Barley Root Tips Research Articles

Cadmium-induced reactive oxygen species generation, changes in morphogenic responses and activity of some enzymes in barley root tip are regulated by auxin

AbstractMorphogenic responses, ROS generation and changes in enzymes activity induced by the short-term exposure of barley roots to Cd were very similar to those of triiodobenzoic acid (TIBA) treatment. Both Cd and TIBA markedly increased the amount of IAA in root apex. In addition, the treatment of roots with TIBA before Cd treatment had an additive effect on morphogenic responses, changes in enzymes activity and ROS generation. In turn, the application of auxin efflux/influx inhibitor 1-naphthoxyacetic acid (1-NOA) before Cd treatment significantly reduced the Cd-induced ROS generation, root growth inhibition and enzymes activity in spite of the elevated level of IAA in the root apex. The auxin signalling inhibitor p-chlorophenoxyisobutyric acid (PCIB) nearly fully eliminated some of these Cd-induced responses. These results indicated that the short-term Cd treatment-induced ROS generation, morphogenic responses and activity of some enzymes can be attributed to the disturbance of IAA homeostasis in root apex. While auxin efflux inhibitor TIBA evoked similar response to Cd, auxin efflux/influx inhibitor 1-NOA attenuated and auxin signalling inhibitor PCIB blocked these Cd-induced responses suggesting that apart from the elevated level of IAA in root apex its enter and accumulation inside of until closely undetermined cells or tissues is a prerequisite for the induction of characteristic root responses to short-term Cd exposure.

Enhanced nitric oxide generation in root transition zone during the early stage of cadmium stress is required for maintaining root growth in barley

The objective of this study was to analyse the role of nitric oxide (NO) in relation to reactive oxygen species (ROS) production and root growth inhibition in the early response of barley root tip to cadmium. We used cell-permeable NO-binding dye DAF-2DA for the NO localization and cell-impermeable dye DAR-4M for the quantification of NO in barley root apex. A considerable increase in NO production was detected in the transition zone of root tips already 30 min after the immersion of barley roots into 15 μM Cd. This Cd-induced NO generation further increased during the recovery period and reached its maximum level 2 h after the short-term treatment. At this stage an enhanced Cd-induced NO generation was observed in the meristematic, transition and in the beginning of elongation zone in comparison with control roots. Inhibitor of NO synthase activity markedly reduced root growth in control seedlings and had a synergistic growth inhibitory effect with Cd. On the contrary, this Cd-induced root growth inhibition was markedly alleviated by the NO donor. Exposure of roots to Cd markedly increased the production of ROS in the proximal elongation zone of root. While NO did not affect ROS generation, inhibition of its synthesis slightly decreased ROS generation in both control and Cd-treated roots. Our results revealed that enhanced NO production is a very early response of barley root to Cd stress and it is involved in the regulation of root growth mitigating Cd-induced root growth inhibition.

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.

Primary stress response induced by different elements is mediated through auxin signalling in barley root tip

Short-term exposure (15 min) of barley roots to different chemical elements revealed that Cd, Cu, Hg and Pb were the most toxic ones causing a marked root growth inhibition even at µM concentrations. Gd, La, Al, Cr, As, Zn, Ni and Se inhibited root growth to a similar extent only at mM concentrations. Despite the high 20 mM concentration, Co caused only a slight, while Mn, Mg or Ca did not evoke any root growth inhibition. Elements at concentrations inhibiting root growth caused a considerable accumulation of indole-3-acetic acid in the root apex. While Cr, As and Zn inhibited, Cd, Cu, Hg, Pb, Gd, La and Al markedly stimulated the generation of reactive oxygen species in the beginning of differentiation zone. Auxin signalling inhibitor alleviated or prevented root growth inhibition, reactive oxygen species generation and the stimulation of lipoxygenase and glutathione peroxidase activity by various elements, indicating a key role of auxin signalling in the stress response of barley root tip. On the other hand, it did not affect or even had an additive effect on dehydroascorbate reductase and ascorbic acid oxidase activity in combination with different elements. Our results indicate that the primary response of barley roots to the presence of various chemical elements during the short-term treatment is not a specific but rather a general adaptive stress response enabling the plant to survive adverse conditions.

Proteomic Analysis of Barley Cell Nuclei Purified by Flow Sorting

Many proteins are present in the nucleus; some are involved with its structural and functional organization, some with gene expression, and some with cell division. The plant nuclear proteome has not been well explored. Its characterization requires extraction methods which minimize both the artifactual alteration of the proteins and the extent of contamination with non-nuclear proteins. The conventional multi-step fractionation procedure is both laborious and prone to contamination. Here, we describe a single-step method based on flow sorting. The method allows the separation of G1, S and G2 phase nuclei and minimizes the risk of contamination by non-nuclear proteins. Preliminary results obtained using G1 phase cell nuclei from barley root tips indicate that flow sorting coupled with a protein/peptide separation and mass spectrometry will permit a comprehensive characterization of the plant nuclear proteome.

