The osmoadaptive response of the wine yeast Saccharomyces cerevisiae K1-V1116 during icewine fermentation

The adapted metabolic response of commercial wine yeast under prolonged exposure to concentrated solutes present in Icewine juice is not fully understood. Presently, there is no information regarding the transcriptomic changes in gene expression associated with the adaptive stress response ofwine yeast during Icewine fermentation compared to table wine fermentation. To understand how and why wine yeast respond differently at the genomic level and ultimately at the metabolic level during Icewine fermentation, the focus ofthis project was to identify and compare these differences in the wine yeast Saccharomyces cerevisiae KI-Vll16 using cDNA microarray technology during the first five days of fermentation. Significant differences in yeast gene expression patterns between fermentation conditions were correlated to differences in nutrient utilization and metabolite production. Sugar consumption, nitrogen usage and metabolite levels were measured using enzyme assays and HPLC. Also, a small subset of differentially expressed genes was verified using Northern analysis. The high osmotic stress experienced by wine yeast throughout Icewine fermentation elicited changes in cell growth and metabolism correlating to several fermentation difficulties, including reduced biomass accumulation and fermentation rate. Genes associated with carbohydrate and nitrogen transport and metabolism were expressed at lower levels in Icewine juice fermenting cells compared to dilute juice fermenting cells. Osmotic stress, not nutrient availability during Icewine fermentation appears to impede sugar and nitrogen utilization. Previous studies have established that glycerol and acetic acid production are increased in yeast during Icewine fermentation. A gene encoding for a glycerollW symporter (STL1) was found to be highly expressed up to 25-fold in the i Icewine juice condition using microarray and Northern analysis. Active glycerol transport by yeast under hyperosmotic conditions to increase cytosolic glycerol concentration may contribute to reduced cell growth observed in the Icewine juice condition. Additionally, genes encoding for two acetyl CoA synthetase isoforms (ACSl and ACS2) were found to be highly expressed, 19- and II-fold respectively, in dilute juice fermenting cells relative to the Icewine juice condition. Therefore, decreased conversion of acetate to acetyl-CoA may contribute to increased acetic acid production during Icewine fermentation. These results further help to explain the response of wine yeast as they adapt to Icewine juice fermentation. ii

Hyperosmotic Stress and the Impact on Metabolite Formation and Redox Balance in Saccharomyces cerevisiae and Saccharomyces bayanus strains

Wine produced using an appassimento-type process represents a new and exciting innovation for the Ontario wine industry. This process involves drying grapes that have already been picked from the vine, which increases the sugar content due to dehydration and induces a variety of changes both within and on the surface of the grapes. Increasing sugar contents in musts subject wine yeast to conditions of high osmolarity during alcoholic fermentations. Under these conditions, yeast growth can be inhibited, target alcohol levels may not be attained and metabolic by-products of the hyperosmotic stress response, including glycerol and acetic acid, may impact wine composition. The further metabolism of acetic acid to acetylCoA by yeast facilitates the synthesis of ethyl acetate, a volatile compound that can also impact wine quality if present in sufficiently high concentrations. The first objective of this project was to understand the effect of yeast strain and sugar concentration on fermentation kinetics and metabolite formation, notably acetic acid and ethyl acetate, during fermentation in appassimento-type must. Our working hypotheses were that (1) the natural isolate Saccharomyces bayanus would produce less acetic acid and ethyl acetate compared to Saccharomyces cerevisiae strain EC-1118 fermenting the high and low sugar juices; (2) the wine produced using the appassimento process would contain higher levels of acetic acid and lower levels of ethyl acetate compared to table wine; (3) and the strains would be similar in the kinetic behavior of their fermentation performances in the high sugar must. This study determined that the S. bayanus strain produced significantly less acetic acid and ethyl acetate in the appassimento wine and table wine fermentations. Differences in acetic acid and ethyl acetate production were also observed within strains fermenting the two sugar conditions. Acetic acid production was higher in table wine fermented by S. bayanus as no acetic acid was produced in appassimento-style wine, and 1.4-times higher in appassimento wine fermented by EC-1118 over that found in table wine. Ethyl acetate production was 27.6-times higher in table wine fermented by S. bayanus, and 5.2-times higher by EC-1118, compared to that in appassimento wine. Sugar utilization and ethanol production were comparable between strains as no significant differences were determined. The second objective of this project was to bring a method in-house for measuring the concentration of pyridine nucleotides, NAD+, NADP+, NADH and NADPH, in yeast cytosolic extract. Development of this method is of applicative interest for our lab group as it will enable the redox balance of the NAD+/ NADH and NADP+/ NADPH systems to be assessed during high sugar fermentations to determine their respective roles as metabolic triggers for acetic acid production. Two methods were evaluated in this study including a UV-endpoint method using a set of enzymatic assay protocols outlined in Bergmeyer (1974) and a colorimetric enzyme cycling method developed by Sigma-Aldrich® using commercial kits. The former was determined to be limited by its low sensitivity following application to yeast extract and subsequent coenzyme analyses, while the latter was shown to exhibit greater sensitivity. The results obtained from the kits indicated high linearity, accuracy and precision of the analytical method for measuring NADH and NADPH, and that it was sensitive enough to measure the low coenzyme concentrations present in yeast extract samples. NADtotal and NADPtotal concentrations were determined to be above the lower limit of quantification and within the range of the respective calibration curves, making this method suitable for our research purposes.

