Metabolic and Oxidative Changes in the Fern Adiantum raddianum upon Foliar Application of Metals.

Cadmium (Cd) or nickel (Ni) were applied as a foliar spray (1 µM solution over one month) to mimic air pollution and to monitor metabolic responses and oxidative stress in the pteridophyte species. Exogenous metals did not affect the metal content of the soil and had relatively little effect on the essential elements in leaves or rhizomes. The amounts of Cd and Ni were similar in treated leaves (7.2 µg Cd or 5.3 µg Ni/g DW in mature leaves compared with 0.4 µg Cd or 1.2 µg Ni/g DW in the respective control leaves), but Ni was more abundant in rhizomes (56.6 µg Ni or 3.4 µg Cd/g DW), resulting in a higher Cd translocation and bioaccumulation factor. The theoretical calculation revealed that ca. 4% of Cd and 5.5% of Ni from the applied solution per plant/pot was absorbed. Excess Cd induced stronger ROS production followed by changes in SOD and CAT activities, whereas nitric oxide (NO) stimulation was less intense, as detected by confocal microscopy. The hadrocentric vascular bundles in the petioles also showed higher ROS and NO signals under metal excess. This may be a sign of increased ROS formation, and high correlations were observed. Proteins and amino acids were stimulated by Cd or Ni application in individual organs, whereas phenols and flavonols were almost unaffected. The data suggest that even low levels of exogenous metals induce an oxidative imbalance, although no visible damage is observed, and that the responses of ferns to metals are similar to those of seed plants or algae.

High Temperature Alters Secondary Metabolites and Photosynthetic Efficiency in Heracleum sosnowskyi.

Due to global warming, invasive species have spread across the world. We therefore studied the impact of short-term (1 day or 2 days) and longer (7 days) heat stress on photosynthesis and secondary metabolites in Heracleum sosnowskyi, one of the important invasive species in the European Union. H. sosnowskyi leaves exposed to short-term heat stress (35 °C/1 d) showed a decrease in chlorophyll and maximum potential quantum efficiency of photosystem II (Fv/Fm) compared to control, 35 °C/2 d, or 30 °C/7 d treatments. In turn, the high level of lipid peroxidation and increased H2O2 accumulation indicated that the 30 °C/7 d stress induced oxidative damage. The contents of xanthotoxin and bergapten were elevated in the 2 d and 7 d treatments, while isopimpinellin was detected only in the heat-stressed plants. Additionally, the levels of free proline and anthocyanins significantly increased in response to high temperature, with a substantially higher increase in the 7 d (30 °C) treatment. The results indicate that the accumulation of proline, anthocyanins, and furanocoumarins, but not of phenolic acids or flavonols, contributes to protection of H. sosnowskyi plants against heat stress. Further studies could focus on the suppression of these metabolites to suppress the spread of this invasive species.

Long-term impact of cadmium in protonema cultures of Physcomitrella patens.

Antioxidative responses of axenic protonema cultures of the moss Physcomitrella patens exposed to 10 µM Cd over 40 d were studied. Cd treatment suppressed growth by ca. 75% with concomitant browning of some filaments and suppression of chlorophyll autofluorescence but had no impact on tissue water content. Despite this negative growth responses which could be related to enhanced ROS formation (as detected using fluorescence staining reagents for total ROS, hydroperoxides and lipid peroxidation), some metabolites revealed strong elevation by Cd which could contribute to attenuation of long-term Cd stress (elevation of ascorbic, malic and citric acids). Molar ratio of malate to Cd was 12.7 and citrate to Cd 2.5, thus potentially contributing to Cd chelation. Interestingly, GSH/GSSG pool and nitric oxide formation remained unaltered by Cd. Accumulation of Cd reached 82 µg/g DW with bioaccumulation factor of 73. Data indicate that Cd induces elevation of potentially protective metabolites even after prolonged exposure though they do not prevent oxidative stress sufficiently.

Tolerance of Facultative Metallophyte Carlina acaulis to Cadmium Relies on Chelating and Antioxidative Metabolites.

