Nodule Enzyme Research Articles

Rhizobium Inoculant and Seed-Applied Fungicide Effects Improve the Drought Tolerance of Soybean Plants as an Effective Agroecological Solution under Climate Change Conditions.

Rhizobial inoculation in combination with fungicidal seed treatment is an effective solution for improving soybean resistance to modern climate changes due to the maximum implementation of the plant's stress-protective antioxidant properties and their nitrogen-fixing potential, which will contribute to the preservation of the environment. Model ecosystems at different stages of legume-rhizobial symbiosis formation, created by treatment before sowing soybean seeds with a fungicide (fludioxonil, 25 g/L) and inoculation with an active strain of Bradyrhizobium japonicum (titer 109 cells per mL), were subjected to microbiological, biochemical, and physiological testing methods in controlled and field conditions. Seed treatment with fungicide and rhizobia showed different patterns in the dynamics of key antioxidant enzymes in soybean nodules under drought conditions. Superoxide dismutase activity increased by 32.7% under moderate stress, while catalase increased by 90.6% under long-term stress. An increase in the antioxidant enzyme activity induced the regulation of lipoperoxidation processes during drought and after the restoration of irrigation. Regeneration after stress was evident in soybean plants with a combination of fungicide seed treatment and rhizobial inoculant, where enzyme levels and lipoperoxidation processes returned to control plant levels. Applying seed treatment with fungicide and Rhizobium led to the preservation of the symbiotic apparatus functioning in drought conditions. As proof of this, molecular nitrogen fixation by nodules has a higher efficiency of 25.6% compared to soybeans without fungicide treatment. In the field, fungicidal treatment of seeds in a complex with rhizobia inoculant induced prolongation of the symbiotic apparatus functioning in the reproductive period of soybean ontogenesis. This positively affected the nitrogen-fixing activity of soybeans during the pod formation stage by more than 71.7%, as well as increasing soybean yield by 12.7% in the field. The application of Rhizobium inoculant and fungicide to seeds contributed to the development of antioxidant protection of soybean plants during droughts due to the activation of key enzymatic complexes and regulation of lipoperoxidation processes, which have a positive effect on nitrogen fixation and productivity of soybeans. This is a necessary element in soybean agrotechnologies to improve plant adaptation and resilience in the context of modern climate change.

Realization of protective and symbiotic properties of soybeans using fungicide seed treatment

Aim. The elaboration of efficient legume-rhizobial symbiosis systems, involving active strains of nodule bacteria, in the combination with fungicide seed treatment may be an alternative method of providing ecologically friendly nitrogen sources to plants and promoting their tolerance to the external factors, which is relevant for preservation and restoration of envi- ronmental quality. Therefore, the aim of our study was to determine the impact of pre-sowing seed treatment with fungi- cides, which differ in the action spectrum of active substances – Standak Top (fipronil, 250 g/l, thiophanate-methyl, 225 g/l, pyraclostrobin, 25 g/l) and Maxim XL (fludioxonyl, 25 g/l, metalaxyl, 10 g/l), on the intensity of the development of lipid peroxidation processes, the activity of antioxidant enzyme ascorbate peroxidase and nitrogen fixation activity in soybeans on the early stages of forming legume-rhizobial symbiosis. Methods. Microbiological (cultivation of a bacterial culture, seed inoculation), physiological (vegetative experiment), biochemical (spectrophotometric determination of the content of lipid peroxidation products and the activity of ascorbate peroxidase; measuring the nitrogen-fixation activity using a gas chro- matography). Results. It was found that pre-sowing fungicide treatment of soybean seeds and subsequent inoculation with active rhizobia of strain 634b did not result in the change in the content of TBA-active products in roots and root nodules (the values of indices were within the experiment deviation). At the same time, after seed inoculation using rhizobia and treat- ment with Maxim XL, there was an increase in the activity of ascorbate peroxidase in the roots from 20.3 to 30.8 %, and with Standak Top – from 20.0 to 29.8 % during the early stages of ontogenesis till the formation of the third ternate leaf. Here, the activity of the enzyme in root nodules increased by 24.7–40.3 % at the fungicidal effect. Our data demonstrate that the combination of fungicide seed treatment and inoculation with active rhizobia does not induce lipid peroxidation processes, but promotes the initiation of protective antioxidant properties in soybeans. It is accompanied with efficient functioning of the symbiotic apparatus, which is manifested in the increase in nitrogen-fixing activity of nodule bacteria, formed by active rhizobia of strain 634b after the seed treatment with Standak Top – by 98.3 and 78.1 % and after Maxim XL – by 78.6 and 196.2 % respectively, during the stages of the second and third ternate leaves. Conclusions. The pre-sowing soybean seed treatment with fungicides Standak Top and Maxim XL and the subsequent inoculation with active rhizobia of strain 634b does not induce the development of lipid peroxidation processes, but increases the activity of the antioxidant enzyme, ascor- bate peroxidase, in the roots and root nodules, which is accompanied with the efficient work of the symbiotic apparatus on the early stages of determining legume-rhizobial symbiosis. This method of seed treatment may be a novel measure, to use in the technologies of cultivating soybeans to enhance the realization of the symbiotic potential and meet the needs of plants in ecologically friendly nitrogen, and to promote the formation of their tolerance to the corresponding cultivation conditions.

