Detection of lysine acetylated proteins in tea leaves under different N treatments
To assess the physiological changes of the tea leaves under N treatments, we mainly detected the N contents of tea leaves and the leaf maximum photochemical quantum yield of PS II (Fv/Fm). Under N-starvation/resupply, the contents of N in tea leaves changed significantly from 0 N to 3hN (Fig. 1a). The contents of N in tea leaves under N-resupply increased by 27.8% from 0 N to 3hN, but then kept stable from 3hN to 3dN. The leaf maximum photochemical quantum yield of PS II (Fv/Fm) of different treatment groups were significantly influenced by the N contents of tea leaves. Under 3 h N-resupply, Fv/Fm was higher than that of N starvation or 3d N-resupply. And Fv/Fm of 3d N-resupply was higher than that of N starvation (Fig. 1b, Additional file 3: Table S2). In order to research overall acetylated proteins in tea leaves under N treatments, the Western Blotting assay was performed with protein extracts from tea leaves using anti-acetyl-lysine antibody. The results indicated that the multiple lysine-acetylated protein bands of different N treatments were detected and showed stronger reactions to the anti-acetyl-lysine (Fig. 1c). So, protein acetylation occurred in tea leaves under N-starvation/resupply and lysine-acetylated peptides can be affinity enriched for further identification and analysis.
The workflow of experimental procedures used in the study was shown in Fig. 2d. In order to verify the validity of MS data, the quality errors of all confirmed acetylpeptides were checked. The distribution of quality errors was close to zero, and the length of most acetylation peptides was distributed between 7 and 25 (Fig. 2e, f). This confirmed the high accuracy of MS data. In total, 1286 proteins were shown to be acetylated, with 2229 unique acetylated sites in tea leaves under N-starvation/resupply (Additional file 4: Table S3). Up to now, tea leaves had a great quantity of acetylated proteins among the plants reported, reflecting a potentially vital role of this modification in tea plants, which has drawn our attention.
Distribution and motif analysis of lysine acetylation sites
To assess the distribution of acetylation sites on the proteins of tea leaves, we calculated the numbers of modified sites on the acetylated proteins. The average degree of acetylation was 1.7 per protein. The results indicated that 62.1% (799/1286) contained a single acetylated site, 20.8% (268/1286) contained two acetylated sites, and 8.6% (111/1286) contained three acetylated sites (Fig. 2a). It is noteworthy that 59 proteins contained five or more acetylated sites and 4 had at least 10 sites, such as m.49400 contained 10 acetylated sites, m.1156 and m.12353 contained 12 acetylated sites, m.45083 contained 15 acetylated sites (Additional file 5: Table S4). According to the protein ratio results, we found that the numbers of proteins were similarity in the different ratios, but the numbers of proteins located in the 0 were reduced with N-resupply. The longer time for the N-resupply, the numbers of proteins were reduced more significantly (Additional file 6: Figure S1).
To further evaluate the natural properties of acetylated lysines in tea, we investigated the motifs of all identified lysine residues using Motif-x program. A total of 16 conserved motifs in E*KacK, Kac*K, Kac*R, Kac*HK, Kac*N, Kac*S, Kac*T, Kac*D, were extracted from 2180 acetylated peptides. The most common combination was Kac*K, which was represented by 343 (17.4%) of the enrichment motifs. Among these motifs, three distinct types of residues were located upstream/downstream of the acetylated lysine: three positively charged (basic) residues, including lysine (K), arginine (R) or histidine (H), and the two negatively charged residues, including glutamic acid (E) or aspartic acid (D), and two residues with a hydroxyl group, including serine (S) and threonine (T), which were enriched at the + 1 position on the C-terminus side (Fig. 2b, Additional file 7: Table S5, Additional file 8: Table S6).
With respect to the general amino acid composition around an acetylated lysine site, Ice Logo heat maps were used to assess whether specific amino acids were significantly enriched or depleted by identifying the relative frequencies of the amino acids at specific positions surrounding the acetylated site (10 amino acids upstream and 10 amino acids downstream from the modification sites). The amino acid frequencies determined with the heat map were consistent by those determined with Web Logo (Fig. 2c). These amino acids could be divided into two categories: the + 1, + 2 or + 3 positions, which were alkaline residues with long side chains (H, K or R), and the − 1 or − 3 positions, which were residues with long hydrophobic side chains (W, V or A). These results showed that amino acid residues with alkaline and hydrophobic side chains might play an important role in acetylation. These new residues of amino acids in tea leaves would potentially provide acetylated binding sites for future studies.
To understand the local secondary structures in more details, we compared the secondary structures surrounding the acetylated lysines with those surrounding all lysines using the NetSurfP software. Approximately, 36.76% of the acetylated lysines were located in the regions of ordered secondary structures. Among them, 30.67% sites were located in α-helices and 6.09% sites in β-strands. The remaining 63.24% were located in disordered regions of the proteins (Fig. 2d). Nevertheless, it seems that the proteins in tea leaves had no tendency of acetylation according to the similar of distribution patterns between the acetylated lysines and non-acetylated lysines. In addition, we evaluated the surface accessibility of acetylated lysine sites, too. The data indicated that 38.43% of the acetylated lysine sites were exposed to the protein surface, compared with 40.41% of non-acetylated lysine residues (Fig. 2e). Consequently, slight change might be happened in the surface property of proteins because of lysine acetylation.
