Ladislav Tamás

Effect of abiotic stresses on glutathione peroxidase and glutathione S-transferase activity in barley root tips

In the present work we investigated the activity of glutathione S-transferase (GST) and glutathione peroxidase (GPX) in barley root tip and their relation to root growth inhibition induced by different abiotic stresses. Cadmium-induced root growth inhibition is strongly correlated with increased GST and GPX activity. Similarly, strong induction of GPX and GST activity was observed in Hg-treated root tips, where also the highest root growth inhibition was detected. Relationship between increased GST activity and root growth inhibition was also observed during other heavy metal treatments. On the other hand, only a slight increase of GPX activity was observed after application of Pb, Ni, and Zn, while Co did not affect GPX activity. Similarly to Hg and Cd, Cu treatment caused a strong increase in GPX activity. GPX activity in barley root tips was not affected by cold, heat or drought treatment and only a slight increase was observed after salt or H(2)O(2) treatment. Apart from salt treatment, only a weak increase in GST activity was observed during heat, drought and H(2)O(2) stresses, while during cold treatment its activity slightly decreased. Some detected differences in the spatial distribution of GST and GPX activity along the root tip suggests that at least two proteins are responsible for these activities. These proteins play a crucial role not only during stresses, but also in unstressed seedlings in the differentiation processes of root tip. The application of different inhibitors suggests that the main proportion of these activities detected in barley root tip are probably catalysed by GSTs possessing also GPX activity.

Effect of aluminium on oxidative stress related enzymes activities in barley roots

The impact of aluminium stress on activities of enzymes of the oxidative metabolism: superoxide dismutase (SOD), ascorbate peroxidase (APX), peroxidase (POD), NADH peroxidase (NADH-POD) and oxalate oxidase (OXO) was studied in barley (Hordeum vulgare L. cv. Alfor) root tips. SOD appeared to be involved in detoxification mechanisms at highly toxic Al doses and after long Al exposure. POD and APX, H2O2 consuming enzymes, were activated following similar patterns of expression and exhibiting significant correlation between their elevated activities and root growth inhibition. The signalling role of NADH-POD in oxidative stress seems to be more probable than that of OXO, which might be involved in Al toxicity mechanism.

Aluminum-induced cell death of barley-root border cells is correlated with peroxidase- and oxalate oxidase-mediated hydrogen peroxide production

The function of root border cells (RBC) during aluminum (Al) stress and the involvement of oxalate oxidase, peroxidase and H2O2 generation in Al toxicity were studied in barley roots. Our results suggest that RBC effectively protect the barley root tip from Al relative to the situation in roots cultivated in hydroponics where RBC are not sustained in the area surrounding the root tip. The removal of RBC from Al-treated roots increased root growth inhibition, Al and Evans blue uptake, inhibition of RBC production, the level of dead RBC, peroxidase and oxalate oxidase activity and the production of H2O2. Our results suggest that even though RBC actively produce active oxygen species during Al stress, their role in the protection of root tips against Al toxicity is to chelate Al in their dead cell body.

Role of reactive oxygen species-generating enzymes and hydrogen peroxide during cadmium, mercury and osmotic stresses in barley root tip.

The effect of cadmium (Cd) on the expression and activity of NADPH oxidase, peroxidase and oxalate oxidase as well as on the expression of aquaporins and dehydrins was studied in barley root tip. The root tip represented intact apical part of the barley root containing the root cap, meristems and elongation zone. Except stress induced by Cd, barley root tips were analysed after their exposure to phytotoxic concentration of mercury (Hg)-, hydrogen peroxide (H2O2)- or polyethylene glycol (PEG)-induced water stress in order to compare the Cd-induced changes with changes induced by these other stress factors. Cd, Hg, H2O2 and with some exceptions also PEG treatments caused similar alterations in the gene expression of reactive oxygen species (ROS)-generating and water deficiency-related genes, and in the activity of ROS-generating enzymes. These evidences support our opinion that ROS accumulation and water imbalance are the common symptoms of these stress factors and that the elevated production of H2O2 plays, probably as a signal molecule, a key role in the induction of plant responses to abiotic stresses in barley root tip. On the other hand, H2O2 at permanent high concentration is probably the main toxic factor during stress conditions.

Effect of cadmium on diaphorase activity and nitric oxide production in barley root tips.

The effect of Cd on NADPH-diaphorase activity and nitric oxide (NO) production was investigated in barley root tips. The Cd-induced increase of NADPH-diaphorase activity occurred at the elongation zone and increased further in the differentiation zone of barley root tips. This activity was associated primarily with the microsomal membrane fraction of crude extract. In situ analysis revealed that the diaphorase activity was localized in the metaxylem and metaphloem elements and to some cells of the pericycle and parenchyma of root tips. Cd-induced NO generation was observed in pericycle, parenchymatic stelar cells and companion cells of protophloem. The results suggest that the Cd-induced generation of NO functions in Cd toxicity through the ectopic and accelerated differentiation of root tips, causing the shortening of the root elongation zone and a subsequent reduction in root growth.

Root growth inhibition by aluminum is probably caused by cell death due to peroxidase-mediated hydrogen peroxide production.

