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We probed the role of the polyunsaturated fatty acids on the dynamic and functional properties of mitochondrial membranes using the fad2 mutant of Arabidopsis thaliana, deficient in omega-6-oleate desaturase. In mitochondria of this mutant, the oleic acid content exceeded 70% of the total fatty acids, and the lipid/protein ratio was greatly enhanced. As a consequence, local microviscosity, probed by anthroyloxy fatty acid derivatives, was increased by 30%, whereas the lipid lateral diffusion, assayed using 1-pyrenedodecanoic acid, was approximately 4 times increased. Functional parameters such as oxygen consumption rate under phosphorylating and nonphosphorylating conditions and proton permeability of the inner mitochondrial membrane were significantly reduced in fad2 mitochondrial membranes, while the thermal dependence of the respiration was enhanced. Moreover, metabolic control analysis of the respiration clearly showed an enhancement of the control exerted by the membrane proton leaks. Our data suggest that the loss of omega-6-oleate desaturase activity in Arabidopsis cells induced an enhancement of both microviscosity and lipid/protein ratio of mitochondrial membranes, which in turn were responsible for the change in lateral mobility of lipids and for bioenergetic parameter modifications.

In order to isolate cytokinin-binding proteins (CBPs), we have developed new affinity probes constituted of a cytokinin such as zeatin riboside ([9R]Z) conjugated to a carrier protein. These probes were used for detecting CBPs in an ELISA procedure. The efficiency of the cytokinin conjugate in detecting CBPs was controlled with protein model: proteins having an affinity for cytokinin such as the monoclonal anti-[9R]Z antibodies did bind the cytokinin conjugate whereas proteins unable to bind cytokinin such as bovine serum albumin did not. Using these new affinity probes, we showed that CBPs are present in the membrane fraction of in vitro cultured Arabidopsis thaliana cells. The nature of the protein at the detected binding sites was demonstrated by submitting the microsomal proteins to a proteolytic treatment, which was found to eradicate the binding. Free biologically active cytokinins or monoclonal anti-[9R]Z antibodies inhibited the binding, thus showing the specificity of the interaction. The detected CBPs were partially solubilized from the membranes with potassium chloride, indicating their peripheral membrane location. The separation by anion exchange chromatography of solubilized microsomal proteins revealed the existence of two different CBPs. They were present at higher levels in cells during the exponential growth phase.