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.     

Effect of cadmium and temperature on the lipoxygenase activity in barley root tip

An analysis of different cell fractions isolated from barley roots revealed that lipoxygenase (LOX) activity occurred both extra- and intracellulary. Cadmium (Cd)-induced LOX activity was observed in the fraction containing cell walls, plasma membrane and the cytoplasm. High temperature-induced root growth inhibition and elevated LOX activity did not induce lipid peroxidation. In contrast, Cd inhibited root growth and caused both enhanced lipid peroxidation and elevated LOX activity at each of the temperatures analyzed. Spatial distribution studies revealed that the patterns of apoplastic LOX activity were different from those of cytoplasmic activity. Cd-induced intracellular LOX activity increased equally along the barley root tip, while Cd-induced apoplastic LOX activity was associated mainly with the differentiation zone of the barley root tip. Our results suggest the involvement of Cd-induced LOX activity in the premature differentiation of the barley root tip during Cd stress. We hypothesize that the role of LOX in plant metabolic processes in the root may depend on the level of reactive oxygen species in the roots: at physiological concentrations of ROS, LOX may be involved in the processes of root growth, while at the elevated harmful concentrations of ROS induced by different stress conditions, it may be involved in root growth inhibition through ectopic differentiation.     

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.     

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.     

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.     

Root adaptations to cadmium-induced oxidative stress contribute to Cd tolerance in the hyperaccumulator Sedum alfredii

Short-term responses of Sedum alfredii roots to Cd exposure was compared in Cd hyperaccumulator (HE) and nonhyperaccumulating ecotype (NHE). Cadmium exposure significantly inhibited root elongation and induced loss of plasma membrane integrity and lipid peroxidation of roots tips in the NHE, whereas these effects were much less pronounced in the HE plants. A strong accumulation of reactive oxygen species with increasing Cd concentration was noted in the NHE root tips, but not in HE. After Cd exposure, a dose-dependent decrease in oxidized glutathione and marked increase in reduced glutathione and non-protein thiols were observed in root tips of HE, but were not seen in the NHE plants. These results suggest that the HE tolerates high Cd in the environment through the differential adaptations against Cd-induced oxidative stress.     

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.     

Nitric oxide (as sodium nitroprusside) supplementation ameliorates Cd toxicity in hydroponically grown wheat roots

Cadmium (Cd) is a non-redox toxic heavy metal present in the environment and induces oxidative stress in plants. We investigated whether exogenous nitric oxide (NO) supplementation as sodium nitroprusside (SNP) has any ameliorating action against Cd-induced oxidative damage in plant roots and thus protective role against Cd toxicity. Cd treatment (50 or 250 μM) alone or in combination with 200 μM SNP was given to hydroponically grown wheat roots for a short time period of 24 h and then these were shifted to distilled water to observe changes in levels of oxidative markers (lipid peroxidation, H₂O₂ content and electrolyte leakage). Supplementation of Cd with SNP significantly reduced the Cd-induced lipid peroxidation, H₂O₂ content and electrolyte leakage in wheat roots. It indicated a reactive oxygen species (ROS) scavenging activity of NO. However, even upon removal of Cd-treatment solution, the levels of oxidative markers increased during 24 h recovery stage and later at 48 h these decreased. Cd treatment resulted in an upregulation of activities of antioxidant enzymes—superoxide dismutase (SOD, 1.15.1.1), guaiacol peroxidase (GPX, 1.11.1.7), catalase (CAT, 1.11.1.6), and glutathione reductase (GR, 1.6.4.2). SNP supply resulted in a reduction in Cd-induced increased activities of scavenging enzymes. The protective role of exogenous NO in decreasing Cd-induced oxidative damage was also evident from the histochemical localization of lipid peroxidation, plasma membrane integrity and superoxides. The study concludes that an exogenous supply of NO protects wheat roots from Cd-induced toxicity.     

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.     

