Disruption of YCP4 enhances freeze-thaw tolerance in Saccharomyces cerevisiae

This study aimed to identify genes related to freeze-thaw tolerance and elucidate the tolerance mechanism in yeast Saccharomyces cerevisiae as an appropriate eukaryote model. In this study, one tolerant strain exposed to freeze-thaw stress was isolated by screening a transposon-mediated mutant library and the disrupted gene was identified to be YCP4. In addition, this phenotype related to freeze-thaw tolerance was confirmed by deletion and overexpressing of this corresponding gene. This mutant strain showed a freeze-thaw tolerance by reducing the intracellular level of reactive oxygen species and the activation of the MSN2/4 and STRE-mediated genes such as CTT1 and HSP12. Disruption of YCP4 in S. cerevisiae results in increased tolerance to freeze-thaw stress.


Introduction
Freezing and thawing is a type of stress that can cause physiological severe injury, including cell wall and cell membrane damage, DNA and protein degeneration, due to the formation of ice crystals and cell dehydration (Cabrera et al. 2020). Free radicals produced during the freeze-thaw process are also a major cause of cell damage (Naparlo et al. 2019). Although the response of cells to freezing and thawing stress is not known in detail, it is generally accepted that freeze-thaw tolerance is related to factors including growth phase (Werner-Washburne et al. 2012), respiratory metabolism (Park et al. 1997), and lipid composition of the membrane (Zhang et al. 2018). The freeze-thaw stress response is of physiological and industrial importance because it involves other stress response networks, including heat shock and oxidative stress (Cabrera et al. 2020). It has been reported that l-proline can protect yeast cells from damage by freezing, or oxidative stress (Takagi et al. 2005). The yeast Saccharomyces cerevisiae become more resistant to freeze-thaw stress by adding glycerol (Izawa et al. 2004) and accumulation of trehalose as cryoprotectants (Nakamura et al. 2009), however the mechanisms underlying freeze-thaw tolerance has not been completely understood in the eukaryotes. The aim of this study was to identify genes related to a freezethaw tolerance and to elucidate the tolerance mechanism in yeast S. cerevisiae as an appropriate eukaryote model (Manning et al. 2002 Several studies have been reported on freeze-thaw tolerance. Hydrogen peroxide pretreatment could induce freeze-thaw tolerance of yeast cells (Park et al. 1998). Alpha-ketoglutarate enhances freezethaw tolerance in S. cerevisiae (Bayliak et al. 2018). Freeze-tolerant yeast mutant strains were isolated with freeze-thaw cycles after UV irradation (Teunissen et al. 2002) and ethyl methanesulfonate (EMS) processing by evolutionary engineering (Cakar et al. 2005). Some yeast yeast strains with freeze-thaw tolerance have been selected from natural sources (Hahn & Kawai, 1990) and constructed by gene manipulations (Shima et al. 1999). In addition, the genes conferring freeze-thaw sensitivity were identified by genome-wide screening of S. cerevisiae deletion strains and revealed as genes related to vacuole functions and cell wall biogenesis (Ando et al. 2007). An alternative method is to use disruptive mutants for direct isolation of strains tolerant to various stress factors (Yazawa et al. 2007;Yoshikawa et al. 2009). I have previously reported the isolation of an organic solvent and heavy metal-tolerant strain of S. cerevisiae by screening a transposon-mediated mutant library, respectively (Kim 2016(Kim , 2020. A transposonmediated mutant library has advantages as a means of directly selecting freeze-thaw tolerant strains and construction of mutants harboring up-or down-regulated genes by insertion in the regulatory regions, which is particularly important for lethal genes. In this study, by screening a transposon-mediated disruption mutant library in S. cerevisiae L3262, a freeze-thaw resistant strain was obtained and the gene involved was identified.

Strains, culture conditions and transposon mutagenesis
The strains and plasmids used in this study are listed in Table 1. S. cerevisiae deletion library was provided from Seoul National University (Seoul, South Korea). Yeast strains and Escherichia coli DH5α were cultured according to the previous method (Kim 2016). S. cerevisiae genomic library pools with transposonmediated random insertions of mTn3-lacZ/LEU2 were kindly provided by the Yale Genome Analysis Center (New Haven, CT; http:// ygac. med. yale. edu/ mtn/ reage nt/ avail_ reage nts/ lacZ_ LEU2_ info_p. stm). The plasmid DNA from pools of the mTn3-mutagenized genomic library was digested with NotI by the method provided in the Yale Genome Analysis Center and transformed into S. cerevisiae L3262 by the lithium acetate as previously reported (Livak and Schmittgen 2001).

