Allelochemical induces growth and photosynthesis inhibition, oxidative damage in marine diatom Phaeodactylum tricornutum

Journal of Experimental Marine Biology and Ecology 444 (2013) 16–23

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Allelochemical induces growth and photosynthesis inhibition, oxidative damage in marine diatom Phaeodactylum tricornutum Cuiyun Yang a, Jun Zhou a, Sujing Liu a, Ping Fan b,⁎, Wenhai Wang a, Chuanhai Xia a,⁎ a b

Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai 264003, China College of Chemistry, Liaoning University, Shenyang 110036, China

a r t i c l e

i n f o

Article history: Received 24 May 2012 Received in revised form 1 March 2013 Accepted 2 March 2013 Available online xxxx Keywords: Antioxidant enzyme Cellular pigments Growth Hydroquinone Gene transcription Phaeodactylum tricornutum

a b s t r a c t Algal blooms have been occurring in many regions all over the world and allelochemical is considered as one of the important and promising algaecides to control algal blooms. In the present study, we investigated the effects of allelochemical hydroquinone (HQ) on growth, photosynthesis and other physiological levels of Phaeodactylum tricornutum (P. tricornutum). The results showed that HQ (above 3 × 10−7 mol/L) significantly inhibited the growth and specific growth rate of algae. EC50 values were calculated at four different incubation times, i.e. 24, 48, 72 and 96 h. EC50 values increased with the treated-time increasing, which suggested that HQ stress on the algae gradually weakened with time prolonging. The contents of cellular pigments including Chlorophyll a (Chl.a) and carotenoids were significantly decreased by HQ. However, the ratios of carotenoids to Chl.a increased obviously when algae were exposed to 6 and 7 × 10 −7 mol/L of HQ for 72 h, which implied that the ratios of pigments changed in extreme conditions to resist environmental stress. At the same time, HQ also induced the responses of antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), glutathione S-transferase (GST) and non-enzymatic antioxidant reduced glutathione (GSH). Additionally, flow cytometric assays showed that HQ stress altered the permeability of cell membrane and mitochondrial membrane potential in different degrees and HQ significantly inhibited the transcription of photosynthesis and respiration related genes. All these results showed that HQ might have the potential as an algaecide to control marine microalgae. © 2013 Elsevier B.V. All rights reserved.

1. Introduction In recent decades, algal blooms have been occurring in many regions all over the world. Algal blooms reduce the transparency of the water, affect the normal growth of aquatic organisms and decrease the biodiversity. When a large number of algal cells die, the water can emit offensive odor, which is a serious problem for drinking water supplies and recreational economic development. Additionally, algal blooms deplete oxygen in the waters or release many kinds of toxins that may cause illness in humans and other animals (Malbrouck and Kestemont, 2006; Romanowska-Duda et al., 2002; Sinclair et al., 2008). Therefore, the control and elimination of algal blooms have become a significant goal in environmental science. Many methods are currently tested for controlling algal blooms, including physical, chemical and biological methods (Ahn et al., 2003; Prokopkin et al., 2006; Qian et al., 2010). Among them, allelochemical

Abbreviations: CAT, Catalase; Chl.a, Chlorophyll a; FDA, Fluorescein diacetate; GPX, Glutathione peroxidase; GSH, Reduced glutathione; GST, Glutathione S-transferase; HQ, Hydroquinone; P. tricornutum, Phaeodactylum tricornutum; Rh123, Rhodamine 123; SOD, Superoxide dismutase. ⁎ Corresponding authors. Tel.: +86 535 2109173; fax: +86 535 2109000. E-mail addresses: [email protected] (P. Fan), [email protected] (C. Xia). 0022-0981/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jembe.2013.03.005

is considered as one of the most promising biological algaecides because of its higher environmental safety. Many allelochemicals that can inhibit or control algal blooms have been isolated. For example, N-phenyl2-naphthylamine (PNA), a secondary metabolite from root exudates of water hyacinth (Eichhornis crassipes), strongly inhibited the growth of Microcystis aeruginosa and Chlorella vulgaris (Qian et al., 2009, 2010). The extracts from Tibetan hulless barley (Hardeum unlgar Ls.) and golden thread (Coptis chinensis Franch) inhibited the growth of M. aeruginosa (X. Xiao et al., 2010; Zhang et al., 2011). Hong et al. (2008) reported that a new kind of allelochemical ethyl 2-methyl acetoacetate (EMA) in reed (Phragmites communis or Phragmites australis) inhibited the growth of freshwater algae including M. aeruginosa and Chlorella pyrenoidosa, and our reports also demonstrated that EMA inhibited the growth of marine diatom P. tricornutum (Li and Hu, 2005; Yang et al., 2011). From these reports, we find that most allelochemicals tested on algae are mainly used to control freshwater algae, but not marine algae, and the mechanism of allelochemicals inhibition on marine microalgae remains unclear. However, it is very important to know the mechanism of action whenever an allelochemical is proposed as algaecide (Leu et al., 2002). Previous reports showed that the mechanisms of allelochemicals inhibition on algal growth were mainly in four aspects, such as destruction