Functional characterization and subcellular localization of lysine acetylated proteins in tea leaves
GO functional classification of all the acetylated proteins was investigated based on their biological process, molecular function and cellular component so that we can further and better understand the acetylome in tea plants. The results indicated that the largest group of acetylated proteins consisted of many enzymes, such as ATP synthase, ribosomal proteins and malate dehydrogenase [NADP], which were related to metabolism (38%) in the biological process (Fig. 3a). For the molecular function category (Fig. 3b), the acetylated proteins related to catalytic activity and binding functions were identified, accounting for 46 and 41% of all the acetylated proteins, respectively. Regarding the cellular component category (Fig. 3c), most of the acetylated proteins were related to cell (39%), macromolecular complex (24%) and organelle (22%). Further studies showed that the major classes were similarly under the different N treatments, but the numbers of proteins were different under the N-starvation/resupply. For instance, there were 82 acetylated proteins associated with metabolic process. Among them there were 27 acetylated proteins associated with photosynthesis and glycolysis in the 3hN/0 N. There were 80 and 29 acetylated proteins in the 3dN/0 N. And there were 90 and 21 acetylated proteins in the 3dN/3hN. The more detailed information was provided in the Additional file 9: Table S7.
A large proportion of the identified acetylated proteins in tea leaves were located to the cytoplasm (29%) and chloroplast (39%) which were shown in subcellular localization analysis. In further researches, we discovered that 10% of the acetylated proteins presented in chloroplast participated in the process of tricarboxylic acid (TCA) cycle and photosynthesis. As expected, 17% of acetylated proteins, including histones and nonhistones were located in the nucleus, confirming the regulatory role of lysine acetylation in post-transcriptional regulation. Beyond that, we found that some proteins were distributed in the mitochondria (5%), cytoskeleton (2%), plasma membrane (3%) and endoplasmic reticulum (1%) (Fig. 3d). Further studies showed that the numbers of acetylated proteins associated with photosynthesis were different under N-starvation/resupply. For example, there were 7 different acetylated proteins in the 3hN/0 N. There were 5 different acetylated proteins in the 3dN/0 N. And there were 11 different acetylated proteins in the 3dN/3hN. The results showed that the distribution of proteins was a dynamic process that related to metabolized continually and changed momentarily. These data, as well as the results of GO functional classification, showed that lysine acetylated proteins had extensive biological functions in tea leaves.
The enrichment analysis of lysine-acetylated proteins in tea leaves under different N treatments
To complete which types of proteins are preferred targets for lysine acetylation, the analysis of GO, KEGG pathway and protein domains was successfully accomplished (Fig. 4, Additional file 10: Table S8).
GO enrichment analysis based on the biological process showed that photosynthesis, translation and metabolic process were enriched in acetylated proteins in the 3hN/0 N (Fig. 4a). Similarly, the photosynthesis ranked first in the 3dN/3hN (Fig. 4c), followed by the metabolic process and organelle activity. While the metabolic process ranked first in the 3dN/0 N (Fig. 4b), followed by the biosynthetic process. As far as the molecular function category was concerned, the activities of enzyme inhibitor and enzyme regulator were significantly enriched in the 3hN/0 N. However, DNA binding and protein activity were enriched in the 3dN/0 N and 3dN/3hN. There were a few differences among the three different ratios. Consistently, for the cellular components the acetylated proteins were significantly enriched in photosystem and thylakoid part in the 3hN/0 N. But the organelles and DNA complexes were significantly enriched in the 3dN/0 N and 3dN/3hN.
In order to better understand its general functions in tea leaves, these acetylated proteins were mapped to KEGG metabolic pathways. The results showed that the KEGG pathway of the photosynthesis was enriched significantly in the 3hN/0 N and 3dN/3hN (Fig. 4d, f). The metabolic process and biosynthesis were enriched significantly in the 3dN/0 N (Fig. 4e). This indicated that lysine acetylation occurred on many proteins related to photosynthesis in the 3hN/0 N. Meanwhile, the enrichment proteins took part in photosynthesis and flavonoid biosynthesis in the 3dN/3hN. But most acetylated proteins were related to amino acid metabolism and biosynthesis, such as phenylalanine metabolism, linoleic acid metabolism and phenylalanine biosynthesis in the 3dN/0 N. Furthermore, the enzymatic activity and the NAD (P)-binding domain were enriched notably in tea leaves under the N-starvation/resupply.
The analysis of interaction network in lysine acetylated proteins
For the purpose of deeply understanding how these acetylated proteins are related and how the acetylated proteins involved in different pathways crosslink to each other, we chose STRING database and Cytoscape software (https://string-db.org) to construct the PPI (protein-protein interaction) networks for the distinct proteins. We extracted several highly rich interactive clusters from the entire interaction network by means of the MCODE plug-in tool kit.
