篩選阿拉伯芥參與 HIT4 媒介耐熱能力之遺 傳因子

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胡承恩(Cheng-En Hu) 查詢紙本館藏

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論文名稱

篩選阿拉伯芥參與 HIT4 媒介耐熱能力之遺傳因子
(Screening of genetic determinants involved in HIT4-dependent heat tolerance in Arabidopsis)

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摘要(中)

植物無法自由移動，因此在遭遇環境逆境時，需演化出相應的生存能力。為了探討阿拉伯芥中與熱逆境相關的基因，本實驗室先前利用正向遺傳學的策略，篩選出對熱敏感的突變株heat intolerance 4（hit4）。後續研究發現，HIT4蛋白質在常溫下位於細胞核內的染色質中心。而在植物受熱後，HIT4會在染色質中心消散前，由染色質中心轉移到核仁，且在熱逆境結束後，隨著染色質中心的重新形成而回到染色質中心。另一方面，hit4突變蛋白質雖然保有在高溫環境下轉移到核仁的能力，hit4突變株內的染色質中心卻不會因高溫而消散。據此推測，應有其他分子參與在HIT4媒介熱誘導染色質中心消散及植物耐熱能力的反應。為了找出這些潛在的分子，進而完整揭露HIT4在阿拉伯芥中的作用機制，本研究使用TurboID來標記HIT4附近的蛋白質，並以pull down assay與質譜分析（mass spectrometry），來辨認可能與HIT4有交互作用的蛋白質。隨後對這些候選蛋白質進行突變株的篩選，測試其對高溫逆境的耐受能力，並以雙分子螢光互補（BiFC）等實驗，對候選蛋白質進行分析。在候選蛋白質中，PUB49（PLANT U-BOX 49）在正常溫度下均勻散佈在整個細胞核中，但在遭遇熱逆境時，會先聚集到色質中心，並隨著時間推移，與HIT4一樣位移到核仁。BiFC實驗則顯示了兩者之間有交互作用。尤有甚者，pub49突變株也失去對高溫逆境的耐受能力。綜上結果，PUB49即為參與HIT4調節植物耐熱反應的分子之一。

摘要(英)

Plants, unable to move freely, evolve corresponding survival abilities to cope with environmental adversities. To investigate genes related to heat stress in Arabidopsis, our laboratory previously utilized a forward genetic approach to screen for heat-sensitive mutant lines, identifying heat intolerance 4 (hit4). Subsequent studies revealed that the HIT4 protein is localized in the chromocenters of the nucleus at normal temperatures (23°C). Upon heat stress, HIT4 translocates from the chromocenters to the nucleolus before decondensing, and upon cessation of heat stress, it returns to the chromocenters as they reform. Conversely, the hit4 mutant protein, while retaining the ability to translocate to the nucleolus under high temperatures (37°C), does not allow the decondensation of chromocenters in mutant plant nuclei under heat stress. These results suggest the involvement of other molecules in HIT4-mediated heat-induced chromocenter decondensation and plant thermotolerance. To identify these potential molecules and fully elucidate the role of HIT4 in Arabidopsis, this study employed TurbolD to label proteins near HIT4 and utilized pull-down assays and mass spectrometry to identify proteins that may interact with HIT4. Subsequently, mutant strains of these candidate proteins were screened for thermal sensitivity, and candidate proteins were analyzed using techniques such as bimolecular fluorescence complementation (BiFC). Among the candidate proteins, PUB49 (PLANT U-BOX 49) was found to be uniformly distributed throughout the nucleus at normal temperatures but aggregates at the chromocenters upon heat stress, similar to HIT4, and relocates to the nucleolus over time, interacting with HIT4 as indicated by BiFC experiments. Importantly, pub49 mutant plants also lose their tolerance to high-temperature stress. These results collectively indicate that PUB49 is one of the molecules involved in regulating plant thermotolerance together with HIT4 in Arabidopsis.

