First comprehensive proteomics analysis of lysine crotonylation in leaves of peanut (Arachis hypogaea L.)

Lysine crotonylation is an important post‐translational modification process. Most research in this area has been carried out on mammals and yeast, but there has been little research on it in plants. In the current study, large‐scale lysine crotonylome analysis was performed by a combination of affinity enrichment and high‐resolution LC‐MS/MS analysis. Altogether, 6051 lysine crotonylation sites were identified in 2508 protein groups. Bioinformatics analysis showed that lysine‐crotonylated proteins were involved in many biological processes, such as carbon fixation in photosynthetic organisms, biosynthesis of amino acids, ribosomes structure and function. In particular, subcellular localization analysis showed that 43% of the crotonylated proteins were located in the chloroplast. Twenty‐nine crotonylation proteins were associated with photosynthesis and functional enrichment that these proteins were associated with the reaction center, photosynthetic electron transport, and ATP synthesis. Based on these results, further studies to expand on the lysine crotonylome analysis were suggested.


Significance Statement
Peanut (Arachis hypogaea L.) is one of the most important oil crops in the world, and the harvested seeds from this nitrogen-fixing crop have high concentrations of lipids and proteins. Lysine crotonylation is a recently discovered posttranslational modification that has been found to have functional roles in the regulation of gene transcription, regulation of acute kidney injury, and regulation of spermatogenesis, etc. in animals. However, only some global profiling of crotonylation has been carried out on plants with no functional studies. Our research showed that lysine crotonylated proteins were involved in many peanut biological processes, including carbon fixation in photosynthetic organisms, photosynthesis, biosynthesis of amino acids, ribosomes, etc. This dataset is the first comprehensive proteomics analysis of lysine crotonylation in peanut and will serve as an important resource with which to study the biosynthesis and function of lysine crotonylation in peanut and other leguminous plants.
Crotonylation is regulated by crotonyl-CoA via the transcriptional coactivator such as p300, which is also influenced by the concentration of crotonyl-CoA in cells. Therefore, both genetic and environmental factors can be used to regulate histone crotonylation [14,15]. Some writers, erasers, and readers of Kcr have been identified, which enzymatically regulate the dynamic balance of lysine crotonylation between crotonyl transferases and decrotonylases [16]. Lysine crotonylation is involved in the regulation of many biological processes, such as the regulation of gene transcription [17], regulation of acute kidney injury [18], regulation of spermatogenesis [19], regulation of telomere maintenance [20,21], regulation of HIV and cancer latency [13,21], etc.
Peanut (Arachis hypogaea L.) is one of the most important oil crops in the world, and the harvested seeds contain high concentrations of lipids and proteins. The peanut genome was sequenced in 2016, revealing peanut to be a major source of candidate genes for fructification, oil biosynthesis, and allergens, and providing considerable information on plant biology [30]. No research has been published on crotonylated proteins on peanut. In the current study, using a combination of affinity purification and high-resolution liquid chromatographytandem mass spectrometry (LC-MS/MS) analysis, 6051 lysine crotonylation sites were identified in 2508 protein groups. Bioinformatics analysis showed that lysine crotonylated proteins were involved in many biological processes, including carbon fixation in photosynthetic organisms, photosynthesis, biosynthesis of amino acids, ribosome structure and function, etc. This paper describes the first comprehensive proteomics analysis of lysine crotonylation in peanut and will serve as an important resource with which to study the biosynthesis and function of Kcr in peanut and related plants.

