that the heat tolerance of photosynthesis is enhanced upon an increase in the level of saturation of membrane lipids. It is also shown that light does not affect the ...
Plant Physiol. (1994) 104: 563-567
The Unsaturation of Membrane Lipids Stabilizes Photosynthesis against Heat Stress' Zoltan Combos, Hajime Wada', Eva Hideg, and Norio Murata* Department of Regulation Biology, National lnstitute for Basic Biology, Myodaiji, Okazaki, 444 Japan (Z.G., H.W., N.M.); and lnstitute of Plant Physiology, Biological Research Center of the Hungarian Academy of Sciences, H-6701 Szeged, P.O. Box 521, Hungary (Z.G., E.H.)
thylakoid membranes can be achieved by changing the growth temperatures of photosynthetic organisms. Pearcy (1978) and Raison et al. (1982) observed that increases in growth temperature increase the level of saturated fatty acids in membrane lipids and enhance the heat stability of photosynthesis. These results led them to conclude that the saturation of fatty acids increases heat stability. However, such studies fail to correlate definitively the degree of saturation of fatty acids with heat stability, because a change in growth temperature affects not only the fatty-acid saturation but also various other metabolic factors. Santarius and Miiller (1979) observed that, in spinach, the increase in heat tolerance of photosynthesis during the acclimation to high temperature is not associated with changes in the level of saturation of membrane lipids. McCourt et al. (1987) observed that a decrease in the level of sn-1-18:3/sn2-16:3-MGDG and a corresponding increase in the level of sn-1-18:2/sn-2-16:2-MGDG in a mutant strain of Arabidopsis did not affect the heat stability of photosynthesis. Hugly et al. (1989) observed only insignificant differences in the thermal stability of the photosynthetic electron transports from H 2 0 to methyl viologen and H 2 0 to dichlorophenol indophenol in thylakoid membranes from the fudC mutant of Arabidopsis, which contain a reduced level of sn-l-l8:3/sn2-16:3-MGDG and an enhanced level of sn-1-18:l/sn-216:l-MGDG (Browse et al., 1989) compared with those from wild-type leaves. When the thermal stability of the electron transport of thylakoid membranes from the wild-type Arabidopsis was compared with that from the fadB mutant, which contained a reduced level of sn-1-18:3/sn-2-16:3-MGDG and an enhanced level of sn-1-18:3/sn-2-16:0-MGDG, there was no distinct difference (Kunst et al., 1989a, 1989b). In a previous study (Gombos et al., 1991) we also demonstrated that the complete elimination of trienoic fatty acids by mutation of Synechocystis PCC6803 had no effect on the thermal stability of photosynthesis. A11 these studies tend to suggest that heat stability is not associated with the level of saturation of membrane lipids. The oxygen-evolving activity is more sensitive to heat than other photosynthetic activities (Berry and Bjorkman, 1980; Mamedov et al., 1993). Nash et al. (1985) demonstrated that
The effect of the unsaturation of glycerolipids of thylakoid membranes on the heat tolerance of the photosynthetic evolution of oxygen was studied in vivo by mutation and transformation of fatty-acid desaturases in the cyanobacterium Synechocystis PCC6803. The experimental results indicate that elimination of dienoic lipid molecules decreases, to a small but distinct extent, the heat tolerance of photosynthetic oxygen evolution, but that elimination of trienoic lipid molecules has no effect on the heat tolerance. This conclusion contrasts with the previous hypothesis that the heat tolerance of photosynthesis is enhanced upon an increase in the level of saturation of membrane lipids. It is also shown that light does not affect the nature of the effect of lipid unsaturation on the heat tolerance of photosynthesis.
