Comprehensive Analysis of the Lysine Succinylome and Protein Co-modifications in Developing Rice Seeds

Lysine succinylation has been recognized as a post-translational modification (PTM) in recent years. It is plausible that succinylation may have a vaster functional impact than acetylation because of bulkier structural changes and more significant charge differences on the modified lysine residue. Currently, however, the quantity and identity of succinylated proteins and their corresponding functions in cereal plants remain largely unknown. In this study, we estimated the native succinylation occupancy on lysine was between 2% to 10% in developing rice seeds. Eight hundred fifty-four lysine succinylation sites on 347 proteins have been identified by a thorough investigation in developing rice seeds. Six motifs were revealed as preferred amino acid sequence arrangements for succinylation sites, and a noteworthy motif preference was identified in proteins associated with different biological processes, molecular functions, pathways, and domains. Remarkably, heavy succinylation was detected on major seed storage proteins, in conjunction with critical enzymes involved in central carbon metabolism and starch biosynthetic pathways for rice seed development. Meanwhile, our results showed that the modification pattern of in vitro nonenzymatically succinylated proteins was different from those of the proteins isolated from cells in Western blots, suggesting that succinylation is not generated via nonenzymatic reaction in the cells, at least not completely. Using the acylation data obtained from the same rice tissue, we mapped many sites harboring lysine succinylation, acetylation, malonylation, crotonylation, and 2-hydroxisobutyrylation in rice seed proteins. A striking number of proteins with multiple modifications were shown to be involved in critical metabolic events. Given that these modification moieties are intermediate products of multiple cellular metabolic pathways, these targeted lysine residues may mediate the crosstalk between different metabolic pathways via modifications by different moieties. Our study exhibits a platform for extensive investigation of molecular networks administrating cereal seed development and metabolism via PTMs. Lysine succinylation has been recognized as a post-translational modification (PTM) in recent years. It is plausible that succinylation may have a vaster functional impact than acetylation because of bulkier structural changes and more significant charge differences on the modified lysine residue. Currently, however, the quantity and identity of succinylated proteins and their corresponding functions in cereal plants remain largely unknown. In this study, we estimated the native succinylation occupancy on lysine was between 2% to 10% in developing rice seeds. Eight hundred fifty-four lysine succinylation sites on 347 proteins have been identified by a thorough investigation in developing rice seeds. Six motifs were revealed as preferred amino acid sequence arrangements for succinylation sites, and a noteworthy motif preference was identified in proteins associated with different biological processes, molecular functions, pathways, and domains. Remarkably, heavy succinylation was detected on major seed storage proteins, in conjunction with critical enzymes involved in central carbon metabolism and starch biosynthetic pathways for rice seed development. Meanwhile, our results showed that the modification pattern of in vitro nonenzymatically succinylated proteins was different from those of the proteins isolated from cells in Western blots, suggesting that succinylation is not generated via nonenzymatic reaction in the cells, at least not completely. Using the acylation data obtained from the same rice tissue, we mapped many sites harboring lysine succinylation, acetylation, malonylation, crotonylation, and 2-hydroxisobutyrylation in rice seed proteins. A striking number of proteins with