Analysis of the expression of polyamine biosynthesis genes in nodules of the garden pea (Pisum sativum L.) and the effect of exogenous treatment with polyamines on their development

BACKGROUND: Polyamines are acting as signaling molecules during adaptation to stressful environment and as regulators of plant development. In plants, polyamines are represented mainly by putrescine, spermidine and spermine. The concentration of polyamines in symbiotic nodules of some legumes is 510 times higher than in the other organs, which indicates their important role in the formation and functioning of symbiotic nodules.
 MATERIALS AND METHODS: We analyzed the expression of genes encoding polyamine biosynthesis enzymes in symbiotic nodules, as well as the effect of exogenous polyamines on the nodule number and the average nodule weight in wild-type SGE plants and symbiotic pea mutants SGEFix-1 (sym40-1) and SGEFix-2 (sym33-3).
 RESULTS: The comparable expression level of arginine decarboxylase gene (PsADC) was observed in all analyzed nodules, whereas the expression level of ornithine decarboxylase gene (PsODC), was highly increased in nodules of SGEFix-2 (sym33-3) mutant. Treatment of the root system with a 0.1 mM solution of polyamines mixture led to an increase in the average weight of the nodule in wild-type plants and in the SGEFix-2 (sym33-3) mutant plants.
 CONCLUSIONS: It was shown that the main pathway of putrescine synthesis in wild-type pea symbiotic nodules is the arginine pathway, while the ornithine pathway is probably associated with activation of plant defense reactions. Polyamines acting, apparently, through ethylene, affect the functioning of the nodule meristem.

