Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/31542
標題: 一氧化氮與水楊酸在番茄過敏性反應與防禦基因表現中所扮演之角色
Role of Nitric Oxide and Salicylic Acid in Hypersensitive Reaction and Defense Gene Expression of Tomato (Lycopersicon esculentum L.)
作者: 陳懿芬
Chen, I-Fen
關鍵字: nitric oxide radical;一氧化氮自由基;salicylic acid;reactive oxygen species;hypersensitive reaction;水楊酸;激活態氧;過敏性反應
出版社: 植物病理學系
摘要: 
一氧化氮與水楊酸在番茄過敏性反應與防禦基因表現中所扮演之角色
陳懿芬
本研究利用番茄 (Lycopersicon esculentum L.) 懸浮細胞接種青枯病菌 (Ralstonia solanacearum) 無毒力菌株 (Ps61-) 作為模式系統,探討在過敏性反應中與一氧化氮自由基 (NO.) 之產生及其可能參與誘發防禦基因表現有關之關鍵性生化因子。本論文共分成三部份:第一部份是探討鈣離子湧動與蛋白質磷酸化作用在番茄細胞過敏性反應中所扮演的角色。結果顯示:反應系統中 NO 產生為一雙相 (biphasic) 生化過程;第一階段產生量相當低而短暫,第二階段則明顯快速大幅度增加。外加 Ca2+,Ca2+ ionophore-A23187 與 ionomycin 均可促進 NO 產生;反之添加 EGTA,Ca2+ channel blocker LaCl3 及BAPTA 均有抑制作用;外加 thapsigargin 則不影響 NO 產生。此些證據充分顯示細胞外鈣離子源內移的重要性。另外,外加蛋白質激脢 (protein kinase) 抑制劑及 cycloheximide 均可抑制 NO 產生。綜合以上結果顯示:當寄主與病原認知後,鈣離子內湧 (influx) 的增加、蛋白質磷酸化酵素的活化以至酵素de novo生合成等一連串生化代謝改變,對 NO 的產生及過敏性反應之病徵表現至為重要。論文第二部份主要內容探討 NO 與 ROS 之共同作用在過敏性反應細胞死亡中所扮演之關鍵性角色。研究結果證實所測試之寄主病原交互作用模式系統中伴隨雙相 NO 自由基的產生可檢測到雙相O2- 自由基的併行增加。而隨著第二階段激活態氧 (ROS) 與 NO 自由基之快速增加,則可檢測到細胞過敏性死亡明顯提高。又者,此一細胞死亡現象,隨著細胞培養震盪速度加快而愈加嚴重,而外加一氧化氮合成酵素 (NOS) 抑制劑或 NO 與 ROS 之消除劑,則均可減少細胞之死亡。此些證據顯示 ROS 與 NO 之協力作用對過敏性細胞死亡具決定性影響。另外由外加 desferal 可明顯抑制 NO 產生並減少細胞過敏性死亡的結果也再度印證此論點。然而,試驗中又發現,反應過程中 ROS 與 NO 的作用似端受 SA 產生的影響。外加 SA 一方面可減少細胞過敏性死亡,另方面則可降低第二階段O2-. 的產生、促進 H2O2 產生與第二階段 NO 產生。外加 SA 處理可些微抑制 catalase 與 glutathione reductase活性,對 peroxidase 活性則有明顯促進作用。上述 SA 對細胞過敏性死亡的保護效果顯與 NO 生及對 ROS 消除能力的提昇等複合效應有密切關連。本研究的第三部份是以溫室種植番茄植株為材料,探討 NO 與 SA 在系統性防禦基因表現中所扮演的訊號分子角色。既有之結果顯示番茄葉片接種無毒力青枯病菌 Ps61- 菌株後,如同在細胞懸浮培養系統中所見,明顯有雙相氣體 NO. 之產生。在過敏性反應過程中,生體內 SA 含量可增加約 1〜4 倍,同時在接種後 2〜4 小時與 4〜8 小時分別有 PAL 與 PR-1 基因之誘導表現。外加 NO donors 可如同接種 Ps61- 一樣誘導此兩基因表現;外加 cGMP 與 cADPR 也有類似的效果,顯示此兩種在哺乳類動物系統中扮演著次級信息分子角色之分子,在所測試植物系統中亦有同樣的作用。相反地,外加 SA 只能誘導 PR-1 表現,外加 DPI 則可抑制由 Ps61- 所誘導 PAL 與 PR-1 基因活化,顯示活化此二基因表現的重要因子應為超氧自由基 (superoxide) 而非過氧化氫 (H2O2)。又者,由其可誘導未處理葉 PAL 與 PR-1 基因表現之作用,進而顯示 NO 與 SA 可作為長距離移行的訊號分子。由 NO 與 SA 處理葉片後可促進過氧化酵素 (peroxidase) 活性表現之作用性,也再次印證此一看法。綜合本研究三部份之試驗結果顯示,Ps61- 接種所誘發產生的 NO 與 SA 確為參與局部防衛反應與活化系統性抗病性之重要因子。由於 NO 容易偵測與操控,在本研究模式系統中有關 NO 在抗病性生化與生理上重要性之闡明,確為未來植物抗病性研究應用提供一些相當有用的資訊。

Role of Nitric Oxide and Salicylic Acid in Hypersensitive Reaction and Defense Gene Expression of Tomato (Lycopersicon esculentum L.)
