FRET kinase sensor development reveals SnRK2/OST1 activation by ABA but not by MeJA and high CO2 during stomatal closure

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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.

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Data generated or analysed during this study are included in the manuscript and supporting files.

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Li Zhang

Division of Biological Sciences, Cell and Developmental Biology Section, University of California, San Diego, La Jolla, United States

Competing interests
The authors declare that no competing interests exist.
Yohei Takahashi

Division of Biological Sciences, Cell and Developmental Biology Section, University of California, San Diego, La Jolla, United States

For correspondence
ytakahashi@UCSD.EDU

Competing interests
The authors declare that no competing interests exist.
Po-Kai Hsu

Division of Biological Sciences, Cell and Developmental Biology Section, University of California, San Diego, La Jolla, United States

Competing interests
The authors declare that no competing interests exist.
Kollist Hannes

Institute of Technology, University of Tartu, Tartu, Estonia

Competing interests
The authors declare that no competing interests exist.
Ebe Merilo

Institute of Technology, University of Tartu, Tartu, Estonia

Competing interests
The authors declare that no competing interests exist.
Patrick J Krysan

Horticulture Department, University of Wisconsin-Madison, Madison, United States

Competing interests
The authors declare that no competing interests exist.
Julian I Schroeder

Division of Biological Sciences, Cell and Developmental Biology Section, University of California, San Diego, La Jolla, United States

For correspondence
jischroeder@ucsd.edu

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-3283-5972

Funding

National Science Foundation (MCB-1900567)

Julian I Schroeder

National Institutes of Health (GM060396)

Julian I Schroeder

China Scholarship Council

Li Zhang

Japan Society for the Promotion of Science

Yohei Takahashi

Eesti Teadusagentuur (PUT1133)

Kollist Hannes

Eesti Teadusagentuur (PRG719)

Kollist Hannes

Eesti Teadusagentuur (PRG433)

Kollist Hannes

European Regional Development Fund

Ebe Merilo

National Science Foundation (MCB‐1137950)

Po-Kai Hsu

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

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This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.