Symbiotic association between soybean plants and Bradyrhizobium japonicum develops oxidative stress and heme oxygenase-1 induction at early stages

We have previously demonstrated that the induction of heme oxygenase-1 (HO-1) (EC 1.14.99.3) plays a protective role against oxidative stress in leaves and nodules of soybean plants subjected to cadmium, UV-B radiation, and salt stress. Here, we investigated HO-1, localization and their relationship with oxidative stress in different growth stages of soybean plants roots inoculated with Bradyrhizobium japonicum (3, 5, 7, 10, and 20 days post-inoculation) and nodules. After 7 days of inoculation, we observed a 70% increase in thiobarbituric acid-reactive substances that correlates with an enhancement in the gene expression of HO-1, catalase, and superoxide dismutase. Furthermore, the inhibition of HO-1 activity by Zn-protoporphyrin IX produced an increase in lipid peroxidation and a decrease in glutathione content suggesting that, in this symbiotic process, HO-1 may act as a signal molecule that protects the root against oxidative stress. We determined, for the first time, the tissular localization of HO-1 in nodules by electron-microscope examination. These results undoubtedly demonstrated that this enzyme is localized only in the plant tissue and its overexpression may play an important role as antioxidant defense in the plant. Moreover, we demonstrate that, in roots, HO-1 is induced by oxidative stress produced by inoculation of B. japonicum and exerts an antioxidant response against it.     

Phytochelatin synthesis and response of antioxidants during cadmium stress in Bacopa monnieri L.

The phytotoxicity imposed by cadmium (Cd) and its detoxifying responses of Bacopa monnieri L. have been investigated. Effect on biomass, photosynthetic pigments and protein level were evaluated as gross effect, while lipid peroxidation and electrolyte leakage reflected oxidative stress. Induction of phytochelatins and enzymatic and non-enzymatic antioxidants were monitored as plants primary and secondary metal detoxifying responses, respectively. Plants accumulated substantial amount of Cd in different plant parts (root, stem and leaf), the maximum being in roots (9240.11 microg g(-1) dw after 7 d at 100 microM). Cadmium induced oxidative stress, which was indicated by increase in lipid peroxidation and electrical conductivity with increase in metal concentration and exposure duration. Photosynthetic pigments showed progressive decline while protein showed slight increase at lower concentrations. Enzymes viz., superoxide dismutase (SOD, EC 1.15.1.1), guaiacol peroxidase (GPX, EC 1.11.1.7) ascorbate peroxidase (APX, EC 1.11.1.11) and glutathione reductase (GR, EC 1.6.4.2) showed stimulation except catalase (CAT, EC 1.11.1.6) which showed declining trend. Initially, an enhanced level of cysteine, glutathione and non-protein thiols was observed, which depleted with increase in exposure concentration and duration. Phytochelatins induced significantly at 10 microM Cd in roots and at 50 microM Cd in leaves. The phytochelatins decreased in roots at 50 microM Cd, which may be correlated with reduced level of GSH, probably due to reduced GR activity, which exerted increased oxidative stress as also evident by the phenotypic changes in the plant like browning of roots and slight yellowing of leaves. Thus, besides synthesis of phytochelatins, availability of GSH and concerted activity of GR seem to play a central role for Bacopa plants to combat oxidative stress caused by metal and to detoxify it. Plants ability to accumulate and tolerate high amount of Cd through enhanced level of PCs and various antioxidants suggest it to be a suitable candidate for phytoremediation.     

Antioxidant enzymes and isoflavonoids in chilled soybean (Glycine max (L.) Merr.) seedlings

Changes of activity antioxidant enzymes and of levels of isoflavonoids were studied in the roots and hypocotyls of the etiolated soybean (Glycine max (L.) Merr. var. Essor) seedlings, submitted to cold. Prolonged exposure to 1 degrees C inhibited hypocotyl and root elongation and limited their growth after seedlings were transferred to 25 degrees C. Roots were more sensitive to chilling than hypocotyls. At 1 degrees C a gradual increase in MDA concentration in roots but not in hypocotyls was observed. An increase in catalase (CAT, EC 1.11.1.6) and superoxide dismutase (SOD, EC 1.15.1.1) activity in hypocotyls was observed both at 1 degrees C and after transfer of plants to 25 degrees C. In roots, CAT activity increased after 4 days of chilling, while SOD activity only after rewarming. L-Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) activity decreased in roots of chilled seedlings, but did not change in hypocotyls until activity increased after transfer to 25 degrees C. The content of genistein and daidzein increased after 24 h of treatment by low temperature and then decreased with prolonged chilling in hypocotyls and remained high in roots. However, it should be noted that genistin level (genistein glucoside) in chilled hypocotyls is 10 times higher than in roots, despite falling tendency. The role of antioxidant enzymes and isoflavonoids in preventing chilling injury in hypocotyls and roots of soybean seedlings is discussed.     

