Identification and characterization of an animal delta(12) fatty acid desaturase gene by heterologous expression in Saccharomyces cerevisiae

We have cloned a Caenorhabditis elegans cDNA encoding a Delta12 fatty acid desaturase and demonstrated its activity by heterologous expression in Saccharomyces cerevisiae. The predicted protein is highly homologous both to the cloned plant genes with similar function and to the published sequence of the C. elegans omega-3 fatty acid desaturase. In addition, it conforms to the structural constraints expected of a membrane-bound fatty acid desaturase including the canonical histidine-rich regions. This is the first report of a cloned animal Delta(12) desaturase gene. Expression of this cDNA in yeast resulted in the accumulation of 16:2 and 18:2 (linoleic) acids. The increase of membrane fluidity brought about by this change in unsaturation was measured. The production of polyunsaturated fatty acids in yeast cells and the concomitant increase in membrane fluidity was correlated with a modest increase in growth rate at low temperature and with increased resistance to ethanol and oxidative stress.     

Low temperature and light regulate delta 12 fatty acid desaturases (FAD2) at a transcriptional level in cotton (Gossypium hirsutum)

Lipid modifying enzymes play a key role in the development of cold stress tolerance in cold-resistant plants such as cereals. However, little is known about the role of the endogenous enzymes in cold-sensitive species such as cotton. Delta 12 fatty acid desaturases (FAD2), known to participate in adaptation to low temperatures through acyl chain modifications were used in gene expression studies in order to identify parameters of plant response to low temperatures. The induction of microsomal delta 12 fatty acid desaturases at an mRNA level under cold stress in plants is shown here for first time. Quantitative PCR showed that though both delta 12 omega 6 fatty acid desaturase genes FAD2-3 and FAD2-4 identified in cotton are induced under cold stress, FAD2-4 induction is significantly higher than FAD2-3. The induction of both isoforms was light regulated, in contrast a third isoform FAD2-2 was not affected by cold or light. Stress tolerance and light regulatory elements were identified in the predicted promoters of both FAD2-3 and FAD2-4 genes. Di-unsaturated fatty acid species rapidly increased in the microsomal fraction isolated from cotton leaves, following cold stress. Expression analysis patterns were correlated with the observed increase in both total and microsomal fatty acid unsaturation levels suggesting the direct role of the FAD2 genes in membrane adaptation to cold stress.     

Low-temperature-induced desaturation of fatty acids and expression of desaturase genes in the cyanobacterium Synechococcus sp. PCC 7002

Changes in response to temperature of lipid classes, fatty acid composition and mRNA levels for acyl-lipid desaturase genes were studied in the marine unicellular cyanobacterium, Synechococcus sp. PCC 7002. The degree of unsaturation of C₁₈ fatty acids increased in cells grown at lower temperature for all lipid classes, and ω3 desaturation occurred specifically in cells grown at low temperature. While the level of 18:1(9) fatty acids declined, desaturation at the ω3 position of C₁₈ fatty acids increased gradually during a 12-h period after a temperature shift-down to 22°C. However, the mRNA levels of the desA (Δ12 desaturase), desB (ω3 desaturase) and desC (Δ9 desaturase) genes increased within 15 min after a temperature shift-down to 22°C; the desaturase gene mRNA levels also rapidly declined within 15 min after a temperature shift-up to 38°C. Therefore, the elevation of mRNA levels for the desaturase genes is not the rate-limiting event for the increased desaturation of membrane lipids after a temperature shift-down. The rapid, low-temperature-induced changes in mRNA levels occurred even when cells were grown under light-limiting conditions for which the growth rates at 22°C and 38°C were identical. These studies indicate that the ambient growth temperature, and not some other growth rate-related process, regulates the expression of acyl lipid desaturation in this cyanobacterium.     

Glycine betaine protects tomato (Solanum lycopersicum) plants at low temperature by inducing fatty acid desaturase7 and lipoxygenase gene expression

Cold stress is among the environmental stressors limiting productivity, yield and quality of agricultural plants. Tolerance to cold stress is associated with the increased unsaturated fatty acids ratio in the plant membranes which are also known to be substrates of octadecanoid pathway for jasmonate and other oxylipins biosynthesis. Accumulation of osmoprotectant, glycine betaine (GB) is well known to be effective in the protecting membranes and mitigating cold stress effects but, the mode of action is poorly understood. We studied the role of GB in cold stress responses of two tomato cultivated varieties; Gerry (cold stress sensitive) and T47657 (moderately cold stress tolerant) and compared the differences in lypoxygenase-13 (TomLOXF) and fatty acid desaturase 7 (FAD7) gene expression profiles and physiological parameters including relative growth rates, relative water content, osmotic potential, photosynthetic efficiency, membrane leakage, lipid peroxidation levels. Our results indicated that GB might have a role in inducing FAD7 and LOX expressions for providing protection against cold stress in tomato plants which could be related to the desaturation process of lipids leading to increased membrane stability and/or induction of other genes related to stress defense mechanisms via octadecanoid pathway or lipid peroxidation products.     

