Introduction
Marine lineages living intertidally, or in ephemeral pools, are exposed to varying periods of dehydration. Many green algae also live under a high salinity condition in the marine environment which induces an oxidative stress similar to dehydration. Salinity and desiccation are considered as different types of water deficiency. In this regard, salinity stress is recognized as ‘physiological drought’ which may reduce the water potential of algal cells that are still in full contact with water, while desiccation stress could increase an intensive dehydration in the cells (Kirst, Reference Kirst1990). Although both types of stress similarly cause loss of turgor as an important agent for cell elongation and growth; they might induce different changes in the ratio of ions selectively taken up by the cells (Holzinger & Karsten, Reference Holzinger and Karsten2013).
Desiccation leads to oxidative stress and, eventually, generation of reactive oxygen species (ROS) in algae. ROS are a potent cause of destruction to biological macromolecules such as nucleic acids, proteins and lipids (Scheibe & Beck, Reference Scheibe, Beck and Lüttge2011). Numerous studies concerning desiccation tolerance in cyanobacteria, lichens, mosses and plants have been addressed in the literature (Kranner et al., Reference Kranner, Beckett, Hochman and Nash2008; Gechev et al., Reference Gechev, Dinakar, Benina, Toneva and Bartels2013). The ability of organisms to survive in dry conditions is called desiccation tolerance. Desiccation tolerance in algae is a promising field for research, as suggested by Bewley ~40 years ago (Bewley, Reference Bewley1979). Davis (Reference Davis1972) reported desiccation tolerance in vegetative and reproductive cells of 119 species of green algae. Several recent studies have reviewed physiological, biochemical and molecular impacts of desiccation (Tardieu et al., Reference Tardieu, Simonneau and Muller2018; Bremer & Krämer, Reference Bremer and Krämer2019; Oliver et al., Reference Oliver, Farrant, Hilhorst, Mundree, Williams and Bewley2020). However, the mechanisms of desiccation tolerance in green algae, especially microalgae are not fully understood. For instance, Holzinger & Karsten (Reference Holzinger and Karsten2013) have reviewed the mechanisms of desiccation tolerance in green algae at the ultra-structural, physiological and molecular levels. More recently, Lin & Wu (Reference Lin and Wu2014) compared the PEG-induced drought responses of some drought-tolerant and non-tolerant algal species and suggested that the tolerance to drought stress might be indicated by physiological parameters such as the proline level and the enzymatic and non-enzymatic antioxidant system.
Dunaliella as one of the most tolerant eukaryotic and photosynthetic organisms is a suitable microalgal model to clarify the tolerance mechanisms of plant and algal cells to different stresses (Cowan et al., Reference Cowan, Rose and Horne1992). The genus Dunaliella belongs to the Volvocales order and Polyblepharidaceae family. The members of the Dunaliella are unicellular, biflagellate, ovoid or spherical algae without a rigid cell wall. Several researchers have investigated the effects of different abiotic stresses such as heavy metals, salinity, low temperature, acidic environment, UV and oxidative stress on D. salina (Yilancioglu et al., Reference Yilancioglu, Cokol, Pastirmaci, Erman and Cetiner2014; Belghith et al., Reference Belghith, Feki, Athmouni, Ayadi and Bellassoued2016). For instance, a proportional relationship was found between the accumulation of stress metabolites such as proline, glycine betaine, glycerol and total carbohydrates and the salt concentration of the medium ranging from 0.5 to 5.5 M (Mishra et al., Reference Mishra, Mandoli and Jha2008). The synergistic effects of salinity and PEG on the growth of D. salina have been previously reported in a preliminary study (Kumar & Dharmaraj, Reference Kumar and Dharmaraj2003); however, the sole effect of PEG on the growth and physiology of D. salina has not been studied yet. This study aimed to investigate the effect of different concentrations of PEG on the growth, morphology and physiology of D. salina as an attempt to shed some light on the mechanisms underlying the effects of drought stress on this microalga.
Materials and methods
Cell culture and PEG treatment
The alga, D. salina (UTX LB 2538) was obtained from the Culture Collection of Algae in UTEX, USA and maintained and cultivated in a modified Johnson's medium (Johnson et al., Reference Johnson, Johnson, McElrory, Speer and Bruff1968). The cells were grown at 25°C in 500 ml Erlenmeyer flasks containing 250 ml of the medium on a rotary shaker stirred at 110 rpm under continuous white light with a photon flux density of 100 μmol m−2 s−1. In a preliminary experiment, the cells were treated with different concentrations of PEG 6000 (0, 2.5, 5, 7.5, 10, 15, 20, 25 and 30%) to select the appropriate medium for a subsequent experiment. Finally, two concentrations of PEG (2.5 and 5%) and a control medium were used to simulate drought stress treatment in D. salina. For all PEG treatments as well as control, 5 ml of freshly grown cells were inoculated into the medium with a fixed amount of NaCl (1.5 M) and the cultures monitored for 25 days. The PEG-induced osmotic potential was calculated by using the equation previously formulated by Michel & Kaufmann (Reference Michel and Kaufmann1973):
$${\rm \;OP}\lpar {{\rm Mpa}} \rpar = \lpar {-1.18 \times {10}^{{-}2}} \rpar \times C-\lpar {1.18 \times {10}^{{-}4}} \rpar \times C + \lpar {2.67 \times {10}^{{-}4}} \rpar \times C.T + \lpar {8.39 \times {10}^{{-}7}} \rpar \times C^2 \times T$$where C is PEG concentration, and T is temperature.
