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

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THE REGULATION OF PHOSPHATE UPTAKE BY INTACT BARLEY PLANTS by DANIEL DENIS LEFEBVRE B . S c , U n i v e r s i t y of Ottawa, 1977 THESIS SUBMITTED IN PARTIAL FULFILLMENT THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department of Botany) We accept t h i s t h e s i s as conforming to the re q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA 1980 Daniel Denis Lefebvre In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make i t freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. BOTANY Department of The University of British Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date June 12, 1980 ABSTRACT The study of phosphate i n f l u x i n roots of i n t a c t b a r l e y (Hordeum vulgare L. var. Bonanza) revealed the presence of two d i s t i n c t r e g u l a t o r y processes f o r phosphate absorption. One of these processes, which 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 the form of enhanced phosphate uptake, became evident between 11 and 13 days a f t e r germination. At 16 days the "uptake ra t e s of these p l a n t s had reached a maxi-mum value at 2.43umol/g.f.wt./hour which compared to a value of 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 . Simul-taneously, d i f f e r e n c e s between the r e s p e c t i v e treatments were a l s o noted i n growth ra t e s and phosphate pools. A second r e g u l a t o r y process brought about a r a p i d r e d u c t i o n of phosphate i n f l u x upon the p r o v i s i o n of orthophosphate to p l a n t s p r e v i o u s l y starved of phosphate during the phase of enhanced uptake. W i t h i n hours of supplying i n o r g a n i c phosphate to these p l a n t s i n f l u x was reduced by greater than 50% and during t h i s p e r i o d i n f l u x values were l i n e a r l y c o r r e l a t e d w i t h root orthophosphate concentrations. The time s c a l e of t h i s second response i s suggestive 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 i n t e r n a l orthophosphate l e v e l s . Both r e g u l a t o r y systems s t u d i e d represented p h y s i o l o g i c a l adaptations which would b e t t e r enable p l a n t s , under f i e l d c o n d i t i o n s , i i to o b t a i n a s u f f i c i e n t phosphate supply. 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 morphological response such as the production of longer, narrower roots providing, the p l a n t s a greater surface area, presumably f o r greater phosphate absorption. At a l a t e r time, an increased formation of r o o t - h a i r s r e s u l t e d i n even greater surface area m o d i f i c a t i o n . i i i TABLE OF CONTENTS Page ABSTRACT i i ACKNOWLEDGEMENTS v i i i I . INTRODUCTION 1 I I . METHODS 4 1. Hydroponic P l a n t Growth 4 A. Seeds „ 4 B. Choice of Phosphate Levels and Growth Regimes 4 2. Determination of 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 A n a l y s i s 9 4. Determination of the P l a n t s ' Phosphate Concentrations . . . . . . . . . . 10 A. T o t a l Phosphate Concentrations 10 B. Inorganic Phosphate Concentrations . . . . . 10 5. Double l a b e l l e d Uptake Determination 11 6. Determination of Root E x t e r n a l P r o t e i n 13 I I I . RESULTS AND DISCUSSION 15 1. E f f e c t of 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 of Phosphate I n f l u x 25 A. Determination of Root Wash P e r i o d 25 B. Temperature and pH Optimums 27 i v 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 D e p r i v a t i o n 34 A. Phosphate Uptake 34 B. T o t a l Phosphate and Inorganic Phosphate Levels w i t h i n the P l a n t s 40 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 of Enhanced Phosphate Uptake 49 i . Uptake Isotherms 49 i i . Uptake from S t i r r e d and N o n - s t i r r e d Media 53 i i i . D o u b l e - l a b e l l e d Uptake Experiment. . . . 56 i v . Determination of Root E x t e r n a l P r o t e i n . 59 5. Regulation of Rapid Decline of Enhanced Inorganic Phosphate Uptake 61 A. Short-term versus Long-term Uptake 61 B. Phosphate Uptake Rates and P l a n t Phosphorus Levels Following Exposure of P l a n t s to 15uM Phosphate 63 IV. CONCLUSION 74 REFERENCES 77 V LIST OF TABLES page 1. Composition of hydroponic growth media 6 2. Determination of regime r e q u i r e d f o r e l u t i o n of 86 32 a l l Rb and r e t e n t i o n of a l l P from anion-exchange columns 12 3. E o s i n s t a i n i n g technique 14 4. +P and -P growth equations 16 5. Uptake rates 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 54 6. Uptake rates 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 7. Short-term vs. long-term uptake r a t e determination . . 62 v i LIST OF FIGURES Page 1. 5 day old root t i p s 18 2. 10 day old root t i p s 18 3. 15 day old root t i p s 19 4. 20 day old root t i p s 19 5. 25 day old root t i p s 20 6. 30 day old root t i p s 20 7. 40 day old +P root t i p 21 8. 40 day old -P root t i p 21 9. Plant growth vs. age 22 10. Root:shoot r a t i o vs. plant age 23 11. Growth of roots 24 32 12. E f f l u x of P from rapid exchange phase 26 13. Temperature curve of phosphate uptake at pH 6.25 . . 28 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 rate vs. plant age - g.f.wt. analysis . . . 35 17. P^ uptake rate vs. plant age - per plant analysis . . 37 18. P^ and t o t a l phosphorus of -P roots vs. age 42 19. P^ and t o t a l phosphorus of -P shoots vs. age . . . . 42 20. P^ and t o t a l phosphorus of +P roots vs. age 42 21. P. and t o t a l phosphorus of +P shoots vs. age . . . . 42.. v i i LIST OF FIGURES (continued) Page 22. Phosphate concentration i n -P b a r l e y p l a n t s 47 23. Phosphate concentration 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 : Michaelis-Menten p l o t 50 25. Orthophosphate uptake k i n e t i c s : Eadie-Hofstee p l o t . . 51 26. Phosphate uptake rat e vs. time of root exposure to. 15uM orthophosphate 64 27. T o t a l phosphorus con c e n t r a t i o n during the p e r i o d of phosphate l o a d i n g of -P grown b a r l e y p l a n t s - t r e a t e d roots 65 28. T o t a l phosphorus concentration during the p e r i o d of phosphate l o a d i n g of -P grown b a r l e y p l a n t s - t r e a t e d shoots 66 29. Inorganic phosphate con c e n t r a t i o n during the p e r i o d of phosphate lo a d i n g of -P b a r l e y p l a n t s 68 30. Phosphate uptake rat e v s . P^ concentration 69 31. H i l l p l o t (v/Vmax - v against i n t e r n a l P^ concentration) 73 v i i i ACKNOWLEDGEMENTS I would l i k e to thank my graduate a d v i s o r , Dr. Anthony D. M. Glass, Botany Department, U n i v e r s i t y of B r i t i s h Columbia, f o r h i s t e c h n i c a l a s s i s t a n c e and support throughout the course of the graduate work. Thanks i s a l s o extended to my advisory committee c o n s i s t i n g of Dr. Beverly R. Green and Dr. Paul J . H a r r i s o n . S p e c i a l a p p r e c i a t i o n f o r the pati e n c e , understanding, and help I have r e c e i v e d , goes to my dear f r i e n d Dr. L o i s Shepherd and to my parents. This work was supported by the N a t u r a l Sciences arid Engineering Research C o u n c i l of Canada, through a grant to Dr. Glass. - 1 -I . INTRODUCTION There i s now considerable evidence to suggest that the a c t i v e or energy dependent uptake of d i s t i n c t i n o r g a n i c ions by the roots of higher p l a n t s , i s subject to independent negative feedback c o n t r o l (Cram, 1976). This c o n t r o l i s thought to be e l i c i t e d i n response to changes of the i n t e r n a l c oncentration of the p a r t i c u -l a r n u t r i e n t , which u l t i m a t e l y exerts c o n t r o l over the root uptake process. The i n v e s t i g a t i o n of the mechanism un d e r l y i n g the c o n t r o l of uptake would appear, a p r i o r i , to be more complicated i n the cases 2- -of metabolized i o n s , such as SO. , N0„ , H„PO. , e t c . , by v i r t u e 4 3 2 4 of the d i v e r s i t y of the end products of t h e i r metabolism. This may consequently account f o r the more extensive i n v e s t i g a t i o n s of t h i s aspect of uptake of non-metabolized i o n s . Thus, feedback c o n t r o l mechanisms have been proposed f o r potassium (Humphries, 1951; Johansen e t . a l . , 1970; Young e t . a l . , 1970; Glass, 1976; Jensen and P e t t e r s s o n , 1978), N a + (Pitman 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). I n the case of an i o n which i s metabolized, c o n t r o l of i n f l u x might be achieved v i a concentrations of the i o n i t s e l f or one of i t s metabolic products. S u l f a t e uptake f o r example, has been shown to be reduced by p r i o r feeding w i t h the s u l f u r - c o n t a i n i n g amino acids - 2 -cysteine and methionine (Hart and F i l n e r , 1969; F e r r a r i and Renosto, 1972; Cram, 1976). S i m i l a r r e s u l t s have been obtained f o r the i n f l u e n c e of NH^ + and amino acids 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 of phosphate uptake (Humphries, 1951; Bowen, 1970; Barber, 1972; Cartw r i g h t , 1972; Clarkson e t . a l . , 1978) and a n a l y s i s of t o t a l phosphate concentrations i n these p l a n t s reveals that i n f l u x and phosphate content are n e g a t i v e l y c o r r e l a t e d (Barber, 1972; Clarkson e t . a l . , 1978). L i v i n g organisms might be imagined to possess long- and short-term mechanisms by which they are able to modify t h e i r r a t e s of i o n uptake through biomembranes. The " c a r r i e r " systems r e s p o n s i b l e 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 are thought to be composed of p r o t e i n molecules ( M i t c h e l l , 1967). On a long-term b a s i s " c a r r i e r " degradation or sy n t h e s i s could e f f e c t changes of tr 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 enzymatic c o n t r o l mechanism could 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 or energy supply f o r the s p e c i f i c t r a n s p o r t phenomenon i n question. 