Homeostasis of plasma membrane viscosity in fluctuating temperatures

Temperature has a direct effect at the cellular level on an organism. For instance, in the case of biomembranes, cooling causes lipids to lose entropy and pack closely together. Reducing temperature should, in the absence of other factors, increase the viscosity of a lipid membrane. We have investigated the effect of temperature variation on plasma membrane (PM) viscosity. We used dispersion tracking of photoactivated green fluorescent protein (GFP) and fluorescence recovery after photobleaching in wild-type and desaturase mutant Arabidopsis thaliana plants along with membrane lipid saturation analysis to monitor the effect of temperature and membrane lipid composition on PM viscosity. Plasma membrane viscosity in A. thaliana is negatively correlated with ambient temperature only under constant-temperature conditions. In the more natural environment of temperature cycles, plants actively manage PM viscosity to counteract the direct effects of temperature. Plasma membrane viscosity is regulated by altering the proportion of desaturated fatty acids. In cold conditions, cell membranes accumulate desaturated fatty acids, which decreases membrane viscosity and vice versa. Moreover, we show that control of fatty acid desaturase 2 (FAD2)-dependent lipid desaturation is essential for this homeostasis of membrane viscosity. Finally, a lack of FAD2 function results in aberrant temperature responses.     

Thermoadaptive regulation of microsomal desaturase and electron-transport enzyme activities in lipid-manipulated Tetrahymena cells. Extent of unsaturated fatty acid production is dependent on membrane fluidity before temperature down-shift

Exposure of Tetrahymena pyriformis NT-1 to chimyl alcohol (1-O-hexadecyl glycerol) produced a reproducible enhancement in unsaturated fatty acids and a great decrease in order parameter (S), which result from the 2-fold increases of stearoyl-CoA and oleoyl-CoA desaturase activities in microsomes. When the chimyl alcohol-fed cells were shifted from 34 to 15 degrees C (down-shift), unlike the drastic increases in palmitoyl-CoA, stearoyl-CoA and oleoyl-CoA desaturase activities in the native cells, there was only a slight increase in palmitoyl-CoA desaturase activity with a parallel rise in the activity of the terminal component (cyanide-sensitive factor; CSF) of the desaturase system. During cold acclimation, the decrease of order parameter in chimyl alcohol-fed cells was smaller than that in native cells, since the order parameter had already been decreased by the addition of chimyl alcohol before the shift. These results suggest that chimyl alcohol-fed cells are easily able to accomplish temperature acclimation without requiring great modification of fatty acid composition and membrane fluidity, while the non-fed control cells have difficulty doing so.     

Desaturase mutants reveal that membrane rigidification acts as a cold perception mechanism upstream of the diacylglycerol kinase pathway in Arabidopsis cells

Membrane rigidification could be the first step of cold perception in poikilotherms. We have investigated its implication in diacylglycerol kinase (DAGK) activation by cold stress in suspension cells from Arabidopsis mutants altered in desaturase activities. By lateral diffusion assay, we showed that plasma membrane rigidification with temperature decrease was steeper in cells deficient in oleate desaturase than in wild type cells and in cells overexpressing linoleate desaturase. The threshold for the activation of the DAGK pathway in each type of cells correlated with this order of rigidification rate, suggesting that cold induced-membrane rigidification is upstream of DAGK pathway activation.     

Stearoyl-CoA desaturase expression and fatty acid composition in milkfish (Chanos chanos) and grass carp (Ctenopharyngodon idella) during cold acclimation

Desaturation of fatty acids is an important adaptation mechanism for fish to maintain membrane fluidity under thermal stress. To comprehend the temperature adaptation mechanism in fish, we investigated the difference in the changes of stearoyl-CoA desaturase expression and fatty acid composition between milkfish and grass carp under cold acclimation. We find that in both fish the proportions of unsaturated fatty acids at 15 degrees C are all higher than those at 25 degrees C. In milkfish Delta(9)-desaturation index (ratios of 16:1/16:0 and 18:1/18:0) increases significantly in the beginning of cold acclimation at 15 degrees C and decreases afterward, but in grass carp it increases slightly in the beginning of cold acclimation followed by a sustained dramatic increase. Similarly, activity of stearoyl-CoA desaturase in milkfish increases significantly in the beginning, peaks at day 4, and then decreases constantly, but in grass carp it increases gradually in the first week, rises dramatically afterward, and then maintains a very high level. The change of stearoyl-CoA desaturase activity is parallel to the change of Delta(9)-desaturation index in both milkfish and grass carp, but it is one day earlier than Delta(9)-desaturation index in milkfish. The difference of adaptation capability between milkfish and grass carp under cold stress is further evidenced by RT-PCR and Northern blot analysis of stearoyl-CoA desaturase gene expression.     

