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Effects of low temperature on K⁺ nutrition of some barley (Hordeum vulgare. L) varieties De Silva, M. H. Mala Chandani 1983

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EFFECTS OF LOW TEMPERATURE ON K* NUTRITION OF SOME BARLEY (HORDEUM VULGARE. L ) VARIETIES M. H. MALA CHANDANI/DE SILVA B . S c , U n i v e r s i t y of Peradeniya, S r i Lanka, 1978 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF THE FACULTY OF GRADUATE STUDIES Department Of Botany We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA December 1983 by MASTER OF SCIENCE i n © Mala Chandani De S i l v a , 1983 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an advanced degree a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I agree t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e head o f my department o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f BOTANY  The U n i v e r s i t y o f B r i t i s h C o l u m b i a 1956 Main Mall V a n c o u v e r , Canada V6T 1Y3 Date DECEMBER 1983 ABSTRACT The main o b j e c t i v e of the present study was to i n v e s t i g a t e the c a p a c i t y to compensate f o r reduced root a c t i v i t y caused by low temperature. Potassium i n f l u x , net f l u x and t i s s u e K + content in 23 s p r i n g and 2 winter b a r l e y v a r i e t i e s grown at low (5°C) and high (15°C) root temperatures were s t u d i e d . It was apparent that the r a t e s of uptake (at low temperature) of low temperature grown winter and some s p r i n g v a r i e t i e s were a d j u s t e d i n response to continuous low temperature so that a b s o r p t i o n r a t e s approached those of p l a n t s maintained at higher (15°C) temperature. The temperature s e n s i t i v i t y f o r i n f l u x v a r i e d among v a r i e t i e s depending on the growth temperature and i n t e r n a l K + c o n c e n t r a t i o n . Halcyon, a winter v a r i e t y , showed a low temperature s e n s i t i v i t y when grown at low temperature (5°C) and low e x t e r n a l [ K T ] (0.005 mM). The a c c l i m a t i o n p o t e n t i a l s (the r a t i o of K + i n f l u x e s measured at the same temperature f o r the p l a n t s a c c l i m a t e d to d i f f e r e n t temperatures) were higher f o r winter v a r i e t i e s and some s p r i n g (e.g. Fergus) v a r i e t i e s , while most of the s p r i n g v a r i e t i e s e x h i b i t e d a lower p o t e n t i a l to a c c l i m a t e . I t was a l s o apparent that a c c l i m a t i o n of i i i K + uptake to low temperature c o u l d be achieved only i f the e x t e r n a l K + supply was not l i m i t e d . Growth r a t e s of the whole p l a n t , shoots and roots monitored over a short p e r i o d (14 days) r e v e a l e d d i f f e r e n c e s i n the p a r t i t i o n i n g of K + under d i f f e r e n t temperatures (8° and 18°C) and at d i f f e r e n t n u t r i e n t (K 4) regimes (0.1 mM and 0.005 mM) . Despite the r e d u c t i o n in growth r a t e s , roots appeared to maintain a high K + l e v e l when grown at low temperature. N e v e r t h e l e s s , such maintenance was p o s s i b l e only i f the supply of K + was adequate. A low e x t e r n a l K+ content and low temperature reduced the a c c l i m a t i o n c a p a c i t y even i n the winter v a r i e t y , Halcyon. T h e r e f o r e , the importance of K + f e r t i l i z a t i o n f o r p l a n t s to withstand c o l d temperatures i s obvious. Net f l u x e s of Halcyon and Kombar (a v a r i e t y bred for warm s o i l s i n C a l i f o r n i a ) were i n v e s t i g a t e d over a p e r i o d of 24 hours. Incomplete a c c l i m a t i o n of net f l u x e s was observed for both v a r i e t i e s over that p e r i o d . T h i s r e v e a l e d the importance of c o n s i d e r i n g e f f l u x e s as w e l l as i n f l u x e s i n determining a c c l i m a t i o n . The amount of r a d i o a c t i v i t y ( 8 6Rb) d e l i v e r e d to the shoot over a p e r i o d of one hour was used to estimate the t r a n s l o c a t i o n r a t e . T h i s was g r e a t e r f o r the winter v a r i e t y than i n the s p r i n g v a r i e t y . Growth temperature, and to a l e s s e r extent the d u r a t i o n of exposure to low temperature, r e q u i r e d f o r a c c l i m a t i o n appeared to be the f a c t o r s that determined i v a c c l i m a t i o n . Halcyon seemed t o a c c l i m a t e much f a s t e r than Bonanza, a s p r i n g v a r i e t y which showed o n l y a v e r y l i m i t e d c a p a c i t y of a c c l i m a t i o n . TABLE OF CONTENTS ABSTRACT i i LIST OF TABLES v i LIST OF FIGURES x i ACKNOWLEDGEMENTS x i I. INTRODUCTION 1 I I . MATERIALS AND METHODS 13 1. Growth of p l a n t s 13 1.1 Seeds 13 1.2 Seed germination 13 1.3 Choice of growth regimes 14 2. Potassium i n f l u x measurements 18 3. P r e l i m i n a r y survey 20 3.1 Measurement of potassium i n f l u x and t i s s u e potassium c o n c e n t r a t i o n i n b a r l e y v a r i e t i e s ..20 3.2 Determination of temperature c o e f f i c i e n t and a c t i v a t i o n energy 22 4. R e g u l a t i o n of potassium i n f l u x 23 5. Growth s t u d i e s of 3 b a r l e y v a r i e t i e s 24 v i 6. E f f e c t of low e x t e r n a l potassium [K*] on a c c l i m a t i o n of uptake 25 7. Determination of a c c l i m a t i o n of potassium i n f l u x at d i f f e r e n t ages and growth temperatures 26 7.1 E f f e c t of age on a c c l i m a t i o n 26 7.2 E f f e c t of growth temperature p e r t u r b a t i o n ....26 8. Time course of a c c l i m a t i o n by short term exposure to low temperatures 27 I I I . RESULTS AND DISCUSSION 28 1. P r e l i m i n a r y survey 28 1.1 V a r i a t i o n s of potassium i n f l u x and t i s s u e potassium c o n c e n t r a t i o n among 23 s p r i n g b a r l e y and 2 winter b a r l e y v a r i e t i e s 28 1.2 Temperature s e n s i t i v i t y of potassium i n f l u x among b a r l e y v a r i e t i e s 55 1.3 Determination of a c c l i m a t i o n p o t e n t i a l 63 2. Re g u l a t i o n of potassium i n f l u x 67 2.1 E f f e c t of growth temperature 67 2.2 E f f e c t of i n c u b a t i o n ( i n f l u x ) temperature ....72 2.3 C o r r e l a t i o n of i n f l u x e s f o r the i n t e r n a l K + s t a t u s 73 v i i 3. Growth and t i s s u e potassium content in r e l a t i o n to temperature and e x t e r n a l potassium c o n c e n t r a t i o n ...77 3.1 E f f e c t of temperature at low potassium l e v e l s 77 3.2 E f f e c t of potassium supply at two d i f f e r e n t growth temperatures 104 (a) E f f e c t of d i f f e r e n t K* supply at high root temperatures 104 (b) E f f e c t of d i f f e r e n t K* supply under low root temperatures 105 4. Temperature-low n u t r i e n t i n t e r a c t i o n s on i n f l u x at t i s s u e potassium c o n c e n t r a t i o n 108 5. Determination of a c c l i m a t i o n of i n f l u x at d i f f e r e n t ages, growth temperatures and exposure times ..114 5.1 V a r i e t y , growth temperature and age e f f e c t s ..114 5.2 V a r i e t y , growth temperature p e r t u r b a t i o n and exposure time e f f e c t s 116 6. Time course of a c c l i m a t i o n of net f l u x e s and " T r a n s l o c a t i o n " 126 6.1 A c c l i m a t i o n of net f l u x e s ...127 6.2 A c c l i m a t i o n of t r a n s l o c a t i o n s 131 IV. CONCLUSIONS 138 REFERENCES 141 v i i i LIST OF TABLES 1. Composition of hydroponic growth media 15 2. K* i n f l u x at 15°C i n b a r l e y grown at 5°C root temperature 33 3. Root K* content i n b a r l e y grown at 5°C root temperature 35 4. Shoot K* content i n b a r l e y grown at 5°C root temperature 37 5. K* i n f l u x at 15°C i n b a r l e y grown at 15°C root temperature 39 6. Root K* content i n b a r l e y grown at 15°C root temperature 41 7. Shoot K* content i n b a r l e y grown at 15°C root temperature 43 8. Comparison of i n f l u x e s at 5° and 15°C root temperatures 45 9. Comparison of root and shoot [K*] f o r 8 s p r i n g v a r i e t i e s 47 10. Comparison of root and shoot [ K + ] f o r 2 winter v a r i e t i e s 4?-11. Means of i n f l u x and root [K*] of 2 winter v a r i e t i e s 49 12. Comparison of i n f l u x of s p r i n g and winter v a r i e t i e s 51 i x 13. Comparison of root K* of s p r i n g and winter v a r i e t i e s 51 14. Comparison of shoot K" of s p r i n g and winter v a r i e t i e s 53 1 5 . Q 1 0 values f o r K* i n f l u x i n 6 b a r l e y var i e t i e s 56 16. Estimated Q 1 0 of two b a r l e y v a r i e t i e s 56 17. A c t i v a t i o n energies f o r K* i n f l u x 61 18. A c c l i m a t i o n p o t e n t i a l f o r K* i n f l u x 65 19. Equations r e p r e s e n t i n g the c o r r e l a t i o n of i n f l u x to [ K ] 0 75 20. R e l a t i v e growth r a t e s of p l a n t s 98 21. R e l a t i v e growth r a t e s of shoots 100 22. R e l a t i v e growth ra t e s of roots 102 23. K* i n f l u x of 3 v a r i e t i e s grown at low [ K * ] 0 110 24. Root K* content of 3 v a r i e t i e s grown at low [ K * ] o HO 25. Shoot K* content of 3 v a r i e t i e s grown at low [ K * ] o 110 26. Root/shoot r a t i o of 3 v a r i e t i e s grown at low [K* ] 0 112 27. Q 1 0 of 3 v a r i e t i e s grown at low [ K * ] 0 112 28. E f f e c t of v a r i a t i o n s of 3 f a c t o r s on 3 v a r i a b l e s of Halcyon and Gerbel 117 29. E f f e c t of v a r i a t i o n s of 3 f a c t o r s on 3 v a r i a b l e s of Halcyon and Bonanza 122 X 30. K* net f l u x e s at growth temperature and " T r a n s l o c a t i o n " i n 2 v a r i e t i e s 129 31. K* net f l u x e s at 10°C and % of r a d i o a c t i v i t y d e l i v e r e d to shoots i n 2 v a r i e t i e s 132 32. " T r a n s l o c a t i o n " of K* at 10°C i n 2 v a r i e t i e s 134 x i LIST OF FIGURES 1. Block diagram of a feedback system ...5 2. K + i n f l u x at 15°C of 23 v a r i e t i e s grown at 5°-15°C. ...29 3. Root R' content of 23 v a r i e t i e s grown at 5°-15°C 29 4. K* i n f l u x vs. i n t e r n a l K* of Halcyon grown at 15°C. ...68 5. K* i n f l u x vs. i n t e r n a l K* of Halcyon grown at 5°C 68 6 . K* i n f l u x vs. i n t e r n a l K* of Kombar grown at 15°C 70 7. K* i n f l u x vs. i n t e r n a l K* of Kombar grown at 5°C 70 8. Plant weight vs. Age i n Halcyon 80 9. Plant K + vs. Age in Halcyon 80 10. Plant weight vs. Age i n Hector 82 11. Plant K* vs. Age i n Hector 82 1 2 . Plant weight vs. Age i n Bonanza 84 13. Plant K* vs. Age i n Bonanza 84 14. Shoot weight vs. Age i n Halcyon 8 6 15. Shoot K* vs. Age i n Halcyon 8 6 16. Shoot weight vs. Age i n Hector 88 17. Shoot K* vs. Age i n Hector 88 1 8 . Shoot weight vs. Age i n Bonanza 90 19. Shoot K + vs. Age i n Bonanza .....90 20. Root weight vs. Age i n Halcyon 92 21. Root K* vs. Age i n Halcyon 92 22. Root weight vs. Age in Hector 94 23. Root K + vs. Age i n Hector 94 24. Root weight vs. Age i n Bonanza 96 x i i 25. Root K* vs. Age in Bonanza 96 26. K* net f l u x vs. time of a c c l i m a t i o n i n Halcyon 136 27. K* net f l u x vs. time of a c c l i m a t i o n i n Kombar 136 x i i i ACKNOWLEDGEMENTS I extend my s i n c e r e . thanks to my s u p e r v i s o r , Dr. A. D. M. Glass f o r h i s a s s i s t a n c e , i n v a l u a b l e advice and encouragement d u r i n g the course of t h i s study. I wish to thank my a d v i s o r y committee members, Dr. P. J . H a r r i s o n , Dr. B r i a n H o l l and Dr. G. H. N. Towers. Thanks are a l s o extended to Dr. M. Y. S i d d i q i and my c o l l e a g u e s f o r h e l p w i t h v a r i o u s aspects of t h i s study. A s p e c i a l a p p r e c i a t i o n goes f o r Mr. Sarath Abekoon and Ms. A. M. Chan f o r e f f i c i e n t t y p i n g of the manuscript. F i n a n c i a l support through a postgraduate s c h o l a r s h i p from Commonwealth S c h o l a r s h i p s and F e l l o w s h i p Plan i s g r a t e f u l l y acknowledged. L a s t , but not l e a s t , I extend my immeasurable g r a t i t u d e to a l l my f r i e n d s f o r t h e i r moral support and encouragement. 1 I. INTRODUCTION Despite c o n s i d e r a b l e f l u c t u a t i o n s i n the e x t e r n a l environment, e.g., temperature, l i g h t , humidity and ion c o n c e n t r a t i o n , l i v i n g organisms are g e n e r a l l y c o n s i d e r e d to maintain an e s s e n t i a l l y constant i n t e r n a l environment. According to Bernard (1878), the maintenance of such a s t a t e ( r e f e r r e d to as homeostasis) was e s s e n t i a l f o r l i f e , " l a c o n d i t i o n de l a v i e l i b r e " . Environmental v a r i a t i o n s can be viewed as having a p e r t u r b i n g i n f l u e n c e upon the i n t e r n a l constancy, tending to d i s p l a c e the l a t t e r from what might be an " i d e a l " s t a t e . T y p i c a l l y , • p e r t u r b a t i o n s a r i s i n g from exogenous or even endogenous i n f l u e n c e s tend to evoke f o r c e s or processes which oppose or counteract these changes, r e s t o r i n g the organisms to i t s i d e a l c o n d i t i o n . Thus, i d e a l l y , homeostatic mechanisms make the organism independent of the environmental v a r i a t i o n s . However, when sub j e c t e d to extreme c o n d i t i o n s these mechanisms may not be s u c c e s s f u l . Mechanisms which counteract environmental p e r t u r b a t i o n s can be more r a p i d l y achieved at the p h y s i o l o g i c a l and b i o c h e m i c a l l e v e l s . A l t e r a t i o n s of the l a t t e r , in the long-2 ru n , a re almost c e r t a i n l y r e s p o n s i b l e a l s o f o r m o r p h o l o g i c a l and d e v e l o p m e n t a l changes. An i n c r e a s e of r o o t p r o l i f e r a t i o n s and r o o t / s h o o t r a t i o under low n u t r i e n t and low temperature c o n d i t i o n s have been r e p o r t e d by many a u t h o r s ( C h a p i n , 1980; Davi d s o n , 1969; Osmond et a l . , 1980; T h o r n l e y , 1977). These changes b u f f e r the organism through i n c r e a s e d biomass, a m o r p h o l o g i c a l response a c h i e v e d by d i f f e r e n t i a l a l l o c a t i o n of r e s o u r c e s between r o o t s and s h o o t s . E x t e n s i v e e v i d e n c e from some p h y s i o l o g i c a l ( p h o t o s y n t h e t i c and r e s p i r a t o r y p r o c e s s e s ) and d e v e l o p m e n t a l (e.g. l i f e h i s t o r y p a t t e r n s , t i m i n g of f l o w e r i n g ) p r o c e s s e s a l s o i n d i c a t e p l a n t s ' a b i l i t y t o compensate f o r the e n v i r o n m e n t a l p e r t u r b a t i o n s or t o a v o i d the haz a r d s a s s o c i a t e d w i t h u n f a v o u r a b l e c o n d i t i o n s a l t o g e t h e r . The e x t e n t or c a p a c i t y t o w i t h s t a n d adverse e n v i r o n m e n t a l c o n d i t i o n s (e.g. temperature s t r e s s , s a l i n i t y s t r e s s e t c . ) , however, i s det e r m i n e d by the mechanisms developed i n p l a n t s i n the e v o l u t i o n a r y p r o c e s s of s e l e c t i o n . Chapin (1974b) p r o v i d e d a good example on t h i s i s s u e . He examined the i n t e r s p e c i f i c d i f f e r e n c e s i n the c a p a c i t y of p l a n t s t o a b s o r b H 2PO a~ i o n s a t low temperature (5°C). S p e c i e s from h i g h l a t i t u d e s had a much g r e a t e r uptake c a p a c i t y ( e i t h e r at r a t e l i m i t i n g or at r a t e s a t u r a t i n g phosphate c o n c e n t r a t i o n s ) than d i d s p e c i e s from warmer, lower l a t i t u d e s . A g r e a t e r c a p a c i t y f o r " a c c l i m a t i z a t i o n " t o f l u c t u a t i o n s i n temperature would appear t o be of g r e a t e r s e l e c t i v e advantage f o r the h i g h e r l a t i t u d e s p e c i e s . Chapin (1974a) d e f i n e d " a c c l i m a t i z a t i o n " as a method by which organisms compensate f o r 3 (or b u f f e r ) the f l u c t u a t i o n s in the environment. According to Chapin (1974a), s e l e c t i o n for a higher " a c c l i m a t i o n p o t e n t i a l " w i l l occur in the more f l u c t u a t i n g environments, e.g. temperate c l i m a t e s . Hochachka and Somero (1973) have suggested the f o l l o w i n g s t r a t e g i e s whereby changes at the molecular l e v e l may permit an organism to b u f f e r the changes imposed by temperatures. 1) Q u a l i t a t i v e changes i n p r o p e r t i e s of key c o n s t i t u e n t s of the metabolic apparatus, such as new enzymes or d i f f e r e n t l i p i d m ixtures. 2) Q u a n t i t a t i v e changes i n the amount of s p e c i f i c c o n s t i t u e n t s which compensate fo r e f f e c t of temperature on the corresponding r e a c t i o n . 