The crotonylated and succinylated proteins of jujube involved in phytoplasma-stress responses

Abstract

Background: Protein posttranslational modifications (PTMs) are fast and early responses to environmental changes, including pathogen infection. Jujube witches' broom (JWB) is a phytoplasma disease causing great economic loss in jujube production. After phytoplasma infection, the transcriptional, translational, and metabolic levels in jujube were activated, enabling it to survive during phytoplasma invasion. However, no study has yet reported on PTMs in jujube. Lysine crotonylation (Kcr) and lysine succinylation (Ksu) have been popular studies in recent years and their function in plant phytoplasma-stress responses remains unclear.

Results: Here, 1656 crotonylated and 282 succinylated jujube proteins were first identified under phytoplasma-stress, of which 198 were simultaneously crotonylated and succinylated. Comparative analysis revealed that 656 proteins, 137 crotonylated and 43 succinylated proteins in jujube were regulated by phytoplasma infection, suggesting that Kcr was more universal than Ksu. Kcr differentially expressed proteins (DEPs) were related to ribosomes, photosynthetic and carbon metabolism, while Ksu DEPs were mainly involved in carbon metabolism, the TCA cycle and secondary metabolite biosynthesis. The crosstalk network among proteome, crotonylome and succinylome showed that DEPs related to ribosomal, peroxidases and glutathione redox were enriched. Among them, ZjPOD51 and ZjPHGPX2 significantly increased at the protein and Kcr level under phytoplasma-stress. Notably, 7 Kcr sites were identified in ZjPHGPX2, a unique antioxidant enzyme. After inhibitor nicotinamide (NAM) treatment, GPX enzyme activity in jujube seedlings was reduced. Further, site-directed mutagenesis of key Kcr modification sites K130 and/or K135 in ZjPHGPX2 significantly reduced its activity.

Conclusions: This study firstly provided large-scale datasets of Kcr and Ksu in phytoplasma-infected jujube and revealed that Kcr modification in ZjPHGPX2 positively regulates its activity.

Keywords: Crotonylome; Enzyme activity; Inhibitor NAM; Jujube phloem; Phytoplasma-stress; Site-directed mutagenesis; Succinylome; ZjPHGPX2.

Fig. 1

Profile of identified crotonylated and succinylated proteins and sites in jujube under phytoplasma stress. A WB with pan anti-2-hydroxyisobutyryllysine antibody. H1, H2 and H3 were three healthy samples; D1, D2 and D3 were three diseased samples. B WB with pan anti-acetyllysine antibody. C WB with pan anti-succinyllysine antibody. D WB with pan anti-crotonyllysine antibody. E The statistical analysis of the overlap between the Kcr and Ksu proteins. F The statistic of the overlap between the Kcr and Ksu sites

Fig. 2

Distribution and amino acid compositions of the Kcr and Ksu sites. A Distribution of Kcr peptides in one protein. B Distribution of Ksu peptides in one protein. C The intensity map of crotonylation motif shows the relative abundance of ± 10 amino acids flanking the crotonylated lysine site. The colors in the intensity map mean the log10 (the frequencies within crotonyl-21-mers/the frequencies within non-crotonyl-21-mers) (red, enrichment; green, depletion); D The intensity map of succinylation motif shows the relative abundance of ± 10 amino acids flanking the succinylated lysine site. The colors in the intensity map mean the log10 (the frequencies within succinyl-21-mers/the frequencies within non-succinyl-21-mers) (red, enrichment; green, depletion)

Fig. 3

Functional enrichment analysis of crotonylated and succinylated DEPs in jujube under phytoplasma stress. A Predicted subcellular localization analysis of crotonylated and succinylated DEPs. B KOG functional classification chart of proteins corresponding to differentially expressed modification sites. C KEGG enrichment analysis of crotonylated DEPs. D KEGG enrichment analysis of succinylated DEPs. The negative logarithm of Fisher’s exact test P value is shown on the X axes. The number of proteins found in each category was provided after the score

Fig. 4

Crosstalk among JWB-related proteome, crotonylome and succinylome

Fig. 5

ZjPHGPX2 and ZjPOD51 were responsive to phytoplasma-stress. A Heat map analysis of DEPs related to ribosomal, peroxidase and glutathione redox in the healthy and diseased jujube trees. The accessions of proteins and their relative expressions in translation level were listed in Additional file 8: Table S4. B The transcriptional expressions of some DEPs by qRT-PCR. All data are presented as means ± SD from three independent experiments. Statistical significance was determined by independent t-test (*, P < 0.05; **, P < 0.01; ***, P < 0.001). C POD and GPX enzyme activities in healthy and diseased jujube trees (field) and seedlings (tissue culture). The raw data in Fig. 5B and 5C were shown in Additional file 9: Table S5. D WB analysis of ZjPHGPX2 (19 kDa) and ZjPOD51 (35 kDa) in healthy and diseased jujube trees (field) and seedlings (tissue culture) under phytoplasma stress. The original, uncropped gels/blots were shown in Additional file 10: Fig. S5

Fig. 6

Inhibitors NAM significantly reduced the Kcr modification of ZjPHGPX2. A Phenotype of diseased seedlings after NAM treatment. B GPX enzyme activities of jujube seedlings after NAM treatment. The raw data were shown in Additional file 9: Table S5. C The diagram of ion peak area distribution of ZjPHGPX2^K130 crotonylated peptide in healthy (H1, H2, H3) and diseased samples (D1, D2, D3). D WB analysis of ZjPHGPX2^Kcr130 in healthy and diseased jujube trees (field) and seedlings (tissue culture), and diseased seedlings after NAM treatment. The original, uncropped gels/blots were shown in Additional file 11: Fig. S6

Fig. 7

Kcr modification at site K130 and/or K135 positively regulates ZjPHGPX2 activity. A Location of Kcr sites in ZjPHGPX2. B Multiple sequence alignment of PHGPXs among jujube and other 6 species. C The tertiary structure prediction of ZjPHGPX2 constructed by SWISS-MODEL. D Effect of site-directed mutations at K130 and/or K135 of ZjPHGPX2 on GPX activity in E. coli. The different letters above the columns indicate significant differences according to Duncan’s multiple range test (P < 0.05). Values were presented as the mean of three replicates. The raw data were shown in Additional file 9: Table S5. WB analysis of ZjPHGPX2^Kcr130 levels in different mutations. The original, uncropped gels/blots were shown in Additional file 14: Fig. S8