First comprehensive proteome analysis of lysine crotonylation in seedling leaves of Nicotiana tabacum

Abstract

Histone crotonylation is a new lysine acylation type of post-translational modification (PTM) enriched at active gene promoters and potential enhancers in yeast and mammalian cells. However, lysine crotonylation in nonhistone proteins and plant cells has not yet been studied. In the present study, we performed a global crotonylation proteome analysis of Nicotiana tabacum (tobacco) using high-resolution LC-MS/MS coupled with highly sensitive immune-affinity purification. A total of 2044 lysine modification sites distributed on 637 proteins were identified, representing the most abundant lysine acylation proteome reported in the plant kingdom. Similar to lysine acetylation and succinylation in plants, lysine crotonylation was related to multiple metabolism pathways, such as carbon metabolism, the citrate cycle, glycolysis, and the biosynthesis of amino acids. Importantly, 72 proteins participated in multiple processes of photosynthesis, and most of the enzymes involved in chlorophyll synthesis were modified through crotonylation. Numerous crotonylated proteins were implicated in the biosynthesis, folding, and degradation of proteins through the ubiquitin-proteasome system. Several crotonylated proteins related to chromatin organization are also discussed here. These data represent the first report of a global crotonylation proteome and provide a promising starting point for further functional research of crotonylation in nonhistone proteins.

Figure 1

Proteome-wide identification of lysine crotonylation sites in Nicotiana tabacum. (a) Overview of experimental procedures used in the present study. Kcr indicates the crotonylated lysine. (b) Distribution of lysine crotonylation in one protein. (c) Distribution of lysine crotonylation peptides based on their length. (d) Mass error distribution of all crotonylated peptides.

Figure 2

Properties of the lysine crotonylation sites. (a) Sequence probability logos of significantly enriched crotonylation site motifs around the lysine crotonylation sites. (b) Heat map of the amino acid compositions around the lysine crotonylation sites showing the frequency of different types of amino acids around this residue. Red indicates enrichment and green indicates depletion. (c) Probabilities of lysine crotonylation in different protein secondary structures (alpha helix, beta-strand and disordered coil). (d) Predicted surface accessibility of crotonylation sites.

Figure 3

GO classification of the crotonylated proteins based on biology process (a) molecular functional (b) and subcellular localization (c), respectively.

Figure 4

Enrichment analysis of crotonylated proteins. (a) GO-based enrichment analysis of crotonylated proteins in terms of cellular component, molecular function, and biological process. (b) KEGG pathway-based enrichment analysis. (c) Protein domain enrichment analysis. The numbers in X axes represent the value of significant analysis. When the value is greater than 1.3, the p value is less than 0.05, which means the data is statistically significant.

Figure 5

Interaction networks of the crotonylated proteins in tobacco.