關鍵字(中)

★ Heat Intolerance 4 (HIT4)
★ 熱逆境
★ 染色質中心
★ 阿拉伯芥
★ PLANT U-BOX 49 (PUB49)

關鍵字(英)

★ Heat Intolerance 4 (HIT4)
★ Heat stress
★ Chromocenter
★ Arabidopsis
★ PLANT U-BOX 49 (PUB49)

論文目次

摘要 I
Abstract II
誌謝 IV
總目錄 V
表目錄 VII
圖目錄 VIII
一、緒論 1
二、研究材料與方法 5
2-1 植物材料 5
2-1-1 植物品系 5
2-1-2 植物的培養 5
2-1-3 植物熱逆境（heat stress）與熱休克（heat shock）處理條件 6
2-2 質體的製備 6
2-2-1 細菌LB （Luria Broth）培養基製備 6
2-2-2 質體的轉殖、篩選與萃取 7
2-3 植物DNA萃取 9
2-4 建立基因轉殖植物 10
2-4-1 製備農桿菌勝任細胞 10
2-4-2 農桿菌電穿孔轉型 10
2-4-3 阿拉伯芥的農桿菌基因轉殖 10
2-5 阿拉伯芥原生植體的製備與轉殖 11
2-6 蛋白質膠體分析 12
2-6-1 製作SDS-PAGE與蛋白質的電泳分離 12
2-6-2 西方墨點法（western blot） 12
2-6-3 Coomassie Brilliant Blue染色 13
2-6-4 銀染（Silver stain） 13
2-7 Affinity Purification-Mass Spectroscopy (AP-MS) 14
2-8 反轉錄PCR（RT-PCR） 15
2-9 免疫共沉澱(Co-immunoprecipitation, Co-IP) 15
三、結果 17
3-1 尋找與HIT4交互作用的蛋白質 17
3-1-1 TbID-HIT4融合蛋白不會影響HIT4參與的耐熱反應 17
3-1-2 TbID-HIT4 pull down assay 18
3-2 分析候選基因與HIT4之間的關係 18
3-2-1 HEAT SHOCK PROTEIN (HSP) family 19
3-2-2 DEAD BOX RNA HELICASE 1 (DRH1) 19
3-2-3 CHR11與ARP4 20
3-2-4 NUCLEOLIN LIKE 1 (NUC1) 21
3-3 PUB49會參與HIT4誘導的耐熱性 21
3-3-1 PUB49在熱逆境下的位置與HIT4一致 22
3-3-2 PUB49會與HIT4進行交互作用 22
3-3-3 pub49突變株失去對高溫逆境的耐受能力 23
3-3-4 PUB49的突變不影響HIT4轉移到核仁的能力 23
四、討論 24
五、參考文獻 28