The impact of long-term chronic cadmium stress (ChS, 0.1 µM Cd, 85 days) or short-term acute cadmium stress (AS, 10 µM Cd, 4 days) on Carlina acaulis (Asteraceae) metabolites was compared to identify specific traits. The content of Cd was higher under AS in all organs in comparison with ChS (130 vs. 16 µg·g-1 DW, 7.9 vs. 3.2 µg·g-1 DW, and 11.5 vs. 2.4 µg·g-1 DW in roots, leaves, and trichomes, respectively) while shoot bioaccumulation factor under ChS (ca. 280) indicates efficient Cd accumulation. High content of Cd in the trichomes from the AS treatment may be an anatomical adaptation mechanism. ChS evoked an increase in root biomass (hormesis), while the impact on shoot biomass was not significant in any treatment. The amounts of ascorbic acid and sum of phytochelatins were higher in the shoots but organic (malic and citric) acids dominated in the roots of plants from the ChS treatment. Chlorogenic acid, but not ursolic and oleanolic acids, was elevated by ChS. These data indicate that both chelation and enhancement of antioxidative power contribute to protection of plants exposed to long-term (chronic) Cd presence with subsequent hormetic effect.

Nitric oxide affects cadmium-induced changes in the lichen Ramalina farinacea.

Metabolic responses of epiphytic lichen Ramalina farinacea to cadmium (Cd) and/or nitric oxide (NO) scavenger (cPTIO) were studied. Accumulation of Cd and other metallic nutrients was not affected by cPTIO while total and absorbed amounts differed. Cd-induced NO formation was suppressed by cPTIO but ROS signal was synergistically enhanced, confirming that NO is essential to keep ROS under control. This excessive ROS generation could be a reason for depleted amount of all fatty acids, including SFAs, MUFAs and PUFAs. Total content of fatty acids reached 3.89 mg/g DW in control with linoleic (40%), palmitic (24%), oleic (12.8%) and stearic (8%) acids as major compounds: interestingly, shift in relative ratio of saturated (from 40 to 35% of total FAs) versus polyunsaturated fatty acids (from 42 to 48% of total FAs) was observed. Glutathione was suppressed by all treatments but Krebs acids were almost unaffected by cPTIO, indicating no regulatory role of NO in their accumulation. On the contrary, Cd-induced elevation in NO signal was related to increase in ascorbate and proline content while cPTIO suppressed it, indicating a tight relation between NO and these metabolites. Data are compared also with algae and vascular plants to show similarities between various life lineages.

Allantoin attenuates cadmium-induced toxicity in cucumber plants.

Concentration-dependent responses of cucumber plants to cadmium (Cd, 5-15 µM) and/or allantoin (Alla, 10-1000 µM) have been investigated to detect a possible protective role of Alla under Cd excess. After 14 days of exposure, Alla often considerably reversed Cd-induced inhibition of growth and reduction of the content of photosynthetic pigments. Higher Alla doses depleted the Cd amount in shoots, which could be related to citric acid (increase in the shoots but depletion in the roots in Cd+Alla treatments) rather than to phytochelatins (Alla had a negative impact on the phytochelatin accumulation). An increase in the Alla concentration suppressed Cd-induced spatial H2O2 appearance, which does not seem to be related to antioxidative enzymes (low impact of Alla on catalase, ascorbate peroxidase, and guaiacol peroxidase). On the contrary, shoot glutathione and mainly ascorbic acid accumulation strongly increased in Cd+Alla treatments, indicating their prominent role in Alla-induced amelioration of Cd-stimulated oxidative stress and growth retardation. Similarly, phenolic metabolites (total soluble phenols and flavonols) were slightly influenced by Alla and their antioxidative action was not expected. We conclude that Alla-mediated attenuation of Cd-induced toxicity relies on enhanced accumulation of glutathione and ascorbate in the shoot tissue mainly, rather than on elevated antioxidative enzyme activities.

The Impact of Long-and Short-Term Strontium Treatment on Metabolites and Minerals in Glycine max.

The impact of long-term exposure to Sr2+ (LTE, four doses, 43.5 mg Sr2+ per pot, with a total of 174 mg Sr2+ per pot during the entire period of cultivation) and short-term exposure to Sr2+ (STE, one dose, 870 mg Sr2+ per pot four days before harvest) on the content of phytoestrogens and allantoin in soybeans were compared. Sr2+ accumulation, the effect on the concentration of macroelements, and basic physiology were also analyzed. LTE reduced the content of malonyldaidzin and malonylgenistin in the roots (58% and 50% compared to the control, respectively). STE increased the amount of all isoflavones in the stem and genistein in the leaves and decreased the content of malonyldaidzin and malonylgenistin in the leaves (55% and 48% compared to the control, respectively) and roots (69% and 62% of the control, respectively) as well as genistein and coumestrol in the roots (both 50% compared to the control). Sr2+ presence stimulated the accumulation of allantoin in the roots (three-fold higher than in the control), but only STE had similar effects on the shoots. In contrast to LTE, Sr2+ was transported extensively from the roots to the leaves under STE. In comparison to the control, LTE resulted in an increase in the Ca content in the stem by 36%, whereas Ca2+ accumulation in the leaves, stems, and roots increased by 60%, 80%, and 36%, respectively, under STE. Additionally, a significant accumulation of K was found only in the roots of the LTE group. The chlorophyll content did not differ between the treatments. Overall, the production of phytoestrogens and Sr accumulation were affected by both the applied dose and the duration of exposure to Sr.