The NaCl-induced osmotic potential was calculated using the Van't Hoff equation and total osmotic potential was calculated by summing PEG- and NaCl-induced osmotic potentials.
Growth analysis
The cell number was counted using a hemocytometer under an Olympus light microscope at 400×. The dry biomass was recorded after filtration of 10 ml of culture through a pre-weighed 45 mm glass filter, rinsed with 10 mL ammonium formate (0.5 M) and dried in an oven at 70°C until constant.
Specific growth rate was determined using the equation previously used by Levasseur et al. (Reference Levasseur, Thompson and Harrison1993) as follows: K′ = ln(N2/N1)/t2 − t1 (where, N1 and N2 were biomass (mg ml−1) at the first time (t1) (day 5) and the second time (t2) (day 15), respectively). The number of divisions per day (Div./day) and the doubling time of the cells (D.time) were also calculated as indicated below:
$${\rm Div}./{\rm day} = \displaystyle{{{K}^{\prime}} \over {\ln 2}}$$
$${\rm D}.{\rm time} = \displaystyle{1 \over {{\rm Div}./{\rm day}}}$$ImageJ software was used to measure the relative changes of cell surface area under different PEG concentrations compared with the control.
The Speed index of cells, as an indicator of the cell motility, scored from 0 (no motility) to 5 (maximum motility) under the light microscope.
Glycerol determination
Glycerol content was measured according to Chen et al. (Reference Chen, Lao and Jiang2011) method. The algal pellets were collected from 10 ml of the cultures centrifuged at 6000 rpm for 5 min and then were resuspended in 1.0 ml of distilled water and 0.2 ml of chloroform. After homogenizing the suspension for 8 min, the supernatant was harvested by centrifugation at 11,000 rpm for 10 min. Next, 100 μl of the supernatant was mixed with 200 μl distilled water and 1 ml of sodium periodate reagent. After 5-min, 2.5 ml of acetylacetone reagent was added. The suspension was placed in a water bath at 60°C for 30 min. After cooling at room temperature, absorbance was read at 410 nm. Glycerol content was determined using a standard curve for pure glycerol as reference and results were expressed as micrograms of glycerol per 106 cells.
Proline determination
Proline was extracted and measured using the method of Bates et al. (Reference Bates, Waldren and Teare1973) based on the reaction of proline with ninhydrin. About 10 ml of cell culture were centrifuged at 6000 rpm for 5 min. The pellet was homogenized in 3 ml sulphosalicylic acid 3% and then centrifuged at 13,200 rpm for 20 min. A 1:1:1 (v:v:v) solution of the supernatant, ninhydrin acid and glacial acetic acid was boiled for 1 h and transferred on ice for 10 min. The reaction mixture was extracted with 2 ml of toluene by vortexing. The absorbance of the upper phase (coloured solution) was determined at 520 nm using toluene as the blank and L-proline as the standard. Results were expressed as nanomolar of proline per gram fresh weight.
Glycine betaine determination
The amount of glycine betaine (GB) was determined following the method of Sairam & Srivastava (Reference Sairam and Srivastava2002). The cells were harvested by centrifugation of 10 ml of the cultures at 6000 rpm for 5 min and then resuspended in 10 ml of distilled water. The extract was recentrifuged and equal volumes of 2 N H2SO4, and 0.8 ml cold KI–I2 reagent (1.75 g I2 and 2 g KI in 10 ml deionized water) was added to the supernatant and was thoroughly mixed. The homogenized sample was held in ice for 16 h, centrifuged and then 1 ml of supernatant was carefully mixed with 1 ml of 1, 2-dichloroethane. After 3 h of incubation at room temperature, the absorbance was read at 365 nm. The concentration of glycine betaine (GB) was estimated using the known amount of GB as standard and results were expressed as micrograms of GB per 106 cells.
Protein quantification
For protein extraction, 10 ml of cell suspensions were centrifuged at 6000 rpm for 5 min. Cell pellets were homogenized in 80% cold hydrous ethanol and the extract was held at −20°C for 30 min after centrifuging. The precipitated protein pellets were dissolved in 0.1 N NaOH. Total soluble protein content was determined by the Bradford (Reference Bradford1976) method using crystalline bovine serum albumin (BSA) as a standard reference. Results were expressed as micrograms of protein per 106 cells.
Determination of soluble carbohydrates
Total soluble sugars were estimated according to the phenol sulphuric acid method, as described by Kochert (Reference Kochert, Helebust and Craigie1978). For extraction of carbohydrates, 10 ml of 70% ethanol was added to ~0.1 g dry weight of the algal pellet. 1 ml of the extract was brought to a volume of 2 ml with deionized water and then mixed well with 1 ml of phenol solution (5%). After adding 5 ml concentrated sulphuric acid (80%), the optical density of the sample was measured at 480 (rhamnose), 485 (glucose), and 490 (mannose). The concentration of the sugars was calculated by comparison with their standard curves, and results were expressed as micrograms of carbohydrates per 106 cells.