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 could act 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 the mechanisms described above. The present study i s d i r e c t e d toward a b e t t e r understanding of the s i g n a l s and mechanisms which govern phosphate uptake i n b a r l e y (Hordeum vulgare (L.) c v . Bonanza) . - 3 -I n higher p l a n t s i n t e r n a l orthophosphate concentration i s much more s u s c e p t i b l e to f l u c t u a t i o n s i n phosphorus supply than the organic phosphorus concentration ( B i e l e s k i , 1968; Nassery, 1971). Because of the f l e x i b i l i t y of the i n o r g a n i c phosphate l e v e l i t s absolute magnitude w i t h i n p l a n t c e l l s represents a good candidate f o r e f f e c t i v e r e g u l a t i o n of phosphate uptake. With t h i s i n mind the a n a l y s i s of i n o r g a n i c and organic phosphate l e v e l s w i t h i n the pla n t was conducted simultaneously w i t h measure-ments of phosphate uptake rates 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 study, namely b a r l e y , i s one of economic value and r e p r e s e n t a t i v e of the c e r e a l crop p l a n t s r a i s e d i n Canada. - 4 -I I . METHODS 1. Hydroponic P l a n t Growth A. Seeds Seeds of b a r l e y (llordeum vulgare (L.) c v . Bonanza) purchased from B u c k e r f i e l d s , Vancouver, were surface s t e r i l i z e d f o r ten 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 water were germinated i n sand at 27±2C. B. Choice of Phosphate Levels and Growth Regimes Many workers i n previous s t u d i e s have employed phosphate l e v e l s which were u n n a t u r a l l y high (Cartwright, 1972; Clarkson e t . a l . , 1978; B i e l e s k i , 1968; Barber, 1967). I t was considered worth-w h i l e i n the present study to use phosphorus l e v e l s which were more re 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 concentrations ( B i e l e s k i , 1973). These c r i t e r i a are met by a 15uM orthophosphate l e v e l i n hydroponic media. P l a n t s were th 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) or n u t r i e n t s o l u t i o n i n which no phosphorus was s u p p l i e d (-P, see Table 1). Because of the la r g e volume of c i r c u l a t e d growth medium i t was necessary to monitor and top up phosphate l e v e l s to 15yM only twice d a i l y . Both +P and -P growth media were replaced i n f u l l every four days. P l a n t s were grown i n an environmental regime which co n s i s t e d of 16h days at 27±2C and - 5 --2 i r r a d i a n c e of 3.0 mW cm , and 8h n i g h t s at 19±2C. - 6 -Table 1. Composition of hydroponic growth media - Concentrations are uMolar -P 111.2 KNO„ 0.11 MnSO..H„0 3 4 2 83.3 Mg(N0 3) 2 0.11 ZnS0 4.7H 20 27.8 MgS0 4 0.03 CuS0 4.5H 20 55.6 C a ( N 0 3 ) 2 0.03 H 2Mo0 4 2.7 KC1 0.11 FeEDTA 1.4 H 3B0 3 Buffered to pH 6.25 w i t h lOuM N a 3 C i t r a t e : C i t r i c A c i d +P as -P plus 5.0 Na 2HP0 10.0 NaH„P0 - 7 -2. Determination of Phosphate I n f l u x Method A. Root Wash P e r i o d The 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 contain sub-s t a n t i a l i o n r e s e r v o i r s which possess h a l f - l i v e s f o r i o n exchange of the order of 1-3 min (Walker and Pitman, 1976; Cram, 1973; Dainty and Hope, 1959). P r i o r to i n f l u x experiments of r e l a t i v e l y short d u r a t i o n , designed to estimate i n i t i a l plasmalemma f l u x e s , i t i s necessary th e r e f o r e to standardize the c e l l w a l l phosphate st a t u s of roots 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 root free 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 introduce u n c e r t a i n t y to the c a l c u l a t e d f l u x . 32 By loading the root w i t h 15uM P - l a b e l l e d orthophosphate and subsequently t r a n s f e r r i n g these roots to 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 be 32 made. This was done by measuring release of P to the n o n - l a b e l l e d wash medium at i n t e r v a l s of time up to 30 min a f t e r t r a n s f e r . The estimated value 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 can be used to determine the appropriate 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 estimate of plasmalemma i n f l u x i t i s necessary to d i s t i n g u i s h the a c t i v e uptake from passive adsorption i n the c e l l - 8 -w a l l . The h a l f - l i f e f o r c e l l w a l l exchange can be used here to obt a i n the necessary s e p a r a t i o n . B. Phosphate I n f l u x Rates of orthophosphate uptake were determined a f t e r a 5 min prewash i n 50yM CaSO^ at 30C. The uptake s o l u t i o n was 32 i d e n t i c a l to the +P growth medium w i t h P - l a b e l l e d orthophosphoric a c i d and/or RbCl,, added. E i t h e r 10 min (short-term) or 24h (long„ term) uptake periods were used. I n a l l but one experiment 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 aerated. Uptakes were performed at 30C i n s o l u t i o n volumes (1.6 1) where n u t r i e n t deple-t i o n was n e g l i g i b l e . The uptake p e r i o d was followed immediately by a 5 min desorption 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 basket c e n t r i f u g e to remove e x t r a -neous water. These samples were weighed"into glass v i a l s to o b t a i n f r e s h weights and f i n a l l y ashed at 500C. The r e s u l t a n t ashed samples 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 and t h e i r r a d i o a c t i v i t y was determined by Cerenkov counting ( 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 counter. - 9 -3. E f f l u x A n a l y s i s E f f l u x determination was performed a f t e r feeding p l a n t s 32 i n a constant 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 pl a n t s were then placed i n n o n - l a b e l l e d +P s o l u t i o n s at 30C and _2 an i r r a d i a n c e l e v e l of 3.0 mW cm . I n order to estimate e f f l u x uncomplicated 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 , standard procedure i s to 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 the medium back to the cytoplasm i s presumed to be zero. At the end of the e f f l u x a n a l y s i s t o t a l phosphate content as w e l l as i s o t o p i c content remaining i n the root t i s s u e , was determined. This enabled subsequent c a l c u l a t i o n s of the p a t t e r n of e f f l u x according to standard methodology (Walker and Pitman, 1976). - 10 -4. Determination of the P l a n t s ' Phosphate Concentrations A. T o t a l Phosphate Concentrations Fresh root 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 water, and assayed f o r t o t a l phosphorus by the method of E i b l and Lands (1969). Values expressed through-out the text are i n umol/g.f.wt. B. Inorganic Phosphate Concentrations Inorganic phosphorus pools of both shoots and roots were obtained by a v a r i a t i o n of the method of Daley and Vines (1977). Samples were plunged i n t o b o i l i n g water f o r two min, then were r a p i d l y f r o z e n , thawed, and placed i n b o i l i n g baths f o r 5 min, twice i n succession. Using samples of glucose-6-P and ATP, i t was e s t a b l i s h e d that t h i s method,as claimed by H u l e t t (1970), caused no h y d r o l y s i s of organic P bonds. - 11 -5. Double L a b e l l e d Uptake Determination 32 86 P and Rb were used simultaneously to determine phosphate and potassium 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) r e s p e c t i v e l y . The s e p a r a t i o n of these isotopes was obtained by anion-exchange chromatography of the ashed samples. Dowex-lx8-100 was primed i n one hundred times i t s volume of 5M NaOH. 2 ml Pasteur p i p e t t e columns were then poured and washed three times w i t h deionized water. 2 ml of wet Dowex-lx8-100 r e s i n has an exchange c a p a c i t y of 2.8 m i l l i - a q u i y a l e n t s . By the means described i n Table 2 a s u i t a b l e regime was defined f o r use on b i o l o g i c a l samples. The s e p a r a t i o n 86 32 of 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 these isotopes were present i n ca r r i e r s - f r e e s o l u t i o n s or 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 of the n a t u r a l d i s t r i b u t i o n of potassium and phosphate. Samples were counted before and a f t e r exposure to 86 32 the defined regime. I n i t i a l counts gave estimates of Rb + P a c t i v i t y . Counting of the e l u a t e obtained a f t e r anion-exchange 86 32 chromatography gave Rb a c t i v i t y . P counts were obtained by sub-86 32 t r a c t i n g the l a t t e r a c t i v i t y from the combined Rb + P r a d i o -a c t i v i t y . - 12 -Table 2. Determination of regime required f o r e l u t i o n of a l l 86 32 Rb and r e t e n t i o n of a l l P from anion-exchange columns. 10 ml of s o l u t i o n s shown (A to F) were a p p l i e d to the columns and elu a t e c o l l e c t e d 1. without f u r t h e r column washing. 2. a f t e r e l u t i o n w i t h 10 ml H^O. 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 ml of H 20. E f f i c i e n c y of e l u t i o n of Kb or P expressed as % of known counts a p p l i e d to column 8 6Rb Treatment A. C a r r i e r - f r e e B. +0.1M K C. +0.1MK+ 3 2 P + 2.0mM P 1. 75.5 80.7 81.6 2. 24.0 20.3 18.4 3. 0.5 n i l 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 6Rb + 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. Determination 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 technique of W i l l i a m s (1962) was used to determine root e x t e r n a l p r o t e i n . The procedure i s presented i n Table 3. - 14 -Table 3. Eo s i n s t a i n i n g technique Treatment Duration (min) 1. Weighing of root samples 2. 