Josef Hedbavny

Comparison of cadmium and copper effect on phenolic metabolism, mineral nutrients and stress-related parameters in Matricaria chamomilla plants

Cadmium and copper uptake and its consequence for activity of selected enzymes of phenolic metabolism, phenolic acids accumulation, quantity of mineral nutrients and stress-related parameters in Matricaria chamomilla plants exposed to 60xa0μM and 120xa0μM for 7xa0days has been studied. Cu content in the above-ground biomass was ca. 10-fold lower compared to Cd and amount of Cd in the methanol-soluble fraction was lower than in the water-soluble fraction. “Intra-root” Cd represented 68% and 63% of total Cd content at 60xa0μM and 120xa0μM, but no difference was observed in Cu-exposed roots. Cu excess had more pronounced effect on shikimate dehydrogenase, cinnamyl alcohol dehydrogenase, polyphenol oxidase and ascorbate peroxidase activity mainly in the roots. Among eight detected benzoic acid derivatives and four cinnamic acid derivatives, the latter were preferentially accumulated in response to Cd excess. Content of salicylic acid increased in all variants. Amount of superoxide was elevated in both the rosettes (preferentially by Cu) and roots (preferentially by Cd). Accumulation of Ca and Mg was not affected by excess of metals, while potassium decreased in both the rosettes and roots (Cu caused stronger depletion). Amount of Fe increased in the roots in response to both metals (more expressively in Cu-treated ones). Present study using other metabolic parameters (and supplementing our previous studies) has confirmed higher Cu toxicity for chamomile plants, to support its strong pro-oxidant properties. These observations as complex metabolic responses are discussed.

Salicylic acid alleviates NaCl-induced changes in the metabolism of Matricaria chamomilla plants

Influence of 100xa0mM NaCl and 50xa0μM salicylic acid (SA) and their combination on the metabolism of chamomile (Matricaria chamomilla) during 7xa0days was studied. NaCl reduced growth and selected physiological parameters and SA in combined treatment (NaClxa0+xa0SA) reversed majority of these symptoms. Application of SA reduced NaCl-induced increase of Na+ in the rosettes, but not in the roots. Accumulation of total amino acids was stimulated in NaCl-treated roots, especially due to exceptional increase of proline (4.4-fold). Among phenolic acids, accumulation of protocatechuic acid was the most enhanced in NaCl-exposed leaf rosettes (ca. 3-fold) while chlorogenic and caffeic acids in the roots (2.4- and 2.8-fold, respectively). Total soluble phenols increased after NaCl and SA treatments, but root lignin content was not affected. Activity of phenylalanine ammonia-lyase and shikimate dehydrogenase increased in response to NaCl, but cinnamyl alcohol dehydrogenase was not affected and polyphenol oxidase decreased. Stress parameters were elevated by NaCl treatment (superoxide radical and malondialdehyde content, activities of catalase, ascorbate- and guaiacol-peroxidase) and substantially prevented by SA, while accumulation of hydrogen peroxide decreased. Overall, SA showed strong beneficial properties against NaCl-induced negative symptoms. Protective effect of SA was the most visible at the level of guaiacol-peroxidase and through amelioration of stress parameters and mineral nutrient contents.

Significance of phenols in cadmium and nickel uptake

The effects of 2-aminoindane-2-phosphonic acid (AIP), a potent phenylalanine ammonia-lyase (PAL) inhibitor, on the accumulation of cadmium and nickel in chamomile (Matricaria chamomilla) were examined in this study. In vitro assay of AIP effect showed a 90% reduction in PAL activity. In plants cultured for 7 days in Cd or Ni solutions with AIP, PAL activity was higher in both shoots and roots (in comparison with metals without AIP), and was correlated with changes in free phenylalanine content. Individual amino acids were both positively and negatively affected by AIP, with the accumulation of tyrosine and proline showing increases in some variants. Contents of soluble phenols and flavonoids were not considerably affected, while amounts of coumarin-related compounds, cell wall-bound phenols and phenolic acids were substantially reduced in AIP-treated variants. Lignin accumulation decreased in controls and increased in Cd variants in response to AIP. Shoot Cd content was depleted, but shoot Ni was elevated by AIP. Total root content of Cd and Ni decreased in +AIP variants. AIP also caused more expressive changes in hydrogen peroxide and superoxide content in Cd than in Ni variants. Our results indicate that phenols have important roles in the uptake of Cd and Ni. The present findings are discussed in the context of available data regarding AIPs effect on phenols.

