Sugar-binding activity of pea lectin enhances heterologous infection of transgenic alfalfa plants by Rhizobium leguminosarum biovar viciae

Transgenic alfalfa (Medicago sativa L. cv Regen) roots carrying genes encoding soybean lectin or pea (Pisum sativum) seed lectin (PSL) were inoculated with Bradyrhizobium japonicum or Rhizobium leguminosarum bv viciae, respectively, and their responses were compared with those of comparably inoculated control plants. We found that nodule-like structures formed on alfalfa roots only when the rhizobial strains produced Nod factor from the alfalfa-nodulating strain, Sinorhizobium meliloti. Uninfected nodule-like structures developed on the soybean lectin-transgenic plant roots at very low inoculum concentrations, but bona fide infection threads were not detected even when B. japonicum produced the appropriate S. meliloti Nod factor. In contrast, the PSL-transgenic plants were not only well nodulated but also exhibited infection thread formation in response to R. leguminosarum bv viciae, but only when the bacteria expressed the complete set of S. meliloti nod genes. A few nodules from the PSL-transgenic plant roots were even found to be colonized by R. leguminosarum bv viciae expressing S. meliloti nod genes, but the plants were yellow and senescent, indicating that nitrogen fixation did not take place. Exopolysaccharide appears to be absolutely required for both nodule development and infection thread formation because neither occurred in PSL-transgenic plant roots following inoculation with an Exo(-) R. leguminosarum bv viciae strain that produced S. meliloti Nod factor.     

Production and function of jasmonates in nodulated roots of soybean plants inoculated with Bradyrhizobium japonicum

Little is known regarding production and function of endogenous jasmonates (JAs) in root nodules of soybean plants inoculated with Bradyrhizobium japonicum. We investigated (1) production of jasmonic acid (JA) and 12-oxophytodienoic acid (OPDA) in roots of control and inoculated plants and in isolated nodules; (2) correlations between JAs levels, nodule number, and plant growth during the symbiotic process; and (3) effects of exogenous JA and OPDA on nodule cell number and size. In roots of control plants, JA and OPDA levels reached a maximum at day 18 after inoculation; OPDA level was 1.24 times that of JA. In roots of inoculated plants, OPDA peaked at day 15, whereas JA level did not change appreciably. Shoot dry matter of inoculated plants was higher than that of control at day 21. Chlorophyll a decreased more abruptly in control plants than in inoculated plants, whereas b decreased gradually in both cases. Exogenous JA or OPDA changed number and size of nodule central cells and peripheral cells. Findings from this and previous studies suggest that increased levels of JA and OPDA in control plants are related to senescence induced by nutritional stress. OPDA accumulation in nodulated roots suggests its involvement in "autoregulation of nodulation."     

Inoculation with a bacterial consortium alleviates the effect of cadmium overdose in soybean plants

Inoculating plants that have inefficient antioxidant systems with plant-associated bacteria allows them to overcome heavy metal intoxication. We monitored protein oxidation, the activity of plant defense system enzymes, and the phenolics content in soybean (Glycine max L.) during a prolonged exposure to cadmium (Cd). The assistance of the bacterial consortium reduced the bioavailability of Cd in a soil containing 10 times the metal’s Standard Maximum Value (SMV). This reduced the accumulation of Cd in the soybeans’ roots and seeds. At 100 SMV, bacterial inoculation resulted in increased Cd bioavailability, which enhanced cadmium uptake by the soybean plants. At both Cd concentrations, oxidative stress was more prolonged in the soybean’s roots than its leaves. In cadmium-polluted soil, glutathion peroxidase activity changed more rapidly in the roots of plants when they had been inoculated. Inhibition of the peroxidases’ activities strengthened the activity of glutathione-S-transferase; increased the phenolics content in plant roots; and alleviated stress in inoculated soybean plants compared to untreated plants. The bacterial consortium may be recommended for a plant protection at 10 SMV Cd in the soil, and for phytostabilization at 100 SMV.     

