DOI QR코드

DOI QR Code

마이크로어레이를 이용한 애기장대 AtERF71/HRE2 전사인자의 하위 유전자 분석

Analysis of Putative Downstream Genes of Arabidopsis AtERF71/HRE2 Transcription Factor using a Microarray

  • 석혜연 (부산대학교 자연과학대학 분자생물학과) ;
  • 이선영 (부산대학교 자연과학대학 분자생물학과) ;
  • 우동혁 (부산대학교 자연과학대학 분자생물학과) ;
  • 박희연 (부산대학교 자연과학대학 분자생물학과) ;
  • 문용환 (부산대학교 자연과학대학 분자생물학과)
  • Seok, Hye-Yeon (Department of Molecular Biology, Pusan National University) ;
  • Lee, Sun-Young (Department of Molecular Biology, Pusan National University) ;
  • Woo, Dong-Hyuk (Department of Molecular Biology, Pusan National University) ;
  • Park, Hee-Yeon (Department of Molecular Biology, Pusan National University) ;
  • Moon, Yong-Hwan (Department of Molecular Biology, Pusan National University)
  • 투고 : 2012.08.02
  • 심사 : 2012.10.05
  • 발행 : 2012.10.30

초록

애기장대에서 AtERF71/HRE2는 핵에서 전사인자로 작용하여 하위 유전자의 발현을 증가시키는 역할을 수행함으로써 저산소와 삼투 스트레스 반응에 관여할 것으로 여겨지는 유전자이다. 본 연구에서는 AtERF71/HRE2에 의해 직, 간접적으로 발현이 조절되는 하위 유전자를 알아보기 위해 AtERF71/HRE2 과발현체를 대상으로 마이크로어레이 실험을 수행하였다. 야생형에 비해 AtERF71/HRE2 과발현체에서 발현이 2배 이상 증가한 기능이 알려진 유전자는 AtERF71/HRE2 자신을 제외하고 161개였다. 161개 유전자 중 전사인자와 DNA-결합 단백질 등과 같은 전사조절자가 24개로 확인되어, AtERF71/HRE2는 하위 전사조절 유전자의 발현 조절을 통해 더 많은 유전자의 발현을 조절하는 상위 전사인자로서의 기능을 가질 것으로 추정되었다. 161개 유전자 중 15개 유전자를 대상으로 RT-PCR을 수행하여 마이크로어레이 결과의 신뢰성을 검증하였다. Genevestigator 데이터베이스 분석 결과, 161개 유전자 중 51개 유전자는 저산소 및 삼투 스트레스에 의해 발현이 증가하는 것으로 확인되었다. RT-PCR 분석 결과 AtERF71/HRE2 과발현체에서 발현이 증가한 15개 유전자 중 3개 유전자가 저산소에 의해 발현이 증가하였고, 다른 3개 유전자가 삼투 스트레스에 의해 발현이 증가하였으며, 이러한 결과는 이들 유전자가 AtERF71/HRE2에 의해 매개되는 저산소 또는 고염 스트레스 신호전달의 하위 유전자일 수 있음을 의미한다. 또한 본 연구의 마이크로어레이 분석 결과는 AtERF71/HRE2가 저산소 및 삼투 스트레스 반응뿐만 아니라 다른 환경 스트레스 반응과 식물 발달 조절에도 관여할 수 있음을 시사한다.

Arabidopsis AtERF71/HRE2, a transcription activator, is located in the nucleus and is involved in the signal transduction of low oxygen and osmotic stresses. In this study, microarray analysis using AtERF71/HRE2-overexpressing transgenic plants was performed to identify genes downstream of AtERF71/HRE2. A total of 161 different genes as well as AtERF71/HRE2 showed more than a twofold higher expression in AtERF71/HRE2-overexpressing transgenic plants compared with wild-type plants. Among the 161 genes, 24 genes were transcriptional regulators, such as transcription factors and DNA-binding proteins, based on gene ontology annotations, suggesting that AtERF71/HRE2 is an upstream transcription factor that regulates the activities of various downstream genes via these transcription regulators. RT-PCR analysis of 15 genes selected out of the 161 genes showed higher expression in AtERF71/HRE2-overexpressing transgenic plants, validating the microarray data. On the basis of Genevestigator database analysis, 51 genes among the 161 genes were highly expressed under low oxygen and/or osmotic stresses. RT-PCR analysis showed that the expression levels of three genes among the selected 15 genes increased under low oxygen stress and another three genes increased under high salt stress, suggesting that these genes might be downstream genes of AtERF71/HRE2 in low oxygen or high salt stress signal transduction. Microarray analysis results indicated that AtERF71/HRE2 might also be involved in the responses to other abiotic stresses and also in the regulation of plant developmental processes.