C. Yang et al. / Journal of Experimental Marine Biology and Ecology 444 (2013) 16–23

of cell structure, alteration of algal photosynthesis, respiration and enzymatic activities (Berger and Schagerl, 2004; Hong et al., 2009). To scavenge ROS and avoid oxidative damage, algal cells possess a set of cellular defense system via the enzymes and non-enzymatic antioxidants. The antioxidant enzymes include SOD, CAT, GPX, GST, etc., and non-enzymatic antioxidants involve in GSH and ascorbic acid (AsA) pools (Aravind and Prasad, 2005; Horemans et al., 2000; Vranová et al., 2002). The relationship between enzymes and non-enzymatic antioxidants is very complicated. Some enzymes participate in the formation of antioxidants, and some non-enzymatic antioxidants involve in the enzyme cycle to scavenge reactive oxygen species, etc. Zhang et al. (2011) reported that enzyme activities including SOD, CAT and non-enzymatic antioxidant GSH were significantly increased after M. aeruginosa treated with allelochemical berberine isolated from golden thread. Qian et al. (2009) reported that enzyme activities including SOD, CAT and peroxidase (POD) were greatly increased after C. vulgaris treated with allelochemical N-phenyl-2-naphthylamine from water hyacinth (E. crassipes) and the production of ROS disrupted the subcellular structure of C. vulgaris. Nakai et al. (2001) reported that polyphenols notably inhibited the growth of M. aeruginosa and the mechanism might be that polyphenol-autoxidized products such as radicals induce oxidative stress on algae. Pandey et al. (2005) also reported that hydroquinone (HQ) as allelochemical in many plants (e.g., wheat) killed the aquatic weed green musk Chara (Chara zeylanica Willd) by causing massive damage to cellular membrane integrity, loss of metabolic activities and oxidative stress. However, the effect of HQ on marine algae is still unknown, not to mention its mechanism of inhibition on them. Recently, real-time PCR analysis has been applied to measure gene transcription during algal growth in M. aeruginosa (Qian et al., 2010) and Thermosynechococcus elongatus (Kós et al., 2008). By using this technique, we focused on photosynthesis-related genes (psaB, psbA) and respiration-related genes (nad1, cob) to analyze the effect of HQ on photosynthesis and respiration of P. tricornutum. Phenolic compounds are important secondary metabolites produced by plants, and most of them have strong allelopathic effects on plants and algae. They are generally used as plant growth regulators, herbicides and algaecides for controlling freshwater algae (Nakai et al., 2001; Pandey et al., 2005). However, the effects of phenolic compounds on marine microalgae have rarely been considered. Our present study demonstrated that HQ had high inhibitory effects on marine microalgae P. tricornutum, which is a model algal species that widely spreads in marine environment. More importantly, the genome information of P. tricornutum is available, which helps us further study the mechanism of HQ inhibition on marine microalgae. In the current experiment, the effects of HQ on physiological levels were assessed by measuring antioxidant enzymes, non-enzymatic antioxidant GSH, cellular membrane system and the relative transcript abundance of photosynthesis and respiration related gene.

2. Materials and methods 2.1. Reagents and algal cultures Fluorescein diacetate (FDA) and Rhodamine 123 (Rh123) were purchased from Sigma-Aldrich Company. HQ was purchased from Sinopharm Chemical Reagent Co., Ltd (Beijing, China). All other chemicals were analytical or higher grades. The unicellular marine diatom P. tricornutum was provided by the Institute of Oceanology, Chinese Academy of Sciences. The microalgae were grown in axenic conditions, in f/2 medium based on autoclaved natural seawater at 20 °C and light intensity of 48 μmol photons m − 2 s − 1 with a 12:12 h light: dark cycle in the incubator. All cultures were shaken twice a day and cultured to the exponential phase before using in the following experiments.