multiple modifications were shown to be involved in critical metabolic events. Given that these modification moieties are intermediate products of multiple cellular metabolic pathways, these targeted lysine residues may mediate the crosstalk between different metabolic pathways via modifications by different moieties. Our study exhibits a platform for extensive investigation of molecular networks administrating cereal seed development and metabolism via PTMs. Post-translational modifications (PTMs)1 are covalent modifications that transpire during or after protein biosynthesis. Lysine succinylation (Ksu) is an evolutionarily-conserved PTM (1.Hirschey M.D. Zhao Y. Metabolic regulation by lysine malonylation, succinylation, and glutarylation.Mol. Cell Proteomics. 2015; 14: 2308-2315Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, 2.Zhang Z. Tan M. Xie Z. Dai L. Chen Y. Zhao Y. Identification of lysine succinylation as a new post-translational modification.Nat. Chem. Biol. 2011; 7: 58-63Crossref PubMed Scopus (572) Google Scholar) that attaches a succinyl group (-CO-CH2-CH2-CO-) to a protein lysine residue (2.Zhang Z. Tan M. Xie Z. Dai L. Chen Y. Zhao Y. Identification of lysine succinylation as a new post-translational modification.Nat. Chem. Biol. 2011; 7: 58-63Crossref PubMed Scopus (572) Google Scholar). The addition of a succinyl group induces a mass shift of +100.0186 Da and generates a negative charge on lysine residue under physiological pH (2.Zhang Z. Tan M. Xie Z. Dai L. Chen Y. Zhao Y. Identification of lysine succinylation as a new post-translational modification.Nat. Chem. Biol. 2011; 7: 58-63Crossref PubMed Scopus (572) Google Scholar, 3.Papanicolaou K.N. O'Rourke B. Foster D.B. Metabolism leaves its mark on the powerhouse: recent progress in post-translational modifications of lysine in mitochondria.Front. Physiol. 2014; 5: 301Crossref PubMed Scopus (64) Google Scholar). Because succinylation results in a bulkier structural change and more significant charge difference on lysine, one could postulate that it generates a grander impact on the substrate protein's structures and functions compared with well-studied lysine acetylation and methylation (1.Hirschey M.D. Zhao Y. Metabolic regulation by lysine malonylation, succinylation, and glutarylation.Mol. Cell Proteomics. 2015; 14: 2308-2315Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, 2.Zhang Z. Tan M. Xie Z. Dai L. Chen Y. Zhao Y. Identification of lysine succinylation as a new post-translational modification.Nat. Chem. Biol. 2011; 7: 58-63Crossref PubMed Scopus (572) Google Scholar). Succinyl-CoA levels and E2 subunit of α-ketoglutarate dehydrogenase (KGDHC) have emerged as the primary regulators of protein succinylation through nonenzymatic and enzymatic ways (4.Weinert B.T. Schölz C. Wagner S.A. Iesmantavicius V. Su D. Daniel J.A. Choudhary C. Lysine succinylation is a frequently occurring modification in prokaryotes and eukaryotes and extensively overlaps with acetylation.Cell Rep. 2013; 4: 842-851Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar, 5.Wagner G.R. Payne R.M. Widespread and enzyme-independent Nε-acetylation and Nε-succinylation of proteins in the chemical conditions of the mitochondrial matrix.J. Biol. Chem. 2013; 288: 29036-29045Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar, 6.Gibson G.E. Xu H. Chen H.L. Chen W. Denton T.T. Zhang S. Alpha-ketoglutarate dehydrogenase complex-dependent succinylation of proteins in neurons and neuronal cell lines.J. Neurochem. 2015; 134: 86-96Crossref PubMed Scopus (75) Google Scholar), respectively. The succinylation effectiveness of KGDHC is superior compared with succinyl-CoA alone (6.Gibson G.E. Xu H. Chen H.L. Chen W. Denton T.T. Zhang S. Alpha-ketoglutarate dehydrogenase complex-dependent succinylation of proteins in neurons and neuronal cell lines.J. Neurochem. 