Nitrogen stress and the expression of asparagine synthetase in roots and nodules of soybean (Glycine max)

The difficulty of assaying asparagine synthetase (AS) (EC 6.3.5.4) activity in roots of soybean has been circumvented by measuring expression of the AS genes. Expression of three soybean asparagine synthetase (SAS) genes ( SAS1 , SAS2 and SAS3 ) was observed in roots of non-nodulated soybean plants cultivated on nitrate. Expression of these genes was reduced to very low levels within days after submitting the plants to a N-free medium. The subsequent return to a complete medium (containing nitrate) restored expression of all three AS genes. Roots of nodulated plants, where symbiotic nitrogen fixation was the exclusive source of N (no nitrate present), showed very weak expression of all three AS genes, but on transfer to a nitrate-containing medium, strong expression of these genes was observed within 24 h. In nodules, all three genes were expressed in the absence of nitrate. Under conditions that impair nitrogen fixation (nodules submerged in aerated hydroponics), only SAS1 expression was reduced. However, in the presence of nitrate, an inhibitor of N₂ fixation, SAS1 expression was maintained. High and low expressions of AS genes in the roots were associated with high and low ratios of Asn/Asp transported to the shoot through xylem. It is concluded that nitrate (or one of its assimilatory products) leads to the induction of AS in roots of soybean and that this underlies the variations found in xylem sap Asn/Asp ratios. Regulation of nodule AS expression is quite different from that of the root, but nodule SAS1 , at least, appears to involve a product of N assimilation rather than nitrate itself.     

Antioxidant gene-enzyme responses in Medicago truncatula genotypes with different degree of sensitivity to salinity

Antioxidant responses and nodule function of Medicago truncatula genotypes differing in salt tolerance were studied. Salinity effects on nodules were analysed on key nitrogen fixation proteins such as nitrogenase and leghaemoglobin as well as estimating lipid peroxidation levels, and were found more dramatic in the salt-sensitive genotype. Antioxidant enzyme assays for catalase (CAT, EC 1.11.1.6), superoxide dismutase (EC 1.15.1.1), ascorbate peroxidase (EC 1.11.1.11) and guaiacol peroxidase (EC 1.11.1.7) were analysed in nodules, roots and leaves treated with increasing concentrations of NaCl for 24 and 48 h. Symbiosis tolerance level, depending essentially on plant genotype, was closely correlated with differences of enzyme activities, which increased in response to salt stress in nodules (except CAT) and roots, whereas a complex pattern was observed in leaves. Gene expression responses were generally correlated with enzymatic activities in 24-h treated roots in all genotypes. This correlation was lost after 48 h of treatment for the sensitive and the reference genotypes, but it remained positively significant for the tolerant one that manifested a high induction for all tested genes after 48 h of treatment. Indeed, tolerance behaviour could be related to the induction of antioxidant genes in plant roots, leading to more efficient enzyme stimulation and protection. High induction of CAT gene was also distinct in roots of the tolerant genotype and merits further consideration. Thus, part of the salinity tolerance in M. truncatula is related to induction and sustained expression of highly regulated antioxidant mechanisms.     

