Investigation of molecular and metabolic events in response to anoxia-reoxygenation in Arabidopsis

• Main Authors: Kuen-Jin Tsai, 蔡昆縉
• Other Authors: Ming-Che Shih
• Format: Others
• Language: en_US
• Published: 2015
• Online Access: http://ndltd.ncl.edu.tw/handle/85382924999588069689

博士 === 國立臺灣大學 === 植物科學研究所 === 103 === 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.