臺灣博碩士論文加值系統

背景
大多數的C4植物不耐寒，C4植物的生長在低溫下受限制。台灣有五種本土芒草，其中以生長在阿里山1500 m海拔的五節芒 (M. floridulus) 與台灣芒 (M. sinensis var. formasanus)，及塔塔加的高山芒 (M. sinensis var. transmorrisonesis) 最能適應低溫環境。本試驗之研究目的在於探討這些本土芒草在自然生態環境下溫度對其C4光合作用的影響，期望對C4光合作用的耐寒機制有所了解。

結果
結果顯示，不耐寒的C4植物玉米及狼尾草，其光合作用率在冬天大幅降低 (分別為31%及13%)，但最適光合作用溫度仍維持在30℃，而無馴化現象。在自然的生態環境下，冬天生長於原棲息地的五節芒、台灣芒和高山芒之最適光合作用溫度在冬天分別下移5℃、5℃及10℃，顯示有馴化的能力。高山芒的C4光合酵素的phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) 及pyruvate, Pi dikinase (PPDK, EC 2.7.9.1) 活性在冬天比夏天分別提高約3倍及4倍，而台灣芒則分別提高0.5及1.5倍；相反地，玉米及狼尾草等不耐寒的C4植物其PEPC和PPDK酵素活性在冬天大幅降低。耐寒的芒草具有低溫穩定特性之PPDK，而玉米及狼尾草等其他不耐寒之C4植物具有低溫不穩定之PPDK，而且在低溫下基因表達受抑制。除此，RT-PCR的分析發現台灣芒與高山芒遇冷時能大幅提高其PPDK之表達量，而不耐寒之玉米則無此現象。

結論
光合作用能力的維持是植物是否能在低溫底下生存的重要因素之一，C4光合作用最重要的兩個光合酵素為PEPC 和PPDK，這兩個酵素的活性與C4植物的光合作用能力有密切的關係，本研究的結果顯示，台灣芒與高山芒能夠在冬天維持很高的PEPC和PPDK酵素活性，或許與它們的耐寒能力有關，台灣芒與高山芒在低溫生長條件下有較高的PPDK活性可能是因為低溫增進抗寒PPDK的表達量有關。因此維持PEPC和PPDK在低溫下的穩定性與高活性可能是芒屬植物在低溫下維持其C4光合作用與生長的一個重要機制。

Background

Most C4 plants are adapted to warm climate, which limit their growth in the winter and distribution in cooler regions. Several Miscanthus species native to Taiwan, such as M. floridulus (Mf), M. sinensis var. formosanus (Msf) and M. sinensis var. transmorrisonesis (Mst), are known to grow at high altitudes (1500, 1500 and 2600 m, respectively). The main objective of this study was to elucidate the mechanism by which C4 photosynthesis of these Miscanthus species tolerates low temperatures.

Results

Both maize and napiergrass, two warm adapted C4 species, showed a photosynthetic optimum temperature at 30℃, regardless of growth temperature but maize exhibited a greater inhibition of photosynthesis in the winter than napiergrass. In contrast, with the ability to acclimate Mf, Msf and Mst lowered their optimum temperatures for photosynthesis by 5-10℃ in the winter. The activities of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) and pyruvate, Pi dikinase (PPDK, EC 2.7.9.1) were much lower in the winter-grown plants of maize and napiergrass, compared to summer-grown plants. On the other hand, the activities of PEPC and PPDK were 3 and 4 fold higher in Mst and 0.5 and 1.5 fold higher in Msf in the winter-grown plants, respectively. Also, PPDK proteins from all five Miscanthus species native to Taiwan exhibited cold resistance high stability while the proteins of the cold sensitive C4 species showed a severe lability at 0℃. Furthermore, RT-PCR analysis showed that when subjected to low temperatures (16℃day/12℃night), the PPDK transcript of Mst increased by 50% after a brief decline. By contrast, it was severely reduced in maize there was a severe reduction after one day of exposure.

Conclusion

PEPC and PPDK are two key photosynthetic enzymes in the C4 pathway of photosynthesis. Apparently, the ability of both Msf and Mst to maintain high activities of PEPC and PPDK at low temperatures is important for these plants to perform active photosynthesis. The higher activities of PPDK could be due to the capability of these species to express high levels of a cold resistant PPDK at low temperatures.

Table of contents

Abstract
List of figures
List of table
List of abbreviations

I. Introduction………………………………………………………………................1

II. Literature review………………………………………………………………..........3
1. Photosynthetic mechanisms of plants......................................................................3
2. Temperature dependence of C4 photosynthesis…………………………..........9
3. Distribution of Miscanthus species and the native Miscanthus species of Taiwan……………………………………………………………………........19

III. Materials and Methods…………………………………………………………21
1. Plant material……………………………………………………………..21
2. Gas exchange measurements…………………………………………….. 22
3. Enzyme extraction and assays of PEPC and PPDK………………………23
4. Assay of PPDK cold lability……………………………………………....24
5. Protein extraction and western blot analysis………………………….......25
6. RNA extraction and RT-PCR.......................................................................26
7. Isolation of genomic DNA...........................................................................27
8. Southern blot hybridization..........................................................................28

IV. Results

Part I. Field experiments

1. Photosynthetic response to temperature.......................................................29
1.1 Maize (Zea mays)................................................................................29
1.2 Napiergrass (Pennisetum purpureum).................................................31
1.3 Miscanthus floridulus (Mf)..................................................................33
1.4 Miscanthus sinensis var. formasanus (Msf).........................................35
1.5 Miscanthus sinensis var. transmorrisonesis (Mst)...................................37
2. Activity of key C4 photosynthetic enzymes.................................................40
3. Western immunoblot analysis......................................................................44
4. Cold lability of PPDK at 0℃.............................................................................46
5. Copy of PPDK gene in maize and Miscanthus genomes..........................51

Part II. Transfer experiments in controlled environments

1. Photosynthetic enzyme activity................................................................ 53
2. Western immunoblot analysis...................................................................57
3. Expression of PEPC and PPDK genes during cold treatment,
as determined by RT-PCR.........................................................................61

V. Discussion..........................................................................................................65

VI. Conclusion.........................................................................................................73

VII. References..........................................................................................................74

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