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The regulation of phosphate uptake by intact barley plants Lefebvre, Daniel Denis 1980

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THE  REGULATION  UPTAKE  BY  OF  INTACT  PHOSPHATE  BARLEY  PLANTS  by DANIEL  DENIS  LEFEBVRE  B . S c , U n i v e r s i t y o f Ottawa, 1977  THESIS THE  SUBMITTED  IN  REQUIREMENTS MASTER  PARTIAL  FOR OF  THE  FULFILLMENT DEGREE  OF  SCIENCE  in THE  FACULTY  OF  GRADUATE  STUDIES  (Department of Botany) We a c c e p t t h i s t h e s i s as c o n f o r m i n g to the required  standard  THE UNIVERSITY OF BRITISH COLUMBIA 1980 D a n i e l Denis L e f e b v r e  In presenting  this thesis in partial fulfilment of the requirements for  an advanced degree at the University of B r i t i s h Columbia, I agree that the Library shall make i t f r e e l y available for reference  and  study.  I further agree that permission for extensive copying of this thesis for scholarly purposes may by his representatives.  be granted by the Head of my Department or It is understood that copying or publication  of this thesis for f i n a n c i a l gain shall not be allowed without my written permission.  BOTANY Department of The University of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5  Date  June 12,  1980  ABSTRACT The  s t u d y of phosphate i n f l u x i n r o o t s of i n t a c t b a r l e y  (Hordeum v u l g a r e L. v a r . Bonanza) r e v e a l e d d i s t i n c t r e g u l a t o r y p r o c e s s e s f o r phosphate One of these p r o c e s s e s ,  t h e presence o f two absorption.  w h i c h was e l i c i t e d i n response  to phosphate d e p r i v a t i o n , i n t h e form of enhanced phosphate u p t a k e , became e v i d e n t between 11 and 13 days a f t e r  germination.  At 16 days the "uptake r a t e s o f these p l a n t s had reached a m a x i mum v a l u e a t 2.43umol/g.f.wt./hour w h i c h compared t o a v a l u e o f 0.39umol/g.f.wt./hour f o r phosphate s u f f i c i e n t p l a n t s . taneously,  Simul-  d i f f e r e n c e s between t h e r e s p e c t i v e t r e a t m e n t s were  a l s o n o t e d i n growth r a t e s and phosphate p o o l s . A second r e g u l a t o r y p r o c e s s brought about a r a p i d r e d u c t i o n o f phosphate i n f l u x upon t h e p r o v i s i o n of orthophosphate t o p l a n t s p r e v i o u s l y s t a r v e d of phosphate d u r i n g the phase of enhanced u p t a k e .  W i t h i n hours of s u p p l y i n g i n o r g a n i c phosphate  t o these p l a n t s i n f l u x was reduced by g r e a t e r t h a n 50% and d u r i n g t h i s p e r i o d i n f l u x v a l u e s were l i n e a r l y c o r r e l a t e d w i t h orthophosphate c o n c e n t r a t i o n s . response i s s u g g e s t i v e i n t e r n a l orthophosphate  The time s c a l e o f t h i s second  of an a l l o s t e r i c i n h i b i t i o n of i n f l u x by levels.  B o t h r e g u l a t o r y systems s t u d i e d r e p r e s e n t e d adaptations  root  physiological  w h i c h would b e t t e r e n a b l e p l a n t s , under f i e l d  conditions,  ii  to o b t a i n a s u f f i c i e n t phosphate s u p p l y .  Severe phosphorus  .dep.riyatipn e v e n t u a l l y r e s u l t e d i n a m o r p h o l o g i c a l  response s u c h  as the p r o d u c t i o n of l o n g e r , n a r r o w e r r o o t s p r o v i d i n g , the p l a n t s a g r e a t e r s u r f a c e a r e a , presumably f o r g r e a t e r phosphate a b s o r p t i o n . At a l a t e r t i m e , an i n c r e a s e d f o r m a t i o n of r o o t - h a i r s r e s u l t e d i n even g r e a t e r s u r f a c e a r e a m o d i f i c a t i o n .  iii  TABLE  OF  CONTENTS Page  ABSTRACT  i  ACKNOWLEDGEMENTS  i viii  I.  INTRODUCTION  1  II.  METHODS  4  1.  4  2.  H y d r o p o n i c P l a n t Growth  „ 4  A.  Seeds  B.  Choice o f Phosphate L e v e l s and Growth Regimes  D e t e r m i n a t i o n o f Phosphate I n f l u x Method  7  A.  Root Wash P e r i o d  7  B.  Phosphate I n f l u x  8  3.  E f f l u x Analysis  4.  D e t e r m i n a t i o n o f t h e P l a n t s ' Phosphate Concentrations  III.  . . . .  4  9  . . . . . . . . . .  A.  T o t a l Phosphate C o n c e n t r a t i o n s  B.  I n o r g a n i c Phosphate C o n c e n t r a t i o n s  10 10  . . . . .  10  5.  Double l a b e l l e d Uptake D e t e r m i n a t i o n  11  6.  D e t e r m i n a t i o n o f Root E x t e r n a l P r o t e i n  13  RESULTS  AND  DISCUSSION  15  1.  E f f e c t o f Phosphate L e v e l on Growth  15  2.  C h a r a c t e r i z a t i o n o f Phosphate I n f l u x  25  A.  D e t e r m i n a t i o n o f Root Wash P e r i o d  25  B.  Temperature and pH Optimums  27  iv  TABLE OF CONTENTS  (continued) Page  3.  Phosphate E f f l u x K i n e t i c s  29  4.  Enhancement of Phosphate Uptake Rate by Phosphate Deprivation  34  A.  Phosphate Uptake  34  B.  T o t a l Phosphate and I n o r g a n i c  Phosphate  Levels w i t h i n the Plants C.  5.  40  P h y s 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 Enhanced Phosphate Uptake  49  i.  Uptake Isotherms  49  ii.  Uptake from S t i r r e d and N o n - s t i r r e d Media  53  iii.  D o u b l e - l a b e l l e d Uptake E x p e r i m e n t . . . .  56  iv.  D e t e r m i n a t i o n of Root E x t e r n a l P r o t e i n .  59  R e g u l a t i o n of Rapid D e c l i n e of Enhanced I n o r g a n i c Phosphate Uptake  61  A.  S h o r t - t e r m v e r s u s Long-term Uptake  61  B.  Phosphate Uptake Rates and P l a n t Phosphorus L e v e l s F o l l o w i n g Exposure of P l a n t s t o 15uM Phosphate  IV.  CONCLUSION  REFERENCES  63 74 77  V  LIST  OF  TABLES  page 1.  C o m p o s i t i o n of h y d r o p o n i c growth media  2.  D e t e r m i n a t i o n of regime r e q u i r e d f o r e l u t i o n of all  86  Rb and r e t e n t i o n of a l l  6  32 P from a n i o n -  exchange columns  12  3.  E o s i n s t a i n i n g technique  14  4.  +P and -P growth e q u a t i o n s  16  5.  Uptake r a t e s from 2.5uM s o l u t i o n w i t h or without s t i r r i n g  6.  7.  54  Uptake r a t e s of P from a 15yM P s o l u t i o n and K from a l l l y M K s o l u t i o n  57  S h o r t - t e r m v s . l o n g - t e r m uptake r a t e d e t e r m i n a t i o n . .  62  vi  LIST  OF  FIGURES Page  1.  5 day o l d r o o t t i p s  18  2.  10 day o l d r o o t t i p s  18  3.  15 day o l d r o o t t i p s  19  4.  20 day o l d root t i p s  19  5.  25 day o l d r o o t  tips  20  6.  30 day o l d root t i p s  20  7.  40 day o l d +P r o o t t i p  21  8.  40 day o l d -P r o o t  21  9.  P l a n t growth v s . age  22  10.  Root:shoot r a t i o v s . p l a n t age  23  11.  Growth of r o o t s  24  tip  32 12.  E f f l u x of  P from r a p i d exchange phase  26  13.  Temperature  curve o f phosphate uptake at pH 6.25  14.  pH curve of phosphate uptake at 30C  28  15.  T o t a l phosphate e f f l u x k i n e t i c s  31  16.  P^ uptake r a t e v s . p l a n t age - g.f.wt. a n a l y s i s . . .  35  17.  P^ uptake r a t e v s . p l a n t age - p e r p l a n t a n a l y s i s . .  37  18.  P^ and t o t a l phosphorus of -P r o o t s v s . age  42  19.  P^ and t o t a l phosphorus of -P shoots v s . age  20.  P^ and t o t a l phosphorus of +P r o o t s v s . age  21.  P. and t o t a l phosphorus of +P shoots v s . age  . .  . . . .  28  42 42  . . . .  42..  vii  LIST  OF  FIGURES  (continued) Page  22.  Phosphate c o n c e n t r a t i o n i n -P b a r l e y p l a n t s  47  23.  Phosphate c o n c e n t r a t i o n i n +P b a r l e y p l a n t s  47  24.  Orthophosphate uptake k i n e t i c s : M i c h a e l i s - M e n t e n p l o t  50  25.  Orthophosphate uptake k i n e t i c s : E a d i e - H o f s t e e p l o t .  51  26.  Phosphate uptake r a t e v s . time of r o o t exposure t o .  .  15uM o r t h o p h o s p h a t e 27.  64  T o t a l phosphorus c o n c e n t r a t i o n d u r i n g t h e p e r i o d of phosphate l o a d i n g o f -P grown b a r l e y p l a n t s - t r e a t e d roots  28.  65  T o t a l phosphorus c o n c e n t r a t i o n d u r i n g the p e r i o d of phosphate l o a d i n g o f -P grown b a r l e y p l a n t s - t r e a t e d shoots  29.  66  I n o r g a n i c phosphate c o n c e n t r a t i o n d u r i n g the p e r i o d of phosphate l o a d i n g o f -P b a r l e y p l a n t s  68  30.  Phosphate uptake r a t e v s . P^ c o n c e n t r a t i o n  69  31.  H i l l p l o t (v/Vmax - v a g a i n s t i n t e r n a l P^ concentration)  73  viii  ACKNOWLEDGEMENTS  I would l i k e t o thank my graduate a d v i s o r , D r . Anthony D. M. G l a s s , Botany Department, U n i v e r s i t y o f B r i t i s h Columbia, f o r h i s t e c h n i c a l a s s i s t a n c e and s u p p o r t t h r o u g h o u t the c o u r s e o f t h e graduate work. Thanks i s a l s o extended t o my a d v i s o r y committee c o n s i s t i n g o f Dr. B e v e r l y R. Green and Dr. P a u l J . H a r r i s o n . Special appreciation f o r the patience,  understanding,  and h e l p I have r e c e i v e d , goes t o my dear f r i e n d Dr. L o i s Shepherd and  t o my p a r e n t s . T h i s work was s u p p o r t e d by t h e N a t u r a l S c i e n c e s  Engineering Glass.  arid  Research C o u n c i l of Canada, t h r o u g h a grant t o D r .  - 1 -  I.  INTRODUCTION  There i s now c o n s i d e r a b l e e v i d e n c e t o suggest t h a t t h e a c t i v e o r energy dependent uptake o f d i s t i n c t i n o r g a n i c i o n s by the r o o t s o f h i g h e r p l a n t s , i s s u b j e c t t o independent n e g a t i v e feedback c o n t r o l (Cram, 1976).  T h i s c o n t r o l i s thought t o be e l i c i t e d i n  response t o changes of the i n t e r n a l c o n c e n t r a t i o n of the p a r t i c u l a r n u t r i e n t , w h i c h u l t i m a t e l y e x e r t s c o n t r o l over the r o o t uptake process. The i n v e s t i g a t i o n o f t h e mechanism u n d e r l y i n g t h e c o n t r o l of uptake would appear, a p r i o r i , t o be more c o m p l i c a t e d i n t h e cases of m e t a b o l i z e d i o n s , such as SO. 4  2-  , N0„ , H„PO. , e t c . , by v i r t u e 3 2 4  of the d i v e r s i t y of t h e end p r o d u c t s o f t h e i r metabolism.  T h i s may  c o n s e q u e n t l y account f o r t h e more e x t e n s i v e i n v e s t i g a t i o n s o f t h i s a s p e c t o f uptake of n o n - m e t a b o l i z e d i o n s .  Thus, feedback c o n t r o l  mechanisms have been proposed f o r p o t a s s i u m (Humphries, 1951; Johansen e t . a l . , 1970; Young e t . a l . , 1970; G l a s s , 1976; Jensen and P e t t e r s s o n , 1978), N a  +  ( P i t m a n e t . a l . , 1968), C l " (Cram, 1968; 1973; Mott and  Steward, 1972), and B r " ( S u t c l i f f e , 1954; Cseh e t . a l . , 1970).  In  the case o f an i o n w h i c h i s m e t a b o l i z e d , c o n t r o l of i n f l u x might be a c h i e v e d v i a c o n c e n t r a t i o n s of the i o n i t s e l f o r one o f i t s metabolic products.  S u l f a t e uptake f o r example, has been shown t o be  reduced by p r i o r f e e d i n g w i t h the s u l f u r - c o n t a i n i n g amino a c i d s  - 2 -  c y s t e i n e and m e t h i o n i n e (Hart and F i l n e r , 1969; F e r r a r i and Renosto, 1972; Cram, 1976). the i n f l u e n c e of NH^  +  S i m i l a r r e s u l t s have been o b t a i n e d f o r  and amino a c i d s upon n i t r a t e uptake  (Heimer  and F i l n e r , 1970). P l a n t s grown i n low phosphorus  regimes c o n s i s t e n t l y  d i s p l a y e l e v a t e d r a t e s o f phosphate uptake (Humphries, 1951; Bowen, 1970; B a r b e r , 1972; C a r t w r i g h t , 1972; C l a r k s o n e t . a l . , 1978) and a n a l y s i s of t o t a l phosphate c o n c e n t r a t i o n s  i n these p l a n t s  reveals  t h a t i n f l u x and phosphate c o n t e n t a r e n e g a t i v e l y c o r r e l a t e d (Barber,  1972; C l a r k s o n e t . a l . , 1978). L i v i n g organisms might be imagined t o p o s s e s s l o n g - and  s h o r t - t e r m mechanisms by w h i c h t h e y a r e a b l e t o modify t h e i r r a t e s of i o n uptake t h r o u g h biomembranes.  