Identification and analysis of lysine crotonylation sites and crotonylated proteins in peanut leaves
The experimental procedure is presented diagrammatically in Among the 2508 crotonylated proteins, the majority had fewer than three crotonylation sites, with only a few proteins having seven or more crotonylation sites. With respect to the plant species for which crotonylation data have been reported, there were 701 (tobacco), 234 (rice), and 1566 (papaya) conserved crotonylation sites respectively accounting for 11.6%, 3.9%, and 25.9% of the total crotonylation sites in peanut ( Figure 1A,C, Table S11). The MS data validation is shown in Figure S1. Firstly, we checked the mass error values of all the identified peptides. The distribution of mass error was near zero and most of them were less than 0.02 Da, which meant that the mass accuracy of the MS data fitted the requirements ( Figure S1B). Secondly, the lengths of most peptides were distributed between 8 and 20 residues,

Analysis of crotonylation site motifs
To investigate whether there were any patterns of amino acids adjacent to the Kcr sites, we used the Motif-X program. A total of six clearly conserved motifs (with a motif score > 25) were identified (Figure 2A), namely EKcrG, AKcrE, EKcrA, EKcrE, DKcrI, and EKcrV. The statistics of the data are presented in Table S3; a substantial bias in terms of amino acid distribution was observed from the −10 to

Structural analysis of all the crotonylated proteins
To understand the relationship between Kcr and protein structure, the structures of all the crotonylated proteins were analyzed. A total of 46.5% of the crotonylated sites were located in α-helices, 10.6% of the sites were in β-strands, and the remaining sites were in disordered coils. The distribution pattern between the crotonylated lysines and all lysines was very similar ( Figure 2C). In addition, analysis of surface accessibility of crotonylation sites of peanut proteins showed that Kcr did not affect the surface properties of the modified proteins, because the enrichment of crotonylated sites on the protein surface was very similar to that of all lysine residues ( Figure 2D).

Functional enrichment analysis of crotonylated proteins
In order to identify the types of proteins that were involved in lysine crotonylation, we performed GO enrichment and KEGG pathway analysis. GO enrichment analysis included biological process, cellular component, and molecular function classifications ( Figure 4A, Table S6). Enrichment analysis of the biological process grouping indicated that crotonylated proteins were significantly enriched with respect to biosynthetic processes and catabolic processes. In the cellular component category, the crotonylated proteins were significantly enriched with respect to the cytoplasmic component. There were also many crotonylated proteins associated with thylakoids, intracellular non-membrane-bound organelles, and the proteasome core complex.
With respect to enrichment in the molecular function category, most crotonylated proteins were enriched with respect to ligase activity, cofactor binding, structural constituents of ribosomes, oxidoreductase activity, and isomerase activity ( Figure 4A, Table S6).

F I G U R E 4 Enrichment analysis of crotonylated proteins. (A) GO-based enrichment analysis of identified proteins. (B) KEGG pathway-based enrichment analysis. (C) Protein domain enrichment analysis of all identified proteins
The KEGG pathway indicated that Kcr was associated with many proteins involved in carbon fixation in photosynthetic organisms, aminoacyl-tRNA biosynthesis, glutathione (GSH) metabolism, the pentose phosphate pathway, biosynthesis of amino acids, ribosomes, carbon metabolism, proteasomes, photosynthesis, glyoxylate and dicarboxylate metabolism, oxidative phosphorylation, pyruvate metabolism, glycolysis/gluconeogenesis, the citrate cycle (TCA cycle), photosynthesis-antenna proteins, and alanine, aspartate, and glutamate metabolism ( Figure 4B, Table S7). These results implied that crotonylated proteins played important regulatory roles in a wide range of important processes. In line with these results, the ClpP/crotonase-like domain, glutathione S-transferase (GST), Cterminal-like and GST N-terminal domains were clearly enriched among the crotonylated proteins ( Figure 4C, Table S8). showed that a large number of subunits of ribosomes were crotonylated ( Figure 5B). These results showed that crotonylation in proteins may involved in proteins synthesis and processing.

Interactive networks among crotonylated proteins in peanuts
To further investigate the regulatory role of crotonylation in photosynthesis and ribosomes, we used the algorithm from the Cytoscape software to establish protein interaction networks ( Figure 6A, Table S9).
In the peanut photosynthesis network, 26 crotonylated proteins were identified as nodes in the protein interaction database. In the peanut ribosome network, 145 proteins were mapped to the protein interaction database ( Figure 6B, Table S10). These maps provided a view of crotonylated proteins involved in photosynthetic and ribosomeassociated biological processes.

