Chemosphere 112 (2014) 217–224

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Mycorrhizal fungi modulate phytochemical production and antioxidant activity of Cichorium intybus L. (Asteraceae) under metal toxicity a _ P. Rozpa˛dek a,c,⇑, K. We˛zowicz , A. Stojakowska b, J. Malarz b, E. Surówka c, Ł. Sobczyk a, T. Anielska a, _ d, Z. Miszalski c, K. Turnau a,d R. Wazny a

Institute of Environmental Sciences, Jagiellonian University, Kraków, Poland Department of Phytochemistry, Institute of Pharmacology, Polish Academy of Sciences, Kraków, Poland Institute of Plant Physiology, Polish Academy of Sciences, Kraków, Poland d Malopolska Centre of Biotechnology, Jagiellonian University, Kraków, Poland b c

h i g h l i g h t s  Inoculation of Cichorium intybus with AM fungi improves its productivity.  Phenolic and enzymatic antioxidants were examined in inoculated plants.  Toxic metal tolerance was increased in AMF inoculated chicory.  AM fungi improved chicory photochemistry in toxic metal stress conditions.

a r t i c l e

i n f o

Article history: Received 19 December 2013 Received in revised form 4 April 2014 Accepted 6 April 2014

Handling Editor: A. Gies Keywords: Mycorrhiza Hydroxycinnamates Sesquiterpene lactones Cichorium intybus

a b s t r a c t Cichorium intybus (common chicory), a perennial plant, common in anthropogenic sites, has been the object of a multitude of studies in recent years due to its high content of antioxidants utilized in pharmacy and food industry. Here, the role of arbuscular mycorrhizal fungi (AMF) in the biosynthesis of plant secondary metabolites and the activity of enzymatic antioxidants under toxic metal stress was studied. Plants inoculated with Rhizophagus irregularis and non-inoculated were grown on non-polluted and toxic metal enriched substrata. The results presented here indicate that AMF improves chicory fitness. Fresh and dry weight was found to be severely affected by the fungi and heavy metals. The concentration of hydroxycinnamates was increased in the shoots of mycorrhizal plants cultivated on non-polluted substrata, but no differences were found in plants cultivated on metal enriched substrata. The activity of SOD and H2O2 removing enzymes CAT and POX was elevated in the shoots of mycorrhizal plants regardless of the cultivation environment. Photochemical efficiency of inoculated chicory was significantly improved. Our results indicate that R. irregularis inoculation had a beneficial role in sustaining the plants ability to cope with the deleterious effects of metal toxicity. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Vegetables, fruits and herbs are important components of the human diet. Their quality depends on the abundance of antioxidants and other biologically active organic compounds, as well as on the composition of micro-and macronutrients. Common chicory (Cichorium intybus L.), a perennial herb of the Asteraceae family, is utilized as a medicinal plant, coffee substitute, vegetable crop or animal forage. Due to the abundance of biologically active ⇑ Corresponding author at: Institute of Environmental Sciences, Jagiellonian University, Kraków, Poland. Tel.: +48 124251833. E-mail address: [email protected] (P. Rozpa˛dek). http://dx.doi.org/10.1016/j.chemosphere.2014.04.023 0045-6535/Ó 2014 Elsevier Ltd. All rights reserved.

secondary metabolites, chicory has received much attention in recent years. Among secondary metabolites terpenoids (e.g. lactucin-like sesquiterpene lactones) and phenolic compounds (e.g. flavonoids and hydroxycinnamates) are the best described. C. intybus is a cosmopolitan weed tolerating a broad range of climatic and soil conditions. It is also known as an indicator for toxic metal contamination (Simon et al., 1996) and is relatively easy to cultivate under laboratory conditions. Additionally, it interacts with a diversity of microorganisms, including AMF (arbuscular mycorrhiza fungi). Mycorrhizal fungi colonize the root and stimulate growth, plant chemistry, morphology and structure, affect rhizosphere microbial composition and induce resistance to pathogens or attenuate environmental toxicity (Smith and Read,