3) Immediate responses of the e x i s t i n g metabolic systems which c o n t r o l or minimize the p e r t u r b a t i o n caused by a change of temperature. Temperature and c o n c e n t r a t i o n of ions in s o i l s o l u t i o n are two f a c t o r s which e x h i b i t s c o n s i d e r a b l e d i u r n a l , seasonal and annual f l u c t u a t i o n s . Although the e f f e c t of temperature i s e q u a l l y important, f a r l e s s a t t e n t i o n has been d i r e c t e d towards the understanding of mechanisms which compensate fo r temperature f l u c t u a t i o n s , e s p e c i a l l y to those developed in the root system. However, compensations i n 4 response to c o n c e n t r a t i o n have been known s i n c e 1936, when Hoagland and Broyer reported an i n v e r s e r e l a t i o n s h i p between the a v a i l a b i l i t y of n u t r i e n t s and t h e i r a b s o r p t i o n . Cram (1976) has d i s c u s s e d the o p e r a t i o n of p h y s i o l o g i c a l feedback mechanisms r e s p o n s i b l e f o r the r e g u l a t i o n of ion a b s o r p t i o n . Using a language borrowed from c y b e r n e t i c s , the l a t t e r author has d e s c r i b e d the f o l l o w i n g components of a simple negative feedback system for the maintenance of c e l l u l a r c o n c e n t r a t i o n , volume in w a l l - l e s s c e l l s and t u r g o r . A' s i m p l i f i e d block diagram of such a system i s shown in F i g . 1. T h i s type of r e p r e s e n t a t i o n does not n e c e s s a r i l y represent components of the r e a l system but serves to i l l u s t r a t e the p o s s i b l e i n f o r m a t i o n s . The c o n c e n t r a t i o n s of s o l u t e s in a c e l l mainly depend upon "processes" such as t r a n s p o r t of ions across the membranes, breakdown or formation of organic s o l u t e s and growth of the c e l l . The r e s u l t a n t of the a c t i v i t y of such processes i s termed the "output". Hence', the i n t e r n a l c o n c e n t r a t i o n of s o l u t e s , the osmotic pressure and turgor can be regarded as outputs of accumulatory pro c e s s e s . G e n e r a l l y a flow of i n f o r m a t i o n from the "output" to the process i s c o n s i d e r e d e s s e n t i a l so that when the output decreases, the a c t i v i t y of the process can be i n c r e a s e d or v i c e - v e r s a ; i . e . a negative feedback mechanism operates. The output of a negative feedback s i g n a l i s by d e f i n i t i o n a l s o the primary feedback s i g n a l ( F i g . 1). The d i f f e r e n c e between the a c t u a l "output" and a " d e s i r e d " or a " r e f e r e n c e " value ( C R E F = input of i n f o r m a t i o n to a system), termed the " e r r o r " serves as a s i g n a l a c t i n g 5 F i g . 1 Block diagram of a feedback system (from Cram,1976) C = r e f e r e n c e c o n c e n t r a t i o n C t = i n t e r n a l c o n c e n t r a t i o n C 0 = e x t e r n a l c o n c e n t r a t i o n A = area, V = volume $ = a c t i v e f l u x , </ = p a s s i v e f l u x CfiEF two* FIGURE 1 Supply Q-i) 3 CONTROLLED PROCESS <p pASsrve 0ft crivB Cr 1 (pur PUT} P*r/lKRy feej, suck LOOP 7 d i r e c t l y on the process. If the " d e s i r e d " value i s const a n t , then the output should be kept constant and a homeostat would be o p e r a t i n g . If the reference changes, then the output would change and a " t r a c k i n g " or "follow-up" system would be o p e r a t i n g . The a c t i v e t r a n s p o r t of ions, which can be c o n s i d e r e d as a "process" of the above d e s c r i b e d system, i s a l s o i n f l u e n c e d by s e v e r a l exogenous v a r i a b l e s such as: 1) E x t e r n a l ion c o n c e n t r a t i o n , 2) Temperature, 3) pH, 4) Energy supply, e t c . As p r e v i o u s l y mentioned, e f f e c t s of c o n c e n t r a t i o n on ion a b s o r p t i o n have been e x t e n s i v e l y i n v e s t i g a t e d . The e x i s t e n c e of a negative feedback c o n t r o l of the s o r t d e s c r i b e d above for non-metaboiizable (K +, Na +, CI" and Br") and me t a b o l i z a b l e ions (H 2PO„~) have been proposed by many authors (Cram, 1968; Cseh et a l . , 1970; G l a s s , 1976; Humphries, 1951; Jensen and P e t t e r s s o n , 1978; Johansen et a l . , 1970; Lefebvre and Glass,1982; S u t c l i f f e , 1954). However, l i t t l e i s known about the p h y s i o l o g i c a l b a s i s of temperature-nutrient i n t e r a c t i o n . Temperature has d i r e c t and i n d i r e c t e f f e c t s upon ion t r a n s p o r t . I t may have a d i r e c t i n f l u e n c e upon a c t i v i t y and/or the s p e c i f i c a c t i v i t y ( i . e . number of " t r a n s p o r t e r s " / a r e a of root) of " t r a n s p o r t e r s " 8 (Clarkson and Deane-Drummond, 1981) which are suggested to be p r o t e i n s ( M i t c h e l l , 1967). The former authors suggested that these d i r e c t temperature e f f e c t s were most e a s i l y e x p l a i n e d by an i n c r e a s e i n the number of " t r a n s p o r t e r s " . T h i s c o n c l u s i o n was based on the observed enhancement of xylem exudation in d e c a p i t a t e d b a r l e y and rye root systems p r e c o n d i t i o n e d f o r s e v e r a l days at low temperature (8°C). A s i m i l a r type of e x p l a n a t i o n has been given f o r the i n c r e a s e d r a t e of N0 3" uptake observed when roo t s are exposed to low temperatures (8°C) (Clarkson and Deane-Drummond, 1981). Temperature may a l s o i n f l u e n c e t r a n s p o r t i n d i r e c t l y by a l t e r i n g a v a i l a b i l i t y of ions through e f f e c t s on the r a t e of d i f f u s i o n through s o i l s o l u t i o n . It may a l s o a l t e r the energy supply by a f f e c t i n g r a t e s of metabolism. The well-known parameters used to ev a l u a t e the e f f e c t of temperature on the r a t e of a r e a c t i o n are the temperature c o e f f i c i e n t ( Q 1 0 ) and the a c t i v a t i o n energy (E f t) of the r e a c t i o n . Most authors have re p o r t e d an i n c r e a s e d temperature s e n s i t i v i t y of p h y s i o l o g i c a l processes at low temperatures, i n d i c a t i n g a dramatic r e d u c t i o n of t h e i r r a t e s under such c o n d i t i o n s . For example, Carey and Berry (1978) reported a strong i n h i b i t i o n of 8 6Rb uptake i n e x c i s e d corn and b a r l e y roots at temperatures below 10°C ( Q i o = 5 - 8 ) . The apparent a c t i v a t i o n energy ( E ) f o r uptake by these s p e c i e s were changed d r a m a t i c a l l y above and below 10°C ( E a was 6.4 k c a l mol" 1 and 26.5 k c a l mol" 1 above and below 10°C r e s p e c t i v e l y ) . However, the v a l i d i t y of t h i s o b s e r v a t i o n with regard .to what happens under f i e l d c o n d i t i o n s i s q u e s t i o n a b l e 9 s i n c e the p l a n t s used in the l a t t e r study were not exposed to the low temperature regime f o r a s u f f i c i e n t time. The experimental procedure i n v o l v e d , growing the p l a n t s at 28°C followed by a l i m i t e d (20 minute) exposure to reduced temperature duri n g the uptake p e r i o d . I t would t h e r e f o r e be i n t e r e s t i n g to i n v e s t i g a t e whether t h e i r o b s e r v a t i o n s are borne out under c o n d i t i o n s in which the s p e c i e s are a c c l i m a t e d to low temperature. T h i s i s an important c o n s i d e r a t i o n s i n c e the extent of t o l e r a n c e or s e n s i t i v i t y to a p a r t i c u l a r thermal regime appears to be p a r t l y determined by the d u r a t i o n of exposure to such c o n d i t i o n s . Moreover, under n a t u r a l c o n d i t i o n s such dramatic instantaneous drops of ten or more degrees(°C) are r a r e . T h e r e f o r e , e s p e c i a l l y to understand the u n d e r l y i n g mechanisms of low temperature r e g u l a t i o n of ion uptake, c o n s i d e r a t i o n of the f p l l o w i n g f a c t o r s i s e s s e n t i a l . 1) Exposure of p l a n t s (or t i s s u e under c o n s i d e r a t i o n ) to a low temperature for a s u f f i c i e n t p e r i o d p r i o r to measurement of r a t e s of any p h y s i o l o g i c a l process and, 2) To know the e f f e c t of temperatures per  se on the process under c o n s i d e r a t i o n by minimizing the c o m p l i c a t i o n s imposed by other f a c t o r s . In a d d i t i o n , in e v a l u a t i n g the e f f e c t of temperature upon _in v i v o p h y s i o l o g i c a l processes (such as ion t r a n s p o r t ) i t 10 i s e s s e n t i a l to be aware of the p o t e n t i a l complexity of the system. As an example, i f ion uptake i s c o n s i d e r e d , in a d d i t i o n to the d i r e c t e f f e c t of temperature on the ion f l u x , any a l t e r a t i o n of the negative feedback due to the changes of i n t e r n a l c o n c e n t r a t i o n , may a l s o a f f e c t f l u x e s . Changes of i n t e r n a l ion c o n c e n t r a t i o n can a l s o r e s u l t from a l t e r a t i o n s i n the rate of root growth. If the growth rate but not the s p e c i f i c a c t i v i t y of t r a n s p o r t e r s i s decreased, then an i n c r e a s e in c o n c e n t r a t i o n of ions i n s i d e the t i s s u e i s a n t i c i p a t e d . On the other hand, i n c r e a s e d root growth without any i n c r e a s e of the s p e c i f i c a c t i v i t y of t r a n s p o r t e r s would r e s u l t i n a d i l u t i o n of i n t e r n a l ' ion c o n c e n t r a t i o n . These changes caused by a l t e r a t i o n of root growth r a t e may t h e r e f o r e be r e s p o n s i b l e f o r r e g u l a t i n g ion f l u x e s . Recently, S i d d i q i et a l . (1983) have proposed a p o s s i b l e a l t e r a t i o n of r e g u l a t i o n of ion t r a n s p o r t i n a b a r l e y v a r i e t y grown under low temperature c o n d i t i o n s . By o b t a i n i n g the i n f l u x measurements (at 10°C and 20°C) over a wide range of i n t e r n a l [K'J i n s e e d l i n g s grown under v a r i o u s c o n d i t i o n s (10°/10°C, 10°/20°C and 20°/20°C root/shoot temperatures) these authors have estimated the temperature a c c l i m a t i o n of K + uptake f r e e from c o m p l i c a t i o n s a s s o c i a t e d with a l t e r e d i n t e r n a l [ K + ] , T h e r e f o r e , i n studying the a c c l i m a t i o n of ion uptake the c o n t r i b u t i o n of t i s s u e ion content cannot be overlooked. Furthermore, a c c l i m a t i o n of ion t r a n s p o r t may not be c o n s i d e r e d complete i f c o n s i d e r a t i o n i s l i m i t e d only to the uptake of ions. T r a n s l o c a t i o n of ions a l s o play an important 11 r o l e i n i o n t r a n s p o r t . In f a c t , a p p r o x i m a t e l y 80% of absorbed i o n s are t r a n s l o c a t e d t o the s h o o t . T h e r e f o r e , shoot s t a t u s and t r a n s l o c a t i o n are e x t r e m e l y i m p o r t a n t i n d e t e r m i n i n g p a t t e r n of r o o t uptake. The e x t e n t of a c c l i m a t i o n of t h e s e two p r o c e s s e s may not n e c e s s a r i l y be i d e n t i c a l . S t u d i e s on some temperate p l a n t s p e c i e s i n d i c a t e t h a t the main e f f e c t of low temperature may be an i n h i b i t i o n of t r a n s l o c a t i o n r a t h e r • than the a b s o r p t i o n (Humphries, 1951; N i e l s e n e t a l . , 1960; Power et a l . , 1970). Hence i t would be i n t e r e s t i n g t o i n v e s t i g a t e both phenomena u s i n g a c c l i m a t e d and n o n - a c c l i m a t e d p l a n t s . I f the a c c l i m a t i o n of i o n t r a n s p o r t i s not complete due t o i n s u f f i c i e n t time of exposure t o low temperature or i f the p l a n t i s not c a p a b l e of a c c l i m a t i n g at a l l , then the i n t e r n a l c o n c e n t r a t i o n of i o n s may t e n d t o be lower than i n c o n t r o l s ( p l a n t s which a r e grown at r e l a t i v e l y h i g h t e m p e r a t u r e s ) . With the a f o r e m e n t i o n e d i s s u e s i n mind the p r e s e n t study was u n d e r t a k e n ; 1) t o examine the e x t e n t of v a r i a t i o n i n a c c l i m a t i o n of i o n ( K + ) i n f l u x i n a group of b a r l e y v a r i e t i e s . 2) t o i n v e s t i g a t e d i f f e r e n c e s i n t i s s u e c o n t e n t and a c c u m u l a t i o n i n w i n t e r and s p r i n g v a r i e t i e s . 3) t o determine the time c o u r s e of a c c l i m a t i o n which would a l l o w p o s s i b l e 12 mechanisms to be proposed. 4) to i n v e s t i g a t e the e f f e c t of e x t e r n a l [K] on temperature a c c l i m a t i o n of ion (K +) i n f l u x . T h i s study was l i m i t e d to the a b s o r p t i o n .and t r a n s l o c a t i o n of K +, because; 1) There i s strong evidence i n d i c a t i n g a p o s s i b l e r o l e f o r K + in the pr e v e n t i o n of f r o s t damage in crop v a r i e t i e s s u bjected to K* f e r t i l i z a t i o n . 2) K + performs an extremely important f u n c t i o n as an osmoticum in p l a n t s . The e s s e n t i a l r o l e of i n o r g a n i c osmotica ( e s p e c i a l l y K +) i n l a t e f r o s t c o n d i t i o n s has been p o i n t e d out (Beringer and T r o l l d e n i e r , 1978). According to these authors, u n l i k e f a l l or e a r l y winter f r o s t , the l a t e s p r i n g f r o s t u s u a l l y occurs when the t i s s u e s have been a l r e a d y dehardened. Reeves and McBee (1972) a l s o suggested the importance of in o r g a n i c s o l u t e s under such c o n d i t i o n s e s p e c i a l l y because the other p r o t e c t i v e substances (sugars etc.) have been p a r t i a l l y metabolized d u r i n g dehardening. The s p e c i e s used, namely b a r l e y (Hordeum v u l g a r e . L ) , i s c u l t i v a t e d on a commercial s c a l e i n Canada. 13 I I . MATERIALS AND METHODS 1. Growth of p l a n t s 1.1 Seeds Seeds of b a r l e y (Hordeum v u l g a r e . L) were g e n e r o u s l y p r o v i d e d t h r o u g h t h e c o u r t e s y of A g r i c u l t u r e Canada. 1.2 Seed g e r m i n a t i o n Seeds were t h o r o u g h l y washed w i t h c o l d water t o remove the f u n g i c i d e s and i n s e c t i c i d e s and were i m b i b e d w i t h d i s t i l l e d w a ter w i t h a e r a t i o n a t room t e m p e r a t u r e . D u r a t i o n of i m b i b i t i o n v a r i e d between w i n t e r and s p r i n g v a r i e t i e s . W i n t e r v a r i e t i e s showed b e t t e r g e r m i n a t i o n a f t e r o v e r n i g h t i m b i b i t i o n w h i l e 2-4 h o u r s i m b i b i t i o n a p p e a r e d t o be a d e q u a t e f o r s p r i n g v a r i e t i e s f o r a s i m i l a r g e r m i n a t i o n r e s p o n s e . Imbibed s e e d s were t h e n p l a c e d on n y l o n mesh i n P l e x i g l a s d i s c s (8 s e e d s / d i s c ) . These d i s c s were t h e n p l a c e d on homogeneously m o i s t e n e d s t e r i l e sand i n p l a s t i c t r a y s and c o v e r e d w i t h a t h i c k l a y e r of m o i s t s a n d . T r a y s were t h e n c o v e r e d w i t h aluminum f o i l and l e f t a t 22°C f o r 3 d a y s . 14 1.3 Choice of growth regimes Except f o r the temperature and K + c o n c e n t r a t i o n s the method of hydroponic p l a n t growth i s s i m i l a r to that d e s c r i b e d by S i d d i q i and Glass (1982). Before t r a n s f e r , the d i s c s c o n t a i n i n g s e e d l i n g s were g e n t l y washed in running c o l d water (10-15 seconds) to remove the sand. D i s c s having 3 day o l d s e e d l i n g s were t r a n s f e r r e d to the P l e x i g l a s hydroponic tanks of 36 L c a p a c i t y and f i t t e d with c i r c u l a t i o n pumps ( LAUDA , MODEL T-1 , Brinkmann Instruments). The background n u t r i e n t s o l u t i o n was 0.01 mo d i f i e d Johnson's s o l u t i o n ( E p s t e i n , 1972) minus K* (Table 1). The i n i t i a l c o n c e n t r a t i o n of the v a r i o u s elements i s given i n Table 1. The potassium ion c o n c e n t r a t i o n i n the growth medium was 0.1 mM f o r " h i g h - s a l t " p l a n t s and 0.005 mM f o r "low-s a l t " p l a n t s . The r e q u i r e d K + c o n c e n t r a t i o n s were maintained by adding a p p r o p r i a t e amounts of potassium s u l p h a t e , using FMI LAB PUMPS (Model RP-G6) which fed i n t o the tank r e s e r v o i r s c o n t i n u o u s l y . The c o n c e n t r a t i o n of K* i n the tank was measured each day using a flame photometer (Instrumentation Laboratory,443) and the pump speed and (or) c o n c e n t r a t i o n of the potassium sulphate s o l u t i o n were a d j u s t e d a c c o r d i n g l y . The Johnson's s o l u t i o n minus K + was d e l i v e r e d along with K + to a l l tanks in order to maintain i t ' s c o n c e n t r a t i o n c l o s e to 0.01 s t r e n g t h . The s o l u t i o n s were a e r a t e d in a r e s e r v o i r , from which they were c o n t i n u o u s l y c i r c u l a t e d i n t o the compartment c o n t a i n i n g p l a n t s to ensure e f f i c i e n t mixing. The p l a n t s were maintained in a growth cabi n e t on a 16/8 day/night c y c l e and 70% r e l a t i v e humidity. An i r r a d i a n c e at p l a n t l e v e l af 15 Table 1 Composition of 0.01 Johnson's K + medium (from E p s t e i n , 1972). 