H2O2 determination
H2O2 measurement was performed according to the method of Sergiev et al. (Reference Sergiev, Alxieva and Karanov1997). Cell suspension (10 ml) was centrifuged at 6000 rpm for 5 min. The supernatant was removed carefully, and the pellets were resuspended in 2 ml 0.1% (w/v) cold trichloroacetic acid (TCA) in an ice bath. The mixture was centrifuged at 12,000 rpm for 15 min. Next, 1 ml of the supernatant was transferred to a test tube containing 1 ml of 10 mM potassium phosphate buffer (pH 7.0) and 2 ml of 1 M KI. After mixing thoroughly in a vortex mixer (model SA 8, Stuart, Britain), the absorbance of the supernatant was recorded at 390 nm. The concentration of H2O2 was calculated by comparison with a standard curve. A mixture without the supernatant served as a blank, and results were expressed as micromole of H2O2 per 106 cells.
Lipid peroxidation
The level of lipid peroxidation was estimated through the amount of malondialdehyde (MDA). The MDA content was measured according to the method of Heath & Packer (Reference Heath and Packer1968). First, 10 ml suspension culture was centrifuged at 6000 rpm for 5 min, and the resulting pellet was resuspended by pipetting in 3 ml of 0.1% (w/v) trichloroacetic acid (TCA). The homogenate was then centrifuged, and 500 μl supernatant was mixed with 500 μl of 0.5% (w/v) thiobarbituric acid (TBA). The mixture was incubated at 95°C for 30 min and immediately cooled in an ice bath to stop the reaction. The absorbance was read at both 532 and 600 nm. The MDA concentration was calculated using the molar extinction coefficient of 155 mM−1cm−1. Results were reported as nanomoles of MDA per 106 cells.
$${\rm MDA}\,{\rm equivalent} = \lsqb {\lpar {A532-A600} \rpar /155000} \rsqb \times 10^6$$The 532 and 600 nm levels were the maximum absorbances of the TBA-MDA complex and the correction for non-specific turbidity, respectively.
Membrane permeability
Electrolyte leakage (EL) was determined for assaying algal membrane permeability using a conductivity meter (istek, model C65, Korea) according to the method of Dionisio-Sese & Tobita (Reference Dionisio-Sese and Tobita1998). Samples (10 ml) were centrifuged at 6000 rpm for 5 min and the pellets were washed twice with double distilled water. After dissolving the pellets in 10 ml deionized water and holding on a shaker for 6 h, the initial conductivity of the solution was measured (C1). The samples were then boiled for 40 min and cooled to room temperature to measure the second conductivity (C2). The percentage of electrolyte leakage was measured as follows:
$$\percnt {\rm EC} = \displaystyle{{C1} \over {C2}} \times 100$$Enzymes extraction and assay
For assaying antioxidant enzymes, the cells were harvested from 10 ml of culture by centrifugation at 6000 rpm for 5 min. The pellet was homogenized in 1 ml of cold potassium phosphate buffer (50 mM, pH 7.4), containing 1% (w/v) polyvinylpyrrolidone (PVP) in an ice bath with mortar and pestle. The homogenate was centrifuged at 4°C for 15 min at 4000 rpm and the supernatant was used as enzyme extract in order to assay the activities of both catalase (CAT, EC 1.11.1.6) and ascorbate peroxidase (APX, EC 1.11.1.11), spectrophotometrically.
The CAT activity was determined as previously described by Aebi (Reference Aebi and Keating1983). The reaction mixture contained 10 mM H2O2 in 50 mM potassium phosphate buffer (pH 7.4). The reaction was started with the addition of 100 μl enzyme extract to 900 μl reaction mixture. The decline in absorbance at 240 nm was recorded for 1 min, and catalase activity was calculated by following the dissociation of hydrogen peroxide and using an absorbance coefficient of 0.039 mM−1 cm−1 for H2O2. One unit of catalase was defined as the amount of enzyme that is required for the disintegration of 1 μmol of H2O2 in 1 min at 25°C.
Analysis of APX activity was performed based on the Nakano & Asada (Reference Nakano and Asada1981) method. The reaction mixture contained 625 μl phosphate buffer with 0.2 mM EDTA (pH 7.4), 175 μl ascorbic acid (0.5 mM), 50 μl H2O2 (25 mM), 50 μl BSA and 100 μl enzyme extract. The reaction was initiated by the addition of H2O2 (25 mM) and a reduction in absorbance at 290 nm was monitored for 1 min. Enzyme activity was measured by an absorbance coefficient of 2.6 mM−1 cm−1 for ascorbic acid. One unit of ascorbate peroxidase was expressed as the oxidation of 1 μmol ascorbic acid in 1 min at 25°C.
DNA fragmentation test
The effect of PEG on DNA fragmentation was studied as an indicator of damage to the nucleic acid under stress conditions by running and investigating the extracted DNA on an agarose gel. DNA was extracted from the cell suspension (10 ml) using a silica-based technique previously described by Li et al. (Reference Li, Li and Sheen2010). DNA integrity was checked by electrophoresis on 1.5% agarose gel. Lambda DNA/HindIII (Thermo Scientific) was used as a size marker.
