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.
To investigate how the fatty acid composition of membrane lipids influences cell growth and mitochondrial respiration, in particular the expression and capacity of alternative oxidase (AOX), under cold stress, we used the Arabidopsis thaliana fad2 knockout and FAD3+ -overexpressing cultured cells lines affected in extrachloroplastic fatty acid desaturation activities. At 22 degrees C, fad2 mitochondria exhibited a low polyunsaturated fatty acid content and low protein to lipid ratio, while mitochondria from FAD3+ were enriched in linolenic acid and in total membrane protein. As a consequence, both mutants showed a higher membrane microviscosity than the wild type. After exposure to 9 degrees C, FAD3+ mitochondria exhibited lower microviscosity and lower rigidification upon a temperature downshift than fad2. Furthermore, the extent of reduction of cell growth and respiratiory rates in the phosphorylating state was positively related to the cold sensitivity of each cell line, being more pronounced in fad2 that in the wild type, whereas the stability of those parameters reflected the cold resistance of FAD3+. In contrast, an increase in AOX capacity was observed in the three cell lines at 9 degrees C. These inductions were correlated to AOX protein amounts and seem to result from an accumulation of AOX1c transcripts in the three cell lines and of AOX1a transcripts in wild-type and fad2 cells. The fact that there is no direct relationship between the degree of cold tolerance of each cell line and their ability to enhance their AOX capacity suggests that the participation of AOX in the response of Arabidopsis cells to cold stress does not necessarily favor cold tolerance.
Diacylglycerol kinases (DGKs) catalyze the phosphorylation of diacylglycerol into phosphatidic acid. To fulfill their role in many signalling processes, DGKs must be located at, or in, membranes. Most mammalian DGKs are cytosolic and are recruited to membranes upon stimulation, except for e type DGKs that are permanently membrane-associated through a hydrophobic segment. Nothing is known about the mechanism(s) involved in the membrane localization of plant DGKs. By fusion to fluorescent proteins, we show that two DGKs from cluster I in Arabidopsis thaliana possess amino-terminal hydrophobic segments that are sufficient to address them to endoplasmic reticulum membranes.
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