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2008). These characteristics make C. intybus an ideal model in acclimation/tolerance to toxic substances research. Heavy metals, like other abiotic stressors may induce the production of ROS (reactive oxygen species) and cause oxidative stress. ROS, besides their deleterious effects, act as signaling molecules (Foyer and Noctor, 2005; Opdenakker et al., 2012) adjusting cell metabolism to the changing environment. These adjustments determine the quality of the plant material, influencing survival and promoting various adaptations in plants. As shown in previous studies mycorrhizal fungi may provide protection against ROS and influence ROS homeostasis (Liu et al., 2011; Estrada et al., 2013). The protective mechanisms shown in plants include, among others antioxidant enzymes such as SOD, CAT and POX (Strzałka et al., 2010; Foyer and Shigeoka, 2011). With that in mind, shedding light on the mechanisms of plant– microbe interactions can be essential in enhancement of phytoremediation techniques that already were shown to be effective in hazardous site treatment. In the present study we evaluated the interaction between chicory plants and arbuscular mycorrhizal fungi (AMF) under the presence of toxic metals. We have also made an attempt to determine the role of AMF in controlling the abundance of biologically active metabolites and activity of antioxidant enzymes of C. intybus under the presence of toxic metals.

2. Methods 2.1. Plant material Chicory seeds were provided by the Garden of Medicinal Plants of the Institute of Pharmacology, Polish Academy of Sciences in Kraków. Seeds were germinated on a wet filter paper in Petri dishes. Four day-old seedlings were transferred into 400 mL pots containing (1) sterile substratum composed of a mixture of ARO garden soil (Terrasan, Poland), sand, and expanded clay in a 5:4:1 (v/v/v) ratio, (2) sterile substratum composed of a mixture of garden ARO soil, sand, expanded clay and industrial waste substratum from the ZG Trzebionka in a 10:8:2:5 (v/v/v/v) ratio containing: Zn – 12 000 lg g 1, Pb – 2400 lg g 1 and Cd – 100 lg g 1 (for detailed description see Orłowska et al., 2005; Ryszka and Turnau, 2007). For metal extractable concentrations see Table 1. Both substrata were enriched with 25 lg per g of rock phosphate (SIARKOPOL, Poland). Plants were cultivated in the absence (NM, non-mycorrhizal) or presence (M, mycorrhizal) of AM fungal inoculum (ca. 15 g per pot) containing propagules of Rhizophagus irregularis (Blaszk., Wubet, Renker & Buscot) C. Walker & Schüßler = Glomus intraradices (UNIJAG.PL24-1), which were carefully mixed into the upper layer of the substrata prior to seedling transfer. Pots were kept in Sunbags (Sigma) under greenhouse conditions at 20 °C average air temperature, at light intensity ca. 200 lmol m 2 s 1 and 12 h photoperiod for 16 weeks. Plants were irrigated weekly with 30 mL of distilled water per pot. Plants were grown 3 per pot in 4 pots for M plants and 8 pots for control. For one sample 2 plants from each pot were collected.

Table 1 PCA data supplementary to Figs. 4 and 5 showing correlations between secondary metabolites. Element

