DOI QR코드

DOI QR Code

벼에 존재하는 CRL4 복합체 scaffold 유전자의 발현 양상에 대한 연구

Expression Study on the Scaffold Gene of CRL4 Complex in Rice (Oryza sativa L.)

  • Bae, Yoowon (Department of Biology Education, Pusan National University) ;
  • Kim, Hani (Department of Biology Education, Pusan National University) ;
  • Kim, Sang-Hoon (Department of Biology Education, Pusan National University) ;
  • Lee, Jae-Hoon (Department of Biology Education, Pusan National University)
  • 투고 : 2018.06.05
  • 심사 : 2018.08.21
  • 발행 : 2018.10.30

초록

진핵생물에서 유비퀴틴화 과정을 통해 단백질 안정성이 조절되며, E3 ligase는 유비퀴틴화 과정 동안 분해 대상 기질의 결정 및 기질로의 유비퀴틴 전달을 위한 주효소로 작용한다. Multi-subunit E3 ligase의 일종인 cullin4(CUL4)-based E3 ligase (CRL4) 복합체는 식물의 다양한 호르몬, 스트레스와 관련된 세포 내 과정에서 중요한 역할을 하는 것으로 알려져 있다. 호르몬, 스트레스 신호 전달 과정에서 CRL4의 다양한 역할에 대한 보고가 애기장대에서 이루어져 왔음에도 불구하고, 주요 식량 작물인 벼에서의 CRL4 기능에 대한 연구는 매우 미흡한 실정이다. 이에 벼에서 CRL4에 의해 매개되는 세포 내 반응들을 상세히 이해하기 위해, 본 연구에서는 애기장대 cullin4 (CUL4)의 상동 유전자를 벼에서 동정하고, 조직별 벼 CUL4 유전자의 발현 양상과 다양한 식물 호르몬 및 환경 스트레스 처리에 의한 해당 유전자의 발현 양상을 탐색하였다. 벼 CUL4 유전자인 OsCUL4는 앱시스산, 사이토키닌과 같은 식물 호르몬과 가뭄, 고염 스트레스에 의해 발현량이 급격히 상형 조절되는 양상을 보였는데 이는 해당 단백질이 앱시스산 및 사이토키닌에 의해 매개되는 세포 내 반응과 기능적으로 연계되어 있음을 암시한다. 또한, OsCUL4는 CRL4 복합체의 어댑터로 작용하는 OsDDB1과 직접적으로 결합하였는데, 이는 본 연구를 통해 동정한 OsCUL4가 벼에서 실질적으로 CRL4의 scaffold 단백질로 기능할 수 있음을 보여준다. 본 연구를 통해 수행된 OsCUL4 유전자의 발현 양상에 대한 연구는, 벼에서 CRL4 매개 유비퀴틴화 과정이 관여하는 세포 내 반응을 규명하기 위한 시작점으로 활용될 수 있을 것이라 사료된다.

The stability of diverse cellular proteins in eukaryotes is regulated via ubiquitination. Moreover, E3 ligase plays a crucial role in determining substrate specificity and transfers ubiquitins into the substrates during the ubiquitination process. As a type of multi-subunit E3 ligase, cullin4 (CUL4)-based E3 ligase (CRL4) complex is involved in a variety of cellular processes, such as hormonal and stress responses in plants. In spite of several reports on the versatile roles of CRL4 in various signalings in Arabidopsis, CRL4's function in rice has been poorly known. To learn about CRL4-mediated cellular processes in rice in more detail, OsCUL4 that exhibits the highest homology with Arabidopsis CUL4 was isolated, and its expression patterns in various tissues and in response to plant hormones and abiotic stresses were monitored. Exogenous application of ABA or cytokinin increased the transcript levels of the OsCUL4 gene. Moreover, OsCUL4 was significantly upregulated in response to drought and salt stresses. These findings imply that OsCUL4 may be functionally related to ABA- and/or cytokinin-mediated cellular responses. OsCUL4 directly interacted with OsDDB1, an adaptor protein of CRL4, indicating that OsCUL4 can act as a scaffold protein of CRL4. An expression study on the OsCUL4 gene from this report could be used as a starting point to elucidate cellular responses in which a CRL4-mediated ubiquitination process is involved in rice.

