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유채 두 계통에서 저온 스트레스에 반응하는 전사체 발현 비교 분석

Comparative Transcriptome Analysis of the Response of Two Lines of Rapeseed (Brassica napus L.) to Cold Stress

  • 이지은 (농촌진흥청 국립식량과학원 바이오에너지작물연구소) ;
  • 김광수 (농촌진흥청 국립식량과학원 바이오에너지작물연구소) ;
  • 차영록 (농촌진흥청 국립식량과학원 바이오에너지작물연구소) ;
  • 안다희 (농촌진흥청 국립식량과학원 바이오에너지작물연구소) ;
  • 변종원 (농촌진흥청 국립식량과학원 바이오에너지작물연구소) ;
  • 강용구 (농촌진흥청 국립식량과학원 바이오에너지작물연구소)
  • Lee, Ji-Eun (Bioenergy Crop Research Institute, National Institute of Crop Science, RDA) ;
  • Kim, Kwang-Soo (Bioenergy Crop Research Institute, National Institute of Crop Science, RDA) ;
  • Cha, Young-Lok (Bioenergy Crop Research Institute, National Institute of Crop Science, RDA) ;
  • An, Da-Hee (Bioenergy Crop Research Institute, National Institute of Crop Science, RDA) ;
  • Byun, Jong-Won (Bioenergy Crop Research Institute, National Institute of Crop Science, RDA) ;
  • Kang, Yong-Ku (Bioenergy Crop Research Institute, National Institute of Crop Science, RDA)
  • 투고 : 2020.11.25
  • 심사 : 2021.02.03
  • 발행 : 2021.03.01

초록

본 연구에서는 유채 두 계통에 0℃ 이하의 저온 스트레스를 처리하고 이에 따른 proline 함량 및 생존율 변화를 확인하고 이에 따른 저온에서의 유전자 발현 변화를 비교 분석하기 위해 수행되었으며, 결과는 다음과 같다. 1. 0℃ 이하의 저온 스트레스 처리 전 저온 순화 후 유채 'J8634-B-30' 계통에서 proline 함량이 2.02 mM/g FW로 증가하여 처리 전보다 8.7배 증가하였으며, 'EMS26' 계통에서는 0℃ 이하의 저온 스트레스 처리로 인한 proline 함량 변화를 보이지 않았다. 2. 저온 순화 전후 유채 두 계통의 전사체를 분석한 결과, 'J8634-B-30' 계통의 DEG는 발현이 유도된 DEG가 2,784개로, 발현이 억제된 DEG 2,299개보다 많았으며, 'EMS 26' 계통에서는 발현이 유도된 DEG가 2,199개로 발현이 억제된 DEG 3,632개로 적었다. 3. 저온 스트레스 처리에 의한 유채 두 계통의 상위 100개 DEG를 분석한 결과, 'J8634-B-30' 계통에서는 flowering-promoting factor (BnaA10g21640D) 유전자가 강하게 발현되었으며, 특히 proline 생합성 관련 유전자의 발현이 강하게 유도되었다. 'EMS26' 계통의 DEG에서는 식물체 생장과 관련된 유전자가 발현이 억제되었다. 4. 'J8634-B-30' 계통에서는 proline 생합성 관련 P5CSA(BnaA04g22810D, BnaC04g46630D) 유전자의 발현이 유도되었으며, proline 이화작용 관련 PDH (BnaAnng 37880D, BnaC04g31100D) 유전자 발현이 억제되어, 저온 처리에 의한 proline 함량 이 증가한 것으로 판단된다. 5. 저온 반응 관련 생리반응 경로 유전자 중 ABA 호르몬 수용체 PYL5 (BnaAnng40650D), PYL9 (BnaA07g38130D, BnaC06g17940D)는 'J8634-B-30' 계통에서만 발현이 유도되었다. 또한, ICE-CBF-COR 신호 회로 중 원형질막 안정화에 관여하는 COR413 유전자(BnaA08g15470D, BnaC03g61740D)에서도 'J8634-B-30' 계통에서만 발현이 유도되었다. 6. 이러한 저온 스트레스 반응과 관련된 유전자 발현 차이는 초기 저온 처리 후 두 유채 계통의 스트레스 반응에 관여했을 것으로 판단되며, 특이적인 발현 양상을 보인 유전자에 대해 향후 추가적인 기능을 분석하여 내동성 유채 품종 개발에 활용할 수 있을 것으로 판단된다.

