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Expression and Function of a Modified AP2/ERF Transcription Factor from Brassica napus Enhances Cold Tolerance in Transgenic Arabidopsis

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Abstract

One of the most rapid and effective defensive mechanisms plants have for protecting themselves, from a variety of biotic and abiotic stresses, is the regulation of plant signal transcription factors. AP2/ERF factors play an important role in plant development as well as in hormonal regulation and cold response. Directed evolution is a powerful tool to modify proteins, improving their properties, and for studying their structure–function relations. Here, the transgenic Arabidopsis plants over-expressed a mutant gene, BnaERF-B3-hy15-mu3, which encoded for a factor that exhibited more binding activity with the GCC box element than the wild-type gene BnaERF-B3-hy15 encode factor, and exhibited more freezing tolerance than transgenic plants containing the original BnaERF-B3-hy15 gene. Real-time PCR analyses also revealed that the expression levels of several stress-regulated genes were altered in the over-expressed BnaERF-B3-hy15-mu3 transgenic lines. The BnaERF-B3-hy15 responded to exogenous ABA. Using RT-PCR analysis, the expression of BnaERF-B3-hy15 at different stages and stress treatments were also analyzed.

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Abbreviations

ABA:

Abscisic acid

ABRE:

ABA responsive element binding factor

CaMV:

Cauliflower mosaic virus

cor:

Cold responsive

rd:

Responsive drought

CTAB:

Cetyl trimethyl ammonium bromide

AP2:

APETALA2 factor

TF:

Transcription factor

CBF:

C-repeat binding factor

DREB:

Dehydration responsive element binding factor

ERF:

Ethylene responsive element binding factor

RAV:

Related to ABI3/VP

PEG:

Polyethylene glycol

WT:

Wild-type

References

  1. Sun, C. C., Zhao, H., Fang, G. H., Wang, W. R., Li, Y. L., & Qian, X. F. (2002). Breeding of a double low rapeseed variety Huyou No.15 in Brassica napus L. Chinese Journal of Oil Crop Sciences, 24, 65–67.

    Google Scholar 

  2. Chen, W. J., & Zhu, T. (2004). Networks of transcription factors with roles in environmental stress response. Trends in Plant Science, 9, 591–596.

    Article  CAS  Google Scholar 

  3. Mitsuda, N., & Ohme-Takagi, M. (2009). Functional analysis of transcription factors in Arabidopsis. Plant and Cell Physiology, 50, 1232–1248.

    Article  CAS  Google Scholar 

  4. Nakashima, k., Ito, Y., & Yamaguchi-Shinozaki, K. (2009). Transcriptional regulatory networks in response to abiotic stresses in Arabidopsis and grasses. Plant Physiology, 149, 88–95.

    Article  CAS  Google Scholar 

  5. Ashraf, M. (2010). Inducing drought tolerance in plants: Recent advances. Biotechnology Advances, 28, 169–183.

    Article  CAS  Google Scholar 

  6. Riechmann, J. L., & Meyerowitz, E. M. (1998). The AP2/EREBP family of plant transcription factors. Biological Chemistry, 379, 633–646.

    CAS  Google Scholar 

  7. El-Sharkawy, I., Sherif, S., Mila, I., Bouzayen, M., & Jayasankar, S. (2009). Molecular characterization of seven genes encoding ethylene-responsive transcriptional factors during plum fruit development and ripening. Journal of Experimental Botany, 60, 907–922.

    Article  CAS  Google Scholar 

  8. Zhu, Q., Zhang, J., Gao, X., Tong, J., Xiao, L., & Zhang, H. (2010). The Arabidopsis AP2/ERF transcription factor RAP2.6 participates in ABA, salt and osmotic stress responses. Gene, 457, 1–12.

    Article  CAS  Google Scholar 

  9. Sakuma, Y., Liu, Q., Dubouzet, J. G., Abe, H., & Shinozaki, K. (2002). DNA-binding specificity of the ERF/AP2 domain of Arabidopsis DREBs, transcription factors involved in dehydration- and cold-inducible gene expression. Biochemical and Biophysical Research Communications, 290, 998–1009.

    Article  CAS  Google Scholar 

  10. Nakano, T., Suzuki, K., Fujimura, T., & Shinshi, H. (2006). Genome-wide analysis of the ERF gene family in Arabidopsis and rice. Plant Physiology, 140, 411–432.

