Studies On Role And Relationship Of Hydrogen Peroxide And Nitric Oxide In Light/Dark-and Phytohormone-regulated Stomatal Movement Of Vicia Faba

There is now compelling evidence that hydrogen peroxide (H₂O₂) and nitric oxide (NO) function as signalling molecules in the world of plants. In the last decade H₂O₂ and NO were proved to participate in many key plant physiological processes such as growth and development, pathogen defense reaction, programmed cell death (PCD), and stress tolerance. Though previous studies suggest that both H₂O₂ and NO can effect stomatal movement, the role of H₂O₂ and NO in light/dark-, cytokinin- and auxin-regulated stomatal movement has not been investigated, and the interrelationship between H₂O₂ and NO in light/dark-regulated stomatal movement was unknown. In this study, the abaxial epidermis of Vicia faba was used as materials, and role and relationship of H₂O₂ and NO in light/dark-regulated stomatal movement were investigated by epidermal strip bioassay and laserscanning confocal microscopy (LSCM). Additionally, the role of H₂O₂ and NO in cytokinin- and auxin-regulated stomatal movement was studied. The results were as follows:1. Both H₂O₂ and SNP, when applied exogenously in light, induce stomatal closure, and the effects are in a dose- and time-dependent manner. In darkness, the effects of SNP and H₂O₂ on stomatal closure are very unconspicuous. However, the dark-induced stomata closure can be reversed by cPTIO, l-NAME, ASA, CAT and DPI, which have no significant affect on stomatal aperture in light. So It's speculated that the levels of endogenous H₂O₂ and NO in light are different from that in darkness. Concretely, the levels of H₂O₂ and NO in light are low, but in dark that is high. Moreover, comparative high levels of endogenous H₂O₂ and NO are required for full stomatal closure induced by darkness. By means of LSCM based on H₂DCF-DA and DAF-2 DA, the specific molecular probes of H₂O₂ and NO respectively, the work provide evidence that in darkness the endogenous H₂O₂ fluorescence is very striking as well as NO fluorescence over the light. ASA, CAT and DPI can suppress dark-induced H₂DCF-DA fluorescence. Analogously, both cPTIO and l-NAME canprevent dark-induced DAF-2 DA fluorescence. These results support clearly that darkness can induce H₂O₂ and NO generation, and the levels of H₂O₂ and NO in darkness is affirmatively higher than that in light, which are consistent with the results of stomatal bioassays. Furthermore, both DPI and l-NAME, inhibitor of the H₂O₂-generating enzyme NADPH oxidase and arginine analogous NOS inhibitor respectively, can readily reverse stomatal closure and H₂O₂ and NO generation induced by darkness, the studies presume that H₂O₂ and NO generation in guard cells of Viciafaba is likely related to NADPH oxidase and NOS-like enzyme, respectively. 2. Guard cells generated NO in response to H₂O₂ in light, exogenous H₂O₂ increased the level of NO in guard cells, and such NO production was required for full stomatal closure in response to H₂O₂. Both cPTIO and L-NAME reversed largely exogenous H2_O2-induced stomatal closure in light, and exogenous H₂O₂-induced DAF-2 DA fluorescence in guard cells was substantially prevented by cPTIO or L-NAME. Otherwise, the reversibility of darkness-induced NO generation by CAT and DPI indicates that accumulation of endogenous H₂O₂ is also related to NO generation induced by darkness. Interestingly, SNP could induce H₂O₂ accumulation in guard cells under the light as well as NO generation induced by H₂O₂. ASA, CAT and DPI abolished largely stomatal closure induced by SNP, and SNP did not induce H₂O₂ fluorescence increase in the presence of ASA and CAT. In addition, cPTIO or t-NAME could reverse H₂O₂ generation by darkness implying that accumulation of endogenous NO is benefit for H₂O₂ generation induced by darkness. Results of this present study clearly indicate a causal and interdependent relationship between NO and H₂O₂ in light/dark-regulated stomatal movement of V. faba guard cells, and in the process, this cross talking of NO and H₂O₂ may lead to the information of a self-amplification loop about them. Additionally, L-NAME can readily reverse stomatal closure and NO generation induced by H₂O₂, the results support that H₂O₂ induce NO production mediated probably by the action of NOS-like enzyme in V. faba guard cells.3. Within an appropriate concentration of 0-0.6μmol/L 6-BA and KT, or 0-10μmol/L IAA and NAA they could induce stomatal opening in darkness in a dose-dependent manner. In light both auxins and cytokinins reversed effectively stomata closure by exogenous H₂O₂ and SNP(p<0.01). By LSCM it was proved that darkness-induced generation of H₂O₂ and NO in guard cells could be abolished...