Selenium alleviates cadmium toxicity by preventing oxidative stress in sunflower (Helianthus annuus) seedlings

The present study investigated the possible mediatory role of selenium (Se) in protecting plants from cadmium (Cd) toxicity. The exposure of sunflower seedlings to 20 μM Cd inhibited biomass production, decreased chlorophyll and carotenoid concentrations and strongly increased accumulation of Cd in both roots and shoots. Similarly, Cd enhanced hydrogen peroxides content and lipid peroxidation as indicated by malondialdehyde accumulation. Pre-soaking seeds with Se (5, 10 and 20 μM) alleviated the negative effect of Cd on growth and led to a decrease in oxidative injuries caused by Cd. Furthermore, Se enhanced the activities of catalase, ascorbate peroxidase and glutathione reductase, but lowered that of superoxide dismutase and guaiacol peroxidase. As important antioxidants, ascorbate and glutathione contents in sunflower leaves exposed to Cd were significantly decreased by Se treatment. The data suggest that the beneficial effect of Se during an earlier growth period could be related to avoidance of cumulative damage upon exposure to Cd, thus reducing the negative consequences of oxidative stress caused by heavy metal toxicity.     

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.     

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.     

Effects of aluminum on superoxide dismutase and peroxidase activities,and lipid peroxidation in the roots and calluses of soybeans differing in aluminum tolerance

The seedlings of two soybean genotypes, Al-tolerant PI 416937 (PI) and Al-sensitive Young, were cultured in the solution containing 0, 25 or 50 μM Al (AlCl₃·6H₂O) for 24, 36 or 48 h in the hydroponics, and the calluses induced from two genotypes were cultured in medium containing 0, 10, 50 or 100 μM Al for 5, 10 or 15 days, respectively. The effects of Al on growth of seedling roots and calluses, antioxidant enzyme activities of superoxide dismutase (SOD) and peroxidase (POD) and lipid peroxidation were investigated. Under Al stress, PI was more tolerant to Al toxicity than Young at both intact plant and tissue levels and lower concentrations of Al significantly stimulated the root and callus growth of PI. Al application enhanced the activities of SOD and POD and lipid peroxidation in both roots and calluses of two genotypes. Although the differences of SOD activities between two genotypes in response to Al toxicity depended on Al concentration and durations of treatment, SOD activities in the roots of PI were higher than those in the roots of corresponding Young in the presence of Al for 36 or 48 h. Meanwhile, the POD activities in PI roots increased as the Al levels and durations of treatment increased, significantly higher than those in the corresponding Young roots. Moreover, Al-treated PI had significantly lower lipid peroxidation than Young at both root and callus levels. These results suggest that the enhanced antioxidant-related enzyme activities and reduced lipid peroxidation in PI might be one of Al-tolerant mechanisms.     

Alterations of the gene expression, lipid peroxidation, proline and thiol content along the barley root exposed to cadmium

Barley seedlings grown on filter paper moistened with 1mM Cd showed 50% root growth inhibition within 24h of exposure. The amount of cadmium after 24h Cd treatment was highest in the first 2mm-long apical root segment, while it was slightly higher in the fourth segment, 6-8mm behind the root tip, after 48h. In recovery experiments, when Cd-treated plants were transferred onto filter paper moistened with distilled water, a large amount of Cd was localised in the apoplast and considerable cell death was detected even though root growth was renewed. This indicates that cell death is likely an active physiological process that contributes to the removal of Cd from the root during root growth recovery. Elevated lipid peroxidation and thiol contents were detected in all individual segments of Cd-treated barley root. On the other hand, proline accumulation was disturbed during Cd stress, showing a significant decrease in all of the studied segments except the first. Cd-induced alteration in the expression of genes involved in metal signalling and detoxification and in drought and oxidative stress responses indicates that Cd-induced water and oxidative stress is responsible for the root growth inhibition, probably through an accelerated differentiation of root tissues.     

Enhanced zinc consumption prevents cadmium-induced alterations in lipid metabolism in male rats

It has been investigated, based on a rat model of human exposure to cadmium (Cd), whether zinc (Zn) supplementation may prevent Cd-induced alterations in lipid metabolism. For this purpose, the concentrations of free fatty acids (FFA), phospholipids (PL), triglycerides (TG), total cholesterol (TCh), and high and low density lipoprotein cholesterol (HDL and LDL, respectively) as well as the concentrations of chosen indices of lipid peroxidation such as lipid peroxides (LPO), F₂-isoprostane (F₂-IsoP) and oxidized LDL (oxLDL) were estimated in the serum of male Wistar rats administered Cd (5 or 50 mg/l) or/and Zn (30 or 60 mg/l) in drinking water for 6 months. The exposure to 5 and 50 mg Cd/l resulted in marked alterations in the lipid status reflected in increased concentrations of FFA, TCh, LDL, LPO, F₂-IsoP and oxLDL, and decreased concentrations of PL and HDL in the serum. The concentrations of LDL, LPO, F₂-IsoP and oxLDL were more markedly enhanced at the higher Cd dosage. The supplementation with Zn during the exposure to 5 and 50 mg Cd/l entirely prevented all the Cd-induced changes in the serum concentrations of the estimated lipid compounds and indices of lipid peroxidation, except for the F₂-IsoP for which Zn provided only partial protection. Based on the results it can be concluded that Zn supplementation during exposure to Cd may have a protective effect on lipid metabolism consisting in its ability to prevent hyperlipidemia, including especially hypercholesterolemia, and to protect from lipid peroxidation. The findings seem to suggest that enhanced dietary Zn intake during Cd exposure, via preventing alterations in the body status of lipids may, at least partly, protect against some effects of Cd toxicity, including oxidative damage to the cellular membranes and atherogenic action. The paper is the first report suggesting protective impact of Zn against proatherogenic Cd action on experimental model of chronic moderate and relatively high human exposure to this toxic metal.     

