Insufficiency of Copper Ion Homeostasis Causes Freeze-Thaw Injury of Yeast Cells. Saccharomyces cerevisiae

Saccharomyces cerevisiae is exposed to freeze-thaw stress in commercial processes including frozen dough baking. The cell viability and fermentation activity after freeze-thaw were dramatically decreased due to freeze-thaw injury. Because freeze-thaw injury involves complex phenomena, the mechanisms of it are not fully understood. We attempted to analyze the mechanisms of freeze-thaw injury by indirect gene expression analysis during post-thaw incubation after freeze-thaw treatment using DNA microarray profiling. The results showed that a high frequency of the genes involved in the homeostasis of metal ions were up-regulated depending on the freezing period. The phenotype of the deletion mutants of the up-regulated genes extracted by indirect gene expression analysis was assessed. The deletion strains 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. Supplementation with copper ions during post-thaw incubation increased intracellular superoxide dismutase activity. Inverse correlated with intracellular superoxide dismutase activity, intracellular levels of reactive oxygen species were decreased. Moreover, cell viability increased by supplementation with copper ions under specific assessment conditions. This study suggested that insufficiency of copper ion homeostasis may be one of the causes of freeze-thaw injury. Overall design: Total RNA was extracted from the stress-treated yeast cells by using a hot phenol method. Poly(A)+ RNA was enriched from total RNA by using an Oligotex dT30 (Super) mRNA purification kit (Takara Bio, Ohtsu, Japan). cDNA synthesis, cRNA synthesis, and labeling were performed according to the Affymetrix user’s manual (Affymetrix, Santa Clara, USA). Biotinyated cRNA was fragmented and then used as a probe.Affimetrix Yeast Genome 2.0 arrays (Affymetrix) were used as DNA microarrays. All experiments were done in triplicate independently.

酿酒酵母（Saccharomyces cerevisiae）在包括冷冻面团烘焙在内的各类商业化生产流程中，均会遭遇冻融胁迫。冻融损伤会导致酿酒酵母经冻融处理后的细胞活力与发酵活性显著下降。鉴于冻融损伤涉及复杂的生物学过程，其具体分子机制尚未完全明晰。本研究借助DNA微阵列（DNA microarray）谱分析技术，对冻融处理后复苏培养阶段的基因表达情况进行间接分析，以此尝试解析冻融损伤的分子机制。
研究结果显示，参与金属离子稳态调控的基因的上调频率随冷冻时长的延长呈升高趋势。我们对通过间接基因表达分析筛选得到的上调基因的缺失突变体表型进行了评估。结果发现，参与铜离子稳态调控的MAC1与CTR1基因的缺失菌株呈现出冻融敏感性，这表明铜离子稳态是维持酿酒酵母冻融耐受性的必要条件。
在复苏培养阶段添加铜离子，可提升胞内超氧化物歧化酶（superoxide dismutase）活性；而胞内活性氧（reactive oxygen species）水平与超氧化物歧化酶活性呈负相关，即酶活性升高时活性氧水平随之降低。此外，在特定评估条件下，复苏阶段添加铜离子可有效提高细胞活力。
本研究证实，铜离子稳态失衡或许是引发冻融损伤的诱因之一。
实验整体设计：采用热酚法从经胁迫处理的酵母细胞中提取总RNA；使用Oligotex dT30 (Super) mRNA纯化试剂盒（Takara Bio，日本大津）从总RNA中富集Poly(A)+ RNA；参照Affymetrix用户手册（Affymetrix，美国圣克拉拉）完成cDNA合成、cRNA合成与探针标记。将生物素标记的cRNA片段化后作为杂交探针，采用Affymetrix酵母基因组2.0芯片（Affymetrix）作为DNA微阵列。所有实验均独立重复三次。