Osmoregulation in Cotton in Response to Water Stress' I

Plant Physiol. (1981) 67, 484 488 0032-0889/81/67/0484/05/$00.50/0

Osmoregulation in Cotton in Response to Water Stress' I. ALTERATIONS IN PHOTOSYNTHESIS, LEAF CONDUCTANCE, TRANSLOCATION, AND ULTRASTRUCTURE

Received for publication June 13, 1980 and in revised form October 13, 1980

ROBERT C. ACKERSON AND RICHARD R. HEBERT Central Research and Development Department, Experimental Station, E. L du Pont de Nemours and Company, Wilmington, Delaware 19898

ABSTRACT paper in this series defines the role of specific cellular carbohy- drates in stress adaptation (2). Cotton plants subjected to a series of water deficits exhibited stress adaptation In the form of osmoregulation when plants were subjected to a subsequent drying cycle. After adaptation, the leaf water potential coincid- MATERIALS AND METHODS ing with zero turgor was considerably lower than in plants that had never Plant Material. Cotton (Gossypium hirsutum L. Tamcot SP37) experienced a water stress. The relationship between leaf turgor and leaf plants were grown from seed in controlled environment facilities water potential depnded O leaf age. as described (3). When the fifth leaf above the cotyledons was Nonstomatal factors severely limited photosynthesis in adapted plants about 75% expanded, one set of plants was subjected to repetitive at high leaf water potential. Nonetheless, adapted plants maintained pho- water stress cycles while another set was well watered. Each stress tosyPnthesis to a much lower leaf water potential than did control plants, in cycle consisted of allowing plants to dehydrate until midday leaf part because of increased stomatal conductance at low leaf water poten- water potentials reached approximately -20 bars. Dehydration tials. Furthermore, adapted plants continued to translocate recently derived required 24 to 48 h, depending on plant age. Five days ofrecovery photosynthate to lower leafwater potentials, compared with control plants. (plants well watered) were interspersed between successive stress Stress preconditioning modffied cellular ultrastructure. Chloroplasts of cycles. Plants were subjected to a total of five such cycles. Midday fully turgid adapted leaves contained extremely large starch granules, leaf water potentials of control plants ranged from -5 to -8 bars. seemed swollen, and had some breakdown of thylakoid membrane struc- Plants that had been subjected to the five stress cycles are referred ture. In addition, cells of adapted leaves appeared to have smaller vacuoles to as "adapted" plants. This should be construed as referring to and greater nonosmotic cell volume than did control plants. "phenotypic" adaptation, rather than genotypic. Control plants had never been stressed. Five days after the last stress cycle, all plants were subjected to stress. Data were obtained during this dehydration period from leaves at nodes 5 and 8. Leaves at node 5 were 75% expanded at the time of the first stress cycle, whereas the leaves at node 8 were just emerging. After completion of the last stress cycle, the areas Osmoregulation enables plants to withstand temporary or sus- of the leaves at node 5 were similar in adapted and nonadapted tained water deficits (19). Although cellular mechanisms that plants. In adapted plants, the leaves at node 8 were approximately induce or promote osmoregulation are unknown, solute accumu- one-half the size of equivalent leaves in control plants. lation within the cell seems to play a central role in the adaptive Water Potential, Leaf Conductance, and Photosynthesis. Leaf process (10-12, 19, 31, 32). In stress-adapted plants, lowering of water potentials and leaf conductances were obtained using iso- cellular osmotic potential through accumulation of osmotica per- piestic thermocouple psychometry and diffusion porometry as mits turgor maintenance at relatively low leaf water potentials (1, previously described (3). Osmotic potentials were determined 10, 11, 14, 16, 19-21, 31, 32). psychometrically on leaf discs (1.0 cm