Aug 13, 1986 - Department ofAgriculture, Agricultural Research Service Plant Stress and ... analyzed in two photoperiodic stains of field gro cotton (Gosypiam.
Plant Physiol. (1987) 84, 251-254
0032-0889/87/84/025 1/04/$01.00/0
Plant Morphological and Biochemical Responses to Field Water Deficits' II. RESPONSES OF LEAF GLYCEROLIPID COMPOSITION IN COTTON Received for publication August 13, 1986 and in revised form December 10, 1986
RICHARD F. WiLSON*, JOHN J. BuRKE, AND JERRY E. QUISENBERRY United States Department ofAgriculture, Agricultural Research Service Soybean and Nitrogen Fixation Research Unit, North Carolina State University, Raleigh, North Carolina (R.F.W.); and United States Department ofAgriculture, Agricultural Research Service Plant Stress and Water Conservation Research Unit, Texas Tech. University, Lubbock, Texas (J.J.B., J.E.Q.) ABSTRACr
The effects of water deficits on leaf glycerolipid composition were analyzed in two photoperiodic stains of field gro cotton (Gosypiam kirsutlm L) that differ in sensitivity to drought. Leaves from plants grown under dryland cons exhibited inaresed dry weight and specific leaf weight. The average midday leaf water potential in the dryland treatment decreased to -1.9 and -2A mega ls, respcvely, for the X5 and T185 genoypes. Total kaf lipid content of plats exposed to dryland conditons was 5.9 and 7.5% of leaf dry weight for Ain T25 and T185, respectively. The difference in leaf lipid contet between these genotypes was caused by water deficits and was attrbted to loss of both phospholipids and glycolipids in sin T25. Te was no aprent loss of phospholipids due to water deficits in the T185 genotype; however, a signifiant loss of glycolipids was parilly compe ted by a 2-fold increase in triacylglyceroL No change in triacylglycerol was fomud between tratnent in T25 leaves. Water deficit caused a s declie in the reative degree of acyhmluns tion in phospholipids and glycolipids from both genotypes; however, the double bond index for trincylglycerol incesed in both genotypes. It is believed that the observed responses of leaf lipid composition to drylnd dis my bean addil crterio for c types.
and selection of new drougbt-toleant cotton geno-
Drought-tolerant cultivars have been identified within several agronomic crops such as wheat, barley, and cotton (4, 15). A factor common to drought-tolerant genotypes within those species appears to be the ability to maintain leaf turgor at low leaf water potentials. That trait is believed to be achieved through osmoregulation (1). A variety of morphological and biochemical characteristics may contribute to the expression of osmoregulation in drought-tolerant genotypes (2, 18). Although the mechanism of osmoregulation is complex, the adaptive ability to maintain membrane integrity during long periods of water deficit may be an essential biological trait for drought tolerance. In drought-sensitive genotypes, cellular dehydration may cause
'Cooperative investigations of the North Carolina Agriculturl Research Service and the United States Department of Agriculture-Agricultural Research Service at Raleigh, NC and Lubbock, TX. Paper No. 10566 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695-7601.