DISCUSSION
Several recent reports have shown that crotonylation plays an important role in various biological processes in humans, animals [26], plants [25,29], and microorganisms [28]. Crotonylation has been reported in several plants, such as tobacco [25], rice [27], papaya [29], and tea [7], but there has been no report of crotonylation in leguminous plants, such as peanut. In the present study, we used a combination of immunoaffinity purification and high-resolution LC-MS/MS analysis to identify 6051 lysine crotonylation sites in 2508 protein groups.
Most of these proteins had fewer than three crotonylation sites and were involved in diverse biological processes, cellular components, and molecular functions. These data showed that crotonylated proteins played a diverse and very important role in peanut biological functions. Since we focused on qualitative identification of cronotylation sites, these data suggest that crotonylation may be a common protein modification that occurs at many sites but it is not necessarily very abundant.
With respect to the subcellular localization of crotonylated proteins, the distributions reported for rice, papaya, tea, and tobacco were identical, with the order of location frequencies, from high to low, being in the chloroplast, cytosol, nuclear, and mitochondrion. The distribution pattern in peanut was very similar to those in the earlier-described plants, with the first three locations being the same, but the fourth location on the list in peanut was the plasma membrane, rather than the mitochondrion. It is worth noting that the identify of different species may not use the identical experimental approach, there may occur small deviation, so that comparisons between qualitative lists are dependent on the lower limit of identification. Thus, when comparing lists from different samples the presence or absence is a reflection of changes in abundance relative to the low limit of identification.
Absence from a list does not infer non-existence, but analysis and the network of Kcr also showed that many ribosomal proteins were crotonylated. A similar phenomenon has been reported in tobacco [25] and rice [27]. These findings suggest that lysine crotonylation may play important regulatory roles in peanut photosynthesis and ribosome structure and function.
There is considerable research interest in Kac, which has been reported in many plants, such as Arabidopsis [34,35], rice [36,37], wheat [34], and soybean [38]. A number of acetylated proteins have been localized to the chloroplast and have been shown to take part in photosynthesis. Kcr and Kac can occur on the same protein, but they may not have exactly the same modification sites [39]. In rice, there were 21 acetylated proteins and 16 overlapping proteins, which were both crotonylated and acetylated [27,36]. Compared with acetylation, crotonylation has a greater ability to promote gene expression, while the balance between crotonylation and acetylation (another PTM) has functional effects on gene expression [14]. Histone crotonylation reprogrammed the function of the nucleosome, distinguishing it from histone acetylation, by interacting with a specific set of chromatin modifiers [30,[40][41][42]. These lysine modifications can be changed by environmental stresses (e.g., starvation or submergence) or metabolic cues, controlling the relative proportions in histone-acylated plants, which, in turn, control the expression of responsive genes [43].
PTMs impact a diverse array of cellular processes, and Kac is one of the main PTMs studied. It has been reported that Kac plays a central role in the host-pathogen interaction [44]. Research into plant defense signaling has established that modulation of histone acetylation is very important for immune responses in plants. GSH is an important reducing substance commonly present in plant, and plays an important role in the antioxidant defense against the oxidation of membrane lipids by free radicals. The GSH-conjugating enzymes, known as GSTs, are present in different subcellular compartments, and upregu-lation appears to be an evolutionary response by cells to achieve the protection against chemical toxicity and oxidative stress [45][46][47]. Plant GST genes are expressed differently when plants encounter biotic and abiotic stresses. The stress-induced plant growth regulator, nitric oxide, differentially affects the expression level of GSTs [48,49]. The current study showed that there were 19 lysine-crotonylated proteins involved in GSH metabolism ( Figure S2). GST, C-terminal-like and GST N-terminal domains were clearly enriched with respect to crotonylated proteins. These results suggested that crotonylated proteins involved in GSH may play an important role in plant response to biotic and abiotic stresses. There has been some research on Kcr involvement in response to stress in different plant species [7,43]. When checked the respond to stress in biological process, we obtained 222 crotonylated sites were involved (Table S2)

CONFLICT OF INTEREST
The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT
The mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD017675.