Natural and CRISPR /Cas9 Editing Variations of Dual‐ uORFs Synergistically Increase the Nutritional Value of Silage Maize Feed

Abstract

The precise enhancement of nutritional quality in silage maize is a core strategy for increasing livestock production efficiency. Through evolutionary analysis of multiple plant species, we identified two functionally synergistic upstream open reading frames (uORF1 and uORF2) within the 5' untranslated region of the gene encoding GDP-L-galactose phosphorylase (GGP). By leveraging a natural translation-enhancing haplotype of uORF1 (Hap2) and performing CRISPR/Cas9-mediated targeted mutagenesis of the highly conserved uORF2, we successfully engineered an elite dual-uORFs variant. This variant significantly increased vitamin C (Vc) content and concurrently improved key silage quality traits, including crude protein and phosphorus levels, without inducing growth penalties. Transcriptomic profiling further elucidated the molecular mechanisms by which the dual-uORFs variation coordinately regulates Vc biosynthesis and the improvement of silage quality. Our findings deepen the understanding of the conventional paradigm of single-uORF regulation and provide a novel strategy and superior germplasm resources for the precision breeding of high-Vc, high-quality silage maize.