Glycerolipids of thylakoid membranes not only serve as a major constituent of the membrane-forming bilayers, but they also provide hydrophobic ligands to membranous proteins (Doyle and Yu, 1985). It has been suggested that four abundant glycerolipids of thylakoid membranes in the chloroplasts of higher plants and in the cells of cyanobacteria play important roles in maintaining the photosynthetic electron-transport machinery. It has been reported that sulfoquinovosyl diacylglycerol is associated with the ATP synthase (Pick et al., 1987) and that MGDG is bound to the reaction center of PSII (Murata et al., 1990). The degree of unsaturation of acyl residues of glycerolipids determines the physical characteristics of membranes (Chapman, 1975; Quinn, 1988; Quinn et al., 1989) and, consequently, the molecular motions of these lipids in the membranes. Therefore, one can postulate that fatty-acid unsaturation should affect various functions of membrane-bound proteins. It is of interest to examine the way in which the fatty-acid unsaturation of the glycerolipids of thylakoid membranes is related to the thermal tolerance of photosynthesis. Alterations in fatty-acid unsaturation of glycerolipids in Supported by Grants-in-Aid for Scientific Research on Priority Area (Nos. 04273102 and 04273103) to N.M. from the Ministry of Education, Science and Culture, Japan, and by a grant from the Hungarian National Scientific Research Foundation (OTKA, No. T520) to Z.G. and E.H. Present address: Biological Laboratory, Kyushu University, Ropponmatsu, Fukuoka 810, Japan. * Corresponding author; fax 81-564-54-4866.
Abbreviations: Km', kanamycin-resistance gene; MGDG, monogalactosyl diacylglycerol; X:Y,fatty acid containing X carbon atoms with Y double bonds in the cis configuration.
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a direct result of heat inactivation is the release of functional manganese ions from the PSII complex. Recently we developed a nove1 system in which the desaturation of fatty acids can be eliminated in a stepwise manner (Wada and Murata, 1989; Wada et al., 1992). In the present study we addressed the question of whether fatty-acid unsaturation plays a key role in the heat stability of photosynthesis using this cyanobacterial system. We demonstrated that, in contrast to the previous hypothesis, unsaturation of membrane lipids stabilizes, to a small but distinct extent, photosynthesis against heat inactivation. MATERIALS A N D M E T H O D S Organisms and Culture Conditions
Wild-type and Fad6 strains of Synechocystis PCC6803 were obtained as described by Wada and Murata (1989). The transformant of Synechocystis PCC6803, Fad6/desA::Kmr, was obtained as described previously (Wada et al., 1990, 1992). These strains were grown photoautotrophically under illumination from incandescent lamps at an intensity of 0.07 mE m-2 s-' in BG-11 medium (Stanier et al., 1971) supplemented with 20 mM Hepes-NaOH (pH 7.5), with aeration by sterile air that contained 1%C 0 2 (Ono and Murata, 1981). In some cases, the Fad6/desA::Kmr mutant was cultured in the presente of kanamycin at 5 gg mL-'.
Plant Physiol. Vol. 104, 1994 RESULTS
The temperature profiles of the heat inactivation of the photosynthetic activities of intact wild-type, Fad6, and Fad6/ desA::Km' cells were measured in terms of net photosynthesis and the photosynthetic evolution of oxygen, which was monitored with 1,4-benzoquinone as an artificial electron acceptor (Fig. 1). Essentially the same inactivation profiles were observed with the wild type and the Fad6 mutant. The Fad6/desA::Kmr mutant, by contrast, revealed a small but distinct decrease in the heat stability of photosynthesis and oxygen-evolving activity (Fig. 1). The temperatures for 50% inactivation of oxygen-evolving activity were 49.0 f 0.2OC, 49.2 f 0.2OC, and 47.8 f 0.2OC in the wild type, the Fad6 mutant, and the Fad6/desA::Kmr mutant, respectively. These results are compatible with those reported by Mamedov et al. (1993). The effect of light on the heat stability was studied to examine the possibility of cooperation of heat with light. Figure 2 shows that temperatures for 50% inactivation of the
