The Regulatory Domain of SRK2E/OST1/SnRK2.6 Interacts with ABI1 and Integrates Abscisic Acid (ABA) and Osmotic Stress Signals Controlling Stomatal Closure in Arabidopsis

Riichiro Yoshida,Taishi Umezawa,Tsuyoshi Mizoguchi,Seiji Takahashi,Fuminori Takahashi,Kazuo Shinozaki

doi:10.1074/jbc.m509820200

Riichiro Yoshida, Taishi Umezawa + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m509820200

Copy DOI

Abstract

ABI1 and ABI2 encode PP2C-type protein phosphatases and are thought to negatively regulate many aspects of abscisic acid (ABA) signaling, including stomatal closure in Arabidopsis. In contrast, SRK2E/OST1/SnRK2.6 encodes an Arabidopsis SnRK2 protein kinase and acts as a positive regulator in the ABA-induced stomatal closure. SRK2E/OST1 is activated by osmotic stress as well as by ABA, but the independence of the two activation processes has not yet been determined. Additionally, interaction between SRK2E/OST1 and PP2C-type phosphatases (ABI1 and ABI2) is not understood. In the present study, we demonstrated that the abi1-1 mutation, but not the abi2-1 mutation, strongly inhibited ABA-dependent SRK2E/OST1 activation. In contrast, osmotic stress activated SRK2E/OST1 even in abi1-1 and aba2-1 plants. The C-terminal regulatory domain of SRK2E/OST1 was required for its activation by both ABA and osmotic stress in Arabidopsis. The C-terminal domain was functionally divided into Domains I and II. Domain II was required only for the ABA-dependent activation of SRK2E/OST1, whereas Domain I was responsible for the ABA-independent activation. Full-length SRK2E/OST1 completely complemented the wilty phenotype of the srk2e mutant, but SRK2E/OST1 lacking Domain II did not. Domain II interacted with the ABI1 protein in a yeast two-hybrid assay. Our results suggested that the direct interaction between SRK2E/OST1 and ABI1 through Domain II plays a critical role in the control of stomatal closure.

Highlights

abscisic acid (ABA) activated two protein kinases, p44 and p42, in wild-type roots, and p44 was not detected in srk2e, indicating that p44 corresponds to SRK2E (Fig. 1B)
We examined whether low humidity directly activated SRK2E/OST1. 2E-green fluorescent protein (GFP) was clearly activated by low humidity in leaf tissues (Fig. 1E)
Biochemical analysis of the GFP fusion of the kinases with the abi and aba mutations and the yeast two-hybrid analysis enabled us to demonstrate that the ABA-dependent and -independent pathways mediate between low humidity and stomatal closure in Arabidopsis

Summary

Introduction

Reactive oxygen species have been proposed to function as second messengers for ABA signaling in stomata cells and to activate Ca2ϩ channels [9]. SRK2E/ OST1/SnRK2.6 is regarded as a major, positive regulator of ABA signaling in Arabidopsis. We examined whether ABA-dependent or ABA-independent pathways, or both, function in the activation of SRK2E/OST1 to close stomata cells under low humidity stress. An experiment using ABA-insensitive or ABA-deficient mutants revealed that the OS-dependent activation of SRK2E/OST1 was not mediated by ABA, implying that two independent pathways exist and function in SRK2E/OST1 signaling. The possible mechanisms involved in the activation of SRK2E/OST1 in the response to ABA and OS were discussed in relation to stomata closure

Methods

Results

Conclusion