Grabov A, Blatt M R
Laboratory of Plant Physiology and Biophysics, University of London, Wye College, Wye, Kent TN25 5AH, United Kingdom.
Proc Natl Acad Sci U S A. 1998 Apr 14;95(8):4778-83. doi: 10.1073/pnas.95.8.4778.
In higher plants changes and oscillations in cytosolic free Ca2+ concentration ([Ca2+]i) are central to hormonal physiology, including that of abscisic acid (ABA), which signals conditions of water stress and alters ion channel activities in guard cells of higher-plant leaves. Such changes in [Ca2+]i are thought to encode for cellular responses to different stimuli, but their origins and functions are poorly understood. Because transients and oscillations in membrane voltage also occur in guard cells and are elicited by hormones, including ABA, we suspected a coupling of [Ca2+]i to voltage and its interaction with ABA. We recorded [Ca2+]i by Fura2 fluorescence ratio imaging and photometry while bringing membrane voltage under experimental control with a two-electrode voltage clamp in intact Vicia guard cells. Free-running oscillations between voltages near -50 mV and -200 mV were associated with oscillations in [Ca2+]i, and, under voltage clamp, equivalent membrane hyperpolarizations caused [Ca2+]i to increase, often in excess of 1 microM, from resting values near 100 nM. Image analysis showed that the voltage stimulus evoked a wave of high [Ca2+]i that spread centripetally from the peripheral cytoplasm within 5-10 s and relaxed over 40-60 s thereafter. The [Ca2+]i increases showed a voltage threshold near -120 mV and were sensitive to external Ca2+ concentration. Substituting Mn2+ for Ca2+ to quench Fura2 fluorescence showed that membrane hyperpolarization triggered a divalent influx. ABA affected the voltage threshold for the [Ca2+]i rise, its amplitude, and its duration. In turn, membrane voltage determined the ability of ABA to raise [Ca2+]i. These results demonstrate a capacity for voltage to evoke [Ca2+]i increases, they point to a dual interaction with ABA in triggering and propagating [Ca2+]i increases, and they implicate a role for voltage in "conditioning" [Ca2+]i signals that regulate ion channels for stomatal function.
在高等植物中，胞质游离钙离子浓度（[Ca2+]i）的变化和振荡是激素生理学的核心，包括脱落酸（ABA）的激素生理学，脱落酸可指示水分胁迫状况并改变高等植物叶片保卫细胞中的离子通道活性。[Ca2+]i的此类变化被认为是细胞对不同刺激的反应编码，但人们对其起源和功能了解甚少。由于保卫细胞中也会出现膜电压的瞬变和振荡，且这些变化由包括ABA在内的激素引发，我们怀疑[Ca2+]i与电压之间存在耦合及其与ABA的相互作用。我们在完整的蚕豆保卫细胞中通过双电极电压钳将膜电压置于实验控制之下的同时，利用Fura2荧光比率成像和光度法记录[Ca2+]i。在接近-50 mV和-200 mV的电压之间自由振荡与[Ca2+]i的振荡相关，并且在电压钳制下，等效的膜超极化导致[Ca2+]i从接近100 nM的静息值增加，通常超过1 microM。图像分析表明，电压刺激引发了一波高[Ca2+]i，该波在5 - 10秒内从外周细胞质向心扩散，此后在40 - 60秒内松弛。[Ca2+]i的增加显示出接近-120 mV的电压阈值，并且对外部Ca2+浓度敏感。用Mn2+替代Ca2+以淬灭Fura2荧光表明，膜超极化触发了二价离子内流。ABA影响[Ca2+]i升高的电压阈值、其幅度及其持续时间。反过来，膜电压决定了ABA升高[Ca2+]i的能力。这些结果证明了电压引发[Ca2+]i增加的能力，它们指出了在触发和传播[Ca2+]i增加方面与ABA的双重相互作用，并且它们暗示了电压在“调节”[Ca2+]i信号中的作用，这些信号调节气孔功能的离子通道。