키워드

참고문헌

  1. Bernhardt, A., Lechner, E., Hano, P., Schade, V., Dieterle, M., Anders, M., Dubin, M. J., Benvenuto, G., Bowler, C., Genschik, P. and Hellmann, H. 2006. CUL4 associates with DDB1 and DET1 and its downregulation affects diverse aspects of development in Arabidopsis thaliana. Plant J. 47, 591-603. https://doi.org/10.1111/j.1365-313X.2006.02810.x
  2. Biedermann, S. and Hellmann, H. 2010. The DDB1a interacting proteins ATCSA-1 and DDB2 are critical factors for UV-B tolerance and genomic integrity in Arabidopsis thaliana. Plant J. 62, 404-415. https://doi.org/10.1111/j.1365-313X.2010.04157.x
  3. Callis, J. 2014. The ubiquitination machinery of the ubiquitin system. Arabidopsis Book 12, e0174.
  4. Chen, H., Shen, Y., Tang, X., Yu, L., Wang, J., Guo, L., Zhang, Y., Zhang, H., Feng, S., Strickland, E., Zheng, N. and Deng, X. W. 2006. Arabidopsis CULLIN4 forms an E3 ubiquitin ligase with RBX1 and the CDD complex in mediating light control of development. Plant Cell 18, 1991-2004. https://doi.org/10.1105/tpc.106.043224
  5. Dutilleul, C., Ribeiro, I., Blanc, N., Nezames, C. D., Deng, X. W., Zglobicki, P., Palacio Barrera, A. M., Atehortùa, L., Courtois, M., Labas, V., Giglioli-Guivarc'h, N. and Ducos, E. 2016. ASG2 is a farnesylated DWD protein that acts as ABA negative regulator in Arabidopsis. Plant Cell Environ. 39, 185-198.
  6. Gagne, J. M., Downes, B. P., Shiu, S. H., Durski, A. M. and Vierstra, R. D. 2002. The F-box subunit of the SCF E3 complex is encoded by a diverse superfamily of genes in Arabidopsis. Proc. Natl. Acad. Sci. USA. 99, 11519-11524. https://doi.org/10.1073/pnas.162339999
  7. Gingerich, D. J., Gagne, J. M., Salter, D. W., Hellmann, H., Estelle, M., Ma, L. and Vierstra, R. D. 2005. Cullins 3a and 3b assemble with members of the broad complex/tramtrack/bric-a-brac (BTB) protein family to form essential ubiquitin-protein ligases (E3s) in Arabidopsis. J. Biol. Chem. 280, 18810-18821.
  8. Gruber, H., Heijde, M., Heller, W., Albert, A., Seidlitz, H. K. and Ulm, R. 2010. Negative feedback regulation of UV-B-induced photomorphogenesis and stress acclimation in Arabidopsis. Proc. Natl. Acad. Sci. USA. 107, 20132-20137.
  9. Heijde, M. and Ulm, R. 2013. Reversion of the Arabidopsis UV-B photoreceptor UVR8 to the homodimeric ground state. Proc. Natl. Acad. Sci. USA. 110, 1113-1118. https://doi.org/10.1073/pnas.1214237110
  10. Hotton, S. K. and Callis, J. 2008. Regulation of cullin RING ligases. Annu. Rev. Plant Biol. 59, 467-489. https://doi.org/10.1146/annurev.arplant.58.032806.104011
  11. Hua, Z. and Vierstra, R. D. 2011. The cullin-RING ubiquitin-protein ligases. Annu. Rev. Plant Biol. 62, 299-334. https://doi.org/10.1146/annurev-arplant-042809-112256
  12. Iconomou, M. and Saunders, D. N. 2016. Systematic approaches to identify E3 ligase substrates. Biochem. J. 473, 4083-4101.
  13. Irigoyen, M. L., Iniesto, E., Rodriguez, L., Puga, M. I., Yanagawa, Y., Pick, E., Strickland, E., Paz-Ares, J., Wei, N., De Jaeger, G., Rodriguez, P. L., Deng, X. W. and Rubio, V. 2014. Targeted degradation of abscisic acid receptors is mediated by the ubiquitin ligase substrate adaptor DDA1 in Arabidopsis. Plant Cell 26, 712-728. https://doi.org/10.1105/tpc.113.122234
  14. Kim, S. H., Kim, H., Chung, S. and Lee, J. H. 2017. DHU1 negatively regulates UV-B signaling via its direct interaction with COP1 and RUP1. Biochem. Biophys. Res. Commun. 491, 285-290. https://doi.org/10.1016/j.bbrc.2017.07.110
  15. Kim, S. H., Kim, H., Seo, K. I., Kim, S. H., Chung, S., Huang, X., Yang, P., Deng, X. W. and Lee, J. H. 2014. DWD HYPERSENSITIVE TO UV-B 1 is negatively involved in UV-B mediated cellular responses in Arabidopsis. Plant Mol. Biol. 86, 571-583. https://doi.org/10.1007/s11103-014-0247-0
  16. Kim, S. H., Woo, O. G., Jang, H. S. and Lee, J. H. 2018. Characterization and comparative expression analysis of CUL1 genes in rice. Genes Genom. 40, 233-241. https://doi.org/10.1007/s13258-017-0622-8
