Manganese superoxide dismutase 4 interacting with WRKY6 enhances cadmium tolerance in potato plants

We found that the overexpression of the OE-StWRKY6 lines is implicated in the cadmium (Cd) stress response, with co-expressed genes predominantly concentrated in the cellular oxidative stress pathway. As a member of the WRKY III group, StWRKY6 enhances the production of antioxidants in the leaves, thereby improving photosynthesis and carbon metabolism to mitigate the effects of Cd exposure. However, the specific interacting targets and functions of StWRKY6 remain unclear. We hypothesized that the three interacting proteins of StWRKY6 could be developed into biological agents to enhance Cd tolerance in potatoes. To reduce costs, we proposed to truncate the protein, retaining only the core domain, and verify whether it maintains the Cd tolerance function. In this study, based on pull-down results, we constructed recombinant plasmids for the StWRKY6 interacting proteins StMSD4 (manganese superoxide dismutase), StHBP2 (heme-binding protein), and StGTPR11 (GTP-binding nuclear protein). These plasmids were introduced into the Cd-sensitive yeast strain Δycf1 for further functional validation of Cd tolerance. Concurrently, core domain truncation, subcellular localization, transient expression, and other experiments were conducted to detect the related functions of the target protein in reactive oxygen species (ROS) oxidative stress. The results indicated that the OD₆₀₀ value of the Δycf1-OEStMSD4 strain was the highest when subjected to 70 µM Cd treatment. In contrast, the overexpression of StHBP2 and StGTPR11 in the Δycf1 strain demonstrated limited tolerance to Cd. Leaf transient expression of StMSD4 significantly increased ROS-related enzyme activity and decreased malondialdehyde (MDA) content. The subcellular localization of StMSD4 in mitochondria enhances Cd tolerance in leaves through the antioxidative stress system. Unfortunately, the Cd resistance function is lost after the deletion of the core domain of the target protein, indicating that the core domain's Cd resistance warrants further study. The interaction protein StMSD4 of StWRKY6 enhances Cd tolerance in potato leaves through the antioxidant stress system in mitochondria. This finding provides a new theoretical basis for understanding the mechanisms of Cd tolerance in plants.