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significant disorganization of membrane structures (14). Water deficits may provoke swelling and structurl breakdown of thylakoid membranes, disruption of the outer chloroplast envelope, thinner partitions and adhesions within the grana, and decreased chloroplast volume (1, 8, 10). Mechanical and chemical strsses imposed upon other cell membranes during water stress may be equally important in determining cell turgor and physiological function. Hence, differences in membrane lipid composition should be anticipated in the response of drought-tolerant and drought-sensitive genotypes grown under arid conditions. There is a general lack of knowledge concerning the effect of water-deficit upon lipid metabolism in agronomic crops. The relatively few studies that address this topic have shown decreases in percent oil, linolenic acid content, degree of acyl unsaturation, phospholipid and glycolipid content of leaf tissues exposed to long periods of water deficit (4, 5, 8, 14). These changes in leaf lipid composition apparently result from sustained metabolic turnover of glycerolipids coupled with inhibition of phospholipid, glycolipid, and polyunsaturated fatty acid synthesis (13, 20). However, drought-induced changes in leaflipid composition are not as severe in drought-tolerant genotypes compared to drought-sensitive strains of the same species (4-6, 14). Thus, damage to cell membranes would appear to be greater in droughtsensitive genotypes and could affect lower turgor potentials. Another observation is the accumulation of TG,2 a storage lipid, in water strssed leaves of wheat (12), corn (7), and cotton (13). The TG content of leaftissue is normally very low, accounting for perhaps 1 to 5% (w/w) of leaf dry mass. The previously mentioned reports claim a 2- to 5-fold increase in the TG content of water-stesed leaves. Upon relief of the stress, the TG levels return to normal. Such treatments also result in parallel changes in the number and size of lipid bodies found within chloroplasts and the cytoplasm of drought-stressed leaves (7, 10). There is reason to believe that highly unsaturated DG derived from phospholipid and glycolipid metabolism during water stress are utilized in TG synthesis, and cause the observed increase in TG content and acylunsaturation (7, 13, 19). Although the relation between drought tolerance and incrased TG accumulation during water stress is unknown, TG is an efficient means to store
2Ablreviations:
TG, tiacylglycerol; DAP, days
after planing; TL,
totl lipid; TPL, total polar glycerolipids; DO, diacylglycerol FFA, free fatty acid; PL, phospholipids; GL, glycolipids; PC, phosphatidylcholine; PI, phosphatidylinositol; PE, phosphatidylethanolamine; DPG, cardiolipin; MGDG, mongalactosyl diacylglycerol; DGDG, digalyl diacylglycerol; PG, phosphatidylglycerol.
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WILSON ET AL.
fixed carbon for long periods, as compared to starch; and a substantially greater amount of energy may be derived from TG metabolism (12). Therefore, it is conceivable that TG accumulation in leaf tissues during water deficit affords some adaptive advantage for recovery of metabolic activity when drought stress is relieved. Based upon this information, the following study was conducted with two photoperiodic strains of cotton which differed in drought tolerance (2). The response of phospholipid, glycerolipid, and TG composition in leaf tissue to irrigated and dryland growth conditions was evaluated in the drought-tolerant strain, T25 and the drought-sensitive strain, T185. The results of this investigation have demonstrated that changes in leaf lipid composition may be an additional selection criteria for characterization of drought-tolerant cotton germplasm.
MATERIALS AND METHODS Two nonflowering (photoperiodic) cotton (Gossypium hirsutum L.) strains were grown under field irrigation and dryland conditions at Lubbock, TX. These treatments were conducted in the same field about 30 m apart. The soil type was an Acuff fine sandy loam (fine-loamy, mixed, thermic, Aridic Paleustolls).3 The plants in the dryland treatment were grown under a rain-exclusion shelter to divert precipitation. Irrigated treatments were grown under open field conditions and were watered weekly to field capacity with a drip irrigation system. The Texas designations for the strains analyzed in this study are T25 and T185. The strains were planted in five 3 m rows with 1 m between rows. Plants were thinned after emergence to 11 plants m-'. The uppermost fully expanded leaves were harvested for lipid analysis at 106 DAP from the three center rows. Each treatment-strain combination was replicated three times. Measurement of leaf water potential was performed as described by Burke et aL (2). Lipid Analysis. Total lipid content of leaf tissues was determined from ground samples (lO g dry weight) by wide-line NMR (21). Lipids were extracted from samples (10 g fresh weight) by homogenization in 120 ml chloroform:methanol (2:1 v/v) followed by 60 ml methanol. The homogenate was suction filtered to remove cellular debris. The filtrate plus 60 ml deionized H20 was shaken and chilled to 4C until phase separation. After removal of the methanol-water phase, the volume of the chloroform phase (TL) was reduced under vacuum and stored at -20°C in chloroform:methanol (2:1 v/v). The TPL, DG, FFA, and TG constituents of TL were separated on thin layers of Silica Gel GHR (Alltech Inc) using petroleum ether:diethylether:glacial acetic acid (80:20:1, v/v/v) as the developing solvent. Individual PL (phosphatidylinositol, phosphatidylcholine, phosphatidylethanolamine, cardiolipin) and GL (monogalactosyl diacylglycerol, digalactosyl diacylglycerol, and phosphatidylglycerol) constituents of TPL were separated from aliquots of TL by TLC using chloroform:methanol:7 N NH40H (70:20:1.5 v/v/v) as the developing solvent. Each lipid was identified by co-chromatography with authentic standards (Supelco, Inc.) under UV after spray with 2,7-dichlorofluorescin in ethanol. Fatty acid analysis of each lipid type was determined by GC as described previously (11, 21).
