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標題: Characterization of AtRboh I under Hypoxic Stress in Arabidopsis
阿拉伯芥中 AtRboh I 在缺氧逆境下 之特性分析
作者: 林依萱
I-Shiuan Lin
關鍵字: 阿拉伯芥
rboh I
NADPH oxidase
引用: Andrés-Colás N., Perea-García A., Andrés S. M., Garcia-Molina A., Dorcey E., Rodríguez-Navarro S., Pérez-Amador M. A., Puig S., and Peñarrubia L. (2013). Comparison of global responses to mild deficiency and excess copper levels in Arabidopsis seedlings. Metallomics 5: 1234-1246. Asai S., and Yoshioka H. (2009). Nitric oxide as a partner of reactive oxygen species participates in disease resistance to necrotrophic pathogen Botrytis cinerea in Nicotiana benthamiana. Mol. Plant-microb. Interact. 22: 619-629. Ashraf M., and Arfan M. (2005). Gas exchange characteristics and water relations in two cultivars of Hibiscus esculentus under waterlogging. Biol. Plant. 49: 459-462. Bartel B. (1997). Auxin biosynthesis. Annu. Rev. Plant Biol. 48: 51-66. Baxter-Burrell A., Yang Z., Springer P. S., and Bailey-Serres J. (2002). RopGAP4-dependent Rop GTPase rheostat control of Arabidopsis oxygen deprivation tolerance. Science 296: 2026-2028. Blokhina O.,Virolainen E., and Fagerstedt K. V. (2003). Antioxidants, oxidative damage and oxygen deprivation stress: a review. Ann. Bot. 91: 179-194. Bradford M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Chem. 72: 248-254. Chandler J. W. (2009). Local auxin production: a small contribution to a big field. Bioessays 31: 60-70. Cheng Y., Dai X., and Zhao Y. (2004). AtCAND1, a HEAT-repeat protein that participates in auxin signaling in Arabidopsis. Plant Physiol. 135: 1020-1026. Colmer T. D., and Voesenek L. A. C. J. (2009). Flooding tolerance: suites of plant traits in variable environments. Plant Biol. 36: 665-681. Ding X., Cao Y., Huang L., Zhao J., Xu C., Li X., and Wang S. (2008). Activation of the indole-3-acetic acid-amido synthetase GH3-8 suppresses expansin expression and promotes salicylate- and jasmonate-independent basal immunity in rice. Plant Cell 20: 228-40. Domingo C., Andrés F., Tharreau D., Iglesias D. J., and Talón M. (2009). Constitutive expression of OsGH3.1 reduces auxin content and enhances defense response and resistance to a fungal pathogen in rice. Mol. Plant Microbe Interact. 22: 201-210. Finkel T. (2000). Redox-dependent signal transduction. FEBS Lett. 476: 52-54. Foreman J., Demidchik V., Bothwell J. H. F., Mylona P., Miedema H., Torres M. A., Linstead P., Costa S., Brownlee C., Jones J. D. G., Davies J. M., and Dolan L. (2003). Reactive oxygen species produced by NADPH oxidase regulate plant cell growth. Nature 422: 442-446. Friml J. (2010). Subcellular trafficking of PIN auxin efflux carriers in auxin transport. Eur. J. Cell Biol. 89: 231-235. Fujii S., Hayashi T., and Mizuno K. (2010). Sucrose synthase is an integral component of the cellulose synthesis machinery. Plant Cell Physiol. 51: 294-301. Fukao T., and Bailey-Serres J. (2004). Plant responses to hypoxia-is survival a balancing act? Trends Plant Sci. 9: 449-456. Gapper C., and Dolan L. (2006). Control of plant development by reactive oxygen species. Plant Physiol. 141: 341-345. Geigenberger P. (2003). Response of plant metabolism to too little oxygen. Curr. Opin. Plant Biol. 6: 247-256. Goodwin S. B., and Sutter T. R. (2009). Microarray analysis of Arabidopsis genome response to aluminum stress. Biol. Plantarum. 