The " c a r r i e r " systems  responsible  f o r t r a n s p o r t a c r o s s ' n a t u r a l membranes a r e thought t o be composed of p r o t e i n m o l e c u l e s ( M i t c h e l l , 1967). degradation  On a l o n g - t e r m b a s i s  "carrier"  o r s y n t h e s i s c o u l d e f f e c t changes o f t r a n s p o r t r a t e s .  A much more r a p i d , d i r e c t e n z y m a t i c c o n t r o l mechanism c o u l d be e l i c i t e d by a l l o s t e r i c c o n t r o l of " c a r r i e r " a c t i v i t y o r energy f o r the s p e c i f i c t r a n s p o r t phenomenon i n q u e s t i o n .  supply  F l u c t u a t i o n of  n u t r i e n t l e v e l s w i t h i n p l a n t s c o u l d a c t as the s i g n a l f o r n u t r i e n t uptake r e g u l a t i o n through t h e mechanisms d e s c r i b e d above.  The p r e s e n t  study i s d i r e c t e d toward a b e t t e r u n d e r s t a n d i n g o f the s i g n a l s and mechanisms w h i c h govern phosphate uptake i n b a r l e y (Hordeum v u l g a r e (L.) c v . Bonanza) .  - 3 -  I n h i g h e r p l a n t s i n t e r n a l orthophosphate c o n c e n t r a t i o n i s much more s u s c e p t i b l e t o f l u c t u a t i o n s i n phosphorus than t h e o r g a n i c phosphorus c o n c e n t r a t i o n 1971).  supply  ( B i e l e s k i , 1968; N a s s e r y ,  Because o f the f l e x i b i l i t y o f the i n o r g a n i c phosphate  l e v e l i t s a b s o l u t e magnitude w i t h i n p l a n t c e l l s r e p r e s e n t s c a n d i d a t e f o r e f f e c t i v e r e g u l a t i o n of phosphate u p t a k e .  a good  With  t h i s i n mind t h e a n a l y s i s of i n o r g a n i c and o r g a n i c phosphate l e v e l s w i t h i n t h e p l a n t was conducted s i m u l t a n e o u s l y  with  measure-  ments o f phosphate uptake r a t e s under d i f f e r e n t e x t e r n a l phosphate regimes.  The p l a n t chosen f o r t h i s s t u d y , namely b a r l e y , i s one  of economic v a l u e and r e p r e s e n t a t i v e o f the c e r e a l crop p l a n t s r a i s e d i n Canada.  - 4 -  II.  1.  Hydroponic P l a n t  A.  Seeds  METHODS  Growth  Seeds o f b a r l e y (llordeum v u l g a r e  ( L . ) c v . Bonanza)  p u r c h a s e d from B u c k e r f i e l d s , Vancouver, were s u r f a c e for  sterilized  t e n minutes i n 1% h y p o c h l o r i t e and a f t e r s e v e r a l washings  w i t h d i s t i l l e d w a t e r were germinated i n sand a t 27±2C.  B.  C h o i c e o f Phosphate L e v e l s and Growth Regimes Many workers i n p r e v i o u s  s t u d i e s have employed  phosphate  l e v e l s w h i c h were u n n a t u r a l l y h i g h ( C a r t w r i g h t , 1972; C l a r k s o n e t . al.,  1978; B i e l e s k i , 1968; B a r b e r , 1967).  I t was c o n s i d e r e d  worth-  w h i l e i n t h e p r e s e n t s t u d y t o use phosphorus l e v e l s which were more r e p r e s e n t a t i v e of s o i l s o l u t i o n phosphorus c o n c e n t r a t i o n s 1973).  These c r i t e r i a  h y d r o p o n i c media.  (Bieleski,  a r e met by a 15uM orthophosphate l e v e l i n  P l a n t s were t h e r e f o r e grown i n e i t h e r f u l l  n u t r i e n t s o l u t i o n (+P) o r n u t r i e n t s o l u t i o n i n w h i c h no phosphorus was s u p p l i e d (-P, see T a b l e 1 ) . Because of t h e l a r g e volume of c i r c u l a t e d growth medium i t was n e c e s s a r y t o m o n i t o r and top up phosphate l e v e l s t o 15yM o n l y t w i c e d a i l y . media were r e p l a c e d i n f u l l e v e r y f o u r days.  Both +P and -P growth P l a n t s were grown i n  an e n v i r o n m e n t a l regime w h i c h c o n s i s t e d o f 16h days a t 27±2C and  - 5 -  -2 i r r a d i a n c e o f 3.0 mW cm , and 8h n i g h t s a t 19±2C.  - 6 -  T a b l e 1.  C o m p o s i t i o n o f h y d r o p o n i c growth media  - Concentrations  a r e uMolar  -P 111.2 KNO„ 3  0.11 MnSO..H„0 4 2  83.3 M g ( N 0 ) 3  27.8 MgS0  3  4  2  4  3  2.7 KC1  0.11 Z n S 0 . 7 H 0 0.03 CuS0 .5H 0  4  55.6 C a ( N 0 )  1.4 H B 0  2  2  0.03 H M o 0 2  2  4  0.11 FeEDTA 3  B u f f e r e d t o pH 6.25 w i t h lOuM N a C i t r a t e : C i t r i c A c i d 3  +P as -P p l u s 5.0 Na HP0 2  10.0 NaH„P0  - 7 -  2.  Determination  A.  Root Wash P e r i o d The  of Phosphate I n f l u x Method  c e l l w a l l or f r e e space of p l a n t c e l l s c o n t a i n sub-  s t a n t i a l i o n r e s e r v o i r s w h i c h p o s s e s s h a l f - l i v e s f o r i o n exchange o f the o r d e r of 1-3 min D a i n t y and Hope, 1959).  (Walker and P i t m a n , 1976;  Cram,  P r i o r to i n f l u x e x p e r i m e n t s of  short d u r a t i o n , designed to estimate  1973; relatively  i n i t i a l plasmalemma f l u x e s ,  i t i s n e c e s s a r y t h e r e f o r e to s t a n d a r d i z e the c e l l w a l l phosphate s t a t u s of r o o t s grown i n d i f f e r i n g phosphate regimes.  Otherwise  the p o s s i b l e t r a n s f e r of phosphorus w i t h i n the r o o t f r e e space to the uptake s o l u t i o n would change the s p e c i f i c a c t i v i t y of phosphate i n the uptake s o l u t i o n and i n t r o d u c e u n c e r t a i n t y t o the c a l c u l a t e d flux. 32 By l o a d i n g the r o o t w i t h 15uM  P - l a b e l l e d orthophosphate  and s u b s e q u e n t l y t r a n s f e r r i n g these r o o t s t o n o n - l a b e l l e d 15uM  ortho-  phosphate a measure of the h a l f - l i f e of c e l l w a l l exchange can 32  be  made.  T h i s was  done by measuring r e l e a s e of  wash medium a t i n t e r v a l s of time up t o 30 min estimated  P t o the n o n - l a b e l l e d after transfer.  v a l u e of the h a l f - l i f e f o r c e l l w a l l i o n exchange  The  can  be used to determine the a p p r o p r i a t e d u r a t i o n of the s t a n d a r d i z i n g prewash. To o b t a i n an e s t i m a t e o f plasmalemma i n f l u x i t i s n e c e s s a r y t o d i s t i n g u i s h the a c t i v e uptake from p a s s i v e a d s o r p t i o n i n the  cell  - 8 -  wall.  The h a l f - l i f e f o r c e l l w a l l exchange  can be used h e r e t o  o b t a i n the n e c e s s a r y s e p a r a t i o n .  B.  Phosphate  Influx  Rates of orthophosphate uptake were determined a f t e r a 5 min prewash i n 50yM CaSO^ a t 30C.  The uptake s o l u t i o n  was  32 i d e n t i c a l t o the +P growth medium w i t h a c i d and/or  RbCl,, added.  P-labelled orthophosphoric  E i t h e r 10 min ( s h o r t - t e r m ) or 24h (long„  term) uptake p e r i o d s were used.  I n a l l but one e x p e r i m e n t the  uptake s o l u t i o n was v i g o r o u s l y s t i r r e d and a e r a t e d . performed a t 30C i n s o l u t i o n volumes t i o n was n e g l i g i b l e .  Uptakes were  (1.6 1) where n u t r i e n t d e p l e -  The uptake p e r i o d was  followed immediately  by a 5 min d e s o r p t i o n p e r i o d i n c o l d +P s o l u t i o n at 4C.  Thereafter  p l a n t samples were spun i n a b a s k e t c e n t r i f u g e t o remove e x t r a neous w a t e r .  These samples were w e i g h e d " i n t o g l a s s v i a l s t o o b t a i n  f r e s h w e i g h t s and f i n a l l y ashed a t 500C. were d i s s o l v e d i n 10 ml d i s t i l l e d H^O  The r e s u l t a n t ashed  and t h e i r r a d i o a c t i v i t y  samples was  determined by Cerenkov c o u n t i n g ( L a u c h l i , 1969; G l a s s , 1978a) w i t h an Isocap/300 l i q u i d s c i n t i l l a t i o n c o u n t e r .  - 9 -  3.  E f f l u x Analysis E f f l u x d e t e r m i n a t i o n was performed a f t e r f e e d i n g p l a n t s  i n a constant  32 P/P +P n u t r i e n t s o l u t i o n f o r f i v e days.  These  p l a n t s were t h e n p l a c e d i n n o n - l a b e l l e d +P s o l u t i o n s a t 30C and _2 an i r r a d i a n c e l e v e l o f 3.0 mW cm uncomplicated  .  I n order to estimate  by p l a n t r e a b s o r p t i o n of i s o t o p e , s t a n d a r d  efflux procedure  i s t o r e p l a c e the e f f l u x medium w i t h f r e s h n o n - l a b e l l e d s o l u t i o n at r e g u l a r i n t e r v a l s .  By t h i s methodology i s o t o p i c f l u x from t h e  medium back t o the c y t o p l a s m  i s presumed t o be z e r o .  of the e f f l u x a n a l y s i s t o t a l phosphate content  At t h e end  as w e l l as i s o t o p i c  content  remaining  i n t h e r o o t t i s s u e , was d e t e r m i n e d .  enabled  subsequent c a l c u l a t i o n s o f the p a t t e r n o f e f f l u x  t o s t a n d a r d methodology (Walker and P i t m a n , 1976).  This according  - 10 -  4.  D e t e r m i n a t i o n o f the P l a n t s ' Phosphate C o n c e n t r a t i o n s  A.  T o t a l Phosphate C o n c e n t r a t i o n s F r e s h r o o t and shoot samples were weighed,  ashed,  d i s s o l v e d i n 10ml d i s t i l l e d w a t e r , and assayed f o r t o t a l phosphorus by the method of E i b l and Lands (1969).  Values e x p r e s s e d  through-  out the t e x t a r e i n umol/g.f.wt.  B.  I n o r g a n i c Phosphate C o n c e n t r a t i o n s I n o r g a n i c phosphorus p o o l s of b o t h shoots and r o o t s were  o b t a i n e d by a v a r i a t i o n of the method of Daley and V i n e s  (1977).  Samples were plunged i n t o b o i l i n g w a t e r f o r two min, t h e n were r a p i d l y f r o z e n , thawed, and p l a c e d i n b o i l i n g b a t h s f o r 5 min, twice i n succession.  U s i n g samples of glucose-6-P and ATP,  e s t a b l i s h e d t h a t t h i s method,as c l a i m e d by H u l e t t ( 1 9 7 0 ) , no h y d r o l y s i s of o r g a n i c P bonds.  i t was caused  - 11 -  5.  Double L a b e l l e d Uptake D e t e r m i n a t i o n 32  86  P and  Rb were used s i m u l t a n e o u s l y t o determine  phosphate and p o t a s s i u m i n f l u x e s ( L a u c h l i and E p s t e i n , 19 70) respectively.  The s e p a r a t i o n of t h e s e i s o t o p e s was o b t a i n e d by  anion-exchange chromatography o f t h e ashed samples.  Dowex-lx8-100  was primed i n one hundred times i t s volume of 5M NaOH.  2 ml P a s t e u r  p i p e t t e columns were then poured and washed t h r e e times w i t h d e i o n i z e d water.  2 ml o f wet Dowex-lx8-100 r e s i n has an exchange c a p a c i t y o f  2.8 m i l l i - a q u i y a l e n t s .  By t h e means d e s c r i b e d i n T a b l e 2 a s u i t a b l e  regime was d e f i n e d f o r use on b i o l o g i c a l samples. 86 of  The s e p a r a t i o n  32 Rb and  P by t h i s method was e q u a l l y e f f e c t i v e whether t h e s e  i s o t o p e s were p r e s e n t i n c a r r i e r s - f r e e s o l u t i o n s o r i n s o l u t i o n s more r e p r e s e n t a t i v e o f t h e n a t u r a l d i s t r i b u t i o n of p o t a s s i u m and phosphate.  Samples were counted b e f o r e and a f t e r exposure t o 86 32 t h e d e f i n e d regime. I n i t i a l counts gave e s t i m a t e s o f Rb + P activity. C o u n t i n g o f t h e e l u a t e o b t a i n e d a f t e r anion-exchange 86 32 chromatography gave Rb a c t i v i t y . P counts were o b t a i n e d by sub86 32 t r a c t i n g t h e l a t t e r a c t i v i t y from t h e combined activity.  Rb +  P radio-  - 12 -  T a b l e 2.  D e t e r m i n a t i o n o f regime r e q u i r e d  86  f o r elution of a l l  32 Rb and r e t e n t i o n  of s o l u t i o n s  of a l l  P from anion-exchange columns.  shown (A t o F) were a p p l i e d  e l u a t e c o l l e c t e d 1. w i t h o u t f u r t h e r e l u t i o n w i t h 10 ml H^O.  10 ml  t o the columns and  column w a s h i n g .  2. a f t e r  3. a f t e r e l u t i o n w i t h a f u r t h e r 10 m l  of H 0 . 2  E f f i c i e n c y of e l u t i o n of  Kb o r  of known counts a p p l i e d  8 6  Treatment  P e x p r e s s e d as % t o column  Rb  A. C a r r i e r - f r e e  B. +0.1M K  1.  75.5  80.7  81.6  2.  24.0  20.3  18.4  3.  0.5  n i l  C. + 0 . 1 M K +  3 2  P + 2.0mM P  n i l  32 ' D. C a r r i e r - f r e e  E. +2.0mM P  F. +2.0mM P +  8 6  R b + 0.1M K  1.  n i l  n i l  n i l  2.  n i l  n i l  n i l  3.  n i l  n i l  n i l  - 13 -  6.  