臺灣博碩士論文加值系統

以綠豆植株 (Vigna radiata L. cv. TN5) 處理200 mM過氧化氫再經4 oC，36小時的低溫測試以評估植株之低溫耐受性。發現重複處理過氧化氫得到較1次處理過氧化氫佳之低溫抗性，且以間隔3小時重複處理過氧化氫所得到的電解滲漏率最低，相近於經10oC, 48小時冷馴化處理的植株。以2次過氧化氫前處理隨即誘導麩胱甘肽累積，且累積量高於控制組、1次過氧化氫，甚至是冷馴化處理的植株。經過氧化氫處理所誘導綠豆植株之麩胱甘肽累積量不受光照所影響；然而經光照之冷馴化處理植株則明顯降低麩胱甘肽含量。於過氧化氫處理前，以鈣離子螫合劑EGTA或glutathione (GSH) 生合成抑制劑BSO處理綠豆植株，均導致電解滲漏率提高，逆轉其抗寒能力，但無顯著影響麩胱甘肽累積。以細胞內鈣離子抑制劑ruthenium red處理對電解滲漏率麩胱甘肽累積並無顯著影響。經實驗結果暗示，過氧化氫處理綠豆植株所生成的訊號回應中，細胞外鈣離子與麩胱甘肽於其中扮演著重要角色。以冷馴化處理所誘導植株低溫抗寒訊息傳遞中，可能經光依賴（light-dependent）及光不依賴（light-independent）之訊息傳遞調節。

Mung bean seedlings (Vigna radiata L. cv. TN5, a chilling-sensitive cultivar) pretreated twice with 200 mM hydrogen peroxide (H2O2) by a 3-h interval apart followed by chilled at 4oC for 36 h showed a similar electrolyte leakage levels to those of seedlings cold-acclimated at 10oC for 48 h. Double H2O2 treated seedlings showed higher glutathione levels than those of control, single-treated, and even cold-acclimated plants. This H2O2 induced glutathione accumulation in seedlings did not inference by illumination; however, the glutathione levels of cold-acclimated plants diminished significantly. Seedlings treated with buthionine sulfoximine (BSO), a specific inhibitor of glutathione synthesis, prior to H2O2 application reversed the H2O2-induced tolerances and thus showed a higher electrolyte leakage. Combination of distinct treatments with ethylene glycol-bis (β-aminothyl ether)-N,N,Nˊ,Nˊ-tetraacetic acid (EGTA), a specific calcium chelator, although had no significantly influence on the glutathione accumulation. However, the tolerances of cold acclimated and H2O2 treated seedlings do decrease by EGTA. Notably, ruthenium red, inhibitor of Ca2+ flux from intracellular stores, had no obvious influence on both glutathione and electrolyte leakage levels. These observations indicated that extracellular calcium and glutathione accumulation play important roles in the response elicited by H2O2 pretreatment in mung bean seedlings. Also, cold acclimation induced chilling tolerance response could separately mediate via both light-dependent and light-independent pathways.

目錄
封面內頁
簽名頁
授權書1................................................................................................　iii
授權書2.................................................................................................iv
中文摘要................................................................................................v
英文摘要...............................................................................................vi
誌謝...................................................................................................... vii
目錄.....................................................................................................viii
圖目錄...................................................................................................xi
表目錄..................................................................................................xii
附錄圖目錄.........................................................................................xiii

第一章 前言......................................................................................1
第一節 環境逆境與游離基的關係......................................1
第二節 抗氧化防禦系統（antioxidant defense
system）與低溫防禦之關係........................1
第三節 過氧化氫與逆境防禦的關係..................................3
第四節 過氧化氫、鈣離子與低溫防禦之關係................... 4
第二章　材料與方法
第一節 實驗藥品..................................................................6
一、化學藥品及試劑...................................................... 6
第二節 植物材料與生長條件..............................................6
第三節 植物處理方法..........................................................7
第四節 電解質滲漏率測試(electrolyte leakage).................8
第五節 蛋白質測定..............................................................8
第六節 綠豆麩胱甘肽總量分析(total glutathione
assay, TG).................................................................8
第三章　結果
第一節 重複過氧化氫前處理使植物提高低溫耐
受性........................................................................10
第二節 重複過氧化氫前處理使植物累積高量麩
胱甘肽....................................................................12
第三節 以麩胱甘肽生合成抑制劑抑制麩胱甘肽
累積使綠豆植株降低低溫耐受性.........................13
第四節 以EGTA前處理綠豆植株使綠豆植株提
高電解滲漏率但經 ruthenium red 前處理
的植株影響則較小.................................................14
第五節 外生性鈣離子(exogenous calcium)亦誘發
植物生成低溫抗性................................................15
第四章　結論
第一節 重複過氧化氫前處理誘導綠豆植株獲致
同等於冷馴化之低溫耐受性................................17
第二節 重複過氧化氫前處理誘使麩胱甘肽累積，
進而顯著提升植物低溫耐受性............................18
第三節 重複過氧化氫前處理以鈣離子做為二次
訊號傳遞者（second messenger）誘使綠
豆植株生成低溫耐受性........................................20
第四節 總結........................................................................21
參考文獻..............................................................................................23
圖表......................................................................................................31
附錄......................................................................................................42






