    Article  CAS  Google Scholar 

  11. Zhuang, J., Cai, B., Peng, R. H., Zhu, B., Jin, X. F., Xue, Y., et al. (2008). Genome-wide analysis of the AP2/ERF gene family in Populus trichocarpa. Biochemical and Biophysical Research Communications, 371, 468–474.

    Article  CAS  Google Scholar 

  12. Zhuang, J., Peng, R. H., Cheng, Z. M., Zhang, J., Cai, B., Zhang, Z., et al. (2009). Genome-wide analysis of the putative AP2/ERF family genes in Vitis vinifera. Scientia Horticulturae, 123, 73–81.

    Article  CAS  Google Scholar 

  13. Dougherty, M. J., & Arnold, F. H. (2009). Directed evolution: New parts and optimized function. Current Opinion in Biotechnology, 20, 486–491.

    Article  CAS  Google Scholar 

  14. Shivange, A. V., Marienhagen, J., Mundhada, H., Schenk, A., & Schwaneberg, U. (2009). Advances in generating functional diversity for directed protein evolution. Current Opinion in Chemical Biology, 13, 19–25.

    Article  CAS  Google Scholar 

  15. Xiong, A. S., Peng, R. H., Liu, J. G., Zhuang, J., Qiao, Y. S., Xu, F., et al. (2007). High efficiency and throughput system in directed evolution in vitro of reporter gene. Applied Microbiology and Biotechnology, 74, 160–168.

    Article  CAS  Google Scholar 

  16. Xiong, A. S., Peng, R. H., Cheng, Z. M., Li, Y., Liu, J. G., Zhuang, J., et al. (2007). Concurrent mutations in six amino acids in β-glucuronidase improve its thermostability. Protein Engineering, Design and Selection, 20, 319–325.

    Article  CAS  Google Scholar 

  17. Xiong, A. S., Yao, Q. H., Peng, R. H., & Cheng, Z. M. (2010). Directed in vitro evolution of reporter genes based on semi-rational design and high throughput screening. In J. Braman (Ed.), Vitro mutagenesis protocols (3rd ed.). Totowa, NJ, USA: Springer, Humana Press Inc.

    Google Scholar 

  18. Sen, S., Venkata, D. V., & Mandal, B. (2007). Developments in directed evolution for improving enzyme functions. Applied Biochemistry and Biotechnology, 143, 212–223.

    Article  CAS  Google Scholar 

  19. Stemmer, W. P. (1994). Rapid evolution of a protein in vitro by DNA shuffling. Nature, 370, 389–391.

    Article  CAS  Google Scholar 

  20. Zhao, H., Chockalingam, K., & Chen, Z. (2002). Directed evolution of enzymes and pathways for industrial biocatalysis. Current Opinion in Biotechnology, 13, 104–110.

    Article  CAS  Google Scholar 

  21. Otten, L. G., & Quax, W. J. (2005). Directed evolution: Selecting today’s biocatalysts. Biomolecular Engineering, 22, 1–9.

    Article  CAS  Google Scholar 

  22. Whalen, R. G., Kaiwar, R., Soong, N. W., & Punnonen, J. (2001). DNA shuffling and vaccines. Current Opinion in Molecular Therapeutics, 3, 31–36.

    CAS  Google Scholar 

  23. Locher, C. P., Soong, N. W., Whalen, R. G., & Punnonen, J. (2004). Development of novel vaccines using DNA shuffling and screening strategies. Current Opinion in Molecular Therapeutics, 6, 34–39.

    CAS  Google Scholar 

  24. Lassner, M., & Bedbrook, J. (2001). Directed molecular evolution in plant improvement. Current Opinion in Plant Biology, 4, 152–156.

    Article  CAS  Google Scholar 

  25. Dixon, D. P., McEwen, A. G., Lapthorn, A. J., & Edwards, R. (2003). Forced evolution of a herbicide detoxifying glutathione transferase. The Journal of Biological Chemistry, 278, 23930–23935.

    Article  CAS  Google Scholar 

  26. Castle, L. A., Siehl, D. L., Gorton, R., Patten, P. A., Chen, Y. H., Bertain, S., et al. (2004). Discovery and directed evolution of a glyphosate tolerance gene. Science, 304, 1151–1154.

    Article  CAS  Google Scholar 

  27. Zhou, M., Xu, H., Wei, X., Ye, Z., Wei, L., Gong, W., et al. (2006). Identification of a glyphosate-resistant mutant of rice 5-enolpyruvylshikimate 3-phosphate synthase using a directed evolution strategy. Plant Physiology, 140, 184–195.

