Functional divergence and promoter variation of STAY-GREEN genes control chlorophyll accumulation in radish roots

Root color variation in radish (Raphanus sativus) is a significant agronomic trait, yet the molecular mechanisms underlying chlorophyll retention in green-fleshed roots remain to be elucidated. Here, we identify the STAY-GREEN homologs RsSGR1 and RsSGR2 as pivotal factors of green versus white root phenotypes through differential transcriptional regulation. White-rooted lines exhibited negligible chlorophyll content consistent with high transcript levels of RsSGR1 and RsSGR2, whereas green-rooted lines retained high level of chlorophyll with minimal expression of both genes. Although sequence analyses identified polymorphisms in the coding regions, all RsSGR1 and RsSGR2 alleles exhibited conserved structural features, chloroplast localization, and protein dimerization ability. Functional assays revealed organ-specific activity: RsSGR1 promoted stronger chlorophyll degradation than RsSGR2 in cotyledons and leaves, with RsSGR2 attenuating this activity upon co-expression, whereas RsSGR1 and RsSGR2 functioned redundantly in roots. Consistently, promoter activity assays revealed that the RsSGR1 and RsSGR2 alleles from white-rooted lines drove higher transcriptional activity than those from green-rooted lines. This differential activity correlated with specific variations in the distribution and frequency of cis-elements recognized by DNA-binding with one finger (Dof) transcription factors within the promoter regions. Collectively, our findings establish that allele-specific promoter architecture, rather than protein functional divergence, is a key molecular determinant governing chlorophyll catabolism and root color formation in radish.