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Responses of water-stressed Hordeum distichum L. and Cucumis sativus to proline and betaine
Plant Science Letters, 25 (1982) 329--335 Elsevier/North-Holland Scientific Publishers Ltd.
329
RESPONSES O F WATER-STRESSED H O R D E U M D I S T I C H U M L. A N D CUCUMIS SA T I V U S T O PROLINE AND BETAINE
C. ITAI* and L.G. PALEG Department o f Plant Physiology, Waite Agricultural Research Institute, The University of Adelaide, Glen Osmond, South Australia 5064 (Australia)
(Received July 27th, 1981) (Revision received October 28th, 1981) (Accepted November 3rd, 1981)
SUMMARY
Growth during recovery of water-stressed barley plants was enhanced by proline and betaine treatments, while no effect on growth during stress was noted. Cucumber plants were not similarly affected by proline, although it was taken up by the plants.
INTRODUCTION
Increases in the free proline and betaine content as a response to an imposed water stress is evident in the leaves o f m a n y different plant species [ 1 ]. Whether this is part of an adaptive response or merely a detrimental consequence of the stress is still disputed [2]. Several possible functional aspects are ascribed to the accumulation of betaine and proline: osmoregulation [ 3], stabilizing protein solubility [4], preventing inactivation of enzymes [5] as well as a means of conservation of nitrogen compounds [6] or respiratory substrate [7]. A direct approach to assessing the involvement of proline and betaine in stress adaptation is by evaluating the responses of stressed plants to treatm e n t with these compounds; this was done previously by visual assessment [ 8,9 ]. We used a similar approach to study the growth of two species differing in the accumulation o f proline and betaine during stress.
*Permanent address: Department of Biology, Ben-Gurion University, Beer Sheva 84120, Israel. Abbreviation: PEG, polyethylene gi~j~ol.
330 MATERIALS A N D M E T H O D S Cucumis sativus (cv. Marketeer) and Hordeum distichum L. (cv. Clipper) were grown in 6-inch pots in washed sand in 16 h daylength (4.1 mW cm -2) and 25°C/20°C day/night temperature for cucumbers and 20°C/16°C for barley. Plants were watered with culture solution containing 4 mM Ca(NO3)2, 3 mM KNO3, 2 mM KH2PO4, 2 mM MgSO4, 160 ~M FeEDTA, 41 gM H2BO3, 9 pM MnCI2, 0.74 pM ZnSO4, 0.32 pM CuSO4 and 0.14 ~M NaMnO4. Plants were water,stressed by adding polyethylene glycol (PEG) (4000 mol. wt.) to the nutrient solution. To obtain solutions of - 4 and - 1 0 bar, 150 g/1 and 240 g/l, respectively, were added. Proline and betaine (25 raM) (Sigma) pH 6.2 were sprayed on the leaves with a Hill's Supamist sprayer. Control treatments were sprayed with H20. Leaf area was measured with a Paton electronic planimeter and proline determination was carried out according to Singh et al. [10]. RESULTS Nineteen-day-old barley plants (Table I) were watered with P E G solution (-10 bar) for 4 days w h e n 5 0 % of the plants were harvested. The pots of the remaining ones were drained and standard culture solution was added daffy for 4 more days (recovery). Plants were sprayed with 25 m M betaine either on the firstor third day of the stressor immediately before recovery. The betaine treatment affected the growth during recovery but this was not evident at the time of the stress.Only the treatment applied toward stress termination was effective,in contrast to those given before or after stress. All the parameters determined were similarly affected but tilleringwas the most responsive one. In the result of a similar experiment (Table II) in which 21~lay~ld plants were sprayed with 25 m M of either betaine, proline or betaine + proline, no effect of proline by itselfwas noted but it is of interest that the greatest response was to the combination of the two solutes. Comparable experiments with 14~iay-old cucumber plants, stressed for 4 days with P E G solution (-4 bar) and sprayed on the firstday of stress with 25 m M proline, failed to show similar results (Table Ill).Even when the sprays were applied at different times during the stress (data not presented), no effects of proline on the stressed plants could be detected inspire of the fact that proline applied was taken up by the leaves (Table IV). The tissues assayed in this experiment were thoroughly rinsed in water before they were frozen and extracted for the proline assay. The experiment verified,as well, the fact that cucumber does not accumulate proline as a result of water stress.Results of the experiments in which proline was added to the root media indicated also that barley (Table V) and cucumbers (Table VI) reacted differently. In both experiments the plants were stressed for 4 days. It is of interestto note that the effects of stresson cucumber plants are accentuated by proline treatment, while this was not observed with barley.
