The study of ligand binding specificities of the lipid binding proteins : recombinant human a-tocopherol transport protein (a-ttp), supernatant protein factor (spf) and S. cerevisiae Sec 14p for vitamin e (rrr-a-tocopherol) and other hydrophobic ligands.

One of the various functions of proteins in biological systems is the transport of small molecules, for this purpose proteins have naturally evolved special mechanisms to allow both ligand binding and its subsequent release to a target site; a process fundamental to many biological processes. Transport of Vitamin E (a-tocopherol), a lipid soluble antioxidant, to membranes helps in the protection of polyunsaturated fatty acids against peroxidative damage. In this research, the ligand binding characteristics of several members of the CRALTRIO family of lipid binding proteins was examined; the recombinant human a-Tocopherol Transfer Protein (a-TIP), Supernatant Protein Factor (SPF)ffocopherol Associated Protein (TAP), Cellular Retinaldehyde Binding Protein (CRALBP) and the phosphatidylinositol transfer protein from S. cerevisiae Sec 14p. Recombinant Sec 14p was expressed and purified from E. coli for comparison of tocopherol binding to the two other recombinant proteins postulated to traffic a-tocopherol. Competitive binding assays using [3H]-a-tocopherol and Lipidex-l000 resin allowed determination of the dissociation constants ~) of the CRAL-TRIO proteins for a-tocopherol and - 20 hydrophobic ligands for evaluation of the possible biological relevance of the binding interactions observed. The KIs (nM) for RRR-a-tocopherol are: a-TIP: 25.0, Sec 14p: 373, CRALBP: 528 and SPFffAP: 615. This indicates that all proteins recognize tocopherol but not with the same affinity. Sec 14p bound its native ligand PI with a KI of381 whereas SPFffAP bound PI (216) and y-tocopherol (268) similarly in contrast to the preferential binding ofRRR-a-tocopherol by a-TIP. Efforts to adequately represent biologically active SPFff AP involved investigation of tocopherol binding for several different recombinant proteins derived from different constructs and in the presence of different potential modulators (Ca+2, Mg+2, GTP and GDP); none of these conditions enhanced or inhibited a-tocopherol binding to SPF. This work suggests that only aTTP serves as the physiological mediator of a-tocopherol, yet structural homology between proteins allows common recognition of similar ligand features. In addition, several photo-affmity analogs of a-tocopherol were evaluated for their potential utility in further elucidation of a-TTP function or identification of novel tocopherol binding proteins.

Irrigation scheduling for Sovereign Coronation grapevine based upon evapotranspiration calculations and crop coefficients /

Several irrigation treatments were evaluated on Sovereign Coronation table grapes at two sites over a 3-year period in the cool humid Niagara Peninsula of Ontario. Trials were conducted in the Hippie (Beamsville, ON) and the Lambert Vineyards (Niagara-on-the-Lake, ON) in 2003 to 2005 with the objective of assessing the usefulness of the modified Penman-Monteith equation to accurately schedule vine irrigation needs. Data (relative humidity, windspeed, solar radiation, and temperature) required to precisely calculate evapotranspiration (ETq) were downloaded from the Ontario Weather Network. One of two ETq values (either 100 or 150%) were used in combination with one of two crop coefficients (Kc; either fixed at 0.75 or 0.2 to 0.8 based upon increasing canopy volume) to calculate the amount of irrigation water required. Five irrigation treatments were: un irrigated control; (lOOET) X Kc =0.75; 150ET X Kc =0.75; lOOET X Kc =0.2-0.8; 150ET X Kc =0.2-0.8. Transpiration, water potential (v|/), and soil moisture data were collected each growing seasons. Yield component data was collected and berries from each treatment were analyzed for soluble solids (Brix), pH, titratable acidity (TA), anthocyanins, methyl anthranilate (MA), and total volatile esters (TVE). Irrigation showed a substantial positive effect on transpiration rate and soil moisture; the control treatment showed consistently lower transpiration and soil moisture over the 3 seasons. Transpiration appeared accurately reflect Sovereign Coronation grapevines water status. Soil moisture also accurately reflected level of irrigation. Moreover, irrigation showed impact of leaf \|/, which was more negative throughout the 3 seasons for vines that were not irrigated. Irrigation had a substantial positive effect on yield (kg/vine) and its various components (clusters/vine, cluster weight, and berries/cluster) in 2003 and 2005. Berry weights were higher under the irrigated treatments at both sites. Berry weight consistently appeared to be the main factor leading to these increased yields, as inconsistent responses were noted for some yield variables. Soluble solids was highest under the ET150 and ET100 treatments both with Kc at 0.75. Both pH and TA were highest under control treatments in 2003 and 2004, but highest under irrigated treatments in 2005. Anthocyanins and phenols were highest under the control treatments in 2003 and 2004, but highest under irrigated treatments in 2005. MA and TVE were highest under the ET150 treatments. Vine and soil water status measurements (soil moisture, leaf \|/, and transpiration) confirmed that irrigation was required for the summers of 2003 and 2005 due to dry weather in those years. They also partially supported the hypothesis that the Penman-Monteith equation is useful for calculating vineyard water needs. Both ET treatments gave clear evidence that irrigation could be effective in reducing water stress and for improving vine performance, yield and fruit composition. Use of properly scheduled irrigation was beneficial for Sovereign Coronation table grapes in the Niagara region. Findings herein should give growers some strong guidehnes on when, how and how much to irrigate their vineyards.