Agricultural Meteorology, 14(1974) 113--127 © Elsevier Scientific Publishing Company, Amsterdam - - Printed in The Netherlands
TO WATER STRESS*
CHARLES Y. SULLIVAN and JERRY D. EASTIN
Department of Horticulture, and Department of Forestry and Agronomy, University of Nebraska, Lincoln, Nebr. (U.S.A.) (Received October 1, 1973; accepted May 20, 1974)
ABSTRACT Sullivan, C. Y. and Eastin, J. D., 1974. Plant physiological responses to water stress. Agric. Meteorol., 14: 113--127.
Factors involved in physiological responses to water stress are discussed as they may relate to water-use efficiency by plants grown under conditions of drought. Evidence is presented that genotypic differences exist in plant responses to water stress. It is shown that previous exposure to stress conditions, or a natural hardening period, can markedly influence measured responses, such as photosynthesis and stomatal movement. Stage of growth at which the stress occurs also influences the response. A practical technique for measuring desiccation and heat tolerance demonstrates that genetically controlled tolerance factors can be selected for and used in plant breeding programs.
INTRODUCTION The topic of this paper encompasses a very broad subject, and our intent i o n s h e r e w i l l n o t b e a n e x h a u s t i v e r e v i e w a n d m e n t i o n o f as m a n y w a t e r s t r e s s r e s p o n s e s as p o s s i b l e , b u t r a t h e r a s e l e c t i o n o f a r e a s w h i c h w e f e e l h a v e application to our subject of plant modification for more efficient water use, and most of which our own research has touched on. Particular reference will b e g i v e n t o o u r w o r k in r e c e n t y e a r s in N e b r a s k a w i t h g r a i n s o r g h u m a n d drought resistance. Greater drought resistance does not necessarily imply greater water-use efficiency. Although, in many cases they are positively correlated. However, i f w e a r e t o a c c o m p l i s h p l a n t m o d i f i c a t i o n f o r m o r e e f f i c i e n t w a t e r use, w h e n p l a n t s a r e g r o w n u n d e r c o n d i t i o n s o f l i m i t e d w a t e r a v a i l a b i l i t y , i t is
* Support for this project was received in part from the Rockefeller Foundation and from the Office of Water Resources Research, Department of the Interior, under the Public Law 88-379 program. Published with the approval of the Director as Paper No.3765 Journal Series, Nebraska Agr. Exp. Sta.
114 necessary that we have a complete understanding of the physiological responses to water stress including those factors which contribute to conventional drought resistance. Selection pressures can then be placed on those factors contributing to the greatest water-use efficiency. In our project at Nebraska emphasis has been placed on selection of physiological techniques which are applicable in plant-breeding programs. Background will be given here for the de ve l opment of some of these techniques. In order to methodically approach the problem, we have combined some of the classical approaches with more m ode r n techniques. It soon becomes obvious to anyone working with plant responses to water stress that they are very complex, and may change with degree and time of exposure, stage of maturity, previous environmental exposure, etc., with many interactions apparent. For example, drought is frequently accompanied by high temperatures, thus our concern with organ and organelle temperatures and c o n c o m i t a n t heat responses. Transpiration rates may affect not only internal water potentials and resulting responses, but may also affect plant temperatures, and consequently the response may be different than when under water stress only. STRESS TOLERANCE
It is recognized that stress responses may be divided into either avoidance or tolerance mechanisms. Levitt (1972) has reiterated this in his recent book, as shown in Fig.1. This is true not only between widely different plant genera CRODGHTRESISTING F Drought (stress) Avoiding
Drought (stress) Tolerating
Dehydration Toleratin 9
Fig.1. The nature of xerophytism (from Levitt, 1972).
and species, b ut we also have evidence that significant differences do exist among varieties to tolerate, and function metabolically at reduced internal water potentials, or to avoid the stress. For convenience d e h y d r a t i o n or desiccation tolerance has been defined as the equilibruim relative humidity, or water potential, causing 50% killing of the cells (Levitt, 1956, 1964). We also refer to this degree of desiccation as the critical water potential. Specific enzymatic reactions or metabolic processes may also have critical water potentials at which they are severely altered or cease to function. And, if we are attempting to m o d i f y plants for more efficient water use, particularly under
115 a stress c o n d i t i o n , we n e e d s o m e p r a c t i c a l m e a n s o f m e a s u r i n g t h e s e critical levels. An e v a l u a t i o n o f t h e r e l a t i o n b e t w e e n critical levels and general resp o n s e o f t h e p l a n t t o t h e stress is also n e e d e d . T h e m e a s u r e m e n t s m u s t be m a d e in a m a n n e r t h a t can b e used b y t h e p l a n t b r e e d e r , if t h e desired c o m b i n a t i o n s are to be o b t a i n e d . G e n o t y p e d i f f e r e n c e s in r e s p o n s e to w a t e r stress and critical w a t e r p o t e n tials are b e c o m i n g increasingly e v i d e n t in o u r s t u d y o f s o r g h u m . This was shown, f o r e x a m p l e , in one o f o u r e x p e r i m e n t s w i t h t w o s o r g h u m h y b r i d s g r o w n in m i x e d stands in s a n d - n u t r i e n t c u l t u r e in large t a n k s in a g r e e n h o u s e as s h o w n in Fig.2 (Sullivan, 1972). T w o h y b r i d s , RS 6 2 6 , a U.S. regional s o r g h u m , and CSH-1, a s u s p e c t e d d r o u g h t resistant h y b r i d f r o m I n d i a ( H o u s e and R a o , 1966), w e r e p l a n t e d in t w o rows t o t a l i n g 36 plants w i t h e v e r y o t h e r p l a n t t h e s a m e h y b r i d . T h e p u r p o s e of the m i x e d p l a n t i n g was t o i n t e r m i x
