Plant Cell, Tissue and Organ Culture (PCTOC)

, Volume 135, Issue 3, pp 545–558 | Cite as

Overexpression of two cold-responsive ATAF-like NAC transcription factors from fine-stem stylo (Stylosanthes guianensis var. intermedia) enhances cold tolerance in tobacco plants

  • Peng-Lin Zhan
  • Shan-Wen Ke
  • Pan-Yu Zhang
  • Cong-Cong Zhou
  • Bei-Ling Fu
  • Xiang-Qian Zhang
  • Tian-Xiu Zhong
  • Shu ChenEmail author
  • Xin-Ming XieEmail author
Original Article


Stylosanthes (stylo) species are economically important tropical and subtropical forage legumes. They are vulnerable to chilling and frost, and little is known about the genetic and molecular mechanisms of their responses or adaptations to low temperature stress. Two cold-responsive NAC genes, SgNAC1 and SgNAC2, were selected from a whole transcriptome profiling study of fine-stem stylo (S. guianensis var. intermedia) and further investigated for their roles in cold stress tolerance. Bioinformatic analysis indicated that SgNAC1 and SgNAC2 belonged to ATAF subgroup of NAC family, and shared highly conserved N-terminal A–E NAC subdomains and a EVQS[E/x]PK[W/I] motif with ATAF-like NAC transcription factors. Expression profiling, subcellular location and transactivation assay revealed that SgNAC1 and SgNAC2 encode nucleus-localized polypeptides with transactivation activities and responds promptly to cold stress. Phenotypic and physiological changes indicated that the transgenic tobacco plants overexpressing SgNAC1 and SgNAC2 were more tolerant to cold stress than the wild-type plants. Meanwhile, the expression of SgNAC1 and SgNAC2 were significantly enhanced at the early stages of cold treatment, indicating that overexpression of SgNAC1 and SgNAC2 is sufficient to confer cold tolerance to tobacco plants. These results suggest that SgNAC1 and SgNAC2 are promising candidate genes for cold tolerance improvement strategies in stylo.


NAC transcription factor ATAF Cold tolerance Fine-stem stylo Overexpression Expression profiling 



[No apical meristem (NAM), Arabidopsis transcription activation factor (ATAF), cup-shaped cotyledon (CUC)]

MS medium

Murashige and Skoog medium




1-Naphthylacetic acid




Green fluorescent protein


Complementary DNA







YPDA medium

Yeast, peptone, dextrose and adenine medium



Financial support from the National Natural Science Foundation of China (Nos. 31601990, 31272491 and 31802116) is gratefully acknowledged.

Author contributions

XMX conceived and initiated the project. PLZ and SWK designed and performed most of the experiments and data analysis. PLZ and SC wrote the article; XQZ and BLF participated in plasmid construction, plant transformation and physiological determination; SC, TXZ, LL and PYZ conducted the data collection and bioinformatic analysis.


The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

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11240_2018_1486_MOESM3_ESM.docx (35 kb)
Supplementary file s3 (DOCX 31 KB)
11240_2018_1486_MOESM4_ESM.svg (620 kb)
Supplementary file s4 (SVG 620 KB)