Arbuscular mycorrhizal influence on leaf water potential, solute accumulation, and oxidative stress in soybean plants subjected to drought stress

This study investigated several aspects related to drought tolerance in arbuscular mycorrhizal (AM) soybean plants. The investigation included both shoot and root tissues in order to reveal the preferred target tissue for AM effects against drought stress. Non-AM and AM soybean plants were grown under well-watered or drought-stressed conditions, and leaf water status, solute accumulation, oxidative damage to lipids, and other parameters were determined. Results showed that AM plants were protected against drought, as shown by their significantly higher shoot-biomass production. The leaf water potential was also higher in stressed AM plants (-1.9 MPa) than in non-AM plants (-2.5 MPa). The AM roots had accumulated more proline than non-AM roots, while the opposite was observed in shoots. Lipid peroxides were 55% lower in shoots of droughted AM plants than in droughted non-AM plants. Since there was no correlation between the lower oxidative damage to lipids in AM plants and the activity of antioxidant enzymes, it seems that first the AM symbiosis enhanced osmotic adjustment in roots, which could contribute to maintaining a water potential gradient favourable to the water entrance from soil into the roots. This enabled higher leaf water potential in AM plants during drought and kept the plants protected against oxidative stress, and these cumulative effects increased the plant tolerance to drought.     

Rhizobial position as a main determinant in the problem of competition for nodulation in soybean

Selected Bradyrhizobium japonicum strains inoculated on soybean seeds often fail to occupy a significant proportion of nodules when a competitor rhizobial population is established in the soil. This competition problem could result from a genetic/ physiological advantage of the adapted soil population over the introduced inoculant or from a positional advantage, as the soil population already occupies the soil profile where the roots will penetrate, whereas the inoculant remains concentrated around the seeds. Here, we have assessed the contribution of these factors with a laboratory model in which a rhizobial population is established in sterile vermiculite. We observed that the wild-type strain B. japonicum LP 3004 was able to grow in pots with N-free plant nutrient solution-watered vermiculite for six or seven generations with a duplication rate of at least 0.7 day(-1). In addition, the rhizobial population persisted for 3 months with 10(6)-10(7) colony-forming units ml(-1) of the vermiculite-retained solution. N-starved, young rhizobial cultures are more efficient in performing several steps along their early association with soybean roots. However, N starvation during growth of rhizobia used for seed inoculation did not enhance their competitiveness against a 1 month vermiculite-established rhizobial population, which occupied more than 72% of the nodules. When a similarly established rhizobial population was recovered from the vermiculite and homogeneously suspended in plant nutrient solution, these cells were significantly less competitive (29% of nodules occupied) than rhizobia obtained from a fresh, logarithmic culture in a N-poor minimal medium, thus indicating that cell position rather than intrinsic competitiveness was the determinant for nodule occupation.     

The PhyR-σEcfG signalling cascade is involved in stress response and symbiotic efficiency in Bradyrhizobium japonicum

PhyR is an unusual type of response regulator consisting of a receiver domain and an extracytoplasmic function (ECF) sigma factor-like domain. It was recently described as a master regulator of general stress response in Methylobacterium extorquens . Orthologues of this regulator are present in essentially all free-living Alphaproteobacteria. In most of them, phyR is genetically closely linked to a gene encoding an ECF σ factor. Here, we investigate the role of these two regulators in the soybean symbiont Bradyrhizobium japonicum USDA110. Using deletion mutants and phenotypic assays, we showed that PhyR and the ECF σ factor σ^EcfG are involved in heat shock and desiccation resistance upon carbon starvation. Both mutants had symbiotic defects on the plant hosts Glycine max (soybean) and Vigna radiata (mungbean). They induced fewer nodules than the wild type and these nodules were smaller, less pigmented, and their specific nitrogenase activity was drastically reduced 2 or 3 weeks after inoculation. Four weeks after infection, soybean nodule development caught up to a large extent whereas most mungbean nodules remained defective even 5 weeks after infection. Remarkably, both mutants triggered aberrant nodules on the different host plants with ectopically emerging roots. Microarray analysis revealed that PhyR and σ^EcfG control congruent regulons suggesting both regulators are part of the same signalling cascade. This finding was further substantiated by in vitro protein–protein interaction studies which are in line with a partner-switching mechanism controlling gene regulation triggered by phosphorylation of PhyR. The large number of genes of unknown function present in the PhyR/σ^EcfG regulon and the conspicuous symbiotic phenotype suggest that these regulators are involved in the Bradyrhizobium –legume interaction via yet undisclosed mechanisms.     

