Acta Botanica 2-2016 - za web.indd 186 ACTA BOT. CROAT. 75 (2), 2016 Acta Bot. Croat. 75 (2), 186–193, 2016 CODEN: ABCRA 25 DOI: 10.1515/botcro-2016-0034 ISSN 0365-0588 eISSN 1847-8476 The impact of cadmium on photosynthetic performance and secondary metabolites in the lichens Parmelia sulcata, Flavoparmelia caperata and Evernia prunastri Ana Maslać1, Maja Maslać2, Mirta Tkalec1* 1 University of Zagreb, Faculty of Science, Department of Biology, Rooseveltov trg 6, HR-10000 Zagreb, Croatia 2 Oikon Ltd. Institute of Applied Ecology, Trg senjskih uskoka 1–2, HR-10000 Zagreb, Croatia Abstract – Lichens are one of the most common air quality bioindicators. Airborne heavy metal pollution causes various physiological changes in lichens, but sensitivity to metal pollution is species specifi c. In this research, three lichen species (Parmelia sulcata, Flavoparmelia caperata and Evernia prunastri) were ex- posed to cadmium (50 mg L–1) in laboratory conditions. Photosynthetic effi ciency of photosystem II and content of secondary metabolites were determined after one, three and eight days of exposure. In all investi- gated species treatment of lichen thalli with cadmium signifi cantly changed Fv/Fm and RFd only after eight days of exposure. Quantifi cation of metabolites showed a decreased content of the medullary depsidones salazinic acid (in P. sulcata) and protocetraric acid (in F. caperata) but increased content of cortical depside atranorin (in P. sulcata) and dibenzofurane usnic acid (in F. caperata) after cadmium exposure. However, no changes in secondary metabolites were found in E. prunastri. Results show that investigated species are rela- tively resistant to short-term cadmium-exposure and that secondary metabolites could have an important role in the protection of primary metabolism from negative cadmium impacts, at least in some species. Key words: air pollution, heavy metal, HPLC, photosynthesis * Corresponding author, e-mail: mtkalec@zg.biol.pmf.hr Introduction Lichens are perennial, slow-growing organisms that have the ability to take in all necessary nutrients and water directly from the air. Due to the absence of cuticle and roots, they absorb pollutants over their entire surface. More- over, lichens have effi cient mechanisms that take in more nutrients than they need. It has been found that lichens are able to accumulate various metals (cadmium, lead, zinc, etc.) at high levels (Garty 2001, Aprile et al. 2010). Ever since the industrial revolution in the 1800s lichens have been recognized as bioindicators of air quality and, nowa- days, they are commonly used to indicate the presence of air pollutants. More recently, lichens have also been used as biomonitors, in experiments measuring the lichen physio- logical responses to atmospheric pollution over time and providing additional information about the amount and in- tensity of the exposure (Garty et al. 2003, Bačkor and Lop- pi 2009, Paoli et al. 2015a). So far, they have been used as biomonitors of atmospheric pollution from different sourc- es (Loppi et al. 2004, Branquino et al. 2008, Lackovičová et al. 2013, Paoli et al. 2015b). Signifi cant correlations between metal concentrations in the lichen thalli and in their environment have been found in many studies (Garty 2001, Bačkor et al. 2003, Carreras et al. 2005, Dzubaj et al. 2008, Stamenković et al. 2013). However, physiological responses of lichens to metals and their tolerance mechanisms are species-specifi c, ranging from relative resistance to high sensitivity. Exposure of lichens to metals can affect membrane integrity (Garty et al. 2003, Pi- sani et al. 2010), chlorophyll content (Bačkor and Zetíková 2003, Carreras and Pignata 2007), photosynthetic perfor- mance (Karakoti et al. 2014, Paoli et al. 2015a) and second- ary metabolites (Hauck et al. 2013, Gauslaa et al. 2016). Among heavy metals, cadmium (Cd) is considered to be particularly toxic for various lichen species causing adverse physiological changes (Sanità di Toppi et al. 2005). In- creased Cd content in the environment can be attributed to human activities such as battery production, industrial met- allurgical processes, combustion of fossil fuels and emis- sions from motor vehicles (Pacyna 1998). Lichens produce various secondary metabolites that have multiple functions in the interactions of the lichens with the THE IMPACT OF CADMIUM ON LICHENS ACTA BOT. CROAT. 