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332 TABLE II NUMBER AND AREA OF LEAVES, AND DRY WEIGHT OF THE SHOOTS OF BARLEY PLANTS WHICH RECOVERED FOR 4 DAYS FROM A STRESS IMPOSED BY PEG SOLUTION ( - 1 0 BAR) Plants were sprayed with 25 ram of betaine or proline or 12.5 mM proline + 12.5 mM betaine. In this experiment yellow areas of leaves were not included in the area measurement. Treatment values are means of 2 pots each with 8 plants ± S.E. Treatment
Control PEG - 1 0 bar PEG + 25 mM proline PEG + 25 mM betaine PEG + 12.5 mM proline and 12.5 mM betaine
No. of leaves
Leaf area (cm)
Dry wt. (mg) 360 148 173 205
Main
Tiller
5.6 5.6 6.0 6.0
3.6 ± 1.2 0 0 1.6 ± 1.2
4.36 0.18 0.27 1.00
1.8 ± 1.1
2.44 ± 0.95
± 0.5 ± 0.5 ± 0.2 ±0
6.0 ± 0
± 0.53 ± 0.15 ± 0.22 ± 0.55
-+ 53 -+ 30 -+ 38 ± 43
282 ± 86
DISCUSSION The outcome of the experiments with barley does not support the conclusion t h a t p r o l i n e a n d / o r b e t a i n e a c c u m u l a t i o n has n o survival value t o w a t e r - s t r e s s e d p l a n t s a n d t h a t this p h e n o m e n o n is m e r e l y a s y m p t o m o f d r o u g h t injury r a t h e r t h a n an a d a p t i v e r e s p o n s e [ 2 ] . T h e e n h a n c e d r e c o v e r y a n d lack o f r e s p o n s e d u r i n g stress d u e t o b e t a i n e t r e a t m e n t (Tables I a n d I I ) m a k e s it u n l i k e l y t h a t b e t a i n e a n d p r o l i n e are acting m e r e l y as o s m o t i c agents c o n t r i b u t i n g to o s m o r e g u l a t i o n [ 3 ] . R a t h e r it a d v o c a t e s a role f o r t h e s e c o m p a t i b l e solutes e i t h e r t h r o u g h a f f e c t i n g e n z y m a t i c p r o t e i n s [4,5] o r b y c o n s e r v a t i o n o f specific s u b s t r a t e s [ 6 , 7 ] . H o w e v e r , this last possibility s e e m s t o b e m o r e d i r e c t l y r e l a t e d t o t h e r e c o v e r y process. S u p p o r t i n g t h e finding that solute treatment enhanced recovery rather than affected processes d u r i n g stress is t h e o b s e r v a t i o n t h a t high p r o l i n e a c c u m u l a t i o n is r e l a t e d t o t h e ability o f s o r g h u m p l a n t s t o r e c o v e r f r o m w a t e r stress [ 11 ]. I n this r e s p e c t o u r findings also i n d i c a t e t h a k t h e t i m e b e t a i n e is a d m i n i s t e r e d a f f e c t s t h e o u t c o m e . This is p r e s u m a b l y r e l a t e d t o t h e p r o c e s s e s o c c u r r i n g at the time of treatment rather than to the metabolism of betaine which s e e m s t o b e v e r y slow [ 3 ] . O n l y p l a n t s t r e a t e d t o w a r d t h e e n d o f t h e stress p e r i o d , b u t well b e f o r e r e c o v e r y , r e s p o n d e d d u r i n g r e c o v e r y (Table I). T h e large, c o m b i n e d e f f e c t s o f p r o l i n e a n d b e t a i n e m i g h t s t e m f r o m t h e f a c t t h a t b a r l e y a c c u m u l a t e s b o t h d u r i n g w a t e r stress [3] a n d p r o b a b l y indicates t h a t t h e i r role is n o t r e l a t e d t o o s m o r e g u l a t i o n p e r se. O n t h e w h o l e , t h e results in T a b l e s I a n d I I i n d i c a t e t h a t d r o u g h t a d a p t a t i o n in b a r l e y m a y b e p o s i t i v e l y c o r r e l a t e d t o b e t a i n e a n d p r o l i n e a c c u m u l a t i o n d u e t o stress [ 8 - - 1 0 ] . O n t h e o t h e r h a n d , p r o l i n e a n d b e t a i n e m a y n o t p r o d u c e t h e s a m e resp o n s e in all p l a n t s s u b j e c t e d t o d r o u g h t (Tables I I I a n d V I ) . The suggestion
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334 TABLE IV PROLINE CONTENT (~g/mg DRY MATTER) OF PORTIONS OF THE SHOOT OF CUCUMBER PLANTS WHICH WERE TREATED WITH PEG SOLUTION ( - 4 BAR) FOR 3 DAYS AND SPRAYED ONCE WITH 25 mM PROLINE Values are means of 2 samples taken from 2 pots, each of 3 plants.