키워드

참고문헌

  1. Abbasi, A. R., Hajirezaei, M., Hofius, D., Sonnewald, U. and Voll, L. M. 2007. Specific Roles of $\alpha$- and $\gamma$-Tocopherol in Abiotic Stress Responses of Transgenic Tobacco. Plant Physiol. 143, 1720-1738. https://doi.org/10.1104/pp.106.094771
  2. Bao, Y. M., Sun, S. J., Li, M., Li, L., Cao, W. L., Luo, J., Tang, H. J., Huang, J., Wang, Z. F., Wang, J. F. and Zhang, H. S. 2012. Overexpression of the Qc-SNARE gene OsSYP71 enhances tolerance to oxidative stress and resistance to rice blast in rice (Oryza sativa L.). Gene 504, 238-244. https://doi.org/10.1016/j.gene.2012.05.011
  3. Broun, P. 2004. Transcription factors as tools for metabolic engineering in plants. Curr. Opin. Plant Biol. 7, 202-209. https://doi.org/10.1016/j.pbi.2004.01.013
  4. Brownfield, D. L., Todd, C. D. and Deyholos, M. K. 2008. Analysis of Arabidopsis arginase gene transcription patterns indicates specific biological functions for recently diverged paralogs. Plant Mol. Biol. 67, 429-440. https://doi.org/10.1007/s11103-008-9336-2
  5. Dombrecht, B., Xue, G. P., Sprague, S. J., Kirkegaard, J. A., Ross, J. J., Reid, J. B., Fitt, G. P., Sewelam, N., Schenk, P. M., Manners, J. M. and Kazana, K. 2007. MYC2 Differentially Modulates Diverse Jasmonate-Dependent Functions in Arabidopsis. Plant Cell 19, 2225-2245. https://doi.org/10.1105/tpc.106.048017
  6. Dubos, C., Gourrierec, J. L., Baudry, A., Huep, G., Lanet, E., Debeaujon, I., Routaboul, J. M., Alboresi, A., Weisshaar, B. and Lepiniec, L. 2008. MYBL2 is a new regulator of flavonoid biosynthesis in Arabidopsis thaliana. Plant J. 55, 940-953. https://doi.org/10.1111/j.1365-313X.2008.03564.x
  7. Fan, H. F., Dua, C. X. and Guo, S. R. 2010. Nitric oxide enhances salt tolerance in cucumber seedlings by regulating free polyamine content. Environ. Exp. Bot. In Press.
  8. Gong, W., Shen, Y. P., Ma, L. G., Pan, Y., Du, Y. L., Wang, D. H., Yang, J. Y., Hu, L. D., Liu, X. F., Dong, C. X., Ma, L., Chen, Y. H., Yang, X. Y., Gao, Y., Zhu, D., Tan, X., Mu, J. Y., Zhang, D. B., Liu, Y. L., Dinesh-Kumar, S. P., Li, Y., Wang, X. P., Gu, H. Y., Qu, L. J., Bai, S. N., Lu, Y. T., Li, J. Y., Zhao, J. D., Zuo, J., Huang, H., Deng, X. W. and Zhu, Y. X. 2004. Genome-wide ORFeome cloning and analysis of Arabidopsis transcription factor genes. Plant Physiol. 135, 773-782. https://doi.org/10.1104/pp.104.042176
  9. Gutterson, N. and Reuber, T. L. 2004. Regulation of disease resistance pathways by AP2/ERF transcription factors. Curr. Opin. Plant Biol. 7, 465-471. https://doi.org/10.1016/j.pbi.2004.04.007
  10. Haake, V., Cook, D., Riechmann, J. L., Pineda, O., Thomashow, M. F. and Zhang, J. Z. 2002. Transcription Factor CBF4 Is a Regulator of Drought Adaptation in Arabidopsis. Plant Physiol. 130, 639-648. https://doi.org/10.1104/pp.006478
  11. Hao, D., Ohme-Takagi, M. and 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, 26857-26861. https://doi.org/10.1074/jbc.273.41.26857
  12. Hattori, Y., Nagai, K., Furukawa, S., Song, X. J., Kawano, R., Sakakibara, H., Wu, J., Matsumoto, T., Yoshimura, A., Kitano, H., Matsuoka, M., Mori, H. and Ashikari, M. 2009. The ethylene response factors SNORKEL1 and SNORKEL2 allow rice to adapt to deep water. Nature 460, 1026-1030. https://doi.org/10.1038/nature08258