16 Compound Element F i n a l of C o n c e n t r a t i o n element yM C a ( N 0 3 ) £ N 160.00 Ca 80.00 MgS0 4 Mg 10.00 NH^H 2P0 4 P 20.00 C o C l 2 CI 50.00 H 3B0 3 B 0.250 ' MnSO„ ,HaO 4 * Mn 0.020 ZnSC> . 7H,0 Zn 0.020 CuSC- . 5H 0 Cu 0. 005 H,MoO. Mo 0.005 Fe.EDTA Fe 0.200 17 600 MEm"2s"' was provided by " V i t a - L i t e " f l u o r e s c e n t tubes having s p e c t r a l composition s i m i l a r to n a t u r a l s u n l i g h t . The experimental tanks were maintained at low temperatures (5°C unless otherwise s p e c i f i e d ) by a continuous flow of water (5°C) pumped by a Forma S c i e n t i f i c R e f r i g e r a t e d Unit (Model 2325) through s t a i n l e s s s t e e l exchanger c o i l s immersed in the r e s e r v o i r part of the tank. Growth media were maintained at the temperature of the growth c a b i n e t (15°C unless otherwise s p e c i f i e d ) . P l a n t s grown at low temperature (LT) were maintained under these c o n d i t i o n s f o r 2 weeks and high temperature p l a n t s only f o r 1 week (unless otherwise s p e c i f i e d ) p r i o r to h a r v e s t i n g . These p l a n t s appeared to have a t t a i n e d a s i m i l a r " p h y s i o l o g i c a l age" during t h i s p e r i o d ( i . e . approximately s i m i l a r f r e s h weights) (Deane-Drummond and G l a s s , 1983)., 18 2. K + i n f l u x measurement In a l l cases i n t a c t p l a n t s were used. The i n c u b a t i o n temperatures were s e l e c t e d to be c l o s e to the growing temperatures. Three d i s c s c o n t a i n i n g 8 p l a n t s i n each, were used from each treatment and c o n t r o l tank. P r i o r to i n c u b a t i o n in l a b e l e d medium, roots of low and high temperature p l a n t s were r i n s e d f o r 5 minutes in medium, (0.5 mM c a l c i u m sulphate, 0.05 mM potassium sulphate unless otherwise s p e c i f i e d ) , whose composition was i d e n t i c a l to that of the i n c u b a t i o n medium except f o r B 6 R b C l . T h i s pretreatment i s e s s e n t i a l because 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 c o n t a i n s s u b s t a n t i a l ion r e s e r v i o r s which possess h a l f - l i v e s f o r ion exchange i n the order of 1-3 minutes (Walker and Pitman, 1976; Cram, 1973; Dainty and Hope, 1959). T h e r e f o r e , 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 (10 minutes) designed to estimate plasmalemma f l u x e s i t i s necessary to s t a n d a r d i z e the c e l l w a l l K + s t a t u s of roots grown in d i f f e r e n t K + and temperature regimes. Incubation medium having the same i o n i c composition as above was l a b e l e d with 8 6 R b C l (0.13 MCi m o l . " 1 ) . Roots of i n t a c t p l a n t s were immersed f o r 10 minutes in these s o l u t i o n s with continuous a e r a t i o n . Subsequently, ions in the f r e e spaces were desorbed by immersing the roots f o r 5 minutes in non-l a b e l e d i c e - c o l d media which was otherwise s i m i l a r to i n c u b a t i o n media. Roots were then e x c i s e d , spun f o r 20 seconds to remove extraneous water in a basket c e n t r i f u g e , weighed i n t o s c i n t i l l a t i o n v i a l s and ashed f o r 24 hours at 500°C. The ash was d i s s o l v e d i n 10 ml of d i s t i l l e d water and the 19 r a d i o a c t i v i t i e s measured by Cerenkov c o u n t i n g i n a S e a r l e I s o c a p 300 s c i n t i l l a t i o n s p e c t r o p h o t o m e t e r ( L a u c h l i , 1969; G l a s s , 1978). The same aqueous s o l u t i o n s were used t o a n a l y s e the t i s s u e K + by a flame photometer. 20 3. P r e l i m i n a r y s u r v ey 3.1 Measurement of K + i n f l u x and t i s s u e K + c o n c e n t r a t i o n i n 23 s p r i n g and 2 w i n t e r b a r l e y v a r i e t i e s Seeds of twenty-two s p r i n g , two w i n t e r and one summer(warm adapted) b a r l e y v a r i e t i e s ( l i s t e d i n T a b l e s 2-8) were sown, germinated and grown a c c o r d i n g t o the p r e v i o u s l y d e s c r i b e d methods (1.2 and 1.3). Root/shoot temperature regimes of 15°/15°C and 5°/l5°C were used as c o n t r o l and tr e a t m e n t t e m p e r a t u r e s , r e s p e c t i v e l y . F i f t e e n degrees has been r e p o r t e d t o be the optimum temperature f o r r o o t growth of b a r l e y (Power et a l . , 1970). Mean tempe r a t u r e s of s o i l s i n which b a r l e y i s grown ( R e g i n a , Saskatoon) at 5 cm below s o i l s u r f a c e u s u a l l y ranges from 2-6°C d u r i n g f a l l , and l a t e s p r i n g seasons ( r e p o r t of A g r i c u l t u r e Canada). Hence, a r o o t temperature of 5°C was chosen f o r t h i s study and assumed t o be a p p r o p r i a t e i n o r d e r t o s i m u l a t e e a r l y f a l l and l a t e s p r i n g c o n d i t i o n s . D u r i n g e a r l y f a l l , seeds of w i n t e r v a r i e t i e s a r e sown and d u r i n g l a t e s p r i n g most of the o v e r - w i n t e r e d p l a n t s b e g i n t o r e c o v e r from t h e i r dormant s t a t e . The p o t a s s i u m i o n c o n c e n t r a t i o n i n the growth media was m a i n t a i n e d a t 0.10 mM l e v e l , , h e n c e these a r e r e f e r r e d t o as " h i g h - s a l t " p l a n t s . I n f l u x of 8 6 R b was measured a t both 5°C and 15°C f o r each t r e a t m e n t and c o n t r o l . 21 The d a t a c o l l e c t e d on i n f l u x and t i s s u e K + c o n t e n t f o r t w e n t y - t h r e e b a r l e y v a r i e t i e s grown at two d i f f e r e n t t e m p e r a t u r e s were a n a l y s e d u s i n g the f o l l o w i n g t e s t s . 1. S t u d e n t ' s t - t e s t t o compare the mean v a l u e s of each v a r i a b l e i n each v a r i e t y . 2. Spearman's rank c o r r e l a t i o n a n a l y s i s f o r non p a r a m e t r i c d a t a . T h i s a n a l y s i s p e r m i t t e d a comparison of the r e s u l t s of two e x p e r i m e n t s c a r r i e d out under i d e n t i c a l c o n d i t i o n s . 3. Two way a n a l y s i s of v a r i a n c e (ANOVA) was used t o determine the f a c t o r ( i . e . t emperature or v a r i e t i e s r e s p o n s i b l e f o r v a r i a t i o n s among the v a r i a b l e s ( i n f l u x ) . 22 3 .2 Determination of temperature c o - e f f i c i e n t ( Q i 0 ) and a c t i v a t i o n energy ( E R ) To determine the temperature c o e f f i c i e n t ( Q i 0 ) and a c t i v a t i o n energy ( E a) f o r uptake, i n c u b a t i o n s were c a r r i e d out in l a b e l l e d medium at 5° , 10°, 15° and 25°C. The methods of c a l c u l a t i o n of temperature c o e f f i c i e n t and a c t i v a t i o n e n e r g i e s are given i n s e c t i o n I I I , 1 . 2 . 23 4. R e g u l a t i o n of K + i n f l u x In t h i s experiment the method was s i m i l a r to that of S i d d i q i et a l . (1983) except f o r the temperature and v a r i e t i e s used. P l a n t s were grown under two temperature regimes as d e s c r i b e d i n experiment 1. On the day of t r a n s p l a n t i n g , 0.1 mM K + ( p o t a s s i u m sulphate) was added to the growth medium but no f u r t h e r supply of K + was provided u n t i l the day of i n f l u x measurements in order to produce p l a n t s of low K + c o n t e n t s . However, other n u t r i e n t s were c o n t i n u o u s l y s u p p l i e d as d e s c r i b e d i n experiment 1. Potassium ion i n f l u x e s from 0.5 mM c a l c i u m sulphate plus 0.05 mM potassium sulphate s o l u t i o n were measured on the morning of day 10 (from sowing) fo r high temperature p l a n t s (15°/15°C) and i n the morning of day 13 for low temperature (5°/l5°C) p l a n t s at both 5°C and 15°C ( T 0 samples). A f t e r the removal of T 0 samples, s u f f i c i e n t potassium sulphate was added to each tank to b r i n g the K + c o n c e n t r a t i o n to 6 mM. T h e r e a f t e r , K" i n f l u x e s from s o l u t i o n s c o n t a i n i n g 0.1 mM K + (at 5°C and 15°C) were measured at hou r l y i n t e r v a l s u n t i l T 2 a ; K + i n f l u x e s of the l a s t samples (T 2„) were determined the f o l l o w i n g morning. K + content of t i s s u e s of a l l these samples were a l s o determined. 24 5. Growth s t u d i e s of 3 b a r l e y v a r i e t i e s Two s p r i n g b a r l e y v a r i e t i e s (Hector and Bonanza) and one winter v a r i e t y , Halcyon, were chosen f o r t h i s study. Although both are s p r i n g v a r i e t i e s , Hector and Bonanza appeared to show d i f f e r e n t growth responses i n the p r e v i o u s experiments. T h i s experiment was c a r r i e d out to i n v e s t i g a t e the e f f e c t of low root temperature on growth r a t e s of r o o t s and shoots and accumulation of t i s s u e K +. E f f e c t of low e x t e r n a l K + (0.005 mM) at low and high temperature c o n d i t i o n s on root and shoot growth was a l s o i n v e s t i g a t e d . Three days a f t e r sowing, f r e s h weights of e x c i s e d shoots and r o o t s of known numbers of s e e d l i n g s (14-16) were determined. A f t e r l e a v i n g these t i s s u e s at 80°C for 2 days, dry weights were a l s o determined. These measurements were con s i d e r e d as T 0 . T i s s u e K + content at t h i s stage was assumed to be s i m i l a r to the seed re s e r v e s s i n c e no n u t r i e n t was s u p p l i e d d u r i n g the germination p e r i o d . The r e s t of the s e e d l i n g s were t r a n s f e r r e d to growth medium where the c o n d i t i o n s were maintained as d e s c r i b e d above. Growth regimes other than temperature and e x t e r n a l K + c o n c e n t r a t i o n were maintained as i n experiment 3. Seedlings grown at 0.1 mM e x t e r n a l K + c o n c e n t r a t i o n or " h i g h - s a l t " p l a n t s were c o n s i d e r e d as " c o n t r o l s " f o r comparison with the " l o w - s a l t " treatment. The temperature regimes used were 8°/l8°C and 18°/18°C root/shoot f o r treatment and c o n t r o l s , r e s p e c t i v e l y . F r e s h weight and dry weights of .approximately 16 s e e d l i n g s were used on the f o u r t h , e i g h t h and f o u r t e e n t h day a f t e r t r a n s p l a n t i n g . A f t e r ashing these samples t i s s u e K + was determined. 25 6. E f f e c t of low e x t e r n a l [ K + ] on a c c l i m a t i o n of uptake The 3 v a r i e t i e s used in experiment 4 were a l s o used i n t h i s experiment. Seedlings were grown under the p r e v i o u s l y d e s c r i b e d " l o w - s a l t " c o n d i t i o n s . Treatment and c o n t r o l temperature regimes were s i m i l a r to those of experiment 5. Two-week o l d s e e d l i n g s were used f o r i n f l u x measurements and K + determinat i o n . 26 7. Determination of a c c l i m a t i o n of K+ i n f l u x at d i f f e r e n t ages and growth temperatures 7.1 E f f e c t of age on a c c l i m a t i o n Three day o l d s e e d l i n g s of 2 winter b a r l e y v a r i e t i e s (Halcyon and Gerbel) and a s p r i n g b a r l e y v a r i e t y (Bonanza) were t r a n s f e r r e d to tanks maintained under standard c o n d i t i o n s (as d e s c r i b e d p r e v i o u s l y ) except f o r temperature. The temperature regimes used were 8°/l8°C and 18°/18°C root/shoot temperatures. The potassium ion c o n c e n t r a t i o n of the medium was s i m i l a r to that i n experiment 3. I n f l u x r a t e s at both 8°C and 18°C and t i s s u e K + were measured on the s i x t h , tenth and s i x t e e n t h days a f t e r t r a n s p l a n t i n g . 7.2 E f f e c t of growth temperature p e r t u r b a t i o n Half of each set of the s e e d l i n g s grown under the above d e s c r i b e d c o n d i t i o n s were t r a n s f e r r e d to the other growth temperature on the tenth day a f t e r t r a n s p l a n t i n g (13 days o l d s e e d l i n g s ) and allowed to grow at t h i s new temperature regime for 6 more days before 8 6 R b i n f l u x was measured at 8°C and 18°C. The above experiment was a l s o c a r r i e d out with a lengthened exposure to perturbed temperature. In t h i s i n s t a n c e s e e d l i n g s were t r a n s f e r r e d on the s i x t h day to the other growth temperature and allowed to remain f o r another 10 days p r i o r to i n f l u x measurement. 27 8. Time course of a c c l i m a t i o n by short term exposure to low temperatures Two b a r l e y v a r i e t i e s , a s p r i n g v a r i e t y (Kombar) and a winter v a r i e t y (Halcyon) were used in t h i s experiment. Seeds were germinated as d e s c r i b e d p r e v i o u s l y (experiment 1.1). Fo l l o w i n g t r a n s f e r to high temperature (15°/l5°C root/shoot) c o n d i t i o n s , p l a n t s were l e f t to grow f o r 6 days. The other growth regimes were s i m i l a r to those of experiment 1. Then, at a p p r o p r i a t e time i n t e r v a l s (24, 16, 13, 4 and 2 hours p r i o r to i n f l u x measurements), samples were t r a n s f e r r e d to 5°/l5°C root/shoot temperature treatment tanks. A l l the other c o n d i t i o n s were approximately s i m i l a r to "high-temperature" c o n t r o l s . C o n t r o l (0 hour) samples remained at 15°C throughout. Potassium ion i n f l u x e s of a l l the treatments and the c o n t r o l s were determined at the same time, using 0.5 mM calcium sulphate p l u s 0.05 mM potassium sulphate at 10°C. In t h i s experiment p l a n t s were l e f t in the l a b e l l e d medium f o r an hour (compared to 10 minutes i n other experiments). I t was assumed that t h i s d u r a t i o n (1 hour) i s enough f o r the t r a n s p o r t of measurable amounts of 8 6 R b to the shoot. Hence, the f l u x e s c a l c u l a t e d represent the net f l u x e s i n s t e a d of i n f l u x . Only f o r the c o n t r o l s and the 24 hour exposed treatments, K* i n f l u x was a l s o measured at 15°C. Three r e p l i c a t e s each having 5 s e e d l i n g s were used. T i s s u e [ K + ] was determined f o r a l l the samples. 28 I I I . RESULTS AND DISCUSSION 1. P r e l i m i n a r y survey 1.1 V a r i a t i o n s of K + i n f l u x and t i s s u e [ K + ] among 23 s p r i n g b a r l e y and 2 winter b a r l e y v a r i e t i e s . Plasmalemma i n f l u x f o r both low (LT) and high (HT) temperature grown b a r l e y v a r i e t i e s r e v e a l e d s u b s t a n t i a l d i f f e r e n c e s between v a r i e t i e s ( F i g . 2 ) . Potassium i n f l u x i n b a r l e y r o o t s has been demonstrated to be extremely s e n s i t i v e to i n t e r n a l K + s t a t u s (Glass,1978; Pitman et al.,1968; Pitman and Cram,1973). The observed d i f f e r e n c e s between v a r i e t i e s might t h e r e f o r e r e f l e c t d i f f e r e n c e s i n i n t e r n a l [ K + ] ( F i g . 3 ) . The present study re v e a l e d d i f f e r e n c e s among v a r i e t i e s , i n the extent of i n f l u x s e n s i t i v i t y to the i n t e r n a l [ K + ] , at both growth temperatures. Ranks obtained by Spearman's rank c o r r e l a t i o n f o r i n f l u x were not r e l a t e d to those obtained f o r t i s s u e [ K + ] (Tables 2-7). T h i s i n d i c a t e s that the inherent or g e n e t i c e f f e c t i s greater than the temperature f a c t o r on the r e l a t i o n s h i p between i n f l u x and i n t e r n a l [ K 4 ] . S i m i l a r o b s e r v a t i o n s has been r e p o r t e d by other authors in t h e i r s t u d i e s of v a r i e t a l v a r i a t i o n s of K 4 t r a n s p o r t (Glass and P e r l e y , i 9 8 0 ; S i d d i q i and G l a s s , 1 9 8 2 ) . A n a l y s i s of v a r i a n c e 29 F i g , K + i n f l u x f o r r o o t s at 15°C of 23 b a r l e y v a r i e t i e s grown at 5 /15°C(A) and 15/15°C(0) root/shoot temperature. Each p o i n t represent the mean of 24-27 s e e d l i n g s . Standard e r r o r s were l e s s than 10% of the mean v a l u e . V a r i e t y Code 1. Betzes 2. B.T.334 3. Bonanza 4. Compana 5. Conquest 6. E x c e l s i o r 7. F a i r f i e l d 8. Fergus 9. G a i t 10. Hannachan 11. Hector 12. Himalaya 13. Johnston 14. Keystone 15. Klages 16. Klondike 1?.Kombar 18.Lion 19. L a u r i e r 20. Melvin 21. Mingo 22.Odessa 23.011i F i g . 