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.     

Aluminium Stress Affects Nitrogen Fixation and Assimilation in Soybean (Glycine max L.)

Nitrogen fixation and assimilation in nodules and roots were studied in soybean (Glycine max L.) exposed to different levels of aluminium (Al) stress (0, 50, 200 and 500 μM). Al at 500 μM induced oxidative stress, which became evident from an increase in lipid peroxidation accompanied by a concomitant decline in antioxidant enzyme activities and leghaemoglobin breakdown. Consequently, there was also a reduction in nitrogenase activity. However, the leghaemoglobin levels and nitrogenase activity were unexpectedly found to be higher in nodules when the plants were treated with 200 μM Al. Of the enzymes involved in nitrogen assimilation, the activity of glutamate dehydrogenase-NADH was reduced in nodules under Al stress, but it was significantly higher in roots at 500 μM Al as compared to that in the control. In nodules, the glutamine synthetase/glutamate synthase-NADH pathway, assayed in terms of activity and expression of both the enzymes, was inhibited at >50 μM Al; but in roots this inhibitory effect was apparent only at 500 μM Al. No significant changes in ammonium and protein contents were recorded in the nodules or roots when the plants were treated with 50 μM Al. However, Al at ≥200 μM significantly increased the ammonium levels and decreased the protein content in the nodules. But these contrasting effects on ammonium and protein contents due to Al stress were observed in the roots only at 500 μM Al. The results suggest that the effect of Al stress on nitrogen assimilation is more conspicuous in nodules than that in the roots of soybean plants.     

Expression of a class 1 hemoglobin gene and production of nitric oxide in response to symbiotic and pathogenic bacteria in Lotus japonicus

Symbiotic nitrogen fixation by the collaboration between leguminous plants and rhizobia is an important system in the global nitrogen cycle, and some molecular aspects during the early stage of host-symbiont recognition have been revealed. To understand the responses of a host plant against various bacteria, we examined expression of hemoglobin (Hb) genes and production of nitric oxide (NO) in Lotus japonicus after inoculation with rhizobia or plant pathogens. When the symbiotic rhizobium Mesorhizobium loti was inoculated, expression of LjHb1 and NO production were induced transiently in the roots at 4 h after inoculation. In contrast, inoculation with the nonsymbiotic rhizobia Sinorhizobium meliloti and Bradyrhizobium japonicum induced neither expression of LjHb1 nor NO production. When L. japonicus was inoculated with plant pathogens (Ralstonia solanacearum or Pseudomonas syringae), continuous NO production was observed in roots but induction of LjHb1 did not occur. These results suggest that modulation of NO levels and expression of class 1 Hb are involved in the establishment of the symbiosis.     

The High-Affinity Phosphate Transporter GmPT5 Regulates Phosphate Transport to Nodules and Nodulation in Soybean

Legume biological nitrogen (N) fixation is the most important N source in agroecosystems, but it is also a process requiring a considerable amount of phosphorus (P). Therefore, developing legume varieties with effective N(2) fixation under P-limited conditions could have profound significance for improving agricultural sustainability. We show here that inoculation with effective rhizobial strains enhanced soybean (Glycine max) N(2) fixation and P nutrition in the field as well as in hydroponics. Furthermore, we identified and characterized a nodule high-affinity phosphate (Pi) transporter gene, GmPT5, whose expression was elevated in response to low P. Yeast heterologous expression verified that GmPT5 was indeed a high-affinity Pi transporter. Localization of GmPT5 expression based on β-glucuronidase staining in soybean composite plants with transgenic roots and nodules showed that GmPT5 expression occurred principally in the junction area between roots and young nodules and in the nodule vascular bundles for juvenile and mature nodules, implying that GmPT5 might function in transporting Pi from the root vascular system into nodules. Overexpression or knockdown of GmPT5 in transgenic composite soybean plants altered nodulation and plant growth performance, which was partially dependent on P supply. Through both in situ and in vitro (33)P uptake assays using transgenic soybean roots and nodules, we demonstrated that GmPT5 mainly functions in transporting Pi from roots to nodules, especially under P-limited conditions. We conclude that the high-affinity Pi transporter, GmPT5, controls Pi entry from roots to nodules, is critical for maintaining Pi homeostasis in nodules, and subsequently regulates soybean nodulation and growth performance.     