Nodule metabolism in cold stress tolerant and susceptible chickpea cultivars

Early maturing short duration cold-tolerant chickpea cultivars are being introduced to avoid yield loss from spell faced by crops. However, the biochemical basis of the low temperature stress tolerance is not known. In the present study carbon and nitrogen metabolizing enzymes in nodules along with physiological indices were measured in early maturing short duration cold- tolerant chickpea cultivars (ICCV 93929 and ICCV 88503) and in normal cold - susceptible (PBG1 and GPF2) cultivars. The higher biomass in nodules of cold-tolerant cultivars might act as a greater sink for attracting photoassimilates to fuel N2-fixation. The simultaneous increase of sucrose synthase and alkaline invertase in nodules of ICCV 93929 and ICCV 88503 at the stage corresponding to lower temperature, ensures hexose supply for meeting the energy and reducing power demands for active nitrogen fixation. The greater activity of glutamine synthetase observed in nodules of cold tolerant cultivars at stages exposed to lower temperature indicates an efficient assimilation of the symbiotically fixed nitrogen into amino acids. The total protein and amino acid contents were also higher in cold tolerant cultivars. There was a greater and more rapid dry matter accumulation in ICCV 88503 and ICCV 93929 as compared to PBG1 and GPF2 in which biomass accumulation extended up to 70 DAS. Sustainable photosynthetic efficiency and activities of carbon and nitrogen metabolizing enzymes for a longer period in ICCV 93929 and ICCV 88503 even after flowering and during the onset of pod setting indicates that period of active nitrogen fixation is extended in cold tolerant chickpea cultivars due to allocation of photosynthates for longer period. This will lead to more transfer of reduced N as amino compounds towards the shoots which will lead to more nitrogen availability to seeds and ultimately better seed filling. The molecular diversity characterized by using RAPD primers and clustering method revealed a greater similarity between cold tolerant cultivars than between the cold susceptible cultivars.

Mechanisms of physiological adjustment of N2 fixation in Cicer arietinum L. (chickpea) during early stages of water deficit: single or multi-factor controls.

Drought negatively impacts symbiotic nitrogen fixation (SNF) in Cicer arietinumL. (chickpea), thereby limiting yield potential. Understanding how drought affects chickpea nodulation will enable the development of strategies to biotechnologically engineer chickpea varieties with enhanced SNF under drought conditions. By analyzing carbon and nitrogen metabolism, we studied the mechanisms of physiological adjustment of nitrogen fixation in chickpea plants nodulated with Mesorhizobium ciceri during both drought stress and subsequent recovery. The nitrogenase activity, levels of several key carbon (in nodules) and nitrogen (in both nodules and leaves) metabolites and antioxidant compounds, as well as the activity of related nodule enzymes were examined in M.ciceri-inoculated chickpea plants under early drought stress and subsequent recovery. Results indicated that drought reduced nitrogenase activity, and that this was associated with a reduced expression of the nifK gene. Furthermore, drought stress promoted an accumulation of amino acids, mainly asparagine in nodules (but not in leaves), and caused a cell redox imbalance in nodules. An accumulation of organic acids, especially malate, in nodules, which coincided with the decline of nodulated root respiration, was also observed under drought stress. Taken together, our findings indicate that reduced nitrogenase activity occurring at early stages of drought stress involves, at least, the inhibition of respiration, nitrogen accumulation and an imbalance in cell redox status in nodules. The results of this study demonstrate the potential that the genetic engineering-based improvement of SNF efficiency could be applied to reduce the impact of drought on the productivity of chickpea, and perhaps other legume crops.

Short-term supply of elevated phosphate alters the belowground carbon allocation costs and functions of lupin cluster roots and nodules

The legume Lupinus albus is able to survive under low nutrient conditions due to the presence of two specialized below ground organs for the acquisition of nitrogen and phosphate, respectively.In this regard, cluster roots increase phosphate uptake and root nodules acquire atmospheric N₂via biological nitrogen fixation(BNF). Although these organs normally tolerate low phosphate conditions, very little is known about their physiological and metabolic flexibility during short-term changes in phosphate supply. The aim of this investigation was therefore to determine the physiological and metabolic flexibility of these organs during short-term supply of elevated phosphate nutrition. L. albus was cultivated in sand culture for 4 weeks at 0.1 mM phosphate supply, and then supplied with 2 mM phosphate for 2 weeks. Short-term elevated phosphate supply caused increased allocation of carbon and respiratory costs to nodules, at the expense of cluster root function. This alteration was also reflected in the increase in nodule enzyme activities related to organic acid synthesis, such as Phosphoenol-pyruvate Carboxylase (PEPC), Pyruvate Kinase (PK), Malate Dehydrogenase(NADH-MDH) and Malic Enzyme (ME). In cluster roots, elevated phosphate conditions caused a decline in these organic acid synthesizing enzymes. Phosphate recycling via Acid Phosphatase (APase),declined in nodules with elevated phosphate supply, but increased in cluster roots. Our findings suggest that during short-term elevated phosphate supply, there is a great degree of physiological and metabolic flexibility in lupin nutrient acquiring structures, and that these changes are related to the altered physiology of these organs [corrected].