17

2.2. HQ treatment on P. tricornutum The flasks of 250 mL were prepared and each of them contained 150 mL f/2 algal culture medium (it is a very common culture medium for marine algae). HQ concentrations were designed as follows: 0, 1, 2, 3, 4, 5, 6 and 7 × 10−7 mol/L. The medium without HQ was taken as the control and the initial algal density was 1.8 × 105 cells/mL. Algal cells were harvested to determine cell densities, cellular pigments, physiological and biochemical indicators after HQ exposure. All the flasks and culture medium were autoclaved and all experiments were at least three replicates. 2.3. Growth assays The algal cells were counted microscopically with a Haemacytometer (OLYMPUS-CX21, JAPAN). The specific growth rate (μ) was calculated according to the equation as follow: μ = (lnN2 − lnN1) / (t2 − t1), where N1and N2 were the cell numbers of the beginning and the end of the experiment; t2 − t1was time interval, in days. The growth inhibition effect of HQ on algae was calculated according to the equation I = 1 − Nt1 / Nt0, where Nt1 and Nt0 were the cell numbers of time t1 and the beginning of growth phase respectively. The growth inhibition effect (I) showed that HQ treatment reduced the cell numbers in comparison with the control. The treatment time when the maximum inhibition rate occurred was selected to calculate EC50, which was the minimum effective inhibition concentration of HQ necessary to cause 50% growth inhibition compared to the control. Cell density of control group was expressed by 100%, treatment groups were expressed by relative cell densities. This was used to determine EC50 in SigmaPlot 10.0 using Logistic curve fitting based on equation. 2.4. Photosynthetic pigment assays Chlorophyll a (Chl. a) and carotenoid were measured spectrophotometrically (UV-1810, Beijing, PR China) at 665, 649 and 470 nm after cells were extracted with 95% ethanol over night at 4 °C when algal cells were incubated for 72 h and 144 h. The pigment contents were calculated using the following equations (Y. Xiao et al., 2010): Chl:a ¼ 13:95 A665 −6:88A649 Carotenoid ¼ ð1000A470 −2:05Chl:aÞ=245:

2.5. Physiological and biochemical assays Algal cells were harvested by centrifugation at 4 °C and 6000 g for 10 min, then washed with PBS (50 mM, pH 7.8) and centrifuged twice. The cells were resuspended in 2 mL PBS solution and homogenized by an ultrasonic cell pulverizer at 300 W for a total time of 5 min (ultrasonic time: 3 s; rest time: 4 s). Then the homogenate was centrifuged at 6000 g for 10 min at 4 °C. The supernatant, cell-free enzyme extract, was used to analyze the activities of SOD, CAT, GPX, GST and GSH. SOD activity was detected according to the method of Beyer and Fridovich (1987). The total volume of reaction mixture was 3 mL, including 1.5 mL PBS (50 mM, pH 7.8), 0.3 mL methionine solution (130 mM), 0.3 mL nitroblue tetrazolium solution (NBT, 750 μM), 0.3 mL Na2EDTA solution(100 μM), 0.3 mL riboflavin solution(20 μM), 0.25 mL distilled water and 0.05 mL enzyme extract. SOD could inhibit the photochemical reduction of NBT, the assay utilized the negative controls (full-dark without any photochemical reduction of NBT), positive controls (deficiency of enzyme extract, in light with full photochemical reduction of NBT), and treatment groups (in light with enzyme extract on photochemical reduction of NBT) to measure SOD

C. Yang et al. / Journal of Experimental Marine Biology and Ecology 444 (2013) 16–23

activity. The 560 nm absorbance was measured after 20 min irradiance of 40–60 μmol photons m−2 s−1. One unit of SOD activity was defined as the amount of enzyme causing 50% inhibition of the rate of NBT reduction. CAT activity was measured in terms of the decomposition of hydrogen peroxide, which was monitored directly by the decrease in absorbance at 240 nm (Cakmak and Marschner, 1992). The reaction mixture of 3 mL contained 50 mM PBS (pH 7.0) 1.8 mL, 0.2% H2O2 1 mL and the enzyme extract 0.2 mL. One unit of CAT was defined as the decrease of absorbance up to 0.01 at 240 nm in 1 min. GPX activity was measured according to the method of Drotar et al. (1985) using glutathione as a substrate. GPX could promote the reaction of H2O2 and GSH to H2O and oxidized glutathione (GSSG). The decrease rate of GSH could be used to express GPX activity. The determination tube reaction mixture included 400 μL GSH (1 mM), 0.2 mL H2O2 (1.25 mM) and 400 μL enzyme extract. The control tube and blank tube reaction mixtures were measured by using PBS (50 mM, pH 7.0) and distilled water instead of enzyme extract. All the reaction mixtures were incubated at 37 °C for 5 min and then added quickly with 4 mL TCA (50 g/L) to prevent the reaction for 10 min, then centrifugated at 6000 g for 5 min. GSH content in the supernatant was determined according to the formation rate of 5-thio-2-nitrobenzoic acid (TNB) reduced from DTNB with 412 nm absorbance. The reaction mixture included 2.5 mL Na2HPO4 (0.4 mol/L), 0.5 mL DTNB (0.4 g/L) and the supernatant 2 mL for reaction time 1 min. One unit of GPX activity was defined as the decrease of GSH content at 1 μM, by excluding non-enzymatic reaction. GST activity was measured according to the method of Habig and Jakoby (1981) by evaluating the conjugation of GSH with the standard model substrate 1-chloro-2, 4-dinitrobenzene (CDNB). The total volume of reaction mixture was 0.9 mL, including 0.3 mL distilled water, 0.2 mL CDNB (15 mM), 0.2 mL GSH (15 mM) and enzyme extract 0.2 mL. GST activity was expressed by the reaction product of 2, 4-dinitrophenyl glutathione at 340 nm absorbance. GSH contents were detected according to the method of GPX. 2.6. Flow cytometric assays