53: 85-99. Gupta K. J., Zabalza A., and Dongen J. T. V. (2009). Regulation of respiration when the oxygen availability changes. Physiol. Plant. 137: 383-391. Halliday K. J., Martínez-García J. F., and Josse E. M. (2009). Cold Spring Harb Perspect. Biol. 1: a001586. Hamant O., Traas J., and Boudaoud A. (2010). Regulation of shape and patterning in plant development. Curr. Opin. Genet. Dev. 20: 454-459. Hörtensteiner S. (2004). The loss of green color during chlorophyll degradation-a prerequisite to prevent cell death? Planta 219: 191-194. Hörtensteiner S., and Kräutler B. (2011). Chlorophyll breakdown in higher plants. Biochim. Biophys. Acta. 1807: 977-988. Huang S. J., Chang C. L., Wang P. H., Tsai M. C., Hsu P. H., and Chang I. F. (2013). A type III ACC synthase, ACS7, is involved in root gravitropism in Arabidopsis thaliana. J. Exp. Bot. 64: 4343-4360. Iglesias M. J., Terrile M. C., Bartoli C. G., D'Ippólito S., and Casalongué C. A. (2010). Auxin signaling participates in the adaptative response against oxidative stress and salinity by interacting with redox metabolism in Arabidopsis. Plant Mol. Biol. 4: 215-222. Ismond K. P., Dolferus R., Pauw M. D., Dennis E. S., and Good A. G. (2003). Enhanced low oxygen survival in Arabidopsis through increased metabolic flux in the fermentative pathway. Plant Physiol. 132: 1292-1302. Jackson W. T. (1956). The relative importance of factors causing injury to shoots of flooded tomato plants. Amer. J. Bot. 43: 637-639. Jefferson R. A. (1989). The GUS reporter gene system. Nature 342: 837-838. Jones M. A., Raymond M. J., Yang Z., and Smirnoff N. (2007). NADPH oxidase-dependent reactive oxygen species formation required for root hair growth depends on ROP GTPase. J. Exp. Bot. 58: 1261-1270. Junghans U., Polle A., Düchting P., Weiler E., Kuhlman B., Gruber F., and Teichmann T. (2006). Adaptation to high salinity in poplar involves changes in xylem anatomy and auxin physiology. Plant Cell Environ. 29: 1519-1531. Kennedy R. A., Rumpho M. E., and Fox T. C. (1992). Anaerobic metabolism in plants. Plant Physiol. 100: 1-6. Kieffer M., Neve J., and Kepinski S. (2010). Defining auxin response contexts in plant development. Curr. Opin. Plant Biol. 13: 12-20. Klok E. J., Wilson I. W., Wilson D., Chapman S. C., Ewing R. M., Somerville S. C., Peacock W. J., Dolferus R., and Dennis E. S. (2002). Expression profile analysis of the low-oxygen response in Arabidopsis root cultures. Plant Cell 14: 2481-2491. Knaap E., Sauter M., Wilford R., and Kende H. (1996). Identification of a gibberellin-induced receptor-like kinase in deepwater rice. Plant Physiol. 112: 1397-1401. Licausi F., and Perata P. (2009). Low oxygen signaling and tolerance in plants. Adv. Bot. Res. 50: 139-198. Liu F., Toai T. V., Moy L. P., Bock G., Linford L. D., and Quackenbush J. (2005). Global Transcription Profiling Reveals Comprehensive Insights into Hypoxic Response in Arabidopsis. Plant Physiol. 137: 1115-1129. Ljung K., Hul A. K., Kowalczyk M., Marchant A., Celenza J., Cohen J. D., and Sandberg G. (2002). Biosynthesis, conjugation, catabolism and homeostasis of indole-3-acetic acid in Arabidopsis thaliana. Plant Mol. Biol. 50: 309-332. Lohar D. P., Haridas S., Gantt J. S., and VandenBosch K. A. (2007). A transient decrease in reactive oxygen species in roots leads to root hair deformation in the legume-rhizobia symbiosis. New Phytol. 