RESULTS AND DISCUSSION The effect of water deficit upon the water status and development of the two photoperiodic cotton strains (T25 and T1 85) 3 Mention of a trade name does not constitute a guarantee or warranty of the product by the United States Department of Agriculture or the North Carolina Agricultural Research Service and does not imply its approval to the exclusion of other products that may also be suitable.
Plant Physiol. Vol. 84, 1987
was monitored throughout the growing season by Burke et al. (2). Those data demonstrated the severity of the soil water deficit upon growth parameters of the two cotton strains. At 106 DAP, reductions in plant height, leaf area index, plant dry weight, and number of leaves per plant between irrigated and dryland treatments were, in each case, 2-fold greater for strain T185 than strain T25. Although strain T25 had smaller leaves, specific leaf weight was not significantly different between strains at 106 DAP in the irrigated treatment, and increased 1.7- to 1.9-fold in both strains as a result of the dryland treatment (Table I). The midday leaf water status of the two photoperiodic cotton strains at 106 DAP in the dryland treatment demonstrated significant differences in leaf water potential and leaf turgor potential, but no difference in leaf osmotic potential. Hence the differential in leaf water potential between strains exposed to arid conditions (-1.9 MPa for T25 and -2.4 MPa for T185) at 106 DAP was attributed to leaf turgor potential, which was 4- to 5fold greater in strain T25 than strain T185. Furthermore the rate of change in leaf water potential from 50 to 106 DAP in the respective strain-treatment combinations was accelerated only in the T 1 85-dryland experiment (Table I). These data also indicated that strain T1 85 dehydrated more rapidly than strain T25 when grown under severe soil-water deficit. Although it is probable that the apparent decline in water-use efficiency was affected in part by greater disruption of membranes in strain T1 85, the relative leaf lipid content declined only slightly (Table I). In contrast, the relative leaf lipid content of the drought-tolerant strain was significantly lower in the dryland treatment. Actual TL content, mmol (kg dry weight)-', of T25 leaves also was significantly decreased by dryland conditions; whereas, no significant change in TL was found between treatments with strain T185 (Table II). Analysis of the TL constituents from each strain-treatment combination revealed that the TPL content of leaves declined in both strains due to dryland conditions; but to a greater extent in strain T25. Significant breakdown of TPL (membrane glycerolipids) contributed to the increases in DG and FFA content of T25 leaves. Because DG and FFA derived from TPL may be utilized in TG synthesis (19), one might expect an accumulation of TG in strain T25. However, the TG content of T25 leaves was unchanged by the irrigated or dryland treatments. This anticipated response occurred only in T185 leaves, where there was a 2-fold increase in TG in the dryland treatment. In a previous study with cotton (13), TG synthesis from radiolabeled acetate was stimulated 3fold by severe water stress in the drought-sensitive cultivar 'Reba,' but only 1.6-fold in the drought-tolerant cultivar 'Mocosinho.' Thus, TG synthesis in drought-tolerant genotypes could be inhibited by water deficits or metabolic breakdown of TG could be