  1. Ahmad M (2011) Polyamines: natural and engineered abiotic and biotic stress tolerance in plants. Biotechnol Adv 29:300–311CrossRefGoogle Scholar
  2. Ariel FD, Manavella PA, Dezar CA, Chan RL (2007) The true story of the HD-Zip family. Trends Plant Sci 12:419–426. CrossRefPubMedGoogle Scholar
  3. Aslam M, Grover A, Sinha VB et al (2012) Isolation and characterization of cold responsive NAC gene from Lepidium latifolium. Mol Biol Rep 39:9629CrossRefGoogle Scholar
  4. Bais HP, Ravishankar GA (2002) Role of polyamines in the ontogeny of plants and their biotechnological applications. Plant Cell Tissue Organ Cult 69:1–34CrossRefGoogle Scholar
  5. Bao G, Zhuo C, Qian C et al (2015) Co-expression of NCED and ALO improves vitamin C level and tolerance to drought and chilling in transgenic tobacco and stylo plants. Plant Biotechnol J 1–9. CrossRefGoogle Scholar
  6. Bart R, Chern M, Park C-J et al (2006) A novel system for gene silencing using siRNAs in rice leaf and stem-derived protoplasts. Plant Methods 2:13. CrossRefPubMedPubMedCentralGoogle Scholar
  7. Castilhos G, Lazzarotto F, Spagnolo-Fonini L et al (2014) Possible roles of basic helix-loop-helix transcription factors in adaptation to drought. Plant Sci 223:1–7. CrossRefPubMedGoogle Scholar
  8. Chen Q, Quan W, Xiong L, Lou Z (2011) A structural view of the conserved domain of rice stress-responsive NAC1. Protein Cell 2:55–63CrossRefGoogle Scholar
  9. Chen L, Song Y, Li S et al (2012) The role of WRKY transcription factors in plant abiotic stresses. Biochim Biophys Acta Gene Regul Mech 1819:120–128. CrossRefGoogle Scholar
  10. Christianson JA, Dennis ES, Llewellyn DJ, Wilson IW (2010) ATAF NAC transcription factors: regulators of plant stress signaling. Plant Signal Behav 5:428–432CrossRefGoogle Scholar
  11. Cui Y, Wang Q (2006) Physiological responses of maize to elemental sulphur and cadmium stress. Plant Soil Environ 52:523–529CrossRefGoogle Scholar
  12. Delessert C, Wilson IW, Van Der Straeten D et al (2004) Spatial and temporal analysis of the local response to wounding in Arabidopsis leaves. Plant Mol Biol 55:165–181. CrossRefPubMedGoogle Scholar
  13. Delessert C, Kazan K, Wilson IW et al (2005) The transcription factor ATAF2 represses the expression of pathogenesis-related genes in Arabidopsis. Plant J 43:745–757. CrossRefPubMedGoogle Scholar
  14. Ernst HA, Olsen AN, Larsen S, Lo LL (2004) Structure of the conserved domain of ANAC, a member of the NAC family of transcription factors. EMBO Rep 5:297–303CrossRefGoogle Scholar
  15. Fang Y, You J, Xie K et al (2008) Systematic sequence analysis and identification of tissue-specific or stress-responsive genes of NAC transcription factor family in rice. Mol Genet Genom 280:547–563CrossRefGoogle Scholar
  16. Farrant JM (2010) Mechanisms of desiccation tolerance in resurrection plants: a review from the molecular to whole plant physiological level. S Afr J Bot 76:389CrossRefGoogle Scholar
  17. Gill SS, Tuteja N (2010) Polyamines and abiotic stress tolerance in plants. Plant Signal Behav 5:26–33CrossRefGoogle Scholar
  18. Goel D, Singh AK, Yadav V et al (2010) Overexpression of osmotin gene confers tolerance to salt and drought stresses in transgenic tomato (Solanum lycopersicum L.). Protoplasma 245:133–141. CrossRefPubMedGoogle Scholar
  19. Grigg A, Shelton M, Mullen B (2000) The nature and management of rehabilitated pastures on open-cut coal mines in central Queensland. Trop Grassl 34:241–250Google Scholar
  20. Guo W-L, Wang S-B, Chen R-G et al (2015) Characterization and expression profile of CaNAC2 pepper gene. Front Plant Sci 6:1–9. CrossRefGoogle Scholar
  21. Hauck P, Thilmony R, He SY (2003) A Pseudomonas syringae type III effector suppresses cell wall-based extracellular defense in susceptible Arabidopsis plants. Proc Natl Acad Sci USA 100:8577–8582. CrossRefPubMedGoogle Scholar
