Galactinol synthase 4 requires sulfur assimilation pathway to provide tolerance towards arsenic stress under limiting sulphur condition in Arabidopsis

Heavy metalloid stress such as arsenic (As) toxicity and nutrient imbalance constitute a significant threat to plant productivity and development. Plants produce sulfur (S)-rich molecules like glutathione (GSH) to detoxify arsenic, but sulfur deficiency worsens its impact. Previous research identified Arabidopsis thaliana ecotypes Koz2-2 (tolerant) and Ri-0 (sensitive) under low-sulfur (LS) and As(III) stress. Transcriptomic analysis of the contrasting ecotypes revealed that AtGolS4 was highly induced in Koz2-2, suggesting its possible role in LS+As(III) stress response. In this study, AtGolS4 overexpressing lines (AtGolS4OX) in Col-0, AtGolS4 mutant (atgols4), and Ri-0 backgrounds showed lesser root growth reduction under LS+As(III) stress, with lower free sulfate accumulation and higher GSH levels, indicating the possible role of AtGolS4 in regulating antioxidant production to reduce oxidative stress generated due to As(III) stress. Overexpression of AtGolS4 in the AtSULTR1;2 mutant (atsultr1:1^CR, developed through CRISPR/Cas9 approach) background resulted in a sensitive phenotype, suggesting sulfate availability limits sulfur compound production. AtGolS4 promoter analysis revealed absence of sulfur-responsive elements (SURE) but identified MYC2 binding sites, and experiments showed that AtMYC2 likely regulates AtGolS4. Overall, this study highlights AtGolS4 as a key gene for enhancing tolerance to LS and As(III) stress, by increasing the antioxidant capability and sulphate assimilation.