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

Abstract

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 has been suggested as a means by which H2O2 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 the N-terminal region of ETR1 disrupts H2O2 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 two-component 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). Yeast mutants lacking a functional HK that are more susceptible to H2O2 can be complemented with ETR1. The sln1::ssk1 mutant yeast strain TM219 (MATα ura3, leu2, trp1, his3, sln1::URA3, ssk1::LEU2) was grown and maintained in YPD medium (1% yeast extract, 2% bactopeptone, and 2% dextrose). For transformation of TM219, cells from an overnight culture were used to inoculate 100 mL of liquid YPD and incubated at 30°C until log phase. The cells were recovered by centrifugation (4,500g for 5 min), washed in 5 mL of 1 × LiAc/TE mix (100 mm lithium acetate, pH 7.5, 10 mm Tris-HCl, and 1 mm EDTA), and resuspended in 1 mL LiAc/TE. The plasmid DNA to be transformed (<5 μg) and 50 to 100 μg of sonicated salmon sperm DNA were mixed with 100 μL of yeast suspension and 700 μL of sterile 40% PEG4000 and incubated for 30 min at 30°C. The cells were heat shocked for 15 min at 42°C in the presence of 88 μL of DMSO and subjected to a pulse spin before resuspending in 0.2 mL of TE. The transformed cells were selected on appropriate selection plates. The expression of ETR1 in TM219 was confirmed by western blotting using anti-ETR1 (C-terminal) or anti-GST antibodies (Insight Biotechnology, Wembley, UK), as described (Rodriguez et al., 1999). For H2O2 sensitivity tests, log-phase cells were treated with H2O2 (1.25 mm) for 30 min and plated out at various dilutions on YPD agar plates. After incubation for 3 d at 30°C, the number of colonies was counted and percent survival calculated by comparing untreated versus treated cultures. 219vec, TM219 transformed with pYCDE2 vector alone; 219-ETR1, 219 with pYCDE2 containing full-length ETR1; 219-ETR1(1-128), 219 with pYCDE2 containing N-terminal region (1–128) of ETR1 fused to GST; 219-etr1-1(1-128), 219 transformed with pYCDE2 containing the Cys-65 mutation in N-terminal region of ETR1 fused to GST; 219ss, 219 with SLN1 and SSK1 plasmids as a positive control. Data represent the mean ± se from four independent experiments. Sections below indicate the expression of ETR1 in the 219 transformants, as determined by western blotting of yeast membranes isolated as described (Rodriguez et al., 1999), using an anti-ETR1 (C-terminal) antibody for the full-length ETR1 transformant or an anti-GST antibody for the N-terminal transformants. ETR1 is expressed in wild-type Arabidopsis guard cells and is required for H2O2-induced closure. A, RT-PCR of RNA extracted from guard cells (lane 2) and whole leaves (lane 3). Lane 4, Genomic DNA positive control; lane 1, DNA marker (indicated in basepairs). For RNA extractions, frozen leaf material was blended in a Waring blender (3 × 15 s) in water (2.5 g: 20 mL containing 1.5% TRIzol reagent [Invitrogen, Paisley, UK]) and ice, and the homogenate filtered through a 100-μm mesh (Spectramesh, VWR Intern