Ammonium triggered the response mechanism of lysine crotonylome in tea plants

Abstract

Background: Lysine crotonylation, as a novel evolutionarily conserved type of post-translational modifications, is ubiquitous and essential in cell biology. However, its functions in tea plants are largely unknown, and the full functions of lysine crotonylated proteins of tea plants in nitrogen absorption and assimilation remains unclear. Our study attempts to describe the global profiling of nonhistone lysine crotonylation in tea leaves and to explore how ammonium (NH₄⁺) triggers the response mechanism of lysine crotonylome in tea plants.

Results: Here, we performed the global analysis of crotonylome in tea leaves under NH₄⁺ deficiency/resupply using high-resolution LC-MS/MS coupled with highly sensitive immune-antibody. A total of 2288 lysine crotonylation sites on 971 proteins were identified, of which contained in 15 types of crotonylated motifs. Most of crotonylated proteins were located in chloroplast (37%) and cytoplasm (33%). Compared with NH₄⁺ deficiency, 120 and 151 crotonylated proteins were significantly changed at 3 h and 3 days of NH₄⁺ resupply, respectively. Bioinformatics analysis showed that differentially expressed crotonylated proteins participated in diverse biological processes such as photosynthesis (PsbO, PsbP, PsbQ, Pbs27, PsaN, PsaF, FNR and ATPase), carbon fixation (rbcs, rbcl, TK, ALDO, PGK and PRK) and amino acid metabolism (SGAT, GGAT2, SHMT4 and GDC), suggesting that lysine crotonylation played important roles in these processes. Moreover, the protein-protein interaction analysis revealed that the interactions of identified crotonylated proteins diversely involved in photosynthesis, carbon fixation and amino acid metabolism. Interestingly, a large number of enzymes were crotonylated, such as Rubisco, TK, SGAT and GGAT, and their activities and crotonylation levels changed significantly by sensing ammonium, indicating a potential function of crotonylation in the regulation of enzyme activities.

Conclusions: The results indicated that the crotonylated proteins had a profound influence on metabolic process of tea leaves in response to NH₄⁺ deficiency/resupply, which mainly involved in diverse aspects of primary metabolic processes by sensing NH₄⁺, especially in photosynthesis, carbon fixation and amino acid metabolism. The data might serve as important resources for exploring the roles of lysine crotonylation in N metabolism of tea plants. Data were available via ProteomeXchange with identifier PXD011610.

Keywords: Ammonium deficiency/resupply; Camellia sinensis L.; Enzymatic activity; Lysine crotonylation; Primary metabolism.

Fig. 1

The WB analysis and proteome-wide identification of lysine crotonylation sites in tea leaves. a WB analysis of the total protein content of tea leaves showing duplicates at three time-points after NH₄⁺ resupply. b The mass error distributions of crotonylation profiles. c The peptide length distributions of crotonylation profiles

Fig. 2

Functional classification of lysine crotonylome compared to global proteome. a Subcellular localization of lysine crotonylome compared to global proteome. b Molecular function of lysine crotonylome compared to global proteome. Abbreviation: Chlo for chloroplast, Cyto for cytoplasm, Nucl for nucleus, Mito for mitochondria, Plas for plasma membrane, Extr for extracellular, Bind for binding, Cata for catalytic activity, Stru for structural molecule activity, Tran for transporter activity, Anti for antioxidant activity, and Elec for electron carrier activity

Fig. 3

Motif analysis of lysine crotonylated peptides. a Crotonylated sequence motifs and conservation of crotonylation sites. The 0 position K refers to the crotonylation sites. b Number of identified peptides containing crotonylation in each motif. c Heatmap of the amino acid compositions of the crotonylation sites showing the frequency of the different of amino acids around the crotonylation. “+ 1” and “-1” represent the position around the crotonylation

Fig. 4

Enrichment analysis of DCPs in tea leaves after NH₄⁺ resupply. a GO enrichment analysis of specific DCPs at 3hN/DN. b GO enrichment analysis of common DCPs at 3hN/DN and3dN/DN. c GO enrichment analysis of specific DCPs at 3dN/DN. d KEGG pathway enrichment analysis of specific DCPs at 3hN/DN. e KEGG pathway enrichment analysis of common DCPs at 3hN/DN and3dN/DN. f KEGG pathway enrichment analysis of specific DCPs at 3dN/DN

Fig. 5

PPI network of DCPs after NH₄⁺ resupply. a The PPI of specific DCPs at 3hN/DN. b The PPI of common DCPs at 3hN/DN and3dN/DN, c The PPI of specific DCPs at 3dN/DN

Fig. 6

DEPs in involved in primary metabolic processes after NH₄⁺ resupply. a photosynthesis. b Calvin cycle. c glycine, serine and threonine metabolism. The rectangle was divided into three equal parts (the left of rectangle represented specific DCPs at 3hN/DN; the middle of rectangle represented common DCPs at 3hN/DN and 3dN/DN; the right of rectangle represented specific DCPs at 3dN/DN). The color in the rectangle represents the crotonylated proteins were regulated after NH₄⁺ resupply (red indicated up-regulation; yellow indicates mutli-regulation; green indicated down-regulation)