Proteome-Wide Analyses Reveal the Diverse Functions of Lysine 2-Hydroxyisobutyrylation in Oryza sativa

doi:10.1186/s12284-020-00389-1

. 2020 Jun 5;13(1):34.

doi: 10.1186/s12284-020-00389-1.

Proteome-Wide Analyses Reveal the Diverse Functions of Lysine 2-Hydroxyisobutyrylation in Oryza sativa

Chao Xue^{1

2}, Zhongying Qiao³, Xu Chen^{1

2}, Penghui Cao³, Kai Liu^{1

2}, Shuai Liu^{1

2}, Lu Ye^{1

2}, Zhiyun Gong^{4

5}

Affiliations

¹ Jiangsu Key Laboratory of Crop Genetics and Physiology/ Key Laboratory of Plant Functional Genomics of the Ministry of Education/ Jiangsu Key Laboratory of Crop Genomics and Molecular Breeding, Agricultural College of Yangzhou University, Yangzhou, 225009, China.
² Jiangsu Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, Yangzhou, 225009, China.
³ Suzhou Academy of Agricultural Sciences, North of Wangting Town, Suzhou, 215128, China.
⁴ Jiangsu Key Laboratory of Crop Genetics and Physiology/ Key Laboratory of Plant Functional Genomics of the Ministry of Education/ Jiangsu Key Laboratory of Crop Genomics and Molecular Breeding, Agricultural College of Yangzhou University, Yangzhou, 225009, China. zygong@yzu.edu.cn.
⁵ Jiangsu Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, Yangzhou, 225009, China. zygong@yzu.edu.cn.

PMID: 32572646
PMCID: PMC7310055
DOI: 10.1186/s12284-020-00389-1

Proteome-Wide Analyses Reveal the Diverse Functions of Lysine 2-Hydroxyisobutyrylation in Oryza sativa

Chao Xue et al. Rice (N Y). 2020.

. 2020 Jun 5;13(1):34.

doi: 10.1186/s12284-020-00389-1.

Authors

Chao Xue^{1

2}, Zhongying Qiao³, Xu Chen^{1

2}, Penghui Cao³, Kai Liu^{1

2}, Shuai Liu^{1

2}, Lu Ye^{1

2}, Zhiyun Gong^{4

5}

Affiliations

¹ Jiangsu Key Laboratory of Crop Genetics and Physiology/ Key Laboratory of Plant Functional Genomics of the Ministry of Education/ Jiangsu Key Laboratory of Crop Genomics and Molecular Breeding, Agricultural College of Yangzhou University, Yangzhou, 225009, China.
² Jiangsu Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, Yangzhou, 225009, China.
³ Suzhou Academy of Agricultural Sciences, North of Wangting Town, Suzhou, 215128, China.
⁴ Jiangsu Key Laboratory of Crop Genetics and Physiology/ Key Laboratory of Plant Functional Genomics of the Ministry of Education/ Jiangsu Key Laboratory of Crop Genomics and Molecular Breeding, Agricultural College of Yangzhou University, Yangzhou, 225009, China. zygong@yzu.edu.cn.
⁵ Jiangsu Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, Yangzhou, 225009, China. zygong@yzu.edu.cn.

PMID: 32572646
PMCID: PMC7310055
DOI: 10.1186/s12284-020-00389-1

Abstract

Background: Lysine 2-hydroxyisobutyrylation (Khib), a newly identified post-translational modification, is known to regulate transcriptional activity in animals. However, extensive studies of the lysine 2-hydroxyisobutyrylome in plants and animals have yet to be performed.

Results: In this study, using LC-MS/MS qualitative proteomics strategies, we identified 4163 Khib sites on 1596 modified proteins in rice (Oryza sativa) seedlings. Motif analysis revealed 10 conserved motifs flanking the Khib sites, and subcellular localization analysis revealed that 44% of the Khib proteins are localized in the chloroplast. Gene ontology function, KEGG pathway, and protein domain enrichment analyses revealed that Khib occurs on proteins involved in diverse biological processes and is especially enriched in carbon metabolism and photosynthesis. Among the modified proteins, 20 Khib sites were identified in histone H2A and H2B, while only one site was identified in histone H4. Protein-protein interaction (PPI) network analysis further demonstrated that Khib participates in diverse biological processes including ribosomal activity, biosynthesis of secondary metabolites, and metabolic pathways. In addition, a comparison of lysine 2-hydroxyisobutyrylation, acetylation, and crotonylation in the rice proteome showed that 45 proteins with only 26 common lysine sites are commonly modified by three PTMs. The crosstalk of modified sites and PPI among these PTMs may form a complex network with both similar and different regulatory mechanisms.

Conclusions: In summary, our study comprehensively profiles the lysine 2-hydroxyisobutyrylome in rice and provides a better understanding of its biological functions in plants.

Keywords: Histone acylation; Lysine 2-hydroxyisobutyrylation; Oryza sativa; Photosynthesis.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

**Fig. 1**
Workflow for large-scale identification of Khib sites in rice. a Western blotting analysis of total proteins in rice seedling leaves. The gels for SDS-PAGE were stained with Coomassie Blue. b Workflow for identification of Khib modification in rice. Total proteins extracted from seedling leaves were digested to peptides by trypsin. Peptides bearing Khib sites were affinity enriched with a specific Khib antibody and subsequently analyzed by LC-MS/MS.