75 (2), 2016 187 environment. Some of them are located in the upper cortex, while most are located in the medulla (Solhaug et al. 2009). These compounds have antiherbivore, antimicrobial and larvicidal effects, and can protect thalli from high UV irra- diation and oxidative stress. They might also have impor- tant role in metal homeostasis and lichen tolerance to pollu- tion, although the biochemical mechanisms are mostly unknown (Molnár and Farkas 2010). Nevertheless, it is known that lichen substances function in vitro as chelators of cations, including heavy metals (Bačkor and Loppi 2009). Few studies have compared metal pollution and sec- ondary compound concentration, and showed different re- sponses. Levels of medullary compounds in Hypocenomyce scalaris (lecanoric acid) and Cladonia furcata (fumarproto- cetraric acid) were increased by metal pollution (Pawlik- Skowrońska and Bačkor 2011). Białonska and Dayan (2005) found that levels of atranorin, a cortical compound, and medullary compounds physodic and hydroxyphysodic acid decreased, while medullary physodalic acid increased after transplantation of Hypogymnia physodes to the pollut- ed area. On the other hand, Paoli et al. (2015a) showed that the medullary compound caperatic acid decreased and the cortical compound usnic acid increased in Flavoparmelia caperata located close to the landfi ll. The aim of this study was to investigate the effects of Cd-exposure on photosynthetic performance and content of secondary metabolites in three widely distributed epiphytic lichen species Parmelia sulcata, Flavoparmelia caperata and Evernia prunastri in a short-term laboratory experi- ment. Materials and methods Lichen material The foliose lichens Parmelia sulcata Taylor (Fig. 1A) and Flavoparmelia caperata (L.) Hale (Fig. 1B), and the fruticose lichen Evernia prunastri (L.) Ach. (Fig. 1C) were collected from old branches of Quercus sp. in Maksimir Park (45°49’45”N, 16°01’17”E, 160 m above sea level) in Zagreb in October, 2014. Lichens were placed on several layers of fi lter paper wetted with distilled water, and then acclimated in a growth chamber for a week. For each spe- cies individual thalli were sprayed with 5 mL of Cd solution in a concentration of 50 mg L–1, which should cause toxic effects in a short time. Lichens sprayed only with distilled water were used as control. All lichens were kept in the growth chamber under fl uorescent light (60 μmol photons m–2 s–1, photoperiod of 16 h day/8 h night) at 22 ± 2 °C. Chlorophyll fl uorescence and secondary metabolite content were determined after one, three and eight days of Cd-ex- posure. All treatments were done in triplicate. Chlorophyll fl uorescence parameters Light-induced chlorophyll fl uorescence parameters at continuous saturating white light were measured using a chlorophyll fl uorometer (Qubit Systems Inc., Canada) in 30 min pre-darkened thalli. Low intensity red light was used to determine the minimal fl uorescence level (F0), and then continuous saturating light ca. 1500 μmol m–2 s–1 was ap- plied. Upon irradiation, the fl uorescence increased from F0 to the maximum Fm and then declined to steady state fl uo- rescence (Fs) during seven minutes. Maximum quantum yield of PSII (Fv/Fm), a widely used indicator of photosyn- thetic effi ciency of photosystem II, was calculated as (Fm – F0)/Fm (Maxwell and Johnson 2000). Additionally, the fl uo- rescence decrease ratio (RFd), a parameter directly related to the rate of photosynthesis (Lichtenthaler et al. 2005), was calculated as (Fm – Fs)/Fs, according to Lichtenthaler et al. (2005). High-performance liquid chromatography Lichen secondary metabolites were extracted from lyophilised thalli (18 mg) which were suspended in 1.5 mL acetone and incubated for 1 h at 4 °C. The samples were centrifuged for 15 min at 4 °C and 15 000 g. The superna- tant was separated with a pipette into a separate tube and centrifuged again for 30 min at 4 °C and 30 000 g. The su- pernatants