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 barley root tip swelling induced by cadmium, auxin or hydrogen peroxide

Lipoxygenases (EC 1.13.11.12) catalyse the formation of hydroperoxy derivates by oxygenation of polyunsaturated fatty acids. They act as signal molecules, triggering several developmental processes and defence responses under stress conditions. Incubation of Cd-, IAA- or H2O2-short-term treated seedlings in the presence of LOX inhibitors efficiently inhibited both Cd-, IAA- or H2O2-induced LOX activity and root swelling in a concentration dependent manner, suggesting a key role of LOX or LOX signalling pathway in radial expansion of root cells. Application of antioxidants (ascorbate or N-acetyl cysteine) to the treated seedlings at low 2mM concentration did not affect the Cd-, IAA- or H2O2-induced LOX activity and root swelling. At higher, 10mM concentration antioxidants markedly inhibited root growth, significantly increased the activity of LOX and evoked the radial expansion of root cells leading to root swelling with well developed root hairs already in control roots. By contrast, the lipophilic antioxidant trolox, a scavenger of hydroperoxides, severely inhibited the development of Cd-, IAA- or H2O2-induced root swelling, indicating that not directly LOX, but probably oxylipins, products of LOX pathway, are involved in the induction of root swelling in barley root tip. The results of this study suggest a strong connection between abiotic stress-induced alteration in redox and hormone status caused root growth inhibition and LOX pathway mediated radial expansion of root tip cells.

Auxin signalling is involved in cadmium-induced glutathione-S-transferase activity in barley root

Transient exposure of barley roots to Cd, IAA or H2O2 for 30 min resulted in a significant root growth inhibition. Cd significantly increased the GST activity of roots 6 h after the end of short-term treatment. This increase was more relevant in root segment containing differentiation zone than in root segment just immediately behind the root apex. In contrast to Cd treatment, the short-term exposure of barley roots to IAA resulted in a significant increase of GST activity along the whole root tip and this increase was detectable already 3 h after the treatment with 10 μM IAA. Similarly to IAA, exogenously applied 10 mM H2O2 for 30 min caused significant increase of GST activity along the whole root tip 6 h after the treatment. This increase was already detectable 3 h after the exposure, but only in the differentiation zone of root tip. Auxin influx or signalling inhibitor considerable decreased the Cd- or IAA-induced GST activity in barley root tips. The strong activation of GST even after a brief exposure of barley roots to Cd support the crucial role of GST in the Cd-induced stress response in which presumably IAA and H2O2 play an important signalling role including the activation of GST.

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.

Impact of cadmium and hydrogen peroxide on ascorbate-glutathione recycling enzymes in barley root

We analyse the effect of Cd and H<sub>2</sub>O<sub>2</sub> short-term treatments on the activity of ascorbate-glutathione recycling enzymes in barley root tip. Even a short transient exposure of barley roots to low 15 µmol Cd concentration caused a marked approximately 70% root growth inhibition. Higher Cd concentrations caused root growth cessation during the first 6 h after short-term Cd treatment. Similarly, a marked root growth inhibition was also detected after the short-term exposure of barley seedlings to H<sub>2</sub>O<sub>2</sub>. Our results indicate that root ascorbate pool is more sensitive to Cd treatment than glutathione pool. Rapid activation of dehydroascorbate reductase and monodehydroascorbate reductase is the important component of stress response to the Cd-induced alterations in barley root tips. H<sub>2</sub>O<sub>2</sub> is probably involved in the Cd-induced activation of monodehydroascorbate reductase, but it is not involved in the Cd-induced increase of dehydroascorbate reductase activity.

Genotoxicity evaluation of drinking water and rates of population morbidity in North Kazakhstan oblast

The genotoxicity assay of drinking water in regional centers of North Kazakhstan oblast is carried out by a cytogenetic ana-telophase method using barley root tips. It is shown that drinking water of North Kazakhstan oblast is able to cause cytogenetic damages. A direct correlation between genotoxic properties of drinking water and the rates of congenital abnormalities, malignant neoplasms (by one of the most common localization in the oblast – a stomach cancer), gastric and duodenal ulcers among the population are revealed.

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

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 (30min) of barley roots with a low 10μM Cd concentration induced significant H2O2 production in the elongation and differentiation zone of the root tip 3h after treatment. This elevated H2O2 production was accompanied by root growth inhibition and probably invoked root swelling in the elongation zone of the root tip. By contrast, a high 60μM Cd concentration induced robust H2O2 production in the elongation zone of the root tip already 1h after short-term treatment. This robust H2O2 generation caused extensive cell death 6h after short-term treatment. Similarly to low Cd concentration, exogenously applied H2O2 caused marked root growth inhibition, which at lower H2O2 concentration was accompanied by root swelling. The auxin signaling inhibitor p-chlorophenoxyisobutyric acid effectively inhibited 10μM Cd-induced root growth inhibition, H2O2 production and root swelling, but was ineffective in the alleviation of 60μM Cd-induced root growth inhibition and H2O2 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.

