Abrasion, transport and distribution of sediment in selected streams of Southern Ontario and Western New York /

The rate of decrease in mean sediment size and weight per square metre along a 54 km reach of the Credit River was found to depend on variations in the channel geometry. The distribution of a specific sediment size consist of: (1) a transport zone; (2) an accumulation zone; and (3) a depletion zone. These zones shift downstream in response to downcurrent decreases in stream competence. Along a .285 km man-made pond, within the Credit River study area, the sediment is also characterized by downstream shifting accumulation zones for each finer clast size. The discharge required to initiate movement of 8 cm and 6 cm blocks in Cazenovia Creek is closely approximated by Baker and Ritter's equation. Incipient motion of blocks in Twenty Mile Creek is best predicted by Yalin's relation which is more efficient in deeper flows. The transport distance of blocks in both streams depends on channel roughness and geometry. Natural abrasion and distribution of clasts may depend on the size of the surrounding sediment and variations in flow competence. The cumulative percent weight loss with distance of laboratory abraded dolostone is defined by a power function. The decrease in weight of dolostone follows a negative exponential. In the abrasion mill, chipping causes the high initial weight loss of dolostone; crushing and grinding produce most of the subsequent weight loss. Clast size was found to have little effect on the abrasion of dolostone within the diameter range considered. Increasing the speed of the mill increased the initial amount of weight loss but decreased the rate of abrasion. The abrasion mill was found to produce more weight loss than stream action. The maximum percent weight loss determined from laboratory and field abrasion data is approximately 40 percent of the weight loss observed along the Credit River. Selective sorting of sediment explains the remaining percentage, not accounted for by abrasion.

The Catharanthus roseus 16-hydroxytabersonine O-methyltransferase involved in vindoline biosynthesis /

Madagascar periwinkle (Catharanthus roseus) produces the well known and remarkably complex dimeric anticancer alkaloids vinblastine and vincristine that are derived by coupling vindoline and catharanthine monomers. This thesis describes the novel application of carborundum abrasion (CA) technique as a tool for large scale isolation of leaf epidermis enriched proteins. This technique was used to facilitate the purification to apparent homogeneity of 16-hydroxytabersonine-16-0-methyltransferse (l60MT) that catalyses the second step in the 6 step pathway that converts tabersonine into vindoline. This versatile tool was also used to harvest leaf epidermis enriched mRNAs that facilitated the molecular cloning of the 160MT. Functional expression and biochemical characterization of recombinant 160MT enzyme showed that it had a very narrow substrate specificity and high affinity for 16-hydroxytabersonine, since other closely related monoterpene indole alkaloids (MIAs) did not act as substrates. In addition to allowing the cloning of this gene, CA technique clearly showed that 160MT is predominantly expressed in Catharanthus leaf epidermis, in contrast to several other OMTs that appear to be expressed in other Catharanthus tissues. The results provide compelling evidence that most of the pathway for vindoline biosynthesis including the 0- methylation of 16-hydroxytabersonine occurs exclusively in leaf epidermis, with subsequent steps occurring in other leaf cell types. Small molecule O-methyltransferases (OMTs) (E.C. 2.1.1.6.x) catalyze the transfer of the reactive methyl group of S-adenosyl-L-methionine (SAM) to free hydroxyl groups of acceptor molecules. Plant OMTs, unlike their monomeric mammalian homologues, exist as functional homodimers. While the biological advantages for dimer fonnation with plant OMTs remain to be established, studies with OMTs from the benzylisoquinoline producing plant, Thalictrum tuberosum, showed that co-expression of 2 recombinant OMTs produced novel substrate specificities not found when each rOMT was expressed individually (Frick, Kutchan, 1999) . These results suggest that OMTs can fonn heterodimers that confer novel substrate specificities not possible with the homodimer alone. The present study describes a 160MT model based strategy attempting to modify the substrate specificity by site-specific mutagenesis. Our failure to generate altered substrate acceptance profiles in our 160MT mutants has lead us to study the biochemical properties ofhomodimers and heterodimers. Experimental evidence is provided to show that active sites found on OMT dimers function independently and that bifunctional heterodimeric OMTs may be fonned in vivo to produce a broader and more diverse range of natural products in plants.

Localization and functional characterization of iridoid biosynthetic genes in Catharanthus roseus /

