Barley Root Tips Research Articles

Multi-omics analysis unveils early molecular responses to aluminum toxicity in barley root tip

Multi-omics analysis unveils early molecular responses to aluminum toxicity in barley root tip

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

Main conclusionNitric 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.

Arsenic Cytotoxicity: A bioassay using Barley

Barley, Hordeum vulgare (2n=14) has been used by many cytologists, to determine cytotoxicity of environmental pollutants like heavymetals, industrial effluents, agricultural chemicals etc. In the present study, Arsenic(in the form of arsenic trioxide of concentrations100,250, 500,750and1000ppb), cytotoxicity was analyzed on Barley root tip mitosis, to understand its toxic effects on cells and chromosomes of a plant-basedsystem. The results show that Arsenic can induce various Mito depressive and clastogenic chromosomal aberrations like chromosomal breakages, disturbed spindle organization and other cellular metabolic defects. Generally the cytological anomalies were found to be dose-dependent.

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

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 ifPRXs 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

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, 30min, 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.

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.

Mild cadmium stress induces auxin synthesis and accumulation, while severe cadmium stress causes its rapid depletion in barley root tip

The aim of this study was to investigate the effect of 4-phenoxyphenylboronic acid (PPBo), an effective inhibitor of YUCCA enzyme on mild, moderate and severe Cd stress-induced responses in the root tips of young barley seedlings. While mild and moderate Cd stresses increased, severe Cd stress decreased the root IAA level. PPBo post-treatment strongly inhibited the Cd-induced IAA accumulation without affecting the Cd level in roots and mitigated the mild Cd stress-induced root growth inhibition, root swelling and lipoxygenase and glutathione peroxidase activity. In addition, PPBo reduced the H2O2 and NO generation under mild but had no effect under severe Cd stress, indicating that both H2O2 and NO act downstream of IAA in root responses to mild Cd stress. In turn, moderate or severe Cd stress strongly reduced the expression of IAA synthesis and cell cycle genes in the barley root tip. Therefore, we can conclude that IAA accumulation in the barley root tips is a distinctive feature of mild and moderate Cd stresses, which is involved in both root growth reorientation and defense response activation. PPBo completely reversed this Cd-induced IAA accumulation and stress responses, indicating that Cd induces IAA synthesis via indole-3-pyruvic acid biosynthesis pathway. On the contrary, severe Cd stress reduced IAA level was coupled with high ROS in the root tips and root growth arrest.

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.

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.

Ca2+ involved in GABA signal transduction for phenolics accumulation in germinated hulless barley under NaCl stress

In this study, in order to investigate the role of Ca2+ in GABA signal transduction involved in phenolics accumulation in barley seedlings under NaCl stress, the seedlings were treated with exogenous GABA and its synthesis inhibitor, 3-mercaplopropionic acid (3-MP), as well as Ca2+ channel blockers La3+, Ca2+ chelator EGTA, and Ca2+ release channel inhibitor 2-aminoethoxydiphenyl borate (2-APB). The results showed that GABA significantly enhanced phenolics, calcium and calmodulin content. It also induced Ca2+ influx in barley root tips cells, and altered the distribution of Ca2+, making calcium precipitates more uniform and intensive. While, 3-MP treatment led to opposite changes, which suggested that GABA was essential for calcium content increase. In addition, accumulation of phenolics was inhibited by LaCl3, EGTA and 2-APB treatments, and this inhibition could be alleviated partly by exogenous GABA. Taken together, Ca2+ was involved in GABA signal transduction for phenolics accumulation in barley seedlings under NaCl stress.

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.

Analysis of aluminum toxicity in Hordeum vulgare roots with an emphasis on DNA integrity and cell cycle.

