A CDC25 homologue from rice functions as an arsenate reductase

Enzymatic reduction of arsenate to arsenite is the first step in arsenate metabolism in all organisms studied. The rice genome contains two ACR2-like genes, OsACR2.1 and OsACR2.2, which may be involved in regulating arsenic metabolism in rice. Here, we cloned both OsACR2 genes and expressed them in an Escherichia coli strain in which the arsC gene was deleted and in a yeast (Saccharomyces cerevisiae) strain with a disrupted ACR2 gene. OsACR2.1 complemented the arsenate hypersensitive phenotype of E. coli and yeast. OsACR2.2 showed much less ability to complement. The gene products were purified and demonstrated to reduce arsenate to arsenite in vitro, and both exhibited phosphatase activity. In agreement with the complementation results, OsACR2.1 exhibited higher reductase activity than OsACR2.2. Mutagenesis of cysteine residues in the putative active site HC(X)(5)R motif led to nearly complete loss of both phosphatase and arsenate reductase activities. In planta expression of OsACR2.1 increased dramatically after exposure to arsenate. OsACR2.2 was observed only in roots following arsenate exposure, and its expression was less than OsACR2.1.     

NaCl-induced expression of glutathione reductase in roots of rice (Oryza sativa L.) seedlings is mediated through hydrogen peroxide but not abscisic acid

Reactive oxygen species (ROS) play an important role in NaCl stress. Plants tolerant to NaCl stress may evolve certain strategies to remove these ROS, thus reducing their toxic effects. Therefore, the expression patterns of the gene family encoding glutathione reductase (GR, EC 1.6.4.2) were analyzed in roots of etiolated rice (Oryza sativa L.) seedlings in response to NaCl stress. Semi-quantitative RT-PCR was applied to quantify the mRNA levels for one cytosolic (OsGR2) and two chloroplastic (OsGR1 and OsGR3) isoforms of glutathione reductase identified in the rice genome. The expression of OsGR2 and OsGR3 but not OsGR1 was increased in rice roots treated with 150 mM NaCl. The Rab16A is an abscisic acid (ABA)-responsive rice gene. Increasing concentrations of ABA, from 1 to 12 μM, progressively increased the expression of OsRab16A in rice roots. In the present study, the ABA level was judged by the expression of OsRab16A in rice roots. Treatment with 150 mM NaCl induced the expression of OsRab16A, and the expression increased with increasing concentrations of ABA, which suggests that ABA may be involved in this response in rice roots. In fact, exogenous application of ABA enhanced the expression of OsGR2 and OsGR3 in rice roots. On inhibiting ABA accumulation with sodium tungstate (Tu), an inhibitor of ABA biosynthesis, the expression of OsGR2 and OsGR3 was still induced by NaCl; therefore, NaCl-triggered expression of OsGR2 and OsGR3 in rice roots is not mediated by accumulation of ABA. However, NaCl treatment could induce H₂O₂ production in rice roots, and H₂O₂ treatment resulted in enhanced OsGR2 and OsGR3 induction. On inhibiting the NaCl-induced accumulation of H₂O₂ with diphenylene iodonium, the expression of OsGR2 and OsGR3 was also suppressed. Moreover, the increase in H₂O₂ level was prior to the induction of OsGR2 and OsGR3 in NaCl-treated rice roots. Thus, H₂O₂, but not ABA, is involved in regulation of OsGR2 and OsGR3 expression in NaCl-treated rice roots.     

The physiological mechanism of enhanced oxidizing capacity of rice (Oryza sativa L.) roots induced by phosphorus deficiency

