3.2 Overexpression of MdASMT9 stimulated ROS scavenging under heat stress
High levels of ROS, such as H2O2 and O2, can damage macromolecules and membrane lipid structures, leading to oxidative damage with deleterious effects on plants (Mittler et al. , 2011). Therefore, we stained the leaves with 3,3’-diaminobenzidine (DAB) and nitro blue tetrazolium (NBT) separately to detect H2O2 and O2–, accumulation, respectively. Results showed that heat stress caused ROS accumulation in all genotypes; however,MdASMT9 -OE lines exhibited fewer brown areas and blue spots on their leaves compared to the WT plants (Figure 2A). Quantitative measurements of H2O2 and O2– were consistent with these observations, showing that after 8h of heat treatment, the MdASMT9 -OE lines had lower ROS levels than the WT plants (Figure 2B and 2C). Moreover, activities of antioxidant enzymes (SOD, CAT, and POD) were quantified. Exposure to 48°C for 8h increased SOD, POD, and CAT activities, with OE-3 and OE-4 exhibiting a greater increase than WT plants (Figure 2D-F). These findings indicate that MdASMT9 -OE apple plants showed less oxidative damage than WT plants when exposed to high-temperature stress.