Global Proteomic Analysis Reveals Widespread Lysine Succinylation in Rice Seedlings

Abstract

Lysine succinylation (Ksu) is a dynamic and reversible post-translational modification that plays an important role in many biological processes. Although recent research has analyzed Ksu plant proteomes, little is known about the scope and cellular distribution of Ksu in rice seedlings. Here, we report high-quality proteome-scale Ksu data for rice seedlings. A total of 710 Ksu sites in 346 proteins with diverse biological functions and subcellular localizations were identified in rice samples. About 54% of the sites were predicted to be localized in the chloroplast. Six putative succinylation motifs were detected. Comparative analysis with succinylation data revealed that arginine (R), located downstream of Ksu sites, is the most conserved amino acid surrounding the succinylated lysine. KEGG pathway category enrichment analysis indicated that carbon metabolism, tricarboxylic acid cycle (TCA) cycle, oxidative phosphorylation, photosynthesis, and glyoxylate and dicarboxylate metabolism pathways were significantly enriched. Additionally, we compared published Ksu data from rice embryos with our data from rice seedlings and found conserved Ksu sites between the two rice tissues. Our in-depth survey of Ksu in rice seedlings provides the foundation for further understanding the biological function of lysine-succinylated proteins in rice growth and development.

Keywords: lysine succinylation; post-translational modification; rice seedlings.

Figure 1

Representative MS/MS spectra of succinylpetides from three proteins: (a) succinylated peptide _LVYTNDQGEIVK(su)GVCSNFLCDLKPGSDVK_ with a succinylated site at K177 from the chloroplast protein petH-ferredoxin-NADP(+) reductase (Q0DF89); (b) succinylated peptide _IVSSIEQK(su)EEGR_ with a succinylated site at K70 from the 14-3-3-like protein GF14c (Q6ZKC0); and (c) succinylated peptide _DNIQGITK(su)PAIR_ with a succinylated site at K31 from histone H4(Q7XUC9).

Figure 2

Motif analysis of all identified lysine succinylation (Ksu) sites. (a) Succinylation motifs and conservation of succinylation sites. The size of each letter corresponds to the frequency of that amino acid residue. (b) Heat map of the amino acid compositions of the succinylated sites showing the frequency of different amino acids surrounding the succinylated lysine.

Figure 3

(a) GO Classification of succinylated proteins based on second-level terminology. (b) Subcellular localization of the identified succinylated proteins from GO analysis.

Figure 4

Protein domain enrichment analysis of succinylated proteins in rice seedlings.

Figure 5

Protein succinylation regulates diverse metabolic pathways in rice. (a) KEGG pathway-based enrichment analysis of the identified proteins. (b,c) Key enzyme with succinyl post-translational modifications (PTM) in photosynthesis pathways.

Figure 6

Comparing lysine succinylated proteins between rice embryo and seedling. (a,b) Venn diagram of succinylated proteins and sites that overlapped in embryos and seedlings; 80 protein contained Ksu sites in both embryos and seedlings, and only 122 Ksu sites had conserved motifs. (c) Subcellular localization of the overlapping succinylated and acetylated proteins in rice embryos and seedlings.

Figure 7

Lysine succinylation and acetylation of individual protein sites in rice: (a,b) Venn diagram of overlapping PTM proteins and sites in rice embryos; 134 proteins contained both lysine acetylation (Kac) and Ksu sites in the embryo, and 131 sites overlapped. (c) Subcellular localization of overlapping succinylated and acetylated proteins in rice emybros. (d,e) Venn diagram of overlapping PTM proteins and sites in rice seedling; 124 proteins contained both Kac and Ksu sites, and 104 sites overlapped. (f) Subcellular localization of overlapping succinylated and acetylated proteins in rice seedling.