Extracellular nitric oxide sustains root surface redox activity and growth under sudden flooding-induced hypoxic conditions in barley root tips.

MAIN CONCLUSION: Nitric oxide sustains root tip surface redox activity and restricts lipid peroxidation-triggered cell death in the root tips. In order to gain more insight into the involvement of nitric oxide (NO) in plant response to sudden flooding-induced hypoxic stress, we studied the effect of two NO donors, sodium nitroprusside and S-nitroso-L-glutathione, on short-term partial submergence-induced root growth inhibition, alteration in root surface redox activity, lipid peroxidation and cell death in two barley cultivars (cv.) at their early seedling stage. The short-term hypoxic stress induces root growth arrest in cv. Karmel, accompanied by increased lipid peroxidation and cell death. By contrast, in cv. Slaven, short-term hypoxic conditions cause only reduced root growth rate, associated with elevated extracellular NO level in the root tips. The root tip surface redox activity decreases with the increasing timespan of hypoxic conditions in both cultivars; however, this decrease in redox activity started earlier and was greater in the cv. Karmel in comparison with cv. Slaven. Application of NO donors during hypoxic stress sustains the root redox activity and eliminates the hypoxia-induced lipid peroxidation, accompanied by a partial restoration of root growth after short-term hypoxic stress. These results suggest that extracellular NO plays a key role in maintaining the root tip surface redox activity and in the restriction of lipid peroxidation and cell death under short-term hypoxic stress in the root tips of barley seedlings.

Inhibition of peroxidases and oxidoreductases is crucial for avoiding false-positive reactions in the localization of reactive oxygen species in intact barley root tips.

MAIN CONCLUSION: NBT and HE may be efficiently used for the detection of superoxide, while DCDHF-DA and DHR123 for the detection of peroxynitrite in intact barley root tips, only if PRXs and oxidoreductases are inhibited to avoid false-positive reactions. Strong peroxidase (PRX) and oxidoreductase activities were observed in the barley root tips that were markedly inhibited by NaN3. Rapid and strong nitro-blue tetrazolium chloride (NBT) reduction is associated mainly with the vital functions of root cells but not with superoxide formation. In turn, the inhibition of root surface redox activity by NaN3 strongly reduced the formation of formazan, but its slight accumulation, observed in the root elongation zone, was a result of NADPH oxidase-mediated apoplastic superoxide formation. A longer staining time period with NBT was required for the detection of antimycin A-mediated superoxide formation inside the cells. This antimycin A-induced superoxide was clearly detectable by hydroethidine (HE) after the inhibition of PRXs by NaN3, and it was restricted into the root transition zone. TEMPOL, a superoxide scavenger, strongly inhibited both NBT reduction and HE oxidation in the presence of NaN3. Similarly, the DCDHF-DA and DHR123 oxidation was markedly reduced after the inhibition of apoplastic PRXs by NaN3 and was detectable mainly in the root transition zone. This fluorescence signal was not influenced by the application of pyruvate but was strongly reduced by urea, a peroxynitrite scavenger. The presented results suggest that if the root PRXs and oxidoreductases are inhibited, both NBT and HE detect mainly superoxide, whereas both DCDHF-DA and DHR123 may be efficiently used for the detection of peroxynitrite in intact barley root tips. The inhibition of PRXs and oxidoreductases is crucial for avoiding false-positive reactions in the localization of reactive oxygen species in the intact barley root tip.

Early gene expression response of barley root tip to toxic concentrations of cadmium.

