Contribution of lowered unsaturation levels of chloroplast lipids to high temperature tolerance of photosynthesis in Chlamydomonas reinhardtii

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P l l t J l ~ ~t~m~tmY Journalof Photochemistryand PhotobiologyB: Biology36 (1996) 333-337

ELSEVIER

Contribution of lowered unsaturation levels of chloroplast lipids to high temperature tolerance of photosynthesis in Chlamydomonas reinhardtii N o r i h i r o S a t o a, K i n t a k e S o n o i k e b, A k i h i k o K a w a g u c h i c, M i k i o T s u z u k i a'* • School of Life Science. Tokyo Universityof Pharmacy and Life Science. Hachi~ji. Tokyo. 192.03. Japan b Department of Botany. The Universityof Tokyo. Hongo. Bunkyo-lm. Tokyo. 113. Japan c Department of Biology. College of Arts and Sciences. The Universityof Tokyo. Komaba. Meguro-ku. Tokyo. 1.53.Japan

Abstract A mutant of Chlamydomonas reinhardtii designated as hf-9 is impaired in fatty acid desaturation of chloroplasts, and showed lowered unsaturation levels of chloroplast lipids, as compared with the parent (Sato et al., Eur. J. Biochem., 230 ( 1995) 987-993.~ The effects of temperature on photosynthesis were compared between hf.9 and the parent for investigation whether ¢r not unsaturation levels of chloroplast lipids are correlated with the thermal properties of photosynthesis. Growth rates determined by turbidity were higher in the parent than in hf-9 at both 10 and 24 °C, while similar for the parent and hf-9 at 39 °{2.The cells grown at 24 °(2 revealed that both activities of CO2-dependent oxygen evolution and photosystem I! were higher in the parent than in I~f-9in the range between 7 and 40 °C. In contrast, hf.9 surpassed tim parent in both activities at 45 °C. Optimal teml:eratures for both activities were at around 35 °C and 40 °C in the parent and hf-9, respectively. Incubation of the cells at 41 and 45 °C demonstrated that the activity of photosystem II in hf-9 was more tolerant to the high temperatures than that in the parent. These results suggest that lowered unsaturation levels of chloroplast lipids contributed to high temperature tolerance of photosystem II, and eventually to that of photosynthesis. Keywords: Chlamydomonas reinhardtii; Chloroplastlipids;Desaturation:Hightemper-alinetolerance;PhotosystemIt

1. Introduction Rates of photosynthesis in plants, algae, and cyanobacteria are affected by various environmental factors such as temperatures and light intensities. In photosynthetic reactions, photosystem II in thylakoid membranes is especially damaged at extremely high and low temperatures [1-3]. Recently, PSI was shown to be suceptible to low temperature

[4,51. Biomembranes are constructed from specific lipids and proteins which are responsible for special functions. However, information on the physiological roles of lipids in biomembranes is poor. Thylakoid membranes, the functions of which are very sensitive to temperature-stress, mainly contain monogalactosyl diacylglycerol (MGDG), digalactosyl diacylglycerol (DGDG), sulfoquinovosyl diacylglycerol, and phosphatidylglycerol as lipid components. MGDG and Abbleviations: Chl. chlorophyll:CO2-PS, CO2-dependentoxygen evolution; DGDG,digalactosyldiacylglycerol;LHCll, light-harvestingcomplex of photosystem!1; MGDG,monogalactosyldiacylglycerol:PSI, photosysternI; PSll, photosystemI!; Fattyacidsare denotedby thenumbersof carbon atoms and doublebonds * Con'espondingauthor. Fax: + 8 ! 42676672I.

DGDG are characteristic of highly unsaturated fatty acids such as linolenic acid. Since the physical behavior of membrane lipids is largely affected by unsaturation levels of constituent fatty acids, the temperature-dependent properties of photosynthesis have been studied in mutants and transformants with altered unsaturation levels of chloroplast lipids [6]. Concerning low temperature, the physiological significance of highly unsaturated fatty acids in photosynthesis is postulated to be connected to DI protein turnover [7,8]. In the case of high temperature, a mutant of Synechocystis PCC6803 defective in A 12 desauration, with reduced contents of dienoic acids and loss of trienoic acids in thylakoid membranes, showed no alteration in thermal stability of photosynthesis [9]. This indicated that unsaturated fatty acids, the contents of which were lowered, have little effect on the thermal stability of photosynthesis in cyanobacteria. In contrast, two respective mutants of chloroplast desaturation of Arabidopsis thaliana, with reduced contents of highly unsaturated fatty acids of chloroplasts, showed enhanced stability of PSII in relation to heat [ 10, I ! ]. We previously isolated a mutant of a green alga, Chlamydomonas reinb~rdtii designated as hf-9, which was impaired in o,,6 desaturation of chloroplasts [ 121. This mutant showed

