Plasma membrane protein OsMCA1 is involved in regulation of hypo-osmotic shock-induced Ca(2+ )influx and modulates generation of reactive oxygen species in cultured rice cells

BACKGROUND: Mechanosensing and its downstream responses are speculated to involve sensory complexes containing Ca(2+)-permeable mechanosensitive channels. On recognizing osmotic signals, plant cells initiate activation of a widespread signal transduction network that induces second messengers and triggers inducible defense responses. Characteristic early signaling events include Ca(2+ )influx, protein phosphorylation and generation of reactive oxygen species (ROS). Pharmacological analyses show Ca(2+ )influx mediated by mechanosensitive Ca(2+ )channels to influence induction of osmotic signals, including ROS generation. However, molecular bases and regulatory mechanisms for early osmotic signaling events remain poorly elucidated. RESULTS: We here identified and investigated OsMCA1, the sole rice homolog of putative Ca(2+)-permeable mechanosensitive channels in Arabidopsis (MCAs). OsMCA1 was specifically localized at the plasma membrane. A promoter-reporter assay suggested that OsMCA1 mRNA is widely expressed in seed embryos, proximal and apical regions of shoots, and mesophyll cells of leaves and roots in rice. Ca(2+ )uptake was enhanced in OsMCA1-overexpressing suspension-cultured cells, suggesting that OsMCA1 is involved in Ca(2+ )influx across the plasma membrane. Hypo-osmotic shock-induced ROS generation mediated by NADPH oxidases was also enhanced in OsMCA1-overexpressing cells. We also generated and characterized OsMCA1-RNAi transgenic plants and cultured cells; OsMCA1-suppressed plants showed retarded growth and shortened rachises, while OsMCA1-suppressed cells carrying Ca(2+)-sensitive photoprotein aequorin showed partially impaired changes in cytosolic free Ca(2+ )concentration ([Ca(2+)](cyt)) induced by hypo-osmotic shock and trinitrophenol, an activator of mechanosensitive channels. CONCLUSIONS: We have identified a sole MCA ortholog in the rice genome and developed both overexpression and suppression lines. Analyses of cultured cells with altered levels of this putative Ca(2+)-permeable mechanosensitive channel indicate that OsMCA1 is involved in regulation of plasma membrane Ca(2+ )influx and ROS generation induced by hypo-osmotic stress in cultured rice cells. These findings shed light on our understanding of mechanical sensing pathways.