2 d i s t i l l e d water r i n s e s 3. 0.1 N HC1 1.0 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 0.5 6. 5 d i s t i l l e d water r i n s e s 7. 0.1 M KC1 + K0H (pH = 0.5 13.0) 3 ml. volume 8. O p t i c a l Density measure-ment at 520 nm. I I I . RESULTS AND DISCUSSION 1. E f f e c t of Phosphate L e v e l on Growth Fresh weights 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 Figure 9 ) . There were no d i f f e r e n c e s between +P and -P seedlings up to day 11 (a = 0.05), beyond which stage 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 rates w h i l e the phosphate starved p l a n t s increased 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 = 0.05, see Table 4 ) . By day 20 the r a t i o of +P to -P p l a n t weights was i n excess of two but no q u a l i t a t i v e morphological d i f f e r e n c e s were apparent between the treatments. The diameters of roots i n the +P and -P p l a n t s are shown i n Figures 1 to 8. Phosphate s t r e s s has been p r e v i o u s l y shown to r e s u l t i n decreased root diameters (Bowen e t . a l . , 1974). Increased r o o t - h a i r development became apparent i n -P roots only w e l l a f t e r day 20 (see Figures 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 s i g n i f i -cant (a = 0.05) root:shoot r a t i o increases (see Figure 10) when compared to +P p l a n t s . By day 20 f o r example, -P p l a n t s had favoured root growth to such an extent that the root:shoot r a t i o e q u a l l e d 2 compared to 0.5 f o r +P p l a n t s . This p r e f e r e n t i a l growth enabled the -P p l a n t s to form almost as much root mass as the f u l l y nourished p l a n t s (Figure 11). I n the s o i l environment where phosphate supply may be l i m i t e d and d e p l e t i o n zones sharply l o c a l i z e d due to the - 16 -Table 4. +P and -P growth equations A. +P ex p o n e n t i a l growth equation from day 5 to 20, y = 0 . 1 0 7 2 e 0 , 1 7 4 3 x , r = 0.9967 B. -P l i n e a r growth equation from days 12 to 20. y = -1.1667 + 0.13983x, r = 0.9679 y = p l a n t s i z e (g.f.wt./plant) x = age of 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, increased surface area and s o i l e x p l o r a t i o n would be an adaptive advantage (Harley, 1969). Growth st u d i e s performed w i t h numerous species under various phosphorus regimes have shown p o s i t i v e c o r r e l a t i o n s between growth and P^ supply. The magnitude of growth response i s depen-dent upon the species i n v o l v e d (Clarkson, 1967; P i g g o t t , 1971; Rorison, 1968; Asher and Loneragan, 1967; Bradshaw e t . a l . , 1960). Root to 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 (Hackett, 1968; Asher and -Loneragan, 1967), but t h i s i s not always the case (Troughton, 1977). Increased root growth, however, might have occurred i f Troughton had used lower l e v e l s of phosphorus (Bradshaw e t . a l . , 1960). The root:shoot r a t i o s of a number of grasses grown at d i f f e r e n t P - l e v e l s are comparable to the b a r l e y data obtained (Figure 10). Because r o o t - h a i r formation i s retarded i n hydroponic c u l t u r e (Bole, 1973) , i t i s of p a r t i c u l a r i n t e r e s t that r o o t - h a i r s were observed to develop under severe P - d e f i c i e n c y and only once has t h i s phenomenon been p r e v i o u s l y reported (Brewster e t . a l . , 1976). Root-hairs enable p l a n t s to increase 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 process (Barley and R o v i r a , 1970). - 18 -Figure 1. 5 day o l d root t i p s (8 times l i f e - s i z e ) F igure 2. 10 day o l d root t i p s (8 times l i f e - s i z e ) Figure 4. 20 day o l d root t i p s (8 times l i f e - s i z e ) - 20 -Figure 5. 25 day o l d root t i p s (10 times l i f e - s i z e ) Figure 6. 30 day o l d root t i p s , +P above, -P below C26 times l i f e - s i z e ) - 21 -Figure 8. 40 day old -P root t i p (30 times l i f e - s i z e ) - 22 -Figure 9. P l a n t growth vs. age 5 10 15 20 AGE (days) - 23 -Figure 10. Root: shoot r a t i o vs. p l a n t age 10 15 20 Age (days) - 24 -Figure 11. Growth of roots 1.5r A +P A - P A A A ' A A ^ A A A A A A A A 10 15 20 AGE (days) - 25 -2. C h a r a c t e r i z a t i o n of Phosphate I n f l u x A. Determination of Root Wash P e r i o d 32 The k i n e t i c s of P re l e a s e from roots exposed to l a b e l l e d medium f o r 10 min are shown i n Figure 12. Greater than 96% of 32 the rapidly-exchanging P f r a c t i o n had e f f l u x e d by 5 min. In subsequent experimentation t h e r e f o r e , a 5 min wash p e r i o d was adopted as standard procedure wherever i t was necessary to estimate 32 i n t r a c e l l u l a r , as opposed to e x t r a c e l l u l a r phosphate or P-phosphate content. Figure 12. E f f l u x of 32p f r o m R a p i d Exchange Phase 550 P e Time (min) - 27 -B. Temperature and pH Optimums. Transport of phosphate i n t o the symplasm from the e n v i r o n -ment i s probably an enzymatic process ( E p s t e i n , 1976) and the " c a r r i e r s " which c a t a l y z e v e c t o r i a l phenomena are the r e f o r e s u s c e p t i b l e to changes i n environmental temperature and pH. These p h y s i c a l para-meters could f e a s i b l y a l t e r the r a t e of phosphate uptake by modifying the molecular c o n f i g u r a t i o n of s p e c i f i c enzymes or by an e f f e c t on the general energy metabolism i n t o t o (Boyer, 1970). The temperature at which b a r l e y roots express t h e i r optimum P-uptake rat e i s approximately 30C (see Figure 13). The optimum pH f o r phosphate uptake w i t h i n a reasonable s o i l range of 5.0 to 7.5 was 6.25 (Fi g u r e 14). P l a n t s i n general p r e f e r e n t i a l l y absorb mono-va l e n t ions over d i v a l e n t or t r i v a l e n t ions ( E p s t e i n , 19 76) and Hagen and Hopkins (1955) have hypothesized that phosphate uptake i s d i r e c t l y p r o p o r t i o n a l to the amount of monovalent orthophosphate present i n the medium as determined by medium pH. Although pH does 2-c 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 to be the only e f f e c t of pH upon orthophosphate uptake i n the present study. Dunlop and Bowling's (1978) study w i t h white c l o v e r i n d i -cated that pH may a f f e c t phosphate uptake i n ways other than i t s c o n t r o l on the absolute amount of a v a i l a b l e monovalent orthophosphate. A l l phosphate f l u x determinations were subsequently performed at 30C i n media b u f f e r e d to pH = 6.25 by 10uM N a ^ c i t r a t e and c i t r i c a c i d . - 28 -Figure 14. pH Curve of Phosphate Uptake at 30C 6.0 r 5 ? 5 .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 nourished f i v e day o l d Bonanza b a r l e y p l a n t s are shown i n Figure 15. The data 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 three phases of e f f l u x . Considerations of the s i z e and the k i n e t i c constants of exchange f o r these phases have l e d workers to the conclusion that these phases represent a s e r i e s arrangement of the c e l l w a l l , cytoplasm, and vacuolar f r a c t i o n s (Walker and Pitman, 1976). Most work i n root 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 d e a l t w i t h non-metabolized ions such as K +, Na +, C l , e t c . (Cram, 1973; Walker and Pitman, 1976). A p r i o r i i t was a n t i c i p a t e d that the k i n e t i c s of phosphate e f f l u x would be complex. Not only would the P^ f r a c t i o n be expected to show the standard t r i p h a s i c p a t t e r n but organic f r a c t i o n s could be expected to overlay t h i s b a s i c form. By contrast the s t r i k i n g ' s i m i l a r i t y to p r e v i o u s l y reported k i n e t i c s l e a d to the c o n c l u s i o n that the observed k i n e t i c s d e s c r i b e the exchange of a s i n g l e P species - most probably i n o r g a n i c phos-phate si n c e under the present c o n d i t i o n s of growth i t would represent the major P - f r a c t i o n . Furthermore the enzymatic r e a c t i o n s which would r e l e a s e l a b e l l e d orthophosphate from metabolized forms are probably not l i m i t i n g exchange. I f such re 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 process would almost c e r t a i n l y be much more complex. As such i t i s reasonable to b e l i e v e that the three phases represent c e l l w a l l , cytoplasmic and vacuolar exchange of P^. - 30 -C a l c u l a t i o n of the P-content of the f r a c t i o n w i t h the lowest exchange r a t e gave a maximum pool s i z e of 11.97ymol/g.f.wt. (see Figure 15). Although t h i s value 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), the m a j o r i t y of the exchanging phosphate probably a r i s e s from the vacuoles of c e l l s and the root xylem i f a comparison can be made, to non-metabolized ions ( 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 values which place vacuolar 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 exchanging phase has a magnitude of 1.27ymol/g j f ,#wt. and an exchange h a l f - l i f e of approximately 45 min which i s comparable to the h a l f - l i f e of cytoplasmic exchange f o r other ions (Walker and Pitman, 1976). The f r e e space phase contained 2.38ymol/g,jf..wt. arid had a h a l f - l i f e of approximately 2 min which i s again s i m i l a r .to values obtained f o r other ions (Walker and Pitman, 1976). These data provide c o n f i r m a t i o n that a f i v e min wash pe r i o d i s s u f f i c i e n t to 32 release the bulk of f r e e space P, as w e l l as v a l i d a t i n g the use of a 10 min f l u x p e r i o d to estimate plasmalemma i n f l u x . These three phases are c o n s i s t e n t w i t h the b e l i e f that only a s i n g l e phosphate species i s being exchanged and that compart-mental b a r r i e r s are the plasmalemma and tono p l a s t . The terms " p o o l " and "compartment" as defined 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 of a compound are m e t a b o l i c a l l y - 31 ^ Figure 15. T o t a l phosphate e f f l u x k i n e t i c s 1.20 r 1.05 I 1 «. 