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Overexpression of a Mei (Prunus mume) CBF gene confers tolerance to freezing and oxidative stress in Arabidopsis

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Abstract

Mei (Prunus mume Sieb. et Zucc.) is an important ornamental plant in China, but low temperature severely restricts its application in North China. The C-repeat binding factor/Dehydration responsive element binding factors (CBF/DREB1) pathway has important roles in plant responses to cold stress. However, knowledge on the function of CBF/DREB1 genes from Mei and its regulatory roles in freezing and other abiotic stresses is limited. Three PmhCBF genes, designated as PmhCBFa, PmhCBFb and PmhCBFc respectively, were isolated and characterized from Prunus mume ‘Huahudie’. Gene expression analysis revealed that PmhCBFa and PmhCBFb were more induced than PmhCBFc by cold stress, and other stress treatments including H2O2, Mannitol, NaCl and Abscisic Acid also induced the expressions of PmhCBFa/b/c at a relatively lower level. Compared to PmhCBFa and PmhCBFb, PmhCBFc appeared to be more triggered by H2O2 and showed broad regulation potential in all the abiotic stresses. Overexpression of PmhCBFc in Arabidopsis improved freezing and oxidative stress tolerance, and reduced the inhibition of PSII, but delayed development and flowering. The superoxide dismutase (SOD, EC 1.11.1.6) and peroxidase (POD, EC 1.11.1.7) activities were also elevated in the transgenic Arabidopsis plants during freezing-thawing cycles. Our results suggested that PmhCBFc responded to reactive oxygen species (ROS) and functioned in freezing tolerance by enhancing the capacities for ROS scavenging.

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Acknowledgments

This work was supported by the grants from the National Natural Science Foundation of China (No. 31070624) and the National 863 Project of China (No. 2011AA100207). We thank all the colleagues in our laboratory for constructive discussion and technical support.

Author contributions

M. Z. B. and J. W. Z. designed research, T. P., C. G. and J. Y. conducted all the sampling, RNA extraction and cDNA synthesis. T. P., M. X. and J. Z. detected the activities of anti-oxidative enzymes and analyzed the data. T. P. also performed the remaining experiments and drafted the manuscript. J. W. Z. contributed for the discussion of the results and revision of the manuscript. All authors approved the manuscript for final submission.

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  1. Key Laboratory of Horticultural Plant Biology, Ministry of Education, College of Horticulture and Forestry Sciences, Huazhong Agricultural University, Wuhan, 430070, People’s Republic of China

    Ting Peng, Cong Guo, Jie Yang, Min Xu, Jing Zuo, Manzhu Bao & Junwei Zhang

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  1. Ting Peng
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  2. Cong Guo
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  3. Jie Yang
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Correspondence to Junwei Zhang.

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Supplementary material 1 (DOCX 13 kb)

11240_2016_1004_MOESM2_ESM.pdf

Fig. S1 The deduced amino sequence alignment of CBF proteins from Prunus mume and Arabidopsis thaliana. The CBF sequences and their GenBank accession numbers are PmhCBFa (KU587042), PmhCBFb (KU587043), PmhCBFc (KU587044) from P. mume ‘Huahudie’ and PmCBFa (ADF43033.1), PmCBFb (ADF43034.1), PmCBFc (ADF43035.1) from P. mume ‘Xuemei’; AtDREB1B (AT4G25490.1), AtDREB1C (AT4G25470.1) and AtDREB1A (AT4G25480.1) from A. thaliana. The putative nuclear localization signal (NLS), AP2/ERF domain, DSAWR- and LWSY- motif are indicated by solid lines. (PDF 2579 kb)

11240_2016_1004_MOESM3_ESM.pdf

Fig. S2 Phylogenetic relationships of P. mume ‘Huahudie’ and CBF/DREB proteins from other plants. The proteins aligned included 16 CBF protein sequences, 3 CBFs from P. mume ‘Huahudie’ (in red box): PmhCBFa (KU587042), PmhCBFb (KU587043), PmhCBFc (KU587043); 3 CBFs from P. mume ‘Xue Mei’: PmCBFa (ADF43033.1), PmCBFb (ADF43034.1), PmCBFc (ADF43035.1); 2 CBFs from P. persica: PpDREB1 (ABR19831.1), PpCBF1 (ADU03762.1); 2CBFs from P. avium: PaCIGA (BAC20183.1), PaCIGB (BAC20184.1); 2CBFs from P. dulics: PdCBF1 (AFL48190.1), PdCBF2 (AFL48191.1); 1 CBF from P. canescens x P. cerasus, PcxPcDREB1 (ACF94686.1); 3 DREB1s from A. thaliana AtDREB1B (AT4G25490.1), AtDREB1C (AT4G25470.1) and AtDREB1A (AT4G25480.1). (PDF 190 kb)

11240_2016_1004_MOESM4_ESM.pdf

Fig. S3 PmhCBFc overexpression in transgenic Arabidopsis under normal condition. Transcript level of PmhCBFc was analyzed by qRT-PCR using AtEF1α as internal control. Error bars show the standard deviation based on three replicates. (PDF 74 kb)

11240_2016_1004_MOESM5_ESM.pdf

Fig. S4 Phenotypic evaluation of PmhCBFc overexpression Arabidopsis plants under drought and high salinity stress. a, c Effects on seed germination in response to drought stress (a) and high salinity stress (c). The average of three independent experiments is shown. b, d Effects on seedling response to drought stress (b) and high salinity stress (d). WT and transgenic plants were germinated on 1/2 MS agar plates for 5 days, then transferred to a new MS agar plate supplemented with 1.5 μM MV or 300 mM mannitol or 160 mM NaCl for 15 days. Repeated experiments showed similar tendencies. (PDF 146 kb)

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Peng, T., Guo, C., Yang, J. et al. Overexpression of a Mei (Prunus mume) CBF gene confers tolerance to freezing and oxidative stress in Arabidopsis. Plant Cell Tiss Organ Cult 126, 373–385 (2016). https://doi.org/10.1007/s11240-016-1004-7

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