Analysis of Lipids and Fatty Acids
The lipids were extracted from the intact cells by the method of Bligh and Dyer (1959). Analyses of lipids and fatty acids were performed as described by Wada and Murata (1989). Measurement of Photosynthetic Activities
Photosynthetic evolution of oxygen by intact cells was measured with a Clark-type oxygen electrode (Gombos et al., 1991), either with no exogenously added electron acceptor and donor, or with 1 m~ 1,4-benzoquinone and 1 m K3Fe(CN)6as electron acceptors. Light was provided from an incandescent lamp, after passage through a red optical filter (R62; Hoya Glass Co., Tokyo, Japan), at an intensity of 3.5 mE m-'s-'. The light treatment of cells was camed out as described by Gombos et al. (1992). The Chl concentration of cells was adjusted to about 10 gg Chl mL-I, as detennined by the method of Amon et al. (1974). Flash dependence of the evolution of oxygen was measured with an unmodulated bare-platinum oxygen electrode (Vass et al., 1990). A suspension of cells at a concentration that corresponded to 50 gg Chl mL-' was preilluminated with a train of 50 short flashes, which was followed by 5 min of dark adaptation. The evolution of oxygen was induced by a series of flashes of 3 ms duration and a frequency of 1 Hz, which were provided by a xenon flash lamp (1539-A xenon flash; GenRad, Concord, MA). Signals from the electrode were detected with a custom-built amplifier and monitored with a multichannel analyzer set at 2.5 to 10 ms/point (ICA KFKI, Budapest, Hungary).
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The temperature profiles of heat inactivation of photosynthetic activities in Synechocystis PCC6803 grown at 34°C. Cells of the wild-type, Fad6, and Fad6/desA::Kmrstrains were incubated in BG-11 medium at t h e designated temperatures in darkness for 60 min and photosynthetic activities were measured at 34°C in the light at an intensity of 3.5 mE m-2 s-' and a t a Chl concentration of 10 pg mL-'. A, Activity of net photosynthesis without exogenously added acceptors of electrons; the activity for the arbitrary unit, 100, corresponds to 360, 380, and 350 pmol O2 (mg Chl)-' h-' in the wild-type, Fad6, and Fad6/desA::Kmr cells, respectively. B, Oxygenevolving activity with 1,4-benzoquinoneadded as an acceptor of electrons; t h e activity for the arbitrary unit, 100, corresponds to 490, 480, and 500 kmol O2 (mg Chl)-' h-' in the wild-type, Fad6, and Fad6/desA::Kmrcells, respectively. T h e values were obtained from results of three independent experiments. O, Wild type; O, Fad6; O, Fad6/desA::Kmr. Figure 1.
Membrane Lipids and Heat Stability of Photosynthesis
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inactivation of photosynthetic oxygen-evolving activity. Wild-type, Fad6, and Fad6/desA::Kmrcells grown at 34°C were incubated at various temperatures under light of designated intensity for 40 min. Cells were suspended in BG-11 medium at a concentration of Chl that corresponded to 4 pg mL-'. The photosynthetic evolution of oxygen with 1,4-benzoquinoneas the acceptor of electrons was measured at 34°C. The values were obtained from results of three independent experiments. O, Wild type; O, Fad6; O, Fad6/ desA::Km'. evolution of oxygen fel1 with increases in light intensity. Nevertheless, the extent of the effect of light on the heat inactivation appeared to be the same for the wild type, the Fad6 mutant, and the Fad6/desA::Kmr mutant. These observations suggest that the differences in terms of the heat stability of photosynthesis among the three strains were not affected by light. To obtain more direct information about the effect of lipid unsaturation on the heat stability of the evolution of oxygen, we measured the oxygen