diameter), frozen in liquid Although many plants partially adapt to water deficits by N2, and thawed. Turgor pressure was calculated as the difference osmoregulation, thereby maintaining some growth during between total leaf water potential and osmotic potential. Osmotic drought, adaptation is normally associated with reduced growth potentials were not corrected for the dilution of cell sap with and productivity (9, 19). Two physiological processes necessary apoplastic water that occurs during freezing (22). Leaf resistances for growth and productivity are photosynthesis and translocation. were converted to conductances by taking the reciprocal of total Both are important in generation and distribution of osmotically leafresistance obtained by assuming individual surface resistances active solutes. In most plants, water stress reduces photosynthesis act in parallel. (6) and movement of assimilates out of the leaf (24, 28, 33, 34). Apparent photosynthetic rates were determined using the CO2 The purpose of the present study was to examine the process of pulse-labeling technique described by Naylor and Teare (26). osmoregulation in cotton focusing on photosynthesis, transloca- Translocation. Sections of intact plants consisting of the vege- tion, and leafcarbohydrate status and their responses to leafwater tative apex and four to five monopodial and two to three sympo- status. dial branches were excised under water and the stems were placed In this paper we describe photosynthesis, translocation, and in distilled H20. The plants were enclosed in a Plexiglas chamber cellular ultrastructure in relation to osmoregulation. A companion containing a fan to facilitate mixing, and air containing about 320 PI 1-' CO2 was passed through the chamber (open system). Dew I Contribution 2803 from the Central Research and Development De- point of the air was adjusted to between 24 and 26 C. Plants were partment, E. I. du Pont de Nemours and Co. allowed to photosynthesize for 1 h at 27 C with PAR of 850 ,IE 484 Plant Physiol. Vol. 67, 1981 WATER STRESS ADAPTATION IN COTTON 485 m-2 s-' provided by a Xenon arc lamp. This PAR was equivalent to that during the growth period in the controlled environment chamber (3). After 1 h, the chamber was sealed and 5 to 10 ,uCi "CO2 were injected into the chamber. The pulse period was 15 to 20 min, followed by 15 to 20 min chase with air (320,d 1-1). After the chase period, discs from leaves at node 6 or 7 were removed aa and their radioactivity was counted. The percentage ofradioactiv- Ca ity remaining in the leaves after selected time intervals was deter- j mined. In each experiment, translocation was determined in a c fully turgid plant (stems in and a in 2 H20) wilting plant (stems 0.7 ai M mannitol after the pulse and chase periods). Excised, rather - C than intact plants, were used to dehydrate the control and adapted L plants at about the same rate. LLJi Leaf and Cellular Ultrastructure. Leaf samples were fixed in I (,:, 10 phosphate-buffered 5% glutaraldehyde (pH 7.0) for 2 h. Samples UI o CONTROL r = 0.94* * ILA were rinsed with buffer and treated with phosphate-buffered r. 8 * ADAPTED r = 0.88** osmium tetroxide (2%) for 1.5 h. Tissue was dehydrated by a 6- . graded series of ethanol and embedded in Spurr. Thin sections NODE 5 were cut and stained with lead citrate and uranyl acetate. Sections -jJ were examined on a Zeiss EM 10 at 60 kv. 4 2 RESULTS AND DISCUSSION 0 A series of brief water deficits reduced growth of cotton (Fig. 1). After each successive stress cycle the lead conductances offully -2 _ .I control 0 -4 -8 -12 -16 -20 turgid and adapted plants were similar (Fig. 1). Following LEAF WATER the first three stress cycles, photosynthetic rates of adapted plants POTENTIAL, bars were lower than those of control plants. However, immediately FIG. 2. Relationship between leaf water potential and leaf pressure after the last two stress cycles, adapted leaves photosynthesized potential of cotton leaves at the eighth (upper) and fifth (lower) node more rapidly than control