Proteome-wide identification of non-histone lysine methylation in tomato during fruit ripening

INTRODUCTION: Histone and non-histone methylations are important post-translational modifications in plants. Histone methylation plays a crucial role in regulating chromatin structure and gene expression. However, the involvement of non-histone methylation in plant biological processes remains largely unknown. METHODS: The methylated substrates and methylation sites during tomato fruit ripening were identified by LC-MS/MS. Bioinformatics of lysine methylated proteins was conducted to analyze the possible role of methylated proteins. The effects of methylation modification on protein functions were preliminarily investigated by site-directed mutation simulation. RESULTS: A total of 241 lysine methylation (mono-, di- and trimethylation) sites in 176 proteins were identified with two conserved methylation motifs: xxxxxxExxx_K_xxxExxxxxx and xxxxxxExxx_K_xxxxxxxxxx. These methylated proteins were mainly related to fruit ripening and senescence, oxidation reduction process, signal transduction, stimulus and stress responses, and energy metabolism. Three representative proteins, thioredoxin (Trx), glutathione S-transferase T1 (GST T1), and NADH dehydrogenase (NOX), were selected to investigate the effect of methylation modifications on protein activity. Mimicking demethylation led to decreased Trx activity but increased GST T1 and NOX activities. In addition, RT-qPCR exhibited that the expression of many genes that encode proteins subjected to methylation was upregulated during fruit ripening. CONCLUSION: Our study suggests that tomato fruit ripening undergo non-histone lysine methylation, which may participate in the regulation of fruit ripening. It is the first report of methyl proteome profiling of non-histone lysine in horticultural crops.