Barley is one of the cereals that are most sensitive to aluminum (Al). Al in acid soils limits barley growth and development and, as a result, its productivity. The inhibition of root growth is a widely accepted indicator of Al stress. Al toxicity is affected by many factors including the culture medium, pH, Al concentration and the duration of the treatment. However, Al can act differently in different species and still Al toxicity in barley deserves study. Since the mechanism of Al toxicity is discussed we cytogenetically describe the effects of different doses of bioavailable Al on the barley nuclear genome—mitotic activity, cell cycle profile and DNA integrity. At the same time, we tested an established deep-water culture (DWC) hydroponics system and analyzed the effects of Al on the root system parameters using WinRHIZO software. We demonstrated the cytotoxic and genotoxic effect of Al in barley root cells. We showed that Al treatment significantly reduced the mitotic activity of the root tip cells and it also induced micronuclei and damaged nuclei. The DNA-damaging effect of Al was observed using the TUNEL test. We define the inhibitory influence of Al on DNA replication in barley. Analysis with the labelling and detection of 5-ethynyl-2‘-deoxyuridin (EdU) showed that the treatment with Al significantly decreased the frequency of S phase cells. We also demonstrated that Al exposure led to changes in the cell cycle profile of barley root tips. The delay of cell divisions observed as increased frequency of cells in G2/M phase after Al treatment was reported using flow cytometry.

Heavy metal-induced reactive oxygen species and cell death in barley root tip

Transient exposure of roots to Cd, Pb, Hg or Cu for 30min reduced root growth in a concentration-dependent manner. While the lower concentrations of metals evoked a marked root growth inhibition accompanied by a visible radial root expansion, the higher concentrations caused root growth arrest without the radial expansion of root cells. At lower metal concentrations, evoking mild stress, the amount of reactive oxygen species (ROS) increased linearly after the treatments. They probably function in both stress signalization and metabolic adaptation processes without damaging the root cells. In turn, at high metal concentrations, causing moderate or severe stress, their production has a biphasic character. The amount of ROS, mainly superoxide, increased rapidly in a metal dose-dependent manner, causing a marked cell death at the site of their generation in the root tips. After this transient burst, ROS generation increased again, together with highly ROS, during the destructive processes associated with cell death in the root tips. Our results show that despite the different origin of ROS induced by these heavy metals, the rate of their generation, depending on the metal concentration, determines the development of typical mild stress-activated morphogenic changes or severe stress-caused cell death within a few minutes after the exposure of roots to toxic level of metals.

Depletion of extracellular calcium increases cadmium toxicity in barley root tip via enhanced Cd uptake-mediated superoxide generation and cell death

Whereas severe Cd stress (150 µM Cd) causes root growth arrest as a consequence of marked superoxide generation leading to extensive cell death in the root tips, mild Cd stress (15 µM Cd) evokes morphogenic responses, such as reduced root elongation and radial root expansion, resulting in shorter and thicker roots. Similar to the low Cd concentration-caused mild stress, treatment of roots with either Ba to remove exchangeable or EDTA to remove both exchangeable and tightly bound cations, including Ca and Mg, from the apoplast, induced root growth inhibition and swelling. However, pre-treatment of roots with Ba had a synergistic effect on the development of these mild Cd stress-induced morphogenic responses, but without the development of any other symptoms in the root tips. In turn, EDTA pre-treatment markedly increased the toxicity of Cd in barley root tips via enhanced Cd uptake-mediated superoxide generation, which evoked extensive cell death in the transition zone of root tips identically to the high Cd concentration-induced severe stress. While the mild stress-induced responses were alleviated by the inhibition of auxin signalling pathway, the severe stress-induced symptoms were prevented by Ca, but not Mg, supplementation or by the inhibition of Cd uptake into the root symplasm. Therefore, the appropriate concentration of Ca in the apoplast is crucial to prevent the rapid accumulation of Cd in the symplasm, which above a certain threshold level leads to the huge superoxide generation and cell death.

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.