D e t e r m i n a t i o n of Root E x t e r n a l P r o t e i n The e o s i n p r o t e i n s t a i n i n g t e c h n i q u e of W i l l i a m s  (1962) was  used to determine r o o t e x t e r n a l p r o t e i n .  i s p r e s e n t e d i n T a b l e 3.  The  procedure  - 14 -  T a b l e 3.  Eosin s t a i n i n g technique  Treatment  D u r a t i o n (min)  1.  Weighing of r o o t samples  2.  2 d i s t i l l e d water r i n s e s  3.  0.1 N HC1  4.  5 d i s t i l l e d water r i n s e s  5.  0.2% e o s i n  6.  5 d i s t i l l e d water r i n s e s  7.  0.1 M KC1 + K0H  1.0  0.5  (pH =  13.0) 3 m l . volume 8.  O p t i c a l D e n s i t y measurement a t 520  nm.  0.5  III.  1.  RESULTS  AND  DISCUSSION  E f f e c t of Phosphate L e v e l on Growth F r e s h w e i g h t s of b a r l e y p l a n t s were monitored, f o r a 20  day growth p e r i o d (see F i g u r e 9 ) . +P  There were no d i f f e r e n c e s between  and -P s e e d l i n g s up t o day 11 (a = 0.05), beyond w h i c h s t a g e the  f u l l y nourished  p l a n t s e x h i b i t e d e x p o n e n t i a l growth r a t e s w h i l e  the  phosphate s t a r v e d p l a n t s i n c r e a s e d t h e i r mass at a l i n e a r r a t e (Treatments s i g n i f i c a n t l y d i f f e r e n t at 12 days and o l d e r , a = see T a b l e 4 ) .  By day 20 the r a t i o of +P  t o -P p l a n t w e i g h t s  i n excess of two but no q u a l i t a t i v e m o r p h o l o g i c a l apparent between the t r e a t m e n t s .  The  0.05, was  d i f f e r e n c e s were  diameters of r o o t s i n the  and -P p l a n t s are shown i n F i g u r e s 1 t o 8.  +P  Phosphate s t r e s s has  been p r e v i o u s l y shown t o r e s u l t i n decreased r o o t d i a m e t e r s (Bowen et.  a l . , 1974).  Increased  r o o t - h a i r development became apparent i n  -P r o o t s o n l y w e l l a f t e r day  20 (see F i g u r e s 7 and  8).  Beyond day 13 -P p l a n t s demonstrated s t a t i s t i c a l l y cant (a = 0.05)  root:shoot  compared t o +P p l a n t s .  r a t i o i n c r e a s e s (see F i g u r e 10) when  By day 20 f o r example, -P p l a n t s had  r o o t growth t o such an e x t e n t t h a t the r o o t : s h o o t compared t o 0.5  f o r +P p l a n t s .  may  be  favoured  r a t i o equalled  T h i s p r e f e r e n t i a l growth e n a b l e d  -P p l a n t s t o form almost as much r o o t mass as the f u l l y p l a n t s (Figure 11).  signifi-  the  nourished  I n the s o i l environment where phosphate  l i m i t e d and d e p l e t i o n zones s h a r p l y l o c a l i z e d due  2  supply  t o the  - 16 -  T a b l e 4.  A.  +P and -P growth e q u a t i o n s  +P e x p o n e n t i a l growth e q u a t i o n from day 5 t o 20,  y = 0.1072e  B.  0 , 1 7 4 3 x  ,  r = 0.9967  -P l i n e a r growth e q u a t i o n from days 12 t o 20.  y = -1.1667 + 0.13983x,  y = plant size  r = 0.9679  (g.f.wt./plant)  x = age o f p l a n t s (days)  - 17  r  r e l a t i v e i m m o b i l i t y of phosphate, i n c r e a s e d s u r f a c e a r e a and  soil  e x p l o r a t i o n would be an a d a p t i v e advantage ( H a r l e y , 1969). Growth s t u d i e s performed w i t h numerous s p e c i e s under v a r i o u s phosphorus regimes have shown p o s i t i v e c o r r e l a t i o n s growth and P^ s u p p l y .  between  The magnitude of growth response i s depen-  dent upon the s p e c i e s i n v o l v e d ( C l a r k s o n , 1967; P i g g o t t ,  1971;  R o r i s o n , 1968; Asher and Loneragan, 1967; Bradshaw e t . a l . , 1960). Root t o shoot r a t i o s o f t e n i n c r e a s e i n p l a n t s grown i n low phosphate environments ( H a c k e t t , 1968; Asher and Loneragan, -  i s not always the case (Troughton, 1977).  1967), b u t t h i s  I n c r e a s e d r o o t growth,  however, might have o c c u r r e d i f Troughton had used l o w e r l e v e l s of phosphorus  (Bradshaw e t . a l . , 1960).  The r o o t : s h o o t r a t i o s of  a number of g r a s s e s grown a t d i f f e r e n t P - l e v e l s a r e comparable t o the  b a r l e y data obtained (Figure 10).  Because r o o t - h a i r f o r m a t i o n  i s r e t a r d e d i n h y d r o p o n i c c u l t u r e ( B o l e , 1973) , i t i s of p a r t i c u l a r i n t e r e s t t h a t r o o t - h a i r s were observed t o develop under s e v e r e P - d e f i c i e n c y and o n l y once has t h i s phenomenon been p r e v i o u s l y r e p o r t e d ( B r e w s t e r e t . a l . , 1976).  Root-hairs enable p l a n t s to  i n c r e a s e t h e i r P-uptake from the s o i l environment where a v a i l a b l e phosphate i s s t r o n g l y l o c a l i z e d and d i f f u s i o n i s o f t e n l i m i t i n g the  uptake p r o c e s s ( B a r l e y and R o v i r a , 1970).  - 18 -  F i g u r e 1.  5 day o l d r o o t t i p s  F i g u r e 2.  10 day o l d r o o t t i p s  (8 times  (8 times  life-size)  life-size)  Figure  4.  20 day o l d r o o t t i p s (8 times  life-size)  - 20 -  F i g u r e 5.  F i g u r e 6.  25 day o l d r o o t t i p s  (10 times  life-size)  30 day o l d r o o t t i p s , +P above, -P below C26 times l i f e - s i z e )  - 21  Figure  8.  -  40 day o l d -P r o o t t i p (30 times  life-size)  - 22 -  F i g u r e 9. P l a n t growth v s .  5  age  10  15 AGE  (days)  20  - 23 -  F i g u r e 10.  Root: shoot r a t i o v s . p l a n t  10  age  15  Age  (days)  20  - 24 -  F i g u r e 11.  Growth of r o o t s  1.5r  A  +P  A  -P  A A  A ' A  A A  A  A  ^  A  A  A  A A  10  15 AGE  (days)  20  - 25 -  2.  C h a r a c t e r i z a t i o n of Phosphate  A.  D e t e r m i n a t i o n o f Root Wash P e r i o d The k i n e t i c s of  Influx  32 P r e l e a s e from r o o t s exposed t o l a b e l l e d  medium f o r 10 min a r e shown i n F i g u r e 12.  G r e a t e r than 96% of  32 the r a p i d l y - e x c h a n g i n g  P f r a c t i o n had e f f l u x e d by 5 min.  In  subsequent e x p e r i m e n t a t i o n t h e r e f o r e , a 5 min wash p e r i o d was adopted as s t a n d a r d p r o c e d u r e wherever i t was n e c e s s a r y t o e s t i m a t e 32 i n t r a c e l l u l a r , as opposed t o e x t r a c e l l u l a r phosphate o r content.  P-phosphate  F i g u r e 12. E f f l u x o f 32p f 550  r  o  m  R  a  p  id  Exchange Phase  P  e  Time (min)  - 27 -  B.  Temperature  and pH Optimums.  T r a n s p o r t of phosphate i n t o the symplasm from the e n v i r o n ment i s p r o b a b l y an e n z y m a t i c p r o c e s s ( E p s t e i n , 1976) and the " c a r r i e r s " w h i c h c a t a l y z e v e c t o r i a l phenomena a r e t h e r e f o r e s u s c e p t i b l e t o changes i n e n v i r o n m e n t a l temperature and pH.  These p h y s i c a l p a r a -  meters c o u l d f e a s i b l y a l t e r the r a t e of phosphate uptake by m o d i f y i n g the m o l e c u l a r c o n f i g u r a t i o n of s p e c i f i c enzymes o r by an e f f e c t on the g e n e r a l energy m e t a b o l i s m i n t o t o ( B o y e r , 1970).  The  temperature a t w h i c h b a r l e y r o o t s e x p r e s s t h e i r optimum P-uptake r a t e i s a p p r o x i m a t e l y 30C (see F i g u r e 13).  The optimum pH f o r  phosphate uptake w i t h i n a r e a s o n a b l e s o i l range of 5.0 6.25  ( F i g u r e 14).  t o 7.5  was  P l a n t s i n g e n e r a l p r e f e r e n t i a l l y absorb mono-  v a l e n t i o n s o v e r d i v a l e n t or t r i v a l e n t i o n s ( E p s t e i n , 19 76) and Hagen and Hopkins (1955) have h y p o t h e s i z e d t h a t phosphate uptake i s d i r e c t l y p r o p o r t i o n a l t o the amount of monovalent p r e s e n t i n t h e medium as determined by medium pH.  orthophosphate A l t h o u g h pH does  2c o n t r o l H^PO^  :HP0^  r a t i o s , t h i s r e l a t i o n s h i p d i d not appear t o  be the o n l y e f f e c t of pH upon orthophosphate uptake i n t h e p r e s e n t study.  Dunlop and B o w l i n g ' s (1978) s t u d y w i t h w h i t e c l o v e r i n d i -  c a t e d t h a t pH may  a f f e c t phosphate uptake i n ways o t h e r than i t s  c o n t r o l on the a b s o l u t e amount of a v a i l a b l e monovalent  orthophosphate.  A l l phosphate f l u x d e t e r m i n a t i o n s were s u b s e q u e n t l y performed at  30C i n media b u f f e r e d t o pH = 6.25 by 10uM N a ^ c i t r a t e and  acid.  citric  - 28 -  Figure 14. 6.0 r  5  p  ?  5  H  Curve of Phosphate Uptake at 30C  .6.0  6.5  7.0  7.5  8.0  8.5  - 29  3.  ~  Phosphate E f f l u x K i n e t i c s The  e f f l u x k i n e t i c s of w e l l n o u r i s h e d  Bonanza b a r l e y p l a n t s are shown i n F i g u r e 15.  f i v e day The  data  old revealed  a r a t h e r s t r a i g h t f o r w a r d s e p a r a t i o n i n t o t h r e e phases of e f f l u x . Considerations for  of the s i z e and  the k i n e t i c c o n s t a n t s  of exchange  t h e s e phases have l e d workers t o the c o n c l u s i o n t h a t t h e s e  phases r e p r e s e n t  a s e r i e s arrangement of the c e l l w a l l ,  and v a c u o l a r f r a c t i o n s (Walker and P i t m a n , 1976).  Most work i n  r o o t i o n exchange p r e v i o u s l y p u b l i s h e d , however, has n o n - m e t a b o l i z e d i o n s such as K , +  W a l k e r and P i t m a n , 1976).  Na , +  dealt with  C l , e t c . (Cram,  A p r i o r i i t was  cytoplasm,  1973;  a n t i c i p a t e d that  k i n e t i c s of phosphate e f f l u x would be complex. P^ f r a c t i o n be e x p e c t e d to show the s t a n d a r d  Not  the  o n l y would the  triphasic pattern  but o r g a n i c f r a c t i o n s c o u l d be e x p e c t e d t o o v e r l a y t h i s b a s i c form. By  c o n t r a s t the s t r i k i n g ' s i m i l a r i t y t o p r e v i o u s l y  k i n e t i c s l e a d t o the c o n c l u s i o n t h a t the observed k i n e t i c s the exchange of a s i n g l e P s p e c i e s - most p r o b a b l y phate s i n c e under the p r e s e n t the major P - f r a c t i o n .  reported describe  i n o r g a n i c phos-  c o n d i t i o n s of growth i t would  represent  Furthermore the e n z y m a t i c r e a c t i o n s w h i c h  would r e l e a s e l a b e l l e d orthophosphate from m e t a b o l i z e d p r o b a b l y n o t l i m i t i n g exchange.  forms are  I f such r e a c t i o n s were r e s t r i c t i n g  e f f l u x the exchange p r o c e s s would almost c e r t a i n l y be much more complex.  As such i t i s r e a s o n a b l e  represent  c e l l w a l l , cytoplasmic  t o b e l i e v e t h a t the t h r e e phases  and v a c u o l a r exchange of  P^.  - 30 -  C a l c u l a t i o n o f t h e P-content o f t h e f r a c t i o n w i t h t h e l o w e s t exchange (see F i g u r e 1 5 ) .  r a t e gave a maximum p o o l s i z e o f 11.97ymol/g.f.wt. A l t h o u g h t h i s v a l u e i n c l u d e s non-exchanging  components such as s t a b l e DNA ( B i e l e s k i , 1973), t h e m a j o r i t y o f the e x c h a n g i n g phosphate p r o b a b l y a r i s e s from t h e v a c u o l e s o f c e l l s and t h e r o o t xylem i f a comparison can be made, t o nonm e t a b o l i z e d i o n s ( J e s c h k l e , 1973).  This estimate i s e n t i r e l y  c o n s i s t e n t w i t h the l i t e r a t u r e v a l u e s w h i c h p l a c e v a c u o l a r P - l e v e l s i n t h i s range ( B i e l e s k i , 1973). The second f a s t e s t e x c h a n g i n g phase has a magnitude o f 1.27ymol/g j f , wt. and an exchange h a l f - l i f e o f a p p r o x i m a t e l y 45 min #  w h i c h i s comparable t o t h e h a l f - l i f e o f c y t o p l a s m i c exchange f o r o t h e r i o n s (Walker and P i t m a n , 1976). The f r e e space phase c o n t a i n e d 2.38ymol/g,jf..wt. arid had a h a l f - l i f e o f a p p r o x i m a t e l y 2 min w h i c h i s a g a i n s i m i l a r .to v a l u e s o b t a i n e d f o r o t h e r i o n s (Walker and P i t m a n , 1976).  These d a t a  p r o v i d e c o n f i r m a t i o n t h a t a f i v e min wash p e r i o d i s s u f f i c i e n t t o 32 r e l e a s e t h e b u l k o f f r e e space  P, as w e l l as v a l i d a t i n g t h e use  of a 10 min f l u x p e r i o d t o e s t i m a t e plasmalemma i n f l u x . These t h r e e phases a r e c o n s i s t e n t w i t h the b e l i e f t h a t o n l y a s i n g l e phosphate s p e c i e s i s b e i n g exchanged  and t h a t compart-  mental b a r r i e r s a r e the plasmalemma and t o n o p l a s t .  