Freezing and thawing conditions
Yeast cells were cultured on YPD medium at 30 °C to exponential phase, were harvested by centrifugation (5000×g) and washed with distilled water. After dilution with 10 mM potassium phosphate buffer (pH 7.0) to an optical density (OD) at 600 nm of 1.0, 0.5 ml were transfered into 1.5 ml effendorf tubes and frozen at − 20 °C for 1, 3, and 7 days and then thawed at 0 °C for 40 min. Survival was determined by diluting cells into YPD medium at room temperature and plating on YPD plate at 30 °C for 2 days. Data was expreesed as a percentage of the colony-forming unit (CFU) after freeze-thaw stress treatments compared with no stress conditions.

Isolation of freeze-thaw tolerant mutants and identification of disrupted genes
Yeast transformants were treated under freeze-thaw condition and cultured on YPD plates by spot assay (Kim 2016). Mutant strains that grew faster than wild-type strain were selected. All the experiments for selection of freeze-thaw tolerant strains were examined in triplicate. To reveal the disruption sites, genomic DNA fragments containing transposon were rescued as plasmids that were amplified in Escherichia coli following the procedures described at http:// ygac. med. yale. edu/ mtn/ reage nt/ avail_ reage nts/ lacZ_ LEU2_ info_p. stm. Rescued plasmids were sequenced using a primer derived from the lacZ sequence of transposon (Livak and Schmittgen 2001). The transposon inserted genes were determined through the Saccharomyces Genome Database.

Gene cloning
The open reading frames of identified genes from S. cerevisiae L3262 were cloned into pRS316-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for overexpression experiments (Kim 2016). The ORFs with their own promoter and terminator sequences from S. cerevisiae L3262 were constructed into pRS316 for complementation experiments (Table 1) (Kim 2016).

Quantitative reverse transcription PCR (qRT-PCR)
All yeast strains were cultivated in YPD medium under freeze-thaw condition. Quantitative reverse transcription PCR (qRT-PCR) was performed using gene specific primers (Table 1) according to the previous method (Kim 2016).

Measurement of intracellular reactive oxygen species (ROS)
The level of intracellular ROS was measured according to the reported method (Kim et al. 2012) using 2′7′-dichlorodihydrofluorescein diacetate (DCF; Molecular probes, Eugene, USA). Exponentially growing yeast cells were treated under freeze-thaw condition. Measurement of intracellular ROS was done as previously described (Kim et al. 2012). In short, aliquots of cells were removed at various times over a 12-h period. Cells were washed with 1 ml of distilled water and incubated with 10 μM CM-H2DCFDA in 0.1 ml phosphate buffered saline (PBS) for 30 min. After washing with PBS, cells were visualized by fluorescence microscopy. Finally, the fluorescence intensities of at least 100 cells were measured and averaged using NIH image J, version 1.61.

Statistics
All data are presented as the mean ± standard error of the mean (SEM). Statistical analyses were performed with GraphPad Prism 5.0 (GraphPad Software, Inc. San Diego, CA, USA). Comparisons between multiple groups were performed with one-way analysis of variance with Bonferroni post-hoc test. The P-value of less than 0.05 was considered significant.

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 was selected following a spot assay (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 is shown in Fig. 2. The insertion site was located in the ORF of YCP4 in TN-F ( Table 2). The effect of disrupting YCP4 on freezethaw tolerance was confirmed using the single-gene deletion library of S. cerevisiae BY4741. When the culture of ΔYCP4 was spot-assayed in a YPD medium under freeze-thaw condition, it showed an improved growth compared to the BY4741 strain (Fig. 3A).
YCP4 is 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 flavodoxinlike proteins, had an increased response to oxidative stress (Cardona et al. 2011). In addition, YCP4 has been studied in regulation of the tumor suppressor PTEN (Kim 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, Results indicate 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. 3B). In addition, the ORF of YCP4 was amplified by PCR, constructed into pRS316-GAPDH for an overexpression, and transformed into L3262.
The yeast strain overexpressing (L3262/YCP4) was more sensitive to freeze-thaw stress than other strains (Fig. 3C). In addition, the gene dosage of YCP4 was examined by qRT-PCR. The expression of YCP4 was higher under the control of the GAPDH than by 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. Several studies have been reported that oxidative stress is a major cause of yeast cell damage, which is responsible for accumulation of ROS during freezethaw stress (Nakagawa et al. 2013;Park et al. 1998). Therefore, it was investigated whether TN-F mutation 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 addition, 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, it was 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. The 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 STREmediated 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 freezethaw tolerance in higher eukaryotic organisms.  The effect was determined by quantitative reverse transcription PCR. Values were normalized to glyceraldehyde 3-phosphate dehydrogenase before calculating changes and represented relative to the value of L3262 or TN-F untreated with freeze-thaw (no stress) respectively. Values are presented as means ± standard error of the mean (SEM) of three independent experiments. *P < 0.05 compared to L3262 treated under freeze-thaw stress conditions