Influence of linoleic acid on growth, oxidative stress and photosynthesis of the cyanobacterium Cylindrospermopsis raciborskii

ABSTRACT

In this study, the effects of linoleic acid on the growth, photosynthetic activities and oxidative system of the invasive cyanobacterium Cylindrospermopsis raciborskii were investigated. The results indicated that linoleic acid decreased the growth rate and chlorophyll a concentration in C. raciborskii after 48 h incubation. The maximum photochemical efficiency (F_v/F_m) of photosystem II (PSII) and maximum relative electron transport rate (ETR_max) decreased significantly at concentrations higher than 1 mg L⁻¹. However, non-photochemical quenching (NPQ) significantly increased when cultured at concentrations of 0.5, 1, 2 and 4 mg L⁻¹. PSII electron transfer rate and chlorophyll a fluorescence transients appeared to be affected after the cells were exposed to higher concentrations of linoleic acid; the density of the reaction centres (RCs), quantum energy flux ratios and the performance index on absorption basis decreased, but absorption flux per RC and dissipated energy flux per RC increased. Malondialdehyde content, superoxide dismutase and catalase activities were enhanced when exposed to 2 and 4 mg L⁻¹ of linoleic acid. This suggests that the blockage of electron transport on the donor and acceptor sides of PSII is the mechanism responsible for the toxicity of linoleic acid in PSII of C. raciborskii.

KEYWORDS:

• Allelopathic
• Cylindrospermopsis raciborskii
• linoleic acid
• JIP test
• PSII

Introduction

The cyanobacterium Cylindrospermopsis raciborskii is a nitrogen-fixing filamentous species found in many eutrophic freshwater lakes and reservoirs worldwide (Padisák 1997). It has been described as an invasive species because of its increased occurrence in temperate regions (Sinha et al. 2012). This causes many problems, such as oxygen depletion, deterioration in water quality and decline in water transparency (Paerl et al. 2001). Moreover, C. raciborskii produces diverse toxins such as cylindrospermospin and paralytic shellfish poisons that are harmful to zooplankton, fish, shellfish and humans (Ohtani et al. 1992; Lagos et al. 1999; Fastner et al. 2003). Therefore, control of Cylindrospermopsis blooms is crucial for the maintenance of water supply safety.

Previous studies have demonstrated that chemical algicides (Hrudey et al. 1999) can efficiently control cyanobacterial blooms. However, subsequent secondary pollution was observed to influence the ecosystem of the water bodies due to a toxic to other aquatic biology via the algicides or their degradation products (Anderson 1997). Therefore, biological control was considered a promising strategy for controlling cyanobacterial blooms (Choi et al. 2005). Recently, aquatic macrophytes have been proven to produce many algicidal compounds such as eugenin (Saito et al. 1989), ellagic acid (Nakai et al. 2000), nonanoic acid (Nakai et al. 2005), pyrogallic acid (Dziga et al. 2007) and N-phenyl-2-naphthylamine (Qian et al. 2009). These compounds showed different allelopathic effects on growth and photosynthesis in cyanobacteria (Saito et al. 1989; Nakai et al. 2005; Dziga et al. 2007). Among these compounds, polyunsaturated fatty acids were thought to be potential promising algicides due to their significantly allelopathic effect on inhibiting several phytoplanktons at low concentrations (Alamsjah et al. 2005) with little negative affect on marine organisms (Alamsjah et al. 2008). Linoleic acid isolated from Typha latifolia (Aliotta et al. 1990) is a common polyunsaturated fatty acid. Alamsjah et al. (2008) indicated that linoleic acid was significantly active against the growth of Chattonella antiqua, Chattonella marina, Fibrocapsa japonica, Heterosigma akashiwo and Karenia mikimotoi, as well as moderately effective against Heterocapsa circularisquama, Prorocentrum minimum and Prorocentrum sigmoides. A previous study has shown that linoleic acid inhibited the growth of Microcystis aeruginosa by affecting O^2– generation, which induced lipid peroxidation of the cell membrane, changed the penetrability of the cell membrane, resulted in damage to DNA and some macromolecules, inhibited the synthesis of the chlorophyll a and PsbA protein of PSII (D1 proteins) and destroyed algal cells (Zhang et al. 2009). However, there is little information on the effects of linoleic acid on species that belong to the genus Cylindrospermopsis.