2015; 134: 86-96Crossref PubMed Scopus (75) Google Scholar). SIRT5 and SIRT7 are mammalian sirtuins of class III family histone deacetylases, and they were identified as the key enzymes for lysine desuccinylation in cells and tissues (7.Rardin M.J. He W. Nishida Y. Newman J.C. Carrico C. Danielson S.R. Guo A. Gut P. Sahu A.K. Li B. SIRT5 regulates the mitochondrial lysine succinylome and metabolic networks.Cell Metab. 2013; 18: 920-933Abstract Full Text Full Text PDF PubMed Scopus (440) Google Scholar, 8.Park J. Chen Y. Tishkoff D.X. Peng C. Tan M. Dai L. Xie Z. Zhang Y. Zwaans B.M. Skinner M.E. SIRT5-mediated lysine desuccinylation impacts diverse metabolic pathways.Mol. Cell. 2013; 50: 919-930Abstract Full Text Full Text PDF PubMed Scopus (621) Google Scholar, 9.Peng C. Lu Z. Xie Z. Cheng Z. Chen Y. Tan M. Luo H. Zhang Y. He W. Yang K. The first identification of lysine malonylation substrates and its regulatory enzyme.Mol. Cell Proteomics. 2011; 10Abstract Full Text Full Text PDF Scopus (512) Google Scholar, 10.Li L. Shi L. Yang S. Yan R. Zhang D. Yang J. He L. Li W. Yi X. Sun L. SIRT7 is a histone desuccinylase that functionally links to chromatin compaction and genome stability.Nat. Commun. 2016; 7: 12235Crossref PubMed Scopus (212) Google Scholar). Apart from desuccinylation, SIRT5 exhibits broader activities for demalonylation and deglutarylation, but it demonstrates low activity for deacetylation (8.Park J. Chen Y. Tishkoff D.X. Peng C. Tan M. Dai L. Xie Z. Zhang Y. Zwaans B.M. Skinner M.E. SIRT5-mediated lysine desuccinylation impacts diverse metabolic pathways.Mol. 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Lu Z. Zhang Y. Wan X. Chen Y. Cha Y.H. Lin H. Identification of lysine succinylation substrates and the succinylation regulatory enzyme CobB in Escherichia coli.Mol. Cell Proteomics. 2013; 12: 3509-3520Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). Prior research has suggested that lysine succinylation is a pervasive modifier among histone and nonhistone proteins (2.Zhang Z. Tan M. Xie Z. Dai L. Chen Y. Zhao Y. Identification of lysine succinylation as a new post-translational modification.Nat. Chem. Biol. 2011; 7: 58-63Crossref PubMed Scopus (572) Google Scholar, 4.Weinert B.T. Schölz C. Wagner S.A. Iesmantavicius V. Su D. Daniel J.A. Choudhary C. Lysine succinylation is a frequently occurring modification in prokaryotes and eukaryotes and extensively overlaps with acetylation.Cell Rep. 2013; 4: 842-851Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar, 7.Rardin M.J. He W. Nishida Y. Newman J.C. Carrico C. Danielson S.R. Guo A. Gut P. Sahu A.K. Li B. SIRT5 regulates the mitochondrial lysine succinylome and metabolic networks.Cell Metab. 2013; 18: 920-933Abstract Full Text Full Text PDF PubMed Scopus (440) Google Scholar, 13.Colak G. Xie Z. Zhu A.Y. Dai L. Lu Z. Zhang Y. Wan X. Chen Y. Cha Y.H. Lin H. Identification of lysine succinylation substrates and the succinylation regulatory enzyme CobB in Escherichia coli.Mol. Cell Proteomics. 2013; 12: 3509-3520Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 14.Kosono S. Tamura M. Suzuki S. Kawamura Y. Yoshida A. Nishiyama M. Yoshida M. Changes in the Acetylome and Succinylome of Bacillus subtilis in Response to Carbon Source.PLoS ONE. 2015; 10: e0131169Crossref PubMed Scopus (94) Google Scholar, 15.Li X. Hu X. Wan Y. Xie G. Li X. Chen D. Cheng Z. Yi X. Liang S. Tan F. Systematic identification of the lysine succinylation in the protozoan parasite Toxoplasma gondii.J. Proteome Res. 2014; 13: 6087-6095Crossref PubMed Scopus (82) Google Scholar, 16.Xie Z. Dai J. 