Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/22423
標題: 冰花訊息傳導相關mcSNF1在鹽逆境下基因表現及蛋白累積之分析
Analyses of gene expression and protein accumulation of a signal transduction-related mcSNF1 in halophyte Mesembryanthemum crystallinum under salt stress
作者: 楊邡郁
Yang, Fang-Yu
關鍵字: mcSNF1;冰花
出版社: 生命科學系所
引用: 洪郁惠 (1999) 冰花耐鹽相關基因之分離及表現分析,中興大學植物學研究所碩士論文。 周映孜 (2002) 鹽逆境下高等植物鉀鈉離子平衡及相關基因表現之分析,中興大學植物學研究所碩士論文。 王詠中 (2006) 耐鹽植物冰花SNF-1基因之分離與分析,中興大學生命科學系學士論文。 Alepuz, P.M., Cunningham, K.W., and Estruch, F. (1997). Glucose repression affects ion homeostasis in yeast through the regulation of the stress-activated ENA1 gene. Mol. Microbiol. 26, 91-98. Bhalerao, R. P., Salchert, K., Bako, L., Okresz, L., Szabados, L., Muranaka, T., Machida, Y., Schell, J., Koncz, C. (1999). Regulatory interaction of PRL1 WD protein with Arabidopsis SNF1-like protein kinases. Proc. Natl. Acad. Sci. USA 96, 5322-5327 Borland, A., Elliott, S., Patterson, S., Taybi, T., Cushman, J., Pater B., and Barnes, J. (2006). Are the metabolic components of crassulacean acid metabolism up-regulated in response to an increase in oxidative burden? J. Exp. Botany 57, 319-328. Boudsocq, M., Barbier-Brygoo, H., and Laurière, C. (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. Celenza, J. L., and Carlson, M. (1989). Mutational analysis of the Saccharomyces cerevisiae SNF1 protein kinase and evidence for functional interaction with the SNF4 protein. Mol. Cell. Biol. 9, 5034-5044. Cooper, J. A., and MacAuley, A. (1988). Potential positive and negative autoregulation of p60c-src by intermolecular autophosphorylation. Proc. Natl. Acad. Sci. USA 85, 4232-4236. Crute, B. E., Seefeld, K., Gamble, J., Kemp, B. E., and Witters, L. A. (1998). Functional domains of the alpha1 catalytic subunit of the AMP-activated protein kinase. J. Biol. Chem. 273, 35347-35354. Estruch, F., Treitel, M. A., Yang, X., and Carlson, M. (1992). N-terminal mutations modulate yeast SNF1 protein kinase function. Genetics 132, 639-650. Farras, R., Ferrando, A., Jasik, J., Kleinow, T., Okresz, L., Tiburcio, A., Salchert, K., delPozo, C., Schell, J., Koncz, C. (2001). SKP1-SnRK protein kinase interactions mediate proteasomal binding of a plant SCF ubiquitin ligase. EMBO J. 20, 2742-2756 Gaber, R.F., Styles, C.A., and Fink, G.R. (1988). TRK1 encodes a plasma membrane protein required for high-affinity potassium transport in Saccharomyces cerevisiae. Mol. Cell. Biol. 8, 2848-2859. Ganster, R. W., McCartney, R. R., and Schmidt, M. C. (1998). Identification of a calcineurin-independent pathway required for sodium ion stress response in Saccharomyces cerevisiae. Genetics 150, 31-42 Gong, D., Guo, Y., Schumaker, K.S., and Zhu, J. K. (2004). The SOS3 family of calcium sensors and SOS2 family of protein kinases in Arabidopsis. Plant physiol. 134, 919-926 Goossens, A., de La Fuente, N., Forment, J., Serrano, R. and Portillo, F. (2000). Regulation of yeast H+-ATPase by protein kinases belonging to a family dedicated to activation of plasma membrane transporters. Mol. Cell. Biol. 20, 7654-7661. Guo, Y., Qiu, Q. S., Quintero, F. J., Pardo, J. M., Ohta, M., Zhang, C., Schumaker, K. S., and Zhu, J. K. (2004). Transgenic evaluation of activated mutant alleles of SOS2 reveals a critical requirement for its kinase activity and C-terminal regulatory domain for salt tolerance in Arabidopsis thaliana. Plant cell 16, 435-449 Halford, N. G., and Hardie, D. G. (1998). SNF1-related protein kinases: global regulators of carbon metabolism in plants? Plant Mol. Biol. 37, 735-748. Halford, N. G., Hey, S., Jhurreea, D., Laurie, S., McKibbin, R. S., Paul, M., and Zhang, Y. (2003). Metabolic signalling and carbon partitioning: role of Snf1-related (SnRK1) protein kinase. J. Exp. Botany 54, 467-475. Halford, N. G., Hey, S., Jhurreea,D., Laurie, S., McKibbin,R. S., Zhang, Y., and