Apex Young Mature
Control
+ PEG
+ PEG + spray
1.76 0.57 0.74
1.79 0.30 0.41
2.33 2.06 0.25
TABLE V NUMBER AND AREA OF LEAVES OF BARLEY PLANTS TREATED WITH PEG ( - 1 0 BAR) AND PROLINE (25 raM), BOTH ADDED TO THE CULTURE SOLUTION Each treatment was of 2 pots, each of 8 plants and leaf area values are means + S.E. Values in brackets are % of controls.
Control PEG - 1 0 bar PEG - 1 0 bar + proline 25 mM
No. of leaves
Leaf area (cm 2)
3.08 2.66 (86) 2.75 (89)
1.20 ± 0.48 0.77 ± 0.20 (64) 0.74 ± 0.28 (62)
TABLE VI NUMBER AND AREA OF LEAVES OF CUCUMBER PLANTS TREATED WITH PEG SOLUTION AND 25 mM PROLINE, BOTH ADDED TO THE CULTURE SOLUTION Each treatment was of 2 pots, each of 3 plants and leaf area values are means ± S.E. Values in brackets are % of controls.
Control PEG - 4 bar PEG - 4 bar + proline 25 mM
No. of leaves
Leaf area (cm 2)
3.33 3.66 (110) 2.33 (70)
1.36 ± 0.25 0.85 ± 0.20 (63) 0.32 ± 0.10 (24)
t h a t c u c u m b e r s are drought-sensitive b e c a u s e t h e y d o n o t a c c u m u l a t e c o m patible solutes is u n l i k e l y since t r e a t m e n t w i t h p r o l i n e (Table I I I ) did n o t a f f e c t t h e r e s p o n s e o f c u c u m b e r s t o w a t e r stress even t h o u g h it was t a k e n u p (Table IV). F u r t h e r , t h e adverse effects o f p r o l i n e a n d b e t a i n e ( d a t a n o t p r e s e n t e d ) , in c o m b i n a t i o n w i t h P E G s o l u t i o n (Table V I ) , q u e s t i o n s t h e ability o f t h e t w o solutes t o a f f e c t p r o t e i n a n d e n z y m a t i c a c t i v i t y since such a role w o u l d be, a s s u m e d l y , o f a universal n a t u r e . I t c o u l d be a r g u e d t h a t t h e a p p a r e n t l y adverse e f f e c t s s t e m f r o m t o x i c e f f e c t s o f p r o l i n e [ 1 2 ] , or a
335 severe r e d u c t i o n in p H d u e t o m i c r o - o r g a n i s m c o n t a m i n a t i o n [ 1 3 ] t o w h i c h P E G - t r e a t e d c u c u m b e r p l a n t s are m o r e sensitive t h a n n o n - s t r e s s e d o n e s , o r e v e n t o intrinsic d i f f e r e n c e s in t r a n s l o c a t i o n , a c c u m u l a t i o n a n d / o r intracellular l o c a t i o n b e t w e e n e n d o g e n o u s a n d e x o g e n o u s solutes. A t this stage, h o w e v e r , t h e c u c u m b e r results s h o u l d b e t a k e n as s u p p o r t i n g a n d c o m p l i m e n t i n g s t u d i e s in w h i c h n o c o r r e l a t i o n b e t w e e n p r o l i n e a c c u m u l a t i o n a n d d r o u g h t r e s i s t a n c e w a s f o u n d [ 2 , 1 4 ] . I n a n y case, it is clear t h a t p r o l i n e a n d b e t a i n e h a v e a d i r e c t i n v o l v e m e n t in p l a n t a d a p t a t i o n o f a n u m b e r o f species t o w a t e r stress b y a f f e c t i n g p r o c e s s e s leading t o t h e e n h a n c e d r e c o v e r y f r o m stress ( T a b l e s I a n d I I ) . REFERENCES 1 D. Aspinall and L.G. Paleg, Proline accumulation: physiological aspects, in: L.G. Paleg and D. Aspinall (Eds.), Physiology and Biochemistry of Drought Resistance in Plants, Academic Press, Sydney, 1981. 