植物於缺氧再復氧逆境中之訊息傳遞與代謝體之探討

Abstract

淹水是一種常見於低漥或排水不良地區的非生物逆境，經常導致全世界嚴重的農業損害。隨著氣候變遷日益嚴重，暴雨或者是集中降雨發生的頻率頻繁，淹水事件不再只侷限在某些特定區域，且發生機率也越來越高。根據氧氣的供應量，淹水逆境主要可區分為四個時期，分別是：正常供氧、短暫缺氧、無氧、以及復氧。氧氣缺乏(包含短暫缺氧和無氧)的相關文獻在過去數十年來，正快速在累積。各式各樣的適應機制，以及分子訊息傳遞陸陸續續已被揭露。然而，復氧也同樣對植物造成嚴重的傷害，但目前對於復氧的相關研究卻是相當有限。 本博士論文中，我著重在植物於復氧時期的分子機制研究。在第一個部份，我主要藉由厭氧氣缸來精準模擬植物在淹水逆境中缺氧及復氧的反應，並且利用微矩陣晶片全面性地偵測復氧時的植物基因表現。我發現到當植物進入到復氧環境時，會迅速表現與熱反應、脫水反應、細胞基礎代謝、過氧化物清除的相關基因。從此可推論，植物在復氧時可能面臨了大量的過氧化物產生，並且造成細胞膜的破壞從而引發了脫水現象。在這同時，我也觀察到植物會誘導與檸檬酸循環代謝相關的基因，促使植物在復氧時快速補充缺乏的檸檬酸循環代謝中間產物，回復正常的有氧呼吸作用。植物在復氧的過程中，會啟動乙烯與茉莉酸的訊息傳導相關基因。依據過去的研究，已知乙烯的訊息傳遞在缺氧過程中具有重要功能，但乙烯在復氧環境中的角色至今卻是相當模糊。因此，我利用乙烯不敏感的突變株ein2-5以及ein3eil1來了解其扮演角色。從微矩陣分析中，得知乙烯影響了大多數在復氧時的上述反應以及賀爾蒙的交互作用平衡。為了證明其重要性，我進行了表現型測試，相較於野生型，短時間的無氧再復氧的環境處理會造成乙烯不敏感突變株在復氧時期較嚴重傷害，這強烈說明著乙烯訊息在復氧時的不可或缺性。 在第二個部分，我深入探討乙烯在無氧－復氧過程中的訊息傳遞，專注於乙烯首要轉錄因子ETHYLENE INSENSITIVE 3 (EIN3)的下游基因轉錄。根據前人利用EIN3所做的染色質免疫沉澱定序，再與前述所做的微矩陣實驗進行交叉分析，我推導出一系列可能在缺氧－復氧逆境中轉錄的EIN3下游標的基因。在這些基因當中，我選擇了谷氨酸脫氫酶進行進一步的研究。谷氨酸脫氫酶是一個重要的代謝酵素，負責交互轉換谷胺酸跟α-酮戊二酸並影響細胞內的碳與氮含量的平衡。在即時聚合酶鏈式反應的實驗中，經由無氧－復氧處理後，我觀察到較低的谷氨酸脫氫酶基因表現在乙烯不敏感突變株ein2-5以及ein3eil1，且同樣的現象也發生在酵素活性測定實驗。這些結果說明著谷氨酸脫氫酶直接被乙烯訊息傳遞所調控。為了驗證谷氨酸脫氫酶在逆境中的生理角色，我利用了谷氨酸脫氫酶的突變株 gdh1gdh2進行了氣象層析的實驗。全面性的代謝質體分析顯示谷氨酸脫氫酶影響了復氧時丙胺酸的代謝並且造成了檸檬酸循環中間產物的補充缺陷。從液象層析質譜實驗得知，在突變株中，延遲的檸檬酸循環回復造成了生物能量無法在復氧的過程中快速補充。有趣的是，當我將突變株gdh1gdh2處理淹水逆境，或者是厭氧氣缸的無氧逆境，都會在復氧的過程當中開始造成細胞傷害以及葉片萎凋。這顯示，谷氨酸脫氫酶在復氧時的代謝回復過程當中扮演著重要的角色，也同時連結了乙烯訊息傳遞與細胞代謝在缺氧逆境時的相互關係。 總結來說，我的研究不僅開啟了對復氧逆境訊息傳遞的了解，同時也闡述了乙烯訊息傳導在缺氧及復氧時的重要角色。許多的EIN3下游標的基因仍尚待功能分析，相關的潛力研究相信將會在不久後揭露。