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Xu M. Huang Q. Zeng J. Zhou M. Xie J. First succinyl-proteome profiling of extensively drug-resistant Mycobacterium tuberculosis revealed involvement of succinylation in cellular physiology.J. Proteome Res. 2014; 14: 107-119Crossref PubMed Scopus (93) Google Scholar, 21.Jin W. Wu F. Proteome-wide identification of lysine succinylation in the proteins of tomato (Solanum lycopersicum).PloS One. 2016; 11: e0147586Crossref PubMed Scopus (62) Google Scholar, 22.He D. Wang Q. Li M. Damaris R.N. Yi X. Cheng Z. Yang P. Global proteome analyses of lysine acetylation and succinylation reveal the widespread involvement of both modification in metabolism in the embryo of germinating rice seed.J. Proteome Res. 2016; 15: 879-890Crossref PubMed Scopus (99) Google Scholar, 23.Zhen S. Deng X. Wang J. Zhu G. Cao H. Yuan L. Yan Y. First comprehensive proteome analyses of lysine acetylation and succinylation in seedling leaves of Brachypodium distachyon L.Sci. Rep. 2016; 6: 31576Crossref PubMed Scopus (54) Google Scholar, 24.Zhang Y. Wang G. Song L. Mu P. Wang S. Liang W. Lin Q. Global analysis of protein lysine succinylation profiles in common wheat.BMC Genomics. 2017; 18: 309Crossref PubMed Scopus (38) Google Scholar, 25.Shen C. Xue J. Sun T. Guo H. Zhang L. Meng Y. Wang H. Succinyl-proteome profiling of a high taxol containing hybrid Taxus species (Taxus× media) revealed involvement of succinylation in multiple metabolic pathways.Sci. Rep. 2016; 6: 21764Crossref PubMed Scopus (33) Google Scholar). Recently, the advancements in the mass spectrometry technology and succinyl-peptides enrichment methods facilitated the identification of hundreds to thousands of succinylation sites in both prokaryotes and eukaryotes (4.Weinert B.T. Schölz C. Wagner S.A. Iesmantavicius V. Su D. Daniel J.A. Choudhary C. Lysine succinylation is a frequently occurring modification in prokaryotes and eukaryotes and extensively overlaps with acetylation.Cell Rep. 2013; 4: 842-851Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar, 7.Rardin M.J. He W. Nishida Y. Newman J.C. Carrico C. Danielson S.R. Guo A. Gut P. Sahu A.K. Li B. SIRT5 regulates the mitochondrial lysine succinylome and metabolic networks.Cell Metab. 2013; 18: 920-933Abstract Full Text Full Text PDF PubMed Scopus (440) Google Scholar, 13.Colak G. Xie Z. Zhu A.Y. Dai L. Lu Z. Zhang Y. Wan X. Chen Y. Cha Y.H. Lin H. Identification of lysine succinylation substrates and the succinylation regulatory enzyme CobB in Escherichia coli.Mol. Cell Proteomics. 2013; 12: 3509-3520Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 14.Kosono S. Tamura M. Suzuki S. Kawamura Y. Yoshida A. Nishiyama M. Yoshida M. Changes in the Acetylome and Succinylome of Bacillus subtilis in Response to Carbon Source.PLoS ONE. 2015; 10: e0131169Crossref PubMed Scopus (94) Google Scholar, 15.Li X. Hu X. Wan Y. Xie G. Li X. Chen D. Cheng Z. Yi X. Liang S. Tan F. Systematic identification of the lysine succinylation in the protozoan parasite Toxoplasma gondii.J. Proteome Res. 2014; 13: 6087-6095Crossref PubMed Scopus (82) Google Scholar, 17.Yang M. Wang Y. Chen Y. Cheng Z. Gu J. Deng J. Bi L. Chen C. Mo R. Wang X. Succinylome analysis reveals the involvement of lysine succinylation in metabolism in pathogenic Mycobacterium tuberculosis.Mol. Cell Proteomics. 2015; 14: 796-811Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 19.Pan J. Chen R. Li C. Li W. Ye Z. Global Analysis of Protein Lysine Succinylation Profiles and Their Overlap with Lysine Acetylation in the Marine Bacterium Vibrio parahemolyticus.J. Proteome Res. 2015; 14: 4309-4318Crossref PubMed Scopus (76) Google Scholar, 20.Xie L. Liu W. Li Q. Chen S. Xu M. Huang Q. Zeng J. Zhou M. Xie J. First succinyl-proteome profiling of extensively drug-resistant Mycobacterium tuberculosis revealed involvement of succinylation in cellular physiology.J. Proteome Res. 2014; 14: 107-119Crossref PubMed Scopus (93) Google Scholar, 21.Jin W. Wu F. Proteome-wide identification of lysine succinylation in the proteins of tomato (Solanum lycopersicum).PloS One. 2016; 11: e0147586Crossref PubMed Scopus (62) Google Scholar, 22.He D. Wang Q. Li M. Damaris R.N. Yi X. Cheng Z. Yang P. Global proteome analyses of lysine acetylation and succinylation reveal the widespread involvement of both modification in metabolism in the embryo of germinating rice seed.J. Proteome Res. 2016; 15: 879-890Crossref PubMed Scopus (99) Google Scholar, 23.Zhen S. Deng X. Wang J. Zhu G. Cao H. Yuan L. Yan Y. First comprehensive proteome analyses of lysine acetylation and succinylation in seedling leaves of Brachypodium distachyon L.Sci. Rep. 2016; 6: 31576Crossref PubMed Scopus (54) Google Scholar, 24.Zhang Y. Wang G. Song L. Mu P. Wang S. Liang W. Lin Q. Global analysis of protein lysine succinylation profiles in common wheat.BMC Genomics. 