Calcium availability but not its content modulates metal toxicity in Scenedesmus quadricauda.

Impact of calcium nutrition (pre-culture on solid medium with standard or elevated Ca dose, i. e. 0.17 and 4.40mM marked as low and high Ca) on acute metal toxicity (Cd, Mn and Pb, 24h of exposure to 10µM) in freshwater green alga Scenedesmus quadricauda was studied. Surprisingly, Ca content differed only slightly between low and high Ca samples and applied metals rather suppressed its amount. Na content was higher in metal-exposed high Ca samples, indicating that Ca/Na ratio may affect accumulation of metals. Content of heavy metals increased in order Cd < Mn < Pb and high Ca samples contained less metal than low Ca samples at least in absorbed fraction. Accumulation of ascorbic acid and thiols (GSH - glutathione and PC2 - phytochelatin 2) was affected mainly by Cd, GSH also by Mn and PC2 by Pb with often significant differences between low Ca and high Ca samples. Calcium nutrition also affected responses of algae to metals at the level of antioxidative enzyme activities (SOD, APX, and CAT) and elevated values were typically found in high Ca samples while ROS (hydrogen peroxide and superoxide radical) were mainly depleted in Mn treatment. These data confirm that Ca nutrition affects accumulation of metals in algae and metabolic parameters as observed in vascular plants but, unlike them, rather Ca/Na ratio than absolute Ca content seems to regulate the uptake of metals.

Effect of Long-Term Strontium Exposure on the Content of Phytoestrogens and Allantoin in Soybean.

Abiotic stress, including metal excess, can modify plant metabolism. Here we investigated the influence of long-term strontium exposure (12 weeks, 0.5⁻4.0 mM Sr) on the content of phytoestrogens and allantoin as well as the mineral composition in soybean. Seven phytoestrogens were identified in the soybean: daidzin, glycitin, genistin, malonyldaidzin, malonylgenistin, daidzein, and coumestrol. The results showed that both malonyldaidzin and malonylgenistin were dominant phytoestrogens; however, the roots contained a relatively high amount of daidzein. It was found that strontium reduced the phytoestrogen content and decreased the antioxidant capacity. Strontium evoked depletion of the sum of all phytoestrogens by 40⁻70% in the leaves, 25⁻50% in the stems and in the seeds, depending on the strontium concentration. In the roots, 0.5 and 4.0 mM of strontium decreased the total phytoestrogen content by 25 and 55%, respectively, while 2.0 mM of strontium did not exert an effect on their accumulation. On the other hand, strontium ions induced allantoin accumulation mainly in the roots. Strontium was preferentially accumulated in the leaves, with a slight impact on macro- and micro-nutrients. Our research showed strontium-secondary metabolites interaction in the soybean, which can be useful for obtaining a natural pharmaceutical product containing both strontium and phytoestrogens for remediation of postmenopausal osteoporosis.

Visual plant anatomy: From science to education and vice versa.

Plant biology, mainly plant anatomy, is a less attractive area for students at high school and university, but not much research has been devoted to improve this field. We therefore researched into the teaching of root, stem and leaf anatomy combined with the preparation of native microscopic slides and histochemical reaction using two selected dyes (classic phloroglucinol test combined with textile dye 'Duha green' to visualize xylem and phloem, respectively). The use of reagents in teaching had a positive effect on students' knowledge (control/ experimental class) of root (+70%), stem (+70%) and leaf anatomy (+130%) as well as vascular and mechanical tissues (+170%), leading to an overall improvement of knowledge by ca. 100%. Students' ability to identify individual tissues on microscopic slides increased and they also understood the functions of individual tissues after self-preparing and staining slides. However, we identified that some aspects were still problematic for students after the experimental education (e.g. identification of tissue providing secondary growth, significance of sclerenchyma and transpiration). We also attach correct answers for the anatomy test and worksheets used for practical exercises as motivation for wider use to improve students' knowledge of plant anatomy.