Determination of pigments
To determine pigments including chlorophylls (a and b) and β-carotene, 10 ml of the cell suspension was centrifuged for 5 min and the pellet was homogenized in 80% acetone solvent. After centrifugation, the absorbance of the supernatant was measured at 431, 460, 480 and 412 nm. The pigments were estimated by the equations previously derived (Eijckelhoff & Dekker, Reference Eijckelhoff and Dekker1997). The tolerance index (TI) was measured as described by Kohl & Losch (Reference Kohl, Losch and Prasad2004) using the following formula:
$${\rm TI}\lpar \percnt \rpar = \displaystyle{{{\rm chlorophylls}\,{\rm content}\,{\rm of}\,{\rm PEG}\,{\rm treated}\,{\rm cells}} \over {{\rm chlorophylls}\,{\rm content}\,{\rm of}\,{\rm control}}} \times 100$$Statistical analyses
The experimental design was entirely randomized, with three repeats per treatment. The obtained data and difference between the mean values were analysed statistically by conducting ANOVA (analysis of variance) followed by Fisher's LSD test at the P < 0.01 level. Statistical analyses and graphs were produced in Excel and Graph Pad Prism 6 software.
Results
The effect of PEG on algal growth and morphology
The threshold of drought tolerance in D. salina was initially assayed by cultivating the alga in a wide range of PEG doses. Generally, we did n't observe any cell growth or culture greening in PEG doses higher than 10%. With the increasing PEG concentration up to 10%, both the PEG-induced potential and the total osmotic potential of media as indicators of stress intensity decreased from 0.0 to −1.48 Mpa and from −72.32 to −73.80 Mpa, respectively (Table 1). The kinetic growth of D. salina (Figure 1) indicated that all the treatments reached a stationary phase over 24 days of cultures. According to the results, a slight increase in cell growth was observed for 2% PEG-treated cells compared with the PEG-free control. However, increase in PEG up to 5% or more, had significant negative effects on the rate of cell growth. The 2% PEG treated cells and PEG-free control reached their stationary phases on days 20 and 22 of culture with the maximum values of 238 × 104 and 260 × 104 cells ml−1, respectively. The stationary phase of D. salina growth in 5% PEG took place on the day 24 with a maximum number of 125 × 104 cells ml−1. The growth of Dunaliella in 7.5 or 10% PEG was not significant and only plateau-like curves were observed in both cases. As depicted in Table 1, other growth kinetic parameters were also influenced by PEG treatment. Specific growth rates and a number of divisions per day were found to be 4.45 mg day ml−1 and 6.4 for 2% PEG treatment, respectively and were slightly higher than those for control. The doubling time of the cells decreased from 0.19 in control to 0.15 in 2% PEG. PEG treatment higher than 2% reduced the specific growth rate and the number of divisions per day, while it significantly increased the doubling time of the cells.
Fig. 1. The effect of PEG 6000 concentrations (0, 2, 5, 7.5 and 10%) on growth of D. salina. Growth kinetics determined from the number of cells counted every 2 days during 25 days of culture. The cells treated with 2% PEG showed a slightly higher biomass than the PEG-free control. The growth of microalgae was significantly inhibited when the dose of PEG increased more than 2%.
Table 1. The PEG-induced and total osmotic potentials (OPs) of the media with different concentrations of PEG 6000 and their effects on the growth kinetic parameters of microalgae, D. salina
The values shown are means followed by ± SD, N = 3. Different superscript letters indicate significant differences between the means according to Fisher's LSD test at the P < 0.01 level. Div./day and D.time indicate the number of divisions per day and the doubling time of the cells, respectively.
Microscopy studies were performed to investigate the morphology of Dunaliella cells under different doses of PEG stress. Although no clumping cells in PEG-free control or PEG 2% treatment were observed, large cell clumps were noticed in the PEG treatments higher than 2% (Figure 2). On the other hand, the cell sizes increased in the various doses of PEG, such that compared with the control cells, the relative area surface of the cells treated with 2, 5, 7.5 and 10% PEG, raised by 1.21-, 1.51-, 1.69- and 1.72-fold, respectively. The shape of cells changed mainly from normal-ellipsoid in control to more irregular shapes in high concentrations of PEG (Figure 2 and Table 2).
Fig. 2. The morphology of microalga, D. salina cultured under different PEG 6000 concentrations. A: D. salina cultured in PEG-free control medium; B, C, D and E: D. salina cultured in media with 2, 5, 7.5 and 10% PEG, respectively; Optical zoom ×400.
Table 2. The effects of different concentrations of PEG 6000 on morphology parameters and motility of microalgae, D. salina
The values shown are means followed by ± SD, N = 3. Different superscript letters indicate significant differences between the means according to Fisher's LSD test at the P < 0.01 level. The Speed index of cells scored from 0 (no motility) to 5 (maximum motility).
The speed index, as an indicator of the cell motility, was affected by the PEG treatment. The motility of the cells in the control medium was the maximum with a mean speed index of 5, whereas the application of any dose of PEG to the medium proportionally reduced the speed index. The cells treated with 10% PEG had no motility and had the minimum speed index.
Glycerol content increased dramatically during PEG treatment
Intracellular glycerol content of D. salina raised from 6.73 ± 0.51 in control up to 16.74 ± 2.63 μg/106 cells in 5% PEG treatment. Based on the results, a logarithmic relationship existed between the cellular accumulation of glycerol and the elevation of PEG concentration (Figure 3A). The maximum value of glycerol was observed in cells treated with 5% PEG, which was about 1.8- and 2.5-fold more than those in PEG-free control and 2% PEG, respectively. By increasing PEG from 0 to 2%, a slight increase in glycerol was observed in the cells; however, more PEG treatment up to 5% led to a logarithmic enhancement of this compound in Dunaliella.
