Introduction

Lichens are a stable and self-supporting association between fungi (mycobionts) and photoautotrophic algae or cyanobacteria (photobionts). Interactions within a lichen thallus are, however, complex, and lichens may be considered more like an ecosystem or community than a typical organism (Fahselt 2008).

Lichens produce many unique compounds that are considered to have important biological and ecological roles. These roles include antimicrobial activity, allelopathy, antiherbivory, chelating of heavy metals, light screening, and other proposed functions not yet sufficiently supported by experimental evidence under laboratory conditions (Lawrey 1986; Pöykkö et al. 2005; Latkowska et al. 2006; Hauck et al. 2009; Solhaug et al. 2009). Secondary metabolites may be responsible for the ecological success of lichenization, allowing lichens frequently to be the dominant organisms in environments characterized by extreme ecological conditions. Lichen compounds are secreted by the fungal partner, and they are deposited on the surface of hyphae, as well as on lichen algae, typically constituting 0.1 to 5.0% (w/w) of thallus dry weight (Fahselt 1994).

Usnic acid, a yellowish pigment and dibenzofuran derivative, is a common lichen metabolite, especially abundant in members of the genera Alectoria, Usnea, or Xanthoparmelia. The antibiotic, antiviral, antimycotic, antiprotozoal, antiherbivoral, antiproliferative, anti-inflammatory, analgesic, antipyretic, allelopathic, and UV protecting effects of usnic acid have been stated previously (for review see Cocchietto et al. 2002).

There is little doubt that the presence of secondary metabolites, including usnic acid, has significant influence on lichen survival. Lichens are slow growing and long lived organisms, and due to the toxic effect of usnic acid to a wide range of organisms, their thalli are relatively well protected against pathogens and herbivores. However, usnic acid potentially can be toxic to cells that come in direct contact with it, even the algal partner of the lichen that produces it. Crystals of lichen cortical secondary metabolites may be deposited directly on the surface of photobiont cells (Sarret et al. 1998). Previous studies have suggested that the presence of usnic acid may regulate photobiont populations (Bačkor et al. 1998; Buďová et al. 2006). Production of usnic acid may be a critical step of maintaining the required ratio of mycobiont to photobiont biomass in thalli for mutualistic symbiosis.

The mechanisms of the phytotoxic effects of usnic acid on plants, including on its own photobionts are not understood sufficiently; however, inhibition of chlorophyll a fluorescence (FV/FM), decrease of carotenoid and chlorophyll levels in the cells, and decrease of viability have been demonstrated as responses of plants to the presence of usnic acid (Cardarelli et al. 1997; Endo et al. 1998; Buďová et al. 2006). Decrease of biomass production also has been demonstrated (Bačkor et al. 1998; Buďová et al. 2006; Lechowski et al. 2006).

Concentrations of secondary metabolites around photobiont cells are high, and lichen photobionts are in direct contact with cortical metabolites produced by the mycobiont (Takahagi et al. 2008). Although lichen compounds may act as allelochemicals and are responsible for maintaining balance between bionts inside lichen thalli, photobionts must be protected from the toxicity of these compounds. We hypothesized that lichen photobionts have evolved protective mechanisms against phytotoxicity of secondary metabolites through co-evolution with lichen mycobionts. To test this hypothesis, we evaluated the potential phytotoxic effect of usnic acid on the aposymbiotically grown lichen photobiont Trebouxia erici. For assessment of the potential differences in phytotoxicity of usnic acid to free-living algae without evolutionary experience with the presence of lichen secondary metabolites, Scenedesmus quadricauda also was chosen.