Plants show various responses to phosphorus (P) deficiency. Root oxidizing capacity enhancement is one of adaptive mechanisms for rice (Oryza sativa L.) to P deficiency. However, it remains unclear how P deficiency enhances the root oxidizing capacity. In this study, rice seedlings were treated in P-deficient nutrient solution for different periods. Variations of reactive oxygen species (ROS), antioxidant enzyme activity, root lignin content, root porosity, root oxygen release, total oxidative substances and root structural changes in rice roots in response to P-sufficient and P-deficient treatments were investigated. Results indicated that P deficiency induced the production of H₂O₂ and O ₂ ^·? in roots significantly, which reached their maximum after 1- to 2-day P-deficient treatment. Interestingly, the endogenous total oxidative substances kept stable in rice roots. P deficiency increased the activities of peroxidase and superoxide dismutase by 89.5 and 51.8 % after 4-day P-deficient treatment, respectively. Moreover, one-day P deficiency elevated lignin accumulation. Root porosity of rice seedling under 2-day P-deficient treatment was 19.8 % higher than that under P-sufficient treatment. P deficiency also enhanced the release of both O₂ and total oxidative substances after 1- to 4-day P deficiency. In addition, results from electronic microscopy indicated that the thickness of root cell wall tended to increase after 2-day P-deficient treatment. Taken together, our results suggested that P-deficiency-induced enhancement of root oxidizing capacity in rice roots was probably associated with ROS production, antioxidant enzyme activity increment in root tissues, and the release of O₂ and oxidative substances from root inside to rhizosphere.     

Uptake by rice seedlings and in-plant degradation of atrazine as influenced by the oxidative stress induced by added arsenic or phosphate deficiency

The uptake and degradation of atrazine (ATR) by rice seedlings (Oryza sativa L.) was investigated with and without arsenate and phosphate nutrient in the cultured solution over a period of 48 h. The hydrogen peroxide (H₂O₂) contents in plants under different treatments were measured to evaluate the oxidative stress of the plant cell and its influence on the plant uptake and degradation of ATR. Results indicated that the ATR levels and main degradation products, deethylatrazine (DEA) and deisopropylatrazine (DIA), in plants varied significantly in different treatments. Added arsenate in solution increased the level of DEA and the ratios of DEA to the total (ATR, DEA, and DIA) in roots, while it either increased or decreased the H₂O₂ content in roots. Added arsenate increased the ratios of degradation products to the total in shoots, which corresponded to the 110%–285% increase of the H₂O₂ content. In phosphate-deficient systems, the H₂O₂ contents in shoots increased significantly, especially when exposed to a low level of ATR while the ratios of DIA and DEA to the total in shoots increased. The oxidative stress in rice seedlings induced by arsenic coexisting with ATR and by phosphate deficiency affected the plant uptake and degradation of ATR.     

The involvement of hydrogen peroxide in abscisic acid-induced activities of ascorbate peroxidase and glutathione reductase in rice roots

We have monitored the changes in antioxidant enzyme activities and H₂O₂ concentrations in roots of rice (Oryza sativa L., cv. Taichung Native 1) seedlings treated with exogenous abscisic acid(ABA). Decrease in superoxide dismutase (SOD) and catalase (CAT) activities was observed in rice roots in the presence of ABA. However, ascorbate peroxide (APX) and glutathione reductase (GR) activities were increased after the ABA treatment. ABA treatment resulted in an increase in H₂O₂ concentrations in rice roots. Pre-treatment with dimethylthiourea, a chemical trap for H₂O₂, and diphenyleneiodonium chloride (DPI), a well known inhibitor of NADPH oxidase, inhibited ABA-induced accumulation of H₂O₂ and ABA-induced activities of APX and GR. ABA-induced accumulation of H₂O₂ was found to be prior to ABA-induced activities of APX and GR. Our results suggest that H₂O₂ is involved in ABA-induced APX and GR activities in rice roots.     

Arbuscular mycorrhizas alter root system architecture of Citrus tangerine through regulating metabolism of endogenous polyamines