Fig. 3. The effect of drought stress on compatible solutes and protein production in D. salina cultured under different PEG 6000 concentrations (0, 2 and 5%). The amounts of glycerol (A), proline (B) and glycine betaine (C) significantly increased by exposing the algae to higher content of PEG. The content of protein (D) was not meaningfully different among the treatments. Different lower case letters indicate significant differences between the means according to Fisher's LSD test at the P < 0.01 level, Means ± SEs, N = 3.
Significant accumulations of proline and glycine betaine were observed under PEG treatment in D. salina
The findings showed that drought stress rising due to PEG treatment intensified higher production of compatible osmolytes such as proline and glycine betaine in Dunaliella compared with control (Figure 3B, C). A considerable increase was detected in the proline content of D. salina cells after increasing PEG. The highest accumulation of proline was in 5% PEG treated cells with a mean of 4 nmol g−1 FW, being ~4- and 2-fold more than those in control or PEG 2% treated cells, respectively.
The results also indicated that another vital osmolyte, glycine betaine, substantially increased with a mean from 100.6 μg/106 cells in control up to 145.2 and 225.8 μg/106 cells in PEG 2 and 5%, respectively. The highest value of glycine betaine was again found for the 5% PEG-treated cells, which were ~2.2-fold more than the value of the control.
A relatively constant amount of protein was produced in D. salina resulting from PEG treatment
Treatment with 2% or 5% PEG compared with the control, did not change protein content in D. salina cells meaningfully (Figure 3D). The protein content of the control (518 ± 103 mg g−1 FW) only was 1.18- and 1.20-fold more than those in 2% (439 ± 34 mg g−1 FW) and 5% PEG (406 ± 40 mg g−1 FW), respectively.
Soluble carbohydrates increased in D. salina over PEG treatment period
According to our data, treating the cells with different concentrations of PEG led to increasing trends in soluble carbohydrates, including glucose, mannose and rhamnose (Figure 4). In this context, higher concentrations of PEG induced more sugar accumulations in Dunaliella, such that the highest mean content of glucose, mannose and rhamnose was obtained in 5% PEG-treated cells, which were 18.80, 10.05 and 10.82 μg/106 cells, respectively.
Fig. 4. The effect of drought stress on carbohydrates accumulation in D. salina cultured under different PEG 6000 concentrations (0, 2 and 5%). The amounts of glucose, rhamnose and mannose significantly were increased by exposing the algae to the higher content of PEG. Different lower case letters indicate significant difference between the means according to Fisher's LSD test at the P < 0.01 level, Means ± SEs, N = 3.
Very large H2O2 production resulting from PEG-induced drought stress in D. salina
The amount of hydrogen peroxide significantly increased in PEG-treated cells, especially in 5% PEG treatment. As indicated in Figure 5A, a slight increase in H2O2 was observed upon exposure of Dunaliella cells to 2% PEG, however by changing the PEG concentration from 2% to 5%, a vast amount of H2O2 accumulation was observed in the cells. The mean value of H2O2 content in PEG 5%-treated cells was 0.066 μmol/106 cells which showed a 33-fold increase relative to untreated cells (0.002 μmol/106 cells).
Fig. 5. The effect of drought stress on the amounts of H2O2 (A), malondialdehyde (MDA) (B) and electrolyte leakage (C) in D. salina cultured under different PEG 6000 concentrations (0, 2 and 5%). The amount of H2O2 and MDA dramatically increased after exposing the algae to the higher content of PEG, especially 5%. However, there was no significant difference in the electrolyte leakage among the treatments. Different lower case letters indicate significant differences between the means according to Fisher's LSD test at the P < 0.01 level, Means ± SEs, N = 3.
PEG treatment-induced lipid peroxidation in D. salina
The MDA content as an indicator of lipid peroxidation level is shown in Figure 5B. A negligible amount of MDA was seen in the PEG-free control. However, a PEG-dependent increase in MDA contents was also observed, such that the higher the PEG level applied, the more MDA content increased. These results demonstrated that compared with control, application of 2 and 5% PEG induced 25- and 74-fold increase of lipid peroxidation, respectively.
PEG induced leakage in the cell membrane
As another part of the article shows, it was found that the PEG treatment of Dunaliella induced significant leakage in the cell membrane; however, there were no meaningful differences in electrolyte leakage of cells treated with 2% or 5% PEG (Figure 5C). Generally, PEG-induced drought stress increased leakage in D. salina cells up to 30% compared with control.
Antioxidant enzyme activities
As seen in Figure 6A and B, following PEG treatment, enzymatic antioxidant systems of D. salina, including catalase (CAT) and ascorbate peroxidase (APX) were strongly affected. The data indicated that in comparison with control, by increasing PEG from 2% to 5%, the mean activity of CAT rose by ~2.0- to 3.9-fold, respectively (Figure 6A). A similar trend was also revealed for APX. The mean activity of this enzyme increased 1.7-fold in 2% PEG and up to 2.9-fold in 5% PEG compared with control (Figure 6B).