Effect of Nitrogen and Sulphur Nutrition on Nitrogen assimilating Enzymes in Soybean Roots and Nodules

Soybean is an important legume crop with high protein content but deficient in sulphur (S) containing amino acids viz methionine and cysteine. Soybean protein quality can be improved by nutrient fertilization. Present studies report the effect of nitrogen (N) and S nutrition on nodulation, activities of ammonia assimilating enzymes and biochemical parameters in soybean roots and nodules. Nitrogen as urea @ 31.25 kg ha−1 and sulphur as gypsum @ 20 kg ha−1 alone significantly increased nodule number, fresh and dry weight per nodule, root length and nodular leghemoglobin content whereas total soluble proteins, free amino acids, glucose and sucrose content did not vary significantly in nodules and roots. Treatment of N or S significantly increased glutamate dehydrogenase activity in cytosol and bacteroidal fractions from 70 to 90 days after sowing whereas aspartate aminotransferase, glutamate synthase, glutamine synthetase and alanine aminotransferase activities showed non-significant variations in roots at different stages of development under the influence of N or S alone or their combination. Results suggested that N and S could improve nodulation and vegetative growth in soybean but ammonia assimilating enzyme activities in roots/nodules did not vary significantly.

Influence of arbuscular mycorrhiza on osmotic adjustment compounds and antioxidant enzyme activity in nodules of salt-stressed soybean (Glycine max)

The influence of the colonization with arbuscular mycorrhizal (AM) fungus, Glomus mosseae (Nicolson and Gerdemann), on characteristics of growth, osmotic adjustment compounds and activity of antioxidant enzymes in nodules of salt-stressed soybean (Glycine max (L.) Merr.) was studied in this experiment. The pot experiment was arranged as a factorial in randomized complete block design with four replications at greenhouse of College of Agriculture, Tehran University, Iran. Results indicated that the contents of glycine betaine and proline in nodules were higher in inoculated than in non-inoculated plants. AM fungal colonization increased the activities of superoxide dismutase, catalase, and peroxidase in the nodules. The results indicate that the AM fungus is capable of alleviating the damage caused by salt stress on symbiotic nitrogen fixation of soybean plants by increasing the accumulation of compatible osmolytes and by increased antioxidant enzyme activity. Consequently, arbuscular mycorrhiza formation highly enhanced the salinity tolerance of soybean plant, which increased symbiotic nitrogen fixation and promoted plant growth.

Atividade de enzimas fermentativas em raízes e nódulos de soja sob condições de hipóxia e pós-hipóxia

O objetivo deste trabalho foi avaliar a atividade de enzimas fermentativas em raízes e nódulos de soja (Glycine max) sob condições de hipóxia e pós-hipóxia. Os genótipos Fundacep 53 RR e BRS Macota foram cultivados em vasos de 3L contendo vermiculita como substrato em associação com Bradyrhizobium elkanii em casa de vegetação. No estádio R2, o sistema radicular das plantas foi submetido à hipóxia pelo encaixe de um segundo vaso sem perfurações e inundado com solução nutritiva diluída a 1/3 da concentração original, por 8 dias. Posteriormente, foram retirados os vasos sem perfurações para avaliação da recuperação. O material vegetal foi coletado no 2º, 4º, 6º e 8º dias após a inundação ou recuperação, juntamente com os controles para a dosagem da atividade das enzimas álcool desidrogenase (ADH), piruvato descarboxilase (PDC) e lactato desidrogenase (LDH) em raízes e nódulos. Os dados foram submetidos à ANOVA e comparados pelo teste de Tukey a 5% de probabilidade. As atividades da ADH, PDC e LDH foram fortemente estimuladas com a inundação, especialmente em nódulos, e diminuíram com a recuperação, em ambos os genótipos. Fundacep 53 RR respondeu de forma mais efetiva aos efeitos dos tratamentos.