Methods and Materials

Organisms and Culture Conditions

Free-living alga Scenedesmus quadricauda (Turp.) Bréb. (UTEX 76) and aposymbiotically grown lichen photobiont Trebouxia erici Ahmadjian (UTEX 911) were used. Both species belong to Chlorophyta, a division of green algae. Algae were cultivated on agar medium (1.5%) previously developed for cultivation of lichen algae by Ahmadjian (1993). This was Bold’s Basal Medium (BBM 3N) plus 10 g casein acid hydrolyzate and 20 g glucose per liter with the pH adjusted to 6.5 (Bačkor et al. 2004). Cultures were maintained at 22°C under a 16:8 hr, L:D photoperiod and 30 µmol.m−2.s−1 artificial irradiance (“cool white” tubes).

Toxicity Assay

Algae were cultivated on the surface of Whatman CF/C filters (glass fiber filter disks, 25 mm diam). Glass fiber filter disks were used instead of the cellulose-acetate disks, previously used for lichen photobiont cultivation (Bačkor et al. 2003, 2004). Glass fiber filter disks may be used directly for disruption in the mortar of algal cells grown on their surfaces. Crystals of usnic acid on the surface of fibers of the disk simulate the situation in natural lichens, where extracellular secondary metabolites located on the surface of hyphae are in the contact with algal cells in the photobiont layer. Briefly, cells from both algal species (approximately two inoculation loops) grown on stock solid Trebouxia media were transferred into 50 ml of liquid Trebouxia media in an Erlenmeyer flask. Cells were suspended by gentle stirring on a magnetic stirrer for 1 hr. Cultures were maintained for 5 d in a cultivation room under conditions described previously, with daily stirring on a magnetic stirrer for about 1 hr. Homogeneity of the algal suspension was verified microscopically, and the number of cells was calculated by using a standard haemocytometer. Cell density of cultures was adjusted to approximately 106 cells ml−1 of medium before quantitative cultivation of algae.

For cultivation of both algal species, sterilized Whatman CF/C filters (25 mm diam) were subjected to three different pretreatments. Usnic acid (0.01 and 0.1 mg / disk) dissolved in acetone (volume 30 μl) was applied by automatic pipette on the surface of disks, while the same volume of acetone was applied to control disks. We used (+)-usnic acid enantiomer (Sigma-Aldrich). After evaporation of acetone for 4 hr, one disk was transferred to the surface of solid Trebouxia medium in a separate Petri dish, (6 cm diam), and 20 µl of algal suspension were inoculated into the center of each disk. Disk pores allowed supplemental nutrient media to pass through the disk and permitted growth to be determined easily from changes in biomass (Bačkor et al. 2003, 2004). The total mass of cultures was calculated by subtracting the mean fresh weight (fw) of a Whatman CF/C disk saturated by identical medium, from the fw of a disk supporting algal cultures after 14 d. Each treatment was replicated eighteen times for both algal species assessed.

Pigment Analysis and Measurement of Chlorophyll a Integrity

The influence of usnic acid on the algae was determined by using cultures grown on Whatman CF/C disks. Weighed disks were extracted in the dark for 1 hr at 65°C in 5 ml of dimethyl sulfoxide (DMSO). To maximize chlorophyll extraction, cell aggregates were homogenized with a mortar, and glass fibers of disks facilitated disruption of cell walls of algae. After cooling to ambient temperature, the absorbance of the extract, as a reflection of turbidity, was determined at 750 nm with a spectrophotometer to be certain that it was always less than 0.01. Absorbance of extracts was read at 665, 649, 435, and 415 nm to assess chlorophyll content and the possibility of chlorophyll a degradation (Barnes et al. 1992; Wellburn 1994). To utilize the linear portion of the response curve, extracts from disks with very high cell densities (absorbance at 665 nm higher than 0.8) were diluted with fresh DMSO to fall into the absorbance range 0.2–0.8. To determine the content of “total” carotenoids, absorbance was read at 480 nm. Chlorophyll a, chlorophyll b, chlorophyll a + b, and total carotenoids were calculated by using equations derived from specific absorption coefficients for pure chlorophyll a and chlorophyll b in DMSO. Chlorophyll a/b was used to assess the physiological competence of algal cells.