Fig. 6. The effect of drought stress on the enzymatic antioxidant systems in D. salina cultured under different PEG 6000 concentrations (0, 2 and 5%). The activities of catalase (CAT) (A) and ascorbate peroxidase (APX) (B). MDA were strongly increased after exposing the algae to the higher content of PEG, especially 5%. Different lower case letters indicate significant differences between the means according to Fisher's LSD test at the P < 0.01 level, Means ± SEs, N = 3.
Chlorophylls increase did not coincide with significant changes in β-carotene during PEG-induced drought stress
An increase in chlorophyll a and b pigments compared with the control were triggered by drought stress, regardless of the PEG concentrations applied. As depicted in Table 3, the content of chlorophyll a and b was higher in D. salina at 2% or 5% PEG compared with the control. The algae exposed to 2% or 5% of PEG had approximately identical TI.
Table 3. The effect of drought on the accumulation of pigments including chlorophyll a (Chl a), chlorophyll b (Chl b) and β-carotene (β-car) and the ratio of total chlorophylls (TChl) to β-carotene (TChl:β-car) in microalgae, D. salina cultured under different concentrations of PEG 6000. Tolerance Index (TI) also measured on the basis of chlorophylls content
The values shown are means followed by ± SD, N = 3. Different superscript letters indicate significant differences between the means according to Fisher's LSD test at the P < 0.01 level.
No meaningful difference was found in β-carotene content between the control and PEG treatments. Other results also indicated that the ratio of total chlorophyll/β-carotene increased by PEG treatments. However, there was no difference in the ratios between 2% or 5% PEG treatments.
DNA fragmentation
The use of DNA laddering evaluated the effect of PEG on nucleic acid fragmentation, as an indicator of programmed cell death (PCD). A single band from an intact genomic DNA of alga was observed at a size of ~20,000 base pairs (bp) in all DNA samples extracted from the control (Figure 7, lane 1) and PEG-treated cells (Figure 7, lanes 2 and 3). The absence of DNA laddering showed no damage to nucleic acids even at the high concentration of PEG.
Fig. 7. The effect of drought stress on the DNA fragmentation in D. salina cultured under different PEG 6000 concentrations (0, 2 and 5%). M: Lambda DNA/HindIII (Thermo Scientific), lane 1: control, lane 2: algae treated with PEG 2%, lane 3: algae treated with PEG 5%.
Discussion
Although there are numerous reports focused on the defence mechanisms in response to drought stress in higher plants and some species of algae (Gechev et al., Reference Gechev, Dinakar, Benina, Toneva and Bartels2013; Lin & Wu, Reference Lin and Wu2014), to the best of our knowledge, there has been no study about physiological responses of the microalga Dunaliella to drought stress to date. Recently, Lee & Yeh (Reference Lee and Yeh2014) evaluated the effects of PEG concentrations on cell growth, size variation and lipid production of microalgae, Nannochloropsis oculata. The simultaneous effects of salinity and PEG on D. salina growth have been previously studied by Kumar & Dharmaraj (Reference Kumar and Dharmaraj2003); however, the effect solely of different concentrations of PEG on the growth and physiology of this alga remained under-investigated. The effect of different concentrations of PEG on D. salina growth was first studied by measuring specific growth rate, doubling time, cell divisions per day and finally, the growth kinetic. Our overall results showed that although D. salina grew slightly better in 2% PEG than the control, the elevation of the PEG concentrations had adverse effects on cell growth. This was in accordance with the results obtained by Lee & Yeh (Reference Lee and Yeh2014), which demonstrated that growth of the microalgae, N. oculata, could be facilitated only in PEG-6000 concentrations ≤0.5% (w/v). In the present study, PEG concentrations of more than 5% induced significant mortality rates and finally quickly reaching a plateau phase. Lin & Wu (Reference Lin and Wu2014) stated that the total inhibition in the growth of Chlorella vulgaris did not take place even at PEG concentrations of more than 40%. Other microalgae in the same study, including Chlamydomonas reinhardtii, Klebsormidium flaccidum and Leptolyngbya boryana, also continued to grow in PEG-6000 concentrations by more than 20%, 30% and 40%, respectively. The higher inhibition of algal growth under lower PEG concentrations in our study might be related to the effective formation of NaCl-PEG complexes in the culture medium. The surface adsorption of these complexes on the microalgal cell wall would lead to further complex formation caused by cross-linkages, which eventually might result in the production of cell clumping and the reduction of the speed indices of the cells (Chan et al., Reference Chan, Wong and Ng1981). Dunaliella salina cells grown in PEG doses higher than 2% had bigger cell sizes and the majority of them tend to be highly attached and formed larger cell clumps. The PEG tolerance of D. salina in this study was shown to be significantly lower compared with the microalgae tested in Lin & Wu's (2014) study, which might be related to the fact that a higher concentration of NaCl (1.5 M) used in our culture medium, strongly accelerated the effect of PEG by the formation of NaCl-PEG complexes, whereas the maximum concentration of NaCl in Lin and Wu's study did not exceed more than 200 mM. On the other hand, the high concentration of NaCl in combination with 5% PEG induced a massive total osmotic potential of −73.8 Mpa, while this maximum total value in Lin and Wu's study was only −2.7 Mpa.
