ChSCL9 negatively regulates citric acid accumulation via repressing <i>PH4</i> - <i>PH5</i> module in kumquat

Citric acid is the major organic acid affecting citrus fruit taste, which varies widely in the citrus family. An acidless kumquat, a small-fruited citrus species (Citrus crassifolia), has been developed and has become popular on the market. To investigate the molecular basis of the acidless phenotype in Huapi (HP) kumquat, it was crossed with early-flowering, high-acid Hong Kong (HK) kumquat to generate an F1 population, providing a system to dissect the genetic and molecular basis of citric acid accumulation. Organic acid content exhibited a wide and continuous distribution across three consecutive years, which is consistent with quantitative inheritance. Bulked segregant analysis sequencing mapped a major locus, and integration with transcriptomic data identified the GRAS transcription factor SCARECROW-like protein 9 (ChSCL9) as a candidate major-effect gene, showing high expression in HP fruit and low expression in HK fruit. Subcellular localization confirmed that ChSCL9 is a transcription factor. Functional analyses-including CRISPR-Cas9 gene editing, overexpression in kumquat, and RNA interference (RNAi) in citrus juice sacs-demonstrated that ChSCL9 negatively regulates citric acid accumulation. Biochemical experiments showed that ChSCL9 directly represses the expression of the R2R3-MYB gene ChPH4 and the vacuolar P-ATPase gene ChPH5, both of which are key genes for vacuolar acidification, thereby inhibiting citric acid accumulation. These results identify ChSCL9 as a key regulator of citric acid in kumquat, reveal an upstream regulator of PH4, and provide targets for citrus flavor improvement and rational design for citrus breeding.