Molecular Biology Reports

, Volume 41, Issue 1, pp 45–56 | Cite as

Analysis of Brassica napus ESTs: gene discovery and expression patterns of AP2/ERF-family transcription factors



Starting from expressed sequence tag sequences and using the conserved amino acid sequence of the Arabidopsis thaliana AP2/ERF domain as a probe, we used in silico cloning to identify 87 genes that encode putative AP2/ERF transcription factors (TFs) from the Brassica napus. Almost all of the putative AP2/ERF factors from B. napus were similar to genes previously defined as AP2/ERF genes from A. thaliana. Based on the number of AP2-domains and the function of the genes, the AP2/ERF TFs from B. napus were classified into four subfamilies, named the AP2, DREB, ERF, and RAV subfamilies. We then predicted and analyzed cDNA sequences and amino acid sequences, amino acid compositions, physical and chemical characteristics, phylogenetic trees, conserved domain sequences, functional domains, molecular models, and folding states of the proteins they are predicted to encode. Expression analysis showed that four factors, which belonged to the ERF and DREB subfamilies, were induced by abiotic stresses, as well as by hormone treatment. This suggests that those AP2/ERF factors may be involved in signaling pathways responsive to abiotic and biotic stresses. The results from this study, reported herein, form a basis for future functional analyses of B. napus TFs that belong to the AP2/ERF family.


Brassica napus Arabidopsis thaliana AP2/ERF Transcription factor Phylogeny Stress response 



Transcription factor


Abscisic acid


APETALA2 factor


C-repeat binding factor


Dehydration responsive element binding factor


Ethylene responsive element binding factor


Related to ABI3/VP


Polyethylene glycol





The research was supported by the National Natural Science Foundation of China (31200520), Jiangsu Natural Science Foundation (BK2012774), China Postdoctoral Science Foundation (2013M541686) and the Ph.D Programs Foundation of Ministry of Education of China (20120097120031).

Supplementary material

11033_2013_2836_MOESM1_ESM.docx (2.3 mb)
Supplementary material 1 (DOCX 2366 kb)


