AcWRKY70-AcAMR1 modulates ascorbic acid accumulation in kiwifruit

Kiwifruit (Actinidia species) are among the fruits rich in ascorbic acid (AsA), although the AsA content varies significantly across species and cultivars. However, the regulatory mechanisms underlying these differences in AsA content remain largely unexplored. Here, we cloned the AsA negative regulators AeAMR1 and AcAMR1 from Actinidia eriantha 'Maohua' and Actinidia chinensis 'Hongyang', respectively. AcAMR1 exhibited higher expression in 'Hongyang' compared to 'Maohua'. Overexpression of AcAMR1 significantly reduced AsA content by approximately 16.03% in fruits, while silencing AcAMR1 increased AsA content by approximately 14.52% in fruits. Comparative promoter analysis revealed pronounced divergence between the AeAMR1 and AcAMR1 promoters, including differences in multiple cis-elements (W-box, MYB, LTR, MYC, ARE, DRE1, AE-box, and G-box). Yeast one-hybrid (Y1H) screening identified AcWRKY70 as an upstream regulator of AcAMR1. EMSA confirmed that AcWRKY70 directly binds to the AcAMR1 promoter, while GUS and dual-luciferase assays demonstrated that AcWRKY70 represses its activity. Consistently, AcWRKY70 positively regulates AsA levels. Together, these results define an AcWRKY70-AcAMR1 regulatory module that contributes to AsA biosynthesis in kiwifruit, providing genetic targets for breeding AsA-rich cultivars and a mechanistic framework for fruit quality improvement.