were then transferred with a pipette into dark vial glass bottles. Secondary metabolites were analysed by high-perfor- mance liquid chromatography (HPLC) using a Perkin El- mer Series 200 system with a UV/VIS diode-array detector. Analytes were separated on a reverse-phase C18 Brownlee Speri-5 ODS column (5 μm, 250 × 4.6 mm, Perkin Elmer, USA) with pre-column (5 × 4.6 mm). The elution program was modifi ed according to Feige et al. (1993). The mobile phase consisted of 1% (v/v) phosphoric acid (A) and 100% methanol (B). For P. sulcata the elution program was: 0.6 min equilibration with 30% B, 11 min linear gradient from 30% to 70% B, 4 min linear gradient from 70% to 100% B, Fig. 1. Lichens used in the experiment: (A) Parmelia sulcata, (B) Flavoparmelia caperata and (C) Evernia prunastri. MASLAĆ A., MASLAĆ M., TKALEC M. 188 ACTA BOT. CROAT. 75 (2), 2016 10 min isocratic with 100% B and 6 min re-equilibration with 30% B. For E. prunastri and F. caperata the linear gra- dient from 70% to 100% B was 8 min instead of 4 min. The fl ow rate was 0.8 mL min–1, and elution was moni tored at 245 nm. The identifi cation of lichen metabolites was made by comparing retention times in combination with UV spec- tral data with known chromatographic data (Feige et al. 1993, Huneck and Yoshimura 1996). Quantifi cation was per- formed using calibration curves of individual compounds isolated from authentic-source lichens and the results were expressed as mg per gram of dry weight (mg g–1DW). Statistical analysis Results were shown as average ± standard error. Deter- mination of photochemical effi ciency and quantifi cation of secondary compounds content was performed in triplicate. For processing data Microsoft Excel 2010 and Statistica 10 (StatSoft Inc., SAD) were used. The results were compared by analysis of variance (ANOVA) and post hoc Tukey’s test. Differences between means were considered statisti- cally signifi cant at p ≤ 0.05. Results Fluorescence parameters In control lichen species the values of Fv/Fm parameter were around 0.625 during the experiment, except in F. caperata which had a slightly lower value (0.56) at the end of the experiment. However, the difference was not signifi - cant (p > 0.05) compared to the values measured on other days (Fig. 2). Lichens treated with Cd did not show signifi - cant differences (p > 0.05) from corresponding controls af- ter one and three days of exposure, while after eight days of exposure the Fv/Fm values in all species were signifi cantly lower than the values in untreated lichens measured on the same day (p < 0.01) and those measured in Cd-treated li- chens on the other days (p ≤ 0.01). In untreated lichens, the highest RFd value ( 2.4) was measured in F. caperata, but it decreased signifi cantly (p ≤ 0.05) to 1.5 after eight days of experiment (Fig. 3). In the other two species, the RFd values were lower (1.4 for E. prunastri and 1.7 for P. sulcata) and no signifi cant change (p < 0.05) in values was observed during the course of the A B C ab ab ab b ab a ab c 0 0.2 0.4 0.6 0.8 1 0 1 3 8 F v/ F m Days Flavoparmelia caperata Control Cadmium ab ab ab b ab a ab c 0 0.2 0.4 0.6 0.8 1 0 1 3 8 F v/ F m Days Evernia prunastri Control Cadmium a a a a a a a b 0 0.2 0.4 0.6 0.8 1 0 1 3 8 F v/ F m Days Parmelia sulcata Control Cadmium A B C a a a b a a a b 0 1 2 3 4 0 1 3 8 R F d Days Flavoparmelia caperata Control Cadmium ab ab ab a ab ab ab b 0 1 2 3 4 0 1 3 8 R F d Days Evernia prunastri Control Cadmium ab ab ab a ab ab ab b 0 1 2 3 4 0 1 3 8 R F d Days Parmelia sulcata Control Cadmium Fig. 2. Maximum effi ciency of PSII (Fv/Fm) in lichens Parmelia sulcata (A), Flavoparmelia caperata (B) and Evernia prunastri (C) before (0 day), and after one, three and eight days of the expo- sure to cadmium in a concentration of 50 mg L–1. Different letters above the bars denote signifi cantly different results (p ≤ 0.05). Fig. 3. Fluorescence decrease ratio (RFd) in lichens Parmelia sul- cata (A), Flavoparmelia caperata (B) and Evernia prunastri (C) before (0 day), and after one, three and eight days of the exposure to cadmium in a concentration of 50 mg L–1. Different letters above the bars denote signifi cantly different results (p ≤ 0.05). THE IMPACT OF CADMIUM ON LICHENS ACTA BOT. CROAT. 