Pisum Sativum Nodules Research Articles

Comparison of the Formation of Plant-Microbial Interface in Pisum sativum L. and Medicago truncatula Gaertn. Nitrogen-Fixing Nodules.

Different components of the symbiotic interface play an important role in providing positional information during rhizobial infection and nodule development: successive changes in cell morphology correspond to subsequent changes in the molecular architecture of the apoplast and the associated surface structures. The localisation and distribution of pectins, xyloglucans, and cell wall proteins in symbiotic nodules of Pisum sativum and Medicago truncatula were studied using immunofluorescence and immunogold analysis in wild-type and ineffective mutant nodules. As a result, the ontogenetic changes in the symbiotic interface in the nodules of both species were described. Some differences in the patterns of distribution of cell wall polysaccharides and proteins between wild-type and mutant nodules can be explained by the activation of defence reaction or premature senescence in mutants. The absence of fucosylated xyloglucan in the cell walls in the P. sativum nodules, as well as its predominant accumulation in the cell walls of uninfected cells in the M. truncatula nodules, and the presence of the rhamnogalacturonan I (unbranched) backbone in meristematic cells in P. sativum can be attributed to the most striking species-specific features of the symbiotic interface.

Altered plant organogenesis under boron deficiency is associated with changes in high-mannose N-glycan profile that also occur in animals

Boron (B) deficiency affects the development of Pisum sativum nodules and Arabidopsis thaliana root meristems. Both organs show an alteration of cell differentiation that result in the development of tumor-like structures. The fact that B in plants is not only able to interact with components of the cell wall but also with membrane-associated glycoconjugates, led us to analyze changes in high mannose type N-glycans (HMNG). The affinoblots with concanavalin A revealed alterations in the N-glycosylation pattern during early development of nodules and roots under B deprivation. Besides, there is increasing evidence of a B role in animal physiology that brought us to investigate the impact of B deficiency on Danio rerio (zebrafish) development. When B deficiency was induced prior to early cleavage stages, embryos developed as an abnormal undifferentiated mass of cells. Additionally, when B was removed at post-hatching, larvae undergo aberrant organogenesis. Resembling the phenomenon described in plants, alteration of the N-glycosylation pattern occurred in B-deficient zebrafish larvae prior to organogenesis. Overall, these results support a common function of B in plants and animals associated with glycosylation that might be important for cell signaling and cell fate determination during development.

Hendrik Poorter.

Hendrik Poorter.

Analysis of alternative splicing events in the root tips and nodules of Pisum sativum L

Background. Legumes establish symbioses with nitrogen-fixing bacteria from the Rhizobium group. In exchange for nutrients, bacteria provide fixed nitrogen needed to support plant growth. At the moment, information about the involvement of alternative splicing (AS) in the establishment and maintenance this symbiotic relationships is almost absent, but, as it is a powerful mechanism for the regulation of proteome diversity of the cell, it therefore may participate in cellular response to microsymbionts.
 Materials and methods. Alternative splicing was analyzed using the assembly of supertranscripts and alignment of the reads from nodules and root tips to this reference. Target genes expression levels was estimated in tips of non-inoculated roots, and in nodules (2, 4, and 6 weeks post inoculation) with use of RT-qPCR.
 Results.In this study, the analysis of AS events in the nodules and root tips of the pea was carried out. The presence of isoforms of four pea genes (PsSIP1, PsIGN, PsWRKY40, PsPR-10) was confirmed and their expression level was estimated.
 Conclusion. Pea nodules were shown to be more enriched with AS events compared to root tips. Among the functional groups of genes that demonstrate AS events, one of the most enriched functional groups is the pathogens stress response. Intron retention probably leads to degradation of the transcript via NMD-system or to change of the protein function, that modulates the activity of genes in nodules.

Triarabinosylation is required for nodulation-suppressive CLE peptides to systemically inhibit nodulation in Pisum sativum.

