Jozef Kováčik

Nitric oxide signals ROS scavenger-mediated enhancement of PAL activity in nitrogen-deficient Matricaria chamomilla roots: side effects of scavengers.

Owing to the abundance of phenolic metabolites in plant tissue, their accumulation represents an important tool for stress protection. However, the regulation of phenolic metabolism is still poorly known. The regulatory role of reactive oxygen species (ROS) in the activity of phenylalanine ammonia-lyase (PAL) in nitrogen (N)-deficient chamomile roots treated for 24 h was studied using three ROS scavengers [dithiothreitol (DTT), salicylhydroxamic acid, and sodium benzoate]. Scavengers decreased the level of hydrogen peroxide and/or superoxide (and up-regulated ascorbate/guaiacol peroxidase and glutathione reductase), but, surprisingly, stimulated PAL activity. This up-regulation was correlated with increases in nitric oxide (NO) content, total soluble phenols, selected phenolic acids, and, partially, lignin (being expressed the most in DTT-exposed roots). We therefore tested the hypothesis that NO may be involved in these changes. Application of 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO) decreased PAL activity and the accumulation of soluble phenols in all treatments. Exogenous H(2)O(2) and NO also stimulated PAL activity and the accumulation of phenols. We conclude that NO, in addition to hydrogen peroxide, may regulate PAL activity during N deficiency. The anomalous effect of PTIO on NO content and possible mechanism of ROS scavenger-evoked NO increases in light of the current knowledge are also discussed.

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.

Physiological responses of Scenedesmus quadricauda (Chlorophyceae) to UV-A and UV-C light.

Despite intensive research focused on the effects of UV‐B, deeper metabolic responses to UV‐A and UV‐C are still scarce. Besides, especially microalgal species had to develop efficient protective features in comparison with tissue structure of vascular plants. We exposed axenic cultures of Scenedesmus quadricauda (Chlorophyceae) to UV‐A (366 nm) and UV‐C (254 nm) light over 1 h. Both wavelengths stimulated increase in soluble proteins, superoxide radical and hydrogen peroxide, but had a nonsignificant effect on cell viability. Within 17 detected free amino acids, five (including proline) increased in response to UV‐A while only aspartic acid and histidine increased in UV‐C treatment. Total soluble phenols and flavonoids were influenced neither by UV‐A nor by UV‐C while selected flavonols (quercetin and kaempferol) decreased in UV‐A and were not detected in UV‐C treatment. Benzoic acid derivatives increased preferentially after UV‐A illumination (vanillic acid and vanillin) while cinnamic derivatives (caffeic, chlorogenic and p‐coumaric acids) decreased in both UV‐A and UV‐C. It is concluded that UV light stimulated oxidative stress while exposure time was not sufficient to stimulate larger changes in phenolic metabolites. Present findings in the context of available data and with emphasis on phenolics in algae 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.

Physiology of Matricaria chamomilla exposed to nickel excess.

Influence of nickel (Ni) excess on selected physiological aspects of Matricaria chamomilla metabolism after 10 days of presence was studied. Biomass, water content, assimilation pigments and lignin contents were not affected by any of the doses tested. High Ni doses elevated root-soluble proteins. The highest Ni concentration stimulated accumulation of soluble phenolics in both the rosettes and roots, and hydrogen peroxide in the roots. Malondialdehyde content was unaltered, but proline content increased more pronouncedly in the rosettes. Histidine was elevated in the roots, suggesting its involvement in Ni retention. Roots contained 3.4, 7.3 and 6.1 times more Ni than leaf rosettes with 3, 60 and 120 microM treatments, indicating that chamomile is a Ni excluder. Leaf rosettes accumulated 174.1 microg Ni g(-1) DW at 120 microM treatment. The results suggest chamomile tolerance to Ni excess and its considerable accumulation in above-ground biomass (ca. 30% of whole plant Ni content).

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.

Phenolic metabolism of Matricaria chamomilla plants exposed to nickel

We examined accumulation of phenolic acids, total soluble phenolics and flavonoids, and activities of phenolic metabolism-related enzymes (shikimate dehydrogenase (SKDH), phenylalanine ammonia-lyase (PAL), cinnamyl alcohol dehydrogenase (CAD), polyphenol oxidase (PPO)) in Matricaria chamomilla plants exposed to 3, 60 and 120 microM of nickel (Ni) for 10 days. Ni showed low toxicity as indicated by unaltered content of total soluble phenolics in the leaf rosettes. In the roots, the effects of Ni were more visible, including increased total phenolics and PAL activity, but a decrease in PPO activity was observed. CAD activity was not affected by any of the Ni concentrations. Cinnamic acid derivatives were affected more than benzoic acid derivatives. Accumulation of chlorogenic acid, an important antioxidant compound, was enhanced by Ni treatment (ca. 4-fold in 120 microM Ni). Accumulation of protocatechuic acid, a phenol with high chelating strength, even decreased in the leaf rosettes. These observations are discussed in connection to antioxidative properties of phenolic metabolites and previously tested metals (cadmium and copper).