  1. 1.
    Wang HZ (2005) Problem in the development of oilseed industry and it’s countermeasure in China. Chin J Oil Crop Sci 27:100–105Google Scholar
  2. 2.
    Meyerowitz EM, Pruitt RE (1985) Arabidopsis thaliana and plant molecular genetics. Science 229:1214–1218PubMedCrossRefGoogle Scholar
  3. 3.
    Cavell AC, Lydiate DJ, Parkin IA, Dean C, Trick M (1998) Collinearity between a 30-centimorgan segment of Arabidopsis thaliana chromosome 4 and duplicated regions within the Brassica napus genome. Genome 41:62–69PubMedGoogle Scholar
  4. 4.
    Li F, Wu X, Tsang E, Cutler AJ (2005) Transcriptional profiling of imbibed Brassica napus seed. Genomic 86:718–730CrossRefGoogle Scholar
  5. 5.
    Dubouzet JG, Sakuma Y, Ito Y, Kasuga M, Dubouzet EG, Miura S et al (2003) OsDREB genes in rice, Oryza sativa L., encode transcription activators that function in drought-, high-salt- and cold-responsive gene expression. Plant J 33:751–763PubMedCrossRefGoogle Scholar
  6. 6.
    Yamaguchi SK, Shinozaki K (2006) Transcriptional regulatory networks in cellular responses and tolerance to dehydration and cold stresses. Annu Rev Plant Biol 57:781–803CrossRefGoogle Scholar
  7. 7.
    Golldack D, Lüking I, Yang O (2011) Plant tolerance to drought and salinity: stress regulating transcription factors and their functional significance in the cellular transcriptional network. Plant Cell Rep 30:1383–1391PubMedCrossRefGoogle Scholar
  8. 8.
    Padmalatha KV, Dhandapani G, Kanakachari M, Kumar S, Dass A, Patil DP et al (2012) Genome-wide transcriptomic analysis of cotton under drought stress reveal significant down-regulation of genes and pathways involved in fibre elongation and up-regulation of defense responsive genes. Plant Mol Biol 78:223–246PubMedCrossRefGoogle Scholar
  9. 9.
    Liu JG, Zhang Z, Qin QL, Peng RH, Xiong AS, Chen JM et al (2007) Isolated and characterization of a cDNA encoding ethylene-responsive element binding protein (EREBP)/AP2-type protein, RCBF2, in Oryza sativa L. Biotechnol Lett 29:165–173PubMedCrossRefGoogle Scholar
  10. 10.
    Fu XY, Zhang Z, Peng RH, Xiong AS, Liu JG, Wu LJ et al (2007) Isolation and characterization of a novel cDNA encoding ERF/AP2-type transcription factor OsAP25 from Oryza sativa L. Biotechnol Lett 29:1293–1299PubMedCrossRefGoogle Scholar
  11. 11.
    Yao D, Zhang X, Zhao X, Liu C, Wang C, Zhang Z et al (2011) Transcriptome analysis reveals salt-stress-regulated biological processes and key pathways in roots of cotton (Gossypium hirsutum L.). Genomics 98:47–55PubMedGoogle Scholar
  12. 12.
    Nishiyama R, Le DT, Watanabe Y, Matsui A, Tanaka M, Seki M et al (2012) Transcriptome analyses of a salt-tolerant cytokinin-deficient mutant reveal differential regulation of salt stress response by cytokinin deficiency. PLoS ONE 7:e32124PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Fowler S, Thomashow MF (2002) Arabidopsis transcriptome profiling indicates that multiple regulatory pathways are activated during cold acclimation in addition to the CBF cold response pathway. Plant Cell 14:1675–1690PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Yang TW, Zhang LJ, Zhang TG, Zhang H, Xu SJ, An LZ (2005) Transcriptional regulation network of cold-responsive genes in higher plants. Plant Sci 169:987–995CrossRefGoogle Scholar
  15. 15.
    Ito Y, Katsura K, Maruyama K, Taji T, Kobayashi M, Seki M et al (2006) Functional analysis of rice DREB1/CBF-type transcription factors involved in cold-responsive gene expression in transgenic rice. Plant Cell Physiol 47:141–153PubMedCrossRefGoogle Scholar
  16. 16.
    Li T, Xu SL, Oses-Prieto JA, Putil S, Xu P, Wang RJ et al (2011) Proteomics analysis reveals post-translational mechanisms for cold-induced metabolic changes in Arabidopsis. Mol Plant 4:361–374PubMedCrossRefGoogle Scholar
  17. 17.
