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 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 (95% guard cells, as assessed by FDA/DAPI staining; Hey et al., 1997) were then homogenized in TRIzol reagent (1 mL) with glass beads in a Fastprep bead beater (Fisher, Loughborough, UK) to break open the guard cells. RNA was extracted following the TRIzol RNA extraction procedure provided by the manufacturers. Reverse transcription was performed on DNAsed RNA, with PCR primers designed against sequences unique to ETR1; forward primer was ETR1F (5′-GTTTGTGAATCTGATGGAGGG-3′) and reverse primer was ETR1R (5′-GTTGTTTTGTGAATTTCTCG-3′). Genomic DNA was used as a control for the PCR to check that the RT products were from cDNA, and the PCR product subsequently sequenced. B, Western blot of guard cell proteins. Lane 1, Guard cell proteins; lane 2, yeast proteins. Guard cell-enriched epidermal fragments were prepared as above (but minus TRIzol) and the proteins extracted in extraction buffer (100 mm HEPES, pH 7.5, 5 mm EDTA, 5 mm EGTA, 10 mm DTT, 10 mm Na3VO4, 10 mm NaF, 50 mm α-glycerophosphate, 1 mm PMSF, 5 μg mL−1 aprotinin, and 5 μg mL−1 leupeptin) in the Fastprep bead beater, centrifuged for 2 × 20 min at 15,500g at 4°C, and the supernatant concentrated using Microcon (VWR International) spin columns. Yeast proteins were isolated from cells expressing full-length ETR1, as described (Rodriguez et al., 1999). Proteins were prepared for SDS-PAGE by incubating at 37°C for 1 h in SDS buffer without DTT and electrophoresed on a 7.5% SDS-polyacrylamide gel. Western blotting was performed using an anti-ETR1 (C terminus) antibody (Insight Biotechnology) and detected using enhanced chemiluminescence (GE Healthcare, Bucks, UK). C, The loss-of-function etr1-7 mutant is insensitive to H2O2. Arabidopsis leaves were floated for 3 h under continuous illumination (200–250 μE m−2 s−1) in MES/KCl buffer (5 mm KCl/10 mm MES/50 μ m CaCl2, pH 6.15). Once the stomata were fully open, leaves were treated with H2O2 for a further 3 h. The leaves were subsequently homogenized individually in a Waring blender for 30 s and the epidermal fragments collected on a 100-μm nylon mesh (SpectraMesh). Stomatal apertures from epidermal fragments were then measured using a calibrated light microscope attached to an imaging system (Leica QWin software, Leica, Milton Keynes, UK). Stomatal closure response to H2O2 (100 μ m) in wild type (wt); etr1-7; etr1-7 complemented with full-length ETR1 (ETR1etr1-7); etr1-7 complemented with ETR1 truncated at 349 (ETR11-349); etr1-7 complemented with ETR1 containing a mutation in the G2 box of the HK domain (ETR1G2). White bars, control; black bars, H2O2. Data are expressed as mean ± se (n = 60 guard cells) from three independent experiments. The function of various ETR1 domains in guard cell-H2O2 signaling was then assessed by utilizing etr1-7 plants complemented with the HK inactive G2 mutant or a truncated ETR1 (1-349). The mutation in the G2 box of ETR1 results in expression of a protein containing the HK domain, but in which there is no HK activity, whereas the 1-349 mutation results in a truncated protein lacking the HK domain (Gamble et al., 2002). Stomata of both these mutants responded to H2O2 and stomatal closure resulted (Fig. 2), indicating that the N-terminal region of ETR1 is sufficient for this response, and that neither the presence nor function of the HK domain is required for H2O2-induced closure. This is unlike the situation for ethylene signaling, where the presence but not the function of the HK domain in ETR1 is essential for a response (Gamble et al., 2002). Cys-65 of ETR1 is required for H2O2-induced stomatal closure. Leaves of wild-type (○), etr1-7 (×), etr1-1 (▵), and etr1-3 (▿) plants were incubated in the light to induce stomatal opening, followed by exposure to H2O2 at the indicated concentrations, and stomatal apertures measured after 3 h. The data were obtained from four to five independent experiments (n = 100 guard cells per data point). The raw data were analyzed by model selection using Generalized Linear modeling with a Gamma response and inverse link, and the calculated apertures and error bars are shown. In summary, our data demonstrate an unexpected role for ETR1, that of mediating stomatal closure in response to H2O2. Until now, ETR1 has been associated solely with ethylene perception and signaling. Our discovery that ETR1 can, in fact, mediate cellular responses to two different signaling molecules, namely ethylene and H2O2, indicates multiple functions for a single protein, as suggested recently for other plant receptors and enzymes (Szekeres, 2003; Moore, 2004). Moreover, it is possible that ETR1 could act as a central node mediating cross-talk between ethylene and H2O2 signaling, although whether such shared responses occur in other cells in addition to guard cells remains to be determined. We thank E. Schaller (Dartmouth College, Hanover, NH) for the ETR1 full-length construct and for seeds of etr1-7 plants complemented with ETR1 constructs; T. Bleecker's laboratory (University of Wisconsin, Madison, WI) for the ETR1 and etr1-1 N-terminal region constructs; E. Meyerowitz (California Insitute of Technology, Pasadena, CA) for etr1-7 seeds; H. Saito (University of Tokyo, Tokyo) for the TM219 yeast mutant; and J. Gray and colleagues (University of Sheffield, Sheffield, UK) for advice on RNA and protein isolation from Arabidopsis guard cells.