i n g of phosphate uptake  40.0|-i  ,  ,  oL  i  .  24  48  Percentage of the t o t a l plant nucleotide activity i n the stems  0  Hours a f t e r the b e g i n n i n g of phosphate uptake  100.0  Percentage of the t o t a l plant nucleotide activity i n the roots  Hours a f t e r Fig.  8:  D i s t r i b u t i o n of  the b e g i n n i n g of phosphate uptake  nucleotide  P  32 i n young bean p l a n t s .  96  - 43 DISCUSSION A 48 h o u r e x p o s u r e o f t h e r o o t s  o f young bean  t o 100 p.p.m. a m i n o t r i a z o l e d i d n o t a f f e c t  their  ability  to take up phosphate from the n u t r i e n t s o l u t i o n . W o r t a n d Loughman (44)  plants  However,  f o u n d t h a t AT r e d u c e d t h e a b i l i t y  of young b a r l e y p l a n t s t o a b s o r b P  reduction  i nabsorption  increased  l e n g t h o f e x p o s u r e t o AT.  32 and f u r t h e r m o r e , t h i s w i t h an increase of the  The d e c r e a s e i n a b s o r p t i o n o f  32  P  was  greatest  i nbarley plants  t r e a t e d w i t h 100 p.p.m.  AT f o r 96 h o u r s p r i o r t o t h e a b s o r p t i o n and L i n c k  (23)  period.  Herrett  found that t h e treatment o f P - d e f i c i e n t  Canada t h i s t l e p l a n t s w i t h AT r e s u l t e d i n a m a r k e d i n the uptake of f o l i a r a p p l i e d that the a c t i v i t y  reduction  These w o r k e r s f o u n d  o f AT was g r e a t l y r e d u c e d i n p h o s p h o r u s -  d e f i c i e n t p l a n t s a s compared t o p l a n t s a d e q u a t e amount o f p h o s p h a t e .  supplied w i t h an  I f t h i s were a l s o t h e c a s e  i n bean p l a n t s , a phosphate d e f i c i e n c y might tend t o a n t a g o n i z e t h e A T - i n l i i b i t i o n o f P-^ u p t a k e b y t h e r o o t s  of these p l a n t s .  2  However, t h e bean p l a n t s had no v i s i b l e deficiency prior possibility  signs  t o phosphate uptake, thus,  of phosphorus  this  latter  seems u n l i k e l y .  A T - t r e a t e d b e a n p l a n t s r e t a i n more P^2 when r e t u r n e d t o a p h o s p h a t e - f r e e environment than do c o n t r o l p l a n t s . i s analogous t o the e f f e c t of P^  2  plants  by b a r l e y p l a n t s  o f A T - t r e a t m e n t on t h e r e t e n t i o n  (44)  l o s t up t o o n e - t h i r d  This  i n w h i c h i t v/as f o u n d of the a c t i v i t y during  a f t e r t h e b e g i n n i n g of phosphate uptake.  that 3 hours  Bean p l a n t s  lost  - 44 activity stable  steadily  f o r 48 hours and reached a r e l a t i v e l y  state thereafter.  At this  time,  c o n t r o l p l a n t s had  l o s t 53.7% of t h e i r i n i t i a l a c t i v i t y whereas t r e a t e d l o s t only 41%.  plants  As r e p o r t e d by Wort and Loughman, the  i n i t i a l l o s s of P^2  m a  y be  a  rapid  d i f f u s i o n process whereas  the  slower l o s s o c c u r i n g a f t e r 48 hours i s probably a s s o c i a t e d w i t h m e t a b o l i c processes o c c u r i n g w i t h i n the p l a n t . A T - t r e a t e d p l a n t s t r a n s p o r t much more P  to the shoots than do c o n t r o l p l a n t s .  32  from the r o o t s The steady r i s e of 32 P  i n the l e a v e s m i r r o r e d by the steady the r o o t s i n d i c a t e s accumulate P-^ . 