異位表現AtAMY3羧端誘導阿拉伯芥葉生理變化之研究

Abstract

植物白天行光合作用時，會將一部分的光合作用產物合成為暫存性澱粉儲存於葉綠體內，夜間則將澱粉降解供植物體代謝使用。其澱粉合成與降解相關的酵素需在日夜周期間受到適當的調控，以使澱粉得以在日間合成，並於夜間降解。在阿拉伯芥葉綠體中，AtAMY3為唯一的型澱粉水解酶，能夠水解澱粉的-1,4鍵結。過去的研究指出，AtAMY3的蛋白質結構含有不同的功能性區段。其胺端可能含有負調控AtAMY3蛋白質本身的功能，而其羧端則含有水解酵素的活性。先前的研究製造了過量表現AtAMY3胺端及羧端的轉殖株以研究其功能，其中過量表現AtAMY3羧端並帶有黃色螢光蛋白 (EYFP)的轉植株（AMY3C-EY）葉片的澱粉含量大量減少。此轉殖株也表現出許多異常的性狀，包括葉片黃化並帶有網紋、植株矮小、開花期延後等等。本論文希望能探討為何這些性狀會伴隨著AtAMY3羧端的過量表現而產生。 我提出了兩個可能產生這些性狀的假說。第一，這些性狀可能是由AMY3C-EY降解澱粉之後所產生的小分子醣類誘導植物體基因的表現所造成。第二，這些性狀的產生可能是過量表現AMY3羧端後，植物在白天行光合作用卻同時降解澱粉，導致能量過度浪費的結果。為了驗證這些假說，我利用遺傳的方法，在經過輻射照射AMY3C-EY轉殖株所衍生的子代中篩選能夠改變AMY3C-EY親本表現型的突變株，這些植株中的突變基因能夠用以分析假說是否成立。其中的一株突變株，47，植株葉片變綠並且無網紋，實驗結果發現此47中的突變造成喪失合成澱粉所需的AGPase酵素活性，使47無法累積澱粉，因而改變葉片性狀。由47的研究得知，AMY3C-EY葉片黃化並帶有網紋的性狀並非單純緣自於日間暫存性澱粉量的減少，而是澱粉合成後下游代謝改變才造成這些性狀，因此間接支持我所提出的假說。 另一方面，為了研究AMY3C-EY如何改變植物生理，我利用LexA-VP16-ER (XVE)轉錄因子建立了一個可誘導AMY3C-EY表現的系統。藉由塗抹雌二醇(17--estradiol)，可以將XVE轉錄因子限制在特定的植物部位啟動後誘導AMY3C-EY表現。為了研究AMY3C-EY對植物不同發育階段的影響，我將藥劑17--estradiol分別塗在XVE AMY3C-EY轉殖株的頂端分生組織與成熟葉片上。我發現雖然兩種處理都會使相應的植物部位澱粉含量減少，但只有處理藥劑在分生組織後新形成的葉子才會展現黃化及網紋的性狀，已成熟的葉片則不受影響。實驗結果傾向支持AMY3C-EY降解澱粉之後所產生的小分子醣類誘導植物性狀的改變，並且顯示不適當的AtAMY3活性調控只會影響葉綠體的早期發育。至於是否因能量浪費造成性狀改變的假說，我的實驗似乎不完全支持。 綜上所述，我的研究指出AtAMY3羧端的異位表現會影響到葉的早期發育，顯示出適當的AtAMY3水解澱粉活性調控對於阿拉伯芥葉片的重要性。

Forty to fifty percent of photosynthate is synthesized as transitory starch during daytime and then degraded during nighttime in chloroplasts. To achieve the diurnal transitory starch metabolism, enzymes for starch degradation should be properly regulated. In Arabidopsis chloroplast, AtAMY3 is an α-amylase which hydrolyzes α-1,4-glucosidic linkage of starch. It is known that AtAMY3 has an N-terminal domain which may negatively regulate AtAMY3 itself and a C-terminal domain which is responsible for amylolytic activity. Transformants overexpressing a fusion protein of AtAMY3 C-terminal domain and EYFP (AMY3C-EY) were generated, and the starch content in leaves of transformants was greatly reduced. In addition, AMY3C-EY showed distinct phenotypes such as chlorotic and reticulate leaves, dwarf, and late flowering. How altered starch metabolism in AMY3C-EY affects plant growth and produces these phenotypes remains unclear. It is possible that AMY3C-EY phenotypes are induced for two reasons. First, these phenotypes could be induced by accumulation of small glucans released from the AMY3C-EY amylolytic activity. Second, these phenotypes could be caused by energy overconsumption because of constitutive starch degradation during photosynthesis. To test these hypotheses, I isolated mutants from-irradiation mutagenized AMY3C-EY progenies. In these mutants, mutations which rescued AMY3C-EY phenotypes would provide information why AMY3C-EY phenotypes were produced. One of the revertants, 47, was shown to be an adg1 mutant without ADP-glucose pyrophosphorylase activity and starch synthesis. The study of 47 suggested that starch synthesis is needed for producing AMY3C-EY phenotypes, which indirectly supported both the hypotheses. To find out how AMY3C-EY alters plant physiology, I constructed an inducible expression system for AMY3C-EY with LexA-VP16-ER (XVE) chimeric transcription factor. Through controlling the activation of XVE by 17--estradiol, the AMY3C-EY expression could be induced and restricted at certain parts of a plant. 17--Estradiol was applied to shoot apical meristem or mature leaves of XVE AMY3C-EY double transformant. Only newly formed leaves with reduced starch content showed the chlorotic and reticulate phenotypes. This suggests that improper regulation of AtAMY3 affects chloroplast at early development. In summary, this study indicates that ectopic expression of AtAMY3 C-terminal domain affects early chloroplast development, revealing the importance of properly regulated AtAMY3 amylolytic activity for Arabidopsis leaves.