Cell and Developmental Biology Section, Division of Biological Sciences, University of California, San Diego, San Diego, United States.
Institute of Technology, University of Tartu, Tartu, Estonia.
Elife. 2020 May 28;9:e56351. doi: 10.7554/eLife.56351.
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 leaf cells and 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 CO and MeJA, did not increase SNACS FRET ratios. These findings and gas exchange analyses of quintuple/sextuple ABA receptor mutants show that stomatal CO signaling requires basal ABA and SnRK2 signaling, but not SnRK2 activation. A recent model that CO 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.
蔗糖非发酵相关蛋白激酶 2（SnRK2s）在植物非生物胁迫反应中起着关键作用，包括脱落酸（ABA）信号转导。在这里，我们开发了一种用于 SnRK2 激酶活性的遗传编码报告器。这个传感器被命名为 SNACS，它通过 OST1/SnRK2.6 介导的 SNACS 中特定丝氨酸残基的磷酸化，显示出黄色到青色荧光发射比值的增加。ABA 可迅速增加叶片细胞和保卫细胞中的 FRET 效率。有趣的是，蛋白激酶抑制降低了保卫细胞中的 FRET 效率，这为基础 SnRK2 活性在保卫细胞中占主导地位提供了直接的实验证据。此外，与 ABA 不同，气孔关闭刺激物，高浓度的 CO 和 MeJA，并没有增加 SNACS 的 FRET 比值。这些发现和对五重/六重 ABA 受体突变体的气体交换分析表明，气孔 CO 信号需要基础 ABA 和 SnRK2 信号，但不需要 SnRK2 激活。最近的一个模型表明，CO 信号是由 PYL4/PYL5 ABA 受体介导的，但在两个独立的实验室中都无法得到支持。我们报告了一种用于实时活细胞应激信号研究的有效方法。