2 A.D. Hanson, C.E. Nelson, A.R. Pedersen and E.H. Everson, Crop Sci., 19 (1979) 489. 3 R.G. Wyn Jones and R. Storey, Aust. J. Plant Physiol., 5 (1978) 877. 4 B. Schobert and H. Tschesche, Biochim. Biophys. Acta, 541 (1978) 270. 5 L.G. Paleg, T.J. Douglas, V. van Daal and D.B. Keech, Aust. J. Plant Physiol., 8 (1981) 107. 6 N.N. Savitskaya, Biolog. Nauki (Moscow), 19 (1976) 49. 7 C.R. Stewart, S.F. Boggess, D. Aspinall and L.G. Paleg, Plant Physiol., 59 (1977) 930. 8 L.A. Tyankova, C.R. Acad. Bulg. Sci. (Tom), 19 (1966) 847. 9 C. Hubac and D. Guerrier, Oecol. Plant., 7 (1972) 147. 10 T.N. Singh, D. Aspinall and L.G. Paleg, Nature (New Biol.), 236 (1972) 188. 11 A. Blum and A. Ebercon, Crop Sci,, 16 (1976) 428. 12 L.J. Audus and J.H. Quastel, Nature, 160 (1974) 222. 13 R. Storey, Ph.D. Dissertation, University of Wales, 1976. 14 M, Tal, A. Katz, H. Heikin and K. Deh~n, New Phytol., 82 (1978) 349.
329
RESPONSES O F WATER-STRESSED H O R D E U M D I S T I C H U M L. A N D CUCUMIS SA T I V U S T O PROLINE AND BETAINE
C. ITAI* and L.G. PALEG Department o f Plant Physiology, Waite Agricultural Research Institute, The University of Adelaide, Glen Osmond, South Australia 5064 (Australia)
(Received July 27th, 1981) (Revision received October 28th, 1981) (Accepted November 3rd, 1981)
SUMMARY
Growth during recovery of water-stressed barley plants was enhanced by proline and betaine treatments, while no effect on growth during stress was noted. Cucumber plants were not similarly affected by proline, although it was taken up by the plants.
INTRODUCTION
Increases in the free proline and betaine content as a response to an imposed water stress is evident in the leaves o f m a n y different plant species [ 1 ]. Whether this is part of an adaptive response or merely a detrimental consequence of the stress is still disputed [2]. Several possible functional aspects are ascribed to the accumulation of betaine and proline: osmoregulation [ 3], stabilizing protein solubility [4], preventing inactivation of enzymes [5] as well as a means of conservation of nitrogen compounds [6] or respiratory substrate [7]. A direct approach to assessing the involvement of proline and betaine in stress adaptation is by evaluating the responses of stressed plants to treatm e n t with these compounds; this was done previously by visual assessment [ 8,9 ]. We used a similar approach to study the growth of two species differing in the accumulation o f proline and betaine during stress.
*Permanent address: Department of Biology, Ben-Gurion University, Beer Sheva 84120, Israel. Abbreviation: PEG, polyethylene gi~j~ol.