Interaction of nickel with oxidative and antioxidative molecules in Cichorioideae species.

The uptake of nickel (Ni) by Asteraceae/Cichorioideae species Cichorium intybus, Leontodon hispidus and Hieracium aurantiacum exposed to Ni (0.3 or 30 µM) over 14 days and subsequent changes of metabolites were compared in order to identify their phytoaccumulation potential. Hieracium contained the most Ni (194 and 1558 µg Ni/g DW at 30 µM Ni in shoots and roots) but had unchanged amount of antioxidants (vitamin C and thiols) in the shoots and an elevated amount in the roots, which may be the reason for the absence of visible damage. On the contrary, Leontodon reacted by a decrease in antioxidants to an excess of Ni, which can be related to enhanced oxidative stress (an increase in ROS and a decrease in nitric oxide detected by fluorescence microscopy). All roots were anatomically in the secondary state and Ni-induced cell wall thickening (i.e. lignin/suberin deposition) was most visible in Hieracium roots, which also contained 2-times more Ni than the other species. Among essential elements, mainly Fe accumulation was affected by Ni excess. The content of soluble phenols increased while organic acids (malic and citric) decreased sometimes extensively (up to 90%) in individual species. PCA analyses showed that especially ascorbic acid, thiols and phenols affect the separation in the shoots especially with regard to applied concentration of Ni, while these metabolites in the roots clearly separated the species (Cichorium from the others). The data show the highest tolerance to Ni in Hieracium, but the highest phytoaccumulation of Ni was found in Cichorium (626 µg Ni/plant or 122 µg Ni/shoot at a dose of 30 µM Ni).

Mercury Content and Amelioration of Its Toxicity by Nitric Oxide in Lichens.

Mercury (Hg) content measured in five epiphytic lichen species collected in Slovakia mountain forests ranged from 30 to 100 ng/g DW and was species-specific, decreasing in the order Hypogymnia > Pseudevernia > Usnea > Xanthoria > Evernia prunastri (but polluted sites had no impact on Hg amount in Xanthoria). Evernia was therefore used to study the impact of short-term exogenous Hg (100 µM, 24 h) and possible amelioration of Hg toxicity by nitric oxide (NO) donor sodium nitroprusside (SNP). NO was efficiently released from SNP as detected by two staining reagents and fluorescence microscopy and reduced Hg-induced ROS signal and absorption of Hg by thalli of Evernia prunastri. At the same time, NO ameliorated Hg-induced depletion of metabolites such as ascorbic acid and non-protein thiols, but not of free amino acids. The amount of metabolites, including soluble phenols, was reduced by excess Hg per se. On the contrary, NO was unable to restore Hg-stimulated depletion of chlorophyll autofluorescence but mitigated the decline of some macronutrients (K and Ca). Data confirm that accumulation of Hg in the epiphytic lichens is species-specific and that NO is a vital molecule in Evernia prunastri that provides protection against Hg-induced toxicity with considerable positive impact on metabolic changes.

Elemental profile identifies metallurgical pollution in epiphytic lichen Xanthoria parietina and (hypo)xanthine correlates with metals.

The accumulation of 55 elements in lichens under the heap of a former nickel smelter (village Dolná Streda, Slovakia) and at eight sites at different distances from the heap plus six sites throughout Slovakia was studied to determine the elemental profile. The major metals in the heap sludge and in the lichens below the heap (Ni, Cr, Fe, Mn, and Co) were surprisingly low in lichens from both the near and far vicinity of the heap (4-25 km), indicating limited airborne spread. However, two different sites with metallurgical activity (another site near the ferroalloy producer in Orava) typically contained the highest amount of individual elements, including rare earth elements, Th, U, Ag, Pd, Bi and Be, and their separation from other sites was confirmed by PCA and HCA analyses. In addition, the amounts of Cd, Ba and Re were highest at sites without a clear source of pollution and further monitoring is needed. It was also an unexpected finding that the enrichment factor calculated using UCC values was increased (often considerably >10) for 12 elements at all 15 sites, indicating eventual anthropogenic contamination with P, Zn, B, As, Sb, Cd, Ag, Bi, Pd, Pt, Te and Re (and other EF values were locally increased). Metabolic analyses showed a negative correlation between some metals and metabolites (ascorbic acid, thiols, phenols and allantoin), but slightly positive (amino acids) or highly positive correlation with purine derivatives hypoxanthine and xanthine. The data suggest that lichens adapt their metabolism to excessive metal loading and that epiphytic lichens are suitable for identifying metal contamination even at apparently clean sites.

