Abstract
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.
Highlights
Protein phosphorylation of downstream substrates by protein kinases is a central and critical molecular switch for activation of many cell biological processes (Ardito et al, 2017; Stone and Walker, 1995)
The highly active CPK6 protein kinase (Brandt et al, 2012), did not increase the Forster resonance energy transfer (FRET) ratio (Figure 1—figure supplement 1D). These results indicate that SnRK2 activity sensor (SNACS) shows an increase of FRET ratio based on a phosphorylation of the ABA-responsive kinase substrate 1 (AKS1) domain by SnRK2 protein kinases in vitro
Abscisic acid activation of SnRK2 protein kinases is required for abscisic acid (ABA) signal transduction (Cutler et al, 2010; Raghavendra et al, 2010; Zhu, 2016)
Summary
Protein phosphorylation of downstream substrates by protein kinases is a central and critical molecular switch for activation of many cell biological processes (Ardito et al, 2017; Stone and Walker, 1995). In-gel kinase assays are the most common method for measuring protein kinase activities using the (auto-)phosphorylation state of a kinase or a substrate as indicator of the kinase activity (Manning et al, 2002) With this method, it is difficult to track dynamic kinase activity in specific cell types or tissues, and time course measurements in living cells and subcellular analyses are not feasible (Aoki et al, 2012). It is difficult to track dynamic kinase activity in specific cell types or tissues, and time course measurements in living cells and subcellular analyses are not feasible (Aoki et al, 2012) To overcome this drawback, a first Forster resonance energy transfer (FRET) biosensor reporting the activity of cAMP-dependent protein kinase A (PKA) was developed by R.Y. Tsien and colleagues (Zhang et al, 2001). This enhanced affinity between the two domains can trigger the two fluorophores to be in closer proximity to one-another
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