Actinidia eriantha, a kiwifruit species endemic to China, produces fruits with notable nutritional, medicinal, and economic value, particularly due to its high L-ascorbic acid (L-AsA) content. However, the regulatory mechanisms underlying L-AsA accumulation in its fruit remain poorly understood. This study meticulously measured L-AsA levels of fruits in 216 A. eriantha accessions from natural populations and performed a genome-wide association study, through which we identified significantly associated lead single nucleotide polymorphisms and insertion deletions, and characterized a key candidate gene AebHLH89, AePPR, AePP2Ab, and AePHL1 involved in the positive regulation of L-AsA accumulation. Functional experiments showed that overexpression of AebHLH89 significantly enhanced L-AsA accumulation, while its silencing via virus-induced gene silencing markedly decreased L-AsA levels. Yeast one-hybrid assay and dual-luciferase assay preliminary revealed that AebHLH89 could bind to the AeGMP1 promoter and activates its transcription, thereby upregulating the L-AsA biosynthesis pathway and promoting L-AsA synthesis and accumulation. These findings provide valuable genetic resources for molecular marker-assisted breeding in kiwifruit and contribute to germplasm innovation. Simultaneously, the identification of key regulatory genes enhances our understanding of L-AsA metabolism and lays a theoretical foundation for the genetic improvement of kiwifruit.

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