3 Root K + content of 23 b a r l e y v a r i e t i e s grown at 5/15°C ( ) and 15/15°C ( ) root/shoot t e m p e r a t u r e s . ( V a r i e t y code i s s i m i l a r to that of Fig.2) . 1 FIGURE 2 75 70 ~ 65 '*> £ 60 cr> *S 55 E 3 - 50 LU »-z 8 45 40 35 30 25 20 G G G G • • • • o I I I I I • • • • G D • D D G JSL i i i i i i * • • i 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 VARIETY CODE FIGURE 3 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 VARIETY CODE 31 (ANOVA) a l s o r e v e a l e d g r e a t e r s i g n i f i c a n t d i f f e r e n c e s i n i n f l u x between v a r i e t i e s (p<0.0l) than between the two growth temperatures (p=0.05). T h i r t e e n of the twenty-three LT v a r i e t i e s showed a s i g n i f i c a n t l y g r e a t e r i n f l u x at 15°C (Table 8). However, i r r e s p e c t i v e of growth temperature some v a r i e t i e s (Conquest, -Klages and Keystone) showed c o n s i s t e n t l y low i n f l u x while some others (Compana, O l l i and Hector) showed c o n s i s t a n t l y high f l u x e s . Although i t i s d i f f i c u l t to make a general c o n c l u s i o n c o n s i d e r i n g the s p r i n g v a r i e t i e s as a whole, another experiment c a r r i e d out under i d e n t i c a l c o n d i t i o n s (but with fewer v a r i e t i e s and more r e p l i c a t e s ) showed a somewhat general t r e n d (Table 9). LT p l a n t s e x h i b i t e d s i g n i f i c a n t l y lower (p<0.005) f l u x e s when measured at 5°C than at 15°C. However, a s i g n i f i c a n t d i f f e r e n c e was not observed between LT and HT p l a n t s when i n f l u x was measured at 15°C (HT). In t h i s i n stance a s i g n i f i c a n t l y lower (p<0.0l) t i s s u e [ K + ] i n LT p l a n t s was a l s o observed. Under s i m i l a r growth c o n d i t i o n s the trend shown by two winter v a r i e t i e s i s given in Table 10. Although some s i m i l a r i t y was d i s c e r n e d , when the o v e r a l l p i c t u r e i s c o n s i d e r e d the a b s o l u t e v a l u e s for s p r i n g v a r i e t i e s d i f f e r from those of winter v a r i e t i e s . The LT winter v a r i e t i e s show a s i g n i f i c a n t l y g r e a t e r i n f l u x at 5°C while HT winter v a r i e t i e s show a s i g n i f i c a n t l y lower i n f l u x at 15°C (comparison of e and h i n Table 10). I n t e r e s t i n g l y , the root [ K + ] was not d i f f e r e n t as a n t i c i p a t e d f o r LT and HT p l a n t s while the shoot [K +] showed a m a r g i n a l l y s i g n i f i c a n t d i f f e r e n c e . The a b s o lute values observed for 2 winter b a r l e y v a r i e t i e s are given in Table "11.. A s i m i l a r experiment c a r r i e d out using 4 s p r i n g and 2 32 winter b a r l e y v a r i e t i e s r e v e a l e d the r e s u l t s given i n Tables 12, 13 and 14. Fergus appears to act more l i k e a winter v a r i e t y than the other 3 s p r i n g v a r i e t i e s . S i d d i q i et a l . (1983) have a l s o r e p o r t e d Fergus a c h i e v i n g a g r e a t e r temperature a c c l i m a t i o n of K + uptake when grown at low root temperatures. Th e r e f o r e , i t appears that among other s p r i n g v a r i e t i e s , Fergus possesses a g r e a t e r c a p a c i t y f o r temperature a c c l i m a t i o n , at l e a s t f o r K + i o n s . (No s t u d i e s are a v a i l a b l e on uptake of other ions f o r Fergus under these c o n d i t i o n s ) . The p a t t e r n shown between two winter v a r i e t i e s a l s o d i f f e r s s l i g h t l y i n d i c a t i n g v a r i e t a l v a r i a t i o n s . 33 Table 2 I n f l u x r a t e s (Mmol.gfwt.- 1h.- 1) at 15°C, of K + ( 8 6Rb) at 15°C from a 0.1 mM K + s o l u t i o n f o r 23 b a r l e y variet.es grown at 15°/5°C Shoot/root temperature. (Spearman's RHO = 0.1870; p>0.25). Each value represent the mean root i n f l u x of 24-27 s e e d l i n g s 34 Rank No. Ranking f o r 5°C grown V a r i e t y v a r i e t i e s I n f l u x + SE 1 Compana 2.239 + 0.39 2 Hector 2. 057 + 0.09 3 O l l i 1.917 + 0.09 4 Lio n 1 .748 + 0.27 5 F a i r f i e l d 1 .668 + 0.26 6 Betzes 1 .643 + 0.00 7 Kombar 1 .630 + 0.25 8 Johnston 1 . 525 + 0.09 9 Mingo 1.515 + 0.13 1 0 B.T.334 1 .488 + 0.22 • 1 1 Odessa • 1 .437 + 0.12 12 Hannachan 1.314 + 0.07 1 3 Ga i t 1.215 + 0.08 1 4 Klondi ke 1 . 1 95 + 0.03 1 5 E x c e l s i o r 1 . 172 + 0.11 1 6 Melvin 1 . 1 56 + 0.00 1 7 Rlages 1 . 088 + 0.05 18 Fergus 1 . 078 + 0.01 1 9 Himalaya 0.973 + 0.06 20 Keystone 0.974 + 0.08 21 Bonanza 0.850 + 0.04 22 L a u r i e r 0.832 + 0.30 23 > Conquest 0.516 + 0.41 35 T a b l e 3 Root K + ( M m o l . g f w t . " 1 ) c o n c e n t r a t i o n of 23' b a r l e y v a r i e t i e s grown a t 15°/5°C s h o o t / r o o t t e m p e r a t u r e . (Spearman's RHO =0.373; p<0.05). Each v a l u e r e p r e s e n t the mean r o o t K* of 24-27 s e e d l i n g s . 36 Rank No. Ranking f o r 5°C grown Va r i e t y v a r i e t i e s Root [ K + ] 1 Betzes 51 .67 + 5.30 2 Klages 44.43 + -3.5 O l l i 42. 00 + 7.30 3.5 Hector 42. 00 + 1 . 20 4 Johnston 38.92 + 3.40 5 B.T.334 37.62 + 0.09 7 Bonanza 36. 54 + 2.99 8 Lio n 36.25 + 4. 50 9 Conquest 36.22 + 0.95 1 0 Himalaya 34.00 + -1 1 Klond i ke . 33.99 + 0.65 1 2 Keystone 33. 96 + 2.40 1 3 Compana 33.47 + 4.89 1 4 .Odessa 33.00 + 4.80 1 5 Fergus 32.78 + 6.10 1 6 Laur i e r 31 .07 + 1 .60 1 7 F a i r f i e l d 30.30 + 3.84 18 Kombar 29.58 + 1 .80 19 G a i t 28.30 + 4.80 20 Mingo 28. 1 0 + 6. 50 21 Melvin 26.80 + 3.10 22 .Hannachan .21 .06 1 .00 1 37 Table 4 Shoot K + (ymol. gf wt." 1.) c o n c e n t r a t i o n of 23 b a r l e y v a r i e t i e s grown at 15°/5°C shoot/root temperature. (Spearman's RHO 0.272; p= 0.1 ). Each value represent the mean shoot K + of 24-27 s e e d l i n g s . 38 Ranking f o r 5°C grown v a r i e t i e s Rank No. Var i e t y Shoot [ K + ] ± SE 1 Hector 182.03 + 13.10 2 Betzes 177.29 + 29. 00 3 Hannachan 173.20 + 00.65 4 Kombar 171.04 + 8.76 5 Fergus 167.53 + 8.56 6 Ga i t 161.52 + 1.14 7 Melvin 159.46 + 6.29 8 O l l i 153.44 + 10.18 9 Lio n 152.62 + 1 3.28 1 0 . Kl o n d i ke 147.59 + 32.80 1 1 Klages 146.11 + 12.41 1 2 Bonanza 141.80 + 6.20 1 3 Conquest 138.66 + 13.50 1 4 Compana 1 36.87 + 9.30 1 5 B.T.334 1 35.23 + 4 . 40 1 6 Keystone 1 30.48 + 10.30 1 7 Mingo 127.41 + 4.50 18 Odessa 125.98 + 5.19 1 9 E x c e l s i o r 118.11 + 21 .00 20 Johnston 113.96 + 38.30 21 L a u r i e r 92.81 + 15.09 22 F a i r f i e l d 91 .36 + 5.28 23 Himalaya 89.01 + 5.19 39 Table 5 I n f l u x r a t e s of K + ( 8 6Rb)(umol.gfwt." 1h.- 1) at 15°C from a 0.1 mM K + s o l u t i o n f o r 23 b a r l e y v a r i e t i e s grown at 15°/15°C shoot/root temperature. (Spearman's rank c o r r e l a t i o n c o e f f i c i e n t = 0.5365; P < 0.005). Each value represent the mean i n f l u x of 24-27 s e e d l i n g s . 40 Rank No. Ranking f o r 15°C Var i ety grown v a r i e t i e s I n f l u x ± SE 1 Compana 2.131 + 0.35 2 B.T.334 1 .557 + 0.37 3 O l l i 1 .549 + 0.17 4 Betzes 1 . 394 + 0.02 5 Hector 1 .345 + 0.09 6 Johnston 1 .340 + 0.16 7 Mingo 1 . 338 + 0.02 8 F a i r f i e l d 1.318 + 0.08 9 Odessa 1.100 + 0.65 •1 0 Himalaya .1.097 + 0.03 1 1 L a u r i e r 1 .001 + 0.01 1 2 Lio n 0. 992 + 0.04 1 3 Hannachan 0.974 + 0.03 1 4 Bonanza 0.971 + 0.11 1 5 Kombar 0.962 + 0.09 •1 6 Go l t 0.952 + 0.03 1 7 Melvin 0.937 + 0.25 18 Fergus 0.930 + 0.02 1 9 Keystone 0.867 + 0.09 20 Klo n d i ke 0.813 + 0.05 21 Klages 0.771 + 0.04 22 E x c e l s i o r 0.696 + 0.05 23 Conquest 0.590 0.05 j 41 Table 6 Root K + c o n c e n t r a t i o n (nmol.gfwt." 1) of 23 b a r l e y v a r i e t i e s grown at 15°/15°C shoot/root temperature. (52.2% of these r e s u l t s are h i g h l y c o n s i s t a n t ; . Spearman's RHO 0.0296; p>0.25). Each value represent the mean root K* of 24-27 s e e d l i n g s . 42 Rank No. Ranking f o r 15°C grown Var i e t y var i e t i e s Root [ K + ] ± SE 1 Odessa 69.76 + 5.20 2 O l l i 66.72 + 5.70 3 Lion 66.66 + 1 4.70 4 Gait 62. 1 1 + 2.80 5 Betzes 57. 96 + 3.50 6 Hannachan 57. 18 + 14.90 7 Mingo 56.45 + 3.80 8 Kombar 56.32 + 1 .61 9 Compana 55.01 + 1 . 55 10 Klages 51 .03 + 2.41 1 1 B.T.334 50.85 + 4.40 1 2 Keystone 51 .23 + 3.60 1 3 Bonanza 49.31 + 6. 27 1 4 Conquest 48.53 + 2 . 60 1 5 Klondi ke 48.06 + 4.60 1 6 Melvin 47.52 + 3.64 17 Hector 47.39 + 4. 40 18 Fergus 46. 54 + 1 . 96 19 Himalaya 44 . 22 + 4. 90 20 L a u r i e r 39. 32 + 2.90 21 Johnston 35.42 + 6.35 22 E x c e l s i o r 34.65 a. 2.60 23 I F a i r f i e l d 31.21 + 1 .96 43 Table 7 Shoot K + c o n c e n t r a t i o n (/umol. gf wt. ~ 1 ) of 23 b a r l e y v a r i e t i e s grown at 15°/15°C shoot/root temperature. (Spearman's rank c o r r e l a t i o n c o e f f i c i e n t = 0.5287; P<0.005). Each value represent the mean shoot K + of 24-27 s e e d l i n g s . 44 Rank No. Ranking f o r Var i e t y 15°C grown v a r i e t i e s Shoot [ K + ] ± SE 1 Gai t 190.03 + 7.07 2 Lion 170.57 + 5. 30 3 Bonanza 168.20 + 1 1 .90 4 Betzes 159.40 + -5 B.T.334 155.34 + 1 .76 6 Compana 155.25 + 2.60 7 Hannachan 152.73 + 9. 05 8 Conquest 152.42 + 2.00 9 Mingo 144.35 + 3.40 1 0 Kombar 143.57 + 1 0.70 1 1 Hector 137.81 + 11.80 12 Klondi ke 133.29 + 6.60 1 3 E x c e l s i o r 132.26 + 8.90 1 4 Odessa 130.67 + 2.00 1 5 Keystone 129.69 + 4.90 1 6 Melvin 129.28 2.20 1 7 F a i r f i e l d 1 24.04 + 3. 30 1 8 Fergus 123.12 + 18.60 1 9 Johnston 112.78 + 1 6. 59 20 O l l i 112.57 + 4.20 21 Laur i e r 109.64 + 4.30 22 Himalaya 105.01 + 1 2. 50 23 Klages 104.20 8.50 45 Table 8 K + i n f l u x (umol.gfwt." 1h." 1) at 15°C of p l a n t s grown at 5°/l5°C (A) and 15°/15°C (B). P r o b a b i l i t y (p) i s based on the Student's t - t e s t f o r comparison of two means. Each value represent the mean of 24-27 s e e d l i n g s . 46 V a r i e t y I n f l u x ± SE Conquest 0 .516 + 0. 04 Exc e l s i o r 1 . 1 72 + 0. 11 Klages 1 .088 + 0. 05 Klondi ke 1 . 1 95 + 0. 03 Keystone 0 .974 + 0. 08 Fergus 1 .078 + 0. 01 Melvin 1 . 1 56 + 0. 00 Ga i t 1 .215 + 0. 08 Kombar 1 .630 + 0. 25 Bonanza 0 .850 + 0. 04 Hannachan 1 .314 + 0. 07 Lio n 1 .748 + 0. 27 L a u r i e r 0 .832 + 0. 30 Himalaya 0 .973 + 0. 06 Odessa 1 .437 + 0. 1 2 F a i r f i e l d 1 .668 + 0. 26 Mingo 1 .515 + 0. 1 3 Johnston 1 . 525 + 0. 09 Hector 2 .057 + 0. 09 Betzes 1 .643 + 0. 00 O l l i 1 .917 + 0. 09 B.T.334 1 .488 + 0. 22 Compana 2 .329 + 0. 39 0 .590 + 0. 05 0. 004 0 .696 + 0. 05 0. 010 0 .771 + 0. 04 0. 005 0 . 8 1 3 + 0. 05 0. 030 0 .867 + 0. 09 0. 230 0 .930 + 0. 02 0. 005 0 . 937 + 0. 25 0. 1 40 0 .952 + 0. 03 0. 020 0 . 962 + 0. 09 0. 030 0 .971 + 0. 1 1 0. 250 0 . 974 + 0. 03 0. 008 0 .992 + 0. 04 0. 020 1 . 001 4- 0. 01 0. 310 1 .097 + 0. 03 0. 030 1 . 1 00 + 0. 65 0. 070 1 .318 + 0. 08 0. 1 50 1 .338 + 0. 02 0. 090 1 . 340 + 0. 1 6 0. 230 1 . 345 + 0. 09 0. 007 1 .394 + 0. 02 0. 007 1 . 549 + 0. 1 7 0. 220 1 .557 0. 37 0. 450 2 .131 0. 35 0. 380 47 Table 9 Mean values f o r K + i n f l u x ( y m o l . g f w t . " 1 h . " 1 ) , r o o t [ K + ] and shoot [ K + ] ( M H I O I .gf wt. "" 1 ) f o r 8 s p r i n g b a r l e y v a r i e t i e s . P r o b a b i l i t y (p) i s based on Student's t - t e s t (n = 24-27). Table 10 Mean values f o r K * i n f lux (/xmol. g f w t 1 h 1 ) , root [ K + ] and shoot [ K + ] (jumol. gfwt. " 1 ) f o r 2 winter b a r l e y v a r i e t i e s . P r o b a b i l i t y (p) i s based on Student's t - t e s t (n = 24-27). 48 Var i a b l e Growth Temperature Comparison P 5°/l5°C 15°/l5°C of means I n f l u x at 5° a0.536 C0.462 a&b <0 .005 I n f l u x at 15° *1.380 31.300 a&d <0 .001 Root [ K + ] 34.960 46.280 <0 .01 Shoot [ K + ] 102.330 121 .630 <0 .01 V a r i a b l e Growth Temperature Comparison P 5°/l5°C 15°/15°C of means I n f l u x at 5° 6 0.943 3 0 . 1 19 e&f <0.01 I n f l u x at 15° f 2.163 h 0 . 244 e&h <0.01 Root [ K + ] 49.000 55.240 NS Shoot [ K + ] 89.320 113.600 <0.05 49 Table 11 K + ( 8 6 Rb) i n f l u x (/umol.gfwt." 1h.r 1 ) from a 0.1mM K* s o l u t i o n at 15°C, root and shoot [ K + ] ( mnol.gfwt. ~ 1 ) -for 2 winter b a r l e y v a r i e t i e s grown under 5/l5°C root/shoot (A), and 15/15°C root/shoot (B) temperatures (n = 24-27). 50 Var i a b l e V a r i e t y Growth Temperature A B I n f l u x ± SE Halcyon 2.087 ± 0.32 0.230 ± 0.01 G e r b e l 2.238 ± 0.13 0.257 ± 0.006 Root K ± SE Halcyon 53.20 ± 1 .8 52.35 ± 1 . 0 G e r b e l 44.80 ± 1.9 58. 12 ± 2.9 Shoot K ± SE Ha l c y o n 95.46 ± 5.0 1 17.70 ± 3.0 G e r b e l 83.17 ± 6.5 109.50 ± 3.9 51 Table 12 K + ( 8 6 R b ) i n f l u x (^mol.gfwt." 1h." 1) for 6 b a r l e y v a r i e t i e s (4 s p r i n g and 2 winter) grown under 5°C(A) and 15°C(B) root temperature. I n f l u x was measured at a temperature s i m i l a r to root temperature. (Shoot temperature i n both cases was 15°C) (n = 24-27) . Table 13 Root [K +](Mmol.gfwt." 1) f o r 4 s p r i n g b a r l e y v a r i e t i e s and 2 winter b a r l e y v a r i e t i e s . (A-values f o r 5°/l5°C root/shoot temperature. B-values f o r 15°/15°C root/shoot temperature) (n = 24-27). 52 V a r i e t y I n f l u x ± SE p A B Fergus 1 .385 + 0. 08 0 .278 + 0. 01 0. 003 He c t o r 1 . 1 06 + 0. 01 0 .375 + 0. 07 0. 005 Kombar 1 . 180 + 0. 1 0 0 .233 + 0. 01 0. 010 O l l i 0 .977 + 0. 005 0 .266 + 0. 004 0. 000 Halcy o n 0 .823 + 0. 06 0 .230 + 0. 01 0. 006 G e r b e l 1 .063 + 0. 1 1 0 .257 + 0. 006 0. 009 Var i e t y Root [K +]± SE P A B Fergus 41 .65 + 3.0 46.54 + 1 .8 0.25 He c t o r 34.57 + 2.6 44.30 + 2 . 2 0.40 Kombar 29. 58 + 1 .8 44.00 + 2. 1 0.04 O l l i 42.00 + 1 . 4 52. 1 0 + 1 .9 0.02 Halcy o n 53.20 + 1 .8 52.40 + 1 .0 0.44 G e r b e l 44.80 + 1 .9 58. 1 0 + 2.9 0.01 53 Table 14 Shoot [ K + ] ( u m o l . g f w t . " 1 ) f o r 4 s p r i n g b a r l e y v a r i e t i e s and 2 winter b a r l e y v a r i e t i e s . (A-values f o r 5°/l5°C root/shoot temperature, B-values f o r 15°/15°C root/shoot temperature) (n = 24-27) . 54 Va r i e t y Shoot [K +]± SE P A B Fergus 78. 30 ± 4.3 107.90 ± 1 . 9 0.009 Hector 85. 10 ± 3.7 116.40 ± 2 . 1 0.006 Kombar 74. 10 ± 10.2 127.60 ± 3.6 0.002 O l l i 91 . 90 ± 6.8 118.50 ± 3.4 0.014 Halcyon 95. 50 ± 5.0 117.70 ± 3.0 0. 1 00 Gerbel 83. 20 ± 6.5 109.50 ± 3.9 0.004 55 1.2 Temperature s e n s i t i v i t y of K + i n f l u x among v a r i e t i e s The most e x t e n s i v e l y used parameters t o determine the temperature i n h i b i t i o n of a p a r t i c u l a r p r o c e s s a r e , the tempe r a t u r e c o e f f i c i e n t Q 1 0 ( g i v e n as the a c t i v i t y a t (T+10)/T°C) and the a c t i v a t i o n energy, E . The l a t t e r i s m o s t l y used i n e v a l u a t i n g the r a t e s of enzyme c a t a l y z e d r e a c t i o n s a t d i f f e r e n t t e m p e r a t u r e s . Because of the suggested s i m i l a r i t y of c a r r i e r mediated i o n t r a n s p o r t t o enzyme c a t a l y s e d r e a c t i o n s , most a u t h o r s have used the above parameters i n e v a l u a t i n g low temperature i n h i b i t i o n of i o n t r a n s p o r t . T a b l e 15 p r o v i d e s the temperature c o e f f i c i e n t s c a l c u l a t e d u s i n g e q u a t i o n 1, showing a r a t e of a r e a c t i o n as a f u n c t i o n of temperature ( B e r r y and R a i s o n , l 9 8 l ) T l o g Q 1 0 l o g K = + C ( 1 ) 1 0 where, R = r a t e of r e a c t i o n ( i n f l u x i n t h i s i n s t a n c e ) T = a b s o l u t e temperature Q 1 0 = temperature c o e f f i c i e n t Q 1 0 was c a l c u l a t e d u s i n g the s l o p e s of l i n e a r r e g r e s s i o n o b t a i n e d by p l o t t i n g l o g of i n f l u x v e r s u s i n c u b a t i o n t e m p e r a t u r e s (5°,10°,15° and 25°C) f o r both LT and HT p l a n t s . The a c t i v a t i o n e n e r g i e s were c a l c u l a t e d u s i n g the Q 1 0 v a l u e s a c c o r d i n g t o e q u a t i o n 2. 56 Table 15 Temperature c o e f f i c i e n t ( Q 1 0 ) for i n f l u x of K + f o r 4 s p r i n g (1,2,3 and 4) and 2 winter (5,6) b a r l e y v a r i e t i e s . (The r 2 value obtained f o r the i n f l u x as a f u n c t i o n of temperature, i s given i n parentheses) Table 16 Temperature c o e f f i c i e n t s of a s p r i n g b a r l e y v a r i e t y (Kombar) and a winter b a r l e y v a r i e t y (Halcyon) grown under two temperature regimes. A c t u a l Q 1 0 values are given i n rows a and the Q 1 0 estimated a f t e r e l i m i n a t i n g the e f f e c t of low and h i g h root [ K + ] are given i n rows b and c. (The r 2 values f o r the i n f l u x as a f u n c t i o n of temperature are given i n p a r e n t h e s e s ) . 57 Va r i e t y Q 1 0 5°/15°C grown 15°/15°C grown 1 Fergus 1 . 7 8 ( 0 . 9 9 ) 1 . 7 4 ( 0 . 9 8 ) 2 Hector 1.91(0.91) 1 . 8 2 ( 0 . 8 7 ) 3 O l l i 2 . 2 0 ( 0 . 9 6 ) 2 . 2 4 ( 0 . 9 8 ) 4 Kombar 2 . 0 4 ( 0 . 9 9 ) 2 . 0 9 ( 0 . 9 4 ) 5 Halcyon 1 . 7 0 ( 0 . 9 2 ) 1 . 8 6 ( 0 . 9 5 ) 6 Gerbel 2. 1 0 ( 0 . 9 9 ) 1 . 9 5 ( 0 . 9 6 ) Var i e t y Root [ K + ] ± SE umol.g."1 5°/l5°C grown Qi o 15°/l5°C grown Kombar a 2 . 