and finally cells were resuspended with 1 mL PBS (0.1 M, pH 7.8). The fluorescence intensity was monitored by flow cytometry (BD, USA) using an excitation wavelength of 485 nm and an emission wavelength of 530 nm. The mitochondrial membrane potential was measured by the incorporation of a cationic fluorescent dye Rh123 (Liu et al., 2008). The amount of fluorescent dye uptake by cells depended on mitochondrial membrane potential. Rh123 (final concentration in the mixture was 26 μM) was added to the cells suspended in 1 mL PBS (0.1 M, pH 7.8) and the mixture was incubated in an incubator at 20 °C in the dark for 30 min. Then cells were immediately washed three times with PBS (0.1 M, pH 7.8) and finally cells were resuspended with 1 mL PBS. The fluorescence intensity was monitored by flow cytometry (BD, USA) using an excitation wavelength of 480 nm and an emission wavelength of 530 nm.

2.7. RNA extraction, reverse transcription and real-time analysis Ten milliliters of algal culture media was centrifuged at 10000 rpm for 10 min at 4 °C. Cell pellets were frozen at −80 °C until RNA extraction. Total RNA was extracted using the RNAiso kit (TakaRa Company, Dalian, China). According to the method of Qian et al. (2010), reverse transcript

A 60

Agal Density (x105 cells/mL)

18

The permeability of cell membrane was determined by using a fluorescent probe (FDA), according to Vigneault et al. (2000), with slight modifications. FDA (final concentration in the mixture was 6 μM) was added to the cells suspended in 1 mL 0.1 M PBS (pH 7.8) and the mixture was incubated in an incubator at 20 °C in the dark for 10 min. Then cells were immediately washed three times with PBS (0.1 M, pH 7.8)

Control 0.1 uM 0.2 uM 0.3 uM 0.4 uM 0.5 uM 0.6 uM 0.7 uM

50 40 30 20 10 0 0

1

2

3

4

5

6

7

**

**

6

7

Time (d)

B 0.5

Table 1 Sequences of primer pairs in P. tricornutum for real-time PCR. Primers (sense and anti-sense)

18SrRNA Forward 5′-ggtaattccagctccaatagc-3′ Reverse 5′-gatggcggacccaaaacag-3′ psbA Forward 5′-caggtgtattcggtggttctttattct-3′ Reverse 5′-cgagagttgttaaatgaagcgtattgg-3′ psaB Forward 5′-caggtcgtggtggtacttgtgatat-3′ Reverse 5′-cgtagccagcccatgatgtaatttg-3′ Cob Forward 5′-ttgaaactggacaaatagccacactc-3′ Reverse 5′-tgcccaacagtaggaattaagataagg-3′ nad1 Forward 5′-cagaagcagaagcagaattggtatcg-3′ Reverse 5′-gaaacaaccaccctcccagaaaga-3′

Transcription product Internal reference

Photosystem II protein D1

Photosystem I P700 Chlorophyll a apoprotein A2

specific growth rate (u)

Gene name

* **

0.4

** 0.3

0.2

0.1

Cytochrome b

0.0 NADH dehydrogenase subunit 1

0

1

2

3

4

5

HQ concentration (0.1 uM) Fig. 1. Effects of HQ on the growth (A) and specific growth rate (B) of P. tricornutum. All error bars indicated SE of the three replicates. * (p b 0.05) and ** (p b 0.01) indicated significant differences compared to the corresponding controls without HQ.

