Insufficiency of copper ion homeostasis causes freeze-thaw injury of yeast cells as revealed by indirect gene expression analysis - PubMed (original) (raw)

Insufficiency of copper ion homeostasis causes freeze-thaw injury of yeast cells as revealed by indirect gene expression analysis

Shunsuke Takahashi et al. Appl Environ Microbiol. 2009 Nov.

Abstract

Saccharomyces cerevisiae is exposed to freeze-thaw stress in commercial processes, including frozen dough baking. Cell viability and fermentation activity after a freeze-thaw cycle were dramatically decreased due to freeze-thaw injury. Because this type of injury involves complex phenomena, the injury mechanisms are not fully understood. We examined freeze-thaw injury by indirect gene expression analysis during postthaw incubation after freeze-thaw treatment using DNA microarray profiling. The results showed that genes involved in the homeostasis of metal ions were frequently contained in genes that were upregulated, depending on the freezing period. We assessed the phenotype of deletion mutants of the metal ion homeostasis genes that exhibited freezing period-dependent upregulation and found that the strains with deletion of the MAC1 and CTR1 genes involved in copper ion homeostasis exhibited freeze-thaw sensitivity, suggesting that copper ion homeostasis is required for freeze-thaw tolerance. We found that supplementation with copper ions during postthaw incubation increased intracellular superoxide dismutase activity and intracellular levels of reactive oxygen species were decreased. Moreover, cell viability was increased by supplementation with copper ions. These results suggest that insufficiency of copper ion homeostasis may be one of the causes of freeze-thaw injury.

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Figures

FIG. 1.

FIG. 1.

Changes in viable cell numbers (A) and intracellular levels of ROS (B) of strain BY4741 after freeze-thaw treatment. In panel A, viable cell numbers were measured after freeze-thaw treatment for 24 to 96 h. In panel B, the intracellular levels of ROS were measured after postthaw incubation preceded by freeze-thaw treatment for 24 to 96 h. The fluorescence intensity of the cells before freeze-thaw treatment was relatively set as 100%. The values are the means and standard deviations of results from three independent experiments.

FIG. 2.

FIG. 2.

The viabilities of strain BY4741 (as a positive control) and the Δ_mac1_, Δ_ctr1_, and Δ_pro1_ (as a negative control) deletion strains were measured after freeze-thaw treatment for 24 or 48 h. The values are means and standard deviations of results from three independent experiments.

FIG. 3.

FIG. 3.

Changes in intracellular SOD activity (A) and intracellular levels of ROS (B) in BY4741 by supplementation with copper ions during postthaw incubation. In panel A, the intracellular SOD activities were measured after postthaw incubation preceded by freeze-thaw treatment for 24 or 48 h using SD medium containing various copper ion concentrations. The unit of SOD activity of the cells before freeze-thaw treatment was relatively set as 100%. In panel B, the intracellular levels of ROS were measured after postthaw incubation preceded by freeze-thaw treatment for 24 or 48 h using SD medium containing various copper ion concentrations. The fluorescence intensity of the cells before freeze-thaw treatment was relatively set as 100%. The values are the means and standard deviations of results from three independent experiments. Asterisks and double asterisks indicate that there are significant differences between the values from samples supplemented with no copper and the values from respective samples with P values of <0.05 and <0.01, respectively.

FIG. 4.

FIG. 4.

Effects of supplementation with copper ions on cell viability of BY4741 after freeze-thaw treatment. The cell viabilities after freeze-thaw treatment were measured using YPD (A) and SD (B) agar media containing various copper ion concentrations. The viability of cells before freeze-thaw treatment was relatively set as 100%. The data shown are means of more than triplicate measurements from a representative experiment. Error bars represent the standard deviations of the means. Asterisks and double asterisks indicate that there are significant differences between the viabilities of samples supplemented with no copper and the viabilities of respective samples with P values of <0.05 and <0.01, respectively.

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