0.0 * * * • ' • 1.0 2.0 3.0 4.0 Time (h) I I 5.0 —I i I 6.0 7.0 - 32 -i s o l a t e d from one another, whether or not t h i s i s due to t h e i r p h y s i c a l s e paration i n c e l l o r g a n e l l e s . Smith (1966) has shown that i n excess of 90% of the s o l u b l e phosphate-esters such as sugar-phosphates are present i n the cytoplasm. The t e r m i n a l P-groups of ATP turn over w i t h a h a l f - l i f e of 2-20 sec and most P-esters have h a l f - l i v e s of 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; Weigl, 1963). I n a d d i t i o n i f as has been suggested, the greater p r o p o r t i o n of phosphate i s i n the form of orthophosphate, i t i s not unreasonable to assume that the magnitude of P-esters would not l i m i t the exchange of P_^  at the membrane l e v e l . Very l i t t l e i s known about the turnover of P l i p i d s and RNA which cont a i n over 75% of the metabolized phosphate i n p l a n t s ( B i e l e s k i , 19 73). Although the h a l f - l i v e s of these compounds may vary consider-32 32 abl y , i f the magnitude of P-ester to P. conversion i s many f o l d 32 32 higher than that of P - l i p i d s or P-RNA, then the l a t t e r ' s c o n t r i -b u t i o n t o the cytoplasmic exchange phase would be n e g l i g i b l e . DNA 32 i s not expected to c o n t r i b u t e any exchangable P. RNA and P - l i p i d turnover could be of importance i n the slowest exchange phase, however the la r g e vacuolar P^-content ( B i e l e s k i , 1973) would render t h i s u n l i k e l y . I t i s encouraging to observe e s s e n t i a l l y s i m i l a r p atterns of phosphate e f f l u x to those d e s c r i b i n g the e f f l u x of non-metabolized i o n s . The h a l f - l i f e of cytoplasmic exchange may be of importance i n short-term 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 severely - 3 3 -P - d e f i c i e n t p l a n t s where the vacuole i s no longer able to supply P^ to the cytoplasm ( B i e l e s k i , 1968; Crossett and Loughman, 1966; Greenway and Klepper, 1968). - 34 -4. Enhancement of Phosphate Uptake Rate by Phosphate D e p r i v a t i o n A. Phosphate Uptake As f u l l y nourished b a r l e y seedlings aged t h e i r phosphate uptake ra t e s on a per gram f r e s h weight b a s i s d e c l i n e d . A s i m i l a r trend occurred i n -P grown p l a n t s u n t i l they were 12 days o l d (see Figure 16), at which time a r a p i d increase or enhancement of i n o r -ganic phosphate uptake was i n i t i a t e d i n the low P p l a n t s . By day 16 the uptake r a t e of -P p l a n t s was 6.3 times that of +P p l a n t s , however w i t h i n two days a dramatic d e c l i n e became evident. P l a n t s exposed to low amounts of phosphate have o f t e n been shown to express enhanced uptake ra t e s (Barber, 1972; Cartwright, 1972; Clarkson 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 high i n comparison to t h i s study. Clarkson and h i s colleagues (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 before t r a n s f e r r i n g h i s -P p l a n t s to phosphate minus s o l u t i o n s . As such, uptake r a t e s obtained by these researchers were i n some instances an order of magnitude lower than the present r e s u l t s . 4-P p l a n t s i n the present s t u d i e s could almost be described 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 extent of the r e g u l a t o r y response to P-status. The present choice of 15yM P, as p r e v i o u s l y s t a t e d , was an attempt to simulate more n a t u r a l s o i l c o n d i t i o n s . - 36 -Clarkson e t . a l . (1978) obtained four f o l d d i f f e r e n c e s i n uptake between P - d e f i c i e n t and f u l l y nourished treatments on a per gram f r e s h weight b a s i s . 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 the uptake ra t e of -P p l a n t s . When P^-uptake per pl a n t i s p l o t t e d (see Figure 17) s i m i l a r r a t e s are witnessed f o r +P and -P p l a n t s at younger ages, but at day 13 dramatic increases 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 per p l a n t b a s i s r e d u c t i o n i n uptake ra t e s as seen i n the per gram f r e s h weight p r e s e n t a t i o n ( a f t e r day 16) d i d not occur. Rather the rat e s reached a maximum value at day 16, which was sustained u n t i l the te r m i n a t i o n of the experiment at day 20. Throughout the p e r i o d from day 16 to 20 the r a t i o of uptake rates (-P/+P) remained steady at 4. The response of b a r l e y p l a n t s to low phosphorus l e v e l s (-P) i n hydroponic 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 to regulate phosphate uptake rates i n r e l a t i o n to phosphate s t a t u s . The enhance-ment curve of the -P p l a n t s ' uptake r a t e s on a per gram f r e s h weight b a s i s can be d i v i d e d i n t o three regions; a l a g stage, an enhancement stage, and a d e c l i n e stage. The length of the l a g and r a p i d i t y of the enhancement stage might be dependent on the balance of n u t r i e n t s t o r e s w i t h i n the seeds used. Changes i n the phosphate uptake ra t e s can be a t t r i b u t e d to p h y s i o l o g i c a l p r o p e r t i e s of the p l a n t since the uptakes were performed under co n d i t i o n s where phosphate d i f f u s i o n was not l i m i t i n g the phosphorus supply to the roots ( P o l l e and Jenny, 1971). FIGURE 17. Pj Uptake vs. Plant A g e 1.0 c 0.5 c/> o E I -L 1 1 tJ. 1 1 I t 5 7 9 11 13 15 17 19 21 AGE (Days) to ~*4 - 38 -I t i s i n t e r e s t i n g to compare the d i f f e r e n c e s i n the time s c a l e of response i n t h i s experiment to those e x h i b i t e d by K-uptake rat e s i n b a r l e y i n response to K - d e p r i v a t i o n (Glass, 1975), I n 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 to K-minus s o l u t i o n s produced increased 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 to the major osmotic f u n c t i o n of potassium. Enzyme a c t i v a t i o n requirements have been p o s t u l a t e d to be i n the range of 5-10uM, whereas osmotic requirements n e c e s s i t a t e up to 100-200mM K. This l a r g e demand f o r K by contrast to the r e l a t i v e l y low l e v e l s of P required may account f o r the extreme responsiveness i n the c o n t r o l of K-uptake. The r a p i d i t y of r e g u l a t i o n i n the case of K has l e d to the proposal of an a l l o s t e r i c c o n t r o l ( G l a s s , 1976; Pe t t e r s s o n and Jensen, 1978). The time s c a l e of the P-response makes i t d i f f i c u l t to assess the importance of 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 per gram f r e s h weight b a s i s there was a d e c l i n e stage i n P-uptake r a t e s . This may be a t t r i b u t e d to a r a p i d d e c l i n e i n v i g o u r of the 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, at t h i s stage no obvious signs of P - d e f i c i e n c y , other than reduced growth by comparison to +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 te r m i n a t i o n of " c a r r i e r " s y n t h e s i s , e t c . , or morphological e.g. an increased r e l a t i v e root growth. Fresh weight a n a l y s i s i n d i c a t e s that the roots of -P p l a n t s were a c t i v e l y growing during that 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 . This suggests that the vigour of the p l a n t was not s e v e r e l y retarded and that metabolic energy was a v a i l a b l e f o r numerous biochemical processes. I t i s u n l i k e l y that there would have been an energy l i m i t a t i o n upon the a c t i v e uptake processes and t h e r e f o r e the d e c l i n e i n P-uptake per gram f r e s h weight was probably the r e s u l t of a c e s s a t i o n i n net " 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 l e v e l l i n g o f f stages are of i d e n t i c a l d u r a t i o n i n both +P and -P treatments. These simultaneous occurrences r e v e a l t h e i r developmental o r i g i n as d i s t i n c t and independent of the phosphorus s t a t u s . The magnitude of the enhancement of P-uptake, however, could be a t t r i b u t e d to 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 metabolites. Growth patterns already presented showed divergence between +P and -P grown p l a n t s at the p e r i o d between days 11 and 13 which i s concurrent w i t h the divergence i n phosphate uptake rates between the two treatments. Therefore the morphological responses revealed through d i s t i n c t growth patterns 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 to r e s u l t i n e l e v a t e d P-uptake rates (Humphries, 1951; Barber, 1972; Cartwright, 1972; Bowen, 1970; Clarkson e t . a l . , 1978), such simultaneous p h y s i o l o g i c a l and morphological e f f e c t s of phosphate s t a t u s have not been p r e v i o u s l y reported. - 40 -B. T o t a l Phosphate and Inorganic Phosphate Le 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 content of -P r o o t s on a per gram f r e s h weight b a s i s , as expected, d i d occur as the b a r l e y seedlings aged (Figure 18). Such a d e c l i n e was not evident i n the i n o r g a n i c phosphate p o o l (confidence l e v e l a = 0.05), although t h i s p ool represented but a small percentage of the t o t a l P. The shoots of P - d e f i c i e n t p l a n t s dropped i n phosphorus content by over one h a l f between days 11 and 12, and a subsequent steady d e c l i n e followed (see Figure 19). The 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 occurred