Unsaturation of fatty acids in membrane lipids enhances tolerance of the cyanobacterium Synechocystis PCC6803 to low-temperature photoinhibition

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1 Proc. Nati. Acad. Sci. USA Vol. 89, pp , October 1992 Plant Biology Unsaturation of fatty acids in membrane lipids enhances tolerance of the cyanobacterium Synechocystis PCC683 to low-temperature photoinhibition (chilling sensitivity/photosynthesis/polyunsaturated fatty acid) ZOLTAN GOMBOS*t, HAJIME WADA*, AND NORIO MURATA*t *Department of Regulation Biology, National Institute for Basic Biology, and *Department of Molecular Biomechanics, Graduate University of Advanced Studies, Myodaiji, Okazaki 444, Japan Communicated by Olle Bjorkman, July 13, 1992 ABSTRACT Effect of the unsaturation of fatty acids in the glycerolipids of thylakoid membranes on low-temperature photoinhibition of photosynthesis was studied by mutation and transformation of the cyanobacterium Synechocystis PCC683. When grown at 34C, the wild type contained mono-, di-, and triunsaturated lipids; a mutant, designated Fad6, contained mono- and diunsaturated lipids; and a transformant of Fad6, with a disrupted gene for desaturation and designated Fad6/desA::Kmr, contained only monounsaturated lipids. Fad6/desA::Kmr was the most susceptible among these strains to low-temperature photoinhibition of photosynthesis, whereas Fad6 and the wild type were apparently indistinguishable in terms of sensitivity to photoinhibition. This result suggests that the presence of diunsaturated fatty acids is important in protecting against low-temperature photoinhibition. The photoinhibition at room temperature, although much less significant than that at low temperature, was also affected by the unsaturation of fatty acids. By contrast, the photosynthetic transport of electrons, measured at various temperatures, was not affected by changes in extent of fatty acid unsaturation. The relationship between low-temperature sensitivity of plants and extent of unsaturation of fatty acids in membrane lipids has been of interest since the original proposal by Raison (1) and Lyons (2). Chilling-sensitive plants contain high levels of saturated molecular species of phosphatidylglycerol in their thylakoid membranes, whereas in chillingresistant plants, almost all the phosphatidylglycerols in thylakoid membranes contain unsaturated fatty acids (3, 4). Furthermore, during acclimation of plants to low temperature, the fatty acids in their membrane lipids become less saturated (5). Similar alterations in extent offatty acid unsaturation during low-temperature adaptation have been well-documented in cyanobacteria (6-8). However, experimental results to date do not prove the contribution of unsaturation of fatty acids to the low-temperature tolerance because acclimation to low temperature is accompanied not only by unsaturation of fatty acids but also by a number of other metabolic changes. Therefore, such metabolic changes, and not the unsaturation of fatty acids in membrane lipids, could allow plants to be tolerant to low temperatures. To verify whether the unsaturation of fatty acids contributes to low-temperature tolerance, it is necessary to establish a system in which only fatty acid unsaturation is altered without any effect on other metabolic processes. Recently we isolated a gene, desa, for desaturation at the A12 position of fatty acids of membrane lipids from the cyanobacterium Synechocystis PCC683 (9). Using this gene, The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C solely to indicate this fact. we transformed another cyanobacterial strain, Synechococcus PCC7942, which can desaturate fatty acids only at the A9 position (9). Cells transformed with the desa gene could introduce a second double bond in the cis configuration at the A12 position of fatty acids and thereby to synthesize 16:2(9, 12) and 18:2(9,12) fatty acids. Moreover, these cells were more tolerant to low temperature than were wild-type cells. The transformed cells provide direct evidence for the contribution of unsaturation of fatty acids to low-temperature tolerance. Another characteristic feature of low-temperature sensitivity is the impairment of photosynthesis when plants are exposed to light of high intensity at low temperature, a phenomenon