- 學位論文
阿拉伯芥葉綠素去植醇酶群之功能分析
Functional analysis of chlorophyll dephytylases in Arabidopsis thaliana
摘要
葉綠素為光合作用之基本元素，參與將光能量轉化為生物能的生化反應，以滋養地球上的大多數生命。葉綠素（chlorophyll, Chl）在結構上包含了親水性的葉綠酯（chlorophyllide）與疏水性的植醇（phytol），並可藉由去植醇反應（dephytylation）將這兩部分水解開來。葉綠素去植醇反應一直被認為是植物的基礎代謝步驟，參與在不同的葉綠素相關代謝途徑，包括葉綠素之轉換、降解、構型循環及生育酚（tocopherol）生合成等，但其中執行去植醇反應的酶大多未能被確認。最近，一個新穎的葉綠素去植醇酶（CLD1）被發現可能參與葉綠素轉換時的回收再利用。蛋白序列親源分析顯示，CLD1和參與葉片老化時葉綠素降解的脫鎂葉綠素去植醇酶（pheophytinase, PPH）以及另外兩個功能未知的蛋白具有同源性。因為後兩者的重組蛋白均有葉綠素去植醇酶活性，故被命名為CLD2和CLD3。為了解這些去植醇酶的生理作用，我們建立了阿拉伯芥cld1 cld2 cld3（TK）和cld1 cld2 cld3 pph（QK）基因剔除突變株。正常與逆境（強光、或光度變動、或高溫、或無氮源）條件下，相較於野生型，TK的生長不受影響。植物葉綠素主要為Chl a與Chl b兩種構型，Chl b 被認為主要來自Chl a 構型轉化，若此假設成立，中間需經過去植醇反應。比較白化苗照光4小時內的葉綠素累積，Chl b含量在TK中一開始略少但最終接近於野生型，此結果無法支持先前之假設。在黑暗誘導葉片老化過程，TK與野生型之葉綠素降解程度並無顯著差別，表明CLDs並不參與此途徑。此外，TK的生育酚含量顯著低於野生型，進一步分析不同組合的雙突變體，發現主要是因CLD2缺失造成，表明此酵素參與了生育酚的生合成。
並列摘要
Chlorophyll is the basic element of photosynthesis. It participates in the biochemical reaction that converts light energy into biological energy to nourish most life on earth. Chlorophyll (Chl) contains hydrophilic chlorophyllide (chlorophyllide) and hydrophobic phytol (phytol) in structure, and the two parts can be hydrolyzed by dephytylation. Chlorophyll removal of phytol has been considered as a basic metabolic step of plants, participating in different chlorophyll-related metabolic pathways, including chlorophyll turnover, degradation, interconversion cycle and tocopherol biosynthesis. However, the enzymes are responsible for dephytylation mostly remained known. Recently, a novel chlorophyll dephytolase (CLD1) was discovered possibly involved in the chlorophyll turnover. The protein sequence analysis showed that CLD1 has homology with pheophytinase (PPH) involved in chlorophyll degradation during leaf senescence and two other proteins of unknown functions. Due to the latter two recombinant proteins have chlorophyll dephytase activity, they are further named as CLD2 and CLD3. In order to understand the physiological role of these phytolase enzymes, we established Arabidopsis cld1 cld2 cld3 (TK) and cld1 cld2 cld3 pph (QK) gene knockout mutants. Under normal and stress conditions (high light, or changes in luminosity, or high temperature, or nitrogen deficiency), the growth of TK is similar to that of wild-type. Plant chlorophyll mainly has two moieties of Chl a and Chl b. Chl b is considered to be mainly derived from the conversion of Chl a. If this hypothesis is established, the process needs to undergo a phytol removal reaction. Comparing the chlorophyll accumulation of etiolated seedlings within 4 hours of light exposure, the Chl b content in TK was slightly less at first but close to the wild-type in the end and cannot support the previous hypothesis. During the dark-induced leaf senescence process, there is no significant difference in the degree of chlorophyll degradation between TK and wild-type, indicating that CLDs do not participate in this pathway. In addition, the tocopherol content of TK was significantly lower than that of the wild-type. Further analysis of the double mutants of different combinations revealed that it was mainly caused by the lack of CLD2, indicating that this enzyme is involved in the biosynthesis of tocopherols.