Aluminum (Al)stress is a major limitation to crop productivity on acidic soils. To help understand the cellular mechanisms underlying the toxicity and resistance of plants to Al, this thesis involved a large-scale, transcriptomic and proteomic analysis of roots of Arabidopsis thaliana and reports on comparative analysis of transcriptome and proteome of Al stress responses. Using a microarray representing ~93% of the predicted genes in Arabidopsis, a relatively small proportion (3%) of transcripts were detected as Al- responsive. More changes in the transcriptome were detected after long-term (48 h; 1,114 genes), than short-term (6 h; 401 genes) with relatively little overlap of transcripts detected for each time point. These results suggest that Al toxicity is progressive over time and poses some unique challenges to plants. Further, using two dimensional differential in gel electrophoresis (DiGE), 12 (6 h) and 17 (48 h) proteins were found differentially abundant after Al exposure. Most of the identified proteins were involved in primary metabolism and oxidative stress. Cytosolic-malate dehydrogenase (cyt-MDH) was one of the novel Al-responsive protein identified in this study. Transcript abundance of cyt-MDH correlated well with protein abundance, suggesting that cyt-MDH is regulated in part at transcriptional level. Furthermore, homozygous mdh-1 and mdh-2 mutants were more resistant to Al as compared to WT suggesting that regulation of cyt-MDH could play a role in Al resistance. In general, comparative analysis of proteomics data and transcriptomics data showed a poor correlation for both 6 h (r2 = 0.155) and 48 h (r2 = 0.083). The potential role of five class III peroxidases (PER2, PER27, PER34, PER42, PER69) in resistance of roots to Al was explored using quantitative reverse transcriptase PCR and a reverse genetics approach. A diverse range of patterns of transcript abundance was detected using QRT-PCR in response to Al. Furthermore, per2, per21, and per69 mutants showed greater increases in root lengths as compared to WT after Al stress suggesting that regulation of PER might play a role in Al resistance. These results contribute to the identification of candidate genes for the generation of Al-resistant transgenic plants. / Plant Biology

Compound I of myeloperoxidase is capable of both one-electron oxidation and two-electron oxidation reactions. Halides and pseudohalides are the substrates for the two-electron oxidation and other compounds including a large variety of aromatic alcohols and amines can be oxidized via the single electron oxidation pathway. To investigate the catalytic mechanism of myeloperoxidase four structures of complexes of myeloperoxidase were solved. Two of them are complexes with hydroxamic acids and the other two are complexes with nitrite. Hydroxamic acids (salicylhydroxamic acid and benzylhydroxamic acid) can function as structural analogues for the aromatic alcohol and amine substrates of myeloperoxidase. The crystal structures of complexes of MPO with both hydroxamic acids have been solved at 1.85 Å resolution and their binding to myeloperoxidase is compared. The models show similar binding of their hydroxamic acid moieties but different orientations of their aromatic rings. The absence of the hydroxyl group covalently bound to the benzyl group in benzylhydroxamic acid creates an environment that does not permit the same favorable interactions with MPO when compared to salicylhydroxamic acid. These findings could explain the three orders of magnitude difference in the value of the dissociation constants of the two complexes. Nitrite has been shown to bind myeloperoxidase and also to reduce Compound I and Compound II. Crystal structures of the complex between myeloperoxidase and nitrite confirmed the binding of nitrite to the native enzyme both in the distal cavity and the chloride-binding site. The binding in the distal cavity occurred to the heme iron in the nitro mode. In the MPO-cyanide-nitrite ternary complex, nitrite had been shown to bind only at the chloride-binding site. No secondary site for nitrite binding had been seen in the distal cavity when cyanide was liganded to the iron. Overall, this study is the first to show from a crystallographic point of view a comparison in the mode of binding of the two hydroxamic acids to a mammalian peroxidase and also the binding of nitrite to a heme peroxidase.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 76-93).

