Broccoli Plants Over-expressing an ERF Transcription Factor Gene BoERF1 Facilitates Both Salt Stress and Sclerotinia Stem Rot Resistance

  • Ming Jiang
  • Zi-hong Ye
  • Hui-juan Zhang
  • Li-xiang Miao


Ethylene response factors (ERFs) are members of the APETALA2/ERF transcription factor family, and they play important roles in plant growth, development, and multiple environmental stress responses. In our present study, an ERF transcription factor gene designated as BoERF1 was isolated from broccoli, and its expression was induced by both NaCl and Sclerotinia sclerotiorum. Transgenic plants over-expressing BoERF1 were generated by Agrobacterium tumefaciens-mediated transformation, and they exhibited higher seed germination rates and less chlorophyll loss under salt stress as compared to wild-type (WT) broccoli plants, and an approximately two-fold increase in chlorophyll content was observed in three transgenic lines. Over-expression of BoERF1 in broccoli dramatically decreased hydrogen peroxide (H2O2), relative electrical conductivity (REC), and malondialdehyde (MDA) contents, but increased free proline, the activities of catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD), resulting in less cell death in the leaves of transgenic plants. Moreover, broccoli plants over-expressing BoERF1 exhibited significant resistance to Sclerotinia stem rot as compared to the WT line. Qualitative real-time PCR (qRT-PCR) results confirmed that the expression levels of BoERF1 in transgenic lines were higher than those in WT plants, and the peak expression levels were seen at 24 and 12 h with 3.21- and 4.66-fold changes after treatments with S. sclerotiorum and NaCl, respectively. Taken together, our results indicate that BoERF1 acts as a positive regulator in resistance to both salt stress and Sclerotinia stem rot, suggesting its potential utility in molecular breeding of broccoli.


Brassica oleracea var. italica Sclerotinia stem rot Over-expression ERF transcription factor Salt stress 



This work was supported by Public Welfare Technology Research Projects of Zhejiang Province (2016C32091), Taizhou Science and Technology Project (162ny14), and Research Projects of Taizhou University (2017JQ001).

Author Contributions

Conceived and designed the experiments: MJ. Performed the experiments: MJ, HZ, and LM. Analyzed the data: ZY. Wrote the paper: MJ.


  1. Alves MS, Dadalto SP, Gonçalves AB, De Souza GB, Barros VA, Fietto LG (2013) Plant bZIP transcription factors responsive to pathogens: a review. Int J Mol Sci 14(4):7815–7828CrossRefPubMedPubMedCentralGoogle Scholar
  2. Apostolova P, Yordanova R, Popova L (2008) Response of antioxidative defence system to low temperature stress in two wheat cultivars. Gen Appl Plant Physiol 34(3–4):281–294Google Scholar
  3. Ares AM, Nozal MJ, Bernal J (2013) Extraction, chemical characterization and biological activity determination of broccoli health promoting compounds. J Chromatogr A 1313:78–95CrossRefPubMedGoogle Scholar
  4. Arnon DI (1949) Copper enzymes in isolated chloroplasts, polyphenoxidase in Beta vulgaris. Plant physiol 24:1–15CrossRefPubMedPubMedCentralGoogle Scholar
  5. Bartels D, Sunkar R (2005) Drought and salt tolerance in plants. Crit Rev Plant Sci 24(1):23–58CrossRefGoogle Scholar
  6. Bates LS, Waldren RP, Teare ID (1973) Rapid determination of free proline for water stress studies. Plant Soil 39:205–207CrossRefGoogle Scholar
  7. Batool N, Shahzad A, Ilyas N (2014) Plants and salt stress. Int J Agric Crop Sci 7(14):1439–1446Google Scholar
  8. Bayer WF, Fridovich I (1987) Assaying for superoxide dismutase activity: some large consequences of minor changes in condition. Ann Clin Biochem 161:559–566CrossRefGoogle Scholar