2  l o s s of a c t i v i t y from  that the l e a v e s of A T - t r e a t e d  plants  On the other hand, the P^2 content  l e a v e s of c o n t r o l p l a n t s i n c r e a s e s  but l i t t l e  of  the'  and f l u c t u a t i o n s  of a c t i v i t y appear to occur d u r i n g the 96 hour p e r i o d . f l u c t u a t i o n together w i t h the steady the r o o t s and the steady  increase  l o s s of a c t i v i t y  in P^  2  This from  a c t i v i t y i n the  stems suggests that c o n t r o l p l a n t s do not accumulate P ^ i n the l e a v e s .  These r e s u l t s  i n d i c a t e that AT i n h i b i t s  downward t r a n s l o c a t i o n of phosphate compounds. Loughman (44)  Wort and  have found that b a r l e y p l a n t s r e a c t i n a  s i m i l a r manner to A T - t r e a t m e n t . p l a n t s r e q u i r e d an exposure before  2  They found that b a r l e y  to 100 p . p . m . AT of over 4 hours  i n c r e a s e d t r a n s p o r t of phosphate compounds from'the  r o o t s to the l e a v e s occured. When the phosphate compounds were separated i n t o s o l u b l e P-*  2  nucleotides)  ( i n o r g a n i c phosphates,  sugar phosphates,  and a c i d - i n s o l u b l e P^2  n u c l e i c a c i d s and p h o s p h o l i p i d s ) ,  acidand  (phosphoproteins,  the r e s u l t s  indicated  that  -  45  -  both of these f r a c t i o n s are AT-treated  plants.  The  c o n t e n t of the r o o t s  accumulated i n the  a c i d - s o l u b l e and  t o A T - t r e a t m e n t I s an 2  an  and  transport  Further reveals  the  2  i n t h e amount o f  due  2  acid-soluble  which i s translocated rather  o f j u s t one  than  of t h e s e f r a c t i o n s .  c h a r a c t e r i z a t i o n of the a c i d - s o l u b l e f r a c t i o n i n t e r e s t i n g f a c t that  acid-soluble activity i s not  acid-insoluble  i n t r a n s l o c a t i o n of P^  increase  a c i d - i n s o l u b l e P^  increased  of  of t h e s e p l a n t s d e c r e a s e d s t e a d i l y .  Thus, the pronounced i n c r e a s e  P^  leaves  confined  i n the  t o any  acid-soluble activity.  the a c c u m u l a t i o n  leaves  of A T - t r e a t e d  of plants  of the f r a c t i o n s w h i c h c o m p r i s e Instead,  an a c c u m u l a t i o n of I n o r g a n i c  the a c c u m u l a t i o n  the  represents  phosphates, n u c l e o t i d e s ,  and  sugar phosphates. Synthesis phospholipids,  of c e r t a i n o r g a n i c ATP,  p h o s p h a t e compounds  (e.g.,  ribonucleic acid, deoxyribonucleic  acid)  i s e s s e n t i a l f o r normal growth i n p l a n t s of i n o r g a n i c the  photosynthetic  (oxidative phosphorylation)  phosphorylative  o f ATP,  I t has  pool,  ATP.  o f ATP  or i n d i r e c t l y  Iodoacetate,  inhibition  or v i a  mechanism r e s u l t s i n  the  the  r e a c t i o n s r e q u i r i n g a supply  b e e n shown t h a t g r o w t h i s d e p e n d e n t on  continuous synthesis directly  and  a high-energy phosphate, which i s then  u t i l i z e d for mediating v i t a l energy.  Esterification  phosphate v i a reduced p y r i d i n e n u c l e o t i d e s  cytochrome system  generation  (2).  (2).  C e r t a i n growth  e f f e c t t h e m a i n t e n a n c e of  f o r example, b l o c k s  of g l y c o l y s i s .  