Submergence, a common abiotic stress that usually occurs in low-lying regions or poorly drained lands, impacts world agriculture. Owing to worsening climate change, extreme or concentrated rainfall occurs frequently. Submergence is no longer restricted in certain districts and numbers of flood events have increased across the globe. Based on oxygen availability, submergence is divided into four distinct stages: normaxia, transient hypoxia, anoxia and reoxygenation. Studies of oxygen deprivation including transient hypoxia and anoxia have developed rapidly in the past few decades. Increasing knowledge of adaptive mechanisms and signal transductions in response to low oxygen had been shown. In contrast, although reoxygenation also causes severe damages to plants, limited literatures related to reoygenation are presented. In this Ph. D. thesis, I focused on characterizing the molecular events involved in reoxygenation. My work is divided into two parts. In part I, I built-up an anaerobic gas chamber system to mimic anoxia-reoxygenation (A/R), and conducted a microarray assay to globally inspect the gene expression profiles under this condition. I observed that when reoxygenation began, genes of heat response, dehydration and ROS detoxification were highly activated. It was suggested that a burst of ROS and cellular dehydration resulted from cell membrane damage occurred during recovery. Besides, several genes encoding metabolic enzymes were induced during reoxygenation and part of them are required for the replenishment of TCA cycle intermediates. Jasmonic acid and ethylene signaling were also activated during reoxygenation. The roles of ethylene signaling under low oxygen condition have been reported, but it is still unclear in reoxygenation. To understand its function during recovery, two ethylene insensitive mutants ein2-5 and ein3eil1 were used in the microarray assays. The results indicated that ethylene signaling might participate in the regulation of most of above reoxygenation responses and hormone homeostasis. Phenotype testing was applied to verify the importance of ethylene signaling in reoxygenation. Severe damages in ein2-5 and ein3eil1 comparing to the wild-type were found during reoxygenation. These results strongly implied the pivotal role of ethylene signaling during reoxygenation. In part II, I further looked into the ethylene signal transduction during A/R, and focused on ETHYLENE INSENSITIVE 3 (EIN3) driven downstream targets. Through analyses of the microarray data and published Arabidopsis EIN3 ChIP-seq dataset, I inferred the putative EIN3 direct targets under the stress. Among them, GDH2, which encodes one subunit of glutamate dehydrogenase (GDH), was chosen for further study owing to its metabolic role in TCA cycle replenishment. Through qRT-PCR, I showed that both GDH1 and GDH2 were induced during A/R and that the induction was mediated via ethylene signaling. In addition, the results of enzymatic assays showed that the induction level of GDH during A/R decreased in the ethylene insensitive mutants ein2-5 and ein3eil1. Global metabolite analysis indicated that GDH with its deamination activity might regenerate 2-oxoglutarate to facilitate alanine breakdown during reoxygenation. Subsequently, in gdh1gdh2 mutant, impaired TCA cycle replenishment and energy regeneration were shown. Taken together, my research expands the current knowledge of signal transduction of reoxygenation, and demonstrates the important roles of ethylene signaling within this process. The functional characterization of EIN3 targets during low oxygen stress sheds lights on resolving ethylene-triggered adaptive mechanisms. I believe that the pertinent literatures and potential studies should be revealed in the near future.