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N. Sato et oL /Journal of Photochemistryand PhotobiologyB: Biology36 (I 996) 333-337

more deleterious effects on unsaturation levels of chloroplast iipids than the Arabidopsis mutants described above, and thus might be more suitable to elucidate the physiological role of unsaturation levels of chloroplast lipids, hf-9 previously showed decreased photosyntbetic activity at normal temperature, suggesting that high unsaturation levels of chloroplast lipids play a basic role in photosynthesis at normal temperature [ 12], In this study, we examined whether or not hf-9 was affected in the thermal properties of photosynthesis to clarify the correlation of unsaturation levels of chloroplast lipids with thermal stability of photosynthesis in eukaryotes.

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2. Materials a n d methods

2.1. Algal culture Cells of Chlamydomonas reinhardtii 6145c (,~.itl-305) mt- ant~ its mutant, hf-9, were grown photoautotrophically at 24 °C, as described previously [ 13]. Cell growth was d;;termined by OD measured at 750 nm.

2.2. Measurements of photosynthetic activities Cells at the logarithmic growth phase were harvested by centrifugation at 3000 g for 5 min and then were resuspended in 50 mM Tricine, pH 7.5. Two photosynthetic activities, i.e. CO2-dependent oxygen evolution (CO2-PS) rate and PSII activity were measured in these cells at 6/zg Chl m l - t in the same buffer, containing I mM NaHCO3, and 300 /~M pbenzoquinone and 2 mM NH+CI, respectively, in a Clarktype electrode (Rank Brothers, London). Temperature dependency of photosynthetic activities was investigated after 5 min equilibration of the cells in the dark at a designated temperature. Time-course of heat-inactivation of PSII was examined by measurement of PSII activity at 30 °C after the cells were incubated at 41 or 45 °C for certain periods in the dark.

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then transferredto 10°C, 24 °C, and 39 °C for furthergrowth.Cell growth after the transferwas measuredby OD at 750 nm. The OD valuewas 0.034 and 0.041 in the parentand hf-9, respectively,at the the timeof the transfer. 1-1,l0 °C; O. 24 °C: A, 39 °C for the parent;I , 10 °C; O, 24 °C; &, 39 °C for I~f-9. spicuous as compared with that at 24 °C. In contrast, growth rates at 39 °C were similar for the parent and hf-9, resulting in an upward-shift of optimal temperature for cell growth in hf-9. In addition, Chl content was much lower in the parent than in hf-9 after 68 h culture at 39 °C ( 14 and 32/zg Chl m l - ~ in the parent and hf-19, respectively,). These results indicate enhanced high-temperature-tolerance for cell growth in hf-9.

3.2. Temperature-dependency of photosynthetic activities in the parent and hf-9 To examine the effects of the desaturation mutation on temperature dependency of photosynthesis, CO2-PS rates at a designated temperature were determined in the parent and hf-9 after equilibration for 5 min in the dark (Fig. 2). hf-9 showed lower rates of COz-PS than the parent at temperatures between 7 and 40 °C. However, it surpassed the parent in the rate at 45 °C, and showed a shift of optimal temperature for

3. Results " 2oo

3.1. Temperature-dependency of cell growth of rhe parent and hf-9

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A mutant of C. reinhardtii designated as hf-9, which had lesion in o,'6 desaturation of fatty acids of chloroplasts, accumulated 16:1 (7) and 18:1 (9) with decreases in contents of highly unsaturated fatty acids such as 18:3(9,12,15) and 16:4(4,7,10,13) in chloroplast lipids, as compared with the parent [ 12]. Firstly, we investigated the growth of the parent and hf-9 at various temperatures ( 10, 24, and 39 °C, Fig. 1). The growth rate at 24 °C was higher in the parent than hf-9, which corresponded to our previous report [ 12]. The parent also showed a higher growth rate at 10 °C than hf-9, but the depressio• of growth rate in hf-9 at 10 °C was not so con-