accelerated to maintain a relatively constant level of TG in the leaf tissues. Further examination of the TPL constituents of leaf tissue at 106 DAP indicated that dryland conditions reduced PL (PI + PC + PE + DPG) content 1.8-fold in T25, but elicited no change in T185 leaves. With regard to GL content a 1.3- to 1.5-fold decline in MGDG + DGDG + PG occurred in both strains (Table III). Similar responses of PL and GL content to arid growth conditions have been found in leaves of drought-sensitive genotypes of corn (7), wheat and barley (4, 5); and in droughttolerant genotypes of wheat and barley (5) and soybeans (RF Wilson, unpublished data). The concomitant decrease in acylunsaturation of PL and GL from T25 and T185 leaves (Table IV), primarily due to loss of linolenic acid, also supported previous evidence for the inhibition of acyldesaturase activity during water deficit (13, 14). Hence, the increased double bond index for TG probably resulted from metabolism of highly unsaturated DG or FFA derived from PL and GL (7). Thus far, two striking differences in leaf glycerolipid compo-
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PLANT RESPONSES TO WATER DEFICIT Table I. Effect of Drought Stress upon Leaf Characteristics of two Photoperiodic Cotton Strains T185 T25 Statistic at 106 DAP' Dryland Irrigated Dryland Irrigated 28.4 ± 0.3 20.4 ± 0.2 23.7 ± 0.8 20.0 0.3 Leaf dry wt(%) 0.70 ± 0.7 0.55 ± 0.1 0.33 ± 0.4 0.29 ± 0.5 Leaf dry wt (g) 86.5 ± 1.6 132.2 ± 5.5 41.8 ± 1.0 62.2 3.0 Leaf area (cm2) Specific leaf wt (mg 8.1 ± 0.3 4.2 ± 0.1 7.8 ± 0.1 4.6 0.2 cm-2) Leaf water potential -2.38 ± 0.17 -1.60 ± 0.12 -1.89 ± 0.08 -1.34 0.12 (MPa) Rate of change in leaf water potential (MPa -0.026 -0.017 -0.018 -0.016b day ') 7.5 ± 0.4 7.9 ± 0.3 5.9 ± 0.3 7.6 ± 0.5c Leaf lipid content (%) b Linear regression coefficient a DAP, d after planting; data represent mean + SE of three replications. for values between 50 to 106 DAP, correlation coefficients for each analysis ranged between -0.90 to -0.99; c mg lipid (g dry wt)-' (10)2; determined by wide-line NMR. (see Burke et al. [2]). Table II. Effect of Drought Stress upon Leaf Lipid Composition of two Photoperiodic Cotton Strains T185 T25 LSD 0.05 Lipida Dryland Irrigated Dryland Irrigated mmol (kg dry wt)' 33.8 205.2 243.2 139.2 229.1 TPL 5.8 16.6 11.4 28.7 12.4 DG 4.4 6.6 2.1 13.8 0.3 FFA 4.8 32.1 16.6 22.5 20.7 TG 22.8 260.5 273.3 204.2 TL 262.5 a TPL, total polar glycerolipid; DG, diacylglycerol; FFA, free fatty acid; TG, triacylglycerol; TL, total lipid.
Table III. Effect of Drought Stress upon Leaf Total Polar Lipid Composition of two Photoperiodic Cotton Strains Total Fatty Fatty Acid Add 18:3 20:0 18:2 18:1 18:0 16:1 16:0 14:0 Lipid^
(kg ~~~~~~mmol dry wt)h
MO/% mol%o T125 (irrigated) PL GL T25 (dryland) PL GL T185 (irrigated) PL
GL T185 (dryland)
5.1 3.4
28.9 13.6
1.4 3.6
2.3 1.6
5.3 1.8
19.0 5.2
35.7 67.7
2.3
3.1
97.8 131.3
6.1 3.8
36.1 16.7
5.1 4.1
3.8 2.3
6.2 3.1
11.1 6.3
28.6 60.3
3.0 3.4
53.8 85.4
5.7 3.2
29.6 13.7
2.9 3.9
2.6 1.8
5.5
2.1
19.2 4.8
31.6 67.0
2.9 3.5
103.8 139.4
101.6 4.0 22.4 13.2 1.2 4.4 4.6 43.4 6.8 PL 103.6 3.2 55.0 6.1 2.7 1.1 4.5 23.5 3.9 GL a PL (phospholipid) includes phosphatidylinositol, phosphatidylcholine, phosphatidylethanolamine, and cardiolipin; GL (glycolipid) includes monogalactosyl diacylglycerol, digalactosyl diacylglycerol, and phosphab LSD 0.05 between treatments, 17.4 for PL and 18.3 for GL. tidylglycerol.
