阿拉伯芥之油菜素類固醇透過 BZR1 調控缺氮下 花青素之累積 篩選參與抗病反應之阿拉伯芥 NPF 轉運蛋白突變株

Brassinosteroids Regulate Anthocyanin Accumulations via BZR1 under Nitrogen Limitation condition in Arabidopsis Screening for Arabidopsis NPF transporter mutants in Pathogen Responses

駱冠宇(Guan-Yu Louh)

指導教授 ： 王雅筠

國立臺灣大學/生命科學院/植物科學研究所/碩士(2019年)

https://doi.org/10.6342/NTU201903535

摘要

缺氮造成植物生長發育受限制，使植物演化各種對抗缺氮逆境的方式。在地下部，過去大多數研究表明植物可透過根部構型來做抵抗逆境的方式，且缺氮逆境下，會透過荷爾蒙作為訊號來促進根部生長，然而，在地上部的研究極少，儘管地上部花青素的累積，過去被證實參與調控對抗缺氮逆境的一種方式，但其調控方式及訊號尚未闡明。我們研究透過反向遺傳學，以逆境相關荷爾蒙突變株進行篩選，發現油菜素類固醇生合成及受器皆為缺氮逆境下花青素生合成之必要因子。為了瞭解油菜素類固醇如何調控缺氮下花青素之生合成，本研究發現是透過油菜素類固醇的轉錄因子BZR1 來直接抑制目前已知最上層之花青素負調控因子LBD37及LBD38的轉錄表現。同時，油菜素類固醇含量及訊號也扮演植物於缺氮逆境下保留較多重量之關鍵。以及，本研究也提出一個調控機制來說明，油菜素類固醇如何參與到植物的氮源的吸收及利用。總結，在植物地上部，油菜素類固醇在缺氮逆境中的重要性，並且影響花青素產生以及地上部生物量。
NITRATE TRANSPORTER1/PEPTIDE TRANSPORTER family 轉運蛋白已被報導可藉由調節受質之分布並影響許多植物生理功能。雖然許多 NPF 之功能已被研究，但對於 NPF 是否參與及調控生物性逆境仍然未知。本研究，透過分析過去資料，研究在病原感染下之 53 個 NPF 的基因表現量，並選出 11 個 NPF 基因來做感染測試。透過感染結果分析後，選出 NRT1.5/NPF7.3 及 PTR3/NPF5.2 兩組基因做更多測試。透過細菌型番茄斑點病源， Pst DC3000 感染後，其中 nrt1.5/npf7.3 顯示叫野生種 Col-0 較為感病，暗示根部至地上部硝酸鹽運輸可能參與植株的抗病能力。除此之外，NRT1.5/NPF7.3 之轉錄表現也會受 Pst DC3000 及鞭毛 (Flg22) 誘導。但是是否透過根部至地上部之硝酸鹽運輸進而影響抗病，仍需更多研究證實。雙胜肽運輸蛋白 PTR3/NPF5.2 之轉錄表現會受 Pst DC3000、鞭毛 (Flg22)、及水楊酸誘導。雖然 PTR3/NPF5.2 並不影響植株基礎抗性，但在 ptr3 突變株中發現，突變株會失去部分系統性抗病。但 PTR3/NPF5.2 突變株如何參與在系統性抗病仍須要更多研究。總結，本研究發現 NPF 轉運蛋白不只參與在營養分布，也會參與在生物性逆境。然而，詳細功能及如何參與仍需更多研究來證實。

關鍵字

油菜素類固醇 ； 花青素 ； 缺氮

並列摘要

Nitrogen (N) limitation inhibits plant growth and development, leading to plant evolves diverse ways to adapt it. Most researches revealed that plant could modify the root architecture via hormone as a signal to resist N limitation. Nevertheless, the regardless of shoot researches let the shoot part still obscure. Even though anthocyanin accumulation in shoot have been proven which is one of the important strategy to challenge N limitation, the mechanism and signaling are still not elucidated. Here, through reverse genetics, screening of stress-related hormone mutants, the results exhibited that brassinosteroid (BR) biosynthesis and receptor were necessary for anthocyanin production under N limitation. The master transcription factor of BR signaling, BZR1, was then identified as the key hub for anthocyanin production by inhibiting anthocyanin repressors, LBD37 and LBD38, directly. Meanwhile, BR is also required for the biomass maintaining to N limitation by maintaining more biomass. Our investigation propose a model explaining how BR involves in plant nitrogen uptakes and utilizations. In conclusion, our research validated that BR is a key hormone for plant anthocyanin production; for plant biomass maintaining under N limitation, especially in shoot.
NITRATE TRANSPORTER1/PEPTIDE TRANSPORTER family (NPF) has been reported to involve in plant physiological functions through mediating distribution of various substrates. Although the functions of these proteins have been studied, the roles of NPF transporters upon biotic stresses remain unclear. In this study, we first analyzed the transcription level of all 53 genes in NPF family upon pathogen infection from BAR microarray database (e-Northerns w. Expression Browser), and 17 NPF genes showed altered expression patterns. After the pathogenicity test by Pst DC3000 infection, some NPF mutants showed different responses compared to wild-type plants. Two genes were selected to study further, which are NRT1.5/NPF7.3, PTR3/NPF5.2, The nrt1.5/npf7.3 mutants showed more susceptibility to Pst DC3000, suggesting that NRT1.5/NPF7.3 which mediates root-to-shoot nitrate transport is required for plant resistance to Pst DC3000. In addition, the transcription level of NRT1.5/NPF7.3 is induced by both Pst DC3000 and Flg22. Whether root-to-shoot nitrate transport by NRT1.5/NPF7.3 enhances the plant defense responses will be further investigated. The expression of PTR3/NPF5.2, a dipeptide transporter, is induced by Pst DC3000, Flg22, and salicylic acid (SA). Although PTR3 has no effect on local defense responses, the two ptr3 T-DNA insertion mutants lost the systemic acquired resistance (SAR) to Pst DC3000. What is the role of PTR3/NPF5.2 in SAR will be analyzed. Taken together, transport activity of NPF proteins is not only important for nutrient distribution, but also participates in adjust physiological responses upon biotic stresses. However, the detailed functions of how NPF transporters are able to resist pathogen need further examination.

並列關鍵字

brassinosteroid ； anthocyanin ； nitrogen limitation

參考文獻

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