Chinese Journal of Oceanology and Limnology   2016, Vol. 34 Issue(1): 102-108     PDF       
http://dx.doi.org/10.1007/s00343-015-4351-z
Institute of Oceanology, Chinese Academy of Sciences
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Article Information

WANG Ying(王影), ZHAO Xinyu(赵新宇), TANG Xuexi(唐学玺)_L
Antioxidant system responses in two co-occurring green-tide algae under stress conditions
Chinese Journal of Oceanology and Limnology, 2016, 34(1): 102-108
http://dx.doi.org/10.1007/s00343-015-4351-z

Article History

Received Dec. 8, 2014;
accepted in principle Feb. 6, 2015;
accepted for publication Feb. 25, 2015
Antioxidant system responses in two co-occurring green-tide algae under stress conditions
WANG Ying(王影), ZHAO Xinyu(赵新宇), TANG Xuexi(唐学玺)        
Department of Marine Ecology, College of Marine Life Sciences, Ocean University of China, Qingdao 266003, China
ABSTRACT:Green tides have occurred every year from2007 to 2014 in the Yellow Sea. Ulva prolifera (Müller) J. Agardh has been identified as the bloom-forming alga, co-occurring with U. intestinalis. We observed distinct strategies for both algal species during green tides. U. prolifera exhibited a high abundance initially and then decreased dramatically, while U. intestinalis persisted throughout. The antioxidant system responses of these two macroalgae were compared in the late phase of a green tide (in-situ) and after laboratory acclimation. Lipid peroxidation and antioxidant system responses diff ered significantly between the two. Malondialdehyde and hydrogen peroxide contents increased significantly in-situ in U. prolifera, but not in U. intestinalis. In U. prolifera, we observed a significant decrease in total antioxidant ability (T-AOC), antioxidant enzymes (SOD and Apx), and non-enzyme antioxidants (GSH and AsA) in-situ. U. intestinalis showed the same pattern of T-AOC and SOD, but its Gpx, Apx, and GSH responses did not diff er significantly. The results suggest that U. prolifera was more susceptible than U. intestinalis to the harsh environmental changes during the late phase of a Yellow Sea green tide. The boom and bust strategy exhibited by U. prolifera and the persistence of U. intestinalis can be explained by diff erences in enzyme activity and antioxidant systems.
Keywordsantioxidant system     Yellow Sea green tide     U. prolifera     U. intestinalis     algal bloom    

1 INTRODUCTION

Excessive growth of some green algae species has caused the formation of green tides in many parts of the world, including Europe, America, Australia, and Asia(Taylor et al., 2001; Nelson et al., 2003, 2008; Sun et al., 2008; Yabe et al., 2009; Kim et al., 2010). Ulva prolifera and U. intestinalis species with mass occurrences recorded in the eutrophic estuaries of most global oceans(Baeck et al., 2000; Sun et al., 2008). Large-scale green tides in China’s Yellow Sea have occurred every year from2007 to 2014(Sun et al., 2008; Liu et al., 2009; Wang et al., 2010; Luo and Liu, 2011; Zhao et al., 2012; Liu et al., 2013). Previous studies have reported that the dominant species in these green tides is Ulva prolifera(Sun et al., 2008; Ye et al., 2008; Leliaert et al., 2009). U. intestinalis always co-occurs with U. prolifera(Liu et al., 2010), but unlike the ephemeral U. prolifera it maintains a constant biomass during Yellow Sea green tides. Therefore, we suspected that these co-occurring species may have diff erent strategies to cope with environmental stress.