Cadmium (Cd) is readily taken up by the roots of rice seedlings, leading to growth reduction. H₂O₂ is a constituent of oxidative metabolism and is itself a reactive oxygen species. In this study, the participation of H₂O₂ in CdCl₂-inhibited growth of rice roots was investigated. CdCl₂ treatment increased H₂O₂ production in rice roots. CdCl₂ treatment had no effect on the activities of superoxide dismutase, ascorbate peroxidase, and glutathione reductase, but inhibited the activity of catalase (CAT) in rice roots. CdCl₂-inhibited root growth and -increased H₂O₂ content were lessened in the presence of diphenyleneiodonium chloride, an inhibitor of H₂O₂ generating NADPH oxidase. However, this stimulation of root growth in CdCl₂-treated seedlings is small (about 5%). Calcium (Ca) is important in many physiological processes in plants. Attempts were also made to determine whether the action of Ca on CdCl₂-inhibited growth of rice roots is associated with H₂O₂. CaCl₂ application reduced the production of H₂O₂, the decrease in CAT activity, and the inhibition of root growth caused by CdCl₂. The effects of CaCl₂ application could be reversed by exogenous H₂O₂. Our results indicate that the Cd causes a decline in CAT and to a lower extent a stimulation of NADPH oxidase in rice roots, with the subsequent generation of H₂O₂, an agent responsible for growth inhibition.     

NaCl-induced heme oxygenase in roots of rice seedlings is mediated through hydrogen peroxide

Recent findings have suggested that H₂O₂ is an important signaling molecule for regulating plant responses to abiotic stress. H₂O₂ plays a critical role in NaCl stress. Heme oxygenase (HO) is known to play a protective role against oxidative stress. In this study, we examined the possible involvement of H₂O₂ in regulating NaCl-promoted HO activity in rice roots. Treatment with NaCl increased HO activity and H₂O₂ content in rice roots. As well, NaCl could induce OsHO1 mRNA expression. NaCl (150 mM) and NaNO₃ (150 mM) were equally effective in inducing HO activity. However, mannitol at the concentration (276 mM) iso-osmotic with 150 mM NaCl had no effect on HO activity. NaCl-promoted HO activity and OsHO1 expression in rice roots was reduced by NADPH oxidase inhibitors i.e. dipehnyleneiodonium and imidazole. Moreover, exogenous application of H₂O₂ enhanced the activity of HO and the mRNA level of OsHO1. Our data suggest that H₂O₂ production plays a positive role in NaCl- induced HO activity by enhancing its mRNA level in rice roots.     

Arsenate Causes Differential Acute Toxicity to Two P-deprived Genotypes of Rice Seedlings (Oryza sativa L.)

Significant genotypic difference in response to arsenate toxicity in rice (Oryza sativa) was investigated in root elongation, arsenate uptake kinetics, physiological and biochemical response and arsenic (As) speciation. Uptake kinetics data showed that P-deprived genotype 94D-54 had a little higher As uptake than P-deprived 94D-64, but the difference was not large enough to cause acute toxicity in P-deprived 94D-54. There was no difference in tissue P concentrations between the two genotypes under P deficient conditions. In addition, arsenic speciation in plant tissues (using high performance liquid chromatography-inductively coupled plasma mass spectrometry) was not different between P pretreatments and between genotypes. P-deprived genotype 94D-54 suffered much higher stress induced by arsenate toxicity than P-deprived genotype 94D-64, in terms of lipid peroxidation, tissue H₂O₂ concentrations and exosmosis of K, P and As. However, P-deprived 94D-54 also had higher overproduction of enzymatic antioxidants (with higher GPX, SOD, CAT) and NPT (non-protein thiols) than P-deprived 94D-64. It appeared that, the higher sensitivity of P-deprived 94D-54 to arsenate toxicity might cause the overproduction of NPT, thus leading to the depletion of GSH and to the accumulation of H₂O₂. The differential sensitivity of the two genotypes has major implications for breeding rice for As affected paddy soil.     

