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研究生:陳俐婷
研究生(外文):Li-Ting Chen
論文名稱:阿拉伯芥組蛋白去乙醯化酶HDA6和HDA19對於離層酸與逆境反應之功能研究
論文名稱(外文):Role of Arabidopsis histone deacetylases HDA6 and HDA19 in ABA and abiotic stress responses
指導教授:吳克強
指導教授(外文):Keqiang Wu
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:植物科學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2008
畢業學年度:97
語文別:英文
論文頁數:69
中文關鍵詞:阿拉伯芥組蛋白去乙醯化酶離層酸非生物逆境
外文關鍵詞:Arabidopsis thalianahistonehistone deacetylaseABA and abiotic stress
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組蛋白乙醯化(Histone acetylation)與去乙醯化(Histone deacetylation)對於真核生物的轉錄活性非常重要,而組蛋白乙醯化程度是由組蛋白乙醯化酶(Histone acetyltransferase)與去乙醯化酶(Histone deacetylase)所調控。組蛋白乙醯化通常會造成基因表現的提高,而組蛋白去乙醯化則會降低基因表現。近幾年的研究報告指出,組蛋白乙醯化參與於植物對於離層酸(ABA)與高鹽、乾旱、低溫等非生物逆境之反應與抗性當中。然而到目前為止,植物中組蛋白乙醯化與去乙醯化與逆境反應之間的分子機制尚未明朗化。因此在本研究中,我將針對離層酸與高鹽、乾旱、低溫等非生物逆境,來探討組蛋白乙醯化酶HDA6與HDA19在阿拉伯芥中的功能。 HDA6與HDA19屬於RPD3/HDA1家族中的組蛋白去乙醯化酶,兩者具有類似的氨基酸序列,而且兩者在阿拉伯芥不同的生長階段具有相似的表現。在本次研究裡,我使用了hda6的突變株axe1-5和RNA干擾株 (RNA interfering line) CS24039以及 hda19的T-DNA插入株athd1-t1來研究HDA6和HDA19在阿拉伯芥中對於離層酸與非生物逆境反應的訊號傳遞扮演著何種角色。axe1-5是一種splicing mutant,此突變株在HDA6 剪切位(splicing site)上發生點突變。athd1-t1是HDA19的T-DNA插入株,有一T-DNA插入在HDA19的第二個外顯子(exon)中。
axe1-5,CS24039和athd1-t1在種子萌發階段對於離層酸皆表現出高度敏感性,而且與離層酸反應相關的基因都呈現較低的表現量(ABI1, ABI2, KAT1, KAT2 和RD29B)。在高鹽逆境下,axe1-5和CS24039呈現較低的種子萌發率與存活率,但是athd1-t1與野生型植物並無明顯差異。根據RT-PCR的結果,我發現axe1-5和CS24039中與高鹽有所反應的相關基因(MYB2, RD29B, DREB2A和RD29A)的表現量較低,但是在athd1-t1中只有RD29B和DREB2A呈現較低的表現量。從植物對於高鹽逆境的外表現型與相關基因表現量的結果發現,HDA6相較於HDA19在高鹽逆境的機制中扮演著比較重要的功能。同時,我也觀察了植物在低溫逆境中的外表形與相關基因表現的差異,發現HDA6和HDA19的突變株與野生型植物並無明顯差異。根據這個結果可推論出,HDA6與HDA19並無參與於低溫逆境的機制中。
我利用組蛋白免疫沈澱法(Chromatin immunoprecipitation assay)瞭解了與離層酸和高鹽逆境相關基因的組蛋白3(H3)乙醯化與甲基化程度。透過這種實驗方法得知離層酸與高鹽可以提高其相關基因 (ABI1, ABI2, KAT1, KAT2,DREB2A, RD29A和RD29B)的H3乙醯化與甲基化程度,而Col野生型與axe1-5 之間的差異亦可證明HDA6參與其中。由這個結果可推論出離層酸與高鹽環境透過改變H3乙醯化與甲基化程度的方式來增加其離層酸與高鹽逆經相關基因的表現量,而HDA6扮演著重要的角色。
Acetylation and deacetylation of nucleosomal core histones play important roles in regulation of eukaryotic transcriptional activity. Histone acetylation levels are determined by the action of histone acetyltransferases and histone deacetylases. Acetylation of the histone is often associated with increased gene activity, whereas deacetylation of histones is correlated with transcriptional repression. Recent studies indicated that histone acetylation is involved in plant response to ABA and abiotic stresses including salt, drought and cold stresses. However, little is known about the molecular mechanisms of how histone acetylation and deacetylation are involved in stress response in plants. In this study, I focus on ABA, salt, drought and cold stresses to study the role of Arabidopsis histone deacetylases HDA6 and HDA19 in ABA response and abiotic stress signaling. HDA6 and HDA19 are members of RPD3/HDA1 histone deacetylases family, and they have similar amino acid sequence and gene expression patterns in different development stages. A hda6 mutant line, axe1-5, and a HDA6 RNA interfering line, CS24039, as well as a HDA19 T-DNA insertion line, athd1-t1, were used to study the role of HDA6 and HDA19 in ABA and abiotic stress signaling. axe1-5 is a splicing mutant which carries a point mutation in the HDA6 splicing site, athd1-t1 has a T-DNA inserted in second exon of HDA19.