2017; 18: 309Crossref PubMed Scopus (38) Google Scholar, 25.Shen C. Xue J. Sun T. Guo H. Zhang L. Meng Y. Wang H. Succinyl-proteome profiling of a high taxol containing hybrid Taxus species (Taxus× media) revealed involvement of succinylation in multiple metabolic pathways.Sci. Rep. 2016; 6: 21764Crossref PubMed Scopus (33) Google Scholar). The proteome analysis of lysine succinylation has been reported in E. coli (2.Zhang Z. Tan M. Xie Z. Dai L. Chen Y. Zhao Y. Identification of lysine succinylation as a new post-translational modification.Nat. Chem. Biol. 2011; 7: 58-63Crossref PubMed Scopus (572) Google Scholar, 4.Weinert B.T. Schölz C. Wagner S.A. Iesmantavicius V. Su D. Daniel J.A. Choudhary C. Lysine succinylation is a frequently occurring modification in prokaryotes and eukaryotes and extensively overlaps with acetylation.Cell Rep. 2013; 4: 842-851Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar, 13.Colak G. Xie Z. Zhu A.Y. Dai L. Lu Z. Zhang Y. Wan X. Chen Y. Cha Y.H. Lin H. Identification of lysine succinylation substrates and the succinylation regulatory enzyme CobB in Escherichia coli.Mol. Cell Proteomics. 2013; 12: 3509-3520Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar), B.subtilis (14.Kosono S. Tamura M. Suzuki S. Kawamura Y. Yoshida A. Nishiyama M. Yoshida M. Changes in the Acetylome and Succinylome of Bacillus subtilis in Response to Carbon Source.PLoS ONE. 2015; 10: e0131169Crossref PubMed Scopus (94) Google Scholar), V.parahemeolyticus (19.Pan J. Chen R. Li C. Li W. Ye Z. Global Analysis of Protein Lysine Succinylation Profiles and Their Overlap with Lysine Acetylation in the Marine Bacterium Vibrio parahemolyticus.J. Proteome Res. 2015; 14: 4309-4318Crossref PubMed Scopus (76) Google Scholar), M.tuberculosis (17.Yang M. Wang Y. Chen Y. Cheng Z. Gu J. Deng J. Bi L. Chen C. Mo R. Wang X. Succinylome analysis reveals the involvement of lysine succinylation in metabolism in pathogenic Mycobacterium tuberculosis.Mol. Cell Proteomics. 2015; 14: 796-811Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 20.Xie L. Liu W. Li Q. Chen S. Xu M. Huang Q. Zeng J. Zhou M. Xie J. First succinyl-proteome profiling of extensively drug-resistant Mycobacterium tuberculosis revealed involvement of succinylation in cellular physiology.J. Proteome Res. 2014; 14: 107-119Crossref PubMed Scopus (93) Google Scholar), H.sapiens (4.Weinert B.T. Schölz C. Wagner S.A. Iesmantavicius V. Su D. Daniel J.A. Choudhary C. Lysine succinylation is a frequently occurring modification in prokaryotes and eukaryotes and extensively overlaps with acetylation.Cell Rep. 2013; 4: 842-851Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar, 11.Du J. Zhou Y. Su X. Yu J.J. Khan S. Jiang H. Kim J. Woo J. Kim J.H. Choi B.H. Sirt5 is a NAD-dependent protein lysine demalonylase and desuccinylase.Science. 2011; 334: 806-809Crossref PubMed Scopus (962) Google Scholar), M.musculus (4.Weinert B.T. Schölz C. Wagner S.A. Iesmantavicius V. Su D. Daniel J.A. Choudhary C. Lysine succinylation is a frequently occurring modification in prokaryotes and eukaryotes and extensively overlaps with acetylation.Cell Rep. 2013; 4: 842-851Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar, 7.Rardin M.J. He W. Nishida Y. Newman J.C. Carrico C. Danielson S.R. Guo A. Gut P. Sahu A.K. Li B. SIRT5 regulates the mitochondrial lysine succinylome and metabolic networks.Cell Metab. 