Fig.2. Sorghum grown in sand-nutrient cultures. Tanks contained about four inches of roofing gravel in the bottoms with the interspaces of the gravel filled with nutrient at all times when water stress was not desired. t h e r o o t s y s t e m s so t h a t r o o t m o i s t u r e availability w o u l d be the s a m e f o r t h e t w o h y b r i d s . A t a p p r o x i m a t e l y six w e e k s o f age, w a t e r was d i s c o n t i n u e d and w a t e r p o t e n t i a l s w e r e p e r i o d i c a l l y m e a s u r e d on leaf discs o f the s e c o n d and third leaves until t h e average w a t e r p o t e n t i a l o f b o t h h y b r i d s was - 3 3 bar,
116 which was believed to be near the critical level. The tanks were t h e n rewatered and the plants observed for recovery. Table I shows t h a t all o f the CSH-1 plants initially r e c o v e r e d and 90% o f t h e m c o n t i n u e d growth and developm e n t , b u t n o n e o f the RS 626 plants recovered, although t h e y had b o t h reached the same average leaf w a t e r potential. TABLE I Desiccation tolerance of sorghum hybrids CSH-1 and RS 626 Sorghum
Average maximum stress (water potential, bat')
Plants initially recovering after rewatering (%)
Plants continuing growth (%)
CSH-1 RS 626
Desiccation t o l e r a n c e o f RS 626 was obviously m u c h l o w e r than that of CSH-1. B e f o r e desiccation was started, heat t o l e r a n c e tests with leaf discs showed t h a t CSH-1 had significantly higher heat t o l e r a n c e t h a n RS 6 2 6 (Sullivan, 1972). Sullivan et al. ( 1 9 6 8 ) also f o u n d good correlation b e t w e e n field observed d r o u g h t resistance and levels o f h e a t t o l e r a n c e measured on a n u m b e r o f field grown crops, and there have been a n u m b e r of o t h e r r e p o r t s o f positive correlations b e t w e e n heat and desiccation t o l e r a n c e (Julander, 1945; Levitt, 1956, 1972; Kilen and A n d r e w , 1969; Williams et al., 1969). Kaloyereas ( 1 9 5 8 ) related t h e h e a t stability o f c h l o r o p h y l l to d r o u g h t resistance o f pine trees. Table II shows b o t h species and varietal differences in critical desiccation levels o f a n u m b e r o f sorghums and pearl millet. Similarly, Table III shows varietal differences in h e a t t o l e r a n c e of grain sorghums. H e a t and desiccation .tolerance was evaluated b y the electrical c o n d u c t i v i t y m e t h o d with leaf discs (Sullivan and Kinbacher, 1967; K i n b a c h e r et al., 1967; Sullivan et al., 1968; Sullivan, 1972). Critical t e m p e r a t u r e s (50% injury) for a one h o u r e x p o s u r e ranged f r o m 45 ° to 52°C, a d i f f e r e n c e of 7°C. STRESS HARDENING Marked differences have also been f o u n d in the ability of d i f f e r e n t species and varieties to h a r d e n in response to d r o u g h t e x p o s u r e . Levitt et al. ( 1 9 6 0 ) f o u n d t h a t plants t h a t owe their d r o u g h t resistance to m a i n t e n a n c e o f high w a t e r potentials failed to h a r d e n on e x p o s u r e to soil d r o u g h t , including foxtail millet. We have also observed differences in the hardening response o f
117 sorghum varieties upon exposttre to moderate drought, and likewise differences in heat hardening upon exposure to high temperatures (unpublished results). Thus, simple differences in ability to survive or metabolically function when under water stress and to harden when exposed to the stress are gross, and important, considerations to be made in our modification attempts, and they should be measured. When the critical response to stress is established periodic measurements of water potentials in the field will indicate how near the plants are approaching a serious water stress level. T A B L E II Critical w a t e r p o t e n t i a l s (50% recovered f r o m d e s i c c a t i o n ) o f t h e s e c o n d and t h i r d expanded leaves o f field g r o w n s o r g h u m s and pearl millet (all in b l o o m t o grain filling stage; values are m e a n s o f 4 replications) Critical w a t e r p o t e n t i a l (bar)
Sorghum M.35-1 C.K.-60 RS 610 C. 7078 2140 Caprock
-48+3 -36-+1 -36+3 -38-+2 -35-+2 -31+1
Millet H.B.-1 Tift 2 3 A
a* b b b b c
-33-+1 c -29-+2 c
* Values f o l l o w e d by t h e s a m e l e t t e r are n o t significantly d i f f e r e n t at t h e 5% level by D u n c a n ' s new m u l t i p l e range. T A B L E III Heat t o l e r a n c e o f s o r g h u m s as d e t e r m i n e d by t h e c o n d u c t i v i t y m e t h o d w i t h leaf discs* Sampling date
Sorghum: 9084 2140
July 11--20 July 21--31 Aug. 1--10 Aug. 11--20
51.4 -47.6 50.7
-52.0 46.7 49.8
51.6 -48.5 47.9
50.9 50.0 -49.2
50.7 50.4 49.6 --
-51.2 46.2 46.3
-46.2 48.3 46.0
45.0 49.5 46.0 46.0
Aug. 2 1 - - 3 1
* Each value is t h e m e a n o f d u p l i c a t e s a m p l e s o f 20 plants each. The values give the temp e r a t u r e (°C) at 50% injury. ( F r o m Sullivan, 1971.)
