Phytosphingosine-phosphate is a signal for AtMPK6 activation and Arabidopsis response to chilling.

• Long-chain bases (LCBs) are pleiotropic sphingolipidic signals in eukaryotes. We investigated the source and function of phytosphingosine-1-phosphate (PHS-P), a phospho-LCB rapidly and transiently formed in Arabidopsis thaliana on chilling. • PHS-P was analysed by thin-layer chromatography following in vivo metabolic radiolabelling. Pharmacological and genetic approaches were used to identify the sphingosine kinase isoforms involved in cold-responsive PHS-P synthesis. Gene expression, mitogen-activated protein kinase activation and growth phenotypes of three LCB kinase mutants (lcbk1, sphk1 and lcbk2) were studied following cold exposure. • Chilling provoked the rapid and transient formation of PHS-P in Arabidopsis cultured cells and plantlets. Cold-evoked PHS-P synthesis was reduced by LCB kinase inhibitors and abolished in the LCB kinase lcbk2 mutant, but not in lcbk1 and sphk1 mutants. lcbk2 presented a constitutive AtMPK6 activation at 22°C. AtMPK6 activation was also triggered by PHS-P treatment independently of PHS/PHS-P balance. lcbk2 mutants grew comparably with wild-type plants at 22 and 4°C, but exhibited a higher root growth at 12°C, correlated with an altered expression of the cold-responsive DELLA gene RGL3. • Together, our data indicate a function for LCBK2 in planta. Furthermore, they connect PHS-P formation with plant response to cold, expanding the field of LCB signalling in plants.

Genetically modified Arabidopsis thaliana cells reveal the involvement of the mitochondrial fatty acid composition in membrane basal and uncoupling protein-mediated proton leaks.

We investigated the role of membrane fatty acids in basal proton leaks and uncoupling protein (UCP)-dependent proton conductance in Arabidopsis mitochondria. Using wild-type cells, cold-sensitive fad2 mutant cells, deficient in omega-6-oleate desaturase, and cold-tolerant FAD3(+) transformant cells, overexpressing omega-3-linoleate desaturase, we showed that basal proton leak in the non-phosphorylating state was dependent on lipid composition. The extent of membrane proton leak was drastically reduced in the fad2 mutant, containing low amounts of polyunsaturated fatty acids. Conversely, this proton leak was higher in FAD3(+) mitochondria that exhibit a higher polyunsaturated fatty acid content and high protein to lipid ratio. The dependency of membrane leaks upon membrane potential was higher in FAD3(+) and lower in fad2. UCP content was higher in both the fad2 mutant and FAD3(+) transgenic lines compared with wild-type cells and so was the UCP activity, assayed by the reduction of phosphorylation yield (ADP/O) triggered by palmitate as UCP activator. This UCP assay was validated by measurements of UCP-proton leak in the non-phosphorylating state (flux-force relationships between proton flux and membrane potential). The potential uncoupling capacity of the UCP was high enough to allow the loss of respiratory control in the three genotypes. Taken together, the data reported here suggest that the cold tolerance of FAD3(+) cells and the cold sensitivity of fad2 cells are associated with changes in their mitochondrial membrane basal proton leaks, whereas differences in functional expression of UCP are not simply related to cold adaptation in Arabidopsis cells.

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.

Diacylglycerol pyrophosphate is a second messenger of abscisic acid signaling in Arabidopsis thaliana suspension cells.

In plants, the importance of phospholipid signaling in responses to environmental stresses is becoming well documented. The involvement of phospholipids in abscisic acid (ABA) responses is also established. In a previous study, we demonstrated that the stimulation of phospholipase D (PLD) activity and plasma membrane anion currents by ABA were both required for RAB18 expression in Arabidopsis thaliana suspension cells. In this study, we show that the total lipids extracted from ABA-treated cells mimic ABA in activating plasmalemma anion currents and induction of RAB18 expression. Moreover, ABA evokes within 5 min a transient 1.7-fold increase in phosphatidic acid (PA) followed by a sevenfold increase in diacylglycerol pyrophosphate (DGPP) at 20 min. PA activated plasmalemma anion currents but was incapable of triggering RAB18 expression. By contrast, DGPP mimicked ABA on anion currents and was also able to stimulate RAB18 expression. Here we show the role of DGPP as phospholipid second messenger in ABA signaling.

Development of the primary root and mobilisation of reserves in etiolated seedlings of Brassica napus grown on a slowly rotating clinostat.

The effect of the slow rotating clinostat (1 rpm) on the growth of the primary root was studied on Brassica napus seedlings. After 5 d in darkness, the primary root was longer and thinner in seedlings grown on the clinostat than in seedlings grown in the vertical position. However, the breakdown of lipid reserves, sucrose level and transport of 14C-labeled sucrose from the cotyledons to the primary root, were not altered by growth on the clinostat. Moreover, the activity of isocitrate lyase, one of the two enzymes necessary for the conversion of lipids into glucids also was also not modified in the cotyledons of clinorotated seedlings. Thus, there was clear evidence that clinorotation had a direct effect on the growth of the primary root that was independent of the mobilisation of lipid reserves in the cotyledons. As a sink, the primary root had the same strength on the clinostat as in the vertical position, but the reserves were used in a different way. The increase in root elongation on the clinostat could be due to the slight, but continuous, omnilateral gravitropic stimulation due to the rotation of the seedlings about a horizontal axis.

Drought stress affects chloroplast lipid metabolism in rape (Brassica napus) leaves.

Rape (Brassica napus L. var. Bienvenue) is a 16:3 plant which contains predominantly prokaryotic species of monogalactosyldiacylglycerol i.e. sn-1 C18, sn-2 C16 (C18/C16 MGDG). Rape plants were exposed to a restricted water supply for 12 days. Under drought conditions, considerable changes in lipid metabolism were observed. Drought stress provoked a decline in leaf polar lipids, which is mainly due to a decrease in MGDG content. Determination of molecular species in phosphatidylcholine (PC) and MGDG indicated that the prokaryotic molecular species of MGDG (C18/C16) decreased after drought stress while the eukaryotic molecular species (C18/C18) remained stable. Drought stress had different effects on two key enzymes of PC and MGDG synthesis. The in vitro activity of MGDG synthase (EC. 2.4.1.46) was reduced in drought stressed plants whereas cholinephosphotransferase (EC. 2.7.8.2) activity was not affected. Altogether these results suggest that the prokaryotic pathway leading to MGDG synthesis was strongly affected by drought stress while the eukaryotic pathway was not. It was also observed that the molecular species of leaf PC became more saturated in drought stressed plants. This could be due to a specific decrease in oleate desaturase activity.