Thesis (Ph. D.)--West Virginia University, 2001. / Title from document title page. Document formatted into pages; contains xiv, 180 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.

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Lignin is the second most abundant biopolymer on earth and is found in the wood (xylem) of vascular land plants. To transport the hydro-mineral sap, xylem forms specialized conduit cells, called tracheary elements (TEs), which are hollow dead cylinders reinforced with lateral secondary cell walls (SCW). These SCWs incorporate lignin to gain mechanical strength, water impermeability and resistance against pathogens. The aim of this thesis is to understand the spatio-temporal deposition of lignin during TE differentiation and the relationship with its neighbouring cells. In vitro TE differentiating cell cultures of Zinnia elegans and Arabidopsis thaliana are ideal tools to study this process: cells differentiate simultaneously into 30-50% TEs while the rest remain parenchymatic (non-TEs). Live-cell imaging of such TEs indicated that lignification occurs after programmed cell death (PCD), in a non-cell autonomous manner, in which the non-TEs provide the lignin monomers. This thesis confirms that lignification occurs and continues long after TE PCD in both in vitro TE cultures and whole plants using biochemical, pharmacological and cytological methods. The cooperative supply of lignin monomers by the non-TEs was demonstrated by using Zinnia and Arabidopsis in vitro TE cultures. Inhibitor experiments revealed further that the non-TEs supply reactive oxygen species (ROS) to TEs and that ROS are required for TE post-mortem lignification. Characterization of the non-TEs showed an enlarged nucleus with increased DNA content, thus indicating that non-TEs are in fact endoreplicated xylem parenchyma cells (XP). The cooperative lignification was confirmed in whole plants by using knock-out mutants in a lignin monomer synthesis gene, which exhibit reduced TE lignification. The XP specific complementation of these mutants led to nearly completely rescuing the TE lignin reduction. Using microscopic techniques, the spatial distribution of lignin was analyzed in TEs from in vitro cultures and whole plants, revealing that lignification is restricted to TE SCWs in both protoxylem and metaxylem. These specific deposition domains were established by phenoloxidases, i.e. laccases localized to SCWs and peroxidases, present in SCWs and the apoplastic space. Laccases were cell-autonomously produced by developing TEs, indicating that the deposition domains are defined before PCD. Altogether, these results highlight that the hydro-mineral sap transport through TEs is enabled by the spatially and temporally controlled lignification of the SCW. Lignification occurs post-mortem by the supply of monomers and ROS from neighbouring XP cells and is restricted to specific deposition domains, defined by the pre-mortem sequestration of phenoloxidases.

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TRPA1 channels are sensors for noxious stimuli in a subset of nociceptive neurons. TRPA1 channels are also expressed in cells of the mammalian inner ear, but their function in this tissue remains unknown given that Trpa1–/– mice exhibit normal hearing, balance and sensory mechanotransduction. Here we show that non-sensory (supporting) cells of the hearing organ in the cochlea detect tissue damage via the activation of TRPA1 channels and subsequently modulate cochlear amplification through active cellshape changes. We found that cochlear supporting cells of wild type but not Trpa1–/– mice generate inward currents and robust long-lasting Ca2+ responses after stimulation with TRPA1 agonists. These Ca2+ responses often propagated between different types of supporting cells and were accompanied by prominent tissue displacements. The most prominent shape changes were observed in pillar cells which here we show possess Ca2+-dependent contractile machinery. Increased oxidative stress following acoustic overstimulation leads to the generation of lipid peroxidation byproducts such as 4-hydroxynonenal (4-HNE) that could directly activate TRPA1. Therefore, we exposed mice to mild noise and found a longer-lasting inhibition of cochlear amplification in wild type than in Trpa1–/– mice. Our results suggest that TRPA1-dependent changes in pillar cell shape can alter the tissue geometry and affect cochlear amplification. We believe this novel mechanism of cochlear regulation may protect or fine-tune the organ of Corti after noise exposure or other cochlear injuries.

