Our previous work revealed that Acacia mearnsii extract can inhibit the growth of Microcystis aeruginosa, the common species forming toxic cyanobacterial blooms in eutrophic freshwater. In the present study, we demonstrated that this plant extract can significantly increase cell membrane permeability and Ca2+/Mg2+-ATPase activity on the membrane. Long-term exposure to concentrations of 20 ppm A. mearnsii extract led to algal cell membrane leakage or even lysis. Comparison of expression of three photosynthesis-related genes (rbcL, psaB and psbD) in M. aeruginosa with and without plant extract treatment revealed that their expression was remarkably reduced in the presence of the extract. Down-regulation of photosynthesis-related genes could indicate the inhibition of the photosynthetic process. Thus, our results suggested that both photosynthetic systems and membranes of M. aeruginosa are potentially damaged by A. mearnsii extract.

INTRODUCTION

Recently, rapid economic and population growth have intensified the process of eutrophication in freshwater lakes and estuaries worldwide. This is primarily due to the fact that accelerated industrialization and the agricultural modernization process have greatly increased inputs of nitrogen, phosphorus and other nutrients into water bodies. Consequently, eutrophic water bodies result in algal blooms, which damage the sustainability of aquatic ecosystems (Carey et al. 2012; Michalak et al. 2013). Microcystis aeruginosa, a main bloom-forming species, secretes toxins in the water, thereby threatening public health (Paerl & Paul 2012). Thus, effective prevention and control of M. aeruginosa blooms has been a major concern of environmental research in the last decade (Ni et al. 2012).

So far, physical, chemical, biological and engineering repair approaches have been taken to inhibit M. aeruginosa growth in recent years. Compared with chemical and physical methods, natural plant extracts, due to their effective inhibition, low cost, natural availability, no secondary pollution and other advantages, have received much attention (Shao et al. 2013). Recent works have shown that many plant agents (allelochemicals) can effectively inhibit M. aeruginosa blooms (Yang et al. 2009; Ó hUallacháin & Fenton 2010; Shao et al. 2013). Our previous work also demonstrated that the growth of M. aeruginosa can be successfully repressed by A. mearnsii extract (Zhou et al. 2012).

Currently, explanations of the inhibitory mechanism on M. aeruginosa have focused primarily on the oxidative damage caused by oxidized polyphenolics (Shao et al. 2010). Although they may be the most useful explanation, few studies have investigated the physiological responses of cyanobacteria, such as photosynthesis, to these plant extracts. Chlorophyll a is the major constituent of the primary reactions of oxygenic photosynthesis in land plants and green algae (Tanaka & Tanaka 2011). In photosystem II (PSII), chlorophyll a captures the energy of sunlight to excite electrons, which are then transferred from cytochrome to photosystem I (PSI), generating nicotinamide adenine dinucleotide phosphate (NADPH) (Li et al. 2000). The gene psaB (PSI P700 chlorophyll a apoprotein A2) is expressed during electron transfer from plastocyanin to the PSI acceptor side, acting as one of the reaction center subunits of PSI (Sommer et al. 2002). The gene psbD encodes the PSII reaction center chlorophyll protein D2, which is the rate-determining subunit for the assembly of PSII. The gene rbcL encodes the large subunit of a key enzyme, rubisco, in the Calvin cycle (Chen & Melis 2013). Therefore, it is imperative to determine if there are molecular or protein changes in algal cells in the presence of A. mearnsii extract.

To explore the inhibitory mechanism of the A. mearnsii extract on M. aeruginosa further, we examined the physiological response (chlorophyll a content, cellular structure and Ca2+/Mg2+-ATPase activity) and gene expression (photosynthesis-related genes of rbcL, psaB and psbD) of M. aeruginosa in the presence of A. mearnsii extract.

METHODS

Preparation of A. mearnsii extract

The A. mearnsii extract was obtained by using water to leach the materials from black wattle bark, followed by evaporation. Polyphenol content in the extract is about 75%, which was determined according to methods developed by Claderaforteza et al. (1995).

Algal strains and culture

The strain M. aeruginosa (FACHB 942) was purchased from the Freshwater Algae Culture Collection of the Institute of Hydrobiology, Chinese Academy of Sciences (Wuhan, China). This cyanobacteria was incubated in BG11 medium (Stanier et al. 1971) at 25 °C under 140 μmol photons m−2 s−1 of illumination intensity with a photoperiod of 12:12 h (light:dark).

Assessment of the antialgal activities of extract

Algicidal efficacy was evaluated by the US Environmental Protection Agency standard method (Weber et al. 1989) with some modifications. Before the experiment, the algae were inoculated to ensure they were in the exponential growth phase and at initial densities of 105 cells mL−1. The extract was added to nine flasks to achieve three cultures with concentrations of 0, 10 and 20 ppm (three replicates each) and maintained in culture under the conditions described above. Algal growth was monitored with a microscope and hemocytometer by counting cell numbers daily for 5 days.

