**Transient Cold Shock Induces Oxidative Stress Events in Antarctic Fungi**

Nedelina Kostadinova, Ekaterina Krumova, Tzvetanka Stefanova, Vladislava Dishliyska and Maria Angelova *The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, Bulgaria* 

#### **1. Introduction**

74 Oxidative Stress – Environmental Induction and Dietary Antioxidants

Wu, G. & Morris, S.M.Jr. (1998). Arginine Metabolism: Nitric Oxide and Beyond. *The* 

Yamaner, F. (2010). Oxidative Predictors and Lipoproteins in Male Soccer Players. *Turkish* 

*Biochemical Journal,* Vol. 336, No. 1, (November 1998)*,* pp. 1–17

*Journal of Medical Sciences,* Vol. 40, No. 3, pp. 1-8

The Antarctic biota has evolved under the influence of a suite of geological and climatic factors, including the geographic isolation of the landmass and the continental shelves, extremely low temperatures and intense seasonality (Russo et al., 2010). The isolation and environmental history of Antarctica have led to a unique biota. Many groups of organisms became extinct in Antarctica as a result of the extremely cold conditions. Although this continent is the coldest, highest, windiest, driest, wildest and most pristine of all of the continents, it is full of life. In addition to its well-known inhabitants, such as penguins and seals, it also has a diverse and unique range of microbial diversity (Nichols et al., 1999; Vincent, 2000). Microorganisms successfully colonise cold habitats and play a major role in the processes of nutrient turnover at low temperatures. In recent years, a growing attention in research has been devoted to cold-adapted microorganisms. This interest in Antarctic microorganisms stems from several reasons. Antarctica's environmental extremes present conditions in which microorganisms have evolved unique characteristics for survival, which are of great scientific interest. Moreover, the availability of novel Antarctic species, which are generally isolated from extreme environments, opens the door for biotechnological exploration. Investigations of psychrotolerant and psychrophilic microorganisms are also important for human health because microorganisms can cause food spoilage and food-borne diseases. Research on cold shock raises a number of questions: which cellular function is affected most upon cold shock, what makes cell growth stop, and are there well-conserved or common cold shock proteins as in the case of heat-shock proteins? These questions are no less important than those in the case of heat shock (Inouye, 1999).

Cold-adapted microorganisms include both psychrophilic (organisms with an optimal growth temperature at or below 15°C and a maximum growth temperature below 20°C) and psychrotrophic or psychrotolerant (organisms exhibiting the ability to grow at temperatures below 15°C but exhibiting maximum growth rates at temperature optima above 18°C) organisms, and they are often subjected to other extreme environmental parameters (Morgan-Kiss et al., 2006). Antarctic microflora is also represented by mesophilic and thermophilic bacteria that are isolated from geothermal soils and by mesophilic fungi, which

Transient Cold Shock Induces Oxidative Stress Events in Antarctic Fungi 77

The fungal strains, *Penicillium sp.* 161 and *Aspergillus glaucus* 363 (with optimal growth temperatures of 20°C and 25°C, respectively), isolated from Livingston Island (South Shetlands archipelago, Antarctica) (Tosi et al., 2010) were used for the experiments. The strains belong to the Mycological collection at the Institute of Microbiology, Sofia, and they

The composition of the seed and production media has been described previously (Angelova et al., 1996). The cultivation was performed in a 3 L bioreactor, which was ABR-09-developed and constructed by the former Central Laboratory for Bioinstrumentation and Automatisation (CLBA) of the Bulgarian Academy of Sciences. The bioreactor was equipped with temperature, pH and automatic dissolved oxygen (DO) monitoring equipment and a

For the submerged cultivation, 74 ml of seed medium was inoculated with 6 ml of spore suspension at a concentration of 2×108 spores/ml in 500-ml Erlenmeyer flasks. The cultivation was performed at 20C for 48 h for the psychrotolerant strain and at 25C for 24 h for mesophilic strain on a rotary shaker (220 rpm). For the bioreactor cultures, 200 ml of the seed culture was brought into the 3-L bioreactor, which contained 1800 ml of the production medium. The cultures were grown at an optimal temperature with a stirrer speed of 400 rpm and an air flow of 0.5 vvm. During the middle of the exponential phase (24 h for *Penicillium sp.* 161 and 18 h for *A. glaucus* 363), the temperature was reduced to 4 or 10C. This downshift was reached in approximately 40 min. After an incubation of 6 h under cold stress conditions, the temperature was shifted up to the optimal value. The control variants

**2.2 Cell-free extract preparation and isolation of the cytosolic and mitochondrial** 

The cell-free extract, in addition to the cytosolic and mitochondrial fractions, were prepared as previously described (Krumova et al., 2008). All of the steps were performed at 0–4°C.

For the measurement of the O2¯production rate, the method of superoxide dismutaseinhibitable reduction of cytochrome *c* was used (Hassan & Fridovich, 1979) with some modifications. Briefly, the cell suspensions or mitochondrial fractions, which were taken from the control and cold-stressed cultures, were incubated for 60 min at 30°C on a water bath rotary shaker at 150 rpm. The reaction mixtures contained 50 M cytochrome *c*, 2% non-autoclaved glucose, 20 mM NADPH in the presence and absence of 50 g ml-1 of the superoxide dismutase from bovine erythrocytes in a 0.05 M potassium phosphate buffer with a pH of 7.8. The reaction was stopped by cooling in an ice-cold water bath. The cells were removed by centrifugation before measuring absorbance at 550 nm to determine the extent of cytochrome *c* reduction. A molar extinction coefficient of 2.11xl04 was used to calculate the concentration of reduced cytochrome C. For measurement of the hydrogen peroxide production, the method of Pick & Mizel (1981) was used. Briefly, fungal cells

were grown at their optimal temperature during the entire period.

**2. Materials and methods** 

control system.

**fractions** 

**2.3 Determination of ROS** 

**2.1 Fungal strains, culture media and cultivation** 

were maintained at 4°C on beer agar at pH 6.3.

are present as viable propagules that are able to grow actively, at least under Antarctic summer conditions (Pepi et al., 2005; Ruisi, 2007).

Cold-adapted microorganisms have developed several strategies to adapt to low temperatures (Onofri et al., 2007). This adaptation includes the production of cold-active enzymes (Feller & Gerday, 2003; Collins et al., 2008; Gatti-Lafranconi et al., 2010), the modulation of lipid compositions to maintain the fluidity of the cell membrane (Chintalapati et al., 2004; Russell, 2008), the production of RNA chaperones to suppress the formation of undesired secondary structures of RNA (Kwak et al., 2011) and the synthesis of antifreeze (García-Arribas et al., 2007) and cold shock proteins (Horn et al., 2007).

Additionally, low temperatures can induce oxidative stress due to the enhanced generation of reactive oxygen species (ROS), such as the superoxide anion (O2¯), hydrogen peroxide H2O2, and the hydroxyl radical OH (Chattopadhyay, 2002; Gocheva et al., 2009). These ROS are highly damaging to cellular components, including DNA, lipids and proteins (Sies, 1993). To scavenge ROS and prevent damage, all aerobic cells have evolved a complex defence system consisting of both low molecular mass scavengers and high molecular mass antioxidants, particularly, antioxidant enzymes, such as superoxide dismutase (SOD) and catalase (CAT) (Fridovich, 1998). Thus, antioxidant defence may play a significant role in the microbial survival mechanism under extremely cold conditions (Chattopadhyay, 2002; Chattopadhyay et al., 2011).

Terrestrial microfungal communities in the Antarctic are rarely investigated (Ruisi et al., 2007). There are even fewer studies of the relationship between low temperatures and the induction of oxidative stress events. The decrease in temperature gives signal to organism for evoking cold-shock response generating molecules that are required for growing at the lower range of growth temperature. These cold-shock response are also critical for survival and growth at lower temperatures (Ray, 2006). Our previous studies indicated that growth at a low temperature induced oxidative stress in fungal strains isolated from soil samples of three regions of Antarctica: Casey Station, Terra Nova Bay and South Georgia (Gocheva et al., 2005, 2006, 2009). In recent years, we have also isolated filamentous fungi from samples taken from another Antarctic region – the permanent Bulgarian Antarctic base "St. Kl. Ohridski" on Livingston Island in the South Shetland Islands (Maritime Antarctica) – during the Bulgarian Antarctic expedition 2006/07 (Tosi et al., 2010). Published data about the filamentous and larger fungi of Livingston Island are scarce (Gray & Smith 1984; Wirtz et al., 2003). Furthermore, the filamentous fungi from the Bulgarian area on Livingston Island have only been investigated by our research team. Moreover, the studies on the specificity of the cell response to cold-induced oxidative stress between two different thermal classes of Antarctic fungi, psychrophilic (psychrotolerant) and mesophilic, are scant (Gocheva et al., 2009).

This paper is an attempt to enlarge our previous investigation focusing on two Antarctic fungi (psychrotolerant and mesophilic strains) that were isolated from the Bulgarian Antarctic area on Livingston Island. This study was designed to compare the effect of shortterm cold shock on the growth of mycelia, glucose consumption and the level of oxidative stress biomarkers, including ROS, oxidatively damaged proteins, reserve carbohydrates and trehalose metabolising enzymes. Moreover, the Antarctic strains were used to obtain information concerning changes in the activities of two antioxidant enzymes, SOD and CAT, under a short-term cold treatment.

