阿拉伯芥中MTM-like基因功能探討以及其對於錳超氧歧化酶活化機制的研究

Functional Study of Yeast MTM-like Genes in MnSOD Activation of Arabidopsis

林書帆(Shu-Fan Lin)

指導教授 ： 靳宗洛

國立臺灣大學/生命科學院/植物科學研究所/碩士(2012年)

https://doi.org/10.6342/NTU.2012.01158

摘要

錳超氧歧化酶為對抗粒線體中氧化逆境的第一道防線，但對於其活化機制卻是所知甚少。目前已知在酵母菌中，存在著一個名為MTM1(manganese trafficking factor for mitochondrial SOD2)的蛋白質，其為參與運輸物質進出粒腺體的載體家族(Mitochondrial Carrier Family,MCF)成員之一，並發現MTM1對於錳超氧歧化酶的活性，有顯著的影響。在本研究中證實，兩個同屬於阿拉伯芥粒腺體中運送物質載體家族的成員，AtMTM1 (At4g27940)及AtMTM2 (At2g46320)，與酵母菌yMTM1具有相似的功能。利用螢光蛋白標記的方法，我們證實了AtMTM1與AtMTM2均座落在粒線體中。此外，利用雙分子螢光互補系統，更加證實了阿拉伯芥的錳超氧歧化酶(AtMSD1)，與AtMTM1、AtMTM2彼此存在著交互作用的關係。然而，AtMTM1和AtMTM2在不同器官的表現量及其在氧化逆境下的誘導量，均有顯著性的差異。在轉殖株的部分，相較於野生型，當AtMTM2剔除後，其在氧化逆境下根部的生長有延長的現象；當AtMTM1基因靜默時，其在氧化逆境下根部的生長有受阻的狀況。明顯的是，在AtMTM1和AtMTM2雙突變轉植株中，AtMSD1的活性顯著下降，伴隨葉綠體中鐵超氧歧化酶(AtFSD1)活性的上升。由上述結果我們可以得知，阿拉伯芥AtMTM1及AtMTM2均可以影響AtMSD1的活性，但二者功能並不完全相同；除了相互影響其表現量之外，也會參Mn及Fe離子的平衡機制，而此機制的調控則是跨越了粒線體與葉綠體不同胞器間的離子調節。這些結果都顯示植物中MnSOD的活化及離子平衡作用相較於酵母菌來得複雜許多。

關鍵字

阿拉伯芥 ； 超氧歧化酶錳超氧歧化酶金屬輔酶氧化逆境 ； 離子恆定作用

並列摘要

Manganese-containing superoxide dismutase (MnSOD) constitutes the first line of mitochondria defense against ROS, but the mechanism of the MnSOD activation still remains unclear. The MTM1 protein (manganese trafficking factor for mitochondrial SOD2) has been identified in yeast, which is a member of the mitochondrial carrier family (MCF), affecting the yeast mitochondrial MnSOD (SOD2) activity in an uncertain pathway. Two yeast MTM1-like genes, AtMTM1 (At4g27940) and AtMTM2 (At2g46320), were identified by sequence similarity, and both genes also encoded mitochondrial substrate carrier proteins in Arabidopsis (Arabidopsis thaliana). Here, we used genetic and transgenic approaches to study the molecular mechanism underlying the relationship between Arabidopsis MTMs (AtMTMs) and MnSOD (AtMSD1). We confirmed that expressing both AtMTM genes in a yeast MTM1-knockout strain can recover ySOD2 activity, implying AtMTMs functional similarity as with the yeast MTM1. We also found that the protein products of AtMTM1 and AtMTM2 were localized in mitochondria and an interactive relationship existed among AtMTM1, AtMTM2 and AtMSD1. The expression patterns of AtMTM1 and AtMTM2 were significantly different in various organs, and also differ to the response under oxidative stress. Besides, the root length was inhibited in the Atmtm1-RNAi lines but increased in Atmtm2-knockout mutants under oxidative stress. Notably, we observed the MnSOD activity was decreased and associated with an increase of FeSOD activity in the Atmtm1 and Atmtm2 double mutant lines. In summary, both AtMTM1 and AtMTM2 could affect the activity of MnSOD, but the functions between these two MCF proteins were not all the same. In Arabidopsis, they could not only interact to the one another, but participated in a complex mechanism of ion homeostasis. Moreover, the regulation of ionic balance mechanisms may stretch across mitochondria and chloroplast. These results demonstrated that the activation of MnSOD and ion homeostasis in plants were much complicated then in yeast.

並列關鍵字

Arabidopsis ； superoxide dismutase(SOD) ； MnSOD ； metal cofactor ； oxidative stress ； ion homeostasis

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