173: 39-49. Mach J. M., Castillo A. R., Hoogstraten R., and Greenberg J. T. (2001). The Arabidopsis-accelerated cell death gene ACD2 encodes red chlorophyll catabolite reductase and suppresses the spread of disease symptoms. Proc. Natl. Acad. Sci. 98: 771-776. Magneschi L., and Perata P. (2009). Rice germination and seedling growth in the absence of oxygen. Ann. Bot. 103: 181-196. Marino D., Andrio E., Danchin E. G. J., Oger E., Gucciardo S., Lambert A., Puppo A., and Pauly N. (2011). A Medicago truncatula NADPH oxidase is involved in symbiotic nodule functioning. New Phytol. 189: 580-592. Marino D., Dunand C., Puppo A., and Pauly N. (2012). A burst of plant NADPH oxidases. Trends Plant Sci. 17: 9-15. Markakis M. N., Boron A. K., Van Loock B., Saini K., Cirera S., Verbelen J. P., and Vissenberg K. (2013). Characterization of a small auxin-up RNA (SAUR)-like gene involved in Arabidopsis thaliana development. PLoS One 8: e82596. Mathur J. (2004). Cell shape development in plants. Trends Plant Sci. 9: 583-590. Mergemann H., and Sauter M. (2000). Ethylene induces epidermal cell death at the site of adventitious root emergence in rice. Plant Physiol. 124: 609-614. Miller G., Schlauch K., Tam R., Cortes D., Torres M. A., Shulaev V., Dangl J. L., and Mittler R. (2009). The plant NADPH oxidase RBOHD mediates rapid systemic signaling in response to diverse stimuli. Sci. Signal. 2: ra45. Mishra B. S., Singh M., Aggrawal P., and Laxmi A. (2009). Glucose and auxin signaling interaction in controlling Arabidopsis thaliana seedlings root growth and development. PLoS One 4: e4502. Muller I. M., Jensen P. E., and Hansson A. (2007). Oxidative Modifications to Cellular Components in Plants. Annu. Rev. Plant Biol. 58: 459-481. Mühlenbock P., Szechynska-Hebda M., Plaszczyca M., Baudo M., Mateo A., Mullineaux P. M., Parker J. E., Karpinska B., and Karpinski S. (2008). Chloroplast signaling and LESION SIMULATING DISEASE1 regulate crosstalk between light acclimation and immunity in Arabidopsis. Plant Cell 20: 2339-2356. Müller K., Carstens A. C., Linkies A., Torres M. A., and Leubner-Metzger G. (2009). The NADPH-oxidase AtrbohB plays a role in Arabidopsis seed after-ripening. New Phytol. 184: 885-897. Nakano T., Suzuki K., Fujimura T., and Shinshi H. (2006). Genome-wide analysis of the ERF gene family in Arabidopsis and rice. Plant Physiol. 140: 411-432. Neill S., Desikan R., and Hancock J. (2002). Hydrogen peroxide signaling. Curr. Opin. Plant Biol. 5: 388-395. Normanly J. (2010). Approaching cellular and molecular resolution of auxin biosynthesis and metabolism. Cold Spring Harb Perspect. Biol. 2: a001594. Oda T., Hashimoto H., Kuwabara N., Akashi S., Hayashi K., Kojima C., Wong H. L., Kawasaki T., Shimamoto K., Sato M., and Shimizu T. (2010). Structure of the N-terminal regulatory domain of a plant NADPH oxidase and its functional implications. J. Biol. Chem. 285: 1435-1445. Overvoorde P. J., Okushima Y., Alonso J. M., Chan A., Chang C., Ecker J. R., Hughes B., Liu A., Onodera C., Quach H., Smith A., Yu G., and Theologis A. (2005). Functional genomic analysis of the Auxin/indole-3-acetic acid gene family members in Arabidopsis thaliana. Plant Cell 17: 3282-3300. Parelle J., Brendel O., Bodénès C., Berveiller D., Dizengremela P., Joliveta Y., and Dreyera E. (2006). Differences in morphological and physiological responses to water-logging between two sympatric oak species. Ann. For. Sci. 63: 