#### **2. Materials and methods**

76 Oxidative Stress – Environmental Induction and Dietary Antioxidants

are present as viable propagules that are able to grow actively, at least under Antarctic

Cold-adapted microorganisms have developed several strategies to adapt to low temperatures (Onofri et al., 2007). This adaptation includes the production of cold-active enzymes (Feller & Gerday, 2003; Collins et al., 2008; Gatti-Lafranconi et al., 2010), the modulation of lipid compositions to maintain the fluidity of the cell membrane (Chintalapati et al., 2004; Russell, 2008), the production of RNA chaperones to suppress the formation of undesired secondary structures of RNA (Kwak et al., 2011) and the synthesis of antifreeze

Additionally, low temperatures can induce oxidative stress due to the enhanced generation of reactive oxygen species (ROS), such as the superoxide anion (O2¯), hydrogen peroxide H2O2, and the hydroxyl radical OH (Chattopadhyay, 2002; Gocheva et al., 2009). These ROS are highly damaging to cellular components, including DNA, lipids and proteins (Sies, 1993). To scavenge ROS and prevent damage, all aerobic cells have evolved a complex defence system consisting of both low molecular mass scavengers and high molecular mass antioxidants, particularly, antioxidant enzymes, such as superoxide dismutase (SOD) and catalase (CAT) (Fridovich, 1998). Thus, antioxidant defence may play a significant role in the microbial survival mechanism under extremely cold conditions (Chattopadhyay, 2002;

Terrestrial microfungal communities in the Antarctic are rarely investigated (Ruisi et al., 2007). There are even fewer studies of the relationship between low temperatures and the induction of oxidative stress events. The decrease in temperature gives signal to organism for evoking cold-shock response generating molecules that are required for growing at the lower range of growth temperature. These cold-shock response are also critical for survival and growth at lower temperatures (Ray, 2006). Our previous studies indicated that growth at a low temperature induced oxidative stress in fungal strains isolated from soil samples of three regions of Antarctica: Casey Station, Terra Nova Bay and South Georgia (Gocheva et al., 2005, 2006, 2009). In recent years, we have also isolated filamentous fungi from samples taken from another Antarctic region – the permanent Bulgarian Antarctic base "St. Kl. Ohridski" on Livingston Island in the South Shetland Islands (Maritime Antarctica) – during the Bulgarian Antarctic expedition 2006/07 (Tosi et al., 2010). Published data about the filamentous and larger fungi of Livingston Island are scarce (Gray & Smith 1984; Wirtz et al., 2003). Furthermore, the filamentous fungi from the Bulgarian area on Livingston Island have only been investigated by our research team. Moreover, the studies on the specificity of the cell response to cold-induced oxidative stress between two different thermal classes of Antarctic

fungi, psychrophilic (psychrotolerant) and mesophilic, are scant (Gocheva et al., 2009).

This paper is an attempt to enlarge our previous investigation focusing on two Antarctic fungi (psychrotolerant and mesophilic strains) that were isolated from the Bulgarian Antarctic area on Livingston Island. This study was designed to compare the effect of shortterm cold shock on the growth of mycelia, glucose consumption and the level of oxidative stress biomarkers, including ROS, oxidatively damaged proteins, reserve carbohydrates and trehalose metabolising enzymes. Moreover, the Antarctic strains were used to obtain information concerning changes in the activities of two antioxidant enzymes, SOD and CAT,

(García-Arribas et al., 2007) and cold shock proteins (Horn et al., 2007).

summer conditions (Pepi et al., 2005; Ruisi, 2007).

Chattopadhyay et al., 2011).

under a short-term cold treatment.

#### **2.1 Fungal strains, culture media and cultivation**

The fungal strains, *Penicillium sp.* 161 and *Aspergillus glaucus* 363 (with optimal growth temperatures of 20°C and 25°C, respectively), isolated from Livingston Island (South Shetlands archipelago, Antarctica) (Tosi et al., 2010) were used for the experiments. The strains belong to the Mycological collection at the Institute of Microbiology, Sofia, and they were maintained at 4°C on beer agar at pH 6.3.

The composition of the seed and production media has been described previously (Angelova et al., 1996). The cultivation was performed in a 3 L bioreactor, which was ABR-09-developed and constructed by the former Central Laboratory for Bioinstrumentation and Automatisation (CLBA) of the Bulgarian Academy of Sciences. The bioreactor was equipped with temperature, pH and automatic dissolved oxygen (DO) monitoring equipment and a control system.

For the submerged cultivation, 74 ml of seed medium was inoculated with 6 ml of spore suspension at a concentration of 2×108 spores/ml in 500-ml Erlenmeyer flasks. The cultivation was performed at 20C for 48 h for the psychrotolerant strain and at 25C for 24 h for mesophilic strain on a rotary shaker (220 rpm). For the bioreactor cultures, 200 ml of the seed culture was brought into the 3-L bioreactor, which contained 1800 ml of the production medium. The cultures were grown at an optimal temperature with a stirrer speed of 400 rpm and an air flow of 0.5 vvm. During the middle of the exponential phase (24 h for *Penicillium sp.* 161 and 18 h for *A. glaucus* 363), the temperature was reduced to 4 or 10C. This downshift was reached in approximately 40 min. After an incubation of 6 h under cold stress conditions, the temperature was shifted up to the optimal value. The control variants were grown at their optimal temperature during the entire period.

#### **2.2 Cell-free extract preparation and isolation of the cytosolic and mitochondrial fractions**

The cell-free extract, in addition to the cytosolic and mitochondrial fractions, were prepared as previously described (Krumova et al., 2008). All of the steps were performed at 0–4°C.

#### **2.3 Determination of ROS**

For the measurement of the O2¯production rate, the method of superoxide dismutaseinhibitable reduction of cytochrome *c* was used (Hassan & Fridovich, 1979) with some modifications. Briefly, the cell suspensions or mitochondrial fractions, which were taken from the control and cold-stressed cultures, were incubated for 60 min at 30°C on a water bath rotary shaker at 150 rpm. The reaction mixtures contained 50 M cytochrome *c*, 2% non-autoclaved glucose, 20 mM NADPH in the presence and absence of 50 g ml-1 of the superoxide dismutase from bovine erythrocytes in a 0.05 M potassium phosphate buffer with a pH of 7.8. The reaction was stopped by cooling in an ice-cold water bath. The cells were removed by centrifugation before measuring absorbance at 550 nm to determine the extent of cytochrome *c* reduction. A molar extinction coefficient of 2.11xl04 was used to calculate the concentration of reduced cytochrome C. For measurement of the hydrogen peroxide production, the method of Pick & Mizel (1981) was used. Briefly, fungal cells

Transient Cold Shock Induces Oxidative Stress Events in Antarctic Fungi 79

Non-denaturing polyacrylamide gel electrophoresis of the extract was followed by activity staining for each of the enzymes tested. A forty g of total protein was applied to a 10% nondenaturing PAGE. Staining for SOD and CAT was performed as described by

The results obtained in this investigation were evaluated from at least three repeated experiments using three or five parallel runs. The statistical comparison between the controls and the treated cultures was determined by the Student's *t*-test for MIE (mean interval estimation) and by a one-way analysis of variance (ANOVA) followed by Dunnet's

Our previous study indicated that the optimal growth temperatures for both Antarctic strains, the psychrotolerant *Penicillium sp.* 161 and the mesophilic *A. glaucus* 363, are 20C and 25C, respectively (Tosi et al., 2010). The duration of the temperature shift (6 h) was chosen because this length of time was found to be sufficient to give a clear contrast between

**3.1 Response of the fungal growth and glucose consumption to temperature shifts**  Mycelia of the Antarctic strains, which were grown until the middle of the exponential phase at optimal temperature, were shifted to colder temperatures, i.e., 4C and 10C. Figure 1 shows the growth curves of the model strains after the temperature downshift exposure

Within the first 4 h of the beginning of the stress, the growth of the psychrotolerant strain *Penicillium sp.* 161 ceased and the biomass as measured by dry weight decreased sharply in comparison to the control (Fig. 1A). In the next 2 h, growth resumed and the return to the optimal 20C after 6 h allowed the biomass to increase relative to the control levels. A similar trend was demonstrated for the Antarctic mesophilic strain, *A. glaucus* 363, after a shift from 25C to 10C or 4C (Fig. 1B), but the difference in biomass production between the control and treated mycelia was more significant compared to the psychrotolerant strain. Moreover, after 4 h of recovery from either temperature treatment, biomass production was

The concentration of glucose in the culture medium was measured throughout the experiment (Fig. 2). The maximum glucose consumption occurred in cultures incubated at

A comparison of the model strains also shows that the consumption of glucose by the mesophilic strain (Fig. 2B) was faster than that of the psychrotolerant fungus (Fig. 2A). Comparing the curves in Fig. 2A and Fig. 2B, it was possible to verify that the downshift of temperature from the optimal temperature to 10C or 4C caused a significant decrease in glucose consumption. This tendency continued even after the return to the optimal

Beauchamp and Fridovich (1971) and Woodbury et al. (1971), respectively.

**2.6 PAGE analyses** 

**3. Results** 

temperature.

**2.7 Statistical evaluation of the results** 

post-test, with a significance level of 0.05.

the control and the stressed cultures.

and the subsequent restoration of the normal conditions.

restored and the dry weight reached its pre-stress level.

an optimal growth temperature was comparable to the stressed cultures.

treated with temperature were suspended in a 0.05 M potassium phosphate buffer with a pH of 7.8 and containing 50 g ml-1 of horseradish peroxidase type VI-A. After incubation at 30°C for 45 min, the reaction was stopped by the addition of 1N NaOH, and the absorbance was read at 620 nm. For the calculations, a standard curve with H2O2 concentrations (from 5 to 50 M) was used.

#### **2.4 Enzyme activity determination**

SOD activity was measured by the nitro-blue tetrazolium (NBT) reduction method of Beauchamp & Fridovich (1971). The reaction mixture contained 56 M (NBT), 0.01 M methionine, 1.17 M riboflavin, 20 M KCN and 0.05 M phosphate buffer with a pH of 7.8. Superoxide was measured by the increase in absorbance at 560 nm at 30 C after 6 min of incubation from the beginning of the illumination. One unit of SOD activity was defined as the amount of enzyme required for the inhibition reduction of NBT by 50% (A560) and was expressed as units per mg protein [U/mg protein]. Cyanide (5 mM) was used to distinguish between the cyanide-sensitive isoenzyme Cu/Zn-SOD and the cyanide-resistant Mn SOD. The Cu/Zn-SOD activity was obtained as the total activity minus the activity in the presence of 5 mM cyanide.

The catalase activity was determined by monitoring the decomposition of 18 mM H2O2 at 240 nm (Beers & Sizer, 1952). One unit of activity was that which decomposes 1 μmol of H2O2 min-1 mg protein-1 at 25C and a pH of 7.0. Specific activity is given as U/mg protein.

The neutral trehalase (NT) and trehalose-6-phosphate synthase (TPS) enzyme activities were assayed in cell-free extracts by the methods of Müller et al. (1992) and Vandercammen et al. (1989), respectively, as described by El-Bashiti et al. (2005).

#### **2.5 Analytical methods**

The glycogen and trehalose contents were determined following the procedure of Becker (1978) and Vandecamen et al. (1989) and modified by Parrou et al. (1997). The soluble reducing sugars were determined by the Somogy-Nelson method (Somogy 1952).

The protein oxidative damage was measured spectrophotometrically as the protein carbonyl content using the 2,4-dinitrophenylhydrazine (DNPH) binding assay (Hart et al., 1999) slightly modified by Adachi & Ishii (2000). The cell-free extracts were incubated with DNPH for 1 h at 37C; the proteins were precipitated in 10% cold TCA and washed with ethanol:ethylacetate (1:1) to remove any excess of DNPH and finally dissolved in 6 M guanidine chloride with a pH of 2. The optical density was measured at 380 nm, and the carbonyl content was calculated using a molar extinction coefficient of 21 mM-1 cm-1 as nanomoles of DNPH incorporated (protein carbonyls) per mg of protein.