The terms " p o o l "  and "compartment" as d e f i n e d by Oaks and B i d w e l l (1970) denote the s i t u a t i o n where d i f f e r e n t p o r t i o n s o f a compound a r e m e t a b o l i c a l l y  - 31  F i g u r e 15.  1.20  1.05  ^  T o t a l phosphate e f f l u x  kinetics  r  I  1  0.0  *  «.  1.0  * 2.0  *  •  ' 3.0 Time  • 4.0 (h)  I  I  5.0  —I 6.0  i I  7.0  - 32 -  i s o l a t e d from one a n o t h e r , whether o r n o t t h i s i s due t o t h e i r physical separation i n c e l l organelles.  Smith (1966) has shown t h a t  i n excess of 90% o f t h e s o l u b l e p h o s p h a t e - e s t e r s such as s u g a r phosphates  are present i n the cytoplasm.  The t e r m i n a l P-groups of  ATP t u r n over w i t h a h a l f - l i f e of 2-20 s e c and most P - e s t e r s have h a l f - l i v e s o f l e s s than 30 min ( B i e l e s k i , 1968; Johnson and B l u f f , 1967; Loughman, 1960; W e i g l , 1963).  I n a d d i t i o n i f as has been  s u g g e s t e d , t h e g r e a t e r p r o p o r t i o n o f phosphate  i s i n t h e form of  o r t h o p h o s p h a t e , i t i s n o t u n r e a s o n a b l e t o assume t h a t t h e magnitude of P - e s t e r s would n o t l i m i t t h e exchange o f P_^ a t t h e membrane l e v e l . Very l i t t l e i s known about the t u r n o v e r of P l i p i d s and RNA w h i c h c o n t a i n over 75% o f t h e m e t a b o l i z e d phosphate 19 73).  i n plants ( B i e l e s k i ,  A l t h o u g h t h e h a l f - l i v e s o f these compounds may v a r y c o n s i d e r -  a b l y , i f t h e magnitude o f 32 h i g h e r t h a n t h a t of  32 32 P-ester to P. c o n v e r s i o n i s many f o l d 32  P - l i p i d s or  P-RNA, then t h e l a t t e r ' s c o n t r i -  b u t i o n t o t h e c y t o p l a s m i c exchange phase would be n e g l i g i b l e . 32 i s n o t e x p e c t e d t o c o n t r i b u t e any exchangable  P.  DNA  RNA and P - l i p i d  t u r n o v e r c o u l d be o f importance i n t h e s l o w e s t exchange phase, however t h e l a r g e v a c u o l a r P^-content this  ( B i e l e s k i , 1973) would  render  unlikely. I t i s e n c o u r a g i n g t o observe e s s e n t i a l l y s i m i l a r p a t t e r n s  of phosphate ions.  e f f l u x t o those d e s c r i b i n g t h e e f f l u x of n o n - m e t a b o l i z e d  The h a l f - l i f e o f c y t o p l a s m i c exchange may be of i m p o r t a n c e i n  s h o r t - t e r m r e g u l a t i o n of phosphate  uptake p a r t i c u l a r l y i n s e v e r e l y  -  33  -  P - d e f i c i e n t p l a n t s where the v a c u o l e i s no l o n g e r a b l e t o s u p p l y P^ to the cytoplasm  ( B i e l e s k i , 1968; C r o s s e t t and Loughman, 1966;  Greenway and K l e p p e r , 1968).  -  4.  34  -  Enhancement of Phosphate Uptake Rate by Phosphate Deprivation  A.  Phosphate Uptake As f u l l y n o u r i s h e d  b a r l e y s e e d l i n g s aged t h e i r phosphate  uptake r a t e s on a p e r gram f r e s h w e i g h t b a s i s d e c l i n e d . trend occurred  A  similar  i n -P grown p l a n t s u n t i l they were 12 days o l d  F i g u r e 1 6 ) , a t w h i c h time a r a p i d i n c r e a s e or enhancement of g a n i c phosphate uptake was  i n i t i a t e d i n the low P p l a n t s .  16 the uptake r a t e of -P p l a n t s was  6.3  times t h a t of +P  (see inor-  By  day  plants,  however w i t h i n two days a d r a m a t i c d e c l i n e became e v i d e n t . P l a n t s exposed t o low amounts of phosphate have o f t e n been shown t o e x p r e s s enhanced uptake r a t e s ( B a r b e r , 1972;  1972;  Cartwright,  C l a r k s o n e t . a l . , 1978), however the phosphate l e v e l s employed  p r e v i o u s l y were h i g h i n comparison t o t h i s s t u d y . colleagues  Clarkson  and  his  (1978), f o r example, s u p p l i e d b a r l e y p l a n t s w i t h 150uMe...  phosphate f o r a p e r i o d of seven days b e f o r e p l a n t s t o phosphate minus s o l u t i o n s . by t h e s e r e s e a r c h e r s  As s u c h , uptake r a t e s  were i n some i n s t a n c e s  lower t h a n the p r e s e n t r e s u l t s . c o u l d almost be d e s c r i b e d  transferring his  -P obtained  an o r d e r of magnitude  4-P p l a n t s i n the p r e s e n t  studies  as -P by comparison w i t h the former s t u d i e s .  These r e s u l t s i n d i c a t e the e x t e n t of the r e g u l a t o r y response to P-status.  The  p r e s e n t c h o i c e of 15yM  P, as p r e v i o u s l y s t a t e d , was  attempt t o s i m u l a t e more n a t u r a l s o i l c o n d i t i o n s .  an  - 36 -  C l a r k s o n e t . a l . (1978) o b t a i n e d  four f o l d differences  i n uptake between P - d e f i c i e n t and f u l l y n o u r i s h e d per gram f r e s h w e i g h t b a s i s .  t r e a t m e n t s on a  S i m i l a r l y Cartwright's  (1972) work  y i e l d e d a 2.5 f o l d i n c r e a s e i n t h e uptake r a t e o f -P p l a n t s . When P^-uptake p e r p l a n t i s p l o t t e d (see F i g u r e 17) s i m i l a r r a t e s a r e w i t n e s s e d f o r +P and -P p l a n t s a t younger ages, b u t at day 13 d r a m a t i c i n c r e a s e s i n uptake a b i l i t i e s were i n i t i a t e d i n both treatments.  On a p e r p l a n t b a s i s r e d u c t i o n i n uptake r a t e s  as seen i n t h e p e r gram f r e s h w e i g h t p r e s e n t a t i o n not occur.  ( a f t e r day 16) d i d  R a t h e r t h e r a t e s reached a maximum v a l u e a t day 16,  w h i c h was s u s t a i n e d u n t i l t h e t e r m i n a t i o n o f t h e experiment a t day 20.  Throughout  t h e p e r i o d from day 16 t o 20 t h e r a t i o o f uptake  r a t e s (-P/+P) remained s t e a d y a t 4. The response o f b a r l e y p l a n t s t o l o w phosphorus l e v e l s (-P) i n h y d r o p o n i c c u l t u r e c l e a r l y shows t h e i r a b i l i t y t o r e g u l a t e phosphate uptake r a t e s i n r e l a t i o n t o phosphate s t a t u s .  The enhance-  ment c u r v e o f t h e -P p l a n t s ' uptake r a t e s on a p e r gram f r e s h w e i g h t b a s i s c a n be d i v i d e d i n t o t h r e e r e g i o n s ; a l a g s t a g e , an enhancement s t a g e , and a d e c l i n e s t a g e .  The l e n g t h o f t h e l a g and r a p i d i t y o f  the enhancement s t a g e might be dependent on t h e b a l a n c e o f n u t r i e n t s t o r e s w i t h i n t h e seeds used.  Changes i n t h e phosphate uptake r a t e s  can be a t t r i b u t e d t o p h y s i o l o g i c a l p r o p e r t i e s o f t h e p l a n t s i n c e t h e uptakes were performed under c o n d i t i o n s where phosphate was n o t l i m i t i n g t h e phosphorus s u p p l y 1971).  diffusion  t o t h e r o o t s ( P o l l e and Jenny,  FIGURE 17. Pj Uptake vs. Plant A g e 1.0  I  c 0.5  to ~*4  c/>  o E  5  7  9  11 13 AGE (Days)  -L  1  15  1  tJ.  17  1  1  19  I t  21  - 38 -  I t i s i n t e r e s t i n g t o compare t h e d i f f e r e n c e s i n t h e time s c a l e o f response i n t h i s experiment t o t h o s e e x h i b i t e d by K-uptake r a t e s i n b a r l e y i n response t o K - d e p r i v a t i o n  ( G l a s s , 1975),  In  the l a t t e r experiments t r a n s f e r of p l a n t s from K - s u f f i c i e n t t o Kminus s o l u t i o n s produced i n c r e a s e d  i n f l u x w i t h i n hours.  This  may be a t t r i b u t e d t o t h e major o s m o t i c f u n c t i o n o f p o t a s s i u m . Enzyme a c t i v a t i o n r e q u i r e m e n t s have been p o s t u l a t e d  t o be i n t h e  range o f 5-10uM, whereas o s m o t i c r e q u i r e m e n t s n e c e s s i t a t e up t o 100-200mM K.  T h i s l a r g e demand f o r K by c o n t r a s t t o t h e r e l a t i v e l y  low l e v e l s o f P r e q u i r e d may account f o r t h e extreme r e s p o n s i v e n e s s i n t h e c o n t r o l of K-uptake.  The r a p i d i t y o f r e g u l a t i o n i n t h e case  o f K has l e d t o t h e p r o p o s a l o f an a l l o s t e r i c c o n t r o l ( G l a s s , 1976; Pettersson  and J e n s e n , 1978).  The time s c a l e o f t h e P-response  makes i t d i f f i c u l t t o a s s e s s t h e i m p o r t a n c e o f a l l o s t e r i c as opposed to t r a n s c r i p t i o n a l r e g u l a t i o n . On a p e r gram f r e s h w e i g h t b a s i s t h e r e was a d e c l i n e s t a g e i n P-uptake r a t e s .  T h i s may be a t t r i b u t e d t o a r a p i d d e c l i n e  i n v i g o u r o f t h e p l a n t s r e s u l t i n g from P - d e p r i v a t i o n  although, a t  t h i s s t a g e no o b v i o u s s i g n s o f P - d e f i c i e n c y , o t h e r than reduced growth by comparison t o +P p l a n t s , were apparent.  The mechanism  i n v o l v i n g t h i s response might be e i t h e r p h y s i o l o g i c a l e.g. a t e r m i n a t i o n o f " c a r r i e r " s y n t h e s i s , e t c . , o r m o r p h o l o g i c a l e.g. an i n c r e a s e d r e l a t i v e r o o t growth.  Fresh weight a n a l y s i s i n d i c a t e s  t h a t t h e r o o t s of -P p l a n t s were a c t i v e l y growing d u r i n g t h a t time  - 39 -  p e r i o d a f t e r P-uptake per p l a n t had  reached i t s maximum l e v e l .  s u g g e s t s t h a t the v i g o u r of the p l a n t was t h a t m e t a b o l i c energy was processes.  This  not s e v e r e l y retarded  and  a v a i l a b l e f o r numerous b i o c h e m i c a l  I t i s u n l i k e l y t h a t t h e r e would have been an energy  l i m i t a t i o n upon the a c t i v e uptake p r o c e s s e s  and t h e r e f o r e  d e c l i n e i n P-uptake per gram f r e s h weight was  probably  the  the  result  of a c e s s a t i o n i n n e t " c a r r i e r " s y n t h e s i s . On a per p l a n t b a s i s the l a g , enhancement, and o f f s t a g e s are of i d e n t i c a l d u r a t i o n i n b o t h +P These simultaneous  occurrences  and -P  levelling treatments.  r e v e a l t h e i r developmental o r i g i n  d i s t i n c t and independent of the phosphorus s t a t u s .  The  as  magnitude  of the enhancement of P-uptake, however, c o u l d be a t t r i b u t e d t o a c r i t i c a l l e v e l of P^ and/or one or more of i t s numerous m e t a b o l i t e s . Growth p a t t e r n s a l r e a d y p r e s e n t e d +P  showed d i v e r g e n c e  between  and -P grown p l a n t s a t the p e r i o d between days 11 and 13 w h i c h i s  c o n c u r r e n t w i t h the d i v e r g e n c e  i n phosphate uptake r a t e s between  the two t r e a t m e n t s .  the m o r p h o l o g i c a l  Therefore  responses r e v e a l e d  through d i s t i n c t growth p a t t e r n s are p a r a l l e l e d by p h y s i o l o g i c a l d i f f e r e n c e s i n orthophosphate uptake.  Although  phosphate d e f i c i e n c y  has o f t e n been shown t o r e s u l t i n e l e v a t e d P-uptake r a t e s (Humphries, 1951;  Barber,  1972;  C a r t w r i g h t , 1972;  1978), such s i m u l t a n e o u s  Bowen, 1970;  Clarkson et. a l . ,  p h y s i o l o g i c a l and m o r p h o l o g i c a l  phosphate s t a t u s have not been p r e v i o u s l y r e p o r t e d .  e f f e c t s of  - 40 -  B.  T o t a l Phosphate and I n o r g a n i c Phosphate L e v e l s w i t h i n the P l a n t s . A d e c l i n e i n t o t a l phosphate c o n t e n t of -P r o o t s on a p e r  gram f r e s h w e i g h t b a s i s , as e x p e c t e d , d i d o c c u r as the s e e d l i n g s aged ( F i g u r e 18).  Such a d e c l i n e was  i n o r g a n i c phosphate p o o l ( c o n f i d e n c e pool represented  barley  not e v i d e n t  i n the  l e v e l a = 0.05), a l t h o u g h t h i s  but a s m a l l p e r c e n t a g e of the t o t a l P.  The  of P - d e f i c i e n t p l a n t s dropped i n phosphorus c o n t e n t by over h a l f between days 11 and (see F i g u r e 19).  