Light-dependent cold-induced fatty acid unsaturation, changes in membrane fluidity, and alterations in gene expression in Synechocystis

Cold stress causes unsaturation of the membrane lipids. This leads to adjustment of the membrane fluidity, which is necessary for cold acclimation of cells. Here we demonstrate that the cold-induced accumulation of PUFAs in the cyanobacterium Synechocystis is light-dependent. The desA(-)/desD(-) mutant, that lacks the genes for Δ12 and Δ6 desaturases, is still able to adjust the fluidity of its membranes in spite of its inability to synthesize PUFAs and modulate the fatty acid composition of the membrane lipids under cold stress. The expression of cold-induced genes, which are controlled by the cold sensor histidine kinase Hik33, depends on the fluidity of cell membranes and it is regulated by light, though it does not require the activity of the photosynthetic apparatus. The expression of cold-induced genes, which are not controlled by Hik33, does not depend on the membrane fluidity or light. Thus, membrane fluidity determines the temperature dependence of the expression of cold-induced genes that are under control of the Hik33, which might be the sensor of changes in the membrane fluidity. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

In situ molecular identification of the plastid omega3 fatty acid desaturase FAD7 from soybean: evidence of thylakoid membrane localization

omega3 fatty acid desaturases are the enzymes responsible for the synthesis of trienoic fatty acids in plants. These enzymes have been mainly investigated using molecular, biochemical, and genetic approaches but very little is known about their subcellular distribution in plant cells. In this work, the precise subcellular localization of the omega3 desaturase FAD7 was elucidated by immunofluorescence and immunogold labeling using a monospecific GmFAD7 polyclonal antibody in soybean (Glycine max) photoautotrophic cell suspension cultures. Confocal analysis revealed the localization of the GmFAD7 protein within the chloroplast; i.e. signals from FAD7 and chlorophyll autofluorescence showed specific colocalization. Immunogold labeling was pursued on cryofixed and freeze-substituted samples for convenient preservation of antigenicity and ultrastructure of membrane subcompartments. Our data revealed that the FAD7 protein was preferentially localized in the thylakoid membranes. Biochemical fractionation of purified chloroplasts and western analysis of the subfractions further confirmed these results. These findings suggest that not only the envelope, but also the thylakoid membranes could be sites of lipid desaturation in higher plants.     

The desA gene of the cyanobacterium Synechocystis sp. strain PCC6803 is the structural gene for delta 12 desaturase.

The desA gene of the cyanobacterium Synechocystis sp. strain PCC6803 was expressed in Escherichia coli, which does not contain any fatty acid desaturase. The product of the desA gene catalyzed the desaturation of fatty acids at the delta 12 position. This result demonstrates that desA is the structural gene for a delta 12 desaturase.     

A novel derivative pentacyclic triterpene and omega 3 fatty acid

Adjuvant induced arthritis (AIA) is accompanied by marked changes in the levels of lysosomal enzymes, glycoproteins and metabolic turnover of collagen. The role of a pentacyclic triterpene and eicosapentaenoic acid (EPA) derivative--Lupeol-EPA (50 mg/kg body weight--orally) was tested in vivo in rats. The increased activities of lysosomal enzymes and glycoproteins associated with decreased collagen in arthritic animals were significantly altered to nearly that of controls. Indomethacin (3 mg/kg body weight) was used as a reference compound. The therapeutic usefulness of Lupeol-EPA derivative in inflammatory conditions is attractive and deserves further work in this direction.     

Differences in membrane fluidity and fatty acid composition between phenotypic variants of Streptococcus pneumoniae

Phase variation in the colonial opacity of Streptococcus pneumoniae has been implicated as a factor in the pathogenesis of pneumococcal disease. This study examined the relationship between membrane characteristics and colony morphology in a few selected opaque-transparent couples of S. pneumoniae strains carrying different capsular types. Membrane fluidity was determined on the basis of intermolecular excimerization of pyrene and fluorescence polarization of 1,6-diphenyl 1,3,5-hexatriene (DPH). A significant decrease, 16 to 26% (P < or = 0.05), in the excimerization rate constant of the opaque variants compared with that of the transparent variants was observed, indicating higher microviscosity of the membrane of bacterial cells in the opaque variants. Liposomes prepared from phospholipids of the opaque phenotype showed an even greater decrease, 27 to 38% (P < or = 0.05), in the pyrene excimerization rate constant compared with that of liposomes prepared from phospholipids of bacteria with the transparent phenotype. These findings agree with the results obtained with DPH fluorescence anisotropy, which showed a 9 to 21% increase (P < or = 0.001) in the opaque variants compared with the transparent variants. Membrane fatty acid composition, determined by gas chromatography, revealed that the two variants carry the same types of fatty acids but in different proportions. The trend of modification points to the presence of a lower degree of unsaturated fatty acids in the opaque variants compared with their transparent counterparts. The data presented here show a distinct correlation between phase variation and membrane fluidity in S. pneumoniae. The changes in membrane fluidity most probably stem from the observed differences in fatty acid composition.     