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30

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Temperature (°C)

Fig. 2. Temperature-dependencyof CO2-dependentoxygenevolutionin the parent (O) and hf-9 (O). Oxygenevolutionwas measuredin the cells of the patent and hf-9 in the presenceof I mM NaHCO3 at the designated temperatureafter 5 rainequilibrationin the dark.

iV. Saw et al. I Journalof Photochemistryand PhowbiologyB: Biology36 (! 996j 333-337

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Fig, 3, Temperature-dependencyof PSIIactivityin the $ment (O) and hi: 9(O). PSI!activitywas measuredin the cellsof the parent and hr.9withpbenzoquinoneas the electronacceptor,at the designatedtemperatmeafter 5 min equilibrationin the dark, The values are the me,ens for three independent experiments. the rate from 35 °C of the parent to 40 °C. The shift of optimal temperature coincided with high temperature tolerance in hf-9 (Fig. 1). Temperature-dependency of PSII activity of the parent and hf-9 were similar m those observed with CO,-PS: hf-9 showed higher activity than the parent only at 45 °(2 in the tempe|ature range examined, and an upward-shift of optimal temperature for the activity (Fig. 3). Fig. 4(a) shows the temperature dependency of the ratio of CO2-PS to PSII activity in the parent and hf-9. This value gives the degree of supression of PSII activity in the whole chain of photosynthesis. The ratio was 0.34 at 7 °C in the parent, implying that photosynthesis was limited at some point in the process except at PSII, probably at CO2 fixation by RubisCO. Increase in the ratio with elevation of temperature in the parent suggested that the suppression was diminished at higher temperatures. The value was increased up to 0.85 at 45 °C, indicating that PSII reaction proceeded at its almost maximal rate in the whole chain of photosynthesis. These results suggested that PSII activity was responsible for photosynthetic capacity at high temperature in the parent cells. On the contrary, the ratio in hf-9 was between 0.74 and 0.90 at all temperatures examined, indicating that ?SII reaction ~howed the activity to capacity in the whole chain of 1.0

335

photosynthesis. Since both PSH activity and the rate of photosynthesis were lowered in hf-9, PSH may be a main factor to limit photosyntbesis in this lipid mutant. The ratio of laSH activity of hf-9 to that of the parent was shown at temperatures between 7 and 45 °C in Fig. 4(b). The values, almost constant in tbe range from 7 to 30 °C, were increased to 1.6 when elevating temperatme up to 45 °C (Fig. 4 (b)), corresponding to high temperatme tolerance in hf-9 (Fig. I). These resultsalso showed that lowcRd unsaturation levels of chloroplast lipids brought about in PSH activity at low temperatures as well as at normal temperatures.

3.3. Heat-induced innactivation of PSll activity in the parent and hf- 9 The cells of the parent and hf-9 incubated at 41 °C or 45 °C for a given period in the dark were used for measorement of PSII activity at 30 °C, for comparison of the heat-induced inactivation of PSll between tbe parent and hf-9 (Fig. 5). Incubation of the parent at 41 °C resulted in a fast decrease in PSH activity (time required for 50% decrease in PSII activity, tl/2=53 min) for the first 40 min, followed by a slow decrease (t|/2 = 144 mir). In contrast. PSII activity of hf-9 was decreased slowly throughout incubation times (tt/ 2 = 159 min). When the cells were incubated at 45 ~ , both the parent and hf-9 showed rapid decreases (Fig. 4). However, the rate was faster in the parent (tz/2=3 min) than in hf-9 (tt/2 ffig min ), resulting in coraplete loss of the activity in the parent before hf-9. These resu!is demonstrated that PSII activity of hf-9 was more stable in relation to heat than that of the parent.

4. Discussion The mutant, hf-9, showing decreases in unsaturation levels of chloroplast lipids, is suitable for investigation of physiological roles of unsaturated fatty acids in chloroplasts. We previously showed that hf-9 was irapaired in the activities of ~.e~-"fo) 1,61"

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Fig. 4. Temperature-dependencyof the ratioof CO2-dependentoxygenevolutionto i~!I activityin the parent (O) and hf-9 (O) ((a) see Fig. 2)), and of the ratio of PSI!activityof hf-9 to that of the parent ((b), see Fig. 3).