  22. He XJ, Mu RL, Cao WH et al (2005) AtNAC2, a transcription factor downstream of ethylene and auxin signaling pathways, is involved in salt stress response and lateral root development. Plant J 44:903–916CrossRefGoogle Scholar
  23. Hong Y, Zhang H, Huang L et al (2016) Overexpression of a stress-responsive NAC transcription factor gene ONAC022 improves drought and salt tolerance in rice. Front Plant Sci 7:1–19. CrossRefGoogle Scholar
  24. Hu H, You J, Fang Y et al (2010) Characterization of transcription factor gene SNAC2 conferring cold and salt tolerance in rice. Plant Mol Biol 72:567–568CrossRefGoogle Scholar
  25. Huang G-T, Ma S-L, Bai L-P et al (2012a) Signal transduction during cold, salt, and drought stresses in plants. Mol Biol Rep 39:969–987. CrossRefPubMedGoogle Scholar
  26. Huang H, Wang Y, Wang S et al (2012b) Transcriptome-wide survey and expression analysis of stress-responsive NAC genes in Chrysanthemum lavandulifolium. Plant Sci 193–194:18–27CrossRefGoogle Scholar
  27. Humphrey LR (1980) A guide to better pastures for the tropics and sub-tropics, 4th edn. Wright Stephenson and Co. (Australia) Pty. Ltd, MelbourneGoogle Scholar
  28. Jensen MK, Rung JH, Gregersen PL et al (2007) The HvNAC6 transcription factor: a positive regulator of penetration resistance in barley and Arabidopsis. Plant Mol Biol 65:137–150. CrossRefPubMedGoogle Scholar
  29. Jensen MK, Kjaersgaard T, Nielsen MM et al (2010) The Arabidopsis thaliana NAC transcription factor family: structure-function relationships and determinants of ANAC019 stress signalling. Biochem J 426:183–196CrossRefGoogle Scholar
  30. Jiang Y, Deyholos MK (2006) Comprehensive transcriptional profiling of NaCl-stressed Arabidopsis roots reveals novel classes of responsive genes. BMC Plant Biol 6:25. CrossRefPubMedPubMedCentralGoogle Scholar
  31. Jin H, Huang F, Cheng H et al (2013) Overexpression of the GmNAC2 Gene, an NAC transcription factor, reduces abiotic stress tolerance in tobacco. Plant Mol Biol Rep 31:435–442. CrossRefGoogle Scholar
  32. Kato H, Motomura T, Komeda Y et al (2010) Overexpression of the NAC transcription factor family gene ANAC036 results in a dwarf phenotype in Arabidopsis thaliana. J Plant Physiol 167:571–577. CrossRefPubMedGoogle Scholar
  33. Kjaersgaard T, Jensen MK, Christiansen MW et al (2011) Senescence-associated barley NAC (NAM, ATAF1,2, CUC) transcription factor interacts with radical-induced cell death 1 through a disordered regulatory domain. J Biol Chem 286:35418–35429CrossRefGoogle Scholar
  34. Le DT, Nishiyama R, Watanabe Y et al (2011) Genome-wide survey and expression analysis of the plant-specific NAC transcription factor family in soybean during development and dehydration stress. DNA Res 18:263–276. CrossRefPubMedPubMedCentralGoogle Scholar
  35. Li X, Yang X, Hu YX et al (2014) A novel NAC transcription factor from Suaeda liaotungensis K. enhanced transgenic Arabidopsis drought, salt, and cold stress tolerance. Plant Cell Rep 33:767–778. CrossRefPubMedGoogle Scholar
  36. Licausi F, Ohme-Takagi M, Perata P (2013) APETALA2/ethylene responsive factor (AP2/ERF) transcription factors: mediators of stress responses and developmental programs. New Phytol. CrossRefPubMedGoogle Scholar
  37. Liu YG, Chen Y (2007) High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences. Biotechniques 43:649–650, 652, 654 passimCrossRefGoogle Scholar
  38. Liu X, Zhang BY, Hong L et al (2010) Molecular characterization of Arachis hypogaea NAC 2 (AhNAC2) reveals it as a NAC-like protein in peanut. Biotechnol Biotechnol Equip 24:2066–2070. CrossRefGoogle Scholar