    Article  CAS  Google Scholar 

  28. Yamaguchi, S. K., & Shinozaki, K. (2006). Transcriptional regulatory networks in cellular responses and tolerance to dehydration and cold stresses. Annual Review of Plant Biology, 57, 781–803.

    Article  Google Scholar 

  29. Yang, T. W., Zhang, L. J., Zhang, T. G., Zhang, H., & Xu, S. J. (2005). Transcriptional regulation network of cold-responsive genes in higher plants. Plant Science, 169, 987–995.

    Article  CAS  Google Scholar 

  30. Jin, X. F., Zhu, B., Peng, R. H., Jiang, H. H., Chen, J. M., 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 Reports, 43, 567–572.

    Article  CAS  Google Scholar 

  31. Richards, E., Reichardt, M., & Rogers, S. (1998). Preparation of plant DNA using CTAB. In: J. A. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, & K. S. Struhl (Eds.), Current protocol in molecular biology. Current Protocol (pp. 233–237). Boston, MA, USA.

  32. Zhang, X., Henriques, R., & Lin, S. S. (2006). Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method. Nature Protocols, 1, 641–646.

    Article  CAS  Google Scholar 

  33. Livak, K. J., & Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔc(t) method. Methods, 25, 402–408.

    Article  CAS  Google Scholar 

  34. Alferez, F., Pozo, L., & Burns, J. K. (2006). Physiological changes associated with senescence and abscission in mature citrus fruit induced by 5-chloro-3-methyl-4- nitro-1H-pyrazole and ethephon application. Physiologia Plantarum, 127, 66–73.

    Article  CAS  Google Scholar 

  35. Yemm, E. W., & Willis, A. J. (1954). The estimation of carbohydrates in plant extracts by anthrone. Biochemical Journal, 57, 508–514.

    CAS  Google Scholar 

  36. Shinozaki, K., Yamaguchi, S. K., & Seki, M. (2003). Regulatory network of gene expression in the drought and cold stress responses. Current Opinion in Plant Biology, 6, 410–417.

    Article  CAS  Google Scholar 

  37. Yamaguchi, S. K., & Shinozaki, K. (2005). Organization of cis-acting regulatory elements in osmotic- and cold-stress-responsive promoters. Trends in Plant Science, 10, 88–94.

    Article  Google Scholar 

  38. Kizis, D., Lumbreras, V., & Pagès, M. (2001). Role of AP2/EREBP transcription factors in gene regulation during abiotic stress. FEBS Letters, 498, 187–189.

    Article  CAS  Google Scholar 

  39. Gutterson, N., & Reuber, T. L. (2004). Regulation of disease resistance pathways by AP2/ERF transcription factors. Current Opinion in Plant Biology, 7, 465–471.

    Article  CAS  Google Scholar 

  40. Jaglo, K. R., Kleff, S., Amundsen, K. L., Zhang, X., Haake, V., Zhang, J. Z., 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 Physiology, 127, 910–917.

    Article  CAS  Google Scholar 

  41. Savitch, L. V., Allard, G., Seki, M., Robert, L. S., Tinker, N. A., Huner, N. P., et al. (2005). The effect of overexpression of two Brassica CBF/DREB1-like transcription factors on photosynthetic capacity and freezing tolerance in Brassica napus. Plant and Cell Physiology, 46, 1525–1539.

    Article  CAS  Google Scholar 

  42. Gao, M. J., Allard, G., Byass, L., Flanagan, A. M., & Singh, J. (2002). Regulation and characterization of four CBF transcription factors from Brassica napus. Plant Molecular Biology, 49, 459–471.

    Article  CAS  Google Scholar 

  43. Liu, Y., Zhao, T. J., Liu, J. M., Liu, W. Q., Liu, Q., Yan, Y. B., et al. (2006). The conserved Ala37 in the ERF/AP2 domain is essential for binding with the DRE element and the GCC box. FEBS Letters, 580, 1303–1308.

    Article  CAS  Google Scholar 

  44. Zhao, T. J., Sun, S., Liu, Y., Liu, J. M., Liu, Q., Yan, Y. B., 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. The Journal of Biological Chemistry, 281, 10752–10759.

    Article  CAS  Google Scholar 

  45. Zhao, T. J., Liu, Y., Yan, Y. B., Feng, F., Liu, W. Q., & Zhou, H. M. (2007). Identification of the amino acids crucial for the activities of drought responsive element binding factors (DREBs) of Brassica napus. FEBS Letters, 581, 3044–3050.

