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 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 60μM 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 6h 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 10μM Cd-induced root growth inhibition, H(2)O(2) production and root swelling, but was ineffective in the alleviation of 60μM 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.     

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 30 μM Cd. Higher Cd concentrations caused extensive cell death and root growth arrest. Enhanced lipid peroxidation was observed as early as 1 h after the short-term treatment in a Cd concentration-dependent manner. In contrast to lipid peroxidation, the induction of lipoxygenase activity was detected only 3 h after the exposure of roots to 15 or 30 μM Cd. In addition, it was not observed in 60 μM Cd-treated root tips. The highest lipoxygenase activity was detected 6 h after 15 μM 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.     

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.     

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.     

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.     

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

Key message

Abiotic stress-induced superoxide generation depending on its localization, level, duration and presumably also on the action of other signals may lead to different stress responses.

Abstract

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.     

Changes in intracellular and apoplastic peroxidase activity, ascorbate redox status, and root elongation induced by enhanced ascorbate content in Allium cepa L

Onions (Allium cepa L.) treated with external ascorbic acid or with the immediate precursor of its synthesis L-galactono-gamma-lactone show a stimulated elongation rate of the roots and an increase in the number of new radicles appearing at the bulb base. Treatment with both molecules resulted in an enhanced accumulation of ascorbate and dehydroascorbate along the root axis, but the distribution of these redox forms was not uniform along the root, as detected in intracellular (symplastic) and extracellular (apoplastic) compartments. Thus, those radicular zones metabolically more active, such as the meristem and the elongation zone, accumulated the highest amount of both redox forms of ascorbate. On the other hand, ascorbate and L-galactono-gamma-lactone also stimulated cytosolic glucose-6-phosphate dehydrogenase activity and inhibited peroxidase activity as deduced from in vivo and in vitro experiments. Differences were also found when comparing apoplastic and symplastic activities. These results are compatible with the idea of an ascorbate-mediated stimulation of root growth by inhibiting cell wall stiffening and increasing root metabolism.     

Morphological changes in the apex of pea roots during and after recovery from aluminium treatment

We investigated how the pea (Pisum sativum cv. Harunoka) root, upon return to an Al-free condition, recovers from injury caused by exposure to Al. The growing region of the root during and after treatment with Al was examined by marking the root at intervals with India ink. Al-induced cell death was detected by staining with Evans blue. Root growth in 40 μM Al solution relative to that in Al-free solution (RRG) was approximately 45% from 6 h to12 h after the start of the treatment. However, values of RRG from 12 h to 24 h in Al-free solution for recovery or in the same Al solution were about 75% and 35%, respectively, indicating recovery from Al-induced growth inhibition. Images of the root characterized by zonal staining with Evans blue were observed in the sub-apical region (more than 1 mm from the tip) in Al-stressed roots. However, the interval of the stained zone was widened in the root after recovery from Al-induced growth inhibition, though it was narrower and more densely stained with time in the Al-stressed roots. During the recovery, the root apex may resume elongation in a specified region without Al-induced death or injury in cells detected by Evans blue.     

Zonal changes in ascorbate and hydrogen peroxide contents,peroxidase, and ascorbate-related enzyme activities in onion roots

Onion (Allium cepa) roots growing hydroponically show differential zonal values for intra- (symplastic) and extra- (apoplastic) cellular ascorbate (ASC) and dehydroascorbate (DHA) contents and for related enzyme activities. In whole roots, ASC and DHA concentrations were higher in root apex and meristem and gradually decreased toward the root base. Guaiacol peroxidase, ASC peroxidase, monodehydroascorbate oxidoreductase, DHA reductase, catalase, and glutathione reductase activities showed differential activity patterns depending on the zone of the root and their apoplastic or symplastic origin. An in vivo staining of peroxidase activity also revealed a specific distribution pattern along the root axis. Using electron microscopy, hydrogen peroxide was found at different locations depending on the root zone but was mainly located in cell walls from epidermal and meristematic cells and in cells undergoing lignification. A balanced control of all of these molecules seems to exist along the root axis and may be directly related to the mechanisms in which the ASC system is involved, as cell division and elongation. The role of ASC on growth and development in relation to its presence at the different zones of the root is discussed.     