849-859. Park J. E., Park J. Y., Kim Y. S., Staswick P. E., Jeon J., Yun J., Kim S. Y., Kim J., Lee Y. H., and Park C. M. (2007). GH3-mediated auxin homeostasis links growth regulation with stress adaptation response in Arabidopsis. J. Biol. Chem. 282: 10036-10046. Pasternak T., Potters G., Caubergs R., and Jansen M. A. (2005a). Complementary interactions between oxidative stress and auxins control plant growth responses at plant, organ, and cellular level. J. Exp. Bot. 56: 1991-2001. Pasternak T., Rudas V., Potters G., and Jansen M. A. K. (2005b). Morphogenic effects of abiotic stress: reorientation of growth in Arabidopsis thaliana seedlings. Environ. Exp. Bot. 53: 299-314. Peer W. A., Cheng Y., and Murphy A. S. (2013). Evidence of oxidative attenuation of auxin signalling. J. Exp. Bot. 64: 2629-2639. Peng H. P., Lin T. Y., Wang N. N., and Shih M. C. (2005). Differential expression of genes encoding 1-aminocyclopropane-1-carboxylate synthase in Arabidopsis during hypoxia. Plant Mol. Biol. 58: 15-25. Perchepied L., Balagué C., Riou C., Claudel-Renard C., Rivière N., Grezes-Besset B., and Roby D. (2010). Nitric oxide participates in the complex interplay of defense-related signaling pathways controlling disease resistance to Sclerotinia sclerotiorum in Arabidopsis thaliana. Mol. Plant Microbe Interact. 23: 846-860. Perrot-Rechenmann C. (2010). Cellular responses to auxin: division versus expansion. Cold Spring Harb Perspect. Biol. 2: a001446. Potocky M., Jones M. A., Bezvoda R., Smirnoff N., and Zarsky V. (2007). Reactive oxygen species produced by NADPH oxidase are involved in pollen tube growth. New Phytol. 174: 742-751. Pucciariello C., Banti V., and Perata P. (2012). ROS signaling as common element in low oxygen and heat stresses. Plant Physiol. Biochem. 59: 3-10. Rahman M., Grover A., Peacock W. J., Dennis E. S., and Ellis M. H. (2001). Effects of manipulation of pyruvate decarboxylase and alcohol dehydrogenase levels on the submergence tolerance of rice. J. Plant Physiol. 28: 1231-1241. Rawyler A., Arpagaus S., and Braendle R. (2002). Impact of oxygen stress and energy availability on membrane stability of plant cells. Ann. Bot. 90: 499-507. Remans T., Opdenakker K., Smeets K., Mathijsen D., Vangronsveld J., and Cuypers A. (2010). Metal-specific and NADPH oxidase dependent changes in lipoxygenase and NADPH oxidase gene expression in Arabidopsis thaliana exposed to cadmium or excess copper. Funct. Plant Biol. 37: 532-544. Roberts J. K., Callis J., Wemmer D., Walbot V., and Jardetzky O. (1984). Proc. Natl. Acad. Sci. 81: 3379-3383. Sagi M., Davydov O., Orazova S., Yesbergenova Z., Ophir R., Stratmann J. W., and Fluhr R. (2004). Plant respiratory burst oxidase homologs impinge on wound responsiveness and development in Lycopersicon esculentum. Plant Cell 16: 616-628. Song X. G., She X. P., He J. M., Huang C., and Song T. S. (2006). Cytokinin- and auxin-induced stomatal opening involves a decrease in levels of hydrogen peroxide in guard cells of Vicia faba. Funct. Plant Biol. 33: 573-583. Suzuki N., Miller G., Morales J., Shulaev V., Torres M. A., and Mittler R. (2011). Respiratory burst oxidases: the engines of ROS signaling. Curr. Opin. Plant Biol. 14: 691-699. Teale W. D., Paponov I. A., and Palme K. (2006). Auxin in action: signalling, transport and the control of plant growth and development. Nat. Rev. Mol. Cell Biol. 7: 847-859. Tester M., and Langridge P. (2010). Breeding technologies to increase crop production in a changing world. Science 327: 818-822. Tognetti V. B., Mühlenbock P., and Van Breusegem F. (2012). Stress homeostasis-the redox and auxin perspective. Plant Cell Environ. 