published_or_final_version / Botany / Doctoral / Doctor of Philosophy

For the successful development of a hyperaccumulating plant sufficient for use in phytoremediation strategies, a thorough understanding of the mechanism of hyperaccumulation is required. A proteomic survey of the response of plants to metal exposure is a step towards this understanding. The frd3-3 metal accumulating mutant of Arabidopsis thaliana and its non-accumulating wildtype parental ecotype, Columbia, were grown hydroponically in growth chamber experiments and exposed to copper in the growth medium. The responses of the global and copper-targeted proteomes were examined both spatially and temporally. Exposure to copper caused a general increase in protein abundance, however, a prolonged exposure to copper that approached toxicity caused a decrease in protein abundance. The protein species differed between the roots of the two genotypes, with more defense- and stress-related proteins, and fewer transport and storage proteins identified in the mutant when compared to the wildtype. Proteomic evidence suggests that in the mutant the uptake and transport of copper ions to the aerial tissues is regulated. The protein expression patterns over time demonstrate a constitutive expression of defense- and stress-related proteins in the mutant, whereas the wildtype expression was one of induction. The constitutive expression of key defense proteins suggests a state-of-readiness for metal exposure in the mutant. The plant response to reactive oxygen species, as a consequence of copper exposure, is important in the overall metal accumulation mechanism. A suppression of the oxidative burst produced upon exposure to heavy metals is suggested by the proteomic evidence.

For the successful development of a hyperaccumulating plant sufficient for use in phytoremediation strategies, a thorough understanding of the mechanism of hyperaccumulation is required. A proteomic survey of the response of plants to metal exposure is a step towards this understanding. The frd3-3 metal accumulating mutant of Arabidopsis thaliana and its non-accumulating wildtype parental ecotype, Columbia, were grown hydroponically in growth chamber experiments and exposed to copper in the growth medium. The responses of the global and copper-targeted proteomes were examined both spatially and temporally. Exposure to copper caused a general increase in protein abundance, however, a prolonged exposure to copper that approached toxicity caused a decrease in protein abundance. The protein species differed between the roots of the two genotypes, with more defense- and stress-related proteins, and fewer transport and storage proteins identified in the mutant when compared to the wildtype. Proteomic evidence suggests that in the mutant the uptake and transport of copper ions to the aerial tissues is regulated. The protein expression patterns over time demonstrate a constitutive expression of defense- and stress-related proteins in the mutant, whereas the wildtype expression was one of induction. The constitutive expression of key defense proteins suggests a state-of-readiness for metal exposure in the mutant. The plant response to reactive oxygen species, as a consequence of copper exposure, is important in the overall metal accumulation mechanism. A suppression of the oxidative burst produced upon exposure to heavy metals is suggested by the proteomic evidence.

