This study showed that foliar spray of 2 % methanol significantly increased stomatal conductance, photosynthesis, and growth of broad bean (Vicia faba L.). To understand the molecular mechanisms behind the methanol-enhanced photosynthesis of broad bean, a forward suppression subtractive hybridization (SSH) cDNA library was constructed with 2 % methanol-sprayed leaves. A total of 145 methanol-induced genes were identified, and 63 of these had a known function. Clustering analysis indicated that the largest proportion (41.3 %) comprised genes associated with photosynthesis. RT-PCR analysis verified that the transcription of photosynthetic genes, including Rubisco and chlorophyll a/b-binding proteins of photosystem I and II, was upregulated in 2 % methanolsprayed leaves, which might contribute to the molecular mechanism of methanol-enhanced photosynthesis in broad bean. Further analysis suggested that the transcription and expression of 14-3-3 protein (present in the SSH library) and PM H⁺-ATPase encoding genes, PM H⁺-ATPase phosphorylation and its interactions with 14-3-3 proteins as well as stomatal aperture increased in leaves after 2 % methanol sprayed. Simultaneously, the leaf H₂O₂ content decreased in a time-dependent manner. Based on these results, we hypothesize that methanol spray induces the expression of 14-3-3 proteins and PM H⁺-ATPase and simultaneously reduces the H₂O₂ content in leaves. Consequently, the interaction between phosphorylated PM H⁺- ATPase and 14-3-3 proteins is enhanced, and PM H⁺- ATPase and H⁺ pump activities elevated; these functions may constitute the molecular mechanism through which methanol increases stomatal aperture and conductance in broad bean leaves.
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