A phosphoenol pyruvate phosphatase transcript is induced in the root nodule cortex of Phaseolus vulgaris under conditions of phosphorus deficiency.

Although previous studies on N2-fixing legumes have demonstrated the contribution of acid phosphatases to their phosphorus (P) use efficiency under P-deficient growth conditions, localization of these enzymes in bean nodules has not been demonstrated. In this study, phosphoenol pyruvate phosphatase (PEPase) gene transcripts were localized within the nodule tissues of two recombinant inbred lines, RIL115 (P-deficiency tolerant) and RIL147 (P-deficiency sensitive), of Phaseolus vulgaris. Nodules were induced by Rhizobium tropici CIAT899 under hydroaeroponic conditions with a sufficient versus a deficient P supply. The results indicated that PEPase transcripts were particularly abundant in the nodule infected zone and cortex of both RILs. Analysis of fluorescence intensity indicated that nodule PEPase was induced under conditions of P deficiency to a significantly higher extent in RIL147 than in RIL115, and more in the inner cortex (91%) than in the outer cortex (71%) or the infected zone (79%). In addition, a significant increase (39%) in PEPase enzyme activity in the P-deficient RIL147 correlated with an increase (58%) in the efficiency of use in rhizobial symbiosis. It was concluded that nodule PEPase is upregulated under conditions of P deficiency in the P-deficiency-sensitive RIL147, and that this gene may contribute to adaptation of rhizobial symbiosis to low-P environments.

Effect of iron deficiency on the localization of phosphoenolpyruvate carboxylase in common bean nodules

Phosphoenolpyruvate carboxylase (PEPC) plays an important role in nodules, when there is an increase in the demand for energy. This enzyme provides carbon skeletons to sustain amino acid synthesis and malate to support energy required to fix nitrogen. Since PEPC is important for nodules, and there is lack of information about the effect of some nutrient deficiency in the expression and localization of this enzyme in legume nodules, this work focused on the localization of PEPC in nodules under iron deficiency of two common bean cultivars: Flamingo tolerant and Coco blanc sensitive to iron (Fe) deficiency. The results of immunolocalization using polyclonal antibody showed that this enzyme was detected in all regions of nodule sections; but the signal intensity was increased in Fe-deficient nodules as compared to Fe-sufficient ones in the tolerant cultivar, whereas the intensity was less pronounced in nodules of Fe-deficient plants than in those of Fe-sufficient plants for the sensitive cultivar Coco blanc. This work showed that the symbiotic tolerance of Flamingo to iron deficiency was linked to the increase of PEPC enzymes expression. However, the activity of these enzymes supported the energy required in bacteroids to maintain the nitrogenase activity. Keywords: Common bean, immunolocalization, iron deficiency, nodules, phosphoenol pyruvate carboxylase

Cobalt stress affects nitrogen metabolism, photosynthesis and antioxidant system in chickpea (Cicer arietinum L.)

Plants of chickpea were exposed to varied levels of cobalt (Co) and sampled at the 60-day stage. Cobalt at concentration <100 µM significantly increased the number of nodules, their dry mass, leghemoglobin concentration and the activity of nitrogenase. Similarly, the activities of glutamate dehydrogenase, glutamine synthetase and glutamate synthase also exhibited an increase in the presence of Co <100 µM, in nodules and leaves, respectively. The various photosynthetic attributes in leaves and the activity of antioxidative enzymes both in nodules and leaves were inhibited by Co in a concentration-dependent manner. However, the lipid peroxidation and the content of proline exhibited a significant increase in response to Co and were at a maximum in the plants exposed to 250 µM concentration of cobalt. Since most of the parameters showed a significant increase in response to 50 µM cobalt, this concentration may be regarded as a threshold concentration.

