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Overexpression of a Miscanthus lutarioriparius NAC gene MlNAC5 confers enhanced drought and cold tolerance in Arabidopsis

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Abstract

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MLNAC5 functions as a stress-responsive NAC transcription factor gene and enhances drought and cold stress tolerance in transgenic Arabidopsis via the ABA-dependent signaling pathway.

Abstract

NAC transcription factors (TFs) play crucial roles in plant responses to abiotic stress. Miscanthus lutarioriparius is one of Miscanthus species native to East Asia. It has attracted much attention as a bioenergy crop because of its superior biomass productivity as well as wide adaptability to different environments. However, the functions of stress-related NAC TFs remain to be elucidated in M. lutarioriparius. In this study, a detailed functional characterization of MlNAC5 was carried out. MlNAC5 was a member of ATAF subfamily and it showed the highest sequence identity to ATAF1. Subcellular localization of MlNAC5-YFP fusion protein in tobacco leaves indicated that MlNAC5 is a nuclear protein. Transactivation assay in yeast cells demonstrated that MlNAC5 functions as a transcription activator and its activation domain is located in the C-terminus. Overexpression of MlNAC5 in Arabidopsis had impacts on plant development including dwarfism, leaf senescence, leaf morphology, and late flowering under normal growth conditions. Furthermore, MlNAC5 overexpression lines in Arabidopsis exhibited hypersensitivity to abscisic acid (ABA) and NaCl. Moreover, overexpression of MlNAC5 in Arabidopsis significantly enhanced drought and cold tolerance by transcriptionally regulating some stress-responsive marker genes. Collectively, our results indicated that MlNAC5 functions as an important regulator during the process of plant development and responses to salinity, drought and cold stresses.

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Acknowledgments

This study was supported by Grants from the Joint Funds of the National Natural Science Foundation of China (U1432126), the National Key Technology Support Program of China (2013BAD22B01), the National High-Tech Research and Development Program of China (2011AA100209), and the 100 Talents Program of the Chinese Academy of Sciences (to C. Fu).

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The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. College of Life Sciences, Guizhou Normal University, Guiyang, 550001, Guizhou, People’s Republic of China

    Xuanwen Yang & Jingcheng Ran

  2. CAS Key Laboratory of Biofuels and Shandong Provincial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, 266101, People’s Republic of China

    Xuanwen Yang, Xiaoyu Wang, Chunxiang Fu, Ruibo Hu & Gongke Zhou

  3. Laboratory of Basic Biology, Hunan First Normal University, Changsha, 410205, Hunan, People’s Republic of China

    Lu Ji

  4. College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha, 410128, Hunan, People’s Republic of China

    Zili Yi

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  1. Xuanwen Yang
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  3. Lu Ji
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Correspondence to Ruibo Hu or Gongke Zhou.

Additional information

Communicated by Q. Zhao.

Electronic supplementary material

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299_2015_1756_MOESM1_ESM.eps

Supplementary material 1 Multiple sequence alignment of the deduced amino acid sequences of MlNAC5 with ATAF1 orthologous NAC proteins reported in other plant species. The five highly conserved subdomains (A-E) in the NAC domain is boxed and indicted by lines above the sequences. The sequence of putative nuclear localization signal is labeled by a double-headed arrow under the sequence. Alignments were performed using MegAlign of Lasergene software (Version 7.0) and edited in BioEdit (Version 7.0). The accession numbers for the sequences are as follows: AtATAF1 (Arabidopsis thaliana, NP_171677); OsNAC6/SNAC2 (Oryza sativa, AB028185); OsNAC052 (O.sativa, AAT44250); HvNAC6 (Hordeum vulgare, AM500854); GmNAC20 (Glycine max, EU440353), ZmSNAC1 (Zea Mays, 165855636) and CarNAC5 (Cicer arietinum L., FJ477886). (EPS 3408 kb)

299_2015_1756_MOESM2_ESM.jpg

Supplementary material 2 Phylogenetic tree of MlNAC5 and NAC proteins reported from other plant species. The tree was constructed by Neighbor-Joining method with MEGA program (Version6.0) with 1000 bootstrap replications. The percentage of bootstrap values is shown at the branch nodes. Scale bar indicates amino acid substitutions. The corresponding accession numbers are as follows: AtATAF1 (Arabidopsis thaliana, NP_171677); AtATAF2 (A. thaliana, NP_680161); ANAC019 (A. thaliana, NP_175697); ANAC055 (A. thaliana, NP_188169); ANAC072 (A. thaliana, NP_567773); ANAC102 (A. thaliana, NP_201184); ANAC032 (A. thaliana,NP_177869); SNAC1 (Oryza sativa, ABD52007); OsNAC4 (O. sativa,BAA89798); OsNAC5 (O. sativa, AB028184); OsNAC6/SNAC2 (O. sativa, AB028185); OsNAC10 (O. sativa, ABA91266); OsNAC052 (O. sativa, AAT44250); TaNAC2 (Trticum aestivumL. AAU08786); TaNAC67 (T. aestivumL. KF646593); TaNAC69 (T. aestivumL. AAU08785); HvNAC6 (Hordeum vulgare, AM500854); GmNAC11 (Glycine max, EU440354) GmNAC20 (G. max, EU440353), ZmSNAC1 (Zea Mays, 165855636) and CarNAC3 (Cicer arietinumL.FJ356671); CarNAC5 (C. arietinum L., FJ477886) and AhNAC2 (Arachis hypogaea, EU755023). (JPEG 223 kb)

Supplementary material 3 Primers used in this study. (XLSX 13 kb)

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Yang, X., Wang, X., Ji, L. et al. Overexpression of a Miscanthus lutarioriparius NAC gene MlNAC5 confers enhanced drought and cold tolerance in Arabidopsis. Plant Cell Rep 34, 943–958 (2015). https://doi.org/10.1007/s00299-015-1756-2

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