Hydrogen peroxide induces a rapid production of nitric oxide in mung bean (Phaseolus aureus)

Nitric oxide (NO) has recently been identified as an important signaling molecule in plant immune response. The present study aims to investigate the signaling pathway that leads to NO production. Using the NO specific fluorescent dye DAF-2DA, we observed rapid production of NO in mung bean leaves after the addition of 10 mM hydrogen peroxide (H2O2). NO was probably produced by a NOS-like enzyme in plants, as the NO production was inhibited by L-NAME, a NOS inhibitor. The NOS-like activity in the total leaf protein preparation of mung bean (Phaseolus aureus) was elevated 8.3-fold after 10 mM H2O2treatment, as demonstrated using the chemiluminescence NOS assay. The NOS-like activity was BH4dependent: omitting BH4in the reaction mixture of NOS assay reduced the NOS activity by 76%. We also found that the H2O2induced NO production was mediated via calcium ion flux, as it was blocked in the presence of a calcium ion channel blocker, verapamil. Results from the present study identified H2O2as an upstream signal that leads to NO production in plants. H2O2and NO, besides acting as two independent signaling molecules in plant immune response, may interrelate to form an oxidative cell death (OCD) cycle.