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.

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 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 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.

Alterations of the gene expression, lipid peroxidation, proline and thiol content along the barley root exposed to cadmium.

Barley seedlings grown on filter paper moistened with 1mM Cd showed 50% root growth inhibition within 24h of exposure. The amount of cadmium after 24h Cd treatment was highest in the first 2mm-long apical root segment, while it was slightly higher in the fourth segment, 6-8mm behind the root tip, after 48h. In recovery experiments, when Cd-treated plants were transferred onto filter paper moistened with distilled water, a large amount of Cd was localised in the apoplast and considerable cell death was detected even though root growth was renewed. This indicates that cell death is likely an active physiological process that contributes to the removal of Cd from the root during root growth recovery. Elevated lipid peroxidation and thiol contents were detected in all individual segments of Cd-treated barley root. On the other hand, proline accumulation was disturbed during Cd stress, showing a significant decrease in all of the studied segments except the first. Cd-induced alteration in the expression of genes involved in metal signalling and detoxification and in drought and oxidative stress responses indicates that Cd-induced water and oxidative stress is responsible for the root growth inhibition, probably through an accelerated differentiation of root tissues.

Rhizosphere localized cationic peroxidase from barley roots is strongly activated by cadmium and correlated with root growth inhibition.

The activity of Cd-induced POD isozyme isolated from the surface of intact barley roots growing under some abiotic stress conditions (toxic metals: Al, Co, Cu, Hg; drought, NaCl, extreme temperatures: heat, cold) and compounds activating (2,4-D) or inhibiting (SHAM) POD activity as well as H(2)O(2) and H(2)O(2) scavenger (DTT) was characterized. Strong Cd concentration-dependent accumulation of one cationic POD isozyme was observed on PAGE, which correlated with Cd- and other stress induced root growth inhibition. This isoPOD is basic with isoelectric point about 9 and its localization is restricted only to the apical part of the barley root tip.