G195R mutation disrupts AtSWEET1 structure and impairs sugar uptake in Arabidopsis

Nutrient availability, particularly the carbon (C) to nitrogen (N) ratio, is crucial for regulating plant metabolism, though coordination mechanisms remain unclear. To identify regulators, we screened an Arabidopsis thaliana mutant library under high C/N conditions, where wild-type cotyledons turn reddish-purple due to anthocyanin accumulation. We isolated a mutant that retained green cotyledons, indicating a disrupted C/N response. Genetic analysis revealed a G1091A substitution in the AtSWEET1 gene, resulting in a G195R change. This mutation impairs the AtSWEET1 protein's three-dimensional structure and significantly compromises its ability to transport exogenous sugar from the medium into the plant tissues. The green cotyledon phenotype was fully suppressed by expressing the wild-type AtSWEET1 but not AtSWEET1^G195R. Crucially, only the loss-of-function of AtSWEET1, and no other AtSWEET family members, specifically affected this sugar uptake. Our results demonstrate that G195 is a key residue required for the AtSWEET1 protein's function in intracellular sugar transport, which aids in advancing our understanding of SWEET protein family function.