0 4 ( 0 . 99) 2 . 0 9 ( 0 . 9 4 ) 2 9 . 5 8 ± 1.8 b 1 . 9 5 3 . 09 5 3 . 2 0 ± 1.8 c 1.81 5 . 6 0 Halcyon a 1 . 7 0 ( 0 . 92) 1 . 8 6 ( 0 . 9 5 ) 2 9 . 5 8 ± 1.8 b 1 . 30 2 .51 5 3 . 2 0 ± 1.8 c 2 . 2 6 1 . 22 58 RT (T + 10) -E = In Q 1 0 (2) 3 10 where, R = 8.314 J.mol- 1K- 1 T = a b s o l u t e temperature The number of i n c u b a t i o n temperatures (4) in t h i s experiment was not adequate to o b t a i n the Arrhenius r e l a t i o n s h i p . If there had been more v a l u e s , a b e t t e r comparison of a c t i v a t i o n e n e r g i e s f o r a c c l i m a t e d and non-a c c l i m a t e d p l a n t s would have been obtained. The r e s u l t s of Table 15 r e v e a l s a Q 1 0 2 f o r a l l the v a r i e t i e s . However, an attempt to f i n d the d i r e c t e f f e c t of temperature on i n f l u x , (as d i s t i n c t from the combined e f f e c t of temperature and i n t e r n a l [ K + ] ) r e v e a l e d a d i f f e r e n t p i c t u r e . Here, the Q i 0 s were c a l c u l a t e d using i n f l u x values c o r r e c t e d f o r v a r i a t i o n s of i n t e r n a l K +. The i n f l u x values were estimated by using the a p p r o p r i a t e equations (Table 19) showing the r e l a t i o n s h i p s between i n f l u x and i n t e r n a l [K +] f o r given growth and i n c u b a t i o n temperatures. (The method by which these equations were obtained i s d i s c u s s e d i n the f o l l o w i n g c h a p t e r ) . The Q 1 0 values c a l c u l a t e d for known low (29.58 Mmol.gfwt." 1) and high (53.2 ymol.gfwt." 1) i n t e r n a l [ K + ] are given i n Table 16. The lower value r e p r e s e n t s that obtained f o r 6 r e p l i c a t e s of LT grown Rombar and the higher value was that obtained f o r LT Halcyon (6 r e p l i c a t e s ) . In Halcyon, temperature s e n s i t i v i t y appears to depend both on growth temperatures and on i n t e r n a l R J s t a t u s , while that of :Kombar seem to depend 59 mainly on growth temperature. At low i n t e r n a l [ K + ] , ( 30 Mmol.gfwt." 1 i n t h i s instance) LT Halcyon d i d not show a temperature s e n s i t i v i t y ( Q 1 0 1). I t must be s t r e s s e d that these f i g u r e s are p r e d i c t i o n s of i n f l u x at p a r t i c u l a r temperatures. At low temperature and moderate ambient K + l e v e l s , Kombar f a i l s to achieve high i n t e r n a l [ K + ] . I t was mentioned e a r l i e r ( i n t r o d u c t i o n ) that low temperature has a tendency to decrease the a v a i l a b i l i t y of n u t r i e n t s i n the s o i l s o l u t i o n . Under such c o n d i t i o n s a higher i n t e r n a l K + s t a t u s i s hard to a t t a i n . T h e r e f o r e , a lower temperature s e n s i t i v i t y i n K + i n f l u x at lower i n t e r n a l K + s t a t u s appears to be advantageous f o r Halcyon to t h r i v e i n such adverse c o n d i t i o n s . T h i s may be an inherent c a p a c i t y of Halcyon. I t w i l l be i n t e r e s t i n g t h e r e f o r e to see the v a l i d i t y of t h i s issue over many winter b a r l e y v a r i e t i e s , so that a g e n e r a l i z a t i o n c o u l d be made. It appears, at l e a s t in Halcyon, the r e g u l a t i o n of K + t r a n s p o r t , e s p e c i a l l y at low growth temperatures, occurs through an i n t e r a c t i o n of temperatures and i n t e r n a l K + s t a t u s r a t h e r than the i n t e r n a l [K*] per se. However, Kombar, which i s a v a r i e t y bred f o r warmer c o n d i t i o n s i n C a l i f o r n i a , seem to be h i g h l y temperature s e n s i t i v e at any growth temperature and at any i n t e r n a l K + s t a t u s (Table 1 7 ) . T h e r e f o r e , the aforementioned "temperature -i n t e r n a l [ K + ] " i n t e r a c t i v e r e g u l a t i o n of K + uptake does not seem to occur i n Kombar. Instead, the type of r e g u l a t i o n of K* uptake in Kombar may be s i m i l a r to that d e s c r i b e d by s e v e r a l authors (Glass, 1976; Pitman et a l . , 1968), at high .temperatures, but dim i n i s h e d at low temperatures. 60 The a c t i v a t i o n e n e r g i e s f o r K + uptake c a l c u l a t e d using the Q 1 0 values do not show a marked d i f f e r e n c e between low and high growth temperatures (Table 17). Some authors, however have rep o r t e d a higher a c t i v a t i o n energy f o r ion uptake at low temperature ( Carey and Berry,1978). T h e i r approach in t h i s issue i s q u e s t i o n a b l e i n a more r e a l i s t i c sense, s i n c e the p l a n t s were not exposed to low temperatures p r i o r to i n f l u x measurements. In f i e l d c o n d i t i o n s however, root system do not encounter such dramatic changes i n temperature on a short time s c a l e although shoots may experience such changes in rare i n s t a n c e s (snow storms, heat waves). N e v e r t h e l e s s , when the p l a n t s were allowed to a c c l i m a t e by long term exposure to a low temperature the a c t i v i t y of " t r a n s p o r t e r s " appears to be almost i d e n t i c a l to that of HT p l a n t s . T h i s , i n d i c a t e s that these v a r i e t i e s possess a c a p a c i t y to a c c l i m a t e K + uptake. N e v e r t h e l e s s , the extent of a c c l i m a t i o n seems to d i f f e r among v a r i e t i e s . Halcyon, when grown at low temperature showed a lower a c t i v a t i o n energy (35.35 kJ.mol." 1) f o r uptake than HT p l a n t s (41.31 k J . m o l . " 1 ) . I f t h i s d i f f e r e n c e i s s i g n i f i c a n t (which c o u l d not be t e s t e d i n t h i s s t udy), then " t r a n s p o r t e r s " in Halcyon may be present i n a r e a d i l y a c t i v a t e d s t a t e . More d e t a i l e d d i s c u s s i o n of the r e g u l a t i o n of K* i n f l u x w i l l be d e f e r r e d u n t i l s e c t i o n 2. 61 Table 17 A c t i v a t i o n e n e r g i e s f o r 4 s p r i n g b a r l e y v a r i e t i e s and 2 winter b a r l e y v a r i e t i e s grown at 5° and 15°C root temperatures (n = 24-27 62 Var i e t y A c t i v a t i o n Energy E 5°/l5° grown (k j . m o l . " 1 ) 15°/15° grown Fergus 38.41 36.87 Hector 43.07 39.88 Kombar 47.46 49.06 O l l i 52.46 53.66 Halcyon 35.35 41.34 Gerbel 49.39 44.47 63 1.3 D e t e r m i n a t i o n of a c c l i m a t i o n p o t e n t i a l A d a p t i v e changes t o c h i l l i n g and f r e e z i n g t e m p e r a t u r e s , both i n terms of w i n t e r s u r v i v a l and m e t a b o l i c e f f i c i e n c y are termed a c c l i m a t i o n , a c c l i m a t i z a t i o n or h a r d e n i n g ( L e v i t t , 1980). S i n c e a c c l i m a t i o n i s g e n e t i c a l l y c o n t r o l l e d (Chabot and B i l l i n g s , 1972), d i f f e r e n t s p e c i e s may have d i f f e r e n t a c c l i m a t i o n p o t e n t i a l s (Chapin, 1974). In s t u d y i n g HZPC^_ a b s o r p t i o n , Chapin (1974) deduced a parameter, " a c c l i m a t i o n p o t e n t i a l " , d e f i n e d as the r a t i o of the V m Q X of 5°C-acclimated r o o t s to the v m a x of 20°C-acclimated r o o t s , where f l u x e s were measured at a s t a n d a r d t e m p e r a t u r e . A h i g h a c c l i m a t e d p o t e n t i a l i s c h a r a c t e r i s t i c of a s i t u a t i o n where l a r g e compensatory changes i n the r a t e s of a b s o r p t i o n occur i n response t o changes i n the temperature at which the p l a n t i s growing ( C h a p i n , 1974). In the p r e s e n t s t u d y , i n s t e a d of maximum v e l o c i t y , V m Q , the r a t e of K + a b s o r p t i o n a t 15°C of 11 b a r l e y v a r i e t i e s grown a t 5°C and 15°C was used. However, i t s h o u l d be borne i n mind t h a t the i n t e r n a l K + s t a t u s v a r i e s among v a r i e t i e s , l e a d i n g t o v a r i a t i o n s i n f l u x e s independent of temperature (see S e c t i o n 1.1). Most of the v a r i e t i e s shown i n T a b l e 18 d i d not show a s i g n i f i c a n t d i f f e r e n c e i n t h e i r r o o t [ K 4 ] a t two growth t e m p e r a t u r e s (Table 14). Hence, the a c c l i m a t i o n p o t e n t i a l s o b t a i n e d f o r these v a r i e t i e s ( F e r g u s , H e c t o r , O ' l l i and Halcyon) can be c o n s i d e r e d as due to the e f f e c t s of growth t e m p e r a t u r e . However, when s i g n i f i c a n t d i f f e r e n c e i n r o o t [ K + ] was shown (as f o r Kombar, Table 14), then .estimated i n f l u x f o r known i n t e r n a l [K*] ( d e t a i l s g i v e n i n the f o l l o w i n g c h a p t e r ) was used t c determine the AP ( i n 64 parentheses i n Table 18). A f t e r c o r r e c t i n g the i n f l u x f o r i n t e r n a l [ K + ] , the AP value estimated f o r Kombar (1.09) was much lower than the observed value. Hence, the AP values c a l c u l a t e d using the i n f l u x e s (other than V ) do not ^ max n e c e s s a r i l y represent the a c t u a l a c c l i m a t i o n p o t e n t i a l except f o r those v a r i e t i e s whose root K + contents are not s i g n i f i c a n t l y d i f f e r e n t when grown at low and at high temperatures. Higher AP values were observed i n Fergus, Halcyon and Ge r b e l . T h i s i n d i c a t e s that Fergus may have a ge n e t i c p o t e n t i a l f o r a c c l i m a t i o n of K + f l u x e s s i m i l a r to that of the winter v a r i e t i e s (Halcyon and G e r b e l ) . The low AP of Bonanza and Himalaya i n d i c a t e s t h e i r i n a b i l i t y to compensate f o r the low temperature c o n d i t i o n s . Although AP valu e s are s l i g h t l y g r e a t e r , the r e s t of the v a r i e t i e s a l s o cannot be c o n s i d e r e d as having a g r e a t e r c a p a c i t y to a c c l i m a t e . Chapin (1974) repo r t e d that s p e c i e s i n h a b i t i n g more f l u c t u a t i n g environments show AP values as l a r g e as 11.5 (Eleocar i s p a l u s t r i s ) and 18.29 (S c i r p u s m i c r o c a r p u s ) . 65 Table 18 A c c l i m a t i o n p o t e n t i a l s f o r 8 s p r i n g ( s ) , 2 winter(w), and 1 summer(su) b a r l e y v a r i e t i e s . The valu e s were compared with those obtained from another experiment using Spearman's rank c o r r e l a t i o n f o r non-parametric data. (RHO = 0.744, p < 0.025).. 66 Var i e t y A c c l i m a t i o n P o t e n t i a l Fergus(s) 8.90 He c t o r ( s ) 5.57 Kombar(su) 10.43(1.09) O l l i ( s ) 9.96 Bonanza(s) 0.54 E x c e l s i o r ( s ) 1 .05 Himalaya(s) 0.83 L i o n ( s ) 1 .33 Odessa(s) 1.14 Halcyon(w) 9.07 Gerbel(w) 8.71 67 2. R e g u l a t i o n of K + i n f l u x 2.1 E f f e c t of growth temperatures A strong negative c o r r e l a t i o n between K + i n f l u x and i n t e r n a l K* ' s t a t u s was observed i n Halcyon at both growth temperatures (5°C and 15°C), while Kombar responded in t h i s manner only when grown at high temperatures. When grown at 15°C both v a r i e t i e s responded i n an i d e n t i c a l manner ( F i g . 4 and 6). In c o n t r a s t , at the low growth temperature (5°C) d i s t i n c t d i f f e r e n c e s i n response to i n t e r n a l K + were d i s c e r n e d ( F i g . 5 and 7). The HT p l a n t s (both v a r i e t i e s ) showed a s l i g h t l y g r e a t e r i n f l u x (when measured at 15°C) at any given i n t e r n a l [ K + ] ( p l o t a of F i g . 4 and 6) than the L T , p l a n t s (when measured at 5°C; p l o t b of F i g . 5 and 7). T h i s confirms the f i n d i n g s of S i d d i q i et a l . (1983). Using another b a r l e y v a r i e t y (Fergus), they have r e p o r t e d that K + i n f l u x i n HT p l a n t s ( i n f l u x measured at 20°C) was s l i g h t l y higher than i n f l u x in low temperature-grown p l a n t s ( i n f l u x measured at 10°C). The d i f f e r e n c e s between the i n f l u x (at 5°C and 15°C) measured under steady s t a t e c o n d i t i o n s r e p r e s e n t s the s h o r t f a l l of complete a c c l i m a t i o n ; i . e . the l a r g e r the d i f f e r e n c e s i n f l u x e s , the g r e a t e r the s h o r t f a l l . In Halcyon, the d i f f e r e n c e i n f l u x appear to be n e g l i g i b l e ( F i g . 4a and 5b). However, a c o n s i d e r a b l e d i f f e r e n c e i n i n f l u x under such c o n d i t i o n s was shown by Kombar ( F i g . 6a and 7b). 68 F i g . 4 Regula t i o n of K + i n f l u x by root K* c o n c e n t r a t i o n i n a winter b a r l e y v a r i e t y (Halcyon) grown at 15°C root - 15°C shoot temperature (HT) . I n f l u x measured at 15°C ((D) and at 5°C ( A ) . The l i n e s were f i t t e d a c c o r d i n g to the best r e g r e s s i o n obtained for i n f l u x upon [K] . The r e l a t i o n s h i p obtained i s given in the equations a and b. F i g . 5 . Reg u l a t i o n of K + i n f l u x by root K + c o n c e n t r a t i o n i n a winter b a r l e y v a r i e t y (Halcyon) grown at 5°C root - 15°C shoot temperature (LT) . I n f l u x measured at 15°C ( 0 ) and at 5°C ( A ) . Curve f i t t i n g i s same as i n F i g . 4. 69 FIGURE 4 ROOT [K+] ^imol.gfwt " l) 70 F i g . 6 Reg u l a t i o n of K + i n f l u x by root K + c o n c e n t r a t i o n i n a s p r i n g b a r l e y v a r i e t y (Kombar) grown at 15°C root - 15°C shoot temperature (HT). Symbols i n d i c a t e the s i m i l a r s t a t e as before and curve f i t t i n g i s a l s o as b e f o r e . F i g . 7 Regulat i o n of K + i n f l u x by root K + c o n c e n t r a t i o n i n a s p r i n g b a r l e y v a r i e t y (Kombar) grown at 5°C root - 15°C shoot temperature (LT). Symbols i n d i c a t e the s i m i l a r s t a t e as i n F i g . 4 and 5. Curve f i t t i n g i s same as b e f o r e . 71 FIGURE 6 4. 5 r 0-. Y .119 - 88 48.x (r\ 0-89) X • b: Y -2 33 . • 968.19. X (r-o-89) 1 4-) * 3. 0 cn • • E 3-X ID | 1. 5 • \ . U. + n. n-^—— A i 15 20 25 30 35 40 45 ROOT [K*] yjmol. gf wt -1) FIGURE 7 25 35 " 45 ROOT [K*] y j m o l . g f . t ~1> i 55 72 2.2 E f f e c t of i n c u b a t i o n ( i n f l u x ) temperature When grown at low temperatures, Halcyon demonstrated a s i m i l a r response to i n t e r n a l K + r e g a r d l e s s of the temperatures (5°C and 15°C) at which i n f l u x was measured ( F i g . 5). I n f l u x at both temperatures was s t r o n g l y i n f l u e n c e d by i n t e r n a l K +. That i s , at l e a s t i n the short term, f o r Halcyon i t appears that the r e g u l a t o r y e f f e c t of i n t e r n a l K + i s e s s e n t i a l l y independent of temperature. A measure of the responses to i n t e r n a l K + i s given by the slope of the r e g r e s s i o n of i n f l u x on i n t e r n a l [ K + ] . For Halcyon, the s l o p e s of LT grown p l a n t s are almost i d e n t i c a l to those of HT grown p l a n t s (1.45 and 1.53 f o r i n f l u x measured at 15°C, and 1.63 and 1.65 f o r i n f l u x measured at 15°C i n F i g . 4 and 5). Such responses were not observed i n Kombar ( F i g . 7). I n f l u x at 5°C i n LT grown Kombar d i d not appear to e x h i b i t a strong negative c o r r e l a t i o n (slope=0.35) with i n t e r n a l K + s t a t u s . However, a s l i g h t l y g r e a t e r negative c o r r e l a t i o n (slope=0.48) between i n f l u x and i n t e r n a l [ K + ] was shown by LT Kombar when i n f l u x was measured at 15°C. T h e r e f o r e , under such c o n d i t i o n s , ( e s p e c i a l l y LT steady s t a t e ) , r e g u l a t i o n of K + t r a n s p o r t appears to be independent of the i n t e r n a l K + s t a t u s . A s i m i l a r s i t u a t i o n to the low temperature e f f e c t s on feedback c o n t r o l was r e p o r t e d to occur i n high e x t e r n a l c o n c e n t r a t i o n s (Glass and Dunlop, 1978). According to these authors, when K + i n f l u x i s measured at high e x t e r n a l c o n c e n t r a t i o n s (>10 mM), the d i f f e r e n c e s between low-K + and high-K + p l a n t s appears to decrease with i n c r e a s i n g e x t e r n a l K + i n b a r l e y and r y e g r a s s . 73 However, the responses shown by HT Kombar ( F i g . 6) at both i n c u b a t i o n temperatures were s i m i l a r to those of other HT p l a n t s ; i . e . the negative feedback c o n t r o l operates i n Kombar only when grown at high temperatures. G l a s s (1976) proposed an a l l o s t e r i c r e g u l a t i o n of K + i n f l u x whereby K* i n f l u x was c o n t r o l l e d by i n t e r n a l [ K + ] . Systems having a l l o s t e r i c r e g u l a t i o n should e x h i b i t almost instantaneous responses to exogenous f a c t o r s such as c o n c e n t r a t i o n and temperature. T h i s c o n t r a s t s with the time r e q u i r e d f o r responses based upon t r a n s c r i p t i o n and t r a n s l a t i o n a l events. Cohen (1968) reported " c o l d " as one of many f a c t o r s (high i o n i c s t r e n g t h , changes i n pH etc.) which d e s e n s i t i z e a l l o s t e r i c s i t e s hence "uncoupling" the r e g u l a t i o n . However, LT or HT Kombar d i d not show any instantaneous changes of the responses at d i f f e r e n t i n c u b a t i o n temperatures (5°C and 15°C). T h e r e f o r e , i n Kombar, growth temperature r a t h e r than the i n c u b a t i o n temperature appears to be determining the response (or lac k of response) to i n t e r n a l K +. Hence the e f f e c t of temperature on a l l o s t e r i c r e g u l a t i o n d e s c r i b e d by Cohen (1968) does not appear to be a p p l i c a b l e to the r e g u l a t o r y system of K + uptake, at l e a s t i n Kombar. 