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1.2

remained lower than that of the control. When algae were treated with HQ (1, 2,3, 4, 5, 6 and 7 × 10 −7 mol/L) for 96 h, the growth inhibition (I) were 18.6%, 22.6%, 31.5%, 54.5%, 68.5%, 83.7% and 90.2%, respectively. Besides, the results of specific growth rate also indicated that HQ inhibited the growth of P. tricomutum (Fig. 1B). However, lower concentrations of HQ (1 and 2 × 10 −7 mol/L) did not significantly decrease the specific growth rate of algal cells compared to that of control and only when algae were treated with higher HQ concentrations (above 3 × 10 −7 mol/L), where the specific growth rate obviously inhibited versus that of control (p b 0.05 or p b 0.01). To reflect the inhibition effects of HQ on the growth of P. tricornutum, EC50 was calculated by logistic curve fitting equation by selecting the different exposure time (Fig. 2). The EC50, 24h of HQ on P. tricornutum was 1.86 × 10 − 7 mol/L (p = 0.0001), and for 48 h, 72 h, and 96 h, they were 3.16 × 10 − 7 mol/L (p b 0.0001), 3.51 × 10 − 7 mol/L (p = 0.0001) and 3.74 × 10 − 7 mol/L (p b 0.0001), respectively.

Algal density (100%)

1.0

.8

.6

.4

.2

24 h 48 h 72 h 96 h

0.0 0

1

19

2

3

4

5

6

7

3.2. Effects of HQ on cellular pigments in P. tricornutum

HQ concentration (0.1 u M) Fig. 2. Inhibitory effect of HQ on the growth of P. tricornutum. EC50, 24h was 1.86 × 10−7 mol/L (p = 0.0001); EC50, 48h was 3.16 × 10−7 mol/L (p b 0.0001); EC50, 72h was 3.51 × 10−7 mol/L (p = 0.0001); EC50, 96h was 3.74 × 10−7 mol/L (p b 0.0001). All error bars indicated SE of the three replicates.

and real-time PCR analyze for two target photosynthesis-related genes (psaB, psbA) and two target respiration-related genes (nad1, cob). Primer pairs for psaB, psbA, nad1, and cob are listed in Table 1. The following PCR protocol was used with two steps: one denaturation step at 95 °C for 10 s and 40 cycles of 95 °C for 5 s, followed by 60 °C for 30 s. 18 S rRNA was used as a housekeeping gene to normalize the expression changes. The relative gene expression among the treatment groups was quantified by the 2−ΔΔCt method (Livak an3d Schmittgen, 2001).

The contents of cellular pigments including Chl.a and carotenoid were significantly decreased after HQ exposure at concentrations above 4 × 10−7 mol/L (p b 0.01) (Table 2). With increasing exposure time to HQ, the increase in the contents of Chl.a and carotenoid was observed. Although the contents of Chl.a and carotenoid were decreased by HQ compared to the control with increasing exposure concentration, the ratio of carotenoid to Chl.a remained little change between the treatment groups and control. When algal cells were exposed to HQ for 72 h, the higher the HQ concentrations (6 and 7 × 10−7 mol/L), the more obvious its inhibition effects on the ratio of carotenoid to Chl.a (p b 0.01). While for 144 h exposure, the ratio showed significant increase only under HQ of 2 × 10−7 mol/L, and the ratios of other treatment groups kept almost no difference from that of the control. 3.3. Effects of HQ on SOD, CAT, GPX, GST activities and GSH contents in P. tricornutum

2.8. Statistics All data shown in this study were the means ± SE of three replicates and were evaluated by using one-way analysis of variance (ANOVA) followed by least significant difference test (LSD), p b 0.01 and p b 0.05 (Origin 7.5 for Windows). 3. Results 3.1. Effects of HQ on the growth of algal cells Fig. 1 showed the effects of HQ on the growth and specific growth rate of Phaeodactylum tricomutum. The results demonstrated that HQ inhibited algal growth and the inhibition effect increased with increase in HQ concentrations. Although algal cell densities of all treatment groups gradually increased with the time (Fig. 1A), they

Cellular enzymatic activities including SOD, CAT, GR, GPX, GST and non-enzymatic antioxidant GSH contents were shown in Fig. 3. The activity of SOD increased gradually with the treated concentrations of HQ increasing after 48 h exposure. The significant increases in SOD activities were observed at concentrations 5 and 7 × 10−7 mol/L of HQ for 48 h exposure (p b 0.01). The activity values were 9.66 times and 12.09 times of the control, respectively. However, with increasing exposure time to HQ, SOD activities decreased, and after 96 h exposure, all treatment groups showed no significant difference from that of the control. Different from SOD, CAT activities showed a peak with the increase of treatment concentrations when algal cells were treated with HQ for 48 h. A significant increase in CAT activity was observed at concentration of 5 × 10 −7 mol/L after HQ exposure. The values of CAT activities were 1.51 times (p b 0.05), 7.98 times