Previous reports have indicated that when macroalgae are subjected to environmental stress they can accumulate reactive oxygen species(ROS) and experience oxidative stress(Collén and Davison, 1999; Cohen and Fong, 2004; Liu et al., 2010; Luo and Liu, 2011). Free radicals such as hydrogen peroxide(H2 O2), superoxide radicals(O2• ˉ), singlet oxygen(1 O2), and hydroxyl radicals(OH•)disrupt normal metabolism through peroxidizing membrane lipids and denaturing proteins and nucleic acids(Mackerness et al., 1999). Macroalgae have evolved antioxidant defense mechanisms to cope with potential damage from ROS. Previous studies have shown that higher antioxidant content and antioxidant enzyme activity are associated with higher stress tolerance in various algae(Cohen and Fong, 2004; Liu et al., 2010; Luo and Liu, 2011). In the late phase of Yellow Sea green tides, highly dynamic environmental conditions, such as excess irradiation or aberrant temperatures, create stressful conditions for macroalgae. We deduced that diff erent oxidative stress tolerances in U. prolifera and U. intestinalis contribute to their diff erent strategies for coping with environmental stress.

Enzymatic activity and antioxidant content reflect the status of an organelle’s oxidative machinery and may be taken as an indicator of cell acclimation and tolerance(Apel and Hirt, 2004; Ross and Van Alstyne, 2007). We compared the enzymatic activity and antioxidant contents of co-occurring U. prolifera and U. intestinalis in the late phase of a Yellow Sea green tide(early August 2010)to investigate if there was a relationship between algae’s the different oxidative stress tolerances and their strategies for coping with environmental stressors.

2 MATERIAL AND METHOD 2.1 Site descriptions Field

observations were carried out along the rocky intertidal shores around Cape Taiping(36.049 2°N, 120.353 6°E), Qingdao, China, an area in which green tides have occurred for eight consecutive years(2007-2014). Multiple natural macroalgal assemblages are found in the intertidal zone, either attached or free-floating. Free-floating U. prolifera is the dominant Ulva species, while the attached U. intestinalis co-occurs during green tide periods.

2.2 Macroalgal cultures

U. prolifera and U. intestinalis thalli were collected from coastal Qingdao in June 2010, during a bloom period. These were gently rinsed in sterile seawater and thoroughly cleaned with a brush under a magnifier to remove attached sediment, small grazers, and epiphytes. The thalli were lab acclimated by culturing in sterile seawater enriched with f/2 medium(Guillard, 1975), at a constant 20°C and a light intensity of 72 μmol photons/(m2·s), in a 12 h:12 h light: dark cycle using a GXZ-280 C intelligent illumination incubator(Ningbo Jiangnan Instrument Factory, Zhejiang, China). Germanium dioxide(GeO2)at a concentration of 0.5 mg/L was added to the cultures to suppress diatom growth(Lotze et al., 2000). The culture medium was completely renewed every 2 days. Laboratory acclimation lasted for 7 days. The experiments followed the conditions and procedures described above unless otherwise noted.

2.3 Experimental system set-up

To compare the antioxidant system of the two species and in-situ to laboratory acclimated(control)algae, five healthy thalli were r and omly sampled in the late phase of a Yellow Sea green tide(early August 2010). Thalli were treated in-situ and used directly to determine the enzymatic activity and antioxidant content, and then transferred to the laboratory for acclimation. After acclimation, the antioxidant systems of these same thalli were re-tested.

The samples(0.2-0.3 g)were ground in liquid nitrogen and extracted with 1.5 mL of 0.05 mol/L potassium phosphate buff er(pH 7.0)containing 0.000 1 mol/L EDTA(Na2ethylenediaminetetraacetic acid). The extracts were centrifuged at 1 000× g for 15 min because the crude enzymatic extraction was used for lipid peroxidation, antioxidant enzymatic activity, and non-enzymatic antioxidant assays.

2.4 Lipid peroxidation and H2O2 content analysis

As an indication of oxidative stress, malondialdehyde(MDA)formation as an index of lipid peroxidation was studied. The MDA content was measured using thiobarbituric acid(TBA). To do this, 0.2 mL extract was homogenized in 1 mL of 10% trichloroacetic acid(TCA). The homogenates were centrifuged at 1 000× g at 4°C for 10 min. Two-mL of 0.6% TBA was added to each 2-mL aliquot of the supernatant. The mixtures were heated at 95°C for 40 min and then quickly cooled in an ice bath. After centrifugation at 1 000× g at 4°C for 10 min, the absorbance of the supernatant was recorded at 532 nm. The results are expressed as nmol MDA per mg of total soluble protein(nmol MDA/mg pro.).