    Article  CAS  Google Scholar 

  46. Gao, S. Q., Chen, M., Xia, L. Q., Xiu, H. J., Xu, Z. S., Li, L. C., et al. (2009). A cotton (Gossypium hirsutum) DRE-binding transcription factor gene, GhDREB, confers enhanced tolerance to drought, high salt, and freezing stresses in transgenic wheat. Plant Cell Reports, 28, 301–311.

    Article  CAS  Google Scholar 

  47. Chinnusamy, V., Gong, Z., & Zhu, J. K. (2008). Abscisic acid-mediated epigenetic processes in plant development and stress responses. Journal of Integrative Plant Biology, 50, 1187–1195.

    Article  CAS  Google Scholar 

  48. Nambara, E., & Marion, P. A. (2005). Abscisic acid biosynthesis and catabolism. Annual Review of Plant Biology, 56, 165–185.

    Article  CAS  Google Scholar 

  49. Bari, R., & Jones, J. D. (2009). Role of plant hormones in plant defence responses. Plant Molecular Biology, 69, 473–488.

    Article  CAS  Google Scholar 

  50. Haake, V., Cook, D., Riechmann, J. L., Pineda, O., Thomashow, M. F., & Zhang, J. Z. (2002). Transcription factor CBF4 is a regulator of drought adaptation in Arabidopsis. Plant Physiology, 130, 639–648.

    Article  CAS  Google Scholar 

  51. Nunes, M. E. S., & Smith, G. R. (2003). Electrolyte leakage assay capable of quantifying freezing resistance in rose clover. Crop Science, 43, 1349–1357.

    Article  Google Scholar 

  52. Qin, F., Sakuma, Y., Li, J., Liu, Q., Li, Y. Q., Shinozaki, K., et al. (2004). Cloning and functional analysis of a novel DREB1/CBF transcription factor involved in cold-responsive gene expression in Zea mays L. Plant and Cell Physiology, 45, 1042–1052.

    Article  CAS  Google Scholar 

  53. Zhang, G., Chen, M., Li, L., Xu, Z., Chen, X., Guo, J., et al. (2009). Overexpression of the soybean GmERF3 gene, an AP2/ERF type transcription factor for increased tolerances to salt, drought, and diseases in transgenic tobacco. Journal of Experimental Botany, 60, 3781–3796.

    Article  CAS  Google Scholar 

  54. Yamaguchi, S. K., & Shinozaki, K. (1993). Characterization of the expression of a desiccation-responsive rd29 gene of Arabidopsis thaliana and analysis of its promoter in transgenic plants. Molecular and General Genetics, 236, 331–340.

    Article  Google Scholar 

  55. Yamaguchi, S. K., & Shinozaki, K. (1993). Arabidopsis DNA encoding two desiccation-responsive rd29 genes. Plant Physiology, 101, 1119–1120.

    Article  Google Scholar 

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Acknowledgments

The research was supported by the Priority Academic Program Development of Jiangsu Higher Education Institutions; New Century Excellent Talents in University (NCET); National key Project of Transgenic Crops of China (2009ZX08002-011B). The authors would like to thank Dr. Jeffrey Charrois and Andrea Bailey for providing the critical reading the manuscript and valuable suggestions.

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Authors and Affiliations

  1. State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Horticulture, Nanjing Agricultural University, 1 Weigang, 210095, Nanjing, China

    Ai-Sheng Xiong & Feng Wang

  2. Biotechnology Research Institute, Shanghai Academy of Agricultural Sciences, 201106, Shanghai, China

    Hai-Hua Jiang, Ri-He Peng, Xiao-Fen Jin, Bo Zhu & Quan-Hong Yao

  3. Life Science, Alberta Innovates-Technology Futures/Alberta Research Council, Vegreville, AB, T9C 1T4, Canada

    Jing Zhuang & Jian Zhang

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  1. Ai-Sheng Xiong
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Correspondence to Ai-Sheng Xiong or Quan-Hong Yao.

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Xiong, AS., Jiang, HH., Zhuang, J. et al. Expression and Function of a Modified AP2/ERF Transcription Factor from Brassica napus Enhances Cold Tolerance in Transgenic Arabidopsis . Mol Biotechnol 53, 198–206 (2013). https://doi.org/10.1007/s12033-012-9515-x

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