The effects of temperature on nickel tocicity in goldfish (carassius auratus L.)

Although it is widely assumed that temperature affects pollutant toxicity, few studies have actually investigated this relationship. Moreover, such research as has been done has involved constant temperatures; circumstances which are rarely, if ever, actually experienced by north temperate, littoral zone cyprinid species. To investigate the effects of temperature regime on nickel toxicity in goldfish (Carassius auratus L.), 96- and 240-h LCSO values for the heavy metal pollutant, nickel (NiCI2.6H20), were initially determined at 2DoC (22.8 mg/L and 14.7 mg/L in artificially softened water). Constant temperature bioassays at 10°C, 20°C and 30°C were conducted at each of 0, 240-h and 96-h LCSO nickel concentrations for 240 hours. In order to determine the effects of temperature variation during nickel exposure it was imperative that the effects of a single temperature change be investigated before addressing more complex regimes. Single temperature changes of + 10°C or -10°C were imposed at rates of 2°C/h following exposures of between 24 hand 216 h. The effects of a single temperature change on mortality, and duration of toxicant exposure at high and low temperatures were evaluated. The effects of fluctuating temperatures during exposure were investigated through two regimes. The first set of bioassays imposed a sinewave diurnal cycle temperature (20.±.1DOC) throughout the 10 day exposure to 240-h LeSO Ni. The second set of investigations approximated cyprinid movement through the littoral zone by imposing directionally random temperature changes (±2°C at 2-h intervals), between extremes of 10° and 30°C, at 240-h LC50 Ni. Body size (i.e., total length, fork length, and weight) and exposure time were recorded for all fish mortalities. Cumulative mortality curves under constant temperature regimes indicated significantly higher mortality as temperature and nickel concentration were increased. At 1DOC no significant differences in mortality curves were evident in relation to low and high nickel test concentrations (Le., 16 mg/L and 20 mg/L). However at 20°C and 30°C significantly higher mortality was experienced in animals exposed to 20 mg/L Ni. Mortality at constant 10°C was significantly lower than at 30°C with 16 mg/L and was significantly loWer than each of 2DoC and 39°C tanks at 20 mg/L Ni exposure. A single temperature shift from 20°C to 1DoC resulted in a significant decrease in mortality rate and conversely, a single temperature shift from 20°C to 30°C resulted in a significant increase in mortality rate. Rates of mortality recorded during these single temperature shift assays were significantly different from mortality rates obtained under constant temperature assay conditions. Increased Ni exposure duration at higher temperatures resulted in highest mortality. Diurnally cycling temperature bioassays produced cumulative mortality curves approximating constant 20°C curves, with increased mortality evident after peaks in the temperature cycle. Randomly fluctuating temperature regime mortality curves also resembled constant 20°C tanks with mortalities after high temperature exposures (25°C - 30°C). Some test animals survived in all assays with the exception of the 30°C assays, with highest survival associated with low temperature and low Ni concentration. Post-exposure mortality occurred most frequently in individuals which had experienced high Ni concentrations and high temperatures during assays. Additional temperature stress imposed 2 - 12 weeks post exposure resulted in a single death out of 116 individuals suggesting that survivors are capable of surviving subsequent temperature stresses. These investigations suggest that temperature significantly and markedly affects acute nickel toxicity under both constant and fluctuating temperature regimes and plays a role in post exposure mortality and subsequent stress response.