H2O2 contents were determined based on the decomposition of H2O2 by peroxidase as described by Okuda et al.(1991). H2O2 content was expressed as nmol horseradish peroxidase per g of fresh weight(nmol/g FW).

2.5 Antioxidant system analysis

Total soluble protein of the crude extract for antioxidant enzyme activities(protein g/mL, pro. g/mL) and total antioxidant ability(T-AOC)were determined following the methods described by Wang et al.(2012).

2.6 Antioxidant enzyme activity analysis

Superoxide dismutase(SOD)was measured using the xanthine oxidase-cytochrome c reduction method(Mishra et al., 1993). One unit of SOD was defined as the amount of enzyme required for a 50% inhibition of cytochrome c reduction. Glutathione peroxidase(Gpx)activity was measured in a coupled enzyme system in which the formed oxidized glutathione(GSSG)was converted to its reduced form by glutathione reductase(Lawrence and Burk, 1976). The Gpx activity was calculated according to the method described by Wang et al.(2012). Enzyme activities were expressed as units per mg of total soluble protein(U/mg pro.).

Ascorbate peroxidase(Apx)was measured according to the method described by Nakano and Asada(1981). The oxidation of ascorbate was initiated by adding 50 μL supernatant to 950 μL potassium phosphate buff er(pH 7.0, 0.05 mol/L)containing 0.000 1 mol/L EDTA, 0.5 mol/L ascorbate, and 0.000 1 mol/L H2O2. Thirty seconds into the reaction, the decrease in absorbance at 290 nm was measured. One unit of Apx activity determined the amount necessary to decompose 1 μmol of ascorbate per min.

2.7 Antioxidant content analysis

Total glutathione(GSH, reduced and oxidized)content was measured using 5, 5′-dithio-bis(2-nitrobenzoic acid)(DTNB). To do this, 0.5 mL algal extract was combined with 2 mL of 5%(v/v)5-sulphosalicylic acid containing 1%(w/v)polyvinylpyrrolidone 40. The homogenates were centrifuged at 1 000× g at 4°C for 10 min. One milliliter of supernatant was mixed with 100 μL of 0.006 mol/L DTNB, 175 μL distilled water, and 25 μL of 266 U/mL glutathione reductase. The increased rate of absorbance at 420 nm was quantified using a st and ard curve. Total glutathione content was expressed as mg GSH per mg of total soluble protein(mg GSH/mg pro.)

Ascorbate(AsA)content was measured according to the method described by Foyer et al.(1983). AsA content was determined by recording the decrease in absorbance at 265 nm. AsA contents were expressed as nmol ascorbate oxidase per g of fresh weight(nmol/g FW).

2.8 Statistical analysis

The data were compared by two-way ANOVA. The data were tested with two sampling phases(in-situ and after 7 days acclimation; the “phase”), and species(the “species”). These were treated as fixed factors. All values cited in this paper were obtained from fully independent samples. Data were initially examined using Levene’s test for homogeneity and the Shapiro-Wilk test for normality. The Student-Newman-Keuls post-hoc multiple comparison test and Duncan’s post-hoc test were used if ANOVA indicated a significant eff ect. Diff erences between treatment means were considered significant if P<0.05. Data were analyzed using IBM SPSS Statistics 19(SPSS Inc., Chicago, USA).

3 RESULT

Antioxidant system parameters, lipid peroxidation, and H2O2 content of U. prolifera and U. intestinalis sampled in-situ and after 7 days acclimation(control)exhibited highly significant species eff ects and treatment eff ects(Table 1, two-way ANOVA, P<0.01). MDA was not significantly aff ected by the combined eff ects of species and developing phase(F1,16 =3.9, 0.01< P<0.05), and GSH did not diff er between either species or developing phase(F1,16 = 2.075, P = 0.157>0.05). The antioxidant enzymatic activity(SOD and Gpx)of both species showed more highly significant species than treatment eff ects, and antioxidant contents(GSH and AsA)were more aff ected by treatments(in-situ and control).

