Effect of Aluminium on Oxidative Stress Related Enzymes Activities in Barley Roots

The impact of aluminium stress on activities of enzymes of the oxidative metabolism: superoxide dismutase (SOD), ascorbate peroxidase (APX), peroxidase (POD), NADH peroxidase (NADH-POD) and oxalate oxidase (OXO) was studied in barley (Hordeum vulgare L. cv. Alfor) root tips. SOD appeared to be involved in detoxification mechanisms at highly toxic Al doses and after long Al exposure. POD and APX, H₂O₂ consuming enzymes, were activated following similar patterns of expression and exhibiting significant correlation between their elevated activities and root growth inhibition. The signalling role of NADH-POD in oxidative stress seems to be more probable than that of OXO, which might be involved in Al toxicity mechanism. This revised version was published online in July 2006 with corrections to the Cover Date.     

Role of reactive oxygen species-generating enzymes and hydrogen peroxide during cadmium, mercury and osmotic stresses in barley root tip

The effect of cadmium (Cd) on the expression and activity of NADPH oxidase, peroxidase and oxalate oxidase as well as on the expression of aquaporins and dehydrins was studied in barley root tip. The root tip represented intact apical part of the barley root containing the root cap, meristems and elongation zone. Except stress induced by Cd, barley root tips were analysed after their exposure to phytotoxic concentration of mercury (Hg)-, hydrogen peroxide (H₂O₂)- or polyethylene glycol (PEG)-induced water stress in order to compare the Cd-induced changes with changes induced by these other stress factors. Cd, Hg, H₂O₂ and with some exceptions also PEG treatments caused similar alterations in the gene expression of reactive oxygen species (ROS)-generating and water deficiency-related genes, and in the activity of ROS-generating enzymes. These evidences support our opinion that ROS accumulation and water imbalance are the common symptoms of these stress factors and that the elevated production of H₂O₂ plays, probably as a signal molecule, a key role in the induction of plant responses to abiotic stresses in barley root tip. On the other hand, H₂O₂ at permanent high concentration is probably the main toxic factor during stress conditions.     

Expression of defence-related peroxidases Prx7 and Prx8 during abiotic stresses in barley roots

The expression of defence-related peroxidases Prx7 and Prx8 in barley roots grown under selected abiotic stress conditions (toxic metals: Cd, Al, Co, Cu, Hg; drought, salinity, extreme temperatures: heat, cold) and compounds activating (2,4-D) or inhibiting (SHAM) POD activity as well as H₂O₂ and H₂O₂ scavenger (DTT) was characterized. Strong Cd concentration dependent expression of Prx8 peroxidase gene was observed, which correlated with root growth inhibition induced by Cd- and some other stress factors (heavy metals, heat and salinity). Application of H₂O₂ did not cause changes in expression of Prx8, but H₂O₂ scavenger (DTT) as well as the inhibitor (SHAM) and the activator (2,4-D) of PODs induced increase in Prx8 expression. Our results demonstrate that root growth inhibition during any disturbance of active oxygen species (AOS) in root tissue is correlated with up-regulation of Prx8 gene expression in barley roots.     

Cadmium-induced microsomal membrane-bound peroxidases mediated hydrogen peroxide production in barley roots

The effect of cadmium on microsomal membrane-bound peroxidases and their involvement in hydrogen peroxide production was studied in barley roots. One anionic and two cationic peroxidases were detected, which were strongly activated by Cd treatment. Positive correlation was found between root growth inhibition and increased peroxidase, NADH oxidase activity and H₂O₂ generation in root microsomal membrane fraction of Cd-treated barley roots.     

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 H₂O₂ evoked root growth inhibition and radial cell expansion in barley root tip too.     