2013; 18: 920-933Abstract Full Text Full Text PDF PubMed Scopus (440) Google Scholar, 8.Park J. Chen Y. Tishkoff D.X. Peng C. Tan M. Dai L. Xie Z. Zhang Y. Zwaans B.M. Skinner M.E. SIRT5-mediated lysine desuccinylation impacts diverse metabolic pathways.Mol. Cell. 2013; 50: 919-930Abstract Full Text Full Text PDF PubMed Scopus (621) Google Scholar, 11.Du J. Zhou Y. Su X. Yu J.J. Khan S. Jiang H. Kim J. Woo J. Kim J.H. Choi B.H. Sirt5 is a NAD-dependent protein lysine demalonylase and desuccinylase.Science. 2011; 334: 806-809Crossref PubMed Scopus (962) Google Scholar), S. cerevisiae (4.Weinert B.T. Schölz C. Wagner S.A. Iesmantavicius V. Su D. Daniel J.A. Choudhary C. Lysine succinylation is a frequently occurring modification in prokaryotes and eukaryotes and extensively overlaps with acetylation.Cell Rep. 2013; 4: 842-851Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar), and T.gondii (15.Li X. Hu X. Wan Y. Xie G. Li X. Chen D. Cheng Z. Yi X. Liang S. Tan F. Systematic identification of the lysine succinylation in the protozoan parasite Toxoplasma gondii.J. Proteome Res. 2014; 13: 6087-6095Crossref PubMed Scopus (82) Google Scholar). This vital aspect has substantially extended our understanding of protein succinylation. Plant succinylomes have also been reported in tomato seedlings (21.Jin W. Wu F. Proteome-wide identification of lysine succinylation in the proteins of tomato (Solanum lycopersicum).PloS One. 2016; 11: e0147586Crossref PubMed Scopus (62) Google Scholar), rice germinating embryos (22.He D. Wang Q. Li M. Damaris R.N. Yi X. Cheng Z. Yang P. Global proteome analyses of lysine acetylation and succinylation reveal the widespread involvement of both modification in metabolism in the embryo of germinating rice seed.J. Proteome Res. 2016; 15: 879-890Crossref PubMed Scopus (99) Google Scholar), B. distachyon seedling leaves (23.Zhen S. Deng X. Wang J. Zhu G. Cao H. Yuan L. Yan Y. First comprehensive proteome analyses of lysine acetylation and succinylation in seedling leaves of Brachypodium distachyon L.Sci. Rep. 2016; 6: 31576Crossref PubMed Scopus (54) Google Scholar), common wheat (24.Zhang Y. Wang G. Song L. Mu P. Wang S. Liang W. Lin Q. Global analysis of protein lysine succinylation profiles in common wheat.BMC Genomics. 2017; 18: 309Crossref PubMed Scopus (38) Google Scholar), and hybrid Taxus species (25.Shen C. Xue J. Sun T. Guo H. Zhang L. Meng Y. Wang H. Succinyl-proteome profiling of a high taxol containing hybrid Taxus species (Taxus× media) revealed involvement of succinylation in multiple metabolic pathways.Sci. Rep. 2016; 6: 21764Crossref PubMed Scopus (33) Google Scholar). However, possible protein lysine co-modification by various moieties has not been explored in plants. Rice is one of the most significant cereals as it serves as the staple food for over half of the world's population (26.Bhullar N.K. Gruissem W. Nutritional enhancement of rice for human health: the contribution of biotechnology.Biotechnol. Adv. 2013; 31: 50-57Crossref PubMed Scopus (125) Google Scholar). In rice grain, most nutrients are stored in the form of starch, lipid, and protein, which extensively contribute to grain nutritional value, milling properties, appearance, and cooking quality (27.Chen Y. Wang M. Ouwerkerk P.B. Molecular and environmental factors determining grain quality in rice.Food Energy Secur. 2012; 1: 111-132Crossref Scopus (94) Google Scholar). The content and composition of storage starch and protein are directly associated with seed development. Recently, PTMs of lysine acetylation (28.Meng X. Lv Y. Mujahid H. Edelmann M.J. Zhao H. Peng X. Peng Z. Proteome-wide lysine acetylation identification in developing rice (Oryza sativa) seeds and protein co-modification by acetylation, succinylation, ubiquitination, and phosphorylation.BBA-Proteins Proteomics. 2018; 1866: 451-463Crossref PubMed Scopus (25) Google Scholar), malonylation (29.Mujahid H. Meng X. Xing S. Peng X. Wang C. Peng Z. Malonylome analysis in developing rice (Oryza sativa) seeds suggesting that protein lysine malonylation is well-conserved and overlaps with acetylation and succinylation substantially.J. Proteomics. 