Rapeseed is a typical winter crop, and its freezing stress tolerance is a major feature for winter survival. Therefore, it is important to comprehend clearly the physical and molecular mechanisms of rapeseed under freezing stress conditions. This study investigates the physical and transcriptome changes of two rapeseed lines, 'J8634-B-30' and 'EMS26', under cold acclimation and freezing temperature treatments. The proline content of 'J8634-B-30' at 5 ℃ increased 8.7-fold compared to that before treatment, and there was no significant change in that of 'EMS26' RNA-sequencing analysis revealed 5,083 differentially expressed genes (DEGs) of 'J8634-B-30' under cold acclimation condition. Among the genes, 2,784 (54.8%) were up-regulated and 2,299 (45.2%) were down-regulated. The DEGs of 'EMS26' under cold acclimation condition were 5,831 genes, and contained 2,199 up-regulated genes (37.7%) and 3,632 down-regulated genes (62.3%). Among them, only DEGs annotated in the cold response-related signaling pathways were selected, and their expression in the two rapeseed lines was compared. Comparative DEGs analysis indicated that cold response related signaling pathways are proline metabolism and ABA (Abscisic acid) signaling. And ICE (Inducer of CBF expression) - CBF (C-repeat-binding factor) - COR (Cold-regulated) signaling were the significantly differentially expressed transcripts in the two rapeseed lines. The major induced transcripts of 'J8634-B-30' induced P5CS (Δ'-pyrroline-5-carboxylate synthetase), which is related to proline biosynthesis, PYL (pyrabactin resistance-like protein, ABA receptor) and COR413 (cold-regulated 413 plasma membrane 1). In conclusion, these result provide a foundation for understanding the mechanisms of freezing stress tolerance in rapeseeds. Further functional studies should be performed on the freezing stress-related genes identified in this study, which can contribute to the transgenic and molecular breeding for freezing stress tolerance in rapeseed.

키워드

참고문헌

  1. Amasino, R. 2004. Vernalization, competence, and the epigenetic memory of winter. Plant Cell. 16 : 2553-2559. https://doi.org/10.1105/tpc.104.161070
  2. Anders, S. and W. Huber. 2010. Differential expression analysis for sequence count data. Nature Precedings, 1-1.
  3. Anders, S., P. T. Pyl, and W. Huber. 2015. HTSeq-a Python framework to work with high-throughput sequencing data. Bioinformatics 31 : 166-169. https://doi.org/10.1093/bioinformatics/btu638
  4. Bolger, A. M., M. Lohse, and B. Usadel. 2014. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 30 : 2114-2120. https://doi.org/10.1093/bioinformatics/btu170
  5. Borba, A. R., T. S. Serra, A. Gorska, P. Gouveia, A. M. Cordeiro, I. Reyna-Llorens, J. Knerova, P. M. Barros, I. A. Abreu, and M. M. Oliveira. 2018. Synergistic binding of bHLH transcription factors to the promoter of the maize NADP-ME gene used in C4 photosynthesis is based on an ancient code found in the ancestral C3 state. Mol Biol Evol. 35 : 1690-1705. https://doi.org/10.1093/molbev/msy060
  6. Boudsocq, M., H. Barbier-Brygoo, and C. Lauriere. 2004. Identification of nine sucrose nonfermenting 1-related protein kinases 2 activated by hyperosmotic and saline stresses in Arabidopsis thaliana. J Biol Chem. 279 : 41758-41766. https://doi.org/10.1074/jbc.M405259200
  7. Burbulis, N., V. Jonytiene, R. Kupriene, and A. Blinstrubiene. 2011. Changes in proline and soluble sugars content during cold acclimation of winter rapeseed shoots in vitro. J Food Agric Environ. 9 : 371-374.
