菸草毛狀根的生長與其尼古丁高量累積之研究

ﱋﰾﱡﱢﱣﱤﱥﱦﱧﱨﱩﱪﱫﱬﱭﱮﰥﰦﰧﰩﱯﱰﱱﱲﱌﰥﰦ 毛狀根是植物受到根毛農桿菌感染產生的特化組織，常用於根生理的研究中。此外，毛狀根具有快速生長與高次級代謝物累積的特性，故可用於生產植物的次級代謝物。目前人們對於毛狀根誘發、生長調控與次級代謝物累積之機制仍然未知。在本研究論文中，我們利用菸草、能感染菸草的根毛農桿菌 A4，以及菸草著名的次級代謝物尼古丁作為研究模型，希望透過研究毛狀根的生長與次級代謝物累積之機制，來增進毛狀根的應用性。在生長調控的部分，我們聚焦於根毛農桿菌 A4 之 TL-DNA 中的四個 rol 基因，分別是 rolA、rolB、rolC 和 rolD。目前已知這些基因對於促進毛狀根形成有關，但他們參與在形成或是維持毛狀根型態中所扮演的角色仍未知。本篇研究中，我們在農桿菌中分別剔除了四個 rol 基因，並利用這些 rol 缺陷農桿菌來誘導毛狀根。我們發現，當農桿菌缺少 rolB 或 rolC 時，會延遲發根並減少發根率。而缺少 rolA 或 rolD 時沒有這個現象。此外，由缺少 rolB 或 rolC 農桿菌感染所得的毛狀根會產生較少的側根數，且無法在長時間繼代中存活。而 rolA 或 rolD 時則不具有這些現象。我們認為 rolB 和 rolC 是主要調控生長的因子。藉由寡核苷酸微陣列分析，缺少 rolB 或 rolC 的毛狀根中脂質轉移蛋白與活性氧分子相關基因表現量顯著低於由野生型農桿菌感染生成的毛狀根。我們也發現側根越多的毛狀根，就會擁有越高的脂質轉移蛋白表現量。我們也比較了由野生型農桿菌感染產生的毛狀根與菸草原生根的轉錄體差異，發現這些脂質轉移蛋白的表現量確實在毛狀根中大量累積。除脂質轉移蛋白外，我們利用 ROS 染劑對缺陷 rolB 或 rolC 的毛狀根染色，發現缺失 rolB 或 rolC 時會有較低量的活性氧分子。藉此我們推論在毛狀根的生長調控中，脂質轉移蛋白與活性氧分子含量的改變是重要的因子。在次級代謝物的調控方面，我們發現隨機挑選出來的菸草毛狀根含有的尼古丁量都遠大於菸草原生根，而且尼古丁的含量與新菸鹼具有正相關，顯示在毛狀根中的次級代謝路徑可能整體被提高：尼古丁的代謝路徑以及儲存相關的運送蛋白在毛狀根中大量表現，造成尼古丁的累積。此外，我們發現生長較快速的菸草毛狀根含有較多的尼古丁，顯示毛狀根的生長與尼古丁的累積可能受到相關因子調控。而尼古丁在毛狀根中的累積非受到茉莉花酸訊息傳遞路徑的誘導，但依舊提升由茉莉花酸路徑啟動之乙烯反應因子 189 和 199 的表現量，進而增加關鍵酵素腐胺-N-甲基轉移酶與 N-甲基腐胺氧化酶的表現。我們的發現提供了一個簡單篩選高次級代謝物毛狀根的方法：測量毛狀根的生長速率。我們認為，這個結果可以大幅促進毛狀根在次級代謝物生產的研究與其應用性。