  13. Hess, N., Klode, M., Anders, M. and Sauter, M. 2011. The hypoxia responsive transcription factor genes ERF71/HRE2 and ERF73/HRE1 of Arabidopsis are differentially regulated by ethylene. Physiol. Plant 143, 41-49. https://doi.org/10.1111/j.1399-3054.2011.01486.x
  14. Hinz, M., Wilson, I. W., Yang, J., Buerstenbinder, K., Llewellyn, D., Dennis, E. S., Sauter, M. and Dolferus, R. 2010. Arabidopsis RAP2.2: an ethylene response transcription factor that is important for hypoxia survival. Plant Physiol. 153, 757-772. https://doi.org/10.1104/pp.110.155077
  15. Ibrahim, R. K., Bruneau, A. and Bantignies, B. 1998. Plant O-methyltransferases: molecular analysis, common signature and Classification. Plant Mol. Biol. 36, 1-10. https://doi.org/10.1023/A:1005939803300
  16. Kagaya, Y., Ohmiya, K. and Hattori, T. 1999. RAV1, a novel DNA-binding protein, binds to bipartite recognition sequence through two distinct DNA-binding domains uniquely found in higher plants. Nucleic Acids Res. 27, 470-478. https://doi.org/10.1093/nar/27.2.470
  17. Khan, M. N., Siddiqui, M. H., Mohammad, F. and Naeem, M. 2012. Interactive role of nitric oxide and calcium chloride in enhancing tolerance to salt stress. Nitric Oxide 27, 210-218. https://doi.org/10.1016/j.niox.2012.07.005
  18. Kim, S. J. and Bassham, D. C. 2011. TNO1 Is Involved in Salt Tolerance and Vacuolar Trafficking in Arabidopsis. Plant Physiol. 156, 514-526. https://doi.org/10.1104/pp.110.168963
  19. Knight, H. and Knight, M. R. 2001. Abiotic stress signalling pathways: specificity and cross-talk. Trends Plant Sci. 6, 262-267. https://doi.org/10.1016/S1360-1385(01)01946-X
  20. Lexer, C. and Fay, M. F. 2005. Adaptation to environmental stress: a rare or frequent driver of speciation? J. Evol. Biol. 18, 893-900. https://doi.org/10.1111/j.1420-9101.2005.00901.x
  21. Licausi, F., van Dongen, J. T., Giuntoli, B., Novi, G., Santaniello, A., Geigenberger, P. and Perata, P. 2010. HRE1 and HRE2, two hypoxia-inducible ethylene response factors, affect anaerobic responses in Arabidopsis thaliana. Plant J. 62, 302-315. https://doi.org/10.1111/j.1365-313X.2010.04149.x
  22. Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473-497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
  23. Nakano, T., Suzuki, K., Fujimura, T. and Shinshi, H. 2006. Genome-Wide Analysis of the ERF Gene Family in Arabidopsis and Rice. Plant Physiol. 140, 411-432. https://doi.org/10.1104/pp.105.073783
  24. Park, H. Y., Seok, H. Y., Woo, D. H., Lee, S. Y., Tarte, V. N., Lee, E. H., Lee, C. H. and Moon, Y. H. 2011. AtERF71/HRE2 transcription factor mediates osmotic stress response as well as hypoxia response in Arabidopsis. Biochem. Biophys. Res. Commun. 414, 135-141. https://doi.org/10.1016/j.bbrc.2011.09.039
  25. Perata, P. and Voesenek, L. A. 2007. Submergence tolerance in rice requires Sub1A, an ethylene-response-factor-like gene. Trends Plant Sci. 12, 43-46. https://doi.org/10.1016/j.tplants.2006.12.005
  26. Rushton, P. J. and Somssich, I. E. 1998. Transcriptional control of plant genes responsive to pathogens. Curr. Opin. Plant Biol. 1, 311-315. https://doi.org/10.1016/1369-5266(88)80052-9
