Cameleon calcium indicator reports cytoplasmic calcium dynamics in Arabidopsis guard cells
Cameleon calcium indicator reports cytoplasmic calcium dynamics in Arabidopsis guard cells
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DOI：
10.1046/j.1365-313x.1999.00574.x
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发表时间：
1999-09-01
期刊：
影响因子：
7.2
通讯作者：
Schroeder, JI
中科院分区：
文献类型：
--
作者：
Allen, GJ;Kwak, JM;Schroeder, JI
细胞质游离钙（[Ca 2 +]（cyt））是植物细胞内受外界刺激诱导的第二信使，气孔保卫细胞内[Ca 2 +]（cyt）的变化受多种信号转导途径的调控。测量保卫细胞中的[Ca 2 +]（cyt）以前需要使用侵入性和技术上困难的微注射技术加载钙敏感染料。为了避免这些问题，我们已经在拟南芥中组成型表达了pH非依赖性的、基于绿色荧光蛋白的钙指示剂黄色cameleon 2.1（Miyawaki等人，1999; Proc.Natl. Acad. Sci. USA 96，2135-2140）。这种黄色的钙指示剂在保卫细胞中表达，主要在细胞质中积累。黄色cameleon 2.1的荧光比率成像允许拟南芥保卫细胞中[Ca 2 +]（cyt）的时间依赖性测量。应用细胞外钙或激素脱落酸（阿坝）诱导重复的[Ca 2 +]（细胞色素）瞬变保卫细胞。[Ca2+]（cyt）的变化可以在校正叶绿体自发荧光的校准程序后半定量地确定。细胞外钙诱导的重复性[Ca 2 +]（cyt）瞬变峰值高达约1.5 μ M，而ABA诱导的[Ca 2 +]（cyt）瞬变峰值高达约0.6 μ M。这些值是类似的刺激诱导的[Ca 2 +]（细胞色素）的变化，以前报道的植物细胞中使用比率染料或水母发光蛋白。在一些警卫细胞灌注低细胞外KCl浓度，自发钙瞬变观察。由于黄Cameleon 2.1在所有保卫细胞中均有表达，因此首次在单气孔的两个保卫细胞中独立测定了[Ca ~（2+）]（cyt）。ABA诱导的，钙诱导的或自发的[Ca 2 +]（cyt）的增加不一定同步的两个保卫细胞。总体而言，这些数据表明，基于GFP的骆驼钙指示剂适合于测量保卫细胞中的[Ca 2 +]（cyt）变化，并且能够在拟南芥细胞中以高水平的再现性测量[Ca 2 +]（cyt）动态模式。这一技术进步与细胞生物学和分子遗传学方法相结合，将成为解剖植物细胞信号转导途径的宝贵工具。
Cytoplasmic free calcium ([Ca2+](cyt)) acts as a stimulus-induced second messenger in plant cells and multiple signal transduction pathways regulate [Ca2+](cyt) in stomatal guard cells. Measuring [Ca2+](cyt) in guard cells has previously required loading of calcium-sensitive dyes using invasive and technically difficult micro-injection techniques. To circumvent these problems, we have constitutively expressed the pH-independent, green florescent protein-based calcium indicator yellow cameleon 2.1 in Arabidopsis thaliana (Miyawaki et al., 1999; Proc. Natl. Acad. Sci. USA 96, 2135-2140). This yellow cameleon calcium indicator was expressed in guard cells and accumulated predominantly in the cytoplasm. Fluorescence ratio imaging of yellow cameleon 2.1 allowed time-dependent measurements of [Ca2+](cyt) in Arabidopsis guard cells. Application of extracellular calcium or the hormone abscisic acid (ABA) induced repetitive [Ca2+](cyt) transients in guard cells. [Ca2+](cyt) changes could be semi-quantitatively determined following correction of the calibration procedure for chloroplast autofluorescence. Extracellular calcium induced repetitive [Ca2+](cyt) transients with peak values of up to approximately 1.5 mu M, whereas ABA-induced [Ca2+](cyt) transients had peak values up to approximately 0.6 mu M. These values are similar to stimulus-induced [Ca2+](cyt) changes previously reported in plant cells using ratiometric dyes or aequorin. In some guard cells perfused with low extracellular KCI concentrations, spontaneous calcium transients were observed. As yellow cameleon 2.1 was expressed in all guard cells, [Ca2+](cyt) was measured independently in the two guard cells of single stomates for the first time. ABA-induced, calcium-induced or spontaneous [Ca2+](cyt) increases were not necessarily synchronized in the two guard cells. Overall, these data demonstrate that that GFP-based cameleon calcium indicators are suitable to measure [Ca2+](cyt) changes in guard cells and enable the pattern of [Ca2+](cyt) dynamics to be measured with a high level of reproducibility in Arabidopsis cells. This technical advance in combination with cell biological and molecular genetic approaches will become an invaluable tool in the dissection of plant cell signal transduction pathways.