yield after repeated flashes of light in heat-treated cells (Fig. 3). Heat treatment at 44OC decreased the oxygen yield in both wild-type and Fad6/desA::Kmr cells, but there were no changes in the patterns of oscillation of the yield. This result suggests that the heat treatment entirely destroyed part of the oxygen-evolving manganese complex but left the remaining part fully operative in both wild-type and Fad6ldesA::Km' cells. However, the results in Figure 3 indicate that the wild-type cells were more resistant to heat than the Fad6/desA::Km' cells; after treatment at 44OC for 40 min, the wild-type cells retained 50% of their oxygen-evolving activity, whereas the Fad6/desA::Kmr cells lost 75% of their activity. DISCUSSION
In the present study, our aim was to determine, using genetically engineered strains of Synechocystis PCC6803, whether the heat stability of photosynthesis is correlated with unsaturation of membrane lipids. Since the sn-2 position of the glycerol moiety of Synechocystis PCC6803 is esterified exclusively by 16:0, it is possible to calculate the number of
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lntensity of light during incubation (mEin m - * S I )
Figure 2. The effect of light intensity on the temperature for 50°/o
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Figure 3. Changes in the pattern of oxygen yield for a sequence of flashes. Wild-type and Fad6/desA::Km' cells grown at 34°C were incubated in BG-11 medium at 36°C (A), 40°C (B), 44°C (C), and 46°C (D) in darkness for 40 min. The Chl concentration during the heat treatment and during the measurement of oxygen yield was 50 pg mL-'. O, Wild type; O, Fad6/desA::Kmr.
unsaturated bonds in lipid molecules (Murata et al., 1992). Figure 4 shows the major molecular species estimated in this way. In the wild-type cells, sn-1-18:3/sn-2-16:0 and sn-l18:2/sn-2-16:0 account for 35 and 24%, respectively, whereas the Fad6 mutant contains sn-1-18:2/sn-2-16:0 at a leve1 equivalent to 50% of the total molecular species but no sn-1-18:3/sn-2-16:0. In the Fad6/desA::Kmr mutant, sn-l18:l/sn-2-16:0 accounts for 85% of the total molecular species, and there are no polyunsaturated species. In a11 three
'""I 16 0/16 O
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18 3/16 O
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Figure 4. Estimated composition in terms of molecular species of membrane lipids in Synechocystis PCC6803. Values were calculated on the basis of the fatty-acid composition of total lipids (Wada et al., 1992). For calculations, we assumed that the sn-2 position of the glycerol moiety was esterified exclusively by 16:O (Murata et al., 1992).
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strains, the saturated molecule 16:0/16:0 remained at a constant level of about 15%. The heat stability of the photosynthetic machinery in the Fad6 and the wild-type cells cannot be distinguished (Fig. 1). The photosynthetic machinery in the Fad6/desA::Kmr mutant, which does not contain any polyunsaturated lipid molecules, exhibited a small but distinct decrease in heat tolerance compared with that in the wild-type a n d the Fad6 mutant cells. The present study demonstrates that unsaturation of lipid molecules does indeed stabilize photosynthesis. This conclusion stands in contrast to the previous hypothesis, based on physiological studies (Raison et al., 1982), that the saturation of lipid molecules stabilizes the photosynthetic evolution of oxygen against heat inactivation. Since this contradiction might be the result of different experimental conditions, such as the presence or absence of light, we examined the effect of lipid unsaturation on the heat stability of photosynthesis under light of various intensities. With increases in light intensity, the inactivation of photosynthesis was accelerated (Fig. 2). Nevertheless, the effect of the unsaturation on the inactivation of photosynthesis was not altered