In response to various stresses such as salt stress, drought stress, cold stress, heavy metal toxicity and UV or gamma radiation, cells tend to accumulate a considerable amount of compatible solutes like low-molecular-weight compounds such as proline, glycine betaine and glycerol as well as other kinds of sugars. Solutes accumulation is well known to be a species-dependent character, such that the resistant plants generally produce larger amounts of compatible compounds in response to stresses (Delauney et al., Reference Delauney, Hu, Kavi Kishor and Verma1993; Molinari et al., Reference Molinari, Marur, Daros, De Campos, Carvalho, Filho, Pereira and Vieira2007; Lin & Wu, Reference Lin and Wu2014; Toghyani et al., Reference Toghyani, Karimi, Hosseini Tafreshi and Talei2020). The compatible solutes could help osmotic and volume control and act as an osmoprotectant (Molinari et al., Reference Molinari, Marur, Daros, De Campos, Carvalho, Filho, Pereira and Vieira2007), a redox buffer (Hare & Cress, Reference Hare and Cress1997), protecting enzymes and proteins against destruction (Matysik et al., Reference Matysik, Alia, Bhalu and Mohanty2002), a regulator of cytosolic acidity (Venekemp, Reference Venekemp1989), a remover of free radicals (Matysik et al., Reference Matysik, Alia, Bhalu and Mohanty2002) and a membrane stabilizer (Lin & Wu, Reference Lin and Wu2014). Additionally, solutes such as proline are considered to play an essential role in the defence mechanism of stressed cells by providing carbon, nitrogen and an energy source (Kuznetsov & Shevykova, Reference Kuznetsov and Shevykova1999). The amounts of proline have been previously reported to increase in D. salina cells under salinities up to 5.5 M (Mishra et al., Reference Mishra, Mandoli and Jha2008). According to our results, D. salina indicated a high ability to accumulate more intracellular proline contents in response to drought stress, which was highly correlated with the increased doses of PEG. This was compatible with the results of Delauney et al. (Reference Delauney, Hu, Kavi Kishor and Verma1993) and Molinari et al. (Reference Molinari, Marur, Daros, De Campos, Carvalho, Filho, Pereira and Vieira2007) for higher plants and Lin & Wu (Reference Lin and Wu2014) for soil algae and cyanobacteria. In the current study, it was also found that the proline accumulation co-occurred with a significant PEG-induced H2O2 generation, especially in 5% PEG-treated cells. This compound was produced 33-fold more than in untreated cells. The compatible solutes such as proline could protect proteins, especially enzymes, from denaturing under stress conditions (Anaraki et al., Reference Anaraki, Hosseini Tafreshi and Shariati2018). In such a condition, the activity of antioxidant enzymes, CAT and APX, simultaneously rose with a positive correlation with H2O2 generation and proline accumulation. These enzymes seemingly act as scavenger and detoxifier of stress-generated ROS, such as hydrogen peroxide (Arora et al., Reference Arora, Sairam and Srivastava2002) and have been demonstrated to protect the plant cells from programmed cell death (PCD), especially under stress conditions (Tattini et al., Reference Tattini, Traversi, Castelli, Biricolti, Guidi and Massai2009). In this study, the absence of DNA fragmentation in D. salina cells showed no occurrence of PCD under PEG treatment, which could be related to the significant activation of antioxidant enzymes of the alga.
Several species of marine algae have been reported to accumulate glycine betaine as a stabilizing osmolyte (Mishra et al., Reference Mishra, Mandoli and Jha2008). In the present study, the amount of glycine betaine increased in Dunaliella cells in proportion to the amount of the PEG level. Mishra et al. (Reference Mishra, Mandoli and Jha2008) reported a glycine betaine accumulation of about 5.7 μg per 105 cells during the culture of D. salina in salt concentrations up to 5.5 M. According to our results as the first report regarding the effect of drought stress on glycine betaine accumulation in Dunaliella, cells are grown under 1.5 M NaCl and 5% PEG contained about 22.6 μg per 105 cells glycine betaine, which suggests that glycine betaine contributes to PEG-induced stress. Glycine betaine reportedly functioning as an osmoprotectant caused a greater acclimation capacity of plants under drought stress. This osmolyte is mainly accumulated in chloroplasts and especially has essential roles in protecting the thylakoid membrane of chloroplasts; thus it might affect photosynthesis (Rogers et al., Reference Rogers, Colmer, Frost, Henry, Cornwall, Hulm, Hughers, Nichols and Craig2009).
