Role of NADPH oxidase generated O2− in seed germination and cold stress response of Brassica napus

NADPH oxidase, a key enzyme responsible for reactive oxygen species (ROS) production in plants, plays a central role in regulating plant growth and development as well as the transmission of stress signals. However, the regulatory mechanism of ROS mediated by NADPH oxidase in Brassica napus remains unclear. In this study, B. napus 16NTS309 was used as the experimental material, and NADPH oxidase-specific inhibitor (diphenyleneiodonium chloride, DPI) was applied to treat seeds, seedlings, and callus tissues respectively. Combined with nitroblue tetrazolium (NBT) staining, tissue section observation, and superoxide anion (O 2 − ) content determination, the mechanism by which NADPH oxidase-mediated ROS (O 2 − ) regulates seed germination, cell division, and cold stress signal transmission in B. napus was systematically investigated. The results showed that: (1) DPI treatment completely inhibited seed germination (germination rate = 0%), while exogenous application of 0.6% H₂O₂ significantly alleviated this inhibitory effect. Specifically, the seed germination rate, ROS (O 2 − ) content and NADPH oxidase activity increased to 68.34%, 18.52 nmol/g and 423.01 U/g, respectively, which were significantly higher than those in the DPI treatment group. This confirms that NADPH oxidase-mediated ROS plays an important role in seed germination of B. napus ; (2) Under normal growth conditions, ROS (O 2 − ) mainly accumulated in the root tip meristem and the edge of the rapidly dividing region of callus tissues. After DPI treatment, the ROS (O₂⁻) signals in root tissues and callus cells were significantly reduced, indicating that NADPH oxidase-mediated ROS signals are involved in regulating the cell division process of B. napus ; (3) After cold stress treatment, ROS (O 2 − ) signals in B. napus seedlings and callus tissues burst significantly, with contents of 152.42 nmol/g and 512.61 nmol/g, respectively. In contrast, ROS (O 2 − ) levels and NADPH oxidase activity decreased significantly under the combined treatment of cold stress and DPI, confirming that the massive production of ROS under cold stress is closely associated with NADPH oxidase. This study clarifies the key role of NADPH oxidase-mediated ROS in seed germination, cell division, and cold stress response of B. napus , providing an important theoretical basis and technical support for the genetic improvement of cold-tolerant B. napus varieties and the in-depth study of plant ROS signal regulatory mechanisms.