Plant cell expansion depends on the uptake of solutes across the plasma membrane and their storage within the vacuole. In contrast to the well-studied plasma membrane, little is known about the regulation of ion trans...Plant cell expansion depends on the uptake of solutes across the plasma membrane and their storage within the vacuole. In contrast to the well-studied plasma membrane, little is known about the regulation of ion transport at the vacuolar membrane. We therefore established an experimental approach to study vacuolar ion transport in intact Arabidopsis root cells, with multi-barreled microelectrodes. The subcellular position of electrodes was detected by imaging current-injected fluorescent dyes. Comparison of measurements with electrodes in the cytosol and vacuole revealed an average vacuolar membrane potential of -31 inV. Voltage clamp recordings of single vacuoles resolved the activity of voltage-independent and slowly deactivating channels. In bulging root hairs that express the Ca2＋ sensor R-GECO1, rapid elevation of the cytosolic Ca^2＋ concentration was observed, after impalement with microelectrodes, or injection of the Ca^2＋ chelator BAPTA. Elevation of the cytosolic Ca^2＋ level stimulated the activity of voltage- independent channels in the vacuolar membrane. Likewise, the vacuolar ion conductance was enhanced during a sudden increase of the cytosolic Ca^2＋ level in cells injected with fluorescent Ca^2＋ indicator FURA-2. These data thus show that cytosolic Ca^2＋ signals can rapidly activate vacuolar ion channels, which may prevent rupture of the vacuolar membrane, when facing mechanical forces.展开更多
基金This work was supported by a grant from the Deutsche Forschungsgemeinschaft to M.R.G.R. （GK 1342, Project B5）, grants from the NSFC of China （No. 31270306） and the ＂111＂ Project of China （No. B06003）, grants from the Deutsche Forschungsgemeinschaft （FOR 964） to K.S., and by grants from the National Institutes of Health （GM060396） and National Science Foundation （MCB1414339） to Julian Schroeder （University of California, San Diego, USA） for the generation of the R-GECO1 plasmids and initial Ca^2＋ imaging experiments in the Schroeder lab by R.W.We thank Tracey Ann Cuin （University of Wurzburg） for help with preparation of the manuscript. No conflict of interest declared.
文摘Plant cell expansion depends on the uptake of solutes across the plasma membrane and their storage within the vacuole. In contrast to the well-studied plasma membrane, little is known about the regulation of ion transport at the vacuolar membrane. We therefore established an experimental approach to study vacuolar ion transport in intact Arabidopsis root cells, with multi-barreled microelectrodes. The subcellular position of electrodes was detected by imaging current-injected fluorescent dyes. Comparison of measurements with electrodes in the cytosol and vacuole revealed an average vacuolar membrane potential of -31 inV. Voltage clamp recordings of single vacuoles resolved the activity of voltage-independent and slowly deactivating channels. In bulging root hairs that express the Ca2＋ sensor R-GECO1, rapid elevation of the cytosolic Ca^2＋ concentration was observed, after impalement with microelectrodes, or injection of the Ca^2＋ chelator BAPTA. Elevation of the cytosolic Ca^2＋ level stimulated the activity of voltage- independent channels in the vacuolar membrane. Likewise, the vacuolar ion conductance was enhanced during a sudden increase of the cytosolic Ca^2＋ level in cells injected with fluorescent Ca^2＋ indicator FURA-2. These data thus show that cytosolic Ca^2＋ signals can rapidly activate vacuolar ion channels, which may prevent rupture of the vacuolar membrane, when facing mechanical forces.