Cylindrospermopsis raciborskii, a N₂-fixing filamentous cyanobacterium, is attracting increasing concern worldwide due to its ability to produce diverse toxins, including cylindrospermopsin and neurotoxins, as well as other, as yet unidentified, toxins (Lagos et al. 1999; Humpage et al. 2000). Moreover, C. raciborskii is regarded as an invasive cyanobacterial species as a result of the increasing occurrence of population blooms in temperate regions (Padisák 1997; Sinha et al. 2012). Controlling the growth of Cylindrospermopsis in water could, therefore, play a critical role in limiting the occurrence of harmful algal bloom (HAB) conditions. In the present study, two aspects of the possible effect of linoleic acid on Cylindrospermopsis were investigated. First, chlorophyll a concentration, growth rate and oxidative system of C. raciborskii cultured with varying concentrations of linoleic acid were determined. Second, fluorescence kinetic parameters for chlorophyll a on PSII were also monitored when cultured with varying concentrations of linoleic acid.

Materials and methods

Strain and culture conditions

Cylindrospermopsis raciborskii FACHB-1096 was provided by the Freshwater Algae Culture Institute of Hydrobiology, Chinese Academy of Science. The alga was cultured in liquid medium for algae (MA) (Ichimura 1979) at 25 ± 1 °C in an incubator under a constant light intensity of 35 µmol photons·m⁻²·s⁻¹ with a light:dark cycle of 12:12, and shaken three times every day.

Experimental design

Cylindrospermopsis raciborskii were harvested in the exponential growth phase by centrifugation at 4000 × g for 10 min, washed twice in fresh MA medium, and divided into 18 equal volumes. Linoleic acid solution (Aladdin) was added to the cultures and diluted to the following concentrations: 0.1, 0.5, 1, 2 and 4 mg L⁻¹ on the basis of previous studies (Alamsjah et al. 2005; Zhang et al. 2009) and a preliminary experiment. Cultures without linoleic acid were used as negative controls. All treatments were carried out in triplicate and cultured for 48 h under the same conditions described above. The initial concentration of C. raciborskii in each treatment was 10⁴ cells·mL⁻¹.

Chlorophyll ɑ concentration and mean growth rate

Growth of the cultures was monitored daily by measuring cell optical density at 680 nm (OD₆₈₀) with a UV visible spectrophotometer (UV2550; Shimadzu Corporation). Cell density number was determined using a hemocytometer chamber under a Nikon CE-I microscope (Nikon Corp.). The number of cell filaments was obtained by: first, measuring the size and cell number of different individuals in order to count the mean cell number per unit; second, determining the size of each filament; and, finally, converting individual size to cell number. Chlorophyll a was extracted with 90% acetone and measured according to the method described by Nusch (1980). Mean growth rate (μ) was calculated using the following equation:where C_t2 and C_t1 are cell density concentrations at times t₂ and t₁, respectively.

Photosynthetic efficiency of PSII measurement

After 48 h culture, cells were used to measure chlorophyll fluorescence parameters with a pulse-amplitude-modulated fluorescence monitoring system (Phyto-PAM). After the cells were dark-adapted for 20 min, F₀ (original fluorescence) and F_m (maximum fluorescence) were measured under a low measuring light and a saturation light pulse, respectively. F₀′ (minimal fluorescence of light-adapted cells) and F_m′ (maximum fluorescence of light-adapted cells) were measured without dark adaptation under a low measuring light and a saturation light pulse, respectively. Maximum photochemical efficiency of PSII (F_v/F_m), relative electron transport rate (rETR) and non-photochemical quenching (NPQ) were calculated as follows: F_v/F_m = (F_m–F₀)/F_m (Ting & Owens 1992); rETR = (F_m′–F_s)/F_m′ × 0.42 × photosynthetic photo flux density (Schreiber et al. 1998); and NPQ = F_m/F_m′ – 1 (Kromkamp & Forster 2003). Rapid light curves (RLC) were determined after dark adapting cells for 20 min. Fluorescence was measured under a sequence of actinic light levels: 1, 32, 64, 192, 320, 512, 704, 960, 1088 and 1216 μmol photons m⁻² s⁻¹ at 30 s intervals between light levels. Non-linear curve fitting was performed with Origin v6.1 (Microcal Software) according to the methods of Platt et al. (1980).