75 (2), 2016 189 experiment. In lichens treated with Cd, the RFd values were not signifi cantly different (p > 0.05) from those of unex- posed controls after one and three days of exposure. How- ever, the values signifi cantly decreased in E. prunastri (p = 0.03) and P. sulcata (p = 0.05) eight days after Cd-exposure compared to the corresponding control values measured on the same day. In F. caperata, the RFd of Cd-treated thalli was signifi cantly lower (p < 0.01) than RFd values obtained for treated lichens earlier in the experiment, but it was not signifi cantly different (p > 0.05) from the control value measured on the same day. Secondary metabolites In P. sulcata acetone extracts we successfully separated two secondary metabolites and identifi ed them according to their retention times and UV spectral data (Fig. 4). The ma- jor metabolite was depsidone salazinic acid (peak at 24 min) while the minor metabolite was depside atranorin (peak at 36 min). In untreated samples, the content of sala- zinic acid slightly decreased during the exposure experi- ment, from 2.04 to 1.76 mg g–1DW, while the content of atra- norin did not signifi cantly change, with values around 0.3 mg g–1DW (Tab. 1). In Cd-treated P. sulcata, the content of salazinic acid was lower than in the corresponding control, but signifi cantly (p < 0.01) only after three days of expo- sure when it decreased to 1.10 mg g–1DW. In contrast, the content of atranorin was signifi cantly higher (p < 0.01) than in the corresponding controls after one and eight days of Cd-exposure, amounting 0.58 and 0.62 mg g–1DW, respec- tively. In F. caperata samples, HPLC analysis revealed depsi- done protocetraric acid (peak at 20 min), as a major metab- olite, and dibenzofurane usnic acid (peak at 35–36 min), as a minor metabolite (Fig. 5). In control lichens, the contents of metabolites slightly increased during the experiment, from 0.77 to 0.99 mg g–1 DW for protocetraric acid and from 0.22 to 0.52 mg g–1DW for usnic acid (Tab. 1). In Cd-treated lichens, the content of protocetraric acid was slightly higher Fig. 4. Chromatogram of the acetone extract of Parmelia sulcata at 245 nm. Identifi ed peaks: acetone (5 min), salazinic acid (24 min) and atranorin (36 min). UV spectral data of metabolites quantifi ed in the experiment are also shown. Tab. 1. Content of secondary metabolites quantifi ed in untreated (control) and cadmium-treated lichens Parmelia sulcata, Flavoparmelia caperata and Evernia prunastri after one, three and eight days of exposure. Different letters in superscript denote signifi cantly different results (p ≤ 0.05). DW – dry weight. Lichen metabolites (mg g–1DW) Parmelia sulcata Flavoparmelia caperata Evernia prunastri salazinic acid atranorin protocetraric acid usnic acid evernic acid atranorin Treatment Days Control 1 2.04±0.01a 0.27±0.06b 0.77±0.09b 0.22±0.00b 1.08±0.09a 2.11±0.07a 3 1.95±0.23a 0.35±0.07b 0.97±0.09ab 0.53±0.02ab 1.16±0.10a 2.03±0.31a 8 1.76±0.07a 0.26±0.03b 0.99±0.02a 0.52±0.05ab 1.07±0.11a 2.02±0.24a Cadmium 1 1.73±0.03a 0.58±0.02a 0.92±0.01ab 0.29±0.04b 1.05±0.06a 2.01±0.11a 3 1.10±0.18b 0.35±0.08b 0.80±0.02ab 0.42±0.00ab 1.15±0.11a 2.14±0.04a 8 1.57±0.22ab 0.62±0.06a 0.76±0.10b 0.75±0.19a 1.10±0.12a 1.89±0.09a MASLAĆ A., MASLAĆ M., TKALEC M. 190 ACTA BOT. CROAT. 75 (2), 2016 (0.92 mg g–1DW) than in the corresponding control after one day of Cd-exposure. However, it decreased to 0.80 mg g–1DW after three days of Cd-exposure and then to 0.76 mg g–1DW after eight days of exposure, which was signifi cantly lower (p < 0.05) than in corresponding control. Content of usnic acid in Cd-treated F. caperata was mostly similar to control values after one and three days of exposure, but the value increased signifi cantly (p < 0.01) up to 0.75 mg g–1DW after eight days of exposure. In samples of E. prunastri we found depsides evernic acid (peak at 31 min) and atranorin (peak at 36–37 min), as major metabolites, and chloroatranorin (peak at 37–38 min) as a mi- nor metabolite (Fig. 6). We further quantifi ed only the two ma- jor metabolites. In control samples, the contents of evernic acid