An investigation of the dual mechanism of uORF-mediated regulation of GGP1 expression and the effects on drought stress in cotton.

Upstream open reading frames (uORFs) are intrinsic regulatory elements that fine-tune gene expression at the translational level without altering genomic sequences or protein structures. Here, we targeted the conserved uORF that resides in the 5'-untranslated region (5'-UTR) of a GDP-galactose phosphorylase gene (GhGGP1) in cotton (Gossypium hirsutum). Regulation of GGP1 is a key control point of plant L-ascorbic acid biosynthesis. Using a dual-luciferase reporter system, we demonstrate that this uORF represses translation of the downstream GGP1 coding sequence in a feedback manner in response to elevated ascorbate, confirming previous findings in other species. Furthermore, CRISPR/Cas9-mediated disruption of the uORFGhGGP1 generated homozygous uorfGhGGP1 mutants with unchanged transcript levels but markedly increased protein abundance and L-ascorbic acid accumulation. Under drought stress, these mutants exhibit reduced malondialdehyde levels, enhanced total antioxidant capacity, and improved drought tolerance. Mechanistically, we further show that under drought conditions, the transcription factor GhBBM directly binds to the uORF-containing cis-regulatory element, enhancing (GhGGP1) transcription and drought resistance. Together, our findings establish the uORFGhGGP1 as a dual-layer regulatory module that integrates transcriptional activation by GhBBM with translational control of the downstream protein encoding open reading frame, thereby fine-tuning drought stress responses in cotton.