Functional genomics of O-glucosyltransferases from Concord grape (Vitis labrusca)

Grape (Vitis spp.) is a culturally and economically important crop plant that has been cultivated for thousands of years, primarily for the production of wine. Grape berries accumulate a myriad of phenylpropanoid secondary metabolites, many of which are glucosylated in plantae More than 90 O-glucosyltransferases have been cloned and biochemically characterized from plants, only two of which have been isolated from Vitis spp. The world-wide economic importance of grapes as a crop plant, the human health benefits associated with increased consumption of grape-derived metabolites, the biological relevance of glucosylation, and the lack of information about Vitis glucosyltransferases has inspired the identification, cloning and biochemical characterization of five novel "family 1" O-glucosyltransferases from Concord grape (Vitis labrusca cv. Concord). Protein purification and associated protein sequencIng led to the molecular cloning of UDP-glucose: resveratrollhydroxycinnamic acid O-glucosyltransferase (VLRSGT) from Vitis labrusca berry mesocarp tissue. In addition to being the first glucosyltransferase which accepts trans-resveratrol as a substrate to be characterized in vitro, the recombinant VLRSGT preferentially produces the glucose esters of hydroxycinnamic acids at pH 6.0, and the glucosides of trans-resveratrol and flavonols at 'pH 9.0; the first demonstration of pH-dependent bifunctional glucosylation for this class of enzymes. Gene expression and metabolite profiling support a role for this enzyme in the bifuncitonal glucosylation ofstilbenes and hydroxycinnamic acids in plantae A homology-based approach to cloning was used to identify three enzymes from the Vitis vinifera TIGR grape gene index which had high levels of protein sequence iii identity to previously characterized UDP-glucose: anthocyanin 5-0-glucosyltransferases. Molecular cloning and biochemical characterization demonstrated that these enzymes (rVLOGTl, rVLOGT2, rVLOGT3) glucosylate the 7-0-position of flavonols and the xenobiotic 2,4,5-trichlorophenol (TCP), but not anthocyanins. Variable gene expression throughout grape berry development and enzyme assays with native grape berry protein are consistent with a role for these enzymes in the glucosylation of flavonols; while the broad substrate specificity, the ability of these enzymes to glucosylate TCP and expression of these genes in tissues which are subject to pathogen attack (berry, flower, bud) is consistent with a role for these genes in the plant defense response. Additionally, the Vitis labrusca UDP-glucose: flavonoid 3-0-glucosyltransferase (VL3GT) was identified, cloned and characterized. VL3GT has 96 % protein sequence identity to the previously characterized Vitis vinifera flavonoid 3-0-glucosyltransferase (VV3GT); and glucosylates the 3-0-position of anthocyanidins and flavonols in vitro. Despite high levels of protein sequence identity, VL3GT has distinct biochemical characteristics (as compared to VV3GT), including a preference for B-ring methylated flavonoids and the inability to use UDP-galactose as a donor substrate. RT-PCR analysis of VL3GT gene expression and enzyme assays with native grape protein is consistent with an in planta role for this enzyme in the glucosylation of anthocyanidins,but not flavonols. These studies reveal the power of combining several biochemistry- and molecular biology-based tools to identify, clone, biochemically characterize and elucidate the in planta function of several biologically relevant O-glucosyltransferases from Vitis spp.

A comparative study of chitin synthase activity in two Mortierella species

An in vitro investigation of some important factors controlling the activity of chitin synthase in cell-free extracts of two Mortierella species has been carried out. Mixed membrane fractions from mycelial homogenates of Mortierella candelabrum and Mortierella pusilla were found to catalyse the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine into an insoluble product characterized as chitin by its insolubility in weak acid and alkali, and the release of glucosamine and diacetylchitobiose on hydrolysis with a strong acid and chitinase, respectively. Apparent Km values for UDP-GlcNAc were 1.8 mM and 2.0 mM for M. pusilla and ~ candelabrum, respectively. Polyoxin D was found to be a very potent competitive inhibitor with values of the constant of inhibition, Ki' for both species about three orders of magnitude lower than theKm for UDP-GlcNAc. A divalent cation, Mg+2 , Mn+2 or Co+2 , was required for activity. N-acetylglucosamine, the monomer of chitin, stimulated the activity of the enzyme. The crude enzyme preparation of ~ candelabrum, unlike that of ~ pusilla, showed an absolute requirement for both Mg+2 and N-acetylglucosamine. Large differences in response to exogenous proteases were noted in the ratio of active to inactive chitin synthase of the two species. A fifteen fold or greater increase was obtained after treatment with acid protease (from Aspergillussaitoi) as compared to a two- to four-fold activation of the M. pusilla membrane preparation treated similarly. During storage at 4°C over 48 hours, an endogenous activation of chitin synthase of ~ pus ilIa was achieved, comparable to that obtained by exogenous protease treatment. The high speed supernatant of both species inhibited the chitin synthase activity of the mixed membrane fractions. The inhibitor of ~ pus ilIa was effective against the pre-activated enzyme whereas that of M. candelabrum inhibited the activated enzyme. Several possibilities are discussed as to the role of the different factors regulating the enzyme activity. The suggestion is made from the properties of chitin synthase in the two species that in vivo a delicate balance exists between the activation and inactivation of the enzyme which is responsible for the pattern of wall growth of each fungus.