leaves. This increase in photosynthesis above the cotyledonary node. Each data point is the mean of two mea- appeared to be transient inasmuch as fully turgid adapted leaves surements and data from two independent experiments were pooled to consistently had lower rates of photosynthesis than control plants derive the relationships. (0), control plants; (0), adapted plants. Asterisks 5 days after the last stress cycle (Fig. 5). Even though stress indicate significance at the 0.01 level. inhibited growth, plants subjected to water stress preconditioning exhibited signs of adaptation when subjected to a subsequent water stress. Similarly, Cutler and Rains (9) demonstrated greater drought tolerance in cotton subjected to limited irrigation than in 1.4 frequently irrigated cotton, even though limited water availability reduced growth. The adaptation exhibited - 12 by stressed plants appeared to be due to osmoregulation, at least with respect to leaves at node 8. This E 0 8 was inferred from the leafturgor-leaf water potential relationships 0.6 in adapted and control leaves (Fig. 2). Adapted, young leaves X E60.4 o CONTROL (node 8) exhibited greater leafpressure potentials than did control leaves at all leaf water potentials, prior to complete loss of turgor 0.2 * ADAPTED A (Fig. 2). Accordingly, adapted, younger leaves must have accu- 01 mulated solutes during the preconditioning phase of the experi- 100 ° ment. Osmoregulation in these young leaves was similar to that CONTROL observed in slowly adapted sorghum (21). In older leaves (node .- 80 - * ADAPTED 5), approximately the same turgor was observed in control and W 60 adapted plants at high leaf water potentials (2-5 bars). During z 40 dehydration, adapted plants sustained greater pressure potentials than did control plants as leaf water potentials declined (Fig. 2). X- 20 8 Consequently, adapted leaves approached zero turgor at lower 0 leaf water potentials than did control leaves. The pressure poten- 08 tial-leafwater potential relationships in these fully expanded older Z70.6- leaves resembled those observed in fully expanded sorghum leaves that had undergone moderate stress in the field (5). At least two a E 0.4-04 ° CONTROL explanations account for the adaptation response in older leaves. °1 0.2 * ADAPTED C First, solutes could accumulate when adapted leaves undergo 0 water stress, even though the solute status of adapted and control 1 2 3 4 5 leaves are similar when leaves are fully turgid. Alternatively, CYCLE OF STRESS solutes in adapted leaves may be concentrated to a greater extent FIG. 1. Influence ofrepetitive stress cycles on photosynthesis (A), plant if adapted leaves have larger nonosmotic cell volumes, as com- height (B), and leaf conductance (C) on stress-adapted (0) and control pared with control leaves. This is typical in drought-adapted cotton plants (0). Data were obtained from leaves at node 5 2 days after cotton (11). Electron micrographs of fully turgid control and plants had recovered from each period of water stress. Each data point adapted leaves (node 5) indicate that starch accumulated in represents the mean ± SD of four determinations. Data are from well- adapted leaves (Fig. 3). Based on measurements of 75 to 100 cells, watered plants with soil at about "field capacity" in all cases. adapted leaves at node 5 had about 30 to 40o less osmotic volume 486 ACKERSON AND HEBERT Plant Physiol. Vol. 67, 1981

0.8 o CONTROL r .59* 0.6 0 * ADAPTED r=.78** 0 0 0.4 0 05 0.2 NODE 8 0 0 0 0 E c @00 0 U I 0. 0 -4 -8 -12 -16 -20 -24 z 4 I- , . ... . 0.8 - NODE 5 0 CONTROL r -.86** z 0.6 - * *ADAPTED r:.81** - 0 S C. 0.4- 0 0.2- 0 0 _0.0 0 -4 -8 -12 -16 -20 -24 LEAF WATER POTENTIAL, bors FIG. 4. Relationship between leafwater potential and leafconductance of adapted (0) and control (0) cotton leaves at the eighth (upper) and fifth (lower) node above the cotyledonary node. Each conductance value represents the mean of five to six measurements obtained from two experiments. Asterisks indicate significance at the 0.01 (**) or 0.05 (*) level.

