Cadmium activates both diphenyleneiodonium- and rotenone-sensitive superoxide production in barley root tips.

MAIN CONCLUSION: Mild Cd stress-activated diphenyleneiodonium-sensitive superoxide production is utilized in root morphogenic responses, while severe Cd stress-induced robust rotenone-sensitive superoxide generation may lead to cell and root death. In barley, even a few minute exposure of roots to Cd concentration higher than 10 µM evoked a strong superoxide generation in the root transition zone. This superoxide generation was strongly inhibited by the inhibition of mitochondrial electron flow into complex III in the presence of the mitochondrial complex I inhibitor rotenone. Similarly, the superoxide generation induced by antimycin A, an inhibitor of mitochondrial complex III, was considerably reduced by rotenone, suggesting the involvement of complex III also in the severe Cd stress-induced superoxide generation. This severe Cd stress-induced superoxide generation was followed by an extensive cell death in this part of the root tip, which similar to the superoxide generation, was eliminated by rotenone co-treatment. In turn, mild Cd stress-induced diphenyleneiodonium (DPI)-sensitive superoxide generation was observed only in the post-stressed roots, suggesting that it is not directly associated with Cd toxicity. Diphenyleneiodonium, an inhibitor of NADPH oxidase, markedly inhibited the mild Cd stress-induced radial expansion of root apex, indicating that enhanced DPI-sensitive superoxide production is required for rapid isotropic cell growth. Severe Cd stress, probably through the inhibition of complex III, caused a rapid and robust superoxide generation leading to cell and/or root death. By contrast, mild Cd stress did not evoke oxidative stress, and the enhanced DPI-sensitive superoxide generation is utilized in adaptive morphogenic responses.

Salicylic acid alleviates cadmium-induced stress responses through the inhibition of Cd-induced auxin-mediated reactive oxygen species production in barley root tips.

Auxin is a master regulator of root growth by modulating its development under the constantly changing environment. Recently, an antagonistic interaction was suggested between SA and IAA signaling. Therefore, the purpose of this work was to analyze and compare the effect of the indole-3-acetic acid (IAA) signaling inhibitor p-chlorophenoxyisobutyric acid (PCIB) and salicylic acid (SA) as a potential IAA signaling inhibitor on the root growth, enzyme activity and reactive oxygen species (ROS) production in Cd- and IAA-treated barley root tips. Exposure of plants to Cd resulted in a more than threefold increase of IAA content in the root apex even 3h after the treatment. In addition, exogenously applied IAA evoked root responses such as root growth inhibition and swelling, ROS generation and activation of lipoxygenase or glutathione peroxidase identical to those induced by Cd. Furthermore, both Cd- and IAA-induced stress responses were markedly reversed by PCIB or SA post-treatment. Similarly to PCIB, SA did not affect the IAA content of root tips, suggesting the action of SA on the IAA signaling pathway in barley roots. SA probably does not alleviate the Cd toxicity in roots, but rather prevents or partially inhibits the root defense response to the presence of Cd through the inhibition of Cd-induced IAA-mediated ROS generation in roots.

Low Cd concentration-activated morphogenic defence responses are inhibited by high Cd concentration-induced toxic superoxide generation in barley root tip.

Exposure of roots to low Cd concentration induced morphogenic responses including the inhibition of root growth and the radial swelling of root tip. High Cd concentrations within a few minutes caused a robust induction of superoxide generation leading to the cell death and root growth arrest. This toxic superoxide generation blocked the development of low Cd concentration-activated morphogenic responses. While the morphogenic responses of roots to low Cd concentration are induced very rapidly and probably due to the interaction of Cd with the apoplast of root tissue, high Cd concentration-induced superoxide production required the entry of Cd into the symplast. Auxin signaling is involved in the activation of Cd-induced morphogenic defence responses but not in the Cd-induced toxic superoxide generation. These results suggest that oxidative stress is not a primary cause for the Cd-induced morphogenic responses such as growth reduction and radial cell expansion in barley root tips.

Enhanced lipoxygenase activity is involved in the stress response but not in the harmful lipid peroxidation and cell death of short-term cadmium-treated barley root tip.