Table 1 Results of two-way ANOVA on eff ects of culture treatment and species on malondialdehyde (MDA), total antioxidant ability (T-AOC), superoxide dismutase (SOD), glutathione peroxidase (Gpx), ascorbate peroxidase (Apx), glutathione (GSH), and ascorbate (AsA)
3.1 Lipid peroxidation and H2O2 content analysis

MDA content did not diff er significantly between the two control groups(7.1 nmol/mg pro. for U. prolifera and 8.4 nmol /mg pro. for U. intestinalis). A statistically significant increase in MDA concentration in-situ was found in U. prolifera compared to the control treatment(Fig. 1, post-hoc, P<0.05), indicating lipid oxidative damage during the in-situ treatment. MDA concentration in U. intestinalis(Fig. 1, posthoc, P>0.05)did not diff er significantly between the two experimental treatments. In the late phase of the Yellow Sea green tide, MDA concentration in U. prolifera was twice that in U. intestinalis.

Fig. 1 Mean lipid peroxidation presented as MDA values and H 2 O2 content in U. prolifera and U. intestinalis measured in-situ and after 7 days laboratory culture (control)
Values are means±SD ( n =5). Letters above the bars indicate significantly diff erent values (post-hoc, P<0.05).

Similar to MDA concentration, the U. prolifera H2O2 content increased significantly compared with that of the control(Fig. 1, post-hoc, P<0.05), while the U. intestinalis H2O2 content increased only slightly. After 7 days acclimation, the H2O2 content did not diff er significantly between the two species(Fig. 1, post-hoc, P>0.05).

3.2 Antioxidant system analysis

T-AOC can be used as an estimator of total antioxidant capacity in algal defense systems. T-AOC values were lower in U. prolifera than in U. intestinalis and exhibited a marked decrease in-situ compared with the control(Fig. 2).

Fig. 2 Mean T-AOC in U. prolifera and U. intestinalis measured in-situ and after 7 days laboratory culture (control)
Values are means±SD ( n =5). Letters above the bars indicate significantly diff erent values (post-hoc, P<0.05).
3.3 Antioxidant enzyme activity analysis

SOD, Gpx, and Apx activity in U. prolifera diff ered significantly between in-situ and control treatments(Figs.4-6, post-hoc, P<0.05), whereas Gpx and Apx activity in U. intestinalis did not(Fig. 4, post-hoc, P>0.05). SOD, Gpx, and Apx activity in U. prolifera were markedly lower than those in U. intestinalis in both in-situ and control treatments(Figs.3 and 4, post-hoc, P<0.05). These antioxidant enzymes in U. prolifera were more sensitive to experimental treatments than in U. intestinalis.

Fig. 4 Mean Gpx and Apx in U. prolifera and U. intestinalis measured in-situ and after 7 days laboratory culture (control)
Values are means±SD ( n =5). Letters above the bars indicate significantly diff erent values (post-hoc, P<0.05).
3.4 Antioxidant contents analysis

The contents of the two major endogenous nonenzyme antioxidants, GSH and AsA, were lower in U. prolifera compared to U. intestinalis in the late phase of a Yellow Sea green tide(Fig. 5, post-hoc, P<0.05). GSH and AsA content decreased significantly in the U. prolifera in-situ treatment compared with the control(Fig. 5, post-hoc, P<0.05).

Fig. 3 Mean SOD in U. prolifera and U. intestinalis measured in-situ and after 7 days laboratory culture (control)
Values are means±SD ( n =5). Letters above the bars indicate significantly diff erent values (post-hoc, P <0.05).

Fig. 5 Mean GSH and AsA in U. prolifera and U. intestinalis measured in-situ and after 7 days laboratory culture (control)
Values are means±SD ( n =5). Letters above the bars indicate significantly diff erent values (post-hoc, P <0.05).