known as low-temperature photoinhibition (1-13). The question then arises as to whether or not the unsaturation of fatty acids in membrane lipids is related to low-temperature photoinhibition. Somerville and his coworkers (14, 15) tried to address this question by using mutants of Arabidopsis defective in the desaturation of fatty acids. However, the changes in unsaturation of fatty acids in the lipids of the thylakoid membranes of these mutants were incomplete-that is, levels of di- and triunsaturated fatty acids were reduced, but they were not fully eliminated from these mutants. Thus, the relationship between unsaturation of fatty acids and low-temperature tolerance remains to be clarified. Strains of Synechocystis PCC683 that contain membrane lipids with defined levels of unsaturation of fatty acids can now be obtained by mutation and transformation with desa (16). For the present experiments we grew, at the same temperature, the wild type, which contains mono-, di- and triunsaturated fatty acids (17); a mutant, designated Fad6, which contains mono- and diunsaturated fatty acids (17); and a transformant of Fad6, designated Fad6/desA::Kmr, which contains monounsaturated but no polyunsaturated fatty acids (16). Our experimental results suggest that the unsaturation of fatty acids, at the A12 position, in particular, enhances the protection against low-temperature photoinhibition. MATERIALS AND METHODS Organisms and Culture Conditions. Wild-type cells, and derivatives Fad6 and Fad6/desA::Kmr of Synechocystis PCC683, were grown photoautotrophically at 34 C under illumination from incandescent lamps [7 microeinsteins (JLE)/m2 per sec] in BG-11 medium (18)/2 mm Hepes' Abbreviations: BQ, 1,4-benzoquinone; Chl, chlorophyll; DCIP, 2,6- dichlorolindophenol; DPC, diphenylcarbazide; PS, photosystem; E, einstein(s). tpresent address: Plant Physiology Institute, Biological Research Center of the Hungarian Academy of Sciences, H-671 Szeged, P.O. Box 521, Hungary. To whom requests for reprints should be addressed. 9959

2 996 Plant Biology: Gombos et al. NaOH, ph 7.5, with aeration by sterile air/1% CO2 (19). In some cases, Fad6/desA::Kmr was cultured with kanamycin at 5,ug/ml. Analysis of Fatty Acids. Lipids were extracted from the cells by the method of Bligh and Dyer (2). The extracted lipids were subjected to methanolysis with 5% HCl/methanol at 85 C for 2.5 hr. The esterified fatty acids were analyzed with a gas/liquid chromatographic system (GC-7A; Shimadzu, Kyoto) equipped with a hydrogen flame ionization detector and a capillary column (17). Relative amounts of fatty acid methyl esters were calculated by comparing areas under chromatographic peaks with a data processor (C-R 2AX; Shimadzu, Kyoto). Isolation of Thylakoid Membranes. Cells of the wild type and Fad6/desA::Kmr were harvested by centrifugation at 5 x g for 1 min. The cells were washed twice with 1 mm N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid NaOH buffer (ph 7.)/6 mm sucrose/2 mm CaCl2/1 mm MgCl2 (buffer A) and suspended in 5 ml of this buffer. The suspension was mixed with the same volume of glass beads ( mm in diameter, Sigma G-8893). The mixture was agitated three times for 3 min each on a Vortex mixer, with 2-min intervals of cooling in an ice-water bath. Unbroken cells, cell debris, and glass beads were removed by centrifugation at 1 x g for 1 min, and the supernatant was recentrifuged at 27, x g for 3 min. The pelleted thylakoid membranes were resuspended in 5 ml of buffer A and were kept at 4 C until use. Photoinhibition. Cells corresponding to 6,ug of chlorophyll (Chl) were suspended in 3 ml of BG-11 medium. The suspension of cells was illuminated at various temperatures in a thermostated reaction vessel with white light of various intensities supplied by two incandescent lamps (15 W; Toshiba, Tokyo) with aeration with sterile air/1% CO2. Light intensity was regulated in the range of -4 mein/m2 per sec. Measurement of Photosynthetic Activities. Photosynthetic evolution of oxygen by intact cells suspended in BG-11 medium was monitored by means of oxygen exchange with a Clark-type oxygen electrode. The oxygen-evolving activity of intact cells due to the activity of photosystem (PS) II was measured with 1 mm 1,4-benzoquinone (BQ) and 1 mm K3Fe (CN)6 as electron acceptors (19). Light