乙烯長期被認為是主要植物生長調節物質且對於葉片老化過程中扮演重要角色。Ethephon是一個釋出乙烯的化合物，於甘藷切離葉片會加速葉片老化及H2O2含量上升，這些影響於外加還原態榖胱甘肽和抗壞血酸下可以有效的被減緩。Ethephon處理 3天時會增加甘藷葉片內生性總量及還原態榖胱甘肽和抗壞血酸的含量，然而其H2O2含量亦顯著上升。另外外加還原態榖胱甘肽於ethephon處理切離的甘藷葉片24小時，其內生性總量及還原態榖胱甘肽的含量比單獨ethephon處理有極顯著的大量增加，並且於72小時其H2O2含量亦比單獨ethephon處理顯著下降。Ethephon處理切離的甘藷葉片約4小時會快速增加一個小的 H2O2 的頂峰(peak)，且此H2O2 peak會受外加還原態榖胱甘肽前處理而被移除。切離的甘藷葉片前處理NADPH oxidase 抑制劑會減緩ethephon誘導 3天的葉片老化及H2O2含量，且此減緩作用於乙烯處理後前 4小時內是有效的。Ethephon 及一個榖胱甘肽合成酶抑制劑 BSO 可以誘導老化相關基因asparaginyl endopeptidase (SPAE) 及cysteine proteases (SPCP1, SPCP2 及 SPCP3) 的表現，且此基因活化作用受外加還原態榖胱甘肽的抑制。根據這些結果我們結論ethephon處理可能會經由NADPH oxidase快速造成一個小的 H2O2 peak，此H2O2 peak的功能與ethephon 訊息傳遞誘導甘藷葉片老化、H2O2含量上升、及老化相關基因表現可能有關，抗氧化劑例如還原態榖胱甘肽的增加速率是重要的，且會影響甘藷葉片老化、H2O2含量及老化相關基因表現。

Ethylene has long been considered as the main plant growth regulator that plays a key role in the regulation of leaf senescence. In sweet potato, ethephon, an ethylene releasing compound, promoted leaf senescence and H2O2 elevation. These ethephon-mediated effects were alleviated or attenuated by exogenous reduced glutathione and ascorbic acid. Ethephon treatment gradually increased endogenous total and reduced glutathione and ascorbic acid levels in sweet potato detached leaves 3 days after treatment. The H2O2 amount, however, was also increased at 72 h after treatment. Sweet potato detached leaves pretreated with reduced glutathione did significantly increased endogenous total and reduced glutathione levels at 24 h and remarkably decreased H2O2 amount at 72 h after ethephon application compared to that of ethephon alone control. Ethephon caused quick elevation of a small H2O2 peak at about 4 h after application, and the enhancement was eliminated by reduced glutathione pretreatment in treated sweet potato leaves. Pretreatment of diphenylene iodonium (DPI), an NADPH oxidase inhibitor, also repressed leaf senescence and H2O2 elevation at day 3 after ethephon treatment in sweet potato detached leaves, and the attenuation was effective within the first 4 h after ethephon treatment. For senescence-associated gene expression, ethephon and L-buthionine sulfoximine (BSO), an endogenous glutathione synthase inhibitor, did induced asparaginyl endopeptidase (SPAE) and cysteine proteases (SPCP1, SPCP2 and SPCP3) gene expression and the activation was repressed by reduced glutathione pretreatment. Based on these data we conclude that ethephon treatment may cause quick elevation of a small H2O2 peak likely via the NADPH oxidase, which may function as a signal component leading to leaf senescence, H2O2 elevation and senescence-associated gene expression in sweet potato detached leaves. The rate of endogenous antioxidant such as reduced glutathione elevation is also important and affects leaf senescence, H2O2 elevation and senescence-associated gene expression in sweet potato leaves.

