Introduction

Antarctica is a continent with extreme low temperature, nutrient, and high ultraviolet (UV) radiation. Temperature is one of the major factors affecting the survival of Antarctic organisms. Antarctic organisms are able to thrive at low temperature and have developed a variety of adaptation strategies to grow and multiply (Morgan-Kiss et al. 2006). By understanding the molecular mechanisms underlying how a range of Antarctic organisms respond to climate change in Antarctica will enable the predictions as how they will adapt to global climate change. Cold and heat adaptation studies have been carried out on some Antarctic organisms such as algae (Hu et al. 2008; Hwang et al. 2008; Chong et al. 2010), plants (Gidekel et al. 2003), fish (Hofmann et al. 2000; Chen et al. 2008), marine bivalve (Park et al. 2008; Kim et al. 2009), insect (Rinehart et al. 2006), fungi (Zucconi et al. 1996; Tosi et al. 2005; Vishniac 2006; Gocheva et al. 2009), and bacteria (Médigue et al. 2005).

Several reports have indicated that heat shock induces the production of heat shock proteins (HSPs) (Lindquist and Craig 1988; Watson 1990) and the disaccharide trehalose (Attfield 1987; Hottiger et al. 1989). HSPs mainly function as molecular chaperones with respect to protein folding and assembly, whereas trehalose is a thermoprotectant that stabilizes cell membranes and increases the temperature stability of cellular proteins to enable the maintenance of cell structures (Hottiger et al. 1989; Iwahashi et al. 1995). On the other hand, cells are known to respond to cold stress by the elite and rapid production of cold shock proteins (CSPs) (Ermolenko and Makhatadze 2002), cryoprotectant (Borges et al. 2002), antifreeze proteins (AFPs) (Davies et al. 2002) and by activating the desaturase system to maintain membrane fluidity (Deming 2002).

The Antarctic organisms have evolved under the influence of the geological and climatic factors, including geographic isolation of the landmass and the continental shelves, extremely low temperatures, and intense seasonality (Clarke and Crame 1992). Antarctic organisms living in the extreme cold habitats do not always respond in the same way as their temperate or tropical counterparts. Although the heat shock response is thought to be nearly universal among organisms, exception is found in several organisms such as the Antarctic teleost fish, Trematomus bernacchii (Hofmann et al. 2000) and Antarctic ciliate, Euplotes focardii (LaTerza et al. 2004). Additionally, some Antarctic notothenioids living in subzero temperatures have been shown to lose some of their traits during the process of evolution. For instance, the loss of hemoglobin and myoglobin gene expression abilities in the Antarctic icefish (family Channichthyidae) (Cocca et al. 1997; Sidell et al. 1997; Somero et al. 1998). These reports suggested that some of the living organisms do not response in a similar fashion to temperature shift. Adaptation studies on a diverse population of living organisms from different habitats will help us to better understand the cells response to temperature shifts.

Glaciozyma antarctica PI12 (previously known as Leucosporidium antarcticum) is a psychrophilic yeast strain which was isolated from a marine environment in Antarctica (Fell et al. 1969). Recently, Turchetti et al. (2011) have proposed to reclassify this yeast from L. antarcticum to G. antarctica. Besides, G. antarctica has also been isolated from various locations in Antarctica (Fell et al. 1969; Donachie 1995; Connell et al. 2008; Law, personal communication). Turkiewicz et al. (2005) reported that G. antarctica 171 has an optimum growth temperature at around 15 °C and is able to tolerate higher temperatures up to 20 °C. Thus far, only two thermal stress studies have been carried out on the Antarctic yeasts focusing on the effect of heat shock on the production of HSPs using the proteomic approach (Deegenaars and Watson 1997; Deegenaars and Watson 1998). This project was set out to expand our knowledge on the cold adaptation mechanisms employed by Antarctic yeast by determining the expression patterns of 14 thermal stress-related genes, namely afp4, hsp70, hsp90, hsp100, nth1, tps1, tps2, fad, prx, gst, grxA, grxB, cat, and MnSOD using the qPCR when G. antarctica PI12 is exposed to cold shock (0 °C), mild cold shock (5 °C), and heat shock (22 °C) conditions.

