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

We investigated the signaling role of hydrogen peroxide (H(2)O(2)) in regulating the ascorbate (AsA) level after exogenous methanol (MeOH) application. The endogenous H(2)O(2) 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 (50mM) triggered the synthesis of H(2)O(2) 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 H(2)O(2) generated by MeOH application is a product of MeOH detoxification through alcohol oxidase and NADPH oxidase activation. In this chain, H(2)O(2) acts as a secondary messenger for the activation of AsA-related genes. Our results reveal the signaling function of H(2)O(2) and cellular AsA homeostasis in Oncidium orchids in response to MeOH stimulation. A mechanism for the MeOH effect on AsA production is suggested.