330 MATERIALS A N D M E T H O D S Cucumis sativus (cv. Marketeer) and Hordeum distichum L. (cv. Clipper) were grown in 6-inch pots in washed sand in 16 h daylength (4.1 mW cm -2) and 25°C/20°C day/night temperature for cucumbers and 20°C/16°C for barley. Plants were watered with culture solution containing 4 mM Ca(NO3)2, 3 mM KNO3, 2 mM KH2PO4, 2 mM MgSO4, 160 ~M FeEDTA, 41 gM H2BO3, 9 pM MnCI2, 0.74 pM ZnSO4, 0.32 pM CuSO4 and 0.14 ~M NaMnO4. Plants were water,stressed by adding polyethylene glycol (PEG) (4000 mol. wt.) to the nutrient solution. To obtain solutions of - 4 and - 1 0 bar, 150 g/1 and 240 g/l, respectively, were added. Proline and betaine (25 raM) (Sigma) pH 6.2 were sprayed on the leaves with a Hill's Supamist sprayer. Control treatments were sprayed with H20. Leaf area was measured with a Paton electronic planimeter and proline determination was carried out according to Singh et al. [10]. RESULTS Nineteen-day-old barley plants (Table I) were watered with P E G solution (-10 bar) for 4 days w h e n 5 0 % of the plants were harvested. The pots of the remaining ones were drained and standard culture solution was added daffy for 4 more days (recovery). Plants were sprayed with 25 m M betaine either on the firstor third day of the stressor immediately before recovery. The betaine treatment affected the growth during recovery but this was not evident at the time of the stress.Only the treatment applied toward stress termination was effective,in contrast to those given before or after stress. All the parameters determined were similarly affected but tilleringwas the most responsive one. In the result of a similar experiment (Table II) in which 21~lay~ld plants were sprayed with 25 m M of either betaine, proline or betaine + proline, no effect of proline by itselfwas noted but it is of interest that the greatest response was to the combination of the two solutes. Comparable experiments with 14~iay-old cucumber plants, stressed for 4 days with P E G solution (-4 bar) and sprayed on the firstday of stress with 25 m M proline, failed to show similar results (Table Ill).Even when the sprays were applied at different times during the stress (data not presented), no effects of proline on the stressed plants could be detected inspire of the fact that proline applied was taken up by the leaves (Table IV). The tissues assayed in this experiment were thoroughly rinsed in water before they were frozen and extracted for the proline assay. The experiment verified,as well, the fact that cucumber does not accumulate proline as a result of water stress.Results of the experiments in which proline was added to the root media indicated also that barley (Table V) and cucumbers (Table VI) reacted differently. In both experiments the plants were stressed for 4 days. It is of interestto note that the effects of stresson cucumber plants are accentuated by proline treatment, while this was not observed with barley.
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332 TABLE II NUMBER AND AREA OF LEAVES, AND DRY WEIGHT OF THE SHOOTS OF BARLEY PLANTS WHICH RECOVERED FOR 4 DAYS FROM A STRESS IMPOSED BY PEG SOLUTION ( - 1 0 BAR) Plants were sprayed with 25 ram of betaine or proline or 12.5 mM proline + 12.5 mM betaine. In this experiment yellow areas of leaves were not included in the area measurement. Treatment values are means of 2 pots each with 8 plants ± S.E. Treatment
Control PEG - 1 0 bar PEG + 25 mM proline PEG + 25 mM betaine PEG + 12.5 mM proline and 12.5 mM betaine
No. of leaves
Leaf area (cm)
Dry wt. (mg) 360 148 173 205
Main
Tiller
5.6 5.6 6.0 6.0
3.6 ± 1.2 0 0 1.6 ± 1.2
4.36 0.18 0.27 1.00
1.8 ± 1.1
2.44 ± 0.95
± 0.5 ± 0.5 ± 0.2 ±0
6.0 ± 0
± 0.53 ± 0.15 ± 0.22 ± 0.55
-+ 53 -+ 30 -+ 38 ± 43
282 ± 86