All plants are exposed to abiotic and biotic stresses and have developed intricate signalling responses to survive. They respond to cell-structure disruption caused by herbivore probing and feeding by the formation of callose. Callose is a linear homopolymer made up of β-1,3-linked glucose residues with some β-1,6-branches. Plant responses to abiotic or biotic stress share events such as phosphorylation, membrane depolarization, calcium influx and the release of reactive oxygen species such as hydrogen peroxide. These events lead to the up-regulation of several pathways leading to biosynthesis of signalling molecules such as salicylic acid, jasmonate, abscisic acid and ethylene pathways. The aim of this study was to determine the most suitable proteomic approach for identifying proteins and signalling pathways involved in cereal response to aphid infestation. An in silico approach was first evaluated in which the 5ʹ upstream regulatory region of proteins belonging to the family of callose synthases was scanned for cis-regulatory elements in order to identify which callose synthases are possibly expressed in plants during biotic or abiotic stresses. Bioinformatics tools were used in the identification of twelve Arabidopsis and ten rice callose synthase coding regions. Genome sequences for rice and Arabidopsis were scanned for the 2000 bp 5ʹ region upstream of the start codon of each callose synthase coding region. PlantCare, PLACE and Athena software were used to identify putative cis-regulatory elements present in the 2000 bp 5ʹ upstream sequences. The majority of cis-acting elements identified were involved in drought and high temperature responses and only one cis-acting element was involved in wound stress. These results therefore indicated a probable role for plant callose synthases in drought stress responses rather than in biotic stress responses. Genevestigator analysis of Arabidopsis results of micro-array experiments indicated that AtGSL10 is highly up-regulated, with AtGSL1, 3, 5, 6, 7, 8, 11 and 12 showing medium up-regulation and AtGSL2, 4 and 9 no up-regulation during aphid infestation of Arabidopsis plants, implicating a possible role for AtGSL10 in the plant response to aphid infestation. An LC/MS/MS approach was used to identify specific signalling pathways involved in wheat resistance or stress response to aphid infestation. Eight proteins were identified as being up-regulated during aphid feeding in wheat, and 11 proteins were identified as possibly involved in the wheat resistance mechanism against aphid infestation. Several proteins were also identified as constitutively expressed proteins, during normal conditions and aphid infestation. Most pathways identified with proteins up-regulated in the resistance mechanisms of TugelaDN plants, were related to energy metabolism and located in the chloroplast. Evaluation of two dimensional gel electrophoresis to identify phosphoproteins differentially regulated in wheat during aphid infestation, revealed the up-regulation of three proteins namely photosystem II oxygen-evolving complex protein 2, HVUNKNOWN from Hordeum vulgare subsp vulgare and HSKERAT9 NID from Homo sapiens. Additional 57 proteins were partially identified as involved in the stress response but due to low protein levels, the percentage of matching peptides to these proteins was below the required confidence level. Although these protein identifications were below the confidence level, it is interesting to note that several of the proteins are known stress response proteins, and therefore could serve as potential targets for future investigations. In conclusion, the down and up-regulation of wheat proteins after aphid feeding reported in this study suggest that several signalling pathways are involved in the cereal stress response to aphid feeding. In silico approaches require knowledge or identification of potential proteins whereas 2D and LC/MS can identify numerous proteins still unknown to be involved in specific stress responses. The 2D approach is also limited in that the proteins of interest may be in low abundance and therefore not detected in the gels due to the presence of high abundant proteins. Therefore the best approach to identify proteins and signalling pathways involved in the stress response of wheat to aphid infestation, is the LC/MS/MS approach, as this proved to be the most sensitive and robust, identifying the most proteins with a high degree of confidence.

WRKY transcription factors (TFs) belong to a large family of regulatory proteins in plants that modulate many plant processes. Extensive studies have been conducted on WRKY-mediated defense response in Arabidopsis thaliana and many crop species. This study aims to investigate the potential roles and contributions of WRKY TFs regulation in improving defense response in the resynthesized Arabidopsis allotetraploids (Arabidopsis suecica) from two related autotetraploid progenitors, Arabidopsis thaliana (At4) and Arabidopsis arenosa (Aa). Upon infection by Pseudomonas syringae (Pst), the allotetraploids has showed enhanced resistance against the pathogen when compared to the parents. Rapid induction of WRKY18, WRKY40, WRKY38, WRKY53, WRKY6; MAP kinase pathway related genes, WRKY33, PAD3; SA-pathway related genes, ICS1, EDS1, PBS3, MYB31; was evident in response to Pst and salicylic acid treatment in the allotetraploids. Cleaved amplified polymorphic sequences analysis further revealed that the AtWRKY18, AaWRKY40, AtWRKY33, and AtWRKY60 alleles expressed at higher levels when compared to their respective homoeologs in the allotetraploids, suggesting potential altered protein-protein interaction networks in the hybrids. Therefore, a split-luciferase complementation assay was used to characterize and quantify protein-protein interaction among these homoeologous WRKYs in the allotetraploids. Results showed that preferential protein-protein interactions exist for the cis-interacting AtWRKY18/AtWRKY18 homodimer or trans-interacting AtWRKY18/AaWRKY40 heterodimer when compared to the respective interacting complexes. In addition, differential affinities of WRKY18 and WRKY40 homo- and hetero- dimers toward the W-boxes at the WRKY60 promoter were observed. In the allotetraploids, PR1 expression was repressed under basal state when compared to the progenitors. Although PR1 is expressed at a higher level in A. thaliana, its expression fold change was higher and faster in the all otetraploids upon salicylic acid treatment. Transient expression of WRKY18 or WRKY40 homodimer in various combinations induced differential expression of PR1 gene in their respective wrky18 and wrky40 Arabidopsis thaliana mutants. In contrast, similar PR1 induction by homodimer in various combinations was observed when they were transiently expressed in the allotetraploids. In addition, transgenic AtWRKY18 overexpression plant displayed enhanced disease resistance against Pst when compared to AaWRKY18 overexpression lines. Such enhanced disease resistance was found to associate with the higher expression of PR1 and PR2 in AtWRKY18 transgenic lines. Moreover, differential Pst-induced expression of the direct targets (ICS1, EDS1 and PBS3) of WRKY18 in the Arabidopsis AtWRKY18 and AaWRKY18 overexpressors supported a biological difference between the At and Aa homodimers in mediating the targets regulation, thus contributing to the difference in disease responses. Overall, our findings suggested that the rapid differential alleles expression and altered protein-protein or protein-DNA interactions of WRKY transcription factors could contribute to the improved defense in the allotetraploids, providing a molecular basis of for heterotic phenotype development in hybrids.

