α-AMYLASE3在阿拉伯芥葉澱粉代謝中功能之研究

Abstract

葉澱粉在白天合成於葉綠體中，而在晚上降解以供應植物體所需的糖分。在阿拉伯芥中，AMY3是唯一位於葉綠體內的α-澱粉水解酶。AMY3的胺端有葉綠體導引訊息及與GWD1 (GLUCAN WATER DIKINASE)胺端相似的區段，而在羧端有α-澱粉水解酶活性區段。在一多澱粉突變株sex4 (starch excess 4)中，AMY3的蛋白質含量及酵素活性相較於野生型植株有減少的現象，然而在RNA含量上卻無差異，顯示SEX4透過後轉錄修飾調控AMY3蛋白質穩定度。為了解AMY3在葉澱粉代謝中所扮演的角色及其與SEX4之間的關係，分別構築具有葉綠體導引訊息和AMY3胺端或羧端區段與螢光蛋白(EYFP)序列結合的載體，使其於植物體中表現，並進一步分析轉殖株的蛋白質、澱粉及醣類含量。實驗結果顯示，AMY3N-EYFP與AMY3C-EYFP皆可透由葉綠體導引訊息進入葉綠體，但只有AMY3C-EYFP具水解澱粉的活性。內生性AMY3之蛋白質含量在P35S:AMY3C-EYFP/Col和P35S:AMY3C-EYFP/sex4轉殖株中較之在野生型植株中略微減少，而其在P35S:AMY3N-EYFP/Col和P35S:AMY3N-EYFP/sex4轉殖株中卻有上升的現象。此外在P35S:AMY3C-EYFP/- P35S:AMY3N-EYFP/-和 P35S:AMY3C-EYFP/- P35S:AMY3N-EYFP/- sex4/sex4轉殖株中，內生性AMY3及AMY3N-EYFP蛋白質含量有不同的變化。這些結果顯示AMY3的胺端會受到SEX4影響，而其羧端可能會參與在AMY3蛋白質的降解調控機制中。另一方面，在P35S:AMY3C-EYFP/Col和P35S:AMY3C-EYFP/sex4轉殖株中，有澱粉含量下降和葡萄糖含量上升的現象，然而在P35S:AMY3N-EYFP/Col和P35S:AMY3N-EYFP/sex4轉殖株中卻無此現象，顯示AMY3的羧端在植物體中確實會參與葉澱粉的降解，但其水解澱粉的活性會受本身蛋白質的胺端影響。綜上而言，本研究指出AMY3的胺端可能為SEX4穩定AMY3蛋白質的作用區段且參與其自身蛋白質水解澱粉活性的調控。

The Arabidopsis AMY3 gene encodes a plastidial α-amylase with protein features including the chloroplast targeting peptide (TP), GWD1 N-terminal like domain (CBD) and α-amylase (AAD) domains expanding from its N- to C-terminus. Previous studies show that the expression of AMY3 is posttranscriptionally attenuated in a starch-excess mutant, sex4 (starch excess 4). To investigate the role of AMY3 in leaf starch metabolism and to clarify if AMY3 is regulated by its protein features through a SEX4 dependent mechanism, I generated constructs of P35S:AMY3N-EYFP and P35S:AMY3C-EYFP which would express recombinant proteins TP-CBD-EYFP (AMY3N-EYFP) and TP-AAD-EYFP (AMY3C-EYFP) in plant cells, respectively. Interestingly, the chlorotic and reticulate leaves, dwarf, and late-flowering phenotypes were found only in P35S:AMY3C-EYFP/Col and P35S:AMY3C-EYFP/sex4 plants. The amount of endogenous AMY3 protein in P35S:AMY3C-EYFP/Col and P35S:AMY3C-EYFP/sex4 plants was slightly lower than that of wild-type plants, whereas that of P35S:AMY3N-EYFP/Col and P35S:AMY3N-EYFP/sex4 plants was higher. Moreover, there were different changes in protein contents of AMY3 and AMY3N-EYFP in P35S:AMY3C-EYFP/- P35S:AMY3N-EYFP/- and P35S:AMY3C-EYFP/- P35S:AMY3N-EYFP/- sex4/sex4 plants. These results suggest that the N-terminus of AMY3 is the domain subject to the action of SEX4 and the C-terminus of AMY3 may play a role in AMY3 protein turnover. Reduced starch contents and increased glucose contents in the P35S:AMY3C-EYFP/Col and P35S:AMY3C-EYFP/sex4 lines compared to those of wild-type plants indicate that the C-terminal AAD of AMY3 involves in leaf starch degradation and its activity is confined by the N-terminus of AMY3. I hypothesize that the N-terminal domain of AMY3 may regulate AMY3 function by confining the amylolytic activity of the C-terminal catalytic domain and is the targeted domain for SEX4 regulation.