Immediate and enduring effects of corticosterone administration during adolescence and adulthood on anxiety and depressive behavior in male rats

Previous research has shown that the stress hormone corticosterone can increase depressive and anxiety-like behavior in rats as well as dampen the HPA response to a novel stressor (Kalynchuk et aI., 2004; Johnson et aI., 2006). Several studies have also shown that adolescence is a period of increased sensitivity to the negative effects of stressors (reviewed in McCormick et aI., 2010), which are often the result of exposure to corticosterone, and yet there is no research to date examining the effects of corticosterone administration during adolescence. The purpose of these experiments is to determine both the immediate and enduring effects of prolonged exposure to corticosterone in adolescence and adulthood on anxiety-like behavior, depressive behavior, and the HPA response. In Experiment 1 adolescent and adult rats were administered an injection of 40 mg/kg of corticosterone or vehicle daily for 16 days. Ha l f of the rats were then tested on the elevated plus maze (EPM) one day after their last injection, and the following day were tested on the forced swim test (FST). After the FST, which is a stressor, blood samples were collected at three time points, and the plasma concentrations of corticosterone were determined using a radioimmunoassay. The remaining rats were left undisturbed for three weeks, and then underwent the same testing as the first group. Corticosterone treatment had little effect on anxiety-like and depressive behavior, but it did alter the HPA response to the FST. In those rats tested soon after the period of injections, corticosterone dampened the HPA response as compared to vehicle treated rats in both adolescent and adult treated rats. For the adolescent treated rats that were tested several weeks later, corticosterone treatment increased HPA response as compared to the vehicle treated rats, but the same was not true for the adult treated rats. I t was hypothesized that the lack of behavioral effects of the corticosterone treatment may be the result of the vehicle injections inducing a stress response and thereby both groups would have similarly altered behavior. In Experiment 2 rats were administered corticosterone dissolved in their drinking water with 2.5% ethanol, or jus t the 2.5% ethanol or plain water, to determine the effects of corticosterone treatment without a stressor present. The regular drinking water was replaced with treated water for 16 days either during adulthood or adolescence, and as before, rats were either tested in the FST one day after the water was removed or three weeks later. Again there was no effect of treatment on depressive behavior. Similar to what was observed in Experiment 1, corticosterone treatment dampened the HPA response to a stressor for the rats tested soon after the treatment period. However, in Experiment 2 there was no effect of treatment on HPA response in those rats tested several weeks after they were treated. These results indicate that corticosterone can have a lasting effect on the HPA when administered in adolescence by injections but not in drinking water, which is likely because of the different schedules of exposure and rates of absorption between the two administration methods.

Starvation Induces Expression of the Plant-Adhesin Gene, Mad2, of the Entomopathogenic Fungus Metarhizium robertsii.

Metarhizium robertsii is an entomopathogenic fungus that is additionally plant rhizosphere competent. Two adhesin-encoding gens, Mad1 and Mad2, are involved in insect pathogenesis or plant root colonization, respectively. This study examined differential expression of the Mad genes for M robertsii grown on a variety of insectand plant-related substrates. Mad1 was up regulated in response to insect cuticles and up regulation of Mad2 resulted from root exudates, tomato stems and non-preferred carbohydrates. A time course analysis that compared water, minimal media, and nutrient rich broth revealed Mad2 gene expression increased as nutrient availability decreased. The regulation of Mad2 compared to known stress-related genes (Hsp30, Hsp70 and ssgA) under various stresses (nutrient, pH, osmotic, oxidative, temperature) revealed Mad2 to be generally up regulated by nutrient starvation only. Examination of the Mad2 promoter region revealed two copies of a stress-response element (S TRE) known to be regulated under the general stress response pathway.