Compared with wild-type, axe1-5, CS24039 and athd1-t1 displayed higher sensitivity to ABA during the seed germination stage, and lower expression level of ABA-responsive genes (ABI1, ABI2, KAT1, KAT2 and RD29B). In high salinity stress, axe1-5 and CS24039 displayed lower germination rates and survival rates compared with wild-type. However, athd1-t1 has no significant difference with wild-type in high salinity stress. Compared with wild-type, axe1-5 and CS24039 displayed lower expression of salt-responsive genes (MYB2, RD29B, DREB2A and RD29A) under salt stress, but athd1-t1 displayed lower expression of RD29B and DREB2A only under salt stress. These results suggested that HDA6 might plat an important role in salt stress signaling pathway. Phenotypic comparison and RT-PCR analysis of cold stress responsive genes indicated that there was no significant difference between wild types, hda6 and hda19 mutants, suggesting that HDA6 and HDA19 are not involved in cold stress signaling pathways.
I further analyzed the H3 acetylation and methylation levels of the salt and ABA response genes by chromatin immunoprecipitation assay. It was found that ABA and salt can increase the H3 acetylation and methylation level of ABA and salt response genes (ABI1, ABI2, KAT1, KAT2, DREB2A, RD29A and RD29B), and wild-type and axe1-5 displayed different level of H3 acetylation and methylation. These results suggested that ABA and salt can induces the response gene expression by altering the H3 acetylation and methylaiton level, and HDA6 is required for this process.
Table of Contents
Abstract..................................................I
Abbreviations...........................................III
Table of Contents......................................VIII
List of Figures..........................................XI
List of Tables.........................................XIII
Introduction..............................................1
1.Histone acetylation.....................................1
2.Abiotic stress responses and signaling pathways in plants...................................................12
3.Histone acetylation and plant abiotic stress response.................................................15
Materials and methods....................................18
Results..................................................35
1.HDA6 and HDA19 mutants were more sensitive to ABA and NaCl in seed germination.................................35
2.axe1-5 and HDA6 RNAi plants were more sensitive to salt
.........................................................38
3.axe1-5 and HDA6 RNAi plants were more sensitive to drought stress...........................................40
4.No significant difference was found among wild-type, HDA6 and HDA19 mutantsin cold response...................40
5.HDA6 and HDA19 mutants displayed decreased expression of ABA-responsive genes.....................................42
6.HDA6 and HDA19 mutants displayed lower expression of salt stress-responsive genes.............................44
7.HDA6 and HDA19 did not affect gene expression involved in cold and drought stress pathways......................47
8.ABA affected the level of histone acetylation and methylation of some ABA-responsive genes.................48
9.Salt affected the level of histone acetylation and methylation of some salt-responsive genes................51
Discussion...............................................54
1.HDA6 and HDA19 are both involved in ABA response in Arabidopsis..............................................54
2.HDA6 and HDA19 may have distinct functions in abiotic stress response..........................................55
3.ABA and salt can affect the histone acetylation and methylation and HDA6 is required for this process........56
References...............................................60
Abe H., Yamaguchi-Shinozaki K., Urao T., lwasaki T., Hosokawa D., and Shinozaki K. (1997). Role of Arabidopsis MYC and MYB homologs in drought and abscisic acid- regulated gene expression. Plant Cell 9: 1859-1868.