4 DISCUSSION

Our study measured the antioxidant enzyme levels of two algae species in-situ during a green tide and after 7 days of laboratory acclimation. Some inhibiting eff ects present in the field, such as nutrient limitation and herbivory were eliminated after laboratory acclimation. The aim of acclimation was to measure optimal performance under non-stress conditions; thallus measurements after 7 days of laboratory acclimation were used as a control. Many case studies on Ulva species have used the production of reactive oxygen species( Ross and Van Alstyne, 2007)to indicate the influence of environmental stress on growth performance, and have reported that antioxidant systems in these species are sensitive to stress(Lu et al., 2006; Liu et al., 2010; Luo and Liu, 2011). The present study further confirmed that the U. prolifera and U. intestinalis antioxidant systems are sensitive to the environmental stressors present during the late phase of a Yellow Sea green tide.

The extent of lipid peroxidation indicated that the level of environmental stress induced diff erent amounts of oxidative stress in each species. MDA content was the parameter that directly indicated lipid peroxidation, and the content of the ROS H 2 O2 was taken as a proxy for oxidative damage as indicated by the concomitant increase of the extent of lipid peroxidation(Apel and Hirt, 2004; Lu et al., 2006; Luo and Liu, 2011). In the present study, the MDA and H 2 O2 contents increased significantly in the late phase of the Yellow Sea green tide in U. prolifera, but not in U. intestinalis(Figs.1, 2). There was no significant diff erence between the two species after 7 days laboratory acclimation. This indicates that the basic level of lipid peroxidation was the same in both species, and oxidative stress was induced when the algal samples were exposed to environmental stress conditions. U. prolifera was more aff ected by the environmental stressors present during the late phase of a Yellow Sea green tide.

The U. prolifera antioxidant systems exhibited more pronounced responses to oxidative stress induced by environmental stressors than those in U. intestinalis. In U. prolifera, significant decreases in T-AOC, antioxidant enzymes(SOD and Apx), and non-enzyme antioxidants(GSH and AsA)were observed in the thallus compared with the controls(Figs.2-5). U. intestinalis T-AOC and SOD exhibited the same pattern, but Gpx, Apx, and GSH responses did not diff er statistically when exposed to the same stressors. Combined with the lipid peroxidation results, the diff erent responses of the antioxidant systems to the same stressors suggest that the U. intestinalis antioxidant system eff ectively prevents the damaging eff ects of ROS; however, it was insufficient in U. prolifera. Some important case studies on the stress response in Ulva species have also indicated that increased availability of antioxidants and antioxidant enzyme activity are important measures of an algae’s ability to cope with oxidative stress(Choo et al., 2004; Lu et al., 2006; Luo and Liu, 2011). Additionally, in U. prolifera T-AOC, antioxidant enzymes(SOD, Gpx, and Apx), non-enzyme antioxidant(GSH and AsA)activity and content were markedly lower than those in U. intestinalis both in-situ and in controls(Figs.2-5). The ephemeral strategy of U. prolifera and the persistence of U. intestinalis can be explained by the highly dynamic and harsh environment of a late phase Yellow Sea green tide because U. prolifera is more susceptible to environmental stress than U. intestinalis. The same stress in the late phase of a Yellow Sea green tide could cause U. prolifera greater damage and produce ROS beyond its ability to remove them. Antioxidant activity in U. prolifera is insufficient to prevent and repair the damaging eff ects of ROS.

5 CONCLUSION

The antioxidant system responses of U. prolifera and U. intestinalis were compared both in-situ during the late phase of a Yellow Sea green tide(early August 2010) and following laboratory acclimation. The lipid peroxidation and antioxidant system response diff ered significantly between the two macroalgae. In U. prolifera a significant decrease in total antioxidant ability(T-AOC), antioxidant enzymes(SOD and Apx), and non-enzyme antioxidants(GSH and AsA)was observed in the thallus of algae in-situ compared with the control. U. intestinalis T-AOC and SOD exhibited the same pattern, but Gpx, Apx, and GSH responses did not diff er statistically when exposed to the same stressors. Our results suggest that U. prolifera was more susceptible than U. intestinalis to the harsh environmental changes during the late phase of a Yellow Sea green tide, and that antioxidant activity in U. prolifera is insufficient to prevent the damaging eff ects of oxidative stress. The ephemeral boom and bust cycle in U. prolifera and the persistence of U. intestinalis can be explained by diff erent antioxidant capacities.

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