Table 2 Effect of HQ on cellular pigments of P. tricornutum. HQ concentration (10−7 mol/L)

Chl.a (μg/L) 72 h

0 1 2 3 4 5 6 7

0.62 0.56 0.57 0.51 0.32 0.31 0.15 0.12

Carotenoid (μg/L) 144 h

± ± ± ± ± ± ± ±

0.04 0.01 0.14 0.10 0.04⁎⁎ 0.05⁎⁎ 0.03⁎⁎ 0.07⁎⁎

All data indicated the mean ± SE of the three replicates. ⁎ p b 0.05 indicated significant differences. ⁎⁎ p b 0.01 indicated significant differences.

1.81 1.45 1.36 1.25 0.91 0.88 0.47 0.43

± ± ± ± ± ± ± ±

72 h 0.25 0.42 0.79 0.33 0.14⁎⁎ 0.07⁎⁎ 0.10⁎⁎ 0.14⁎⁎

0.35 0.29 0.30 0.29 0.19 0.19 0.11 0.11

Carotenoid/Chl.a 144 h

± ± ± ± ± ± ± ±

0.03 0.04 0.07 0.05 0.03⁎⁎ 0.03⁎⁎ 0.03⁎⁎ 0.06⁎⁎

1.04 0.88 0.95 0.86 0.63 0.61 0.39 0.39

± ± ± ± ± ± ± ±

72 h 0.21 0.21 0.46 0.36 0.09⁎ 0.08⁎ 0.08⁎⁎ 0.13⁎⁎

0.57 0.52 0.58 0.57 0.60 0.62 0.81 1.28

144 h ± ± ± ± ± ± ± ±

0.04 0.07 0.04 0.10 0.09 0.11 0.09⁎ 0.32⁎

0.58 0.60 0.90 0.52 0.69 0.70 0.83 0.77

± ± ± ± ± ± ± ±

0.11 0.14 0.03⁎ 0.09 0.10 0.10 0.17 0.17

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(p b 0.01) and 2.36 times respectively (p b 0.01) versus that of the control with increasing exposure concentrations from 3 to 7 × 10−7 mol/L. GPX activities in algal cells significantly increased with the treated concentrations increasing (p b 0.01) after 48 h exposure to HQ, and the values after 48 h exposure were obviously higher than that of 96 h exposure during all the experiment, which was similar to that of SOD and CAT. The effects of HQ on GST activities followed a similar manner as that of GPX (Fig. 3D). When algal cells were exposed to HQ for 48 h, GST activities also increased remarkably with the treated concentrations increasing. The activity values were 1.83 times (p b 0.05),

16.31 times (p b 0.01) and 12.07 times (p b 0.01) respectively than that of the control. But after 96 h exposure, the activities of GST showed only a slightly increase with increasing the treatment concentrations and all the treatment groups were not significantly different from the control except the highest HQ concentration exposure (7 × 10 − 7 mol/L). GSH was one of the important non-enzymatic antioxidants, protecting cells against the external stress. Different from the above activities of antioxidant enzymes, intracellular GSH contents did not change significantly between the treatment groups and the control. Only after 96 h exposure

c

A

e

B

c

0h 48 h 96 h

10

CAT activity (U/105 cells)

SOD activity (U/106 cells/mL)

12

8 6 4 2

a

a

a

0h 48 h 96 h

60

40

af 20

a

a

80

b

0

0

3

5

c 0

7

h

g

0

3

5

7

HQ concentration (0.1 uM)

HQ concentration ( 0.1uM)

C

ad

a c

b

b

a

b

a b

a

a

d 8

d

D

d

7

0h 48 h 96 h

20

GST activity (U/106 cells)

GPX activity (U/104 cells)

25

15

10

a

a

5

a

a

0

0

h

g

f 3

5

4 3 2 1

a

HQ concentration (0.1 uM)

b 0

a

a c 3

f

ef

e

e 0

7

d

5

a

b

5

7

HQ concentration (0.1 uM)

E GSH content (umol/106 cells)

6

c e

0h 48 h 96 h

b

0h 48 h 96 h

0.06

ab

ab 0.04

bc abd

a

abd

abd

abd

a 0.02

ad

d 0.00

0

3

5

7

HQ concentration (0.1 uM) Fig. 3. Effect of HQ on the activities of SOD, CAT, GPX, GST and GSH contents of P. tricornutum. All error bars indicated SE of the three replicates. Letters represent whether there are differences between values (p b 0.05).