Abe H., Urao T., Ito T., Seki M., Shinozaki K., and Yamaguchi-Shinozaki K. (2003). Arabidopsis AtMYC2 (bHLH) and AtMYB2 (MYB) function as transcriptional activators in abscisic acid signaling. Plant Cell 15: 63–78.

Anderson J.A., Rhuprika S.S., Kochiank L.C., Lucasl W.J., and Gaber R.F. (1992). Functional expression of a probable Arabidopsis thaliana potassium channel in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 89: 3736-3740.

Aufsatz W. Mette M.F., van der Winden J., Matzke1 M., and Matzke A.J.M. (2001). HDA6, a putative histone deacetylase needed to enhance DNA methylation induced by doublestranded RNA. The EMBO Journal 21: 6832-6841.

Baker S.S., Wilhelm K.S., and Thomashow M.F. (1994). The 5''-region of Arabidopsis thaliana corl5a has cis-acting elements that confer cold-, drought- and ABA-regulated gene expression. Plant Molecular Biology 24: 701-713.

Benhamed M., Bertrand C., Servet C., and Zhou D.-X. (2006). Arabidopsis GCN5, HD1, and TAF1/HAF2 interact to regulate histone acetylation required for light-responsive gene expression. Plant Cell 18: 2893–2903.

Berger S.L. (2002). Histone modifications in transcriptional regulation. Current Opinion in Genetics and Development 12: 142–148.

Bertrand C., Bergounioux C., Domenichini S., Delarue M., and Zhou D.-X. (2003). Arabidopsis histone acetyltransferase AtGCN5 regulates the floral meristem activity through the WUSCHEL/AGAMOUS pathway. The Journal of Biological Chemistry. 278: 28246–28251.

Chinnusamy V., Ohta M., Kanrar S., Lee B.-h., Hong X., Agarwal M., and Zhu J.-K. (2003). ICE1: a regulator of cold-induced transcriptome and freezing tolerance in Arabidopsis. Genes & Development 17:1043–1054.

Deng W., Liu C.Y., Pei Y., Deng X., Niu LF., and Cao XF. (2007). Involvement of the histone acetyltransferase AtHAC1 in the regulation of flowering time via repression of FLOWERING LOCUS C in Arabidopsis. Plant Physiology 143: 1660–1668.

Domagalska M.A., Schomburg F.M., Amasino R.M., Vierstra R.D., Nagy F., and Davis S.J. (2007). Attenuation of brassinosteroid signaling enhances FLC expression and delays flowering. Development 134: 2841-2850.

Dong C.-H., Agarwal M., Zhang Y., Xie Q., and Zhu J.-K. (2006). The negative regulator of plant cold responses, HOS1, is a RING E3 ligase that mediates the ubiquitination and degradation of ICE1. Proc. Natl. Acad. Sci. USA 103: 8281–8286.

Earely K. Lawrence R.J., Pontes O., Reuther R., Enciso A.J., Silva M., Neves N., Gross m., Viegas W., and Pikaard C.S. (2006). Erasure of histone acetylation by Arabidopsis HDA6 mediates large-scale gene silencing in nucleolar dominance. Genes & Development 20: 1283-1293.

Earley K.W., Shook M.S., Brower-Toland B., Hicks L. and Pikaard C.S. (2007). In vitro specificities of Arabidopsis co-activator histone acetyltransferases: implications for histone hyperacetylation in gene activation. Plant Journal 52:615-626.

Finkelstein R.R., Gampala S.S.L., and Rock C.D. (2002). Abscisic acid signaling in seeds and seedlings. Plant Cell Supplement: 15-45.

