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).

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.

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.

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.

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.

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).

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.

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.

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.

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.

Basic physiology and biochemistry in environmental/experimental plant studies: How to quantify and interpret metabolites correctly.

As a reviewer of ca. 50 manuscripts per year submitted to various journals, I often come across questionable metabolic data (both over- or under-estimated) mainly in the journals from the section of Environmental Sciences of Web of Science. Though the trends of visibly incorrect metabolite values may be informative (changes in response to applied treatments or environmental factors), absolute values must be precise enough to allow inter-specific comparison and eventual subsequent calculations. Technical correctness of quantification and calculation of such data is therefore often questionable. One problem arises when calculating metabolites concentration (often nmol or µmol/g of biomass) and another problem is the impact of altered water content on metabolite level (then trend per gram of fresh or dry biomass will differ). Recent discrepancies I found when searching for the literature prompted me to write this technical note aimed at focusing attention of researchers on these problems. I exclude any conflict of interest when discussing the quoted published studies. I strongly urge interested researchers to verify the correctness of metabolite quantification (extraction, dilution/calculation and alternative methods) and also to study similar literature for comparison in order to prevent the spread of incorrect data in the scientific literature.

Use and misuse of 6-((anthracen-9-yl) methyleneamino)-2H-chromen-2-one for detection of metallic ions.

Liu et al. published the use of 6-((anthracen-9-yl) methyleneamino)-2H-chromen-2-one for visualization of copper or cadmium ions by fluorescence microscopy and cited this method by our previous paper. The fluorescence dye 6-((anthracen-9-yl) methyleneamino)-2H-chromen-2-one mentioned by the authors is not specific for Cu or Cd ions, we never tested it for this aim and some photos presented by the authors indicate even double method mistake as explained below.

Organic nitrogen modulates not only cadmium toxicity but also microbial activity in plants.

It is known that organic nitrogen may modify uptake and toxicity of metals but direct metabolic and microbial comparison of various organic N sources is not available. We therefore studied comparative impact of additional N sources (nitrate, urea or allantoin as 1 mM of N for each compound in addition to 15 mM of inorganic N in the Hoagland solution) on Cd toxicity and microbial activity in common crop cucumber. Organic N significantly elevated the growth, chlorophyll content and photosynthetic activity under Cd excess in comparison with inorganic N though the impact on Cd uptake was negligible. Both organic N compounds also affected accumulation of mineral nutrients, total N, amino acids, and protein content in Cd-stressed plants. Among organic acids, mainly allantoin and partially urea affected accumulation of citrate and tartrate. The most notably, we detected that allantoin was decomposed even within 24 h by microbes into the urea, but it significantly elevated rhizosphere microbial activity. All these data indicate that allantoin is metabolized by plants/microbes into the urea and that it affects microbes mainly in the rhizosphere, which could contribute to amelioration of Cd toxicity.

Calcium has protective impact on cadmium-induced toxicity in lichens.

Eventual protective action of calcium (Ca, 100 or 1000 µM) against cadmium (Cd, 10 or 100 µM) toxicity in common lichen Hypogymnia physodes after 24 h of exposure was studied. Total Cd reached 482 and 2801 µg/g DW in 10 and 100 µM Cd treatments while Ca content reached over 23 mg/g DW in 1000 µM Ca treatment. Ca suppressed Cd accumulation by 23 and 38% in total fraction and completely in absorbed fraction. Fluorescence microscopy of Cd and Ca ions revealed good correlation with quantitative data. Cd stimulated increase in ROS formation and lipid peroxidation as detected using fluorescent reagents and quantification of H2O2 while co-application of Ca suppressed these effects. Formation of nitric oxide was mainly affected by cadmium. Cd depleted amount of amino acids but proteins or phenols remained unaffected by Cd or Ca. On the contrary, sum of thiols, reduced glutathione and ascorbic acid were depleted by Cd but reversed mainly by higher Ca dose. Among organic acids, only Cd-induced depletion of citric acid content was reversed by Ca. Data indicate that ameliorative effect of Ca under Cd excess in lichens is comparable with effect in plants and metabolic responses in various life lineages are discussed.

The opposite nitric oxide modulators do not lead to the opposite changes of metabolites under cadmium excess.

Responses of common medicinal herb Matricaria chamomilla to short-term cadmium (Cd) exposure (48 h) alone or in combination with nitric oxide (NO) scavenger (PTIO) or donor (SNP) were studied. Modulators revealed expected impact on NO formation (depletion under PTIO but elevation under SNP) and confirmed anomalous impact on Cd accumulation (stimulation by both modulators). Changes of proline and free amino acids in the roots and fatty acids (mainly α-linolenic acid) in the shoots indicate rather relation to elevated Cd uptake than to altered NO formation. On the contrary, root profile of fatty acids revealed the most prominent changes in response to PTIO (elevation of monounsaturated and polyunsaturated fatty acids) which confirm that NO depletion stimulates their biosynthesis. Soluble phenols were elevated by SNP co-application in the roots while accumulation of chlorogenic acid and umbelliferone revealed clear regulation by NO (i.e. enhancement in response to SNP and depletion in response to PTIO). Data indicate that quantities of some metabolites are affected by accumulated Cd or NO formation and that the opposite NO modulators do not automatically lead to the opposite changes of plant metabolites.

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.

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.

Nitrogen nutrition modulates oxidative stress and metabolite production in Hypericum perforatum.

Impact of various nitrate concentrations (14.12 mM, 3.53 mM, no nitrate) or ammonium presence (14.12 mM) on physiological and metabolic changes in Hypericum perforatum after 14 days of cultivation was monitored. Nitrate deficiency suppressed growth of shoots but stimulated root growth while ammonium suppressed root growth: concomitant changes of ascorbic acid and glutathione supported these growth changes, e.g., unaltered level in roots under nitrate deficiency but depleted in ammonium treatment. Soluble proteins and water content were more suppressed by nitrate deficiency but total ROS, nitric oxide formation, and antioxidative enzyme activities (APX and SOD) indicate higher sensitivity of plants to ammonium. Though both extreme treatments (NO3- deficiency or ammonium) stimulated accumulation of total soluble phenols and affected PAL activity (in comparison with full or 1/4× nitrate dose), major phenols (chlorogenic acid and three flavonoids) were elevated mainly by NO3- deficiency. At the level of specific metabolites, NO3- deficiency had stimulatory impact on pseudohypericin (but not hypericin) content while hyperforin decreased. Expression of earlier putative gene of hypericin biosynthesis (hyp-1) showed rather partial correlation with pseudohypericin amount. Data indicate that depletion of NO3- is useful to obtain Hypericum plants with higher amount of health-positive secondary metabolites.