Paul, M. J. (2004). Highly conserved protein kinases involved in the regulation of carbon and amino acid metabolism. J. Exp. Botany, 55, 35-42. Hasegawa, P. M., Bressan, R. A., Zhu, J. K., and Bohnert, H. J. (2000). Plant cellular and molecular response to high salinity. Ann. Rev. Plant Physiol. Plant Mol. Biol. 51, 463-499 Hedbacker, K., Hong, S. P., and Carlson, M. (2004). Pak1 protein kinase regulates activation and nuclear localization of Snf1-Gal83 protein kinase. Mol. Cell. Biol. 24, 8255-8263. Hong, S. P., Leiper, F. C., Woods, A., Carling, D., and Carlson, M. (2003). Activation of yeast Snf1 and mammalian AMP-activated protein kinase by upstream kinases. Proc. Natl. Acad. Sci. U.S.A. 100, 8839-8843. Hrabak, E. M., Chan, W. M., Gribskov, M., Harper, J. F., Choi, J. H., Halford, N., Kudla, J., Luan, S., Nimmo, H. G., Sussman, M. R., Thomas, M., Walker-Simmons, K., Zhu, J. K., and Harmon, A. C. (2003). The Arabidopsis CDPK-SnRK superfamily of protein kinases. Plant physiol. 13, 666-680. Ishitani, M., Liu, J., Halfter, U., Kim, C. S., Shi, W., and Zhu, J. K. (2000). SOS3 function in plant salt tolerance requires N-myristoylation and calcium-binding. Plant Cell 12, 1667–1677. Jambunathan, N. and McNellis, T. W. (2003). Regulation of Arabidopsis COPINE 1 gene expression in response to pathogens and abiotic stimuli. Plant Physiol. 132, 1370-81 Jou, Y. T., Chou, P. H.,He, M. G., Hung, Y. H., and Yen, H. E. (2004). Tissue-specific expression and functional complementation of a yeast potassium-uptake mutant by a salt-induced ice plant gene mcSKD1. Plant Mol. Biol. 54, 881-893. Jou, Y. T. Chiang,C. P., Jauh, G. Y., and Yen, H. E. (2006). Functional characterization of ice plant SKD1, an AAA-Type ATPase associated with the endoplasmic reticulum-Golgi network, and its role in adaptation to salt stress. Plant Physiol. 141, 135-146. Ko, C.H., and Gaber, R.F. (1991). TRK1 and TRK2 encode structurally related K+ transporters in Saccharomyces cerevisiae. Mol. Cell. Biol. 11, 4266-4273. Kobayashi, Y., Yamamoto, S., Minami, H., Kagaya, Y., Hattori, T. (2004). Differential activation of the rice sucrose nonfermenting1-related protein kinase 2 family by hyperosmotic stress and abscisic acid. Plant Cell 16, 1163-1177. Kuchin, S., Vyas,V. K., Kanter, E., Hong, S. P. and Carlson , M. (2003). Std1p (Msn3p) positively regulates the Snf1 kinase in Saccharomyces cerevisiae. Genetics 163, 507-514. Leech, A., Nath, N., McCartney, R. R., and Schmidt, M. C. (2003). Isolation of mutations in the catalytic domain of the Snf1 kinase that render its activity independent of the Snf4 subunit. Eukaryot. Cell 2, 265-273 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. Ludin, K., Jiang, R., and Carlson, M. (1998). Glucose-regulated interaction of a regulatory subunit of protein phosphatase 1 with the Snf1 protein kinase in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 95, 6245-6250. McCartney, R. R., and M. C. Schmidt. (2001). Regulation of Snf1 kinase. Activation requires phosphorylation of threonine 210 by an upstream kinase as well as a distinct step mediated by the Snf4 subunit. J. Biol. Chem. 276, 36460-36466. McCartney, R. R., Rubenstein, E. M., and Schmidt, M. C. (2005). Snf1 kinase complexes with different beta subunits display stress-dependent preferences for the three Snf1-activating kinases. Curr. Genet. 47, 335-344. Nath, N., R. R. McCartney, and M. C. Schmidt. (2003). Yeast Pak1 kinase associates with and activates Snf1. Mol. Cell. Biol. 23, 3909-3917. Perier, F., Coulter, K. L., Liang, H., Radeke, C. M., Gaber, R. F., and Vandenberg, C. M. (1994). Identification of a novel mammalian member of the NSF/CDC48p/Pas1p/TBT-1 family through heterologous expression in yeast. FEBS. Lett. 351, 286-290. Portillo, F. (2000). Regulation of plasma membrane H+-ATPase in fungi and plants. Biochim. Biophys. Acta 1469, 31-42. Portillo, F., Muletb, J. M., Serrano R. (2005). A role for the non-phosphorylated form of yeast Snf1: tolerance to toxic cations and activation of potassium transport. FEBS. Lett. 579, 512-516 Qi, Z., and Spalding, P. E. (2004). Protection of plasma membrane K+ transport by the salt overly sensitive 1 Na+-H+ antiporter during salinity stress. Plant Physiol. 