Materials and methods

Strain and culture conditions

Glaciozyma antarctica PI12 was isolated from sea ice collected in the vicinity of Casey Station (66°21′25″S; 110°37′09″E), Antarctica. It was identified based on its LSU rRNA and ITS sequences which have been deposited in the GenBank database under accession numbers: JX896955 and JX896956. G. antarctica PI12 was routinely cultivated in yeast peptone dextrose (YPD) broth (Difco) medium.

Growth performance under different temperatures

The optimum growth temperature of G. antarctica PI12 was pre-determined in this work in order to select temperatures that will trigger cold and heat shock responses. G. antarctica PI12 was grown in YPD broth medium at temperatures ranging from 5 to 15 °C (with an interval of 2 or 3 °C) under shaking at 210 rpm. Three replicates were prepared for each incubation temperature. The optical density of the culture at 600 nm (OD600) was measured at an interval of 24 h until it reached the stationary phase. The growth rate was calculated using OD600 results.

Exposure of yeast culture to thermal stress

Glaciozyma antarctica PI12 was grown in YPD broth medium in 12 conical flasks at the optimal temperature 12 °C to mid log phase. Subsequently, these cultures (3 replicates each) were exposed to 0, 5, and 12 °C for 24 h to give the slow growing yeast sufficient time to response and at 22 °C for 1 h. After the exposure period, the cultures were snap frozen in liquid nitrogen and stored at −80 °C prior to RNA extraction.

RNA extraction and cDNA preparation

The total RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. Extracted RNA was treated with RNase-free DNase (Qiagen) to remove traces of genomic DNA. Subsequently, the sample was purified using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. The first-strand cDNA synthesis was performed using the SuperScript™ III First-Strand Synthesis System for RT-PCR (Invitrogen) using a reaction mixture comprising, 1 μg total RNA, 50 μM Oligo dT20 primer, and 200 U SuperScript III reverse transcriptase. The mixture was incubated at 50 °C for 50 min. DNA contamination assay was conducted using PCR amplification. The primers used were 5′-CGCACGTAACCGTGGTATC-3′ and 5′-TAGAGCTGGGTAGCCGAGAA-3′ that targeted the intron of the hsp70 gene.

Primer design

Specific primers were designed based on the annotated expressed sequence tag (EST) of G. antarctica PI12 generated by Izwan and Munir (personal communication). Nineteen pairs of specific primers were designed to target the reference genes and target genes (Table 1) using Primer3 software, with the length of the amplicons varying between 90 and 150 bp. The primers were synthesized by First-Base Laboratories (Singapore).

Table 1 Description, primer sequences, amplicon length, and PCR efficiency for five candidate reference genes and 14 target genes
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qPCR

The iScript™ One-Step RT-PCR kit with SYBR® Green (Bio-rad) was used for qPCR. qPCR was carried out in 25 μl mixture containing, 12.5 μl 2× SYBR Green RT-PCR reaction mix, 5 μM primers, 500 ng total RNA, 0.5 μl iScript reverse transcriptase, and 10 μl nuclease-free water. The PCR conditions were 50 °C for 10 min and 95 °C for 5 min, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s. Data were collected at 60 °C. The melt curve was determined under the following conditions: 95 °C for 1 min, 55 °C for 1 min followed by 80 cycles at 55 °C for 10 s at an increment of 0.5 °C per cycle. All reactions were performed in triplicate using the iCycler (Bio-rad). A set of negative control was included in each run. The qPCR products were sequenced and aligned against the sequences in the NCBI for annotation purposes.

qPCR data analysis

The correlation coefficient, R2, and the primer efficiencies were determined using the iCycler software based on the standard curve. The standard curve was generated using five dilution series. The stability of five reference genes, act3, act4, IdhD, tub2, and gdh was analyzed using the geNorm (version 3.4) (Vandesompele et al. 2002) and NormFinder (Andersen et al. 2004) analysis tools to determine the most suitable reference genes for this work. The fold changes in expression were calculated using the gene expression macro software version 1.1 (Bio-Rad Laboratories). It was normalized against the act4 and IdhD reference genes, and 12 °C was used as calibrator condition. The macro was derived based on the algorithms developed by Vandesompele et al. (2002).