Protein was estimated by the Lowry procedure (Lowry, 1951) using a solution of bovine serum albumin as a standard.

The dry weight determination was performed on samples of mycelia harvested throughout the culture period. The culture fluid was filtered through a Whatman (Clifton, USA) No. 4 filter. The separated mycelia were washed twice with distilled water and dried to a constant weight at 105C.

#### **2.6 PAGE analyses**

78 Oxidative Stress – Environmental Induction and Dietary Antioxidants

treated with temperature were suspended in a 0.05 M potassium phosphate buffer with a pH of 7.8 and containing 50 g ml-1 of horseradish peroxidase type VI-A. After incubation at 30°C for 45 min, the reaction was stopped by the addition of 1N NaOH, and the absorbance was read at 620 nm. For the calculations, a standard curve with H2O2 concentrations (from 5

SOD activity was measured by the nitro-blue tetrazolium (NBT) reduction method of Beauchamp & Fridovich (1971). The reaction mixture contained 56 M (NBT), 0.01 M methionine, 1.17 M riboflavin, 20 M KCN and 0.05 M phosphate buffer with a pH of 7.8. Superoxide was measured by the increase in absorbance at 560 nm at 30 C after 6 min of incubation from the beginning of the illumination. One unit of SOD activity was defined as the amount of enzyme required for the inhibition reduction of NBT by 50% (A560) and was expressed as units per mg protein [U/mg protein]. Cyanide (5 mM) was used to distinguish between the cyanide-sensitive isoenzyme Cu/Zn-SOD and the cyanide-resistant Mn SOD. The Cu/Zn-SOD activity was obtained as the total activity minus the activity in the presence

The catalase activity was determined by monitoring the decomposition of 18 mM H2O2 at 240 nm (Beers & Sizer, 1952). One unit of activity was that which decomposes 1 μmol of H2O2 min-1 mg protein-1 at 25C and a pH of 7.0. Specific activity is given as U/mg protein. The neutral trehalase (NT) and trehalose-6-phosphate synthase (TPS) enzyme activities were assayed in cell-free extracts by the methods of Müller et al. (1992) and Vandercammen et al.

The glycogen and trehalose contents were determined following the procedure of Becker (1978) and Vandecamen et al. (1989) and modified by Parrou et al. (1997). The soluble

The protein oxidative damage was measured spectrophotometrically as the protein carbonyl content using the 2,4-dinitrophenylhydrazine (DNPH) binding assay (Hart et al., 1999) slightly modified by Adachi & Ishii (2000). The cell-free extracts were incubated with DNPH for 1 h at 37C; the proteins were precipitated in 10% cold TCA and washed with ethanol:ethylacetate (1:1) to remove any excess of DNPH and finally dissolved in 6 M guanidine chloride with a pH of 2. The optical density was measured at 380 nm, and the carbonyl content was calculated using a molar extinction coefficient of 21 mM-1 cm-1 as

Protein was estimated by the Lowry procedure (Lowry, 1951) using a solution of bovine

The dry weight determination was performed on samples of mycelia harvested throughout the culture period. The culture fluid was filtered through a Whatman (Clifton, USA) No. 4 filter. The separated mycelia were washed twice with distilled water and dried to a constant

reducing sugars were determined by the Somogy-Nelson method (Somogy 1952).

nanomoles of DNPH incorporated (protein carbonyls) per mg of protein.

(1989), respectively, as described by El-Bashiti et al. (2005).

to 50 M) was used.

of 5 mM cyanide.

**2.5 Analytical methods** 

serum albumin as a standard.

weight at 105C.

**2.4 Enzyme activity determination** 

Non-denaturing polyacrylamide gel electrophoresis of the extract was followed by activity staining for each of the enzymes tested. A forty g of total protein was applied to a 10% nondenaturing PAGE. Staining for SOD and CAT was performed as described by Beauchamp and Fridovich (1971) and Woodbury et al. (1971), respectively.

#### **2.7 Statistical evaluation of the results**

The results obtained in this investigation were evaluated from at least three repeated experiments using three or five parallel runs. The statistical comparison between the controls and the treated cultures was determined by the Student's *t*-test for MIE (mean interval estimation) and by a one-way analysis of variance (ANOVA) followed by Dunnet's post-test, with a significance level of 0.05.

### **3. Results**

Our previous study indicated that the optimal growth temperatures for both Antarctic strains, the psychrotolerant *Penicillium sp.* 161 and the mesophilic *A. glaucus* 363, are 20C and 25C, respectively (Tosi et al., 2010). The duration of the temperature shift (6 h) was chosen because this length of time was found to be sufficient to give a clear contrast between the control and the stressed cultures.

#### **3.1 Response of the fungal growth and glucose consumption to temperature shifts**

Mycelia of the Antarctic strains, which were grown until the middle of the exponential phase at optimal temperature, were shifted to colder temperatures, i.e., 4C and 10C. Figure 1 shows the growth curves of the model strains after the temperature downshift exposure and the subsequent restoration of the normal conditions.

Within the first 4 h of the beginning of the stress, the growth of the psychrotolerant strain *Penicillium sp.* 161 ceased and the biomass as measured by dry weight decreased sharply in comparison to the control (Fig. 1A). In the next 2 h, growth resumed and the return to the optimal 20C after 6 h allowed the biomass to increase relative to the control levels. A similar trend was demonstrated for the Antarctic mesophilic strain, *A. glaucus* 363, after a shift from 25C to 10C or 4C (Fig. 1B), but the difference in biomass production between the control and treated mycelia was more significant compared to the psychrotolerant strain. Moreover, after 4 h of recovery from either temperature treatment, biomass production was restored and the dry weight reached its pre-stress level.

The concentration of glucose in the culture medium was measured throughout the experiment (Fig. 2). The maximum glucose consumption occurred in cultures incubated at an optimal growth temperature was comparable to the stressed cultures.

A comparison of the model strains also shows that the consumption of glucose by the mesophilic strain (Fig. 2B) was faster than that of the psychrotolerant fungus (Fig. 2A). Comparing the curves in Fig. 2A and Fig. 2B, it was possible to verify that the downshift of temperature from the optimal temperature to 10C or 4C caused a significant decrease in glucose consumption. This tendency continued even after the return to the optimal temperature.

Transient Cold Shock Induces Oxidative Stress Events in Antarctic Fungi 81

To further characterise the fungal cell response to cold stress, the generation of ROS in both intact cells and mitochondrial fractions was determined. Table 1 shows the effect of decreased temperatures on the superoxide anion level in the model Antarctic strains. Elevated O2¯ content was found for all of the temperatures tested. In the psychrotolerant strain, the temperature downshift to 10ºC resulted in an increase in the O2¯ level (approximately 116% and 124% for the intact cells and mitochondria, respectively),

The next diminution in the temperature to 4ºC caused a more significant increase in the O2¯ content in the intact cells (196%) and the mitochondrial fraction (143%). Furthermore, the mesophilic strain that was treated by cold stress to 10ºC and 4ºC showed extremely high O2¯ levels, especially in the mitochondria (410% and 660% of the control, respectively). In both of the strains, the exposure to the above-mentioned low temperatures had a less pronounced

Control 3.590.03 5.240.03 4.700.02 0.550.01 10ºC 4.150.04 6.500.01 6.780.01 2.250.01 4ºC 7.050.18 7.470.05 11.10.07 3.630.02 Table 1. Increase in O2¯generation in the intact cells or mitochondrial fractions of *Penicillium*

Control 5.680.18 12.110.10 11.391.10 6.400.28 10ºC 6.220.40 15.000.37 13.721.09 9.790.33 4ºC 9.180.11 21.570.44 14.610.56 11.580.43

These results also demonstrated a more active process of ROS generation in the mitochondrial fractions than in the intact cells, and in the mesophilic *A. glaucus* 363

The reaction of proteins with oxygen radicals leads to the appearance of carbonyl groups in polypeptide chains (Davies and Goldberg 1987). Thus, carbonyl formation is a marker for protein oxidation. We investigated whether the exposure of Antarctic strains to a temperature downshift causes oxidative damage to proteins during the stress period and in

Table 2. Increase in H2O2 generation in the intact cells or mitochondrial fractions of

O2¯ [M/mg d.w. per h] *Penicillium* sp. 161 *A. glaucus* 363

Н2О2 [mM/mg d.w. per h] *Penicillium* sp. 161 *A. glaucus* 363

fraction Intact cells Mitochondrial

fraction Intact cells Mitochondrial

fraction

fraction

induction in the H2O2 levels compared to the superoxide anion content (Table 2).

Intact cells Mitochondrial

Intact cells Mitochondrial

*Penicillium* sp. 161 and *A. glaucus* 363 treated by low temperatures

compared to the psychrotolerant *Penicillium* sp. 161.

**3.3 Cold shock causes protein oxidation** 

sp. 161 and *A. glaucus* 363 treated by low temperatures

**3.2 Effect of cold stress on ROS generation** 

compared with the control.

Variant

Variant

Fig. 1. Effect of cold shock on biomass production by *Penicillium sp.* 161 (A) and *A. glaucus* 363 (B) ▲ – growth at optimal temperature; – downshift from optimal temperature to 4C; - downshift from optimal temperature to 10C. Bars represent SD of means. The effect of treatment was significant for the temperature treatment (P ≤ 0.05)

Fig. 2. Glucose consumption in *Penicillium sp.* 161 (A) and *A. glaucus* 363 (B) during cold shock and following recovery period. () – growth at optimal temperature; ()– downshift from optimal temperature to 4C; () - downshift from optimal temperature to 10C. Bars represent SD of means. The effect of treatment was significant for the temperature treatment (P ≤ 0.05)

#### **3.2 Effect of cold stress on ROS generation**

80 Oxidative Stress – Environmental Induction and Dietary Antioxidants

**0,4**

Fig. 1. Effect of cold shock on biomass production by *Penicillium sp.* 161 (A) and *A. glaucus* 363 (B) ▲ – growth at optimal temperature; – downshift from optimal temperature to 4C; - downshift from optimal temperature to 10C. Bars represent SD of means. The effect of

**0,6**

**0,8**

**1,0**

**1,2**

**1,4**

**A B**

**0 2 4 6 8 10**

**Stress**

**Time [h]**

**0 2 4 6 8 10 12**

**Stress**

**0 2 4 6 8 10**

**Time [h]**

treatment was significant for the temperature treatment (P ≤ 0.05)

**0 2 4 6 8 10 12**

**Stress**

**20**

**Time [h] Time [h]**

Fig. 2. Glucose consumption in *Penicillium sp.* 161 (A) and *A. glaucus* 363 (B) during cold shock and following recovery period. () – growth at optimal temperature; ()– downshift from optimal temperature to 4C; () - downshift from optimal temperature to 10C. Bars represent SD of means. The effect of treatment was significant for the temperature treatment

**25**

**30**

**35**

**40**

B

**20**

**25**

**30**

**Residual glucose content (mg/ml)**

(P ≤ 0.05)

**[mg/ml]**

**35**

**40**

A

**Stress**

**0,4**

**0,6**

**0,8**

**Biomass content [g/100 ml]**

**1,0**

**1,2**

**1,4**

To further characterise the fungal cell response to cold stress, the generation of ROS in both intact cells and mitochondrial fractions was determined. Table 1 shows the effect of decreased temperatures on the superoxide anion level in the model Antarctic strains. Elevated O2¯ content was found for all of the temperatures tested. In the psychrotolerant strain, the temperature downshift to 10ºC resulted in an increase in the O2¯ level (approximately 116% and 124% for the intact cells and mitochondria, respectively), compared with the control.