between days 9 and 11. The i n o r g a n i c phosphate content of -P roots remained constant w h i l e a steady d e c l i n e i n t o t a l phosphate was present through-out the i n v e s t i g a t i o n p e r i o d . At as e a r l y as day 5 the P^-content was at i t s minimum l e v e l and s i n c e P. i s considered the most f l e x i b l e l of P-pools i n higher p l a n t s ( B i e l e s k i , 1973) i t i s not s u r p r i s i n g that the P . - l e v e l had reached a minimum before the d e c l i n e i n the root organic phosphate was evident. The subsequent d e c l i n e i n organic phosphate could be r e s p o n s i b l e f o r the enhancement of phosphate uptake, however i t i s impossible to d i s c e r n to what extent a l l o s t e r i c and t r a n s c r i p t i o n a l processes were i n v o l v e d . Obviously no a l l o s t e r i c c o n t r o l could be a t t r i b u t e d to 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 experiment. Discounting the - 41 ^ p o s s i b i l i t y of developmental uptake processes which, would be independent of the p l a n t s ' P'-status, the timing of the enhancement of phosphate uptake must of n e c e s s i t y be c o n t r o l l e d by the l e v e l s of one or more organic phosphates. Although p o s i t i v e c o r r e l a t i o n s between phosphate absorption and ATP l e v e l s have been reported i n h i g h e r ^ p l a n t s ( L i n and Hanson, 1974) negative c o r r e l a t i o n s between uptake and organic phosphate content of roots have not. On^fche other hand, as already discussed, there does appear to be a develop-mental c o n t r o l over P-uptake and as such the absolute magnitude of a phosphate s p e c i e s , P^ i n c l u d e d , could govern the extent of P-uptake enhancement. The t o t a l amount of phosphorus per gram f r e s h weight decreased s l o w l y i n the roots of P - s u f f i c i e n t p l a n t s w h i l e a subse-quent i n c r e a s e i n P^-content ensued (see Figure 20). T o t a l phosphate i n the +P shoots increased r a p i d l y between days 5 and 7, and then decreased g r a d u a l l y throughout the d u r a t i o n of the experiment (Figure 20). 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 the dramatic 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 the +P shoots which occured between days 11 and 12. This was followed by a l e v e l l i n g o f f of the P^-concentration at approximately 8umol/g.f.wt. The l a c k of such a s h i f t i n phosphate pool composition could be the s i g n a l f o r increased P-uptake i n -P r o o t s . I n the present study, where a l l determinations of P-uptake were performed w i t h i n t a c t p l a n t s , the shoot's phosphate s t a t u s could e f f e c t change i n the r o o t s ' phosphate uptake r a t e s . -P shoots d i d - 42 -Figures 18 to 21 . P^ and t o t a l phosphorus of aging b a r l e y p l a n t s 3.0 7.0 u 3. 0.0 M S 5.0 —1 O o '•S 3-0 2.0 1.0 5 7 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 i i i ? T T T i T i T 9 U 13 15 17 19 5 7 9 11 13 15 17 19 Time (Days) Time (Days) Figure -18. P i (•) and P t o t a l (*) of -P Figure 19. Pi (•) and P t o t a l (*) of -P roots vs. Age. shoots vs. Age. 20.0 r 15.0 10.0 o a. 5.0 . • - • • • • • • • • 20.0 15.0 10.0 5.0 I I I I I I I I I 7 9—^(Day's 3) 1 5 1 7 1 9 5 7 9,, 1 1 13 , 15 17 19 . Time (Days) Time Figure 20. Pi (•) and P t o t a l ( A) of +P Figure 21. Pt (•) and P C o t a l (*) of +P roots vs. Age. shoots vs. Age. - 43 -show a large decrease i n t o t a l phosphorus at the time of i n i t i a t i o n of enhanced P-uptake (Days 11 and 12). There i s a l s o a drop i n these shoots' P_^-levels between days 9 and 11. These occurrences could t r i g g e r c o n t r o l through t r a n s l o c a t i o n of hormones from the shoot to the uptake organ. Several studies have shown that hormones ap p l i e d to t i s s u e s increase t h e i r i o n uptake ra t e s (Luttge and Higinbotham, 1979). Exc i s e d maize r o o t s , however, d i d not i n c r e a s e 86 36 i n Rb or C l uptake when auxin was a p p l i e d (van Steveninck, 1974), 2-and auxin induced only a 20% s t i m u l a t i o n of SO^ uptake i n beetroot s l i c e s ( N e i r i n c k x , 1968). P u r s u i t s i n the hormonal c o n t r o l of root P^-uptake, per se, have been neglected. Where hormones have been e x t e r n a l l y a p p l i e d to t i s s u e s i t i s not c l e a r whether increased i o n i n f l u x was the cause or the r e s u l t of increased 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 root growth at the time of i n i t i a t i o n of increased P^-uptake r a t e s . However, the -P growth media contained no phosphate, and t h e r e f o r e increased growth could not be due to orthophosphate accumulation. P l a n t growth i s thought to be mediated through i n t e r -a c t i o n s between the major p l a n t hormones. Since growth i m p l i e s a greater 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 not s u r p r i s i n g to f i n d that increased growth rates i n response to hormonal treatments are a s s o c i a t e d w i t h higher r a t e s of i n o r g a n i c i o n uptake. In t h i s manner, the hormonal i n f l u e n c e upon i o n absorption i s an i n d i r e c t , general e f f e c t p r o v i d i n g no opportunity f o r the c o n t r o l of - 44 -uptake of s p e c i f i c i o n s . Nevertheless on a short-term b a s i s there are c l e a r i n d i c a t i o n s that 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 tr 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 + 86 32 K tr a n s p o r t i n t o guard c e l l s (van Steveninck, 1976). Rb and P d o u b l e - l a b e l l e d experiments performed at various p l a n t ages i n d i c a t e d complete independence of t h e i r i n f l u x (see Table 6) and i t would be d i f f i c u l t to a t t r i b u t e t h i s to the general hormonal e f f e c t s discussed. I n t h i s study the shoot was designated to comprise the seed and green t i s s u e of the s e e d l i n g . The r a t h e r l a r g e ortho-phosphate pool which was suddenly formed i n the +P shoots i s more re p r e s e n t a t i v e of the P^ i n higher p l a n t s than are the l e v e l s witnessed at the younger b a r l e y ages ( B i e l e s k i , 1973). The breakdown of p h y t i n , s t a b l e orthophosphate uptake and t r a n s l o c a t i o n , and a net conversion of shoot organic-P could a l l c o n t r i b u t e to t h i s i n c r e a s e i n ortho-phosphate con c e n t r a t i o n . Phosphate s t o r e d as p h y t i n could have broken down to r e l e a s e l a r g e amounts of phosphorus a v a i l a b l e f o r the photosynthesizing t i s s u e i n the t r a n s l o c a t e d form of orthophosphate (Morrison, 1965; Selvendran, 1970). C o r r e l a t i o n s between p h y t i n hydro-l y s i s and P^ increases have been demonstrated i n seedlings starved f o r phosphorus (Mukherji e t . a l . , 1971; E r g l e and Guinn, 1959). The a c t i o n of phytase i s retarded when phosphate i s su p p l i e d to the 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 although these s t u d i e s were terminated at 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 during t h i s p e r i o d u n t i l at the t e r m i n a t i o n of the study they were almost exhausted. Hence i t i s u n l i k e l y that a sudden breakdown of p h y t i n could have occurred at day 11 of the present study (Figure 21). P^ uptake from the 15uM P growth media could have accounted f o r approximately one t h i r d of the i n c r e a s e , i . e . i f a l l P_^  taken up was t r a n s l o c a t e d unchanged to the leaves. Polyphosphates have been found i n r e l a t i v e l y low l e v e l s i n a few higher p l a n t s ( M i y a c h i , 1961; Nassery, 1969; Tewari and Singh, 1964; Vagabov and Kulaev, 1964) and t h e r e f o r e they could be only -a minor source f o r orthophosphate. Organic P sources are the best candidates f o r breakdown to supply the observed P ^ - l e v e l s i n the +P shoots beyond day 11. Expression 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 gives 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 . Although 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 constant throughout the f i r s t 18 days of growth, a drop i n t o t a l phosphorus which was not p a r a l l e l e d i n the i n o r g a n i c pool occurred between day 18 and 20 (see Figure 22). This i m p l i e s that phosphorus had e f f l u x e d i n t o the environment and t h i s may have been due to minor losses of plasmalemma i n t e g r i t y . I n d i r e c t evidence, r e c e n t l y put forward by Menge e t . a l . (1978), suggests that leakage of root 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 f u n g a l a s s o c i a t i o n s . The f a c t that the number of these a s s o c i a t i o n s i n a - 46 -given 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 the a v a i l a b l e phosphate l e v e l may a l s o be ex p l a i n e d , however, by the reduced s u r v i v a l rates of m y c o r r h i z a l spores under hig h l e v e l s of 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). These rhizosphere a s s o c i a t i o n s , although apparently impossible to form i n hydroponic c u l t u r e are b e l i e v e d to be e s s e n t i a l f o r proper 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 are o f t e n i n excess of 0.5uM ( B i e l e s k i , 1973) and could t h e r e f o r e account f o r some of the -P p l a n t s ' phosphate l o s s (-P medium was changed every 4 days). A r a p i d increase i n the l e v e l s of i n o r g a n i c phosphate occurred i n the +P treatment between days 11 and 12 (see Figure 23), which can be a t t r i b u t e d to the shoot phenomenon which has already been discussed at length. The increase i n phosphate i n f l u x i n both +P and -P p l a n t s on a per 