at 3.5 mein/m2 per sec was provided from an incandescent lamp combined with a red optical filter (VR62; Hoya Glass Co., Tokyo). The light-induced reduction of 2,6-dichloroindophenol (DCIP) was measured by monitoring the absorbance change at 59 nm with a spectrophotometer (UV-3; Shimadzu, Kyoto) used in the split-beam mode (21). The activity was measured in buffer A/2,uM DCIP. The intensity of red actinic light was 1.6 mein/m2 per sec. The concentration of Chl was determined by the method of Arnon et al. (22). RESULTS Fatty Acid Composition. Fatty acids of total glycerolipids were analyzed for the wild type, the mutant, and the transformant of Synechocystis PCC683, each grown at 34 C (Table 1). The most abundant fatty acids in the wild type were Table 1. Major fatty acids from total lipids of wild-type Synechocystis PCC683, Fad6 mutant, and Fad6/desA::Kmr transformant, grown at 34 C Fatty acid, mol. % Strain 16: 18:1(9) 18:2(9,12) 18:3(6,9,12) Wild type Fad Fad6/desA::Kmr Proc. Natl. Acad Sci. USA 89 (1992) 16:, 18:1(9), 18:2(9,12), and 18:3(6,9,12). The major fatty acids in Fad6 were 16:, 18:1(9), and 18:2(9,12), indicative of a defect in desaturation of fatty acids at the A6 position (17). The transformant, Fad6/desA::Kmr, was defective in desaturation at the A6 and A12 positions (16). Consequently this strain did not contain polyunsaturated fatty acids and possessed only 16: and 18:1(9) as the most abundant fatty acids. These results suggest that Fad6/desA::Kmr contained only monounsaturated lipids, that Fad6 contained mono- and diunsaturated lipids, and that the wild type contained mono-, di- and triunsaturated lipids (16). Using these three strains, we studied the effects of unsaturation of fatty acids on the photoinhibition at low temperatures with minimum apparent interference by other factors. Temperature Dependence of the Photosynthetic Evolution of Oxygen. The photosynthetic activities of wild-type, Fad6, and Fad6/desA::Kmr cells, grown at 34WC, were compared at 18C, 25C, and 34C (Table 2). Under saturating light, the oxygen-evolving activity of the photosynthetic machinery, with BQ as the electron acceptor, depended highly on temperature. In all strains the photosynthetic activity at 34C was five times higher than that at 18'C, whereas the activity from H2 to BQ at 34C was about four times higher than that at 18TC. However, despite the considerable differences in unsaturation of fatty acids among the three strains, no significant differences between them in either their photosynthetic or their electron-transport activities were seen at the three temperatures at which measurements were made. Effect of Temperature on Photoinhibition. Fig. 1 presents the time course of photoinhibition of wild-type and Fad6/desA::Kmr cells at 1C, 2TC, and 3C, when cells were exposed to light of 2.5 mein/m2 per sec. At 1 C photoinhibition of photosynthesis occurred very rapidly in both the wild type and Fad6/desA::Kmr (Fig. 1A). However, Fad6/desA::Kmr lost its photosynthetic activity much more rapidly than did the wild type. At 2 C, the rate of photoinhibition of photosynthesis decreased markedly in the wildtype strain (Fig. 1B). Nevertheless, Fad6/desA::Kmr was inhibited more rapidly than the wild type. At 3 C the wild type was resistant to photoinhibition for as much as 2 hr of illumination (Fig. 1C). Fad6/desA::Kmr still experienced photoinhibition, but the photoinhibition occurred less rapidly than at 1 C and 2 C. The results for Fad6 were the same as for the wild type at the three temperatures (data not shown). These observations suggest that Fad6/desA::Kmr is much more susceptible to photoinhibition than are the wild type and Fad6. Table 2. Oxygen-evolving activity of intact cells of wild-type Synechocystis PCC683, Fad6, and Fad6/desA::Kmr, grown at 34C Orevolving activity,,umol of 2 per mg Temperature of of Chl per hr measurement, C Wild type Fad6 Fad6/desA::Kmr Photosynthesis Electron transport from H2 to BQ Cells were suspended in BG-11 medium at a concentration that corresponded to 1,lg of Chl per ml. Before measurements ofoxygen evolution, cells were incubated in darkness for 5 min at the temperature at which measurements were made. The oxygen-evolving activity was determined with measurement for 3 min. Concentrations of BQ and K3Fe(CN)6 were both 1. mm.