  9. Bjellqvist B, Hughes GJ, Pasquali C, Paquet N, Ravier F, Sanchez JC, Frutiger S, Hochstrasser D (1993) The focusing positions of polypeptides in immobilized pH gradients can be predicted from their amino acid sequences. Electrophoresis 14(10):1023–1031CrossRefPubMedGoogle Scholar
  10. Bradford MM (1976) A rapid and sensitive method for microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefPubMedGoogle Scholar
  11. Cakmak I, Marschner H (1992) Magnesium deficiency and high light intensity enhance activities of superoxide dismutase, ascorbate peroxidase, and glutathione reductase in bean leaves. Plant Physiol 98:1222–1227CrossRefPubMedPubMedCentralGoogle Scholar
  12. Cartea ME, Francisco M, Soengas P, Velasco P (2010) Phenolic compounds in Brassica vegetables. Molecules 16(1):251–280CrossRefPubMedGoogle Scholar
  13. Cui L, Feng K, Wang M, Wang M, Deng P, Song W, Nie X (2016) Genome-wide identification, phylogeny and expression analysis of AP2/ERF transcription factors family in Brachypodium distachyon. BMC Genomics 17(1):636CrossRefPubMedPubMedCentralGoogle Scholar
  14. Dong N, Liu X, Lu Y, Du L, Xu H, Liu H, Xin Z, Zhang Z (2010) Overexpression of TaPIEP1, a pathogen-induced ERF gene of wheat, confers host-enhanced resistance to fungal pathogen Bipolaris sorokiniana. Funct Integr Genomics 10(2):215–226CrossRefPubMedGoogle Scholar
  15. Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull 19:11–15Google Scholar
  16. Esfandiari A, Saei A, McKenzie MJ, Matich AJ, Babalar M, Hunter DA (2017) Preferentially enhancing anti-cancer isothiocyanates over glucosinolates in broccoli sprouts: how NaCl and salicylic acid affect their formation. Plant Physiol Biochem 115:343–353CrossRefPubMedGoogle Scholar
  17. Fan W, Hai M, Guo Y, Ding Z, Tie W, Ding X, Yan Y, Wei Y, Liu Y, Wu C, Shi H, Li K, Hu W (2016) The ERF transcription factor family in cassava: genome-wide characterization and expression analyses against drought stress. Sci Rep 6:37379CrossRefPubMedPubMedCentralGoogle Scholar
  18. Guo H, Ecker JR (2004) The ethylene signaling pathway: new insights. Curr Opin Plant Biol 7(1):40–49CrossRefPubMedGoogle Scholar
  19. Hao D, Ohme-Takagi M, Sarai A (1998) Unique mode of GCC box recognition by the DNA-binding domain of ethylene-responsive element binding factor (ERF domain) in plant. J Biol Chem 273:26857–26861CrossRefPubMedGoogle Scholar
  20. Hasanuzzaman M, Nahar K, Alam MM, Roychowdhury R, Fujita M (2013) Physiological, biochemical, and molecular mechanisms of heat stress tolerance in plants. Int J Mol Sci 14(5):9643–9684CrossRefPubMedPubMedCentralGoogle Scholar
  21. Hoang XLT, Nhi DNH, Thu NBA, Thao NP, Tran LP (2017) Transcription factors and their roles in signal transduction in plants under abiotic stresses. Curr Genomics 18(6):483–497CrossRefPubMedGoogle Scholar
  22. Huang PY, Catinot J, Zimmerli L (2016) Ethylene response factors in Arabidopsis immunity. J Exp Bot 67(5):1231–1241CrossRefPubMedGoogle Scholar
  23. Hussain A, Mustafa G (2016) Genome-wide analysis of ethylene responsive factor in maize: an in silico approach. Appl Ecol Environ Res 14(5):177–200CrossRefGoogle Scholar
  24. Jiang M, Miao LX, He C (2012) Overexpression of an oil radish superoxide dismutase gene in broccoli confers resistance to downy mildew. Plant Mol Biol Rep 30(4):966–972CrossRefGoogle Scholar
  25. Joshi R, Wani SH, Singh B, Bohra A, Dar ZA, Lone AA, Pareek A, Singla-Pareek SL (2016) Transcription factors and plants response to drought stress: current understanding and future directions. Front Plant Sci 7:1029CrossRefPubMedPubMedCentralGoogle Scholar
  26. Kim YH, Jeong JC, Park S, Lee HS, Kwak SS (2012) Molecular characterization of two ethylene response factor genes in sweetpotato that respond to stress and activate the expression of defense genes in tobacco leaves. J Plant Physiol 169(11):1112–1120CrossRefPubMedGoogle Scholar