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 at the same time (see Figure 17). These phenomena per se cannot be a t t r i b u t e d to 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 to be developmental i n nature. Crop p l a n t s t u d i e s have i n d i c a t e d that t h e i r P as w e l l as N and K l e v e l s vary i n a manner which can be timed to d i s t i n c t morphological stages w i t h i n the p l a n t s ' development (Mengel, 1969; Mengel and K i r k b y , 1978). I t th e r e f o r e f o l l o w s that the i n i t i a t i o n of elevat e d phosphate uptake i n the present study might a l s o be of a predetermined nature, however the extent of t h i s i n c r e a s e appears to be n e g a t i v e l y r e l a t e d to 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 -Figure 22. Phosphate concentration i n -P barl e y p l a n t s 3.0 r o 2.0 a 1.0 Total P I n o r g a n i c P o D o . o o o D g a • o 10 15 Age (days) 20 Figure 23. Phosphate concentration i n +P ba r l e y plants AO L * 5 Z ' I 2 0 Y 10 Total P A A • Inorganic P J S • • ' , -5 10 15 20 Age (days) - 48 -of the extent of enhancement i s the concentration of the r o o t s ' i n t e r n a l orthophosphate or organic phosphate at the beginning and during the P - i n f l u x enhancement p e r i o d . The r a t i o s of P. (+P / ° i ro o t s / -P ) are 10.4 and 15.7 at days 11 and 16 r e s p e c t i v e l y ; the roots same r a t i o s f o r organic phosphate are 1.5 and 2.4. Clarkson e t . a l . (1978) obtained a +P/-P root 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 of 3.9. Because P^ i s much more f l e x i b l e i n magnitude than the organic phosphates i t could act 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 of 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 Michaelis-Menten or Eadie-Hofstee (see Figures 24 and 25) i n d i c a t e that the phosphate uptake Vmax of -P p l a n t s had increased (-P Vmax = 1.25nmol/.g.f.wt./hour; +P Vmax = 0.32umol/g.f.wt./hour), whereas there was no s i g n i f i c a n t (a = 0.05) increase i n the -P roots 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 the Km (-P Km = 2.37uM; +p Km = 3.00yM) of the phosphate uptake system occurred. The r a t i o of -P to +P V max's i s approximately 4 and the -P p l a n t s i n these isotherm experiments had not reached t o t a l P-uptake enhancement (see Figure 16). The high P-concentration used by many researchers (Nissen, 1973) and the m u l t i p h a s i c nature of the orthophosphate uptake isotherms (Barber, 1972; Nissen, 1973) make i t d i f f i c u l t to compare Km and Vmax val u e s . Because of the 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 , only n a t u r a l s o i l P-concentrations were used i n the present study f o r i n f l u x isotherm determinations. In 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) working w i t h l i c h e n s obtained comparable Km and Vmax values to the -P t r e a t e d b a r l e y roots of t h i s study, whereas Carter and L a t h w e l l (1967) working w i t h corn found values s i m i l a r to the +P treatment. These workers' r e s u l t s can be a t t r i b u t e d to the phosphorus n u t r i t i o n of t h e i r experimental p l a n t s . Barber, using excised b a r l e y roots ( a n a l y s i s by Nissen, 1973) Figure 24., Orthophosphate uptake k i n e t i c s : Miehaelis-Menten p l o t Figure 25. Orthophosphate uptake k i n e t i c s : Eadie-Hofstee p l o t - 52 -obtained, i n phosphate d e f i c i e n t p l a n t s , approximately a 4 f o l d Vmax increase over that of w e l l nourished p l a n t s and h i s -P Km values were lower than i n h i s +P treatments, though as i n the present study the d i f f e r e n c e s of Km were not s t a t i s t i c a l l y s i g n i f i -cant. Cartwright (1972) a l s o showed an increase i n the 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 , although no changes i n Vmax were reported. This could be due to the n u t r i e n t status of the p l a n t s employed as w e l l as the P-concentration range over which i n f l u x determinations were performed. At higher e x t e r n a l concentrations of i n o r g a n i c ions 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 which are apparent at lower concentrations, may be l o s t (Glass and Dunlop, 1978). This combined w i t h the m u l t i p h a s i c nature of i o n uptake i n pl a n t s would suggest that an isotherm determined over a greater range of i o n concentration might overlay the p a t t e r n of uptake which occurs at the lower P-concentrations such as those used i n the present study. The present study together w i t h those c i t e d now c l e a r l y show that increased P-status may be ass o c i a t e d w i t h both decreases of Vmax values f o r P-uptake as w e l l as increases of Km although i n the present 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 not observed. - 53 -i i . 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 determinations so f a r described were performed i n w e l l s t i r r e d media and the 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 supply to the root was reduced t o a minimum. D i f f u s i o n 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 roots are bathed i n n o n - s t i r r e d s o l u t i o n s of concentration below lOuM ( P o l l e and Jenny, 1971; B o l e , 1977). I n such an environment an increase i n the root area r e l a t i v e to i t s weight would c o n t r i b u t e to an increased uptake r a t e . By comparison of uptake rates 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 i n t o p h y s i o l o g i c a l and morphological c o n t r i b u t i o n s to enhanced P^-uptake rates (see Table 5 ) . The f a c t that the -P to +P uptake r a t i o s were c o n s i s t e n t l y lower under the n o n - s t i r r e d compared to the s t i r r e d c o n d i t i o n s might be explained by presuming that increased uptake r a t e s i n -P p l a n t s a r i s e from increased " 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 co n d i t i o n s where uptake rates are d i f f u s i o n l i m i t e d (the n o n - s t i r r e d treatment) the overlap 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 to more adversely reduce uptake rates than i n the +P p l a n t s . An analogy can be drawn to the phenomenon of gas exchange at the stomata of leaves (Heath, 1975). As Raven (1977) has pointed out, however, sp e c u l a t i o n s regarding " 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 the 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 these p u t a t i v e molecules. - 54 -Table 5. Uptake rates 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 without (WS) s t i r r i n g , (umoles/g.f.wt./h.) Ratios are shown. Day S r a t i o -P/+P WS r a t i o -P/+P 8 -P 744.14 304.30 2.22 1.46 +P 335.46 207.75 11 -P 693.33 225.12 2.47 1.27 +P 281.49 176.70 14 -P 548.16 161.84 2.73 1.24 +P 201.61 130.52 - 55 -The seedlings employed i n t h i s and the remaining experiments of the t h e s i s a t t a i n e d optimum P-uptake rates at e i g h t days of age, not 16 as i n the previous p l a n t l e t s . D i f f e r e n c e s i n the age needed to reach optimum uptake rates does not appear to be s o l e l y dependent upon phosphate sto r e s (see Figures 18, 19, 27 and 28), and other n u t r i e n t s , although not i n v e s t i g a t e d , may be i n v o l v e d . - 56 -i i i . 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 metabolic c y c l i n g of energy through a l i m i t a t i o n upon energy coupling which occurs 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. The concentrations of both the ATP precursors ADP and m e t a b o l i c a l l y a v a i l a b l e ortho-phosphate r e l y on a source of phosphorus which could be w i t h i n the c e l l as a storage pool 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 these sources were depleted i t i s p l a u s i b l e that 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 increase i n phosphatase a c t i v i t y could prolong the p l a n t s a b i l i t y to maintain high l e v e l s of energy co u p l i n g . I n f a c t high phosphatase a c t i v i t i e s commonly occur 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 that 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 endergonic i o n uptake processes might be expected to 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 " c a r r i e r s " or changes i n the number of s p e c i f i c " c a r r i e r s " during the course of P - s t a r v a t i o n . I n order to 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 during P - d e f i c i e n c y (Figure 16) was due to l i m i t a t i o n upon energy resources f o r a c t i v e t r a n s p o r t i t was decided to examine the uptake of another a c t i v e l y transported i o n i c s p e c i e s , namely K +. By means of d o u b l e - l a b e l l e d uptake experiments the uptake 86 32 of Rb and P was determined simultaneously during 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 isotope of choice f o r - 57 -Table 6. Uptake rates of P from a 15uM P s o l u t i o n and K from a l l l u M K s o l u t i o n . (nmoles/g.f.wt./h.) K concentrations of the roots are also presented, (mmoles/g.f.wt.) Day P uptake K uptake K concentration 8 -P 2678.12 889.12 0.092±0.021 +P 886.48 1080.64 0.067±0.008 11 -P 2247.08 410.27 0.080±0.005 +P 867.75 9.74.55 0.079±0.003 14 -P 1259.39 618.56 0.077±0.013 +P 699.80 901.59 0.085±0.006 - 58 -the determination of potassium uptake i n higher p l a n t s ( L a u c h l i and E p s t e i n , 1970). While the w e l l nourished p l a n t s g r a d u a l l y reduced t h e i r uptake ra t e s of both ions over the time p e r i o d i n v e s t i g a t e d , there was no such r e l a t i o n s h i p between the r e s p e c t i v e i o n uptakes i n the -P p l a n t s . From day 8 to day 14 phosphate uptake rates i n the -P