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N. Sato et al. / Journal ~f ,Photocheraistryand Photobiology B: Biology 36 (1996) 333-337

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were incubated at 41 *(2 ( a ) and 45 *(2 ( b ) in

the dark for a givenperiod,and thenusedfor measurementof PSI!activityat 30 *(2. PSI and PSII at 25 *(2,with little effect on protein components of Chl-protein complexes responsible for these activities [ 14]. -'~,e activities may be affected by unsaturation levels of chloroplast lipids bound to these complexes [ 14]. In the present paper, we showed that lowered lipid unsaturation of chloroplasts brought about decreased PSII activity also at low temperatures, but increased PSII activity at high temperature such as 45 *(2. In hf-9, optimal temperature for cell growth was elevated, resulting in comparable growth rates at 39 °(2 for the parent and hf-9 (Fig. 1 ). In addition, Chl content was lower in the parent than in hf-9 after 68 h culture at 39 0(2. hf-9 exhibited a high rate of CO2-PS at 45 °(2, ar.a an upward-shift of optimal temperature for the rate as compared with the parent (Fig. 2 ), probably owing to the alteration in the thermal property of PSII (Figs. 3 and 4). This result is in accordance with a slower decrease in PSII activity at both 41 and 45 *C in hf-9 than in the parent, indicating enhanced thermal stability of PSII activity in hf-9 (Fig. 5). Thus, it is concluded that hf-9 acquired high temperature tclerance of PSII, and eventually of photosynthesis, owing to decreased unsaturation levels of chloroplast lipids. Analysis of the thermal property of photosynthesis in a mutant ofA. thaliann impait'ed in chloroplast a,6 desaturation suggested that lowered unsatmation levels ofchiorolast iipids were responsible for enhanced high-temperature-toleranceof thylakoid membrane functions such as association of LHCII to PSII [ 10]. hf-9 indicated other phases of high temperature tolerance of photosynthesis such as no appearance of lowered Chl contents at 39 °(2, and elevation of optimal temperature for growth and photosynthetic activities, as compared with the parent (Figs. I and 2 and 3 ). The mutational effect of agi desaturation on the contents of highly unsaturated fatty acids such as 18:3 ( 9,12,15 ) was less remarkable in theArabidopsis mutant than in hf-9, owing to the presence of a pathway, in A. thaliana, to supply 18:2(9,12) synthesized at ER to chloroplasts for further desaturation [ 15]. We consider that a more pronounced decrease in unsaturation levels of chloroplast lipids in hf-9, in which such a pathway is lacking, is responsible for the remarkable phenotypes of high temperature tolerance of photosymhesis.

In contrast, a mutant of Synechocystis PCC6803 defective in A 12 desaturation, leading to a decreased contents ofdienoic acids and a complete loss of trienoic acids, showed no alteration in the thermal stability of PSII and whole chain photosynthesis in relation to heat [9]. This indicated that highly unsaturated fatty acids are not involved in high temperature tolerance in cyanobacteria, which is in contrast to the conclusion deduced from the data reported in this paper. Unsataration levels of thylakoid membrane iipids are usually higher in green plants than in cyanobacteria [ 16]. The physical properties of boundary lipids and/or lipid matrix, which are affected by their unsaturation levels, may therefore bring about distinct effects of unsaturation levels on thermal stability of PSII between green plants and cyanobacteria. Otherwise, conformation of the PSII complex itself may be adjusted to higher temperatures in cyanobaeteria, hf-9, showing a pronounced high-temperature-tolerance of photosynthesis, will be a suitable tool for elucidation of the mechanism enhancing thermal stability in green plants. Saturation of pea thylakoid membranes in situ with catalytic hydrogenation increased the thermal stability of thylakoid membrane ultrastrocture, in parallel with enhancement of the thermal stability of PSII [ 17 ]. These authors suggested that hydrogenation of chloroplast lipids coincided with a reduced tendency of non-bilayer forming lipids such as monogalactosyl diacylglycerol to phase-separate at high temperatures, and finally with the enhanced thermal stability of PSll. The alteration in phase behavior of chloroplast lipids with decreased numbers of double bonds may also be associated to high temperature tolerance of PSII in vivo (Fig. 3). On the contrary, we observed high unsaturation levels of lipids bound to PSI1 from a wild type of C. reinhardtii [ 13]. We consider that such lipids contribute to the conformation of the PSII complex yielding full activity through interaction with this complex. Physical properties of these boundary iipids with lowered unsaturatioo levels may be suited to keep a proper conformation of PSII at high temperaturos. Transformants of photosynthetic organisms, tolerant to cold temperature, were produced through introduction of genes for synthesis of highly unsaturated fatty acids for thy-

