Evidence And Mechanism Of Protein Tyrosine Phosphatases Regulating Stomatal Movement Of Vicia Faba L

Although both Ser/Thr and Tyr phosphorylation play a crucial role in cellular regulation in animals, only Ser/Thr phosphorylation has been proved to be an important regulatory mechanism in plant cells. Indeed, a large number of Ser/Thr kinases and phosphatases have been identified in plant and shown to be important in various processes of plant growth and development. By contrast, no typical tyrosine kinase has been identified to date from a higher plant. It was thought that protein tyrosine phosphatases (PTPases) might not exist either until recently, when several members of the PTPases family were characterized from Arabidopsis and orther higher plants. But the role of PTPases in plants signaling processes is still unknown. It is important to clarify their functions in plants.Stomata, located in the plant epidermis, consist of a pair of guard cells and the pore they enclose. Stomata open and close through turgor changes driven by massive ion fluxes, which occur mainly through the guard cell plasma membrane and tonoplast. The guard cell has become a model system for the study of signal transduction. Guard cells signaling is considered to be intriguing, not only because stomata play an important role in the plant's response to its variable environment, but also because the guard cell is mechanically separate from the surrounding cells, and because guard cell have sensitive reversible reactions to internal and external stimuli. To test the evidence and mechanism of PTPases regulating stomatal movement, epidermal peels and leaves of Vicia faba were used as the material in this study.The H+-ATPase at the plasma membrane of guard cells plays a critical role in the stomata opening, however, there are some conflicting results regarding the effectiveness of the plasma-membrane H+-ATPase inhibitor, vanadate, in inhibiting stomata opening. We observed that 2mmol/L vanadate hardly inhibited light-stimulated stomata opening in epidermal peels of Vicia faba L.; but significantly inhibited dark- and ABA-induced stomatal closure. These results cannot be explained with the previous finding that H+-ATPase was inhibited by vanadate. In view of the fact that vanadate is also an inhibitor of PTPases, which exist in plants, but their role of in plant signaling processes is unknown. Therefore, we investigated whether the stomatal movement regulated by vanadate is through the regulation of PTPase. As expected, phenylarsine oxide (PAO), a specific inhibitor of PTPase, has very similar effects and even more effective than vanadate. Stomatal closure induced by ABA and dark were prevented by PAO, which added to closed stomata promoted reopening. The addition of PAO to open stomata had no effect on aperture. Zn2+, as an inhibitor of PTPases, can also partially inhibit ABA- and dark-induced stomatal closure. These results not only provide a novel explanation for conflicting results about vanadate modulating stomatal movement, but also provide further evidence for the involvement of PTPases in modulating signal transduction of stomatal movement.We determined that the presence of PTPases activity in the epidermal peel of Vicia faba L. The extracted PTPases showed a high activity with an optimum pH of 6.0 to 7.0. At a relatively high pH (e.g.pH>7.5), the PTPases activity decreased significantly. NaVO₃ and PAO inhibited the activity of PTPases. Ca2+ and Mg2+ had no effect on the PTPases activity. On the contrary, Zn2+ could significantly inhibit the PTPases activity. The redox condition of medium can mediate the activity of PTPases: both PAO and H2O2 effectively inhibited the activity of PTPases in the epidermal peel of Viciafaba L. DTT could not relieve the inhibition of PAO on activity of PTPases, but the inhibition of H2O2 on activity of PTPases could be reversed by DTT. That's to say, PTPases in the epidermal peel of Vicia faba is reversibly inactivated by H2O2.The targets and substrate of PTPases must be some phosphotyrosyl proteins (PYP) that will be important steps toward understanding the role of PTPases in the plant-signaling network. Usi...