N-acetyl-cysteine alleviates Cd toxicity and reduces Cd uptake in the two barley genotypes differing in Cd tolerance

A hydroponic experiment was carried out to study the physiological mechanisms of N-acetyl cysteine (NAC) in mitigating cadmium (Cd) toxicity in two barley (Hordeum vulgare L.) genotypes, Dong 17 (Cd-sensitive) and Weisuobuzhi (Cd-tolerant). Addition of 200 μM NAC to a culture medium containing 5 μM Cd (Cd + NAC) markedly alleviated Cd-induced growth inhibition and toxicity, maintained root cell viability, and dramatically depressed O ₂ ^·? and ·OH, and malondialdehyde accumulation, significantly reduced Cd concentration in leaves and roots, especially in the sensitive genotype Dong 17. External NAC counteracted Cd-induced alterations of certain antioxidant enzymes, e.g., brought root superoxide dismutase and glutathione reductase, leaf/root peroxidase and glutathione peroxidase activities of the both genotypes down towards the control level, but elevated Cd-stress-depressed leaf catalase in Dong 17 and root ascorbate peroxidase activities in both genotypes. NAC counteracted Cd-induced alterations in amino acids and microelement contents. Furthermore, NAC significantly reduced Cd-induced damage to leaf/root ultrastructure, e.g. the shape of chloroplasts in plants treated with Cd + NAC was relatively normal with well-structured thylakoid membranes and parallel pattern of lamellae but less osmiophilic plastoglobuli compared with Cd alone treatment; nuclei of root cells were better formed and chromatin distributed more uniformly in both genotypes. These results suggested that under Cd stress, NAC may protects barley seedlings against Cd-induced damage by directly and indirectly scavenging reactive oxygen species and by maintaining stability and integrity of the subcellular structure.     

Do pH changes in the leaf apoplast contribute to rapid inhibition of leaf elongation rate by water stress? Comparison of stress responses induced by polyethylene glycol and down‐regulation of root hydraulic conductivity

We have dissected the influences of apoplastic pH and cell turgor on short-term responses of leaf growth to plant water status, by using a combination of a double-barrelled pH-selective microelectrodes and a cell pressure probe. These techniques were used, together with continuous measurements of leaf elongation rate (LER), in the (hidden) elongating zone of the leaves of intact maize plants while exposing roots to various treatments. Polyethylene glycol (PEG) reduced water availability to roots, while acid load and anoxia decreased root hydraulic conductivity. During the first 30 min, acid load and anoxia induced moderate reductions in leaf growth and turgor, with no effect on leaf apoplastic pH. PEG stopped leaf growth, while turgor was only partially reduced. Rapid alkalinization of the apoplast, from pH 4.9 ± 0.3 to pH 5.8 ± 0.2 within 30 min, may have participated to this rapid growth reduction. After 60 min, leaf growth inhibition correlated well with turgor reduction across all treatments, supporting a growth limitation by hydraulics. We conclude that apoplastic alkalinization may transiently impair the control of leaf growth by cell turgor upon abrupt water stress, whereas direct hydraulic control of growth predominates under moderate conditions and after a 30-60 min delay following imposition of water stress.     

Abiotic stress�Cinduced 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.     

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

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.     

Changes in Growth Pattern,Enzymatic Activities Related to Ascorbate Metabolism,and Hydrogen Peroxide in Onion Roots Growing Under Experimentally Increased Ascorbate Content

Onion (Allium cepa L.) roots treated with external ascorbate or with the immediate precursor of its synthesis, L-galactono-γ-lactone, increased root development measured as an increase in fresh and dry weights after 48-h treatments compared to controls. Also, treatments induced changes in extracellular (apoplastic) and cytosolic (symplastic) enzyme activities related to ascorbate metabolism and antioxidant protection, such as ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, glutathione reductase, and catalase. Finally, we have found that both chemicals induced increased content of hydrogen peroxide in well-differentiated zones of the root, and local increases in meristematic and elongation zones were detected by cytochemistry as well. The results are discussed on the basis of changes in the root growth rate and other physiologic processes mediated by ascorbate in higher plants.     

Cadmium reduces zinc uptake but enhances its translocation in the cadmium-accumulator, <Emphasis Type="Italic">Carpobrotus rossii</Emphasis>, without affecting speciation

Background and aims

Interactions between Cd and Zn occur in soils and plants but are inconsistent. This study examined how Cd/Zn interactions influence the growth of Carpobrotus rossii (Haw.) and the accumulation of Cd and Zn in plants.

Methods

Plants were grown in nutrient solutions containing 5–100 μM Zn and 0, 5 or 15 μM Cd. Plant growth and tissue concentrations were measured, and the speciation of Zn within the plant tissues determined using synchrotron-based X-ray absorption spectroscopy.

Results

There was an additive negative interaction between Cd and Zn on root growth. Only the highest level of Zn (100 μM) decreased Cd concentrations in root and shoot tissues (by 40–64%), whilst 100 μM Zn enhanced Cd translocation at 5 μM Cd but decreased it at 15 μM Cd. In contrast, both 5 and 15 μM Cd decreased Zn concentrations in root and shoot tissues but increased Zn translocation by 30–90%. This interaction was not associated with changes in Zn speciation within the plants, with most Zn associated with oxalate (48–87%).

Conclusions

The presence of Zn and Cd resulted in an additive negative effect on root growth, but an antagonistic pattern in their accumulation in shoots of C. rossii.