3 Plant Biology: Gombos et al. Proc. Natl. Acad. Sci. USA 89 (1992) C._ c Duration of incubation in the light (min) Photoinhibition of photosynthesis. Cells were suspended in BG-11 medium at a concentration that corresponded to 2 jig Chl per ml FIG. 1. and exposed to a light intensity of 2.5 mein/m2 per sec for designated periods of time at 1C (A), 2'C (B), and 3C (C). o, Wild type; o, Fad6/desA::Kmr. Photosynthetic activity at 34C was measured as oxygen evolution, and the activity at 1% corresponded to 32 and 31 /Amol of 2 per mg of Chl per hr for the wild-type and Fad6/desA::Kmr cells, respectively. Data were obtained from results of three independent experiments. Effect oflight Intensity on Photoinhibition. Fig. 2 shows the effect of light intensity on photoinhibition of the wild type, Fad6, and Fad6/desA::Kmr. The difference in susceptibility to photoinhibition between Fad6/desA::Kmr and the other two strains was more pronounced at 1C and 2C than at 3'C (Fig. 2A and B). Fad6/desA::Kmr completely lost its photosynthetic activity at 1.5 and 2.5 mein/m2 per sec at 1'C and 2'C, respectively. However, both the wild type and Fad6 preserved relatively high levels of photosynthetic activity under these conditions. At 3'C (Fig. 2C) neither the wild type nor Fad6 lost its photosynthetic activity when exposed to light intensities as high as 4 mein/m2 per sec. By contrast, at 1'C and 2C, photosynthesis was completely inactivated at a light intensity of 4 mein/m2 per sec. In contrast to the wild type and Fad6, Fad6/desA::Kmr suffered complete photoinhibition of photosynthesis at 4 mein/m2 per sec at 3TC. At lower light intensities, such as.3 and.6 14 A 1C 12_ 1 meiti/m2 per sec, a slight enhancement of the photosynthetic evolution of oxygen was observed in the wild type and Fad6 mutant at 2'C and 3'C (Figs. 2 B and C) but was not observed at 1C (Fig. 2A). In these experiments, however, Fad6/desA::Kmr transformant was cultured with kanamycin at 5 gg/ml. To examine the effect of kanamycin in the growth medium on photoinhibition of Fad6/desA::Kmr, cells cultured in the presence of kanamycin at 5 ug/ml and in its absence were compared after light exposure of.3,.6, and 4 mein/m2 per sec at 1 C, 2 C, and 3 C. No significant differences were seen with respect to photoinhibition between cells grown with and without kanamycin (data not shown). Determination of the Site of Photoinhibition. To determine the site of photoinhibition, thylakoid membranes were isolated from wild-type and Fad6/desA::Kmr cells before and after photoinhibition. Photosystem (PS) II activity of the c. 1 x Light intensity (mein/m2lsec) FIG. 2. Effects of light intensity on photoinhibition of photosynthesis. Cells were suspended in BG-11 medium at a concentration that corresponded to 2,ug of Chl per ml and were exposed to light at the designated intensity for 6 min at 1 C (A), 2 C (B), and 3 C (C). o, Wild type; A, Fad6 mutant; and o, Fad6/desA::Kmr transformant. Photosynthetic activity at 34 C was measured as oxygen evolution, and the activity at 1%o corresponded to 32, 33, and 31,umol of 2 per mg of Chl per hr for wild-type, Fad6, and Fad6/desA::Kmr cells, respectively. Data were obtained from results of three independent experiments.

4 9962 Plant Biology: Gombos et al. isolated thylakoid membranes was measured by monitoring the reduction of DCIP with and without diphenylcarbazide (DPC), an electron donor that bypasses the transport of electron at the water-oxidizing site of PS II (Table 3). For thylakoid membranes from both strains, examined before and after photoinhibitory treatment, DPC did not enhance reduction of DCIP. These observations suggest that the site of photoinhibition in both the wild type and Fad6/desA::Kmr transformant was located somewhere between an electron carrier that is reduced by DPC and one that is oxidized by DCIP and was not located at the oxygen-evolving site. Most probably the photochemical reaction center was destroyed by the photoinhibition (23, 24). DISCUSSION In our study we investigated the effects of unsaturation of fatty acids of membrane lipids on the tolerance to low temperature of the cyanobacterium Synechocystis PCC683. We used three strains, wild type, Fad6, and Fad6/desA:: Kmr, which contained unsaturated fatty acids with stepwise and discretely deleted double bonds (16). Unsaturated fatty acids of the wild type were mono-, di- and triunsaturated; those of Fad6 mutant were mono- and diunsaturated, and