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TdCBL6, a calcineurin B-like gene from wild emmer wheat (Triticum dicoccoides), is involved in response to salt and low-K+ stresses

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Abstract

The calcineurin B-like proteins (CBLs), a unique family of calcium sensors in plants, have been shown to be involved in abiotic stresses, such as salt, drought and cold. Although extensive studies and remarkable progress have been made in Arabidopsis (Arabidopsis thaliana) CBLs, very little is known about the role of CBL genes in wheat. In this study, a CBL gene, designated TdCBL6, was cloned and characterized from wild emmer wheat (Triticum dicoccoides), the progenitor of cultivated wheat. Sequence alignment revealed that TdCBL6 shares high sequence homology with rice OsCBL6. Phylogenetic analysis also revealed that TdCBL6 protein has the closest evolutionary relationship with rice OsCBL6 protein. TdCBL6 transcription was induced by NaCl, polyethylene glycol and abscisic acid. Further differential expression analysis revealed that TdCBL6 expression was much higher in the salt-tolerant line than in the salt-sensitive line when they were subjected to salt treatment. Transgenic Arabidopsis ectopic expression of the TdCBL6 gene displayed higher levels of photosynthetic efficiency (Fv/Fm) and lower ion leakage (EL) than wild-type (WT) plants under NaCl stress conditions. Moreover, TdCBL6-overexpressing lines showed low-K+ (LK)-sensitive phenotypes compared with WT plants. Further experiments revealed that ectopic expression of TdCBL6 resulted in reduction of H2O2 content, and affected expression of K+-responsive/H2O2-regulated genes under LK stress. Taken together, we demonstrated that heterologous expression of TdCBL6 in Arabidopsis confers salt tolerance by reducing membrane injury and improving photosynthetic efficiency, and that the TdCBL6 gene may be involved in response to LK stress by regulating the reactive oxygen species-mediated LK signaling pathway.

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Acknowledgments

This work was supported by the China National Science Foundation (NSFC) Grant Nos. 31030055 and 31272194, the CAS Strategic Priority Research Program Grant No. XDA05130403, the Hubei Province National Science Foundation Sciences Grant No. ZRY1326 and the outstanding young talent program of CAS Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture (Grant No. Y452341X01).

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    Authors and Affiliations

    1. Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Institute/Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan, 430074, Hubei, China

      Liang Chen, Jing Ren, Yongkuan Zhang, Jibiao Fan, Ke Chen, Shuqian Liu, Jinming Fu & Junhua Peng

    2. Graduate University of Chinese Academy of Sciences, 19 Yuquan Road, Beijing, 100049, China

      Jing Ren, Jibiao Fan & Shuqian Liu

    3. College of Horticulture, Agricultural University of Hebei, Baoding, 071001, China

      Haiyan Shi

    4. Huiyang Sun Yat-Sen Middle School, Huizhou, 516211, Guangdong, China

      Yu You

    5. Institute of Evolution, University of Haifa, 31905, Mount Carmel, Haifa, Israel

      Eviatar Nevo

    6. Department of Soil and Crop Sciences, Colorado State University, Fort Collins, CO, 80523, USA

      Junhua Peng

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    1. Liang Chen
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    Correspondence to Jinming Fu or Junhua Peng.

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    Liang Chen and Jing Ren have contributed equally to this work.

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    11032_2015_229_MOESM1_ESM.docx

    Supplementary Fig. S1 Sequence comparison of TdCBL6 protein with other known CBL6 proteins. Numbers on the right indicate the protein length in amino acids. EF hands are indicated gray and shown by the dotted line above the alignments. The amino acid residues identical among the sequences are highlighted in black. Accession numbers of the proteins in GenBank: TdCBL6, KC849259; BdCBL6, XM_003563091; OsCBL6, DQ201200. (DOCX 17 kb)

    11032_2015_229_MOESM2_ESM.tif

    Supplementary Fig. S2 Phylogenetic relationship of TdCBL6 protein with the other CBL proteins in plants. The minimum evolution tree was constructed in MEGA3.1 from 1,000 bootstrap replicates. The accession numbers of the known CBL proteins in GenBank are as follows: BdCBL1 (XM_003574302), BdCBL2 (JX567947), BdCBL4 (XM_003567977), BdCBL6 (XM_003563091), BdCBL7 (XM_003572631), BdCBL9 (XM_003569206), ZmCBL6 (NM_001157734), SbCBL (XM_002442815), OsCBL1-OsCBL10 (DQ201195, DQ201196, DQ201197, DQ201198, DQ201199, DQ201200, DQ201201, DQ201202, DQ201203, DQ201204), AtCBL2 (NM_124981), AtCBL3 (AF076253), AtCBL6(NM_117730). (TIFF 5119 kb)

    11032_2015_229_MOESM3_ESM.tif

    Supplementary Fig. S3 RT-PCR analysis of differential expression of salt stress-responsive TdCBL6 gene in leaves of wild emmer wheat ST213 and SS312 under 250 mM NaCl treatment. Total RNAs were isolated from leaves of wild emmer wheat ST213 or SS312 treated with 250 mM NaCl for 6 h, respectively. CK, untreated leaves (control). The experiments were repeated three times, and the similar results were obtained. Independent t-tests demonstrated that there was very significant difference of TdCBL6 gene expression (*P value < 0.01) between CK and NaCl condition, or between ST213 and SS312 under salt treatment. (TIFF 359 kb)

    Supplementary Table S1 Primer pairs used in gene-specific RT-PCR analysis. (DOCX 12 kb)

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    Chen, L., Ren, J., Shi, H. et al. TdCBL6, a calcineurin B-like gene from wild emmer wheat (Triticum dicoccoides), is involved in response to salt and low-K+ stresses. Mol Breeding 35, 50 (2015). https://doi.org/10.1007/s11032-015-0229-1

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