María Rosario de Felipe

Oxidative stress occurs during soybean nodule senescence

Abstract. Several markers of oxidative stress were measured in 2- to 10-week-old soybean (Glycine max [L.] Merr.) nodules. There were increases in peroxides, protein carbonyls and modified DNA base concentrations with nodule age. The catalytic iron content also increased significantly during nodule ageing. Iron contained in the peribacteroid space was effective in promoting lipid peroxidation and this might contribute to the degradation of the peribacteroid membrane in senescing nodules. The concentration of the oxidized forms of glutathione and homoglutathione increased significantly during nodule development and the concentration of reduced glutathione and homoglutathione decreased during senescence. Taken together, these results are consistent with the development of oxidative stress in senescing nodules. Significant DNA and protein damage also occurred in the first days of nodule development, suggesting that an earlier period of oxidative stress might occur in the period over which the symbiosis becomes established.

Stress tolerance, genetic analysis and symbiotic properties of root-nodulating bacteria isolated from Mediterranean leguminous shrubs in Central Spain.

Nine root-nodulating bacterial isolates were obtained from the leguminous shrubs Spartium junceum, Adenocarpus hispanicus, Cytisus purgans, Cytisuslaburnuum, Retama sphaerocarpa and Colutea arborescens in areas of Central Spain. A poliphasic approach analyzing phenotypic, symbiotic and genetic properties was used to study their diversity and characterize them in relation to Mediterranean conditions. Stress tolerance assays revealed marked variations in salinity, extreme pH and cadmium tolerance compared with reference strains, with the majority showing salinity, alkalinity and Cd tolerance and three of them growing at acid pH. Variation within the 16S rRNA gene was examined by amplified 16S rDNA restriction analysis (ARDRA) and direct sequencing to show genetic diversity. Phylogeny confirmed the close relationship of four isolates with Bradyrhizobium canariense, three with Phylobacterium myrsinacearum, one with Rhizobium rhizogenes and another with Mesorhizobium huakuii. The cross inoculation tests revealed wide spectra of nodulation. This is the first report of P. myrsinacearum being able to nodulate these leguminous shrubs, and also the first time reported the association between B.canariense, R. rhizogenes and M. huakuii and C. laburnuum, C. purgans and C. arborescens, respectively. These results suggested that native rhizobia could be suitable candidates as biofertilizers and/or inoculants of leguminous shrubs with restoration or revegetation purposes in Mediterranean areas.

Nuclear DNA Endoreduplication and Expression of the Mitotic Inhibitor Ccs52 Associated to Determinate and Lupinoid Nodule Organogenesis

Lotus japonicus determinate nodules differ greatly from indeterminate nodules in their organogenesis and morphological characteristics, whereas Lupinus albus lupinoid nodules share features of determinate and indeterminate nodules. The mitotic inhibitor Ccs52A is essential for endoreduplication and ploidy-dependent cell enlargement during symbiotic cell differentiation in Medicago truncatula indeterminate nodules. ccs52A homolog genes were isolated from lupin and lotus nodules; the deduced Ccs52A proteins showed high sequence similarity with other Cdh-1-type activators of the anaphase-promoting complex and were grouped with A-type Ccs52 proteins from different plants. In lupin, ccs52A expression was restricted to the earlier stages of nodule development, whereas ccs52A transcripts accumulated in lotus nodule primordia and, to a lesser extent, in mature nodules. Nodule development in Lupinus albus involved a progressive increase in nuclear and cellular size and ploidy level; similarly, Lotus japonicus nodules contained polyploid nuclei and enlarged cells in the infected zone. Nevertheless, in situ hybridization experiments showed the highest ccs52A expression in the inner cortex cells of the lupin nodule primordium, probably associated to the increased size of these cells in mature nodules. In view of our results, Ccs52A-mediated endoreduplication appears to be a universal mechanism required for nodule cell differentiation during the establishment of nitrogen-fixing symbioses.