Legumes form root nodules to house beneficial nitrogen-fixing rhizobia bacteria. However, nodulation is resource demanding; hence, legumes evolved a systemic signalling mechanism called autoregulation of nodulation (AON) to control nodule numbers. AON begins with the production of CLE peptides in the root, which are predicted to be glycosylated, transported to the shoot, and perceived. We synthesized variants of nodulation-suppressing CLE peptides to test their activity using petiole feeding to introduce CLE peptides into the shoot. Hydroxylated, monoarabinosylated, and triarabinosylated variants of soybean GmRIC1a and GmRIC2a were chemically synthesized and fed into recipient Pisum sativum (pea) plants, which were used due to the availability of key AON pathway mutants unavailable in soybean. Triarabinosylated GmRIC1a and GmRIC2a suppressed nodulation of wild-type pea, whereas no other peptide variant tested had this ability. Suppression also occurred in the supernodulating hydroxyproline O-arabinosyltransferase mutant, Psnod3, but not in the supernodulating receptor mutants, Pssym29, and to some extent, Pssym28. During our study, bioinformatic resources for pea became available and our analyses identified 40 CLE peptide-encoding genes, including orthologues of nodulation-suppressive CLE peptides. Collectively, we demonstrated that soybean nodulation-suppressive CLE peptides can function interspecifically in the AON pathway of pea and require arabinosylation for their activity.

Isolation of Rhizobium from root nodules of Pisum Sativum and its use as biofertilizer

Biofertilizers are substances which contain living microorganisms which when applied to a soil promote the growth by increasing the supply or availability of primary nutrients to the host plant. Biofertilizers are usually prepared as a carrier based inoculants containing effective microorganism which would show a good relationship with the host plant. This makes it easy to handle with wide range of acceptance by the host. Rhizobia are a special type of bacteria which live in root nodules in symbiotic association and fix atmospheric free nitrogen and make it available to the plants. In this study Rhizobium is isolated and cultured from the root nodules of Pisum sativum (pea plant) in a selective media i.e. YEMA (Yeast Extract Mannitol Agar medium with Congo Red). The isolate was found with poor absorption of congo red and appeared as whitish gummy colony which was biochemically tested. The isolated strain was then mixed with carrier and applied on pea. Uninoculated soil was used as control. The growth of plants was observed at regular time intervals. The growth parameters observed were germination of seed, shoot initiation, root initiation, root length and shoot length. The seed started to germinate by day 2. After 20 days the plant with the biofertilizer showed a shoot length of 11.5cm and root length of 18cm while control showed 7cm and 9cm respectively. This report showed that application of isolated rhizobial strain enhanced the growth of plant. The isolated strain can be used as biofertilizer.

Micromonospora ureilytica sp. nov., Micromonospora noduli sp. nov. and Micromonospora vinacea sp. nov., isolated from Pisum sativum nodules.

A diversity study on the presence of strains representing the genus Micromonospora in Pisum sativum nodules collected from Cañizal (Spain) has provided evidence of the high number of isolates that might represent novel species. In the present work, we have characterized three of these isolates: GUI23T, GUI43T and GUI63T. Phenotypic and genotypic analyses confirmed that all strains represent novel species of the genus Micromonospora with the following proposed names: Micromonospora ureilytica sp. nov., type strain GUI23T (=CECT 9022T=DSM 101692T), Micromonospora noduli sp. nov., type strain GUI43T (=CECT 9020T=DSM 101694T), and Micromonospora vinacea sp. nov., type strain GUI63T (=CECT 9019T=DSM 101695T).

Sitona lineatus (Coleoptera: Curculionidae) Larval Feeding on Pisum sativum L. Affects Soil and Plant Nitrogen.