Cadmium disrupts apoplastic ascorbate redox status in barley root tips

Using a short-term Cd treatment (5–30 min), we analysed the effect of Cd on apoplastic ascorbate redox status and their regeneration during the recovery period in barley root tips. Root growth inhibition induced by 15 μM Cd was detectable after 5 min of exposure and increased in a time-dependent manner up to 15 min of exposure. High 30 μM Cd concentration completely inhibited root growth during the first 6 h after short-term treatment. In parallel with Cd-induced root growth inhibition, a rapid decrease of apoplastic ascorbate dehydroascorbate ratio was observed immediately after short-term treatments. During the recovery from 15 μM Cd short-term treatment, apoplastic ascorbate was rapidly regenerated to the control level in the first root segment containing meristem and elongation zone. In contrast to 15 μM Cd treatment, in 30 μM Cd-treated roots apoplastic ascorbate level was sustained at a significantly lower level compared to control roots. We confirmed that a decrease of apoplastic ascorbate/dehydroascorbate ratio in the elongation zone was associated with root growth inhibition or arrest.

How cobalt facilitates cadmium- and ethylene precursor-induced growth inhibition and radial cell expansion in barley root tips

Abstract Significant root growth inhibition was observed during the very short 5 minute exposure time of barley roots to the low 10 μM concentration of cadmium. In addition to the cadmium-induced root growth inhibition, considerable radial expansion of roots was observed as a characteristic symptom of transient short-term exposure of roots to cadmium. The cadmium-induced radial expansion of roots was observed mainly the cortical cells of elongation zone that were twice as large as in control roots. Similarly as in cadmium-treated roots, short-term treatment with ACC significantly inhibited root growth and caused a marked radial expansion of cortical cells. The ethylene synthesis inhibitor cobalt significantly alleviated both the cadmium- and ethylene precursor-induced root growth inhibition and radial root expansion. The results indicate that ethylene probably plays a crucial role in the short-term cadmium-induced inhibition of root growth and radial cell expansion of barley root tips, which are the very early symptoms of cadmium toxicity.

Impact of short-term cadmium treatment on catalase and ascorbate peroxidase activities in barley root tips

The aim of this study was to analyse the relationship between root growth inhibition and alterations in catalase (CAT) and ascorbate peroxidase (APX) contents and activities in barley root tips during the recovery after short-term Cd stress. Significant root growth inhibition was observed after a relatively short-term (30 min) exposure of barley roots to low 15 μM Cd concentration. In seedlings treated with 30 μM Cd root growth was renewed 8-9 h after Cd treatment. By contrast, seedlings exposed to 60 μM Cd failed to restore root growth. The CAT activity increased after 15 μM Cd treatment, whereas, higher Cd concentrations inhibited CAT activity. However, APX activity was not affected by Cd treatment. The content of APX1 transcript increased while content of APX2 decreased 3 h after short-term treatment in a concentration dependent manner. While the expression of CAT1 was upregulated after 15 and 30 μM Cd treatment, it was strongly downregulated by 60 μM Cd. By contrast, CAT2 was upregulated in a concentration dependent manner. These results suggest that increased CAT activity is crucial for restoration of root growth after moderate Cd stress while at severe Cd stress its inhibition may lead to the irreversible damages.

Abiotic stress–induced inhibition of root growth and ascorbic acid oxidase activity in barley root tip is associated with enhanced generation of hydrogen peroxide

Using short-term treatments, the aim of this study was to analyze the role of hydrogen peroxide in the regulation of AAO activity during Cd, Cu or IAA treatments in barley root tips. For analysis individual barley root segments were obtained by the gradual cutting of each root from the tip to the base 1, 2, 3 or 6 h after short-term treatments. Already a short 30 min exposure of barley roots to Cd induced significant root growth inhibition in a Cd concentration dependent manner, which was accompanied by a marked reduction of AAO activity. At Cu concentration which had no effect on the root growth a significant increase in AAO activity was observed. This increased AAO activity was detected only in ionically-bound CW fraction. In contrast, Cu at higher concentration and IAA inhibited both ionically-bound CW AAO isozymes. Prompt inhibition of AAO activity immediately after short-term treatment was observed only in the case of H2O2 treatment suggesting that H2O2 may act as an inhibitor of AAO. This was further supported by the observation that all Cd-, Cu- or IAA-induced root growth and AAO activity inhibition in barley roots was connected with an elevated production of H2O2.

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.

Elevated indole-3-acetic acid peroxidase activity is involved in the cadmium-induced hydrogen peroxide production in barley root tip

All of the analyzed heavy metals significantly inhibited root growth, but in addition to Cd exposure an elevated IAA-POD activity was detected under Co, Cu and Hg treatment, while Ni and Pb inhibited its activity. The Cd-induced IAA-POD activity increased from the root apex towards to the mature region of root tips. However similar or even more severe root growth inhibition was observed by exogenously applied IAA, IAA-POD activity was activated only at high IAA concentrations. Elevated Cd-induced H2O2 production was detected both in the absence or in the presence of IAA in the reaction mixture, but in the case of IAA as a possible substrate for PODs the production of H2O2 increased markedly just in the Cd-treated roots. Exogenously applied H2O2 also activated IAA-POD activity. Our results indicated that in the development of Cd toxicity syndrome the production of ROS during IAA degradation by elevated IAA-POD activity plays a crucial role, mainly under severe Cd stress.