Targeting HO-1 by Epigallocatechin-3-Gallate Reduces Contrast-Induced Renal Injury via Anti-Oxidative Stress and Anti-Inflammation Pathways

Both oxidative stress and inflammation are involved in the pathogenesis of contrast-induced nephropathy (CIN). Epigallocatechin-3-gallate (EGCG), a purified catechin from green tea, has antioxidant and anti-inflammatory effects. However, it is unknown whether or not EGCG is effective in treating CIN. Our present study found that intravenous administration of EGCG, either before or just after the establishment of CIN, had a protective effect, determined by normalization of serum creatinine and blood urea nitrogen levels, improvement in renal histopathological scoring and alleviation of apoptosis, accompanied by decreased oxidative stress and inflammation. Because EGCG is a potent inducer of the antioxidant heme oxygenase-1 (HO-1), we studied HO-1 signaling in CIN. HO-1 levels were increased in CIN; treatment with EGCG further increased HO-1 levels, accompanied by an increase in Nrf2, a regulator of antioxidant proteins. Interestingly, blockade of HO-1 with protoporphyrin IX zinc(II) (ZnPP) prevented the protective effect of EGCG on CIN. ZnPP also blocked the ability of EGCG to increase the activity of an antioxidant (superoxide dismutase), and decrease markers of oxidative stress (myeloperoxidase and malondialdehyde) and inflammation (myeloperoxidase and IL-1β), indicating that HO-1 is the upstream molecule that regulates the EGCG-mediated protection. To determine further the role of HO-1 on the EGCG-mediated inhibition of inflammation, we studied the effect of EGCG on the NLRP3 inflammasome, an upstream signaling of IL-1β. EGCG down-regulated NLRP3 expression, which was blocked by ZnPP, indicating that HO-1 links EGCG with NLRP3. Therefore, EGCG, via up-regulation of HO-1, protects against CIN by amelioration of oxidative stress and inflammation.     

Cadmium induced oxidative stress in soybean plants also by the accumulation of δ-aminolevulinic acid

Cadmium toxicity has been extensively studied in plants, however its biochemical mechanism of action has not yet been well established. To fulfil this objective, four-weeks-old soybean nodulated plants were treated with 200 μM Cd²⁺ for 48 h. δ-aminolevulinic acid dehydratase (ALA-D, E.C. 4.2.1.24) activity and protein expression, as well as δ-aminolevulinic acid (ALA) and porphobilinogen (PBG) concentrations were determined in nodules, roots and leaves. In vitro experiments carried out in leaves were performed using leaf discs to evaluate the oxidant and antioxidant properties of ALA and S-adenosyl-l-methinone (SAM), respectively. Oxidative stress parameters such as thiobarbituric acid reactive substances (TBARS) and GSH levels as well as superoxide dismutase (SOD, E.C. 1.15.1.1), and guaiacol peroxidase (GPOX, E.C. 1.11.1.7) were also determined. Cadmium treatment caused 100% inhibition of ALA-D activity in roots and leaves, and 72% inhibition in nodules whereas protein expression remained unaltered in the three studied tissues. Plants accumulated ALA in nodules (46%), roots (2.5-fold) and leaves (104%), respect to controls. From in vitro experiments using leaf discs, exposed to ALA or Cd²⁺, it was found that TBARS levels were enhanced, while GSH content and SOD and GPOX activities and expressions were diminished. The protective role of SAM against oxidative stress generated by Cd²⁺ and ALA was also demonstrated. Data presented in this paper let us to suggest that accumulation of ALA in nodules, roots and leaves of soybean plants due to treatment with Cd²⁺ is highly responsible for oxidative stress generation in these tissues.     

Involvement of heme oxygenase as antioxidant defense in soybean nodules

Objective: We have previously demonstrated that the inducible form of heme oxygenase plays a critical role in protecting against oxidative stress in mammals. To gain further insight into the functions of this enzyme in plants, we have tested its activity and expression in soybean nodules subjected to cadmium (Cd) stress.