Adults of Sitona lineatus (pea leaf weevil, PLW) feed on foliage of several Fabaceae species but larvae prefer to feed on nodules of Pisum sativum L. and Vicia faba L. Indirectly, through their feeding on rhizobia, weevils can reduce soil and plant available nitrogen (N). However, initial soil N can reduce nodulation and damage by the weevil and reduce control requirements. Understanding these interactions is necessary to make integrated pest management recommendations for PLW. We conducted a greenhouse study to quantify nodulation, soil and plant N content, and nodule damage by weevil larvae in relation to soil N amendment with urea, thiamethoxam insecticide seed coating and crop stage. PLWs reduced the number of older tumescent (multilobed) nodules and thiamethoxam addition increased them regardless of other factors. Nitrogen amendment significantly increased soil available N (>99% nitrate) as expected and PLW presence was associated with significantly lower levels of soil N. PLW decreased plant N content at early flower and thiamethoxam increased it, particularly at late flower. The study illustrated the complexity of interactions that determine insect herbivory effects on plant and soil nutrition for invertebrates that feed on N-fixing root nodules. We conclude that effects of PLW on nodulation and subsequent effects on plant nitrogen are more pronounced during the early growth stages of the plant. This suggests the importance of timing of PLW infestation and may explain the lack of yield depression in relation to this pest observed in many field studies. Also, pea crops in soils with high levels of soil N are unlikely to be affected by this herbivore and should not require insecticide inputs.

Rhizobium anhuiense sp. nov., isolated from effective nodules of Vicia faba and Pisum sativum.

Four rhizobia-like strains, isolated from root nodules of Pisum sativum and Vicia faba grown in Anhui and Jiangxi Provinces of China, were grouped into the genus Rhizobium but were distinct from all recognized species of the genus Rhizobium by phylogenetic analysis of 16S rRNA and housekeeping genes. The combined sequences of the housekeeping genes atpD, recA and glnII for strain CCBAU 23252(T) showed 86.9 to 95% similarity to those of known species of the genus Rhizobium. All four strains had nodC and nifH genes and could form effective nodules with Pisum sativum and Vicia faba, and ineffective nodules with Phaseolus vulgaris, but did not nodulate Glycine max, Arachis hypogaea, Medicago sativa, Trifolium repens or Lablab purpureus in cross-nodulation tests. Fatty acid composition, DNA-DNA relatedness and a series of phenotypic tests also separated these strains from members of closely related species. Based on all the evidence, we propose a novel species, Rhizobium anhuiense sp. nov., and designate CCBAU 23252(T) ( = CGMCC 1.12621(T) = LMG 27729(T)) as the type strain. This strain was isolated from a root nodule of Vicia faba and has a DNA G+C content of 61.1 mol% (Tm).

A genetic discontinuity in root-nodulating bacteria of cultivated pea in the Indian trans-Himalayas.

Evolutionary relationships of 120 root-nodulating bacteria isolated from the nodules of Pisum sativum cultivated at 22 different locations of the trans-Himalayan valleys of Lahaul and Spiti in the state of Himachal Pradesh of India were studied using 16S rRNA gene PCR-RFLP, ERIC-PCR, sequencing of 16S rRNA, atpD, recA, nodC and nifH genes, carbon-source utilization pattern (BIOLOG™), and whole-cell fatty acid profiling. The results demonstrated that all isolates belonged to Rhizobium leguminosarum symbiovar viciae (Rlv). Isolates from the two valleys were clearly separated on the basis of ERIC fingerprints, carbon-source utilization pattern, and whole-cell fatty acid methyl esters. Phylogenetic analysis of atpD, recA, nodC and nifH genes revealed a common Rlv sublineage in Spiti valley. Lahaul valley isolates were represented by three sequence types of atpD and recA genes, and four sequence types of nodC and nifH genes. Genotypes from the two valleys were completely distinct, except for two Lahaul isolates that shared nodC and nifH sequences with Spiti isolates but were otherwise more similar to other Lahaul isolates. Isolates from the two highest Spiti valley sites (above 4000 m) had a distinctive whole-cell fatty acid profile. Spiti valley isolates are closely related to Rlv sublineages from Xinjiang and Shanxi provinces in China, while Lahaul valley isolates resemble cosmopolitan strains of the western world. The high mountain pass between these valleys represents a boundary between two distinct microbial populations.