118 PROTEIN STABILITY Stability of various enzymes and structural proteins under stress may contribute to differences in stress response. To mention only a few such cases, Todd and Yoo (1964) desiccated wheat leaves over CaC12 and stored turgid controls for the same length of time and found a decrease in phosphatase and peroxidase activity with increased desiccation. Peptidase activity also decreased slightly with drying. Saccarase activity decreased in both desiccated samples and in the stored turgid samples. Huffaker et al. (1970) found that the activities of nitrate reductase and PEP carboxylase decreased in water-stressed barley. However, there was little effect on phosphoribulokinase and ribulose1,5-diphosphate carboxylase. The activities of nitrate reductase and PEP carboxylase recovered completely 24 h after rewatering. Shearman et al. (1972) reported that PEP carboxylase activity of sorghum leaf extracts and isolated chloroplasts remained relatively high as water stress increased, even to - 2 5 atmospheres leaf water potential. Bardzik et al. (1971) found that phenylalanine ammonialyase and nitrate reductase were markedly reduced by water deficits of 10 to 20% in maize, but NADH-oxidase was unaffected by the stress. Protein stability and maintenance of membrane integrity under stress was explained partly by Levitt (1962) in his sulfhydryl-disulfide hypothesis, particularly for frost hardiness, but was also projected to drought and heat tolerance. Gaff (1966) showed that titratable disulfide content of cabbage leaf proteins steadily increased as water stress increased. Levitt (1965) demonstrated that intermolecular disulfide bonds occurred when thiolated gelatin was dehydrated. Sullivan and Kinbacher (1967) showed that disulfide bonds of Fraction [ protein, or crude carboxydismutase, extracted from Tepary bean leaves repositioned during heat hardening in such a manner as to increase the stability of the extracted protein to heat denaturation. It was further found that when the disulfide bonds were reduced, or broken with mercaptoethanol, that the stability of the hardened reverted to that of the unhardened. Sullivan et al. (1968) further showed that this drought resistant Tepary bean, originating from the southwest U.S. and Mexico, had greater heat tolerance than some other commercially grown dry bean varieties. Similarly Kinbacher et al. (1967) reported that purified malic dehydrogenase extracted from heat hardened Tepary bean leaves had greater thermal stability than that from unhardened plants. Again heat and drought tolerance were related. There have been numerous reports of protein breakdown accompanying drought stress dating back to the 1920s and 1930s (Mothes, 1928; Petrie and Wood, 1938). Wilson (1968) found increased protein breakdown and accelerated leaf senescence in maize with increased water stress. Sullivan and Levitt (1959) reported that the tip half of excised succulent leaves increased in soluble nitrogen and decreased in protein or insoluble nitrogen with the
119 soluble nitrogen transported to the base of the leaves where it was apparently resynthesized in callus tissue and root growth. However, evidence by others indicates that the more resistant may retain their ability to synthesize protein, or that water stress impedes protein synthesis (Hsiao, 1970; Sturani et al., 1968). Todd and Basler (1965) concluded in their studies with wheat protoplasmic constituents that drought injury was more likely due to destruction of cellular components by hydrolytic enzymes than by coagulation of proteins. Regardless of the mechanisms, it appears that cytoplasmic proteins are more stable to denaturation, coagulation, or hydrolysis in plants more resistant to water stress, and certain enzymes may be leas susceptible to inactivation by the stress, while others are apparently unaffected, at least until extreme stress occurs. Our test for desiccation and heat tolerance by the electrical conductivity method (Sullivan, 1972), modified from Dexter et al. (1930), is presumably based on cellular membrane degradation with resulting exosmosis of soluble electrolytes. As previously indicated, a positive correlation was found between this test and stability of a relatively pure soluble leaf protein and with a purified enzyme. PHOTOSYNTHESIS In crop production, our attention is usually not directed toward survival mechanisms, but primarily to growth and yield. Therefore, the effects of water stress on assimilation processes, particularly photosynthesis, have received considerable attention. Schneider and Childers ( 1941 ), Gaastra ( 1959), Brix (1962), Troughton and Slatyer (1969), Boyer (1970), to mention a few, concluded that decreased photosynthesis with increased water stress was due primarily to increased stomatal diffusive resistance. Slatyer (1969) in reviewing the literature to date concluded that the evidence was increasing in favor of stomatal closure as the primary mechanism by which stress leads to reduced net photosynthesis. He also noted that stomatal closure generally leads to increased leaf temperatures, and that indirect effects of water stress on photosynthesis through this means may outweigh the direct effects of dehydration. On the other hand, various attempts have been made to determine whether there are direct effects of water stress on photosynthesis at the sight of CO2 fixation and reduction. Plaut (1971) working with isolated spinach chloroplasts reduced the osmotic potential with sorbitol and found that the combined CO2 fixation activities of ribose-5-isomerase, ribose-5-kinase, and ribulose-l,5-dicarboxylase were decreased, but NADP reduction and. therefore, oxygen evolution were not decreased. However, as mentioned previously, Huffaker et al. (1970) showed little effect o f water stress on the activity of phosphoribulokinase and ribulose-l,5-diphosphate carboxylase in barley, although, PEP carboxylase decreased with increased stress. Shearman et al.