The ratios of absorbances at 435 and 415 nm (A 435/A 415), termed the phaeophytinization quotient, were calculated as a reflection of the ratio of chlorophyll a to phaeophytin a and to provide an indication of integrity of photobiont chlorophyll. Each treatment was replicated three times.

Activity of Photosystem II

Chlorophyll a fluorescence was measured in algae grown on Whatman CF/C disks on the surface of Trebouxia agar medium. While still on the surface of the medium in Petri dishes (to minimize desiccation), algae were dark-adapted for 30 min prior to measurement. The potential quantum yield of photosystem II (PSII) was measured with a Plant Stress Meter (PSM Mark II, Biomonitor, SCI AB), with a sensor 5 mm diam, and results were expressed as FV/FM calculated as the maximal fluorescence (FM) less the minimal fluorescence (F0), divided by FM of dark adapted plants: (FM−F0) / FM = FV/FM. Chlorophyll fluorescence parameters were taken from three separate positions on each disk, and the mean value used as one observation. Each treatment was replicated three times.

Content of Soluble Proteins

Algae grown on Whatman CF/C disks (controls, as well as with addition of usnic acid on the surface of disks) were homogenized with disks in an ice-cold mortar in phosphate buffer (50 mM). After centrifugation at 15,000 g at 4°C for 20 min, the water-soluble protein content of supernatants was measured using the Bradford (1976) method. Supernatants (100 μl) were pipetted into 900 μl of Bradford assay kit (Biorad) in a spectrophotometric cuvette and mixed. After 10 min, absorbance of samples was spectrophotometrically measured at 595 nm. Bovine serum albumin was used as calibration standard. Each treatment was replicated three times.

Detection of Oxidative Status

Hydrogen peroxide and superoxide were measured in homogenates with potassium phosphate buffer prepared for assay for determination of soluble proteins. Hydrogen peroxide was quantified by the TiCl4 method (Kováčik and Bačkor 2007). Superoxide was estimated according to Elstner and Heupel (1976) by monitoring formation of nitrite from hydroxylamine at 530 nm. Reaction mixture contained 0.27 ml of potassium phosphate buffer, 0.03 ml of 10 mM hydroxylamine, 0.3 ml of supernatant, 0.3 ml of 17 mM sulphanilamide, 0.3 ml of 7 mM α-naphthylamine, and 0.3 ml of diethyl ether. Each treatment was replicated three times.

Cell Size

Algae grown on Whatman CF/C disks from each treatment were separated gently from the surface of the disks by a scalpel. Cells were suspended immediately in 5 mM HEPES buffer, pH 6.5 and viewed under a light microscope at 800×. Cell size parameters (width and length in the case of S. quadricauda cells, or length in the case of T. erici spherical cells) were measured with an eyepiece micrometer. Twenty individual cells from each treatment and both algal species were evaluated.

Statistical Analysis

The one-way analysis of variance and Tukey’s pairwise comparisons (MINITAB Release 11, 1996) were applied to determine significance (P < 0.05) of differences in all measured parameters. In addition, two-way ANOVA with interactions (SPSS version 16, 2008) was applied to evaluate species effect (species), usnic acid effect (UA), and interactions between species effect and UA effect (species × UA) where applicable.

Results

Biomass production of the control Scenedesmus cultures was almost five times higher after 2 weeks of cultivation when compared to control Trebouxia cultures (Table 1). Presence of usnic acid on the disks significantly affected biomass production of both algal cultures in a dose dependent manner. However, a 0.01 mg/disk of usnic acid decreased biomass production of Scenedesmus cultures by a factor of 4, and a 0.1 mg/disk decreased biomass production by a factor of 11, while in Trebouxia culture biomass production was decreased only at the highest tested usnic acid dose of a 0.1 mg/disk by a factor of approximately 2.2 (Table 1). Two-way ANOVA for biomass production: species (F = 63.25, P < 0.001), UA (F = 52.98, P < 0.001), species × UA (F = 40.98, P < 0.001).