Osmotic stress-induced changes in cell wall peroxidase activity and hydrogen peroxide level in roots of rice seedlings

The changes in activity of peroxidase (POD) extracted from the cellwalls and the level of H₂O₂ in rice seedling rootstreatedwith mannitol and their correlation with root growth were investigated.Increasing concentrations of mannitol from 92 to 276 mM, which isiso-osmotic with 50 to 150 mM NaCl, progressively reduced rootgrowth and increased POD activities extracted from the cell walls of riceroots.The reduction of growth was also correlated with an increase inH₂O₂ level. Both diamine oxidase (DAO) and NADHperoxidase(NADH-POD) are known to be responsible for the generation ofH₂O₂. Mannitol treatment increased DAO but not NADH-PODactivities in roots of rice seedlings, suggesting that DAO contributes to thegeneration of H₂O₂ in the cell walls of mannitol-treatedroots. An increase in the level of H₂O₂ and the activityof POD extracted from the cell walls of rice roots preceded root growthreduction caused by mannitol. An increase in DAO activity coincided with anincrease in H₂O₂ in roots caused by mannitol. Since DAOcatalyses the oxidation of putrescine, the demonstration that mannitolincreasesthe activity of DAO in roots is consistent with those that mannitol decreasesthe level of putrescine. In conclusion, cell-wall stiffening catalysed by PODispossibly involved in the regulation of root growth reduction caused bymannitol.     

Arginine decarboxylase ADC2 enhances salt tolerance through increasing ROS scavenging enzyme activity in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

In this study, we investigated the possible role of hemin in alleviating zinc (Zn), lead (Pb) and chromium (Cr) toxicity in rice seedlings grown hydroponically by analyzing the morphological and physiological parameters. Our results showed that exposure of rice seedlings to excess Zn, Pb or Cr could cause severe leaf chlorosis, inhibit photosynthetic activity and consequently suppress plant growth. The concentration of O₂ ^?? and H₂O₂ significantly increased and the activities of antioxidative enzymes decreased in roots of rice seedlings under metal exposure. The combined treatments (hemin?+?ZnSO₄, hemin?+?Pb(NO₃)₂ and hemin?+?K₂Cr₂O₇), on the other hand, significantly enhanced the photosynthesis- and plant growth-related parameters compared with their corresponding heavy-metal-stress alone. Combined treatments dramatically stimulated the activities of superoxide dismutase (SOD), ascorbic peroxidase (APX) and glutathione reductase (GR) as well as the concentrations of ascorbic acid (AsA) and glutathione (GSH) as compared with the metal- stress alone. The concentrations of reactive oxygen species (ROS, e.g. O₂ ^?? and H₂O₂) were significantly reduced in the metal plus hemin treatments. Hemin addition also reduced metal accumulation in rice seedlings especially in root tissues. These findings suggest that hemin-elevated levels of antioxidants, activities of antioxidative enzymes and hemin-reduced accumulation of heavy-metal could confer resistance against Zn, Pb, and Cr stress in rice seedlings, resulting in improved pigments accumulation, photosynthetic attributes and plant growth.     

Cell wall peroxidase activity,hydrogen peroxide level and NaCl-inhibited root growth of rice seedlings

The changes in cell-wall peroxidase (POD) activity and H₂O₂ level in roots of NaCl-stressed rice seedlings and their correlation with root growth were investigated. Increasing concentrations of NaCl from 50 to 150 mM progressively reduced root growth and increased ionically bound cell-wall POD activity. NaCl had no effect on covalently bound cell-wall POD activities. The reduction of root growth by NaCl is closely correlated with the increase in H₂O₂ level. Exogenous H₂O₂ was found to inhibit root growth of rice seedlings. Since ammonium and proline accumulation are associated with root growth inhibition caused by NaCl, we determined the effects of NH₄Cl or proline on root growth, cell-wall POD activity and H₂O₂level in roots. External application of NH₄Cl or proline markedly inhibited root growth, increased cell-wall POD activity and increased H₂O₂ level in roots of rice seedlings in the absence of NaCl. An increase in cell-wall POD activity and H₂O₂ level preceded inhibition of root growth caused by NaCl, NH₄Cl or proline. NaCl or proline treatment also increased NADH-POD and diamine oxidase (DAO) activities in roots of rice seedlings, suggesting that NADH-POD and DAO contribute to the H₂O₂ generation in the cell wall of NaCl- or proline-treated roots. NH₄Cl treatment increased NADH-POD activity but had no effect on DAO activity, suggesting that NADH-POD but not DAO is responsible for H₂O₂ generation in cell wall of NH₄Cl-treated roots.     

