The Effect Of Plasma Membrane H~+-ATPase On Calcium Channel In PM Of Vicia Faba Guard Cells

Guard cells play an important role in photosynthesis and transpiration,by using their special cell wall structure.Exogenous stimuli may activate H⁺-ATPase in plasma membrane (PM) through signal molecules, and then accelerated H⁺ efflux will lead plasma membrane hyperpolarization and increasing of ion concentration in cytoplasm, finally stomatal movement is induced. A great deal of evidence indicated that both exogenous and endogenous stimuli can stimulate the elevation of cytoplasmic Ca2+ in guard cell, which will act as the second messenger in their signal transduction. In this paper, leaf epidermis of Vicia faba were used as main material, the effect of H⁺-ATPase on stomatal movement were investigated by using fusicoccin(FC, agonist of PM H+-ATPase) and sodium vanadate(antagonist of PM H+-ATPase). By using two-step enzyme digestion, protoplasts with high viability were isolated from leaf epidermis effect of FC and sodium vanadate on calcium channel in plasma membrane of guard cells was investigated by using whole cell patch clamping.The stomatal aperture was bigger after adding fusicoccin into the bath solution, while the aperture was smaller after being treated by sodium vanadate. The result is consistent with our previous results by using Arabidopsis as material. Ca²⁺-permeable channel was detected in guard cell plasma membrane by using patch clamp recording, this channel possesses higher voltage-dependent manner. It could be activated when membrane voltage was hyperpolarized to -100mV and the current increasing continuously with the increasing of the cell membrane hyperpolarization. Tail current analysis suggested the reversal potential of inward current was about 0mV. When guard cell protoplast was treated with fusiccocin, Ca²⁺ current increased, and sodium vanadate strongly suppress the activity of Ca²⁺ channel. These results suggest that proton pump is a positive regulator which accommodates stomatal movement by enhancing Ca²⁺ influx through a voltage-dependent, hyperpolarization-activated, inward-recitified Ca²⁺-permeable channel in plasma membrane of Vicia faba guard cell.