| Brassinosteroids (BRs) are a group of phytohormones that regulate various biological processes in plants. However, up to date, whether and how they control stomatal movement is not fully clear. Here, Arabidopsis(Arabidopsis thaliana) wild type and relevant mutants were used as materials, and the roles of G protein a subunit (Ga), H2O2 and NO and the relationship between them during the BR inhibition of stomatal opening were investigated by means of stomatal bioassay and using laser-scanning confocal microscopy. The results were as follows:1.0.1-5 μM of epibrassinolide (EBR), a bioactive BR, inhibited stomatal opening, the optimum EBR concentration was 0.5 μM.2.0.5 μM EBR inhibited stomatal opening in wild type (Ws), the effect was prevented in wild type (Ws) treated with pertussis toxin (PTX), an inhibitor of Ga, or abolished in gpα1-1 and gpα12 mutants. These data indicates that Ga mediates the inhibitory effect of BR on stomatal opening.3. Ascorbic acid (ASA), an important reducing substrate for H2O2 removal, catalase (CAT), a H2O2 scavenger enzyme, diphenylene iodonium (DPI), an inhibitor of NADPH oxidase, prevented the inhibitory effect of EBR on stomatal opening in leaves of wild type (Col-0) and EBR-induced H2O2 production in guard cells of wild type (Col-0). Similarly, the inhibitory effect of EBR on stomatal opening and its inductive effect on H2O2 production were abolished in mutants AtrbohF and AtrbohD/AtrbohF, but were not in AtrbohD. These data provides evidence that AtrbohF-dependent H2O2 production is involved in BR inbition of stomatal opening.4. c-PTIO, a NO scavenger and tungstate, an inhibitor of nitrate reductase, prevented the inhibitory effect of EBR on stomatal opening in leaves of wild type (Col-0) and EBR-induced NO synthesis in guard cells of wild type (Col-0). Likewise, EBR failed to inhibite stomatal opening and to induce NO synthesis in mutants Nial-2 and Nial-2/Nia2-5, but it did these in Nia2-1. These results support that NO sourced from Nial is an important intermediate signal in BR inbition of stomatal opening.5. CTX not only inhibited stomatal opening in the leaves of wild-type (Ws), but also induced H2O2 production in guard cells of wild-type (Ws). However, these effects were prevented by ASA, CAT and DPI. EBR failed to induce H2O2 production in guard cells of gpα1-1 and gpα1-2 mutants, which is consistent with the inability of EBR to inhibit stomatal opening in these mutants. H2O2 inhibited stomatal opening in mutants gpα1-1 and gpα1-2, but CTX failed to induce H2O2 production, and to inhibit stomatal opening, in mutants AtrbohF and AtrbohD/F. These data indicate that Ga mediates the inhibitory effect of BR on stomatal opening via inducing AtrbohF-dependent H2O2 production.6. CTX not only inhibited stomatal opening in the leaves of wild-type (Ws), but also induced NO synthesis in guard cells of wild-type (Ws), these effects of CTX were abolished by c-PTIO and Na2WO4. EBR did not induce NO synthesis in guard cells of gpα1-1 and gpα1-2 mutants, which is similar to the effect of EBR on stomatal opening in these mutants. SNP inhibited stomatal opening in mutants gpα1-1 and gpal-2, but CTX neither induce NO synthesis nor inhibit stomatal opening in mutants Nial-2 and Niα1-2/Nia2-5. These results show that Ga mediates BR inhibition of stomatal opening via activating Nial-catalyzed NO synthesis.7. H2O2 failed to restore the defect of Niα1-2 and Nial-2/Nia2-5, but SNP rescued the lesion of AtrbohF and AtrbohD/F, in EBR inhibition of stomatal opening. EBR induced H2O2 production in guard cells of Niα1-2 and Niα1-2/Nia2-5, but did not stimulate NO synthesis in guard cells of AtrbohF and AtrbohD/F. These data suggest that H2O2 mediates the inhibitory effect of BR on stomatal opening via triggering NO synthesis.To sum up, our results suggest that BR triggers a signaling pathway involving activation of Ga, H2O2 generation, and NO synthesis in the inhibition of stomatal opening. |
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