The  12, and  a subsequent s t e a d y d e c l i n e  followed  between days 9  11. The  constant  i n o r g a n i c phosphate c o n t e n t of -P r o o t s remained  w h i l e a s t e a d y d e c l i n e i n t o t a l phosphate was  out the i n v e s t i g a t i o n p e r i o d . was  one  i n o r g a n i c phosphorus amounts i n -P shoots a l s o  dropped s i g n i f i c a n t l y (a = 0.05), but t h i s o c c u r r e d and  shoots  present through-  At as e a r l y as day 5 the P^-content  at i t s minimum l e v e l and s i n c e P. i s c o n s i d e r e d  the most f l e x i b l e  l  of P-pools i n h i g h e r p l a n t s ( B i e l e s k i , 1973) t h a t the P . - l e v e l had  i t i s not s u r p r i s i n g  reached a minimum b e f o r e  r o o t o r g a n i c phosphate was  evident.  The  the d e c l i n e i n the  subsequent d e c l i n e i n  organic  phosphate c o u l d be r e s p o n s i b l e f o r the enhancement of phosphate u p t a k e , however i t i s i m p o s s i b l e  t o d i s c e r n t o what e x t e n t a l l o s t e r i c  t r a n s c r i p t i o n a l p r o c e s s e s were i n v o l v e d .  Obviously  no  and  allosteric  c o n t r o l c o u l d be a t t r i b u t e d t o the i n t e r n a l orthophosphate whose amount was  e s s e n t i a l l y constant  throughout the e x p e r i m e n t .  Discounting  the  - 41  ^  p o s s i b i l i t y of d e v e l o p m e n t a l uptake p r o c e s s e s which, would be independent o f t h e p l a n t s ' P'-status, the t i m i n g of t h e enhancement of phosphate uptake must o f n e c e s s i t y be c o n t r o l l e d by t h e l e v e l s of one o r more o r g a n i c phosphates.  Although p o s i t i v e  correlations  between phosphate a b s o r p t i o n and ATP l e v e l s have been r e p o r t e d i n h i g h e r ^ p l a n t s ( L i n and Hanson, 1974) n e g a t i v e c o r r e l a t i o n s between uptake and o r g a n i c phosphate c o n t e n t of r o o t s have n o t .  On^fche  o t h e r hand, as a l r e a d y d i s c u s s e d , t h e r e does appear t o be a d e v e l o p m e n t a l c o n t r o l over P-uptake and as such the a b s o l u t e magnitude of a phosphate s p e c i e s , P^ i n c l u d e d , c o u l d govern t h e e x t e n t of P-uptake enhancement. The t o t a l amount of phosphorus p e r gram f r e s h w e i g h t d e c r e a s e d s l o w l y i n the r o o t s of P - s u f f i c i e n t p l a n t s w h i l e a subsequent i n c r e a s e i n P^-content ensued ( s e e F i g u r e 2 0 ) .  Total  phosphate  i n t h e +P s h o o t s i n c r e a s e d r a p i d l y between days 5 and 7, and then d e c r e a s e d g r a d u a l l y throughout the d u r a t i o n of the experiment 20).  (Figure  A p o s s i b l e s i g n a l f o r the r e g u l a t i o n of P-uptake i s t h e  d r a m a t i c 17 f o l d i n c r e a s e i n orthophosphate l e v e l s w i t h i n t h e +P s h o o t s w h i c h o c c u r e d between days 11 and 12.  T h i s was  f o l l o w e d by  a l e v e l l i n g o f f o f t h e P ^ - c o n c e n t r a t i o n at a p p r o x i m a t e l y 8umol/g.f.wt. The l a c k of s u c h a s h i f t i n phosphate p o o l c o m p o s i t i o n c o u l d be the s i g n a l f o r i n c r e a s e d P-uptake i n -P  roots.  I n t h e p r e s e n t s t u d y , where a l l d e t e r m i n a t i o n s of P-uptake were performed w i t h i n t a c t p l a n t s , t h e s h o o t ' s phosphate s t a t u s c o u l d e f f e c t change i n t h e r o o t s ' phosphate uptake r a t e s .  -P s h o o t s d i d  - 42 -  F i g u r e s 1 8 t o 21 .  P^ and t o t a l phosphorus o f a g i n g b a r l e y plants  3.0  8.0  7.0  7.0  u . 0.0  6.0  3  M  S  5.0  5.0  —1  O  4.0 o '•S 3-0  3.0  2.0  2.0  1.0  1.0  5  7  9  U 13 15 17 19 Time (Days) Figure -18. P i (•) and P l (*) of -P roots vs. Age. t  20.0  o  t  a  r  5  i i i ? T T T i T i T  7  9 11 13 15 17 19 Time (Days) Figure 19. P i (•) and P (*) of -P shoots v s . Age. t o t a l  20.0  15.0  15.0  10.0  10.0  o  a. 5.0  5.0  .  • - ••••••  5  7  • •  9,, 1 1 13 , 15 17 19 Time (Days) Figure 20. P i (•) and P t a l ( ) of +P roots vs. Age. A  t o  .  I  I  —Time ^(Day's ) Figure 21. Pt (•) and P shoots v s . Age. 7  9  3  II II 1 5  C o t a l  1  7  I  1 9  I I  (*) of +P  - 43 -  show a l a r g e d e c r e a s e i n t o t a l phosphorus a t the time of i n i t i a t i o n of enhanced P-uptake these shoots'  (Days 11 and 1 2 ) .  There i s a l s o a drop i n  P_^-levels between days 9 and 11.  These o c c u r r e n c e s  c o u l d t r i g g e r c o n t r o l t h r o u g h t r a n s l o c a t i o n of hormones from the shoot t o the uptake organ.  S e v e r a l s t u d i e s have shown t h a t hormones  a p p l i e d t o t i s s u e s i n c r e a s e t h e i r i o n uptake r a t e s ( L u t t g e  and  H i g i n b o t h a m , 1979).  increase  86 in  E x c i s e d maize r o o t s , however, d i d not  36 Rb or  C l uptake when a u x i n was a p p l i e d (van S t e v e n i n c k , 2-  and a u x i n i n d u c e d o n l y a 20% s t i m u l a t i o n of SO^ s l i c e s ( N e i r i n c k x , 1968).  1974),  uptake i n b e e t r o o t  P u r s u i t s i n the hormonal c o n t r o l o f r o o t  P^-uptake, p e r s e , have been  neglected.  Where hormones have been e x t e r n a l l y a p p l i e d t o t i s s u e s i t i s n o t c l e a r whether i n c r e a s e d i o n i n f l u x was the cause or the r e s u l t of i n c r e a s e d  growth r a t e s .  -P p l a n t s demonstrated a  p r e f e r e n t i a l r o o t growth a t t h e time o f i n i t i a t i o n of i n c r e a s e d P^-uptake r a t e s .  However, the -P growth media c o n t a i n e d no phosphate,  and t h e r e f o r e i n c r e a s e d growth c o u l d not be due t o o r t h o p h o s p h a t e accumulation.  P l a n t growth i s thought t o be mediated t h r o u g h i n t e r -  a c t i o n s between the major p l a n t hormones.  S i n c e growth i m p l i e s a  g r e a t e r demand f o r a l l n u t r i e n t s i n c l u d i n g i n o r g a n i c i o n s , i t i s n o t surprising to f i n d that increased  growth r a t e s i n response t o hormonal  treatments are a s s o c i a t e d w i t h higher rates of i n o r g a n i c i o n uptake. I n t h i s manner, the hormonal i n f l u e n c e upon i o n a b s o r p t i o n i n d i r e c t , g e n e r a l e f f e c t p r o v i d i n g no o p p o r t u n i t y  i s an  f o r the c o n t r o l of  - 44 -  uptake o f s p e c i f i c i o n s .  Nevertheless  on a s h o r t - t e r m  basis  there  are c l e a r i n d i c a t i o n s t h a t hormones such as IAA and ABA may i n f l u e n c e s p e c i f i c i o n t r a n s p o r t systems e.g. H  +  e x t r u s i o n ( C l e l a n d , 1973) and  + K t r a n s p o r t i n t o guard c e l l s ( v a n S t e v e n i n c k ,  1976).  86 32 Rb and P  d o u b l e - l a b e l l e d e x p e r i m e n t s performed a t v a r i o u s p l a n t ages i n d i c a t e d complete independence o f t h e i r i n f l u x ( s e e T a b l e 6) and i t would be d i f f i c u l t t o a t t r i b u t e t h i s t o t h e g e n e r a l hormonal e f f e c t s discussed. I n t h i s s t u d y t h e shoot was d e s i g n a t e d seed and green t i s s u e o f t h e s e e d l i n g .  t o comprise t h e  The r a t h e r l a r g e  ortho-  phosphate p o o l w h i c h was suddenly formed i n t h e +P s h o o t s i s more r e p r e s e n t a t i v e o f t h e P^ i n h i g h e r p l a n t s than a r e t h e l e v e l s w i t n e s s e d at t h e younger b a r l e y ages ( B i e l e s k i , 1973).  The breakdown o f p h y t i n ,  s t a b l e o r t h o p h o s p h a t e uptake and t r a n s l o c a t i o n , and a n e t c o n v e r s i o n of shoot o r g a n i c - P  could a l l contribute t o t h i s increase i n ortho-  phosphate c o n c e n t r a t i o n .  Phosphate s t o r e d as p h y t i n c o u l d have  broken down t o r e l e a s e l a r g e amounts o f phosphorus a v a i l a b l e f o r t h e photosynthesizing  t i s s u e i n t h e t r a n s l o c a t e d form o f orthophosphate  ( M o r r i s o n , 1965; S e l v e n d r a n , 1970).  C o r r e l a t i o n s between p h y t i n h y d r o -  l y s i s and P^ i n c r e a s e s have been demonstrated i n s e e d l i n g s f o r phosphorus  starved  ( M u k h e r j i e t . a l . , 1971; E r g l e and Guinn, 1959).  The  a c t i o n o f p h y t a s e i s r e t a r d e d when phosphate i s s u p p l i e d t o t h e seedlings  ( B i a c h e t t i and S a r t i r a n a , 1967; S a r t i r a n a and B i a c h e t t i ,  1967) and a l t h o u g h these s t u d i e s were t e r m i n a t e d  a t 6 days growth,  - 45  -  p h y t i n l e v e l s d e c l i n e d g r a d u a l l y d u r i n g t h i s p e r i o d u n t i l at t e r m i n a t i o n o f the study they were almost exhausted.  the  Hence i t i s  u n l i k e l y t h a t a sudden breakdown o f p h y t i n c o u l d have o c c u r r e d day  11 of the p r e s e n t  study  ( F i g u r e 21).  P^ uptake from the 15uM  growth media c o u l d have accounted f o r a p p r o x i m a t e l y the i n c r e a s e , i . e . i f a l l P_^ t a k e n up was the l e a v e s .  one  N a s s e r y , 1969;  Singh,  and  o n l y -a minor source f o r o r t h o p h o s p h a t e . best candidates the +P  t h i r d of  P o l y p h o s p h a t e s have been found i n r e l a t i v e l y low  Vagabov and K u l a e v , 1964)  P  t r a n s l o c a t e d unchanged t o  i n a few h i g h e r p l a n t s ( M i y a c h i , 1961; 1964;  at  Tewari  levels and  t h e r e f o r e they c o u l d  O r g a n i c P s o u r c e s are  be the  f o r breakdown t o s u p p l y the observed P ^ - l e v e l s i n  s h o o t s beyond day Expression  11.  of i n o r g a n i c and t o t a l phosphate l e v e l s on a  per p l a n t b a s i s g i v e s f u r t h e r i n s i g h t i n t o phosphorus n u t r i t i o n . A l t h o u g h the t o t a l phosphorus and i n o r g a n i c phosphate amounts w i t h i n the -P p l a n t s remained r e l a t i v e l y c o n s t a n t  throughout the f i r s t  days of growth, a drop i n t o t a l phosphorus w h i c h was i n the i n o r g a n i c p o o l o c c u r r e d between day  not  paralleled  18 and 20 (see F i g u r e  T h i s i m p l i e s t h a t phosphorus had e f f l u x e d i n t o the environment t h i s may  have been due  I n d i r e c t evidence,  18  22). and  t o minor l o s s e s of plasmalemma i n t e g r i t y .  r e c e n t l y put f o r w a r d by Menge e t . a l . (1978),  s u g g e s t s t h a t l e a k a g e of r o o t m a t e r i a l s i n phosphorus d e f i c i e n t p l a n t s may  be a p r e r e q u i s i t e f o r the i n i t i a t i o n of r o o t - m y c o r r h i z a l  associations.  The  fungal  f a c t t h a t the number of these a s s o c i a t i o n s i n a  - 46 -  g i v e n s o i l environment does c o r r e l a t e n e g a t i v e l y w i t h t h e a v a i l a b l e phosphate l e v e l may a l s o be e x p l a i n e d , however, by t h e reduced s u r v i v a l r a t e s o f m y c o r r h i z a l s p o r e s under h i g h l e v e l s o f P - a p p l i c a t i o n i n modern a g r i c u l t u r a l p r a c t i c e s .(Ducey, 1980). a s s o c i a t i o n s , although apparently  impossible  These  rhizosphere  t o form i n h y d r o p o n i c  c u l t u r e a r e b e l i e v e d t o be e s s e n t i a l f o r p r o p e r p l a n t phosphorus n u t r i t i o n i n low phosphorus s o i l s (Gerdemann, 1968).  P l a n t phosphate  compensation p o i n t s a r e o f t e n i n excess of 0.5uM ( B i e l e s k i , 1973) and  c o u l d t h e r e f o r e account f o r some of t h e -P p l a n t s ' phosphate l o s s  (-P medium was changed e v e r y 4 d a y s ) .  A rapid increase i n the  l e v e l s o f i n o r g a n i c phosphate o c c u r r e d  i n t h e +P treatment between  days 11 and 12 ( s e e F i g u r e 2 3 ) , w h i c h can be a t t r i b u t e d t o t h e shoot phenomenon w h i c h has a l r e a d y been d i s c u s s e d  at length.  The i n c r e a s e i n phosphate i n f l u x i n b o t h +P and -P p l a n t s on a p e r p l a n t b a s i s s t a r t e d and f i n i s h e d a t t h e same time ( s e e F i g u r e 1 7 ) . These phenomena p e r se cannot be a t t r i b u t e d t o n u t r i t i o n a l s t a t u s and t h e r e f o r e appear t o be d e v e l o p m e n t a l i n n a t u r e .  