2.3 C o r r e l a t i o n of i n f l u x e s f o r the i n t e r n a l K + s t a t u s As d e s c r i b e d i n the methods (Chapt. II Sec. 4), p l a n t s which were maintained at low e x t e r n a l [ K + ] were subsequently provided with an adequate amount of e x t e r n a l K + 74 (6 mM i n the growth medium) fo r the purpose of i n c r e a s i n g i n t e r n a l K + l e v e l s . At known time i n t e r v a l s i n f l u x e s and t i s s u e K* content was obtained as d e s c r i b e d i n Chapt. II Sec. 2. I n f l u x was p l o t t e d a g a i n s t the corresponding i n t e r n a l [ K + ] . The best f i t s f o r r e g r e s s i o n of i n f l u x on i n t e r n a l [K] were given by power r e g r e s s i o n s . The r e l a t i o n s h i p s shown are i l l u s t r a t e d i n F i g u r e s 4, 5, 6 and 7. The equations obtained f o r d i f f e r e n t growth and uptake temperatures are given i n Table 19. These equations were used to estimate the i n f l u x (Y) at known i n t e r n a l K + l e v e l s (X) at a p p r o p r i a t e growth temperatures and uptake temperatures (see Chapt.III Sec. 1.2). 75 Table 19 •Equations d e r i v e d from the r e g r e s s i o n of i n f l u x at two d i f f e r e n t temperatures on i n t e r n a l [ K + ] (nmol.gfwt." 1) of two b a r l e y v a r i e t i e s grown at two temperatures. 76 V a r i e t y Growth Uptake Regression % r Temp.°C Temp.°C equations Halcyon 1 5 1 5 y = 211.761 x" 1 a 5 0.97 5 y=l72.730 X " 1 6 3 0.84 5 1 5 y=231.650 X " 1 5 3 0.93 5 y=286.900 X " 1 e 5 0.99 Kombar 1 5 1 5 y= 88.480 X " 1 1 9 0.89 5 y=968.190 x- 2 2 3 0.89 5 1 5 y= 9.871 x-° 4 8 0.95 5 y=286.900 x-° 3 5 0.71 77 3. Growth and t i s s u e K + content of 3 ba r l e y v a r i e t i e s in r e l a t i o n to temperature and e x t e r n a l [ K + ] Many authors have emphasized the in c r e a s e i n root/shoot weight r a t i o which occurs when roo t s are su b j e c t e d to low temperatures (see Berry and Raison, 1981). Compensating changes i n the root/shoot weight r a t i o are a l s o observed when n u t r i e n t a v a i l a b i l i t y i s a l t e r e d at a constant temperature (Davidson, 1969; Thornley, 1977; Chapin, 1980). In the present study two e x t e r n a l K + l e v e l s (0.005 mM, "low s a l t " and 0.1 mM, " h i g h - s a l t " ) and two temperatures (8° and 18°C) were used (shoots were maintained at 18°C) to determine the e f f e c t s of such c o n d i t i o n s on p a r t i t i o n i n g of K + between root and shoot. 3.1 E f f e c t of temperature at low K + l e v e l s Two s p r i n g b a r l e y s (Hector, Bonanza) and one winter b a r l e y (Halcyon) were the s u b j e c t s f o r i n v e s t i g a t i o n . P l a n t s were maintained at 0.005 mM K +. Although the e x t e r n a l K + supply was at l i m i t i n g l e v e l s , p l a n t s showed a b e t t e r growth response at 18°C than at 8°C ( F i g . 8, 10, 12a and b ) . Q 1 0 values c a l c u l a t e d f o r growth were 1.7, 1.9 and 1.7 f o r Halcyon, Hector and Bonanza r e s p e c t i v e l y . The r e l a t i v e growth r a t e of p l a n t s (RGR), c a l c u l a t e d u sing equation 3, v a r i e d among v a r i e t i e s and between temperatures (Table 20). 78 lnw 2 _ lnw, RGR = , (3) T 2 - T, where, w, and w2 = f r e s h weight of p l a n t at T, (time) and T 2 . HT p l a n t s g e n e r a l l y showed a decrease in RGR with time, while LT e x h i b i t e d e i t h e r an i n c r e a s i n g or a constant RGR. In g e n e r a l , a low root temperature reduced the growth of the p l a n t as a whole in a l l 3 v a r i e t i e s . When the p l a n t K* s t a t u s i s c o n s i d e r e d , a l l v a r i e t i e s demonstrated the same response ( F i g . 9, 11, 13a and b), namely no s i g n i f i c a n t d i f f e r e n c e i n p l a n t K* in response to v a r i a t i o n in growth temperature u n t i l day 8. Beyond t h i s time, a r a p i d i n c r e a s e of K + was observed, from day 8-14 and t h i s was more conspicuous in HT p l a n t s . T h e r e f o r e , although the growth rate of. HT p l a n t s was higher than that of LT p l a n t s , there were only small d i f f e r e n c e s i n the K + s t a t u s of the two groups of p l a n t s . As mentioned e a r l i e r , i f the low root temperature tends to i n c r e a s e the root/shoot r a t i o , a b e t t e r understanding on p a r t i t i o n i n g the resources c o u l d be obtained by c o n s i d e r i n g the growth r a t e s and K* l e v e l s of shoots and r o o t s s e p a r a t e l y . Shoot growth r a t e s of these 3 v a r i e t i e s f o l l o w e d a p a t t e r n which was s i m i l a r to whole p l a n t growth r a t e s ( F i g . 14, 16, 18a and b ) . The RGR of shoots v a r i e d among v a r i e t i e s and a l s o between temperatures (Table 21). The p a t t e r n shown for shoot K + was s i m i l a r to that f o r p l a n t K + ( F i g . 8, 10, 12a and 12b). The root growth r a t e s of Halcyon and Bonanza were e s s e n t i a l l y i d e n t i c a l u n t i l the e i g h t h day 79 beyond which some d i s s i m i l a r i t i e s i n growth ra t e s were apparent ( F i g . 20, 24a and b ) . Roots of LT Hector showed no i n c r e a s e of root biomass f o r the p e r i o d o"f 14 days (Table 22). In f a c t , a n o t i c e a b l e decrease of biomass was observed. Root K + contents observed f o r LT p l a n t s was e i t h e r s i m i l a r (Halcyon) or much great e r (Hector or Bonanza) than - those - of HT p l a n t s . G e n e r a l l y , 'the' r a t e s of shoot and root growth appeared to be reduced when the p l a n t s were sub j e c t e d to low temperature and lo.w'nutrient', l e v e l s . Such c o n d i t i o n s a l s o seem to reduce the shoot K + s t a t u s while the same c o n d i t i o n s appeared to i n c r e a s e root K + l e v e l . Thus, in attempting to r a t i o n a l i z e root K + s t a t u s three f a c t o r s should be c o n s i d e r e d . These are, root growth r a t e s ( M ) , uptake r a t e s (v) and r a t e of t r a n s l o c a t i o n ( T ) . If u +T > v, then a r e d u c t i o n i n root K + w i l l r e s u l t . I f u+T < v, then the re v e r s e ( i n c r e a s e d root K +) w i l l f o l l o w . Based on these assumptions root K + l e v e l s i n LT roots can be e x p l a i n e d . Low temperature and low n u t r i e n t s t a t u s appeared to decrease whole p l a n t and shoot growth r a t e s to a g r e a t e r extent than that of r o o t . T h e r e f o r e , i f the c o n t r i b u t i o n of u i s assumed to be n e g l i g i b l e , then a comparison of T and v should be made. Under LT and low n u t r i e n t c o n d i t i o n s , i f T < v then an i n c r e a s e of root K* i s a n t i c i p a t e d . T h i s seemed to be the case f o r a l l 3 v a r i e t i e s under the above mentioned c o n d i t i o n s . 80 F i g u r e s 8 and 9 Mean p l a n t weight ( F i g . 8) and p l a n t K + ( F i g . 9) f o r Halcyon grown at two temperatures and two [ K + ] 0 . • — 0 0.1 mM and 8°C A • A 0.1 mM and 18°C 0 . o 0.005 mM and 8°C ^ % 0.005 mM and 18°C FIGURE 9 120 r AGE (days) 82 F i g u r e s 10 and 11 Mean pl a n t weight ( F i g . 10) and pl a n t K + ( F i g . 11) f o r Hector grown at two temperatures and two [ K + ] 0 . A — A 0.1 mM and 18°C O — O 0.005 mM and 8°C ' * 0.005 mM and 18°C 0. 00 12 15 AGE (days) 84 F i g u r e s 12 and 13 Mean pl a n t weight ( F i g . 12) and p l a n t K + ( F i g . 13) f o r Bonanza grown at two temperatures and one [ K + ] 0 . © — O 0.005 mM and 8°C — * 0.005 mM and 18°C FIGURE 12 .25 -b .20 CD * . 15 a /-\ PLANT . lOfl .05 0. 00 i 1 \ ) 3 6 9 AGE (days) 12 15 TIGURE 13 70 r AGE (days) 86 F i g u r e s 14 and 15 Mean shoot weight ( F i g . 14) and shoot K + ( F i g . 15) f o r Halcyon grown at two temperatures and two [ K + ] 0 . Q — Q 0.1 mM and 8°C A A 0.1 mM and 18°C O — O 0.005 mM and 8°C — * • 0.005 mM and 18°C 87 FIGURE 14 . 25 r AGE (days) FIGURE 15 AGE (days) 88 F i g u r e s 16 and 17 Mean shoot weight ( F i g . 16) and shoot K * ( F i g . 17) f o r Hector grown at two temperatures and two [ K + ] 0 . A — A 0.1 mM and 18°C O 0.005 mM and 8°C * — * 0.005 mM and 18°C 89 FIGURE 16 . 2 5 r AGE (g) FIGURE 17 160 r AGE (days) 90 F i g u r e s 18 and 19 Mean shoot weight ( F i g . 18) and shoot K + ( F i g . 19) f o r Bonanza grown at two temperatures and one [ K + ] 0 . O — O 0.005 mM and 8°C * — * 0.005 mM and 18°C 91 FIGURE 18 . 150 r . 125 I AGE (days) FIGURE 19 100 r AGE (days) 92 F i g u r e s 20 and 21 Mean root weight ( F i g . 20) and root K + ( F i g . 21) f o r Halcyon grown at two temperatures and two [ K + ] 0 . • — 0 0.1 mM and 8°C A — A 0.1 mM and 18°C O — O 0.005 mM and 8°C * — * 0.005 mM and 18°C 93 FIGURE 20 FIGURE 21 i * L i -en o E o o or 94 F i g u r e s Mean root weight ( F i g . 22) and grown at two temperatures and A — A 0 — O 22 and 23 root K + ( F i g . 23) f o r Hector two [ K + ] 0 . 0.1 mM and 18°C 0.005 mM and 8°C 0.005 mM and 18°C 95 FIGURE 22 . 15 r b cn . 10 - d 1— - . /^ j a ROOT .05; o. nn t? — -e • i 0 3 6 9 AGE (days) 12 15 FIGURE 23 60 r n 3 6 9 12 15 AGE (days) 96 F i g u r e s 24 and 25 Mean root weight ( F i g . 24) and root K + ( F i g . 25) f o r Bonanza grown at two temperatures and one [ K + ] 0 . Q — O 0.005 mM and 8°C * " ~ * 0.005 mM and 18°C 97 FIGURE 24 125 0. 000 AGE (days) FIGURE 25 4 J * Ci-"o E + O O oc 98 Table 20 R e l a t i v e growth rate of plants. ( g . g _ 1 d " 1 ) of 3 b a r l e y v a r i t i e s grown under two temperatures (8 and 18°C) and two n u t r i e n t (K*) c o n c e n t r a t i o n s (0.1 mM and 0.005 mM). (- v a r i e t y not used i n t h i s p a r t of the experiment).(n = 16). 99 RGR(PLANT) (g.g-'d- 1) Va r i e t y Age(days) [ K ] 0 0.005 mM [ K ] 0 0 . 1 mM 8°C 18°C 8°C 18°C Halcyon 4 0.026 0.103 0.015 0.130 8 0.043 0.050 0.017 0. 1 93 1 4 0. 054 0.040 0.084 0.029 Hector 4 .0.010 0.163 - 0. 1 68 8 0.010 0.012 - 0. 1 55 1 4 0.000 0.051 - 0.002 Bonanza 4 0. 055 0.001 - -8 0.683 0.001 - -14 0.000 0.002 - -100 Table 21 R e l a t i v e growth rate of shoots ( g . g ^ d - 1 ) of 3 b a r l e y v a r i e t i e s . Growth c o n d i t i o n s are s i m i l a r to those described, i n Table 20 (n = 16). 101 RGR(SHOOT) (g.g- 1d~ 1) V a r i e t y Age(days) [ K ] 0 0.005 mM [ K ] 0 0 . 1 mM 8°C 18°C 8°C 18°C Halcyon 4 0.021 0.173 0.037 0.367 8 0.085 0. 045 0.040 0. 1 90 1 4 0.072 0.050 0.211' 0.040 Hector 4 0.025 • 0.253 - 0. 1 73 8 0.023 0.023 - 0.463 1 4 0.024 0.046 - 0.390 Bonanza 4 0.039 0.001 - -8 0.486 0.095 - -1 4 0.000 0.024 - -102 T a b l e 22 R e l a t i v e growth r a t e of r o o t s (g.g-'d" 1) of 3 b a r l e y v a r i e t i e s . Growth c o n d i t i o n s are s i m i l a r t o those d e s c r i b e d i n T a b l e 20 (n=16). 103 RGR(ROOT) (g.g- 1d- 1) V a r i e t y Age(days) [ K ] 0 0.005mM [ K ] 0 0. 1 mM 8°C 1 8°C 8°C 18°C Halcyon 4 0.004 0.070 0.020 0.010 8 0.042 0.050 0.023 0.250 1 4 0.018 0. 1 50 0.051 0.011 Hector 4 0.000 0.120 - 0.057 8 0.000 0.062 - 0.128 1 4 0.000 0.110 - 0.001 Bonanza 4 0.001 0.001 - -8 0. 1 97 0.010 - -1 4 0.001 0.004 - -104 3.2 E f f e c t of K + supply at two d i f f e r e n t growth temperatures Changes i n n u t r i e n t supply (K +) e i t h e r at HT or LT c o n d i t i o n s were c o n s i d e r e d s e p a r a t e l y . 3.2a E f f e c t of d i f f e r e n t K* supply at high root temperature (18°C) Re p r e s e n t a t i v e s p r i n g and winter v a r i e t i e s were compared. Both " h i g h - s a l t " and " l o w - s a l t " p l a n t s appeared to have high growth r a t e s d u r i n g the f i r s t few days (4) as shown in F i g u r e s 8 and 10 ( p l o t s b and d) and i n RGR Table 1. Pla n t K + s t a t u s i n both v a r i e t i e s at " h i g h - s a l t " l e v e l showed a r a p i d i n c r e a s e d u r i n g the i n i t i a l 8 days beyond which no in c r e a s e (or decrease) was shown. By c o n t r a s t , no in c r e a s e of K* l e v e l s were observed f o r e i t h e r v a r i e t i e s d u r i n g the i n i t i a l 8 days in " l o w - s a l t " p l a n t s . Beyond 8 days, a dramatic i n c r e a s e of p l a n t K + l e v e l s were d i s c e r n e d . During the i n i t i a l days, the demand f o r n u t r i e n t s seem to be met only in "high-s a l t " p l a n t s , r e s u l t i n g i n a simultaneous i n c r e a s e of growth and t i s s u e K + l e v e l . However, i n " l o w - s a l t " p l a n t s d u r i n g the i n i t i a l r a p i d l y growing stage, the demand f o r n u t r i e n t s may not be s a t i s f i e d , r e s u l t i n g i n a low K + s t a t u s i n a l l the t i s s u e s ( p l o t b i n F i g . 9, 11, 15, 17, 21 and 23). The i n c r e a s e of K + l e v e l beyond 8 days may be achieved by decreased growth r a t e . T h i s p a t t e r n was e x h i b i t e d i n both v a r i e t i e s . T h e r e f o r e , i n HT c o n d i t i o n s both s p r i n g and winter v a r i e t i e s appeared to respond in a s i m i l a r manner. 105 3.2b E f f e c t of d i f f e r e n t K + supply under low root temperature (8°C) U n f o r t u n a t e l y , data fo r t h i s comparison i s a v a i l a b l e only f o r the v a r i e t y , Halcyon. A s i g n i f i c a n t d i f f e r e n c e in growth r a t e s of whole p l a n t s , shoots and roots was not observed between " l o w - s a l t " and " h i g h - s a l t " c o n d i t i o n s . However, the t i s s u e K + l e v e l s of " h i g h - s a l t " p l a n t s appeared to be c o n s i d e r a b l y g r e a t e r than that of " l o w - s a l t " p l a n t s . T h i s i n d i c a t e s t h a t , i r r e s p e c t i v e of ambient temperature, Halcyon i s able to maintain a high t i s s u e K + l e v e l i f the e x t e r n a l supply i s adequate. In g e n e r a l , a l l 3 v a r i e t i e s were unable to achieve a high K + s t a t u s when simultaneously s u b j e c t e d to low temperature and low n u t r i e n t c o n d i t i o n s . At HT and high n u t r i e n t supply, a l l 3 v a r i e t i e s showed a s i m i l a r response i n growth and t i s s u e K + s t a t u s . Under HT and low n u t r i e n t c o n d i t i o n s , the responses were again s i m i l a r i n both winter and s p r i n g v a r i e t i e s . U n f o r t u n a t e l y , data f o r a s p r i n g v a r i e t y was l a c k i n g to compare the responses at low temperature and high n u t r i e n t s t a t u s . In f a c t , a more favo u r a b l e response by winter v a r i e t i e s under the l a t t e r mentioned c o n d i t i o n s c o u l d be a n t i c i p a t e d . T h e r e f o r e , f u r t h e r s t u d i e s are r e q u i r e d to c l a r i f y t h i s p o i n t . In g e n e r a l , d i f f e r e n c e s in temperature appeared to a f f e c t growth r a t e s of whole p l a n t s or t h e i r p a r t s to a g r e a t e r extent than d i d the d i f f e r e n c e s in e x t e r n a l K+- supply. 106 However, i f the n u t r i e n t s were f l u c t u a t i n g at a constant temperature p l a n t s d i d not appear to have the c a p a c i t y to maintain s i m i l a r t i s s u e l e v e l s . For example, a l l three v a r i e t i e s were unable to compensate f o r the l i m i t e d n u t r i e n t supply. However, as long as e x t e r n a l K + supply i s not l i m i t i n g , a l l 3 v a r i e t i e s appeared to have the c a p a c i t y to maintain a c e r t a i n K + l e v e l , although v a r i a t i o n s i n magnitude may e x i s t among v a r i e t i e s . In r o o t s , maintenance of a higher K + l e v e l was always a s s o c i a t e d with lowering of root growth r a t e . T h i s can be well e x p l a i n e d a c c o r d i n g to the suggestions of Szaniawski (1983). He r e p o r t e d that more energy i s a l l o c a t e d f o r maintenance than for growth at low temperatures. Hence i n s t e a d of more growth, roots may be p a r t i t i o n i n g more energy to maintain t h e i r normal f u n c t i o n . Increased r e s p i r a t o r y r a t e s i n c o l d a c c l i m a t e d roots (Carey and Berry, 1978) i n d i c a t e i n c r e a s e d energy l i b e r a t i o n . Part of t h i s energy t h e r e f o r e , may be used for a l t e r a t i o n i n s t r u c t u r e of membranes (see L e v i t t , 1980) r e s u l t i n g i n an i n c r e a s e d number of t r a n s p o r t s i t e s (Clarkson and Deane-Drummond, 1981). T h i s may compensate for reduced a b s o r p t i o n area of r o o t s . Lowering of shoot growth r a t e a l s o lowers the t i s s u e K + l e v e l . In a l l 3 v a r i e t i e s , at l e a s t i n shoots, growth rate determines the demand and i n r o o t s , growth rate determines the supply. For example, when the root growth rate was d i m i n i s h e d by low temperature and when the e x t e r n a l K + a v a i l a b i l i t y i s l i m i t e d , supply to the shoot a l s o was d i m i n i s h e d , r e s u l t i n g i n a higher K + l e v e l i n the r o o t s . E r d e i et a l . (1983) a l s o r e p o r t e d an 107 accumulation of K + i n r o o t s by winter wheat by the end of the w i n t e r . The complete a c c l i m a t i o n f o r low temperature c o u l d t h e r e f o r e be achieved, only i f the e x t e r n a l K + supply i s not l i m i t e d . The importance of K + f e r t i l i z a t i o n to i n c r e a s e the c o l d t o l e r a n c e i s t h e r e f o r e , obvious. 