Relationship between photosynthetic capacity, nitrogen assimilation and nodule metabolism in alfalfa ( Medicago sativa) grown with sewage sludge

Sewage sludge has been used as N fertilizer because it contains some of inorganic N, principally as nitrate and ammonium ions. However, sewage sludge addition to legumes could result in impaired nodule metabolism due to the presence of inorganic N from sludge. A greenhouse experiment was conducted to examine the effects of sewage sludge on growth, photosynthesis, nitrogen assimilation and nodule metabolism in alfalfa ( Medicago sativa L. cv. Aragón). Plants were grown in pots with a mixture of perlite and vermiculite (2:1, v/v). The experiment included three treatments: (1) plants inoculated with rhizobia and amended with sewage sludge at rate of 10% (w/w) (RS); (2) plants inoculated with rhizobia without any amendment (R); and (3) non-inoculated plants fed with ammonium nitrate (N). N 2-fixing plants had lower growth and sucrose phosphate synthase activity but higher photosynthesis than nitrate-fed plants because they compensated the carbon cost of the rhizobia. However, sewage sludge-treated plants evidenced a loss of carbon sink strength due to N 2 fixation by means of decreased photosynthetic capacity, leaf chlorophylls and N concentration in comparison to untreated plants. Sewage sludge did no affect nodulation but decreased nodule enzyme activities involved in carbon and N metabolisms that may lead to accumulation of toxic N-compounds.

Modulation of symbiotic efficiency and nodular antioxidant enzyme activities in two Phaseolus vulgaris genotypes under salinity

To analyse nodular expression of antioxidant enzymes depending on plant genotype and salinity, two Phaseolus vulgaris genotypes, tolerant BAT477 and sensitive COCOT, were inoculated with the reference strain Rhizobium tropici CIAT899 and grown under 25 and 50 mM NaCl. Plant growth, nodulation and nitrogen fixing activity measured by the acetylene reducing activity (ARA) as an indicator of nitrogenase (E.C. 1.7.9.92) activity were more affected by salt concentrations in COCOT than in BAT477, particularly with 50 mM NaCl. Electrophoresis analysis of antioxidant enzymes in nodules, roots and free-living rhizobia showed that only catalase (CAT E.C. 1.11.1.6) isoenzymes varied with genotype. The sensitive genotype showed lower antioxidant enzyme activities than tolerant genotype and it was more affected by salinity. In the tolerant genotype catalase and ascorbate peroxidase (APX, E.C. 1.11.1.11) were inhibited by salt stress, whereas superoxide dismutase (SOD, E.C. 1.15.1.1) and peroxidase (POX, E.C. 1.11.1.7) were activated by salinity. Statistical analysis allowed suggesting that tolerance to salinity is associated with a differential regulation of distinct superoxide dismutase and peroxidase activities.

PHYSIOLOGICAL INTERACTION OF SOYBEAN PLANTS AND NEW TN-5 MUTANTS OF BRADYRHIZOBIUM JAPONICUM

The influence of soybean seeds inoculation with Tn-5 mutants of Bradyrhizobium japonicum with polar symbiotic properties on symbiosis efficiency, photosynthetic intensity and activity of antioxidant enzymes in root nodules of host plant was studied. Most of the selected mutants were highly virulent, but as was shown there were no considerable correlation between studied parameters. The direct relationship of nitrogen fixation activity of roots nodules and photosynthesis intensity of host plant was established. The two types of dynamics of theses processes were determined with their maximum values in blooming and flowering stages, respectively.&#x0D;

Phosphatase and phytase activities in nodules of common bean genotypes at different levels of phosphorus supply