Activation of nitrate reductase by extracts from corn scutella

NADH-nitrate reductase (NR) from the primary leaves and root tips of corn seedlings (var. W64A × W182E) were activated by extracts from corn scutella. The activator extracted in potassium phosphate buffer (pH 7.5) or 80% (v/v) ethanol and fractionated by Dowex 1 (acetate) and Dowex 50 (H⁺) resins was recovered in the cationic fraction. The activator was not detected in extracts from shoots, roots, or endosperm of the seedlings. It activated the nitrate-induced cytochrome c reductase of NR complex but had slight inhibitory effects on the activities of FMNH₂-NR and reduced methylviologen-NR. In addition the activator inhibited the activities of purified NR-inactivating proteins from corn roots (var. Wf9 × 38-11) and rice cell cultures.     

Induction of heat resistance in wheat seedlings by exogenous calcium,hydrogen peroxide,and nitric oxide donor: functional interaction of signal mediators

Functional interactions of calcium ions, hydrogen peroxide, and nitric oxide as signal mediators in root cells of wheat (Triticum aestivum L.) seedlings upon induction of their heat resistance was studied with use of inhibitor-based analysis. Treatment of the seedlings with hydrogen peroxide or a combination of calcium chloride with ionophore A23187 significantly increased their content of nitric oxide, which peaked 0.5–1 h after the start of the treatment. CaCl₂ or exogenous NO donor (sodium nitroprusside, SNP) transitorily increased the hydrogen peroxide level in the roots. Seedlings pretreatments with calcium chelator (EGTA), blocker of Ca²⁺ channels (LaCl₃), inhibitor of phospholipase C (neomycin), or antagonist of cyclic adenosine-5'-diphosphatribose formation (nicotinamide) more or less prevented the rise in the nitric oxide content in roots caused by exogenous H₂O₂; the SNP-induced rise in hydrogen peroxide was also damped down. However, the seedlings pretreatment with antioxidants ionol or dimethylthiourea did not hinder the increase in the NO level, which was caused by exogenous Ca²⁺. The inhibitors of NO synthase (N^G-nitro-L-arginine methyl ester, L-NAME) or nitrate reductase (sodium tungstate) did not interfere in the accumulation of H₂O₂ in root tissues stimulated by exogenous calcium. Calcium antagonists diminished the seedlings heat resistance increased by hydrogen peroxide or SNP. Antioxidants and inhibitors of NO synthase or nitrate reductase weakened the calcium-stimulated enhancement in the seedlings heat resistance. It was concluded that calcium may activate NO- and H₂O₂-generating enzymatic systems as well as participate in the transduction of signals of these mediators into genetic apparatus and in the formation of physiological reactions underlying the enhanced heat resistance.     

Reactive oxygen species, ascorbate–glutathione pool, and enzymes of their metabolism in drought-sensitive and tolerant indica rice (Oryza sativa L.) seedlings subjected to progressing levels of water deficit

Water deficit for rice is a worldwide concern, and to produce drought-tolerant varieties, it is essential to elucidate molecular mechanisms associated with water deficit tolerance. In the present study, we investigated the differential responses of nonenzymatic antioxidants ascorbate (AsA), glutathione (GSH), and their redox pool as well as activity levels of enzymes of ascorbate–glutathione cycle in seedlings of drought-sensitive rice (Oryza sativa L.) cv. Malviya-36 and drought-tolerant cv. Brown Gora subjected to water deficit treatment of ?1.0 and ?2.1 MPa for 24–72 h using PEG-6000 in sand cultures. Water deficit caused increased production of reactive oxygen species such as O₂?^?, H₂O₂, and HO? in the tissues, and the level of production was higher in the sensitive than the tolerant cultivar. Water deficit caused reduction in AsA and GSH and decline in their redox ratios (AsA/DHA and GSH/GSSG) with lesser decline in tolerant than the sensitive seedlings. With progressive level of water deficit, the activities of monodehydroascorbate reductase, dehydroascorbate reductase, ascorbate peroxidase (APX), and glutathione transferase increased in the seedlings of both rice cultivars, but the increased activity levels were higher in the seedlings of drought-tolerant cv. Brown Gora compared to the sensitive cv. Malviya-36. Greater accumulation of proline was observed in stressed seedlings of tolerant than the sensitive cultivar. In-gel activity staining of APX revealed varying numbers of their isoforms and their differential expression in sensitive and tolerant seedlings under water deficit. Results suggest that an enhanced oxidative stress tolerance by a well-coordinated cellular redox state of ascorbate and glutathione in reduced forms and induction of antioxidant defense system by elevated activity levels of enzymes of ascorbate–glutathione cycle is associated with water deficit tolerance in rice.     