and atranorin did not change during experimental period, with the values amounting 1.10 for evernic acid and 2.05 mg g–1DW for atranorin (Tab. 1). Treatment with Cd did not cause any sig- nifi cant change in the content of the investigated metabolites. Fig. 5. Chromatogram of the acetone extract of Flavoparmelia caperata at 245 nm. Identifi ed peaks: acetone (5 min), protocetraric acid (19.5 min) and usnic acid (36 min). UV spectral data of metabolites quantifi ed in the experiment are also shown. Fig. 6. Chromatogram of the acetone extract of Evernia prunastri at 245 nm. Identifi ed peaks: acetone (5 min), evernic acid (31 min), usnic acid (35.5 min), atranorin (36.5 min) and chloroatranorin (37.5 min). UV spectral data of metabolites quantifi ed in the experiment are also shown. THE IMPACT OF CADMIUM ON LICHENS ACTA BOT. CROAT. 75 (2), 2016 191 Discussion Lichens, due to their morphology and physiology, re- ceive all nutrients from the atmosphere, including heavy metals. In this study, the effects of short-term Cd exposure on photosynthetic performance and secondary metabolites content were studied using the epiphytic lichen species P. sulcata, E. prunastri and F. caperata. These lichen species had already been used as bioindicators and/or biomonitors of atmospheric pollution from different sources (Loppi et al. 2004, Lackovičová et al. 2013, Stamenković et al. 2013). In the last 20 years, chlorophyll fl uorescence measure- ments were successfully employed in various lichen studies investigating heavy metal pollution (Bačkor et al 2010, Ka- rakoti et al. 2014, Paoli et al. 2015a). The maximum quan- tum yield of PSII (Fv/Fm), besides being an important indi- cator of photosynthetic effi ciency, can indicate the vitality of a lichen photobiont (Paoli et al. 2015a). In all three li- chen species investigated in our study, the Fv/Fm values of untreated thalli were mostly in accordance with values characteristic for lichens (0.63–0.76), which are lower than those found in plants (0.74–0.83) (Jensen 2002). In Cd- treated thalli signifi cantly lower Fv/Fm values, indicating damage of the photosynthetic apparatus, were observed. However, a decrease was observed only eight days after the exposure suggesting the relative resistance of the lichen photobiont to short-term exposure to high Cd concentra- tion. Bačkor et al. (2010) also reported relatively low toxic- ity of Cd, compared to other metals, e.g. Cu in lichens Peltigera rufescens and Cladina arbuscula 24 h after the exposure. Karakoti et al. (2014) showed that the thallus of lichen Pyxine cocoes, which contained different amounts of various metals did not show a decrease in Fv/Fm. In contrast, a decrease of Fv/Fm value was observed in the epiphytic fru- ticose lichen Ramalina lacera containing higher amounts of Ba, Ni, S, V and Zn after exposure to anthropogenic pollu- tion (Garty et al. 2000). It seems that sensitivity of photo- synthetic effi ciency to different metals varies between dif- ferent lichen species. A delay in the Cd-effect on photosynthetic performance observed in our study might suggest that metal toxicity effect could depend on time of exposure. It has been found that prolonged exposure to lead (Pb) leads to an additional decrease of Fv/Fm in Flavoparme- lia caperata (Garty 2002). In our study, we also employed a fl uorescence decrease ratio (RFd), a parameter which, when measured at saturation irradiance is directly correlated to the net CO2 assimilation rate (Lichtenthaler et al. 2005). The RFd values in control lichens were mostly similar to RFd values found in lichen Anaptychia ciliaris (Valladares et al. 1995). In Cd-treated lichen species, RFd showed the same trend as Fv/Fm parameter, confi rming tolerance of a photo- synthetic process to Cd in the investigated species. Interest- ingly, prolonged time in laboratory conditions caused a de- crease of both, Fv/Fm and especially RFd in untreated thalli of F. caperata, suggesting that this species is sensitive to the environmental conditions that are not natural to it. Most secondary compounds combinations in lichens are species-specifi c and therefore are widely used in lichen tax- onomy and systematics (Molnár and