Glycine Max Root Nodules Research Articles

Microrna Regulation of Nodule Zone-Specific Gene Expression In Soybean

Microrna Regulation of Nodule Zone-Specific Gene Expression In Soybean

Biochemical and molecular investigation of non-rhizobial endophytic bacteria as potential biofertilisers.

This study was performed to isolate non-rhizobial endophytic bacteria from the root nodules of Glycine max (soybean), Vigna radiata (mung bean) and Vigna unguiculata (cowpea). The bacteria were characterized for plant growth promoting properties such as indole acetic acid production, phosphate and zinc solubilisation, nitrogen fixation and hydrogen cyanide production. Phylogenetic identification was performed using the Neighbour-Joining method on16S rRNA gene sequences. The impact of salt tolerant isolates on some properties of wheat cv. Chamran was evaluated by a completely randomised factorial design. Nine isolates having some characteristics related to plant growth promotion were identified as Staphylococcus hominis 7E, Streptomyces sp. 11E, Bacillus sp. 13E, Acinetobacter sp. 19E, from mung bean, Bacillus endophyticus 1E from cowpea, Staphylococcus hominis 9E, Bacillus endophyticus 14E, Brevundimonas sp. 16E and Kocuria sp. 26E from soybean nodules. Isolates 7E and 19E caused maximum growth inhibition of Fusarium on PDA plate. All isolates were able to grow at salinity levels of mixtures containing up to 400mM of NaCl, CaCl2 and MgCl2, but their growth was inhibited by increasing salinity level. Only the growth of isolate 14E increased at three levels of salinity compared with control. Some isolates, i.e. 7E, 14E, 19E and 26E had higher colony diameter at 45°C after 48h of incubation compared to the growth at 30 and 40°C. Inoculation of soil with isolate 1E and isolate 26E caused to ameliorate salinity stress in wheat and increased the weight of 1000-grains as compared with non-inoculated treatments.

Isolation and evaluation of endophytic bacteria from root nodules of Glycine max L. (Merr.) and their potential use as biofertilizers

Aim of study: To isolate and characterize endophytic bacteria inhabiting soybean root nodules collected from two tropical cropping systems in Mexico, and to evaluate the bacterial effects in soybean plants under controlled conditions.Area of study: The study was carried out at two locations (San Antonio Cayal and Nuevo Progreso municipalities) of Campeche State, Mexico.Material and methods: Two experimental stages were performed: 1) isolation, morphological and biochemical characterization, and molecular identification of endophytic bacteria from root-nodules of four soybean varieties grown at field conditions; and 2) evaluation of the effects of endophytic isolates on soybean growth and nodule development, and the effects of bacterial co-inoculation on soybean plants, under controlled conditions.Main results: Twenty-three endophytic bacteria were isolated from root nodules, and identified as Agrobacterium, Bradyrhizobium, Rhizobium, Ensifer, Massilia, Chryseobacterium, Enterobacter, Microbacterium, Serratia, and Xanthomonas. Under controlled conditions, Rhizobium sp. CPO4.13C or Agrobacterium tumefaciens CPO4.15C significantly increased the plant height (46% and 41%, respectively), whereas Bradyrhizobium sp. CPO4.24C promoted the nodule formation (36 nodules/plant). The co-inoculation of B. japonicum USDA110 and Bradyrhizobium sp. CPO4.24C enhanced plant growth, height (33.87 cm), root nodulation (69 nodules/plant) and N-fixation (3.10 µmol C2H4 h-1 plant-1) in comparison to the negative control.Research highlights: Results suggest that the native Bradyrhizobium sp. CPO4.24C may be used as a biofertilizer directed to developing sustainable soybean cropping at tropical regions.

Distinct properties of two glutamine synthetase isoforms in soybean root nodules

Glutamine synthetase (GS) in nodule cytosol plays a major role in the assimilation of the ammonium produced by biological nitrogen fixation. To characterize the GS protein family in Glycine max root nodules, the catalytical properties of 2 GS1 isoenzymes (GS1β1 and GS1γ1) were compared in this study. Although, GmGS1β1 and GmGS1γ1 have very similar kinetic characteristics, they also exhibit distinct enzymatic properties in terms of thermal stability and the transferase to synthetase activity (GSt/GSs) ratios. The results demonstrated that GmGS1γ1, which displayed lower thermal stability and GSt/GSs ratios than GmGS1β1, might be considered as superior isoform to participate in the efficient assimilation of ammonia only when it is needed. Also, it is proposed that the difference of enzymatic properties between isoforms contribute to their differential roles in ammonia assimilation under variable internal and external environments.

Bradyrhizobium ottawaense sp. nov., a symbiotic nitrogen fixing bacterium from root nodules of soybeans in Canada.