Genome-wide Association Mapping Identifies a New Arsenate Reductase Enzyme Critical for Limiting Arsenic Accumulation in Plants

Inorganic arsenic is a carcinogen, and its ingestion through foods such as rice presents a significant risk to human health. Plants chemically reduce arsenate to arsenite. Using genome-wide association (GWA) mapping of loci controlling natural variation in arsenic accumulation in Arabidopsis thaliana allowed us to identify the arsenate reductase required for this reduction, which we named High Arsenic Content 1 (HAC1). Complementation verified the identity of HAC1, and expression in Escherichia coli lacking a functional arsenate reductase confirmed the arsenate reductase activity of HAC1. The HAC1 protein accumulates in the epidermis, the outer cell layer of the root, and also in the pericycle cells surrounding the central vascular tissue. Plants lacking HAC1 lose their ability to efflux arsenite from roots, leading to both increased transport of arsenic into the central vascular tissue and on into the shoot. HAC1 therefore functions to reduce arsenate to arsenite in the outer cell layer of the root, facilitating efflux of arsenic as arsenite back into the soil to limit both its accumulation in the root and transport to the shoot. Arsenate reduction by HAC1 in the pericycle may play a role in limiting arsenic loading into the xylem. Loss of HAC1-encoded arsenic reduction leads to a significant increase in arsenic accumulation in shoots, causing an increased sensitivity to arsenate toxicity. We also confirmed the previous observation that the ACR2 arsenate reductase in A. thaliana plays no detectable role in arsenic metabolism. Furthermore, ACR2 does not interact epistatically with HAC1, since arsenic metabolism in the acr2 hac1 double mutant is disrupted in an identical manner to that described for the hac1 single mutant. Our identification of HAC1 and its associated natural variation provides an important new resource for the development of low arsenic-containing food such as rice.     

Cadmium toxicity and translocation in rice seedlings are reduced by hydrogen peroxide pretreatment

A hydroponic experiment was carried out to study the role of hydrogen peroxide (H₂O₂) in enhancing tolerance and reducing translocation of cadmium (Cd) in rice seedlings. Plant growth (length and biomass of shoot and root) was significantly repressed by Cd exposure. However, pretreatment with 100 μM H₂O₂ for 1d mitigated Cd stress by inducing enzyme activities for antioxidation (e.g., superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (GPX), ascorbate peroxidase (APX)) and detoxification (e.g., glutathione S-transferase (GST)) as well as by elevating contents of reduced glutathione (GSH) and ascorbic acid (AsA). As a result, H₂O₂ and malondialdehyde (MDA) content decreased in plants and the seedling growth was less inhibited. On the other hand, H₂O₂ pretreatment decreased Cd concentration in shoots, thus lowered the ratio of Cd concentration in shoots and roots (S/R), indicating that H₂O₂ may affect Cd distribution in rice seedlings. The improved Cd tolerance is partly due to an enhanced antioxidative system that efficiently prevents the accumulation of H₂O₂ during Cd stress. Increased Cd sequestration in rice roots may contribute to the decline of Cd translocation.     