Table 1 Biomass production (mg fw/disk), chlorophyll integrity (435/415), chlorophyll a/b ratio (A/B), total carotenoid/total chlorophyll ratio (CAR/CHL) and chlorophyll a fluorescence (FV/FM) of 2 week old Secenedesmus and Trebouxia cultures cultivated on disks with addition of usnic acid (0, 0.01 and 0.1 mg)
Full size table

The highest tested usnic acid doses significantly decreased chlorophyll a integrity in Scenedesmus cultures, while in Trebouxia cultures none of the tested usnic acid concentrations significantly altered chlorophyll integrity (Table 1). Two-way ANOVA for chlorophyll a integrity: species (F = 40.75, P < 0.001), UA (F = 10.84, P = 0.006), species × UA (F = 23.75, P < 0.001).

Similarly, the highest tested usnic acid doses significantly decreased chlorophyll a/b of Scenedesmus cultures. In the Trebouxia cultures, we did not observe significant changes of chlorophyll a/b under any of the tested concentrations (Table 1). Two-way ANOVA for chlorophyll a/b: species (F = 10.26, P = 0.002), UA (F = 1.88, P = 0.194), species × UA (F = 24.11, P < 0.001).

Total carotenoid/total chlorophyll ratio was stable in both algal cultures regardless of usnic acid application (Table 1). Two-way ANOVA for total carotenoid/total chlorophyll ratio: species (F = 2.32, P = 0.139), UA (F = 0.13, P = 0.716), species × UA (F = 4.81, P = 0.03).

When calculated per unit of fresh weight (mg/g fw) of algal biomass, we observed a significant decrease of chlorophyll a, chlorophyll b, chlorophyll a + b, and total carotenoids in Scenedesmus cultures with the presence of a 0.1 mg/disk of usnic acid (Table 2). In the Trebouxia cultures, we did not observe any decrease in these parameters when calculated per unit fresh weight (Table 2). Two-way ANOVA for chlorophyll a: species (F = 11.04, P = 0.002), UA (F = 20.69, P < 0.001), species × UA (F = 6.73, P = 0.01). Two-way ANOVA for chlorophyll b: species (F = 3.91, P = 0.049), UA (F = 14.81, P = 0.002), species × UA (F = 0.44, P = 0.651). Two-way ANOVA for chlorophyll a + b: species (F = 8.74, P = 0.004), UA (F = 19.48, P < 0.001), species × UA (F = 4.15, P = 0.042). Two-way ANOVA for total carotenoids: species (F = 9.08, P = 0.004), UA (F = 14.93, P = 0.002), species × UA (F = 8.14, P = 0.005).

Table 2 Chlorophyll a content (A, mg/g fw), chlorophyll b content (B, mg/g fw), chlorophyll a + b content (A + B, mg/g fw) and content of total carotenoids (TOT CAR, mg/g fw) of 2 week old Secenedesmus and Trebouxia cultures cultivated on disks with addition of usnic acid (0, 0.01 and 0.1 mg)
Full size table

Usnic acid decreased the FV/FM ratio of Scenedesmus cultures. Presence of a 0.1 mg/disk decreased the FV/FM ratio more than four times, while in Trebouxia cultures the FV/FM ratio decreased only 1.15 times at the highest tested usnic acid dose of 0.1 mg/disk (Table 1). Two-way ANOVA for FV/FM: species (F = 91.20, P < 0.001), UA (F = 55.38, P < 0.001), species × UA (F = 49.48, P < 0.001).

Increase of usnic acid on the disk did not cause a significant decrease in the soluble protein content of algal cultures calculated per unit fresh weight according to one-way ANOVA (Table 3). Two-way ANOVA for soluble protein content: species (F = 0.492, P = 0.623), UA (F = 29.12, P < 0.001), species × UA (F = 5.60, P = 0.019). According to two-way ANOVA, significant UA, as well as species × UA effects were observed.

