Engineering uORFs in wheat improves abiotic stress tolerance without growth penalties

Developing climate-resilient crops is critical for ensuring global food security, yet agricultural productivity is frequently constrained by the trade-off between stress tolerance and growth. This challenge is particularly acute in allohexaploid wheat, a staple crop susceptible to abiotic stresses, underscoring the demand for precise gene regulation. Here, we established a simple, efficient, and transgene-free method for regulating gene expression in allohexaploid wheat by disrupting or creating de novo upstream open reading frames (uORFs) via genome editing. Using ribosome profiling, we identified 1199 actively translated uORFs across the wheat genome and developed a dual-luciferase screening pipeline to pinpoint potent translational repressors of positive regulators of abiotic stress. CRISPR/Cas9-mediated disruption of the uORFs in TaCIPK14 and TaGGP1 elevated downstream protein accumulation, thereby enhancing drought, cold, and salt tolerance of plants. Furthermore, base editing was employed to generate de novo uORFs in TaMADS26, effectively downregulating gene expression and improving drought resilience. Notably, both the transgene-free uorf_TaGGP1-ACG and uORF_TaMADS26 mutants conferred pronounced yield advantages under drought conditions without incurring growth penalties under well-watered conditions. This study provides a predictable and precise way for modulating gene expression, enabling the coordinated improvement of stress resilience and agronomic performance in wheat.