DNA replication after mutagenic treatment in Hordeum vulgare

The temporal and spatial properties of DNA replication in plants related to DNA damage and mutagenesis is poorly understood. Experiments were carried out to explore the relationships between DNA replication, chromatin structure and DNA damage in nuclei from barley root tips. We quantitavely analysed the topological organisation of replication foci using pulse EdU labelling during the S phase and its relationship with the DNA damage induced by mutagenic treatment with maleic hydrazide (MH), nitroso-N-methyl-urea (MNU) and gamma ray. Treatment with mutagens did not change the characteristic S-phase patterns in the nuclei; however, the frequencies of the S-phase-labelled cells after treatment differed from those observed in the control cells. The analyses of DNA replication in barley nuclei were extended to the micronuclei induced by mutagens. Replication in the chromatin of the micronuclei was rare. The results of simultanous TUNEL reaction to identify cells with DNA strand breaks and the labelling of the S-phase cells with EdU revealed the possibility of DNA replication occurring in damaged nuclei. For the first time, the intensity of EdU fluorescence to study the rate of DNA replication was analysed.

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

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 10µM 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.

Early generation of nitric oxide contributes to copper tolerance through reducing oxidative stress and cell death in hulless barley roots.

The objective of this study was to investigate the specific role of nitric oxide (NO) in the early response of hulless barley roots to copper (Cu) stress. We used the fluorescent probe diaminofluorescein-FM diacetate to establish NO localization, and hydrogen peroxide (H2O2)-special labeling and histochemical procedures for the detection of reactive oxygen species (ROS) in the root apex. An early production of NO was observed in Cu-treated root tips of hulless barley, but the detection of NO levels was decreased by supplementation with a NO scavenger, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO). Application of sodium nitroprusside (a NO donor) relieved Cu-induced root inhibition, ROS accumulation and oxidative damage, while c-PTIO treatment had a synergistic effect with Cu and further enhanced ROS levels and oxidative stress. In addition, the Cu-dependent increase in activities of superoxide dismutase, peroxidase and ascorbate peroxidase were further enhanced by exogenous NO, but application of c-PTIO decreased the activities of catalase and ascorbate peroxidase in Cu-treated roots. Subsequently, cell death was observed in root tips and was identified as a type of programed cell death (PCD) by terminal deoxynucleotidyl transferase dUTP nick end labeling assay. The addition of NO prevented the increase of cell death in root tips, whereas inhibiting NO accumulation further increased the number of cells undergoing PCD. These results revealed that NO production is an early response of hulless barley roots to Cu stress and that NO contributes to Cu tolerance in hulless barley possibly by modulating antioxidant defense, subsequently reducing oxidative stress and PCD in root tips.

Cadmium localization and its toxic effects on root tips of barley

Cadmium localization and its toxic effects on root tips of barley

Extracellular glutathione recycling by γ-glutamyl transferase in barley root tip exposed to cadmium

A considerable amount of extracellular glutathione (eGSH) is detectable in intact root tips, but even the immersion of roots into water markedly decreased its detectability, suggesting its localization into the rhizosphere. While in water-treated seedlings the amount of eGSH was rapidly restored during the recovery period, in Cd-treated roots its amount was restored much more slowly. Enhanced γ-glutamyl transferase (GGT) activity was detected in 15μM Cd-treated root tips 1h after the end of short-term treatment, reaching its maximum level 2h after the treatment. Thereafter it decreased to the level of control roots. By contrast, higher Cd concentrations inhibited the activity of GGT and more strongly inhibited the restoration of eGSH pool in root tips. Depletion of eGSH was accompanied by inhibition of GGT activity, whereas its elevated level slightly activated it. We showed that elevated eGSH, induced either by exogenously applied GSH or by GGT activity inhibition, triggered enhanced ROS generation and marked root growth inhibition in both control and Cd-treated seedlings. In addition, the toxicity of the Cd–GSH complex was more serious than the toxicity of Cd alone. Our results indicated that just recycling of eGSH or/and Cd/eGSH complex by GGT but not its level in the apoplast plays role in the sensing of toxic compounds and in the activation of defence responses. On the other hand, above a certain threshold concentration both eGSH and Cd/GSH complex due to their high toxicity can impair defence responses, causing detrimental damages in root tissues.