Cd Pb Zn

Content Total

Extr. in NH4NO3

Extr. in Ca(NO3)2

108.15 2372.03 12 067

2.12 0.19 114.74

1.35 1.972 3.81

2.2. Mycorrhizal evaluation Estimation of mycorrhizal parameters was performed by the modified method described by Phillips and Hayman (1970). Briefly, after washing in tap water, roots were softened in 10% KOH for 24 h, washed in water, acidified in 5% lactic acid for 1 h at room temperature and stained in 0.01% aniline blue in pure lactic acid for 24 h. After staining the roots were stored in pure lactic acid, cut into 1 cm pieces, mounted on slides in glycerol and analyzed. Mycorrhizal frequency (F), relative (M) and absolute (m) mycorrhizal root length, relative (A) and absolute (a) arbuscular richness were assessed according to Trouvelot et al. (1986; en.u-bourgogne.fr.). 2.3. Phytochemical analysis 2.3.1. Standards and reagents Standards were purchased from Sigma–Aldrich Co. (St. Louis, MO, USA), Roth (Karlsruhe, Germany) and isolated in the Phytochemisty Department of the Institute of Pharmacology PAS in Kraków (for details see Supplementary data). 2.3.2. Quantification of hydroxycinnamates Air dried, pulverized plant shoots and roots (0.1 g) was extracted with 10 mL of 70% methanol (MeOH) at room temperature for 3 h on a rotary shaker (100 rpm). The mixture was filtered and residue re-extracted with 10 mL of the fresh solvent. The extracts were pooled together and evaporated under reduced pressure. The dry residue was re-dissolved in 70% MeOH (1 mL) and centrifuged (11 340 g, 5 min) prior to HPLC analysis. Analytical RP-HPLC separations were performed with the Agilent 1200 Series HPLC system (Agilent Technologies, USA) equipped with a diode array detector (DAD). Chromatographic separations of hydroxycinnamates were carried out at 25 °C, on a Zorbax Eclipse XDB-C18 column, 4.6  150 mm (Agilent Technologies, USA), with a mobile phase consisting of H2O/HCOOH/CH3COOH 99/0.9/0.1 (solvent A) and MeCN/MeOH/HCOOH/CH3COOH 89/10/0.9/0.1 (solvent B), at a flow rate of 1 mL min 1, with 5 lL injections. Gradient elution conditions described by Spitaler et al. (2006) were applied. 2.3.3. Quantification of lactucin-like sesquiterpene lactones Air dried pulverized plant shoots and roots (0.1 g) was extracted twice with 10 mL of MeOH at room temperature. The pooled extracts were evaporated in vacuo and the residue was dissolved in 70% MeCN (1 mL), left to stand overnight at 4 °C, centrifuged (11 340 g, 5 min) and analyzed by RP-HPLC. Samples (5 lL) were injected into a Purospher RP-18e (3  125 mm, particle size 5 lm) column (Merck, Darmstadt, Germany) and eluted with a mobile phase consisting of water and MeCN, at a flow rate of 1 mL min 1, at 40 °C. Gradient elution conditions described by Grass et al. (2006) were applied. All analysis were performed in 5 replicates. 2.3.4. SOD, CAT and total POX activity assays 2.3.4.1. Protein extraction and quantification. Total protein was extracted from plant shoots as described by Miszalski et al. (1998). Protein content was quantified according Bradford (1976) using BSA as a standard. 2.3.4.2. Enzyme analysis. SOD activity was determined according to Beauchamp and Fridovich (1973) 20 lg of total protein was separated in the non-continues Laemmli buffer system (Laemmli, 1970). For total peroxidase (POX) activity 20 lg of proteins from crude extracts were separated in the non-continues Laemmli buffer system (Laemmli, 1970) and stained according to Ros Barcelo et al. (1987). SOD and POX activity measurements were performed in 4 replicates. Densitometric analysis was performed with imageJ (NIH).

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CAT activity was measured according to Aebi (1984) with modifications described by Rozpa˛dek et al. (2013). Measurements were performed in 6 replicates. 2.4. Chlorophyll a fluorescence measurements Chlorophyll fluorescence measurements were performed with the FluorCAM imaging system (PSI, Brno, Czech Republic) according to the instruction manual. Measurements were carried out on dark adapted mature leaves lacking visible damages in the middle of the photoperiod, directly before harvest. Measurements were performed in 5 replicates. 2.5. Chlorophyll and flavonoid content Chlorophyll and flavonoid content was determined with the Dualex (Dynamax) according to the manufacturers instruction. Measurements were performed 5 in replicates.

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and non-polluted substratum we performed Principal Component Analysis (PCA). Two-way ANOVA was performed on scores from the first and second principal axis. Two-way-ANOVA was also carried out in case of biomass, chlorophyll, (POX, CAT, POX, fluorescence) and mycorrhizal colonization. 3. Results 3.1. Mycorrhizal colonization Roots of chicory from P (polluted) and NP (non-polluted) substrata inoculated with R. irregularis, showed no statistically significant differences in mycorrhizal colonization (F% 100, M% 93–95, A% 60–88). The roots were in both cases almost fully colonized by the mycelium (F% = 100) that grew between cells (forming Arum type mycorrhiza) or from the cell to cell forming the intermediate type between Arum and Paris. In plants cultivated without the AM inoculum no mycorrhizal structures were observed. The presence of other fungal symbionts (such as DSE or Olpidum spp.) was negligible.

