Extracellular calmodulin (ExtCaM) exerts multiple functions in animals and plants, but the mode of ExtCaM action is not well understood. In this paper, we provide evidence that ExtCaM stimulates a cascade of intracellular signaling events to regulate stomatal movement. Analysis of the changes of cytosolic free Ca 21 ([Ca 21 (McAinsh et al., 1995;Grabov and Blatt, 1998). Accumulating evidence indicates that many stimuli enhance [Ca 21 ] cyt increase in guard cells (Rudd and Franklin-Tong, 2001); however, the upstream components of calcium signaling are not well understood.Heterotrimeric G proteins composed of a-, b-, and g-subunits are a key intracellular signaling molecule in eukaryotic cells. The activation by G-protein-coupled receptor results in conformation change of the Ga protein due to GTP binding and the separation of Ga from the Gbg dimer. GTP hydrolysis by GTPase activity of Ga results in the reassociation of Ga with Gbg (Jones and Assmann, 2004). In plants, G protein has been found to be involved in ion-channel regulation (Aharon et al., 1998;Wang et al., 2001), control of seed germination (Ullah et al., 2002), pollen tube elongation (Ma et al., 1999), and responses to ABA . Genome sequencing revealed the existence of only one prototypical Ga (GPA1) in Arabidopsis (Arabidopsis thaliana; Ma et al., 1990). It was reported that Ga-subunit-null mutants, gpa1-1 and gpa1-2, were insensitive to ABA inhibition of whole-cell inward K 1 currents and pH-independent ABA-activation of anion channels , suggesting Ga is a key component in ABA signaling. It is unknown whether Ga-subunit participates in calcium signaling in ABA regulation of guard cell responses.Recently, reactive oxygen species (ROS) has been shown to be an important second messenger in signaling to developmental processes, such as polar growth of Fucus rhizoid cells (Coelho et al., 2002) and cell elongation in root growth (Demidchik et al., 2003), responses to environmental stresses (BaxterBurrell et al., 2002), and guard cell movement (Pei et al., 2000). Evidence indicates that homeostasis of ROS depends on the activity of several enzymes involved in ROS generation as well as the activity of ROS scavenging enzymes (for review, see Mittler, 2002). Recently, guard cell-specific NADPH oxidases AtrbohD (Arabidopsis respiratory burst oxidase homologs D) and AtrbohF have been identified, and the double mutants of atrbohD/F are impaired in ABA-induced ROS generation, [Ca 21 ] cyt increases, and stomatal closing (Kwak et al., 2003), suggesting that AtrbohD and AtrbohF NADPH oxidases and ROS play an important 1 This work was supported by the Major State Basic Research Program of China (grant no. G1999011704) and by the National Science Foundation of China (grant no. 30370129).2 These authors contributed equally to the paper. * Corresponding author; e-mail xcwang@cau.edu.cn; fax 86-10-62733450.Article, publication date, and citation information can be found at www.plantphysiol.org/cgi

Extracellular calmodulin (ExtCaM) regulates stomatal movement by eliciting a cascade of intracellular signaling events including heterotrimeric G protein, hydrogen peroxide (H 2 O 2 ), and Ca 2+ . 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 Ga-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 (cGa). In addition, cGa 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 H 2 O 2 . Reduced H 2 O 2 levels in guard cells blocked the stomatal response of cGa lines, whereas exogenously applied H 2 O 2 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 H 2 O 2 -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 H 2 O 2 production and subsequent AtNOA1-dependent NO accumulation.Plant guard cells control opening and closure of the stomata in response to phytohormones (e.g. abscisic acid [ABA]) and various environmental signals such as light and temperature, thereby regulating gas exchange for photosynthesis and water status via transpiration . Cytosolic calcium ([Ca 2+ ] i ) has been shown to be a key second messenger that changes in response to multiple stimuli in guard cells (McAinsh et al., 1995;Grabov and Blatt, 1998;Wood et al., 2000). A large proportion of Ca 2+ is localized in extracellular space. It has been shown that external Ca 2+ concentration ([Ca 2+ ] o ) promotes stomatal closure and induces oscillation in [Ca 2+ ] i in guard cells (MacRobbie, 1992;McAinsh et al., 1995;Allen et al., 2001 Calmodulin is a well-known Ca 2+ sensor that is activated upon binding of Ca 2+ . It has been shown that calmodulin exists not only intracellularly but also extracellularly in many plant species