sition caused by drought-stress have been identified in the two upon microsomal and chloroplastic PL has been shown by Webb photoperiodic cotton strains, T25 and T185. Under dryland and Williams (19, 20). Chetal et al. (6) also have demonstrated conditions the PL content of T25 leaves was severely decreased that the PL content of chloroplasts from drought-tolerant and compared to strain T185; and TG content of T185 leaves was drought-sensitive wheat and barley cultivars was not affected by significantly increased compared to strain T25. It is believed that water stress. In cotton, Ferrari-Iliou et al. (8) found a lower these two characteristics, respectively, may be correlated with proportion of intact chloroplasts in drought-stressed leaves. Indrought-tolerant and drought-sensitive cotton genotypes. Con- tact chloroplasts from the stressed tissues contained reduced sidering the findings of several other reports, the loss of PL in levels of GL; however, PL content was not altered. Therefore, strain T25 could be due to accelerated PL hydrolysis from specific one might conclude that the loss of PL in drought stressed T25 membranes rather than to inhibition of PL synthesis. Evidence leaves was primarily associated with cell wall or microsomal for separate PL hydrolase mechanisms which act independently membranes.
WILSON ET AL.
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Table IV. Effect ofDrought Stress upon the Fatty Acid Composition of Leaf Lipidsfrom two Photoperiodic Cotton Strains Lipid Treatment TL GL TG PL
151.8 119.3
218.9 200.7
157.9 184.2
be useful as additional selection criteria in breeding programs for the improvement of drought-tolerance in cotton. Steps have already been taken to utilize this approach in the development of new drought-tolerant germplasm resources. LITERATURE CITED
double bond indexa T25 Irrigated Dryland T185
Plant Physiol. Vol. 84, 1987
187.4 171.3
Irrigated Dryland
182.9 157.0 216.6 141.6 148.7 179.9 184.4 102.6 12.7 11.7 10.5 16.2 LSD 0.05 aDouble bond index, summation mol% (16:1 + 18:1 + 18:2[2] +
18:3[3]). With reduced cell volume caused by long periods of water deficit (1), a decline in PL content could be beneficial to alleviate physical stress or compaction of membrane structure. Such an adaptive mechanism could enhance turgor potential and the ability to export photosynthate. A high rate of cell elongation under stress, a smaller proportion of spongy parenchyma in the lamina, and a higher proportion of vascular tissue in the petiole are three important criteria for selection of drought-tolerant genotypes (18). Quisenberry et al. (15) have already shown that maintenance of leaf turgor is essential for cell elongation in photoperiodic strains of cotton; and that strain T25 exhibits a higher number of vascular bundles in petioles than strain T1 85 (16). Hence, the decline in PL content of T25 leaves during water deficit could reflect a decrease in the proportion of spongy parenchyma cells. In cotton genotypes adapted to dryland conditions, starch accumulates in chloroplasts during the day and is metabolized at night similar to plants grown under irrigation (1). There was no excess accumulation of TG in those tissues. On the other hand, Huber et al. (9) have shown that starch, sucrose levels, and export of photosynthate were severely lowered by water-deficit in drought-sensitive species. Such conditions would be conducive to the storage of fixed carbon as TG within the cells. Therefore, excess accumulation of TG during drought in strain T1 85 probably resulted from reduced translocation of photosynthate to other plant organs, and indicated a disfunction of normal metabolic processes. The accumulation of heat shock proteins in strain T1 85 (3) also signified a distortion of normal protein synthesis. Hence, problems associated with nitrogen deficiency (17) may further limit the performance of strain T185 under dryland conditions. In conclusion, it is believed that this investigation of the response of lipid composition in leaves of the two photoperiodic cotton strains, T25 and T185, has provided evidence that may