1.2 0 0 CONTROL 1.0 0 * ADAPTED

I 0.8 * 0 0 "' 0.6 *2X 0 E 0.4 NODIE 8 * 0.2 0 0

E 0- .. . .I.I c 0 -4 -8 -12 -16 -20 a w 1.2 z 1.0 NODE 5 0 CONTROL C') ADAPTED 0.8- *~~~~ 3' 0.6- FIG. 3. Transmission electron micrographs of control (upper) and 0.4 *-0 adapted (lower) cotton leaves. Micrographs are from paradermal sections. -~~~~~~~ Magnification is x 11,250. Vac, vacuole; C, chloroplast; SG, starch granule. 0.20.2 per cell than control leaves (based on total cell volume and vacuole 0 -4 -8 -12 -16 -20 volume). A substantial portion of the nonosmotic volume may LEAF WATER POTENTIAL, bars well have been occupied by starch granules inasmuch as estimates FIG. 5. Relationship between leafphotosynthesis and leafwater poten- from electron micrographs indicated that the volume of starch tial of leaves at the eighth (upper) and fifth (lower) node above the granules increased 65-fold during adaptation. Consequently, the cotyledonary node. Each Zvalue represents the mean of five to six deter- adaptation response observed in leaves at node 5 may well have minations from two expernments. (0), control leaves; (0), adapted leaves. been due to concentration of solutes as a result of starch-induced decreases in cellular osmotic volume. In a subsequent paper (2), greater degree of turgor to lower leaf water potentials than did kinetics of starch and soluble sugar accumulation are discussed in control leaves, stomata of adapted leaves remained partially open detail and data will be presented suggesting that both active at low leaf water potentials. Older, fully expanded leaves had accumulation and concentration of solutes are important mecha- lower leaf conductances than younger leaves at the same water nisms in the type of adaptation observed in older cotton leaves. potentials. Jordan et al. (22) demonstrated that stomatal response Similarly, osmoregulation in younger leaves (node 8) is primarily of cotton to leaf water potential depended uniquely on leaf age. due to solute accumulation, whereas concentrating effects attrib- Older adapted leaves exhibited leaf conductances somewhat utable to starch are minimal (2). These data suggest that the higher than did nonadapted counterparts (Fig. 3). Although leaf manner in which osmoregulation or adaptation is expressed de- conductance tends to decline with leaf age even without water pends on leaf age and perhaps the degree of expansion at the time stress (15), the higher leaf conductances of adapted leaves at node of stress. 5 (relative to control leaves) suggest that adaptation partially Stomata of adapted leaves became less sensitive to low leaf overcame the effect of age. Young leaves (node 8) had approxi- water potentials (Fig. 4). This response is typical of many species mately the same leaf conductance at high leaf water potentials in that have been conditioned to water stress in growth chambers or adapted and control plants (Fig. 4). the field (5, 7, 9, 21, 23, 27). In both adapted and control leaves, Adapted leaves exhibited lower rates ofphotosynthesis than did stomatal closure was essentially complete when leaves approached control leaves at relatively high leaf water potentials (Fig. 5). This zero turgor (Figs. 2 and 4). Because adapted leaves maintained a occurred in young and old leaves even though leaf conductances Plant Physiol. Vol. 67,1981 WATER STRESS ADAPTATION IN COTTON 487 stressed (29). In essence, adapted plants utilized in this study mimic closely responses that moderately stressed field-grown 6O[0 plants exhibit with respect to photosynthesis, translocation, and F 40S leaf conductance in relation to leaf water potential (4, 29). 