Relationship between gibberellin, ethylene and nodulation in Pisum sativum.

• Gibberellin (GA) deficiency resulting from the na mutation in pea (Pisum sativum) causes a reduction in nodulation. Nodules that do form are aberrant, having poorly developed meristems and a lack of enlarged cells. Studies using additional GA-biosynthesis double mutants indicate that this results from severe GA deficiency of the roots rather than simply dwarf shoot stature. • Double mutants isolated from crosses between na and three supernodulating pea mutants exhibit a supernodulation phenotype, but the nodule structures are aberrant. This suggests that severely reduced GA concentrations are not entirely inhibitory to nodule initiation, but that higher GA concentrations are required for proper nodule development. • na mutants evolve more than double the amount of ethylene produced by wild-type plants, indicating that low GA concentrations can promote ethylene production. The excess ethylene may contribute to the reduced nodulation of na plants, as application of an ethylene biosynthesis inhibitor increased na nodule numbers. However, these nodules were still aberrant in structure. • Constitutive GA signalling mutants also form significantly fewer nodules than wild-type plants. This suggests that there is an optimum degree of GA signalling required for nodule formation and that the GA signal, and not the concentration of bioactive GA per se, is important for nodulation.

The competition between Rhizobium leguminosarum bv. viciae strains progresses until late stages of symbiosis

The competition potential of 14 Rhizobium leguminosarum bv. viciae isolates originating from nodules of Pisum sativum was estimated. Genotypic analyses of the isolates revealed a high level of chromosomal and plasmid content diversity. The isolates tagged with a plasmid-bearing constitutively expressed gusA gene were used to inoculate vetch (Vicia villosa) in competition experiments carried out under laboratory conditions. Soil extract containing autochthonous rhizobial population was used as competitor for gus-tagged strains, and the competition was studied by: (i) estimation of Gus+ root nodules on whole root systems, (ii) the pattern of individual nodule colonization by Gus+/Gus− rhizobia, and (iii) the number of Gus+/Gus− bacteria recovered from individual nodules. Several patterns of nodule colonization by Gus+/Gus− bacteria were found. Some nodules identified as Gus+ contained gus-tagged bacteria only in the young and saprophytic zones, while the symbiotic zone was occupied by unmarked soil rhizobia. In other Gus+ nodules, despite the visible colonization of the entire nodule by gus-marked bacteria, a high number of Gus− soil-derived rhizobia were recovered. The results suggest that rhizobial strains compete with each other also in the late stage of nodule development. Therefore, they may use different strategies to reach the late saprophytic zone of the nodule, which serves as an optimal environment for massive proliferation.

Ligands of boron in Pisum sativum nodules are involved in regulation of oxygen concentration and rhizobial infection.

Boron (B) is an essential nutrient for N(2)-fixing legume-rhizobia symbioses, and the capacity of borate ions to bind and stabilize biomolecules is the basis of any B function. We used a borate-binding-specific resin and immunostaining techniques to identify B ligands important for the development of Pisum sativum-Rhizobium leguminosarum 3841 symbiotic nodules. arabinogalactan-extensin (AGPE), recognized by MAC 265 antibody, appeared heavily bound to the resin in extracts derived from B-sufficient, but not from B-deficient nodules. MAC 265 stained the infection threads and the extracellular matrix of cortical cells involved in the oxygen diffusion barrier. In B-deprived nodules, immunolocalization of MAC 265 antigens was significantly reduced. Leghaemoglobin (Lb) concentration largely decreased in B-deficient nodules. The absence of MAC 203 antigens in B-deficient nodules suggests a high internal oxygen concentration, as this antibody detects an epitope on the lipopolysaccharide (LPS) of bacteroids typically expressed in micro-aerobically grown R. leguminosarum 3841. However, B-deprived nodules did not accumulate oxidized lipids and proteins, and revealed a decrease in the activity of the major antioxidant enzyme ascorbate peroxidase (APX). Therefore, B deficiency reduced the stability of nodule macromolecules important for rhizobial infection, and for regulation of oxygen concentration, resulting in non-functional nodules, but did not appear to induce oxidative damage in low-B nodules.