Catharanthus roseus is the sole biological source of the medicinal compounds vinblastine and vincristine. These chemotherapeutic compounds are produced in the aerial organs of the plant, however they accumulate in small amounts constituting only about 0.0002% of the fresh weight of the leaf. Their limited biological supply and high economical value makes its biosynthesis important to study. Vinblastine and vincristine are dimeric monoterpene indole alkaloids, which consists of two monomers vindoline and catharanthine. The monoterpene indole alkaloids (MIA's) contain a monoterpene moiety which is derived from the iridoid secologanin and an indole moiety tryptamine derived from the amino acid tryptophan. The biosynthesis of the monoterpene indole alkaloids has been localized to at least three cell types namely, the epidermis, the laticifer and the internal phloem assisted parenchyma. Carborundum abrasion (CA) technique was developed to selectively harvest epidermis enriched plant material. This technique can be used to harvest metabolites, protein or RNA. Sequencing of an expressed sequence tagged (EST) library from epidermis enriched mRNA demonstrated that this cell type is active in synthesizing a variety of secondary metabolites namely, flavonoids, lipids, triterpenes and monoterpene indole alkaloids. Virtually all of the known genes involved in monterpene indole alkaloid biosynthesis were sequenced from this library.This EST library is a source for many candidate genes involved in MIA biosynthesis. A contig derived from 12 EST's had high similarity (E'^') to a salicylic acid methyltransferase. Cloning and functional characterization of this gene revealed that it was the carboxyl methyltransferase imethyltransferase (LAMT). In planta characterization of LAMT revealed that it has a 10- fold enrichment in the leaf epidermis as compared to the whole leaf specific activity. Characterization of the recombinant enzyme revealed that vLAMT has a narrow substate specificity as it only accepts loganic acid (100%) and secologanic acid (10%) as substrates. rLAMT has a high Km value for its substrate loganic acid (14.76 mM) and shows strong product inhibition for loganin (Kj 215 |iM). The strong product inhibition and low affinity for its substrate may suggest why the iridoid moiety is the limiting factor in monoterpene indole alkaloid biosynthesis. Metabolite profiling of C. roseus organs shows that secologanin accumulates within these organs and constitutues 0.07- 0.45% of the fresh weight; however loganin does not accumulate within these organs suggesting that the product inhibition of loganin with LAMT is not physiologically relevant. The limiting factor to iridoid and MIA biosynthesis seems to be related to the spatial separation of secologanin and the MIA pathway, although secologanin is synthesized in the epidermis, only 2-5% of the total secologanin is found in the epidermis while the remaining secologanin is found within the leaf body inaccessable to alkaloid biosynthesis. These studies emphasize the biochemical specialization of the epidermis for the production of secondary metabolites. The epidermal cells synthesize metabolites that are sequestered within the plant and metabolites that are secreted to the leaf surface. The secreted metabolites comprise the epidermome, a layer separating the plant from its environment.

An investigation of geological and geochemical characteristics of late-Quaternay sediments in the Georgian Bay Region, Southern Ontario

Core samples of postglacial sediments and sediment surface samples from Shepherd Lake on the Bruce Peninsula, Harts Lake on the Canadian Shield, and two cores from Georgian Bay (core P-l in the western deep part and core P-7 in the eastern shallow part) have been analyzed for pH, grain size distribution, water content, bulk density, loss on ignition at 4500C and 11000 C, major oxides (Si02 ,A1203,!FeO,MgO,CaO, Na20,K20,Ti02 ,MnO and P205) and trace elements (Ba,Zr,Sr,y,S, Zn,Cu,Ni,Ce and Rb). The sediment in Georgian Bay are generally fine grained (fine silt to very fine silty clay) and the grain size decreases from the Canadian Shield (core p-7) towards the Bruce Peninsula (core P-l) along the assumed direction of sediment transport. This trend coincides with a decrease in sorting coefficient and an increase in roundness. Other physical characteristics, such as water content, bulk density and loss on ignition are positively correlated with the composition of sediments and their compaction, as well as with the energy of the depositional environment. Analyses of sediment surface samples from Shepherd Lake and Harts Lake indicate the influence of bedrock and surficial deposits in the watershed on pH condition that is also influenced by the organic matter content and probably I ! I man's activities. Organic matter content increases significantly in the surface sediment in these small lakes as a result of either natural eutrophication or anthropogenic organic loading. The extremely high organic matter content in Shepherd Lake sediment indicates rapid natural eutrophication in this closed basin and high biological productivity during postglacial time, probably due to high nutrient levels and shallow depth. The chemical composition of the Canadian Shield bedrock is positively correlated with the chemical characteristics of predominantly inorganic lake sediments that were derived from the Shield rocks by glacial abrasion and by postglacial weathering and erosion of both bedrock and surficial deposits. High correlation coefficients were found between organic matter in lake sediments and major oxides (Si02,AI203,.~FeO, MgO,CaO,K20 and MnO) , as well as some trace elements (Ba,Y, S,Zn,Cu,Ni and Rb). The chemical composition of sediments in Harts Lake and core P-7 in Georgian Bay on the Canadian Shield differs from the chemistry of sediments in Shepherd Lake and core P-l in Georgian Bay on the Bruce Peninsula. The difference between cores P-l and P-7 is indicated by values of Si02 , AI203 ,:LFeo,Mgo,CaO,Ba,Zr,Sr,y and S, and also by the organic matter content. This study indicates that the processes of sediment transport, depositional environment, weathering of the rocks and surficial deposits in the watershed, as well as chemical composition of source rocks all affect the chemical characteristics of lake sediments. The stratigraphic changes and variations in lake sediment chemistry with regard to major oxides, trace elements, and organic matter content are probably related to the history of glacial and postglacial lake stages of the Georgian Bay Region and, therefore, the geochemical data can make a useful contribution to a better understanding of the Late-Quaternary history of the Great Lakes.