NO And H₂O₂ Are Involved In Ethylene Regulated Stomatal Movement Of Arabidopsis

Stomatal are specialized epidermal structures which regulate gas and water vapor exchange between plants and the environment. Stomatal closure and opening play an important role in regulating transpiration and photosynthesis. Despite the well-known association between ethylene (Eth) and other hormones in stomatal movements, the effects of ethylene alone on stomatal movements are not clear and there are no reports of the potential signalling pathways that might be regulated by ethylene in guard cells. In this study, the wild Arabidopsis thaliana, nitric oxide (NO) synthesis mutants, ETR1 mutants and polyamine oxidase (PAO) mutants were taken as the material, through plant physiology, cell biology and molecular biology approaches to monitor stomatal movement regulated by ethylene and detect the level of NO or hydrogen peroxide (H2O2), the activities of relative enzymes and the expression pattern of PAO. In order to confirm that ETR1, NO and H2O2 were involved in the signal transduction pathway of ethylene regulated stomatal movement.The results showed that treatment with ethephon (0.0004%,0.004%,0.04%,0.4%) resulted in a time-and dose-dependent stomatal closure under light. Ethylene was shown to enhance nitric oxide levels and, corresponding, nitrate reductase (NR) activity. The NO scavenger or the NR inhibitor reduced ethylene-induced stomatal closure and NO accumulation. Moreover, ethylene was able to induce NO generation and stomatal closure in Atnoal/rif1 plants, but failed to induce NO and stomatal closure in the NR-deficient mutant. It suggested that NO production was nitrate reductase-associated during ethylene induced stomatal closure in Arabidopsis thaliana.Treatment of 0,004% ethephon, the H2O2 level significantly increased in both leaves and guard cells. The H2O2 scavenger, NADPH oxidase and cell wall peroxidases inhibitor blocked ethylene-induced stomatal closure, supporting that both NADPH oxidase and cell wall peroxidase activities are implicated in ethylene-induced H2O2 production in Arabidopsis. Moreover, ethylene-induced NO accumulation and NR activity decreased when H2O2 was compromised, implying that nitrate reductase-derived NO may represent a novel downstream component of H2O2 signaling cascade during ethylene-induced stomatal movement in Arabidopsis thaliana.On the basis of biochemical and pharmacological experimental results, we can conclude that H2O2 was involved in the signal transduction pathway of ethylene induced stomatal closure. NADPH oxidase and cell wall peroxidases, two well known enzymes in H2O2 synthesis, might be responsible for the generation of H2O2 in ethylene-induced stomatal closure in Arabidopsis. Here, the role of PAO during ethylene-induced H2O2 production was investigated. The date clearly showed that ethylene in combined with PAO inhibitors was failed to induce H2O2 accumulation and stomatal closure in Arabidopsis thaliana, supporting the involvement of PAO in ethylene-induced H2O2 production. Quantitative PCR analysis showed that the AtPAO2 and AtPAO4 transcript were induced by ethylene in Arabidopsis thaliana. To further investigate function of AtPAO2 and AtPAO4 in ethylene-induced H2O2 accumulation, transgenic Arabidopsis thaliana were produced to overexpress AtPAO2 or AtPAO4 and T-DNA-inserted mutants were confirmed. Stomatal closure and H2O2 were significantly induced in these Arabidopsis thaliana transgenic lines with AtPAO2 or AtPAO4 expression and T-DNA-inserted mutants. But these transgenic lines exhibited enhanced induction as compared with wild-type, implying that AtPAO2 and AtPAO4 were involved in ethylene-induced H2O2 production, and AtPAO2 and AtPAO4 might be functional redundancy in ethylene-regulated stomatal movement.In order to prove the mechanism of the ethylene receptor ETR1 in ethylene-induced stomatal closure, the effects of ethephon on NO, H2O2 levels and stomatal aperture in wild-type or ETR1 mutants Arabidopsis were analyzed. The results showed that ethephon was failed to induce NO and H2O2 accumulation as well as stomatal closure in the ETR1 mutant, indicating that ETR1 involved in ethylene-induced stomatal closure.Based on these results, we propose a new signal route in Arabidopsis thaliana guard cells: Eth→ETR1→PAO→H2O2→NR→NO→stomatal closure. These discoveries provide some new evidence for mechanism of ethylene-induced stomatal closure, and important research values for the future study of stomatal movement was presented in this research.