Compared 3hN with 0 N, there were 108 acetylated proteins in interaction which mapped to the protein interaction database (Fig. 5a, Additional file 11: Table S9). Thereinto, 62 were up-regulated, including psaN, psbO, psbS, rbcL, rbcS and IDH1, and 46 were down-regulated, including GAPA, psbB, psaA, AGXT, accD and GLDC. They were clustered into 8 groups. The top group (Cluster I) consisted of photosynthesis-related proteins. These acetylated proteins could be roughly classified into chlorophyll a/b binding protein domain and NAD (P)-binding domain, of which 10 highly correlated acetylated proteins were retrieved, including GAPB, psbO, psbQ and PsaN (Fig. 5b, Additional file 12: Table S10). Whereas Cluster II consisted of the proteins involved in ribosome, of which 9 interconnected clusters of acetylated proteins were retrieved (Fig. 5c, Additional file 13: Table S11). In the Cluster I, the acetylated protein GAPB interacted with the acetylated ribosome protein RPL15. Meanwhile, numerous photosynthesis proteins interacted with GAPB, and many ribosome proteins interacted with RPL15. Additionally, in the enrichment analysis of KEGG pathway, several acetylated proteins concentrated in photosynthetic pathways and one typical pathway was shown in Fig. 6a. The almost all the core parts of photosynthesis, such as photosystems (I and II), cytochrome b6f complex, electron transports, and ATP synthases, were acetylated in several different subunits.
Compared 3dN with 0 N, there were 117 acetylated proteins in interaction which were mapped to the protein interaction database (Fig. 5d, Additional file 14: Table S12). Thereinto, 54 were up-regulated, including psaN, psbO, GAPDH, GLDC, COX6B and PGK, and 63 were down-regulated, including GAPB, petC, LHCA3, ANR, ENL and psaD. They were clustered into 8 groups. The top group (Cluster I) consisted of photosynthesis-associated proteins, in which 13 highly interconnected clusters of acetylated proteins were retrieved, including PGK, GAPB, petC, psaN and psbO (Fig. 5e, Additional file 15: Table S13). These acetylated proteins could be classified into three major groups according to their functions in photosynthesis, namely chlorophyll a/b binding proteins, NAD (P)-binding proteins and ATPase proteins. Whereas Clusters II consisted of proteins involved in ribosome (Fig. 5f, Additional file 16: Table S14), in which 9 interconnected acetylated proteins were retrieved. In this network, 20 lysine acetylated proteins were identified with the node degree over 10, of which PGK, GAPB, petC and ALDO had the highest degree. Among them, the acetylated protein GAPB had the highest degree and interacted with photosynthesis proteins. Meanwhile, numerous ATP binding proteins and many NAD (p) binding proteins interacted with GAPB. Moreover, in our KEGG pathway enrichment analysis, one representative pathway was shown in Fig. 6b. The almost all the core parts of oxidative phosphorylation, such as NADH dehydrogenase, Cytochrome c oxidase/reductase and ATPase, were acetylated in several individual subunits. There were a few differences from the 3hN/0 N.
Compared 3dN with 3hN, there were 118 acetylated proteins in interaction which were mapped to the protein interaction database (Fig. 5g, Additional file 17: Table S15). Thereinto, 46 were up-regulated, including psaN, psbO, GAPDH, GLDC, COX6B and PGK, and 72 were down-regulated, including GAPB, petC, LHCA3, ANR, ENO and psaD. They were also clustered into 8 groups. The top group (Cluster I) identified above consisted of ribosome-associated proteins, in which 18 highly interconnected acetylated proteins were retrieved (Fig. 5i, Additional file 18: Table S16). Whereas Cluster II consisted of the proteins involved in photosynthesis, in which 13 interconnected acetylated proteins were retrieved, including psbQ, psbO, LHCB6, LHCA1 and LHCA3 (Fig. 5h, Additional file 19: Table S17). These acetylated proteins could be roughly attributed to chlorophyll a/b binding proteins. As shown in the Fig. 5g, 46 lysine acetylated proteins were identified with the node degree over 10, of which 11 were over 20 node degrees, of which subunit ribosomal protein, ATPase and petC had the highest degree. Among them, the acetylated protein petC had the highest degree and interacted with NAD (p)-binding proteins. Meanwhile, numerous ATP binding proteins and many primary metabolism proteins interacted with petC. In the KEGG pathway enrichment analysis, several pathways related to photosynthetic were enriched and two representative pathways were shown in Fig. 6c, d. The almost all the core parts of light reaction were acetylated in several different subunits, such as photosystems (I and II), cytochrome b6f complex, electron transports and ATP synthases, indicated that these lysine acetylated proteins had close relationship in photosystem. Besides, photosynthesis antenna proteins (LHCA1, LHCA3 and LHCB6) were also acetylated in lysine sites. It can be seen clearly that, almost all the acetylated proteins had close interaction in the photosynthesis and metabolism in tea leaves under N-starvation/resupply.