Precision engineering of upstream open reading frames for translational regulation and crop improvement.

Upstream open reading frames (uORFs) are pervasive cis-regulatory elements in the 5'untranslated regions (UTRs) of eukaryotic mRNAs that modulate translation of downstream main open reading frames (mORFs) by shaping ribosome scanning, initiation, and reinitiation. By operating downstream of transcription, uORFs enable fine-scale control of protein abundance, offering a distinctive regulatory layer for precise tuning of gene expression in plants. Recent advances in ribosome profiling, translation initiation-site mapping, peptidomics, computational annotation, and genome editing have expanded the discovery, functional validation, and targeted manipulation of plant uORFs. In this review, we summarize current understanding of uORF-mediated translational regulation, discuss computational and experimental approaches for candidate identification and functional validation, and highlight genome-editing strategies used to modify endogenous uORFs in crops. We further synthesize representative studies in which uORF editing has been used to modulate metabolic traits, plant development, nutritional quality, and responses to biotic and abiotic stresses. Collectively, available evidence suggests that uORF manipulation can support quantitative and context-dependent trait modulation, in some cases preserving basal gene function and reducing growth fitness penalties. We further discuss outstanding challenges, including transcript isoform complexity, context-dependent translational effects, and the need for validation across diverse genetic and environmental backgrounds. Overall, uORF engineering represents a promising strategy for translational fine-tuning of plant gene expression, with potential implications for crop improvement and sustainable agriculture.