**Fig. 2**
Motif analysis of all identified Khib sites. a Conserved motifs identified around the Khib sites. The size of each letter reflects the frequency of that amino acid residue in that position. b Frequency for each motif occurring in peptides with Khib modification. c Heat-map of the amino acid compositions of the Khib sites, showing the enrichment (red) and depletion (green) of amino acids in each position (from − 10 to + 10) flanking the Khib sites

**Fig. 3**
GO functional classification and subcellular localization of Khib modified proteins. a Classification of Khib proteins based on biological process, molecular function, and cellular component. b Subcellular localization of Khib proteins

**Fig. 4**
KEGG pathway and Protein domain enrichment analyses of 2-hydroxyisobutyrylated proteins

**Fig. 5**
Khib proteins enriched in carbon metabolism and photosynthesis pathways in rice. The 2-hydroxyisobutyrylated enzymes or proteins identified are highlighted in red

**Fig. 6**
Comparison of the 2-hydroxyisobutyrylome with the lysine acetylome and lysine crotonylome in rice leaves. a The overlapped number of Khib proteins among Khib, Kac, and Kcr. b The overlapped number of Khib sites among Khib, Kac, and Kcr. c Khib, Kac, and Kcr sites on the representative protein Rubisco activase (P93431). d Comparison of histone H3 and H4 with Khib, Kac, and Kcr sites between rice and human

**Fig. 7**
The three most enriched clusters in the PPI networks of Khib proteins. The network of Khib protein interactions (listed with protein ID names) as analyzed using Cytoscape. a Ribosome. b Carbon metabolism. c Photosynthesis. The size of the dots represents the number of Khib sites in each figure

**Fig. 8**
The crosstalk PPI network of the overlapped proteins with Khib modification

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References

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[1] Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT, Harris MA, Hill DP, Issel-Tarver L, Kasarskis A, Lewis S, Matese JC, Richardson JE, Ringwald M, Rubin GM, Sherlock G, Consortium GO Gene ontology: tool for the unification of biology. Nat Genet. 2000;25(1):25–29. doi: 10.1038/75556. - DOI - PMC - PubMed

[2] Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT, Harris MA, Hill DP, Issel-Tarver L, Kasarskis A, Lewis S, Matese JC, Richardson JE, Ringwald M, Rubin GM, Sherlock G, Consortium GO Gene ontology: tool for the unification of biology. Nat Genet. 2000;25(1):25–29. doi: 10.1038/75556. - DOI - PMC - PubMed

[3] Ashikari M, Sakakibara H, Lin SY, Yamamoto T, Takashi T, Nishimura A, Angeles ER, Qian Q, Kitano H, Matsuoka M. Cytokinin oxidase regulates rice grain production. Science. 2005;309(5735):741–745. doi: 10.1126/science.1113373. - DOI - PubMed

[4] Ashikari M, Sakakibara H, Lin SY, Yamamoto T, Takashi T, Nishimura A, Angeles ER, Qian Q, Kitano H, Matsuoka M. Cytokinin oxidase regulates rice grain production. Science. 2005;309(5735):741–745. doi: 10.1126/science.1113373. - DOI - PubMed

[5] Bader GD, Hogue CW. An automated method for finding molecular complexes in large protein interaction networks. BMC Bioinformatics. 2003;4:2. doi: 10.1186/1471-2105-4-2. - DOI - PMC - PubMed

[6] Bader GD, Hogue CW. An automated method for finding molecular complexes in large protein interaction networks. BMC Bioinformatics. 2003;4:2. doi: 10.1186/1471-2105-4-2. - DOI - PMC - PubMed

[7] Chen Y, Sprung R, Tang Y, Ball H, Sangras B, Kim SC, Falck JR, Peng JM, Gu W, Zhao YM. Lysine propionylation and butyrylation are novel post-translational modifications in histones. Mol Cell Proteomics. 2007;6(5):812–819. doi: 10.1074/mcp.M700021-MCP200. - DOI - PMC - PubMed

[8] Chen Y, Sprung R, Tang Y, Ball H, Sangras B, Kim SC, Falck JR, Peng JM, Gu W, Zhao YM. Lysine propionylation and butyrylation are novel post-translational modifications in histones. Mol Cell Proteomics. 2007;6(5):812–819. doi: 10.1074/mcp.M700021-MCP200. - DOI - PMC - PubMed

[9] Chen ZJ, Tian L. Roles of dynamic and reversible histone acetylation in plant development and polyploidy. Biochim Biophys Acta. 2007;1769(5–6):295–307. doi: 10.1016/j.bbaexp.2007.04.007. - DOI - PMC - PubMed

[10] Chen ZJ, Tian L. Roles of dynamic and reversible histone acetylation in plant development and polyploidy. Biochim Biophys Acta. 2007;1769(5–6):295–307. doi: 10.1016/j.bbaexp.2007.04.007. - DOI - PMC - PubMed

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Proteome-Wide Analyses Reveal the Diverse Functions of Lysine 2-Hydroxyisobutyrylation in Oryza sativa

Affiliations

Proteome-Wide Analyses Reveal the Diverse Functions of Lysine 2-Hydroxyisobutyrylation in Oryza sativa

Authors

Affiliations

Abstract

Conflict of interest statement

Figures

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References

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