Sixteen strains of symbiotic bacteria from root nodules of Glycine max grown in Ottawa, Canada, were previously characterized and placed in a novel group within the genus Bradyrhizobium. To verify their taxonomic status, these strains were further characterized using a polyphasic approach. All strains possessed identical 16S rRNA gene sequences that were 99.79 % similar to the closest relative, Bradyrhizobium liaoningense LMG 18230T. Phylogenetic analysis of concatenated atpD, glnII, recA, gyrB, rpoB and dnaK genes divided the 16 strains into three multilocus sequence types that were placed in a highly supported lineage distinct from named species of the genus Bradyrhizobium consistent with results of DNA–DNA hybridization. Based on analysis of symbiosis gene sequences (nodC and nifH), all novel strains were placed in a phylogenetic group with five species of the genus Bradyrhizobium that nodulate soybeans. The combination of phenotypic characteristics from several tests including carbon and nitrogen source utilization and antibiotic resistance could be used to differentiate representative strains from recognized species of the genus Bradyrhizobium. Novel strain OO99T elicits effective nodules on Glycine max, Glycine soja and Macroptilium atropurpureum, partially effective nodules on Desmodium canadense and Vigna unguiculata, and ineffective nodules on Amphicarpaea bracteata and Phaseolus vulgaris. Based on the data presented, we conclude that our strains represent a novel species for which the name Bradyrhizobium ottawaense sp. nov. is proposed, with OO99T ( = LMG 26739T = HAMBI 3284T) as the type strain. The DNA G+C content is 62.6 mol%.

Ensifer sojae sp. nov., isolated from root nodules of Glycine max grown in saline-alkaline soils

Thirteen bacterial isolates from root nodules of soybean grown in saline-alkaline soils in the Chinese province of Hebei were identified as a unique group in the genus Ensifer based upon BOX-PCR patterns, sequencing analyses of 16S rRNA and housekeeping genes and DNA-DNA hybridization. Phenotypically, positive tests for acid production and negative results for reduction in litmus milk and sensitivity to 50 µg ampicillin ml(-1), as well as some other features, could differentiate the novel group from defined species of the Ensifer-Sinorhizobium group. The novel group had symbiotic gene sequences (nodC and nifH) that were identical or very similar to those of Ensifer (Sinorhizobium) fredii, and formed effective nodules with Glycine max (soybean), Vigna unguiculata and Glycine soja. Based upon the consensus of these analyses, a novel species, Ensifer sojae sp. nov., is proposed, with CCBAU 05684(T) ( = LMG 25493(T) = HAMBI 3098(T)) as the type strain. The DNA G+C content of strain CCBAU 05684(T) was 60.9 mol% (T(m)).

Effect of 2,4-D on NR, NiR and Leghaemoglobin Synthesis in Root Nodules Formed by Bradyrhizobium japonicum in Glycine max.

The present study revealed that although very low concentrations (≤5μg/ml) of 2, 4-Dichlorophenoxyacetic acid (2, 4-D), a common herbicide, stimulated nitrate reductase (NR), nitrite reductase (NiR) and leghaemoglobin, in root nodules of Glycine max, higher cocentrations (>5μg/ml) were inhibitory. The results have also shown that treatment of rhizobia with different concentrations of 2, 4-D also affected NR and NiR of cytosol fraction of root nodules. This shows that there is a relationship between plant NR, NiR and the bacteroids in the root nodules.

Cyclic [beta]-1,6 -1,3 Glucans Are Synthesized by Bradyrhizobium japonicum Bacteroids within Soybean (Glycine max) Root Nodules.

We have previously reported that free-living cultures of Bradyrhizobium species produce novel oligosaccharides that are cyclic, contain between 10 and 13 glucose residues, and are linked by [beta]-1,6 and [beta]-1,3 glycosidic bonds (K.J. Miller, R.S. Gore, R. Johnson, A.J. Benesi, V.N. Reinhold [1990] J Bacteriol 172: 136-142). In the present study, we show that these glucans are also synthesized by bacteroids of Bradyrhizobium japonicum USDA 110 within Glycine max root nodules.