Summary.The effect of aluminum on hydrogen peroxide production and peroxidase-catalyzed NADH oxidation was studied in barley roots germinated and grown between two layers of moistened filter paper. Guaiacol peroxidase activity significantly increased after 48 h and was approximately two times higher after 72 h in Al-treated roots. The oxidation of NADH was also significantly increased and, like guaiacol peroxidase activity, it was two times higher in Al-treated roots than in controls. Elevated H2O2 production was observed both 48 and 72 h after the onset of imbibition in the presence of Al. Separation on a cation exchange column allowed the detection of two peaks with NADH peroxidase and H2O2 production activity. However, a difference between control and Al-treated plants was found only in one fraction, in which four times higher guaiacol peroxidase activity and five times higher NADH peroxidase activity were expressed and about three times more H2O2 was produced. One anionic peroxidase and three cationic peroxidases were detected in this fraction by native polyacrylamide gel electrophoresis. The anionic peroxidase was activated in the Al-treated root tips and also oxidized NADH but was detectable only after a long incubation time. Two of the cationic peroxidases were capable of oxidizing NADH and producing a significant amount of H2O2, but only one of these was activated by Al stress. The role of these peroxidases during Al stress in barley root tips is discussed.

Rapid and simple method for Al-toxicity analysis in emerging barley roots during germination

The results demonstrate the benefits of using filter-paper-based system for cultivation the germinating barley seeds for Al toxicity or Al tolerance analyses. Due to the high affinity of filter paper to Al monomeric forms, milimolar Al concentrations were required to cause similar Al toxicity symptoms of roots as micromolar Al concentrations in hydroponics: 1 mM Al had no effects on the emerging barley roots, 2 mM Al was moderately toxic but roots showed good recovery, 4 mM Al was highly toxic and 8 mM Al even lethal. Screening of eight barley cultivars revealed different rank of their tolerance to Al. The root growth inhibition positively correlated with the Al concentration in root tips.

Inhibition of Al-induced root elongation and enhancement of Al-induced peroxidase activity in Al-sensitive and Al-resistant barley cultivars are positively correlated

The quantitative changes in peroxidase activity and composition of anionic and cationic isoperoxidases were investigated in roots of two barley cultivars differing in Al resistance. Root growth of Al-resistant cv. Bavaria was in lesser extent reduced by Al treatment (23% after 24 h Al-treatment), whereas 40% reduction of the root growth was observed in Al-sensitive cv. Alfor. The strong root growth inhibition in Al-sensitive cv. Alfor correlated with a 6-fold enhancement of peroxidase activity by Al treatment. Al-induced enhancement of peroxidase activity was found also in roots of Al-resistant cv. Bavaria, but this increase was only half of the Al-sensitive cv. Alfor. Comparison of peroxidase isoenzyme composition of Al-treated and non-treated roots revealed that activity of at least five anionic and four cationic isoperoxidases was stimulated by Al treatment. Three of anionic isoperoxidases (aPOD2-4) were selectively induced only in the Al-sensitive cv. Alfor. A possible involvement of peroxidases in root-growth inhibition is discussed.

Effect of cadmium and temperature on the lipoxygenase activity in barley root tip

An analysis of different cell fractions isolated from barley roots revealed that lipoxygenase (LOX) activity occurred both extra- and intracellulary. Cadmium (Cd)-induced LOX activity was observed in the fraction containing cell walls, plasma membrane and the cytoplasm. High temperature-induced root growth inhibition and elevated LOX activity did not induce lipid peroxidation. In contrast, Cd inhibited root growth and caused both enhanced lipid peroxidation and elevated LOX activity at each of the temperatures analyzed. Spatial distribution studies revealed that the patterns of apoplastic LOX activity were different from those of cytoplasmic activity. Cd-induced intracellular LOX activity increased equally along the barley root tip, while Cd-induced apoplastic LOX activity was associated mainly with the differentiation zone of the barley root tip. Our results suggest the involvement of Cd-induced LOX activity in the premature differentiation of the barley root tip during Cd stress. We hypothesize that the role of LOX in plant metabolic processes in the root may depend on the level of reactive oxygen species in the roots: at physiological concentrations of ROS, LOX may be involved in the processes of root growth, while at the elevated harmful concentrations of ROS induced by different stress conditions, it may be involved in root growth inhibition through ectopic differentiation.

Impact of the auxin signaling inhibitor p-chlorophenoxyisobutyric acid on short-term Cd-induced hydrogen peroxide production and growth response in barley root tip

Short-term treatment (30 min) of barley roots with a low 10 μM Cd concentration induced significant H(2)O(2) production in the elongation and differentiation zone of the root tip 3h after treatment. This elevated H(2)O(2) production was accompanied by root growth inhibition and probably invoked root swelling in the elongation zone of the root tip. By contrast, a high 60 μM Cd concentration induced robust H(2)O(2) production in the elongation zone of the root tip already 1h after short-term treatment. This robust H(2)O(2) generation caused extensive cell death 6 h after short-term treatment. Similarly to low Cd concentration, exogenously applied H(2)O(2) caused marked root growth inhibition, which at lower H(2)O(2) concentration was accompanied by root swelling. The auxin signaling inhibitor p-chlorophenoxyisobutyric acid effectively inhibited 10 μM Cd-induced root growth inhibition, H(2)O(2) production and root swelling, but was ineffective in the alleviation of 60 μM Cd-induced root growth inhibition and H(2)O(2) production. Our results demonstrated that Cd-induced mild oxidative stress caused root growth inhibition, likely trough the rapid reorientation of cell growth in which a crucial role was played by IAA signaling in the root tip. Strong oxidative stress induced by high Cd concentration caused extensive cell death in the elongation zone of the root tip, resulting in the cessation of root growth or even in root death.