in the light. In contrast to the results of Santarius a n d Miiller (1979) and our observations, Kunst et al. (1989a) and Hugly et al. (1989) inferred that the thermal tolerance of photosynthesis was enhanced by mutation of chloroplastic desaturation of membrane lipids in Arubidopsis. However, changes in the level of unsaturation of membrane lipids in their mutants were only partia1 because the cytoplasmic pathway for the supply of lipids to chloroplasts was fully active (Browse et al., 1989; Kunst et al., 1989b). Moreover, changes in the thermal tolerance were insignificant. Therefore, the relationship between the unsaturation of membrane lipids a n d the thermal tolerance was not clear in their reports. Using the Pd-catalyzed hydrogenation of membrane lipids of pea thylakoids, Thomas et al. (1986) observed that the thermal stability of PSII was enhanced after hydrogenation of up to 90% of the total double bonds of lipids in membranes in which the fully saturated lipid molecules were present at a substantial level. After hydrogenation of up to 40% of the total double bonds, which may correspond to changes within physiological conditions, the thermal stability was not altered at all. However, it has been observed in higher plants (Schreiber a n d Berry, 1977; Santarius a n d Miiller, 1979; Havaux, 1992) a n d cyanobacteria (Nishiyama et al., 1993) that heat stability of photosynthesis increases with changes in environmental factors such as temperature, water stress, a n d light. The extent of increases in heat stability due to growth temperature ranges from 3OC to 6OC and is much larger than that caused by the modification of the unsaturation of membrane lipids in the present study. We previously demonstrated (Nishiyama et al., 1993) that the thylakoid membranes isolated from cyanobacterial cells that h a d been acclimated to nonlethal high temperature retained heat stability of photosynthetic electron transport. These observations suggest that unknown biochemical factors associated with the thylakoid membranes are responsible for the enhanced thermal stability of photosynthesis in cells grown at high temperature.
Plant Physiol. Vol. 104, 1994
Received August 6, 1993; accepted November 9, 1993. Copyright Clearance Center: 0032-0889/94/104/0563/05. LITERATURE ClTED
Arnon DI, McSwain BD, Tsujimoto HY, Wada K (1974) Photochemical activity and components of membrane preparations from blue-green algae. I. Coexistence of two photosystems in relation to chlorophyll a and remova1 of phycocyanin. Biochim Biophys Acta 357: 231-245 Berry J, Bjorkman O (1980) Photosynthetic response and adaptation to temperature in higher plants. Annu Rev Plant Physiol 31: 491-543 Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol37: 911-917 Browse J, Kunst L, Anderson S, Hugly S, Somerville C (1989) A mutant of Arabidopsis deficient in the chloroplast 16:1/18:1 desaturase. Plant Physiol 90: 522-529 Chapman D (1975) Phase transition and fluidity characteristics of lipids and cell membranes. Q Rev Biophys 8: 185-235 Doyle MF, Yu C-A (1985) Preparation and reconstitution of a phospholipid-deficient cytochrome b6-f complex from spinach chloroplasts. Biochem Biophys Res Commun 131: 700-706 Gombos Z, Wada H, Murata N (1991) Direct evaluation of effects of fatty-acid unsaturation on the thermal properties of photosynthetic activities, as studied by mutation and transformation of Synechocystis PCC6803. Plant Cell Physiol 32: 205-21 1 Gombos Z, Wada H, Murata N (1992) Unsaturation of fatty acids in membrane lipids enhances the tolerance of the cyanobacterium Synechocystis PCC6803 to low-temperature photoinhibition. Proc Natl Acad Sci USA 8 9 9959-9963 Havaux M (1992) Stress tolerance of photosystem I1 in vivo. Plant Physiol 100 424-432 Hugly S, Kunst L, Browse J, Somerville C (1989) Enhanced thermal tolerance of photosynthesis and altered chloroplast structure in a mutant of Arabidopsis deficient in lipid desaturation. Plant