The next diminution in the temperature to 4ºC caused a more significant increase in the O2¯ content in the intact cells (196%) and the mitochondrial fraction (143%). Furthermore, the mesophilic strain that was treated by cold stress to 10ºC and 4ºC showed extremely high O2¯ levels, especially in the mitochondria (410% and 660% of the control, respectively). In both of the strains, the exposure to the above-mentioned low temperatures had a less pronounced induction in the H2O2 levels compared to the superoxide anion content (Table 2).


Table 1. Increase in O2¯generation in the intact cells or mitochondrial fractions of *Penicillium* sp. 161 and *A. glaucus* 363 treated by low temperatures


Table 2. Increase in H2O2 generation in the intact cells or mitochondrial fractions of *Penicillium* sp. 161 and *A. glaucus* 363 treated by low temperatures

These results also demonstrated a more active process of ROS generation in the mitochondrial fractions than in the intact cells, and in the mesophilic *A. glaucus* 363 compared to the psychrotolerant *Penicillium* sp. 161.

#### **3.3 Cold shock causes protein oxidation**

The reaction of proteins with oxygen radicals leads to the appearance of carbonyl groups in polypeptide chains (Davies and Goldberg 1987). Thus, carbonyl formation is a marker for protein oxidation. We investigated whether the exposure of Antarctic strains to a temperature downshift causes oxidative damage to proteins during the stress period and in

Transient Cold Shock Induces Oxidative Stress Events in Antarctic Fungi 83

by a 1.7-fold increase in trehalose content (Fig. 4A and 4B). It is noteworthy that higher initial levels of both of the reserve carbohydrates were detected in the mesophilic strain, *A. glaucus* 363. The stress conditions (4°C or 10°C) caused an additional sizeable rise in glycogen (1.4- and 1.3-fold, respectively) and trehalose (2.2- and 1.9-fold, respectively) content (Fig. 4C and 4D). The data demonstrated a clear dependence on both the degree of cold shock and the thermal characteristics of the model strain. Four hours after the removal of the temperature stress, the levels of trehalose and glycogen returned to the control values with the exception of glycogen at 4C. In the recovery phase, both strains maintained the

**0 2 4 6 8 10**

**Stress**

**0 2 4 6 8 10 0,2**

**Stress**

**0**

**Time [h] Time [h]**

Our results indicated that trehalose metabolism plays an important role in the response to cold stress (Fig. 5). In both strains used, the trehalose accumulation (see Fig. 4) paralleled a significant increase in the activity of TPS for up to 4 h, after which time the activity decreased (Fig. 5A and 5C). When *A. glaucus* 363 cells were exposed to a temperature shift to 4C or 10C, the TPS activity rapidly increased approximately 4- and 2.5-fold compared to the control culture, respectively. At the same conditions, *Penicillium* sp. 161 cultures demonstrated 2.4-fold higher activity than the control. The mesophilic strain demonstrated strong temperature shift-dependent changes in TPS activity, while no temperature dependence was observed in the psychrotolerant strain. Curiously, the enhanced levels of TPS in both of the Antarctic fungi coincided with an increase in NT activity (Fig. 5B and 5D). These increases were approximately 1.6- and 1.3-fold in the psychrotolerant fungus and 2.2-

Fig. 4. Glycogen (A, C) and trehalose (B, D) content in *Penicillium* sp. 161 (A, B) and *A. glaucus* 363 (C, D) upon optimal temperature () and upon temperature downshift from optimal to 4°C () or 10°C (). Bars represent SD of means. The effect of treatment was

**2**

**4**

**6**

**8**

**D**

**B**

**Trehalose [mg/g dry mass]**

**0,4 0,6 0,8 1,0 1,2 1,4**

high glycogen amount that had accumulated during the stress conditions.

**0 2 4 6 8 10 <sup>5</sup>**

**Stress**

**0 2 4 6 8 10 <sup>10</sup>**

**3.5 Changes in TPS and NT activities during cold stress conditions** 

**Stress**

significant for the temperature treatment (P ≤ 0.05)

**A**

**20**

**30**

**40**

**50**

**C**

**Glycogen [mg/g dry mass]**

the post-stress recovery phase (Fig. 3). The level of protein carbonyl content in the control variants of both Antarctic strains did not change notably during the 6 h of stress. However, after the temperature downshift, the amount of carbonyl groups in the cell proteins increased with the duration of the exposure compared to the control. The increase was dependent on the grade of the temperature shift used in the experiment and less on the temperature requirements of the strains. Both of the strains demonstrated a sharp elevation in oxidatively damaged proteins compared to the control immediately after the start of the treatment.

Fig. 3. Oxidative damage of proteins in *Penicillium* sp. 161 (A) and *A. glaucus* 363 (B), upon optimal temperature () and upon temperature downshift from optimal to 4°C () or 10°C (). Bars represent SD of means. The effect of treatment was significant for the temperature treatment (P ≤ 0.05)

As seen in Fig. 3, the maximum values of the carbonyl content in the mesophilic strain during the downshift to 10ºC and 4ºC were 1.3- and 1.6-fold higher than in the control, respectively. A similar trend was found for the psychrotolerant strain (1.2- and 1.3-fold, respectively), but it showed a comparably lower sensitivity to cold temperatures. Although the content of the damaged proteins decreased significantly during the recovery phase, its value remained considerably higher than in the control variants.

#### **3.4 Accumulation of reserve carbohydrates under conditions of cold shock**

Another physiological consequence of low temperature exposure in both of the Antarctic fungi was the accumulation of reserve carbohydrates through stress. The production of glycogen and trehalose after a temperature downshift to 4C or 10C was detected. As is shown in Fig. 4, there was a sharp increase in the glycogen and trehalose content after the beginning of the stress. In the experiments with the psychrotolerant strain, *Penicillium* sp. 161, glycogen was accumulated up to 1.2 - 1.75 times the basal level and was accompanied

the post-stress recovery phase (Fig. 3). The level of protein carbonyl content in the control variants of both Antarctic strains did not change notably during the 6 h of stress. However, after the temperature downshift, the amount of carbonyl groups in the cell proteins increased with the duration of the exposure compared to the control. The increase was dependent on the grade of the temperature shift used in the experiment and less on the temperature requirements of the strains. Both of the strains demonstrated a sharp elevation in oxidatively damaged proteins compared to the control immediately after the start of the

**0 2 4 6 8 10 12**

**Stress**

value remained considerably higher than in the control variants.

**3.4 Accumulation of reserve carbohydrates under conditions of cold shock** 

**0 2 4 6 8 10 12**

**Stress**

**6**

**Time [h] Time [h]**

Fig. 3. Oxidative damage of proteins in *Penicillium* sp. 161 (A) and *A. glaucus* 363 (B), upon optimal temperature () and upon temperature downshift from optimal to 4°C () or 10°C (). Bars represent SD of means. The effect of treatment was significant for the temperature

As seen in Fig. 3, the maximum values of the carbonyl content in the mesophilic strain during the downshift to 10ºC and 4ºC were 1.3- and 1.6-fold higher than in the control, respectively. A similar trend was found for the psychrotolerant strain (1.2- and 1.3-fold, respectively), but it showed a comparably lower sensitivity to cold temperatures. Although the content of the damaged proteins decreased significantly during the recovery phase, its

Another physiological consequence of low temperature exposure in both of the Antarctic fungi was the accumulation of reserve carbohydrates through stress. The production of glycogen and trehalose after a temperature downshift to 4C or 10C was detected. As is shown in Fig. 4, there was a sharp increase in the glycogen and trehalose content after the beginning of the stress. In the experiments with the psychrotolerant strain, *Penicillium* sp. 161, glycogen was accumulated up to 1.2 - 1.75 times the basal level and was accompanied

**9**

**12**

**15**

**B**

**6**

treatment (P ≤ 0.05)

**9**

**12**

**Protein carbonyl content [nM/mg protein]**

**15**

**A**

treatment.

by a 1.7-fold increase in trehalose content (Fig. 4A and 4B). It is noteworthy that higher initial levels of both of the reserve carbohydrates were detected in the mesophilic strain, *A. glaucus* 363. The stress conditions (4°C or 10°C) caused an additional sizeable rise in glycogen (1.4- and 1.3-fold, respectively) and trehalose (2.2- and 1.9-fold, respectively) content (Fig. 4C and 4D). The data demonstrated a clear dependence on both the degree of cold shock and the thermal characteristics of the model strain. Four hours after the removal of the temperature stress, the levels of trehalose and glycogen returned to the control values with the exception of glycogen at 4C. In the recovery phase, both strains maintained the high glycogen amount that had accumulated during the stress conditions.