1. ACKERSON RC, RR HERBERT 1981 Osmoregulation in cotton in response to water stress. 1. Alterations in photosynthesis, leaf conductance, translocation, and ultrastructure. Plant Physiol 67: 48"488 2. BuRKE JJ, PE GAMBLE, JL HATFIELD, JE QUISENBERRY 1985 Plant morphological and biochemical responses to field water deficits. I. Responses of glutathione reductase activity and paraquat sensitivity. Plant Physiol 79: 415-419 3. BURKE JJ, JL HATFIELD, RR KLEIN, JE MULLEr 1985 Accumulation of heat shock proteins in field grown cotton. Plant Physiol 78: 394-398 4. CHETAL S, DS WAGLE, HS NAINAWATEE 1980 Phospholipid changes in wheat and barley leaves under water stress. Phytochemistry 19: 1393-1395 5. CHETAL S, DS WAGLE, HS NAINAWATEE 1982 Alteration in glycolipids of wheat and barley leaves under water stress. Phytochemistry 21: 51-53 6. CHETAL S, DS WAGLE, HS NAINAWATEE 1983 Phospholipid changes in wheat and barley chloroplast under water stress. Plant Sci Lett 29: 273-278 7. DOUGLAS TJ, LG PALEG 1981 Lipid composition of Zea mays seedlings and water stress induced changes. J Exp Bot 32: 499-508 8. FERRARI-ILIou R, AT PHAM THI, JV DASILVA 1984 Effect of water stress on the lipid and fatty acid composition of cotton chloroplasts. Physiol Plant 62: 219-224 9. HUBER SC, HH ROGERS, FL MOWRY 1984 Effects of water stress on photosynthesis and carbon partitioning in soybean plants grown in the field at different CO2 levels. Plant Physiol 76: 244-249 10. MAROTI I, Z TUBA, M CSIK 1984 Changes in chloroplast ultrastructure and carbohydrate level in Festuca, Achillea, and Sedum during drought and recovery. J Plant Physiol 1 16: 1-10 11. MARTIN BA, RF WILSON 1984 Subcellular localization of triacylglycerol synthesis in spinach leaves. Lipids 19: 117-121 12. MURPHY GTP, ML PARKER 1984 Lipid composition and carbon turnover of wheat leaf oleosomes. J Exp Bot 35: 348-355 13. PHAM THI AT, C BORREL-FLooD, JV DASILVA, AM JUSTIN, P MAZLIAK 1985 Effects of water stress on lipid metabolism in cotton leaves. Phytochemistry 24: 723-727 14. PHAM THI AT, C FLOOD, JV DASILVA 1982 Effects of water stress on lipid and fatty acid composition of cotton leaves. In JFGM Wintermans, PJC Kuiper, eds, Biochemistry and Metabolism of Plant Lipids. Elsevier Biomedical Press, Amsterdam, pp 451-454 15. QUISENBERRY JE, GB CARTWRIGHT, BL MCMICHAEL 1984 Genetic relationship between turgor maintenance and growth in cotton germplasm. Crop Sci 24: 479-482 16. QUISENBERRY JE, CW WENDT, JD BERLIN, BL MCMICHAEL 1985 Potential for using leaf turgidity to select drought tolerance in cotton. Crop Sci 25: 294-299 17. RADIN JW, LL PARKER 1979 Water relations of cotton plants under nitrogen deficiency. I. Dependence upon leaf structure. Plant Physiol 64: 495-498 18. VIDAL A, J-C POGNONEC 1984 Effet de l'alimentation en eau sur quelques caracteres morphologiques et anatomiques des feuilles de soja (Glycine max L. Merrill). Agronomie 4: 967-975 19. WEBB MS, JP WILLIAMS 1984 Changes in the lipid and fatty acid composition of Viciafaba mesophyll protoplasts induced by isolation. Plant Cell Physiol 25: 1541-1550 20. WEBB MS, JP WILLIAMS 1984 Changes in lipid and fatty acid metabolism of Viciafaba mesophyll protoplasts. Plant Cell Physiol 25: 1551-1559 21. WILSON RE, JW BURTON, CA BRIM 1981 Progress in the selection for altered fatty acid composition in soybeans. Crop Sci 21: 788-791
