Current Injection Provokes Rapid Expansion of the Guard Cell Cytosolic Volume and Triggers Ca(2+) Signals.
Current Injection Provokes Rapid Expansion of the Guard Cell Cytosolic Volume and Triggers Ca(2+) Signals.
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DOI：
10.1016/j.molp.2016.02.004
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发表时间：
2016-03
期刊：
影响因子：
27.5
通讯作者：
Lena J. Voss;R. Hedrich;M. Roelfsema
中科院分区：
文献类型：
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作者：
Lena J. Voss;R. Hedrich;M. Roelfsema
高分辨率显微镜为使用荧光报告染料和蛋白质进行详细的单细胞研究打开了大门。我们使用共聚焦旋转圆盘显微镜来监测烟草和拟南芥保卫细胞中的荧光染料和荧光蛋白Venus。使用多管微电极注入染料并施加电压脉冲，从而引起细胞内钙离子水平的瞬时上升。电压脉冲也引起了荧光黄和金星分布的变化，这表明保卫细胞胞浆体积可逆地增加。细胞内的动态体积变化是由电流注入离子引起的。通过用Cs+阻断K+摄取通道来降低钳制电流，强烈地抑制了胞浆体积的变化。CS+不仅抑制胞浆的扩张，而且抑制超极化引起的胞浆内钙浓度的升高。当钙离子通透质膜通道被La3+同时阻断时，电压诱导的钙信号完全消失。这表明，超极化引起细胞内钙离子浓度升高有两种机制：(1)激活电压依赖性钙离子通透通道；(2)渗透诱导胞浆膨胀，导致胞浆内钙离子释放。
High-resolution microscopy opens the door for detailed single-cell studies with fluorescent reporter dyes and proteins. We used a confocal spinning disc microscope to monitor fluorescent dyes and the fluorescent protein Venus in tobacco andArabidopsisguard cells. Multi-barreled microelectrodes were used to inject dyes and apply voltage pulses, which provoke transient rises in the cytosolic Ca2+level. Voltage pulses also caused changes in the distribution of Lucifer Yellow and Venus, which pointed to a reversible increase of guard cell cytosolic volume. The dynamic cytosolic volume changes turned out to be provoked by current injection of ions. A reduction of the clamp current, by blocking K+uptake channels with Cs+, strongly suppressed the cytosolic volume changes. Cs+not only inhibited the expansion of the cytosol, but also inhibited hyperpolarization-induced elevations of the cytosolic Ca2+concentration. A complete loss of voltage-induced Ca2+signals occurred when Ca2+-permeable plasma membrane channels were simultaneously blocked with La3+. This shows that two mechanisms cause hyperpolarization-induced elevation of the cytosolic Ca2+-concentration: (i) activation of voltage-dependent Ca2+-permeable channels, (ii) osmotically induced expansion of the cytosol, which leads to a release of Ca2+from intracellular stores.