參考文獻

Benoit, M., Layat, E., Tourmente, S., & Probst, A. V. (2013). Heterochromatin dynamics during developmental transitions in Arabidopsis - a focus on ribosomal DNA loci. Gene, 526(1), 39-45. https://doi.org/10.1016/j.gene.2013.01.060
Bontinck, M., Van Leene, J., Gadeyne, A., De Rybel, B., Eeckhout, D., Nelissen, H., & De Jaeger, G. (2018). Recent Trends in Plant Protein Complex Analysis in a Developmental Context. Front Plant Sci, 9, 640. https://doi.org/10.3389/fpls.2018.00640
Branon, T. C., Bosch, J. A., Sanchez, A. D., Udeshi, N. D., Svinkina, T., Carr, S. A., Feldman, J. L., Perrimon, N., & Ting, A. Y. (2018). Efficient proximity labeling in living cells and organisms with TurboID. Nature Biotechnology, 36(9), 880-887. https://doi.org/10.1038/nbt.4201
Clapier, C. R., Iwasa, J., Cairns, B. R., & Peterson, C. L. (2017). Mechanisms of action and regulation of ATP-dependent chromatin-remodelling complexes. Nature Reviews Molecular Cell Biology, 18(7), 407-422. https://doi.org/10.1038/nrm.2017.26
Du, J., Gao, Y., Zhan, Y., Zhang, S., Wu, Y., Xiao, Y., Zou, B., He, K., Gou, X., & Li, G. (2016). Nucleocytoplasmic trafficking is essential for BAK 1‐and BKK 1‐mediated cell‐death control. The Plant Journal, 85(4), 520-531.
Du, J. L., Zhang, S. W., Huang, H. W., Cai, T., Li, L., Chen, S., & He, X. J. (2015). The Splicing Factor PRP31 Is Involved in Transcriptional Gene Silencing and Stress Response in Arabidopsis. Mol Plant, 8(7), 1053-1068. https://doi.org/10.1016/j.molp.2015.02.003
Feder, M. E., & Hofmann, G. E. (1999). Heat-shock proteins, molecular chaperones, and the stress response: evolutionary and ecological physiology. Annu Rev Physiol, 61, 243-282. https://doi.org/10.1146/annurev.physiol.61.1.243
Fransz, P., De Jong, J. H., Lysak, M., Castiglione, M. R., & Schubert, I. (2002). Interphase chromosomes in Arabidopsis are organized as well defined chromocenters from which euchromatin loops emanate. Proceedings of the National Academy of Sciences, 99(22), 14584-14589.
Godwin, J., & Farrona, S. (2022). The Importance of Networking: Plant Polycomb Repressive Complex 2 and Its Interactors. Epigenomes, 6(1). https://doi.org/10.3390/epigenomes6010008
Godwin, J., Govindasamy, M., Nedounsejian, K., March, E., Halton, R., Bourbousse, C., Wolff, L., Fort, A., Krzyszton, M., López Corrales, J., Swiezewski, S., Barneche, F., Schubert, D., & Farrona, S. (2024). The UBP5 histone H2A deubiquitinase counteracts PRCs-mediated repression to regulate Arabidopsis development. Nat Commun, 15(1), 667. https://doi.org/10.1038/s41467-023-44546-8
Groves, N. R., Biel, A., Moser, M., Mendes, T., Amstutz, K., & Meier, I. (2020). Recent advances in understanding the biological roles of the plant nuclear envelope. Nucleus, 11(1), 330-346. https://doi.org/10.1080/19491034.2020.1846836
Guo, J., Cai, G., Li, Y. Q., Zhang, Y. X., Su, Y. N., Yuan, D. Y., Zhang, Z. C., Liu, Z. Z., Cai, X. W., Guo, J., Li, L., Chen, S., & He, X. J. (2022). Comprehensive characterization of three classes of Arabidopsis SWI/SNF chromatin remodelling complexes. Nat Plants, 8(12), 1423-1439. https://doi.org/10.1038/s41477-022-01282-z
Han, D., Chen, C., Xia, S., Liu, J., Shu, J., Nguyen, V., Lai, J., Cui, Y., & Yang, C. (2021). Chromatin-associated SUMOylation controls the transcriptional switch between plant development and heat stress responses. Plant Commun, 2(1), 100091. https://doi.org/10.1016/j.xplc.2020.100091
Heitz, E. (1928). “Das” Heterochromatin der Moose. Bornträger.