35: 321-333. Tognetti V. B., Van Aken O., Morreel K., Vandenbroucke K., Van de Cotte B., De Clercq I., Chiwocha S., Fenske R., Prinsen E., Boerjan W., Genty B., Stubbs K. A., Inzé D., Van Breusegem F. (2010). Perturbation of indole-3-butyric acid homeostasis by the UDP-glucosyltransferase UGT74E2 modulates Arabidopsis architecture and water stress tolerance. Plant Cell. 22: 2660-2679. Vanderauwera S., Zimmermann P., Rombauts S., Vandenabeele S., Langebartels C., Gruissem W., Inzé D., and Van Breusegem F. (2005). Genome-wide analysis of hydrogen peroxide-regulated gene expression in Arabidopsis reveals a high light-induced transcriptional cluster involved in anthocyanin biosynthesis. Plant Physiol. 139: 806-821. Vidoz M. L., Loreti E., Mensuali A., Alpi A., and Perata P. (2010). Hormonal interplay during adventitious root formation in flooded tomato plants. Plant J. 63: 551-562. Wang S., Bai Y., Shen C., Wu Y., Zhang S., Jiang D., Guilfoyle T. J., Chen M., and Qi Y. (2010). Auxin-related gene families in abiotic stress response in Sorghum bicolor. Funct. Integr. Genomics. 10: 533-546. Wintermans J. F., and de Mots A. (1965). Spectrophotometric characteristics of chlorophylls a and b and their pheophytins in ethanol. Biochim. Biophys. Acta. 109: 448-453. Woodward A. W., and Bartel B. (2005). Auxin: regulation, action, and interaction. Ann. Bot. 95: 707-735. Xu T., Wen M., Nagawa S., Fu Y., Chen J. G., Wu M. J., Perrot-Rechenmann C., Friml J., Jones A. M., and Yang Z. (2010). Cell surface- and rho GTPase-based auxin signaling controls cellular interdigitation in Arabidopsis. Cell 143: 99-110. Xu K., Xu X., Fukao T., Canlas P., Maghirang-Rodriguez R., Heuer S., Ismail A. M., Bailey-Serres J., Ronald P. C., and Mackill D. J. (2006). Sub1A is an ethylene-response-factor-like gene that confers submergence tolerance to rice. Nature 442: 705-708. Yang C. Y. (2014). Hydrogen peroxide controls transcriptional responses of ERF73/HRE1 and ADH1 via modulation of ethylene signaling during hypoxic stress. Planta 239: 877-885. Yang Z., and Fu Y. (2007). ROP/RAC GTPase signaling. Curr. Opin. Plant Biol. 10: 490-494. Yang C. Y., and Hong C. P. (2015). The NADPH oxidase Rboh D is involved in primary hypoxia signalling and modulates expression of hypoxia-inducible genes under hypoxic stress. Environ. Exp. Bot. 115: 63-72. Yang C. Y., Hsu F. C., Li J. P., Wang N. N., and Shih M. C. (2011). The AP2/ERF transcription factor AtERF73/HRE1 modulates ethylene responses during hypoxia in Arabidopsis. Plant Physiol. 156: 202-212. Zabalza A., Dongen J. T. V., Froehlich A., Oliver S. N., Faix B., Gupta K. J., Schmälzlin E., Igal M., Orcaray L., Royuela M., and Geigenberger P. (2009). Regulation of respiration and fermentation to control the plant internal oxygen concentration. Plant Physiol. 149: 1087-1098. Zeng Y., Wu Y., Avigne W. T., and Koch K. E. (1999). Rapid repression of maize invertases by low oxygen. Invertase/sucrose synthase balance, sugar signaling potential, and seedling survival. Plant Physiol. 121: 599-608. Zhao Y. (2010). Auxin biosynthesis and its role in plant development. Annu. Rev. Plant Biol. 61: 49-64.