DISCUSSION The outcome of the experiments with barley does not support the conclusion t h a t p r o l i n e a n d / o r b e t a i n e a c c u m u l a t i o n has n o survival value t o w a t e r - s t r e s s e d p l a n t s a n d t h a t this p h e n o m e n o n is m e r e l y a s y m p t o m o f d r o u g h t injury r a t h e r t h a n an a d a p t i v e r e s p o n s e [ 2 ] . T h e e n h a n c e d r e c o v e r y a n d lack o f r e s p o n s e d u r i n g stress d u e t o b e t a i n e t r e a t m e n t (Tables I a n d I I ) m a k e s it u n l i k e l y t h a t b e t a i n e a n d p r o l i n e are acting m e r e l y as o s m o t i c agents c o n t r i b u t i n g to o s m o r e g u l a t i o n [ 3 ] . R a t h e r it a d v o c a t e s a role f o r t h e s e c o m p a t i b l e solutes e i t h e r t h r o u g h a f f e c t i n g e n z y m a t i c p r o t e i n s [4,5] o r b y c o n s e r v a t i o n o f specific s u b s t r a t e s [ 6 , 7 ] . H o w e v e r , this last possibility s e e m s t o b e m o r e d i r e c t l y r e l a t e d t o t h e r e c o v e r y process. S u p p o r t i n g t h e finding that solute treatment enhanced recovery rather than affected processes d u r i n g stress is t h e o b s e r v a t i o n t h a t high p r o l i n e a c c u m u l a t i o n is r e l a t e d t o t h e ability o f s o r g h u m p l a n t s t o r e c o v e r f r o m w a t e r stress [ 11 ]. I n this r e s p e c t o u r findings also i n d i c a t e t h a k t h e t i m e b e t a i n e is a d m i n i s t e r e d a f f e c t s t h e o u t c o m e . This is p r e s u m a b l y r e l a t e d t o t h e p r o c e s s e s o c c u r r i n g at the time of treatment rather than to the metabolism of betaine which s e e m s t o b e v e r y slow [ 3 ] . O n l y p l a n t s t r e a t e d t o w a r d t h e e n d o f t h e stress p e r i o d , b u t well b e f o r e r e c o v e r y , r e s p o n d e d d u r i n g r e c o v e r y (Table I). T h e large, c o m b i n e d e f f e c t s o f p r o l i n e a n d b e t a i n e m i g h t s t e m f r o m t h e f a c t t h a t b a r l e y a c c u m u l a t e s b o t h d u r i n g w a t e r stress [3] a n d p r o b a b l y indicates t h a t t h e i r role is n o t r e l a t e d t o o s m o r e g u l a t i o n p e r se. O n t h e w h o l e , t h e results in T a b l e s I a n d I I i n d i c a t e t h a t d r o u g h t a d a p t a t i o n in b a r l e y m a y b e p o s i t i v e l y c o r r e l a t e d t o b e t a i n e a n d p r o l i n e a c c u m u l a t i o n d u e t o stress [ 8 - - 1 0 ] . O n t h e o t h e r h a n d , p r o l i n e a n d b e t a i n e m a y n o t p r o d u c e t h e s a m e resp o n s e in all p l a n t s s u b j e c t e d t o d r o u g h t (Tables I I I a n d V I ) . The suggestion
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334 TABLE IV PROLINE CONTENT (~g/mg DRY MATTER) OF PORTIONS OF THE SHOOT OF CUCUMBER PLANTS WHICH WERE TREATED WITH PEG SOLUTION ( - 4 BAR) FOR 3 DAYS AND SPRAYED ONCE WITH 25 mM PROLINE Values are means of 2 samples taken from 2 pots, each of 3 plants.
Apex Young Mature
Control
+ PEG
+ PEG + spray
1.76 0.57 0.74
1.79 0.30 0.41
2.33 2.06 0.25
TABLE V NUMBER AND AREA OF LEAVES OF BARLEY PLANTS TREATED WITH PEG ( - 1 0 BAR) AND PROLINE (25 raM), BOTH ADDED TO THE CULTURE SOLUTION Each treatment was of 2 pots, each of 8 plants and leaf area values are means + S.E. Values in brackets are % of controls.
Control PEG - 1 0 bar PEG - 1 0 bar + proline 25 mM
No. of leaves
Leaf area (cm 2)
3.08 2.66 (86) 2.75 (89)
1.20 ± 0.48 0.77 ± 0.20 (64) 0.74 ± 0.28 (62)
TABLE VI NUMBER AND AREA OF LEAVES OF CUCUMBER PLANTS TREATED WITH PEG SOLUTION AND 25 mM PROLINE, BOTH ADDED TO THE CULTURE SOLUTION Each treatment was of 2 pots, each of 3 plants and leaf area values are means ± S.E. Values in brackets are % of controls.