the  inhibitors the  ATP  sugar u t i l i z a t i o n  This r e s u l t s i n a reduction  Arsenate substitutes for inorganic  of  phosphate i n  of the  by  - 46 oxidation f r o m ATP  of  t r i o s e phosphate thereby u n c o u p l i n g  synthesis  incorporation seedlings  and  (2).  (10).  Arsenate i n h i b i t s  (2,4-dinitophenol)  incorporation  nucleotides  (44).  are  H o w e v e r , DNP  of i n o r g a n i c  responsible  on  or p h o t o s y n t h e t i c f o r the  does not  incorporation  Loughman (44)  and  suggested that  o f ATP.  the  acid-soluble  o f p h o s p h a t e i n t o one  whereby  to  the AT  o r more o f  the  or phosphoprotein f r a c t i o n s . t h o s e of Wort  t h e i r work w i t h b a r l e y  plants.  and They have  of phosphate i n c o r p o r a t i o n  t h e d i v e r s i o n o f p h o s p h a t e t o the  soluble  which  AT-treatment  a c i d - i n s o l u b l e f r a c t i o n by A T - t r e a t m e n t may  known, t h e r e  not  p r i n c i p l e e f f e c t of  i n agreement w i t h  u p w a r d t r a n s l o c a t i o n of  the  However, A T - t r e a t m e n t d o e s i n -  Thus, t h e  inhibition  Though t h e  pro-  affect  phosphorylation  production  phosphorylated.  These f i n d i n g s a r e  not  e f f e c t on t h e  the n u c l e i c a c i d f r a c t i o n .  t r a n s f e r of phosphate from  the  other  suggests t h a t AT-treatment does  nucleic acid, phospholipid,  the  no  and  phosphate i n t o sugar phosphates  acid-insoluble fraction. is  thereby  seem t o a f f e c t t h o s e g l y c o l y t i c r e a c t i o n s  sugars are hibit  This  oxidative  does not  has  t h e s e g r o w t h i n h i b i t o r s , AT  nucleotides.  affect  uptake,  uncouples  of p h o s p h a t e i n t o ATP  of p h o s p h a t e e n t e r i n g  incorporation and  also  f r o m o x i d a t i v e m e t a b o l i s m and  r e d u c e s the  Unlike  the  oxidation  t u r n o v e r o f r a d i o p h o s p h o r u s i n mung b e a n  DNP  phosphorylation  portion  -  into  account f o r  system r e s p o n s i b l e  for  activity.  a c t u a l mechanism o f p r o t e i n  synthesis  is  i s much e v i d e n c e t o s u g g e s t t h a t b o t h a  ribonucleic acid  (RNA)  and  a ribonucleoprotein  are  Involved are  -  I n t h e sequence o f r e a c t i o n s whereby amino a c i d s  incorporated  inhibit  47  into proteins  (18,39).  B e c a u s e AT may  t h e i n c o r p o r a t i o n o f p h o s p h a t e i n t o e i t h e r one o r  b o t h o f t h e s e compounds, t h e n t h e I n h i b i t i o n o f g r o w t h due t o A T - t r e a t m e n t may a c t u a l l y b e a n i n h i b i t i o n o f p r o t e i n synthesis. postulated has  Sund  (41)  a n d W o r t a n d Loughman (44)  have a l s o  t h a t AT i n t e r f e r e s w i t h p r o t e i n s y n t h e s i s .  found that c e r t a i n purine  precursors,  purines  or purine  r i b o s i d e s when a d d e d t o t o m a t o p l a n t s s i m u l t a n e o u s l y low  concentrations  o f AT w i l l p a r t i a l l y a l l e v i a t e  I n h i b i t i o n due t o A T - t r e a t m e n t .  Of t h e p u r i n e s ,  g u a n i n e and h y p o x a n t h i n e were e f f e c t i v e .  Growth  with  growth adenine, inhibition  o f G h l o r e l l a p y r e n o i d o s a due t o A T - t r e a t m e n t i s a l s o by one  the a d d i t i o n of purines  (4.2).  