  17. Lee, J. H. and Kim, W. T. 2011. Regulation of abiotic stress signal transduction by E3 ubiquitin ligases in Arabidopsis. Mol. Cells 31, 201-208. https://doi.org/10.1007/s10059-011-0031-9
  18. Lee, J. H., Terzaghi, W., Gusmaroli, G., Charron, J. B., Yoon, H. J., Chen, H., He, Y. J., Xiong, Y. and Deng, X. W. 2008. Characterization of Arabidopsis and rice DWD proteins and their roles as substrate receptors for CUL4-RING E3 ubiquitin ligases. Plant Cell 20, 152-167. https://doi.org/10.1105/tpc.107.055418
  19. Lee, J. H., Terzaghi, W. and Deng, X. W. 2011. DWA3, an Arabidopsis DWD protein, acts as a negative regulator in ABA signal transduction. Plant Sci. 180, 352-357. https://doi.org/10.1016/j.plantsci.2010.10.008
  20. Lee, J. H., Yoon, H. J., Terzaghi, W., Martinez, C., Dai, M., Li, J., Byun, M. O. and Deng, X. W. 2010. DWA1 and DWA2, two Arabidopsis DWD protein components of CUL4-based E3 ligases, act together as negative regulators in ABA signal transduction. Plant Cell 22, 1716-1732. https://doi.org/10.1105/tpc.109.073783
  21. Lee, J. H. 2016. Structure and biological function of plant CRL4, and its involvement in plant cellular events. J. Life Sci. 26, 364-375. https://doi.org/10.5352/JLS.2016.26.3.364
  22. Li, D., Zhang, L., Li, X., Kong, X., Wang, X., Li, Y., Liu, Z., Wang, J., Li, X. and Yang, Y. 2018. AtRAE1 is involved in degradation of ABA receptor RCAR1 and negatively regulates ABA signalling in Arabidopsis. Plant Cell Environ. 41, 231-244. https://doi.org/10.1111/pce.13086
  23. Onate-Sanchez, L. and Vicente-Carbajosa, J. 2008. DNA-free RNA isolation protocols for Arabidopsis thaliana, including seeds and siliques. BMC Res. Notes 1, 93.
  24. Petroski, M. D. and Deshaies, R. J. 2005. Function and regulation of cullin-RING ubiquitin ligases. Nat. Rev. Mol. Cell Biol. 6, 9-20.
  25. Rice, P., Longden, I. and Bleasby, A. 2000. EMBOSS: The European Molecular Biology Open Software Suite. Trends Genet. 16, 276-277. https://doi.org/10.1016/S0168-9525(00)02024-2
  26. Seo, K. I., Lee, J. H., Nezames, C. D., Zhong, S., Song, E., Byun, M. O. and Deng, X. W. 2014. ABD1 is an Arabidopsis DCAF substrate receptor for CUL4-DDB1-based E3 ligases that acts as a negative regulator of abscisic acid signaling. Plant Cell 26, 695-711. https://doi.org/10.1105/tpc.113.119974
  27. Tamura, K., Stecher, G., Peterson, D., Filipski, A. and Kumar, S. 2013. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30, 2725-2729.
  28. Vierstra, R. D. 2009. The ubiquitin-26S proteasome system at the nexus of plant biology. Nat. Rev. Mol. Cell Biol. 10, 385-397. https://doi.org/10.1038/nrm2688
  29. Wang, S., Liu, J., Feng, Y., Niu, X., Giovannoni, J. and Liu, Y. 2008. Altered plastid levels and potential for improved fruit nutrient content by downregulation of the tomato DDB1-interacting protein CUL4. Plant J. 55, 89-103.
  30. Xu, G., Ma, H., Nei, M. and Kong, H. 2009. Evolution of F-box genes in plants: different modes of sequence divergence and their relationships with functional diversification. Proc. Natl. Acad. Sci. USA. 106, 835-840. https://doi.org/10.1073/pnas.0812043106
  31. Zang, G., Zou, H., Zhang, Y., Xiang, Z., Huang, J., Luo, L., Wang, C., Lei, K., Li, X., Song, D., Din, A. U. and Wang, G. 2016. The De-Etiolated 1 homolog of Arabidopsis modulates the ABA signaling pathway and ABA biosynthesis in rice. Plant Physiol. 171, 1259-1276.
  32. Zhang, C., Guo, H., Zhang, J., Guo, G., Schumaker, K. S. and Guo, Y. 2010. Arabidopsis cockayne syndrome A-like proteins 1A and 1B form a complex with CULLIN4 and damage DNA binding protein 1A and regulate the response to UV irradiation. Plant Cell 22, 2353-2369.
  33. Zhang, Y., Feng, S., Chen, F., Chen, H., Wang, J., McCall, C., Xiong, Y. and Deng, X. W. 2008. Arabidopsis DDB1-CUL4 ASSOCIATED FACTOR1 forms a nuclear E3 ubiquitin ligase with DDB1 and CUL4 that is involved in multiple plant developmental processes. Plant Cell 20, 1437-1455. https://doi.org/10.1105/tpc.108.058891