H₂O₂-Responsive Hormonal Status Involves Oxidative Burst Signaling and Proline Metabolism in Rapeseed Leaves

Drought alters the level of endogenous reactive oxygen species (ROS) and hormonal status, which are both involved in the regulation of stress responses. To investigate the interplay between ROS and hormones in proline metabolism, rapeseed (Brassica napus L.) plants were exposed to drought or exogenous H₂O₂ (Exo-H₂O₂) treatment for 10 days. During the first 5 days, the enhanced H₂O₂ concentrations in drought treatment were associated with the activation of superoxide dismutase (SOD) and NADPH oxidase, with enhanced ABA and SA levels, while that in Exo-H₂O₂ treatment was mainly associated with SA-responsive POX. During the latter 5 days, ABA-dependent ROS accumulation was predominant with an upregulated oxidative signal-inducible gene (OXI1) and MAPK6, leading to the activation of ABA synthesis and the signaling genes (NCED3 and MYC2). During the first 5 days, the enhanced levels of P5C and proline were concomitant with SA-dependent NDR1-mediated signaling in both drought and Exo-H₂O₂ treatments. In the latter 5 days of drought treatment, a distinct enhancement in P5CR and ProDH expression led to higher proline accumulation compared to Exo-H₂O₂ treatment. These results indicate that SA-mediated P5C synthesis is highly activated under lower endogenous H₂O₂ levels, and ABA-mediated OXI1-dependent proline accumulation mainly occurs with an increasing ROS level, leading to ProDH activation as a hypersensitive response to ROS and proline overproduction under severe stress.