2018; 170: 88-98Crossref PubMed Scopus (23) Google Scholar), and 2-hydroxyisobutyrylation (30.Meng X. Xing S. Perez L.M. Peng X. Zhao Q. Redoña E.D. Wang C. Peng Z. Proteome-wide analysis of lysine 2-hydroxyisobutyrylation in developing rice (Oryza sativa) seeds.Sci. Rep. 2017; 7: 17486Crossref PubMed Scopus (39) Google Scholar) have been reported in developing rice seeds. In this report, we successfully identified 854 lysine succinylation sites across 347 proteins with a false discovery rate (FDR) of ≤ 1% in developing rice seeds. Our results indicate that lysine succinylation is a highly conserved modification. It frequently occurs in the rice proteome with a preference on carbon metabolic pathways, starch biosynthetic pathways, and the major seed storage proteins. Further, many proteins involved in crucial metabolic processes were revealed to embrace various modifications in lysine residues. This analysis provides a comprehensive view of lysine modifications in developing rice seeds. Rice (Oryza sativa L. japonica cv. Nipponbare) leaves and roots were sampled from 20-day-old seedlings grown in an incubator at 28 °C (16-h-day/8-h-night). The flowers, pollen, 7, 15, and 21 days post-anthesis (dpa) developing rice seeds and mature rice seeds were collected from rice plants grown in a greenhouse of the Department of Biochemistry and Molecular Biology, Mississippi State University, MS. The cultured cells are rice (Oryza sativa L. japonica cv. Nipponbare) NB2P suspension cell cultures, which were maintained as reported (31.Lee T.-J. Shultz R.W. Hanley-Bowdoin L. Thompson W.F. Establishment of rapidly proliferating rice cell suspension culture and its characterization by fluorescence-activated cell sorting analysis.Plant Mol. Biol. Rep. 2004; 22: 259-267Crossref Scopus (21) Google Scholar). Proteins were isolated using a phenol extraction method (28.Meng X. Lv Y. Mujahid H. Edelmann M.J. Zhao H. Peng X. Peng Z. Proteome-wide lysine acetylation identification in developing rice (Oryza sativa) seeds and protein co-modification by acetylation, succinylation, ubiquitination, and phosphorylation.BBA-Proteins Proteomics. 2018; 1866: 451-463Crossref PubMed Scopus (25) Google Scholar, 29.Mujahid H. Meng X. Xing S. Peng X. Wang C. Peng Z. Malonylome analysis in developing rice (Oryza sativa) seeds suggesting that protein lysine malonylation is well-conserved and overlaps with acetylation and succinylation substantially.J. Proteomics. 2018; 170: 88-98Crossref PubMed Scopus (23) Google Scholar, 30.Meng X. Xing S. Perez L.M. Peng X. Zhao Q. Redoña E.D. Wang C. Peng Z. Proteome-wide analysis of lysine 2-hydroxyisobutyrylation in developing rice (Oryza sativa) seeds.Sci. Rep. 2017; 7: 17486Crossref PubMed Scopus (39) Google Scholar, 32.Hurkman W.J. Tanaka C.K. Solubilization of plant membrane proteins for analysis by two-dimensional gel electrophoresis.Plant Physiol. 1986; 81: 802-806Crossref PubMed Google Scholar, 33.Xing S. Meng X. Zhou L. Mujahid H. Zhao C. Zhang Y. Wang C. Peng Z. Proteome profile of starch granules purified from rice (Oryza sativa) endosperm.PloS One. 2016; 11: e0168467Crossref PubMed Scopus (26) Google Scholar). The ground plant organ/tissue was mixed with an extraction buffer (0.9 m sucrose, 0.5 m Tris-HCl pH 8.7, 0.05 m EDTA, 0.1 m KCl, and 2% β-mercaptoethanol), combined with the subsequent addition of an equal volume of saturated phenol (pH 8.0), and homogenization for 30 min at 4 °C. The phenol phase was recovered from homogenate by centrifugation at 5000 × g for 15 min at 4 °C. The phenol extraction procedure was repeated three times. The final collection of phenol was mixed with five volumes of precipitation buffer (methanol with 0.1 m ammonium acetate and 1% β–mercaptoethanol) and resided overnight at −80 °C for precipitation. The crude protein was obtained by centrifugation at 15,000 × g for 15 min at 4 °C. Afterward, the protein pellet was washed three times with cold precipitation buffer, accompanied by three-time additional washes with ice-cold 70