Francis N.J., and Kingston R.E. (2001). Mechanisms of transcription memory. Nature Review Molecular Cell biology 2: 409-421.

Gendrel A.-V., Lippman Z., Martienssen R., and Colot V. (2004). Profiling histone modification patterns in plants using genomic tilling microarrays. Nature Methods 2: 213-218.

Grozinger C.M., Chao E.D., Blackwell H.E., Moazed D., and Schreiber S.L. (2001). Identification of a class of small molecule inhibitors of the sirtuin family of NAD- dependent deacetylases by phenotypic screening. The Journal of Biological Chemistry 276: 38837-38843.

Habu Y., Kakutani T., and Paszkowski J. (2001). Epigenetic developmental mechanisms in plants: molecules and targets of plant epigenetic regulation. Current Opinion in Genetics and Development 11: 215-220.

Ishitani M., Xiong L., Lee H., Stevenson B., and Zhu J.-K. (1998). HOS1, a genetic locus involved in cold-responsive gene expression in Arabidopsis. Plant Cell 10: 1151-1161.
Jenuwein T. and Allis C.D. (2001). Translating the Histone Code. Science 293: 1074-1080.

Kang J.-Y. Choi H.-i., Im M.-y., and Kim S.Y. (2002). Arabidopsis basic leucine zipper proteins that mediate stress-responsive abscisic acid signaling. Plant Cell 14: 343–357.

Kim K.-C., Lai Z., Fan B., and Chen Z. (2008). Arabidopsis WRKY38 and WRKY62 transcription factors interact with histone deacetylase 19 in basal defense. Plant Cell Physiology 49: 1580-1588.

Kuo M.-H. and Allis C.D. (1998). Roles of histone acetyltransferases and deacetylases in gene regulation. BioEaasys 20: 615-626.

Lee B.-h., Henderson D.A., and Zhu J.-K. (2005). The Arabidopsis cold-responsive transcriptome and its regulation by ICE1. Plant Cell 17:3155-3175.

Leung J., Merlot S., and Giraudat J. (1997). The Arabidopsis ABSCISIC ACID-INSENSlTIVE2 (AB12) and ABI genes encode homologous protein phosphatases 2C involved in abscisic acid signal transduction. Plant Cell 9:759-771.

Liu Q., Kasuga M., Sakuma Y., AbeH., Miura S., Yamaguchi-Shinozaki K., and Shinozaki K. (1998). Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought- and low- temperature-responsive gene Expression, respectively, in Arabidopsis. Plant Cell 10: 1391–1406.

Li J., Wang X.-Q., Watson M.B., Assmann S.M. (2000). Regulation of abscisic acid-induced stomatal closure and anion channels by guard cell AAPK kinase. Science 287: 300-303.

Long J.A., Ohno C., Smith Z.R., Meyerowitz E.M. (2006). TOPLESS regulates apical embryonic fate in Arabidopsis. Science 312: 1520-1523.

Luger K., Mader A.W., Richmond R.K., Sargent D.F., and Richmond T.J. (1997). Crystal structure of the nucleosome core particle at 2.8A° resolution. Nature 389: 251-260.

Mao Y., Pavangadkar K.A., Thomashow M.F., Triezenberg S.J. (2006). Physical and functional interactions of Arabidopsis ADA2 transcriptional coactivator proteins with the acetyltransferase GCN5 and with the cold-induced transcription factor CBF1. Biochimica et Biophysica Acta 1759:69-79.

Merlot S., Gosti1 F., Guerrier1 D., Vavasseur A., and Giraudat J. (2001). The ABI1 and ABI2 protein phosphatases 2C act in a negative feedback regulatory loop of the abscisic acid signalling pathway. The Plant Journal 25: 295-303.

Meyer K., Leube M.P., and Grill E. (1994). A protein phosphatase 2C involved in ABA signal transduction in Arabidopsis thaliana. Science 264: 1452-1455.
Meyer P. (2001). Chromatin remodeling. Current Opinion in Plant Biology 4:457–462.

