Proteome and Phosphoproteome of Tomato Fruit Identify REDUCED CHLOROPLAST COVERAGE 1a as A Ripening Regulator.

Fruit ripening in tomato (Solanum lycopersicum) has been extensively studied at the transcriptomics level. However, comprehensive profiling of the tomato fruit proteome and phosphoproteome remains limited. In this study, we performed large-scale proteome and phosphoproteome profiling of tomato (Ailsa Craig) fruits across five ripening stages using tandem mass tags (TMT)-based quantitative proteomics. Our analysis quantified over 8800 proteins and 20,000 high-confidence phosphorylation sites. Ripening-associated phosphorylation and dephosphorylation events were identified in diverse ripening regulators, including transcription factors, ethylene biosynthesis and signaling proteins, and epigenetic modifiers. Weighted gene co-expression network analysis (WGCNA) revealed a tetratricopeptide repeat protein, REDUCED CHLOROPLAST COVERAGE 1a (REC1a), as a key regulator of fruit ripening. Parallel reaction monitoring (PRM)-based targeted proteomic analysis validated the expression profiles of REC1a and its three phosphorylation sites. Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated knockout of REC1a resulted in reduced lycopene accumulation and slower chlorophyll degradation, highlighting its role in the chloroplast-to-chromoplast transition, which is critical for fruit pigmentation during ripening. Quantitative proteomic analyses of rec1a mutants demonstrated reduced levels of Clp proteases and chaperones, proteins known to regulate plastid transitions. Additionally, co-immunoprecipitation and split-luciferase complementation assays revealed that REC1a interacts with the eukaryotic translation initiation factor subunits eIF2α and eIF2Bβ, suggesting its role in regulating protein synthesis during ripening. This study provides the most comprehensive quantitative proteome and phosphoproteome atlas of tomato fruits to date and identifies REC1a as a novel regulator of plastid development, offering new insights into the molecular mechanisms underlying fruit ripening.