  27. Sakuma, Y., Liu, Q., Dubouzet, J. G., Abe, H., Shinozaki, K. and 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, 998-1009. https://doi.org/10.1006/bbrc.2001.6299
  28. Sawa, M., Nusinow, D. A., Kay, S. A. and Imaizumi, T. 2007. FKF1 and GIGANTEA Complex Formation Is Required for Day-Length Measurement in Arabidopsis. Science 318, 261-265. https://doi.org/10.1126/science.1146994
  29. Semchuk, N. M., Lushchak, O. V., Falk, J., Krupinska, K. and Lushchak, V. I. 2009. Inactivation of genes, encoding tocopherol biosynthetic pathway enzymes, results in oxidative stress in outdoor grown Arabidopsis thaliana. Plant Physiol. Biochem. 47, 384-390. https://doi.org/10.1016/j.plaphy.2009.01.009
  30. Senthil-Kumar, M., Hema, R., Suryachandra, T. R., Ramegowda, H. V., Gopalakrishna, R., Rama, N., Udayakumar, M. and Mysore, K. S. 2010. Functional characterization of three water deficit stress-induced genes in tobacco and Arabidopsis: An approach based on gene down regulation. Plant Physiol. Biochem. 48, 35-44. https://doi.org/10.1016/j.plaphy.2009.09.005
  31. Stockinger, E. J., Gilmour, S. J. and Thomashow, M. F. 1997. Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit. Proc. Natl. Acad. Sci. USA. 94, 1035-1040. https://doi.org/10.1073/pnas.94.3.1035
  32. Sutter, J. U., Campanoni, P., Tyrrell, M. and Blatt, M. R. 2006. Selective Mobility and Sensitivity to SNAREs Is Exhibited by the Arabidopsis KAT1 $K^{+}$ Channel at the Plasma Membrane. Plant Cell 18, 935-954. https://doi.org/10.1105/tpc.105.038950
  33. Tang, M., Sun, J., Liu, Y., Chen, F. and Shen, S. 2007. Isolation and functional characterization of the JcERF gene, a putative AP2/EREBP domain-containing transcription factor, in the woody oil plant Jatropha curcas. Plant Mol. Biol. 63, 419-428. https://doi.org/10.1007/s11103-006-9098-7
  34. Thomashow, M. F. 1999. PLANT COLD ACCLIMATION: Freezing Tolerance Genes and Regulatory Mechanisms. Annu. Rev. Plant Physiol. Plant Mol. Biol. 50, 571-599. https://doi.org/10.1146/annurev.arplant.50.1.571
  35. Uemura, T., Satob, M. H. and Takeyasu, K. 2005. The longin domain regulates subcellular targeting of VAMP7 in Arabidopsis thaliana. FEBS Lett. 579, 2842-2846. https://doi.org/10.1016/j.febslet.2005.04.022
  36. Wu, L., Chen, X., Ren, H., Zhang, Z., Zhang, H., Wang, J., Wang, X. C. and Huang, R. 2007. ERF protein JERF1 that transcriptionally modulates the expression of abscisic acid biosynthesis-related gene enhances the tolerance under salinity and cold in tobacco. Planta 226, 815-825. https://doi.org/10.1007/s00425-007-0528-9
  37. Xu, K., Xu, X., Fukao, T., Canlas, P., Maghirang-Rodriguez, R., Heuer, S., Ismail, A. M., Bailey-Serres, J., Ronald, P. C. and Mackill, D. J. 2006. Sub1A is an ethyleneresponse- factor-like gene that confers submergence tolerance to rice. Nature 442, 705-708. https://doi.org/10.1038/nature04920
  38. Zhang, G., Chen, M., Li, L., Xu, Z., Chen, X., Guo, J. and Ma, Y. 2009. Overexpression of the soybean GmERF3 gene, an AP2/ERF type transcription factor for increased tolerances to salt, drought, and diseases in transgenic tobacco. J. Exp. Bot. 60, 3781-3796. https://doi.org/10.1093/jxb/erp214