Miura K., Jin J.B., and Hasegawa P.M. (2007). Sumoylation, a post-translational regulatory process in plants. Current Opinion in Plant Biology 10: 495–502.

Mao Y., Pavangadkar K.A., Thomashow M.F., and Triezenberg S.J. (2006). Physical and functional interactions of Arabidopsis ADA2 transcriptional coactivator proteins with the acetyltransferase GCN5 and with the cold-induced transcription factor CBF1. Biochimica et Biophysica Acta 1759: 69–79.

Murfett J., Wang X.-J., Hagen G., and Guilfoyle T.J. (2001). Identification of Arabidopsis histone deacetylase HDA6 mutants that affect transgene expression. Plant Cell 13: 1047–1061. The Journal of Biological Chemistry 276: 53215–3221.

Narlikar G.J., Fan H.-Y., and Kingston R.E. (2002). Cooperation between complexes that regulate chromatin structure and transcription. Cell 108: 475-487.

Pandey R., Muller A., Napoli C.A., Selinger D.A., Pikaard C.S., RichardsE.J., Bender J., Mount D.W., and Jorgensen R.A. (2002). Analysis of histone acetyltransferase and histone deacetylase families of Arabidopsis thaliana suggests functional diversification of chromatin modification among multicellular eukaryotes. Nucleic Acids Research 30: 5036-5055.

Philippar K., Ivashikina N., Ache P., Christian M., Luthen H., Palme K., and Hedrich R. (2004). Auxin activates KAT1 and KAT2, two K+-channel genes expressed in seedlings of Arabidopsis thaliana. The Plant Journal 37: 815-827.

Pilot G., Lacombe B., Gaymard F., Che´rel I., Boucherez J., Thibaud J.-B., and Sentenac H. (2001). Guard cell inward K+ channel activity in Arabidopsis involves
expression of the twin channel subunits KAT1 and KAT2. The Journal of Biological Chemistry. 276: 3215-3221.

Probst A.V., Fagard M., Proux F., Mourrain P., Boutet S., Earley K., Lawrence R.J., Pikaard C.S., Murfett J., Furner I., Vaucheret H., and Scheida O.M. (2004). Arabidopsis histone deacetylase HDA6 is required for maintenance of transcriptional gene silencing and determines nuclear organization of rDNA repeats. Plant Cell 16: 1021–1034.

Reyes J.C. (2006). Chromatin modifiers that control plant development. Current Opinion in Plant Biology 9: 21–27.

Sheen J. (1998). Mutational analysis of protein phosphatase 2C involved in abscisic acid signal transduction in higher plants. Proc. Natl. Acad. Sci. USA 95:975-980.

Shinozaki K. and Yamaguchi-Shinozaki K. (2007). Gene networks involved in drought stress response and tolerance. Journal of Experimental Botany 58: 221-227.

Silverstein R.A. and Ekwall K. (2005). Sin3:a flexible regulator of global gene expression and genome stability. Current Genetics 47: 1-17.

Sokol A. Kwiatkowska A. Jerzmanowski A. Prymakowska-Bosak M. (2007). Up-regulation of stress-inducible genes in tobacco and Arabidopsis cells in response to abiotic stresses and ABA treatment correlates with dynamic changes in histone H3 and H4 modifications. Planta 227:245-254.

Song C.-P., Agarwal M., Ohta M., Guo Y., Halfter U., Wang P., and Zhua J.K. (2005). Role of an Arabidopsis AP2/EREBP-type transcriptional repressor in abscisic Acid and drought stress responses. Plant Cell 17: 2384–2396.

Song C.-P. and Galbraith D.W. (2006). AtSAP18, an orthologue of human SAP18, is involved in the regulation of salt stress and mediates transcriptional repression in Arabidopsis. Plant Molecular Biology 60: 241-257.

Sridha S., and Wu K. (2006). Identification of AtHD2C as a novel regulator of abscisic acid responses in Arabidopsis. The Plant Journal 46: 124–133.

