Genetically Modified Arabidopsis thaliana Cells Reveal the Involvement of the Mitochondrial Fatty Acid Composition in Membrane Basal and Uncoupling Protein-Mediated Proton Leaks

Abstract

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.