Measurement of cellular cGMP in plant cells and tissues using the endogenous fluorescent reporter FlincG
Measurement of cellular cGMP in plant cells and tissues using the endogenous fluorescent reporter FlincG
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DOI：
10.1111/j.1365-313x.2010.04418.x
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发表时间：
2011-01-01
期刊：
影响因子：
7.2
通讯作者：
Maathuis, Frans J. M.
中科院分区：
文献类型：
--
作者：
Isner, Jean-Charles;Maathuis, Frans J. M.
环核苷酸cGMP在植物的生长发育和对生物和非生物胁迫的响应中起着重要的作用。迄今为止，可用于测量植物中cGMP的技术受到低空间和时间分辨率的限制。此外，组织破坏是必要的。为了克服这些缺点，我们使用了delta-FlincG荧光蛋白来创建内源性cGMP传感器，其可以在活植物细胞中以高分辨率的时间和空间报告细胞cGMP水平。在瞬时和稳定表达细胞中的δ-FlincG显示出约200 nm的cGMP解离常数，使其具有约20-2000 nm的动态范围。先前显示改变植物细胞中cGMP的刺激（一氧化氮和赤霉酸）在单细胞和根组织中诱发明显的荧光信号，提供了delta-FlincG在生理相关背景下报告细胞cGMP变化的证据。
P>The cyclic nucleotide cGMP has been shown to play important roles in plant development and responses to abiotic and biotic stress. To date, the techniques that are available to measure cGMP in plants are limited by low spatial and temporal resolution. In addition, tissue destruction is necessary. To circumvent these drawbacks we have used the delta-FlincG fluorescent protein to create an endogenous cGMP sensor that can report cellular cGMP levels with high resolution in time and space in living plant cells. delta-FlincG in transient and stably expressing cells shows a dissociation constant for cGMP of around 200 nm giving it a dynamic range of around 20-2000 nm. Stimuli that were previously shown to alter cGMP in plant cells (nitric oxide and gibberrellic acid) evoked pronounced fluorescence signals in single cells and in root tissues, providing evidence that delta-FlincG reports changes in cellular cGMP in a physiologically relevant context.