Cadmium and nickel uptake are differentially modulated by salicylic acid in Matricaria chamomilla plants.

Chamomile (Matricaria chamomilla) is a widely used medicinal plant which also accumulates heavy metals in its above-ground organs. We investigated the effect of the important plant signaling molecule, salicylic acid (SA), on the accumulation of Ni or Cd, by exposing plants over 7 days to 60 microM solutions of individual heavy metals with or without 50 microM SA. Special emphasis was focused on phenolic metabolism-related parameters, not only because of their importance for growth and stress tolerance but also because phenolics are potent antioxidants in human diet. In combined treatments, SA stimulated an increase in soluble proteins of roots and reduced their water content. SA reduced total Cd in the shoot and increased Ni. Total and intraroot Ni decreased in Ni + SA treatment, while in the case of Cd, only intraroot content decreased in Cd + SA treatment, being correlated with cell wall-bound phenolic acids and lignin. SA was strongly accumulated in roots from the Ni + SA treatment, being correlated with an increase in hydrogen peroxide. In both Cd + SA and Ni + SA treatments, SA enhanced phenylalanine ammonia-lyase activity and accumulation of total soluble phenols, particularly in the roots. Here, we report for the first time that soluble phenols may be involved in Cd shoot-to-root translocation. In the case of Ni, it seems that phenols serve as a root barrier in order to prevent Ni from reaching the above-ground organs. The effects of SA on phenolic metabolism, and the signaling role of ROS in the accumulation of phenols, are discussed.

Nitrate deficiency reduces cadmium and nickel accumulation in chamomile plants.

The effect of nitrogen (nitrate) deficiency (-N) on the accumulation of cadmium (Cd) and nickel (Ni) in chamomile ( Matricaria chamomilla ) plants was studied. Elimination of N from the culture medium led to decreases in N-based compounds (free amino acids and soluble proteins) and increases in C-based compounds (reducing sugars, soluble phenols, coumarins, phenolic acids, and partially flavonoids and lignin), being considerably affected by the metal presence. Proline, a known stress-protective amino acid, decreased in all -N variants. The activity of phenylalanine ammonia-lyase was stimulated only in -N control plants, whereas the activities of polyphenol oxidase and guaiacol peroxidase were never reduced in -N variants in comparison with respective +N counterparts. Among detected phenolic acids, chlorogenic acid strongly accumulated in all N-deficient variants in the free fraction and caffeic acid in the cell wall-bound fraction. Mineral nutrients were rather affected by a given metal than by N deficiency. Shoot and total root Cd and Ni amounts decreased in -N variants. On the contrary, ammonium-fed plants exposed to N deficiency did not show similar changes in Cd and Ni contents. The present findings are discussed with respect to the role of phenols and mineral nutrition in metal uptake.

Unexpected behavior of some nitric oxide modulators under cadmium excess in plant tissue.

Various nitric oxide modulators (NO donors - SNP, GSNO, DEA NONOate and scavengers – PTIO, cPTIO) were tested to highlight the role of NO under Cd excess in various ontogenetic stages of chamomile (Matricaria chamomilla). Surprisingly, compared to Cd alone, SNP and PTIO elevated Cd uptake (confirmed also by PhenGreen staining) but depleted glutathione (partially ascorbic acid) and phytochelatins PC2 and PC3 in both older plants (cultured hydroponically) and seedlings (cultured in deionised water). Despite these anomalous impacts, fluorescence staining of NO and ROS confirmed predictable assumptions and revealed reciprocal changes (decrease in NO but increase in ROS after PTIO addition and the opposite after SNP application). Subsequent tests using alternative modulators and seedlings confirmed changes to NO and ROS after application of GSNO and DEA NONOate as mentioned above for SNP while cPTIO altered only NO level (depletion). On the contrary to SNP and PTIO, GSNO, DEA NONOate and cPTIO did not elevate Cd content and phytochelatins (PC2, PC3) were rather elevated. These data provide evidence that various NO modulators are useful in terms of NO and ROS manipulation but interactions with intact plants affect metal uptake and must therefore be used with caution. In this view, cPTIO and DEA NONOate revealed the less pronounced side impacts and are recommended as suitable NO scavenger/donor in plant physiological studies under Cd excess.

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 3xa0months 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.

Chromium uptake and consequences for metabolism and oxidative stress in chamomile plants.

