Selection of freeze-thaw tolerant mutant strain
Approximately 2000 transposon mediated mutants were tested for freeze-thaw tolerance. One strain that grew faster than wild-type strain was finally selected under freeze-thaw condition. As shown in Fig. 1A, one mutant, designated as TN-F, was more tolerant to freeze-thaw stress than wild type (L3262) and TN-F grew much faster than wild-type strain (Fig. 1B). These results indicate that disrupted gene in TN-F caused tolerance to freeze-thaw stress.
Identification of disrupted gene and contribution to freeze-thaw tolerance
The disrupted gene has been shown in Fig. 2. The insertion site was located in the ORF of YCP4 in TN-F (Table 2). The effect of disrupting YCP4on freeze-thaw tolerance was confirmed using the single-gene deletion library of S. cerevisiae BY4741. When the culture of DYCP4was spot-assayed in a YPD medium under freeze-thaw condition, it showed an improved growth compared to the BY4741 strain (Fig. 3A). YCP4is known as one of flavodoxin-like proteins and is predicted to be palmitolyated, a post-translational modification typical of membrane-binding proteins involved in signal transduction (Roth et al. 2006). There have been no reports connecting YCP4 to a freeze-thaw tolerance in eukaryotic microorganisms. However, it has been reported that the double null mutant of YCP4 along with RFS1, one of flavodoxin-like proteins, had an increased response to oxidative stress (Cardona et al. 2011). In additon, YCP4 have been studied in regulation of the tumor suppressor PTEN (Kim et al. 2011) and regulated the expression of many genes during the late stages of growth (Cardona et al. 2011), but its exact function has not been revealed yet. In this study, the disruption of YCP4 clearly showed an improved the tolerance to freeze-thaw stress. Therefore, I suggest that YCP4 may play a role in response and in the regulation of genes related to freeze-thaw stress, although further studies will be required to elucidate the mechanisms related to freeze-thaw tolerance.
Confirmation of tolerance by complementation
Since Mutant strain TN-F acquired a freeze-thaw tolerance by disruption of specific gene, complementation of the relevant gene would create a freeze-thaw sensitive phenotype to the yeast cells. To verify this hypothesis, the respective gene was endogenously expressed by cloning the amplified DNA fragments estimated to include ORF, their own promoter, and their own terminator (ORF ± 700 bp) into pRS316 and transformed into TN-F. The reference strain was obtained by transforming pRS316 containing nothing. The tolerance of the complemented strain was significantly reduced compared with that of the reference strain (Fig. 1B). In addition, the ORF of YCP4 was amplified by PCR, constructed into pRS316-GAPDH for an overexpression, and transformed into L3262. The overexpressed yeast strain (L3262/YCP4) was were more sensitive to freeze-thaw stress (Fig. 1C). In addition, the gene dosage of YCP4 was examined by qRT-PCR. The expression patterns of YCP4 are higher by the control of the GAPDH than by that of their own promoter (Fig. 4). Therefore, these results suggest that the sensitive phenotype to freeze-thaw stress may be determined by the expression level of the YCP4 gene.
Intracellular ROS at freeze-thawstress
Several studies have been reported that oxidative stress is a major cause of yeast cell damage, which is responsible for accumulation of ROS during freeze-thaw stress (Nakagawa et al. 2013; Park et al. 1998). Therefore, I investigated whether TN-F could reduce the intracellular level of ROS that accumulated upon exposure to freeze-thaw stress. Compared to the control strain (L3262), the level of ROS signal was approximately 55% lower in the TN-F mutant under freeze-thaw stress condition (Fig. 5), indicating that the reduction in ROS was associated with the freeze-thaw tolerant phenotype. Next, it has been reported that the overexpression of the transcriptional activator, MSN2 conferred the tolerance to freeze-thaw stress (Sasano et al. 2012). In contrast, the disruption of the MSN2 gene caused sensitivity to freeze-thaw stress in a laboratory yeast strain (Izawa et al. 2007). In addtion, it has been known that the stress-response transcription factors, including the MSN2/4 bind to stress response elements (STRE) within the promoters of stress-mediated genes such as CTT1, HSP12 under various stress (Causton et al. 2001; Martínez-Pastor et al. 1996). Thus, I compared the transcript levels of MSN2, MSN4,CTT1, and HSP12 between the L3262 and TN-F strains. As shown in Fig. 6, the mRNA levels of MSN2, MSN4,CTT1, and HSP12 in TN-F increased compared with those in the control strain (L3262) under freeze-thaw stress. Without any freeze-thaw stress, the mRNA patterns of corresponding genes in all strains were not changed. In S. cerevisiae,double deletion mutant △msn2 △msn4 has hypersensitive phenotype to carbon source starvation, heat shock, osmotic and oxidative stresses (Estruch and Carlson 1993). In addition, it has been reported that the expression of CTT1 gene was increased in double deletion mutant △ycp4 △rfs1, referred to as oxidative stress tolerance (Cardona et al. 2011). In this study, the disruption of YCP4 increased freeze-thaw tolerance with the activation of MSN2/4 and STRE-mediated genes such as CTT1 and HSP12.Therefore, these results suggest that the disruption of YCP4 may contribute to a freeze-thaw tolerance through ROS scavenging by the expression of the MSN2/4 and STRE-mediated genes such as CTT1 and HSP12.Further studies on an YCP4-mediated metabolic regulation are required to elucidate the mechanisms of freeze-thaw tolerance. In conclusion, the characteristics of YCP gene will contribute to the application of freeze-thaw processes including frozen dough baking and cryopreservation and to provide clues on freeze-thaw tolerance in higher eukaryotic organisms.