Malondialdehyde, catalase and superoxide dismutase assays

Forty-eight hours after inoculation, a 40 mL algal sample was centrifuged at 5000 × g at 4 °C. The algae cell was ground in liquid nitrogen and extracted in 4 mL of 0.05 mol L⁻¹ potassium phosphate buffer (pH 7.8). The extract was centrifuged for 5 min at 5000 × g at 4 °C. The supernatant was used for a lipid peroxidation product (malondialdehyde [MDA]), and antioxidant enzymes (superoxide dismutase [SOD] and catalase [CAT]) analyses. MDA was assayed using the thiobarbituric acid method (Uchimaya & Mihara 1978). CAT and SOD activities were determined according to the methods published by Choo et al. (2004) and Wu et al. (2007), respectively. Total soluble protein (TSP) content was determined using the Coomassie Blue dye-binding assay (Bradford 1976), with bovine serum albumin as the standard protein.

Chlorophyll fluorescence measurement

Forty-eight hours after inoculation, all samples were dark-adapted for 20 min before chlorophyll a fluorescence measurements. Polyphasic chlorophyll a fluorescence transients were measured using the Handy-Plant Efficiency Analyzer (Handy-PEA, Hansatech) with an actinic light of 3000 µmol quanta·m⁻²·s⁻¹. The fluorescence signals were recorded within a period of 10 µs to 2 s. Normalised chlorophyll a fluorescence transient curves were plotted on a logarithmic time scale, known as the JIP test (Strasser et al. 1995).

The following data were directly obtained from the chlorophyll a fluorescence kinetic curves: F₀, initial fluorescence was determined at 50 μs when all PSII reaction centres (RCs) were open; F_J and F_I, fluorescence intensity at step J (2 ms) and step I (30 ms), respectively; F_m, maximum fluorescence at step P when all RCs were closed; and F₃₀₀, fluorescence at 300 μs. Several parameters, selected to quantify the PSII behaviour of C. raciborskii, were calculated from the above mentioned original data, as shown in Table 1 (Strasser & Strasser 1995).

Table 1. JIP test parameters with explanations and formulae (Strasser & Strasser 1995) calculated using data extracted from the O-J-I-P fast fluorescence transient.

Parameters from the JIP test	Details
Quantum efficiencies or flux ratios or yields
ψ0 ≡ ET0/TR0 = (1 – VJ)	Probability (t = 0) that a trapped exciton moves an electron into the electron transport chain beyond QA^−
φE0 ≡ ET0/ABS = [1 – (F0/Fm)] • ψ0	Quantum yield of electron transport (t = 0)
φP0 ≡ TR0/ABS = [1 – (F0/Fm)]	Maximum quantum yield of primary photochemistry (t = 0)
Specific fluxes or specific activities
ABS/RC = M0 • (1/VJ) • (1/φP0)	Absorption flux per RC
DI0/RC = (ABS/RC) – (TR0/RC)	Dissipated energy flux per RC (t = 0)
Density of reaction centres
RC/CS0 = φP0 • (VJ/M0) • (ABS/CS0)	Absorption flux per CS (t = 0)
Performance indexes
PIABS = RC/ABS • [φP0/(1 – φP0)] • [ψ0/(1 – ψ0)]	Performance index on absorption basis

Statistical analysis

All treatments were carried out in triplicate. Mean and standard deviation (SD) values were calculated for each treatment. All analyses were performed using SPSS v16.0 (IBM) and Origin software v8.6 (OriginLab Corp.). Significance of the differences between the control and various treatments was determined using one-way analysis of variance (ANOVA) and the t-test. Statistical significance was accepted when the P value was less than 0.05.