Fig. 4. Glycogen (A, C) and trehalose (B, D) content in *Penicillium* sp. 161 (A, B) and *A. glaucus* 363 (C, D) upon optimal temperature () and upon temperature downshift from optimal to 4°C () or 10°C (). Bars represent SD of means. The effect of treatment was significant for the temperature treatment (P ≤ 0.05)

#### **3.5 Changes in TPS and NT activities during cold stress conditions**

Our results indicated that trehalose metabolism plays an important role in the response to cold stress (Fig. 5). In both strains used, the trehalose accumulation (see Fig. 4) paralleled a significant increase in the activity of TPS for up to 4 h, after which time the activity decreased (Fig. 5A and 5C). When *A. glaucus* 363 cells were exposed to a temperature shift to 4C or 10C, the TPS activity rapidly increased approximately 4- and 2.5-fold compared to the control culture, respectively. At the same conditions, *Penicillium* sp. 161 cultures demonstrated 2.4-fold higher activity than the control. The mesophilic strain demonstrated strong temperature shift-dependent changes in TPS activity, while no temperature dependence was observed in the psychrotolerant strain. Curiously, the enhanced levels of TPS in both of the Antarctic fungi coincided with an increase in NT activity (Fig. 5B and 5D). These increases were approximately 1.6- and 1.3-fold in the psychrotolerant fungus and 2.2-

Transient Cold Shock Induces Oxidative Stress Events in Antarctic Fungi 85

**0 2 4 6 8 10**

**Time [h]**

**Stress**

**0**

**0 2 4 6 8 10**

**Stress**

**Time [h]**

Fig. 6. SOD activities in cultures of *Penicillium* sp. 161 (A) and *A. glaucus* 363 (B) upon optimal temperature () and upon temperature downshift from optimal to 4°C () or 10°C (). Bars represent SD of means. The effect of treatment was significant for the temperature

There were significant differences in the expression of Mn-SOD and Cu/Zn-SOD among strains of both the psychrotolerant and mesophilic thermal classes (Fig. 7). Under optimal conditions, the psychrotolerant strain produced mostly Mn-SOD and less Cu/Zn-SOD (Fig. 7A), while in the mesophilic fungus, the Cu/Zn-SOD content was found to be significantly higher than Mn-containing enzyme (Fig. 7B). The cold shock treatment caused an enhanced level of both isoenzymes, but the increased total SOD activity was due to the Cu/Zn-SOD

To confirm the levels of Mn-SOD and Cu/Zn-SOD activity in the fungal cultures, the enzymatic activity of these antioxidant enzymes was determined using the native gel electrophoresis technique (Fig. 8). The non-denaturing PAGE showed that the cell-free extracts of the psychrophilic strain contained two distinct bands with SOD activity, Cu/Znand Mn-SOD. At least three clear SOD isoforms were observed after cell-free extract

These isoforms were identified as one Mn-SOD and two Cu/Zn-SOD, which were named I and II in order of increasing migration. The temperature treatment did not change the isoenzyme profile of either strain. At the same time, there was a temperature degree-

Figure 9 presents the data on the changes in CAT activity. In contrast to SOD, the psychrophilic strain showed a higher CAT level (1.5-fold) than that of the mesophilic strain.

proteins of the mesophilic strain were electrophoresed on a 10% native PAGE.

dependent increase in Cu/Zn- and Mn-containing SOD (Figs. 8B and 8C).

**<sup>6</sup>** <sup>B</sup>

**0**

treatment (P ≤ 0.05)

isoform for both strains.

**1**

**2**

**3**

**SOD activity [U/mg protein]**

**4**

**5**

A

**10**

**20**

**30**

**40**

**50**

and 1.7-fold in the mesophilic strain after cold shock at 4C or 10C, respectively, over the control value. We also found that the exposure to cold stress increased the NT levels in a time- and temperature shift-dependent manner. During the recovery phase, both strains showed a significant reduction in TPS activity, and the enzyme values reached those of the control cultures. In contrast, NT activity was maintained 4 h after the return to the optimal temperature.

Fig. 5. Cold stress induced trehalose metabolism. TPS (A, C) and NT (B, D) accumulation in *Penicillium* sp. 161 (A, B) and *A. glaucus* 363 (C, D) upon optimal temperature () and upon temperature downshift from optimal to 4°C () or 10°C (). Bars represent SD of means. The effect of treatment was significant for the temperature treatment (P ≤ 0.05)

#### **3.6 Activation of antioxidant enzyme defence**

When the mycelia, taken from middle of the exponential phase at the optimal temperature, were subjected for 6 h to 4C or 10C, the antioxidant enzyme defence was activated. The time courses of SOD activities for both of the strains during the cold shock treatment are shown in Fig. 6.

The results showed that transient exposure to 4C and 10C resulted in elevated total superoxide scavenging activity compared with the control variant, and the increase was in a dose- and time-dependent manner until 10 h, i.e., 4 h after the temperature recovery. The mesophilic strain had an approximately 7-fold higher SOD activity than the psychrotolerant strain. Moreover, the antioxidant cell response of *Penicillium* sp. 161 included an approximately 1.4- and 1.2- fold increase in SOD activity compared with the control culture at 4C and 10C, respectively (Fig. 6A). The fungal cultures of *A. glaucus* 363 also demonstrated a temperature-dependent response that was higher at 4C compared with the condition at 10C. The maximum increase was approximately 1.6- and 1.3-fold in the variants that were exposed to 4C and 10 C, respectively (Fig. 6B).

and 1.7-fold in the mesophilic strain after cold shock at 4C or 10C, respectively, over the control value. We also found that the exposure to cold stress increased the NT levels in a time- and temperature shift-dependent manner. During the recovery phase, both strains showed a significant reduction in TPS activity, and the enzyme values reached those of the control cultures. In contrast, NT activity was maintained 4 h after the return to the optimal

**0 2 4 6 8 10 <sup>0</sup>**

**0 2 4 6 8 10 <sup>0</sup>**

**4**

**Time [h] Time [h]**

Fig. 5. Cold stress induced trehalose metabolism. TPS (A, C) and NT (B, D) accumulation in *Penicillium* sp. 161 (A, B) and *A. glaucus* 363 (C, D) upon optimal temperature () and upon temperature downshift from optimal to 4°C () or 10°C (). Bars represent SD of means.

When the mycelia, taken from middle of the exponential phase at the optimal temperature, were subjected for 6 h to 4C or 10C, the antioxidant enzyme defence was activated. The time courses of SOD activities for both of the strains during the cold shock treatment are

The results showed that transient exposure to 4C and 10C resulted in elevated total superoxide scavenging activity compared with the control variant, and the increase was in a dose- and time-dependent manner until 10 h, i.e., 4 h after the temperature recovery. The mesophilic strain had an approximately 7-fold higher SOD activity than the psychrotolerant strain. Moreover, the antioxidant cell response of *Penicillium* sp. 161 included an approximately 1.4- and 1.2- fold increase in SOD activity compared with the control culture at 4C and 10C, respectively (Fig. 6A). The fungal cultures of *A. glaucus* 363 also demonstrated a temperature-dependent response that was higher at 4C compared with the condition at 10C. The maximum increase was approximately 1.6- and 1.3-fold in the

The effect of treatment was significant for the temperature treatment (P ≤ 0.05)

variants that were exposed to 4C and 10 C, respectively (Fig. 6B).

**0 2 4 6 8 10 <sup>0</sup>**

**0 2 4 6 8 10 <sup>0</sup>**

**<sup>D</sup> <sup>C</sup>**

**3.6 Activation of antioxidant enzyme defence** 

**Trehalose-6-P Synthase [nmol UDP/min/mg protein]**

**A**

**8**

**12**

**Neutral trehalase [mol trehalose/min/mg protein]**

**4**

**8**

**12**

**B**

temperature.

shown in Fig. 6.

Fig. 6. SOD activities in cultures of *Penicillium* sp. 161 (A) and *A. glaucus* 363 (B) upon optimal temperature () and upon temperature downshift from optimal to 4°C () or 10°C (). Bars represent SD of means. The effect of treatment was significant for the temperature treatment (P ≤ 0.05)

There were significant differences in the expression of Mn-SOD and Cu/Zn-SOD among strains of both the psychrotolerant and mesophilic thermal classes (Fig. 7). Under optimal conditions, the psychrotolerant strain produced mostly Mn-SOD and less Cu/Zn-SOD (Fig. 7A), while in the mesophilic fungus, the Cu/Zn-SOD content was found to be significantly higher than Mn-containing enzyme (Fig. 7B). The cold shock treatment caused an enhanced level of both isoenzymes, but the increased total SOD activity was due to the Cu/Zn-SOD isoform for both strains.

To confirm the levels of Mn-SOD and Cu/Zn-SOD activity in the fungal cultures, the enzymatic activity of these antioxidant enzymes was determined using the native gel electrophoresis technique (Fig. 8). The non-denaturing PAGE showed that the cell-free extracts of the psychrophilic strain contained two distinct bands with SOD activity, Cu/Znand Mn-SOD. At least three clear SOD isoforms were observed after cell-free extract proteins of the mesophilic strain were electrophoresed on a 10% native PAGE.

These isoforms were identified as one Mn-SOD and two Cu/Zn-SOD, which were named I and II in order of increasing migration. The temperature treatment did not change the isoenzyme profile of either strain. At the same time, there was a temperature degreedependent increase in Cu/Zn- and Mn-containing SOD (Figs. 8B and 8C).

Figure 9 presents the data on the changes in CAT activity. In contrast to SOD, the psychrophilic strain showed a higher CAT level (1.5-fold) than that of the mesophilic strain.

Transient Cold Shock Induces Oxidative Stress Events in Antarctic Fungi 87

**2**

**4**

**6**

**8**

**10**

**12**

B

**0 2 4 6 8 10 <sup>0</sup>**

**Stress**

**Time [h]**

**0 2 4 6 8 10 <sup>0</sup>**

**Stress**

**Time [h]**

Fig. 9. CAT activities in cultures of *Penicillium* sp. 161 (A) and *A. glaucus* 363 (B) upon optimal temperature () and upon temperature downshift from optimal to 4°C () or 10°C (). Bars represent SD of means. The effect of treatment was significant for the temperature

The low-temperature treatment resulted in a significant increase in CAT activity. The trend of the change was similar to that for SOD (a dose- and time-dependent manner). In all of the variants, the enzyme level exceeded the control value. The highest percentage of increased CAT activity (approximately 2-fold) was found in the psychrotolerant strain after the temperature downshift from 20°C to 4°C. The overexpression of CAT under the cold shock conditions was confirmed by PAGE analysis (Fig. 10). When the protein extracts were separated by native electrophoresis, one CAT isoenzyme was observed in both *Penicillium*

Fig. 10. CAT activity in cultures of *Penicillium* sp. 161 (A) and *A. glaucus* 363 (B) evaluated by polyacrylamide gel electrophoresis (10% gel): lane 1, Control; lane 2, cultures treated with

It is noteworthy that the tendency of increased SOD and CAT activities compared to the

The evaluation of oxidative stress events during low-temperature treatments provides information on the relationship between oxidative stress and cold shock in lower eukaryotes

control variant continued even after the return to the optimal temperature.

**2**

**4**

**6**

**CAT activity [U/mg proten]**

treatment (P ≤ 0.05)

sp. 161 and *A. glaucus* 363.

10°C; lane 3, cultures treated with 4°C

**4. Discussion** 

**8**

**10**

**12**

A

Fig. 7. Effect of low temperatures on isoenzyme profiles of *Penicillium* sp. 161 (A) and *A. glaucus* 363 (B) 4 h after the treatment: total SOD (hatchet bars); Cu/Zn-SOD (light grey bars); Mn-SOD (dark grey bars). Bars represent SD of means. The effect of treatment was significant for the temperature treatment (P ≤ 0.05).