A one-way analysis of variance (ANOVA) was performed to test whether there was a significant evolution in gene expression levels under all the thermal stress conditions. When one-way ANOVA was significant, the tukey HSD method (n = 3, P < 0.05) was used to locate significant differences. SigmaStat® Version 3.0.1 software (SPSS Inc.) was used to perform this statistical analysis. Genes were recorded as significantly down- or up-regulated if the fold change was at least 1.5-fold lower or higher than the calibrator condition at 12 °C, and the P value should be less than 0.05.

Nucleotide sequence accession numbers

The EST sequences were deposited in NCBI GenBank and were assigned the following accession numbers: JF412496 (tub2), JF412497 (act3), JF412498 (act4), JF412499 (gdh), JF412500 (IdhD), JF412501 (nth1), JF412502 (afp4), JF412503 (tps1), JF412504 (tps2), JF412505 (hsp70), JF412506 (hsp90), JF412507 (hsp100), JF412508 (prx), JF412509 (gst), JF412511 (grxA), JF412512 (grxB), JF412513 (cat), JF412514 (MnSOD).

Results

Determination of optimal growth temperature

The growth curve revealed that the optimal growth temperature of G. antarctica PI12 was 12 °C since G. antarctica PI12 had the highest growth rate (0.6 day−1) at this temperature (Fig. 1). The growth rate of G. antarctica PI12 decreased when it was grown at 1 °C lower or higher than 12 °C and drastically decreased at higher temperatures.

Fig. 1
figure 1

Growth rate curve of G. antarctica PI12 at five different growth temperatures (5, 8, 10, 12, and 15 °C) in YPD medium. OD600 was used for growth rate calculation

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Gene expression analysis

An exposure time of 1 h to 22 °C was found to be sufficient for the induction. An exposure of more than 1 h dramatically decreased the amount of RNA recovered (Fig. 2). In addition, G. antarctica PI12 grown to an OD600nm of not more than 1.5 because beyond this, there was a significant reduction and degradation of RNA (data not shown). The act4 and IdhD genes were used as the reference genes in this analysis because they had the lowest expression stability values (M). This means that their expressions are stable when compared to the other three reference gene candidate across all the thermal conditions tested (Table 2).

Fig. 2
figure 2

Total RNA isolated from G. antarctica PI12 after exposed to 22 °C for different time ranges. M ssRNA ladder (NEB), 1–30, 2–60, 3–90 min

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Table 2 Expression stability values (M) of five candidate reference genes as calculated by NormFinder and geNorm
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The expression level of the 14 genes of G. antarctica PI12 exposed to 0, 5, and 22 °C was compared to the expression level at 12 °C (optimal growth temperature) in order to identify genes that were up- or down-regulated. Statistical analysis showed that the gene expression levels of three genes (hsp100, gst, and tps1) did not significantly change after exposure to all three thermal stress conditions. As stated above, only the genes with fold change ≥1.5-fold and p value <0.05 are considered as significantly expressed.

The expression of afp4 increased 15.3-fold and 2.9-fold at 0 and 5 °C, respectively. However, it was not induced under heat shock condition at 22 °C. The transcript level of the fad gene had increased by 1.8-fold when the cells were exposed to cold shock. The expression of hsp70 significantly increased 3.2-fold when the cells were exposed to heat shock at 22 °C. Meanwhile, the expression of the hsp90 and hsp100 genes were maintained at constant levels with the fold change equal to one when the cells were exposed to 22 °C (Fig. 3). There was no significant induction of the three genes, tps1, tps2, and nth1 required for the biosynthesis and degradation of trehalose under all the three thermal stress conditions.

