Project Scientist University of California, San Diego
Protein kinases are dynamically and specifically regulated in diverse signal transduction networks in plants. Abscisic acid (ABA) activates sucrose non-fermenting-1-related protein kinase 2s (SnRK2s) that are critical for orchestrating abiotic stress tolerance responses in plants. However, the existing methods for measuring SnRK2 kinase activity, in-gel kinase assays, have drawbacks and do not permit spatial or temporal resolution. Here, we developed a genetically-encoded FRET-based reporter for activity of SnRK2 protein kinases which mediate ABA signal transduction. This sensor shows an increase in the ratio of yellow to cyan fluorescence emission by phosphorylation of a defined serine residue. ABA rapidly increases FRET efficiency in N. benthamiana leaf cells and Arabidopsis guard cells. Interestingly, protein kinase inhibition decreases FRET efficiency in guard cells, suggesting a reversibility of this sensor and providing experimental evidence for a basal activity of SnRK2 protein kinases in guard cells. Moreover, in contrast to ABA, the stomatal closing stimuli, elevated CO2 and MeJA, did not increase the FRET ratios. These findings and gas exchange analyses of quintuple or sextuple ABA receptor mutants show that stomatal CO2 signaling proceeds effectively in these mutants, but requires basal SnRK2 signaling. However, CO2 and MeJA do not further activate ABA signaling and SnRK2 kinases. These advances illustrate the dynamics of ABA, MeJA and CO2 signaling cross talk and provide a potent approach for real-time live-cell investigations of abiotic stress signaling.
Coauthors: Li Zhang – University of California, San Diego;Po-Kai Hsu – University of California, San Diego;Hannes Kollist – University of Tartu;Ebe Merilo – University of Tartu;Patrick Krysan – University of Wisconsin-Madison;Julian Schroeder – University of California, San Diego