120 (1972) failed to find a r e d u c t i o n in PEP c a r b o x y l a s e in isolated s o r g h u m chloroplasts. We f o u n d t h a t e n d o g e n o u s o x y g e n e v o l u t i o n b y cell free s o r g h u m leaf h o m o g e n a t e s was r e d u c e d f r o m 15 t o 49% b y decreasing t h e o s m o t i c p o t e n tial f r o m - 5 . 4 to - 1 1 . 4 bar w i t h C a r b o w a x 600, and f u r t h e r r e d u c e d b y 58 to 92% b y decreasing the o s m o t i c p o t e n t i a l t o - - 3 1 bar, as s h o w n in T a b l e IV. F o r this p r e p a r a t i o n 10 g fresh weight o f leaves were h o m o g e n i z e d with a razor blade e q u i p p e d O m i n i m i x for 10 sec at 40% of line voltage and 2 × 10 TABLE IV Effects of decreasing osmotic potential on the photosynthetic activity (oxygen evolution; moles 02 mg-1 chlorophyll h-~ ) of cell free extracts of six sorghum types Osmotic potent. (bar)
5.,i -11.4 % reduc. -31.1 % reduc.
65.5 55.7 15.0 27.2 58.5
59.2 46.8 20.9 20.6 65.2
77.6 50.6 34.8 32.9 57.6
74.1 ¢8.6 34.4 28.6 61..1
139.2 71.3 48.8 11.7 91.6
131.3 86.9 33.8 50.4 61.6
sec at full speed in 50 ml o f 0.05M H E P E S b u f f e r , p H 6.8, 0 . 0 0 1 M MgC12, 0 . 0 0 2 M edta, and C a r b o w a x 600 to give the desired o s m o t i c value. T h e h o m o g e n a t e was filtered t h r o u g h 8 layers of cheese-cloth. T h e r e was no e l e c t r o n a c c e p t o r or d o n o r added in t h e s e e x p e r i m e n t s , and since Walter et al. ( 1 9 6 8 ) have s h o w n t h a t the ratio of CO2 f i x a t i o n to o x y g e n e v o l u t i o n is u n i t y in intact chloroplasts, it is suggested t h a t CO2 f i x a t i o n was similarly a f f e c t e d in these e x p e r i m e n t s . Santarius and H e b e r (1967), Santarius and Earnst (1967), and Santarius ( 1 9 6 7 ) f o u n d t h a t ATP synthesis, N A D P r e d u c t i o n , and P G A r e d u c t i o n were n o t r e d u c e d b y d e s i c c a t i o n until 50 to 90% o f t h e p l a n t w a t e r was lost, and the Hill reaction was scarcely affected. Nir and P o l j a k o f f - M a y b e r ( 1 9 6 7 ) w o r k i n g with c h l o r o p l a s t s isolated f r o m swiss chard f o u n d 32 and 85% reduct i o n in the Hill r e a c t i o n w h e n 29 and 61%, respectively, of t h e leaf w a t e r was lost. P h o t o p h o s p h o r y l a t i o n was also r e d u c e d b y the stress t r e a t m e n t . B o y e r and B o w e n ( 1 9 7 0 ) r e p o r t e d i n h i b i t i o n of o x y g e n e v o l u t i o n with p e a and sunflower c h l o r o p l a s t s even at m o d e r a t e stress. F r y ( 1 9 7 0 ) w o r k i n g w i t h chloroplasts isolated f r o m c o t t o n plants s h o w e d t h a t the Hill r e a c t i o n was r e d u c e d b e t w e e n 32 and 55% at - 2 8 bar w a t e r p o t e n t i a l , b u t T o d d and Basler ( 1 9 6 5 ) failed to find a r e d u c t i o n in the Hill r e a c t i o n o f c h l o r o p l a s t s isolated f r o m w a t e r stressed w h e a t plants. H o w e v e r , in n u m e r o u s e x p e r i m e n t s with s o r g h u m , c o r n and pearl millet
121 we have failed to find a r e d u c t i o n in the Hill r e a c t i o n with chloroplasts isolated f r o m plants grown and d r o u g h t e d naturally in the soil (Sullivan and Eastin, 1969). Table V shows t h e results o f several e x p e r i m e n t s w i t h sorghum. T h e d i f f e r e n c e in results o b t a i n e d b y d i f f e r e n t investigators m a y be d u e to the p l a n t material or t o p r o c e d u r e s , b u t in c o m p a r i n g the results we have TABLE V Effects of drought on the Hill reaction by chloroplasts isolated from grain sorghum (field grown plants)* 0 2 evolution (g-moles per mg chlo. per h)
Leaf water potentials (bar)
30.4 38.5 38.1 29.9 26.3 30.2
33.5 37.4 39.9 29.9 15.0 30.8
- 7.5 - 7.5 -12o0 -11.8 -13.8 -12.1
-19.8 -20.2 -22.4 -16.2 -18.3 -24.7
* All values are means of duplicate determinations. n o t e d t h a t t h o s e r e p o r t i n g an i m p a i r m e n t o f the Hill r e a c t i o n by d r o u g h t i n g usually o b t a i n e d t h e i r results with p o t t e d plants or desiccated excised leaves. In e i t h e r case, desiccation was r a t h e r rapid. We t h e n c o n d u c t e d an experim e n t with excised leaves f r o m field grown plants, desiccated at c o n s t a n t relative h u m i d i t y in a g r o w t h c h a m b e r , and as s h o w n in Table VI we also f o u n d a r e d u c t i o n in the Hill reaction w h e n t h e r e was a rapid decrease in w a t e r potential. This response suggests t h a t r e d u c t i o n in p h o t o s y n t h e s i s by i n t a c t plants m a y be influenced b y the previous t r e a t m e n t o f the plants, and some c a u t i o n is p r o b a b l y in o r d e r in i n t e r p r e t i n g absolute values o f w a t e r p o t e n t i a l s causing r e d u c t i o n in p h o t o s y n t h e s i s . F o r e x a m p l e , B o y e r ( 1 9 7 0 ) f o u n d p h o t o s y n thesis in intact corn plants decreased at leaf w a t e r p o t e n t i a l s below - 3 . 