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Overexpression of the transcription factor MdbHLH33 increases cold tolerance of transgenic apple callus

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Abstract

As cold stress greatly affects plant growth and development, understanding the mechanisms underlying cold tolerance in plants is important. In this study, we analyzed the expression levels of apple (Malus domestica) MdbHLH33 and MdCBF1–5 by semi-quantitative PCR after exposure to 4 °C for different amounts of time and generated evolutionary trees for MdbHLH33 and the MdCBFs. Overexpressing MdbHLH33 pro-GUS in ‘Orin’ callus, indicated that transgenic callus had higher GUS activity and was more deeply stained at 4 °C than at 25 °C. Subcellular localization showed that MdbHLH33 was located in the nucleus. Overexpressing MdbHLH33 in ‘Orin’ callus increased the expression level of MdCBF2, MdCOR15A-1, and MdCOR15A-2, and resulted in increased cold tolerance. EMSA and Chip-PCR analysis showed that MdbHLH33 could bind the LTR cis-acting element found in the MdCBF2 promoter. Overexpressing MdCBF2 in ‘Orin’ callus indicated that MdCBF2 could also increase the expression level of MdCOR15A-1 and MdCOR15A-2 and improve cold tolerance; we also found that transgenic callus overexpressing MdCBF2 had reduced MdCBF1 and MdCBF5 expression and increased MdCBF3 and MdCBF4 expression. Overall, these results show that MdbHLH33 can regulate the expression of MdCBF2 and improve the cold tolerance of transgenic callus.

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Abbreviations

bHLH:

Basic helix–loop–helix

GUS:

β-Glucuronidase

EMSA:

Electrophoretic mobility shift assays

Chip-PCR:

Chromatin immunoprecipitation-polymerase chain reaction

ICE:

Inducer of CBF expression

CBF:

C-repeat-binding factor

CORs:

Cold-responsive genes

MES:

4-Morpholineethanesulfonic acid

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (31572091) and National Key Research and Development Project of China (2016YFC0501505). We thank Liwen Bianji, Edanz Group China (http://www.liwenbianji.cn), for editing the English text of a draft of this manuscript.

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Authors and Affiliations

  1. National Key Laboratory of Crop Biology, College of Horticulture Science, Shandong Agricultural University, Tai’an, Shandong, China

    Haifeng Xu, Nan Wang, Yicheng Wang, Shenghui Jiang, Hongcheng Fang, Jing Zhang, Mengyu Su, Weifang Zuo, Lin Xu, Zongying Zhang & Xuesen Chen

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  1. Haifeng Xu
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  2. Nan Wang
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  3. Yicheng Wang
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  4. Shenghui Jiang
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  7. Mengyu Su
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  8. Weifang Zuo
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  9. Lin Xu
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  10. Zongying Zhang
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  11. Xuesen Chen
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Contributions

XC and HX: conceived and designed the experiments. HX and NW: performed the experiments. HX: analyzed the data. YW, SJ, HF, JZ, MS, WZ, LX, ZZ: contributed reagents/materials/analysis tools. HX and XC: wrote the paper.

Corresponding author

Correspondence to Xuesen Chen.

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We declare that we have no conflict of interest.

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Communicated by Sergio J. Ochatt.

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Xu, H., Wang, N., Wang, Y. et al. Overexpression of the transcription factor MdbHLH33 increases cold tolerance of transgenic apple callus. Plant Cell Tiss Organ Cult 134, 131–140 (2018). https://doi.org/10.1007/s11240-018-1406-9

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  • DOI: https://doi.org/10.1007/s11240-018-1406-9

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