Hydrogen peroxide mediates the expression of ascorbate-related genes in response to methanol stimulation in Oncidium

We investigated the signaling role of hydrogen peroxide (H2O2) in regulating the ascorbate (AsA) level after exogenous methanol (MeOH) application. The endogenous H2O2 and AsA levels as well as the expression of related genes were monitored after MeOH treatment of cultures of Oncidium protocorm-like bodies (PLB). A high MeOH concentration was deleterious and caused irreversible consumption of endogenous AsA. However, a low MeOH concentration (50 mM) triggered the synthesis of H2O2 and was effective in enhancing the expression of AsA-biosynthetic genes of the Smirnoff–Wheeler and galacturonate (GalUA) pathways. The increased expression of these genes could be blocked by the addition of hydroxylamine, an inhibitor of alcohol oxidase (EC: 1.1.3.13), and diphenyleneiodonium chloride (DPI), an inhibitor of NADPH oxidase (EC: 1.6.3.1). Thus, the H2O2 generated by MeOH application is a product of MeOH detoxification through alcohol oxidase and NADPH oxidase activation. In this chain, H2O2 acts as a secondary messenger for the activation of AsA-related genes. Our results reveal the signaling function of H2O2 and cellular AsA homeostasis in Oncidium orchids in response to MeOH stimulation. A mechanism for the MeOH effect on AsA production is suggested.