Crop  p l a n t s t u d i e s have i n d i c a t e d t h a t t h e i r P as w e l l as N and K l e v e l s v a r y i n a manner w h i c h can be timed t o d i s t i n c t m o r p h o l o g i c a l  stages  w i t h i n t h e p l a n t s ' development (Mengel, 1969; Mengel and K i r k b y , 1978).  I t therefore follows that the i n i t i a t i o n of elevated  uptake i n t h e p r e s e n t s t u d y might a l s o be o f a p r e d e t e r m i n e d  phosphate nature,  however t h e e x t e n t o f t h i s i n c r e a s e appears t o be n e g a t i v e l y r e l a t e d t o i n t e r n a l phosphorus l e v e l s .  A p o s s i b l e candidate f o r the c o n t r o l  - 47 -  F i g u r e 22. 3.0  Phosphate c o n c e n t r a t i o n i n -P b a r l e y p l a n t s  r Total P  o  2.0  a  1.0  Inorganic P o  F i g u r e 23.  AO  D  o . o o o D g a 10 15 Age (days)  •  o 20  Phosphate c o n c e n t r a t i o n i n +P b a r l e y p l a n t s  L  *  5 Z ' Total P A  I  2 0  Y  A  10 •  J 5  S  •  • ' , 10 Age (days)  Inorganic P  15  20  - 48 -  of t h e e x t e n t of enhancement i s t h e c o n c e n t r a t i o n o f the r o o t s ' i n t e r n a l o r t h o p h o s p h a t e o r o r g a n i c phosphate a t t h e b e g i n n i n g and d u r i n g the P - i n f l u x enhancement p e r i o d . ° -P  roots  The r a t i o s of P. (+P / i roots/  ) a r e 10.4 and 15.7 a t days 11 and 16 r e s p e c t i v e l y ; t h e  same r a t i o s f o r o r g a n i c phosphate a r e 1.5 and 2.4.  Clarkson e t . a l .  (1978) o b t a i n e d a +P/-P r o o t t o t a l phosphorus r a t i o of 2.4 and a -P/+P i n f l u x r a t i o o f 3.9.  Because P^ i s much more f l e x i b l e i n  magnitude than the o r g a n i c phosphates i t c o u l d a c t as a more f i n e l y tuned r e g u l a t o r y s i g n a l f o r P-uptake i n r o o t s .  - 49 -  C.  P h y s 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 Enhanced P-uptake.  i.  Uptake  Isotherms  The k i n e t i c p l o t s , e i t h e r M i c h a e l i s - M e n t e n  or Eadie-Hofstee  (see F i g u r e s 24 and 25) i n d i c a t e t h a t t h e phosphate uptake Vmax o f -P p l a n t s had i n c r e a s e d (-P Vmax = 1.25nmol/.g.f.wt./hour; +P Vmax = 0.32umol/g.f.wt./hour), whereas t h e r e was no s i g n i f i c a n t  (a = 0.05)  i n c r e a s e i n t h e -P r o o t s a f f i n i t y f o r phosphate, i . e . no s i g n i f i c a n t decrease  i n t h e Km (-P Km = 2.37uM; +p Km = 3.00yM) of t h e phosphate  uptake system o c c u r r e d . The  r a t i o o f -P t o +P V max's i s a p p r o x i m a t e l y 4 and t h e  -P p l a n t s i n these i s o t h e r m experiments enhancement ( s e e F i g u r e 16).  had n o t reached  t o t a l P-uptake  The h i g h P - c o n c e n t r a t i o n used by  many r e s e a r c h e r s ( N i s s e n , 1973) and t h e m u l t i p h a s i c n a t u r e o f the orthophosphate  uptake i s o t h e r m s  ( B a r b e r , 1972; N i s s e n , 1973)  make i t d i f f i c u l t t o compare Km and Vmax v a l u e s .  Because o f t h e  m u l t i p h a s i c uptake p a t t e r n i n p l a n t s , o n l y n a t u r a l s o i l  P-concentrations  were used i n t h e p r e s e n t study f o r i n f l u x i s o t h e r m d e t e r m i n a t i o n s . I n t h i s n a t u r a l range ( c i r c a lOuM), F a r r a r (1976) w o r k i n g w i t h l i c h e n s o b t a i n e d comparable Km and Vmax v a l u e s t o t h e -P t r e a t e d b a r l e y r o o t s of t h i s s t u d y , whereas C a r t e r and L a t h w e l l (1967) w o r k i n g w i t h c o r n found v a l u e s s i m i l a r  t o t h e +P t r e a t m e n t .  These w o r k e r s '  can be a t t r i b u t e d t o t h e phosphorus n u t r i t i o n o f t h e i r plants.  results  experimental  B a r b e r , u s i n g e x c i s e d b a r l e y r o o t s ( a n a l y s i s by N i s s e n , 1973)  F i g u r e 24.,  Orthophosphate uptake k i n e t i c s : M i e h a e l i s - M e n t e n p l o t  F i g u r e 25.  Orthophosphate uptake k i n e t i c s : E a d i e - H o f s t e e p l o t  - 52 -  o b t a i n e d , i n phosphate d e f i c i e n t  p l a n t s , approximately  a 4 fold  Vmax i n c r e a s e over t h a t o f w e l l n o u r i s h e d p l a n t s and h i s -P Km v a l u e s were lower than i n h i s +P t r e a t m e n t s , present cant.  though as i n t h e  study t h e d i f f e r e n c e s o f Km were n o t s t a t i s t i c a l l y s i g n i f i Cartwright  (1972) a l s o showed an i n c r e a s e i n t h e a f f i n i t y  f o r phosphate by -P b a r l e y p l a n t s , a l t h o u g h no changes i n Vmax were reported.  T h i s c o u l d be due t o t h e n u t r i e n t s t a t u s o f t h e p l a n t s  employed as w e l l as t h e P - c o n c e n t r a t i o n d e t e r m i n a t i o n s were p e r f o r m e d .  range o v e r w h i c h i n f l u x  At higher e x t e r n a l concentrations  o f i n o r g a n i c i o n s d i f f e r e n c e s i n uptake r a t e s between p l a n t s w h i c h are apparent a t lower c o n c e n t r a t i o n s , may be l o s t ( G l a s s and Dunlop, 1978).  T h i s combined w i t h t h e m u l t i p h a s i c n a t u r e o f i o n u p t a k e i n  p l a n t s would suggest t h a t an i s o t h e r m determined over a g r e a t e r range o f i o n c o n c e n t r a t i o n might o v e r l a y t h e p a t t e r n o f uptake w h i c h o c c u r s a t t h e lower P - c o n c e n t r a t i o n s the p r e s e n t The  such as those used i n  study. present  study t o g e t h e r w i t h those c i t e d now c l e a r l y  show t h a t i n c r e a s e d P - s t a t u s may be a s s o c i a t e d w i t h b o t h d e c r e a s e s of Vmax v a l u e s f o r P-uptake as w e l l as i n c r e a s e s o f Km  although  i n t h e p r e s e n t study s i g n i f i c a n t Km d i f f e r e n c e s were n o t o b s e r v e d .  - 53 -  ii.  Uptake from S t i r r e d and N o n - s t i r r e d Media  A l l uptake d e t e r m i n a t i o n s  so f a r d e s c r i b e d were performed  i n w e l l s t i r r e d media and t h e r e f o r e the d i f f u s i o n a l l i m i t a t i o n on phosphate s u p p l y t o the r o o t was reduced t o a minimum.  Diffusion  i s the l i m i t i n g " f a c t o r f o r i o n uptake when r o o t s a r e bathed i n n o n - s t i r r e d s o l u t i o n s of c o n c e n t r a t i o n below lOuM ( P o l l e and Jenny, 1971; B o l e , 1977).  I n such an environment an i n c r e a s e i n the r o o t  a r e a r e l a t i v e t o i t s w e i g h t would c o n t r i b u t e t o an i n c r e a s e d rate.  uptake  By comparison of uptake r a t e s i n a d i f f u s i o n and a non-  d i f f u s i o n l i m i t i n g system i n s i g h t can be gained and m o r p h o l o g i c a l  into physiological  c o n t r i b u t i o n s t o enhanced P^-uptake  r a t e s (see  Table 5 ) . The f a c t t h a t the -P t o +P uptake r a t i o s were c o n s i s t e n t l y l o w e r under the n o n - s t i r r e d compared t o the s t i r r e d  conditions  might be e x p l a i n e d by presuming t h a t i n c r e a s e d uptake r a t e s i n -P p l a n t s a r i s e from i n c r e a s e d " c a r r i e r " s y n t h e s i s and i n c o r p o r a t i o n i n t o the plasmalemma.  Under c o n d i t i o n s where uptake r a t e s a r e  d i f f u s i o n l i m i t e d ( t h e n o n - s t i r r e d t r e a t m e n t ) the o v e r l a p of d e p l e t i o n zones ( i n -P p l a n t s ) would be a n t i c i p a t e d t o more a d v e r s e l y reduce uptake r a t e s than i n the +P p l a n t s .  An a n a l o g y can be drawn  t o the phenomenon o f gas exchange a t the stomata o f l e a v e s 1975).  (Heath,  As Raven (1977) has p o i n t e d o u t , however, s p e c u l a t i o n s  r e g a r d i n g " c a r r i e r " s y n t h e s i s w i l l remain tenuous u n t i l t h e i s o l a t i o n and c h a r a c t e r i z a t i o n of t h e s e p u t a t i v e  molecules.  - 54 -  T a b l e 5.  Uptake r a t e s from 2.5yM P s o l u t i o n w i t h (S) s t i r r i n g  or w i t h o u t (WS) s t i r r i n g ,  Day  8  S  -P  (umoles/g.f.wt./h.)  r a t i o -P/+P  744.14  R a t i o s a r e shown.  WS  11  335.46  -P  693.33  1.46 207.75 225.12  2.47 14  +P  281.49  -P  548.16  1.27 176.70 161.84  2.73 +P  201.61  -P/+P  304.30 2.22  +P  ratio  1.24 130.52  - 55 -  The s e e d l i n g s employed i n t h i s and the  remaining  e x p e r i m e n t s of the t h e s i s a t t a i n e d optimum P-uptake r a t e s a t e i g h t days of age, not 16 as i n the p r e v i o u s p l a n t l e t s .  Differences i n  the age needed t o r e a c h optimum uptake r a t e s does n o t appear t o be s o l e l y dependent upon phosphate s t o r e s (see F i g u r e s 18, 19, 27 and 2 8 ) , and o t h e r n u t r i e n t s , a l t h o u g h involved.  n o t i n v e s t i g a t e d , may  be  - 56  iii.  -  D o u b l e - l a b e l l e d Uptake Experiment  Phosphorus d e p r i v a t i o n can a l t e r the m e t a b o l i c c y c l i n g of energy t h r o u g h a l i m i t a t i o n upon energy c o u p l i n g w h i c h v i a the s y n t h e s i s and h y d r o l y s i s of ATP. b o t h the ATP  p r e c u r s o r s ADP  The  occurs  concentrations  of  and m e t a b o l i c a l l y a v a i l a b l e o r t h o -  phosphate r e l y on a s o u r c e of phosphorus w h i c h c o u l d be w i t h i n the c e l l as a s t o r a g e p o o l and/or w i t h i n the r o o t s ' e x t e r n a l environment. Even i f t h e s e s o u r c e s were d e p l e t e d  i t i s p l a u s i b l e t h a t an  increase  i n the r a p i d i t y of phosphate c y c l i n g through perhaps an i n c r e a s e i n phosphatase a c t i v i t y c o u l d p r o l o n g h i g h l e v e l s of energy c o u p l i n g .  the p l a n t s a b i l i t y t o  maintain  I n f a c t h i g h phosphatase a c t i v i t i e s  commonly o c c u r i n P - d e f i c i e n t p l a n t s ( B i e l e s k i , 1973). I n the event t h a t p l a n t ATP  s u p p l i e s became lowered as  a r e s u l t of phosphate d e p r i v a t i o n , the r e l a t i v e e f f e c t upon a l l the organism's e n d e r g o n i c i o n uptake p r o c e s s e s might be expected t o be of s i m i l a r magnitude b a r r i n g a l l o s t e r i c changes w i t h i n the or changes i n the number of s p e c i f i c " c a r r i e r s " d u r i n g the of P - s t a r v a t i o n .  "carriers"  course  I n o r d e r t o s p e c i f i c a l l y a s c e r t a i n whether the  d e c l i n e i n P- uptake d u r i n g P - d e f i c i e n c y ( F i g u r e 16) was l i m i t a t i o n upon energy r e s o u r c e s  due  f o r a c t i v e t r a n s p o r t i t was  to decided  t o examine the uptake of a n o t h e r a c t i v e l y t r a n s p o r t e d i o n i c s p e c i e s , namely K . +  86 of  Rb and  By means of d o u b l e - l a b e l l e d uptake experiments the uptake 32 P was  determined s i m u l t a n e o u s l y d u r i n g t h i s p e r i o d of 86 P-uptake d e c l i n e i n -P p l a n t s . Rb i s the i s o t o p e of c h o i c e f o r  - 57 -  T a b l e 6.  Uptake r a t e s of P from a 15uM P s o l u t i o n and K from  a llluM K solution.  (nmoles/g.f.wt./h.)  r o o t s are a l s o p r e s e n t e d ,  Day  8  11  14  K concentrations  of the  (mmoles/g.f.wt.)  P uptake  K uptake  K  concentration  -P  2678.12  889.12  0.092±0.021  +P  886.48  1080.64  0.067±0.008  -P  2247.08  410.27  0.080±0.005  +P  867.75  9.74.55  0.079±0.003  -P  1259.39  618.56  0.077±0.013  +P  699.80  901.59  0.085±0.006  - 58  the d e t e r m i n a t i o n  -  of p o t a s s i u m uptake i n h i g h e r p l a n t s ( L a u c h l i  and E p s t e i n , 1970).  W h i l e the w e l l n o u r i s h e d  plants  gradually  reduced t h e i r uptake r a t e s of b o t h i o n s o v e r the time p e r i o d i n v e s t i g a t e d , t h e r e was ion  no such r e l a t i o n s h i p between the  uptakes i n the -P p l a n t s .  