IV. Sate et al. I Journal of Photochemistry and Photobiology B: Biology 36 (1996) 333-337

iakoid m e m b r a n e s [ 18,19]. Recently, genes for chloroplast o~6 desaturase, which is impaired in }~¢-9, were cloned in higher plants such as Brassica napus [20]. To confer high temperature tolerance to this otganism, it is desirable to depress expression o f the gene on!y at high temperatures, since lesion in chloroplast o~6 desat.uration brought about d a m a g e to photosynthesis at low and normal temperatures in C. reinhardtii (Fig. 2). A transformant with a stable PSII both at normal and at elevated temperatures could be obtained by expression o f chloroplast o~6 desaturase in hf-9 at normal growth temperature, or by anti-sense expression o f the s a m e gene in the wild type at high temperature.

Acknowledgements W e t h a r k Mr. J. W r i g h t for critical reading o f this m a n u script. This investigation was partly supported by the Asahi Glass Foundation and by a grant-in-aid for scientific research from Ministry o f Education, Sciences, Sports and Culture o f Japan (05804047).

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[121N. Sate, M. Tsuzuki, Y. Matsnda, 1". ~ T. Osafun¢ and A. Kawaguchi, Isolation and c h a n ~ = r i ~ of n'anats affected in lipid metabolism of CIdamydomon~ reiMmrdtii. Fur. J. Bioch~. 230 (1995) 987-993. [13] N. Sate, K. ,~noil~, M. TsuzuH and A. KawagucM, Impai~l photos~tem ll in a nmCmtof Ch/amydomonas re/m/mrddidefective in sulfoquinovosyl diacylglycetol, Eur. J. Biocher~, 234 (1995) 1623. [ 14] N. Sate, K. Sonc~ke,M. Tsuzuki and A. Kawaguchi, ¢haractesisticsof a mutant of Ch/amydomonasre/n/mrdt/i impmiredia fatty a~'id d e s a l t , Biochim. Biophys. Acta, 1274 (1996) 112118. [ 15] J. Browse~L. Kanst, S. Anderson, S. Hngiy and C.R. Somerville, A mutant of Arabidopsis deficient in the chlomplest 16:i118:1 desamra~ Plant Physiot.. 90 (1989) 522-529. [16] H.D. Z e t a , E. Heinz, A. Radunz, M. Linsheid mKI R. Pesch, Combination and posilioe~ dislribation of fatty acids in lipids from blue-groan aloe. Arch. Microbiol.. 119 (19"/8) 157-162. [17] P.G. Thoma~ PJ. Domlny, L. Vigh, A.R. Mansomina, PJ. Quinn and W.P. Wimams, hg'nmnd thermal stability of pigrama-laog'in complexes of pea tlfflekoids following catalytic h~h~mmtion of n~.mbranelipide, B/ocMm.Biophy$. ,~ea, 849 (1986) 131-140. [18] H. Wada, 7,. Gombos and N. Mureta, F.,nhancmnm~of chilling t ~ of a cyanol~gterium liy genetic manipulation of fatty asid de,rotation. Nacre, 347 (1990) 200-203. [ 19] N. Muram, O. IsMzaki-Hisldzawa,S. Hignshi, H. Heffashi,Y. Teemlka and !. Nishid~ Gel~iically engin~red ahera~on in the chilling sensitivity of plants, Nature. 356 (1992) 710-713. [ 20] W.D. Hitz, TJ. Carlson, J.R. Booth, AJ. Kinney, K.L. Steccaand N.S. Yadav. Cloning of a higher-plato pl,~id ~ fatty acid d n s ~ . ~ e eDNA and its exlm~ssienin a cyanol~ct~um, Plant Physio£, 105 (1994) 635-.641.