Elevated oxalate oxidase activity is correlated with Al-induced plasma membrane injury and root growth inhibition in young barley roots

In order to characterise the possible mechanisms involved in Al toxicity some functional characteristics were analysed in young barley (Hordeum vulgare L.) seedlings cultivated between moistened filter paper. Transfer of germinated barley seeds into hydroponic culture system caused significant stress, which was manifested by root-growth inhibition and elevated Evans blue uptake of root tips. Hydroponics caused stress unabled the analysis of Al-induced stress in the young barley roots during the first day of cultivation. Several (3–4) days are required for adaptation of barley seedlings to hydroponics in spite of strong aeration of the medium. Using filter paper compared to cultivation in solution application of much higher Al concentrations were required to inhibit root growth. Al-induced root growth inhibition, Al uptake, damage of plasma-membrane (PM) permeability of root cells, as well as elevated oxalate oxidase - OxO (EC 1.2.3.4) activity were significantly correlated. While 1 mM Al concentration had no effect on barley roots growing on filter paper, 5 to 100 mM Al concentration inhibited root growth, enhanced cell death and induced oxalate oxidase activity with increasing intensity. The time course analysis of OxO gene expression and OxO activity showed that 10 mM Al increased OxO activity as soon as 3 h after exposure of roots to Al reaching its maximum at about 18 h after Al application. These results indicate that expression of OxO is activated very early after exposure of barley to Al, suggesting its role in oxidative stress and subsequent cell death caused by Al toxicity in plants.     

Cadmium induces premature xylogenesis in barley roots

The effect of Cd on H₂O₂ production, peroxidase (POD) activity and root hair formation were analyzed in barley root. Cd causes a strong H₂O₂ burst in the root region 0–6 mm behind the root tip. POD activity was activated in root tip and raised toward the root base in Cd treated roots. In situ analyses showed that both elevated H₂O₂ production and POD activity are localized in the early metaxylem vascular bundles. Cd induces root hair formation in the region 2 to 4 mm behind the root tip that was not detected in control roots. These results suggest that Cd-induced root growth inhibition is at least partially the consequence of Cd-stimulated premature root development involving xylogenesis and root hair formation, which is correlated with shortening of root elongation zone and therefore with root growth reduction.     

Proteome analysis of roots of wheat seedlings under aluminum stress

The root apex is considered the first sites of aluminum (Al) toxicity and the reduction in root biomass leads to poor uptake of water and nutrients. Aluminum is considered the most limiting factor for plant productivity in acidic soils. Aluminum is a light metal that makes up 7 % of the earth’s scab dissolving ionic forms. The inhibition of root growth is recognized as the primary effect of Al toxicity. Seeds of wheat cv. Keumkang were germinated on petridish for 5 days and then transferred hydroponic apparatus which was treated without or with 100 and 150 μM AlCl₃ for 5 days. The length of roots, shoots and fresh weight of wheat seedlings were decreased under aluminum stress. The concentration of K⁺, Mg²⁺ and Ca²⁺ were decreased, whereas Al³⁺ and P₂O₅ ^? concentration was increased under aluminum stress. Using confocal microscopy, the fluorescence intensity of aluminum increased with morin staining. A proteome analysis was performed to identify proteins, which are responsible to aluminum stress in wheat roots. Proteins were extracted from roots and separated by 2-DE. A total of 47 protein spots were changed under Al stress. Nineteen proteins were significantly increased such as sadenosylmethionine, oxalate oxidase, malate dehydrogenase, cysteine synthase, ascorbate peroxidase and/or, 28 protein spots were significantly decreased such as heat shock protein 70, O-methytransferase 4, enolase, and amylogenin. Our results highlight the importance and identification of stress and defense responsive proteins with morphological and physiological state under Al stress.     

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.     

