Thesis (Ph. D.)--University of Toronto, 2008. / Includes bibliographical references.

This dissertation focuses on dioecious angiosperms with a genetic system of sex determination based on a pair of heteromorphic sex chromosomes. Silene latifolia and S. dioica (Caryophyllaceae), with their X/Y mechanism and heterogametic males, have become model organisms for the study of genetic sex determination in angiosperms. Random amplified polymorphic DNA (RAPD) markers provide a valuable tool for the study of the genetic nature of the male determining Y chromosome. We first embarked on a search for Y chromosome RAPD markers using the breeding technique of bulked segregant analysis, obtaining 101 Y chromosome markers which together, were shown to characterize the two species. Genetic mapping placed one of these Y chromosome molecular markers in the pseudoautosomal region of the sex chromosomes. This finding provides a tool for the study of recombination rates among sex chromosomes and relative rates of evolution of X and Y chromosomes. Finally, we investigated the molecular nature of a highly conserved Y chromosome marker and looked for homologous sequences in other related dioecious and hermaphroditic species. The marker of choice had been found to be conserved across males from four species of Silene, section Elisanthe. It was cloned, sequenced and analyzed by Southern hybridization. This Y chromosome marker is a high copy sequence that shares homology to female DNA. Forward and reverse primers were designed to amplify the male specific band only. The amplification pattern of the resulting sequence characterized amplified region (SCAR) in related taxa provide evidence for: (1) a common ancestry of the Y chromosomes of dioecious Silene, section Elisanthe, (2) a different branch of the evolution of dioecy in section Otites, (3) the distant relationship of the hermaphrodite S. noctiflora to the dioecious members of section Elisanthe and (4) homology between the Y chromosome of dioecious Silene dioica and autosomes of hermaphroditic S. flos-cuculi.

Arsenic is an acute poison and its contamination in soil and water is widespread. Crambe abyssinica accumulates significantly higher levels of arsenic as compared to other species of the Brassicaceae. Being a non-food, high biomass crop that is naturally tolerant to heavy metals, crambe has significant potential for phytoremediation of arsenic. In order to identify the pathways involved in arsenic metabolism and detoxification in C. abyssinica, differentially expressed genes in response to arsenic exposure were isolated employing a PCR-Select Suppression Subtraction Hybridization approach. A total of 105 differentially expressed subtracted cDNAs were sequenced which were found to represent 38 genes. Those genes encode proteins functioning as antioxidants, metal transporters, reductases, enzymes involved in the protein degradation pathway, and several novel uncharacterized proteins. The differential expression of transcripts corresponding to the subtracted cDNAs was confirmed by the semi-quantitative RT-PCR. ^ Arabidopsis homologs of two uncharacterized proteins from this subtracted cDNA library were further characterized for their role in As detoxification in plants. One of these two genes, AtChaC2-1 functions as a gamma-glutamyl cyclotransferase as evident from in vivo studies in yeast as well as in Arabidopsis. It plays a significant role in glutathione homeostasis and participates in gamma-glutamyl cycle to recycle Glu. T-DNA insertion AtChaC2-1 mutant plants were tolerant to arsenic toxicity due to the elevated glutathione contents. AtChaC2-1 over-expression lines were also tolerant to As presumably due to more active gamma-glutamyl cycle and an efficient Glu recycling. Furthermore, AtChaC2-1 overexpression increased the N utilization efficiency as it decreased the de novo synthesis of Glu and thereby N assimilation. ^ A second gene, AtMATE21, is an efflux protein of MATE family of secondary transporters. Heterologous expression in yeast RM1 mutant strain decreased the As accumulation in yeast presumably by efficient effluxing of As from yeast cells. Arabidopsis plants with T-DNA insertional mutation in the AtMATE21 locus were sensitive to arsenate. The AtMATE21 over-expression lines were more tolerant to arsenate and accumulated a significantly higher amount of arsenic in the aboveground parts. Both AtChaC2-1 and AtMATE21 genes have significant potential to be utilized for developing plant-based strategies for arsenic mitigation in the environment.^