    Qin QL, Liu JG, Zhang Z, Peng RH, Xiong AS, Yao QH et al (2007) Isolation, optimization, and functional analysis of the cDNA encoding transcription factor RdreB1 in Oryza sativa L. Mol Breeding 19:329–340CrossRefGoogle Scholar
  18. 18.
    An D, Yang J, Zhang P (2012) Transcriptome profiling of low temperature-treated cassava apical shoots showed dynamic responses of tropical plant to cold stress. BMC Genomics 13:64PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    Gutterson N, Reuber TL (2004) Regulation of disease resistance pathways by AP2/ERF transcription factors. Curr Opin Plant Biol 7:465–471PubMedCrossRefGoogle Scholar
  20. 20.
    Sherif S, Paliyath G, Jayasankar S (2012) Molecular characterization of peach PR genes and their induction kinetics in response to bacterial infection and signaling molecules. Plant Cell Rep 31:697–711PubMedCrossRefGoogle Scholar
  21. 21.
    Elliott RC, Betzner AS, Huttner E, Oakes MP, Tucker WQ, Gerentes D et al (1996) AINTEGUMENTA, an APETALA2-like gene of Arabidopsis with pleiotropic roles in ovule development and floral organ growth. Plant Cell 8:155–168PubMedCentralPubMedGoogle Scholar
  22. 22.
    Liu J, Li J, Wang H, Fu Z, Liu J, Yu Y (2011) Identification and expression analysis of ERF transcription factor genes in petunia during flower senescence and in response to hormone treatments. J Exp Bot 62:825–840PubMedCrossRefGoogle Scholar
  23. 23.
    Krizek BA (2011) Aintegumenta and Aintegumenta-Like6 regulate auxin-mediated flower development in Arabidopsis. BMC Res Notes 4:176PubMedCentralPubMedCrossRefGoogle Scholar
  24. 24.
    Okamuro JK, Caster B, Villarroel R, Van Montagu M, Jofuku KD (1997) The AP2 domain of APETALA2 defines a large new family of DNA binding proteins in Arabidopsis. Proc Natl Acad Sci USA 94:7076–7081PubMedCrossRefGoogle Scholar
  25. 25.
    Riechmann JL, Meyerowitz EM (1998) The AP2/EREBP family of plant transcription factors. Biol Chem 379:633–646PubMedGoogle Scholar
  26. 26.
    Wessler SR (2005) Homing into the origin of the AP2 DNA binding domain. Trends Plant Sci 10:54–56PubMedCrossRefGoogle Scholar
  27. 27.
    Mizoi J, Shinozaki K, Yamaguchi-Shinozaki K (2012) AP2/ERF family transcription factors in plant abiotic stress responses. Biochim Biophys Acta 1819:86–96PubMedCrossRefGoogle Scholar
  28. 28.
    Sakuma Y, Liu Q, Dubouzet JG, Abe H, Shinozaki K, Yamaguchi-Shinozaki K (2002) DNA-binding specificity of the ERF/AP2 domain of Arabidopsis DREBs, transcription factors involved in dehydration- and cold-inducible gene expression. Biochem Biophys Res Commun 90:998–1009CrossRefGoogle Scholar
  29. 29.
    Nakano T, Suzuki K, Fujimura T, Shinshi H (2006) Genome-wide analysis of the ERF gene family in Arabidopsis and rice. Plant Physiol 140:411–432PubMedCentralPubMedCrossRefGoogle Scholar
  30. 30.
    Riano PDM, Ruzicic S, Dreyer I, Mueller RB (2007) PlnTFDB: an integrative plant transcription factor database. BMC Bioinform 8:42CrossRefGoogle Scholar
  31. 31.
    Sharoni AM, Nuruzzaman M, Satoh K, Shimizu T, Kondoh H, Sasaya T et al (2011) Gene structures, classification and expression models of the AP2/EREBP transcription factor family in rice. Plant Cell Physiol 52:344–360PubMedCrossRefGoogle Scholar
  32. 32.
    Zhuang J, Cai B, Peng RH, Zhu B, Jin XF, Xue Y et al (2008) Genome-wide analysis of the AP2/ERF gene family in Populus trichocarpa. Biochem Biophys Res Commun 371:468–474PubMedCrossRefGoogle Scholar
  33. 33.
    Zhuang J, Peng RH, Cheng ZM, Zhang J, Cai B, Zhang Z et al (2009) Genome-wide analysis of the putative AP2/ERF family genes in Vitis vinifera. Sci Hortic 123:73–81CrossRefGoogle Scholar
  34. 34.
    Licausi F, Giorgi FM, Zenoni S, Osti F, Pezzotti M, Perata P (2010) Genomic and transcriptomic analysis of the AP2/ERF superfamily in Vitis vinifera. BMC Genomics 11:719PubMedCentralPubMedCrossRefGoogle Scholar
  35. 35.
    Hu L, Liu S (2011) Genome-wide identification and phylogenetic analysis of the ERF gene family in cucumbers. Genet Mol Biol 34:624–633PubMedCrossRefGoogle Scholar