Chlorophyll a concentration of cell suspensions was determined spectrophotometrically after extraction with 90% methanol (Jeffrey & Humphrey 1975). Firstly, each M. aeruginosa culture was centrifuged, and the pellet was washed twice with distilled water. After suspension in 90% acetone-distilled water (v/v), each pellet was then sonicated to break the cell walls and extracted for 24 h in the dark at 4 °C. Finally, the suspension was centrifuged, and chlorophyll a content in the supernatant was measured by absorbance. Three replicates of each treatment were analyzed for each experiment.

Assay of membrane permeability and Ca2+/Mg2+-ATPase activity

Membrane permeability was assayed according to Huang et al. (2013). After incubation with A. mearnsii extract, cells of M. aeruginosa were centrifuged for 15 min at 3500 × g. Collected cells were then washed with distilled water three times to remove medium contamination, after which they were suspended in ultrapure water for 2 h. Next, the cell suspension was centrifuged for 15 min at 3500 × g, and its absorbance at 264 nm was measured in the clear supernatant. Ca2+/Mg2+-ATPase activity was measured using the Kits from Nanjing Jiancheng Biological Engineering (China, Nanjing). Mean values were calculated from three independent replicates of three groups of samples.

Transmission electron microscopy (TEM) analysis

A transmission electron microscope Philips CM12 (Philips, Eindhoven, The Netherlands) was used to assess changes in cellular structure caused by A. mearnsii extract. A standard technique was used for preparing samples and performing electron microscopy (Shao et al. 2011).

Real-time polymerase chain reaction (PCR)

For the determination of gene expression, M. aeruginosa was exposed to 10 and 20 ppm A. mearnsii extract for 1, 2, 3 and 4 days, respectively. After the incubation period, 20 mL of algal culture were centrifuged at 10,000 × g for 5 min at 4 °C to collect cells. Total RNA was extracted using TRIZOL reagent (Invitrogen, Carlsbad, CA, USA) following manufacturer instructions. Each cDNA was synthesized using 1 μg of total RNA with the PrimeScriptTM RT reagent Kit with gDNA Eraser (TaKaRa Bio, Dalian, China). Real-time PCR was performed on a CFX Connect Real-Time PCR Detection System (Bio-Rad) using iQ SYBR® Green Supermix (Bio-Rad, Hercules, CA, USA). Data were obtained using the CFX Manager software (Bio-Rad). Primers for rbcL, psaB, psbD and 16s rRNA were designed in previous studies (Qian et al. 2012). All quantitative reverse transcriptase PCR (qRT-PCR) experiments were performed with three technical replicates and three independent biological repetitions; representative results were shown.

RESULTS

Algicidal effect of A. mearnsii extract on the growth of M. aeruginosa

Two concentrations (10 and 20 ppm) of A. mearnsii extract were prepared in the culture medium to evaluate its algicidal activity. Over 5 days, cell density increased from 3.6 × 105 to 3.1 × 106 in the control, significantly more than when A. mearnsii extract was added. These results showed clear inhibition of M. aeruginosa growth in the presence of the extract; moreover, 20 ppm had a stronger effect than 10 ppm (Figure 1(a)). On the other hand, the results of chlorophyll a measurement also indicated a similar inhibitory pattern of photosynthetic activity to that of cell density. A small decrease in chlorophyll a occurred after one day in culture with the extract. By the fourth day of treatment, chlorophyll a content was about 85% and 51% of that of the control in the 10 and 20 ppm treatments, respectively (Figure 1(b)).

Figure 1

Effects of different concentrations of A. mearnsii extract on the inhibition of growth of M. aeruginosa. (a) Changes in cell density. (b) Changes in chlorophyll a concentration. The error bars are the mean ± standard deviation.

Figure 1

Effects of different concentrations of A. mearnsii extract on the inhibition of growth of M. aeruginosa. (a) Changes in cell density. (b) Changes in chlorophyll a concentration. The error bars are the mean ± standard deviation.

Effects on cellular microstructure

TEM images illustrated the direct damage of the extract on algal cell structure. Before treatment, algal cells were plump and spherical shapes with a smooth exterior (Figure 2(a)). However, after adding A. mearnsii extract, the cellular membrane and internal structures began to appear damaged, and after 2 days exposure to 20 ppm extract, the internal structures were obviously depressed or distorted (Figure 2(b)). As exposure time extended to 4 days, more serious cellular damage was observed as some cells were ruptured and even lysed with holes emergence (Figure 2(c)).

Figure 2

Ultrastructure of M. aeruginosa incubated with 20 ppm A. mearnsii extract. (a) Control; (b) after 2 days; (c) after 4 days.

Figure 2

Ultrastructure of M. aeruginosa incubated with 20 ppm A. mearnsii extract. (a) Control; (b) after 2 days; (c) after 4 days.