Physiological responses of Matricaria chamomilla to cadmium and copper excess.

Physiological responses of Matricaria chamomilla plants exposed to cadmium (Cd) and copper (Cu) excess (3, 60, and 120 μM for 7 days) with special emphasis on phenolic metabolism were studied. Cu at 120 μM reduced chamomile growth, especially in the roots where it was more abundant than Cd. Notwithstanding the low leaf Cu amount (37.5 μg g−1 DW) in comparison with Cd (237.8 μg g−1 DW) at 120 μM, it caused reduction of biomass accumulation, Fv/Fm ratio and soluble proteins. In combination with high accumulation of phenolics, strong reduction of proteins and high GPX activity in the roots, this supports severe redox Cu properties. In terms of leaf phenylalanine ammonia‐lyase (PAL) activity, it seems that Cd had a stimulatory effect during the course of the experiment, whereas Cu was found to stimulate it after 7‐day exposure. The opposite trend was visible in the roots, where Cd had a stimulatory effect at high doses but Cu mainly at the highest dose. This supports the assumption of different PAL time dynamics under Cd and Cu excess. A dose of 60 and 120 μM Cu led to 2‐ and 3‐times higher root lignin accumulation while the same Cd doses increased it by 33 and 68%, respectively. A Cu dose of 120 μM can be considered as limiting for chamomile growth under conditions of present research, while resistance to high Cd doses was confirmed. However, PAL and phenolics seemed to play an important role in detoxification of Cd‐ and Cu‐induced oxidative stress.

Manganese-induced oxidative stress in two ontogenetic stages of chamomile and amelioration by nitric oxide.

Impact of manganese (Mn(2+)) excess (100, 500 and 1000 μM over 7 days) on two ontogenetic stages (7-week-old plants and 7-day-old seedlings) of Matricaria chamomilla was compared. Mn excess depressed growth of seedlings (but not germination) and stimulated oxidative stress (ROS and lipid peroxidation) in both plants and seedlings. Growth inhibition could be evoked by higher Mn uptake and higher translocation factor in seedlings than in plants. Total thiols staining revealed elevation in almost all treatments. In 7-week-old plants, activity of peroxidases increased slightly and rather decreased under high Mn doses. Superoxide rather than hydrogen peroxide contributed to visualized ROS presence. Fluorescence of nitric oxide (NO) showed stimulation in plants but decrease in seedlings. Impact of exogenous nitric oxide donor (sodium nitroprusside/SNP) was therefore tested and results showed amelioration of 1000 μM Mn-induced oxidative stress in seedlings (decrease in H2O2 and increase in NO content while antioxidative enzyme activities were variably affected) concomitantly with depleted Mn accumulation. It is concluded that NO participates in tolerance to Mn excess but negative effects of the highest SNP dose were also observed. Extensive fluorescence microscopy is also explanatively discussed.

Hexavalent chromium damages chamomile plants by alteration of antioxidants and its uptake is prevented by calcium.

Toxicity of low (3μM) and high (60 and 120μM) concentrations of hexavalent chromium/Cr(VI) in chamomile plants was studied. Fluorescence staining confirmed reduction of Cr(VI) to Cr(III). Cr was mainly accumulated in the roots with translocation factor <0.007. Notwithstanding this, both shoots and roots revealed increase in oxidative stress and depletion of glutathione, total thiols, ascorbic acid and activities of glutathione reductase and partially ascorbate peroxidase mainly at 120μM Cr. Though some protective mechanisms were detected (elevation of nitric oxide, enhancement of GPX activity and increase in phenols and lignin), this was not sufficient to counteract the oxidative damage. Consequently, soluble proteins, tissue water content and biomass production were considerably depleted. Surprising increase in some mineral nutrients in roots (Ca, Fe, Zn and Cu) was also detected. Subsequent experiment confirmed that exogenous calcium suppressed oxidative symptoms and Cr uptake but growth of chamomile seedlings was not improved. Alteration of naturally present reductants could be a reason for Cr(III) signal detected using specific fluorescence reagent: in vitro assay confirmed disappearance of ascorbic acid in equimolar mixture with dichromate (>96% at pH 4 and 7) while such response of glutathione was substantially less visible.