Genetic diversity of root-nodulating bacteria isolated from pea (Pisum sativum) in subtropical regions of China.

Diversity of 42 isolates from effective nodules of Pisum sativum in different geographical regions of China were studied using 16S rRNA gene RFLP patterns, 16S rRNA sequencing, 16S-23S rRNA intergenic spacer (IGS) region RFLP patterns and G-C rich random amplified polymorphic DNA (RAPD). The isolates were distributed in two groups on the basis of their 16S rRNA gene RFLP patterns. The 16S rRNA gene sequences of strains from 16S rRNA gene RFLP patterns group I were very closely related (identities higher than 99.5%) to Rhizobium leguminosarum USDA 2370. Group II consisting of WzP3 and WzP15 was closely related to Rhizobium etli CFN42. The analysis of the 16S-23S IGS RFLP patterns divided the isolates into 18 genotypes and four groups. Group I was clustered with R. leguminosarum USDA2370. Group II consisted of YcP2, YcP3 and CqP7. The strains of group III were distributed abroad. Group IV consisted of WzP3, WzP15 and R. etli CFN42. RAPD divided the isolates into nine clusters in which group IV only consisted of YcP2 and the strains of group V and IX were from Wenzhou and Xiantao, respectively. This assay demonstrated the geographical effect on genetic diversity of pea rhizobia.

Boron dependent membrane glycoproteins in symbiosome development and nodule organogenesis

During the last two decades, we have analyzed the roles of boron (B) in the development of the legume-rhizobia symbiosis and nodule organogenesis. As in other plant tissues, B is needed for the maintenance of nodule cell wall structure. Moreover, several symbiotic events including rhizobial infection, nodule cell invasion and symbiosome development that involve membrane related functions (i.e. vesicle targeting, secretion, or cell surface interactions) are affected by B deficiency. Using anti-rhamnogalacturonan II (anti-RGII) antiserum and immunological techniques, we recently described membrane glycoproteins (RGII-glycoproteins) developmentally regulated in Pisum sativum nodules, which are not detected by the antibody in B-deficient nodules. RGII-glycoproteins appeared related with development processes involving extensive membrane synthesis, like symbiosome maturation or cell growth, both of them negatively affected by B deficiency. Here, we suggest that, besides maintaining cell wall structure, B is both stabilizing components of the membrane glycocalyx and promoting interactions between cell surfaces glycoconjugates that are important during the establishment of the symbiosis and during nodule development. Moreover, we hypothesize that B is playing a similar role during plant or animal embryogenesis and development.

Modeling Embryonic Development in <I>Sitona lineatus</I> (Coleoptera: Curculionidae) in Fluctuating Temperatures

First-instar larvae of Sitona lineatus L. (Coleoptera: Curculionidae) penetrate and feed on the nodules of Pisum sativum, which causes nitrogen fixation to stop. A model, based on experiments conducted at constant temperatures and the Sharpe and DeMichele formula, was derived to predict mean egg hatch times as a function of temperature. Variability was introduced through the use of the Weibull distribution around the mean. The model was validated in artificial conditions under naturally varying air temperatures. The fit between actual and predicted times was good. The data generated herein was compared with that of Andersen's, which was collected 70 yr ago in the area of Munich, Germany. No biologically significant differences between the parameters derived from the two sets of data were identified. The model can be used to accurately predict egg hatch under natural conditions.

Developmental downregulation of rhizobial genes as a function of symbiosome differentiation in symbiotic root nodules of Pisum sativum.