'STRESSED 201- TURGID A CONTROL Acknowledgments-The technical assistance of W. Hartenstine and M. Ferrari > 0'o the course of this is We Drs. V. 0 2 3 4 5 6 throughout study gratefully acknowledged. thank Wittenbach, D. Krieg, and J. Radin for helpful suggestions. We thank E. Sparre for assistance in preparation of this manuscript. < 100 80 LITERATURE a 60 _ CITED 40- o STRESSED 1. AcEvEIDo E, E FEREREs, TC HSIAo, DW HENDERsoN 1979 Diurnal growth a URIDB water 20 TURGID ADAPTED trends, potentials, and osmotic adjustment of maize and sorghum in the field. Plant Physiol 64: 476-480 -Ool 2 3 4 5 6 2. ACKERSON RC 1981 Osmoregulation in cotton in response to water stress. II. Leaf carbohydrate status in relation to osmotic adjustment. Plant Physiol 67: U) 0 489-493 3. ACKERSON RC 1980 Stomatal response ofcotton to water stress and abscisic acid 4~~~~~~~~~~~ as affected by water stress history. Plant Physiol 65: 455-459 _j -6 4. AcKERSON RC, DR KRIEG, CL HARING, N CHANG 1977 Effects of plant water F CONTROL ADAPTED Zz -9g9 status and stomatal activity, photosynthesis and nitrate reductase activity of °TURGID oTURGID o -12 * STRESSED * STRESSED field grown cotton. Crop Sci 17: 81-84 5. ACKERSON RC, DR KRIEG, FJM SUNG 1979 Osmoregulation and leaf conductance of field grown sorghum genotypes. Crop Sci 20: 10-14 6. BOYER JS 1976 Water deficits and photosynthesis. In TT Kozlowski, ed, Water w Deficits and Plant Growth, Vol 4. Academic Press, New York, pp 153-190 7. BROWN KW, WR JORDON, JC THOMAS 1976 Water stress induced alterations of 1 2 3 4 5 6 the stomatal response to decreases in leaf water potential. Physiol Plant 37: 1- TIME, h 5 8. BUNCE JA 1977 Nonstomatal inhibition ofphotosynthesis at low water potentials FIG. 6. Relationship between retention of 14C activity and time for in intact plants ofspecies from a variety ofhabitats. Plant Physiol 59: 348-350 control (A) and adapted (B) cotton plants, that were turgid or water- 9. CumER JM, DW RAINS 1977 Effects of irrigation history on responses of cotton stressed; leaf water potentials are given in C. Data were obtained from to subsequent water stress. Crop Sci 17: 329-335 10. CUTLER JM, DW RAINS 1978 Effects of water stress and hardening on the four different pairs ofplants for control and adapted plants. Data represent internal water relations and osmotic constituents ofcotton leaves. Physiol Plant the mean + SD from four plants. 42: 261-268 11. CUTLER JM, DW RAINs, RS LooMIs 1977 Role ofchanges in solute concentration were similar or slightly higher in adapted leaves (Fig. 4). These in maintaining favorable water balance in field grown cotton. Agron J 69: 773- 779 data strongly imply that nonstomatal processes limited photosyn- 12. CUTLER JM, DW RAINs, RS LooMIs 1977 The importance of cell size in the thesis in adapted leaves. Photosynthesis can be inhibited through water relations of plants. Physiol Plant 40: 255-260 stress-induced perturbations of photosynthetic partial processes 13. DA SILVA IV, AW NAYLOR, PJ KRAmaR 1974 Some ultrastructural and enzymatic that are not directly linked to CO2 diffusion (6). Decreases in leaf effects of water stress in cotton (Gossypium hirsutum L) leaves. Proc Natl Acad are USA 71: 3243-3247 mesophyll conductance commonly observed when leaf water 14. DAVIES FS, AN LAKSo 1979 Diurnal and seasonal changes in leafwater potential potentials decline (7, 8). Furthermore, the accumulation of pho- components and elastic properties in response to water stress in apple trees. tosynthetic end products may act to reduce photosynthetic rates, Physiol Plant 46: 109-114 perhaps by metabolic regulation or by altering internal CO2 15. DAVIS SD, CHM vAN BAVEL, KJ McCREE 1977 Effect of leaf aging upon stomatal resistance in bean plants. Crop Sci 17: 640-645 diffusion (25, 30). Large starch granules were observed in chloro- 16. FEREREs E, E AcEDvEo, DW HENDERSON, TC HsiAo 1978 Seasonal changes in plasts of fully turgid, stress-adapted plants (Fig. 3). Chloroplast water potential and turgor maintenance in sorghum and maize under water structure is often disrupted by water stress (13, 17, 18). Although stress. Physiol Plant 44: 261-267 some breakdown of thylakoid structure was noted in chloroplasts 17. GELEs KL, MF BEARDSELL, D COHEN 1974 Cellular and ultrastructural changes in mesophyll and bundle sheath cells ofmaize in response to water stress. Plant of adapted leaves, this seemed to be minimal (Fig. 3). However, Physiol 54: 208-212 accumulation ofstarch in chloroplasts ofadapted leaves may have 18. GILS KL, D COHEN, MF BEARDsEL 1976 Effects of water stress on the induced physical changes in the chloroplasts, thus accounting for ultrastructure of leaf cells of Sorghum bicolor. Plant Physiol 57: 11-14 lower rates of photosynthesis in adapted leaves at high water 19. HSIAo TC, E ACEVEDO, E FERERES, DW HENDERSON 1976 Stress metabolism. Water stress, growth and osmotic adjustment. Phil Trans R Soc London [B] potentials (Fig. 3). 