5 bar. A l t h o u g h s o y b e a n s were u n a f f e c t e d until w a t e r p o t e n t i a l s were below - 1 1 bar. Plants were g r o w n in well w a t e r e d pots in g r o w t h c h a m b e r s at 29°C (days) and 21°C (nights) at a b o u t o n e - f o u r t h full sunlight and 70% relative h u m i d i t y . Blum and Sullivan ( 1 9 7 2 ) f o u n d decreased p h o t o s y n t h e s i s at - 5 to - 6 bar leaf w a t e r p o t e n t i a l with leaf sections f r o m seven sorghum varieties u n d e r c o n t r o l l e d e n v i r o n m e n t a l c o n d i t i o n s and with high light intensities. T h e leaves were t a k e n f r o m irrigated field grown plants. Desiccation o f t h e leaf sections was c o n t r o l l e d b y lbwering the a t m o s p h e r i c h u m i d i t y , and was r a t h e r rapid. S h e a r m a n et al. ( 1 9 7 2 ) , o n the o t h e r h a n d , f o u n d net c a r b o n d i o x i d e exchange in intact s o r g h u m plants r e m a i n e d at a fairly steady state
122 until a stress of about - 1 9 to - 2 0 bar leaf water potential occurred. In their case, plants were grown in large pots under relatively low light intensity and desiccation was rather slow. Hultquist (1973), also working with our group at Nebraska, had similar results with sorghum grown in tanks in a greenhouse. Photosynthesis at the floret initiation stage did not decrease until about - 1 5 bar leaf water potential and the compensation point was reached at about -20 bar. T A B L E VI E f f e c t s o f r e d u c e d leaf w a t e r p o t e n t i a l s o n t h e Hill r e a c t i o n by c h l o r o p l a s t s isolated f r o m grain s o r g h u m * Hrs. desiccated
q~ t, (bar)
02 e v o l u t i o n ( u - m o l e s m g-t chlo. h - l )
0 1 2 3
- 2.1 -22.0 -38.2 -45.8
33.1 27.2 25.1 16.0
* E x c i s e d leaves d e s i c c a t e d at 40% R.H., 25°C.
Downton and Slatyer (1972) showed that the optimum temperature for photosynthesis by cotton plants depended on the daytime temperature in which they had been grown. And, the light intensity in which plants are grown effects their photosynthesis response to subsequent light intensities (Bj5rkman, 1972). Smith (1973) found an increase in environmental temperature of three weeks old sorghum seedlings from 25 ° to 43°C for an average of 4 h per day, for 4 consecutive days, resulted in an average of 7% significant increase in yield of 4 varieties over controls treated the same except they did not receive this small temperature increase for a few hours in the early age of their life. There was no significant difference in bloom date or other morphological differences that could be detected between the treated and control plants. In view of these responses to previous temperature and light regimes, it is reasonable to expect similar effects of previous water potential status on photosynthesis and other physiological responses to water stress. Blum and Sullivan (1972) showed that there are distinct varietal differences in sorghum in response of photosynthesis to water stress with leaf sections. Photosynthesis by a drought resistant sorghum from India, M.35-1, was reduced less at a lower leaf water potential than a U.S. hybrid, RS 610, also known somewhat for its drought resistance. Previous desiccation and heat tolerance tests with leaf discs showed that the variety M.35-1 had greater tolerance than RS 610 (Sullivan and Blum, 1970). The question is still unanswered, however, as to what extent and under what conditions water stress affects the photochemical apparatus directly, and to what extent diffusive resistance is responsible for the decrease. Although Redshaw and Meidner
123 (1972) recently concluded that stomatal resistance could account for only one-half of the reduction in photosynthesis accompanying water stress in tobacco. STOMATAL RESPONSE It is known that some species close their stomata sooner at the onset of water stress than others and this is u n d o u b t e d l y a water-conserving mechanism. Many of the millets are known for their drought resistance, and Sullivan and Blum (1970) reported that pearl millets under water stress closed their stomata at significantly higher water potentials than several sorghums. Pallas and Bertrand (1966), Sanchez-Diaz et al. (1969), and Sanchez-Diaz and Kramer (1971) showed that during soil drying the stomata of corn closed at higher water potentials than those of sorghum, but corn lost a much greater percentage of the total leaf water at higher leaf water potentials. Cuticular diffusive resistance was greater in sorghum than in corn (Sanchez-Diaz et al., 1969}. One might expect leaf temperatures to be higher when the stomata are closed, and to tolerate higher temperatures the plants must need greater heat tolerance mechanisms. Pearl millet and corn were found to have greater heat tolerance than sorghum (Sullivan, 1972), which was in line with our prediction. Sullivan (1972) and Blum (A. Blum, personal communication, 1970) independently found that sorghum stomata closed when plants were exposed to water stress for the first time but failed to close