A comparative study of in vitro chitin synthase activity in mucoraceous hosts of a mycoparasite

A comparative study of in vitro chitin synthase activity in mucoraceous hosts of a mycoparasite: Chitin synthase, the enzyme responsible for the synthesis of chitin in fungal cell wall was extracted from young hyphae of Choanephora cucurbitarum and Phascolomyces articulosus, susceptible and resistant hosts, respectively, to the mycoparasite, Piptocephalis virginiana. Crude enzyme was identified and characterized by measuring the incorporation of the substrate [14C]-UDP-N-acetylglucosamine, into chitin. Most activity occurred in mixed membrane fraction. Inhibition of activity with Polyoxin D and activation with proteases, N-acetyl-glucosamine and magnesium and other ions was observed. Properties of the crude enzyme preparation such as cofactor requirement, Vmax , apparent Km value for UDP-GlcNAc, inhibition by Polyoxin D, response to pH and to temperature, and stability at 4°C were determined. Enzyme activity from both fungi displayed basically the same features as the corresponding enzymes reported from other mucoraceous fungi. However, the two preparations from P. articulosus and C. cucurbitarum differed from each other in their expressed activity (i.e., the preparations from ~ articulosus exhibited higher latency and higher specific chitin synthase activity than the corresponding preparations from ~ cucurbitarum). Trypsin was effective in activation only over a narrow concentration range. Acid protease was the most effec.tive activator. En.dogenous protease estimation indicated higher protease activity in C. cucurbitarum than in P. articulosus. The suggestion is made that regulation of chitin synthase activities may be related to host resistance in the mycoparasitic system.

Office of the City Clerk, St. Catharines Records, 1928-1974, n.d.

The site of present-day St. Catharines was settled by 3000 United Empire Loyalists at the end of the 18th century. From 1790, the settlement (then known as "The Twelve") grew as an agricultural community. St. Catharines was once referred to Shipman's Corners after Paul Shipman, owner of a tavern that was an important stagecoach transfer point. In 1815, leading businessman William Hamilton Merritt abandoned his wharf at Queenston and set up another at Shipman's Corners. He became involved in the construction and operation of several lumber and gristmills along Twelve Mile Creek. Shipman's Corners soon became the principal milling site of the eastern Niagara Peninsula. At about the same time, Merritt began to develop the salt springs that were discovered along the river which subsequently gave the village a reputation as a health resort. By this time St. Catharines was the official name of the village; the origin of the name remains obscure, but is thought to be named after Catharine Askin Robertson Hamilton, wife of the Hon. Robert Hamilton, a prominent businessman. Merritt devised a canal scheme from Lake Erie to Lake Ontario that would provide a more reliable water supply for the mills while at the same time function as a canal. He formed the Welland Canal Company, and construction took place from 1824 to 1829. The canal and the mills made St. Catharines the most important industrial centre in Niagara. By 1845, St. Catharines was incorporated as a town, with the town limits extending in 1854. Administrative and political functions were added to St. Catharines in 1862 when it became the county seat of Lincoln. In 1871, construction began on the third Welland Canal, which attracted additional population to the town. As a consequence of continual growth, the town limits were again extended. St. Catharines attained city status in 1876 with its larger population and area. Manufacturing became increasingly important in St. Catharines in the early 1900s with the abundance of hydro-electric power, and its location on important land and water routes. The large increase in population after the 1900s was mainly due to the continued industrialization and urbanization of the northern part of the city and the related expansion of business activity. The fourth Welland Canal was opened in 1932 as the third canal could no longer accommodate the larger ships. The post war years and the automobile brought great change to the urban form of St. Catharines. St. Catharines began to spread its boundaries in all directions with land being added five times during the 1950s. The Town of Merritton, Village of Port Dalhousie and Grantham Township were all incorporated as part of St. Catharines in 1961. In 1970 the Province of Ontario implemented a regional approach to deal with such issues as planning, pollution, transportation and services. As a result, Louth Township on the west side of the city was amalgamated, extending the city's boundary to Fifteen Mile Creek. With its current population of 131,989, St. Catharines has become the dominant centre of the Niagara region. Source: City of St. Catharines website http://www.stcatharines.ca/en/governin/HistoryOfTheCity.asp (January 27, 2011)

Receipt to S.D. Woodruff from Gibb and Co.

Receipt to S.D. Woodruff from Gibb and Co. of Montreal, Merchants, Tailors and Gentlemen’s Haberdashers for a cloth sack coat, July 1876.