KEY MESSAGE: Already a short-term Cd treatment induces changes in gene expression in barley root tips via IAA and ROS signaling during mild and severe Cd stress, respectively. Even a short, 30 min, Cd treatment of roots induced a considerable alteration in gene expression in the barley root tips within an hour after the treatments. The very early activation of MYB1 transcription factor expression is partially regulated by auxin signaling in mildly stressed seedlings. An increase in allene oxide cyclase and NADPH oxidase expression was a distinguishing feature of root tips response to mild Cd stress and their expression is activated via IAA signaling. Meanwhile, early changes in the level of dehydrin transcripts were detected in moderately and severely stressed root tips, and their induction is related to altered ROS homeostasis in cells. The early activation of glutathione peroxidase expression by mild Cd stress indicates the involvement of IAA in the signaling process. In contrast, early ascorbate peroxidase expression was induced only with Cd treatment causing severe stress and ROS play central roles in its induction. The expression of cysteine protease was activated similarly in both mildly and severely Cd-stressed roots; consequently, both increased IAA and ROS levels take part in the regulation of cysteine protease expression. The Cd-evoked accumulation of BAX Inhibitor-1 mRNA was characteristic for moderately and severely stressed roots. Whereas decreased IAA level did not affect its expression, rotenone-mediated ROS depletion markedly reduced the Cd-induced expression of BAX Inhibitor-1. An early increase of alternative oxidase levels in the root tip cells indicated that the reduction of mitochondrial superoxide generation is an important component of barley root response to severe Cd stress.

Structural traits of leaf epidermis correspond to metal tolerance in Rumex acetosella populations growing on metal-contaminated soils.

The pseudometallophyte Rumex acetosella L. occupies habitats with normal and high soil concentrations of zinc (Zn), lead (Pb), and copper (Cu). It remains unclear if the plants respond to the toxic metals by altering their morphology and increasing the resilience of their cells. We compared plants growing on soils contaminated with Zn/Pb (populations Terézia, Lintich), or Cu (populations Spania Dolina, Staré Hory), with those from non-contaminated soil (Dúbravka) in Slovakia, and analysed leaf structure, physiology, and metal contents by light and electron microscopy, element localization by energy-dispersive X-ray analysis (EDX) in scanning electron microscope, and by specific fluorescence dyes. In control population, the epidermis of the amphistomatic leaves of R. acetosella contained capitate glandular trichomes, consisting of four head (secretory), two stalk, and two basal cells. The ultrastructure of secretory cells revealed fine wall ingrowths bordered by plasma membrane protruding into the cytoplasm. The metallicolous populations had higher contents of Zn and Cu in the epidermal and glandular cells, and a higher density of both stomata and trichomes. Extensive cell wall labyrinth was present in the trichome secretory cells. Their abnormal number and elevated metal contents might indicate effects of heavy metals, especially of Cu, on mitosis and cell plate formation. Differences in leaf physiology were indicated by significantly higher cytoplasmic tolerance to Zn and Cu in metallicolous populations and by structural properties of glandular heads suggesting secretion of toxic metals. Our findings are suggestive of plant reactions to metal stress, which facilitate the populations to occupy the metal-contaminated sites.

Indole-3-butyric acid priming reduced cadmium toxicity in barley root tip via NO generation and enhanced glutathione peroxidase activity.

MAIN CONCLUSION: Activation of GPX and enhanced NO level play a key role in IBA-mediated enhanced Cd tolerance in young barley roots. Application of exogenous indole-3-acetic acid (IAA) or an IAA precursor improves the tolerance of plants to heavy metals. However, the physiology of these tolerance mechanisms remains largely unknown. Therefore, we studied the priming effect of indole-3-butyric acid (IBA), an IAA precursor, on mild and severe cadmium (Cd) stress-induced responses in roots of young barley seedlings. IBA, similarly to mild Cd stress, significantly increased the glutathione peroxidase (GPX) activity in the apexes of barley roots, which remained elevated after the IBA pretreatment as well. IBA pretreatment-evoked high nitric oxide generation in roots effectively reduced the high superoxide level under the severe Cd stress, leading to less toxic peroxynitrite accumulation accompanied by markedly reduced Cd-induced cell death. On the other hand, the IBA-evoked changes in IAA homeostasis resulted in root growth reorientation from longitudinal elongation to radial swelling. However, the application of an IAA signaling inhibitor, following the activation of defense responses by IBA, was able to promote root growth even at high concentrations of Cd. Based on the results, it can be concluded that the application of IBA, as an effective activator of Cd tolerance mechanisms in young barley roots, and the subsequent use of an IAA signaling inhibitor for the inhibition of root morphogenic responses induced by altered auxin metabolism, results in a high degree of root Cd tolerance, helping it to withstand even the transient exposure to lethal Cd concentration without the absolute inhibition of root growth.