273: 479-500 Retention of photosynthetically derived assimilates (predomi- 20. JoNEs MM, HM RAWSON 1979 Influence of rate of development of leaf water nantly sugars) would be an effective way of accumulating solutes, deficits upon photosynthesis, leaf conductance, water use efficiency, and os- thereby facilitating turgor maintenance. In control plants, declin- motic potential in sorghum. Physiol Plant 45: 103-111 leaf water inhibited of 21. JomEs MM, NC TuRNER 1978 Osmotic adjustment in leaves of sorghum in ing potentials strongly translocation re- response to water deficits. Plant Physiol 61: 122-126 cently synthesized assimilates (Fig. 6). Several studies have dem- 22. JORDAN WR, KW BROWN, JC THOMAS 1975 Leaf age as a determinant in onstrated that water stress inhibits loading of assimilates into stomatal control of water loss from cotton during water stress. Plant Physiol conducting tissue (24, 27, 33, 34). In contrast, adapted leaves 56: 595-599 continued to export recently derived photosynthate rapidly even 23. McCREE KJ 1974 Changes in the stomatal response characteristics produced by water stress during growth. Crop Sci 14: 273-278 as leaf water potentials declined (Fig. 6). Because turgor mainte- 24. MuNNs R, CJ PEARSON 1974 Effect of water deficit on translocation of carbo- nance in adapted plants was associated with maintenance of hydrate in Solanum tuberosum Aust J Plant Physiol 1: 529-537 photosynthesis at low leaf water potentials (Figs. 2 and 5), the 25. NAFZIGER ED, HR KOLLER 1976 Influence of leaf starch concentration on CO2 supply ofphotosynthetic assimilates may explain continued export assimilation in soybean. Plant Physiol 57: 560-563 26. NAYLOR DG, ID TEARE 1974 An improved rapid field method to measure of sugars in adapted leaves even as leaf water potentials declined. photosynthesis with CO2. Agron J 67: 404-406 In adapted leaves, sugars (sucrose) of photosynthetic origin could 27. O'TOOLE JC, RK CROOKSTON, KJ TREHARNE, JC OZBUN 1976 Mesophyll not act as osmotically active constituents in situ, at least in ex- resistance and carboxylase activity. A comparison under water stress conditions. panding leaves. Translocation ofphotosynthate occurred in cotton Plant Physiol 57: 465-468 28. SosEBEERE, HH WIEBE 1971 Effects of water stress and clipping on photosyn- at leaf water potentials approaching -33 bars, whereas photosynthate translocation in two grasses. Agron J 63: 14-17 thesis declined to a more significant extent as plants became 29. SUNG FJM, DR KRIEG 1979 Relative sensitivity of photosynthetic assimilation 488 ACKERSON AND HEBERT Plant Physiol. Vol. 67, 1981

and translocation of "4C to water stress. Plant Physiol 64: 852-856 32. TURNER NC, JE BEGG, ML TONNET 1978 Osmotic adjustment of sorghum and 30. THORNE JH, HR KoLLER 1974 Influence ofassimilate demand on photosynthesis, sunflower crops in response to water deficits and its influence on the water diffusive resistance, translocation and carbohydrate levels of soybean leaves. potentials at which stomata close. Aust J Plant Physiol 5: 597-608 Plant Physiol 54: 201-207 33. WARDLAW IF 1967 The effect of water stress on translocation in relation to photosynthesis and growth. I. Effect during grain development in wheat. Aust 31. TuRNER NC, JE BEGG, HM RAWSON, SD ENGLISH, AB HEARN 1978 Agronomic J Biol Sci 20: 25-39 and physiological response ofsoybean and sorghum crops to water deficits. III. 34. WARDLAw IF 1969 The effect of water stress on translocation in relation to Components of water potential, leaf conductance, CO2 photosynthesis and photosynthesis and growth. II. Effect during leaf development in Lolium adaptation to water deficits. Aust J Plant Physiol 5: 179-194 temulentum L. Aust J Biol Sci 22: 1-16