GmSTOP1-3 regulates flavonoid synthesis to reduce ROS accumulation and enhance aluminum tolerance in soybean

Aluminum (Al) toxicity is a significant limiting factor for crop production in acid soils. The functions and regulatory mechanisms of transcription factor STOP1 (Sensitive to Proton Rhizotoxicity 1) family genes in Al-tolerance have been widely studied in many plant species, except for soybean. Here, expression of GmSTOP1-3 was significantly enhanced by Al stress in soybean roots. Overexpression of GmSTOP1-3 resulted in enhanced root elongation and decreased Al content, which was accompanied by increased antioxidant capacity under Al treatment. Furthermore, RNA-seq identified 498 downstream genes of GmSTOP1-3, including genes involved in flavonoid biosynthesis. Among them, the expression of chalcone synthase (GmCHS) and isoflavone synthase (GmIFS) were highly enhanced by GmSTOP1-3 overexpression. Further quantitative flavonoid metabolome analysis showed that overexpression of GmSTOP1-3 significantly increased the content of naringenin chalcone, naringenin, and genistein in soybean roots under Al treatment, which positively correlated with the expression level of the genes relative to flavonoid biosynthesis. Notably, genistein had a significant positive correlation with the expression levels of GmIFS. Combination of Dual Luciferase Complementation (LUC) and Electrophoretic Mobility Shift Assays (EMSA) revealed that GmSTOP1-3 directly bound to the promoters of GmCHS/GmIFS and activated both genes' transcription. Taken together, these results suggest that GmSTOP1-3 enhances soybean Al tolerance partially through regulating the flavonoid synthesis.