Results

Cell growth and chlorophyll a inhibition

After 48 h of incubation in different concentrations of linoleic acid, mean growth rate (μ) and chlorophyll a concentration for C. raciborskii were observed (Figure 1). Compared with the controls, chlorophyll a concentration deteriorated significantly with increasing linoleic acid concentrations (P < 0.05, ANOVA; Figure 1A). After 48 h of exposure, mean growth rate (μ) was significantly decreased by 58% and 85% in the 2 and 4 mg L⁻¹ treatments, respectively (P < 0.05, ANOVA; Figure 1B) when compared with the control (P < 0.05, ANOVA; Figure 1B). Additionally, EC₅₀ of linoleic acid for C. raciborskii was 1.288 ± 0.454 mg L⁻¹.

Figure 1. Parameters observed when Cylindrospermopsis raciborskii was exposed to 0, 0.1, 0.5, 1, 2 and 4 mg·L⁻¹ of linoleic acid for 48 h. A, Chlorophyll a concentration; B, mean growth rate (μ).

Error bars represent ± SD for triplicate treatments.

*Indicates a P value less than 0.05; **indicates a P value less than 0.01.

Rapid light curve and photosynthetic parameters of PSII

RLC behaviours in C. raciborskii were dramatically different at different concentrations of linoleic acid (Figure 2). A marked decrease in the initial slope of the RLC (α), the maximal rETR through PSII (ETR_max) and the maximum quantum yield (F_v/Fm) was found following incubation at 1, 2 and 4 mg L⁻¹ of linoleic acid (P < 0.05, ANOVA; Table 2). However, NPQ markedly increased at 0.5, 1, 2 and 4 mg L⁻¹, in contrast to the 0 and 0.1 mg L⁻¹ treatments (P < 0.05, ANOVA; Table 2).

Figure 2. Rapid light curves of Cylindrospermopsis raciborskii exposed to 0, 0.1, 0.5, 1, 2 and 4 mg L⁻¹ of linoleic acid for 48 h. Values shown are the means of three replicates ± SD.

Table 2. Changes in photosynthetic parameters following different concentration treatments of linoleic acid in C. raciborskii.

Linoleic acid concentration (mg L^−1 )	α	ETRmax	NPQ	Fv/Fm
0	0.173 ± 0.006	88.033 ± 5.301	0.019 ± 0.008	0.119 ± 0.008
0.1	0.178 ± 0.006	91.267 ± 3.180	0.012 ± 0.002	0.102 ± 0.012
0.5	0.171 ± 0.002	89.367 ± 0.524	0.040 ± 0.014	0.111 ± 0.017
1	0.023 ± 0.003**	9.067 ± 1.452**	0.052 ± 0.003*	0.060 ± 0.003**
2	0.010 ± 0.002**	1.867 ± 0.437**	0.088 ± 0.018**	0.019 ± 0.001**
4	0.008 ± 0.001**	4.200 ± 2.200**	0.094 ± 0.007**	0.028 ± 0.007**

Values shown are the means of three replicates ± SD.

*In the same column indicates significant differences (P < 0.05).

**In the same column indicates extremely significant differences (P < 0.01).

Chlorophyll a fluorescence

Fluorescence induction kinetics of chlorophyll a in cells treated with increasing concentrations of linoleic acid for 48 h are shown in Figure 3. Effect of linoleic acid on the fast-phase fluorescence induction curve (O-J-I-P) was found to be concentration-dependent in C. raciborskii. Similar chlorophyll a fluorescence transient curves were found at treatments of 0, 0.1 and 0.5 mg L⁻¹ of linoleic acid; however, phase I and phase P disappeared from the curves when C. raciborskii was exposed to 1, 2 and 4 mg L⁻¹ of linoleic acid. Additionally, a K phase (an imbalance between the electron flow leaving the RC to the acceptor side and the electron flow coming to the RC from the donor side) was found at 2 and 4 mg L⁻¹ concentrations of linoleic acid.

Figure 3. Relative variable fluorescence (OJIP transient curve) of Cylindrospermopsis raciborskii after 48 h of incubation with 0, 0.1, 0.5, 1, 2 and 4 mg L⁻¹ of linoleic acid.