C. Yang et al. / Journal of Experimental Marine Biology and Ecology 444 (2013) 16–23

to HQ, the significant increases in GSH content were observed at concentrations of 5 and 7 × 10−7 mol/L (p b 0.05). 3.4. Effects of HQ on membrane systems in P. tricornutum Effects of HQ on membrane systems including cellular membrane permeability and mitochondrial membrane potential were shown in Fig. 4. As can be seen, HQ increased the permeability of cellular membrane in P. tricornutum. The significant increases were observed in concentrations of 1 and 5 × 10−7 mol/L after 48 h exposure. When algal cells were exposed to HQ for 96 h, the permeability significantly increased with the HQ concentrations increasing except the highest concentration (7 × 10 −7 mol/L) exposure. The values representing the permeability were 1.82 times, 2.61 times, 3.27 times and 2.08 times of the control at the concentrations of 1, 3, 5 and 7 × 10−7 mol/L, respectively. The mitochondrial membrane potential did not follow a regular trend. After 48 h exposure, the mitochondrial membrane potential was significantly decreased after HQ exposure at the concentration of 3 × 10−7 mol/L. When algal cells were exposed to HQ for 96 h, lower HQ concentrations (below 7 × 10−7 mol/L) slightly decreased the potential, while higher HQ concentration (7 × 10−7 mol/L) resulted in a significant increase in mitochondrial membrane potential (p b 0.05).

21

energy for microcystin synthesis. Since the given low doses were the same, it seemed that cells treated with HQ needed to harvest more light for resisting the extreme stress by altering the ratios of pigments, which might act as a strategy for algal cells against environmental stress. In general, a literature survey reveals that exogenous substances induce cellular antioxidant responses to different organisms. For example, Zhang et al. (2011) found that berberine isolated from golden thread significantly increased SOD activities after M. aeruginosa exposure for 24 h and with time extending, SOD activities obviously decreased versus that of control, while GSH contents remained continually increasing with exposure concentrations and times increasing. Our results were similar to the report above that SOD activities decreased with increasing the exposure time. Other enzyme activities including CAT, GPX and GST showed the same tendency as SOD and increased with increasing HQ concentrations and decreased with exposure times extending. However, changes in GSH contents are not significant, which implied that GSH might not be a major antioxidant to resist HQ stress on P. tricornutum. This discovery precisely echoed our previous reports that GSH system seemed not to be sensitive to exogenous stress compared to that of antioxidant enzymes (Yang et al., 2011). All of these results showed that HQ induced oxidative stress on marine diatom and cells regulated antioxidant enzyme activities to remove the stress.

3.5. Effect of HQ on the transcription of photosynthesis and respiration related gene

A 300

FDA fluorescense (%)

HQ greatly affected the transcription of photosynthesis and respiration related genes (Fig. 5). HQ at 0.3 μM inhibited the transcription of psbA by 0.4-fold compared to the control after 24 h exposure, but it stimulated the transcription after 48 h exposure. The transcription of psaB was stimulated by 0.3 μM HQ after 24 h exposure, but it significantly inhibited the transcription after 48 h exposure. Other higher concentrations of HQ (0.5, 1 μM) obviously inhibited the transcription of photosynthesis related gene (psbA and psaB) after 24 h and 48 h exposure. The transcription of respiration related genes (nad1 and cob) was stimulated by 0.3 μM HQ after 24 h exposure, and with the increase of HQ concentration, the transcription level decreased. HQ at 1 μM significantly inhibited the transcription of nad1 and cob after 24 and 48 h exposure.

48 h 96 h **

250

200

** 150

*

*

100

4. Discussion

0

1

2

3

4

5

6

7

HQ concentration (0.1 uM)

B

*

140

Rho123 fluorescense (%)

In present work, we studied the effect of allelochemical HQ on marine diatom P. tricornutum and confirmed that HQ significantly inhibited the growth of marine diatom and induced changes in cellular physiological levels. To further reflect the inhibition effect of HQ on marine algae, we calculated the values of EC50 with the results being shown in Fig. 2, which demonstrated that the values increased with increasing exposure time. In the meantime, the algal growth was reestablished, which suggested that cells gradually adapted to HQ stress by changing the intracellular metabolism or transfer HQ into non-toxic substance. Similar patterns could also be found in other exogenous substances on the growth of algae (Hong et al., 2009; Qian et al., 2010), for which exogenous substances only extended its lag phase of growth, and with treated time increasing, algae gradually returned to normal proliferation. Cellular pigment is another indicator reflecting algal cell growth. Our results showed that the contents of Chl.a and carotenoids decreased with HQ concentration increasing, which also showed concentrationdependent inhibitory effects of HQ on algae. However, the ratios of carotenoids to Chl.a only changed remarkably at relatively higher HQ concentration exposure in our experiment. The photosynthetic pigments are of consequence due to their low harvesting function. Y. Xiao et al. (2010) reported that M. aeruginosa adjusted their complement of pigments upward to increase low harvesting efficiency and thereby consumed high

**

48 h 96 h

120 100 80 60 40

*

20 0

1

2

3

4

5

6

7

HQ concentration (0.1 uM) Fig. 4. Effects of HQ on membrane systems in P. tricornutum. A: cellular membrane permeability; B: mitochondrial membrane potential. All error bars indicated SE of the three replicates. * (p b 0.05) and ** (p b 0.01) indicated significant differences.

