Scientific Correspondence A Role for ETR1 in Hydrogen Peroxide Signaling in Stomatal Guard Cells

Signaling through the redox active molecule hydrogen peroxide (H2O2) is important for several processes in plants, such as stomatal closure, root growth, gravitropism, and responses to pathogen challenge (Neill et al., 2002; Laloi et al., 2004). Although oxidative modification of reactive Cys residues within proteins hasbeen suggested as ameans bywhichH2O2 signaling can activate responses such as gene expression and reversible protein phosphorylation (Cooper et al., 2002; Danon, 2002), the linkage of H2O2 perception to intracellular signaling remains to be elucidated. Here, we report genetic and physiological data that demonstrate a previously uncharacterized function for the Arabidopsis (Arabidopsis thaliana) ethylene receptor ETR1, that of mediating H2O2 signaling in stomatal guard cells. Stomata in the loss-of-function etr1-7 mutant do not close in response to H2O2, and mutation of a Cys residue in theN-terminal region of ETR1 disruptsH2O2 signaling in both plants and in yeast (Saccharomyces cerevisiae). Large-scale analyses of H2O2-modulated gene expression in Arabidopsis and tobacco have shown that expression of genes encoding elements of both twocomponent signal transduction pathways and ethylene signaling are up-regulated by exogenous H2O2 (Desikan et al., 2001; Vandenabeele et al., 2003), suggesting that these phenomena may be linked. His kinases (HKs) are part of two-component systems that transduce environmental signals into cellular responses. Some of them are known to function as cytokinin and ethylene receptors in plants (Hwang et al., 2002). Hybrid HKs consist of an N-terminal signal input domain (with some having hydrophobic transmembrane regions, such as ETR1), a HK domain, and a C-terminal response regulator domain. During typical HK signaling, the HK domain is autophosphorylated on a His residue, with subsequent transfer of the phosphate group onto an Asp residue in the response regulatory domain of the same protein. A subsequent relay of phosphotransfer reactions occurs downstream of HK, effecting various signaling processes (Hwang et al., 2002). However, HK activity may not be required for all downstream responses (Wang et al., 2003). In yeast, two-component signaling systems function as H2O2 sensors (Singh, 2000; Buck et al., 2001). As part of a study to determine potential functions for plant HKs in H2O2 signaling, we focused on the ethylene receptor ETR1. ETR1 is a well-characterized hybrid HK in Arabidopsis and one for which extensive genetic, physiological, and biochemical analyses have demonstrated its function as an ethylene receptor (Guo and Ecker, 2004). Although ETR1 does have HK activity, such activity is not required for ethylene responses (Wang et al., 2003). The yeast TM219 mutant lacking a functional SLN1-SSK1 twocomponent system has enhanced susceptibility to growth inhibition by H2O2 (Singh, 2000). This system was used to determine if ETR1 could function in yeast to mediate oxidative stress responses. Transformation of TM219 with SLN1 and SSK1 together increased survival following exposure to H2O2 to a level comparable to that of the wild type (Fig. 1). Transformation of TM219 with full-length ETR1 resulted in a similar effect (Fig. 1), indicating that ETR1 can indeed function in yeast to mediate H2O2 responses. ETR1 is membrane-located in yeast (Fig. 1), but the particular membrane has not been identified. To determine if the N-terminal sensing domain of ETR1 was required forH2O2 responsiveness, TM219was transformedwith N-terminal constructs (containing the first 128 amino acids) of ETR1 containing either a wild-type Cys-65 or a Cys-65Tyr mutation in the second hydrophobic domain of ETR1 (as in the etr1-1 mutant). Only the construct containing the Cys-65 residue was able to increase survival following exposure to H2O2 (Fig. 1), indicating that the N-terminal domain of ETR1 is sufficient and that the Cys-65 residue is required for rescuing sensitivity to H2O2 in yeast. The mechanism by which ETR1 can restore H2O2 perception in TM219 is not known, although these data indicate that the HK domain of ETR1 is not required, but that the Cys-65 is essential. 1 This work was supported by the Biotechnology and Biological Sciences Research Council, UK. * Corresponding author; e-mail [email protected]; fax (44)117–32–82904. www.plantphysiol.org/cgi/doi/10.1104/pp.104.056994.