JIP test

Six parameters, including the density of the RCs (RC/CS₀), absorption flux per RC (ABS/RC), dissipated energy flux per RC (DI₀/RC), maximum quantum yield of primary photochemistry (ϕP₀) and the probability that an absorbed photon will move an electron into the electron transport chain further than Q_A⁻ (ϕE₀), as well as the performance index on absorption basis (PI_ABS, which deviated from the JIP test) are shown in Figure 4. ϕE₀ and RC/CS₀ showed a significant decrease with increasing concentrations of linoleic acid (P < 0.05, ANOVA). When exposed to 4 mg L⁻¹ of linoleic acid, ϕE₀ and RC/CS₀ decreased by 81.1% and 87.3%, respectively (Figure 4A). ϕP₀ and PI_ABS showed the same trends under linoleic acid stress: ϕP₀ and PI_ABS were 6.4 and 42.6 times lower than those of the control at 2 mg L⁻¹ (P < 0.05, ANOVA). Interestingly, ϕP₀, ϕE₀, PI_ABS and RC/CS₀ showed a slight increase at 0.5 mg L⁻¹ when compared with those at 0.1 mg L⁻¹.

Figure 4. Changes in the JIP test parameters expressed as a percentage of the control when Cylindrospermopsis raciborskii was exposed to various concentrations of linoleic acid for 48 h. A, Absorption flux per RC (ABS/RC) and dissipated energy flux per RC (DI₀/RC); B, RC parameters (RC/CS₀), parameters related to Q_A reduction (φP₀ and φE₀) and performance index based on absorption (PI_ABS). For more information, see Table 1.

*Indicates a P value less than 0.05; **indicates a P value less than 0.01.

ABS/RC and DI₀/RC showed a significant increase with increasing concentrations of linoleic acid (P < 0.05, ANOVA; Figure 4B). At 2 mg L⁻¹ of linoleic acid, ABS/RC and DI₀/RC were 7.81 and 8.69 times higher than that of the control, respectively. However, ABS/RC and DI₀/RC decreased slightly at 4 mg L⁻¹, in contrast to the values at 2 mg L⁻¹.

Lipid peroxidation and antioxidant enzyme activities

After 48 h of exposure to various concentration of linoleic acid, antioxidant enzyme (SOD and CAT) activities showed a similar trend (Figure 5A–B). Compared with the control, there were no significant differences in SOD and CAT activities when the specimens were incubated with 0.1, 0.5 and 1 mg L⁻¹ of linoleic acid. A remarkable increment in SOD activity was found at 2 and 4 mg L⁻¹ (P < 0.05, ANOVA; Figure 5A). However, CAT activity significantly increased at 1, 2 and 4 mg L⁻¹ (P < 0.05, ANOVA; Figure 5B). CAT activity was 1.91, 2.01 and 3.08 times higher at 1, 2 and 4 mg L⁻¹, respectively, when compared with the controls. Lipid peroxidation, expressed as MDA concentration, showed that MDA levels generally increased with increasing concentrations of linoleic acid (Figure 5C). Compared with the control, MDA concentration was significantly higher when the concentration of linoleic acid was greater than 0.5 mg L⁻¹ (P < 0.05, ANOVA). MDA concentrations were 2.63, 2.95, 2.84 and 2.52 times higher than the control when exposed to 0.5, 1, 2 and 4 mg L⁻¹ of linoleic acid.

Figure 5. Parameters for Cylindrospermopsis raciborskii incubated with 0, 0.1, 0.5, 1, 2 and 4 mg L⁻¹ of linoleic acid for 48 h. A, Superoxide dismutase (SOD) activity; B, catalase (CAT) activity; C, malondialdehyde (MDA) concentration. Error bars represent ± SD for triplicate treatments.

*Indicates a P value less than 0.05; **indicates a P value less than 0.01.