2.6. Statistical analysis

3.2. Plant biomass

To determine differences in selected metabolite concentrations between mycorhizal plants, non-mycorhizal and grown in polluted

Cultivation of M and NM chicory on control substrate (devoid of heavy metals) had no significant effect on shoot fresh and dry mass

Fig. 1. Mean fresh (A,C) and dry (B,D) weight of mycorrhizal (M) and non-mycorrhizal (NM) Cichorium intybus L. shoots (A,B) and roots (C,D) grown on control (NP) and enriched in heavy metals (P) substrate. Capital letters above bars indicate statistically significant differences between NM and M plants. Capitol letters next to bars indicate statistically significant differences between plants grown in NP and P soil. Tuckey post-hoc test (where significant interaction acquired) results are indicated by letters above bars.

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(Fig. 1A and B), however, it significantly (p = 0.01) affected fresh and dry biomass of the roots (Fig. 1C and D). In M and NM plants cultivated on polluted substratum, differences were found in the fresh (p = 0.02) and dry (p = 0.02) weight of shoots (Fig. 1A and B). Shoot fresh weight of plants cultivated on polluted substratum was significantly higher compared to that of mycorrhizal plants from control soil while the opposite was found in the case of fresh and dry weight of mycorrhizal roots (Fig. 1A–D). 3.3. Chlorophyll and total flavonoids Inoculated and non-inoculated plants cultivated on control substratum did not differ in chlorophyll content regardless of the substratum. Differences in total flavonoid concentrations were statistically insignificant, although plants from polluted soil were slightly enriched in flavonoids (data not shown). 3.4. Hydroxycinnamates and sesquiterpene lactones The Principal Component Analysis (PCA) of secondary metabolite concentrations (Supplementary Table) in plants cultivated in P and NP, NM and M plants (shoots and roots) revealed significant differences between patterns (Fig. 2A). The first axis (differences between plants grown on polluted and non-polluted substrata) represents 34.5% variability, while the second (differences between inoculated and non-inoculated plants) 19.3%. Significant differences were found between plants cultivated in both types of the substratum (p = 0.0001) and in the presence/absence of mycorrhizal fungi (p = 0.006) and its interaction (p = 0.04). The concentration of secondary metabolites differed in shoots of nonmycorrhizal, and mycorrhizal plants grown on polluted and nonpolluted substrata, however, the difference between mycorrhizal and non-mycorrhizal plants were significant only on non-polluted

substrata (Fig. 2B). No statistically significant differences were found in case of the 2nd axis. Hydroxycinnamic acid derivatives (the longest arrows): caftaric acid (CTA), cichoric acid (DCTA) and uCA were selected as predictors in interaction analysis with ANOVA. Concentrations of the selected hydroxycinnamates showed similar patterns and were correlated (Table 3 Supplementary data). The concentration of the examined compounds was lower in non-mycorrhizal plants cultivated on control substratum (lacking toxic metals), while non-mycorrhizal plants cultivated on polluted substrata had similar or even lower concentrations than non-mycorrhizal plants from non-polluted substrata. There was no significant increase of the hydroxycinnamate concentrations in plants from toxic metal supplemented soil in mycorrhizal plant shoots in comparison to non-mycorrhizal chicory (Table 3). In the PCA analysis of secondary metabolite concentrations in roots both axis represent 69.4% (axis one – 38.4 and axis two – 30.9% ) variability (Fig. 3A). According to the first axis inoculation of chicory had no significant effects on the content of the examined metabolites. For scores from axis 2 no differences were found between non-mycorrhizal roots from polluted and non-polluted substratum, while mycorrhizal plants grown on both types of substrata significantly differed from non-mycorrhizal. Sesquiterpene lactones (including 8-deoxylactucin (8-DeoxyLC), lactucopicrin (Lpicr), and jacquinelin (Jacq) were strong predictors of axis 1 (Fig. 5). These compounds were present in higher concentrations in samples cultivated on polluted soil. In the case of 8-DeoxyLC NM and M samples polluted soil showed a significant difference in comparison to NM and M samples from control soil (no toxic metal addition). In the case of other metabolites examined differences were significant between M and NM samples from control substratum and M samples from polluted soil (Fig. 3B and C). Hydroxycinnamic acid derivatives such as CTA, DCTA and 3,5DCQA were strong predictors in reference to the second axis.