From day  8 t o day  uptake r a t e s i n the -P p l a n t s were h a l v e d ,  14 phosphate  and w h i l e the p o t a s s i u m  uptake decreased c o n s i d e r a b l y by day 11, i t had by day  respective  increased  again  14. Because of the independence of the two  i t appears t h a t energy s u p p l y  i o n uptake r a t e s  t o the " c a r r i e r s " w h i c h might be  e x p e c t e d t o a c t s i m i l a r l y upon a c t i v e uptake p r o c e s s e s does not seem t o be the o v e r r i d i n g cause f o r r e d u c t i o n i n phosphate uptake r a t e s and t h e r e f o r e a more l i k e l y cause of the P-uptake d e c l i n e i s a net l o s s of a c t i v e P - " c a r r i e r s " on the r o o t I n b o t h the +P  surface.  and -P p l a n t s a r e l a t i o n s h i p between  P-uptake and p o t a s s i u m c o n t e n t of the r o o t s i s d i f f i c u l t t o a s c e r t a i n . Cram ( i n p r e s s ) , on the o t h e r hand showed d e c i s i v e l y t h a t KC1 t o c a r r o t d i s c s r e s u l t e d i n a d e c r e a s e d phosphate a b s o r p t i o n .  fed Franklin  (1969) showed t h a t adsorbed c a t i o n s i n c r e a s e d phosphate uptake by b a r l e y r o o t s , whereas c o n v e r s e l y , reported  a p p l i e d phosphate has n e v e r been  t o augment K-uptake r a t e s .  I n the p r e s e n t study +P  c o n s i s t e n t l y showed g r e a t e r K-uptake r a t e s than -P p l a n t s and may  be a t t r i b u t e d t o the g r e a t e r growth r a t e s of the +P  plants this  plants.  - 59 -  iv.  Determination  o f Root E x t e r n a l P r o t e i n  Because o f t h e u b i q u i t o u s m e t a b o l i c  r o l e s i n which  phosphorus i s i n v o l v e d i t was o f i n t e r e s t t o d e t e r m i n e i f d e p r i v a t i o n o f t h i s e s s e n t i a l n u t r i e n t would r e s u l t i n t h e reduced s y n t h e s i s o f p r o t e i n and i n p a r t i c u l a r p r o t e i n s w h i c h would be exposed t o t h e e x t e r i o r o f t h e r o o t .  Proteins oriented w i t h i n the  r o o t plasmalemma c o u l d b e r e s p o n s i b l e f o r m i n e r a l uptake and f o r the s t r u c t u r a l i n t e g r i t y o f t h e membrane i t s e l f . Eosin i s a p r o t e i n s p e c i f i c s t a i n which  penetrates  b i o l o g i c a l membranes a t a v e r y slow r a t e and as such can be used t o measure r e l a t i v e amounts o f r o o t e x t e r n a l p r o t e i n ( W i l l i a m s , 1962). At 10 days o f age t h e s t a i n i n g v a l u e f o r -P p l a n t s ' r o o t s was 20.82±5.04 O.D.,-™ u n i t s / g . f .wt. 520nm 35.28±9.06.  and t h a t o f +P r o o t s was  The d i a m e t e r s o f t h e main r o o t s o f b o t h +P and -P  p l a n t s were 0.45±0.05mm ( s e e F i g u r e 2 ) .  A t 30 days o f age p r o t e i n  v a l u e s were 16.61±3.86 and 16.53±1.78 f o r t h e -P and +P r o o t s r e s p c t i v e l y and w h i l e t h e +P r o o t d i a m e t e r had n o t changed from 0.45±0.05mm, t h e -P r o o t w i d t h had d e c r e a s e d t o 0.20±0.02mm  (Figure  6). The  p r i m a r y r o o t ^ s s u r f a c e a r e a t o volume r a t i o might be  t a k e n as r e p r e s e n t a t i v e o f t h e e n t i r e r o o t system, i n w h i c h case t h e c a l c u l a t i o n o f s u r f a c e a r e a p e r g.f.wt. r o o t r e v e a l e d t h a t p e r u n i t a r e a a t b o t h days 10 and 30 t h e r e was a p p r o x i m a t e l y much e x t e r n a l p r o t e i n on -P as on +P r o o t s .  0.6 times as  P - d e f i c i e n t p l a n t s seem  - 60 -  t o have produced and s u s t a i n e d much l e s s plasmalemma and c e l l w a l l protein.  T h i s combined w i t h t h e apparent i n c r e a s e i n d e n s i t y o f  phosphate uptake s i t e s ( s e e s t i r r e d v s . n o n - s t i r r e d experiment) would suggest t h a t t h e -P p l a n t s p o s s e s s e d a s u b s t a n t i a l enrichment i n t h e membrane p r o t e i n s r e s p o n s i b l e f o r phosphate u p t a k e .  Such  an enrichment c o u l d be e x p l o i t e d as a s o u r c e f o r o b t a i n i n g a p u r i f i c a t i o n o f the phosphate " c a r r i e r " i n q u e s t i o n , a f e a t n o t y e t accomplished i n p l a n t s .  The low p r o t e i n l e v e l may a l s o be p a r t l y  r e s p o n s i b l e f o r t h e l o s s o f phosphate from the r o o t s o f t h e 20 day o l d -P p l a n t s ( s e e F i g u r e 2 2 ) .  A l e a k a g e o f phosphate c o u l d have  r e s u l t e d from a d e c r e a s e d r o o t I n t e g r i t y caused by an i n a d e q u a t e p r o d u c t i o n o f s t r u c t u r a l membrane p r o t e i n s .  - 61 -  5.  Regulation  o f R a p i d D e c l i n e of Enhanced P^-uptake  A.  S h o r t - t e r m v e r s u s Long-term Uptake -P p l a n t s w h i c h demonstrated enhanced P-uptake r a t e s i n  short-term  determinations  (10 min i n f l u x p e r i o d s )  d i d n o t show as  pronounced an e l e v a t e d uptake r a t e when a 24h uptake p e r i o d was 32 used ( s e e T a b l e 7 ) . D e p l e t i o n o f  P - l a b e l l e d uptake media was  n e g l i g i b l e i n b o t h s h o r t - and l o n g - t e r m e x p e r i m e n t s and cannot t h e r e f o r e have been r e s p o n s i b l e f o r t h i s e f f e c t .  When -P p l a n t s  were exposed t o +P media f o r 24h p r i o r t o s h o r t - t e r m mination  the v a l u e s  o f P-uptake r e v e a l e d  phosphate had r e s u l t e d i n a c o n s i d e r a b l e  uptake d e t e r -  t h a t t h e s u p p l i e d 15uM reduction i n their P-influx,  d r o p p i n g the r a t e t o a l e v e l s i m i l a r t o t h a t o f t h e +P p l a n t s . S h o r t - t e r m s t u d i e s o f +P and -P p l a n t s a t days 7 and 8 i n d i c a t e d t h a t no d e v e l o p m e n t a l d e c l i n e i n uptake had o c c u r r e d . uptake r a t e s which C l a r k s o n and h i s c o l l e a g u e s  The low  (1978) o b t a i n e d i n  t h e i r low phosphate p l a n t s may be a t t r i b u t e d , a t l e a s t i n p a r t , t o the 24h i n f l u x d e t e r m i n a t i o n s obtained  employed.  Differences i n the values  f o r P - i n f l u x through t h e use of e i t h e r s h o r t - o r l o n g -  term assays i n d i c a t e d t h a t t h e r e g u l a t i o n o f phosphate uptake i n -P p l a n t s was e x t r e m e l y s e n s i t i v e t o i n t e r n a l P - l e v e l s .  This c o n t r o l  mechanism, by v i r t u e o f t h e r a p i d i t y of i t s r e s p o n s e , c o u l d be f u r t h e r i n v e s t i g a t e d o n l y through t h e use of t h e s h o r t - t e r m  assays  i n w h i c h m i n i m a l exposure t o phosphate r e s u l t s i n a measure of t h e i n i t i a l P-uptake r a t e .  T a b l e 7.  S h o r t - t e r m ( s . t . ) v s . l o n g - t e r m ( l . t . ) uptake  rate  determination  Plant Status at Assay Employed  Uptake  (ymol./g.f.wt./h)*  B e g i n n i n g o f Assay  A.  7 day -P  s.t.  2.047±0.226  B.  7 day +P  s.t.  0.699±0.144  C.  8 day -P  s.t.  2.587±0.481  D.  8 day +P  s.t.  0.616±0.104  E.  7 day -P p l u s s.t.  0.65L+0.016  1 day +P F.  7 day -P  l'.t.  0.73210.052  G.  7 day +P  l . t .  0.579±0.039  *Mean and s t a n d a r d d e v i a t i o n o f f o u r r e p l i c a t e s .  -  B.  -  63  P-uptake Rates and P l a n t Phosphorus L e v e l s Exposure of P l a n t s t o 15uM  Following  Phosphate  I n o r d e r t o determine the r a p i d i t y of the -P p l a n t s ' response t o the 15yM obtained  i n f l u x estimates  a f t e r these p l a n t s had been f e d 15uM  time p e r i o d s . 15uM  P supplied, short-term  F i g u r e 26 p r e s e n t s  were  P for increasing  the time course of the e f f e c t of  phosphate upon -P p l a n t s ' uptake r a t e s .  The  r a t e s began  d e c r e a s i n g w i t h i n l h w h i c h i n c i d e n t a l l y i s c l o s e t o the h a l f - l i f e of c y t o p l a s m i c P-exchange ( e s t i m a t e d at 45 min, The  d e c l i n e was  t i o n was  see E f f l u x K i n e t i c s ) .  r a p i d u n t i l 6 h o u r s , beyond w h i c h no f u r t h e r r e d u c -  evident.  The  uptake d e t e r m i n a t i o n s  f o r times 10 t o  24h  were c a r r i e d out on the second day of the 2 day e x p e r i m e n t , however the e l e v a t e d v a l u e s at 12, 16 and 20h  cannot be due  t o the p l a n t s '  d i u r n a l c y c l e s i n c e a l l p r o c e d u r e s were performed w i t h i n the 6 c e n t r a l hours of the l i g h t p e r i o d . +P  and -P p l a n t s a t 0 and The  Rate d e t e r m i n a t i o n  of  non-treated  24h r e v e a l e d o n l y m i n i m a l changes.  t o t a l phosphorus content  per gram f r e s h w e i g h t of  the  t r e a t e d p l a n t s d i d not show a n e t g a i n over the 24h f e e d i n g p e r i o d ( F i g u r e s 27 and and  28).  R a p i d growth, c a u s i n g d i l u t i o n of absorbed P  thus maintenance of P a t a r e l a t i v e l y c o n s t a n t  t h i s phenomenon.  The  l e v e l , best  f a c t t h a t P-content decreased i n the  non-  t r e a t e d -P p l a n t s over the same time p e r i o d i s i n a c c o r d w i t h explanation.  The  explains  this  i n o r g a n i c phosphorus component of b o t h the shoots  and r o o t s changed c o n s i d e r a b l y as phosphate was  s u p p l i e d t o -P  plants  - 64 -  F i g u r e 26.  Phosphate uptake r a t e v s . time of r o o t exposure t o 15uM orthophosphate  2.0 t-  1  5-0  •  ..  i  •  .  .  .  i  10.0 15.0 Time (hours)  .  .  .  .  •  20.0  .  .  .  .  »  25.0  F i g u r e 27.  5  T o t a l phosphorus c o n c e n t r a t i o n d u r i n g t h e p e r i o d o f phosphate l o a d i n g of -P grown b a r l e y p l a n t s  • Treated roots • +P r o o t s A +P shoots O -P r o o t s A - P shoots  16  U-l CO CD 0)  o  A  12  ON  s 3  •  03  3  O  x; to o  •  •  • O  o H  12 Time (hours)  16  20  24  F i g u r e 28.  T o t a l phosphorus c o n c e n t r a t i o n d u r i n g the p e r i o d o f phosphate l o a d i n g of -P grown b a r l e y p l a n t s  • Treated shoots • +P r o o t s A+P shoots O-P r o o t s A - P shoots  O A  8  12 Time (hours)  16  - 20  24  -  67  -  and a l t h o u g h no obvious P-uptake c o n t r o l s i g n a l can be a t t r i b u t e d t o the s h o o t s , t h e r e appears t o be a r e l a t i o n s h i p between the P ^ - c o n t e n t of the r o o t and the P - i n f l u x (see F i g u r e 29).  The  uptake r a t e p l o t t e d a g a i n s t the r o o t ' s i n t e r n a l orthophosphate c o n c e n t r a t i o n ( F i g u r e 30, f e e d i n g curve) b r i n g s t o l i g h t a c o r r e l a t i o n w h i c h was  maintained  negative  u n t i l 6h of exposure t o 15yM  A f t e r 6h the i n t e r n a l phosphate showed a d e c l i n e t o  P.  approximately  0.75umol/g.f.wt. (not shown i n F i g u r e 30) w h i l e the P-uptake r a t e s remained at r e l a t i v e l y low v a l u e s .  T h i s s u g g e s t s t h a t beyond 6h  some c o n t r o l mechanism o t h e r t h a n d i r e c t P_^ feedback upon uptake had a l s o p r e s e n t e d o f the +P  itself.  Moreover, the i n o r g a n i c phosphate l e v e l  r o o t s at the end of the 24h f e e d i n g p e r i o d was  i n the t r e a t e d r o o t s and r e g u l a t o r y p r o c e s s e s  may  t i m e be a c t i v e l y s t a b i l i z i n g the b e t t e r n o u r i s h e d Evidence t h e r e f o r e supports  the p o s s i b i l i t y of two  lower  than  s t i l l at t h i s root's  physiology.  c o n t r o l mechanisms:  1)  i n w h i c h a r a p i d feedback o c c u r s at the e a r l y s t a g e ,  and  2)  a s l o w e r mechanism which might a c t through the d e g r a d a t i v e r e d u c t i o n i n the number of a c t i v e phosphate " c a r r i e r s " . Also presented  i n F i g u r e 30 are the r o o t i n o r g a n i c  phosphate l e v e l s and P-uptake r a t e s o f 11 t o 16 day o l d -P p l a n t s d u r i n g the -P phosphate uptake enhancement phase (see F i g u r e  16).  I n t h i s case i n t e r n a l P ^ - l e v e l s d i d not c o r r e l a t e n e g a t i v e l y w i t h the enhanced uptake and as such i n t e r n a l orthophosphate c o u l d not have a c t e d as an a l l o s t e r i c r e g u l a t o r of the i n f l u x p r o c e s s .  