Crosstalk among nitric oxide, calcium and reactive oxygen species during triterpenoid biosynthesis in Betula platyphylla.

We analysed NO, reactive oxygen species (ROS) and Ca2+ crosstalk during triterpenoid biosynthesis in white birch (Betula platyphylla Suk.) cells. Cells were pretreated with diphenyleneiodonium, sodium diethyldithiocarbamate (DDTC) or catalase (CAT), or a Ca2+ channel blocker or chelator before sodium nitroprusside treatment. Changes in triterpenoid, malondialdehyde and proline levels, cell viability, and CAT, ascorbate peroxidase and peroxidase activity were recorded. Furthermore, enzyme gene expression levels related to triterpene biosynthesis, endogenous signalling and antioxidase activity, and cell apoptosis and death rates were measured. Sodium nitroprusside elevated ROS and Ca2+ levels. Oleanolic acid levels in cells pretreated with diphenyleneiodonium and CAT reduced significantly, but it increased with DDTC pretreatment. ROS inhibition downregulated BpDXR, BpCALM and BpNIA expression. Oleanolic acid, BpMnSOD expression, and CAT, ascorbate peroxidase and peroxidase activities reduced when the Ca2+ signalling pathway was blocked. The apoptosis rates of cells pretreated with DDTC and CAT decreased significantly; cell death rates also reduced in groups Ca2+ pretreated with channel blocker and chelator . Thus ROS and Ca2+ participate in triterpenoid biosynthesis, cell apoptosis and death induced by exogenous NO application. Further, NO causes oxidative stress and restricts the level of intracellular ROS through the Ca2+ signalling pathway.