  36. 36.
    Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739PubMedCrossRefGoogle Scholar
  37. 37.
    Bailey TL, Boden M, Buske FA, Frith M, Grant CE, Clementi L, Ren J, Li WW, Noble WS (2009) MEME SUITE: tools for motif discovery and searching. Nucleic Acids Res 37:W202–W208PubMedCentralPubMedCrossRefGoogle Scholar
  38. 38.
    Wilkinson DL, Harrison RG (1991) Predicting the solubility of recombinant proteins in Escherichia coli. Bio/Technol 9:443–448CrossRefGoogle Scholar
  39. 39.
    Liu Y, Zhao TJ, Liu JM, Liu WQ, Liu Q, Yan YB et al (2006) The conserved Ala37 in the ERF/AP2 domain is essential for binding with the DRE element and the GCC box. FEBS Lett 580:1038–1303Google Scholar
  40. 40.
    Jaglo KR, Kleff S, Amundsen KL, Zhang X, Haake V, Zhang JZ et al (2001) Components of the Arabidopsis C-repeat/dehydration-responsive element binding factor cold-response pathway are conserved in Brassica napus and other plant species. Plant Physiol 127:910–917PubMedCentralPubMedCrossRefGoogle Scholar
  41. 41.
    Gao MJ, Allard G, Byass L, Flanagan AM, Singh J (2002) Regulation and characterization of four CBF transcription factors from Brassica napus. Plant Mol Biol 49:459–471PubMedCrossRefGoogle Scholar
  42. 42.
    Zhao TJ, Liu Y, Yan YB, Feng F, Liu WQ et al (2007) Identification of the amino acids crucial for the activities of drought responsive element binding factors (DREBs) of Brassica napus. FEBS Lett 581:3044–3050PubMedCrossRefGoogle Scholar
  43. 43.
    Zhao TJ, Sun S, Liu Y, Liu JM, Liu Q, Yan YB et al (2006) Regulating the drought-responsive element (DRE)-mediated signaling pathway by synergic functions of trans-active and trans-inactive DRE binding factors in Brassica napus. J Biol Chem 281:10752–10759PubMedCrossRefGoogle Scholar
  44. 44.
    Allen MD, Yamasaki K, Ohme TM (1998) A novel mode of DNA recognition by a beta-sheet revealed by the solution structure of the GCC-box binding domain in complex with DNA. EMBO J 17:5484–5496PubMedCrossRefGoogle Scholar
  45. 45.
    Ye R, Yao QH, Xu ZH, Xue HW (2004) Development of an efficient method for the isolation of factors involved in gene transcription during rice embryo development. Plant J 38:348–357PubMedCrossRefGoogle Scholar
  46. 46.
    Ou B, Yin KQ, Liu SN, Yang Y, Gu T, Wing Hui JM et al (2011) A high-throughput screening system for Arabidopsis transcription factors and its application to Med25-dependent transcriptional regulation. Mol Plant 4:546–555PubMedCrossRefGoogle Scholar
  47. 47.
    Wehner N, Hartmann L, Ehlert A, Böttner S, Oñate-Sánchez L, Dröge-Laser W (2011) High-throughput protoplast transactivation (PTA) system for the analysis of Arabidopsis transcription factor function. Plant J 68:560–569PubMedCrossRefGoogle Scholar
  48. 48.
    Zhuang J, Sun CC, Zhou XR, Xiong AS, Zhang J (2011) Isolation and characterization of an AP2/ERF-RAV transcription factor BnaRAV-1-HY15 in Brassica napus L. HuYou15. Mol Biol Rep 38:3921–3928PubMedCrossRefGoogle Scholar
  49. 49.
    Ohme TM, Shinshi H (1995) Ethylene-inducible DNA binding proteins that interact with an ethylene-responsive element. Plant Cell 7:173–182Google Scholar
  50. 50.
    Weigel D (1995) The APETALA2 domain is related to a novel type of DNA binding domain. Plant Cell 7:388–389PubMedCentralPubMedGoogle Scholar
  51. 51.
    Balaji S, Babu MM, Iyer LM, Aravind L (2005) Discovery of the principal specific transcription factors of Apicomplexa and their implication for the evolution of the AP2-integrase DNA binding domains. Nucleic Acids Res 33:3994–4006PubMedCentralPubMedCrossRefGoogle Scholar
  52. 52.
    Kizis D, Lumbreras V, Pagès M (2001) Role of AP2/EREBP transcription factors in gene regulation during abiotic stress. FEBS Lett 498:187–189PubMedCrossRefGoogle Scholar
  53. 53.