The  - 68 -  -69 -  Figure  30.  Phosphate uptake r a t e v s . ?±  2.4  concentration  O  During uptake enhancement  •  During r a p i d d e c l i n e due to phosphate l o a d i n g  2.0  1.6 60  Enhancement curve, l i n e a r f i t  0)  1.2  Feeding curve, l i n e a r f i t  r= 0.968  r= 0.985  0 O  CD  to  4-1 Cu  I  0.8  •rH  0.4  0.2  0.4  0.6  Inorganic  Phosphate  0.8  l.o  (umoles/g.f.wt.)  1.2  - 70 -  i n t e r n a l P ^ - c o n c e n t r a t i o n remained a t a minimum l e v e l throughout the enhancement s t a g e and hence i n h i b i t i o n o f uptake by i n t e r n a l orthophosphate w o u l d , as such, a l s o be a t a minimum.  I t follows  t h a t a p o s s i b l e mechanism f o r t h e c o n t r o l o f enhanced P-uptake i s through t h e a d d i t i o n o f new " t r a n s p o r t e r p r o t e i n s " , each o f w h i c h was f r e e from a l l o s t e r i c i n h i b i t i o n .  T h i s agrees w i t h t h e argument,  a l r e a d y p u t f o r w a r d , t h a t phosphate uptake enhancement r e s u l t e d from an i n c r e a s e d d e n s i t y o f " c a r r i e r s " on the r o o t s u r f a c e .  It  does remain p o s s i b l e t h a t d u r i n g t h e enhancement phase of P-uptake a c o n c o m i t a n t d e c l i n e i n one o r more o r g a n i c phosphates  resulted  i n t h e r e l e a s e of an a l l o s t e r i c i n h i b i t i o n of t h e uptake p r o c e s s . T h i s was n o t f u r t h e r i n v e s t i g a t e d i n t h e p r e s e n t s t u d y . S e v e r a l s t u d i e s i n v o l v i n g ions which are not metabolized have demonstrated n e g a t i v e r e l a t i o n s h i p s between t h e r a t e o f uptake of a g i v e n i o n and i t s i n t e r n a l c o n c e n t r a t i o n .  The i o n  i t s e l f would i n t h e s e cases be t h e most e f f i c i e n t d i r e c t feedback s i g n a l f o r uptake. Rapid d e c l i n e s i n R b  +  uptake o c c u r r e d when c o r n was  s u p p l i e d w i t h p o t a s s i u m ( L e i g h and Wyn J o n e s , 1973) and i n c r e a s i n g p o t a s s i u m n u t r i t i o n caused d e c r e a s i n g R b  +  uptake r a t e s i n s u n f l o w e r  ( P e t t e r s s o n , 1975) and Lemna minor (Young e t . a l . , 1970).  Allosteric  c o n t r o l o f r u b i d i u m and p o t a s s i u m uptake has been r e p o r t e d i n b a r l e y and s u n f l o w e r ( G l a s s , 1976; 1977; 1978b; P e t t e r s s o n and J e n s e n , 1978; 1979; Jensen and P e t t e r s s o n , 1978).  C l uptake d e c r e a s e s as i n t e r n a l  - 71  CI  l e v e l s increase  and  Br  -  i n c a r r o t t i s s u e s and  i s absorbed more s l o w l y  i n Br  barley roots  f e d beets  (Cram,  (Sutcliffe,  1973) 1954)  and wheat (Cseh e t . a l . , 1970). By v i r t u e of t h e i r metabolism the study of the maintenance of s u l f u r , n i t r o g e n , and  phosphate l e v e l s i n p l a n t s  is  Ivan Smith's work (1975) w i t h c u l t u r e d tobacco c e l l s  complicated.  indicated  t h a t s u l f a t e uptake r a t e s c o r r e l a t e d n e g a t i v e l y w i t h i n t e r n a l s u l f a t e l e v e l s . I f methionine and  c y s t e i n e were a p p l i e d e x t e r n a l l y ,  s u l f a t e uptake r a t e s d e c l i n e d i n tobacco (Hart and barley  ( F e r r a r i and  Renosto, 1972), however c o n v e r s i o n  S-compounds to s u l f a t e may Very l i t t l e NO^ 1973)  e v i d e n c e has  precede the given  been r e p o r t e d  l e v e l s r e g u l a t e NO^ and  F i l n e r , 1969) of  and  the  e f f e c t (Smith, 1975).  to suggest t h a t i n t e r n a l  uptake i n h i g h e r  plants  (Smith, 1973;  such s t u d i e s are rendered more d i f f i c u l t  Cram,  because of  a b i l i t y of n i t r a t e to induce n i t r a t e r e d u c t a s e a c t i v i t y  the  (Jackson e t .  a l . , 1976). The rates points  l i n e a r r e l a t i o n s h i p between r o o t P_^-level and to an a l l o s t e r i c c o n t r o l mechanism.  The  sigmoidal  c h a r a c t e r i s t i c of a l l o s t e r i c mechanisms (Ferdinand,  1976)  present  within  itself  i n the case of i n o r g a n i c m e t a b o l i t e s  Minimum and maximumsP^-levels may sions i n t o organic  forms and  linear relationship. i log  The K  be  P-uptake  may  curve  not  the  plant.  governed by r e v e r s i b l e conver-  hence c o n t r o l may  appear only as  l i n e a r transformation  i Vmax-v . = log — + n log  of the H i l l  I_ I | S|  a equation:  - 72 was  employed t o e v a l u a t e the H i l l c o e f f i c i e n t (n) or the degree of  c o o p e r a t i v i t y p r e s e n t i n the uptake p r o c e s s v a l u e o f 1.81 l e v e l ) was  ( G l a s s , 1976).  ±0.29 ( s i g n i f i c a n t l y d i f f e r e n t from 1.0  o b t a i n e d from the s l o p e o f the l i n e a r  shown i n F i g u r e 31.  An n  at a =  0.01  relationship  I f no c o o p e r a t i v i t y were p r e s e n t n would have  a v a l u e o f 1. n v a l u e s have e r r o n e o u s l y been c l a i m e d , by enzymolog i s t s , to r e p r e s e n t the number of a l l o s t e r i c m o d i f i e r s i t e s when i t i s a c t u a l l y a measure o f the degree of c o o p e r a t i v i t y by the enzymes i n v o l v e d ( F e r d i n a n d , 1976).  possessed  As such, a l t h o u g h  the  number of r e g u l a t o r y s i t e s p e r P - " c a r r i e r " was not a s c e r t a i n e d , c o n t r o l of orthophosphate  uptake appeared t o be a c o o p e r a t i v e  p r o c e s s w i t h r e s p e c t t o the r o o t s ' i n t e r n a l orthophosphate The  level.  r a p i d 'shutdown' o f phosphate uptake c o u l d a l s o be  e x p l a i n e d by more c o m p l i c a t e d p h y s i o l o g i c a l p r o c e s s e s .  These  mechanisms would of n e c e s s i t y i n v o l v e i n d i r e c t feedback o c c u r r i n g through i n t e r m e d i a t e s such as hormones and/or o r g a n i c phosphates. Because of the need f o r added b i o c h e m i c a l s t e p s i n such r e g u l a t o r y processes  t h e i r o c c u r r e n c e might seem u n l i k e l y , e s p e c i a l l y i n l i g h t  of the p o s s i b i l i t y of a more d i r e c t orthophosphate phosphate  uptake.  feedback  upon  - 73 -  F i g u r e 31. .  H i l l p l o t (v/Vmax - v a g a i n s t P i concentration)  -0.8  -0.4  o.O  Log P i c o n c e n t r a t i o n (umoles/g.f.wt.)  internal  IV.  CONCLUSION  A n a l y s i s o f s h o r t - t e r m phosphate  uptake i n i n t a c t b a r l e y  c v . Bonanza has p r o v i d e d c o n s i d e r a b l e i n s i g h t i n t o two p r o c e s s e s w h i c h appear t o be e l i c i t e d by d i s t i n c t c o n t r o l mechanisms. The enhancement o f p o t e n t i a l phosphate  uptake  rates  through P - d e p r i v a t i o n t a k e s p l a c e o v e r a p e r i o d o f days w h i c h would be ample time f o r m e t a b o l i c p r o c e s s e s such as p r o t e i n s y n t h e s i s or d e g r a d a t i o n t o o c c u r .  There i s e v i d e n c e f o r an enrichment i n  P-uptake s i t e s on t h e -P p l a n t s ' r o o t s u r f a c e .  T h i s enrichment may  be the end e f f e c t o f numerous b i o c h e m i c a l p r o c e s s e s which r e s u l t i n an a d a p t i v e response t o P - d e p r i v a t i o n .  ultimately  Control signals  may be e l i c i t e d t h r o u g h growth p a t t e r n s , o r i n o r g a n i c or o r g a n i c phosphate  l e v e l s o f e i t h e r t h e p l a n t s ' shoots o r r o o t s .  Because of  the apparent d e v e l o p m e n t a l p r o c e s s g o v e r n i n g P-uptake r a t e s , the r o o t a b s o l u t e P ^ - c o n c e n t r a t i o n c o u l d c o n t r o l the e x t e n t o f enhanced uptake. The d e c l i n e i n p o t e n t i a l phosphate  uptake r a t e s revealed-  when phosphate was s u p p l i e d t o p l a n t s p o s s e s s i n g e l e v a t e d i n f l u x , r a t e s , s u g g e s t s the o c c u r r e n c e o f two r e g u l a t o r y systems.  The r a p i d  d e c l i n e p r o c e s s s t a r t s w i t h i n one h o u r , and p r o t e i n d e g r a d a t i o n t h e r e f o r e i s n o t l i k e l y t o have been i t s cause.  This process  appears  t o be a l l o s t e r i c a l l y c o n t r o l l e d by the r o o t s ' i n t e r n a l o r t h o p h o s phate c o n c e n t r a t i o n . The time n e c e s s a r y t o e l i c i t d e c l i n e i s  -  75  -  s i m i l a r t o the c y t o p l a s m i c P-exchange h a l f - l i f e and hence P - f l u x e s may The  vacuolar  be i n v o l v e d i n the t r i g g e r i n g of P-uptake d e c r e a s e s .  p r o c e s s which o c c u r s a f t e r 8 hours p r e t r e a t m e n t w i t h  phosphate i s d i s t i n c t from t h a t of the p r e v i o u s  ortho-  time p e r i o d because  i n the l a t t e r p e r i o d the r e d u c t i o n of P-uptake by P^ f a i l e d  to  demonstrate p r o p o r t i o n a l i t y t o P ^ - l e v e l s as i n the former p e r i o d . At the l o n g e r exposure times t o phosphate s u p p l y reduced P-uptake may  be a c h i e v e d by " c a r r i e r " The  degradation.  f l e x i b i l i t y of the p h y s i o l o g i c a l component of phosphate  a b s o r p t i o n r a t e s enables h o m e o s t a t i c c o n t r o l of phosphate concentrations within barley plants. I n the s o i l environment a v a i l a b l e phosphate a c t i v i t y i s b u f f e r e d by a d s o r p t i o n t o s o i l p a r t i c l e s .  This p h y s i c a l a s s o c i a t i o n  i s a l s o r e s p o n s i b l e f o r the l i m i t e d m o b i l i t y of P i n s o i l s .  The  growth of p l a n t r o o t s i n t o a r e g i o n of h i g h phosphate such as i n the d r i l l i n g and banding of P i n a g r i c u l t u r a l p r a c t i c e might l e a d t o e x c e s s i v e P - a b s o r p t i o n w i t h subsequent d e l e t e r i o u s e f f e c t s such as 'burning'  of a e r i a l p a r t s .  T h i s a p p l i e s not o n l y t o p l a n t s adapted  t o low-P s o i l s but even i n a g r i c u l t u r a l l y i m p o r t a n t crop p l a n t s ( B h a t t i and Loneragan, 1970a, b; Green e t . a l . , 1973a, b; 1978).  Siddiqi,  T h e r e f o r e the c a p a c i t y t o 'shutdown' P-uptake f a i r l y r a p i d l y  i n response t o i n c r e a s e d P - a v a i l a b i l i t y i s d e c i d e d l y i m p o r t a n t under natural conditions. P-uptake may  Those cases c i t e d of t i s s u e damage from excess  r e f l e c t e i t h e r an i n a b i l i t y t o r e g u l a t e , as i n p l a n t s  - 76  -  adapted t h r o u g h e v o l u t i o n t o low-P environments ( e . g . h e a t h p l a n t s ) , or an i n a b i l i t y t o respond r a p i d l y enough as i n the case o f  crop  p l a n t s a c c l i m a t e d t o low-P regimes. P h y s i o l o g i c a l adaptations  f o r i n c r e a s e d P-uptake are  l e s s energy consuming than m o r p h o l o g i c a l n e c e s s i t y , r e q u i r e growth. d i f f e r e n c e may  a d a p t a t i o n s w h i c h of  In nutrient limited conditions this  be c r i t i c a l .  B a r l e y p l a n t s grown i n -P media r e v e a l e d  i n c r e a s e s i n main r o o t s u r f a c e a r e a o n l y w e l l a f t e r the  physio-  l o g i c a l uptake r a t e s had maximized on a per p l a n t b a s i s .  Increased  r o o t - h a i r development occurs a t a s t i l l l a t e r age as perhaps a last resort.  M o r p h o l o g i c a l a d a p t a t i o n s may  surface only a f t e r  severe P - d e p r i v a t i o n , e n a b l i n g the r o o t s t o , i n e f f e c t ' s e a r c h ' f o r l o c a l i z e d s o i l phosphorus s o u r c e s , however t h e r e remains the p o s s i bility  t h a t the h y d r o p o n i c environment used i n t h i s s t u d y u n n a t u r a l l y  r e t a r d e d r o o t morphogenesis. The work p r e s e n t e d  i n t h i s t h e s i s has  tended t o  concentrate  upon the p h y s i o l o g i c a l b a s i s of p l a n t a d a p t a t i o n t o P - d e p r i v a t i o n . 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