Fig. 8. Isoenzyme profiles of SOD in *Penicillium* sp. 161 and *A. glaucus* 363 cells : standards (A); SOD activity in *Penicillium* sp. 161 (B) and in *A. glaucus* 363 (C) cells evaluated by polyacrylamide gel electrophoresis (10% gel): lane 1, standard Cu/Zn-SOD from bovine erythrocytes; lane 2, standard Mn-SOD from *Escherichia coli*; lanes 3, 4, and 5, SOD in the *Penicillium* sp. 161 sp cells; lanes 6, 7 and 8, SOD in the *A. glaucus* 363 cells, cultivated at control temperatures (lanes 3 and 6), at downshift to 10C (lanes 4 and 7) and downshift to 4C.

significant for the temperature treatment (P ≤ 0.05).

downshift to 4C.

**A**

**Control**

Fig. 7. Effect of low temperatures on isoenzyme profiles of *Penicillium* sp. 161 (A) and *A. glaucus* 363 (B) 4 h after the treatment: total SOD (hatchet bars); Cu/Zn-SOD (light grey bars); Mn-SOD (dark grey bars). Bars represent SD of means. The effect of treatment was

Fig. 8. Isoenzyme profiles of SOD in *Penicillium* sp. 161 and *A. glaucus* 363 cells : standards (A); SOD activity in *Penicillium* sp. 161 (B) and in *A. glaucus* 363 (C) cells evaluated by polyacrylamide gel electrophoresis (10% gel): lane 1, standard Cu/Zn-SOD from bovine erythrocytes; lane 2, standard Mn-SOD from *Escherichia coli*; lanes 3, 4, and 5, SOD in the *Penicillium* sp. 161 sp cells; lanes 6, 7 and 8, SOD in the *A. glaucus* 363 cells, cultivated at

control temperatures (lanes 3 and 6), at downshift to 10C (lanes 4 and 7) and

**10oC 4oC**

**Cu/Zn- or Mn-SOD [U/mg protein]**

Fig. 9. CAT activities in cultures of *Penicillium* sp. 161 (A) and *A. glaucus* 363 (B) upon optimal temperature () and upon temperature downshift from optimal to 4°C () or 10°C (). Bars represent SD of means. The effect of treatment was significant for the temperature treatment (P ≤ 0.05)

The low-temperature treatment resulted in a significant increase in CAT activity. The trend of the change was similar to that for SOD (a dose- and time-dependent manner). In all of the variants, the enzyme level exceeded the control value. The highest percentage of increased CAT activity (approximately 2-fold) was found in the psychrotolerant strain after the temperature downshift from 20°C to 4°C. The overexpression of CAT under the cold shock conditions was confirmed by PAGE analysis (Fig. 10). When the protein extracts were separated by native electrophoresis, one CAT isoenzyme was observed in both *Penicillium* sp. 161 and *A. glaucus* 363.

Fig. 10. CAT activity in cultures of *Penicillium* sp. 161 (A) and *A. glaucus* 363 (B) evaluated by polyacrylamide gel electrophoresis (10% gel): lane 1, Control; lane 2, cultures treated with 10°C; lane 3, cultures treated with 4°C

It is noteworthy that the tendency of increased SOD and CAT activities compared to the control variant continued even after the return to the optimal temperature.

#### **4. Discussion**

The evaluation of oxidative stress events during low-temperature treatments provides information on the relationship between oxidative stress and cold shock in lower eukaryotes

Transient Cold Shock Induces Oxidative Stress Events in Antarctic Fungi 89

chain, resulting in ROS accumulation in cucumber leaves. For fish living at low temperatures, the increased polyunsaturation of mitochondrial membranes should raise rates of mitochondrial respiration which would in turn enhance the formation of ROS,

Consistent with previous reports (Şahin & Gümüşlü, 2004; Manfredini et al., 2005; Nyström, 2005; Li et al., 2009), the present study demonstrated the coexistence of enhanced ROS activity and oxidatively damaged proteins in Antarctic fungal models. ROS can modify proteins leading to the formation of additional carbonyl groups caused by the oxidation of specific amino acid residues or the protein backbone (Davies & Goldberg, 1987). The level of proteins with formed additional carbonyl groups, which is thought to be an excellent marker for protein oxidation, has been shown to be related to oxidative stress in various studies. Low temperature stress was shown to induce ROS accumulation in cells (Suzuki & Mittler, 2006; Ouellet, 2007), which leads to a production of oxidised proteins. A similar response was observed in hyper-oxygenated cultures of white-rot fungus *Phanerochaete chrysosporium* (Belinky et al., 2003), heavy metal treatment (Krumova et al., 2009; Belozerskaya et al., 2010), addition of oxidant species (Angelova et al., 2005) and temperature exposure (Kim et al., 2006; Li et al., 2008; Gocheva et al., 2009), for example. In the temperature-induced protein carbonylation, Li et al. (2008) speculated that respiratory enzymes and those within mitochondria may be especially vulnerable because oxygenderived free radicals from the respiratory chain are generally held to be the main source of

The psychrotolerant strain *Penicillium* sp. 161 demonstrated a lower level of oxidatively damaged proteins than that in the mesophilic strain *A. glaucus* 363 during the exposure to 4°C or 10°C. At the same time, our unpublished data demonstrate the availability of coldacclimation proteins (Caps) in *Penicillium* sp. 161 and cold shock proteins (Csps) in *A. glaucus* 363. According to D'Amico et al. (2006), the existing distinctions between the mesophilic and the psychrophilic cold shock response include the lack of repression of housekeeping protein synthesis and the presence of Caps in psychrophiles. The authors considered that many of the Csps involved in various cellular processes, such as transcription, translation, protein folding and the regulation of membrane fluidity and observed in mesophiles (D'Amico et al., 2006, as cited in Phadtare, 1994), act as Caps in psychrophiles, and are constitutively rather than transiently expressed at low temperatures. Furthermore, this differential regulation of expression indicates that a temperature-sensory system exists in psychrophiles, and thermosensors at the cell membrane level, which sense changes in fluidity, have been reported (D'Amico et al., 2006, as cited in Ray et al., 1994).

Trehalose and glycogen accumulation in microbial cells when they are subjected to unfavourable growth conditions could also be used as stress indicators (Parrou et al., 1997; Türkel, 2006; Ocón et al., 2007; Iturriaga et al., 2009; Gonçalves et al., 2011). Both carbohydrates are important storage compounds in fungal vegetative cells and spores (Robinson, 2001; Rúa et al., 2008). In Antarctic fungi, *Penicillium* sp. 161 and *A. glaucus* 363, that were exposed to cold stress, a reduction in glucose consumption coincided with a significant increase in the trehalose and glycogen content (Fig. 4). These results agree with earlier studies regarding the microbial response against different types of abiotic stress (Ostrovskii et al., 2003; Jules et al., 2008; Robinson, 2001). Our previous studies have

increase proton leak and favour peroxidation of these membranes (Guderley, 2004).

the oxidative damage seen during stress.

such as filamentous fungi. Very little information is available on the oxidative cell response of microorganisms from cold habitats (Gocheva et al., 2006, 2009; Tosi et al., 2010; Chattopadhyay et al., 2011; Nichols et al., 1999). Even less is known concerning filamentous fungi from the Bulgarian Antarctic area. We choose to compare the cell responses between fungi belonging to two different thermal classes, psychrotolerant and mesophilic, that are able to grow in a wide range of temperatures. According to many reports on fungal biodiversity, these fungi are prevalent in Antarctica (Onofri et al., 2007, 2008; Arenz & Blanchette, 2011). Our results confirmed that the downshift of temperature induces typical events of oxidative stress. The exposure of both fungal cultures to 4°C or 10°C caused a statistically significant reduction in biomass production and glucose consumption compared to the control. Despite their genetic adaptation to survive in the harsh Antarctic environment, the sharp decrease in temperature resulted in a cessation of growth, which was more pronounced in the mesophilic fungus than in the psychrophilic fungus. A possible explanation or cause for this discrepancy is that most of the Antarctic mycoflora is metabolically active whenever a combination of favourable abiotic conditions occurs during the short growing summer season (Ruisi et al., 2007). However, Hébraud M. & Potier (1999) assumed that, as opposed to mesophiles, one or more regulatory factors exists in coldadapted microorganisms prior to cold shock that allows for the maintenance of functional translational machinery at low temperatures. However, despite this continuous protein synthesis, the growth of psychrotrophic strains ceases transiently after a cold shock, and additional regulatory mechanisms may exist that allow for growth resumption at low temperatures.

Additionally, the data for all cold shock variants reveal enhanced ROS generation. These ROS can be generated via a variety of extra-environmental conditions, including low temperature exposure (Belozerskaya & Gessler 2007; Gessler et al., 2007; Gómez-Toribio et al., 2009; Gocheva et al., 2009; Krumova et al., 2009). As is known, the unstressed fungal cells produce O2¯ and H2O2, presumably due to a single electron reduction of 2% of the consumed oxygen as has been previously suggested (Joseph-Horne et al., 2001). Our direct assay showed a significant increase in O2¯ under cold exposure to 4°C and 10°C in dosedependent manner. A similar but less-pronounced tendency was found for H2O2 levels. This increase was more extreme in experiments with mitochondria than in those with intact cells. Thus, cold stress likely imposes an oxidative burden of which O2¯ is a major component. A similar direct analysis of ROS content in fungal cells has not often been reported. Chattopadhyay et al. (2011) reported that the production of free radicals in the Antarctic bacterium *Pseudomonas fluorescens* MTCC 667 was higher at low temperature compared to that at 22°C. When cells are exposed to low temperature, the rate of enzymatic reactions decreases, leading to a decrease in the demand for ATP and an accumulation of electrons at certain points in the respiratory chain. The situation promotes a sudden increase in the production of a number of ROS, which remove the burden of the excess reducing potential (Chattopadhyay, 2002). According to Zhang et al. (2003), the growth temperature downshift increases ROS levels in *Saccharomyces cerevisiae.* The authors reported that H2O2 is a major component in low temperature-exposed yeast cells. In contrast to heterotrophic organisms, most plants possess photosynthetic systems that, when out of control, may produce a lot of ROS and other reactive species (Lushchak, 2011). According to Hu et al.(2008), cold-stress inhibited net photosynthetic rate *(P*N) and cytochrome respiratory pathway but enhanced the photosynthetic electron flux to O2 and over-reduction of respiratory electron transport

such as filamentous fungi. Very little information is available on the oxidative cell response of microorganisms from cold habitats (Gocheva et al., 2006, 2009; Tosi et al., 2010; Chattopadhyay et al., 2011; Nichols et al., 1999). Even less is known concerning filamentous fungi from the Bulgarian Antarctic area. We choose to compare the cell responses between fungi belonging to two different thermal classes, psychrotolerant and mesophilic, that are able to grow in a wide range of temperatures. According to many reports on fungal biodiversity, these fungi are prevalent in Antarctica (Onofri et al., 2007, 2008; Arenz & Blanchette, 2011). Our results confirmed that the downshift of temperature induces typical events of oxidative stress. The exposure of both fungal cultures to 4°C or 10°C caused a statistically significant reduction in biomass production and glucose consumption compared to the control. Despite their genetic adaptation to survive in the harsh Antarctic environment, the sharp decrease in temperature resulted in a cessation of growth, which was more pronounced in the mesophilic fungus than in the psychrophilic fungus. A possible explanation or cause for this discrepancy is that most of the Antarctic mycoflora is metabolically active whenever a combination of favourable abiotic conditions occurs during the short growing summer season (Ruisi et al., 2007). However, Hébraud M. & Potier (1999) assumed that, as opposed to mesophiles, one or more regulatory factors exists in coldadapted microorganisms prior to cold shock that allows for the maintenance of functional translational machinery at low temperatures. However, despite this continuous protein synthesis, the growth of psychrotrophic strains ceases transiently after a cold shock, and additional regulatory mechanisms may exist that allow for growth resumption at low