圖目錄

圖1. 多重過氧化氫前處理對於綠豆植株低溫耐受性的影響
..................................................................................................31
(A) 2次過氧化氫前處理的時間間隔對於綠豆葉面之
電解滲率的影響................................................................31
(B) 多重過氧化氫前處理所誘導的低溫耐受性.....................32
(C) 過氧化氫誘導綠豆植株低溫耐受性之去冷馴化
(deacclimation)分析.........................................................33
圖2. 綠豆植株於4oC 光照 (圖 2A) 或黑暗下(圖 2B)麩胱
甘肽之含量變化.........................................................................34
圖3. BSO前處理對於綠豆葉子其麩胱甘肽含量 (圖3A and
3C) 及電解滲漏率(圖3B and 3D) 的影響..............................35
圖4. EGTA（圖4C及圖4D）及ruthenium red（圖4A及圖
4B）對綠豆植株電解滲漏率與麩胱甘肽含量之影響..............36
圖5. 氯化鈣前處理對電解滲漏率（圖5A）與麩胱甘肽的累
積量（圖5B）之影響...................................................................37
圖6. 以外生性鈣離子前處理可能誘發的訊號傳遞路徑.................38
圖7. 由2次過氧化氫前處理可能誘發的訊號傳遞路徑..................39
圖8. 外生性過氧化氫與冷馴化處理所引發可能的訊號傳遞
路徑............................................................................................40


表目錄

表1. 比較各前處理所生成的逆境記憶、4 oC低溫耐受
時間、麩胱甘肽累積量及是否依賴鈣離子的訊息
傳遞路徑....................................................................................41




附錄圖目錄

附錄1. 由過氧化氫所誘導的可能訊號傳遞路徑.............................41
附錄2. 將含有轉殖Aequorin之菸草轉殖株以2次過氧化
氫處理後分析其所造成的冷光強度.....................................42