Effects on membrane and Ca2+/Mg2+-ATPase activity

To further confirm the effect of extract on cell viability, a membrane leakage assay was conducted. During the treatment of the extract, algal membrane permeability firstly increased followed by subsequent reduction. Moreover, membrane permeability increased more rapidly with treatment of the 20 ppm extract than with 10 ppm (Figure 3(a)). Because variation in membrane permeability may result in the change of membrane ATPase activity, Ca2+/Mg2+-ATPase activity was measured and was found to be influenced by the plant extract in a strongly time-dependent and dose-dependent remarkable manner. Compared with controls, Ca2 +/Mg2+-ATPase activity was obviously increased when treated with the extract. At the fourth day of treatment, the activity went up to 36% and 230% relative to the control in the 10 and 20 ppm extract, respectively (Figure 3(b)).

Figure 3

Effects of different concentrations of A. mearnsii extract on (a) membrane permeability and (b) Ca2+/Mg2+-ATPase activity. The error bars are the mean ± standard deviation. The single and double asterisks represent significant difference determined by the Student's t test at p < 0.05, p < 0.01, respectively.

Figure 3

Effects of different concentrations of A. mearnsii extract on (a) membrane permeability and (b) Ca2+/Mg2+-ATPase activity. The error bars are the mean ± standard deviation. The single and double asterisks represent significant difference determined by the Student's t test at p < 0.05, p < 0.01, respectively.

Effects on gene expression profiles

To explore the potential targets of the inhibitory process of extract on M. aeruginosa, the expression of photosynthesis-related genes of rbcL, psaB and psbD was analyzed. The expression of both rbcL and psbB decreased significantly after 24 h extract treatment and correlated positively with the extract concentration and exposure time (Figure 4). Expression of psaD was slightly reduced after 24 h exposure, but decreased significantly as exposure time extended to 48 and 72 h.

Figure 4

Expression of psaB, psbD and rbcL in M. aeruginosa treated with different concentrations of A. mearnsii extract. 16s rRNA was used as the endogenous control. Relative amounts of treatment samples were normalized with respect to the control.

Figure 4

Expression of psaB, psbD and rbcL in M. aeruginosa treated with different concentrations of A. mearnsii extract. 16s rRNA was used as the endogenous control. Relative amounts of treatment samples were normalized with respect to the control.

DISCUSSION

This study investigated the inhibitory mechanisms of A. mearnsii extract on M. aeruginosa. TEM analysis revealed that consistent with previous reports (Jachlewski et al. 2013; Pakrashi et al. 2013), the extract-induced changes in cell membrane and internal structures ultimately resulted in membrane rupture and organelle destruction (Figure 2). Membrane rupture and organelle destruction could reflect local weakness or damage in the cell membrane, which led to altering membrane permeability (Germain et al. 2007), as the extract was shown to increase cell membrane permeability (Figure 3(a)). Previous studies have also shown that treatment with some chemicals resulted in both damage to cellular structure and the increase of membrane permeability (Hong et al. 2008; Kong et al. 2013). Increased membrane permeability may be due to the serious damage that the extract caused to cell membrane structure, inducing leakage of intracellular matter and even causing cell death (Sreedharan et al. 2013). Furthermore, in view of the change in membrane permeability, we examined Ca2+/Mg2+-ATPase activity on the membrane. Exposure to A. mearnsii extract increased Ca2+/Mg2+-ATPase activity of M. aeruginosa (Figure 3(b)). Similar variation in ATPase activities has also been found when the cyanobacteria or algae are exposed to stress (Saxena et al. 2002; Chen et al. 2008). Ca2+/Mg2+-ATPases act as carrier enzymes that maintain intracellular ion gradients, and changes in these gradients modulate signal transduction to regulate the rate of protein synthesis and cell growth (Pande et al. 2005). Hence, these studies indicate that the extract inhibits growth of M. aeruginosa by targeting its cell membranes.

Apart from the morphology changes, physiological effects of extracts on M. aeruginosa were also found. Chlorophyll a content in the treated cells was less than in the control, and there was a significant dose effect on the degree of inhibition (Figure 1(b)). It was reported that the chlorophyll a content decreased when cyanobacterial growth was inhibited by plant extracts (Park et al. 2006; Shao et al. 2010). In light of the decrease in chlorophyll a, we examined changes in the expression of photosynthesis-related genes. qPCR demonstrated decreased expression of psaB, psbD and rbcL after exposure to the extract. This finding was in accordance with the conclusion that they are sensitive to environmental stress (Qian et al. 2012; Zhang et al. 2013). The products of these genes participate in electron transport; thus, their expression inhibition may block electron transport. In other words, down-regulated of transcription could cause the insufficiency of PsaB, PsbD and RbcL, which results in decrease of energy production. Therefore, negative impacts on the reaction center of photosynthesis may be another imperative reason for the toxicity of A. mearnsii extract on M. aeruginosa.

In conclusion, this study demonstrated toxicity of A. mearnsii extracts on M. aeruginosa by inhibition of algal growth. Two concentration extracts induced severe damage to cellular structure, altered cell membrane permeability and affected mRNA expression of photosynthesis-related genes. Thus, by demonstrating the effective inhibition of M. aeruginosa growth, our results indicate that extracts of A. mearnsii may be a useful agricultural byproduct for controlling cyanobacterial blooms.

ACKNOWLEDGEMENT

This work was supported by grants from the National Natural Science Foundation of China (Nos 51109147 and 31300996).

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