108 4. Temperature-low n u t r i e n t i n t e r a c t i o n s on i n f l u x and t i s s u e [ K + ] One winter and two s p r i n g b a r l e y v a r i e t i e s were used in t h i s study, which sought to i n v e s t i g a t e the i n f l u e n c e of ambient [ K + ] on a c c l i m a t i o n . The n u t r i e n t s o l u t i o n which was normally maintained at 0.1 mM K + was reduced in t h i s experiment to 0.005 mM. Two temperature regimes were employed, i . e . p l a n t s were maintained at 8°C (root and shoot), LT, or 18°C(root and shoot), HT, and i n f l u x measurements were obtained at 0.1 mM K +. T h i s study r e v e a l e d a s i m i l a r t r e n d in i n f l u x and i n t i s s u e [ K + ] f o r a l l 3 v a r i e t i e s (Tahles 23, 24, and 25). P l a n t s exposed to low temperature c o n d i t i o n s showed s i g n i f i c a n t l y lower f l u x e s than HT p l a n t s . However, root [ K + ] in LT (8°C) v a r i e t i e s was s i g n i f i c a n t l y g r e a t e r than that of HT (18°C) v a r i e t i e s . The shoot demand appeared to d i f f e r at d i f f e r e n t shoot temperatures. A l l 3 v a r i e t i e s showed a g r e a t e r root/shoot weight r a t i o when grown at low temperature (Table 26). T h i s i s in accordance with the f i n d i n g s of other authors (Davidson, 1969; Chapin, 1980). Q 1 0 v a l u e s c a l c u l a t e d only by using i n f l u x at 8° and 18°C are given i n Table 27. Although the absolute values d i f f e r only s l i g h t l y , the trend shown by Q 1 0 agrees with the Q 1 0 c a l c u l a t e d using estimated f l u x f o r known [ K + ] (Table 17). In Halcyon, a higher i n t e r n a l K + s t a t u s i n LT p l a n t s and a lower K + s t a t u s i n HT p l a n t s always r e s u l t in a g r e a t e r temperature s e n s i t i v i t y . However, the temperature s e n s i t i v i t y shown by LT p l a n t s was 50% l e s s than that by HT p l a n t s . The other two v a r i e t i e s showed a 109 s l i g h t l y d i f f e r e n t response. The s e n s i t i v i t y i n these two v a r i e t i e s appears to be independent of growth temperature and i n t e r n a l K + s t a t u s ; i . e . at any temperature and any [K] the s e n s i t i v i t y i s the same. T h i s agrees w e l l with the f i n d i n g s for Kombar. T h i s i n d i c a t e s the inherent i n a b i l i t y of Hector and Bonanza to cope with the f l u c t u a t i o n s i n environmental temperatures, e s p e c i a l l y under low n u t r i e n t c o n d i t i o n s . 110 Table 23 K + i n f l u x (/imol. gf wt. " 1 h. " 1 ) of 3 b a r l e y v a r i e t i e s (Halcyon, Hector and Bonanza) at 8°C f o r 8°C grown p l a n t s (A), and at 18°C f o r 18°C grown p l a n t s (B). K + of the growth medium was' 0.005 mM. P r o b a b i l i t y (p) i s based on Student's t - t e s t . Table 24 Root [ K + ] (Mmol.gfwt" 1) of 3 b a r l e y v a r i e t i e s grown at 8°C (A) and 18°C (B). P r o b a b i l i t y (p) i s based on Student's t - t e s t . Table 25 Shoot [ K + ] (Mmol.gfwt" 1) of 3 b a r l e y v a r i e t i e s grown at 8°C (A) and 18°C (B). P r o b a b i l i t y ( p ) i s based on Student's t - t e s t . I l l Var i e t y I n f l u x ± SE Le v e l of • A B s i g n i f i c a n c e Halcyon 1.379 + 0.03 4 .179 ± 0 .68 « 0 . 0 1 Hector 1.955 ± 0.05 3. 528 ± 0. 1 4- « 0 . 0 0 5 Bonanza 1.215 ± 0.12 1 .371 ± 0 .22 NS V a r i e t y RootfK + ]± SE Le v e l of A B s i g n i f i c a n c e Halcyon 44. 21 ± 3.2 30.49 ± 2. 9 0.005 Hector 45. 19 ± 3.8 25,. 52 ± 2. 2 <<0.005 Bonanza 41 . 04 ± 3.7 26.21 ± 2. 4 « 0 . 0 5 Var i e t y S h o ot[K +] L e v e l of A B s i g n i f icance Halcyon 84. 39 ± 4. 0 1 28 . 33 ± 1 . 5 <<0.0001 Hector 84. 63 ± 1 . 6 124.54 ± 3. 5 <<0.0001 Bonanza 79. 67 + 6. 0 126.89 ± 3. 6 <<0.0001 Table 26 Root/shoot r a t i o of 3 b a r l e y v a r i e t i e s grown at 8°C (A) and 18°C (B), Table 27 Q 1 0 f o r 3 b a r l e y v a r i e t i e s grown at 8°C (A) and 18°C (B). 113 V a r i e t y Root/shoot A B Halcyon 0.52 0.24 Hector 0.53 0.20 Bonanza 0.52 0.25 V a r i e t y A Q 1 0 B Halcyon 2.4 4.0 Hector 1 .5 1 .9 Bonanza 1 .7 1.5 114 5. Determination of a c c l i m a t i o n of K + i n f l u x at d i f f e r e n t ages, growth temperatures and exposure times One winter (Halcyon) and two s p r i n g (Hector, Bonanza) b a r l e y were s t u d i e d in t h i s experiment. Data was analyzed using m u l t i v a r i a t e a n a l y s i s of v a r i a n c e (MANOVA), to determine v a r i a t i o n s of 3 f a c t o r s ; age, growth temperature and exposure time on 3 i n t e r r e l a t e d v a r i a b l e s ; K + i n f l u x , root K + and shoot K +. Such s t a t i s t i c a l treatment enables the c l a r i f i c a t i o n of the e f f e c t of.each f a c t o r alone on the v a r i a b l e s , t h e i r i n t e r a c t i o n s on the v a r i a b l e s and the degree to which co-v a r i a n c e of the v a r i a b l e s c o r r e l a t e to the f a c t o r s . As an example, i f the e f f e c t of changes i n temperature on K + i n f l u x and t i s s u e K* are to be determined, i n a d d i t i o n to d i r e c t and i n t e r a c t i o n a l e f f e c t s of temperature, t h i s a n a l y s i s p r o v i d e s an o p p o r t u n i t y to determine the extent to which the f l u c t u a t i o n s of temperature can cause v a r i a t i o n s w i t h i n v a r i a b l e s . To s i m p l i f y the s t a t i s t i c a l a n a l y s i s , the data obtained was t r e a t e d under two c a t e g o r i e s . Sources of v a r i a n c e were somewhat d i f f e r e n t i n these two c a t e g o r i e s . 5.1 E f f e c t of v a r i e t i e s , growth temperature and age on R + i n f l u x and t i s s u e content E f f e c t s of the above three f a c t o r s on three i n t e r r e l a t e d v a r i a b l e s ( f l u x e s , root K + and shoot K +) of two winter b a r l e y v a r i e t i e s (Halcyon and Gerbel) were s t u d i e d . I t 115 was hypothesized that, there would be no s i g n i f i c a n t e f f e c t s of temperature on f l u x e s i f the p l a n t s had a c c l i m a t e d . The s i m p l e s t univariate-one-way ANOVA im p l i e d the f o l l o w i n g . As a n t i c i p a t e d , no s i g n i f i c a n t d i f f e r e n c e s in i n f l u x or t i s s u e K + l e v e l s e x i s t e d between the v a r i e t i e s ( i . e . no v a r i e t a l v a r i a t i o n s - i n i n f l u x e s ) . E f f e c t of age, (10 and 16 days in t h i s i n s t a n c e ) on v a r i a t i o n s of the three v a r i a b l e s was a l s o not s i g n i f i c a n t . However, temperature had a strong i n f l u e n c e on i n f l u x and shoot K* content but only to a l e s s e r extent on root K + (Table 28). HT p l a n t s had a s i g n i f i c a n t l y lower (p<0.05) i n f l u x (0.557 /umol. g - 1 h" 1 ) at 18°C than the LT at 8°C (1.859 y m o l . g " 1 h ~ 1 ) . Mean shoot K + of HT p l a n t s was s i g n i f i c a n t l y g r e a t e r than that of LT p l a n t s . The mean root K + l e v e l s had. a m a r g i n a l l y s i g n i f i c a n t d i f f e r e n c e .(p=0.05) (54.15 and 64.12 /zmoKg - 1 i n LT and HT p l a n t s , r e s p e c t i v e l y ) . The s i g n i f i c a n t l y g r e a t e r f l u x e s i n LT p l a n t s (at 8°C) over and above those of HT p l a n t s may t h e r e f o r e , i n d i c a t e a complete a c c l i m a t i o n of f l u x e s . Since the shoot K" l e v e l i n LT p l a n t s (122.97 Mmol.g" 1) were s i g n i f i c a n t l y lower than that of HT p l a n t s (151.96 ymol.g" 1), a c c l i m a t i o n of t r a n s l o c a t i o n cannot be c o n s i d e r e d as completed. An argument may be advanced s t a t i n g that the d i f f e r e n c e s i n f l u x e s may be due to d i f f e r e n c e s i n shoot K + s t a t u s . If the shoot [ K + ] can i n s t a n t a n e o u s l y ( p l a n t s were exposed to the l a b e l l e d medium only f o r 10 minutes) a f f e c t the i n f l u x e s , then an involvement of a "hormonal" f a c t o r on r e g u l a t i o n of R+ t r a n s p o r t c o u l d be a n t i c i p a t e d . The degree to which temperature and other f a c t o r s 116 are r e s p o n s i b l e f o r the v a r i a t i o n s i n the v a r i a b l e s i s shown as a percentage of the t o t a l , i n parentheses (Table 28). I t i s c l e a r that temperature v a r i a t i o n s represent the major f a c t o r r e s p o n s i b l e f o r d i f f e r e n c e s among v a r i a b l e s . As mentioned e a r l i e r , no s i g n i f i c a n t e f f e c t of age (10 and 16 days) on i n f l u x was observed. However, the e f f e c t of age-temperature i n t e r a c t i v e f a c t o r s appeared to be h i g h l y s i g n i f i c a n t on i n f l u x and shoot K + content (Table 28). E f f e c t of temperature alone on i n f l u x a l s o i n d i c a t e d a c c l i m a t i o n . It i s t h e r e f o r e evident that a c c l i m a t i o n f o r f l u x e s but not f o r t r a n s l o c a t i o n had taken place i n both v a r i e t i e s d u r i n g 10 days. 5.2 E f f e c t of growth temperature " p e r t u r b a t i o n " and the t i s s u e [ K +] In t h i s instance growth temperature was perturbed a f t e r the p l a n t s had experienced an 8°C or 18°C temperature f o r 6 days. F o l l o w i n g the t r a n s f e r from 8° to 18°C and 18° to 8°C, p l a n t s were maintained under the new growth temperatures f o r another 6 or 10 days. The new temperature regime w i l l be henceforth r e f e r r e d to as "perturbed temperature", P T l f f o r the 8° to 18°C t r a n s f e r and PT 2 f o r the 18° to 8°C t r a n s f e r . The d u r a t i o n of exposure to such c o n d i t i o n s w i l l be r e f e r r e d to as "exposure time" (ET). In t h i s experiment, winter b a r l e y (Halcyon) and s p r i n g b a r l e y (Bonanza) were compared. The o b j e c t i v e of t h i s experiment was to examine the a b i l i t y of these v a r i e t i e s to 117 Table 28 E f f e c t of v a r i a t i o n s i n 3 f a c t o r s ( v a r i e t y , growth temperature and age) on 3 i n t e r r e l a t e d v a r i a b l e s (K + i n f l u x (/jmol .gf wt. " 1 h . 1 ) , root K + and shoot K + (/amol. gf wt. - 1 ) ) . S t a t i s t i c a l s i g n i f i c a n c e a p p l i e s to a p r o b a b i l i t y of p < 0.05. F i g u r e s given in parentheses represent the percentage c o n t r i b u t i o n of each f a c t o r to the v a r i a t i o n s i n the measured parameters (NS - not s i g n i f i c a n t ) . ( n = 24-27). 118 Source of Variance I n f l u x V a r i a b l e s Root K Shoot K 1. V a r i e t y Halcyon 1 .054 58.21 140.65 Gerbel 1 .363 60.06 134.28 P 0 . 34 0.72 0. 47 (NS) (NS) (NS) 2. Age (days) 1 0 1 .247 63.38 146.87 16 1 . 1 69 54.89 128.06 P 0.81 0.09 0.02 (NS) (NS) (20) 3 Growth Temp. 8°C 1 .859 54.15 122.97 18°C 0. 557 64. 1 2 151.96 P 0.0 0.05 0.02 (75) (16) (48) 4. Var * Temp.(°C) H * 8 1.515 61 .67 128.20 G * 8 2.204 46.64 117.75 H * 18 0.593 54.75 153.10 G * 18 0.521 73.49 150.82 P 0.0 0.001 0.002 (85) (64) (86) 119 T a b l e 28 (co n t ' d ) 5. Temp.(°C) * Age 8 * 1 0 1 .651 56.86 138.15 18 * 10 0.844 69.91 155.60 8 * 1 6 2.068 51.45 107.80 18 * 1 6 0.27 58.33 148.32 P 0.00 0.06 0.00 (86) (NS) (75) V * A NS 1 NS NS V * T * A NS NS NS 120 cope w i t h the changes of growth t e m p e r a t u r e s i n terms of K + t r a n s p o r t . A l s o the e f f e c t of d u r a t i o n of exposure t o " p e r t u r b e d " t e m p e r a t u r e s on K* i n f l u x and t i s s u e K + l e v e l s was d e t e r m i n e d . T h i s would p r o v i d e i n f o r m a t i o n r e g a r d i n g the time r e q u i r e d f o r a c c l i m a t i o n . I f the p l a n t s can cope e q u a l l y w e l l w i t h e i t h e r " p e r t u r b a t i o n " , then a s i g n i f i c a n t e f f e c t of these " p e r t u r b a t i o n s " on i n f l u x e s and t i s s u e K + l e v e l s cannot be a n t i c i p a t e d . T a b l e 31 p r o v i d e s the r e s u l t s of MANOVA. As f o r the p r e v i o u s case f l u x e s i n the t w o - v a r i e t i e s d i d not show a s i g n i f i c a n t d i f f e r e n c e (Table 29). The r o o t K + of Halcyon was s i g n i f i c a n t l y g r e a t e r (65.42 /amol.g" 1) than t h a t of Bonanza (44.27 j i . m o l . g ~ 1 ) . V a r i e t a l v a r i a t i o n s between the shoot K + was not observed (133.51 and 133.60 Atmol.g" 1 f o r Halcyon and Bonanza r e s p e c t i v e l y ) t h e r e f o r e , i n f l u x seemed t o have a c c l i m a t e d i n Halcyon f o r a g r e a t e r e x t e n t than i n Bonanza. As a n t i c i p a t e d the " p e r t u r b a t i o n " a l o n e had no s i g n i f i c a n t e f f e c t on any of the measured parameters (Table 4 ) . T h i s i n d i c a t e d t h a t any e f f e c t of " p e r t u r b a t i o n " on the K i n f l u x and t i s s u e K + c o n t e n t c o u l d be compensated f o r by a c c l i m a t i o n . However, the p e r i o d r e q u i r e d f o r such compensations appeared t o v a r y a c c o r d i n g t o the type of p e r t u r b a t i o n i n t r o d u c e d . For example, the f l u x e s of p l a n t s exposed from 8° t o 18°C (PT,) f o r 6 days a r e q u i t e s i m i l a r t o those of p l a n t s exposed from 18° t o 8°C ( P T 2 ) f o r t e n days, (1.0847 and 1.1551 umol.g"1h"1 f o r PT, and PT 2 r e s p e c t i v e l y ) . These f l u x e s were c o n s i d e r a b l y g r e a t e r than those of p l a n t s exposed t o PT, f o r 10 days and PT 2 f o r 6 days (0.468 and 0.273 m n o l . g - 1 ' f o r PT, and PT 2 121 r e s p e c t i v e l y ) . For example, when t r a n s f e r r e d from low t o h i g h t e m p e r a t u r e , i n i t i a l h i g h f l u x e s (6 days, 1.085) f o l l o w e d by a l o w e r i n g of f l u x e s (10 days, 0.468) was o b s e r v e d . An i n v e r s e t r e n d t o the above was shown by p l a n t s t r a n s f e r r e d from h i g h t o low t e m p e r a t u r e . A tendency t o de c r e a s e r o o t K + l e v e l w i t h i n c r e a s i n g time of exposure t o the " p e r t u r b e d " temperature was a l s o d i s c e r n e d . However, shoot K + c o n t e n t d i d not show a s i g n i f i c a n t d i f f e r e n c e w i t h i n c r e a s e d exposure t o " p e r t u r b e d " t e m p e r a t u r e . T h i s i n d i c a t e d a l o s s of r o o t K + t o the medium under any p e r t u r b a t i o n . The r e d u c t i o n i n r o o t K + c o n t e n t w i t h time can be e x p l a i n e d i n terms of i n f l u x e s and e f f l u x e s . A p e r t u r b a t i o n of 8° t o 18°C appeared t o cause a decrease i n r o o t K + w i t h i n c r e a s i n g time (57.21 Mmol.g" 1 i n 6 days and 40.82 Mmol.g" 1 i n 10 d a y s ) . T h i s may be caused by the d e c r e a s e d i n f l u x w i t h i n - c r e a s i n g time under such p e r t u r b a t i o n (1.085 and 0.467 jumol.g' 1 h" 1 i n 6 days and 10 days r e s p e c t i v e l y ) . On the o t h e r hand, a l t h o u g h a s i g n i f i c a n t i n c r e a s e i n i n f l u x (0.273 and 1.155 Aimol. g" 1 h~ 1 i n 6 days and 10 days r e s p e c t i v e l y ) was obser v e d i n p l a n t s t r a n s f e r r e d from 18° t o 8°C, a d e c r e a s e i n r o o t K + l e v e l was s t i l l d i s c e r n a b l e . T h i s may have been caused by g r e a t e r e f f l u x e s s i n c e the p o s s i b i l i t y of t r a n s l o c a t i o n t o the r o o t can be r u l e d out due t o the obser v e d d e c r e a s e i n shoot K + l e v e l (160.05 and 138.34 Mmol.g" 1 i n 6 and 10 days, r e s p e c t i v e l y ) . However, e f f l u x a n a l y s i s was not c a r r i e d out i n the p r e s e n t s t u d y . 