Discussion

Allelopathic compounds identified from many aquatic macrophytes have been proposed as a method for controlling the growth of undesired phytoplankton that is a threat to aquatic ecosystems (Huang et al. 2004). In the present study, after inoculation with treatments of 1, 2 and 4 mg L⁻¹ linoleic acid, chlorophyll a and growth rate (μ) significantly declined in C. raciborskii in contrast to the control (Figure 1). Similar results have also been reported by Zhang et al. (2009), who showed that the chlorophyll a content of M. aeruginosa decreased with increasing concentrations of linoleic acid. These results suggest that the ability of C. raciborskii cells to synthesise chlorophyll was reduced in the presence of linoleic acid. Chlorophyll content is closely associated with the photosynthetic characteristics of plants (Niu et al. 2012). Lower chlorophyll content reflects a decrease in the antenna size of the photosynthetic RC complexes (Björkman 1981), suggesting that damage to the photosynthetic system may have occurred when C. raciborskii was exposed to linoleic acid.

The photosynthetic electron transport chain in photosynthetic system II (PSII) is an important target site for allelopathic inhibition in cyanobacteria (Liu et al. 2015). In this study, we investigated the RLCs of C. raciborskii in response to different concentrations of linoleic acid (Figure 2). The initial slope of the RLC (α), F_v/F_m and ETR_max of C. raciborskii were dramatically reduced at concentrations of 1, 2 and 4 mg L⁻¹ (Figure 2, Table 2), indicating that the ability of C. raciborskii to use light dramatically decreased at these concentrations of linoleic acid, while the extent of photoinhibition markedly increased at 1, 2 and 4 mg L⁻¹. NPQ is a major photoprotective mechanism in which the excess light energy absorbed by the light-harvesting chlorophyll–protein complexes (LHCs) of PSII is dissipated as heat (Demmig-Adams & Adams 1996, 2000; Horton et al. 1996). In this study, we found that NPQ increased upon exposure to high concentrations of 0.5, 1, 2 and 4 mg L⁻¹ (Table 2), suggesting that excess energy trapped by the RCs is dissipated as heat to avoid the damaging effects of high concentrations of linoleic acid.

To obtain further insight into the mechanisms involved in the influence of linoleic acid on the photosynthetic system, a fast chlorophyll a fluorescence transient curve was used to detect and estimate the status of PSII (Figure 3). Phase I and phase P disappeared from the chlorophyll a fluorescence induction curves when C. raciborskii was exposed to 1, 2 and 4 mg L⁻¹ of linoleic acid. Strasser et al. (1995) proposed that phase O corresponds to a situation where all molecules of the primary electron acceptor of PSII (Q_A) are in the oxidised state, Q_A is reduced to Q_A^– in the transition from phase O to J, and the secondary electron acceptor of PSII (Q_B) is reduced when phase J transforms to phase P in the polyphasic chlorophyll fluorescence transients. This indicated that a high concentration of linoleic acid might inhibit electron transport at the acceptor side of PSII, leading to the accumulation of P680⁺ (Govindjee 1995; Srivastava et al. 1997). Furthermore, in this study, a K phase was found at 2 and 4 mg L⁻¹ of linoleic acid. Strasser (1997) suggested that the occurrence of a K step may be attributable to an imbalance between the electron flow leaving the RC to the acceptor side and the electron flow coming to the RC from the donor side. The K step has also been observed to appear when the oxygen-evoling complex (OEC) is damaged (Srivastava et al. 1997; Strasser 1997). Therefore, the K step in this study suggested that the OEC of C. raciborskii might be damaged or the electron flow might be imbalanced when exposed to 2 and 4 mg L⁻¹ of linoleic acid, supported by a decrease value of ETR_max at high concentration.

