Natural variation and uORF engineering complementary routes to improve ascorbic acid accumulation in tomato.

Ascorbic acid (AsA) determines fruit nutritional quality and antioxidant capacity in tomato, but available genetic resources for its improvement are limited. Here, we integrated natural variation analysis with upstream open reading frames (uORFs) editing to identify and engineer determinants of AsA accumulation. A natural variant in GGP2 was significantly associated with fruit AsA content, leading to the development of a diagnostic molecular marker for allele discrimination. Targeted editing of uORFs in 3 AsA biosynthetic genes, GGP1, GME2, and GMP3, enhanced translation of their main ORFs without altering transcript abundance, as confirmed by dual-luciferase assays and RT-qPCR. In both T1 and T2 generations, uORF-edited lines accumulated significantly higher levels of total and reduced AsA in fruits and leaves than wild type. The edited lines also showed improved tolerance to methyl viologen-induced oxidative stress, with reduced chlorosis, higher chlorophyll retention, lower reactive oxygen species accumulation and decreased malondialdehyde content. Notably, uORF editing caused only minimal transcriptional perturbation of the broader AsA pathway and no detectable penalties in plant growth, fruit development, or yield. Collectively, our results demonstrate that both natural allelic variation in GGP2 and engineered uORF editing of key biosynthetic genes can effectively enhance tomato AsA content, establishing uORF editing as a promising strategy for nutritional quality improvement with few agronomic trade-offs.