Targeted mutagenesis of acyl-lipid desaturases in Synechocystis: evidence for the important roles of polyunsaturated membrane lipids in growth, respiration and photosynthesis.

Tasaka, Yasushi; Gombos, Zoltán; Nishiyama, Yoshitaka; Mohanty, Prasanna; Ohba, Tetsuhiko; Ohki, Kazuo; Murata, Norio

doi:10.1002/j.1460-2075.1996.tb01033.x

Cited by 223 publications

(155 citation statements)

References 43 publications

(34 reference statements)

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“…Lipids are mostly involved in the responses to temperature stresses by the altered level of unsaturation of their fatty acids (38), but the induction of the desaturase system responsible for the altered unsaturation is additionally light-regulated (39). Unsaturated lipids are further needed in the repair of the PSII reaction centers after photoinhibitory destruction (40).…”

Section: Discussionmentioning

confidence: 99%

Low-temperature-induced accumulation of xanthophylls and its structural consequences in the photosynthetic membranes of the cyanobacterium Cylindrospermopsis raciborskii : An FTIR spectroscopic study

Várkonyi

Masamoto

Debreczeny

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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The effects of the growth temperature on the lipids and carotenoids of a filamentous cyanobacterium, Cylindrospermopsis raciborskii, were studied., The relative amounts of polyunsaturated glycerolipids and myxoxanthophylls in the thylakoid membranes increased markedly when this cyanobacterium was grown at 25°C instead of 35°C. Fourier transform infrared spectroscopy was used to analyze the low-temperature-induced structural alterations in the thylakoid membranes. Despite the higher amount of unsaturated lipids there, conventional analysis of the symCH2 band (characteristic of the lipid disorder) revealed more tightly arranged fatty-acyl chains for the thylakoids in the cells grown at 25°C as compared with those grown at 35°C. This apparent controversy was resolved by a two-component analysis of the symCH2 band, which demonstrated very rigid, myxoxanthophyll-related lipids in the thylakoid membranes. When this rigid component was excluded from the analysis of the thermotropic responses of the symCH2 bands, the expected higher fatty-acyl disorder was observed for the thylakoids prepared from cells grown at 25°C as compared with those grown at 35°C. Both the carotenoid composition and this rigid component in the thylakoid membranes were only growth temperature-dependent; the intensity of the illuminating light during cultivation had no apparent effect on these parameters. We propose that, besides their well-known protective functions, the polar carotenoids in particular may have structural effects on the thylakoid membranes. These effects should be exerted locally-by forming protective patches, in-membrane barriers of low dynamics-to prevent the access of reactive radicals generated in either enzymatic or photosynthetic processes to sensitive spots of the membranes.

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Section: Discussionmentioning

confidence: 99%

Low-temperature-induced accumulation of xanthophylls and its structural consequences in the photosynthetic membranes of the cyanobacterium Cylindrospermopsis raciborskii : An FTIR spectroscopic study

Várkonyi

Masamoto

Debreczeny

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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“…The appearance of a visible phenotype may also be prevented by residual 20:4. Deletions of several desaturases in Synechocystis became critical only if the ∆6-and ∆12-desaturase were knocked out together, whereas a reduction in trienoic acids without affecting dienoic acids was not critical (Tasaka et al, 1996).…”

Section: Functional Analysis Of Ppdes6 In P Patens and S Cerevisiaementioning

confidence: 99%

Identification of a novel D6‐acyl‐group desaturase by targeted gene disruption in Physcomitrella patens

et al. 1998

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SummaryThe moss Physcomitrella patens contains high levels of arachidonic acid. For its synthesis from linoleic acid by desaturation and elongation, novel ∆5-and ∆6-desaturases are required. To isolate one of these, PCR-based cloning was used, and resulted in the isolation of a full-length cDNA coding for a putatively new desaturase. The deduced amino acid sequence has three domains: a N-terminal segment of about 100 amino acids, with no similarity to any sequence in the data banks, followed by a cytochrome b 5 -related region and a C-terminal sequence with low similarity (27% identity) to acyl-lipid desaturases. To elucidate the function of this protein, we disrupted its gene by transforming P. patens with the corresponding linear genomic sequence, into which a positive selection marker had been inserted. The molecular analysis of five transformed lines showed that the selection cartridge had been inserted into the corresponding genomic locus of all five lines. The gene disruption resulted in a dramatic alteration of the fatty acid pattern in the knockout plants. The large increase in linoleic acid and the concomitant disappearance of γ-linolenic and arachidonic acid in all knockout lines suggested that the new cDNA coded for a ∆6-desaturase. This was confirmed by expression of the cDNA in yeast and analysis of the resultant fatty acids by GC-MS. Only the transformed yeast cells were able to introduce a further double bond into the ∆6-position of unsaturated fatty acids. To our knowledge, this is the first report of a successful gene disruption in a multicellular plant resulting in a specific biochemical phenotype.