Table 3 Content of soluble proteins (PROT, mg/g fw), content of hydrogen peroxide (PEROX, µmol/g fw), content of superoxide (SUPEROX, µg/g fw) and cell size (WIDTH and LENGTH, µm) of 2 week old Secenedesmus and Trebouxia cultures cultivated on disks with addition of usnic acid (0, 0.01 and 0.1 mg)
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The highest tested usnic acid dose of 0.1 mg/disk caused a significant increase of hydrogen peroxide content in Scenedesmus cultures (Table 3). In the cultures of Trebouxia, we also observed an increase of hydrogen peroxide content at a dose of 0.1 mg/disk, however, the increase was not strong enough to be significant (Table 3). Two-way ANOVA for hydrogen peroxide content: species (F = 16.16, P < 0.001), UA (F = 11.80, P = 0.004), species × UA (F = 1.96, P = 0.183).

The highest tested usnic acid dose of 0.1 mg/disk caused a significant increase of superoxide in cultures of both algal species tested (Table 3). Two-way ANOVA for superoxide content: species (F = 15.49, P < 0.001), UA (F = 0.42, P = 0.526), species × UA (F = 0.21, P = 0.815).

Both tested usnic acid doses caused significant increases of width and length of Scenedesmus cells. In Trebouxia cultures, we did not observe any significant increase of cell size at any of the tested usnic acid concentrations (Table 3). Two-way ANOVA for width: species (F = 6.70, P = 0.002), UA (F = 8.53, P = 0.005), species × UA (F = 3.86, P = 0.026).

Discussion

Influence of Usnic Acid on Algal Growth

Usnic acid appears to have phytotoxic effects on vascular plants (Cardarelli et al. 1997; Lechowski et al. 2006) as well as on the algal partner of lichens (Bačkor et al. 1998; Buďová et al. 2006). In the present study, we found that control cultures of free-living alga Scenedesmus grew better when compared to aposymbiotically grown control cultures of Trebouxia. Presence of usnic acid on the surface of fibers caused a dose-dependent decrease of the growth of both algal cultures, however, the decrease was more pronounced in cultures of Scenedesmus compared to cultures of Trebouxia. Lower toxicity of usnic acid for photobiont cells may be a result of co-evolution of Trebouxia photobionts with filamentous fungi over a long period of time, perhaps hundreds of millions of years (Yuan et al. 2005). Usnic acid possesses antimitotic effects that have been demonstrated in taxonomically diverse organisms (Cardarelli et al. 1997). Surprisingly, presence of usnic acid in this study caused a significant increase of cell size of Scenedesmus cultures. This may be a result of the effect of usnic acid on the spindle apparatus during mitosis (Al-Bekairi et al. 1991). Although a similar trend was observed in the Trebouxia cells, the effect was not strong enough to be significant.

Influence of Usnic Acid on Algal Metabolic Processes

The mechanisms of phytotoxic effects of usnic acid still are mostly unknown. It has been demonstrated that viability of the protoplasts of Nicotiana tabacum decreases due to the presence of usnic acid (Cardarelli et al. 1997). Buďová et al. (2006) demonstrated that viability of the Trebouxia erici photobiont cells decreased due to increased doses of usnic acid.