completely when droughted subsequently to an initial soil drying and rewatering cycle. Glover (1959) reported that sorghum stomata remained slightly open all day during a severe stress, whereas corn stomata were open only for a short period in the morning. Stomatal activity also returned to normal quicker in sorghum than in corn following rewatering. Fischer et al. (1970) reported that stomatal opening of previously desiccated tobacco and broad bean was depressed in light compared to nondesiccated controls and complete recovery required 2 to 5 days after rewatering. Thus, in evaluating stomatal response to water stress it is also necessary to be aware of the previous growth conditions. The response of abscisic acid (ABA) accumulation in water stressed leaves and its effect on stomatal opening is w o r t h y of mention at this time (Wright, 1969, 1972; Wright and Hiron, 1969, 1972). It has been shown that endogenous ABA content may increase with increased stress and concomitant with increased diffusion resistance, and upon rewatering the ABA level may decline slowly, in some cases over a period of several days. The stomata apparently do not function normally until the ABA content of the leaves returns to its original level. STAGE OF GROWTH It is known that responses to water stress may also change with stages of growth and maturity (Salter and Goode, 1967). Sullivan (1972) found that
124 leaf tissue of several sorghum varieties was less susceptible to desiccation injury at severe water stress levels (about - 3 3 bar) prior to anthesis than at the post-anthesis stage. Similarly, Hultquist (1973) recently found that sorghum stomata lost ability to respond to reduced leaf water potentials at the bloom stage. At this stage photosynthesis continued relatively unimpaired until about -24 bar leaf water potential, but further desiccation resulted in sudden death of the plants, presumably when water potentials dropped below the cellular critical point. He also found that two hybrids responded differently in their apparent efforts to keep the developing inflorescence alive during water stress. As shown by ~4C labeling, one hybrid (RS 626) translocated a greater percent of the photosynthate from the leaves to the lower part of the shoot and roots, whereas, the other hybrid (DeKalb C42y) transported more to the developing panicle. This pattern continued in the latter hybrid until stress became so severe that the whole plant died. In RS 626 part of the developing panicle sometimes died during the stress, but upon rewatering the peduncle would often exert with a partial panicle continuing to develop. This partial panicle development never occurred with the hybrid C42y. Eastin (1972) found with a number of grain sorghum hybrids grown under dryland conditions that the average grain filling stage was reduced by 19.5% and the average yield reduced by 24.5%. Under near normal conditions about one-third to two-thirds of the variability in yield related to the length of grain filling period, but under severe stress the r 2 values for both grain filling stage and yield and yield and days to half-bloom was nonsignificant. It appeared that plant efficiency or assimilation rate factors became more important and the time factor diminished in importance as water stress increased. APPLICATION OF DESICCATION AND HEAT TOLERANCE TESTS Information gained from results of drought and heat tolerance tests, such as the leaf disc technique mentioned in this paper, has led us to the conclusion that it is possible and practical to select for germplasm with desirable stress tolerance characteristics. An example of its applicability is the results of Jurado-Tovar (1972). He selected individual corn plants by the leaf disc method for high and low heat tolerance from a cross between Hays Golden and Latente, two relatively drought resistant varieties. The following year seed from the selected plants was planted and the plants tested for both desiccation and heat tolerance. Significant differences in both heat and drought tolerance were found between the high and low selections. Measurements of chlorophyll stability index also correlated well with desiccation and heat tolerance as determined by the disc method. Jensen (1971), also using the leaf disc heat tolerance method, reported large and significant differences between selected cool and warm season genotype corn lines and also in crosses between these lines. These encouraging results show that we can manipulate these genetically controlled characteristics in some cases even in one year of selection.
125 W h i l e w e h a v e t o u c h e d o n j u s t a few p h y s i o l o g i c a l r e s p o n s e s t o w a t e r stress a n d failed t o e v e n m e n t i o n m a n y o t h e r i m p o r t a n t o n e s , w e h a v e a t t e m p t e d t o s h o w t h a t t h e r e are s o m e r e s p o n s e s t h a t c a n b e m e a s u r e d a n d selected which will p e r m i t m o d i f i c a t i o n of plants to the m o s t desirable p h y s i o l o g i c a l r e s p o n s e s t o w a t e r stress.