Materials and methods: Four-weeks-old soybean nodulated plants were treated with different cadmium chloride concentrations (0, 50 and 200 μM) during 48 h. Oxidative stress parameters such as TBARS content, GSH levels and antioxidant enzyme activities were measured as well as heme oxygenase activity and expression. Besides, the effect of biliverdin and Zn-protophorphyrin IX were analized.

Results: Treatment with 200 μM Cd during 48 h caused a 67% increase in TBARS content, whereas GSH decreased 44%, and total superoxide dismutase, gluthatione reductase and guaiacol peroxidase were also inhibited 54, 20 and 60%, respectively. A total of 200 μM Cd produced the overexpression of heme oxygenase-1, as well as a 10-fold enhancement of its activity. Co-administration of biliverdin (10 μM) completely prevented the effects caused by Cd. Treatment with Zn protoporphyrin IX, a strong inhibitor of heme oxygenase, expectedly decreased heme oxygenase-1 activity to half. When the inhibitor was given together with Cd, completely prevented the enzyme induction and oxidative stress parameters were significantly enhanced.

Conclusion: Taking together, these results are indicating that heme oxygenase plays a protective role against oxidative cell damage in soybean nodules.     

Phosphatidylcholine levels in Bradyrhizobium japonicum membranes are critical for an efficient symbiosis with the soybean host plant

Phosphatidylcholine (PC), the major membrane phospholipid in eukaryotes, is found in only some bacteria including members of the family Rhizobiaceae. For this reason, it has long been speculated that rhizobial PC might be required for a successful interaction of rhizobia with their legume host plants in order to allow the formation of nitrogen-fixing root nodules. A major pathway for PC formation in prokaryotes involves a threefold methylation of the precursor phosphatidylethanolamine (PE). Here, we report on the isolation of a Bradyrhizobium japonicum gene (pmtA) encoding the phospholipid N-methyltransferase PmtA. Upon expression of the bradyrhizobial pmtA gene in Escherichia coli, predominantly monomethylphosphatidylethanolamine was formed from PE. PmtA-deficient B. japonicum mutants still produced low levels of PC by a second methylation pathway. The amount of PC formed in such mutants (6% of total phospholipids) was greatly decreased compared with the wild type (52% of total phospholipids). Root nodules of soybean plants infected with B. japonicum pmtA mutants showed a nitrogen fixation activity of only 18% of the wild-type level. The interior colour of the nodules was beige instead of red, suggesting decreased amounts of leghaemoglobin. Moreover, ultrastructure analysis of these nodules demonstrated a greatly reduced number of bacteroids within infected plant cells. These data suggest that the biosynthesis of wild-type amounts of PC are required to allow for an efficient symbiotic interaction of B. japonicum with its soybean host plant.     

The purple acid phosphatase GmPAP21 enhances internal phosphorus utilization and possibly plays a role in symbiosis with rhizobia in soybean

Induction of secreted and intracellular purple acid phosphatases (PAPs; EC 3.1.3.2) is widely recognized as an adaptation of plants to phosphorus (P) deficiency. The secretion of PAPs plays important roles in P acquisition. However, little is known about the functions of intracellular PAP in plants and nodules. In this study, we identified a novel PAP gene GmPAP21 in soybean. Expression of GmPAP21 was induced by P limitation in nodules, roots and old leaves, and increased in roots with increasing duration of P starvation. Furthermore, the induction of GmPAP21 in nodules and roots was more intensive than in leaves in both P‐efficient genotype HN89 and P‐inefficient genotype HN112 in response to P starvation, and the relative expression in the leaves and nodules of HN89 was significantly greater than that of HN112 after P deficiency treatment. Further functional analyses showed that over‐expressing GmPAP21 significantly enhanced both acid phosphatase activity and growth performance of hairy roots under P starvation condition, indicating that GmPAP21 plays an important role in P utilization. Moreover, GUS expression driven by GmPAP21 promoter was shown in the nodules besides roots. Overexpression of GmPAP21 in transgenic soybean significantly inhibited nodule growth, and thereby affected plant growth after inoculation with rhizobia. This suggests that GmPAP21 is also possibly involved in regulating P metabolism in nodules. Taken together, our results suggest that GmPAP21 is a novel plant PAP that functions in the adaptation of soybean to P starvation, possibly through its involvement in P recycling in plants and P metabolism in nodules.     

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.