Fruits are unique to flowering plants and play a central role in seed maturation and dispersal.Molecular dissection of fruit ripening has received considerable interest because of the biological and dietary significance of fruit.To better understand the regulatory mechanisms underlying fruit ripening,we report here the comprehensive analysis of the nuclear proteome in tomato fruits.Nuclear proteins were isolated from tomatoes in different stages of ripening(mature green,breaker,orange,and red ripe),and subjected to iTRAQ(isobaric tags for relative and absolute quantification)analysis.The proteins that changed abundance across ripening stages are involved in various cellular processes.We additionally evaluated the changes in the nuclear proteome in the ripening-deficient mutant ripening inhibitor(rin)carrying a mutation in the transcription factor RIN.A set of proteins were identified and particular attention was paid to SIUBC32 and PSMD2,the components of ubiquitin-proteasome pathway.Through chromatin immunoprecipitation and gel mobility shift assay,we provide evidence that RIN directly bound to the promoters of SIUBC32 and PSMD2.Moreover,loss of RIN function affected protein ubiquitination in nuclei.SIUBC32 encodes an E2 ubiquitin-conjugating enzyme and genome-wide survey of the E2 gene family in tomatoes identified five more E2s as the direct targets of RIN.Two E2s were demonstrated to be involved in the regulation of fruit ripening based on virus-induced gene silencing assays.Our results uncover the novel function of protein ubiquitination and identify specific E2s as regulator in fruit ripening.These findings contribute to the unraveling of the gene regulatory networks that control fruit ripening.