Role of NADPH oxidase-derived reactive oxygen species in Melanoxylon brauna germination under heat stress

Abstract In plasma membranes, the generation of reactive oxygen species (ROS) is primarily mediated by the activation of NADPH oxidases (NOXs). This study investigated the role of NOX-derived ROS in the germination of Melanoxylon brauna under optimal (25 °C) and heat stress (40 °C) conditions. Seeds were treated with diphenyleneiodonium (DPI), a NOX inhibitor, and evaluated after 48 and 96 h for germination rate, germination speed index, internal anatomy, hydrogen peroxide (H 2 O 2 ) content, and the activity of amylases and antioxidant enzymes. Internal anatomical changes were examined by light microscopy, whereas H 2 O 2 levels and enzyme activities were determined spectrophotometrically. Under optimal conditions, DPI treatment reduced germination rate and speed by 27% and 35.4%, respectively, and hindered seed coat loosening. In addition, DPI lowered H 2 O 2 content and suppressed both amylase and antioxidant enzyme activities. Under heat stress, seeds failed to germinate regardless of treatment; however, ROS inhibition further decreased H 2 O 2 levels and reduced the activities of superoxide dismutase and ascorbate peroxidase. Collectively, these results indicate that in M. brauna , heat stress is the dominant factor impairing germination, and inhibition of NOX-derived ROS does not mitigate—and may even aggravate—the deleterious effects of elevated stress.