Additionally, the data for all cold shock variants reveal enhanced ROS generation. These ROS can be generated via a variety of extra-environmental conditions, including low temperature exposure (Belozerskaya & Gessler 2007; Gessler et al., 2007; Gómez-Toribio et al., 2009; Gocheva et al., 2009; Krumova et al., 2009). As is known, the unstressed fungal cells produce O2¯ and H2O2, presumably due to a single electron reduction of 2% of the consumed oxygen as has been previously suggested (Joseph-Horne et al., 2001). Our direct assay showed a significant increase in O2¯ under cold exposure to 4°C and 10°C in dosedependent manner. A similar but less-pronounced tendency was found for H2O2 levels. This increase was more extreme in experiments with mitochondria than in those with intact cells. Thus, cold stress likely imposes an oxidative burden of which O2¯ is a major component. A similar direct analysis of ROS content in fungal cells has not often been reported. Chattopadhyay et al. (2011) reported that the production of free radicals in the Antarctic bacterium *Pseudomonas fluorescens* MTCC 667 was higher at low temperature compared to that at 22°C. When cells are exposed to low temperature, the rate of enzymatic reactions decreases, leading to a decrease in the demand for ATP and an accumulation of electrons at certain points in the respiratory chain. The situation promotes a sudden increase in the production of a number of ROS, which remove the burden of the excess reducing potential (Chattopadhyay, 2002). According to Zhang et al. (2003), the growth temperature downshift increases ROS levels in *Saccharomyces cerevisiae.* The authors reported that H2O2 is a major component in low temperature-exposed yeast cells. In contrast to heterotrophic organisms, most plants possess photosynthetic systems that, when out of control, may produce a lot of ROS and other reactive species (Lushchak, 2011). According to Hu et al.(2008), cold-stress inhibited net photosynthetic rate *(P*N) and cytochrome respiratory pathway but enhanced the photosynthetic electron flux to O2 and over-reduction of respiratory electron transport

temperatures.

chain, resulting in ROS accumulation in cucumber leaves. For fish living at low temperatures, the increased polyunsaturation of mitochondrial membranes should raise rates of mitochondrial respiration which would in turn enhance the formation of ROS, increase proton leak and favour peroxidation of these membranes (Guderley, 2004).

Consistent with previous reports (Şahin & Gümüşlü, 2004; Manfredini et al., 2005; Nyström, 2005; Li et al., 2009), the present study demonstrated the coexistence of enhanced ROS activity and oxidatively damaged proteins in Antarctic fungal models. ROS can modify proteins leading to the formation of additional carbonyl groups caused by the oxidation of specific amino acid residues or the protein backbone (Davies & Goldberg, 1987). The level of proteins with formed additional carbonyl groups, which is thought to be an excellent marker for protein oxidation, has been shown to be related to oxidative stress in various studies. Low temperature stress was shown to induce ROS accumulation in cells (Suzuki & Mittler, 2006; Ouellet, 2007), which leads to a production of oxidised proteins. A similar response was observed in hyper-oxygenated cultures of white-rot fungus *Phanerochaete chrysosporium* (Belinky et al., 2003), heavy metal treatment (Krumova et al., 2009; Belozerskaya et al., 2010), addition of oxidant species (Angelova et al., 2005) and temperature exposure (Kim et al., 2006; Li et al., 2008; Gocheva et al., 2009), for example. In the temperature-induced protein carbonylation, Li et al. (2008) speculated that respiratory enzymes and those within mitochondria may be especially vulnerable because oxygenderived free radicals from the respiratory chain are generally held to be the main source of the oxidative damage seen during stress.

The psychrotolerant strain *Penicillium* sp. 161 demonstrated a lower level of oxidatively damaged proteins than that in the mesophilic strain *A. glaucus* 363 during the exposure to 4°C or 10°C. At the same time, our unpublished data demonstrate the availability of coldacclimation proteins (Caps) in *Penicillium* sp. 161 and cold shock proteins (Csps) in *A. glaucus* 363. According to D'Amico et al. (2006), the existing distinctions between the mesophilic and the psychrophilic cold shock response include the lack of repression of housekeeping protein synthesis and the presence of Caps in psychrophiles. The authors considered that many of the Csps involved in various cellular processes, such as transcription, translation, protein folding and the regulation of membrane fluidity and observed in mesophiles (D'Amico et al., 2006, as cited in Phadtare, 1994), act as Caps in psychrophiles, and are constitutively rather than transiently expressed at low temperatures. Furthermore, this differential regulation of expression indicates that a temperature-sensory system exists in psychrophiles, and thermosensors at the cell membrane level, which sense changes in fluidity, have been reported (D'Amico et al., 2006, as cited in Ray et al., 1994).

Trehalose and glycogen accumulation in microbial cells when they are subjected to unfavourable growth conditions could also be used as stress indicators (Parrou et al., 1997; Türkel, 2006; Ocón et al., 2007; Iturriaga et al., 2009; Gonçalves et al., 2011). Both carbohydrates are important storage compounds in fungal vegetative cells and spores (Robinson, 2001; Rúa et al., 2008). In Antarctic fungi, *Penicillium* sp. 161 and *A. glaucus* 363, that were exposed to cold stress, a reduction in glucose consumption coincided with a significant increase in the trehalose and glycogen content (Fig. 4). These results agree with earlier studies regarding the microbial response against different types of abiotic stress (Ostrovskii et al., 2003; Jules et al., 2008; Robinson, 2001). Our previous studies have

Transient Cold Shock Induces Oxidative Stress Events in Antarctic Fungi 91

The observations by PAGE analysis suggest that in *A. glaucus* 363, Cu/Zn-SODI is primarily involved in the fungal line of defence under the conditions of temperature downshift. In contrast, Mn-SOD activity was considerably higher than Cu/Zn-SODI in *Penicillium* sp. 161, possibly because of a higher level of O2¯, which was generated in mitochondria during the cold stress (Table 1)**.** Moreover, the PAGE results confirmed the significant role of CAT induction in the cold-stress response, especially in the psychrotolerant strain (Fig. 10).

Our results provide further evidence indicating a relationship between cold shock and oxidative stress. Growth at low temperature clearly induced oxidative stress events in both of the Antarctic fungal strains tested, which consisted of enhanced levels of oxidatively damaged proteins, the accumulation of reserve carbohydrates and increased activity of the antioxidant enzyme defence. Despite the significant induction of antioxidant enzymes, exposure to low temperature is damaging and appears to exceed the antioxidant defence. Although the tested strains were isolated from Antarctic soil samples, they both demonstrated a different degree of oxidative stress damage and strategy of antioxidant defence. The psychrotolerant strain *Penicillium* sp. 161. more successfully confronted the challenge of low temperature, which slows metabolite flux, than the mesophilic *A. glaucus* 363. As shown by Methé et al. (2005), comparative genome analyses suggests that the psychrophilic lifestyle is most likely conferred not by a unique set of genes, but by a collection of synergistic changes in the overall genome content and amino acid composition. Analysis of the relationship between oxidative stress and cold stress in different thermal classes of Antarctic microorganisms will improve our understanding, at the molecular level, of the strategies and mechanisms that facilitate fungal cell survival in harsh environments, and the necessary foundation for practical applications. Future study using comparative molecular analysis of genes coding for proteins with antioxidant enzyme activity in psychrotolerant and mesophilic strains by the bioinformatics approach will enhance the knowledge of cold stress signaling mechanisms in eukaryotic microorganisms. From a biotechnological point of view, new information of cold stress response of Antarctic fungi can help further our efforts to develop an effective technology for production of cold-active enzyme SOD. Such SOD can be very important antioxidant in cryopreservation processes (Rossi et al., 2001), cryosurgery and organ transplantation (Sun et al., 2006), *in vitro* fertilization (Agarwal et al., 2006) etc. Cold-active SOD can be used in all cosmetic formulations, to promote, at least, younger looking skin (Lods et al., 2000; Diehl, 2009).

This work was supported by the NCSI of the Ministry of Education and Science, Bulgaria

Abrashev, R.I., Pashova, S. B., Stefanova, L.N., Vassilev, S.V., Dolashka-Angelova, P.A. &

Angelova, M.B. (2008). Heat-shock-induced oxidative stress and antioxidant response in *Aspergillus niger* 26. *Canadian Journal of Microbiology*, Vol. 54, No. 12,

(grant DO02-172/08 and grant VU-B-205/06) to which sincere thanks are owed.

(December 2008), pp. 977-983, ISSN 1480-3275

**5. Conclusions** 

**6. Acknowledgment** 

**7. References** 

confirmed the role of these reserve carbohydrates in the survival strategies of filamentous fungi under stress conditions such as heavy metal toxicity (Krumova et al., 2009), heat shock (Abrashev et al., 2008), oxidative stress-agent treatment (Angelova et al., 2005) and low temperature exposure (Gocheva et al., 2009). In contrast, heat stress led to a significant decrease in the glycogen and trehalose content of *Pichia anomala* (Parrou et al., 1997) and *P. angusta* cells (Türkel, 2006), respectively. However, the nitrogen starvation-induced biosynthesis and the accumulation of trehalose and glycogen in the same strain, *P. angusta*, suggested that both stress responses do not have an overlapping signal transduction pathway in the activation of trehalose and glycogen biosynthesis in this *Pichia* species (Türkel, 2006). Jules et al. (2008) confirmed an interconnection between trehalose and glycogen in yeast cell carbon storage management. They found that glycogen was accumulated earlier and faster, which indicated the presence of a fine-tuning control during periods of stress. In contrast, our results showed that both reserve carbohydrates started to increase simultaneously, immediately after the temperature downshift (Fig. 4), and glycogen maintained its higher level after the recovery to the optimal temperature longer than trehalose did.