The JIP test can provide information on the fluxes of photons, excitons, electrons and further metabolic events, and it has proven to be a sensitive probe for PSII behaviour under environmental stress (Han et al. 2008). In this study, ϕE₀ represents the efficiency with which a trapped exciton can move an electron into the electron transport chain (Strasser et al. 2004). Linoleic acid caused a decrease in ϕE₀ (Figure 4A), suggesting the probability that an electron residing on Q_A^–, which will enter the transport chain, is reduced. This showed that the electron transport of PSII was blocked because of Q_A^– accumulation. These findings suggest a less efficient electron transfer after Q_A^– to Q_B resulting from disassociation of Q_B from thylakoids and reduction of the Q_B pools (Ran et al. 2015). The decline in the density of active photosynthetic RCs (RC/CS₀) further demonstrated that inactivation of the RCs was increased at high concentrations of linoleic acid. ϕP₀ and PI_ABS showed the same trends under linoleic acid stress: ϕP₀ and PI_ABS were 6.4 and 42.6 times lower than those of the control at 2 mg L⁻¹ (P < 0.05, ANOVA). Interestingly, ϕP₀, ϕE₀, PI_ABS and RC/CS₀ showed a slight increase at 0.5 mg L⁻¹ when compared with those at 0.1 mg L⁻¹. Strasser et al. (2004) showed that a decrease in ϕP₀ resulted in the block of trapping probability of RC. The decreased ϕP₀ implied that the photosynthetic efficiency of C. raciborskii was inhibited. PI_ABS, one of the most sensitive parameter in the JIP test, represents the overall expression of three functional steps: energy absorption; energy trapping; and energy conversion into electron transport (Srivastava et al. 1999). A significant decrease in PI_ABS (Figure 4A) and a marked increase in NPQ at high concentrations of linoleic acid (Table 2) imply visible vitality losses in PSII (Lange et al. 2004).

Increased ABS/RC, especially at the highest concentrations of linoleic acid (1, 2 and 4 mg L⁻¹; Figure 4B), illustrates that there has been a decrease in the antenna size of PSII RC and thus can result from an increase in the number of LHCs per RC or to a partial inactivation of RCs to form non-Q_A-reducing or ‘heat sink’ or ‘silent’ RCs (Eullaffroy et al. 2009). This possible inactivation of PSII RCs was confirmed by the increase of total dissipation through active RCs (DI₀/RC), which indicates that extra energy received has to be dissipated to avoid possible photodamage to remaining active RCs (Figure 4B). Since no decrease in the light reactions leading to the energy flux as trapping per active RC was observed, we concluded that energy dissipation was increasing via a relative increase in LHCs per RC which favour the dissipation of excess energy through non-photochemical pathways (thermal dissipation processes).

Oxidative damage caused by allelochemicals might be a lethal mechanism for algae (Hong et al. 2009). In this study, MDA content in C. raciborskii significantly increased at high concentrations of linoleic acid (Figure 5C), reflecting that oxidative damage occurred in C. raciborskii. To reduce oxidative damage, cellular responses of antioxidant enzymes need to be induced after stress caused by allelochemicals (Gniazdowska & Bogatek 2005; Singh et al. 2006; Hong et al. 2008). In this study, SOD and CAT activities showed a significant increase at 2 and 4 mg L⁻¹ of linoleic acid, indicating that C. raciborskii cells encountered oxidative stress and that they have to remove reactive oxygen species (ROS) when exposed to linoleic acid (Figure 5A and B). Similar results were also obtained by Wu et al. (2013), who observed that SOD and CAT activities were enhanced by pyrogallic acid stress. However, it was apparent that enhanced SOD and CAT activities were not enough to prevent the sudden increase in H₂O₂ content and consequent formation of ROS, leading to extensive lipid peroxidation, as indicated by enhanced MDA values at high concentrations of linoleic acid (Yu et al. 2003; Li & Hu 2005; Hong et al. 2008).

Conclusion

In summary, the results of this study suggest that high concentrations (2 and 4 mg L⁻¹) of linoleic acid significantly inhibited growth and changed the oxidative system and PSII activity of C. raciborskii, indicating that damage to the oxidative and photosynthetic systems could be two important mechanisms underlying the allelopathic effect of linoleic acid on C. raciborskii. In addition, Q_A^– accumulation that resulted in the blockage of electron transport in PSII was the mechanism responsible for the toxicity of linoleic acid in the PSII of C. raciborskii.

Acknowledgement

Associate Editor: Dr Joanne Clapcott.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

The study was supported by Fundamental Research Funds for the Central Universities (Grant number XDJK2016C111), the National Natural Science Foundation of China (Grant number 31170372), and the Natural Science Foundation Project of China SWU (Grant number SWNUB2011).