Characterization of symbiosis specific forms of alpha-glucosidase from Glycine max root nodules

Summary Three forms of α-glucosidase (EC 3.2.1.20) were isolated from soybean ( Glycine max L. Merr. cv. Maple Arrow) root nodules and two of them were further purified by a procedure including fractionation with PEG-8,000 followed by Q-Sepharose, S-Sepharose, Chromatofocusing and Superose 6 column chromatography. Chromatofocusing of the plant soluble fraction yielded two different isoforms, which were designated as I and II. Their isoelectric points were 7.3 (I) and 6.5 (II) as determined by native isoelectric focusing. The α-glucosidase I was further purified 502-fold to a specific activity of 1.8 units (mgprotein) -1 . In the peribacteroid space only one form, designated III, was resolved by chromatofocusing. The isoelectric point was 7.2 estimated by native isoelectric focusing. The α-glucosidase III was purified 86-fold to a specific activity of 3.1 units (mg protein) -1 . The α-glucosidases I and II had similar properties. The pH-optima were 5.2 (I) and 4.8 (III). The subunit molecular weights for α-glucosidase I and α-glucosidase III were estimated by SDS-PAGE to be 65 kDa and 66 kDa, respectively. Both enzymes hydrolyzed maltose, maltotriose, isomaltose and dextrin. Starch was only weakly hydrolyzed and neither a-glucosidase acted on sucrose. K m values for maltose were 0.91 mM (I) and 0.67 mM (III), and for PNPG 0.37 mM (I) and 0.76 mM (III). Ag + and Fe 2+ were the most potent inhibitors of both enzymes. The functions of these enzymes in the symbiotic compartments are discussed.

Immunofluorescent Staining of Microtubules in Plant Tissues: Improved Embedding and Sectioning Techniques using Polyethylene Glycol (PEG) and Steedman's Wax

AbstractMethods for the indirect immunofluorescent staining of microtubules in embedded and sectioned plant tissues are described and compared. Root tips of Vicia faba, Saccharum officinale, Allium cepa, and root nodules of Glycine max were fixed using conventional methods, embedded in polyethylene glycol or Steedman's wax, sectioned with a glass knife on a rotary microtome, and dewaxed in water or alcohol. The addition of dithiothreitol (DTT), dehydrating at low temperatures and reducing the infiltrations times were found to reduce background fluorescence in Allium cepa. Steedman's wax yields a block that is similar to paraffin and is easier to section than PEG. Routine methods for indirect immunofluorescence were used to stain sections for tubulin/microtubules. The major microtubule arrays of mitotic cells are illustrated in this paper. The principal advantage of this technique is the preservation of cell‐to‐cell continuity in multicellular tissues. This method provides a much needed technique for the study of the cytoskeleton during growth and differentiation of plant tissues.

Isolation and enzymological characterization of infected and uninfected cell protoplasts from root nodules of Glycine max

Infected and uninfected cell protoplasts were isolated from soybean (Glycine max L. Merr. cv. Akisengoku) root nodules and purified by the use of nylon mesh filters and discontinuous Percoll gradients. Activities of the enzymes involved in carbon and nitrogen metabolism were measured in cytoplasmic fractions of purified protoplasts, as well as in the bacteroids isolated from infected cell protoplasts and in the cortical tissues after enzymatic digestion of the central zone of the nodules.A high degree of purity of both infected and uninfected cells was demonstrated by microscopic observations and assays of β‐hydroxybutyrate dehydrogenase (EC 1.1.1.30) and uricase (EC 1.7.3.3) activities and leghemoglobin contents.As a whole, higher specific activities of enzymes of glycolysis were found in the cortical and uninfected cells than in the infected cells. The activities of glycolytic enzymes were extremely low in the bacteroids. Invertase (EC 3.2.1.26) was highly localized in the cortex. However, activity of sucrose synthase (EC 2.4.1.13) was highest in the cytosol of infected cells. Alcohol dehydrogenase (EC 1.1.1.1) and lactate dehydrogenase (EC 1.1.1.27) activities were much higher in uninfected than in infected cells. Specific activities of enzymes for nitrogen assimilation, that is, glutamine synthetase (EC 6.3.1.2), glutamate synthase (EC 1.4.1.14), aspartate (EC 2.6.1.1) and alanine (EC 2.6.1.2) aminotransferase were several‐fold higher in uninfected cells than in the infected cells.The results are discussed in relation to the possible cellular organization of carbon and nitrogen metabolism in soybean root nodules.

Properties of glutamine synthetase of Bacteroids from root nodules of Glycine max

Glutamine synthetase (GS) (EC 6.3.1.2) Isozyme I has been purified from bacteroids of root nodules of Glycine max. The purified GSI which had a molecular weight of 760 000 contained 12 subunits each of about 63 000 daltons. The GS had K m -values of 3.3 mM for hydroxylamine and 11.1 mM for l-glutamine for the γ-glutamyl transferase reaction. The K m -values for the biosynthetic reaction were 12.9 and 14.3 mM for l-glutamine and ATP, respectively. The K m -values of 2.8 and 8.9 mM were determined for NH 4 Cl from a biphasic plot. The inhibition of γ-glutamyl transferase activity by l-glutamate and NH 4 Cl was competitive for l-glutamine and non-competitive for hydroxylamine. When hydroxylamine and l-glutamine were the substrates, NH 4 Cl resulted in K i -values of 18 and 10 mM, respectively. Triphosphate nucleotides viz, ITP, UTP, CTP and ATP (48–92%) and diphosphate nucleotides (20–85%) viz. ADP, IDP, UDP, CDP and GDP, restricted either transferase or biosynthetic activities. Alanine, serine and glycine, respectively, inhibited GS activity by 78, 63 and 50%. Different pairs of amino acids in various combinations resulted in a cumulative inhibition of the enzyme.