  8. Chalhoub, B., F. Denoeud, S. Liu, I. A. Parkin, H. Tang, X. Wang, J. Chiquet, H. Belcram, C. Tong, and B. Samans. 2014. Early allopolyploid evolution in the post-Neolithic Brassica napus oilseed genome. Science 345 : 950-953. https://doi.org/10.1126/science.1253435
  9. Chen, H. H., P. H. Li, and M. L. Brenner. 1983. Involvement of abscisic acid in potato cold acclimation. Plant Physiol. 71 : 362-365. https://doi.org/10.1104/pp.71.2.362
  10. Chinnusamy, V., J. Zhu, and J. K. Zhu. 2007. Cold stress regulation of gene expression in plants. Trends Plant Sci. 12 : 444-451. https://doi.org/10.1016/j.tplants.2007.07.002
  11. Chopra, R., G. Burow, C. Hayes, Y. Emendack, Z. Xin, and J. Burke. 2015. Transcriptome profiling and validation of gene based single nucleotide polymorphisms (SNPs) in sorghum genotypes with contrasting responses to cold stress. BMC Genomics. 16 : 1-11. https://doi.org/10.1186/1471-2164-16-1
  12. de Abreu-Neto, J. B., A. C. Turchetto-Zolet, L. F. V. de Oliveira, M. H. Bodanese Zanettini, and M. Margis-Pinheiro. 2013. Heavy metal-associated isoprenylated plant protein (HIPP): characterization of a family of proteins exclusive to plants. FEBS J. 280 : 1604-1616. https://doi.org/10.1111/febs.12159
  13. Deuschle, K., D. Funck, H. Hellmann, K. Daschner, S. Binder, and W. B. Frommer. 2001. A nuclear gene encoding mitochondrial Δ1-pyrroline-5-carboxylate dehydrogenase and its potential role in protection from proline toxicity. Plant J. 27 : 345-356. https://doi.org/10.1046/j.1365-313x.2001.01101.x
  14. Guo, X., L. Zhang, G. Dong, Z. Xu, G. Li, N. Liu, A. Wang, and J. Zhu. 2019. A novel cold-regulated protein isolated from Saussurea involucrata confers cold and drought tolerance in transgenic tobacco (Nicotiana tabacum). Plant Sci. 289 : 110246. https://doi.org/10.1016/j.plantsci.2019.110246
  15. Guy, C.L. 1990. Cold acclimation and freezing stress tolerance: role of protein metabolism. Annu Rev Plant Biol. 41 : 187-223. https://doi.org/10.1146/annurev.pp.41.060190.001155
  16. Hayat, S., Q. Hayat, M. N. Alyemeni, A. S. Wani, J. Pichtel, and A. Ahmad. 2012. Role of proline under changing environments: a review. Plant Signal Behav. 7 : 1456-1466. https://doi.org/10.4161/psb.21949
  17. Hrabak, E. M., C. W. Chan, M. Gribskov, J. F. Harper, J. H. Choi, N. Halford, J. Kudla, S. Luan, H. G. Nimmo, and M. R. Sussman. 2003. The Arabidopsis CDPK-SnRK superfamily of protein kinases. Plant Physiol. 132 : 666-680. https://doi.org/10.1104/pp.102.011999
  18. Hu, C., A. J. Delauney, and D. Verma. 1992. A bifunctional enzyme (delta 1-pyrroline-5-carboxylate synthetase) catalyzes the first two steps in proline biosynthesis in plants. PNAS. 89 : 9354-9358. https://doi.org/10.1073/pnas.89.19.9354
  19. Jeong, Y. J., Y. H. Choy, H. J. Joo, J. H. Hwang, Y. J. Byun, Y. M. Lee, J. S. Lee, Y. S. Jang, and D. H. Lee. 2012. Identification and analysis of cold stress-inducible genes in Korean rapeseed varieties. J Plant Biol. 55 : 498-512. https://doi.org/10.1007/s12374-012-0382-6
  20. Kim, B. M. 2015. Studied on physiological properties and seletion of low-temperature resistant plant of Brassica napus L. Unpublished master's dissertation. Mokpo National University. Mokpo.
  21. Kim, D., B. Langmead, and S. L. Salzberg. 2015. HISAT: a fast spliced aligner with low memory requirements. Nat Methods. 12 : 357-360. https://doi.org/10.1038/nmeth.3317
  22. Koster, K. L. and D. V. Lynch. 1992. Solute accumulation and compartmentation during the cold acclimation of Puma rye. Plant Physiol. 98 : 108-113. https://doi.org/10.1104/pp.98.1.108
  23. Lee, K. R., S. I. Sohn, J. H. Jung, S. H. Kim, K. H. Roh, J. B. Kim, M. C. Suh, and Kim, H. U. 2013. Functional analysis and tissue-differential expression of four FAD2 genes in amphidiploid Brassica napus derived from Brassica rapa and Brassica oleracea. Gene. 531 : 253-262. https://doi.org/10.1016/j.gene.2013.08.095
  24. Levitt, J., 1980. Responses of Plants to Environmental Stresses: Chilling, Freezing and High Temperature Stresses, 2nd Edn, Vol. 1. NY: Academic Press. New York. 0160-9327.
