The two major tobacco plasma membrane H+-ATPases, PMA2 and PMA4 are differentially phosphorylated on their penultimate threonine

Transport of many ions and metabolites in the plant cell depends on the plasma membrane H +-ATPase, which couples ATP hydrolysis and proton pumping outside the cell. This enzyme is activated by phosphorylation of the penultimate residue, a Thr, and subsequent binding of regulatory 14-3-3 proteins. However, the presence of several H+-ATPase isoforms with partly overlapping expression patterns has so far precluded determining whether they all are activated in the same manner. In Nicotiana plumbaginifolia, PMA2 and PMA4 are the most broadly expressed plasma membrane H+-ATPases with a largely overlapping expression pattern. To follow their respective regulatory properties, two strategies were used: 1/ two monoclonal antibodies able to detect the phosphorylated (penultimate Thr) form of each of these isoforms; 2/ N. tabacum transgenic plants expressing 6-His tagged PMA2 or PMA4, thus allowing their individual purification. Once these tools validated, we searched for growth or environmental conditions that might differentially affect PMA2 and PMA4 activation. Fusicoccin, a fungal toxin, increased phosphorylation of both PMA2 and PMA4 as well as their binding to 14-3-3 proteins. Transfer of suspension cells from the stationary phase to a fresh medium strongly triggered PMA2 and PMA4 phosphorylation, 14-3-3 binding and plasma membrane ATPase activity. At day 3 of culture, about one-sixth of PMA2 and up to half of PMA4 was found as a phosphorylated form. H+-ATPase dephosphorylation was then progressively observed with, however, a faster decline for PMA2 than for PMA4. PMA2 and PMA4 phosphorylation in suspension cells was found to be affected to different extents according to the isoform by various treatments such as addition of organic acids, erythrosine B, N-ethylmaleimide, oxidative phosphorylation inhibitors and cold stress. For instance, cold stress of cells at stationary phase resulted in dephosphorylation of PMA2 but increased phosphorylation of PMA4. Cold-induced PMA2 dephosphorylation was shown to be Ca2+-dependent and inhibited by calyculin A, a potent PP1/PP2A phosphatase inhibitor. In conclusion, these results validate the tools allowing characterization of individual H+-ATPases. Different behaviors of PMA2 and PMA4 regarding their activation were identified, thus highlighting the importance of studying single isoforms.Doctorat en sciences agronomiques et ingénierie biologique (AGRO 3)--UCL, 200