摘要: 當植物遭受環境逆境時,其體內之荷爾蒙及氧化還原狀態之平衡皆受到 影響,以使其能因應逆境得以生存,在此過程中,往往伴隨著活性氧族 (Reactive oxygen species, ROS)的產生。高等植物可透過 NADPH 氧化酶之作 用 生 成 超 氧 陰 離 子 (superoxide anion radical, O2.-) 進 而 產 生 過 氧 化 氫 (hydrogen peroxide, H2O2)等活性氧族,NADPH 氧化酶又稱為 RBOHs (respiratory burst oxidase homologues)。前人研究顯示,阿拉伯芥中十個 Rboh 基因家族成員中,有五個成員基因表現會受缺氧逆境所誘導,分別為 AtRboh A、B、D、G 及 I,其中 AtRboh I 轉錄子在缺氧逆境下 3 小時開始受誘導, 到 9 小時達到最高量。為深入探討缺氧訊號路徑下 AtRboh I 之功能,我們 利用阿拉伯芥 T-DNA 插入之 rboh I 突變株與野生型,進行生理及分子特性 分析。在缺氧逆境下之存活率結果顯示,rboh I 突變株之存活率低於野生型。 葉綠素含量試驗顯示,回復缺氧逆境處理後,rboh I 突變株幼苗葉綠素含量 較野生型低。利用 AtRboh I pro ::GUS 轉殖株偵測 AtRboh I 啟動子在不同組織 之表現情形,發現 AtRboh I 啟動子表現於幼苗之根部維管束組織、葉脈、毛 狀體、花柱頂部及果莢兩端,並受機械損傷所誘導表現。GUS 活性分析結 果 顯 示 , 受 缺 氧 逆 境 誘 導 之 GUS 活 性 在 添 加 生 長 素 運 移 抑 制 劑 (1-naphthylphthalamic acid , NPA)處理下,誘導情形會受抑制。進一步利用即 時定量聚合酶連鎖反應 (real-time quantitative polymerase chain reaction, q-PCR)分析缺氧訊號、乙烯生合成之相關基因及生長素反應基因,結果顯示 在缺氧逆境下,缺氧訊號相關基因 AtHRE1 、 AtADH1、AtLDH 及 AtSUS1 在 rboh I 突變株中之表現量皆下降,而在添加 NPA 處理下,僅 AtSUS1 之表現 量上升; 而在缺氧逆境下,生長素反應基因 At1g19840、At3g23030 及 At5g19140 表現量在 rboh I 突變株中之表現量皆上升;在缺氧逆境並添加 NPA 處理下,乙烯生合成之關鍵酵素基因 AtACS7 及 AtACS8 於 rboh I 突變 株中之表現量皆上升。綜合以上實驗結果顯示,於缺氧逆境下,AtRboh I 參與調控乙烯生合成及缺氧相關基因之表現,及缺氧訊號下與生長素訊號路 徑之交互作用,進而影響植株之缺氧耐受性。
Environmental stresses can cause accumulation of reactive oxygen species (ROS) in higher plants which have developed adaptive mechanisms by influence homeostasis of redox state and phytohormones. The NADPH oxidase family lead to the production of apoplastic superoxide (O2.–), then rapidly catalyzed to hydrogen peroxide (H2O2). The NADPH oxidase has ten known members in Arabidopsis which is named respiratory burst oxidase homologues (AtRboh A-J). The transcript levels of five AtRboh genes (AtRboh A, B, D, G and I) were increased under hypoxia. In particular, the transcript levels of AtRboh I were induced after 3 h hypoxia treatment and highest transcript levels after 9 h hypoxia treatment. To further investigate the function of AtRboh I under hypoxic signaling pathways, we used two independent AtRboh I-knockout lines to assess molecular function in Arabidopsis thaliana. Under submergence condition, the AtRboh I displayed reduced survival rate compared with wild-type. Furthermore, the chlorophyll content of AtRbohI-knockout lines displayed lower than that detected in wild-type. Histochemical analysis results presented that GUS expression was detected at vascular tissue of roots and leaves, trichomes, top of column and both ends of siliques in AtRboh I pro ::GUS transgenic plants under normoxic condition, also induced by wounding treatment. The GUS activity assay was showed that AtRboh I promoter was induced by hypoxia, but reduced in hypoxia combined with auxin transport inhibitor (1-naphthylphthalamic acid, NPA) treatment. Quantitative-PCR analyses were showed that hypoxia-inducible AtHRE1, AtADH1, AtLDH and AtSUS1 expression were reduced in AtRboh I-knockout lines under hypoxia, by contrast, AtSUS1 expression increased under hypoxia combined with NPA treatment. Expression of auxin responsive genes At1g19840, At3g23030 and At5g19140 were increased under hypoxia. Moreover, expression of the ethylene biosynthetic genes AtACS7 and AtACS8 were increased under hypoxia combined with NPA treatment. Taken together, our results demonstrate that AtRboh I plays an important role in modulating genes expression of ethylene biosynthesis and down-stream of hypoxia signaling, and interplays of hypoxia signaling and auxin-mediated signaling pathways under hypoxic stress, to give further effect on hypoxia tolerance in plants.
文章公開時間: 2018-07-29
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