This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Research article
yuanzhen lin
South China Agricultural University
Corresponding Author
ORCiD: https://orcid.org/0000-0001-9042-8016
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background
ICE1 (inducer of CBF expression 1), a MYC-like bHLH transcriptional activator, plays an important role in plant under cold stress via regulating transcriptional expression of downstream cold-responsive genes. Ubiquitination-proteasome pathway mediated by high expression of osmotically responsive gene1 (HOS1) can effectively induce the degradation of ICE1 and decrease the expression of expression of CBFs and their downstream genes under cold stress response in Arabidopsis , but the knowledge about ubiquitination regulation of ICE1 by HOS1 is still unknown in woody plants.
Results
The complete EcaICE1 gene and a new E3 ubiquitin ligase gene EcaHOS1 were amplified from the tissue culture seedlings of Eucalyptus camaldulensis . Yeast two-hybrid (Y2H) and BiFC assay results showed that EcaICE1 can interact with EcaHOS1 protein in the nucleus, and further Y2H assay demonstrated that the 126-185 amino acid region at the N-terminus of EcaICE1 protein was indispensable for its interaction with EcaHOS1 protein. Moreover, we found that the amino acids at positions 143, 145, 158 and 184 within the key interaction region were the potential phosphorylation sites of EcaICE1 based on bioinformatics analysis, and that only the substitution of Serine (Ser) 158 by Alanine (Ala) blocked the protein-protein interactions between EcaICE1 and EcaHOS1 by Y2H and β-galactosidase assays using site-direct mutagenesis. Overexpression of EcaICE1 and its mutations in Arabidopsis could significantly increase POD and SOD activities with a reduction for MDA content and up-regulate four cold-responsive genes ( CBF3 , KIN1 , COR15 and COR47A ) in the transgenic lines.
Conclusion
We first reported that EcaICE1 could interact with EcaHOS1 protein in Eucalyptus , and identified Ser 158 of EcaICE1 as the key phosphorylation site for its interaction with EcaHOS1 protein.
Keywords
EcaICE1, Key Phosphorylation Sites, cold stress, Arabidopsis
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This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Research article
yuanzhen lin
South China Agricultural University
Corresponding Author
ORCiD: https://orcid.org/0000-0001-9042-8016
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 22 Jan, 2020
No community comments so far
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background
ICE1 (inducer of CBF expression 1), a MYC-like bHLH transcriptional activator, plays an important role in plant under cold stress via regulating transcriptional expression of downstream cold-responsive genes. Ubiquitination-proteasome pathway mediated by high expression of osmotically responsive gene1 (HOS1) can effectively induce the degradation of ICE1 and decrease the expression of expression of CBFs and their downstream genes under cold stress response in Arabidopsis , but the knowledge about ubiquitination regulation of ICE1 by HOS1 is still unknown in woody plants.
Results
The complete EcaICE1 gene and a new E3 ubiquitin ligase gene EcaHOS1 were amplified from the tissue culture seedlings of Eucalyptus camaldulensis . Yeast two-hybrid (Y2H) and BiFC assay results showed that EcaICE1 can interact with EcaHOS1 protein in the nucleus, and further Y2H assay demonstrated that the 126-185 amino acid region at the N-terminus of EcaICE1 protein was indispensable for its interaction with EcaHOS1 protein. Moreover, we found that the amino acids at positions 143, 145, 158 and 184 within the key interaction region were the potential phosphorylation sites of EcaICE1 based on bioinformatics analysis, and that only the substitution of Serine (Ser) 158 by Alanine (Ala) blocked the protein-protein interactions between EcaICE1 and EcaHOS1 by Y2H and β-galactosidase assays using site-direct mutagenesis. Overexpression of EcaICE1 and its mutations in Arabidopsis could significantly increase POD and SOD activities with a reduction for MDA content and up-regulate four cold-responsive genes ( CBF3 , KIN1 , COR15 and COR47A ) in the transgenic lines.
Conclusion
We first reported that EcaICE1 could interact with EcaHOS1 protein in Eucalyptus , and identified Ser 158 of EcaICE1 as the key phosphorylation site for its interaction with EcaHOS1 protein.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
This is a list of supplementary files associated with this preprint. Click to download.
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