Response of the plant root to aluminum stress: Analysis of the inhibition of the root elongation and changes in membrane function

Pea root elongation was strongly inhibited in the presence of a low concentration of Al (5 μM). In Al-treated root, the epidermis was markedly injured and characterized by an irregular layer of cells of the root surface. Approximately 30% of total absorbed Al accumulated in the root tip and Al therein was found to cause the inhibition of whole root elongation. Increasing concentrations of Ca²⁺ effectively ameliorated the inhibition of root elongation by Al and 1 mM of CaCl₂ completely repressed the inhibition of root elongation by 50 μM Al. The ameliorating effect of Ca²⁺ was due to the reduction of Al uptake. H⁺-ATPase and H⁺-PPase activity as well as ATP and PPidependent H⁺ transport activity of vacuolar membrane vesicles prepared from barley roots increased to a similar extent by the treatment with 50 μM AlCl₃. The rate of increase of the amount of H⁺-ATPase and H⁺-PPase was proportional to that of protein content measured by immunoblot analysis with antibodies against the catalytic subunit of the vacuolar H⁺-ATPase and H⁺-PPase of mung bean. The increase of both activities was discussed in relation to the physiological tolerance mechanism of barley root against Al stress.     

Boron supply alleviates Al-induced inhibition of root elongation and physiological characteristics in rapeseed (Brassica napus L.)

Aluminum (Al) toxicity is one of the major problems affecting crop production. Boron (B) is an essential micronutrient for higher plants. In the present study, we investigated the alleviation of Al-induced inhibition of root growth and physiological characteristics by B in rapeseed. The rapeseeds were grown in different Al concentrations (0 and 300?μM), and for every concentration, two B treatments (2.5 and 25?µM as H₃BO₃) were applied. The results showed that Al toxicity under low B drastically inhibited root growth. The supply of B improved root length, photosynthesis, root activity, total chlorophyll by 60.15%, 104.7%, 102%, and 106.3%, respectively under Al toxicity. This further resulted in improvement of peroxidase, catalase, and ascorbate peroxidase activities while decreasing malondialdehyde, H₂O_2, and Al contents in roots and leaves. It might be supposed that B alleviates Al toxicity by less mobilization of Al in plant parts and through improving antioxidant enzyme activities.     

Root growth inhibition by aluminum is probably caused by cell death due to peroxidase-mediated hydrogen peroxide production

Summary. The effect of aluminum on hydrogen peroxide production and peroxidase-catalyzed NADH oxidation was studied in barley roots germinated and grown between two layers of moistened filter paper. Guaiacol peroxidase activity significantly increased after 48h and was approximately two times higher after 72h in Al-treated roots. The oxidation of NADH was also significantly increased and, like guaiacol peroxidase activity, it was two times higher in Al-treated roots than in controls. Elevated H₂O₂ production was observed both 48 and 72h after the onset of imbibition in the presence of Al. Separation on a cation exchange column allowed the detection of two peaks with NADH peroxidase and H₂O₂ production activity. However, a difference between control and Al-treated plants was found only in one fraction, in which four times higher guaiacol peroxidase activity and five times higher NADH peroxidase activity were expressed and about three times more H₂O₂ was produced. One anionic peroxidase and three cationic peroxidases were detected in this fraction by native polyacrylamide gel electrophoresis. The anionic peroxidase was activated in the Al-treated root tips and also oxidized NADH but was detectable only after a long incubation time. Two of the cationic peroxidases were capable of oxidizing NADH and producing a significant amount of H₂O₂, but only one of these was activated by Al stress. The role of these peroxidases during Al stress in barley root tips is discussed.Correspondence and reprints: Institute of Botany, Slovak Academy of Sciences, Dúbravská cesta 14, 845 23 Bratislava, Slovakia.     

