Global transcriptome and gene co-expression network analyses reveal regulatory and non-additive effects of drought and heat stress in grapevine.

Despite frequent co-occurrence of drought and heat stress, the molecular mechanisms governing plant responses to these stresses in combination have not often been studied. This is particularly evident in non-model, perennial plants. We conducted large scale physiological and transcriptome analyses to identify genes and pathways associated with grapevine response to drought and/or heat stress during stress progression and recovery. We identified gene clusters with expression correlated to leaf temperature and water stress and five hub genes for the combined stress co-expression network. Several differentially expressed genes were common to the individual and combined stresses, but the majority were unique to the individual or combined stress treatments. These included heat-shock proteins, mitogen-activated kinases, sugar metabolizing enzymes, and transcription factors, while phenylpropanoid biosynthesis and histone modifying genes were unique to the combined stress treatment. Following physiological recovery, differentially expressed genes were found only in plants under heat stress, both alone and combined with drought. Taken collectively, our results suggest that the effect of the combined stress on physiology and gene expression is more severe than that of individual stresses, but not simply additive, and that epigenetic chromatin modifications may play an important role in grapevine responses to combined drought and heat stress.

Conformational dynamics of Na+/K+- and H+/K+-ATPase probed by voltage clamp fluorometry.

We used the method of site-directed fluorescence labeling in combination with voltage-clamp fluorometry for time-resolved recording of localized conformational transitions of the Na(+)/K(+)- and H(+)/K(+)-ATPase. Therefore, single cysteine mutations were introduced into the extracellular TM5-TM6 loop of the sheep Na(+)/K(+)-ATPase alpha(1)-subunit devoid of other extracellular cysteines. Upon expression in Xenopus oocytes and covalent attachment of tetramethylrhodamine-maleimide (TMRM) as a reporter fluorophore, Cys-mutant N790C showed large fluorescence changes of up to 5% in response to extracellular K(+) that were completely abolished by ouabain. When voltage jumps were applied under Na(+)/Na(+)-exchange conditions, we observed fluorescence changes that paralleled the transient currents originating from the E(1)P<-->E(2)P transition. These fluorescence changes were also completely inhibited by ouabain, as were the voltage jump-induced transient currents. Transient fluorescence changes could also be measured as a function of increasing K(+) concentrations, that is, under turnover conditions. As a result, the distribution between E(1) and E(2) states can be determined at any time and membrane potential. Very similar fluorescence signals were obtained for rat gastric H(+)/K(+)-ATPase upon expression in oocytes, when a single cysteine was introduced at a position homologous to N790 in Na(+)/K(+)-ATPase for attachment of the fluorophore. As to the high sequence similarity among P-type ATPases within the TM5 helix and the TM5-TM6 loop region, our results enable new means of kinetic investigation for these pumps under physiological conditions in living cells.