Thesis (Ph.D.)--York University, 2003. Graduate Programme in Biology. / Typescript. Includes bibliographical references. Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://wwwlib.umi.com/cr/yorku/fullcit?pNQ99195

published_or_final_version / Chemistry / Master / Master of Philosophy

Histone modifications and histone variants are of importance in many biological processes. Whether they play some roles in regulating soybean salinity stress response is unknown. Previously, no study of histone modifications and histone variants in soybean were reported. In this study, I elucidated that in soybean leaves, mono-, di- and tri-methylation at Lysine (K) 4, 27 and 36, and acetylation at Lysine 14, 18 and 23 were present in histone H3. Moreover, H3K27 methylation and H3K36 methylation usually excluded each other. Although H3K79 methylation was not reported in Arabidopsis, they were detected in soybean. In soybean histone H4, Lysine 8 and 12 were acetylated. In addition, the variants of histone H3 and H4 and their modifications were also determined. The variants of histone H3 were different at positions of A31F41S87S90 (histone variant H3.1) and T31Y41H87 L90 (histone variant H3.2), respectively. Lysine 4 and 36 methylation were only detected in histone H3.2, suggesting that histone variant H3.2 might associate with actively transcribing genes. The two variants of histone H4 (H4.1 and H4.2) were different at amino acid 60. Moreover, I also found that the abundance of most of the histone modifications and histone variants did not change under the salinity stress except that H3K79 methylation would be up-regulated by the salinity stress. / In a parallel study, a PHD (plant homeodomain) finger domain containing protein, GmPHD1, was able to decipher the 'code' underlying H3K4 methylation. GmPHD1 was ubiquitously expressed in soybean and its expression increased upon salinity stress. GmPHD1 could bind to histone H3K4 methylation, with the preference to H3K4 dimethylation. It could then recruit several proteins, which were GmGNAT1, GmElongin A, and GmISWI. The interaction between GmPHD1 and GmGNAT1 was regulated by the self-acetylation of GmGNAT1. GmGNAT1 could also acetylate histone H3; GmElongin A was a transcription elongation factor; and GmISWI was a chromatin remodeling protein. Our data also indicated that the GmPHD1 located at the promoter of several soybean salt stress inducible genes. Therefore, the GmPHD1 recruited proteins to remodel the chromatin structure and facilitate the transcription of those salt stress inducible genes. Moreover, GmGNAT1 exhibited the preference to acetylate histone H3K14, therefore representing a kind of histone crosstalk between H3K4 methylation and H3K14 acetylation. / Proteomics studies with 2-DE revealed that salt treatment may affect soybean photosynthesis and chloroplast formation. Comparison between the proteomic profiles of salt tolerant soybean variety (wild type) and salt sensitive soybean variety (cultivated, Union) indicated that protein levels in the detoxification and defense pathway as well as energy metabolism were higher in the wild type soybean, while the process of protein metabolism was less active. In addition, proteomic profiles of the cultivated soybean roots at different developmental stages were also compared to identify proteins related to soybean development. The expression of proteins which play critical roles in detoxification and defense pathways were higher at the seedling stage, especially the proteins which regulated the formation of ROS. / Soybean is an important economic crop and its production can be severely affected by salinity stress. At present, the soybean response to salinity stress is not clear. In my studies, I tried to understand this process from the perspective of proteomics and epigenetics, especially histone modifications. / Wu, Tao. / Advisers: Njai Sai Ming; Lam Hon Ming. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 126-151). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Thesis (M.S.)--University of Hawaii at Manoa, 2006. / Includes bibliographical references (leaves 103-108). / 108 leaves, bound col. ill. 29 cm

Prof. I.A. Dubery