those of Fad6/desA::Kmr transformant were only monounsaturated. Because =9%o of the total lipids of cyanobacteria originate from the thylakoid membranes (25), the fatty acid composition of the total lipids should be close to that of the thylakoid membrane lipids. By comparing the lowtemperature sensitivity of these strains, we could study the relationship between extent of unsaturation of fatty acids in lipids of thylakoid membranes and low-temperature sensitivity Ṫhe photoinhibition of intact cells is accelerated at low temperature (26). This phenomenon has not been well explained, although some plausible mechanisms for it have been proposed without concrete evidence (27). We found that the photoinhibition of all strains of Synechocystis PCC683 was more pronounced at temperatures lower than the growth temperature. The photoinhibition at low temperatures was accelerated by elimination of a second double bond from individual membrane lipids. However, the photoinhibition at 3 C was faster in Fad6/desA::Kmr transformant than in the wild type and Fad6 mutant. Therefore, at both low and normal temperatures the extent of unsaturation of fatty acids is probably related to the tolerance to photoinhibition, although this effect is more pronounced at low temperature. When the wild type, Fad6 mutant, and Fad6/desA::Kmr transformant were compared in terms of the effect of low Table 3. Effect of photoinhibition on photosynthetic transport of electrons through PS II in thylakoid membranes isolated from the wild type and Fad6/desA::Kmr of Synechocystis PCC683 Electron transport,,umol of DCIP reduced per mg of Chl per hr Electron Before After transport photoinhibition photoinhibition Wild type H2-. DCIP DPC - DCIP Fad6/desA::Kmr H2-* DCIP DPC - DCIP Before isolation of thylakoid membranes, cells were exposed to a light intensity of 1.5 mein/m2 per sec in BG-11 medium for 6 min at 2C. This treatment resulted in 2%6o and 8% photoinhibition, respectively, of the photosynthetic activity of intact cells (see Fig. 2B). The thylakoid membranes were suspended in buffer A at a concentration that corresponded to 5 Ag of Chl per ml. Proc. Natl. Acad Sci. USA 89 (1992) temperature and light intensity on photoinhibition, the most striking difference was seen between Fad6 and Fad6/desA:: Kmr strains. This result emphasizes the importance of the second double bond at the A12 position of fatty acids in tolerance to photoinhibition. The third double bond at the A6 position is much less important or unimportant in tolerance to photoinhibition. In a previous study (16) we compared growth rates of the wild type, Fad6 mutant, and Fad6/desA::Kmr transformant at 22C and 34C under isothermal growth conditions. When these strains were grown at a light intensity of7 uein/m2 per sec, the growth rates at 34C did not differ. Nevertheless, at 22C at the same light intensity, Fad6/desA::Kmr did not grow, or grew only very slowly, whereas the wild type and Fad6 mutant grew well. This difference in growth rate at low temperature may be related to the accelerated photoinhibition at low temperature, particularly in Fad6/desA::Kmr transformant. When photosynthetic activities of cyanobacteria grown at different temperatures are compared under isothermal conditions, those of cells grown at lower temperature are greater than those of cells grown at high temperature (28). A similar phenomenon is seen in higher plants (29). The change in photosynthetic activity with growth temperature has been regarded as a result of alterations in unsaturation of fatty acids due to growth temperature. However, our study (Table 2) clearly shows that the change in unsaturation offatty acids does not affect photosynthesis and electron transport from H2 to BQ in strains with different degrees of unsaturation of fatty acids, when all strains are grown at the same temperature. From these observations it can be deduced that the growth temperature-dependent change in photosynthetic activity is regulated by an unknown factor unrelated to the extent of fatty acid unsaturation. Photoinhibition has been proposed to be induced by light energy not used by the photochemical reaction (27). The present study suggests that photoinhibition is accelerated by the elimination of unsaturation of fatty acids, even though alterations in the extent ofunsaturation do not affect the rates of photosynthesis and electron transport from H2 to BQ (Table 2). In other words, the light energy not trapped