Li, N., Euring, D., Cha, J. Y., Lin, Z., Lu, M., Huang, L. J., & Kim, W. Y. (2020). Plant Hormone-Mediated Regulation of Heat Tolerance in Response to Global Climate Change. Front Plant Sci, 11, 627969. https://doi.org/10.3389/fpls.2020.627969
Maejima, Y., & Sadoshima, J. (2014). SUMOylation: a novel protein quality control modifier in the heart. Circ Res, 115(8), 686-689. https://doi.org/10.1161/circresaha.114.304989
Mair, A., Xu, S.-L., Branon, T. C., Ting, A. Y., & Bergmann, D. C. (2019). Proximity labeling of protein complexes and cell-type-specific organellar proteomes in Arabidopsis enabled by TurboID. eLife, 8, e47864. https://doi.org/10.7554/eLife.47864
Mesihovic, A., Iannacone, R., Firon, N., & Fragkostefanakis, S. (2016). Heat stress regimes for the investigation of pollen thermotolerance in crop plants. Plant Reprod, 29(1-2), 93-105. https://doi.org/10.1007/s00497-016-0281-y
Okanami, M., Meshi, T., & Iwabuchi, M. (1998). Characterization of a DEAD box ATPase/RNA helicase protein of Arabidopsis thaliana. Nucleic Acids Res, 26(11), 2638-2643. https://doi.org/10.1093/nar/26.11.2638
Pavet, V., Quintero, C., Cecchini, N. M., Rosa, A. L., & Alvarez, M. E. (2006). Arabidopsis displays centromeric DNA hypomethylation and cytological alterations of heterochromatin upon attack by Pseudomonas syringae. Molecular Plant-Microbe Interactions, 19(6), 577-587.
Pawloski, L. C., Deal, R. B., McKinney, E. C., Burgos-Rivera, B., & Meagher, R. B. (2005). Inverted repeat PCR for the rapid assembly of constructs to induce RNA interference. Plant Cell Physiol, 46(11), 1872-1878. https://doi.org/10.1093/pcp/pci191
Petricka, J. J., & Nelson, T. M. (2007). Arabidopsis nucleolin affects plant development and patterning. Plant Physiol, 144(1), 173-186. https://doi.org/10.1104/pp.106.093575
Sangwan, V., Orvar, B. L., Beyerly, J., Hirt, H., & Dhindsa, R. S. (2002). Opposite changes in membrane fluidity mimic cold and heat stress activation of distinct plant MAP kinase pathways. Plant J, 31(5), 629-638. https://doi.org/10.1046/j.1365-313x.2002.01384.x
Savchenko, G. E., Klyuchareva, E. A., Abramchik, L. M., & Serdyuchenko, E. V. (2002). Effect of Periodic Heat Shock on the Inner Membrane System of Etioplasts. Russian Journal of Plant Physiology, 49(3), 349-359. https://doi.org/10.1023/A:1015592902659
Schöffl, F., Prandl, R., & Reindl, A. (1999). Molecular responses to heat stress. Molecular responses to cold, drought, heat and salt stress in higher plants, 83(93).
Struk, S., Jacobs, A., Sánchez Martín-Fontecha, E., Gevaert, K., Cubas, P., & Goormachtig, S. (2019). Exploring the protein-protein interaction landscape in plants. Plant Cell Environ, 42(2), 387-409. https://doi.org/10.1111/pce.13433
Tessadori, F., Chupeau, M.-C., Chupeau, Y., Knip, M., Germann, S., van Driel, R., Fransz, P., & Gaudin, V. (2007). Large-scale dissociation and sequential reassembly of pericentric heterochromatin in dedifferentiated Arabidopsis cells. Journal of cell science, 120(7), 1200-1208.
Tessadori, F., Chupeau, M. C., Chupeau, Y., Knip, M., Germann, S., van Driel, R., Fransz, P., & Gaudin, V. (2007). Large-scale dissociation and sequential reassembly of pericentric heterochromatin in dedifferentiated Arabidopsis cells. J Cell Sci, 120(Pt 7), 1200-1208. https://doi.org/10.1242/jcs.000026
Tessadori, F., Schulkes, R. K., Driel, R. v., & Fransz, P. (2007). Light‐regulated large‐scale reorganization of chromatin during the floral transition in Arabidopsis. The Plant Journal, 50(5), 848-857.