136, 2548-2555 Ramos, J., Alijo, R., Haro, R. and Rodriguez-Navarro, A. (1994). TRK2 is not a low-affinity potassium transporter in Saccharomyces cerevisiae. J. Bacteriol. 176, 249-252. Rolland, F., Baena-Gonzalez, E., and Sheen, J. (2006). Sugar sensing and signaling in plants: conserved and novel mechanisms. Annu. Rev. Plant Biol. 57, 675-709 Ruslana Radchuk, Volodymyr Radchuk, Winfriede Weschke, Ljudmilla Borisjuk, and Hans Weber. (2006). Repressing the expression of the sucrose nonfermenting-1-related protein kinase gene in pea embryo causes pleiotropic defects of maturation similar to an abscisic acid-insensitive phenotype. Plant Physiol. 140, 263-278. Sanz, P., G. R. Alms, T. A. J. Haystead, and M. Carlson. (2000). Regulatory interactions between the Reg1-Glc7 protein phosphatase and the Snf1 protein kinase. Mol. Cell. Biol. 20, 1321-1328. Sanz, P. (2003). Snf1 protein kinase: a key player in the response to cellular stress in yeast. Biochem. Soci. Trans. 31, part 1 Sato, K., Aoto, M., Mori, K., Akasofu, S., Tokmakov, A. A., Sahara, S., and Fukami, Y. (1996). Purification and characterization of a src-related p57 protein-tyrosine kinase from Xenopus oocytes. Isolation of an inactive form of the enzyme and its activation and translocation upon fertilization. J. Biol. Chem. 271, 13250-13257 Schmidt, M. C., McCartney, R. R., Zhang, X., Tillman, T. S., Solimeo, H., Wölfl, S., Almonte, C., and Watkins, S. C. (1999). Std1 and Mth1 proteins interact with glucose sensors to control glucose-regulated gene expression in Saccharomyces cerevisiae. Mol. Cell. Biol. 19, 4561-4571. Schmidt, M. C., and McCartney, R. R. (2000). Subunits of Snf1 kinase are required for kinase function and substrate definition. EMBO J. 19, 4936-4943. Serrano, R. (1991). Transport across yeast vacuolar and plasma membrane. In Broach, J.R., Jones, E.W. and Pringle, J.R. eds, The Molecular and Cellular Biology of the Yeast Saccharomyces: Genome, Dynamics, Protein Synthesis and Energetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 523-585, Sutherland, C. M., Hawley, S. A., McCartney, R. R., Leech, A., Stark, M. J., Schmidt, M. C., and Hardie, D. G. (2003). Elm1p is one of three upstream kinases for the Saccharomyces cerevisiae SNF1 complex. Curr. Biol. 13, 1299-1305. Treitel, M. A., Kuchin, S., and Carlson, M. (1998). Snf1 protein kinase regulates phosphorylation of the Mig1 repressor in Saccharomyces cerevisiae. Mol. Cell. Biol. 18, 6273-6280. Umezawa, T., Yoshida, R., Maruyama, K., Yamaguchi-Shinozaki, K. and Shinozaki, K. (2004). SRK2C, a SNF1-related protein kinase 2, improves drought tolerance by controlling stress-responsive gene expression in Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 101, 17306-17311. Vincent, O., and Carlson, M. (1998). Sip4, a Snf1 kinase-dependent transcriptional activator, binds to the carbon source-responsive element of gluconeogenic genes. EMBO J. 17, 7002-7008. Vincent, O., Townley, R., Kuchin, S., and Carlson, M. (2001). Subcellular localization of the Snf1 kinase is regulated by specific β subunits and a novel glucose signaling mechanism. Genes Dev. 15, 1104-1114. Xiong, L., and Zhu, J.K. (2002). Molecular and genetic aspects of plant responses to osmotic stress. Plant Cell Environ. 25, 131–139. Yang, X., R. Jiang, and M. Carlson. (1994). A family of proteins containing a conserved domain that mediates interaction with the yeast SNF1 protein kinase complex. EMBO J. 13,5878–5886. Yen, H. E., Wu, S. M., Hung, Y. H., and Yen, S. K. (2000). Isolation of three salt-induced low-abundance cDNA from light-grown callus of Mesembryanthemum crystallinum by suppression subtractive hybridization. Physiol. Plant. 110, 402-409. Zhu, J. K. (2001). Plant salt tolerance. Trends Plant Sci. 6, 66-71