     

Characterization of mercury-induced stress biomarkers in Fagopyrum tataricum plants

The effect of mercury stress on antioxidant enzymes, lipid peroxidation, photosynthetic pigments, hydrogen peroxide content, osmolytes, and growth parameters in Tartary buckwheat were investigated. The effect of Hg-exposure was found to be time (15 and 30 days) and concentration (0, 25, 50, and 75 μM) dependent. Hg was readily absorbed by seedlings with higher content in roots and it resulted in reduction of root and shoot length. The root and shoot Hg uptakes were significantly and directly correlated with each other. However, the fresh mass and biomass increased up to 50 μM Hg-treatment at both time periods. A significant positive correlation was observed between biomass accumulation with relative water content. Hg levels were positively correlated with the production of hydrogen peroxide in leaves as evidenced by 3, 3-diaminobenzidine (DAB)-mediated tissue fingerprinting. The osmolyte levels in general were elevated except for proline and protein which showed a decline at 75 μM Hg-treatment at 30-days. Amongst the photosynthetic pigments, chlorophyll showed a decline while as carotenoid and anthocyanin levels were elevated. The activity of antioxidant enzymes such as ascorbate peroxidase (APX), guaiacol peroxidase (POD), glutathione reductase (GR), Glutathione-s-transferase (GST) and superoxide dismutase (SOD) were positively correlated with Hg-treatment except SOD, which declined at 75 μM Hg-treatment in 30-days old seedlings. Catalase (CAT) activity showed a positive correlation up to 50 μM Hg-treatment but at 75 μM Hg-stress it decreases at both 15 and 30 days.     

Differential response of wheat roots to Cu, Ni and Cd treatment: oxidative stress and defense reactions

Wheat seedlings cv. Zyta were treated with Cu, Ni and Cd at the concentrations causing approximately 50 % root growth inhibition, i.e. 12.5, 50 and 60 μM, respectively. Tissue metal accumulation, membrane permeability, lipid peroxidation, protein oxidation, concentration of thiol compounds as well as protease, glutathione S-transferase (GST) and peroxidase (POD) activities were studied in roots after 7 days of metal exposure. The metals showed different concentrations in root tissues with Cu and Cd being accumulated to the smallest and to the greatest extent, respectively. Membrane permeability was significantly enhanced by Cu and Ni but not by Cd treatment. All metals induced similar increase in protein oxidation, while significant enhancement of lipid peroxidation was observed only in the case of Cu treatment. The detected thiol compounds: cysteine (Cys), homocysteine (Hcy), γ-glutamylcysteine (γ-GluCys) and glutathione (GSH) were differently influenced by the metal treatment. Ni appeared to be the most effective inductor of GSH accumulation while both Cu and Ni similarly increased Cys content in the roots. Accumulation of γ-GluCys was found in response to Cu and Cd applications. Concentration of Hcy was enhanced by Cd treatment but exposure to Ni decreased its content below the level of detection. The activity of GST was considerably elevated by Cd and Ni treatments, while POD activity was increased only in response to Cu application. Our study showed that wheat roots differently responded to treatment with metals used at the concentrations having similar impact on growth.     

Exogenous Nitric Oxide (as Sodium Nitroprusside) Ameliorates Polyethylene Glycol-Induced Osmotic Stress in Hydroponically Grown Maize Roots

The present study was designed to examine whether exogenous sodium nitroprusside (SNP) supplementation has any ameliorating action against PEG-induced osmotic stress in Zea mays cv. FRB-73 roots. Twenty percent or 40 % polyethylene glycol (PEG6000; ?0.5 MPa and ?1.76 MPa, respectively) treatment alone or in combination with 150 and 300 μM SNP was applied to hydroponically grown maize roots for 72 h. Although only catalase (CAT) activity increased when maize roots were exposed to PEG-induced osmotic stress, induction of this antioxidant enzyme was inadequate to detoxify the extreme levels of reactive oxygen species, as evidenced by growth, water content, superoxide anion radical (O ₂ ^?? ), hydroxyl radical (OH^?) scavenging activity, and TBARS content. However, supplementation of PEG-exposed specimens with SNP significantly alleviated stress-induced damage through effective water management and enhancement of antioxidant defense markers including the enzymatic/non-enzymatic systems. Exogenously applied SNP under stress resulted in the up-regulation of glutathione peroxidase (GPX), glutathione S-transferase (GST), ascorbate peroxidase (APX), glutathione reductase (GR), total ascorbate, and glutathione contents involved in ascorbate–glutathione cycle. On the other hand, growth rate, osmotic potential, CAT, APX, GR, and GPX increased in maize roots exposed to both concentrations of SNP alone, but activities of monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase decreased. Based on the above results, an exogenous supply of both 150 and 300 μM SNP to maize roots was protective for PEG-induced toxicity. The present study provides new insights into the mechanisms of SNP (NO donor) amelioration of PEG-induced osmotic stress damages in hydroponically grown maize roots.