Adams, D.O., and Liyanage, C. 1991. Modification of an enzymatic glutathione assay for determination of total glutathione in grapevine tissues. Am. J. Enol. Vitic. 42: 137-140.
Anderson, J.V., Chevone, B.I., and Hess, J.L. 1992. Seasonal variation in the antioxidant system of eastern white pine needles. Plant Physiol. 98: 501-508.
Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram Quan ties of protein utilizing the principle of protein-dye bindings. Anal. Biochem. 72: 248-254.
Chandra, S., Stennis, M., and Low, P.S. 1997. Measurement of Ca2+ fluxes during elicitation of the oxidative burst in aequorin-transformed tobacco cells. J. Biol. Chem. 272: 28274-28280.
Fadzillah, N.M., Gill, V., Finch, R.P., and Burdon, R.H. 1996. Chilling, oxidative stress and antioxidant responsives in shoot cultures of rice. Planta 199: 552-556.
Frohnmeyer, H., Bowler, C., Zhu, I.K., Yamagata, H., Schafer, E., and Chua, N.H. 1998. Different roles for calcium and calmodulin in phytochrome- and UV-regulated expression of chalcone synthase. Plant J. 13: 763-772.
Gong, M., van der Luit, A.H., Knight, M.R., and Trewavas, A.J.
1998. Heat-Shock-Induced Changes in Intracellular Ca2+ Level Tobacco Seedlings in Relation to Thermotolerance. Plant Physiol. 116: 429-437.
Hariyadi, P., and Parkin, K.L. 1993. Chilling-induced oxidative stress in cucumber (Cucumis sativus L. cv. Calypso) seedlings. J. Plant Physiol. 141: 733-738.
Hodges, D.M., Andrews, C.J., Johnson, D.A. and Hamilton, R.I. 1996. Antioxidant compound responses to chilling stress in differentially sensitive inbred maize lines. Physiol. Plant. 98: 685-92.
Hu, X., Bidney, L.D., Yalpani, N., Duvick, J.P., Crasta, O., Folkerts, O. and Lu, G. 2003. Overexpression of a gene encoding hydrogen peroxide-generating oxalate oxidase evokes defense responses in sunflower. Plant Physiol. 133: 170-181.
Hung, S.H., Yu, C.W., and Lin, C.H. 2005. Hydrogen peroxide functions as a stress signal in plants. Bot. Bull. Acad. Sin. 46: 1-10.
Inzé, D., and Van Montagu, M. 1995. Oxidative stress in plants. Curr. Opin. Biotech. 6: 153-158.
Karpinski, S., Reynolds, H., Karpinska, B., Wingsle, G., Creissen, G., Mullineaux, P.M. 1999. Systemic signaling and acclimation in response to excess excitation energy in Arabidopsis. Science 284: 654-657.
Knight, M.R., Campbell, A.K., Smith, S.M., and Trewavas, A.J. 1991. Transgenic plant aequorin reports the effects of cold shock and elicitors on cytoplasmic calcium. Nature 352: 524-526.
Kingston-Smith, A.H., and Foyer, C.H. 2000. Overexpression of Mn-superoxide dismutase in maize leaves leads to increased monodehydroascorbate reductase, dehydroascorbate reductase and glutathione reductase activities. J. Exp. Bot. 51: 1867-1877.
Knight, M.R., Smith, S.M., and Trewavas, A.J. 1992. Wind-induced plant motion immediately increases cytosolic calcium. Proc. Natl. Acad. Sci. USA 89: 4967-4971.
Knight, H., Trewavas, A.J., and Knight, M.R. 1996. Cold calcium signaling in Arabidopsis involves two cellular pools and a change in calcium signature after acclimation. Plant Cell 8: 489-503.
Knight, H., Trewavas, A.J., and Knight, M.R. 1997. Calcium signalling in Arabidopsis thaliana responding to drought and salinity. Plant J. 12: 1067-1078.
Kocsy, G., Ballmoos, P., Suter, M., Ruegsegger, A., Galli, U., Szalai, G., Galiba, G., and Brunold, C. 2000. Inhibition of glutathione synthesis reduces chilling tolerance in maize. Planta 211: 528-536.
Kocsy, G., Brunner, M., Ruegsegger, A., Stamp, P., and Brunold, C. 1996. Glutathione synthesis in maize genotypes with different sensitivities to chilling. Planta 198: 365-370.
Kocsy, G., Galiba, G., and Brunold, C. 2001a. Role of glutathione in adaptation and signalling during chilling and cold acclimation in plants. Physiol. Plant. 113: 158-164.
Kocsy, G., von Ballmoos, P., Ru¨ egsegger, A., Szalai, G., Galiba, G., and Brunold, C. 2001b. Increasing the glutathione content in a chilling-sensitive maize genotype using safeners increased protection against chilling-induced injury. Plant Physiol. 127: 1147-1156.
Kovtun, Y., Chiu, W.L., Tena, G., and Sheen, J. 2000. Functional analysis of oxidative stress-activated mitogen-activated protein kinase cascade in plants. Proc. Natl. Acad. Sci. USA 97: 2940-2945.
Lee, D.H., and Lee, C.B. 2000. Chilling stress-induced changes of antioxidant enzymes in the leaves of cucumber: in gel enzyme activity assays. Plant Sci. 159: 75-85.
May, M.J., Vernoux, T., Leaver, C., Van Montagu, M., and Inze, D. 1998. Glutathione homeostasis in plants: implications for environmental sensing and plant development. J. Exp. Bot. 49: 649-667.
Meinhard, M., and Grill, E. 2001. Hydrogen peroxide is a regulator of ABI1, a protein phosphatase 2C from Arabidopsis. FEBS Lett. 508: 443-446.
