The Effect Of H～+-ATPase In Plasma Membrane On Activity Of Calcium Channel In Plasma Membrane Of Arabidopsis Thaliana Guard Cells

Guard cell regulates photosynthesis and transpiration, which play an important role in the life of plant, with their special cell wall structure. H⁺-ATPase in plasma membrane can be activated by exogenous stimuli through signaling molecules in plasma membrane, and then accelerate H⁺ efflux, which will cause plasma membrane hyperpolarization and changing ion concentration in cytoplasm and finally lead to stomatal movement.A great deal of research results 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 Arabidopsis thaliana (WS ecotype) were used as main material, guard cell protoplasts with high activity were isolated, the relation between H⁺-ATPase and calcium channel in plasma membrane of guard cells was investigated with whole cell patch clamping.Since Arabidopsis thaliana guard cell protoplast is quite small (6⁹μm) , it had brought lots of difficulty to protoplast isolating and patch clamping. Based on pervious research results, considering the character of material, guard cell protoplasts were isolated successfully by digesting the ripped epidermis with two-step enzyme digestion. The activity of isolated protoplasts were measured with FDA, the result showed that activated protoplasts were isolated and they could be used in patch clamp experiment. 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 under -100mV and membrane current increased 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 H⁺-ATPase activator fusiccocin, Ca²⁺ current was increasing.The result of patch clamp experiment suggested that a voltage-dependent, hyperpolarization-activated, Ca²⁺-permeable, inward-recitified conductance existed inplasma membrane of Arabidopsis thaliana guard cell protoplasts. Plasma H+-ATPase could accelerate Ca2+ influx from extracellular matrix of guard cells by activating Ca2+- permeable channel and lead the increasing of [Ca2+]cyt, which will then regulate stomatal movment.