Impact of antimycin A and myxothiazol on cadmium-induced superoxide, hydrogen peroxide, and nitric oxide generation in barley root tip.

In order to gain more insight into the involvement of mitochondrial complex III in the Cd-induced stress, we studied the effect of complex III inhibitors, antimycin A (AA), and myxothiazol (MYXO), on the Cd-induced ROS and NO generation in the barley root tip. Short-term exposure of barley roots to either MYXO or AA provoked a dose-dependent increase in both H2O2 and NO formation. In contrast to H2O2 generation, an enhanced superoxide formation in the transition zone of the root was a characteristic feature of AA-treated roots. MYXO and AA co-treatment had an additive effect on the amount of both H2O2 and NO formed in roots. On the other hand, AA-induced superoxide formation was markedly reversed in roots co-treated with MYXO. Both AA and MYXO exacerbated the Cd-mediated H2O2 or NO generation in the root tip. On the contrary, while AA also exacerbated the Cd-induced superoxide generation, MYXO dose-dependently attenuated it. These data provide strong evidence that ROS generation, a very early symptom of Cd toxicity in roots, is originated in mitochondria. Cd, similarly to AA, generates superoxide by blocking the mitochondrial electron transport chain (ETC) at complex III. In turn, the site of Cd-induced NO generation is not associated with complex III, but ROS formed in mitochondria at this third complex of ETC are probably responsible for enhanced NO generation in barley root under Cd stress.

Enhanced nitric oxide generation mitigates cadmium toxicity via superoxide scavenging leading to the formation of peroxynitrite in barley root tip.

The aim of this study was to observe the possible function of increased superoxide and NO production in the response of barley root tip to the harmful level of Cd. While superoxide generation was detected only in the transition zone, the formation of NO was observed in the apical elongation zones of the control root tips. However, the root region with the superoxide generation was also associated with peroxynitrite specific fluorescence signal. Superoxide, H2O2 and peroxynitrite generation increased with Cd treatment in a dose-dependent manner. In turn, NO level increased at low 10-20 µM but decreased at high 50-60 µM Cd concentrations in comparison with the control. While co-treatment of roots with rotenone markedly attenuated the Cd-induced superoxide generation and lipid peroxidation, it increased the level of NO in the root tips. Although rotenone did not influence the Cd-induced increase of GPX activity at 10-30 µM Cd concentrations, it markedly reversed the high 40-60 µM Cd concentrations-induced decline of GPX activity. Cd-induced cell death was associated with robust superoxide generation, but not with a high level of peroxynitrite. The Cd-evoked inhibition of root growth was significantly reversed by a strong antioxidant N-acetyl cysteine but not by a peroxynitrite scavenger uric acid, suggesting that similarly to Cd-induced cell death, an imbalance in the ROS homeostasis and not an enhanced level of peroxynitrite is responsible for the Cd-induced root growth inhibition. Based on these findings, it can be assumed that NO acts mainly in the regulation of superoxide level in the tips of root. Under Cd stress, the enhanced NO level is involved in the scavenging of highly toxic superoxide through the formation of peroxynitrite, thus reducing the superoxide-mediated cell death in barley root.

L-NAME decreases the amount of nitric oxide and enhances the toxicity of cadmium via superoxide generation in barley root tip.