Prey-induced changes in the accumulation of amino acids and phenolic metabolites in the leaves of Drosera capensis L.

Effect of prey feeding (ants Formica fusca) on the quantitative changes in the accumulation of free amino acids, soluble proteins, phenolic metabolites and mineral nutrients in the leaves of carnivorous plant Drosera capensis was studied. Arginine was the most abundant compound in Drosera leaves, while proline was abundant in ants. The amount of the majority of amino acids and their sum were elevated in the fed leaves after 3 and 21 days, and the same, but with further enhancement after 21 days, was observed in ants. Accumulation of amino acids also increased in young non-fed leaves of fed plants. Soluble proteins decreased in ants, but were not enhanced in fed leaves. This confirms the effectiveness of sundew's enzymatic machinery in digestion of prey and suggests that amino acids are not in situ deposited, but rather are allocated within the plant. The content of total soluble phenols, flavonoids and two selected flavonols (quercetin and kaempferol) was not affected by feeding in Drosera leaves, indicating that their high basal level was sufficient for the plant's metabolism and prey-induced changes were mainly N based. The prey also showed to be an important source of other nutrients besides N, and a stimulation of root uptake of some mineral nutrients is assumed (Mg, Cu, Zn). Accumulation of Ca and Na was not affected by feeding.

Biochemistry in toxicological studies also needs precision.

Mehdizadeh et al. (2021) reported the impact of biochar on cadmium toxicity in Ocimum ciliatum. As far as the conclusions may be correct (and the positive impact of various biochars is known in numerous experimental setups/species), several numerical mistakes reported in results are not acceptable in any scientific journal. It seems that reviewers and handling editor overlooked these problems and biochemical aspects of this work (along with the impact of biochar on Cd accumulation) can be cited only with great doubts about the correctness of the results. Generally, it is a challenge for reviewers and publishers, mainly in the actual time of a huge load of submissions, not to overlook basic technical mistakes. It is also a challenge for the authors to study literature and to verify uncertain data.

Calcium-enriched biochar modulates cadmium uptake depending on external cadmium dose.

The impact of calcium-enriched biochar (BC, containing Ca, Al, Fe and P as dominant elements in the range of 6.9-1.3% with alkaline pH) obtained from sewage sludge (0.1 or 0.5% in the final soil) on cadmium-induced toxicity (final dose of 1.5 mg Cd/kg in control and 4.5 or 16.5 mg Cd/kg soil in low and high Cd treatment) was tested in medicinal plant Matricaria chamomilla. Low Cd dose had typically less negative impact than high Cd dose at the level of minerals and metabolites and the effect of BC doses often differed. Contrary to expectations, 0.5% BC with a high Cd dose increased Cd accumulation in plants about 2-fold. This was reflected in higher signals of reactive oxygen species, but especially the high dose of BC increased the amount of antioxidants (ascorbic acid and non-protein thiols), minerals and amino acids in shoots and/or roots and usually mitigated the negative effect of Cd. Surprisingly, the relationship between BC and soluble phenols was negative at high BC + high Cd dose, whereas the effect of Cd and BC on organic acids (mainly tartaric acid) differed in shoots and roots. Interestingly, BC alone applied to the control soil (1.5 mg total Cd/kg) reduced the amount of Cd in the plants by about 30%. PCA analyses confirmed that metabolic changes clearly distinguished the high Cd + high BC treatment from the corresponding Cd/BC treatments in both shoots and roots. Thus, it is clear that the effect of biochar depends not only on its dose but also on the amount of Cd in the soil, suggesting the use of Ca-rich biochar both for phytoremediation and safer food production.

Nickel uptake in hydroponics and elemental profile in relation to cultivation reveal variability in three Hypericum species.