Fig. 3
figure 3

Fold changes for 14 thermal stress-related genes after exposed to cold shock (0 °C), mild cold shock (5 °C), and heat shock (22 °C) conditions. a AFP and fatty acid desaturase genes, b HSPs genes, c trehalose biosynthesis and degradation genes, d oxidative stress-related genes. Gene expression was quantified using the gene expression macro software (Bio-rad). The growth condition at 12 °C was used as the calibrator condition, and act4 and IdhD gene were used as internal control. Asterisk indicates significant difference (P < 0.05)

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The expression profiles of the oxidative stress-related genes varied. Under cold shock condition, up-regulation was observed for the MnSOD gene (1.9-fold), whereas down-regulation was observed for the grxA gene (−2.0-fold). The expression of the prx gene had increased by 1.8-fold when the cells were exposed to heat shock at 22 °C. On the other hand, the cat gene was down-regulated 1.8-fold under the same condition (Fig. 3). The grxB gene was down-regulated 3.2-fold under the mild cold shock condition at 5 °C when compared to 12 °C (Fig. 3).

Discussion

Turkiewicz et al. (2005) have reported that the optimal growth temperature of G. antarctica strain 171 was 15 °C. However, G. antarctica strain PI12 used in this study grew best at 12 °C in YPD medium and its growth rate declined at 11 or 13 °C indicating that it is very sensitive to small temperature change. The slight differences are possibly due to strains variation among G. antarctica isolates originated from different geographic locations. Howarth and Ougham (1993), Cairns et al. (1995) reported that a 10 °C increase above the growth temperature of an organism is required to elicit heat shock response. Hence, G. antarctica PI12 was exposed to 22, 10 °C above the optimum growth temperature to induce heat shock response.

The 14 genes assayed in this work were previously found to respond to thermal stress (Fulco and Fuji 1980; Deegenaars and Watson 1998; Porankiewiz et al. 1998; Methé et al. 2005; Hu et al. 2008; Gocheva et al. 2009) in mesophilic or psychrophilic organisms. They were the (1) afp4 gene encoding antifreeze protein (AFP) which is important to provide protection to cells in freezing temperatures (Davies et al. 2002), fad gene encoding the fatty acid desaturase enzyme which is involved in the maintenance of membrane fluidity (Fulco and Fuji 1980); (2) hsp70, hsp90, and hsp100 genes encoding heat shock proteins (HSPs) that act as molecular chaperones for protein folding (Gething and Sambrook 1992); (3) nth1 gene encoding neutral trehalase act as trehalose degradation enzyme, and tps1 and tps2 genes that are involved in the biosynthesis of trehalose (Kopp et al. 1993; Murata et al. 2006); and (4) prx, gst, grxA, grxB, cat, and MnSOD genes encoding anti-oxidant enzymes that are important for reduction in oxidative stress caused by accumulation of ROS (Berlett and Stadtman 1997; Toledano et al. 2003).

The expression profiles of this Antarctic yeast G. antarctica PI12 exposed to cold shock (0 °C), mild cold shock (5 °C), and heat shock (22 °C) were compared to the thermal stress responses of other Antarctic organisms to assess the similarity and differences between them. Firstly, our study showed that AFP and fatty acid desaturase played an important role for cold adaptation in G. antarctica PI12. AFPs have been discovered in a wide range of Antarctic organisms such as algae (Hu et al. 2008; Gwak et al. 2009), fish (Hofmann et al. 2000), plant (Bravo and Griffith 2005), and bacteria (Gilbert et al. 2004). AFPs prevent the formation of ice crystals and lower the freezing temperature of the water without altering its melting point (Davies et al. 2002). Exposure of G. antarctica PI12 to 0 and 5 °C caused significant induction of afp4. Similar results have been noted for two Antarctic algae, Chlorella vulgaris NJ-7 and Chaetoceros neogracile (Hu et al. 2008; Gwak et al. 2009). Although the amino acid sequences of AFP or IBP (ice binding protein) were very diverse among different organisms but the amino acid sequence of AFP4 of G. antarctica PI12 was 93 % similar with IBP of Arctic yeast, Leucosporidum sp. AY30 (Lee et al. 2010; Park et al. 2011). Turchetti et al. (2011) have stated that this Arctic yeast probably belonged to G. watsonii rather than G. antarctica based on the few characteristics described by Lee et al. (2010). Nevertheless, the AFPs produced by G. antarctica PI12 and G. watsonii AY30 which under the same genus of Glaciozyma probably share some similarities in their functional sites and protein domains.