  27. Kristal AR, Lampe JW (2002) Brassica vegetables and prostate cancer risk: a review of the epidemiological evidence. Nutr Cancer 42(1):1–9CrossRefPubMedGoogle Scholar
  28. Kumar S, Tamura K, Nei M (2004) MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5:150–163CrossRefPubMedGoogle Scholar
  29. Kumar D, Yusuf MA, Singh P, Sardar M, Sarin NB (2014) Histochemical detection of superoxide and H2O2 accumulation in Brassica juncea seedlings. Bio-protocol 4: e1108CrossRefGoogle Scholar
  30. Lata C, Mishra AK, Muthamilarasan M, Bonthala VS, Khan Y, Prasad M (2014) Genome-wide investigation and expression profiling of AP2/ERF transcription factor superfamily in foxtail millet (Setaria italica L.). PLoS ONE 9(11), e113092CrossRefPubMedPubMedCentralGoogle Scholar
  31. Li C, Han LB, Zhang XZ (2012) Enhanced drought tolerance of tobacco overexpressing OjERF gene is associated with alteration in proline and antioxidant metabolism. J Am Soc Hortic Sci 137(2):107–113Google Scholar
  32. 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 199(3):639–649CrossRefPubMedGoogle Scholar
  33. Liu Z, Kong L, Zhang M, Lv Y, Liu Y, Zou M, Lu G, Cao J, Yu X, 2013. Genome-wide identification, phylogeny, evolution and expression patterns of AP2/ERF genes and cytokinin response factors in Brassica rapa ssp. pekinensis. PLoS ONE 8(12), e83444CrossRefPubMedPubMedCentralGoogle Scholar
  34. Liu D, Xin M, Zhou X, Wang C, Zhang Y, Qin Z (2017) Expression and functional analysis of the transcription factor-encoding gene CsERF004 in cucumber during Pseudoperonospora cubensis and Corynespora cassiicola infection. BMC Plant Biol 17:96CrossRefPubMedPubMedCentralGoogle Scholar
  35. Livak KJ, Schmittgen TG (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−∆∆C T method. Methods 25:402–408CrossRefPubMedGoogle Scholar
  36. Lv YY, Fu SX, Chen S, Zhang W, Qi CK (2016) Ethylene response factor BnERF2-like (ERF2.4) from Brassica napus L. enhances submergence tolerance and alleviates oxidative damage caused by submergence in Arabidopsis thaliana. Crop J 4(3):199–211CrossRefGoogle Scholar
  37. Maehly AC, Chance B (1954) The assay of catalases and peroxidases. Methods Biochem Anal 1:357–424PubMedGoogle Scholar
  38. Memelink J (2009) Regulation of gene expression by jasmonate hormones. Phytochemistry 70(13–14):1560–1570CrossRefPubMedGoogle Scholar
  39. Mittova V, Guy M, Tal M, Volokita M (2004) Salinity up-regulates the antioxidative system in root mitochondria and peroxisomes of the wild salt-tolerant tomato species Lycopersicon pennellii. J Exp Bot 55:1105–1113CrossRefPubMedGoogle Scholar
  40. Mizoi J, Shinozaki K, Yamaguchi-Shinozaki K (2012) AP2/ERF family transcription factors in plant abiotic stress responses. Biochim Biophys Acta 1819(2):86–96CrossRefPubMedGoogle Scholar
  41. Müller M, Munné-Bosch S (2015) Ethylene response factors: a key regulatory hub in hormone and stress signaling. Plant Physiol 169(1):32–41CrossRefPubMedPubMedCentralGoogle Scholar
  42. Nakano T, Suzuki K, Fujimura T, Shinshi H (2006) Genome-wide analysis of the ERF gene family in Arabidopsis and rice. Plant Physiol 140(2):411–432CrossRefPubMedPubMedCentralGoogle Scholar
  43. Owis AI (2015) Broccoli; the green beauty: a review. J Pharm Sci Res 7(9):696–703Google Scholar
  44. Pegoraro C, Farias D, Mertz LM, Santos RS, Maia LC, Rombaldi CV, Oliveira AC (2013) Ethylene response factors gene regulation and expression profiles under different stresses in rice. Theor Exp Plant Physiol 25(4):261–274CrossRefGoogle Scholar
  45. Phillips T, Hoopes L (2008) Transcription factors and transcriptional control in eukaryotic cells. Nat Educ 1(1):119Google Scholar
  46. Phukan UJ, Jeena GS, Shukla RK (2016) WRKY transcription factors: molecular regulation and stress responses in plants. Front Plant Sci 7:760CrossRefPubMedPubMedCentralGoogle Scholar
