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CELL BIOLOGY AND SIGNAL TRANSDUCTION

A Signaling Pathway Linking Nitric Oxide Production to Heterotrimeric G Protein and Hydrogen Peroxide Regulates Extracellular Calmodulin Induction of Stomatal Closure in Arabidopsis^1,[W]

Hebei Key Laboratory of Molecular and Cellular Biology, College of Life Science, Hebei Normal University, Shijiazhuang 050016, China (J.-H.L., Y.-Q.L., H.-F.L., Y.B., Y.-L.C.); School of Basic Medical Sciences, Hebei Medical University, Shijiazhuang 050017, China (P.L.); and National Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100094, China (X.-C.W.)

Extracellular calmodulin (ExtCaM) regulates stomatal movement by eliciting a cascade of intracellular signaling events including heterotrimeric G protein, hydrogen peroxide (H₂O₂), and Ca²⁺. However, the ExtCaM-mediated guard cell signaling pathway remains poorly understood. In this report, we show that Arabidopsis (Arabidopsis thaliana) NITRIC OXIDE ASSOCIATED1 (AtNOA1)-dependent nitric oxide (NO) accumulation plays a crucial role in ExtCaM-induced stomatal closure. ExtCaM triggered a significant increase in NO levels associated with stomatal closure in the wild type, but both effects were abolished in the Atnoa1 mutant. Furthermore, we found that ExtCaM-mediated NO generation is regulated by GPA1, the G-subunit of heterotrimeric G protein. The ExtCaM-dependent NO accumulation was nullified in gpa1 knockout mutants but enhanced by overexpression of a constitutively active form of GPA1 (cG). In addition, cG Atnoa1 and gpa1-2 Atnoa1 double mutants exhibited a similar response as did Atnoa1. The defect in gpa1 was rescued by overexpression of AtNOA1. Finally, we demonstrated that G protein activation of NO production depends on H₂O₂. Reduced H₂O₂ levels in guard cells blocked the stomatal response of cG lines, whereas exogenously applied H₂O₂ rescued the defect in ExtCaM-mediated stomatal closure in gpa1 mutants. Moreover, the atrbohD/F mutant, which lacks the NADPH oxidase activity in guard cells, had impaired NO generation in response to ExtCaM, and H₂O₂-induced stomatal closure and NO accumulation were greatly impaired in Atnoa1. These findings have established a signaling pathway leading to ExtCaM-induced stomatal closure, which involves GPA1-dependent activation of H₂O₂ production and subsequent AtNOA1-dependent NO accumulation.

² These authors contributed equally to the article.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Yu-Ling Chen (yulingchen{at}mail.hebtu.edu.cn).

^[W] The online version of this article contains Web-only data.

www.plantphysiol.org/cgi/doi/10.1104/pp.109.137067

^* Corresponding author; e-mail yulingchen{at}mail.hebtu.edu.cn.

Received February 15, 2009; accepted March 19, 2009; published March 25, 2009.