Apart from the AFP, fatty acid desaturase (FAD) is important to maintain the membrane fluidity in G. antarctica PI12 under cold shock conditions by converting pre-existing saturated fatty acids to the unsaturated forms (Vigh et al. 1998). Induction of desaturases by cold has been described in an Antarctic algae, C. vulgaris NJ-7. This alga possesses two fatty acid desaturase genes, CvFAD2 and CvFAD6, induced at different temperatures. CvFAD2 mRNA increased when the alga was transferred from 25 to 15 °C or 4 °C, whereas CvFAD6 was only up-regulated under extreme low temperature at 4 °C. The fatty acid desaturase gene, fad, in G. antarctica PI12 was up-regulated at 0 °C but not under mild cold shock condition at 5 °C which has the similar expression pattern to CvFAD6. These results suggested that the fatty acid desaturase genes in some Antarctic organisms have evolved in such a way that they are induced only under extreme low temperature (≤4 °C).

The classical heat shock response involving a strong up-regulation of HSP70 production has been demonstrated in most of the Antarctic organisms examined to date with exception in some Antarctic marine organisms such as fish (Hofmann et al. 2000; Place and Hofmann 2005; Clark et al. 2008a), ciliate (LaTerza et al. 2001, LaTerza et al. 2004), sea star, and gammarid (Clark et al. 2008b). They have possibly lost their ability to respond to heat shock due to evolution after living in the cold environment for a long period of time. G. antarctica PI12 was found to harbor a gene encoding the HSP70 family protein too. The amino acid sequence of hsp70 of G. antarctica PI12 consisted three heat shock protein 70 family signatures, signature 1 (IDGTTYS), signature 2 (IFDLGGGTFD), and signature 3 (IVIVGGSTRIP) indicating that that it belongs to the HSP70 family. Piano et al. (2005) have pointed out that the GGMP repeats are present in the bivalve heat shock cognate 70 (HSC70) but absent in the heat shock protein 70 (HSP70), so the presence of GGMP tetrapeptide repeats in the amino acid sequence probably can be used to differentiate the HSC70 and HSP70. However, the hsp70 of G. antarctica PI12 carries only a single copy of the GGMP amino acid sequence. Hence, it is not known whether hsp70 of G. antarctica PI12 is a cognate or inducible gene based solely on the amino acid alignment analysis. Nevertheless, based on the qPCR results of this study, it was found that the hsp70 of G. antarctica PI12 was heat-inducible at 22 °C and this indicates that hsp70 is an inducible HSP70.

HSP genes were probably not important for cold adaptation since none of them were induced at 0 and 5 °C. Incidentally, low temperature is well known to reduce the probability of protein misfolding and aggregation (King et al. 1996), therefore possibly reducing the need for HSP chaperones. In fact, it has been shown that the induced expression of HSPs in Eschericia coli is harmful to cells at 4 °C to the extent that it reduces cell viability at this temperature (Kandror and Goldberg 1997). Additionally, it was reported that cold repression of HSPs is beneficial to the Antarctic pscyhrophilic bacteria Pseudoalteromonas haloplanktis and Exiguobacterium sibiricum (Piette et al. 2011).