Glycerol production is considered as one of the most essential features of Dunaliella upon different stress conditions. A positive correlation was reported between glycerol production and salt concentration by Arun and Singh in D. salina and D. tertiolecta. They demonstrated that glycerol was induced in the cells with maximum values of 30.5 pg cell−1 for D. salina and 19.36 ± 0.13 pg cell−1 for D. tertiolecta achieved at 30% and 25% NaCl concentration, respectively. So, they concluded that under low concentration of salt (around 10%), negligible amounts of glycerol would be accumulated in D. salina with a mean value of about 5.46 pg cell−1 (Arun & Singh, Reference Arun and Singh2013). In the present study, a similar level of salinity (8%) induced similar amounts of glycerol in D. salina in a PEG-free control with a a mean value of 6.74 pg cell−1. However, when the cells exposed to 8% NaCl (1.5 M) were treated with 2% or 5% PEG, the mean amount of glycerol significantly increased to 9.27 or 16.74 pg cell−1, respectively. This indicates that D. salina has a strong ability to adjust its intracellular osmotic potential when coping with high levels of PEG. Although this is the first report of importance of glycerol accumulation in D. salina upon drought stress, other studies have widely reported the higher accumulation of intracellular glycerol in Dunaliella as a significant mechanism for adjusting osmotic potential against abiotic stress including temperature, metal toxicity, nutrient depletion, as well as different ranges of salinity (Chitlaru & Pick, Reference Chitlaru and Pick1991; Phadwal & Singh, Reference Phadwal and Singh2003; Mishra et al., Reference Mishra, Mandoli and Jha2008). It was also indicated that glycerol acts as a ‘compatible solute’ to protect enzymes against inactivation or inhibition under stress conditions (Chitlaru & Pick, Reference Chitlaru and Pick1991). In addition to low-molecular solutes, for the first time, we reported that the levels of soluble sugars (glucose, mannose and rhamnose) were also elevated under PEG-induced drought conditions in D. salina. Mishra et al. (Reference Mishra, Mandoli and Jha2008) previously reported that the total sugars significantly increased up to 3-fold when D. salina was exposed to high salinity stress (5.5 M NaCl). It has been concluded that the accumulation of soluble sugars in plant cells plays an essential role in response to various abiotic stresses during vegetative and reproductive development.
The alga, D. salina has a well-known ability to accumulate a large amount of β-carotene in the chloroplast (up to 14% of dry weight) to protect the photosynthetic pigments and chlorophylls against photo-damage, especially if the alga is exposed to extreme stress conditions (Aasen et al., Reference Aasen, Eimhjellen and Liaaen-Jensen1969). It was previously indicated that β-carotene synthesis was significantly induced up to several fold by the various ranges of stresses (Phadwal & Singh, Reference Phadwal and Singh2003; Hosseini Tafreshi & Shariati, Reference Hosseini Tafreshi and Shariati2008). Lin & Wu (Reference Lin and Wu2014) reported that during PEG treatment, some microalgae such as C. vulgaris and L. boryana accumulated a high amount of carotenoids; meanwhile, some other species had limited ability to synthesize additional carotenoids in such conditions. Carotenoids are known to effectively quench the singlet excited chlorophylls (1Chl*) and chlorophyll triplets. The excess excitation energy resulting from stress could finally be dissipated via the xanthophyll cycle (Das & Roychoudhury, Reference Das and Roychoudhury2014). The algal carotenoids such as astaxanthin have previously been recognized as the potent light-attenuating molecules that were able to weaken desiccation intensity (Oliver et al., Reference Oliver, Farrant, Hilhorst, Mundree, Williams and Bewley2020). The carotenoids are also known to have protective contributions to stabilizing the plasma membrane against membrane lipid peroxidation (Havaux, Reference Havaux1998). Our results showed that the PEG-induced drought did n't trigger β-carotene accumulation in D. salina. Therefore, the accumulation of carotenoids such as β-carotene probably plays no critical role in drought stress in this microalga, which could explain the high degree of lipid peroxidation and a high percentage of EL in D. salina under PEG treatment. Both the content of chlorophylls and the ratio of total chlorophyll/β-carotene increased in the cells treated with PEG. It has been previously shown that chlorophyll content is significantly induced by salinities of more than 3.0 M in D. tertiolecta and D. salina (Mishra et al., Reference Mishra, Mandoli and Jha2008). A similar tolerance index (TI) between the cells treated with 2% or 5% PEG showed a remarkable ability of D. salina to protect its pigments at a steady-state under a high degree of drought. Additionally, the protein content in PEG-treated cells remained relatively constant in response to stress and this might be correlated again with a high degree of Dunaliella tolerance to drought stress.
Conclusion
The present study demonstrated that the green microalga, D. salina, intrinsically employed various strategies during coping with drought stress, including increase of a range of osmolytes and stimulation of the activity of some antioxidant enzymes. A significant amount of osmolytes such as proline, glycerol, glycine betaine and soluble sugars accumulated in Dunaliella during drought stress which might show that these solutes acted as osmoprotectants to protect the enzymes and other proteins from stress-related injuries. This might be supported by a higher activity of antioxidant enzymes such as catalase and ascorbate peroxidase under PEG-induced drought compared with PEG-free conditions. The microalgae may also consume PEG to break it down and use it as an alternative source of carbon during stress which should be further studied in detail. On the other hand, it was concluded from our study that in contrast to salinity stress, PEG-induced drought did not stimulate a higher carotenoid accumulation in D. salina. Therefore, the responsive mechanisms against these two stressors at least in part may be different in this microalga. Overall, the present study provided an insight into how eukaryotic algal cells can tolerate high intensities of drought stress. Future investigations could be directed towards a full consideration of protective mechanisms of microalgae against drought stress at the genomics, proteomics and metabolomics levels. This may help us to discover gene or protein candidates with potential of reducing the negative effect of drought on eukaryotic cells.
Acknowledgements
This work was supported by graduate studies of the University of Kashan under grant No. 572212/01. Special thanks to Mrs Ejtahed, head of the biotechnology lab in the University of Kashan, for her critical offers of help and suggestions.
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
