Transcription start site aware catalogue of microRNA encoded peptides in tomato (Solanum lycopersicum)

Plant microRNAs (miRNAs) are key regulators of gene expression, and primary miRNA transcripts (pri-miRNAs) can encode short regulatory peptides known as microRNA-encoded peptides (miPEPs), which enhance transcription of their associated miRNAs. However, comprehensive and accurately defined miPEP catalogues remain limited in crop species, including tomato ( Solanum lycopersicum ). Here, we generated a curated tomato miPEP catalogue using a transcription start site (TSS)-aware pipeline that integrates upstream sequence extension, promoter prediction, and stringent open reading frame (ORF) selection. This approach identified 107 candidate tomato miPEPs spanning diverse genomic contexts and peptide lengths, providing a systematic framework for miPEP annotation in plants. Physicochemical analyses showed that tomato miPEPs differ markedly from the tomato proteome, displaying higher isoelectric points, increased charge per residue, and depletion of acidic residues. Comparative analyses with published candidate miPEP sets from poplar and grapevine revealed the same general trend relative to their corresponding proteomes, indicating a recurrent shift toward basic, positively charged peptides. Cross-dataset comparisons further showed that normalized charge density is comparatively stable, whereas isoelectric point, acidic residue content, and especially peptide length vary more strongly among species and datasets. Structural prediction using AlphaFold2 indicated variable confidence across candidates, and transcriptomic analyses provided expression support for selected loci. Together, these results define a curated and biologically plausible set of small peptides and support a recurrent physicochemical profile for plant MIR-associated upstream peptides, consistent with potential interactions with negatively charged biomolecules and with the proposed roles in pri-miRNA regulation.