Using gentian and petunia as the experimental systems, potential alternative post-harvest treatments for cut flowers were explored in this project. Pulsing with GA₃ (1 to 100 µM) or sucrose (3%, w/v) solutions delayed the rate of senescence of flowers on cut gentian stems. The retardation of flower senescence by GA₃ in both single flower and half petal systems was accompanied by a delay in petal discoloration. The delay in ion leakage increase or fresh weight loss was observed following treatment with 5 or 10 µM GA₃ of the flowers at the unopen bud stage. Ultrastructural analysis showed that in the cells of the lower part of a petal around the vein region, appearance of senescence-associated features such as degradation of cell membranes, cytoplasm and organelles was faster in water control than in GA₃ treatment. In particular, degeneration of chloroplasts including thylakoids and chloroplast envelope was retarded in response to GA₃ treatment. In the cells of the top part of a petal, more carotenoids-containing chromoplasts were found after GA₃ application than in water control. In petunia, treatment with 6% of ethanol or 0.3 mM of STS during the flower opening stage was effective to delay senescence of detached flowers. The longevity of isolated petunia petals treated with 6% ethanol was nearly twice as long as when they were held in water. Senescence-associated petal membrane damage, weight decline, ovary growth and decrease in protein and total RNA levels were counteracted in ethanol-treated petals. The accumulation of ROS, particularly superoxide and hydrogen peroxide, was also inhibited or delayed by ethanol application. Anti-senescence mechanisms, particularly the changes of oxidative / antioxidant metabolism involved in petal senescence, were investigated. In gentian, activities of AP and SOD but not POD in the GA₃-treated petals were significantly higher than those of the control. In isolated petunia petals, the decreased trends of antioxidative SOD and AP activities during senescence were apparently prevented in response to ethanol treatment although the levels of ascorbate and photo-protective carotenoids were not affected. Furthermore, by optimizing a range of critical PCR parameters such as primer combinations, cDNA concentrations and annealing temperatures, a reliable protocol has been established for quantifying the expression level of Cu-Zn SOD gene in petunia petals using SYBR Green I based real-time RT-PCR. A 228 bp gene fragment of Cu-Zn SOD was isolated from petunia (var. 'hurrah') using RT-PCR. It was found that the mRNA level (relative to 18S rRNA level) of Cu-Zn SOD decreased significantly after 6 days in water. However, there was about a 55-fold increase in Cu-Zn mRNA level after 6 days of ethanol treatment when compared to water-treated petals. Similarly, down-regulation of the mRNA level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was also observed during senescence of petunia petals. Increased vase life of petunia petals by ethanol treatment was correlated with promotion of GAPDH expression by a factor of about 16 on day 6. Taking together, the anti-senescence effects of GA₃ and ethanol are at least partially associated with an increased efficiency of petal system utilizing ROS since the selected antioxidants were significantly maintained when compared to the corresponding values for the control.

The study aimed to investigate the following: 1. Investigate a putative glutathione peroxidase gene (Glyma17g34110) within Glycine max by an in silico analysis and spatial expression. 2. Determine the effects of exogenously applied nitric oxide on the expression of Glyma17g34110. 3. Investigate the antioxidant mechanism with attention to Glyma17g34110,reactive oxygen species and cell death in the response to salt stress. 4. Establish whether Glyma17g34110 is a glutathione peroxidase or thioredoxindependent peroxidase gene. / Magister Scientiae - MSc

The production of reactive oxygen species (ROS) is prominent in all aerobic metabolisms including plants. For this reason, the redox homeostasis of the production and scavenging of these intermediates is imperative for growth, development and survival during unfavourable conditions. In this study, a putative glutathione peroxidase gene (Glyma17g34110) from Glycine max (soybean) was identified and analyzed. The successful characterisation of Glyma17g34110 provided evidence of it being a glutathione peroxidase using glutathione as its preferred electron donor and substrate. Furthermore, it is known that antioxidant enzymes such as GPX exist in various tissues, performing a diverse set of functions. By a bioinformatic analysis of Glyma17g34110 and its promoter region, it was indicated that Glyma17g34110 could be a putative chloroplast protein that could play an important role in photosynthesis.One of the major factors affecting plant growth and development worldwide is abiotic stresses such as salinity. In the presence of salinity the production of harmful ROS is increased, resulting in detrimental reactions with important biological features (DNA, protein and lipid membranes), leading to cell death. The analysis of Glyma17g34110 under salt stress revealed that it is a salt sensitive gene and thus, the down-regulation of Glyma17g34110 could be due to the lack of known defence and response cis-acting elements present in the promoter region. Furthermore, it was proven in previous studies that the application of exogenous nitric oxide (NO) increases the activity of antioxidant enzymes. In this thesis it was observed that the presence of exogenously applied NO increased the expression of Glyma17g34110 tremendously in all soybean tissues (leaves, roots and nodules) investigated.Studies have found numerous cis-acting elements to be NO responsive, however, none of these elements were found in the promoter region upstream of glyma17g34110. This suggests that novel cis-acting elements could be present in the promoter region of Glyma17g34110.Thus, increasing the expression of Glyma17g34110 during salinity in the presence of NO, as well as the identification of these novel cis-acting elements, could lead to the enhancement of the defence mechanisms against ROS, which could lead to increasing plant tolerance to stress. / >Magister Scientiae - MSc