Rapid reduction of arsenate in the medium mediated by plant roots

Microbes detoxify arsenate by reduction and efflux of arsenite. Plants have a high capacity to reduce arsenate, but arsenic efflux has not been reported. Tomato (Lycopersicon esculentum) and rice (Oryza sativa) were grown hydroponically and supplied with 10 microm arsenate or arsenite, with or without phosphate, for 1-3 d. The chemical species of As in nutrient solutions, roots and xylem sap were monitored, roles of microbes and root exudates in As transformation were investigated and efflux of As species from tomato roots was determined. Arsenite remained stable in the nutrient solution, whereas arsenate was rapidly reduced to arsenite. Microbes and root exudates contributed little to the reduction of external arsenate. Arsenite was the predominant species in roots and xylem sap. Phosphate inhibited arsenate uptake and the appearance of arsenite in the nutrient solution, but the reduction was near complete in 24 h in both -P- and +P-treated tomato. Phosphate had a greater effect in rice than tomato. Efflux of both arsenite and arsenate was observed; the former was inhibited and the latter enhanced by the metabolic inhibitor carbonylcyanide m-chlorophenylhydrazone. Tomato and rice roots rapidly reduce arsenate to arsenite, some of which is actively effluxed to the medium. The study reveals a new aspect of As metabolism in plants.     

Shade light interaction with salicylic acid in regulating growth of sun (alpine) and shade (prairie) ecotypes of Stellaria longipes

Magnesium (Mg) deficiency in plants is a widespread problem, affecting productivity and quality in agriculture. The mechanism of Mg deficiency inducing antioxidant enzyme activities has not been elucidated in rice. We examined the relationship among abscisic acid (ABA), H₂O₂, and antioxidant enzymes in the leaves of rice seedlings grown under conditions of Mg deficiency. The expression of OsRab16A, an ABA responsive gene, was used to determine the content of ABA. Mg deficiency resulted in increased ABA content in leaves of rice seedlings. The production of H₂O₂ was examined by 3,3-diaminobenzidine staining and a colorimetric method. Mg deficiency also induced H₂O₂ production in leaves, which was blocked by dipehnyleneiodonium chloride (DPI), an NADPH oxidase inhibitor. Tungstate (Tu), an ABA biosynthesis inhibitor, was effective in reducing Mg deficiency-increased ABA content, as well as Mg deficiency-induced H₂O₂ production. Both Tu and DPI were effective in reducing Mg deficiency-induced activities of superoxide dismutase, ascorbate peroxidase, glutathione reductase, and catalase in the leaves. Mg deficiency-induced ABA accumulation may trigger increased production of H₂O₂, which may involve plasma-membrane NADPH oxidase, and, in turn, up-regulates the activities of antioxidant enzymes in leaves of rice seedlings.     

Antioxidant response of wheat roots to drought acclimation

Wheat (Triticum aestivum L.) seedlings of a drought-resistant cv. C306 were subjected to severe water deficit directly or through stress cycles of increasing intensity with intermittent recovery periods. The antioxidant defense in terms of redox metabolites and enzymes in root cells and mitochondria was examined in relation to membrane damage. Acclimated seedlings exhibited higher relative water content and were able to limit the accumulation of H₂O₂ and membrane damage during subsequent severe water stress conditions. This was due to systematic up-regulation of superoxide dismutase, ascorbate peroxidase (APX), catalase, peroxidases, and ascorbate–glutathione cycle components at both the whole cell level as well as in mitochondria. In contrast, direct exposure of severe water stress to non-acclimated seedlings caused greater water loss, excessive accumulation of H₂O₂ followed by elevated lipid peroxidation due to the poor antioxidant enzyme response particularly of APX, monodehydroascorbate reductase, dehydroascorbate reductase, glutathione reductase, and ascorbate–glutathione redox balance. Mitochondrial antioxidant defense was found to be better than the cellular defense in non-acclimated roots. Termination of stress followed by rewatering leads to a rapid enhancement in all the antioxidant defense components in non-acclimated roots, which suggested that the excess levels of H₂O₂ during severe water stress conditions might have inhibited or down-regulated the antioxidant enzymes. Hence, drought acclimation conferred enhanced tolerance toward oxidative stress in the root tissue of wheat seedlings due to both reactive oxygen species restriction and well-coordinated induction of antioxidant defense.