<i>CYTOKININ-RESPONSIVE GATA FACTOR 1</i> negatively regulates ascorbic acid biosynthesis in non-heading Chinese cabbage

Abstract Ascorbic acid (AsA), an essential nutrient for human health predominantly obtained from fresh vegetables and fruits, demonstrates considerable genetic complexity in its accumulation mechanisms. This study investigates the genetic regulation of AsA biosynthesis in non-heading Chinese cabbage (NHCC), a crucial leafy vegetable in China. Through comprehensive quantitative trait locus (QTL) analysis across multiple environments, we identified 19 AsA-associated QTLs distributed over ten linkage groups, explaining 3.15-18.04% of phenotypic variance. An environmentally stable QTL (qAsA.A01.1) was prioritized for further investigation. Integrated QTL mapping and comparative transcriptome analysis revealed 297 candidate genes, among which the GATA transcription factor CYTOKININ-RESPONSIVE GATA FACTOR 1 (BcCGA1) emerged as a candidate regulator through allelic variation analysis, gene description analysis, and association analysis. Functional validation via gene silencing and overexpression confirmed the suppressive role of BcCGA1 in AsA biosynthesis. Mechanistic studies established that BcCGA1 plays a direct transcriptional repressor of AsA biosynthesis by binding to the promoters of GDP-L-GALACTOSE PHOSPHORYLASE c (BcGGP.c), GDP-MANNOSE PYROPHOSPHORYLASE b (BcGMP.b), and KONJAC c (BcKJC.c). Furthermore, we identified an interaction between BcCGA1 and the chloroplast-localized protein PS II OXYGEN-EVOLVING COMPLEX 1 (BcPSBO1), which exhibits dual regulatory effects. While BcPSBO1 attenuated BcCGA1's DNA-binding capacity in vitro, it paradoxically enhanced transcriptional repression of target genes in vivo. Our findings elucidate a sophisticated regulatory framework involving transcriptional and protein interaction mechanisms that substantially advances our understanding of AsA accumulation genetics and provides a theoretical basis for nutrient-enhanced NHCC cultivars.