目錄…………………………………………………………………………I 圖目錄……………………………………………………………………IV 表目錄……………………………………………………………………VI 縮寫表……………………………………………………………………VII 謝誌………………………………………………………………………IX 中文摘要…………………………………………………………………XI 英文摘要…………………………………………………………………XIII 第壹章、前言………………………………………………………………1 第貳章、文獻探討…………………………………………………………3 第2-1節 紫外線…………………………………………………….…..4 2-1-1. 紫外線……………………………………………………….4 2-1-2. 紫外線的分類……………………………………………….5 2-1-3. 紫外線對人體健康的益處………………………………….6 2-1-4. 紫外線對人體的傷害……………………………………….7 第2-2節 自由基…………………………………………………………8 2-2-1. 自由基的介紹……………………………………………….8 2-2-2. 自由基的來源……………………………………………….8 2-2-3. 紫外線與自由基…………………………………………….9 2-2-4. 自由基種類………………………………………………….9 第2-3節 抗氧化酵素訊息的傳遞路徑………………………………..12 2-3-1. Nf2…………………………………………………………..12 2-3-2. Keap1………………………………………………………..12 2-3-3. ARE………………………………………………………….13 2-3-4. Nrf2的活化機制…………………………………………….14 第2-4節 細胞抗氧化的防禦系統……………………………………17 2-4-1. 酵素型抗氧化防禦系統…………………………………..18 2-4-2. 非酵素型抗氧化防禦系統………………………………...24 第參章、研究動機與實驗設計架構圖……………………………………...25 第3-1節 硏究動機……………………………………………………..26 第3-2節 實驗設計架構………………………………………………..27 第肆章、實驗材料與方法……………………… …………………………28 第4-1節 實驗材料……………………………………………………...29 第4-2節 實驗儀器……………………………………………………...32 第4-3節 實驗方法……………………………………………………...33 4-3-1. 細胞培養…………………………………………………...33 4-3-2. 存活率試驗………………………………………………...36 4-3-3. ROS產量測定………………………………………………38 4-3-4. 細胞毒性的測定…………………………………………...40 4-3-5. Comet assay彗星試驗………………………………………41 4-3-6. 細胞GSH含量測定………………………………………..44 4-3-7. 細胞凋亡試驗……………………………………………...46 4-3-8. 細胞總蛋白質萃取………………………………………...48 4-3-9. 細胞核與細胞質之蛋白質萃取…………………………...49 4-3-10. 蛋白質定量……………………………………………….51 4-3-11. 西方墨點分析…………………………………………….52 4-3-12. 免疫螢光染色…………………………………………….56 4-3-13. 轉染作用………………………………………………….58 4-3-14. 冷光酵素報導基因分析………………………………….60 4-3-15. RNA干擾…………………………………………………62 4-3-16. 統計分析………………………………………………….64 第伍章、實驗結果與圖表…………………………………………………65 第5-1節 ERG對HaCaT細胞的抗光氧化能力………………………66 5-1-1. ERG以及UVA對HaCaT細胞的細胞毒性影響…………66 5-1-2. ERG對UVA誘導活性氧物種含量之影響………………..66 5-1-3. ERG對UVA誘導的細胞膜損傷之影響…………………..67 5-1-4. ERG對UVA誘導的DNA損傷之影響……………………67 5-1-5. ERG對UVA所誘導之氧化傷害的影響…………………..68 5-1-6. ERG對UVA引發之細胞凋亡的影響……………………..68 第5-2節 ERG誘導之抗氧化系統的機制探討………………………..79 5-2-1. ERG對抗氧化酵素的影響…………………………………79 5-2-2. ERG活化Nrf2路徑探討……………………………………80 5-2-3. ERG影響Nrf2入核的表現………………………………..80 5-2-4. ERG對抗氧化酵素轉錄的影響……………………………81 5-2-5. 以siNrf2探討ERG的保護機制和Nrf2之間的關係……81 第陸章、討論………………………………………………………………95 第6-1節 ERG對於UVA刺激HaCaT細胞造成的細胞損傷之影響..97 第6-2節 ERG對抗氧化基因的表現之影響………………………….97 第6-3節 ERG與UVA影響Nrf2-Keap1 pathway……………………98 第6-4節 Nrf2對ERG所抑制UVA造成之光氧化傷害的影響……..98 第柒章、結論……………… …………………………………………..100 參考文獻 ………………….……………………………………………..102