Fig. 2. PCA diagram (A) illustrating the relationships between the content of the examined secondary metabolites from chicory shoots between different groups. Samples are grouped in accordance to the content of a particular metabolite. Each polygonal figure represents a different group of samples. (B) Two-way ANOVA analysis for scores from the first principal axis and for uCA (C); the variable with the highest differentiating effect (with highest loading). ANOVA results for the second axis showed no significant differences, thus weren’t shown in the diagram. The numerical notation on the axis indicates the percent of the variance account for the principal axis. CTA - caftaric acid, 5CQA - chlorogenic acid, DCTA - cichoric acid, uCA - unidenti-fied derivative of caffeic acid, 3,5-DCQA - 3,5-dicaffeoylquinic acid; (B), 1 - lactucin (LC), 2 - 8-deoxylactucin glucoside (CrepA), 3 - jacquinelin glucoside (CrepB), 4 - 8-deoxylactucin (8-DeoxyLC), 5 - jacquinelin (Jacq), 6 - lactucopicrin/11b,13-dihydrolactucopicrin (Lpicr). Marks as in Fig. 1.

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Fig. 3. PCA diagram (A) illustrating the relationships between the content of the examined secondary metabolites from chicory roots between different groups. Samples are grouped in accordance to the content of a particular metabolite. Each polygonal figures represents a different group of samples. Two-way ANOVA analysis for scores from the first principal axis (B) and for the second principal axis (C), for 8-DeoxyLC (D) and CTA (E); variables with the highest differentiating effects (with highest loading for first and second axis respectively). The numerical notation on the axis indicates the percent of the variance account for the principal axis. CTA - caftaric acid, 5-CQA - chlorogenic acid, DCTA - cichoric acid, uCA - unidenti-fied derivative of caffeic acid, 3,5-DCQA - 3,5-dicaffeoylquinic acid; (B), 1 - lactucin (LC), 2 - 8-deoxylactucin glucoside (CrepA), 3 jacquinelin glucoside (CrepB), 4 - 8-deoxylactucin (8-DeoxyLC), 5 - jacquinelin (Jacq), 6 - lactucopicrin/11b,13-dihydrolactucopicrin (Lpicr). Marks as in Fig. 1.

According to the ANOVA, the concentration of the examined substances did not differ in samples from inoculated plants from polluted soil. 3.5. Antioxidant enzymes CAT activity measured spectrophotometrically (Fig. 4A) was significantly increased in inoculated plants, while the addition of toxic metals to the substratum did not affect enzyme activity. Total peroxidase activity was significantly higher in inoculated plants compared to non-inoculated. Metal toxicity did not affect POX activity (Fig. 4B). SOD activity was significantly increased in non-inoculated plants from polluted substratum compared to plants grown in control soil. Additionally, the presence of mycorrhiza resulted in higher SOD activity regardless of the presence/absence of toxic metals (Fig. 4D) 3.6. Photochemical activity The presence of heavy metals and/or R. irregularis in the substratum affected photochemistry in chicory. Fv/Fm values increased in M plants, but pollution decreased Fv/Fm values in both M and NM plants (Fig. 5B). PSII efficiency significantly decreased in NM plants under toxic metal stress, whereas no differences were reported in the presence of mycorrhiza (Fig. 5C). The NPQ parameter, indicating the ability to dissipate excess light energy showed a decreased value in mycorrhizal plants in comparison to non-mycorrhizal plants (Fig. 5A). 4. Discussion Even though significant progress in the development of technologies allowing the employment of chicory in commercial

