阿拉伯芥粒線體錳運輸蛋白AtMTM1和AtMTM2對錳超氧化物歧化酶活化之重要性

Importance of Mitochondrial MnSOD Activation Mediated by Mn Transporters AtMTM1 and AtMTM2

胡樹萱(Shu-Hsuan Hu)

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/博士(2021年)

https://doi.org/10.6342/NTU202200333

摘要

酵母菌粒線體錳運輸蛋白yMTM1 (manganese tracking factor for mitochondrial Mn superoxide dismutase) 為活化錳超氧化物歧化酶 (Mn superoxide dismutase; MnSOD) 所需的粒線體內膜運輸蛋白，屬於粒線體運輸蛋白族群。此研究確認阿拉伯芥AtMTM1與AtMTM2與酵母菌yMTM1為功能相似的同源蛋白，兩者皆可恢復酵母菌MTM1突變株的MnSOD活性。藉由表達AtMnSOD-3xFLAG於AtMTM1與AtMTM2雙基因突變原生質體系統，證實AtMnSOD活化需要AtMTM1與AtMTM2。本研究亦顯示AtMnSOD、AtMTM1及AtMTM2在粒腺體有交互作用。AtMTM1、AtMTM2及AtMnSOD 之表現可被methyl viologen (MV)與處理金屬離子所誘導。此外，AtMTM1與AtMTM2涉及Mn與Fe平衡、根長及開花時間。MnSOD-overexpression 和MnSOD-knockdown植株以氧化逆境誘導劑MV、NaCl、H2O2及tert-butyl hydroperoxide (t-BH) 處理後, 所得結果顯示阿拉伯芥MnSOD對植物根部生長之控制與清除自由基之能力極為重要。先前研究已報導Fe抑制大腸桿菌E. coli MnSOD活性，酵母菌yMTM1突變體Fe含量會增加而MnSOD活性會降低，這些性狀皆可經由Fe2+螯合劑bathophenanthroline disulphonate (BPS) 處理後恢復。然而，本研究顯示AtMTM1與AtMTM2 單突變以及雙突變株原生質體之 MnSOD活性卻被BPS抑制，意指Fe平衡的改變經由AtMTM1與AtMTM2影響AtMnSOD的活性。感應耦合電漿光學發射光譜儀 (ICP-OES) 分析顯示AtMTM1突變株根部與AtMTM2突變株莖部之Fe/Mn比值異常，指出AtMTM1在根部與AtMTM2在莖部對於維持Fe/Mn平衡之重要性。總結來說，AtMTM1與AtMTM2運輸蛋白皆會參與粒線體AtMSOD活化，而且兩運輸蛋白之基因表達隨著植物組織與氧化逆境而異，意指運輸蛋白AtMTM1與AtMTM2有分工合作之現象。目前研究亦顯示MSOD活性受Mn與Fe離子平衡影響。另外一方面，AtMTM1與AtMTM2與植物組織內Fe/Mn平衡有關，至於這兩個運輸蛋白與鐵離子之間的交互作用仍待進一步研究。

關鍵字

含金屬酶 ； 粒線體運輸蛋白族群 ； 錳超氧化物歧化酶 ； 錳運輸蛋白 ； 活性氧 ； 超氧化物歧化酶 ； 鐵/錳比值

並列摘要

The manganese (Mn) tracking factor for mitochondrial Mn superoxide dismutase (MnSOD) has been annotated as yMTM1 in yeast, which belongs to the mitochondrial carrier family. In previous study, we confirmed that Arabidopsis AtMTM1 and AtMTM2 are functional homologs of yMTM1, as they can restore yeast MnSOD activity in yMTM1-mutant cells. Transient expression of AtMnSOD-3xFLAG in the AtMTM1 and AtMTM2-double mutant protoplasts confirmed that AtMTM1 and AtMTM2 are required for AtMnSOD activation. Our study revealed that AtMnSOD interacts with AtMTM1 and AtMTM2 in the mitochondria. The expression levels of AtMTM1, AtMTM2, and AtMnSOD respond positively to methyl viologen (MV) and metal ion treatment. AtMTM1 and AtMTM2 are involved in Mn and Fe homeostasis, root length, and flowering time. AtMnSOD-overexpressing and AtMnSOD-knockdown mutant plants treated with the oxidative stressors including MV, NaCl, H2O2, and tert-butyl hydroperoxide (t-BH) showed that Arabidopsis MnSOD activity was crucial for root-growth control and superoxide scavenging ability. In addition, it has been reported that E. coli MnSOD activity is inhibited by Fe and that MTM1-mutated yeast cells exhibit elevated Fe content and decreased MnSOD activity, which can be restored by the Fe2+-specific chelator, bathophenanthroline disulfonate (BPS). However, we showed that MnSOD activity was inhibited by BPS in AtMTM1 and AtMTM2 single- and double-mutant protoplasts, implying that altered Fe homeostasis affected MnSOD activation through AtMTM1 and AtMTM2. Importantly, the analysis of inductively coupled plasma-optical emission spectrometry (ICP-OES) revealed an abnormal Fe/Mn ratio in the roots of AtMTM1 mutant and shoot of AtMTM2 mutant under MV stress, indicating the importance of AtMTM1 in roots and AtMTM2 in shoots for maintaining Fe/Mn balance. In conclusion, both AtMTM1 and AMTM2 involve in AtMnSOD activation, and their expressions depend on plant tissue and oxidative stresses, indicating the synergistic effect of these transporter. This research also revealed MnSOD activity are affected by Mn and Fe homeostasis. In addition, AtMTM1 and AtMTM2 transporters maintain Fe/Mn balance in Arabidopsis, and it’s worthy to study the relationship of Fe and these two transporters.

並列關鍵字

metalloenzyme ； mitochondrial carrier family ； MnSOD ； Mn transporter ； reactive oxygen species ； superoxide dismutase ； Fe/Mn ratio

參考文獻

1. Apel, K.; Hirt, H. Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu. Rev. Plant Biol. 2004, 55, 373-399.

2. Fridovich, I. Superoxide dismutases. Ann. Rev. Biochem. 1975, 44, 147-159.

3. Halliwell, B. Free radicals, antioxidants, and human disease: curiosity, cause, or consequence? Lancet 1994, 344, 721-724.

4. Bowler, C.; Camp, W.V.; Montagu, M.V.; Inzé, D. Superoxide dismutase in plants. Crit. Rev. Plant Sci. 1994, 13, 199-218.

5. Hassan, H.M.; Sun, H.C. Regulatory roles of Fnr, Fur, and Arc in expression of manganese-containing superoxide dismutase in Escherichia coli. Proc. Natl. Acad. Sci. USA 1992, 89, 3217-3221.