REFERENCES Bardzik, J. M., Marsh Jr., H. V. and Havis, J. R., :1971. Effects of water stress on the activities of three enzymes in maize seedlings. Plant Physiol., 47:828--831. BjSrkman, O., 1972. Photosynthetic adaptation to contrasting light climates. In: Carnegie Inst. Yearb. 71, Annual report of the Director, Department of Plant Biology, Stanford, pp.82--85. Blum, A. and Sullivan, C. Y., 1972. A laboratory method for monitoring net photosynthesis in leaf segments under controlled water stress. Experiments with sorghum. Photosynthetica, 6 : 18--23. Boyer, J. S., 1970. Differing sensitivity of photosynthesis to low leaf water potentials in corn and soybean. Plant Physiol., 46:236--239. Boyer, J. S. and Bowen, Barbara L., 1970. Inhibition of oxygen evolution in chloroplasts isolated from leaves with low water potentials. Plant Physiol., 45:612--615. Brix, H., 1962. The effect of water stress on the rates of photosynthesis and respiration in tomato plants and loblolly pine seedlings. Physiol. Plant., 15:10--20. Dexter, S. T., Tottingham, W. E. and Graber, L. F., 1930. Preliminary results in measuring the hardiness of plants. Plant Physiol., 5:215--223. Downton, J. and Slatyer, R. O., 1972. Temperature dependence of photosynthesis in cotton. Plant Physiol., 50:518--522. Eastin, J. D., 1972. Efficiency of grain dry matter accumulation in grain sorghum. Proc. Ann. Corn Sorghum Res. Conf., 27th, Chicago, pp.7--17. Fischer, R. A., Hsiao, T. C. and Hagan, R. M., 1970. After-effect of water stress on stomatal opening potential. J. Exp. Bot., 21 : 371--404. Fry, K. E., 1970. Some factors affecting the Hill reaction activity in cotton chloroplasts. Plant Physiol., 45:465--469. Gaastra, P., 1959. Photosynthesis of crop plants as influenced by light, carbon dioxide, temperature, and stomatal diffusion resistance. Meded. Landbouwhogesch. (Wageningen), 59:1--68. Gaff, D. F., 1966. The sulfhydryl-disulfide hypothesis in relation to desiccation injury of cabbage leaves. Aust. J. Biol. Sci., 19:291--299. Glover, J., 1959. The apparent behaviour of maize and sorghum stomata during and after drought. J. Agric. Sci. (London), 153:412--416. House, L. R. and Rao, N. G. P., 1966. Breeding hybrid sorghums in India. Indian J. Genet., 26A:86--97. Hsiao, T. C., 1970. Rapid changes in levels of polyribosomes in Zea mays in response to water stress. Plant Physiol., 46 : 281--285. Huffaker, R. C., Radin, T., Kleinkopf, G. E. and Cox, E. L., 1970. Effects of mild water stress on enzymes of nitrate assimilation and of the carboxylative phase of photosynthesis in barley. Crop Sci., 10:471--474. Hultquist, J. H., 1973. Physiologic and Morphologic Investigations of Grain Sorghum (Sorghum bicolor (L.) Moench), I. Vascularization: II. Response to Internal Drought Stress. Ph.D. thesis, University of Nebraska, Lincoln, Nebr., 140 pp. Jensen, S. D., 1971. Breeding for drought and heat tolerance in corn. Proc. Ann. Corn Sorghum Res. Conf., 26th, Chicago, pp.198--208.
126 Julander, O., 1945. Drought resistance in range and pasture grasses. Plant Physiol., 20: 573--599. Jurado-Tovar, A., 1972. Application of Laboratory Techniques for Selection of Heat and Drought Tolerance in a Synthetic Population of Corn. M. S. Thesis, The University of Nebraska, Lincoln, Nebr., 46 pp. Kaloyereas, S. A., 1958. A new method of determining drought resistance. Plant Physiol., 33:232--233. Kilen, T. C. and Andrew, R. H., 1969. Measurement of drought resistance in corn. Agron. J., 61:669--672. Kinbacher, E. J., Sullivan, C. Y. and Knull, H. R., 1967. Thermal stability of malic dehydrogenase from heat hardened Phaseolus acutifolius "Tepary Buff". Crop Sci., 7:148 --151. Levitt, J., 1956. Hardiness of Plants. Academic Press, New York and London, 278 pp. Levitt, J., 1962. A sulfhydryl-disulfide hypothesis of frost injury and resistance in plants. J. Theoret. Biol., 3:355--391. Levitt, J., 1964. Drought. In: Forage Plant Physiology and Soil Range Relationships. American Society of Agronomy, Madison, Wise., pp.57--66. Levitt, J., 1965. Thiogel -- a model system for demonstrating intermolecular disulfide bond formation on freezing. Cryobiology, 1 : 312--316. Levitt, J., 1972. Responses of Plants to Environmental Stresses. Academic Press, New York and London, 697 pp. Levitt, 3., Sullivan, C. Y. and Krull, E., 1960. Some problems in drought resistance. Bull. Res. Conc. Israel, 8D:173--180. Mothes, K., 1928. Die Wirkung des Wassermangels auf den Eiweissumsatz in hoheren Pflanzen. Ber. Deut. Botan. Ges., 46:59--67. Nit, I. and Poljakoff-Mayber, A., 1967. Effect of water stress on the photochemical activity of chloroplasts. Nature, 213:418--419. Pallas, d. E. and Bertrand, A. R., 1966. Research in plant transpiration: 1963. Prod. Res. Rept. No.89, ARS, USDA, Georgia Agric. Expt. Sta. and Meteorol. Dept. U.S. Army Elect. Res. Dev. Activ., 25 pp. Petrie, A. H. K. and Wood, J. G., 1938. Studies on the nitrogen metabolism of plants, I. The relation between the content of proteins, amino acids and water in the leaves. Ann. Bot. (London), 2:33--60. Plaut, Z., 1971. Inhibition of photosynthetic carbon dioxide fixation in isolated spinach chloroplasts exposed to reduced osmotic potentials. Plant Physiol., 48:591--595. Redshaw, A. J. and Meidner, H., 1972. Effects of water stress on the resistance to uptake of carbon dioxide in tobacco. J. Exp. Bot., 23:229--240":. Salter, P. J. and Goode, J. E., 1967. Crop Responses to Water at Different Stages of Growth. Commonwealth Agricultural Bureaux, Farnham Royal, Bucks, England, 246 pp. Sanchez-Diaz, M. F. and Kramer, P. J., 1971. Behavior of corn and sorghum under water stress and during recovery. Plant Physiol., 48:613--616. Sanchez-Diaz, M. F., Morey, M. and Gonzalez-Bernaldez, 1969. Reaeciones fisiologicas de las hojas al aire mediante el uso de porometros. An. Edaf. Agrobiol., 28:747--754. Santarius, K. A., 1967. Das Verhalten yon CO 2 -Assimilation, NADP- und PGS-Reduktion und ATP-Synthese intakter Blattzellen in Abh~/ngigkeit yore Wassergehalt. Planta, 7 3: 228--242. Santarius, K. A. and Ernst, R., 1967. Das Verhalten yon Hill-Reaktion und Photophosphorylierung Isolierter Chloroplasten in Abh~ngigkeit vom Wassergehalt. I. Wasserentzug mittels konzentrierter LSsungen. Planta, 73:91--108. Santarius, K. A. and Heber, U., 1967. Das Verhalten yon Hill-Reaktion und Photophosphorylierung Isolierter Chloroplasten in Abh~/ngigkeit vom Wassergehalt, II. Wasserentzug fiber CaCl 2 . Planta, 73:109--137. Schneider, G. W. and Childers, N. F., 1941. Influence of soil moisture on photosynthesis, respiration, and transpiration of apple leaves. Plant Physiol., 16:565--583.