  39. Lu S, Wang X, Guo Z (2013) Differential responses to chilling in Stylosanthes guianensis (Aublet) Sw. and Its mutants. Agron J 105:377–382CrossRefGoogle Scholar
  40. Mizoi J, Shinozaki K, Yamaguchi-Shinozaki K (2012) AP2/ERF family transcription factors in plant abiotic stress responses. Biochim Biophys Acta Gene Regul Mech 1819:86–96. CrossRefGoogle Scholar
  41. Nakashima K, Tran LSP, Van Nguyen D et al (2007) Functional analysis of a NAC-type transcription factor OsNAC6 involved in abiotic and biotic stress-responsive gene expression in rice. Plant J 51:617–630. CrossRefPubMedGoogle Scholar
  42. Nakashima K, Takasaki H, Mizoi J et al (2012) NAC transcription factors in plant abiotic stress responses. Biochim Biophys Acta 1819:97–103CrossRefGoogle Scholar
  43. Noble AD, Orr DM, Middleton CH, Rogers LG (2000) Legumes in native pasture - asset or liability? a case history with stylo. Trop Grassl 34:199–206Google Scholar
  44. Nogueira FTS, Schlögl PS, Camargo SR et al (2005) SsNAC23, a member of the NAC domain protein family, is associated with cold, herbivory and water stress in sugarcane. Plant Sci 169:93–106CrossRefGoogle Scholar
  45. Nuruzzaman M, Sharoni AM, Kikuchi S (2013) Roles of NAC transcription factors in the regulation of biotic and abiotic stress responses in plants. Front Microbiol 4:1–16. CrossRefGoogle Scholar
  46. Oh SK, Lee S, Yu SH, Choi D (2005) Expression of a novel NAC domain-containing transcription factor (CaNAC1) is preferentially associated with incompatible interactions between chili pepper and pathogens. Planta 222:876–887. CrossRefPubMedGoogle Scholar
  47. Ohnishi T, Sugahara S, Yamada T et al (2005) OsNAC6, a member of the NAC gene family, is induced by various stresses in rice. Genes Genet Syst 80:135–139. CrossRefPubMedGoogle Scholar
  48. Olsen AN, Ernst HA, Leggio LL, Skriver K (2005) NAC transcription factors: structurally distinct, functionally diverse. Trends Plant Sci 10:79–87CrossRefGoogle Scholar
  49. Ooka H, Satoh K, Doi K et al (2003) Comprehensive analysis of NAC family genes in Oryza sativa and Arabidopsis thaliana. DNA Res 10:239–247CrossRefGoogle Scholar
  50. Peng H, Yu X, Cheng H et al (2010) Cloning and characterization of a novel NAC family gene CarNAC1 from chickpea (Cicer arietinum L.). Mol Biotechnol 44:30–40CrossRefGoogle Scholar
  51. Puranik S, Sahu PP, Srivastava PS, Prasad M (2012) NAC proteins: regulation and role in stress tolerance. Trends Plant Sci 17:369–381. CrossRefPubMedGoogle Scholar
  52. Qu Y, Duan M, Zhang Z et al (2016) Overexpression of the Medicago falcata NAC transcription factor MfNAC3 enhances cold tolerance in Medicago truncatula. Environ Exp Bot 129:67–76CrossRefGoogle Scholar
  53. Rushton DL, Tripathi P, Rabara RC et al (2012) WRKY transcription factors: key components in abscisic acid signalling. Plant Biotechnol J 10:2–11CrossRefGoogle Scholar
  54. Seki M, Urano K (2007) Regulatory metabolic networks in drought stress responses. Curr Opin Plant Biol 10:296–302CrossRefGoogle Scholar
  55. Shan W, Kuang JF, Lu WJ, Chen JY (2014) Banana fruit NAC transcription factor MaNAC1 is a direct target of MaICE1 and involved in cold stress through interacting with MaCBF1. Plant Cell Environ 37:2116–2127. CrossRefPubMedGoogle Scholar
  56. Shao H, Wang H, Tang X (2015) NAC transcription factors in plant multiple abiotic stress responses: progress and prospects. Front Plant Sci 6:902. CrossRefPubMedPubMedCentralGoogle Scholar
  57. Shen H, Yin Y, Chen F et al (2009) A bioinformatic analysis of NAC genes for plant cell wall development in relation to lignocellulosic bioenergy production. BioEnergy Res 2:217–232CrossRefGoogle Scholar
  58. Shen C, Wang S, Bai Y et al (2010) Functional analysis of the structural domain of ARF proteins in rice (Oryza sativa L.). J Exp Bot 61:3971–3981. CrossRefPubMedPubMedCentralGoogle Scholar