Chromium Cr(III) toxicity toward chamomile metabolism and oxidative stress-related parameters after 7 days of exposure was studied. Cr preferentially accumulated in the roots and evoked extensive both dose-dependent and dose-independent increase in fluorescence signals of ROS, NO and thiols. Superoxide increased mainly at the highest Cr dose, whereas H2O2 accumulation revealed a discontinuous trend in relation to external Cr supply, and this could be owing to variation in activities of peroxidases. Glutathione and ascorbate quantification, using LC-MS/MS equipment, revealed strong stimulation despite low shoot Cr amounts. Phenolic enzyme activities, except for PAL, were depressed by Cr presence, whereas phenolic metabolites were stimulated, indicating various time dynamics. Among free amino acids, their sum and even proline decreased in the roots, whereas soluble proteins increased. Mineral nutrients showed negligible responses with only Zn and Cu being depleted in both shoots and roots. Cr staining using Cr(III)-specific (naphthalimide-rhodamine) and metal nonspecific (Phen Green) dyes indicated that the former correlated well with AAS quantification of Cr amount. Use of Phen Green is also discussed. These data indicate that Cr-induced oxidative stress is not simply a function of exposure time and applied concentration. Microscopic observations in terms of oxidative stress and chromium uptake are presented here for the first time.

Tolerance of Silene vulgaris to copper: population-related comparison of selected physiological parameters.

Tolerance of three Slovak populations of Silene vulgaris [from the localities “Harmanec” (total soil Cu 34.1 mg kg−1), “Lubietová”; (total soil Cu 831.6 mg kg−1) and “Špania dolina” (total soil Cu 1368.7 mg kg−1) referred as SV1, SV2, and SV3, respectively] and Silene dioica (SD; from the locality “Špania dolina”) exposed to 60 μM Cu for 7 days has been studied. Respective controls with 0.21 μM Cu were also cultured. SV3 and SD contained higher total shoot Cu than SV1. Methanol‐soluble shoot Cu represented 60, 59, 59, and 55% from total shoot Cu and roots contained 16.7‐, 20.4‐, 9.9‐ and 28.9‐fold more Cu than shoots in SV1, SV2, SV3, and SD, respectively. Soluble proteins were the least affected in SV3 and SD when Cu‐exposed and control plants were compared. Root hydrogen peroxide and superoxide showed similar trend and were correlated with enhancement of ascorbate‐ and guaiacol‐peroxidase activities. Malondialdehyde accumulation increased in SV1 and SV2. Phenylalanine ammonia‐lyase activity and total soluble phenols were higher in SV3 Cu‐exposed plants compared to SV1 and SV2. Shikimate dehydrogenase activity was enhanced in all Cu‐exposed populations. Cinnamyl alcohol dehydrogenase activity and root lignin content were not affected and polyphenol oxidase activity was not detected. Within 14 detected free amino acids, majority of them decreased preferentially in the roots. Only serine increased in shoots and decreased in roots of all populations. Potassium content was not affected in SV3. Na, Ca, and Fe increased in roots of Cu‐exposed plants of all populations, while Mg was the least affected and Zn increased in SV2 and SV3 shoots and roots. This study revealed the highest tolerance in population from the locality with the highest soil Cu content. Present findings as complex metabolic responses to Cu stress with special emphasis on phenolic metabolism are discussed.

Effect of aluminium uptake on physiology, phenols and amino acids in Matricaria chamomilla plants

Chamomile is a widely used medicinal plant and, as observed in our previous studies, also accumulates some metals in its above-ground biomass. We therefore tested selected metabolic responses after treatments with 60 and 120 microM Al for 7 days. Shoot Al content was not elevated in comparison with control (12.3-14.1 microg g(-1) DW) while total root Al increased strongly, reaching 2680 and 4400 microg g(-1) DW in 60 and 120 microM treatments, respectively. Intra-root Al represented 83.6 (60 microM treatment) and 75.8% (120 microM treatment) of total root Al. Soluble proteins were not significantly affected. Free amino acids were almost unaffected in shoots while in roots the highest content was found in 60 microM Al. Ascorbate- and guaiacol-peroxidase activities were the highest in 60 microM Al-exposed roots. On the other hand, phenylalanine ammonia-lyase activity, total soluble phenols, flavonoids, a sum of 13 phenolic acids and partially two flavonols (quercetin and kaempferol) increased in the shoots. Present study has shown lower Al toxicity and unaltered shoot Al content seems to be the most positive outcome in comparison with previously tested metals (Cd, Ni and Cu). Our results indicate that phenols in shoots and free amino acids in roots are influenced by Al excess in chamomile plants. Possible mechanisms in the context of available literature are suggested and discussed.