  47. Podsędek A (2007) Natural antioxidants and antioxidant capacity of Brassica vegetables: a review. Food Sci Technol 40(1):1–11Google Scholar
  48. Pogány M, von Rad U, Grün S, Dongó A, Pintye A, Simoneau P, Bahnweg G, Kiss L, Barna B, Durner J (2009) Dual roles of reactive oxygen species and NADPH oxidase RBOHD in an Arabidopsis-Alternaria pathosystem. Plant Physiol 151:1459–1475CrossRefPubMedPubMedCentralGoogle Scholar
  49. Pré M, Atallah M, Champion A, De Vos M, Pieterse CM, Memelink J (2008) The AP2/ERF domain transcription factor ORA59 integrates jasmonic acid and ethylene signals in plant defense. Plant Physiol 147(3):1347–1357CrossRefPubMedPubMedCentralGoogle Scholar
  50. Rejeb IB, Pastor V, Mauch-Mani B (2014) Plant responses to simultaneous biotic and abiotic stress: molecular mechanisms. Plants 3(4):458–475CrossRefPubMedPubMedCentralGoogle Scholar
  51. Riechmann JL, Heard J, Martin G, Reuber L, Jiang C, Keddie J, Adam L, Pineda O, Ratcliffe OJ, Samaha RR, Creelman R, Pilgrim M, Broun P, Zhang JZ, Ghandehari D, Sherman BK, Yu G (2000) Arabidopsis transcription factors: genome-wide comparative analysis among eukaryotes. Science 290:2105–2110CrossRefPubMedGoogle Scholar
  52. Roy S, 2016. Function of MYB domain transcription factors in abiotic stress and epigenetic control of stress response in plant genome. Plant Signal Behav 11(1), e1117723CrossRefPubMedGoogle Scholar
  53. Saher S, Piqueras A, Hellin E, Olmos E (2004) Hyperhydricity in micropropagated carnation shoots: the role of oxidative stress. Physiol Planta 120:152–161CrossRefGoogle Scholar
  54. 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 290(3):998–1009CrossRefPubMedGoogle Scholar
  55. Scott O, Galicia-Connolly E, Adams D, Surette S, Vohra S, Yager JY (2012) The safety of Cruciferous plants in humans: a systematic review. J Biomed Biotechnol 2012:503241CrossRefPubMedPubMedCentralGoogle Scholar
  56. Sergiev I, Alexieva V, Karanov E (1997) Effect of spermine, atrazine and combination between them on some endogenous protective systems and stress markers in plants. C R Acad Bulg Sci 51:121–124Google Scholar
  57. Sharma P, Jha AB, Dubey RS, Pessarakli M (2012) Reactive oxygen species, oxidative damage, and antioxidative defense mechanism in plants under stressful conditions. J Bot 2012:217037Google Scholar
  58. Shu Y, Liu Y, Zhang J, Song L, Guo C (2016) Genome-wide analysis of the AP2/ERF superfamily genes and their responses to abiotic stress in Medicago truncatula. Front Plant Sci 6:1247CrossRefPubMedPubMedCentralGoogle Scholar
  59. Singh AK, Dubey RS (1995) Changes in chlorophyll a and b contents and activities of photosystems I and II in rice seedlings induced by NaCl. Photosynthetica 31:489–499Google Scholar
  60. Singh K, Foley RC, Oñate-Sánchez L (2002) Transcription factors in plant defense and stress responses. Curr Opin Plant Biol 5(5):430–436CrossRefPubMedGoogle Scholar
  61. Song X, Wang J, Ma X, Li Y, Lei T, Wang L, Ge W, Guo D, Wang Z, Li C, Zhao J, Wang X (2016) Origination, expansion, evolutionary trajectory, and expression bias of AP2/ERF superfamily in Brassica napus. Front Plant Sci 7:1186PubMedPubMedCentralGoogle Scholar
  62. Sun ZM, Zhou ML, Dan-Wang, Tang YX, Lin M, Wu YM (2016) Overexpression of the Lotus corniculatus soloist gene LcAP2/ERF107 enhances tolerance to salt stress. Protein Pept Lett 23(5):442–449CrossRefPubMedGoogle Scholar
  63. Suzuki N, Rivero RM, Shulaev V, Blumwald E, Mittler R (2014) Abiotic and biotic stress combinations. New Phytol 203(1):32–43CrossRefPubMedGoogle Scholar
  64. Thompson JD, Gibson TJ, Plewniak FM, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882CrossRefPubMedPubMedCentralGoogle Scholar
