Onion (Allium cepa L.) is an example of a species that accumulates very high levels of reduced sulfur (S)-containing compounds, particularly in the bulb as alk(en)yl-L-cysteine-sulfoxides (ACSOs) and it is these compounds, or their derivatives, that confers the distinct odour and pungent flavour. In common with higher plants, the S assimilation pathway in onion begins with the activation of uptaken sulfate (SO4 2-) to 5'-adenylylsulfate (APS), a reaction catalysed by ATP sulfurylase (ATPS; EC 2.7.7.4). Then, APS is reduced to sulfide (S2-) in a two-step process catalysed by the enzymes APS reductase (APSR; EC 1.8.4.9) and sulfite reductase (SiR; EC 1.8.7.1). To complete the reductive assimilation pathway, S2- is incorporated into the amino acid skeleton of O-acetylserine (OAS) to form cysteine, and this reaction is catalyzed by OAS (thiol)-lyase (OAS-TL; EC 4.2.99.8). While the regulation of the pathway is quite well defined in the plant model Arabidopsis, much less is known about its regulation in S accumulating species such as onion. The primary aim of this thesis, therefore, was to characterise the enzymes of the S assimilation pathway in onion, with a particular emphasis on ATPS. As part of this charaterisation two genotypes of onion were compared. These comprised a mild genotype, 'Texas Grano 438' (TG) with a lower level of S-containing compounds in the bulb tissues, and 'W202A' (W), a cultivar with a higher level of S containing compounds in the bulb tissues. As well, comparisons were made between seedlings (typically harvested at 7 weeks) and plants at a designated mature stage (at bulbing; typically after 4 months growth), and for plants grown in S-sufficient (S+) media or S-deficicnt (S-) media, as appropriate. In terms of plant growth, S-deprivation generally had a negative influence for both genotypes, with significant reductions in total biomass (measured as fresh weight) for TG at both the seedlings and mature stages. ATPS activity and accumulation were shown to be present in all tissues examined (leaf, root, bulb) as well as the chloroplasts, with highest activity measured in the roots, particularly in seedlings. ATPS activity and accumulation were also compared between the two genotypes (TG and W) with ATPS activity and accumulation higher in W, particularly at the seedling stage. In terms of the influence of S supply, in general higher ATPS activity was measured in chloroplast, leaf and root extracts from plants of both genotypes grown in the S- media, at the seedling stage. In roots of mature plants of both genotypes, a significant increase in activity was measured in response to S-deprivation, while in chloroplasts isolated from mature plants of both genotypes, highest activity was measure in those grown in the S+ media. Finally diurnal variations were observed in chloroplast, leaf and root extracts of both genotypes with a general trend of an increase in ATPS activity and accumulation a few hours after illumination and upon the onset of the dark period. Although a single gene coding for ATPS is presumed to be present in onion, the enzyme was characterized as two electrophoretic forms using 1D-PAGE during western analyses following fractionation of chloroplasts by anion exchange chromatography and also as an alignment of spots using 2D-PAGE. As protease inhibitors were routinely included in the extraction buffers, these forms suggest the occurrence of ATPS isoforms that may arise as a consequence of post-translational modifications. The regulation of ATPS by one mechanism of post-translational modification, phosphorylation, was therefore investigated using several techniques including the detection of a shift in molecular mass, a change in enzyme activity or pI (as determined by 2D-PAGE) and the capability to bind to 14-3-3 proteins using affinity chromatography. Following treatments of chloroplast extracts to promote either the phosphorylation (P+) or the dephosphorylation (P-) of proteins, no molecular mass change or change in activity was observed. However, after fractionation by 2D-PAGE, differences in the spot alignment of ATPS were visualized, suggesting that ATPS is a phosphoprotein. The enzyme was detected in pull-downs after affinity chromatography, suggesting that ATPS may also interact with 14-3-3 proteins (although this needs to be confirmed unequivocally). A model is advanced, therefore, in which upon phosphorylation, no variation in ATPS activity occurs but a change in the surface charged and possibly a change in conformation of the protein does occur to make the enzyme competent to interact with 14-3-3 proteins.

Mercury (Hg) is a global pollutant and potent neurotoxin that bioaccumulates in aquatic and terrestrial food webs as monomethylmercury (MeHg). Anaerobic microbes are largely responsible for MeHg production, which depends on the bioavailability of inorganic Hg substrates to methylators. Hg redox cycling pathways such as Hg reduction play a key role in determining Hg’s availability in the environment. Although abiotic photochemical Hg reduction typically dominates in oxic surface environments, Hg reduction pathways mediated by photosynthetic and anaerobic microbes are thought to play an important role in anoxic habitats where light is limited and MeHg production occurs. Currently, the physiological mechanisms driving phototrophic and anaerobic Hg reduction remain poorly understood. The main objective of my thesis is to provide mechanistic details on novel anaerobic and phototrophic Hg reduction pathways. I used a combination of physiological, biochemical and trace Hg analytical techniques to study Hg reduction pathways in a variety of anaerobic and photosynthetic bacteria. I demonstrated that Hg redox cycling was directly coupled to anoxygenic photosynthesis in aquatic purple non-sulphur bacteria that reduced HgII when cells incurred a redox imbalance. I discovered that terrestrial fermentative bacteria reduced Hg through pathways that relied on the generation of reduced redox cofactors. I also showed that sulphur assimilation controlled Hg reduction in an anoxygenic phototroph isolated from a rice paddy. In addition, I developed methods to explore cryptic anaerobic Hg redox cycling pathways using Hg stable isotope fractionation. At its core, my thesis underscores the intimate relationship between cell redox state and microbial Hg reduction and suggests a wide diversity of microbes can participate in anaerobic Hg redox cycling.