Cold stress regulates lipid metabolism via AMPK signalling in Cherax quadricarinatus

Lipids play an important role in protecting poikilotherms from cold stress, but relatively little is known about the regulation of lipid metabolism under cold stress, especially in crustaceans. In the present study, red-clawed crayfish Cherax quadricarinatus was employed as a model organism. Animals were divided into four temperature groups (25, 20, 15 and 9 °C) and treated for 4 weeks, with the 25 °C group serving as a control. The total lipid content in the hepatopancreas as well as the triglyceride, cholesterol and free fatty acid levels in the hemolymph were determined. Lipids stored in the hepatopancreas and hemolymph decreased with decreasing temperature, with changes in the 9 °C group most pronounced, indicating that lipids are the main energy source for crayfish at low temperatures. Furthermore, enzyme activity of lipase, fatty acid synthase, acetyl-CoA carboxylase, and lipoprotein esterase, and gene expression analysis of fatty acid synthase gene, acetyl-CoA carboxylase gene and carnitine palmitoyltransferase gene showed that the digestion, synthesis and oxidation of lipids in the hepatopancreas were inhibited under low temperature stress, but expression of sphingolipid delta-4 desaturase (DEGS) was increased, indicating an increase in the demand for highly unsaturated fatty acids at low temperatures. Analysis of the expression of genes related to the AMP-activated protein kinase (AMPK) signalling pathway revealed that the adiponectin receptor gene was rapidly upregulated at low temperatures, which may in turn activate the expression of the downstream AMPKα gene, thereby inhibiting lipid anabolism.     

Sterol manipulation that modulates the alteration in membrane fluidity of Tetrahymena pyriformis during temperature acclimation

Our previous results [Umeki and Nozawa (1983) Biochem. Biophys. Res. Commun. 113, 96-101] suggested that ergosterol-replaced Tetrahymena cells (ergosterol-cells) accomplish an adaptive modification of fatty-acid composition by a preferential increase in palmitoyl-CoA desaturase activity, which is principally due to the increased content of the terminal component (cyanide-sensitive factor) of the desaturase system. The present study was designed to obtain information as to how the membrane fluidity of ergosterol-cells is changed during cold temperature acclimation. The order parameter (S) of liposomes prepared from ergosterol-cell lipids was reduced more rapidly after a temperature shift-down than that of control liposomes prepared from native cells containing tetrahymanol. These results indicate that, unlike native cells containing tetrahymanol, ergosterol-cells strive to accomplish cold temperature acclimation by undergoing a great modification of membrane fluidity because of the altered microsomal desaturase activity.     

斑马鱼elovl8缺失对抗冷胁迫下存活率及脂肪代谢的影响

研究利用CRISPR/Cas9技术构建了斑马鱼elovl8a-/-、elovl8b-/-和DKO(elovl8a和elovl8b)敲除模型,通过组织学观察、实时荧光定量PCR和脂肪酸组成分析等实验方法,探究了脂肪酸延长酶8(elovl8)缺失对斑马鱼抗冷胁迫能力的影响.结果表明elovl8缺失导致斑马鱼在低温环境下的存...     

水稻OsFAD2、OsFAD6的克隆及其家族成员对非生物胁迫的响应

植物中的不饱和脂肪酸由脂肪酸去饱和酶(Fatty acid desaturase, FAD)合成, 它在植物的生长发育以及植物非生物胁迫方面起着重要的作用。文章采用RT-PCR方法, 从水稻日本晴(Oryza sativa L.)中克隆了分别与FAD2、FAD6同源的脂肪酸脱氢酶序列, 命名为OsFAD2和OsFAD6。OsFAD2的ORF为1 167 bp, 推测其编码蛋白含有388个氨基酸, 等电点为8.17, 分子量为52.24 kDa, C端有内质网定位序列; OsFAD6的ORF长度为1 365 bp, 推测编码454个氨基酸的蛋白质序列, 分子量44.35 kDa, 等电点为9.24, 推测N端38个氨基酸为叶绿体导肽。两者都具有膜整合脂肪酸去饱和酶特有的3个组氨酸簇。RT-PCR分析表明, OsFAD2和OsFAD6在水稻所有器官中都表达, 在叶中表达量为最高。在水稻FAD基因家族中, 叶中OsFAD2、OsFAD6 的mRNA对低温不响应, 而OsFAD7和OsFAD8的 mRNA在低温下上升。水稻叶中OsFAD2、OsFAD6、OsFAD3和OsFAD7的mRNA表达具有昼夜节律性, 在光照下表达量低, 而在随后的黑暗中表达量高, OsFAD6和OsFAD7 mRNA表达的昼夜节律性可能与水稻幼苗叶片中NADPH量的改变有关。