Impact of alternative splicing on Arabidopsis proteome

Limited proteomic evidence makes it unclear to what extent alternative splicing (AS) isoforms are translated and functionally relevant in eukaryotes. Here, we present a comprehensive proteomic analysis in plants using large-scale data mining, extensive fractionation of AspN-and trypsin-digested proteomes, and both label-free and TMT labeling. In total, we identified 471,196 peptides from 22,479 proteins by searching against Araport11, revealing 32,110 isoform-specific peptides. Using an integrated proteogenomic workflow coupled with SUPPA, we classified these peptides into 2,442 AS events, 879 of which involved intron retention (IR). Further analysis of unannotated events revealed 91 additional IRs that are translated, confirming that retained introns can give rise to peptides. AlphaFold modeling predicted the structural and functional impacts of these isoforms. Our dataset improved existing gene model annotations. By comparing wild-type plants with the AS mutant acinus pinin, we found that IR regulates transcript and protein abundance nonlinearly. Phenotypic assays confirmed the functional consequences, including reduced chlorophyll, impaired growth, and increased anthocyanin. Take together, our results provide large-scale proteomic evidence that AS isoforms are translated in plants, demonstrating how AS diversifies the proteome, regulates protein abundance, and affects growth and development.