Stockinger E.J., Mao Y., Regier M.K., Triezenberg S.J. and Thomashow M.F. (2001). Transcriptional adaptor and histone acetyltransferase proteins in Arabidopsis and their interactions with CBF1, a transcriptional activator involved in cold-regulated gene expression. Nucleic Acid Reasarch 29: 1524-1533.

Sutter J.-U., Sieben C., Hartel A., Eisenach C., Thiel G., and Blatt M.R. (2007). Abscisic acid triggers the endocytosis of the Arabidopsis KAT1 K+ channel and its recycling to the plasma member. Current Biology 17: 1398-1402.

Tanaka M., Kikuchi A., and Kamada H. (2008). The Arabidopsis histone deacetylases HDA6 and HDA19 contribute to the repression of embryonic properties after germination. Plant Physiology 146: 149–161.

Thomashow M.J. (1999). PLANT COLD ACCLIMATION: Freezing tolerance genes and regulatory mechanisms. Annual Review of Plant Physiology and Plant Molecular Biology 50: 571–99.

Tian L., Wang J., Fong M.P., Chen M., Cao H., Gelvin S.B. and Chen Z.J. (2003). Genetic control of developmental changes induced by disruption of Arabidopsis histone deacetylase 1 (AtHD1) expression. Genetics 165: 399-409.
Ueno Y., Ishikawa T., Watanabe K., Terakura S., Iwakawa H., Okada K., Machida C., and Machida Y. (2007). Histone deacetylases and ASYMMETRIC LEAVES2 are involved in the establishment of polarity in leaves of Arabidopsis. Plant Cell 19:445-457.

Uno Y., Furihata T., Abe H., Yoshida R., Shinozaki K., and Yamaguchi-Shinozaki K. (2000). Arabidopsis basic leucine zipper transcription factors involved in an abscisic acid-dependent signal transduction pathway under drought and high-salinity conditions. Proc. Natl. Acad. Sci. USA 97: 11632-11637.

Vlachonasios K.E., Thomashow M.F., and Triezenberg S.J. (2003). Disruption mutations of ADA2b and GCN5 transcriptional adaptor genes dramatically affect Arabidopsis growth, development, and gene expression. Plant Cell 15: 626–638.

Verbsky M.L., and Richards E.J. (2001). Chromatin remodeling in plants. Current Opinion in Plant Biology 4:494–500.

Wu K., Zhang L., Zhou C., Yu C-W. and Chaikam V. (2008). HDA6 is required for jasmonate response, senescence and flowering in Arabidopsis. Journal of Experimental Botany 59: 225–234.

Xu C.-R., Liu C., Wang Y.-L., Li L.-C., Chen W.-Q., Xu Z.-H., and Bai S.-N. (2005). Histone acetylation affects expression of cellular patterning genes in the Arabidopsis root epidermis. Proc. Natl. Acad. Sci. USA 102: 14469–14474.

Yamaguchi-Shinozaki K. and Shinozaki K. (1994). A nove1 cis-Acting element in an Arabidopsis gene is involved in responsiveness to drought, low temperature, or high-salt stress. Plant Cell 6: 251-264.
Yamaguchi-Shinozaki K. and Shinozaki K. (2005). Organization of cisacting regulatory elements in osmotic- and cold-stress-responsive promoters. Trends in Plant Science 10: 88–94.

Yamaguchi-Shinozaki K. and Shinozaki K. (2006). Transcriptional regulatory networks in cellular responses and tolerance to dehydration and cold stresses. Annual Review of Plant Biology 57: 781-803.

Zhou C., Zhang L., Duan J., Miki B., and Wu K. (2005). HISTONE DEACETYLASE19 is involved in jasmonic acid and ethylene signaling of pathogen response in Arabidopsis. Plant Cell 17: 1196–1204.

Zhu J., Jeong J.C., Zhu Y., Sokolchik I., Miyazaki S, Zhu J.-K., Hasegawa P.M., Bohnert H.J., Shi H., Yun D.J., and Bressan R.A. (2008). Involvement of Arabidopsis HOS15 in histone deacetylation and cold tolerance. Proc. Natl. Acad. Sci. USA 105: 4945-4950.
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