The fatty acid desaturase 3 gene encodes for different FADS3 protein isoforms in mammalian tissues

In 2000, Marquardt et al. (A. Marquardt, H. Stöhr, K. White, and B. H. F. Weber. 2000. cDNA cloning, genomic structure, and chromosomal localization of three members of the human fatty acid desaturase family. Genomics. 66: 176–183.) described the genomic structure of the fatty acid desaturase (FADS) cluster in humans. This cluster includes the FADS1 and FADS2 genes encoding, respectively, for the Δ5- and Δ6-desaturases involved in polyunsaturated fatty acid biosynthesis. A third gene, named FADS3, has recently been identified but no functional role has yet been attributed to the putative FADS3 protein. In this study, we investigated the FADS3 occurrence in rat tissues by using two specific polyclonal antibodies directed against the N-terminal and C-terminal ends of rat FADS3. Our results showed three potential protein isoforms of FADS3 (75 kDa, 51 kDa, and 37 kDa) present in a tissue-dependent manner. The occurrence of these FADS3 isoforms did not depend on the mRNA level determined by real-time PCR. In parallel, mouse tissues were also tested and showed the same three FADS3 isoforms but with a different tissue distribution. Finally, we reported the existence of FADS3 in human cells and tissues but different new isoforms were identified. To conclude, we showed in this study that FADS3 does exist under multiple protein isoforms depending on the mammalian tissues. These results will help further investigations to determine the physiological function of FADS3.     

Alteration of the omega-3 fatty acid desaturase gene is associated with reduced linolenic acid in the A5 soybean genotype

Reducing the linolenic acid (18?:?3^{ω? 3,6,9}) concentration of soybean [Glycine max (L.) Merr.] oil may lessen the need for chemical hydrogenation and enhance flavor stability. Soybean genotypes A5 and A23 have reduced linolenic acid concentration compared with current cultivars. Seed linolenic acid is synthesized primarily by the ω-3 fatty acid desaturase located in the microsomes. The objective of this research was to study whether this enzyme has a role in reducing the fatty acid levels in the soybean genotypes A5 and A23. DNA from A5 and A23 was analyzed by gel-blot hybridization with a cDNA encoding the ω-3 fatty acid desaturase. A5 and lines selected from it have a DNA fragment missing compared to A23 and lines with normal linolenic acid concentration. Seventy F_4:5 lines from a population segregating for linolenic acid concentration were scored for presence or absence of the fragment. The absence of the fragment was significantly (P?0.0001) associated with a reduced linolenic acid level and accounted for 67% of the variation for linolenic acid in the population. These results suggest that the reduced linolenic acid concentration in A5 was at least partially the result of a full or partial deletion of a microsomal ω-3 desaturase gene. No DNA polymorphisms were found for the desaturase gene in A23, so no mutations could be studied in this line.     

A TPR-family membrane protein gene is required for light-activated heterotrophic growth of the cyanobacterium Synechocystis sp. PCC 6803

The unicellular cyanobacterium Synechocystis sp. PCC6803 can grow heterotrophically in complete darkness, given that a brief period of illumination is supplemented every day (light-activated heterotrophic growth, LAHG), or under very weak (<0.5 micromol m(-2) s(-1)) but continuous light. By random insertion of the genome with an antibiotic resistance cassette, mutants defective in LAHG were generated. In two identical mutants, sll0886, a tetratricopeptide repeat (TPR)-family membrane protein gene, was disrupted. Targeted insertion of sll0886 and three downstream genes showed that the phenotype was not due to a polar effect. The sll0886 mutant shows normal photoheterotrophic growth when the light intensity is at 2.5 micromol m(-2) s(-1) or above, but no growth at 0.5 micromol m(-2) s(-1). Homologs to sll0886 are also present in cyanobacteria that are not known of LAHG. sll0886 and homologs may be involved in controlling different physiological processes that respond to light of low fluence.     