p l a n t s were halved, and w h i l e the potassium uptake decreased considerably by day 11, i t had increased again by day 14. Because of the independence of the two i o n uptake ra t e s i t appears that energy supply to the " c a r r i e r s " which might be expected to act s i m i l a r l y upon a c t i v e uptake processes does not seem to 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 rates 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 root s u r f a c e . I n both the +P 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 potassium content of the roots i s d i f f i c u l t to a s c e r t a i n . Cram ( i n p r e s s ) , on the other hand showed d e c i s i v e l y that KC1 fed to c a r r o t d i s c s r e s u l t e d i n a decreased phosphate abso r p t i o n . F r a n k l i n (1969) showed that adsorbed ca t i o n s increased phosphate uptake by b a r l e y r o o t s , whereas conversely, a p p l i e d phosphate has never been reported to augment K-uptake r a t e s . In the present study +P p l a n t s c o n s i s t e n t l y showed greater K-uptake ra t e s than -P p l a n t s and t h i s may be a t t r i b u t e d to the greater growth rates of the +P p l a n t s . - 59 -i v . Determination of Root E x t e r n a l P r o t e i n Because of the ubiquitous metabolic r o l e s i n which phosphorus i s i n v o l v e d i t was of i n t e r e s t to determine i f d e p r i v a -t i o n of 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 the reduced s y n t h e s i s of 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 which would be exposed to the e x t e r i o r of the r o o t . P r o t e i n s o r i e n t e d w i t h i n the root plasmalemma could be r e s p o n s i b l e f o r mineral 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 of the membrane i t s e l f . E o s i n 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 at a very slow r a t e and as such can be used to measure r e l a t i v e amounts of root 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 of age the s t a i n i n g value f o r -P p l a n t s ' roots was 20.82±5.04 O.D.,-™ u n i t s / g . f .wt. and that of +P roots was 520nm 35.28±9.06. The diameters of the main roots of both +P and -P p l a n t s were 0.45±0.05mm (see Figure 2 ) . At 30 days of age p r o t e i n values were 16.61±3.86 and 16.53±1.78 f o r the -P and +P roots r e s p c t i v e l y and w h i l e the +P root diameter had not changed from 0.45±0.05mm, the -P root width had decreased to 0.20±0.02mm (Figure 6 ) . The primary root^s surface area to volume r a t i o might be taken as r e p r e s e n t a t i v e of the e n t i r e root system, i n which case the c a l c u l a t i o n of surface area per g.f.wt. root revealed that per u n i t area at both days 10 and 30 there was approximately 0.6 times as 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 . P - d e f i c i e n t p l a n t s seem - 60 -to have produced and sustained much l e s s plasmalemma and c e l l w a l l p r o t e i n . This combined w i t h the apparent increase i n d e n s i t y of phosphate uptake s i t e s (see s t i r r e d vs. n o n - s t i r r e d experiment) would suggest that the -P p l a n t s possessed a s u b s t a n t i a l enrichment i n the membrane p r o t e i n s r e s p o n s i b l e f o r phosphate uptake. Such an enrichment could be e x p l o i t e d as a source 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 of the phosphate " c a r r i e r " i n question, a f e a t not yet accomplished i n p l a n t s . The low p r o t e i n l e v e l may also be p a r t l y r e s p o n s i b l e f o r the l o s s of phosphate from the roots of the 20 day o l d -P p l a n t s (see Figure 22). A leakage of phosphate could have r e s u l t e d from a decreased root I n t e g r i t y caused by an inadequate production of s t r u c t u r a l membrane p r o t e i n s . - 61 -5. Regulation of Rapid D e c l i n e of Enhanced P^-uptake A. Short-term versus Long-term Uptake -P p l a n t s which demonstrated enhanced P-uptake r a t e s i n short-term determinations (10 min i n f l u x periods) d i d not show as pronounced an elevat e d uptake rat e when a 24h uptake p e r i o d was 32 used (see Table 7). Dep l e t i o n of P - l a b e l l e d uptake media was n e g l i g i b l e i n both s h o r t - and long-term experiments and cannot t h e r e f o r e have been re 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 to +P media f o r 24h p r i o r to short-term uptake d e t e r -mination the values of P-uptake revealed that the su p p l i e d 15uM phosphate had r e s u l t e d i n a considerable r e d u c t i o n i n t h e i r P - i n f l u x , dropping the rate to a l e v e l s i m i l a r to that of the +P p l a n t s . Short-term s t u d i e s of +P and -P p l a n t s at days 7 and 8 i n d i c a t e d that no developmental d e c l i n e i n uptake had occurred. The low uptake rates which Clarkson and h i s colleagues (1978) obtained 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 , at l e a s t i n p a r t , to the 24h i n f l u x determinations employed. D i f f e r e n c e s i n the values obtained f o r P - i n f l u x through the use of e i t h e r s h o r t - or lo n g -term assays i n d i c a t e d that the r e g u l a t i o n of phosphate uptake i n -P p l a n t s was extremely s e n s i t i v e to 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 of the r a p i d i t y of i t s response, could be f u r t h e r i n v e s t i g a t e d only through the use of the short-term assays i n which minimal exposure to phosphate r e s u l t s i n a measure of the i n i t i a l P-uptake r a t e . Table 7. Short-term ( s . t . ) v s . long-term ( l . t . ) uptake r a t e determination P l a n t Status at Assay Employed Uptake (ymol./g.f.wt./h)* Beginning of 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 plus 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 standard d e v i a t i o n of four r e p l i c a t e s . - 6 3 -B. P-uptake Rates and P l a n t Phosphorus Levels F o l l o w i n g Exposure of P l a n t s to 15uM Phosphate I n order to determine the r a p i d i t y of the -P p l a n t s ' response to the 15yM P s u p p l i e d , short-term i n f l u x estimates were obtained a f t e r these p l a n t s had been fed 15uM P f o r i n c r e a s i n g time p e r i o d s . Figure 26 presents the time course of the e f f e c t of 15uM phosphate upon -P p l a n t s ' uptake r a t e s . The r a t e s began decreasing w i t h i n l h which i n c i d e n t a l l y i s c l o s e to the h a l f - l i f e of cytoplasmic P-exchange (estimated at 45 min, see E f f l u x K i n e t i c s ) . The d e c l i n e was r a p i d u n t i l 6 hours, beyond which no f u r t h e r reduc-t i o n was e v i d e n t . The uptake determinations f o r times 10 to 24h were c a r r i e d out on the second day of the 2 day experiment, however the e l e v a t e d values at 12, 16 and 20h cannot be due to 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 procedures 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 . Rate determination of non-treated +P and -P p l a n t s at 0 and 24h revealed only minimal changes. The t o t a l phosphorus content per gram f r e s h weight of the t r e a t e d p l a n t s d i d not show a net gain over the 24h feeding p e r i o d (Figures 27 and 28). Rapid growth, causing d i l u t i o n of absorbed P and thus maintenance of P at a r e l a t i v e l y constant l e v e l , best e x p l a i n s t h i s phenomenon. The f a c t that 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 accord w i t h t h i s e x p l a n a t i o n . The i n o r g a n i c phosphorus component of both the shoots and roots changed considerably as phosphate was s u p p l i e d to -P p l a n t s - 64 -Figure 26. Phosphate uptake ra t e vs. time of root exposure to 15uM orthophosphate 2.0 t-1 • . . i • . . . i . . . . • . . . . » 5-0 10.0 15.0 20.0 25.0 Time (hours) Figure 27. T o t a l phosphorus concentration during the p e r i o d of phosphate lo a d i n g of -P grown b a r l e y p l a n t s 5 U-l CO CD 0) o s 3 03 3 O x; to o 16 12 • Treated roots • +P roots A +P shoots O -P roots A - P shoots A ON • • • • O o H 12 Time (hours) 16 20 24 Figure 28. T o t a l phosphorus concentration during the p e r i o d of 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 roots A+P shoots O-P roots A-P shoots O A 8 12 16 - 20 24 Time (hours) - 67 -and although 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 to the shoots, there appears to be a r e l a t i o n s h i p between the P^-content of the root and the P - i n f l u x (see Figure 29). The uptake ra t e p l o t t e d against the root's i n t e r n a l orthophosphate concentration (Figure 30, feeding curve) b r i n g s to l i g h t a negative c o r r e l a t i o n which was maintained u n t i l 6h of exposure to 15yM P. 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 to approximately 0.75umol/g.f.wt. (not shown i n Figure 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 . This suggests that beyond 6h some c o n t r o l mechanism other than d i r e c t P_^  feedback upon uptake had a l s o presented i t s e l f . Moreover, the i n o r g a n i c phosphate l e v e l of the +P roots at the end of the 24h feeding p e r i o d was lower than i n the t r e a t e d roots and r e g u l a t o r y processes may s t i l l at t h i s time 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 nourished root's physiology. Evidence the r e f o r e supports the p o s s i b i l i t y of two c o n t r o l mechanisms: 1) i n which a r a p i d feedback occurs at the e a r l y stage, and 2) a slower mechanism which might act through the degrada-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 " . A lso presented i n Figure 30 are the root i n o r g a n i c phosphate l e v e l s and P-uptake ra t e s of 11 to 16 day o l d -P p l a n t s during the -P phosphate uptake enhancement phase (see Figure 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 could not have acted 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 process. The - 68 --69 -Figure 30. Phosphate uptake rate vs. ?