Changes in composition of assimilation pigments, as well as in chlorophyll a fluorescence of aposymbiotically grown lichen photobionts previously have been found to be excellent markers for assessment of the influence of xenobiotics, including metals (Bačkor et al. 2003). One of the most frequently used parameters in lichen stress physiology is chlorophyll degradation, expressed as the phaeophytinization quotient, which reflects the ratio of chlorophyll a to phaeophytin a (Bačkor and Loppi 2009). This is defined as the ratio of optical densities at 435 nm and 415 nm (OD435/OD415). In healthy lichens and photobionts, the OD435/OD415 ratio is about 1.4, and the presence of xenobiotics, including heavy metals, may cause a marked decrease of this value (Ronen and Galun 1984; Bačkor and Loppi 2009). In the present study, none of the tested usnic acid concentrations caused a significant decrease of phaeophytinization quotient in Trebouxia cultures, while the highest tested dose of 0.1 mg/disk caused a significant decrease of the phaeophytinization quotient in the cultures of Scenedesmus. An identical trend was observed for the ratio of chlorophyll a/b, where only the highest tested dose of usnic acid in the cultures of Scenedesmus caused a significant decrease; chlorophyll a of Scenedesmus cells was more sensitive than chlorophyll b. However, when we compared changes of total chlorophyll content to total carotenoid content due to presence of usnic acid, we did not find significant differences in either algal species.

Irrespective of the previously noted parameters, we observed a decrease in the content of chlorophyll a, chlorophyll b, chlorophyll a + b, and total carotenoids of Scenedesmus calculated per unit of fresh weight. Although chlorophyll a and chlorophyll b differ only in the composition of side chain (chlorophyll a contains a methyl group and chlorophyll b a formyl group), decrease of chlorophyll a was notable. Previous studies suggested that plants exposed to pollutants, including acid rain, heavy metals, and herbicides respond by decrease of chlorophyll a rather than chlorophyll b (Hendry et al. 1987). Degradation of chlorophyll a here was accompanied by increased phaeophytinization. Han et al. (2004) demonstrated that usnic acid is toxic to cultured hepatocytes and disrupts electron transport in mitochondria and induces oxidative stress in the cells. A previous study suggested that production of the reactive oxygen species in the cells of the lichen photobiont Trebouxia erici caused increase of membrane lipid peroxidation (Bačkor et al. 2007).

The reduced FV/FM values measured in our usnic acid treated cultures indicate damage to PSII. Endo et al. (1998) demonstrated a decrease of chlorophyll a fluorescence in spinach leaves due the presence of lichen-derived secondary metabolites. However, we found a differential sensitivity of algae to usnic acid as cultures of Scenedesmus were nearly killed (FV/FM values lower than 0.2) after higher tested doses of usnic acid, while cultures of Trebouxia were decreased only slightly. It has been found previously that PSII in the cells of lichen photobionts is relatively protected against the phytotoxicity of lichen secondary compounds (Buďová et al. 2006; Takahagi et al. 2008).

Xenobiotics, including metals, may alter the composition of soluble proteins in cultures of lichen photobionts (Bačkor and Fahselt 2008). Although we observed a significant decrease of protein content due to usnic acid when analyzed for individual disks (data not shown), we did not observe significant changes in soluble protein content calculated per unit of algal biomass either in Scenedesmus or in Trebouxia cultures. Caviglia et al. (2001) demonstrated that usnic acid in lichen Parmelia soredians may act as an antioxidant that detoxifies reactive oxygen species produced by the application of the herbicide Paraquat. However, Han et al. (2004) demonstrated that usnic acid itself may induce oxidative stress in the cells. In the present study, we found that the highest tested dose of usnic acid significantly increased hydrogen peroxide content in cultures of Scenedesmus, as well as superoxide radicals in cultures of both tested algal species.

In summary, we confirmed that usnic acid has a phytotoxic effect on algae, but that algae are differentially sensitive, and that the lichen photobiont Trebouxia is significantly less affected than Scenedesmus. These conclusions are based on analyses of physiological processes including biomass production, composition of assimilation pigments (mostly by decrease of chlorophyll a content and increase of its phaeophytinization), chlorophyll a fluorescence, soluble proteins, and reactive oxygen species. Higher tolerance of Trebouxia cultures to usnic acid may be an adaptation resulting from the long term co-evolution of these algae with fungi that produce secondary metabolites. These also may act as allelochemicals that control cell division of photobiont cells inside lichen thalli, thus regulating balance between symbionts forming lichens.