Effect of heavy metals and temperature on the oxalate oxidase activity and lignification of metaxylem vessels in barley roots

The effect of Cd on oxalate oxidase (OxO) activity and its localisation were analysed in barley root. In Cd-treated roots OxO activity was strongly induced in the region 2–4 mm behind the root tip and in the area toward the root base. In situ analyses showed that Cd-induced OxO activity was localised to the cell wall (CW) of early metaxylem vascular bundles and surrounding parenchyma cells and was accompanied by lignification of metaxylem vessels. OxO activation was also observed during treatment with other heavy metals (HMs), salt treatment and at elevated non-optimal temperature. In contrast to HM activation of OxO and lignification, high temperature and NaCl indeed activated OxO but did not induce lignification of metaxylem vessels. These results suggest that oxalate oxidase as an H₂O₂-generating enzyme is activated in response to several stresses, however the ectopic lignification of metaxylem vessels is activated specifically by HMs. This HM-induced premature root xylogenesis due to ectopic lignification of metaxylem vessels probably causes shortening of the root elongation zone and therefore a reduction in root growth.     

Effect of heavy metals and temperature on the oxalate oxidase activity and lignification of metaxylem vessels in barley roots

The effect of Cd on oxalate oxidase (OxO) activity and its localisation were analysed in barley root. In Cd-treated roots OxO activity was strongly induced in the region 2–4 mm behind the root tip and in the area toward the root base. In situ analyses showed that Cd-induced OxO activity was localised to the cell wall (CW) of early metaxylem vascular bundles and surrounding parenchyma cells and was accompanied by lignification of metaxylem vessels. OxO activation was also observed during treatment with other heavy metals (HMs), salt treatment and at elevated non-optimal temperature. In contrast to HM activation of OxO and lignification, high temperature and NaCl indeed activated OxO but did not induce lignification of metaxylem vessels. These results suggest that oxalate oxidase as an H₂O₂-generating enzyme is activated in response to several stresses, however the ectopic lignification of metaxylem vessels is activated specifically by HMs. This HM-induced premature root xylogenesis due to ectopic lignification of metaxylem vessels probably causes shortening of the root elongation zone and therefore a reduction in root growth.     

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.     

Salinity induced the changes of root growth and antioxidative responses in two wheat cultivars

This study aimed to investigate the inhibitory mechanism of root growth and to compare antioxidative responses in two wheat cultivars, drought-tolerant Ningchun and drought-sensitive Xihan, exposed to different NaCl concentrations. Ningchun exhibited lower germination rate, seedling growth, and lipid peroxidation than Xihan when exposed to salinity. The loss of cell viability was correlated with the inhibition of root growth induced by NaCl stress. Moreover, treatments with H₂O₂ scavenger dimethylthiourea and catalase (CAT) partly blocked salinity-induced negative effects on root growth and cell viability. Besides, the enhancement of superoxide radical and H₂O₂ levels, and the stimulation of CAT and diamine oxidase (DAO) as well as the inhibition of glutathione reductase (GR) were observed in two wheat roots treated with salinity. However, hydroxyl radical content increased only in Xihan roots under NaCl treatment, and the changes of soluble peroxidase (POD), ascorbate peroxidase (APX), superoxide dismutase (SOD), and cell-wall-bound POD activities were different in drought-tolerant Ningchun and drought-sensitive Xihan exposed to different NaCl concentrations. In conclusion, salinity might induce the loss of cell viability via a pathway associated with extracellular H₂O₂ generation, which was the primary reason leading to the inhibition of root growth in two wheat cultivars. Here, it was also suggested that increased H₂O₂ accumulation in the roots of drought-tolerant Ningchun might be due to decreased POD and GR activities as well as enhanced cell-wall-bound POD and DAO ones, while the inhibition of APX and GR as well as the stimulation of SOD and DAO was responsible for the elevation of H₂O₂ level in drought-sensitive Xihan roots.     

