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.

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 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.