The over-expression of antioxidant genes, MnSOD and prx, in this work suggests that the oxidative stress was induced in G. antarctica PI12 under cold shock and heat shock conditions. However, the antioxidant genes induced under heat shock and cold shock conditions are different. This probably has something to do with the thermal stability of enzymes under different thermal conditions. Our results showed that MnSOD is the only gene up-regulated at 0 °C but not at 5 °C. Similar data were reported that cold exposure significantly increased activities of superoxide dismutase (SOD) in Antarctic plants (Arora et al. 2002), algae (Collén and Davison 2001), and animals (Ansaldo et al. 2000; Abele et al. 2001; Heise et al. 2006). Meanwhile, one of the glutaredoxin gene, grxA in G. antarctica PI12, was down-regulated under cold shock condition. In contrast, Hwang et al. (2008) reported that the glutaredoxin gene was up-regulated under cold shock condition at 4 and 10 °C in C. neogracile. Meanwhile, Gidekel et al. (2003) demonstrated that the glutaredoxin gene was also up-regulated at 4 °C in the Antarctic monocot plant, Deschampsia antarctica.

Similar with cold shock conditions, there was only one antioxidant gene, prx that was significantly up-regulated under heat shock conditions. A similar observation was reported for the Antarctic bivalve Laternula elliptica whereby two of its peroxiredoxins were up-regulated significantly when exposed to 10 °C (Park et al. 2008). Kim et al. (2009) reported that the pi-class glutathione S-transferase of L. elliptica was also up-regulated upon heat treatment. However, the glutathione S-transferase gene, gst in G. antarctica PI12, was not induced at 22 °C. This indicated that the expression of gst in G. antarctica PI12 is possibly triggered by temperatures higher than 22 °C.

Besides, the results in this work revealed that the trehalose accumulation under thermal stress conditions in G. antarctica PI12 was possibly caused by the repression of nth1 but not due to over-expression of the tps1 and tps2 genes. tps1 and tps2 which are involved in the biosynthesis of trehalose are normally up-regulated when cells are exposed to cold shock (Inouye and Phadtare 2004; Kandror et al. 2004; Murata et al. 2006) or heat shock (Attfield 1987; Bell et al. 1992 and Vuorio et al. 1993). Deegenaars and Watson (1998) reported that G. antarctica has the greatest intrinsic level of trehalose when exposed to heat shock when compared to three other psychrophilic yeasts. However, our study showed that there is no significant induction of tps1 and tps2 under heat shock and cold shock conditions in G. antarctica PI12. Hence, we have speculated that the accumulation of trehalose in G. antarctica PI12 under stress conditions is not caused by the over-expression of tps1 and tps2 but is due to other factors. Neutral trehalase is an enzyme involved in the hydrolysis of trehalose and which degrades excess amounts of the disaccharide in the cell (Kopp et al. 1993). When cells are exposed to heat shock or cold shock, trehalose is allowed to accumulate as a way to protect the G. antarctica PI12 cells by down-regulating the nth1 gene as found in this work. Similar findings were reported for fungi (Ocón et al. 2007) and yeast (Kim et al. 2011) exposed to stresses. These organisms were found to accumulate trehalose through down-regulation of trehalase gene, nth1.

The results of this work lead us to conclude G. antarctica PI12 possibly avoids ice crystallization and maintains the membrane fluidity in the cells by over-expressing the AFP and fatty acid desaturase genes under cold stress (Lee et al. 2010; Park et al. 2011). At the same time, the antioxidant enzyme, MnSOD, is highly up-regulated to neutralize the oxidative stress. When the condition is in the reverse, G. antarctica PI12 produces large amounts of HSP70 as chaperone for protein folding. Antioxidant gene prx is over-expressed under heat shock condition. These findings are interesting and warrant a more extensive analysis to provide a better understanding of what is happening in the cells when they are exposed to heat or cold shocks. As sequencing using the next generation sequencing is becoming more affordable, it will be ideal to conduct RNA sequencing to look at the transcriptome profiles of G. antarctica PI12 under these thermal stress conditions.