22

C. Yang et al. / Journal of Experimental Marine Biology and Ecology 444 (2013) 16–23

psbA

1.6

1.2

Control 0.3 uM 0.5 uM 1 uM

0.8 **

**

0.4 **

**

**

Relative transcription abundance

Relative transcriptional abundance

** **

psaB

3.2

Control 0.3 uM 0.5 uM 1 uM

2.4

1.6

0.8

**

* *

**

0.0

0.0 24h

48h

24h

48h

Time

**

nad1

2.4

Control 0.3 uM 0.5 uM 1 uM

1.6

0.8

* **

Relative transcriptional abundance

Time

Relative transcriptional abundance

**

cob Control 0.3 uM 0.5 uM 1 uM

1.2

0.8

**

**

0.4

**

**

0.0

0.0 24h

48h

24h

Time

48h

Time

Fig. 5. Effect of HQ on the transcription of photosynthesis (psbA, psaB) and respiration (nad1, cob) related genes in P. tricornutum. All error bars indicated SE of the three replicates. * (p b 0.05) and ** (p b 0.01) indicated significant differences.

The selected permeable cell membrane forms the first barrier that separates the cells from exogenous substances exposure. The permeability status of the cell membrane could play an important role in mediating the adverse effects of environmental stress. Our results showed that HQ increased the cell membrane permeability of P. tricornutum, which might be one of the important reasons for inhibiting cell growth. Liu et al. (2008) have ever reported that perfluorinated alkyl acids inhibited the growth of C. vulgaris, and increased cell membrane permeability and the enhancement of permeability could pose a serious risk to single-celled alga that is exposed directly to other more toxic pollutants in the environment. Mitochondrial membrane potential is involved in a number of cellular function and plays central roles in the oxidative energy metabolism as well as apoptosis by integrating death signals (Brenner and Kroemer, 2000; Lee et al., 2004). Moreover, Liu et al. (2008) also reported that perfluorinated alkyl acids increased mitochondrial membrane potential. However, in our experiment, mitochondrial membrane potential in the algae did not change in regular, which indicated that the inhibition of HQ on algae might not be enough to cause changes in mitochondrial membrane potential. Therefore, cellular apoptosis was not found in the following experiment by assaying with flow cytometry (unpublished data). Flow cytometry has been widely applied to study the effects of exogenous substances on animal cellular suspensions. However, few literatures have referred to using a flow cytometric technique to detect the physiological and molecular biological indicators on algae, especially on marine algae. And most of them are only related to freshwater algae, for example, Rioboo et al. (2009) reported that terbutryn, a triazine herbicide, inhibited the proliferation of freshwater C. vulgaris

by using flow cytometry. As far as we know, our study was the first to detect the characteristics of cell membrane of marine diatom. HQ also affected the transcription of photosynthesis and respiration related genes. With the increase of HQ concentration, the transcription abundance decreased significantly. These results showed that HQ might interfere with the function of photosynthetic and respiratory electron transport chain, which was similar to the reports of Qian et al. (2009, 2010) that N-phenyl-2-naphthylamine as a kind of allelochemical inhibited the transcription abundance of photosynthetic genes. So it can influence the growth and pigment contents of algae indirectly. The present results showed that HQ inhibited the growth of P. tricornutum and decreased cellular pigment contents and the transcription of photosynthesis and respiration related genes. At the same time, HQ altered cellular membrane system, induced oxidative stress. To suppress the HQ stress, cellular antioxidant enzymes and non-enzymatic antioxidant were activated, which suggested that one of the allelopathic mechanisms responsible for the inhibition was related to HQ-induced oxidative damage in P. tricornutum.

Acknowledgments The work was financially supported by the National Science and Technology Support Program (2011BAC02B04), the Important Directional Projects in the Knowledge Innovation Program of the Chinese Academy of Sciences (KZCX2-YW-Q07-04), and Yantai Science and Technology Development Project (2011063).

C. Yang et al. / Journal of Experimental Marine Biology and Ecology 444 (2013) 16–23

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