本研究利用SDS-PAGE膠體活性染色方法從甘藷完全展開的成熟葉片中辨識出一個分子量接近72 kDa的主要過氧化氫酶。其活性於pH8-12間較pH小於8時為高，且受β-mercaptoethanol及3-amino-1,2,4-triazole所抑制。然而其活性於5-450C間受溫度的影響較小。組織專一性表現顯示其活性主要於葉中被偵測到，且活性由未完全成熟的L2發育階段葉片開始增加，至完全展開成熟的L3發育階段葉片時具有最高的活性，然後於部份黃化的L4發育階段的老化葉片梢微減少，至完全黃化的L5發育階段的老化葉片幾乎偵測不到，類似於未展開未完全成熟的L1發育階段葉片一樣具有最低的活性。葉片中過氧化氫酶的活性與H2O2含量適成反比關聯性。黑暗及ethephon(釋放乙烯的化合物)處理6至24小時亦會誘導增加此過氧化氫酶的活性，於處理24至48小時後此過氧化氫酶的活性又漸漸減少；處理葉片中過氧化氫酶的活性亦與H2O2含量適成反比關聯性。葉片中過氧化氫酶的活性受EGTA抑制，且外加CaCl2能回復此抑制作用。Chlorpromazine 抑制calmodulin活性,亦會抑制葉片中過氧化氫酶的活性及增加H2O2量，且外加純化的SPCAM Calmodulin 融合蛋白質能回復此抑制作用。這些結果顯示葉片中過氧化氫酶的活性受 external calcium ion (Ca2+)及 calmodulin調控。依據這些實驗數據我們結論甘藷葉片具有一個主要的過氧化氫酶，其活性於成熟葉片時為最高，於自然及誘導的老化葉片則顯著下降，此過氧化氫酶的活性受external calcium ion (Ca2+)及 calmodulin 調控，且與甘藷葉片於生長發育過程或環境逆境下清除活化氧族H2O2可能有關。

A major sweet potato leaf-type catalase was detected and identified from fullyexpandedmature leaves using in-gel activity staining assay with native- andSDS-PAGEs. The putative catalase activity band was inhibited by a catalaseinhibitor 3-amino-1,2,4-triazole. The major leaf-type catalase activity wasoptimal over 8, and was significantly repressed by β-mercaptoethanol. However,its activity was much less affected by temperature within the range of 5 to 450C.Temporal and spatial expression showed that it was specifically detected inleaves, but not in roots and stems. Its activity increased from the immature L2leaves, and reached the maximal at the fully-expanded mature L3 leaves, thenslightly decreased in partial yellowing senescent L4 leaves, and was almost notdetected in completely yellowing L5 leaves similar to folding unopenedimmature L1 leaves. The catalase level showed approximately inversecorrelation with the H2O2 amounts in leaves of different developmental stages.Dark and ethephon, an ethylene-releasing compound, also temporarily enhancedthe catalase activities from 6 h to 24 h, however, the enhanced activitydecreased from 24 h to 48 h in detached leaves after treatment. The catalaselevel also showed approximately negative correlation with the H2O2 amounts intreated leaves. The major leaf-type catalase activity was repressed by EGTA,and the repression can be reversed by exogenous CaCl2. The major leaf-typecatalase activity was also repressed by calmodulin inhibitor chlorpromazine,and the repression can be reversed by exogenous purified SPCAM calmodulinfusion protein. Chlorpromazine-treated leaves also elevated H2O2 amount.Based on these data we conclude that a major leaf-type catalase with maximalactivity in L3 leaf was identified in sweet potato. Its activity was temporarilyenhanced by dark and ethephon, and was modulated by external calcium ion(Ca2+) and calmodulin. A possible physiological role and function in associationwith cellular H2O2 homeostasis in cope with developmental and environmentalcues in sweet potato leaves is suggested.

Written Examination Paper…………………………………………………………………….i
Acknowledgement……………………………………………………………………….……ii
Chinese Abstract……………………………………………………………………………...iii
English Abstract………………………………………………………………………………iv
Chapter 1. Introduction……………………………………………………………………..…1
1.1 Sweet potato……………………………………………………………………………1
1.2 Leaf senescence…………………………………………………………………….......1
1.3 Ethylene…………………………………………………………………………….......2
1.4 Catalase……………………………………………………………………………........4
1.5 Calcium and calmodulin function in plant……………………………………………..6
Chapter 2. Materials and Methods...………………………………………………………..…8
2.1 Plant materials…………………………………………………………………….…....8
2.2 Detection and identification of sweet potato catalases from leaves…………….……...8
2.3 Effects of β-mercaptoethanol, pH and temperature on in vitro catalase
activities……………………………………………………………………………….........9
2.4 Temporal and spatial expression of catalase..……………………………………….....9
2.5 Ethephon and dark treatment………………………………………….…………........10
2.6 EGTA treatment...………………………….…………………………………..……..10
2.7 Treatment of calmodulin inhibitor (Chlorpromazine) and calmodulin fusion
protein…………………………………………………………………………….……….11
2.8 Construction, overexpression and purification of SPCAM fusion protein from E.
coli……………………………….………………………………………………………..11
2.9 Leaf morphology………………………………………….…………………………..13
2.10 Measurement of chlorophyll content………………………………….………….....13
2.11 Measurement of photochemical Fv/Fm…………………………………….………..13
2.12 Measurement of H2O2 amount………………………………….……………………13
2.13 Analysis of catalase activity……………………………………………….………...14
2.14 Statistic Analysis……………………………………………………15
Chapter 3. RESULTS………………………………………………………16
3.1 Identification and characterization of sweet potato catalase in leaves………………..16
3.2 Temporal and spatial expression of sweet potato catalase………………………..…..16
3.3 The major leaf-type catalase activity is temporarily enhanced by dark………………17
3.4 The major leaf-type catalase activity is temporarily enhanced by ethephon…………18
3.5 The major leaf-type catalase activity requires external Ca2+ ion……………………..19
3.6 The leaf-type catalase activity is modulated by calmodulin.……………………...…..19
Chapter 4. DISCUSSION..…………………………………………………………………21
Chapter 5. CONCLUSION………………………………………………………..…..……..26
LETERATURE CITED..…………………………………………………………………….27

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