• The expression of nodA and dctA genes of Rhizobium leguminosarum bv. viciae has been studied in mutant nodules of pea (Pisum sativum L.), blocked at the following developmental stages: infection thread development inside the nodule (Itn); infection droplet differentiation (Idd); bacteroid differentiation after endocytosis (Bad); and nodule persistence (Nop). • With the use of reporter fusions to these symbiotic bacterial genes it was shown that both nodA and dctA were expressed at all developmental stages, with a pattern similar to that of constitutive, symbiosis-unrelated genes. • As well as two constitutively expressed genes, both nodA and dctA genes seemed to be subjected to gradual downregulation in nodule bacteria, correlating with the stage of bacteroid differentiation reached. No such effect was observed for the symbiotic, oxygen-regulated fixN gene. The bacteroid development stage also appeared to be related to the ability of bacteria that have been subjected to endocytosis to resume free-living vegetative growth. • The results support the suggestion that bacteroid differentiation into a nitrogen-fixing, organelle-like form, is a gradual process involving several stages, each controlled by different plant genes.

Involvement of Ammonia in Maintenance of Cytosolic Glutamine Synthetase Activity in Pisum sativum Nodules

Involvement of Ammonia in Maintenance of Cytosolic Glutamine Synthetase Activity in Pisum sativum Nodules

Water-deficit effects on carbon and nitrogen metabolism of pea nodules

Two experiments were carried out to investigate the effects of water-deficit stress on carbon and nitrogen metabolism of Pisum sativum nodules. In the first experiment, leaf ψW was allowed to reach −1.0 MPa over a period of 14 d whilst in the second experiment −1.5 MPa was reached during the same time period. Nodule activities of phosphoenol pyruvate carboxylase, glutamine synthetase, alkaline invertase, pyruvate decarboxylase, alcohol dehydrogenase, uridine pyrophosphorylase, and malate dehydrogenase activities were not affected by water-deficit stress. In the first experiment (−1.0 MPa), sucrose synthase (SS), an enzyme which hydrolyses sucrose to support nodule metabolism, declined by 50% in activity and about 25% in content, according to Western immunoblot data. In the second experiment (−1.5 MPa), SS activity decreased by 75% together with glutamate synthase and aspartate aminotransferase which declined by 60% and 40%, respectively. Coincident with the decline of these activities, a dramatic increase in the nodule content of sucrose and a slight increase in the levels of total free amino acids were found. It has been recently suggested that the decline in SS activity and, therefore, a reduced potential to metabolize sucrose may be an important factor contributing to the overall response of soybean nodules to water stress. These results suggest that this observation may be also correct for temperate legumes with indeterminate nodules. However, in this latter case, the activity of some enzymes involved in nitrogen assimilation (glutamate synthase and aspartate aminotransferase) were also affected by water-deficit stress.

Water-deficit effects on carbon and nitrogen metabolism of pea nodules

Two experiments were carried out to investigate the effects of water-deficit stress on carbon and nitrogen metabolism of Pisum sativum nodules. In the first experiment, leaf ψW was allowed to reach −1.0 MPa over a period of 14 d whilst in the second experiment −1.5 MPa was reached during the same time period. Nodule activities of phosphoenol pyruvate carboxylase, glutamine synthetase, alkaline invertase, pyruvate decarboxylase, alcohol dehydrogenase, uridine pyrophosphorylase, and malate dehydrogenase activities were not affected by water-deficit stress. In the first experiment (−1.0 MPa), sucrose synthase (SS), an enzyme which hydrolyses sucrose to support nodule metabolism, declined by 50% in activity and about 25% in content, according to Western immunoblot data. In the second experiment (−1.5 MPa), SS activity decreased by 75% together with glutamate synthase and aspartate aminotransferase which declined by 60% and 40%, respectively. Coincident with the decline of these activities, a dramatic increase in the nodule content of sucrose and a slight increase in the levels of total free amino acids were found. It has been recently suggested that the decline in SS activity and, therefore, a reduced potential to metabolize sucrose may be an important factor contributing to the overall response of soybean nodules to water stress. These results suggest that this observation may be also correct for temperate legumes with indeterminate nodules. However, in this latter case, the activity of some enzymes involved in nitrogen assimilation (glutamate synthase and aspartate aminotransferase) were also affected by water-deficit stress.