Computational prediction-combined proteogenomics unveils widespread non-AUG translation initiation events in plants.

Non-AUG translation initiation can generate N-terminally extended proteoforms, contributing to proteome complexity and regulatory diversity. While well characterized in mammals, its identification in plants remains limited, hindering both functional investigations and cross-species comparisons. Here, we applied a computational prediction-combined proteogenomic strategy to systematically explore non-AUG translation initiation events in the monocots maize and rice and the dicot soybean, identifying 879 transcripts potentially producing 3 938 N-terminally extended proteoforms. These events exhibited both conserved and lineage-specific mechanistic features, including stable RNA secondary structures flanking upstream translation initiation sites (uTISs), codon and sequence context preferences between monocot and dicot species, and a lack of evolutionary conservation. Plant N-terminal extensions were predicted to encode diverse targeting signals, implicating them in subcellular localization and functional diversification. Comparative analysis revealed both conserved trends and plant-specific features relative to humans. Collectively, this study provides a foundational resource and conceptual framework to advance understanding of plant non-AUG translation within a cross-kingdom evolutionary context. It also offers new opportunities to elucidate the roles of non-AUG translation in regulatory networks, proteome diversification, and adaptive biological functions across eukaryotic systems.