Farkas 2010). In li- chen species analysed in this study almost all lichen sub- stances specifi c for particular species (Nash et al. 2002) were successfully detected: salazinic acid and atranorin in P. sulcata; evernic acid, atranorin and chloroatranorin in E. prunastri; and protocetraric acid and usnic acid in F. caper- ata. We did not fi nd only two compounds, consalazinic acid, a minor metabolite in P. sulcata and caperatic acid, a minor metabolite in F. caperata. Caperatic acid is an aliphatic acid which is not detectable by the HPLC method used in this study. Lichen metabolites play an important role in tolerance of lichens to metal pollution (Bačkor and Loppi 2009). Some reports say that compounds located in the medulla (e.g. depsidones) might be chelators of cations (Solhaug et al. 2009). In this study, we found that exposure to Cd in foliose lichens decreased content of the medullary depsidones salazinic acid (in P. sulcata) and protocetraric acid (in F. caperata), but increased the content of cortical depside atranorin (in P. sulcata) and dibenzofurane usnic acid (in F. caperata), whereas metabolites (all depsides) of fruticose lichen E. prunastri did not change after Cd treat- ment. These results are in accordance with Lackovičová et al. (2013) and Paoli (2015a) suggestion that the ratio be- tween cortical and medullary secondary metabolites can in- crease in lichen samples in polluted environment. However, there are some opposite fi ndings that report increased med- ullary compounds and decreased cortical compounds con- tent after heavy metal exposure. For example, Białonska and Dayan (2005) found increased levels of medullary dep- sidone physodalic acid but decreased levels of cortical dep- side atranorin in Hypogymnia physodes transplanted to in- dustrial areas with high emissions of heavy metals. Also, Pawlik-Skowrońska and Bačkor (2011) obtained higher amounts of the medullary compounds, i.e. depside lecanor- ic acid in Hypocenomyce scalaris and depsidone fumarpro- tocetraric acid in Cladonia furcata at a mining site polluted with Pb and Zn. Results of several studies suggested that lichen metabolites control metal homeostasis by promoting the uptake of certain metal cations and/or reducing the ad- sorption of others that could possibly be toxic (Molnár and Farkas 2010). For example, physodalic acid from Hypo- gymnia physdodes, increase the Fe2+ uptake and decrease the uptake of Cu2+, Mn2+ and Na+ (Hauck and Huneck 2007, Hauck 2008). Dibenzofurane usnic acid and divaricatic acid were both found to increase the intracellular uptake of Cu2+ in Evernia mesomorpha and Ramalina menziesii (Hauck et al. 2009), but reduce the Mn2+ uptake. Recently, UV spectroscopic studies and X-ray diffraction analyses showed that the complexation of metal ions with lichen substances is widespread (Bačkor and Fahselt 2004, Hauck et al. 2009). The most recent study by Gauslaa et al. (2016) reported that medullary metabolites in fruticose (Ramalina farinacea, Usnea dasypoga) were reduced in polluted sites, but were not in foliose lichens (Parmelia sulcata, Lobaria pulmonaria) whereas cortical metabolites did not change in any species. Moreover, L. pulmonaria experienced strong reduction in viability in polluted sites despite increased content of medullary compound stictic acid which has a possibility of heavy metal chelating. All these results taken together might suggest that the function of lichen secondary MASLAĆ A., MASLAĆ M., TKALEC M. 192 ACTA BOT. CROAT. 75 (2), 2016 metabolites in metal homeostasis depend on several factors including chemical structure of compounds (depsides/dep- sidones/dibenzofuranes), their location (cortex/medulla) as well as lichen form of growth (foliose/fruticose) and type of metal. Moreover, Valencia-Islas et al. (2007) suggested that increased content of cortical usnic acid in Ramalina asahi- nae could contribute to the antioxidant protection against air pollution. It seems that although our knowledge about the importance of lichen metabolites has increased in the last few years, their biological roles and interactions have not yet been entirely understood. 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