水稻穀胱甘肽影響根部發育與生長素極性分布之研究

Study the Effect of Glutathione on Rice Root Development and Auxin Polar Distribution

吳映樺(Ying-Hua Wu)

指導教授 ： 洪傳揚

國立臺灣大學/生物資源暨農學院/農業化學研究所/碩士(2019年)

https://doi.org/10.6342/NTU201902234

摘要

植物體內的穀胱甘肽 (GSH) 在平衡氧化還原狀態、抵抗非生物逆境及傳遞分子訊息等方面皆扮演關鍵的角色。實驗室過去的研究發現弱化GR1 表達會抑制水稻側根生成及伸長，本研究進一步釐清 GSH 含量以及氧化還原狀態對水稻幼苗根系發育與 Auxin 傳輸之影響。試驗中對水稻幼苗外加 GSH 或 GSSG 處理都會顯著縮短水稻種子根主根長度，並同時減少側根數量和長度，分析 OsGR1弱化表現與大量表現的水稻植株根部，也發現兩轉殖株的主根均較野生型短，其中大量表現型主根最短且側根數量和密度也最低，這些結果顯示GSH / GSSG 比例的改變，會抑制種子根主根和側根生長；以受 Auxin 誘導之 DR5::GUS 轉殖水稻進行組織化學染色分析，結果顯示對照組的 GUS 主要分佈在靠近主根和側根根冠、根尖及延長部，而無論外加 GSH 或 GSSG 都會擴大 GUS 在種子根內之分佈位置；以 GSH 處理 GR1-Ri x DR5::GUS 雜交水稻，能增加主根和側根的 Auxin 分佈且原來生長受抑制的側根能恢復至野生型的水準，顯示 GSH 氧化還原狀態在根部內生長素的傳輸及分佈中扮演關鍵的角色。另一方面，外加 GSH 生合成抑制劑 BSO 會抑制內生 GSH 總生成量及側根生長，而不會影響主根伸長。但隨著 BSO濃度增加，GUS 分佈位置逐漸縮小，最後侷限於近延長部與其附近側根中。同時 GUS 的活性也隨著 BSO 處理濃度增加而逐漸降低，中柱兩側的向基運輸 (Basipatel transport) 明顯減弱並造成主根蜷曲、側根發育嚴重缺陷，顯示 GSH 生合成總量對根部內生長素的傳輸及分佈佔有相當重要的地位，而大量表現GR1 (GR1-OE x DR5::GUS) 能減緩 BSO 對 GUS 分佈及側根長度的抑制。藉由RT-PCR分析 Auxin 相關基因的表現，結果發現水稻內不同 GSH 總量或 GSH / GSSG 比例對運輸載體基因 (OsAUX1 和 OsPINs) 和生合成酵素基因 (OsYUCCA5) 都有迥異的表現差異。以上結果說明水稻內 GSH 含量多寡與氧化還原比例都會影響根系內 Auxin 的生合成、運輸與分佈，進而調控主根和側根的生長與發育。

關鍵字

穀胱甘肽 ； 穀胱甘肽還原酶 ； 根部發育 ； 生長素 ； 水稻

並列摘要

Glutathione plays a critical role in plant, for example, balancing redox status, resisting abiotic stress, and signaling molecular message. Based on our past research, we found that knockdown of OsGR1 inhibited lateral root formation and elongation. This study clarified the effect of GSH level and redox ratio on root development and auxin transport in rice seedling. In seminal root, the primary root length shortened, lateral root number and length decreased significantly after treating with GSH and GSSG. Phenotyping analysis showed that OsGR1 transformation lines [GR1-Ri (RNA interference) and GR1-OE (Overexpression)] had poorer root development than wild type, especially in overexpression line. These outcomes implied that the changes of GSH / GSSG ratio inhibited primary and lateral root growth. The auxin-inducible promoter DR5 was applied to trace the presence of auxin. In transgenic rice line (DR5::GUS), the distribution of GUS was just located near root caps and root tips of primary and lateral roots. However, regardless of treating with GSH or GSSG, the GUS distributed in whole seminal root evenly. The poor auxin distribution and lateral root length of GR1-knockdown line (GR1-Ri x DR5::GUS) could be recovered by treating with 2 mM GSH, which means that GSH redox status plays an important role in transportation and distribution of auxin. On the other hand, the primary root length wasn’t affected by buthionine sulfoximine (BSO), a GSH biosynthesis inhibitor, but the GSH-lacking event caused a severe inhibition on lateral root number and length. The GUS distribution was confined near root tips and GUS activity was decreased as BSO concentration increased. Meanwhile, the basipetal transport around the stele in root weakened significantly, and caused curly primary root and defective development of lateral root. It showed that GSH content is essential to auxin transport and distribution as well. Overexpression of GR1 alleviated the inhibition on auxin distribution and lateral root growth caused by BSO. The expression of auxin-related genes under different GSH contents and GR expression levels was analyzed through RT-PCR, the results showed that auxin influx and efflux transport carrier genes, OsAUX1 and OsPINs etc., and auxin biosynthesis gene, OsYUCCA5, expressed in various levels compared with control or wild type. Overall, these results concluded that both GSH level and GSH / GSSG ratio influenced auxin biosynthesis, transport and distribution, thereby regulate the development of primary and lateral roots of seminal root in rice seedling.

並列關鍵字

Glutathione ； glutathione reductase ； root development ； auxin ； rice (Oryza sativa L.)

參考文獻

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