Root growth inhibition and radial root swelling were the characteristic symptoms of barley root tips after the short-term exposure of roots to 15 and 30µM Cd. Higher Cd concentrations caused extensive cell death and root growth arrest. Enhanced lipid peroxidation was observed as early as 1h after the short-term treatment in a Cd concentration-dependent manner. In contrast to lipid peroxidation, the induction of lipoxygenase activity was detected only 3h after the exposure of roots to 15 or 30µM Cd. In addition, it was not observed in 60µM Cd-treated root tips. The highest lipoxygenase activity was detected 6h after 15µM Cd treatment in the meristematic and elongation zone of root tip and was probably associated with the radial expansion of cells. Our results indicate that the upregulation of lipoxygenase is an important component of stress response in barley roots to toxic Cd. It is probably involved in the morphological stress response of root tips or/and in the alleviation of Cd-induced toxic alterations in plant cell membranes, but it is not responsible for the Cd-induced harmful lipid peroxidation and cell death.

Glutathione peroxidase expression and activity in barley root tip after short-term treatment with cadmium, hydrogen peroxide and t-butyl hydroperoxide.

The purpose of this study was to analyse the alterations of glutathione peroxidase (GPX) expression and activity during the recovery period after a short-term treatment of barley root tip with cadmium (Cd) and hydrogen peroxide (H(2)O(2)). The transcript level of GPX increased as early as 1 h and GPX activity 3 h after short-term treatment independently of Cd concentration. In 15 µM Cd-treated roots, its expression reached a peak within 2 h and sustained until 3 h, after which it gradually declined. After 6 h of short-term Cd treatment, the activity of GPX was the highest in the 15-µM Cd-treated roots. At higher Cd concentrations, the activity of GPX was lower than in 15 µM Cd-treated roots, but still higher than in control roots. A considerable increase in H(2)O(2) production was observed even after only 1 h of short-term exposure of roots to 30 and 60 µM Cd, while after 15 µM Cd exposure, its production increased 3 h after the treatment. Lipid peroxidation increased even 1 h after short-term treatment in a Cd concentration-dependent manner. A considerable decrease of GPX activity was observed after the exposure of roots to H(2)O(2) or t-butyl hydroperoxide in a concentration-dependent manner despite that its expression increased even 1 h after short-term treatment. Presumable, under high acute Cd stress, rapid accumulation of H(2)O(2) leads to the disturbance of basal metabolic processes affecting also GPX activity. In contrast, high GPX activity under moderate Cd stress maintains cell function despite the high rate of H(2)O(2) metabolism in root tip.

Superoxide production induced by short-term exposure of barley roots to cadmium, auxin, alloxan and sodium dodecyl sulfate.

KEY MESSAGE: Abiotic stress-induced superoxide generation depending on its localization, level, duration and presumably also on the action of other signals may lead to different stress responses. The purpose of this study was to analyze the alterations in superoxide generation and morphogenesis following short-term Cd, IAA and alloxan treatments, during stress and recovery period in barley root tips. At low Cd concentration the transient accumulation of superoxide in the epidermal cells was accompanied by root growth inhibition and radial expansion of cortical cells in the elongation zone of root tips. These morphological changes were very similar to the externally applied IAA-induced responses. However, the role of superoxide generated in the epidermal cells by low concentration of Cd and IAA is probably alone not sufficient for the induction of these processes. SDS as an activator of NOX activity caused a strong accumulation of superoxide in the epidermal cells along the whole root apex but without any changes in root morphology and growth. On the other hand, higher Cd concentrations as well as alloxan stimulated the generation of superoxide in the cortical tissue of the elongation zone of root tip, which was accompanied by the induction of cell death. Our results suggest that enhanced superoxide generation, depending on its localization, level, duration and presumably also on the action of other signals, may lead to altered root morphology (15 µM Cd or IAA), root growth inhibition (alloxan), transient root growth cessation (30 µM Cd) or to the death of cells/root at higher (60 µM) Cd concentrations.

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.

Short-term aluminium-induced changes in barley root tips.