applications has been made in recent years, there still is a pressing need to improve ecological cultivation to obtain high-quality material (Wang and Cui, 2011). So far, efforts to improve cultivation methods employing symbiotic organisms such as mycorrhizal fungi have been insufficient. Such technologies may reduce the use of chemicals in agriculture. As demonstrated in this study the introduction of AMF into chicory cultivation under greenhouse conditions increases plant fitness and the content of biologically active compounds. A similar phenomenon was already documented with several other herbs (Jurkiewicz et al., 2010; Zubek et al., 2012). Phenolic compounds have many distinct functions in plants (Sawa et al., 1999; Andrade et al., 2013). They may play a key role in the crosstalk between plants and symbiotic fungi (Scervino et al., 2005a, 2005b). As shown in in vitro studies plant flavonoids affect spore germination, growth of hyphae and stimulate root colonization (Larose et al., 2002; Vierheilig and Piché, 2002). The accumulation of flavonoids in plants was shown to be dependent on the developmental stage of the symbiosis as well as on species of mycorrhizal fungus (Larose et al., 2002). Changes in gene expression of flavonoid biosynthetic pathway proteins were reported during the initial phases of the interaction (Liu et al., 2003) and during colonization (Harrison and Dixon, 1994). In the present study flavonoids were analyzed in the advanced stage of mycorrhiza formation and a significant increase of metabolites was reported in the shoots and roots of chicory. Hydroxycinnamates and sesquiterpene lactones were found to be important in defense against herbivores, insects and pathogens (Fontana et al., 2009). Moreover, an antimicrobial phytoalexin (sesquiterpene) is produced by chicory after inoculation with the bacterium Pseudomonas cichorii (Monde et al., 1990). There are numerous reports indicating ROS and RNS (reactive nitrogen species) scavenging properties of plant hydroxycinnamates (Pellati et al., 2004; Olmos et al., 2008). Moreover, hydroxycinnamates were found to determine resistance of chrysanthemum (Dendranthema grandi-

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Fig. 4. The activity of antioxidant enzymes: CAT (A), POX (B) and SOD (C) in shoots of mycorrhizal (M) and non-mycorrhizal (NM) Cichorium intybus L. grown on control (NP) and enriched in heavy metals (P) substrate. Marks as in Fig. 1.

flora) against western flower thrip (Frankliniella occidentalis) (Leiss et al., 2009) and were active as infection inhibitors against the fungus Alternaria alternata (Kodoma et al., 1998). Here, in the shoots of mycorrhizal plants an accumulation of caffeic acid derivatives such as caftaric acid (CTA), cichoric acid (DCTA) and DCQA was observed. In the roots of mycorrhizal plants the concentration of sesquiterpene lactones including 8-DeoxyLC, Jacq and LPikr was increased. The examined compounds are not only potent antioxidants, but also posses hepatoprotective, anti-inflammatory, analgesic, sedative and anti-malarial properties (Basnet et al., 1996; Bischoff et al., 2004; Cavin et al., 2005; Wesołowska et al., 2006; An et al., 2008). Thus, the use of mycorrhiza in chicory cultivation can improve its dietary properties. In this report we have presented evidence for the beneficial role of AMF in increasing plant fitness under toxic metal stress. Extreme conditions (heat shock, chilling, high light, toxic metals) induces accumulation of phenolics in lettuce (Sakihama and Yamasaki, 2002; Oh et al., 2009). Toxic metals, like other abiotic and biotic stress agents can affect physiological and biochemical processes and generate reactive oxygen species (ROS) (Halliwell and Gutteridge, 1984). Phenols play a dual role in maintaining redox homeostasis. Their antioxidant properties are associated with their ROS scavenging potential (Sawa et al., 1999; Sakihama et al., 2002). They can also serve as potent metal chelators and restrain the Fenton reaction (Cheng et al., 2003). Under certain conditions (i.e. low pH, presence of heavy metals) phenols behave as oxidizing agents damaging proteins and nucleic acids (Sakihama et al., 2002; Cheng et al., 2003). Toxic metals such as Al, Zn and Cd have been found to induce phenoxyl radical production and lipid peroxidation (Sakihama et al., 2002). According to the present study the

