Keinath Nana F, Waadt Rainer, Brugman Rik, Schroeder Julian I, Grossmann Guido, Schumacher Karin, Krebs Melanie
Centre for Organismal Studies, Plant Developmental Biology, University of Heidelberg, Im Neuenheimer Feld 230, 69120 Heidelberg, Germany.
Centre for Organismal Studies, Plant Developmental Biology, University of Heidelberg, Im Neuenheimer Feld 230, 69120 Heidelberg, Germany; Division of Biological Sciences, Cell and Developmental Biology Section, University of California San Diego, 92093 La Jolla, USA.
Mol Plant. 2015 Aug;8(8):1188-200. doi: 10.1016/j.molp.2015.05.006. Epub 2015 May 19.
Intracellular Ca(2+) transients are an integral part of the signaling cascade during pathogen-associated molecular pattern (PAMP)-triggered immunity in plants. Yet, our knowledge about the spatial distribution of PAMP-induced Ca(2+) signals is limited. Investigation of cell- and tissue-specific properties of Ca(2+)-dependent signaling processes requires versatile Ca(2+) reporters that are able to extract spatial information from cellular and subcellular structures, as well as from whole tissues over time periods from seconds to hours. Fluorescence-based reporters cover both a broad spatial and temporal range, which makes them ideally suited to study Ca(2+) signaling in living cells. In this study, we compared two fluorescence-based Ca(2+) sensors: the Förster resonance energy transfer (FRET)-based reporter yellow cameleon NES-YC3.6 and the intensity-based sensor R-GECO1. We demonstrate that R-GECO1 exhibits a significantly increased signal change compared with ratiometric NES-YC3.6 in response to several stimuli. Due to its superior sensitivity, R-GECO1 is able to report flg22- and chitin-induced Ca(2+) signals on a cellular scale, which allowed identification of defined [Ca(2+)]cyt oscillations in epidermal and guard cells in response to the fungal elicitor chitin. Moreover, we discovered that flg22- and chitin-induced Ca(2+) signals in the root initiate from the elongation zone.
细胞内钙离子瞬变是植物中病原体相关分子模式（PAMP）触发的免疫反应信号级联的一个组成部分。然而，我们对PAMP诱导的钙离子信号的空间分布的了解有限。对钙离子依赖性信号传导过程的细胞和组织特异性特性进行研究，需要多功能的钙离子报告基因，这些报告基因能够从细胞和亚细胞结构以及从整个组织中在几秒到几小时的时间段内提取空间信息。基于荧光的报告基因覆盖了广泛的空间和时间范围，这使得它们非常适合用于研究活细胞中的钙离子信号传导。在本研究中，我们比较了两种基于荧光的钙离子传感器：基于Förster共振能量转移（FRET）的报告基因黄色变色龙NES-YC3.6和基于强度的传感器R-GECO1。我们证明，与比率型NES-YC3.6相比，R-GECO1在对几种刺激的反应中表现出显著增加的信号变化。由于其卓越的灵敏度，R-GECO1能够在细胞水平上报告鞭毛蛋白22和几丁质诱导的钙离子信号，这使得我们能够识别表皮细胞和保卫细胞中对真菌激发子几丁质产生的特定细胞质钙离子振荡。此外，我们发现根中鞭毛蛋白22和几丁质诱导的钙离子信号起始于伸长区。