122 Table 29 E f f e c t of v a r i a t i o n s i n 3 .factors ( v a r i e t y , perturbed growth temperature and exposure to p e r t u r b a t i o n on 3 v a r i a b l e s . (PT = "Perturbed" Temperature, ET = Exposure Time). Other d e s c r i p t i o n s are s i m i l a r to those i n Table 28.(n = 24-27) . 123 S o u r c e o f V a r i a n c e I n f l u x V a r i a b l e s R o o t K S h o o t K 1. V a r i e t y H a l c y o n ( H ) 0 . 6 7 0 6 5 . 4 2 131 . 51 B o n a n z a ( B ) 1.041 4 4 . 2 7 1 3 3 . 6 0 P 0 . 97 0 .01 0 . 9 3 (NS) ( 25 ) (NS) 2 . " P e r t u r b a t i o n " 8 18°C(PT,) 0 . 7 7 6 4 9 . 0 2 1 2 3 . 3 4 18 8°C (PT 2 ) 0 . 9 3 5 6 0 . 6 7 1 4 3 . 7 7 P 0 . 4 3 0 . 3 3 0 .11 (NS) (NS) (NS) 3 E x p o s u r e T ime (ET) ( d a y s ) • 6 0 . 8 1 4 7 0 . 7 6 1 3 8 . 6 9 1 0 0 . 8 8 0 4 5 . 2 9 1 3 0 . 4 7 P 0 . 9 3 0 . 001 0 . 7 8 (NS) ( 34 ) (NS) 4 . V a r * P e r t . H * PT , 0 . 8 2 2 61 . 4 5 1 2 0 . 2 2 B * PT, 0 .731 3 6 . 5 9 1 2 6 . 4 6 H * PT 2 0 . 5 1 8 6 9 . 4 0 1 4 6 . 8 0 B * P T 2 1.351 51 . 9 5 1 4 0 . 7 4 P 0 . 1 5 0 .001 0 . 3 6 (NS ) ( 33 ) (NS) 124 Table 29 (cont'd) 5 > n E T „ * II pip I! 6 * P, 10 * P, 6 * P 2 10 * P 2 6. Var * "ET" H * 6 H * 10 B * 6 B * 10 7. Var * "ET" * "PT' H * 6 * PT, H * 10 * PT, H * 6 * PT 2 H * 10 * PT 2 B * 6 * PT! B * 10 * PT1 B * 6 * PT 2 B * 10 * PT 2 1 .085 0.468 0.273 1.155 0.002 (51 ) 0.800 0.539 0.842 1 . 107 0.25 (NS) 1 . 328 0.315 0.273 0.762 0.842 0 . 620 1.351 0.001 (71 ) 57.21 40.82 97.87 48.28 0.01 (66) 87.72 43.13 36.86 46.74 0.39 (NS) 77.56 45.33 97.87 40.83 36.86 36.31 51 .95 0.04 (92) 128.01 118.67 160.05 138.34 0.50 (NS) 146.98 120.03 122.11 137.44 0.75 (NS) 133.92 106.52 160.05 133.54 122.11 130.82 140.74 0.2.1 (NS) 125 In c o n c l u s i o n , ( w i t h some u n c e r t a i n t y ) i t appears t h a t a p e r t u r b a t i o n of growth t e m p e r a t u r e s i n e i t h e r d i r e c t i o n ( d e c r e a s e or an i n c r e a s e ) c o u l d be d e t r i m e n t a l (at l e a s t i n the i n i t i a l days a f t e r the p e r t u r b a t i o n ) f o r the o t h e r w i s e w e l l -b a l a n c e d K + t r a n s p o r t r e g u l a t o r y system. 126 6. Time course of a c c l i m a t i o n of K + net f l u x and " t r a n s l o c a t i o n " A winter v a r i e t y (Halcyon) and a s p r i n g v a r i e t y (Kombar) were used in t h i s experiment. The time of exposure to low temperature (5°C) ranged from 0-24 hours. The p l a n t s exposed to low temperature are he n c e f o r t h r e f e r r e d to as "LT exposed" p l a n t s . Net f l u x e s were measured by a l l o w i n g the p l a n t s to absorb K + from l a b e l l e d medium f o r one hour. In t h i s experiment ashed shoot samples were a l s o used f o r counting of r a d i o a c t i v i t y . These counts p r o v i d e d estimates of the t r a n s l o c a t i o n r a t e s . S e v e r a l authors (Clarkson, 1976; Clarkson and Deane-Drummond, 1981; Deane-Drummond and G l a s s , 1983; S i d d i q i et al.,1983) have r e p o r t e d an enhancement of the c a p a c i t y f o r f l u x e s when roots are exposed to low temperatures for a s u f f i c i e n t p e r i o d of time. T h i s i n c r e a s e was thought to be a r e s u l t of the compensation f o r the reduced root a c t i v i t y , p a r t i c u l a r l y when shoots are maintained at higher temperatures. For example, Clarkson and Deane-Drummond (1981) emphasized the importance of such d i f f e r e n t i a l temperatures, s i n c e they represent the normal s i t u a t i o n encountered by p l a n t s in the s p r i n g and f a l l . The c a u s a t i v e f a c t o r f o r the i n c r e a s e d root a c t i v i t y has been c o n s i d e r e d to be the g r e a t e r demand f o r resources imposed by the shoot (which i s not e x p e r i e n c i n g low temperature). However, S i d d i q i et a l . , (1983) r e c e n t l y emphasized that a d i f f e r e n t i a l temperature i s not e s s e n t i a l f o r 127 such enhancement. E a r l i e r work by Raven and Smith (1978) and Sanders (1981) on the a l g a Chara r e v e a l e d s i m i l a r o b s e r v a t i o n s . Although an enhancement of ion f l u x e s i s q u i t e e v i d e n t , the time of exposure to low temperature r e q u i r e d to a t t a i n adjustments to the new environment i s not q u i t e c l e a r . The k i n e t i c s of these events are important in attempting to i n t e r p r e t the u n d e r l y i n g mechanism(s) of such enhancements. 6.1 A c c l i m a t i o n of net f l u x e s The net f l u x e s of a completely a c c l i m a t e d p l a n t at low temperature should be e i t h e r s i m i l a r to or g r e a t e r than those at high temperature i n HT p l a n t s . U s u a l l y when p l a n t s have been maintained at low temperature and the f l u x e s were subsequently measured at high temperature, an i n c r e a s e over and above c o n t r o l (held at high temperature c o n t i n u o u s l y ) i s a n t i c i p a t e d . Such i n c r e a s e i n net f l u x e s was not observed in Halcyon (3.676 and 3.11 /umol. g~ 1 h~ 1 in HT and LT, r e s p e c t i v e l y ) , while in Kombar the mean net f l u x was higher but not s i g n i f i c a n t l y (2.445 and 2.99 Mmol.g~ 1h~ 1 i n HT and LT, r e s p e c t i v e l y , p>0.05). However, when f l u x e s of HT and LT were measured at low temperature (10°C) s i g n i f i c a n t i n c r e a s e s i n both v a r i e t i e s grown at LT were observed. The argument co u l d be advanced that f l u x e s i n HT p l a n t s at low temperature represent an i n h i b i t i o n ; t h e r e f o r e a s t r a i g h t f o r w a r d way to t e s t the extent of a c c l i m a t i o n of f l u x e s i s to compare the f l u x e s of LT (at low temperature) with those of HT (at HT). Such a comparison made f o r Halcyon re v e a l e d s i g n i f i c a n t l y lower 128 net f l u x e s i n LT p l a n t s (Table 30). T h i s may i n d i c a t e a lack of a c c l i m a t i o n . However, a s i m i l a r comparison in Kombar re v e a l e d a g r e a t e r s i m i l a r i t y of f l u x e s between LT p l a n t s (at 10°C) and HT p l a n t s (at 15°C). T h i s may i n d i c a t e a c e r t a i n degree of a c c l i m a t i o n i n Kombar. Recently, S i d d i q i et a l . (1983), a l s o have reported s i g n i f i c a n t adjustment to low temperature i n another s p r i n g b a r l e y v a r i e t y , namely Fergus. Although not s i g n i f i c a n t , an i n c r e a s e of f l u x e s with i n c r e a s i n g d u r a t i o n of exposure to low temperature was observed i n both v a r i e t i e s (Table 33). Th e r e f o r e , d u r i n g t h i s 24 hours at l e a s t a p a r t i a l a c c l i m a t i o n may have taken p l a c e . In the v a r i e t y Fergus, S i d d i q i et a l . (1983) observed a s i g n i f i c a n t adjustment to low temperature w i t h i n 6 hours. However, such dramatic adjustments i n a short p e r i o d were not observed i n the q u a s i -steady f l u x e s which were measured over a long (>10-2 0 minutes) p e r i o d . In c o n c l u s i o n , both of these v a r i e t i e s appear to r e q u i r e a c e r t a i n p e r i o d of exposure of the order of s e v e r a l hours to low temperature i n order to achieve complete a c c l i m a t i o n . In most of the experiments, where i n f l u x i n s t e a d of net f l u x e s were s t u d i e d , u s u a l l y the LT p l a n t s when exposed to a high temperature (even for short p e r i o d s of 10 minutes i n du r a t i o n ) showed a g r e a t e r f l u x than HT p l a n t s . However, the net f l u x e s do not appear to act i n a s i m i l a r manner. Th e r e f o r e , i t should be borne i n mind that the c o n t r i b u t i o n of e f f l u x e s must be c o n s i d e r e d i n determining a c c l i m a t i o n . 129 Table 30 K + net f l u x e s (jumol .g." 'h.~ 1 ) and " t r a n s l o c a t i o n r a t e " of 2 b a r l e y v a r i e t i e s . LT: p l a n t s exposed to 5°C .for 24 h. HT: p l a n t s growing c o n t i n u o u s l y at 15°C. Fluxes were measured at the r e s p e c t i v e growth temperatures. ( T r a n s l o c a t i o n i s a c t u a l l y an estimate because the s p e c i f i c a c t i v i t y of the cytoplasm counts, r e s p o n s i b l e f o r determining the a c t i v i t y d e l i v e r e d to the xylem i s unknown). 130 V a r i a b l e V a r i e t y HT ' LT P. Net Flux Halcyon 3. 676 ± 0. 30 2. 110 ± 0. 19 0 .009 Kombar 2. 445 ± 0. 1 4 2. 343 ± 0. 19 0 .360 " T r a n s l o c a t i o n " Halcyon 0. 167 ± 0. 02 0. 202 ± 0. 02 0 . 1 90 Kombar 0. 347 ± 0. 008 0. 048 ± 0. 008 0 .000 131 6.2 A c c l i m a t i o n of " t r a n s l o c a t i o n " In the present study the net f l u x e s to roots represent the amount of r a d i o a c t i v i t y accumulated in the root t i s s u e . T h e r e f o r e , i t would be worthwhile to know the f r a c t i o n or percentage of accumulation in the root with regard to t o t a l accumulation. The l a t t e r represent the t o t a l a c t i v i t y i n root and shoot t i s s u e s . Table 31 show the percentage of the r a d i o a c t i v i t y accumulated in root t i s s u e . The p r o p o r t i o n accumulated i n root t i s s u e i s c l e a r l y g r e a t e r than that i n shoots. The "LT-exposed" Halcyon seems to have a lower root f r a c t i o n than the "LT-exposed" Kombar. T h i s i n d i c a t e s that Halcyon has " t r a n s l o c a t e d " a r e l a t i v e l y g r e a t e r amount of isotope to the shoot. Estimated values f o r " t r a n s l o c a t i o n " are given i n Table 32. Incomplete a c c l i m a t i o n of net f l u x e s i n Halcyon may a l s o be a r e s u l t of g r e a t e r e f f l u x e s . However, as i t i s beyond the scope of the present study, the e f f l u x under LT c o n d i t i o n s were not i n v e s t i g a t e d . F i g u r e s 26 and 27 i l l u s t r a t e s the general t r e n d of net f l u x e s and " t r a n s l o c a t i o n " at 10°C shown by LT-exposed p l a n t s . The value obtained f o r HT ( T 0 ) was used as the c o n t r o l . 132 Table 31 K + net f l u x (ymol.g." 1h.~ 1) at 10°C f o r Halcyon and Kombar. T 0=HT. The r e s t represent the time of exposure to low temperature (5„°C). p values are given f o r comparison of T 0 net f l u x with each of LT v a l u e s . A l l f l u x e s were measured at the same time so that any d i u r n a l v a r i a t i o n i n net f l u x e s i s accounted f o r . Of the t o t a l r a d i o a c t i v i t y absorbed, the percentages(%) r e t a i n e d i n the roots i s given under % column. 133 Var i a b l e Time of exposure h r s . N e t f l u x + SE % P Halcyon 0 1 .300 + 0.08 94.5 -2 1.613 + 0.13 91 .9 0.06 4 1 .780 + 0.06 92.42 0.005 1 3 1 .780 + 0.13 91 .75 0.02 .1 6 1.710 + 0.08 91 .3 0.01 24 2.110 + 0. 19 91 .26 0.01 Kombar 0 1 .525 + 0.14 86.45 -2 1 .820 + 0.06 92.67 0.07 4 1.911 + 0.03 95.79 0.06 1 3 2.294 ± 0.36 93.82 0.06 1 6 1 .995 + 0.06 96.75 0.02 24 2.343 + 0.19 97.99 0.01 134 Table 32 Estimated " T r a n s l o c a t i o n " r a t e at 10°C f o r Halcyon and Kombar. T 0=HT. The r e s t represent the time of exposure to low temperature (5°C). p values are given f o r comparison of T 0 " T r a n s l o c a t i o n " with each of LT v a l u e s . A l l f l u x e s were measured at the same time so that any d i u r n a l v a r i a t i o n i n net f l u x e s i s accounted f o r . 135 V a r i a b l e Time of exposure h r s . " T r a n s l o c a t i o n ± SE p Halcyon 0 0.075 + 0.01 -2 0.131 + 0.00 0.01 4 0. 1 46 + 0.01 0.009 1 3 0. 1 60 + 0.00 0.01 1 6 0.103 + 0.00 0.070 24 0.202 + 0.02 0.006 Kombar 0 0.239 + 0.03 -2 0. 1 94 + 0.01 0.14 4 0.084 + 0.01 0.08 1 3 0.151 + 0.03 0.004 1 6 0.067 + 0.00 0.00 24 0.048 + 0.00 0.00 136 F i g . 26 K + net f l u x and " t r a n s l o c a t i o n " of a winter b a r l e y v a r i e t y (Halcyon) exposed to low temperature ( 5 O C ) . ( 0 — " Q athe r e l a t i o n s h i p of net f l u x with time of a c c l i m a t i o n , A A r e l a t i o n s h i p of t r a n s l o c a t i o n r a t e with time of a c c l i m a t i o n . F i g . 27 K* net f l u x and " t r a n s l o c a t i o n " of a s p r i n g b a r l e y v a r i e t y (Kombar) exposed to low temperature (5°C). ( D e s c r i p t i o n i s s i m i l a r to that of F i g . 26). 137 FIGURE 26 TIME OF ACCLIMATION (h.) 138 IV. CONCLUSIONS The i n f l u e n c e of low temperature pretreatment on K + i n f l u x was i n v e s t i g a t e d i n 23 b a r l e y v a r i e t i e s . Most of the v a r i e t i e s responded to low temperature pretreatment by developing an i n c r e a s e d c a p a c i t y f o r K + i n f l u x . However, some of the s p r i n g v a r i e t i e s (e.g. Kombar, Conquest, Klages and Keystone) showed c o n s i s t e n t l y low i n f l u x e s . I r r e s p e c t i v e of the growth • temperature, a high root K + l e v e l was maintained by winter and some s p r i n g (e.g. Fergus) v a r i e t i e s . Increases in both K + i n f l u x and root K + content i n these v a r i e t i e s , exposed to low temperature f o r a c o n s i d e r a b l e time, i n d i c a t e the a b i l i t y of these p l a n t s to a d j u s t to such c o n d i t i o n s . I t was a l s o c l e a r t h at t h i s c a p a c i t y i s more pronounced i n winter v a r i e t i e s . Depending on growth temperature and e x t e r n a l K + supply, the low temperature s e n s i t i v i t y v a r i e d among v a r i e t i e s . Low temperature along with low n u t r i e n t supply had a tendency to decrease the temperature s e n s i t i v i t y of the winter v a r i e t y while most of the s p r i n g v a r i e t i e s d i d not e x h i b i t a change in s e n s i t i v i t y under any c o n d i t i o n s . T h i s reduced low temperature s e n s i t i v i t y to K + uptake, e s p e c i a l l y under K + d e p r i v e d 1 3 9 c o n d i t i o n s may t h e r e f o r e , be an advantage f o r winter v a r i e t i e s to s u r v i v e through the adverse c o n d i t i o n s (Chapin, 1974). As an example, s i n c e the low temperature has a tendency to d i m i n i s h the a v a i l a b i l i t y of n u t r i e n t s , lack of temperature s e n s i t i v i t y under such c o n d i t i o n s may enable the p l a n t s to absorb n u t r i e n t s at a higher r a t e even in f l u c t u a t i n g temperatures. The present study suggested that the usual negative feedback response to i n t e r n a l K* i s not expressed when c e r t a i n v a r i e t i e s (e.g. Kombar) are grown at low temperature. In the winter v a r i e t y , negative feedback c o n t r o l seemed to operate i r r e s p e c t i v e of the growth temperature. T h i s apparent maintenance of the usual r e g u l a t o r y c o n t r o l , under any growth temperatures may be an inherent c a p a c i t y of the winter v a r i e t y , which may enable these p l a n t s to f u n c t i o n 1 normally at low temperatures. Although a c c l i m a t i o n of i n f l u x appeared to take a sh o r t e r time p e r i o d , net f l u x e s , which a l s o i n c l u d e s a term f o r e f f l u x , took a longer time. T h e r e f o r e , v a r i a t i o n s in e f f l u x among v a r i e t i e s may be of some importance i n determining a c c l i m a t i o n . Estimates of K + t r a n s l o c a t i o n i n d i c a t e d that a winter v a r i e t y (Halcyon) was b e t t e r able to t r a n s f e r K + to the shoot under low temperature c o n d i t i o n s than was a s p r i n g v a r i e t y (Kombar). Hence, when the t o t a l t r a n s p o r t process i s con s i d e r e d , Halcyon seemed to have a b e t t e r c a p a c i t y to a c c l i m a t e . 140 Growth r a t e s of 3 b a r l e y v a r i e t i e s monitored over 14 days r e v e a l e d a low temperature r e d u c t i o n r e g a r d l e s s of K + supply. Reduced root growth at high K + supply r e s u l t e d i n a higher K + content of these t i s s u e s . N e v e r t h e l e s s , low n u t r i e n t supply at any growth temperature reduced the t i s s u e K* content. Hence, i t i s c l e a r that the low temperature r e d u c t i o n of root growth can be compensated by i n c r e a s e d uptake r a t e s only i f the e x t e r n a l n u t r i e n t supply i s not l i m i t e d . N e v e r t h e l e s s , t h i s study has e s t a b l i s h e d t h a t , among a group of s p r i n g v a r i e t i e s c o n s i d e r a b l e v a r i a t i o n s e x i s t i n the c a p a c i t y to achieve t h i s a c c l i m a t i o n . I t was evident that temperature v a r i a t i o n s represented one of the major f a c t o r s r e s p o n s i b l e f o r d i f f e r e n c e s i n K + i n f l u x and t i s s u e K + content among v a r i e t i e s . 141 REFERENCES Bernard, C. 1878. 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