Effects of different O2 concentrations on nitrogenase activity, respiration, and O2 diffusion resistance in Lupinus albus L. cv. Multolupa nodules

Summary Nitrogenase, responsible for N 2 fixation in legume nodules, can be denatured by O 2 and functions under microaerobic conditions. However, O 2 is required for ATP production associated with nitrogenase activity; therefore, O 2 concentration within infected cells is highly regulated by a combination of nodule respiration, leghaemoglobin, and an O 2 diffusion barrier. To investigate O 2 diffusion regulation, different concentrations (1 %, 21 %, and 40 % (v/v)) were applied to nodulated root systems of Lupinus albus for 3 and 10 days. The application of 1 % O 2 for 3 days produced a marked decrease in nitrogenase activity, up to 30 % of that of control (21 % O 2 ) plants. The irreversible failure of nitrogenase functioning occurred after 10 days and could not be explained by the small variations in the content of nitrogenase components I and II. The internal O 2 concentration seems to be a limiting factor to N 2 fixation, but nodules can partly maintain their activity if hypoxic conditions are not too prolonged. The nitrogenase inhibition of the 3-day 40 % O 2 treatment was moderate ( 2 by varying the minimum resistance (R min ) of the O 2 diffusion barrier, which was almost doubled from the third day of treatment.

Characterization of rhizobia from legumes of agronomic interest grown in semi-arid areas of Central Spain relates genetic differences to soil properties.

A study of symbiotic bacteria from traditional agricultural legumes from Central Spain was performed to create a collection of rhizobia from soils differing in physicochemical, analytical and/or agroecological properties which could be well‐adapted to the environmental conditions of this region, and be used for sustainable agricultural practices. Thirty‐six isolates were obtained from root‐nodules of fifteen legume species (including Cicer arietinum, Lathyrus sativus, Lens culinaris, Lupinus spp., Medicago sativa, Phaseolus vulgaris, Pisum sativum, and Vicia spp.) from three agriculture areas with soils of different pHs and from a forest area with undisturbed soils. Phenotypical characterization revealed uniformity across the thirty‐six isolates, with important exceptions in terms of environmental tolerance (three isolates survived at high temperatures, three at high salinity and three at acid pH). The molecular analysis of 16S rRNA gene showed a close relationship of twenty‐nine isolates to Rhizobium leguminosarum, one to Rhizobium gallicum, one to Mesorhizobium ciceri, two to Sinorhizobium (Ensifer) meliloti and three to Bradyrhizobium canariense. The sequence analysis of a symbiosis‐specific gene, nod C, showed a correlation with the plant host and grouped twenty‐six isolates with Rhizobium leguminosarum bv. viciae, establishing the diversity in relation to legume‐host. The 16S‐23S rRNA intergenic spacer (IGS) region allowed for intraspecific differentiation, so that strains with equal 16S rRNA were grouped by means of their soil origin. These results indicated that phenotypical and genetically related strains may be widely distributed in this region and that soil abiotic characteristics could have a substantial bearing on the selection of the strains living in each environment. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Molecular characterization and evaluation of mycorrhizal capacity of Suillus isolates from Central Spain for the selection of fungal inoculants

Suillus fungal specimens of pine forests from a Mediterranean area of central Spain (Madrid region) were studied based on molecular and physiological analysis of sporocarps to obtain fungal native inocula to produce mycorrhizal Pinus halepensis Miller in nursery. Variation within the internal transcribed spacer (ITS) region of the ribosomal RNA genes of Suillus was examined by restriction fragment length polymorphism (RFLP) and direct sequencing of polymerase chain reaction products. Ribosomal DNA (rDNA) spacers were amplified from pure cultures obtained from fruit bodies of a range of Suillus species: Suillus bellinii (Inzenga) Watling, Suillus bovinus (Pers.) Kuntze, Suillus collinitus (Fr.) Kuntze, Suillus granulatus (L.) Snell, Suillus mediterraneensis (Jacquet. & Blum) Redeuil, Suillus luteus L. (Gray), and Suillus variegatus (Sw.) Kuntze. Interspecific variation in the length and number of restriction sites of the amplified ITS region was observed. This variation was confirmed by sequencing, which allowed us to identify some isolates. This is the first time that the ITS sequence of S. mediterraneensis is completely described. No intraspecific rDNA variation was observed within isolates of S. collinitus, S. mediterraneensis, and S. luteus. The phylogenetic analysis established the close relationship among these Mediterranean fungal species. As a further step to characterize the different isolates and to understand the relation between genetic and functional diversity, some physiological variables were evaluated. Intraspecific variation in axenic fungal growth and in mycorrhizal capacities was detected, especially within S. collinitus isolates. The fungal isolates stimulated the growth of P. halepensis in different rates. These studies indicated that ITS analysis, in conjunction with mycorrhizal tests, provides suitable combined tools for the analysis of Suillus spp. in a small geographic area for selecting isolates with final afforestation purposes.