目 錄
論文審定書................…………………………..................……………......i
誌謝………………………………………………………………...............ii
圖次………………………………....………………..................................vi
中文摘要………………………………………..............……….….........viii
英文摘要………………………………………..…………………............ix
壹、緒論......................................................................................................1-6
甘藷與葉片老化...............................................................................1-2
乙烯...................................................................................................2-3
抗氧化物...........................................................................................3-4
NADPH oxidase ...............................................................................4-5

貳、材料與方法......................................................................................7-18
I. 實驗材料
A. 甘藷..........................................................................................7
B. 葉片老化相關基因..................................................................7
C. 化學藥品及藥品配製...........................................................7-8
II. 實驗方法
乙烯 (Ethephon) 處理..................................................................9
乙烯及還原態穀胱甘肽(Reduced glutathione, GSH)處理.....9-10
乙烯及抗壞血酸(Ascorbic acid, ASA)處理..........................10-11
乙烯及(Diphenylene iodonium, DPI)處理..................................11
乙烯及(Buthionine sulfoximine, BSO)處理..........................11-12
乙烯，BSO及還原態穀胱甘肽處理......................................12-13
葉綠素含量測量..........................................................................13
光化學反應Fv/Fm測量..............................................................13
H2O2測定................................................................................13-14
穀胱甘肽含量測定......................................................................14
抗壞血酸含量測定......................................................................15
RT-PCR分析..........................................................................15-18
一、Total RNA extraction ..................................................15-17
二、Reverse transcription........................................................17
三、PCR primer序列.........................................................17-18
四、PCR條件..........................................................................18
統計方法......................................................................................18

參、結果..................................................................................................19-26
A. 乙烯誘導甘藷葉片老化及H2O2的增加與抗氧化物質(glutathione和ascorbate)之關聯性探討...............................19-24
B. 乙烯誘導甘藷葉片老化及H2O2含量的增加與NADPH oxidase之關聯性探討........................................................................24-25
C. 乙烯誘導甘藷葉片H2O2含量增加與老化相關基因表現之關聯性探討..........................................................................................26

肆、討論..................................................................................................27-30
A. Ethephon誘導的H2O2含量增加與NADPH oxidase有關...........27
B. 乙烯誘導的葉片老化及大量H2O2含量增加與初期H2O2增加有關係........................................................................................27-28
C. 乙烯誘導的H2O2含量增加可以作為signal誘導老化相關基因表現............................................................................................28-29
D. Ethephon誘導的葉片老化及H2O2含量與內生的glutathione增加速率有關...................................................................................29-30

伍、參考文獻..........................................................................................31-35
圖次
圖一、乙烯處理對甘藷葉片的外觀形態、葉綠素含量及光化學反應Fv/Fm之影響。.............................................................................36
圖二、乙烯處理對甘藷葉片的過氧化氫含量之影響。..........................37
圖三、還原態的榖胱甘肽對乙烯處理的甘藷葉片外觀形態、葉綠素含量及光化學反應Fv/Fm之影響。................................................38
圖四、還原態榖胱甘肽對乙烯處理的甘藷葉片過氧化氫含量之影響。................................................................................................39
圖五、抗壞血酸對乙烯處理的甘藷葉片外觀形態、葉綠素含量及光化學反應Fv/Fm之影響。...............................................................40
圖六、抗壞血酸對乙烯處理的甘藷葉片過氧化氫含量之影響。.........41
圖七、乙烯處理對甘藷葉片的外觀形態、葉綠素含量、光化學反應
Fv/Fm及過氧化氫含量之影響。.................................................42
圖八、乙烯處理對甘藷葉片內生性榖胱甘肽含量的影響。處理時間分別為一天、兩天和三天。............................................................43
圖九、乙烯處理對甘藷葉片內生性的抗壞血酸含量的影響。.............44
圖十、外加還原態榖胱甘肽對乙烯處理的甘薯葉片過氧化氫含量之影響。................................................................................................45
圖十一、外加還原態榖胱甘肽對乙烯處理的甘藷葉片榖胱甘肽含量的影響。..........................................................................................46
圖十二、NADPH氧化酶抑制劑(DPI)對乙烯處理甘藷葉片的外觀形態
、葉綠素含量及光化學反應Fv/Fm之影響。..........................47
圖十三、NADPH氧化酶抑制劑(DPI)及乙烯處理甘藷葉片的過氧化氫含量之影響。..............................................................................48
圖十四、NADPH氧化酶抑制劑(DPI)處理時間對乙烯處理甘藷葉片的外觀形態、葉綠素含量及光化學反應Fv/Fm之影響。.........49
圖十五、NADPH氧化酶抑制劑(DPI)處理時間對乙烯處理甘藷葉片的過氧化氫含量之影響。..............................................................50
圖十六、還原態榖胱甘肽對乙烯或BSO誘導老化相關基因表現之影響。..............................................................................................51

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