Effects Of Al～(3+) And Heat Stresses And Tolerant Mechanisms In Saccharomyces Cerevisiae

Understanding the interaction between life system and environment factors is of great importantance for improving the conditions of human being's existence, living and production. Yeast is a good model organism and a valuable biomaterial in industrial production. Al and heat are important stress factors in environment. The mechanisms about Al toxicity to yeast are yet not to be fully elucidated and confirmed such as the potential role of Al-induced oxidative stress in yeast, and the effective mechanisms of some antioxygen such as GSH, SOD and trehalose on tolerance to Al stress. Although there are many reports about heat shock in yeast, the mechanisms of thormotolerance of thormotorerant strain are also unclear. Therefore, In this study, the interaction between Al, heat shock and Saccharomyces cerevisiae were examined. In order to elucidate and shed some lights on the mechanisms of Al and heat stress on yeast, three couple of yeast strains of CY4, CY8(gshl deletion), LK, LT (mutant of Al-toleranc) , LK, HU-TY-1(thormotolerant mutant) were used in this study and detailed examinations were conducted on the toxicity and absorption of Al in cells, ROS production and lipid peroxidation inducted by Al, the mechanisms of GSH, SOD and trehalose in Al torerance, HSP expression of thormotolerant strain and so on. This study can be categorized into eight sections and major results of each sections are summarized as follows:1. Phenotypes of Al toxicity on Saccharomyces cerevisiae. Experiments indicated that, upon Al treatment, the formation of colony was inhibited and the relative growth of the cell was significantly declined in yeast. Upon exposure to lower Al concentration(≤50μM), the log phase was extended, while in 100μM Al, cells were kept in lag phase. By using FDA-PI staining and fluorescence microscope, it was found that Al treatment resulted in more dead cells and the cells tended to adhere together. Budding cells were more sensitive to Al exposure with higher tendency to die.2. Characterization of the absorption to Al³+ in Saccharomyces cerevisiae cells. Results indicated that, upon exposure to A1C1₃, certain amounts of Al³+ were absorbed in cells of strains CY4 and CY8. The absorbed amounts of Al in mutant CY8 significantly increased with the Alconcentration, however, this is not the case for wide-type CY4. It was also found that the Al absorption capability of CY8 was significantly stronger than that of CY4. The results indicated that absorption of Al into yeast was the main attributor to the Al toxicity, and the capability of Al absorption and Al-tolerance was negatively correlated3. Oxidative stress of Al on cells in Saccharomyces cerevisiae. The ROS values were determined in cells by using fluorescent probe of DCFH-DA. Results indicated that ROS was increased in CY4 and CY8 cells after exposed to Al for 24hrs, and increased for 0.8 and 6.1 folds respectively than contrast after treated with lOOuM Al. At any Al concentrations studied, ROS in CY8 was always more than that in CY4. The lipid peroxidation of membrane and productive ratio of RD (respire defective) mutant were examined and results indicated that the levels of lipid peroxidation (TBARS) of CY8 could be significantly promoted for 55.5% after exposed to 50uM Al, while little change for CY4 as compared to contrast. Upon treatment with lOOuM Al, the productive ratio of RD in strain CY4 and CY8 increased significantly by 0.6 and 2.5 folds than contrasts, respectively, and the productive ratio of RD in strain CY8 was always higher than that of strain CY4. These results suggested that Al stress could induce oxidative reaction in yeast cells and produce ROS, thereby enhancing lipid peroxidation and RD mutant. The deletion of GSH 1 in CY8 strain resulted in higher level of ROS, lipid peroxidation and productive ratio of RD than that of CY4 strain's.4. Roles of GSH and SOD in tolerance to oxidation induced by AL results indicated that Al tolerance of GSH 1-deleted CY8 strain was weaker than that of wild type CY4 strain, suggesting the potential role of GSH win the resistant mechanisms under Al stress. Reduced glutathione (GSH) contents in cells of CY4 decreased for 82% after exposed to 12.5uM Al. After GSH expression were suppressed by CDNB treatment or enhanced by Glu, Cys, Gly incubation, the resistance to Al of CY4 were declined or risen accordingly. The defection of Al tolerance in CY8 strain could be made up after being cultured in GSH media, further suggesting the potential important role of GSH in Al tolerance mechanisms in yeast. The role of GSH is attributed to the antioxidative mechanism of GSH, by which GSH scavenges ROS induced by Al through the reaction of GSH(reduction state) to GSSG(oxidation state), thereby protecting membrane lipids and mitochondria from being destroyed and enhancing the Al tolerance of yeast. Moreover, results alsoindicated that the activity of SOD was significant increased in both strains exposed to 50uM Al, and that of CY8 was always higher than CY4's whether treated with Al or not. This suggested that the oxidant toxicity by Al could be eliminated by both GSH and SOD in wide-type strain, but mainly by SOD in gshl defective strain.5. Characteristics of relative genes expression on GSH and SOD synthesis on Al stess in Saccharomyces cerevisiae. The expression of GSH1 and YAP1 genes on Al stress in CY4 was measured by RT-PCR. Results indicated that the expression levels of both genes on Al stress were significantly higher than contrast. The increased formation of GSH under Al stress further confirms that GSH might play important roles in resistance to oxidative stress by Al in wide-type yeast.6. Isolation of a AMolerant mutant from Saccharomyces cerevisiae and characteristation of ultraweak luminescence. A mutant, designated as LT, with high Al-tolerance was obtained from a Saccharomyce cerevisiae strain (LK) by mutagenesis with diethyl sulfate and UV., and its characteristics of growth and ultraweak luminescence under Al stress were examined. The results showed that the relative growth rate of mutation LT was significantly higher than that of its parental strain LK, and LT strain could grow on LPP plate containing 6mM Al. It was also shown that the emitting proton intensity of LT was higher than that of LK with Al stress. These suggested that ultraweak luminescence might be related to Al-tolerance of yeast.7. Relationship between trehalose and Al tolerance in Saccharomyce cerevisiae. Under 800uM Al stress, trehalose accumulation by LK and LT strains increased by 5-7 folds, and that the mutant LT could accumulate more trehalose than that of parental strain LK. Upon pro-exposure to mild heat shock or cells being in stationary phase, both the trehalose accumulation and Al-tolerance of both strains were enhanced. These suggested that the Al-tolerance may be related to its trehalose accumulation.8. Thormotolerant mechanisms of thermotolerant mutant of Saccharomyce cerevisiae. At high temperature (40°C), the growth rate and fermentation ability of HU-TY-1 were found to be considerably greater than that of the parent. The thermotolerance of both strains increased after mild heat induction, but the increase was much greater for mutant HU-TY-1, as higher as 10 folds (37°C, 20min). Western blotting showed that, either at 23°C or at 37°C, HU-TY-1 couldsynthesizes Hsp70, however, the parent strain LK expressed Hsp70 only after mild heat induction. Two haploids, HZ1 and HZ2, derived from HU-TY-1 maintained their high thermotolerance and ability to constitutively express Hsp70 under control conditions (23°C). Based upon the results of this study , it is suggested that the constitutive synthesis of Hsp70 in HU-TY-1 might play an important role in its higher intrinsic thermotolerance. Besides, pertreatment with ethnol, glucose starving and H2O2 could greatly enhance the yeast's thormotolerance, suggesting that cross tolerance and general stress respons might exist in Saccharomyce cerevisiae.