Denitrification of nitrate to nitrogen gas by washed cells ofRhizobium japonicum and by bacteroids fromGlycine max

Nitrate, nitrite and nitrous oxide were denitrified to N2 gas by washed cells ofRhizobium japonicum CC706 as well as by bacteroids prepared from root nodules ofGlycine max (L.) Merr. (CV. Clark 63). Radiolabelled N2 was produced from either K(15)NO3 or Na(15)NO2 by washed cells ofRh. japonicum CC705 grown with either nitrate only (5 mM) or nitrate (5 mM) plus glutamate (10 mM). Nitrogen gas was also produced from N2O. Similar results were obtained with bacteroids ofG. max. The stoichiometry for the utilization of(15)NO 3 (-) or(15)NO 2 (-) and the produciton of(15)N2 was 2:1 and for N2O utilization and N2 production it was 1:1. Some of the(15)N2 gas produced by denitrification of(15)NO 3 (-) in bacteroids was recycled via nitrogenase into cell nitrogen.

Glutamine synthetase, glutamate synthase and glutamate dehydrogenase in Rhizobium japonicum strains grown in cultures and in bacteroids from root nodules of Glycine max

The growth yields of three strains of Rhizobium japonicum (CB 1809, CC 723, CC 705) in culture solutions containing L-glutamate were about twice those grown with ammonium. The activities of glutamine synthetase (GS; EC 6.3.1.2) and glutamate dehydrogenase (GDH; EC 1.4.1.4) were dependent on the nitrogen source in the medium and also varied with growth. Both NADPH-and NADH-dependent glutamate synthase (GOGAT; EC 1.4.1.13) and NADPH-dependent GDH were found in strains grown with either glutamate or ammonium but NADH-linked GDH was only detected in glutamate-grown cells. Glutamine synthetase was adenylylated in cells grown with NH 4 (+) (90%) and to lesser extent in those grown with L-glutamate (50%). In root nodules produced by the three strains in Glycine max (L.) Merr., the bulk of GS was located in the nodule cytosol (60-85%). The enzyme was adenylylated in bacteroids (43-75%) and in the nodule tissues (52-68%). The enzyme in cell-free extracts of Rh. japonicum (CC 705) grown in culture solutions containing glutamate and in bacteroids (CC 705) was deadenylylated by snake-venom phosphodiesterase. L-methionine-DL-sulfoximine restricted the incoporation of (15)N-labelled (NH4)2SO4 into cells of strains CB 1809 and CC 705, as well as in bacteroids of strain CC 705. It is noteworthy that appreciable activities for GDH were found in the free-living rhizobia grown on glutamate. Thus the presence of an enzyme does not necessarily imply that a particular pathway is operative in assimilating ammonium into cell nitrogen. Based on (15)N studies, the GS-GOGAT pathway of rhizobia (strains CB 1809 and CC 705) is important when grown in culture solutions as well as in bacteroids from root nodules of G. max.

Abscisic acid and gibberellin-like substances in roots and root nodules ofGlycine max

The content of endogenous gibberellin (GA)-like substances of roots and root nodules of SOya, and GA production byRhizobium japonicum cultures, were investigated by a combined thin layer chromatographic (TLC)-dwarf pea epicotyl bioassay technique. GAs were more concentrated in root nodules than in the roots, totalling 1.34 and 0.16 nM GA3 equivalents g−1 dry wt. respectively. GA production byR. japonicum cultures was demonstrated (1.00 nM GA3 equivalentsl−1) and comparison of the GA components of plant and bacterial culture medium extracts, suggested that rhizobial GA production may contribute to the nodule GA content.

Nitrogenase Activity in Protoplasts Isolated from Root Nodules of Glycine max

Root nodule protoplasts, isolated from soybeans under anaerobic conditions, showed nitrogenase activity. Alternatively, rhizobia present in the root nodules ( Rhizobium japonicum ATCC No 10324), kept under the same conditions in pure culture showed no nitrogenase activity. Attempts to culture agglutinated and fusion products of root nodule and normal soybean protoplasts failed due to contaminations of rhizobia set free from burst protoplasts.