Considering that almost all of the genes encoding the enzymes involved in the metabolism of these two reserve carbohydrates contain between one and several copies of the stressresponsive element (STRE), Parrou et al. (1997) suggested the possibility of a link between the potential transcriptional induction of these genes and the accumulation of glycogen and trehalose under different stress conditions. In this study, we evaluated the activities of the trehalose-metabolising enzymes TPS and NT during cold stress conditions. In the used Antarctic strains, cold shock elicits a complex response that involves the activation of both biosynthesis and biodegradation pathways, but not to the same extent. The increase in TPS activity significantly exceeds that of NT activity. This situation leads to a high level of accumulation of trehalose during low temperature exposure. Thus, the intracellular level of trehalose is the result of a well-regulated balance between enzymatic synthesis and degradation. In *Saccharomyces cerevisiae*, an enhanced level of trehalose was the result of the stimulation of trehalose synthase and the inhibition of trehalase by high temperature (Parrou et al., 1997). Moreover, after the stress had ceased, trehalose concentrations in our experiments returned to basal levels, indicating that neutral trehalase activity could have a role in cold shock recovery.

It is well known that the antioxidant systems of microorganisms, including fungi, act as important tolerance mechanisms against abiotic stress. Enhanced SOD and CAT activities have been associated with the induced resistance of fungi to different stress factors (Manfredini et al., 2005; Li et al., 2009; Belozerskaya et al., 2010; Chattopadhyay et al., 2011). Both activities are involved in superoxide radical and hydrogen peroxide scavenging. Our results demonstrated that the exposure to cold shock for 6 h induced the expression of SOD and CAT in a psychrotolerant strain and in a mesophilic strain.

Furthermore, the increase in SOD activity was more pronounced for the mesophilic fungus, while the activation of antioxidant enzyme defence in the psychrotolerant *Penicillium* was primarily due to an increase in CAT activity. One possible explanation for this finding is a much higher level of O2¯ generated upon low temperature stress in the mesophilic strain and higher levels of H2O2 in psychrotolerant cultures (Table 1 and Table 2). To antagonise ROS, *A. glaucus* 363 induces SOD synthesis to a much higher degree, while *Penicillium* sp. 161 enhances CAT levels.

The observations by PAGE analysis suggest that in *A. glaucus* 363, Cu/Zn-SODI is primarily involved in the fungal line of defence under the conditions of temperature downshift. In contrast, Mn-SOD activity was considerably higher than Cu/Zn-SODI in *Penicillium* sp. 161, possibly because of a higher level of O2¯, which was generated in mitochondria during the cold stress (Table 1)**.** Moreover, the PAGE results confirmed the significant role of CAT induction in the cold-stress response, especially in the psychrotolerant strain (Fig. 10).

### **5. Conclusions**

90 Oxidative Stress – Environmental Induction and Dietary Antioxidants

confirmed the role of these reserve carbohydrates in the survival strategies of filamentous fungi under stress conditions such as heavy metal toxicity (Krumova et al., 2009), heat shock (Abrashev et al., 2008), oxidative stress-agent treatment (Angelova et al., 2005) and low temperature exposure (Gocheva et al., 2009). In contrast, heat stress led to a significant decrease in the glycogen and trehalose content of *Pichia anomala* (Parrou et al., 1997) and *P. angusta* cells (Türkel, 2006), respectively. However, the nitrogen starvation-induced biosynthesis and the accumulation of trehalose and glycogen in the same strain, *P. angusta*, suggested that both stress responses do not have an overlapping signal transduction pathway in the activation of trehalose and glycogen biosynthesis in this *Pichia* species (Türkel, 2006). Jules et al. (2008) confirmed an interconnection between trehalose and glycogen in yeast cell carbon storage management. They found that glycogen was accumulated earlier and faster, which indicated the presence of a fine-tuning control during periods of stress. In contrast, our results showed that both reserve carbohydrates started to increase simultaneously, immediately after the temperature downshift (Fig. 4), and glycogen maintained its higher level

Considering that almost all of the genes encoding the enzymes involved in the metabolism of these two reserve carbohydrates contain between one and several copies of the stressresponsive element (STRE), Parrou et al. (1997) suggested the possibility of a link between the potential transcriptional induction of these genes and the accumulation of glycogen and trehalose under different stress conditions. In this study, we evaluated the activities of the trehalose-metabolising enzymes TPS and NT during cold stress conditions. In the used Antarctic strains, cold shock elicits a complex response that involves the activation of both biosynthesis and biodegradation pathways, but not to the same extent. The increase in TPS activity significantly exceeds that of NT activity. This situation leads to a high level of accumulation of trehalose during low temperature exposure. Thus, the intracellular level of trehalose is the result of a well-regulated balance between enzymatic synthesis and degradation. In *Saccharomyces cerevisiae*, an enhanced level of trehalose was the result of the stimulation of trehalose synthase and the inhibition of trehalase by high temperature (Parrou et al., 1997). Moreover, after the stress had ceased, trehalose concentrations in our experiments returned to basal levels, indicating that neutral trehalase activity could have a

It is well known that the antioxidant systems of microorganisms, including fungi, act as important tolerance mechanisms against abiotic stress. Enhanced SOD and CAT activities have been associated with the induced resistance of fungi to different stress factors (Manfredini et al., 2005; Li et al., 2009; Belozerskaya et al., 2010; Chattopadhyay et al., 2011). Both activities are involved in superoxide radical and hydrogen peroxide scavenging. Our results demonstrated that the exposure to cold shock for 6 h induced the expression of SOD

Furthermore, the increase in SOD activity was more pronounced for the mesophilic fungus, while the activation of antioxidant enzyme defence in the psychrotolerant *Penicillium* was primarily due to an increase in CAT activity. One possible explanation for this finding is a much higher level of O2¯ generated upon low temperature stress in the mesophilic strain and higher levels of H2O2 in psychrotolerant cultures (Table 1 and Table 2). To antagonise ROS, *A. glaucus* 363 induces SOD synthesis to a much higher degree, while *Penicillium* sp.

after the recovery to the optimal temperature longer than trehalose did.

and CAT in a psychrotolerant strain and in a mesophilic strain.

role in cold shock recovery.

161 enhances CAT levels.

Our results provide further evidence indicating a relationship between cold shock and oxidative stress. Growth at low temperature clearly induced oxidative stress events in both of the Antarctic fungal strains tested, which consisted of enhanced levels of oxidatively damaged proteins, the accumulation of reserve carbohydrates and increased activity of the antioxidant enzyme defence. Despite the significant induction of antioxidant enzymes, exposure to low temperature is damaging and appears to exceed the antioxidant defence. Although the tested strains were isolated from Antarctic soil samples, they both demonstrated a different degree of oxidative stress damage and strategy of antioxidant defence. The psychrotolerant strain *Penicillium* sp. 161. more successfully confronted the challenge of low temperature, which slows metabolite flux, than the mesophilic *A. glaucus* 363. As shown by Methé et al. (2005), comparative genome analyses suggests that the psychrophilic lifestyle is most likely conferred not by a unique set of genes, but by a collection of synergistic changes in the overall genome content and amino acid composition.

Analysis of the relationship between oxidative stress and cold stress in different thermal classes of Antarctic microorganisms will improve our understanding, at the molecular level, of the strategies and mechanisms that facilitate fungal cell survival in harsh environments, and the necessary foundation for practical applications. Future study using comparative molecular analysis of genes coding for proteins with antioxidant enzyme activity in psychrotolerant and mesophilic strains by the bioinformatics approach will enhance the knowledge of cold stress signaling mechanisms in eukaryotic microorganisms. From a biotechnological point of view, new information of cold stress response of Antarctic fungi can help further our efforts to develop an effective technology for production of cold-active enzyme SOD. Such SOD can be very important antioxidant in cryopreservation processes (Rossi et al., 2001), cryosurgery and organ transplantation (Sun et al., 2006), *in vitro* fertilization (Agarwal et al., 2006) etc. Cold-active SOD can be used in all cosmetic formulations, to promote, at least, younger looking skin (Lods et al., 2000; Diehl, 2009).

### **6. Acknowledgment**

This work was supported by the NCSI of the Ministry of Education and Science, Bulgaria (grant DO02-172/08 and grant VU-B-205/06) to which sincere thanks are owed.

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**5** 

**Changes in Hydrogen Peroxide** 

**Levels and Catalase Isoforms Expression** 

There is evidence that abscisic acid (ABA) has a protective signaling role in freezing stress in plants (Kobayashi et al., 2008), including mosses (Minami et al., 2003). ABA signaling networks and their actions are not totally understood, but H2O2 has been implicated as an intermediary in several ABA responses, where its roles include induction of the antioxidant system (Cho et al., 2009). Mora-Herrera & López-Delgado (2007), using *in vitro* microplants as employed in potato production programs, found freezing tolerance was enhanced by culture on ABA-containing medium. This ABA treatment tripled survival of a -6C incubation in the cold-sensitive cv. Atlantic, while in the more cold-tolerant cv. Alpha, survival improved by two-thirds. In the ABA-treated microplants, they found the H2O2-

Stress tolerance in potato is growing in importance, as increases in potato production by developing countries greatly exceed other major crops (FAO, 2008). The present study used the microplant system to investigate effects of prolonged ABA treatment on catalase, another enzyme important in controlling cellular H2O2. Catalases are tetrameric, hemecontaining oxidoreductases that dismutate H2O2 to water and oxygen. In plants, their peroxisomal location coincides with the cellular site of H2O2 generation by photorespiration or fatty acid -oxidation (Feierabend, 2005)(Scheme 1). Evidence for catalase involvement in

scavenging enzyme ascorbate peroxidase increased in activity.

**1. Introduction** 

**are Induced With Freezing Tolerance by** 

**Abscisic Acid in Potato Microplants** 

Martha E. Mora-Herrera1,2, Humberto López-Delgado1, Ernestina Valadez-Moctezuma3 and Ian M. Scott4 *1Programa Nacional de Papa, Instituto Nacional de Investigaciones* 

*Forestales Agrícolas y Pecuarias, (INIFAP), Metepec* 

*Carr. Tenancingo-Villa Guerrero Km 1.5 Tenancingo,* 

*4Institute of Biological, Environmental and Rural Sciences,* 

*Universidad Autónoma del Estado de México,* 

*Universidad Autónoma Chapingo, Chapingo,* 

 *Aberystwyth University, Ceredigion,* 

*2Centro Universitario Tenancingo,* 

*3Departamento de Fitotecnia,* 

*1,2,3México 4UK* 