  25. Lu, X., J. Li, H. Chen, J. Hu, P. Liu, and B. Zhou. 2017. RNA-seq analysis of apical meristem reveals integrative regulatory network of ROS and chilling potentially related to flowering in Litchi chinensis. Sci Rep. 7 : 1-13. https://doi.org/10.1038/s41598-016-0028-x
  26. Lynch, D. V. and P. L. Steponkus. 1987. Plasma membrane lipid alterations associated with cold acclimation of winter rye seedlings (Secale cereale L. cv Puma). Plant Physiol. 83 : 761-767. https://doi.org/10.1104/pp.83.4.761
  27. McClinchey, S. L., and L. S. Kott. 2008. Production of mutants with high cold tolerance in spring canola (Brassica napus). Euphytica. 162 : 51-67. https://doi.org/10.1007/s10681-007-9554-8
  28. Narusaka, M., M. Seki, T. Umezawa, J. Ishida, M. Nakajima, A. Enju, and K. Shinozaki. 2004. Crosstalk in the responses to abiotic and biotic stresses in Arabidopsis : analysis of gene expression in cytochrome P450 gene superfamily by cDNA microarray. Plant Mol. Biol. 55 : 327-342. https://doi.org/10.1007/s11103-004-0685-1
  29. Ng, L. M., K. Melcher, B. T. Teh, and H. E. Xu. 2014. Abscisic acid perception and signaling : structural mechanisms and applications. Acta Pharmacol Sin. 35 : 567-584. https://doi.org/10.1038/aps.2014.5
  30. Niu, X., T. Luo, H. Zhao, Y. Su, W. Ji, and H. Li. 2020. Identification of wheat DREB genes and functional characterization of TaDREB3 in response to abiotic stresses. Gene. 144514.
  31. Pu, Y., L. Liu, J. Wu, Y. Zhao, J. Bai, L. Ma, J. Yue, J. Jin, Z. Niu, and Y. Fang. 2019. Transcriptome profile analysis of winter rapeseed (Brassica napus L.) in response to freezing stress, reveal potentially connected events to freezing stress. Int J Mol Sci. 20 : 2771. https://doi.org/10.3390/ijms20112771
  32. Ribarits, A., A. Abdullaev, A. Tashpulatov, A. Richter, E. Heberle-Bors, and A. Touraev. 2007. Two tobacco proline dehydrogenases are differentially regulated and play a role in early plant development. Planta. 225 : 1313-1324. https://doi.org/10.1007/s00425-006-0429-3
  33. Ritonga, F. N. and S. Chen. 2020. Physiological and molecular mechanism involved in cold stress tolerance in plants. Plants. 9 : 560. https://doi.org/10.3390/plants9050560
  34. Ruelland, E., M. N. Vaultier, A. Zachowski, and V. Hurry. 2009. Cold signalling and cold acclimation in plants. Adv Bot Res. 49 : 35-150. https://doi.org/10.1016/S0065-2296(08)00602-2
  35. Sah, S. K., K. R. Reddy, and J. Li. 2016. Abscisic acid and abiotic stress tolerance in crop plants. Front Plant Sci. 7 : 571. https://doi.org/10.3389/fpls.2016.00571
  36. Sakai, A. and W. Larcher. 2012. Frost survival of plants: responses and adaptation to freezing stress. Vol. 62. Springer Science & Business Media.
  37. Sakuma, Y., Q. Liu, J. G. Dubouzet, H. Abe, K. Shinozaki, and K. Yamaguchi-Shinozaki. 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
  38. Sanghera, G. S., S. H. Wani, W. Hussain, and N. Singh. 2011. Engineering cold stress tolerance in crop plants. Curr Genomics. 12 : 30. https://doi.org/10.2174/138920211794520178
  39. Saruhashi, M., T. K. Ghosh, K. Arai, Y. Ishizaki, K. Hagiwara, K. Komatsu, Y. Shiwa, K. Izumikawa, H. Yoshikawa, and T. Umezawa. 2015. Plant Raf-like kinase integrates abscisic acid and hyperosmotic stress signaling upstream of SNF1-related protein kinase2. PNAS. 112 : 6388-6396.