  59. Tran LSP, Quach TN, Guttikonda SK et al (2009) Molecular characterization of stress-inducible GmNAC genes in soybean. Mol Genet Genom 281:647–664. CrossRefGoogle Scholar
  60. Tran LS, Nishiyama R, Yamaguchi-Shinozaki K, Shinozaki K (2010) Potential utilization of NAC transcription factors to enhance abiotic stress tolerance in plants by biotechnological approach. GM Crops 1:32–39CrossRefGoogle Scholar
  61. Urano K, Maruyama K, Ogata Y et al (2009) Characterization of the ABA-regulated global responses to dehydration in Arabidopsis by metabolomics. Plant J 57:1065–1078CrossRefGoogle Scholar
  62. Wang Z, Rashotte AM, Moss AG, Dane F (2014) Two NAC transcription factors from Citrullus colocynthis, CcNAC1, CcNAC2 implicated in multiple stress responses. Acta Physiol Plant 36:621–634CrossRefGoogle Scholar
  63. Wang L, Hu Z, Zhu M et al (2017) The abiotic stress-responsive NAC transcription factor SlNAC11 is involved in drought and salt response in tomato (Solanum lycopersicum L.). Plant Cell Tissue Organ Cult 129:161–174. CrossRefGoogle Scholar
  64. Xie Q, Frugis G, Colgan D, Chua NH (2000) Arabidopsis NAC1 transduces auxin signal downstream of TIR1 to promote lateral root development. Genes Dev 14:3024–3036. CrossRefPubMedPubMedCentralGoogle Scholar
  65. Yang J, Guo Z (2007) Cloning of a 9-cis-epoxycarotenoid dioxygenase gene (SgNCED1) from Stylosanthes guianensis and its expression in response to abiotic stresses. Plant Cell Rep 26:1383–1390. CrossRefPubMedGoogle Scholar
  66. Yang SD, Seo PJ, Yoon HK, Park CM (2011) The Arabidopsis NAC transcription factor VNI2 integrates abscisic acid signals into leaf senescence via the COR/RD genes. Plant Cell 23:2155–2168CrossRefGoogle Scholar
  67. Yu X, Liu Y, Wang S et al (2016) CarNAC4, a NAC-type chickpea transcription factor conferring enhanced drought and salt stress tolerances in Arabidopsis. Plant Cell Rep 35:613–627. CrossRefPubMedGoogle Scholar
  68. Yu D, Zhang L, Zhao K et al (2017) VaERD15, a transcription factor gene associated with cold-tolerance in Chinese Wild Vitis amurensis. Front Plant Sci 8:1–13. CrossRefGoogle Scholar
  69. Zhai C, Zhang Y, Yao N et al (2014) Function and interaction of the coupled genes responsible for Pik-h encoded rice blast resistance. PLoS ONE. CrossRefPubMedPubMedCentralGoogle Scholar
  70. Zhang L, Zhao G, Jia J et al (2012) Molecular characterization of 60 isolated wheat MYB genes and analysis of their expression during abiotic stress. J Exp Bot 63:203–214CrossRefGoogle Scholar
  71. Zhong R, Lee C, Ye ZH (2010) Global analysis of direct targets of secondary wall NAC master switches in Arabidopsis. Mol Plant 3:1087–1103CrossRefGoogle Scholar
  72. Zhou B, Guo Z, Liu Z (2005a) Effects of abscisic acid on antioxidant systems of Stylosanthes guianensis (Aublet) Sw. under chilling stress. Crop Sci 45:599–605CrossRefGoogle Scholar
  73. Zhou B, Guo Z, Xing J, Huang B (2005b) Nitric oxide is involved in abscisic acid-induced antioxidant activities in Stylosanthes guianensis. J Exp Bot 56:3223–3228. CrossRefPubMedGoogle Scholar

Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  • Peng-Lin Zhan
    • 1
    • 2
  • Shan-Wen Ke
    • 1
    • 2
  • Pan-Yu Zhang
    • 1
    • 2
  • Cong-Cong Zhou
    • 1
    • 2
  • Bei-Ling Fu
    • 1
    • 2
  • Xiang-Qian Zhang
    • 1
    • 2
  • Tian-Xiu Zhong
    • 1
    • 2
  • Shu Chen
    • 1
    • 2
    Email author
  • Xin-Ming Xie
    • 1
    • 2
    Email author
  1. 1.Department of Grassland Science, College of Forestry and Landscape ArchitectureSouth China Agricultural UniversityGuangzhouChina
  2. 2.Guangdong Engineering Research Center for Grassland ScienceGuangzhouChina

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