The Hypericum species (H. perforatum, H. olympicum, and H. orientale) were cultured in hydroponics with excess nickel (Ni, 1 or 100 µM Ni) to compare the metallic and metabolite content. Identical species were collected outdoor to assess the same parameters (including uranium and lanthanides) with total of 53 elements. The results showed that Ni was less accumulated in shoots in hydroponics (translocation factor of 0.01-0.25) and the highest absolute amount was detected in H. olympicum. Essential elements were typically depleted by Ni excess, but Co and Na increased. Soluble phenols, sum of flavonols and catechin rather increased in response to Ni but quercetin glycosides and free amino acids decreased in the shoots of H. olympicum mainly. Comparison of laboratory and outdoor growing plants showed more phenols in outdoor samples but not in H. olympicum and individual metabolites differed too. Plants cultured in hydroponics contained lower amount of non-essential, toxic and rare earth elements (30-100-fold) and shoot bioaccumulation factor in outdoor samples was low for most elements (<0.01) but not for Cd and Pt. Data reveal that H. olympicum is a potent source of phenolic metabolites whereas H. orientale accumulates many elements (38 out of 53 elements).

Nitrogen modulates strontium uptake and toxicity in Hypericum perforatum plants.

Strontium is an unavoidable element occurring in plants due to its abundance in the soil and similarity with calcium. To mimic natural conditions, impacts of additional inorganic (nitrate) or organic (urea and allantoin) nitrogen sources (1 mM of each N form in addition to 3.53 mM N in the basic cultivation solution) or N deficit on strontium-induced changes (100 µM Sr) in the widely used medicinal plant Hypericum perforatum L. were studied. Though various effects of Sr on primary (stimulation of amino acids but depression of most Krebs acids, ascorbic acid and thiols) and secondary metabolites (stimulation of phenols but no change of pseudo/hypericin) or mineral elements were observed (reduction of Ca amount in both shoots and roots), organic N forms often mitigated negative action of Sr or even combined stimulatory impact was observed. Organic N forms also elevated shoot accumulation of Sr while N deficit reduced it. Additional N forms, rather than Sr itself, modulated reactive oxygen species and nitric oxide formation in the root tissue. Germination experiment showed no toxicity of Sr to H. perforatum up to 1 mM Sr and even stimulated accumulation of amino acids and phenols, indicating similar ontogenetic-related responses.

Allantoin overaccumulation enhances production of metabolites under excess of metals but is not tightly regulated by nitric oxide.

The aln-3 mutant overaccumulating allantoin and respective wild type (WT) strain of Arabidopsis thaliana were exposed to cadmium (Cd) or mercury (Hg) with or without nitric oxide (NO) donor (sodium nitroprusside, SNP) to study cross-talk, metabolic and oxidative changes between these nitrogen sources (organic vs. inorganic). The aln-3 accumulated over 10-fold more allantoin than WT with the effect of Cd and Hg differing in leaf and root tissue: aln-3 contained more ascorbic acid and phytochelatins when treated with Cd or Hg and more Cd in both organs. SNP depleted leaf Cd and root Hg accumulation in aln3 but had a positive impact on the amount of metabolites typically in WT plants, indicating potentially negative relation between allantoin and NO. In agreement, aln-3 roots showed lower NO signals in control or metal treatments, but higher ROS signal, and SNP had more pronounced impact in WT roots. Flavonol glycosides were more abundant in aln-3 and were affected more by metals than by SNP. Malate was the most affected Krebs acid with strong reaction to SNP and Hg treatment. Data indicate that allantoin overaccumulation influences the accumulation of specific metabolites but nitric oxide has a greater impact on the metabolite profile in WT.

Physiological responses of root-less epiphytic plants to acid rain.

Selected physiological responses of Tillandsia albida (Bromeliaceae) and two lichens (Hypogymnia physodes and Xanthoria parietina) exposed to simulated acid rain (AR) over 3 months were studied. Pigments were depressed in all species being affected the most in Tillandsia. Amounts of hydrogen peroxide and superoxide were elevated and soluble proteins decreased only in AR-exposed Hypogymnia. Free amino acids were slightly affected among species and only glutamate sharply decreased in AR-exposed Xanthoria. Slight increase in soluble phenols but decrease in flavonoids in almost all species suggests that the latter are not essential for tolerance to AR. Almost all phenolic acids in Tillandsia leaves decreased in response to AR and activities of selected enzymes (phenylalanine ammonia-lyase, polyphenol oxidase, ascorbate- and guaiacol-peroxidase) were enhanced by AR. In lichens, considerable increase in metabolites (physodalic acid, atranorin and parietin) in response to AR was found but amount of ergosterol was unchanged. Macronutrients (K, Ca, Mg) decreased more pronouncedly in comparison with micronutrients in all species. Xanthoria showed higher tolerance in comparison with Hypogymnia, suggesting that could be useful for long-term biomonitoring.