  40. Savoure, A., S. Jaoua, X. J. Hua, W. Ardiles, M. Van Montagu, and N. Verbruggen. 1995. Isolation, characterization, and chromosomal location of a gene encoding the Δ 1-pyrroline-5-carboxylate synthetase in Arabidopsis thaliana. FEBS letters. 372 : 13-19. https://doi.org/10.1016/0014-5793(95)00935-3
  41. Schroeder, J. I., J. M. Kwak, and G. J. Allen. 2001. Guard cell abscisic acid signalling and engineering drought hardiness in plants. Nature. 410 : 327-330. https://doi.org/10.1038/35066500
  42. Sharma, S., J. G. Villamor, and P. E. Verslues. 2011. Essential role of tissue-specific proline synthesis and catabolism in growth and redox balance at low water potential. Plant Physiol. 157 : 292-304. https://doi.org/10.1104/pp.111.183210
  43. Sheldon, C. C., J. E. Burn, P. P. Perez, J. Metzger, J. A. Edwards, W. J. Peacock, and E. S. Dennis. 1999. The FLF MADS box gene: a repressor of flowering in Arabidopsis regulated by vernalization and methylation. Plant Cell. 11 : 445-458. https://doi.org/10.2307/3870872
  44. Shi, Y., Y. Ding, and S. Yang. 2018. Molecular regulation of CBF signaling in cold acclimation. Trends Plant Sci. 23 : 623-637. https://doi.org/10.1016/j.tplants.2018.04.002
  45. Shinozaki, K. and K. Yamaguchi-Shinozaki. 2000. Molecular responses to dehydration and low temperature: differences and cross-talk between two stress signaling pathways. Curr Opin. Plant Biol. 3 : 217-223. https://doi.org/10.1016/S1369-5266(00)80068-0
  46. Szabados, L. and A. Savoure. 2010. Proline : a multifunctional amino acid. Trends Plant Sci. 15 : 89-97. https://doi.org/10.1016/j.tplants.2009.11.009
  47. Tao, D. L., G. Oquist, and G. Wingsle. 1998. Active oxygen scavengers during cold acclimation of Scots pine seedlings in relation to freezing tolerance. Cryobiology. 37 : 38-45. https://doi.org/10.1006/cryo.1998.2096
  48. Thomashow, M. F. 1999. Plant cold acclimation: freezing tolerance genes and regulatory mechanisms. Annu Rev Plant Biol. 50 : 571-599. https://doi.org/10.1146/annurev.arplant.50.1.571
  49. Tian, X., Z. Wang, X. Li, T. Lv, H. Liu, L. Wang, H. Niu, and Q. Bu. 2015. Characterization and functional analysis of pyrabactin resistance-like abscisic acid receptor family in rice. Rice. 8 : 1-13. https://doi.org/10.1186/s12284-014-0034-1
  50. Trischuk, R. G., B. S. Schiling, M. Wisniewski, and L. V. Gusta. 2006. Freezing stress: systems biology to study cold tolerance, Physiology and Molecular Biology of Stress Tolerance in Plants. Springer, Dordrecht. 131-155.
  51. Verbruggen, N. and C. Hermans. 2008. Proline accumulation in plants: a review. Amino acids. 35 : 753-759. https://doi.org/10.1007/s00726-008-0061-6
  52. Waalen, W. M., K. K. Tanino, J. E. Olsen, R. Eltun, O. A. Rognli, and L. V. Gusta. 2011. Freezing tolerance of winter canola cultivars is best revealed by a prolonged freeze test. Crop Sci. 51 : 1988-1996. https://doi.org/10.2135/cropsci2011.02.0098
  53. Xin, H., N. Xianchao, X. Pan, L. Wei, Y. Min, K. Yu, Q. Lunwen, and H. Wei. 2019. Comparative transcriptome analyses revealed conserved and novel responses to cold and freezing stress in Brassica napus L. G3. 2723-2737.
  54. Xin, Z. and J. Browse. 2000. Cold comfort farm : the acclimation of plants to freezing temperatures. Plant Cell Environ. 23 : 893-902. https://doi.org/10.1046/j.1365-3040.2000.00611.x
  55. Yan, L., A. Loukoianov, G. Tranquilli, M. Helguera, T. Fahima, and J. Dubcovsky. 2003. Positional cloning of the wheat vernalization gene VRN1. PNAS. 100 : 6263-6268. https://doi.org/10.1073/pnas.0937399100