Cadmium (Cd) is one of the major contaminants in agricultural soil, threatening agricultural production and human health. The objectives of this research work were to understand the tolerance mechanism in rice (Oryza sativa L.) genotype with more Cd-tolerance, and the relation between changes of peroxidases activities and peroxidase gene expression profiles after Cd-treated. For more, we analysis about cis-acting elements in the rice peroxidase genes promoter sequences region, gene structure of rice peroxidase genes and phylogenic relation among 9 peroxidase genes which were blasted from 6 Arabidopsis thaliana Cd-induced based on the peroxidase genes protein sequences. We used 9 upland and 32 varieties rice seeds as materials for germination and the growth of seedlings test with 50 or 500 £gM CdCl2 application, respectively. Rice seeds germination is a complex physiological and biochemical process, and is highly affected by cadmium. The results showed that Cd inhibited both the growth of radicles and coleoptiles. At germination stage, Cd highly inhibited the growth of upland rice. Among these rice varieties, Japonica type cultivars are more tolerant to Cd, but Indica type are more sensitive to Cd. Upland rice cultivars are the most sensitive to Cd. At seedling stage, Cd highly inhibited the growth of roots, but slightly inhibited the growth of shoots. To cope with Cd-induced stresses, plants adopt different strategies and posses a variety of defense mechanisms to prevent themselves from Cd damage. Peroxidase (POXs) is an important antioxidative enzyme for defense responses against Cd oxidative stress. The results suggested that different rice variety has a specific peroxidase gene expression pattern by the pI focusing electrophoresis after Cd-treated, and the peroxidase activities are significant differences when these rice varieties faced Cd stress. In this study, we searched the rice databases (japonica type) and cloned the promoter sequences of the indica type of the rice peroxidase genes, pI 4.5 POX and pI 5.1 POX genes. According to the search results about cis-acting elements from PLACE and PlantCARE databases, these cis-acting elements can be divided into three classes, showing as follow¡G1. Transcriptional related; 2. Light regulated related, and 3. Plant hormones- and stress-related. Based on all reported cis-acting elements, there are many types of transcription factors (TFs) involved in regulation of rice pI 4.5 POX and pI 5.1 POX genes expression. These TFs, which can recognize the specific cis-acting elements to regulate the gene expression, and will be induced by stresses and defense-related plant hormones. We found a cis-acting element (CURECORECR) which response to copper in both rice peroxidase genes promoter regions. There is a number of difference between Japonica type and Indica type peroxidase genes, japonica type peroxidase has more cis-acting elements than indica type. Plant class III peroxidases are present in all land plants. All land plant peroxidase genes are with the same putative ancestor of peroxidase genes and are orthologous genes, but they have specific functions of individual perxidase genes owing to their promoter sequences are very divergent. We used 6 Arabidopsis Cd-related peroxidases protein sequences as a starting point for rice peroxidase datas mining. We found 9 rice peroxidase genes have a closely relation among them. For more details about these rice peroxidase genes, searching each one of these rice peroxidases its gene structure on the Rice Genome Annotation Project (RGAP), comparison and their relation. The expressions of these peroxidase genes are very different among them after Cd treatment, and we also found the same cis-acting element (CURECORECR) which response to copper in all 9 rice peroxidase genes promoter regions. There is a number of difference among them.

Cd significantly inhibited the growth of both rice cultivars. The Tainung 67 cultivar is more tolerant to Cd than Taichung 1 cultivar after 48 h incubation in CdCl2 solution. The Cd tolerant cultivar¡XTainung 67¡¦s PODs in roots might synthesize more lignin in Cd-treatments. Meanwhile, the decrease of H2O2 levels is accompanied with the enhancement of POD activity in Cd-treated tissues. PODs here might also remove excess H2O2, thus serving detoxifying role and synthesizing more lignin for protection. In Taichung 1 cultivar, the accumulation of H2O2 in Cd-treated tissues could be due to the less amount of POD enhancement induced by Cd. In response to Cd treatment, the Taichung 1 cultivar also synthesizes little lignin, and therefore is Cd-sensitive.

