Abstract
GDP-L-galactose phosphorylase (GGP) is a key rate-limiting enzyme in the ascorbic acid synthesis pathway, and it plays a crucial role in regulating ascorbic acid metabolism and redox balance. The upstream open reading frame (uORF) in the 5' untranslated region of the GGP gene typically suppresses translation efficiency, reducing vitamin C content. In this study, GGP uORF in rice was identified, and seven GGP uORF allele variants AL1-AL7 were generated by CRISPR/Cas9 technology; then, the effects of these variations on AsA content and osmotic stress resistance were evaluated. The AsA content of the seven mutant plants was 1.80-3.08 times greater than that of the control, and the increase of AL4 mutant was the greatest. Protoplast assays confirmed that the OsGGP uORF inhibits downstream ORF translation. Osmotic stress experiments revealed that in the mutant lines, both the activities of enzymes involved in reactive oxygen species (ROS) scavenging and the proline (Pro) content were significantly increased, while the levels of peroxides were significantly decreased. These results demonstrate that mutation of the GGP uORF significantly increases translation efficiency, AsA content, and the ability of rice to reduce ROS levels, restore osmotic balance, and improve ROS scavenging capacity under osmotic stress. This study reports the regulated osmotic stress of the GGP uORF in monocotyledonous plants for the first time and has created a variety of allelic variation germplasm resources. It provides a novel approach for improving AsA content and tolerance to salt stress in rice and other monocots via genetic approaches.