The short-term exposure of barley roots to low Al concentration caused significant root growth inhibition and radial swelling of roots. During Al treatment, the radial expansion of root cells occurred in root tissues representing elongation zone and meristem. Both low pH and Al treatments caused significant disruption of cell membranes in swollen roots. In contrast to Evans blue uptake callose formation was observed only at higher Al concentrations and was detected in both swollen and adjacent root areas. Similarly to Al, exogenous short-term application of indole-3-acetic acid, polar transport inhibitor triiodobenzoic acid, ethylene precursor 1-aminocyclopropane-1-carboxylic acid or H(2)O(2) evoked root growth inhibition and radial cell expansion in barley root tip too.

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 on the distribution of hydroxyl radical, superoxide and hydrogen peroxide in barley root tip.

In the present study, we investigated the alteration of reactive oxygen species production along the longitudinal axis of barley root tips during Cd treatment. In unstressed barley root tips, H(2)O(2) production decreased from the root apex towards the differentiation zone where again, a slight increase was observed towards the more mature region of root. An opposite pattern was observed for O(2)(*-) and OH(*) generation. The amount of both O(2)(*-) and OH(*) was highest in the elongation zone, decreased in the root apex and at the differentiation zone of root, then increased again towards the more mature region of root. An elevated Cd-induced O(2)(*-) production started in the elongation zone and increased further along the differentiation zone of barley root tip. In contrast, Cd-induced H(2)O(2) production was localised to the root elongation zone and to the beginning of the differentiation zone. In contrast to Cd-induced H(2)O(2) and O(2)(*-) production, Cd reduced OH(*) production along the whole barley root tip. Our results suggest that not only an increase but also the spatial distribution of reactive oxygen species production is involved in the Cd-induced stress response of barley root tip.

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.

Aluminium-induced drought and oxidative stress in barley roots.

The aim of the present study was to examine the relation between Al accumulation in root tissues, root growth inhibition, root water content, cell viability and expression of oxidative and drought stress-related genes in barley roots growing on the filter paper. Al-induced root growth inhibition correlated with Al uptake and cell death. Water content of Al-treated root represented only half of the control one. The expression of the dehydrin gene dhn4, which is a marker for drought stress in plant tissues, was strongly induced during Al stress. Al treatment also induced expression of oxidative stress-related genes such as glutathione peroxidase (gpx), pathogen-related peroxidase (prx8), glutathione reductase (gr) and dehydroascorbate reductase (dhar). The present results suggest correlation between Al uptake, Al-induced drought stress, oxidative stress, cell death and root growth inhibition.

Vascularization, high-volume solution flow, and localized roles for enzymes of sucrose metabolism during tumorigenesis by Agrobacterium tumefaciens.

Vascular differentiation and epidermal disruption are associated with establishment of tumors induced by Agrobacterium tumefaciens. Here, we address the relationship of these processes to the redirection of nutrient-bearing water flow and carbohydrate delivery for tumor growth within the castor bean (Ricinus communis) host. Treatment with aminoethoxyvinyl-glycine showed that vascular differentiation and epidermal disruption were central to ethylene-dependent tumor establishment. CO2 release paralleled tumor growth, but water flow increased dramatically during the first 3 weeks. However, tumor water loss contributed little to water flow to host shoots. Tumor water loss was followed by accumulation of the osmoprotectants, sucrose (Suc) and proline, in the tumor periphery, shifting hexose-to-Suc balance in favor of sugar signals for maturation and desiccation tolerance. Concurrent activities and sites of action for enzymes of Suc metabolism changed: Vacuolar invertase predominated during initial import of Suc into the symplastic continuum, corresponding to hexose concentrations in expanding tumors. Later, Suc synthase (SuSy) and cell wall invertase rose in the tumor periphery to modulate both Suc accumulation and descending turgor for import by metabolization. Sites of abscisic acid immunolocalization correlated with both central vacuolar invertase and peripheral cell wall invertase. Vascular roles were indicated by SuSy immunolocalization in xylem parenchyma for inorganic nutrient uptake and in phloem, where resolution allowed SuSy identification in sieve elements and companion cells, which has widespread implications for SuSy function in transport. Together, data indicate key roles for ethylene-dependent vascularization and cuticular disruption in the redirection of water flow and carbohydrate transport for successful tumor establishment.