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“…PCC 6803 in which the desA and desD genes for the ⌬12 and ⌬6 desaturases, respectively, had been inactivated by targeted mutagenesis (6). The desA Ϫ ͞desD Ϫ cells contain monounsaturated but not polyunsaturated fatty acids, whereas wild-type cells contain polyunsaturated fatty acids such as di-, tri-, and tetraunsaturated fatty acids (7).…”

mentioning

confidence: 99%

“…By comparing these two strains we found that polyunsaturated fatty acids are essential for growth in Synechocystis sp. PCC 6803 at low temperatures (6).…”

mentioning

confidence: 99%

“…Activities of photosystem (PS) II in intact cells were measured with a Clarktype oxygen electrode (Hansatech Instruments, Kings Lynn, U.K.) in the presence of 1.0 mM 1,4-benzoquinone (BQ) or 1.0 mM 2,6-dichlorobenzoquinone (DCBQ) as described previously (6). The light-induced reduction of dichloroindophenol (DCIP) by isolated thylakoid membranes was measured with a dual-wavelength spectrophotometer (UV-300; Shimadzu) as described previously (9).…”

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confidence: 99%

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Genetic engineering of the unsaturation of fatty acids in membrane lipids alters the tolerance ofSynechocystisto salt stress

Allakhverdiev

Nishiyama

Suzuki

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

194

121

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The role of unsaturated fatty acids in membrane lipids in the tolerance of the photosynthetic machinery to salt stress was studied by comparing the desA ؊ ͞desD ؊ mutant of Synechocystis sp. PCC 6803, which contained monounsaturated fatty acids, with the wild-type strain, which contained a full complement of polyunsaturated fatty acids. In darkness, the loss of oxygen-evolving photosystem II activity in the presence of 0.5 M NaCl or 0.5 M LiCl was much more rapid in desA ؊ ͞desD ؊ cells than in wild-type cells. Oxygenevolving activity that had been lost during incubation with 0.5 M NaCl in darkness returned when cells were transferred to conditions that allowed photosynthesis or respiration. Recovery was much greater in wild-type than in desA ؊ ͞desD ؊ cells, and it was prevented by lincomycin. Thus, the unsaturation of fatty acids is important in the tolerance of the photosynthetic machinery to salt stress. It appears also that the activity and synthesis of the Na ؉ ͞H ؉ antiporter system might be suppressed under high-salt conditions and that this effect can be reversed, in part, by the unsaturation of fatty acids in membrane lipids.The regulation of the unsaturation of fatty acids in membrane lipids is important in the acclimation of poikilothermic organisms to changing environmental conditions, in particular, temperature (1-4). The effects of temperature on the desaturation of fatty acids have been extensively studied in cyanobacteria (for reviews, see refs. 2 and 5). When the ambient temperature is shifted from high to low, cyanobacterial cells synthesize fatty acid desaturases that introduce double bonds into the fatty acids of membrane lipids.In a previous study we isolated a desA Ϫ ͞desD Ϫ mutant strain of the cyanobacterium Synechocystis sp. PCC 6803 in which the desA and desD genes for the ⌬12 and ⌬6 desaturases, respectively, had been inactivated by targeted mutagenesis (6). The desA Ϫ ͞desD Ϫ cells contain monounsaturated but not polyunsaturated fatty acids, whereas wild-type cells contain polyunsaturated fatty acids such as di-, tri-, and tetraunsaturated fatty acids (7). By comparing these two strains we found that polyunsaturated fatty acids are essential for growth in Synechocystis sp. PCC 6803 at low temperatures (6).In the present study, we investigated the contribution of the unsaturation of fatty acids in membrane lipids to tolerance to salt stress by comparing desA Ϫ ͞desD Ϫ cells to wild-type cells of Synechocystis sp. PCC 6803. We demonstrated that the unsaturation of fatty acids is associated with the ability of the photosynthetic machinery to tolerate salt stress. MATERIALS AND METHODS Cells, Culture Conditions, and Preparation of ThylakoidMembranes. Wild-type and desA Ϫ ͞desD Ϫ (6) cells of Synechocystis sp. PCC 6803 were grown photoautotrophically in BG-11 medium (pH 7.5) (23), which contained 20 mM Na ϩ ions at 35°C under constant illumination from incandescent lamps (70-80 E⅐m Ϫ2 ⅐s Ϫ1, in which E indicates an einstein, 1 mol of photons) with aeration by sterile air that cont...

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Targeted mutagenesis of acyl-lipid desaturases in Synechocystis: evidence for the important roles of polyunsaturated membrane lipids in growth, respiration and photosynthesis.

Cited by 223 publications

References 43 publications

Low-temperature-induced accumulation of xanthophylls and its structural consequences in the photosynthetic membranes of the cyanobacterium Cylindrospermopsis raciborskii : An FTIR spectroscopic study

Low-temperature-induced accumulation of xanthophylls and its structural consequences in the photosynthetic membranes of the cyanobacterium Cylindrospermopsis raciborskii : An FTIR spectroscopic study

Identification of a novel D6‐acyl‐group desaturase by targeted gene disruption in Physcomitrella patens

Genetic engineering of the unsaturation of fatty acids in membrane lipids alters the tolerance ofSynechocystisto salt stress

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