Exposure of barley roots to mM concentrations of L-NAME for 30 min caused a considerable root growth inhibition in a dose-dependent manner. The inhibition of root growth was higher in seedlings co-treated with Cd and L-NAME, compared with roots treated with Cd alone, despite the fact that L-NAME markedly reduced the uptake of Cd by roots. Treatment of roots with L-NAME evoked a decrease in NO level in both control and Cd-treated root tips only after a relatively long lag period, which overlaps with an increase in superoxide and H2O2 levels and peroxynitrite generation. L-NAME-induced root growth inhibition is alleviated not only by the application of the NO donor SNP but also by the ROS and peroxynitrite scavengers. Our results indicate that L-NAME, a NOS inhibitor in the animal kingdom, indeed evokes NO depletion also in the plant tissues; however, it does not occur due to the action of L-NAME as an inhibitor of NOS or NOS-like activity, but as a consequence of L-NAME-induced enhanced superoxide generation, leading to increased peroxynitrite level in the root tips due to the reaction between superoxide and NO.

Mitochondrial complex II-derived superoxide is the primary source of mercury toxicity in barley root tip.

Enhanced superoxide generation and significant inhibition of succinate dehydrogenase (SDH) activity followed by a strong reduction of root growth were detected in barley seedlings exposed to a 5µM Hg concentration for 30min, which increased further in an Hg dose-dependent manner. While at a 25µM Hg concentration no cell death was detectable, a 50µM Hg treatment triggered cell death in the root meristematic zone, which was markedly intensified after the treatment of roots with 100µM Hg and was detectable in the whole root tips. Generation of superoxide and H2O2 was a very rapid response of root tips occurring even after 5min of exposure to Hg. Application of an NADPH oxidase inhibitor or the inhibition of electron flow in mitochondria by the inhibition of complex I did not influence the Hg-induced H2O2 production. Treatment of roots with thenoyltrifluoroacetone, a non-competitive inhibitor of SDH, markedly reduced root growth and induced both superoxide and H2O2 production in a dose dependent manner. Similar to results obtained in intact roots, Hg strongly inhibited SDH activity in the crude mitochondrial fraction and caused a considerable increase of superoxide production, which was markedly reduced by the competitive inhibitors of SDH. These results indicate that the mitochondrial complex II-derived superoxide is the primary source of Hg toxicity in the barley root tip.

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.

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.

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.

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.

Growth and functional responses of different cultivars of Lotus corniculatus to aluminum and low pH stress.

Aluminum toxicity is an important stress factor in acid soils. Growth, respiration and permeability properties of root cells were studied in five cultivars of Lotus corniculatus subjected to aluminum (Al) or low pH stress. The cultivars showed significant differences in root elongation under stress conditions, which correlated with changes in membrane potential (E(M)) of root cortical cells. A pH drop from 5.5 to 4.0 resulted in significant membrane depolarization and root growth inhibition. The strongest inhibition was observed in cv. São Gabriel (33.6%) and least in cv. UFRGS (25.8%). Application of an extremely high Al concentration (2mM) stopped the root growth in cv. INIA Draco, while inhibition in cv. UFRGS reached only 75%. The E(M) values of cortical cells of Lotus roots varied between -115 and -144mV. Treatment with 250microM of AlCl(3) (pH 4) resulted in rapid membrane depolarization. The extent of the membrane depolarization ranged between 51mV (cv. UFGRS) and 16mV (cv. INIA Draco). The membrane depolarization was followed by a loss of K(+) from Al-treated roots (2mM Al) and resulted in a decrease of the diffusion potential (E(D)). The total amount of K(+) in Al-treated roots dropped from 31.4 to 16.8micromolg(-1) FW in sensitive cv. INIA Draco, or from 26.1 to 22.7micromolg(-1) FW in tolerant cv. UFGRS. The rate of root respiration under control conditions as well as under Al treatment was higher in cv. INIA Draco than in cv. UFRGS. Al-induced inhibition of root respiration was 21-34% of the control.