Tessadori, F., Schulkes, R. K., van Driel, R., & Fransz, P. (2007). Light-regulated large-scale reorganization of chromatin during the floral transition in Arabidopsis. Plant J, 50(5), 848-857. https://doi.org/10.1111/j.1365-313X.2007.03093.x
Trenner, J., Monaghan, J., Saeed, B., Quint, M., Shabek, N., & Trujillo, M. (2022). Evolution and Functions of Plant U-Box Proteins: From Protein Quality Control to Signaling. Annu Rev Plant Biol, 73, 93-121. https://doi.org/10.1146/annurev-arplant-102720-012310
van Zanten, M., Koini, M. A., Geyer, R., Liu, Y., Brambilla, V., Bartels, D., Koornneef, M., Fransz, P., & Soppe, W. J. J. (2011). Seed maturation in <i>Arabidopsis thaliana</i> is characterized by nuclear size reduction and increased chromatin condensation. Proceedings of the National Academy of Sciences, 108(50), 20219-20224. https://doi.org/doi:10.1073/pnas.1117726108
Vierstra, R. D. (2009). The ubiquitin–26S proteasome system at the nexus of plant biology. Nature Reviews Molecular Cell Biology, 10(6), 385-397. https://doi.org/10.1038/nrm2688
Wang, Wu, J.-R., Chang, W.-L., Yeh, C.-H., Ke, Y.-T., Lu, C.-A., & Wu, S.-J. (2013). Arabidopsis HIT4 encodes a novel chromocentre-localized protein involved in the heat reactivation of transcriptionally silent loci and is essential for heat tolerance in plants. Journal of Experimental Botany, 64(6), 1689-1701. https://doi.org/10.1093/jxb/ert030
Wang, H., Dittmer, T. A., & Richards, E. J. (2013). Arabidopsis CROWDED NUCLEI (CRWN) proteins are required for nuclear size control and heterochromatin organization. BMC Plant Biology, 13(1), 200. https://doi.org/10.1186/1471-2229-13-200
Wang, L. C., Wu, J. R., Hsu, Y. J., & Wu, S. J. (2015). Arabidopsis HIT4, a regulator involved in heat-triggered reorganization of chromatin and release of transcriptional gene silencing, relocates from chromocenters to the nucleolus in response to heat stress. New Phytol, 205(2), 544-554. https://doi.org/10.1111/nph.13088
Wang, W., Vinocur, B., Shoseyov, O., & Altman, A. (2004). Role of plant heat-shock proteins and molecular chaperones in the abiotic stress response. Trends in plant science, 9(5), 244-252.
Wiborg, J., O′Shea, C., & Skriver, K. (2008). Biochemical function of typical and variant Arabidopsis thaliana U-box E3 ubiquitin-protein ligases. Biochem J, 413(3), 447-457. https://doi.org/10.1042/bj20071568
Zhang, Q., Wang, Z., Lu, X., Yan, H., Zhang, H., He, H., Bischof, S., Harris, C. J., & Liu, Q. (2023). DDT-RELATED PROTEIN4–IMITATION SWITCH alters nucleosome distribution to relieve transcriptional silencing in Arabidopsis. The Plant Cell, 35(8), 3109-3126. https://doi.org/10.1093/plcell/koad143
Zhang, X., Henriques, R., Lin, S. S., Niu, Q. W., & Chua, N. H. (2006). Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method. Nat Protoc, 1(2), 641-646. https://doi.org/10.1038/nprot.2006.97
Zhang, Y., Sun, Z., Jia, J., Du, T., Zhang, N., Tang, Y., Fang, Y., & Fang, D. (2021). Overview of histone modification. Histone Mutations and Cancer, 1-16.
Zhao, C., Liu, B., Piao, S., Wang, X., Lobell, D. B., Huang, Y., Huang, M., Yao, Y., Bassu, S., Ciais, P., Durand, J. L., Elliott, J., Ewert, F., Janssens, I. A., Li, T., Lin, E., Liu, Q., Martre, P., Müller, C., . . . Asseng, S. (2017). Temperature increase reduces global yields of major crops in four independent estimates. Proc Natl Acad Sci U S A, 114(35), 9326-9331. https://doi.org/10.1073/pnas.1701762114

指導教授

吳少傑(Shaw-Jye Wu)

審核日期

2024-6-24

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