Hydrogen peroxide production by roots and its stimulation by exogenous NADH

H₂O₂ production by roots of young seedlings was monitored using a non-destructive in vivo assay at pH 5.0. A particularly high rate of H₂O₂ production was measured in the roots of soybean (Glycine max L. cv. Labrador) seedlings which were used for further investigation of the physiological and enzymological properties of apoplastic H₂O₂ production. In the soybean root H₂O₂ production can be stimulated 10-fold by exogenous NADH or NADPH. This response displays typical features of a peroxidase-catalyzed oxidase reaction using NAD(P)H as electron donor for the reduction of O₂ to H₂O₂. Comparative measurements showed that the NADH-induced H₂O₂ production of the roots resembles the H₂O₂-forming activity of horseradish peroxidase with respect to NADH and O₂ concentration requirements and sensitivity to inhibition by KCN, NaN₃, superoxide dismutase and catalase. NADH-induced H₂O₂ production can be observed with similar intensity in all regions of the root, in agreement with the distribution of apoplastic peroxidase activity. In contrast, the activity responsible for the basal H₂O₂ production in the absence of exogenous NADH was mainly confined to a short subapical zone of the root and differs from the NADH-induced reaction by insensitivity to inhibition by superoxide dismutase and a strikingly lower requirement for O₂. It is concluded that the basal H₂O₂ production of the root is mediated by an enzyme different from peroxidase, possibly a plasma membrane O₂^?-producing oxidase.     

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.     

The ecotoxicological and interactive effects of chromium and aluminum on growth,oxidative damage and antioxidant enzymes on two barley genotypes differing in Al tolerance

The effects of single or combined stress of aluminum (Al) and chromium (Cr) on plant growth, root dehydrogenase, oxidative stress and antioxidative enzymes were studied using two barley genotypes differing in Al tolerance in a hydroponic experiment. Al or Cr stress decreased plant growth, lowered root dehydrogenase activity and caused oxidative damage, as characterized by increased MDA and H₂O₂ contents. Under Al or Cr stress, the activities of antioxidative enzymes, including superoxide dismutase (SOD), peroxidase (POD), ascorbate peroxidase (APX), glutathione reductase (GR) and catalase (CAT), were dramatically increased in plant tissues. Gebeina, an Al-tolerant genotype, had less oxidative damage than Shang 70-119, an Al-sensitive genotype. The extent of oxidative damage induced by Cr varied with the pH of the culture solution, with lower pH values (4.0) being more severe than higher pH values (6.5). The combination of Cr and Al caused a further decrease in plant growth, a decrease in root dehydrogenase activity and an increase in MDA and H₂O₂ contents as well as the activities of antioxidative enzymes. There was also a marked difference between the two barley genotypes in the extent of increased antioxidative enzyme activity under the Cr and Al stresses.     

Aluminum-induced oxidative stress and changes in antioxidant defenses in the roots of rice varieties differing in Al tolerance

The effects of aluminum (Al) on root elongation, lipid peroxidation, hydrogen peroxide (H₂O₂) accumulation, antioxidant levels, antioxidant enzymatic activity, and lignin content in the roots of the Al-tolerant rice variety azucena and the Al-sensitive variety IR64 were investigated. Treatment with Al induced a greater decrease in root elongation and a greater increase in H₂O₂ and lipid peroxidation as determined by the total thiobarbituric acid-reactive substance (TBARS) level in IR64 than in azucena. Azucena had significantly higher levels of superoxide dismutase, ascorbate peroxidase, glutathione reductase, and glutathione peroxidase GSH POD activity compared with IR64. The concentrations of reduced glutathione (GSH) and ascorbic acid, and the GSH/GSSG ratio (reduced vs. oxidized glutathione) were also higher in azucena than in IR64 in the presence of Al. The addition of 1 mg/L GSH improved root elongation in both varieties and decreased H₂O₂ production under Al stress. By contrast, treatment with buthionine sulfoximine, a specific inhibitor of GSH synthesis, decreased root elongation in azucena and stimulated H₂O₂ production in both varieties. Moreover, Al treatment significantly increased the cytoplasmic activity of peroxidase (POD) as well as the levels of POD bound ionically and covalently to cell walls in the Al-sensitive variety. The lignin content was also increased. Treatment with exogenous H₂O₂ also increased the lignin content and decreased root elongation in IR64. These results suggest that Al induces lignification in the roots of Al-sensitive rice varieties, probably through an increase in H₂O₂ accumulation.