Proteome-wide and lysine crotonylation profiling reveals the importance of crotonylation in chrysanthemum (Dendranthema grandiforum) under low-temperature

Abstract

Background: Low-temperature severely affects the growth and development of chrysanthemum which is one kind of ornamental plant well-known and widely used in the world. Lysine crotonylation is a recently identified post-translational modification (PTM) with multiple cellular functions. However, lysine crotonylation under low-temperature stress has not been studied.

Results: Proteome-wide and lysine crotonylation of chrysanthemum at low-temperature was analyzed using TMT (Tandem Mass Tag) labeling, sensitive immuno-precipitation, and high-resolution LC-MS/MS. The results showed that 2017 crotonylation sites were identified in 1199 proteins. Treatment at 4 °C for 24 h and - 4 °C for 4 h resulted in 393 upregulated proteins and 500 downregulated proteins (1.2-fold threshold and P < 0.05). Analysis of biological information showed that lysine crotonylation was involved in photosynthesis, ribosomes, and antioxidant systems. The crotonylated proteins and motifs in chrysanthemum were compared with other plants to obtain orthologous proteins and conserved motifs. To further understand how lysine crotonylation at K136 affected APX (ascorbate peroxidase), we performed a site-directed mutation at K136 in APX. Site-directed crotonylation showed that lysine decrotonylation at K136 reduced APX activity, and lysine complete crotonylation at K136 increased APX activity.

Conclusion: In summary, our study comparatively analyzed proteome-wide and crotonylation in chrysanthemum under low-temperature stress and provided insights into the mechanisms of crotonylation in positively regulated APX activity to reduce the oxidative damage caused by low-temperature stress. These data provided an important basis for studying crotonylation to regulate antioxidant enzyme activity in response to low-temperature stress and a new research ideas for chilling-tolerance and freezing-tolerance chrysanthemum molecular breeding.

Keywords: APX activity; Biological functions; Chrysanthemum; Crotonylation; Low-temperature; Proteome.

Fig. 1

The phenotype, survival rate, and ROS content of chrysanthemum under low-temperature stress. T refers to chrysanthemums treated first at 4 °C for 24 h and then − 4 °C for 4 h, while CK is chrysanthemum without low-temperature treatment. The value measured without low-temperature treatment is taken as 1, and the relative content refers to the ratio between the low-temperature treatment and the untreated. a The phenotypic changes of chrysanthemum plants under low-temperature; (b) the survival rate of chrysanthemum under low-temperature; (c-d) histochemical staining with DAB and NBT for assessing the accumulation of H₂O₂ and O₂⁻, respectively, under low-temperature. e Relative activity of SOD in chrysanthemum leaves before and after low-temperature treatment; (f) relative activity of POD in chrysanthemum leaves before and after low-temperature treatment; (g) relative activity of CAT in chrysanthemum leaves before and after low-temperature treatment; (h) relative activity of APX in chrysanthemum leaves before and after low-temperature treatment; (i) relative content of glutathione in chrysanthemum leaves before and after low-temperature treatment; (j) relative content of chlorophyll in chrysanthemum leaves before and after low-temperature treatment. Data represent means and standard errors of three replicates. Different letters above the columns indicate significant (P < 0.05) differences according to Duncan’s multiple range test

Fig. 2

Functional classification and enrichment analysis of differentially quantified crotonylated proteins in chrysanthemum under low-temperature. a Cellular component of GO annotation analysis. b Biological process of GO annotation analysis. c Molecular function of GO annotation analysis. d Predicted subcellular localization analysis. e GO enrichment analysis. f KEGG enrichment analysis. The negative logarithm of Fisher’s exact test P value is shown on the X axes. The number of proteins found in each GO class and the number of all proteins present in each GO class were provided in the brackets followed the scores

Fig. 3

Venn diagram of the orthologous crotonylated protein of chrysanthemum, tea, rice, papaya, and tobacco

Fig. 4

Lysine conservation of chrysanthemum compared with other species

Fig. 5

Bioinformatic analysis of lysine crotonylation sites in chrysanthemum under low-temperature. a Plot shows the relative abundance of amino acids flanking crotonylated lysine. The relative abundance was counted and schematically represented by an intensity map. The intensity map shows the enrichment of amino acids in specific positions of crotonylated lysine (10 amino acids upstream and downstream of the crotonylation site). b Probability sequence motifs of crotonylation sites consisting of 10 residues surrounding the targeted lysine residue using Motif-X

Fig. 6

Stacked histogram of crotonylation motifs of chrysanthemum compared with other species

Fig. 7

The amino acid sequence of DgAPX was compared with that of other plants. Note: The black triangles indicated the heme binding site, the substrate binding site, and the K⁺ binding site in turn; the red triangles indicated the lysine crotonylation site on the amino acid sequence of DgAPX; the red rectangles indicated the ascorbate peroxidase conserved domain. AoAPX2 (XP_020276942.1) from Asparagus officinalis; CaAPX1 (NP_001311967.1) from Capsicum annuum; MsAPX2 (AIY27528.1) from Medicago sativa; NtAPX2 (NP_001311803.1) from Nicotiana tabacum; RhAPX1 (ATP66493.1) from Rosa hybrid cultivar; SiAPX2 (NP_001318094.1) from Solanum lycopersicum; SpAPX1 (XP_015079739.1) from Solanum pennellii

Fig. 8

Western blot and APX activity detection. a Western blot and coomassie of wildtype tobacco (WT), tobacco infected with empty carrier (pSuper1300-GFP), infected unmutated tobacco (pSuper1300-DgAPX-GFP), infected simulant decrotonylation tobacco (pSuper1300-DgAPX^K136R-GFP), and infected simulant complete crotonylation tobacco (pSuper1300-DgAPX^K136N-GFP) under a normal temperature (25 °C; 12 h). b APX activity detection of five tobaccos treated under normal temperature (25 °C; 12 h). The different letters above the columns indicate significant (P < 0.05) differences according to Duncan’s multiple range test