127 Shearman, L. L., Eastin, J. D., Sullivan, C. Y. and Kinbacher, E. J., 1972. Carbon dioxide exchange in water stressed sorghum. Crop Sci., 12:406--409. Slatyer, R. O., 1969. Physiological significance of internal water relations to crop yield. In: J. D. Eastin, F. A. Haskins, C. Y. Sullivan and C. H. M. van Bavel (Editors), Physiological Aspects of Crop Yield. Am. Soc. Agron., Crop Sci. Soc. Am., Madison, pp.53-83. Smith, D. H., 1973. Selected studies of heat tolerance in grain sorghum (Sorghum bicolor (L.) Moench). M. S. Thesis, University of Nebr., Lincoln, Nebr., 46 pp. Sturani, E., Cocucci, S. and Marre, E., 1968. Hydration dependent polysome-monosome interconversion in the germinating castor bean endosperm. Plant Cell Physiol., 9:783-795. Sullivan, Y., 1972. Mechanisms of heat and drought resistance in grain sorghum and methods of measurement. In: N. G. P. Rao and L. R. House (Editors), Sorghum in Seventies. Oxford and IBH Publishing Co., New Delhi, India, pp.247--264. Sullivan, C. Y. and Blum, A., 1970. Drought and heat resistance of sorghum and corn. Proc. Ann. Corn Sorghum Res. Conf., 25th, Am. Seed Trade Assoc., Wash. D.C., pp. 55--66. Sullivan, C. Y. and Eastin, J. D., 1969. Effects of heat and drought on the photosynthetic activity of isolated chloroplasts from sorghum, corn and pearl millet. Agron. Abstr. Detroit, p.30. Sullivan, C. Y. and Kinbacher, E. J., 1967. Thermal stability of fraction I protein from heat hardened Phaseolus acutifolius Gray "Tepary Buff". Crop Sci., 7:241--244. Sullivan, C. Y. and Levitt, J., 1959. Nitrogen metabolism as related to drought resistance of Sempervivum glaucum. Proc. Int. Bot. Congr., 9th, Montreal, pp.387--388. Sullivan, C. Y., Eastin, J. D. and Kinbacher, E. J., 1968. Finding the key to heat and drought resistance in grain sorghum. Q. Univ. Nebr., Lincoln, Nebr., Summer Issue 1968. Todd, G. W. and Basler, E., 1965. Fate of various protoplasmic constituents in droughted wheat plants. FOTON, 22:79--85. Todd, G. W. and Yoo, B. Y., 1964. Enzymatic changes in detached wheat leaves as affected by water stress. FOTON, 21:61--68. Troughton, J. H. and Slatyer, R. O., 1969. Plant water status, leaf temperature, and the calculated mesophyll resistance to carbon dioxide of cotton leaves. Aust. J. Biol. Sci., 22:815--828. Walter, D. A., Baldry, C. W. and Cockburn, W., 1968. Photosynthesis by isolated chloroplasts, simultaneous measurement of carbon assimilation and oxygen evolution. Plant Physiol., 43:1419--1422. Williams, T. V., Snell, R. S. and Cress, C. E., 1969. Inheritance of drought tolerance in sweet corn. Crop Sci., 9:19--23. Wilson, J. H., 1968. Water relations of maize, I. Effects of severe soil moisture stress imposed at different stages of growth on grain yields of maize. Rhod. J. Agric. Res., 6:103--105. Wright, S. T. C., 1969. An increase in the "inhibitor-~" content of detached wheat leaves following a period of wilting. Planta (Berl.), 86:10--20. Wright, S. T. C., 1972. Physiological and biochemical responses to wilting and other stress conditions. In: A. R. Rees, K. E. Cockshull, D. W. Hand and R. G. Hurd (Editors), Crop Processes in Controlled Environments. Applied Botany Series, 2. Acad. Press, London, New York, pp.349--361. Wright, S. T. C. and Hiron, R. W. P., 1969. (+)-Abscisic acid, the growth inhibitor induced in detached wheat leaves by a period of wilting. Nature, 224:719--720~ Wright, S. T. C. and Hiron, R. W. P., 1972. The accumulation of abscisic acid in plants during wilting and under other stress conditions. In: D. J. Carr (Editor), Plant Growth Substances. Springer-Verlag, Berlin, Heidelberg and New York, pp.291--298.