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

Abstract

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.

Keywords: acetylation; lysine succinylation; mass spectrometry; plant biology; post-translational modifications; protein modification; rice; seeds; storage nutrient; succinylome.

Graphical abstract

Fig. 1.

Succinylation profile in different rice organs/tissues revealed by Western blots. Molecular weight is labeled on the left. The samples are labeled on the top. M: size marker; 1. suspension cell protein 2: root protein; 3: leave protein; 4: flower protein; 5: pollen protein; 6: protein from 7 dpa seeds; 7: protein from 15 dpa seeds; 8: protein from 21 dpa seeds; 9: protein from mature dry seeds. A, Image of SDS-PAGE stained with Coomassie Brilliant Blue G-250. B, Western blotting image of protein succinylation. The primary antibodies used were rabbit-derived pan anti-succinyl lysine antibody (PTM-401, PTM Biolabs, Chicago, IL). The same amount of proteins (25 μg per lane) were loaded in panels A and B, The original images of SDS-PAGE and Western blotting are shown in supplemental Fig. S1.

Fig. 2.

Estimation of global protein succinylation occupancy in developing rice seeds. A, Molecular weight is labeled on the left. The samples are labeled on the top. M: size marker; 1. Protein (15 μg) dissolved in SDS buffer; 2: Protein (15 μg) dissolved in PBS buffer; 3: Protein (15 μg) dissolved in PBS buffer and treated with 0.5 mm succinyl-CoA; 4: Protein (15 μg) dissolved in PBS buffer and treated with 1 mm succinyl-CoA; 5–12: 60%, 45%, 30%, 15%, 10%, 5%, 2%, 1% of protein loaded comparing to lane 4 (protein source was the same as lane 4). Upper panel: Image of SDS-PAGE stained with Coomassie Brilliant Blue G-250. Lower panel: Western blotting image of protein succinylation. The primary antibodies used were rabbit-derived pan anti-succinyl lysine antibody (PTM-401, PTM Biolabs, Chicago, IL). B, Succinylation intensity for the two major bands in lane 1, 2, 9, 10, 11, and 12 of Western blotting image in panel A. Succinylation intensity was quantified by Image Studio Lite software. The bar plot was created from three technical replicates for succinylation intensity measurement shown in supplemental Fig. S2.

Fig. 3.

Motif and logo-based clustering analyses of the succinylation sites. A, Conserved motifs of succyl-21-mers flanking succinylation sites (“K” at position 0). The size of each letter correlates to the frequency of that amino acid residue occurring in that position. B, Heat map of the amino acid compositions around the succinylation sites. The −log10 (Fisher's exact test p value) for every amino acid in each position (from −10 to +10) is shown. Motif logo-based clustering analyses: GO annotation enrichment (C), KEGG pathway enrichment analysis (D), and domain enrichment analysis (E).

Fig. 4.

Succinylated enzymes in the TCA cycle, glycolysis/gluconeogenesis, and starch biosynthesis pathways. MDH: malate dehydrogenase; CS: citrate synthase; ACO: aconitate hydratase; IDH: isocitrate dehydrogenase; OGDH: 2-oxoglutarate dehydrogenase E1 component; DLD: dihydrolipoamide dehydrogenase; LSC: succinyl-CoA synthetase; DLST: 2-oxoglutarate dehydrogenase E2 component (dihydrolipoamide succinyltransferase); SDHA: succinate dehydrogenase; FH: fumarate hydratase; PGM: phosphoglucomutase; PGI: phosphoglucose isomerase; ALDO: fructose-bisphosphate aldolase; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; TPI: triosephosphate isomerase; PGK: phosphoglycerate kinase; GPML: phosphoglycerate mutase; ENO: enolase; PK: pyruvate kinase; PDHA: pyruvate dehydrogenase; DLAT: pyruvate dehydrogenase E2 component (dihydrolipoamide acetyltransferase); UGP: UDP-glucose pyrophosphorylase; OsSUS: sucrose synthase; OsAGP: ADP-glucose pyrophosphorylase; OsGBSS: granule-bound starch synthase; OsBE: starch branching enzyme; OsSS: starch synthase; OsISA: starch debranching enzyme: isoamylase; OsPUL: starch debranching enzyme: pullulanase; OsPHO: starch phosphorylase; G6PD: glucose-6-phosphate dehydrogenase; PGLS: 6-phosphogluconolactonase; PGD: 6-phosphogluconate dehydrogenase, decarboxylating 2; RPE: Ribulose-phosphate 3-epimerase; RPIA: ribose-5-phosphate isomerase; TKT: transketolase 1. Solid circles in green, red, yellow, blue and pink colors represent enzymes modified by succinylation, crotonylation, acetylation, malonylation, and 2-hydroxyisobutyrylation, respectively.

Fig. 5.

Overlapping rice proteins between lysine succinylation, acetylation, malonylation, crotonylation, and 2-hydroxyisobutyrylation. A, Venn diagram showing the number of proteins overlapping among succinylation, acetylation, malonylation, crotonylation, and 2-hydroxyisobutyrylation. B, Venn diagram showing the number of lysine sites overlapping among succinylation, acetylation, malonylation, crotonylation, and 2-hydroxyisobutyrylation. C, Succinylated, acetylated, malonylated, crotonylated, and 2-hydroxyisobutyrylated sites on representative protein phosphoglycerate kinase (Q6H6C7). su: lysine succinylation; ac: lysine acetylation; cr: lysine crotonylation; mal: lysine malonylation; hib: lysine 2-hydroxyisobutyrylation. D, Heat map represents the frequency of amino acid compositions around (−10 to +10 position) the lysines (K in position 0) of proteins with at least three types of PTMs identified. E, KEGG pathway enrichment analysis (p value < 0.05) for proteins could be modified by at least types of PTMs. −log10(fisher's exact p value) shown as x axis. F, Protein-protein interaction network of rice proteins with at least three PTMs among succinylation, acetylation, malonylation, crotonylation, and 2-hydroxyisobutyrylation. Protein-protein interaction network was built against the STRING database (version 10.5). Identified interactions with confidence score ≥ 0.7 (high confidence) were fetched and visualized by Cytoscape software.