I-Fen Chen
The biochemical determinants leading to hypersensitive response (HR) relating to nitric oxide radical (NO.) production and their possible involvement in eliciting defense gene expression were explored using avirulent Ralstonia solanacearum (Ps61- isolate) challenged-tomato (Lycopersicon esculentum L.) cells in suspension as a model system. The thesis consists of 3 parts. In Part I, the role of calcium influx and protein phosphorylation in nitric oxide radical production during the bacterium-induced hypersensitive reaction of tomato cells was investigated. Production of NO was biphasic-a small and transient initial peak followed by a dramatic increase. NO production was enhanced by the addition of Ca2+ and Ca2+ ionophores and was inhibited by EGTA, LaCl3, and BAPTA, but was not affected by thapsigargin. The production of NO was inhibited by protein kinase inhibitors and by cycloheximide. We conclude from these results that Ca2+ influx, protein kinase activation and de novo enzyme synthesis during host pathogen recognition are important in the production of NO and the HR expression. In Part 2 of the study, the role of NO/ROS combination on HR cell death was investigated. A biphasic surge of O2-1 production was along with NO production. Death of host cells generally became prominent during the second phase and was increased by higher shaking speed. Cell death was reduced by the addition of NOS inhibitors, and NO or ROS scavengers. Desferal inhibited NO production and reduced cell death. The addition of SA reduced cell death and was accompanied by a decrease in phase-2 O2- production, an increase of H2O2 burst, and an increase of phase-2 NO production. A slight inhibition of catalase and glutathione reductase activities and an increase of peroxidase activity were also observed when SA was added to the assay system. The effect of SA was attributed to the potentiation of NO production and the enhanced ROS scavenging ability. In Part 3 of the study, the role of NO and SA as signals for systemic defense gene expression was investigated in intact plants. The biphasic NO production similar to that observed in cell suspension culture system was detected from Ps61--inoculated tomato leaf tissue. During the course of HR, the endogenous level of SA increased approximately 1- to 4-fold, accompanied by the expression of PAL and PR-1 genes 2-4 and 4-8 hours, respectively, after inoculation. Administration of NO donors triggered the expression of these 2 genes similar to that by Ps61-. Similar results were also obtained by the administration of cADPR or cGMP, suggesting the role of secondary signal of these 2 molecules in defense gene expression. In contrast, the application of SA induced only PR-1 expression. The Ps61--induced activation of PAL and PR-1 was suppressed by DPI, suggesting that superoxide, rather than H2O2 was essential for these genes' expression. Moreover, the induction of PAL and PR-1 expression in untreated controlled leaves suggested the role of NO and SA as long distance transported signals for induction of these genes. This view was further strengthened by the evidence that NO and SA both showed effectiveness in inducing the increased activity of peroxidase in treated tissues. It is clear that NO and SA released from Ps61--inoculated tissue do mediate the activation of both local defense and systemic resistance. The biochemical and physiological significance of NO as illustrated in the current model system will provide tools for the exploration of plant resistance to disease infections.
URI: http://hdl.handle.net/11455/31542
Appears in Collections:植物病理學系

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