concentration of phenolic compounds did not differ in mycorrhizal plants cultivated in polluted substratum from non-mycorrhizal plants from polluted substratum. However, it was significantly lower compared to mycorrhizal plants from non-polluted substratum. This may explain why the presence of AMF improves plant metabolic activity as expressed not only by significantly improved growth and better plant tissue hydration but also by the increased activity of antioxidant enzymes. In the presence of the symbiotic fungus a new redox homeostasis is established within the plant that is significantly different from that observed under the control conditions. According to Debiane et al. (2009) oxidative stress induced damage is reduced in in vitro cultivated chicory roots by AMF induced benzo[a]pyrene. In pea plants AMF alleviated arsenic toxicity by reducing oxidative stress and modulating antioxidant mechanisms (Garg and Singla, 2012). In Nicotiana tabacum plants over-expressing CAT, CAT activity, the rate of photosynthesis and plant productivity was increased (Brisson et al., 1998; Zelitch et al., 1991). A similar phenomenon was observed in white cabbage with O3 activated CAT activity (Rozpa˛dek et al., 2013). According to results presented in this paper CAT activity in mycorrhizal plants was increased. This was accompanied by an increase in dry and fresh weight of the shoots. A similar phenomenon was reported for marigold plants inoculated with three different species from the Glomus genus (Liu et al., 2011). The role of ROS, including H2O2 in plant growth has been described previously (Cui et al., 1999; Strzałka et al., 2010). The increase in the activity of H2O2 scavenging enzymes such as CAT and POX allows us to speculate that H2O2 homeostasis was altered in inoculated plants, what led to increased growth. In inoculated plants the activity of these enzymes was significantly elevated compared to NM plants. A

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Fig. 5. (A) The maximal PSII quantum yield (Fv/Fm), (B) Efficiency of PSII (FPSII) and (C) Non-photochemical quenching (NPQ) of Cichorium intybus L. plants cultivated on soil enriched in heavy metals substrate (P) and control (NP), inoculated with G. intradices M and control (NM).

similar phenomenon was previously reported by Ruiz-Lozano (2003). The association between increased biomass (fitness) and increased antioxidant capacity cannot be ruled out. SOD reduces the superoxide anion to H2O2. Elevated CAT and POX activity may had also resulted from the increased SOD-dependent H2O2 production. Antioxidant enzyme activities in toxic metal stressed plants reveal stimulation, no effect and suppression depending on the plant species, metal ion, concentration and exposure duration (Sharma and Dietz, 2008; Liu et al., 2011). No effects on CAT and POX were reported during toxic metal exposition. CAT, POX and SODs’ have been previously shown to be deactivated upon Cd, Zn exposure (Gallego et al., 1996, Schützendübel and Polle 2001). Here metal toxicity resulted in SOD activity elevation in NM plants, suggesting that the other examined enzymes are not sensitive to toxic metal exposure or that there constitutive activity is sufficient enough to deal with the toxicity. It cannot be ruled out that the mode (dose, duration) of toxic meal application was insufficient to induce any changes in the examined antioxidants. Maximal PSII quantum yield (Fv/Fm) and the efficiency of PSII decreased under toxic metal stress in NM plants. In inoculated with R. irregularis chicory Fv/Fm was slightly decreased, but this had no effect on the efficiency, suggesting a protective role of AMF. The NPQ parameter, indicates the ability to dissipate excess light energy, mainly due to the activity of the xanthophyll cycle. Non-photochemical quenching is activated upon light induced acidification of the thylakoid lumen and protonation of specific proteins (Baker, 2008). In inoculated chicory NPQ values are

significantly decreased compared to non-inoculated plants. This may be explained by more efficient C assimilation (increase in biomass of M plants) or the up-regulation of mechanisms regulating the trans-thylakoid H+ gradient. One such mechanism is the activity of the NADPH-MDH dependent malate valve (Scheibe et al., 1990). This issue however, requires more detailed research. 5. Conclusions Taken together, we can confirm that mycorrhiza protects plants against toxic metal stress. The symbiosis between C. intybus plants and R. irregularis facilitates photochemical activity as well as the pro/antioxidant balance and modulates health promoting metabolites of the cultivated plants. Acknowledgments This work was supported by The National Science Center DEC -2011/02/A/NZ9/00137 and the Małopolskie Centre of Biotechnology. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chemosphere. 2014.04.023.

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