Nitrate reductase isozymes in Bradyrhizobium sp. (Lupinus) bacteroids: localisation, biochemical and kinetic characteristics

Summary Bradyrhizobium sp. ( Lupinus ) strains 750 and IM-43B, possessing nitrate reductase activity (NRA) were used to inoculate Lupinus albus cv. Multolupa plants. Bacteroids from root nodules were sonicated and sedimented to separate membranes from cytosolic fractions. Our results confirmed the existence of constitutive NRA mostly in the membranes, but soluble and membrane-bound NR forms were also induced by nitrate, and both of them used NADH as electron donor. Immunolocalisation of NR using a monoclonal antibody showed the presence of a larger number of epitopes in the soluble region than in membrane of bacteroids, and gold particles increased in those nodules grown with nitrate. Chromatographic purification of NR allowed the detection of two active fractions with an average MW of 91 and 362 kD. A further attempt to separate NR fractions in bacteroids and free-living cells grown with nitrate, led us to detect two more native fractions (180 and 720 kD). SDS-PAGE gel electrophoresis from all of the four native fractions revealed the presence of two common bands of around 20 and 70 kD; these results seem to indicate that all of the native enzymes were associations of one single cong 90 kD monomer molecule. Specific NRA increased from the small to the large proteins, suggesting positive cooperation by monomer associations. There was evidence enough to support an interpretation that constitutive and substrate-induced NRs would be formed by a monomeric and its homotetrameric isozyme. On the other hand, three IEF spots were obtained by 2-D gel electrophoresis and by anionic chromatography with pI 7.0, 7.8 and 8.1, when any of the native fractions were analysed. Kinetic studies of the four MW native isoforms showed that the two smallest molecules behaved according to Michaelis-Menten with similar K M , but the 360 and 720 kD fractions fitted much better to straight lines in a Hill plot, indicating allosterism and positive cooperativity.

Review: Dark-induced changes in legume nodule functioning

Exposure of nodulated leguminous plants to prolonged periods of continuous darkness has been used as a convenient tool to investigate host plant control on nitrogen fixing systems in legume root nodules. Foliar dark treatment of plants results in a rapid decline in N2 -fixation in terms of acetylene reduction activity (ARA) and predisposes the nodules to metabolic and structural senescence. After 2 d of darkness, a significant decrease is seen in nitrogenase (N2 -ase) proteins of common bean nodules. The effect of dark treatments on nodule respiration varies with plant species. A variable decrease in nodule carbohydrates is observed in different plant species under dark treatments, but no direct correlation between N2 -ase activity and gross levels of carbohydrates present in the nodules has been detected. Usually nodule leghemoglobin (Lb) shows a decrease of variable intensity depending on plant species. The mRNA of Lb, sucrose synthase and glutamine synthetase shows a significant decline within 24 h of complete darkness. Dark-induced acceleration of proteolytic activity and decreased plant growth are reflected in decreased nodule proteins and accumulation of free amino acids following a drop in ARA. Antioxidants such as ascorbic acid and glutathione, along with the enzymes of their oxidation–reduction cycle, show a considerable decrease in their content and activity in nodules from dark-treated plants. Among H2 O2 scavengers, nodule catalase activity decreases in most of the plants studied, but peroxidase activity shows an increase. Dark-induced adverse effects on N2 -fixation are completely or partially reversible on shifting the plants back to a normal light/dark regime. Significant changes in nodule ultrastructure are induced by dark treatment. Attempts have been made to explain the mechanisms underlying dark-induced changes in nodule functioning.