Copper-treated rice seeding (Oryza sativa cv. Tainung 67 and Taichung native 1) showed significant inhibition in rice root growth, and an enhancement in POD activity. POD within Tainung 67 rice roots might synthesize more lignin in Cu-treated tissue. Meanwhile, the decrease of H2O2 levels is accompanied with the enhancement of POD activity in Cu-treated tissues. The increase in POD activity induced by Cu might remove excess hydrogen peroxide serving a detoxifying role and synthesizing more lignin for protection. In Taichung native 1 rice cultivar, high amounts of H2O2 accumulated in Cu-treated tissues could be due to the less amounts of POD induced by Cu. In response to Cu treatment, the Taichung native 1 cultivar also synthesizes a little lignin, and is more Cu-sensitive. Therefore, The Tainung 67 cultivar is more tolerant to Cu than Taichung native 1.

Cadmium-treated rice seeding (Oryza sativa L. cv. Taichung Native 1 and O. sativa L. cv. Tainung 67) showed inhibition in the growth of rice roots or leaves, and an enhancement in POX activity. In Tainung 67 cultivar, Cd treatment may have influence over cis-regulatory elements in POX promoter region and enhanced transcription of POX or enhance glycosylation of POX. The increase in POX activity induced by Cd might remove excess hydrogen peroxide serving a detoxifying role and synthesizing more lignin for protection. In Taichung Native 1 cultivar, high amounts of H2O2 accumulated in Cd-treated tissues could be due to the less amounts of POX induced by Cd. In response to Cd treatment, the Taichung Native 1 cultivar also synthesizes a little lignin, and is more Cd-sensitive. Therefore, the Tainung 67 cultivar is more tolerant to Cd than Taichung Native 1.

The adverse effect of Cd on growth is apparent from the reduction in root length of the Cd-treated Arabidopsis thaliana roots. The increase of the levels of H2O2 was observed in Cd-treated A. thaliana roots. The lignin biosynthesis related enzymes, POXs and laccases were enhanced during the Cd treatments. The lignin contents slightly increased in Cd-treated A. thaliana roots¡]48 h¡^. The A. thaliana can be tolerant to high concentration of Cd (500 µM), and only part of high levels of H2O2 accumulated in Cd-treated tissues are used by POXs to synthesize the lignin.

The adverse effect of Zn on growth is apparent from the reduction in root length of the Zn-treated Arabidopsis roots. The levels of H2O2 were increased rapidly in Zn-treated Arabidopsis roots. The lignin biosynthesis realated enzymes, peroxidases and laccases were enhanced during the Zn treatments. The lignin contents increased in Zn-treated Arabidopsis roots. Arabidopsis can be tolerant to high concentration of Zn (4 mM), because part of high levels of H2O2 accumulated in Zn-treated tissues are utilized by peroxidases to synthesize the lignin.

Copper (Cu) significantly inhibits the growth of radish (Raphanus sativus) seedlings, even at the concentration of 1 £gM. As far as the relationship between the growth of seedlings and peroxidase (POD) activity was concerned, the reduction of radish seedlings was correlated with the induction of cationic and anionic PODs. The data show that the increase of cationic PODs (pI 8.6 and pI 9.3) and anionic PODs (pI 5.1 and pI 3.5) activities was correlated with the rise of lignin contents in Cu-treated tissues. In our investigation, among the radish root PODs, the cationic pI 8.6 POD isozyme displayed a high affinity (Km of 57.9 £gM) for syringaldazine (an analog of lignin monomer) and the similar value of catalytic efficiency jointly with the anionic pI 5.1 POD, 0.14 £gM-1S-1 and 0.12 £gM-1S-1, respectively. The results suggest that the increase of cationic POD (pI 8.6) induced by Cu treatment might be responsible for the lignification in radish roots.