Physiol 9 0 1134-1142 Kunst L, Browse J, Somerville C (1989a) Enhanced thermal tolerante in a mutant of Arabidopsis deficient in palmitic acid unsaturation. Plant Physiol91: 401-408 Kunst L, Browse J, Somerville C (1989b) A mutant of Arabidopsis deficient in desaturation of palmitic acid in leaf lipids. Plant Physiol 9 0 943-947 Mamedov M, Hayashi H, Murata N (1993) Effects of glycinebetaine and unsaturation of membrane lipids on heat stability of photosynthetic electron-transport and phosphorylation reactions in Synechocystis PCC6803. Biochim Biophys Acta 1142: 1-5 McCourt P, Kunst L, Browse J, Somerville C (1987) The effects of reduced amounts of lipid unsaturation on chloroplast ultrastructure and photosynthesis in a mutant of Arabidopsis. Plant Physiol 8 4 353-360 Murata N, Higashi S-I, Fujimura Y (1990) Glycerolipids in various preparations of photosystem I1 from spinach chloroplasts. Biochim Biophys Acta 1019 261-268 Murata N, Wada H, Gombos Z (1992) Modes of fatty acid desaturation in cyanobacteria. Plant Cell Physiol33 933-941 Nash D, Miyao M, Murata N (1985) Heat inactivation of oxygen evolution in photosystem I1 particles and its acceleration by chloride depletion and exogenous manganese. Biochim Biophys Acta 807: 127-133 Nishiyama Y, Kovacs E, Lee CB, Hayashi H, Watanabe T, Murata N (1993) Photosynthetic adaptation to high temperature associated with thylakoid membranes of Synechococcus PCC7002. Plant Cell Physiol34 337-343 Ono T, Murata N (1981) Chilling susceptibility of the blue-green alga Anacystis nidulans. I. Effect of growth temperature. Plant Physiol67: 176-181 Pearcy R (1978) Effect of growth temperature on the fatty acid composition of the leaf lipids in Atriplex lentiformis (Torr.) Wats. Plant Physiol61: 484-486 Pick U, Weiss M, Gounaris K, Barber J (1987) The role of different thylakoid glycolipids in the function of reconstituted chloroplast ATP synthase. Biochim Biophys Acta 891: 28-39
Membrane Lipids and Heat Stability of Photosynthesis Quinn PJ (1988) Regulation of membrane fluidity in plants. In J Barber, NR Baker, eds, Physiological Regulation of Membrane Fluidity, Vol 3. Alan R Liss, New York, pp 293-321 Quinn PJ, Joo F, Vigh L (1989) The role of unsaturated lipids in membrane structure and stability. Prog Biophys Mo1 Biol 5 3 71-103 Raison JK, Roberts JKM, Berry JA (1982) Correlation between the thermal stability of chloroplast (thylakoid) membranes and the composition and fluidity of their polar lipids upon acclimation of the higher plant, Nerium oleander, to growth temperature. Biochim Biophys Acta 688: 218-228 Santarius KA, Miiller M (1979) Investigation on heat resistance of spinach leaves. Planta 146 529-538 Schreiber U, Berry JA (1977) Heat-induced changes of chlorophyll fluorescence in intact leaves correlated with damage of the photosynthetic apparatus. Planta 136 233-238 Stanier RY, Kunisawa R, Mande1 M, Cohen-Bazire G (1971) Pu-
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rification and properties of unicellular blue-green algae (order Chroococcales). Bacteriol Rev 3 5 171-205 Thomas PG, Dominy PJ, Vigh L, Mansourian AR, Quinn PJ, Williams WP (1986) Increased thermal stability of pigment-protein complexes of pea thylakoids following catalytic hydrogenation of membrane lipids. Biochim Biophys Acta 849 131-140 Vass I, Deak ZS, Hideg E (1990) Charge equilibrium between the water-oxidizing complex and the electron donor tyrosine-D in photosystem 11. Biochim Biophys Acta 1017:63-69 Wada H, Gombos Z, Murata N (1990) Enhancement of chilling tolerance of a cyanobacterium by genetic manipulation of fattyacid desaturation. Nature 347: 200-203 Wada H, Gombos 2, Sakamoto T, Murata N (1992) Genetic manipulation of desaturation of fatty acids in membrane lipids in the cyanobacterium Synechocystis PCC6803. Plant Cell Physiol 33: 535-540 Wada H, Murata N (1989) Synechocystis PCC6803 mutants defective in desaturation of fatty acids. Plant Cell Physiol 3 0 971-978