關鍵字

ﰧﲋﲌﲍﲋﰥﰦ根毛農桿菌；菸草；毛狀根； rol 基因；次級代謝物；尼古丁ﰧﰥﱪﱫﱬﱭﱮﲋﰡﰢﰣﰟ

並列摘要

Hairy root, which resulted from T-DNA transformation of Agrobacterium rhizogenes, is widely used in studying root biology. It is also applied in producing diverse plant secondary metabolites due to its fast-growth and metabolite-accumulating abilities. However, the regulatory mechanisms of hairy root initiation, growth, and metabolite accumulation are largely unknown. To expand the applicability of hairy roots, we used Nicotiana tabacum L. var Wisconsin 38, its pathogen A. rhizogenes A4, and its well-known metabolite nicotine as a study model to unveil the mechanisms that regulate hairy root growth and secondary metabolite accumulation. In the part of growth regulation, we focused on four rol genes, including rolA, B, C, and D, which are located on TL-DNA of A. rhizogenes A4. These rol genes are known to participate in rooting; however, the means by which the rol genes contribute to the initiation and the maintenance of hairy roots remain unknown. In this study, we knocked-out these rol genes in A. rhizogenes A4 respectively, and used for inducing hairy roots. We found that A. rhizogenes lacking rolB or rolC induced hairy roots with less rooting ability than wild-type A. rhizogenes, whereas lacking rolA or rolD showed no significant differences. Moreover, tobacco hairy roots lacking either rolB or rolC exhibited fewer branch roots and lost their growth ability after long-term subculture than wild-type-induced hairy roots, whereas lacking of rolA or rolD did not show significant differences. We considered rolB and rolC involved mainly in the regulation of hairy root growth. Our microarray analysis revealed that the expression of several groups of genes encoding lipid transfer proteins (LTP) and reactive oxygen species (ROS)-related genes was significantly suppressed in rolB- or rolC- deficient hairy roots. We also found that hairy root clones that exhibited greater branching also had higher levels of RolB, RolC, and the microarray-identified LTP genes. In addition, we compared the transcriptomic difference between hairy roots and un-infected intact roots by microarray, and the expression levels of the above mentioned LTP-encoding genes were dramatically higher in the hairy root. Moreover, ROS staining showed that ROS level were lower in rolB- or rolC- deficient hairy roots. We therefore suggest that up-regulating LTP and increasing the level of ROS are important for hairy root growth. In the part of secondary metabolite regulation, we found that tobacco hairy roots accumulate much more nicotine than the intact roots, and the nicotine contents were positively correlated with the amount of another metabolite anabasine, indicating hairy roots had higher secondary metabolic flux. By real-time PCR analysis, hairy roots had more abundant expression of genes encoding enzymes in nicotine biosynthetic pathway and storage transporters, indicating the accumulation of nicotine in hairy roots is via transcriptional regulation. Moreover, hairy roots with a higher growth rate had greater nicotine content, suggesting that growth and nicotine production are regulated synchronically. Nicotine up-regulation in hairy roots was regulated by ethylene response factor (ERF)189 and ERF199 to activate the key enzymes putrescine N-methyltransferase and N-methylputrescine oxidase with a jasmonic acid (JA)-independent signal. However, the possible regulator has not been identified. These findings indicate high secondary metabolites accumulated hairy root clones can be simply selected by measuring their growth rate, which expand the hairy root researches and applications in secondary metabolites.

並列關鍵字

Agrobacterium rhizogenes ； Nicotiana tabacum ； hairy root ； rol genes ； secondary metabolites ； nicotine

參考文獻

Bhattacharjee, S. (2012). The language of reactive oxygen species signaling in plants. J Bot 2012, Article ID 985298, 22 pages. doi:10.1155/2012/985298.

Akiyoshi, D.E., Morris R.O., Hinz, R., Kosuge, T., Garfinkel, D.J., Gordon, M.P. and Nester, E.W. Cytokinin/auxin balance in crown gall tumors is regulated by specific loci in the T-DNA. Proc Natl Acad Sci U S A 80, 407-411.

Aloni, R., Wolf, A., Feigenbaum, P., Avni, A. and Klee, H.J. (1998). The never ripe mutant provides evidence that tumor-induced ethylene controls the morphogenesis of Agrobacterium tumefaciens-induced crown galls on tomato stems. Plant Physiol 117, 841-849.

Altamura, M.M., Capitani, F., Gazza, L., Capone, I. and Costantino, P. (1994). The plant oncogene rolB stimulates the formation of flower and root meristemoids in tobacco thin cell-layers. New Phytol 126, 283-293.

Altamura, M.M., D'Angeli, S. and Capitani, F. (1998). The protein of rolB gene enhances shoot formation in tobacco leaf explants and thin cell layers from plants in different physiological stages. J Exp Bot 49, 1139-1146.

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菸草毛狀根的生長與其尼古丁高量累積之研究

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