Nickel-induced Inhibition of Wheat Root Growth is Related to H2O2 Production, but not to Lipid Peroxidation

Effects of exogenous nickel (Ni: 10 and 200 μM) on growth, mitotic activity, Ni accumulation, H₂O₂ content and lipid peroxidation as well as the activities of various antioxidative enzymes, such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione peroxidase (GSH-Px) were investigated in wheat roots. A considerable Ni accumulation in the roots occurred at both the concentrations. Although Ni at 10 μM did not have any significant effect on root growth, it strongly inhibited the root growth at 200 μM. Mitotic activity in the root tips was not significantly affected by exposure of the seedlings to 10 μM Ni; however, it was almost completely inhibited at 200 μM treatment. Ni stress did not result in any significant changes in CAT and APX activities as well as lipid peroxidation. However, H₂O₂ concentration increased up to 82% over the control in the roots of seedlings exposed to 200 μM Ni. There was a significant decline in both SOD (50%) and GSH-Px (20–30%) activities in the roots when the seedlings were treated with 200 μM Ni. The results indicated that a strong inhibition of wheat root growth caused by Ni stress was not due to enhanced lipid peroxidation, but might be related to the accumulation of H₂O₂ in root tissue.     

Genotype-Dependent Effect of Exogenous Nitric Oxide on Cd-induced Changes in Antioxidative Metabolism, Ultrastructure, and Photosynthetic Performance in Barley Seedlings (Hordeum vulgare)

A greenhouse hydroponic experiment was performed using Cd-sensitive (cv. Dong 17) and Cd-tolerant (Weisuobuzhi) barley seedlings to evaluate how different genotypes responded to cadmium (Cd) toxicity in the presence of sodium nitroprusside (SNP), a nitric oxide (NO) donor. Results showed that 5 μM Cd increased the accumulation of O₂^•−, H₂O₂, and malondialdehyde (MDA) but reduced plant height, chlorophyll content, net photosynthetic rate (P _n), and biomass, with a much more severe response in the Cd-sensitive genotype. Antioxidant enzyme activities increased significantly under Cd stress in the roots of the tolerant genotype, whereas in leaves of the sensitive genotype, superoxide dismutase (SOD) and ascorbate peroxide (APX), especially cytosol ascorbate peroxidase (cAPX), decreased after 5–15 days Cd exposure. Moreover, Cd induces NO synthesis by stimulating nitrate reductase and nitric oxide synthetase-like enzymes in roots/leaves. A Cd-induced NO transient increase in roots of the Cd-tolerant genotype might partly contribute to its Cd tolerance. Exogenous NO dramatically alleviated Cd toxicity, markedly diminished Cd-induced reactive oxygen species (ROS) and MDA accumulation, ameliorated Cd-induced damage to leaf/root ultrastructure, and increased chlorophyll content and P _n. External NO counteracted the pattern of alterations in certain antioxidant enzymes induced by Cd; for example, it significantly elevated the depressed SOD, APX, and catalase (CAT) activities in the Cd-sensitive genotype after 10- and 15-day treatments. Furthermore, NO significantly increased stromal APX and Mn-SOD activities in both genotypes and upregulated Cd-induced decrease in cAPX activity and gene expression of root/leaf cAPX and leaf CAT1 in the Cd-sensitive genotype. These data suggest that under Cd stress, NO, as a potent antioxidant, protects barley seedlings against oxidative damage by directly and indirectly scavenging ROS and helps to maintain stability and integrity of the subcellular structure.