Plants grown at limited P supply can increase the activity of phosphatases in roots to hydrolyse organic-P compounds in the soil thus improving plant P acquisition, but little information is available about the role of these enzymes for internal plant metabolism at limited-P conditions. This work intended to measure the activities of acid phosphatases and phytases in nodules of common bean (Phaseolus vulgaris) genotypes at different levels of P supply. The experiment was carried out in a 5 × 5 factorial design with four replicates, comprising five bean genotypes and five P levels (20, 40, 80, 160 and 320 μmol P plant−1 week−1) in nutrient solution. Root seedlings were inoculated with Rhizobium tropici and plants were grown in 1-l bottles. Nodule samples were detached from 39-day-old plants and enzyme activities were determined in crude extracts. Plants were harvested at the stage of pod setting. Polynomial models fitted to data indicated maximal values at the level of 194 μmol P for shoot mass, at 206 μmol P for nodule mass and at 221 μmol P for shoot N. Whereas shoot mass was 1.7 times lower at 20 than at 160 μmol P, nodule mass was 7.5 times lower. Concentration of P in nodules increased from 40 to 320 μmol P but remained stable between 20 and 40 μmol P, suggesting a minimal threshold concentration of 3 mg P g−1 for nodule growth. Activities of phosphatases and phytases in nodules decreased strongly as P supply was raised from 20 to 80 μmol P, remaining almost stable at higher P levels. Phosphatase activity ranged from 1,154 to 406 nmol min−1 g−1 (nodule fresh mass) from 20 to 80 μmol P respectively, while the phytase activity ranged from 55 to 14 nmol min−1 g−1 from 20 to 80 μmol P. Bean genotypes differed in shoot and nodule mass at the levels of 80 and 160 μmol P, whilst they differed in nodule enzyme activities only at the lowest P level, the relationship between nodule enzyme activities and growth of different bean genotypes was not evident. It is concluded that bean plants at P-deficient conditions increase the activities of phosphatases and phytases in nodules. This may constitute an adaptive mechanism for N2-fixing legumes to tolerate P deficiency, by increasing the utilisation of the scarce P within the nodules.

Symbiotic effectiveness and response to mannitol-mediated osmotic stress of various chickpea–rhizobia associations

Thirty-six symbiotic associations involving six chickpea cultivars against six rhizobial strains were evaluated for symbiotic performance and responses to osmotic stress applied by mannitol (50 mM) in aerated hydroponic cultures. Analyses in different symbioses were focused on biomass production, nodulation, nitrogen fixation, and their modulation under osmotic stress conditions, as well as expression of nodular antioxidant enzymes. Mesorhizobium ciceri reference (835) and local (CMG6) strains, as well as the local (C11) M. mediterraneum allowed the best symbiotic efficiency for all chickpea cultivars. The osmotic stress induces severe decrease ranging 30–50% in aerial biomass and 50–70% for nitrogen fixation. Nevertheless, plants inoculated with M. ciceri (835) and M. mediterraneum (C11) preserve a relatively high growth (4 g plant−1) with nitrogen-fixing activity (25 μmols h−1 plant−1). The bacterial partner was the most important factor of variance of the analysed parameters in osmotic stress or physiological conditions where it gets to 60–85%. The strains allowing the best competent symbioses were proposed for field assays. Under osmotic stress, nodular peroxidase (POX) and ascorbate peroxidase (APX) activities were significantly enhanced. The increase of POX and APX was inversely correlated with the inhibition of aerial biomass production (p = 0.05) and nitrogen-fixing capacity (p = 0.01), suggesting a protective role of these enzymes in nodules. Superoxide dismutase (SOD) was also activated in stressed nodules. However, the spectacular decrease in catalase (CAT) activity discounts its involvement in osmotic stress response.