Treatment with salicylic acid decreases the effect of cadmium on photosynthesis in maize plants

The present study investigated the possible mediatory role of salicylic acid (SA) in protecting photosynthesis from cadmium (Cd) toxicity. Seeds of maize (Zea mays L., hybrid Norma) were sterilized and divided into two groups. Half of the seeds were presoaked in 500muM SA solution for only 6h, after which both groups were allowed to germinate for 3d and were then grown for 14d in Hoagland solution at 22/18 degrees C in a 16/8-h light/dark period and 120mumolm(-2)s(-1) PAR. All seedlings (without H(2)O and SA controls) were transferred to Cd-containing solutions (10, 15, and 25muM) and grown for 14d. The rate of CO(2) fixation and the activity of ribulose 1,5-bisphosphate carboxylase (RuBPC, EC 4.1.1.39) and phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) were measured. Changes in the levels of several important parameters associated with oxidative stress, namely H(2)O(2) and proline production, lipid peroxidation, electrolyte leakage, and the activities of antioxidative enzymes (superoxide dismutase (SOD, EC 1.15.1.1), ascorbate peroxidase (APX, EC 1.11.1.11), catalase (CAT, EC 1.11.1.6), and guaiacol peroxidase (POD, EC 1.11.1.7)) were measured. Exposure of the plants to Cd caused a gradual decrease in the shoot and root dry weight accumulation, with the effect being most pronounced at 25muM Cd. Seed pretreatment with SA alleviated the negative effect of Cd on plant growth parameters. The same tendency was observed for the chlorophyll level. The rate of CO(2) fixation was lower in Cd-treated plants, and the inhibition was partially overcome in SA-pretreated plants. A drop in the activities of RuBPC and PEPC was observed for Cd-treated plants. Pretreatment with SA alleviated the inhibitory effect of Cd on enzyme activity. Proline production and the rates of lipid peroxidation and electrolyte leakage increased in Cd-treated plants, whereas the values of these parameters were much lower in SA-pretreated plants. Treatment of plants with Cd decreased APX activity, but more than doubled SOD activity. Pretreatment with SA caused an increase in both APX and SOD activity, but caused a strong reduction in CAT activity. The data suggest that SA may protect cells against oxidative damage and photosynthesis against Cd toxicity.     

Heme oxygenase exerts a protective role against oxidative stress in soybean leaves

We have previously demonstrated that the induction of heme oxygenase-1 (EC 1.14.99.3) plays a protective role for mammalian cells against oxidative stress. Here, we investigated for the first time the possible role of heme oxygenase-1 as an antioxidant defense in leaves of soybean plants. Treatment with 200 microM Cd during 48 h caused a 70% increase in thiobarbituric acid reactive substances, whereas GSH decreased 67%, guaiacol peroxidase and total superoxide dismutase also inhibited 49% and 46%, respectively. Two hundred micromolar of Cd produced the overexpression of heme oxygenase-1, as well as a 4.5-fold enhancement of its activity. Administration of biliverdin partially prevented the effects caused by Cd. Pretreatment with Zn protoporphyrin IX, a potent inhibitor of heme oxygenase, expectedly decreased heme oxygenase-1 activity to half. When the inhibitor was given before Cd, it completely prevented the enzyme induction increasing the levels of oxidative stress parameters. Collectively, these results indicated that although plant heme oxygenases share little homology to heme oxygenases from non-plant species, they also play an important protective role against oxidative cell damage.     

镉胁迫对萝卜幼苗活性氧产生、脂质过氧化和抗氧化酶活性的影响

镉胁迫使萝卜幼苗超氧阴离子(O2)、过氧化氢(H2O2)和丙二醛(MDA)含量增加;随着镉浓度提高,超氧化物歧化酶(SOD)活性首先明显上升,然后逐渐下降,甚至低于对照;叶片过氧化氢酶(CAT)活性明显增加,根系CAT活性则减少;根系以及较高浓度镉处理后期叶片的谷胱甘肽还原酶(GR)活性均显著增加.由此推测:在胁迫初期可能主要由SOD和CAT发挥抗氧化作用,而在胁迫后期由于抗坏血酸-谷胱甘肽(AsA-GsH)循环途径的激活,还原型谷胱甘肽和植物络合素含量的提高可能在清除活性氧或者直接螯合镉中起作用.     