Noctor, G., Arisi, A.M., Jouanin, L., Kunert, K.J., Rennenberg, H., and Foyer, C.H. 1998. Glutathione: biosynthesis, metabolism and relationship to stress tolerance explored in transformed plants. J. Exp. Bot. 49: 623-647.
Noctor, G., Gomez, L., Vanacker, H., and Foyer, C.H. 2002. Interaction between biosynthesis, compartmentation and transport in the control of glutathione homeostasis and signaling. J. Exp. Bot. 53: 1283-1304.
O’Kane, D., Gill, V., Boyd, P., and Burdon, R. 1996. Chilling, oxidative stress and antioxidant responses in Arabidopsis thaliana callus. Planta 198: 371-377.
Olsen, J.E., Junttila, O., Nilsen, J., Eriksson, M., Martinussen, I., Olsson, O., Sandberg, G., and Moritz, T. 1997. Ectopic expression of phytochrome A in hybrid aspen changes critical daylength for growth and prevents cold acclimation. Plant J. 12: 1339-1350.
Orozco-Cárdenas, M.L., and Ryan, C. 1999. Hydrogen peroxide is generated systemically in plant leaves by wounding and systemin via the octadecanoid pathway. Proc. Natl. Acad. Sci. USA 96: 6553-6557.
Orozco-Cárdenas, M.L., Nárvaez-Vásquez, J., and Ryan, C.A. 2001. Hydrogen peroxide acts as a second messenger for the induction of defense genes in tomato plants in response to wounding, systemin and methyl jasmonate. Plant Cell 13: 179-191.
Pei, Z-M., Murata, Y., Benning, G., Thomine, S., Klusener, B., Allen, G.J., Grill, E., and Schroeder, J.I. 2000. Calcium channels activated by hydrogen peroxide mediate abscisic acid signalling in guard cells. Nature 406: 731-734.
Prasad, T.K., Anderson, M.D., Martin, B.A., and Stewart, C.R. 1994. Evidence for chilling-induced oxidative stress in maize seedlings and a regulatory role for hydrogen peroxide. Plant Cell 6: 65-74.
Prasad, T.K. 1996. Mechanisms of chilling-induced oxidative stress injury and tolerance in developing maize seedlings: changes in antioxidant system, oxidation of proteins and lipids, and protease activities. Plant J. 10: 1017-1026.
Price, A.H., Taylor, A., Ripley, S.J., Griffiths, A., Ttewavas, A.J.,
and Knight, M.R. 1994. Oxidative Signals in Tobacco lncrease Cytosolic Calcium. Plant Cell 6: 1301-1310.
Szalai, G., Janda, T., Bartok, T., and Paldi, E. 1997. Role of light in changes in free amino acid and polyamine contents at chilling temperature in maize (Zea mays L.). Physiol. Plant.101: 434-438.
Tähtiharju, S. 2002. The role of calcium and protein phosphatases in cold signal transduction in Arabidopsis thaliana. University of Helsinki Finland. ISBN 952-10-0478-9.
Takahashi, K., Isobe, M., and Mut, S.O. 1997. An increase in cytosolic calcium ion concentration precedes hypoosmotic shock-induced activation of protein kinases in tobacco suspension culture cells. FEBS Lett. 401: 202-206.
Trewavas, A.J., and Malhó, R. 1997. Signal perception and transduction: the origin of the phenotype. Plant Cell 9: 1181-1195.
Vallelian-Bindschedler, L., Schweizer, P., Mosinger, E. and Metraux, J.P. 1998. Heat-induced resistance to powdery mildew (Blumeria graminis f.sp. hordei) is associated with a burst of active oxygen species. Physiol. Mol. Plant Pathol. 52: 185-199.
Walker, M.A., and McKersie, B.D. 1993. Role of ascorbate-glutathione antioxidant system in chilling resistance of tomato. J. Plant Physiol. 141: 234-239.
Wanner, L.A., and Junttila, O. 1999. Cold-Induced Freezing Tolerance in Arabidopsis. Plant Physiol. 120: 391-399.
Welling, A., Moritz, T., Palva, E.T., and Junttila, O. 2002. Low temperature and short day photoperiod induce distinct cold acclimation pathways in hybrid aspen. Plant Physiol. 129: 1633-1641.
Wingsle, G., and Hallgren, J.E. 1993. Influence of SO2 and NO2 exposure on glutathione, superoxide dismutase and glutathione reductase activities in Scots pine needles. J. Exp. Bot. 44: 463–470.
Yang, T., and Poovaiah, B.W. 2002. A Calmodulin-binding/CGCG Box DNA-binding Protein Family Involved in Multiple Signaling Pathways in Plants. J. Biol. Chem. 277: 45049-45058.
Yoda, H., Yamaguchi, Y., and Sano, H. 2003. Induction of hypersensitive cell death by hydrogen peroxide produced through polyamine degradation in tobacco plants. Plant Physiol. 132: 1973-1981.
Yu, C.W., Murphy, T.M., Sung, W.W., and Lin, C.H. 2002. H2O2 treatment induces glutathione accumulation and chilling tolerance in mung bean. Funct. Plant Biol. 29: 1081-1087.
Yu, C.W., Murphy, T.M., and Lin, C.H. 2003. Hydrogen peroxide-induced chilling tolerance in mung beans mediated through ABA-independent glutathione accumulation. Funct. Plant Biol. 30: 955-963.
Zhang, J., Cui, S., Li, J., Wei, J., and Kirkham, M.B. 1995. Protoplasmic factors, antioxidants responses, and chilling resistance in maize. Plant Physiol. Biochem. 33: 567-575.
Zhang, X., Zhang, L., Dong, F., Gao, J., Galbraith, D.W., Song, C.P. 2001. Hydrogen peroxide is involved in abscisic acid-induced stomatal closure in Vicia faba. Plant Physiol. 126: 1438-1448.