  65. Vasanthi HR, Mukherjee S, Das DK (2009) Potential health benefits of broccoli—a chemico-biological overview. Mini Rev Med Chem 9(6):749–759CrossRefPubMedGoogle Scholar
  66. Verbruggen N, Hermans C (2008) Proline accumulation in plants: a review. Amino Acids 35(4):753–759CrossRefPubMedGoogle Scholar
  67. Wang L, Qin L, Liu W, Zhang D, Wang Y (2014a) A novel ethylene-responsive factor from Tamarix hispida, ThERF1, is a GCC-box- and DRE-motif binding protein that negatively modulates abiotic stress tolerance in Arabidopsis. Physiol Plant 152(1):84–97CrossRefPubMedGoogle Scholar
  68. Wang SJ, Yao WJ, Wei HR, Jiang TB, Zhou BR (2014b) Expression patterns of ERF genes underlying abiotic stresses in Di-Haploid Populus simonii × P. nigra. Sci World J 2014:745091Google Scholar
  69. Wang X, Hou C, Zheng K, Li Q, Chen S, Wang S (2017) Overexpression of ERF96, a small ethylene response factor gene enhances salt tolerance in Arabidopsis. Biol Plant 2017:1–9Google Scholar
  70. Yamaguchi-Shinozaki K, Shinozaki K (1994) A novel cis-acting element in an Arabidopsis gene is involved in responsiveness to drought, low temperature, or high-salt stress. Plant Cell 6:251–264CrossRefPubMedPubMedCentralGoogle Scholar
  71. Yan HW, Hong L, Zhou YQ, Jiang HY, Zhu SW, Fan J, Cheng BJ (2013) A genome-wide analysis of the ERF gene family in sorghum. Genet Mol Res 12(2):2038–2055CrossRefPubMedGoogle Scholar
  72. Zarei A, Körbes AP, Younessi P, Montiel G, Champion A, Memelink J (2011) Two GCC boxes and AP2/ERF-domain transcription factor ORA59 in jasmonate/ethylene-mediated activation of the PDF1.2 promoter in Arabidopsis. Plant Mol Biol 75(4–5):321–331CrossRefPubMedPubMedCentralGoogle Scholar
  73. Zhang H, Huang Z, Xie B, Chen Q, Tian X, Zhang X, Zhang H, Lu X, Huang D, Huang R (2004a) The ethylene-, jasmonate-, abscisic acid- and NaCl-responsive tomato transcription factor JERF1 modulates expression of GCC box-containing genes and salt tolerance in tobacco. Planta 220:262–270CrossRefPubMedGoogle Scholar
  74. Zhang H, Zhang D, Chen J, Yang Y, Huang Z, Huang D, Wang XC, Huang R (2004b) Tomato stress-responsive factor TSRF1 interacts with ethylene responsive element GCC box and regulates pathogen resistance to Ralstonia solanacearum. Plant Mol Biol 55(6):825–834CrossRefPubMedGoogle Scholar
  75. Zhang Z, Yao W, Dong N, Liang H, Liu H, Huang R (2007) A novel ERF transcription activator in wheat and its induction kinetics after pathogen and hormone treatments. J Exp Bot 58(11):2993–3003CrossRefPubMedGoogle Scholar
  76. Zhang M, Li S, Nie L, Chen Q, Xu X, Yu L, Fu C (2015) Two jasmonate-responsive factors, TcERF12 and TcERF15, respectively act as repressor and activator of tasy gene of taxol biosynthesis in Taxus chinensis. Plant Mol Biol 89(4–5):463–473CrossRefPubMedGoogle Scholar
  77. Zhao Y, Chang X, Qi D, Dong L, Wang G, Fan S, Jiang L, Cheng Q, Chen X, Han D, Xu P, Zhang S (2017) A novel soybean ERF transcription factor, GmERF113, increases resistance to Phytophthora sojae infection in soybean. Front Plant Sci 8:299PubMedPubMedCentralGoogle Scholar
  78. Zhou J, Wang J, Bi Y, Wang L, Tang L, Yu X, Ohtani M, Demura T, Zhuge Q (2014) Overexpression of PtSOS2 enhances salt tolerance in transgenic Poplars. Plant Mol Biol Rep 32:185–197CrossRefPubMedGoogle Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Ming Jiang
    • 1
  • Zi-hong Ye
    • 2
  • Hui-juan Zhang
    • 1
  • Li-xiang Miao
    • 3
  1. 1.College of Life SciencesTaizhou UniversityJiaojiangPeople’s Republic of China
  2. 2.Zhejiang Provincial Key Laboratory of Biometrology and Inspection & Quarantine, College of Life SciencesChina Jiliang UniversityHangzhouPeople’s Republic of China
  3. 3.Institute of HorticultureZhejiang Academy of Agricultural SciencesHangzhouPeople’s Republic of China

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