摘要: 
mcSNF1 (sucrose non-fermenting 1) 是利用yeast two-hybrid的方式在耐鹽模式植物冰花 (Mesembryanthemum crystallinum L.)以鹽處理的cDNA library中篩選出與mcSKD1 (suppressor of K+ transport defect 1)有交互作用的基因之一,已知鹽誘導基因mcSKD1參與逆境下intracellular vesicular trafficking之過程,本論文之目的在分析mcSNF1在鹽逆境下之基因表現與蛋白累積的情形,以期對mcSNF1的作用機制有進一步的了解。SNF1蛋白最早在酵母菌中發現,參與糖類代謝並與酵母菌許多抗逆境相關反應,其在植物中的同源蛋白SnRK (SNF1-related protein kinase)蛋白則參與許多植物抗逆境反應例如缺水逆境以及與ABA相關之訊息傳遞過程。
為了確定mcSNF1與鹽逆境之間的關聯性,以RT-PCR分析mcSNF1基因在細胞適應高鹽逆境下的表現,並製備可辨識mcSNF1之anti-AKIN10抗體以偵測mcSNF1蛋白累積之情形。結果指出癒傷組織細胞在長期加鹽處理下mcSNF1基因表現會增加,且蛋白累積提高,顯示在細胞層次mcSNF1為一鹽誘導基因。進一步以共軛焦顯微鏡觀察癒傷組織細胞中mcSNF1蛋白分布情形,結果指出其蛋白分布於細胞膜、細胞質中的小泡構造以及細胞核中。將細胞轉到高鹽環境下mcSNF1分布區域的比例會快速的改變,在細胞膜上的分布會增加,顯示mcSNF1在細胞中可能參與鹽逆境相關之訊息傳導路徑。
植株中的情形與細胞層次則不一樣,RT-PCR結果指出植株中mcSNF1基因是一個持續表現型的基因,且其基因表現不會因為鹽處理而改變。西方點墨法觀察mcSNF1蛋白的累積情形也與基因表現的情形相似。顯示mcSNF1不會受到鹽所誘導而改變基因表現或蛋白累積。以石臘組織切片與免疫呈色標定的方式觀察mcSNF1在葉與根部組織裡的分布情形,發現蛋白沒有專一性的分布,廣泛分布在組織中,顯示植株中mcSNF1似乎不受高鹽誘導而改變累積量,推測在全株的層次可能主要以後轉譯修飾來調控mcSNF1的作用。
由本論文之結果得知mcSNF1在受到高鹽逆境時首先會重新分布在細胞中的位置,由細胞質中之小泡往細胞膜移動,推測mcSNF1蛋白參與鹽逆境相關之訊息傳導過程,而在持續高鹽逆境下基因表現量及蛋白累積量會上升,以適應長期之高鹽環境。

The mcSNF1 (sucrose non-fermenting 1) was identified through the yeast two-hybrid screening of a halophyte ice plant (Mesembryanthemum crystallinum L.) library using mcSKD1 (suppressor of K+ transport defect 1) as a bait. The salt-inducible mcSKD1 protein has been studied in depth that is involved in the intracellular vesicular trafficking in this halophyte. The main focus of this thesis is to examine the gene expression and protein accumulation of mcSNF1, a mcSKD1 interacting protein, in order to access its function in the complex network of salt tolerance in ice plant. The SNF1 was first discovered in yeast that has been shown to play roles in the sugar metabolism and stress responses. The plant homolog SnRK (SNF1-related protein kinase) has also been suggested in participating the stress responses and ABA-mediated signal transduction pathway.
The expression of mcSNF1 at cellular level was examined by RT-PCR and the accumulation of mcSNF1 was examined by Western blotting using an anti-AKIN10 antibody that can specifically recognize mcSNF1. The results showed long-term salt stress induced the expression of mcSNF1 and the accumulation of protein also increased concurrently. As for short-term salt stress, it was found the distribution of mcSNF1 was changed by salt treatment. The re-localization from the cytosolic vesicles to the plasma membrane occurred within hours of salt stress suggesting mcSNF1 is involving in the stress-related signal transduction pathway.
The constitutive expression of mcSNF1 was observed in intact plants and the addition of salt did not significantly change the level of expression as well as protein accumulation. Immunostaining of paraffin sections of leaves and roots did not reveal any tissue-specific localization of mcSNF1. The results suggested that the regulation of mcSNF1 in intact plant mainly occurs at the post-translational level but not at the transcriptional or translational levels.
In conclusion, at the early stage of salt stress, the distribution of mcSNF1 moves from the cytosol to the plasma membrane suggesting this protein is involved in the stress signaling. The amount of mcSNF1 expression increases as the stress persists in order to adapt the prolonged stress environment.
URI: http://hdl.handle.net/11455/22423
其他識別: U0005-2508200615010400
Appears in Collections:生命科學系所

Show full item record
 

Google ScholarTM

Check


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.