by the photochemical reaction is the same in the three strains, but the extent of photoinhibition differs among them. It is likely that the extent of photoinhibition in intact cells is a result of competition between two processes-namely, photochemical damage and recovery of the site targeted by the photoinhibition. It has been suggested that the site of photoinhibition is the photochemical reaction center (23) and that the modification and degradation of D1 protein are involved in this phenomenon (24, 3). At present we do not know how the unsaturation of fatty acids of membrane lipids affects the modification and degradation ofthe D1 protein. In this respect it is worth noting that the PS II reaction center isolated from spinach contains only one molecule of monogalactosyl diacylglycerol, which is constituted with a high level of saturated fatty acids as compared with those of the thylakoid membranes from the same plant (31). The lipids in cyanobacterial reaction centers have not been analyzed. However, the lipids of the reaction center are probably influenced by the extent of unsaturation of fatty acids of membrane lipids because a high rate of exchange of lipid molecules seems to occur between the reaction center and the thylakoid membrane (32). The recovery process after the photochemical damage to the PS II reaction center may involve synthesis of the D1 protein and insertion of the newly synthesized D1 protein into thylakoid membranes (24). The changes in unsaturation of fatty acids in thylakoid membranes may regulate the biosynthesis of D1 protein from the psba gene at the transcriptional and/or the translational level. Alternatively, because the

5 Plant Biology: Gombos et al. change in unsaturation can modify the physicochemical environment of the reaction center, it may affect insertion of the newly synthesized D1 protein. Each of any of these possible mechanisms would affect the recovery from photoinhibition. This work was supported, in part, by Grants-in-Aid for Scientific Research (2444) to N.M. and for Encouragement of Young Scientists (474383) to H.W. from the Ministry of Education, Science and Culture, Japan. 1. Raison, J. K. (1973) J. Bioenerg. 4, Lyons, J. M. (1973) Annu. Rev. Plant Physiol. 24, Murata, N. (1983) Plant Cell Physiol. 24, Murata, N. & Nishida, I. (199) in Chilling Injury ofhorticultural Crops, ed. Wang, C. Y. (CRC, Boca Raton, FL), pp Thompson, G. A., Jr. (198) in The Regulation of Membrane Lipid Metabolism (CRC, Boca Raton, FL) pp %. 6. Sato, N. & Murata, N. (198) Biochim. Biophys. Acta 619, Sato, N., Murata, N., Miura, Y. & Ueta, N. (1979) Biochim. Biophys. Acta 572, Wada, H. & Murata, N. (199) Plant Physiol. 92, Wada, H., Gombos, Z. & Murata, N. (199) Nature (London) 347, Baker, N. R. (1991) Physiol. Plant. 81, Greer, D. H., Berry, J. A. & Bjorkman,. (1986) Planta 168, Oquist, G. (1987) in Progress in Photosynthesis Research, ed. Biggins, J. (Nijhoff, Dordrecht, The Netherlands), Vol. 4, pp Powles, S. B., Berry, J. A. & Bj6rkman,. (1983) Plant Cell Environ. 6, Proc. Natl. Acad. Sci. USA 89 (1992) McCourt, P., Kunst, L., Browse, J. & Somerville, C. R. (1987) Plant Physiol. 84, Somerville, C. & Browse, J. (1991) Science 252, Wada, H., Gombos, Z., Sakamoto, T. & Murata, N. (1992) Plant Cell Physiol. 33, Wada, H. & Murata, N. (1989) Plant Cell Physiol. 3, Stanier, R. Y., Kunisawa, R., Mandel, M. & Cohen-Bazire, G. (1971) Bacteriol. Rev. 35, Ono, T. & Murata, N. (1981) Plant Physiol. 67, Bligh, E. G. & Dyer, W. J. (1959) Can. J. Biochem. Physiol. 37, Mohanty, N., Vass, I. & Demeter, S. (1989) Physiol. Plant. 76, Arnon, D. I., McSwain, B. D., Tsujimoto, H. Y. & Wada, K. (1974) Biochim. Biophys. Acta 357, Arntz, B. & Trebst, A. (1986) FEBS Lett. 194, Kyle, D. J., Ohad, I. & Arntzen, C. J. (1984) Proc. Natl. Acad. Sci. USA 81, Murata, N., Sato, N., Omata, T. & Kuwabara, T. (1981) Plant Cell Physiol. 22, Greer, D. H. & Laing, W. A. (1989) Planta 18, Powles, S. B. (1984) Annu. Rev. Plant Physiol. 35, Ono, T. & Murata, N. (1979) Biochim. Biophys. Acta 545, Berry, J. & Bjorkman,. (198) Annu. Rev. Plant Physiol. 31, Aro, E.-M., Hundal, T., Carlberg, I. & Andersson, B. (199) Biochim. Biophys. Acta 119, Murata, N., Higashi, S.-I. & Fujimura, Y. (199) Biochim. Biophys. Acta 119, Marsch, D. & Watts, A. (1988) in Advances in Membrane Fluidity: Lipid Domains and the Relationship to Membrane Function, eds. Aloia, R. C., Curtain, C. C. & Gordon, L. M. (Liss, New York), Vol. 2, pp