± concentration 6 0 0) CD to 4-1 Cu I •rH 2.4 2.0 1.6 1.2 0.8 0.4 0 Enhancement curve, l i n e a r f i t r= 0.985 O O During uptake enhancement • During rapid decline due to phosphate loading Feeding curve, l i n e a r f i t r= 0.968 0.2 0.4 0.6 0.8 l.o Inorganic Phosphate (umoles/g.f.wt.) 1.2 - 70 -i n t e r n a l P^-concentration remained at a minimum l e v e l throughout the enhancement stage and hence i n h i b i t i o n of uptake by i n t e r n a l orthophosphate would, as such, a l s o be at a minimum. I t f o l l o w s that a p o s s i b l e mechanism f o r the c o n t r o l of enhanced P-uptake i s through the a d d i t i o n of new " t r a n s p o r t e r p r o t e i n s " , each of which 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 . This agrees w i t h the argument, already put forward, that phosphate uptake enhancement r e s u l t e d from an increased d e n s i t y of " c a r r i e r s " on the root s u r f a c e . I t does remain p o s s i b l e that during the enhancement phase of P-uptake a concomitant d e c l i n e i n one or more organic phosphates r e s u l t e d i n the re 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 the uptake process. This was not f u r t h e r i n v e s t i g a t e d i n the present study. Several s t u d i e s i n v o l v i n g ions which are not metabolized have demonstrated negative r e l a t i o n s h i p s between the rate of uptake of a given 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 these cases be the 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 Rb + uptake occurred when corn was su p p l i e d w i t h potassium (Leigh and Wyn Jones, 1973) and i n c r e a s i n g potassium n u t r i t i o n caused decreasing Rb + uptake r a t e s i n sunflower ( P e t t e r s s o n , 1975) and Lemna minor (Young e t . a l . , 1970). A l l o s t e r i c c o n t r o l of rubidium and potassium uptake has been reported i n b a r l e y and sunflower (Glass, 1976; 1977; 1978b; P e t t e r s s o n and Jensen, 1978; 1979; Jensen and P e t t e r s s o n , 1978). C l uptake decreases as i n t e r n a l - 71 -CI l e v e l s increase i n carrot tissues and barley roots (Cram, 1973) and Br i s absorbed more slowly i n Br fed beets ( S u t c l i f f e , 1954) and wheat (Cseh et. 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 , nitrogen, and phosphate l e v e l s i n plants i s complicated. Ivan Smith's work (1975) with cultured tobacco c e l l s indicated that s u l f a t e uptake rates correlated negatively with 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 cysteine were applied e x t e r n a l l y , s u l f a t e uptake rates declined i n tobacco (Hart and F i l n e r , 1969) and barley ( F e r r a r i and Renosto, 1972), however conversion of the S-compounds to s u l f a t e may precede the given e f f e c t (Smith, 1975). Very l i t t l e evidence has been reported to suggest that i n t e r n a l NO^ l e v e l s regulate NO^ uptake i n higher plants (Smith, 1973; Cram, 1973) and such studies are rendered more d i f f i c u l t because of the a b i l i t y of n i t r a t e to induce n i t r a t e reductase a c t i v i t y (Jackson et. a l . , 1976). The l i n e a r r e l a t i o n s h i p between root P_^-level and P-uptake rates points to an a l l o s t e r i c control mechanism. The sigmoidal curve 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) may not present i t s e l f i n the case of inorganic metabolites within the plant. Minimum and maximumsP^-levels may be governed by r e v e r s i b l e conver-sions into organic forms and hence control may appear only as a l i n e a r r e l a t i o n s h i p . The l i n e a r transformation of the H i l l equation: i i Vmax-v . - I _ I log K = log — + n log | S | - 72 -was employed to evaluate 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 present i n the uptake process (Glass, 1976). An n value of 1.81 ±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 at a = 0.01 l e v e l ) was obtained from the slope of the l i n e a r r e l a t i o n s h i p shown i n Figure 31. I f no c o o p e r a t i v i t y were present n would have a value of 1. n values have erroneously been claimed, by enzymolo-g i s t s , to represent 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 of the degree of c o o p e r a t i v i t y possessed by the enzymes i n v o l v e d (Ferdinand, 1976). As such, although the number of r e g u l a t o r y s i t e s per 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 to be a cooperative process w i t h respect to the r o o t s ' i n t e r n a l orthophosphate l e v e l . The r a p i d 'shutdown' of phosphate uptake could a l s o be explained by more complicated p h y s i o l o g i c a l processes. 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 intermediates such as hormones and/or organic phosphates. Because of the need f o r added biochemical steps i n such r e g u l a t o r y processes t h e i r occurrence 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 feedback upon phosphate uptake. - 73 -Figure 31. H i l l p l o t (v/Vmax - v against i n t e r n a l . P i concentration) -0.8 -0.4 o.O Log P i concentration (umoles/g.f.wt.) IV. CONCLUSION A n a l y s i s of short-term phosphate uptake i n i n t a c t b a r l e y c v . Bonanza has provided considerable i n s i g h t i n t o two processes which appear to 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 of p o t e n t i a l phosphate uptake r a t e s through P - d e p r i v a t i o n takes place over a pe r i o d of days which would be ample time f o r metabolic processes such as p r o t e i n synthesis or degradation to occur. There i s evidence f o r an enrichment i n P-uptake s i t e s on the -P p l a n t s ' root surface. This enrichment may be the end e f f e c t of numerous biochemical processes which u l t i m a t e l y r e s u l t i n an adaptive response to P - d e p r i v a t i o n . C o n t r o l s i g n a l s may be e l i c i t e d through growth p a t t e r n s , or i n o r g a n i c or organic phosphate l e v e l s of e i t h e r the p l a n t s ' shoots or r o o t s . Because of the apparent developmental process governing P-uptake rates, the root absolute P^-concentration could c o n t r o l the extent of enhanced uptake. The d e c l i n e i n p o t e n t i a l phosphate uptake rates revealed-when phosphate was s u p p l i e d to p l a n t s possessing elevated i n f l u x , r ates, suggests the occurrence of 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 process s t a r t s w i t h i n one hour, and p r o t e i n degradation the r e f o r e i s not l i k e l y to have been i t s cause. This process appears to 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 ro o t s ' i n t e r n a l orthophos-phate c o n c e n t r a t i o n . The time necessary to e l i c i t d e c l i n e i s - 75 -s i m i l a r to the cytoplasmic P-exchange h a l f - l i f e and hence vacuolar P-fluxes may 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 decreases. The process which occurs a f t e r 8 hours pretreatment w i t h ortho-phosphate i s d i s t i n c t from that of the previous 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 to P ^ - l e v e l s as i n the former p e r i o d . At the longer exposure times to phosphate supply reduced P-uptake may be achieved by " c a r r i e r " degradation. The 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 absorption rates enables homeostatic c o n t r o l of phosphate concen-t r a t i o n s w i t h i n b a r l e y p l a n t s . 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 adsorption to 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 roots i n t o a r e g i o n of high 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 lead to excessive P-absorption 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 . This a p p l i e s not only to p l a n t s adapted to 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 important crop p l a n t s ( B h a t t i and Loneragan, 1970a, b; Green e t . a l . , 1973a, b; S i d d i q i , 1978). Therefore the c a p a c i t y to 'shutdown' P-uptake f a i r l y r a p i d l y i n response to increased P - a v a i l a b i l i t y i s decidedly important under n a t u r a l c o n d i t i o n s . Those cases c i t e d of t i s s u e damage from excess P-uptake may r e f l e c t e i t h e r an i n a b i l i t y to r e g u l a t e , as i n p l a n t s - 76 -adapted through e v o l u t i o n to low-P environments (e.g. heath p l a n t s ) , or an i n a b i l i t y to respond r a p i d l y enough as i n the case of crop p l a n t s acclimated to low-P regimes. P h y s i o l o g i c a l adaptations f o r increased P-uptake are l e s s energy consuming than morphological adaptations which of n e c e s s i t y , r e q u i r e growth. I n n u t r i e n t l i m i t e d c o n d i t i o n s t h i s d i f f e r e n c e may 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 revealed increases i n main root surface area only w e l l a f t e r the p h y s i o -l o g i c a l uptake rates 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 at a s t i l l l a t e r age as perhaps a l a s t r e s o r t . Morphological adaptations may surface only a f t e r severe P - d e p r i v a t i o n , enabling the roots t o , i n e f f e c t 'search' f o r l o c a l i z e d s o i l phosphorus sources, however there remains the p o s s i -b i l i t y that the hydroponic environment used i n t h i s study u n n a t u r a l l y retarded root morphogenesis. The work presented i n t h i s t h e s i s has tended to 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 adaptation to P - d e p r i v a t i o n . 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