Glutathione reductase activity and isoforms in leaves and roots of wheat plants subjected to cadmium stress

The behavior of glutathione reductase (GR, EC 1.6.4.2) activity and isoforms were analyzed in wheat (Triticum aestivum L.) leaves and roots exposed to a chronic treatment with a toxic cadmium (Cd) concentration. A significant growth inhibition (up to 55%) was found in leaves at 7, 14 and 21 days, whereas roots were affected (51%) only after three weeks. Wheat plants grown in the presence of 100microM Cd showed a time-dependent accumulation of this metal, with Cd concentration being 10-fold higher in roots than in leaves. Nevertheless, lipid peroxidation was augmented in leaves in all experiments, but not in roots until 21 days. Cadmium treatment altered neither the GR activity nor the isoform pattern in the leaves. However, GR activity increased 111% and 200% in roots at 7 and 14 days, respectively, returning to control levels after 21 days. Three GR isoforms were found in roots of control and treated plants, two of which were enhanced by Cd treatment at 7 and 14 days, as assessed by activity staining on native gels. The changes in the isoform pattern modified the global kinetic properties of GR, thereby decreasing significantly (2.5-fold) the Michaelis constant (K(m)) value for oxidized glutathione. Isozyme induction was not associated with an enhancement of GR mRNA and protein expression, indicating that post-translational modification could occur. Our data demonstrated that up-regulation of GR activity by the induction of distinctive isoforms occurs as a defense mechanism against Cd-generated oxidative stress in roots.     

Cloning and sequence analysis of a cDNA encoding ferric leghemoglobin reductase from soybean nodules.

A cDNA encoding soybean (Glycine max [L.] Merr) ferric leghemoglobin reductase (FLbR), an enzyme that is postulated to play an important role in maintaining leghemoglobin in its functional ferrous state, has been cloned and characterized. A group of highly degenerate oligonucleotides deduced from the N-terminal amino acid sequence of FLbR was used to prime the polymerase chain reaction (PCR) on soybean nodule mRNA and cDNA. A full-length clone of FLbR cDNA was isolated by screening a lambda gt11 soybean nodule cDNA library using the specific PCR-amplified FLbR cDNA fragment as a probe. The cDNA contained about 1.8 kb and had a coding sequence for 523 amino acids with a predicted molecular mass of 55,729 D, which included a putative 30-residue signal peptide and a 493-residue mature protein. Computer-aided analysis of the deduced FLbR amino acid sequence showed considerable homology (varied from 20-50% with enzymes and species) to dihydrolipoamide dehydrogenase (EC 1.8.1.4), glutathione reductase (EC 1.6.4.2), mercuric reductase (EC 1.16.1.1), and trypanothione reductase (EC 1.6.4.8) in a superfamily of pyridine nucleotide-disulfide oxidoreductases from various organisms. Northern blot analysis using FLbR cDNA as a probe showed that the FLbR gene was expressed in soybean nodules, leaves, roots, and stems, with a greater level of expression in nodules and leaves than in roots and stems. Southern blot analysis of the genomic DNA showed the presence of two homologous FLbR genes in the soybean genome.     

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.     

The role of 5-aminolevulinic acid in the response to cold stress in soybean plants

In this study, the possibility of enhancing cold stress tolerance of soybean plants (Glycine max L.) by exogenous application of 5-aminolevulinic acid (ALA) was investigated. ALA was added to the Hoagland solution at various concentrations ranging from 0 to 40 μM for 12 h. After ALA treatment, the plants were subjected to cold stress at 4°C for 48 h. ALA at low concentrations (5-10 μM) provided significant protection against cold stress compared to non-ALA-treated plants, enhancing chlorophyll content (Chl) as well as relative water content (RWC). Increase of thiobarbituric acid reactive species (TBARS) levels was also prevented, whereas exposure to higher ALA concentrations (15-40 μM) brought about a dose dependent increase of these species, reaching a maximum of 117% in plants pre-treated with 40 μM ALA compared to controls. ALA pre-treatment also enhanced catalase (CAT) and heme oxygenase-1 (HO-1) activities. These findings indicate that HO-1 acts not only as the rate limiting enzyme in heme catabolism, but also as an antioxidant enzyme. The highest cold tolerance was obtained with 5 μM ALA pre-treatment. Results show that ALA, which is considered as an endogenous plant growth regulator, could be used effectively to protect soybean plants from the damaging effects of cold stress by enhancing the activity of heme proteins, e.g., catalase (CAT) and by promoting heme catabolism leading to the production of the highly antioxidant biliverdin and carbon monoxide, without any adverse effect on the plant growth.