FRET kinase sensor development reveals SnRK2/OST1 activation by ABA but not by MeJA and high CO2 during stomatal closure
FRET kinase sensor development reveals SnRK2/OST1 activation by ABA but not by MeJA and high CO2 during stomatal closure
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DOI：
10.7554/elife.56351
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发表时间：
2020-05
期刊：
影响因子：
7.7
通讯作者：
Li Zhang;Yohei Takahashi;P. Hsu;Kollist Hannes;E. Merilo;P. Krysan;J. Schroeder
中科院分区：
文献类型：
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作者：
Li Zhang;Yohei Takahashi;P. Hsu;Kollist Hannes;E. Merilo;P. Krysan;J. Schroeder
蔗糖非发酵1相关蛋白激酶2（SnRK 2）是植物非生物胁迫应答的关键信号分子，其中包括脱落酸（阿坝）信号。在这里，我们开发了一个基因编码的SnRK 2激酶活性的报告。这种传感器，命名为SNACS，显示了增加的比率，黄色到青色荧光发射的OST 1/SnRK2.6介导的磷酸化的一个确定的丝氨酸残基在SNACS。阿坝快速增加N. benthamiana叶细胞和拟南芥保卫细胞。有趣的是，蛋白激酶抑制降低FRET效率在保卫细胞，提供直接的实验证据，基础SnRK 2活性普遍存在于保卫细胞。此外，在阿坝，气孔关闭刺激，CO2和MeJA升高，没有增加SNACS FRET比率。这些研究结果和气体交换分析的五/六倍阿坝受体突变体表明，气孔CO2信号需要基础阿坝和SnRK 2信号，但不SnRK 2激活。最近的一个模型，CO2信号是由PYL 4/PYL 5 ABA受体介导的，不能在这里支持两个独立的实验室。我们报告了一种有效的方法，实时活细胞调查的压力信号。
Sucrose-non-fermenting-1-related protein kinase-2s (SnRK2s) are critical for plant abiotic stress responses, including abscisic acid (ABA) signaling. Here, we develop a genetically encoded reporter for SnRK2 kinase activity. This sensor, named SNACS, shows an increase in the ratio of yellow to cyan fluorescence emission by OST1/SnRK2.6-mediated phosphorylation of a defined serine residue in SNACS. ABA rapidly increases FRET efficiency in N. benthamiana leaf cells and Arabidopsis guard cells. Interestingly, protein kinase inhibition decreases FRET efficiency in guard cells, providing direct experimental evidence that basal SnRK2 activity prevails in guard cells. Moreover, in contrast to ABA, the stomatal closing stimuli, elevated CO2 and MeJA, did not increase SNACS FRET ratios. These findings and gas exchange analyses of quintuple/sextuple ABA receptor mutants show that stomatal CO2 signaling requires basal ABA and SnRK2 signaling, but not SnRK2 activation. A recent model that CO2 signaling is mediated by PYL4/PYL5 ABA-receptors could not be supported here in two independent labs. We report a potent approach for real-time live-cell investigations of stress signaling.