ω-3多聚不饱和脂肪酸脱氢酶的真核表达及鉴定

目的：构建ω-3多聚不饱和脂肪酸脱氢酶真核表达载体,并在293T细胞（人胚肾细胞）中实现表达。方法：通过RT-PCR法扩增得到ω-3多聚不饱和脂肪酸脱氢酶基因fat1,构建重组真核表达载体pCMV－Myc－fat1,用脂质体法转染293T细胞,Western Blot检测fat1的表达,并用间接免疫荧光（IFA）确定其在293T细胞中的定位情况。结果：构建真核表达质粒pCMV－Myc－fat1,转染293T细胞后,可检测到细胞内有fat1的表达并确定其在细胞中的位置。结论：成功构建真核表达质粒pCMV－Myc－fat1,可检测出细胞内有fat1的表达并确定其在细胞膜和细胞质内均有表达,为进行fat1的功能研究奠定了基础。     

A gene required for the regulation of photosynthetic light harvesting in the cyanobacterium Synechocystis 6803

A gene required for the short-term regulation of photosynthetic light harvesting (the state transition) has been identified in the cyanobacterium Synechocystis sp. PCC6803. The open reading frame is designated sll1926 in the complete Synechocystis gene sequence. The deduced amino acid sequence has no homologues in current sequence databases and no recognizable sequence motifs. It encodes a putative integral membrane protein of 16 kDa, which we have designated RpaC (regulator of phycobilisome association C). Fluorescence measurements of an insertional inactivation mutant of rpaC (Deltasll1926) show that it is specifically unable to perform state transitions. Deltasll1926 has approximately wild-type levels of PS1, PS2 and phycobilisomes. Measurements of oxygen evolution and uptake show Deltasll1926 to have no deficiency in electron transport rates. In vitro [gamma-32P]-ATP labelling experiments suggest that RpaC is not the 15 kDa membrane phosphoprotein previously implicated in state transitions. Deltasll1926 grows more slowly than the wild type only at very low light intensities.     

Overexpression of endoplasmic reticulum omega-3 fatty acid desaturase gene improves chilling tolerance in tomato

An endoplasmic reticulum-localized tomato omega-3 fatty acid desaturase gene (LeFAD3) was isolated and characterized with regard to its sequence, response to various temperatures and function in transgenic tomato plants. Northern blot analysis showed that LeFAD3 was expressed in all organs tested and was markedly abundant in roots. Meanwhile, the expression of LeFAD3 was induced by chilling stress (4 °C), but inhibited by high temperature (40 °C). The transgenic plants were obtained under the control of the cauliflower mosaic virus 35S promoter (35S-CaMV). Northern and western blot analyses confirmed that sense LeFAD3 was transferred into tomato genome and overexpressed. Level of linolenic acids (18:3) increased and correspondingly level of linoleic acid (18:2) decreased in leaves and roots. After chilling stress, the fresh weight of the aerial parts of transgenic plants was higher than that of the wild type (WT) plants, and the membrane system ultrastructure of chloroplast in leaf cell and all the subcellular organelles in root tips of transgenic plants kept more intact than those of WT. Relative electric conductivity increased less in transgenic plants than that in WT, and the respiration rate of the transgenic plants was notably higher than that of WT. The maximal photochemical efficiency of PSII (F_v/F_m) and the O₂ evolution rate in WT decreased more than those in transgenic plants under chilling stress. Together with other data, results showed that the overexpression of LeFAD3 led to increased level of 18:3 and alleviated the injuries under chilling stress.     

Role of the Bacillus subtilis fatty acid desaturase in membrane adaptation during cold shock

In our attempt to understand the cold shock response of Bacillus subtilis, we report on the role of the B. subtilis fatty acid desaturase (FA-D) Des during membrane adaptation to low temperatures and demonstrate its importance during cold shock. A des null mutant was constructed and analysed in comparison with its parental strain. Growth studies and large-scale comparative fatty acid (FA) analysis revealed a severe cold-sensitive phenotype of the des deletion mutant during the absence of isoleucine and showed that four unsaturated fatty acid (UFA) species differing in length, branching pattern and position of the double bond are synthesized in B. subtilis JH642 but not in the des null mutant. Apart from the lack of UFA synthesis, the FA-D deletion strain showed a dramatically altered saturated fatty acid (SFA) profile at the onset of the stationary growth phase in the presence of exogenous isoleucine sources. Expression of des integrated in trans at the amyE locus of the des deletion strain not only cured the cold-sensitive phenotype observed for the des mutant but allowed much better growth than in strain JH642 after a shift from 37 degrees C to 15 degrees C. These results show that, during cold shock adaptation, des expression can completely replace the isoleucine-dependent, long-term, FA branching adaptation mechanism. We conclude that the crucial aspect in cold adaptation of the cytoplasmic membrane is not its specific molecular composition but rather its physical status in terms of its fluidity.