    Agarwal PK, Agarwal P, Reddy MK, Sopory SK (2006) Role of DREB transcription factors in abiotic and biotic stress tolerance in plants. Plant Cell Rep 25:1263–1274PubMedCrossRefGoogle Scholar
  54. 54.
    Dietz KJ, Vogel MO, Viehhauser A (2010) AP2/EREBP transcription factors are part of gene regulatory networks and integrate metabolic, hormonal and environmental signals in stress acclimation and retrograde signalling. Protoplasma 245:3–14PubMedCrossRefGoogle Scholar
  55. 55.
    Goldgur Y, Rom S, Ghirlando R, Shkolnik D, Shadrin N, Konrad Z et al (2007) Desiccation and zinc binding induce transition of tomato abscisic acid stress ripening 1, a water stress- and salt stress-regulated plant-specific protein, from unfolded to folded state. Plant Physiol 143:617–628PubMedCentralPubMedCrossRefGoogle Scholar
  56. 56.
    Xu ZS, Chen M, Li LC, Ma YZ (2011) Functions and application of the AP2/ERF transcription factor family in crop improvement. J Integr Plant Biol 53:570–585PubMedCrossRefGoogle Scholar
  57. 57.
    Zhuang J, Anyiaa A, Vidmara J, Xiong AS, Zhanga J (2011) Discovery and expression assessment of the AP2-like genes in Hordeum vulgare. Acta Physiol Plant 33:1639–1649CrossRefGoogle Scholar
  58. 58.
    Li MY, Wang F, Jiang Q, Li R, Ma J, Xiong AS (2013) Genome-wide analysis of the distribution of AP2/ERF transcription factors reveals duplication and elucidates their potential function in Chinese cabbage (Brassica rapa ssp. pekinensis). Plant Mol Biol Rep 31:1002–1011CrossRefGoogle Scholar
  59. 59.
    Zhang Z, Huang R (2010) Enhanced tolerance to freezing in tobacco and tomato overexpressing transcription factor TERF2/LeERF2 is modulated by ethylene biosynthesis. Plant Mol Biol 73:241–249PubMedCrossRefGoogle Scholar
  60. 60.
    Hwang JE, Lim CJ, Chen H, Je J, Song C, Lim CO (2012) Over expression of Arabidopsis dehydration-responsive element-binding protein 2C confers tolerance to oxidative stress. Mol Cells 33:135–140PubMedCrossRefGoogle Scholar
  61. 61.
    Pan Y, Seymour GB, Lu C, Hu Z, Chen X, Chen G (2012) An ethylene response factor (ERF5) promoting adaptation to drought and salt tolerance in tomato. Plant Cell Rep 31:349–360PubMedCrossRefGoogle Scholar
  62. 62.
    Xiong AS, Jiang HH, Zhuang J, Peng RH, Jin XF, Zhu B, Wang F, Zhang J, Yao QH (2013) Expression and function of a modified AP2/ERF transcription factor from Brassica napus enhances cold tolerance in transgenic Arabidopsis. Mol Biotechnol 53:198–206PubMedCrossRefGoogle Scholar
  63. 63.
    Jin XF, Zhu B, Peng RH, Jiang HH, Chen JM, Zhuang J et al (2010) Optimizing the binding activity of the AP2/ERF transcription factor with the GCC box element from Brassica napus by directed evolution. BMB Rep 43:567–572PubMedCrossRefGoogle Scholar
  64. 64.
    Garbuzynskiy SO, Lobanov MY, Galzitskaya OV (2004) To be folded or to be unfolded? Protein Sci 13:2871–2877PubMedCrossRefGoogle Scholar
  65. 65.
    Coeytaux K, Poupon A (2005) Prediction of unfolded segments in a protein sequence based on amino acid composition. Bioinformatics 21:1891–1900PubMedCrossRefGoogle Scholar
  66. 66.
    Fink AL (2005) Natively unfolded proteins. Curr Opin Struct Biol 15:35–41PubMedCrossRefGoogle Scholar
  67. 67.
    Schlessinger A, Punta M, Rost B (2007) Natively unstructured regions in proteins identified from contact predictions. Bioinformatics 23:2376–2384PubMedCrossRefGoogle Scholar
  68. 68.
    Evans PR, Owen DJ (2002) Endocytosis and vesicle trafficking. Curr Opin Struct Biol 12:814–821PubMedCrossRefGoogle Scholar
  69. 69.
    Linding R, Jensen LJ, Diella F, Bork P, Gibson TJ, Russell RB (2003) Protein disorder prediction: implications for structural proteomics. Structure 11:1453–1459PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  1. 1.State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of HorticultureNanjing Agricultural UniversityNanjingChina

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