Growth and nitrogen fixation in Lotus japonicus and Medicago truncatula under NaCl stress: Nodule carbon metabolism

Lotus japonicus and Medicago truncatula model legumes, which form determined and indeterminate nodules, respectively, provide a convenient system to study plant-Rhizobium interaction and to establish differences between the two types of nodules under salt stress conditions. We examined the effects of 25 and 50mM NaCl doses on growth and nitrogen fixation parameters, as well as carbohydrate content and carbon metabolism of M. truncatula and L. japonicus nodules. The leghemoglobin (Lb) content and nitrogen fixation rate (NFR) were approximately 10.0 and 2.0 times higher, respectively, in nodules of L. japonicus when compared with M. truncatula. Plant growth parameters and nitrogenase activity decreased with NaCl treatments in both legumes. Sucrose was the predominant sugar quantified in nodules of both legumes, showing a decrease in concentration in response to salt stress. The content of trehalose was low (less than 2.5% of total soluble sugars (TSS)) to act as an osmolyte in nodules, despite its concentration being increased under saline conditions. Nodule enzyme activities of trehalose-6-phosphate synthase (TPS) and trehalase (TRE) decreased with salinity. L. japonicus nodule carbon metabolism proved to be less sensitive to salinity than in M. truncatula, as enzymatic activities responsible for the carbon supply to the bacteroids to fuel nitrogen fixation, such as sucrose synthase (SS), alkaline invertase (AI), malate dehydrogenase (MDH) and phosphoenolpyruvate carboxylase (PEPC), were less affected by salt than the corresponding activities in barrel medics. However, nitrogenase activity was only inhibited by salinity in L. japonicus nodules.

Nitrogen Fixation Control under Drought Stress. Localized or Systemic?

Legume-Rhizobium nitrogen fixation is dramatically affected under drought and other environmental constraints. However, it has yet to be established as to whether such regulation of nitrogen fixation is only exerted at the whole-plant level (e.g. by a systemic nitrogen feedback mechanism) or can also occur at a local nodule level. To address this question, nodulated pea (Pisum sativum) plants were grown in a split-root system, which allowed for half of the root system to be irrigated at field capacity, while the other half was water deprived, thus provoking changes in the nodule water potential. Nitrogen fixation only declined in the water-deprived, half-root system and this result was correlated with modifications in the activities of key nodule's enzymes such as sucrose synthase and isocitrate dehydrogenase and in nodular malate content. Furthermore, the decline in nodule water potential resulted in a cell redox imbalance. The results also indicate that systemic nitrogen feedback signaling was not operating in these water-stressed plants, since nitrogen fixation activity was maintained at control values in the watered half of the split-root plants. Thus, the use of a partially droughted split-root system provides evidence that nitrogen fixation activity under drought stress is mainly controlled at the local level rather than by a systemic nitrogen signal.

NADPH recycling systems in oxidative stressed pea nodules: a key role for the NADP+ -dependent isocitrate dehydrogenase.

The symbiosis between legumes and rhizobia is characterised by the formation of dinitrogen-fixing root nodules. In natural conditions, nitrogen fixation is strongly impaired by abiotic stresses which generate over-production of reactive oxygen species. Since one of the nodule main antioxidant systems is the ascorbate-glutathione cycle, NADPH recycling that is involved in glutathione reduction is of great relevance under stress conditions. NADPH is mainly produced by glucose 6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.44) from the oxidative pentose phosphate pathway, and also by NADP(+)-dependent isocitrate dehydrogenase (ICDH; EC 1.1.1.42). In this work, 10 microM paraquat (PQ) was applied to pea roots in order to determine the in vivo relationship between oxidative stress and the activity of the NADPH-generating enzymes in nodules. Whereas G6PDH and 6PGDH activities remained unchanged, a remarkable induction of ICDH gene expression and a dramatic increase of the ICDH activity was observed during the PQ treatment. These results support that ICDH has a key role in NADPH recycling under oxidative stress conditions in pea root nodules.