Control PEG - 4 bar PEG - 4 bar + proline 25 mM
No. of leaves
Leaf area (cm 2)
3.33 3.66 (110) 2.33 (70)
1.36 ± 0.25 0.85 ± 0.20 (63) 0.32 ± 0.10 (24)
t h a t c u c u m b e r s are drought-sensitive b e c a u s e t h e y d o n o t a c c u m u l a t e c o m patible solutes is u n l i k e l y since t r e a t m e n t w i t h p r o l i n e (Table I I I ) did n o t a f f e c t t h e r e s p o n s e o f c u c u m b e r s t o w a t e r stress even t h o u g h it was t a k e n u p (Table IV). F u r t h e r , t h e adverse effects o f p r o l i n e a n d b e t a i n e ( d a t a n o t p r e s e n t e d ) , in c o m b i n a t i o n w i t h P E G s o l u t i o n (Table V I ) , q u e s t i o n s t h e ability o f t h e t w o solutes t o a f f e c t p r o t e i n a n d e n z y m a t i c a c t i v i t y since such a role w o u l d be, a s s u m e d l y , o f a universal n a t u r e . I t c o u l d be a r g u e d t h a t t h e a p p a r e n t l y adverse e f f e c t s s t e m f r o m t o x i c e f f e c t s o f p r o l i n e [ 1 2 ] , or a
335 severe r e d u c t i o n in p H d u e t o m i c r o - o r g a n i s m c o n t a m i n a t i o n [ 1 3 ] t o w h i c h P E G - t r e a t e d c u c u m b e r p l a n t s are m o r e sensitive t h a n n o n - s t r e s s e d o n e s , o r e v e n t o intrinsic d i f f e r e n c e s in t r a n s l o c a t i o n , a c c u m u l a t i o n a n d / o r intracellular l o c a t i o n b e t w e e n e n d o g e n o u s a n d e x o g e n o u s solutes. A t this stage, h o w e v e r , t h e c u c u m b e r results s h o u l d b e t a k e n as s u p p o r t i n g a n d c o m p l i m e n t i n g s t u d i e s in w h i c h n o c o r r e l a t i o n b e t w e e n p r o l i n e a c c u m u l a t i o n a n d d r o u g h t r e s i s t a n c e w a s f o u n d [ 2 , 1 4 ] . I n a n y case, it is clear t h a t p r o l i n e a n d b e t a i n e h a v e a d i r e c t i n v o l v e m e n t in p l a n t a d a p t a t i o n o f a n u m b e r o f species t o w a t e r stress b y a f f e c t i n g p r o c e s s e s leading t o t h e e n h a n c e d r e c o v e r y f r o m stress ( T a b l e s I a n d I I ) . REFERENCES 1 D. Aspinall and L.G. Paleg, Proline accumulation: physiological aspects, in: L.G. Paleg and D. Aspinall (Eds.), Physiology and Biochemistry of Drought Resistance in Plants, Academic Press, Sydney, 1981. 2 A.D. Hanson, C.E. Nelson, A.R. Pedersen and E.H. Everson, Crop Sci., 19 (1979) 489. 3 R.G. Wyn Jones and R. Storey, Aust. J. Plant Physiol., 5 (1978) 877. 4 B. Schobert and H. Tschesche, Biochim. Biophys. Acta, 541 (1978) 270. 5 L.G. Paleg, T.J. Douglas, V. van Daal and D.B. Keech, Aust. J. Plant Physiol., 8 (1981) 107. 6 N.N. Savitskaya, Biolog. Nauki (Moscow), 19 (1976) 49. 7 C.R. Stewart, S.F. Boggess, D. Aspinall and L.G. Paleg, Plant Physiol., 59 (1977) 930. 8 L.A. Tyankova, C.R. Acad. Bulg. Sci. (Tom), 19 (1966) 847. 9 C. Hubac and D. Guerrier, Oecol. Plant., 7 (1972) 147. 10 T.N. Singh, D. Aspinall and L.G. Paleg, Nature (New Biol.), 236 (1972) 188. 11 A. Blum and A. Ebercon, Crop Sci,, 16 (1976) 428. 12 L.J. Audus and J.H. Quastel, Nature, 160 (1974) 222. 13 R. Storey, Ph.D. Dissertation, University of Wales, 1976. 14 M, Tal, A. Katz, H. Heikin and K. Deh~n, New Phytol., 82 (1978) 349.