of the pyrimidines, u r a c i l ,  r e v e r s i n g AT-grpwth  A l d r i c h (4) h a s f o u n d  t h a t AT i n t e r f e r e s w i t h  normal  C a r t e r and N a y l o r  i n AT-treated  bean p l a n t s .  t h e s e amino a c i d s c a n p a r t i c i p a t e i n t h e s y n t h e s i s r i n g (11).  2  NH  2  Serine  (15)  i s a sharp r e d u c t i o n i n the f r e e  g l y c i n e and s e r i n e pools  H0CH CHC00H  that  i s also effective f o r  and p y r i m i d i n e metabolism.  h a v e shown t h a t t h e r e  purine  reversed  inhibition.  These r e s u l t s i m p l y purine  Sund  *H NCH C00H o  o  Both of t h e  - 48  -  A T - i n h i b i t i o n of i n c o r p o r a t i o n  of  into acid-  i n s . o l u b l e f r a c t i o n s c o u l d a l s o be due t o a n a l t e r e d phospholipid t h i s problem. riboflavin  (41)  has a l s o  investigated  Knowing t h a t p u r i n e s  not only  stimulated'  metabolism.  production  Sund  b u t a l s o became i n c o r p o r a t e d  t h e r i b o f l a v i n m o l e c u l e , Sund f e l t to that of purines by  riboflavin.  plants tide the  t h a t an e f f e c t comparable  on A T - g r o w t h i n h i b i t i o n m i g h t be p r o d u c e d  T h e r e f o r e , he a d d e d r i b o f l a v i n  simultaneously  w i t h AT.  p r o s t h e t i c group  (coenzyme) o f s e v e r a l  enzymes a n d a r e r i b o f l a v i n  (FAD) a c t a s flavoprotein  d e r i v a t i v e s , these  compounds  investigated.  Whereas t h e p u r i n e s effective  t o tomato  Because f l a v i n mononucleo-  (FMN) a n d f l a v i n a d e n i n e d i n u c l e o t i d e  were a l s o  into  a n d r e l a t e d compounds had b e e n  i n p a r t i a l l y reducing  to AT-treatment, r i b o f l a v i n , marked r e d u c t i o n  the growth i n h i b i t i o n  FMN, a n d FAD b r o u g h t a b o u t a  of the i n h i b i t i o n of p l a s t i d  These a l s o r e d u c e d AT-growth i n h i b i t i o n . that the i n h i b i t i o n  of p l a s t i d  of c h l o r o p h y l l s y n t h e s i s  formation  Sund  formation. concluded  and c o n s e q u e n t l y  b y AT r e s u l t e d f r o m t h e e f f e c t i v e ^  n e s s w i t h w h i c h AT b l o c k e d t h e s y n t h e s i s metabolites,  particularily riboflavin,  f o r normal c h i o r o p l a s t development.  of c e r t a i n  which, a r e n e c e s s a r y  R i b o f l a v i n and i t s  d e r i v a t i v e s a r e necessary f o r normal growth.  Sund h a s  a l s o f o u n d t h a t a l b i n i s t i c c o r n and peas t r e a t e d w i t h h a v e much l e s s r i b o f l a v i n  due  than untreated  tissues.  AT  - 49 Aaronson  (1) h a s a l s o p r o v i d e d  some e v i d e n c e w h i c h  i n d i c a t e s t h a t AT i n t e r f e r e s " w i t h p h o s p h o l i p i d  metabolism.  He f o u n d t h a t c r u d e s o y b e a n l e c i t h i n c o u l d r e v e r s e inhibition  AT-  o f c h l o r o p h y l l s y n t h e s i s i n Ochromonas d a n i c a ,  a phytoflagellate.  I n t h e p r e s e n c e o f i r o n , . l e c i t h i n was  e v e n more e f f e c t i v e  i n reversing AT-inhibition.  I n summary, t h e r e  is-mounting  evidence which  t h a t t h e i n h i b i t i o n by AT o f p l a s t i d  formation  suggests  and growth  i s a c t u a l l y due t o a n i n t e r f e r e n c e b y AT o f n o r m a l p r o t e i n and  phospholipid  synthesis.  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