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[Physiological and agrochemical properties of different symbiotic genotypes of pea (Pisum sativum L.)].

Physiological characters of symbiotic mutants of pea were studied: nodulation, activities of nitrogenase and nitrate reductase, chlorophyll content in leaves and their water-holding capacity, biomass accumulation, and nitrogen forms. The parameters reflecting the genotype state of the macrosymbiont under soil conditions considerably varied. Supernodulation mutants stood out against symbiotic pea genotypes by high contents of chlorophyll and nonprotein nitrogen compounds, high nitrogenase activity, and low nitrate reductase activity. The efficiency of the legume-rhizobium symbiosis was largely mediated by the macrosymbiont genotype. The highest atmospheric nitrogen fixation (50-80%) was observed in the parental pea varieties. Despite the highest nitrogenase activity in the nodules, the supernodulation mutants were inferior to the parental varieties by the nitrogen fixation capacity (40-60%), which was due to their low productivity.

Genotype↗
PubMed2006

Survival, nodulation and N2 fixation ability of root nodule bacteria under different nutritional regimes.

Eleven strains of Rhizobium and five strains of Bradyrhizobium were examined for their viability as well nodulation and nitrogen fixation ability after storage under different conditions for two years. The storage conditions comprised lateritic soil, lateritic soil plus 1% mannitol, lateritic soil plus 0.1% yeast extract, lateritic soil plus 1% mannitol and 0.1% yeast extract, organic soil, organic soil plus 1% mannitol, organic soil plus 0.1% yeast extract, organic soil plus 1% mannitol and 0.1% yeast extract, and sterile distilled water. All the slow growing strains showed better viability than the fast growing strains in any of these conditions. The survived strains maintained their nodulation ability about 50-60% after one year and 40-50% after two years of preservation as compared to control, but the nodulation ability in sterile distilled water was very poor. Acetylene reduction activity in the nodules was found to be 70-90% and 50-70% after 12 and 24 months of preservation, respectively. The strains retained their phenotypic characters like antibiotic resistance and salt tolerance up to their highest survivability in respective nutritional condition.

Alkynes↗
PubMed2002

A plant receptor-like kinase required for both bacterial and fungal symbiosis.

Most higher plant species can enter a root symbiosis with arbuscular mycorrhizal fungi, in which plant carbon is traded for fungal phosphate. This is an ancient symbiosis, which has been detected in fossils of early land plants. In contrast, the nitrogen-fixing root nodule symbioses of plants with bacteria evolved more recently, and are phylogenetically restricted to the rosid I clade of plants. Both symbioses rely on partially overlapping genetic programmes. We have identified the molecular basis for this convergence by cloning orthologous SYMRK ('symbiosis receptor-like kinase') genes from Lotus and pea, which are required for both fungal and bacterial recognition. SYMRK is predicted to have a signal peptide, an extracellular domain comprising leucine-rich repeats, a transmembrane and an intracellular protein kinase domain. Lotus SYMRK is required for a symbiotic signal transduction pathway leading from the perception of microbial signal molecules to rapid symbiosis-related gene activation. The perception of symbiotic fungi and bacteria is mediated by at least one common signalling component, which could have been recruited during the evolution of root nodule symbioses from the already existing arbuscular mycorrhiza symbiosis.

Amino Acid Sequence↗
PubMed2005

Exopolysaccharide structure is not a determinant of host-plant specificity in nodulation of Vicia sativa roots.

Exopolysaccharide (EPS)-deficient strains of the root nodule symbiote Rhizobium leguminosarum induce formation of abortive infection threads in Vicia sativa subsp. nigra roots. As a result, the nodule tissue remains uninfected. Formation of an infection thread can be restored by coinoculation of the EPS-deficient mutant with a Nod factor-deficient strain, which produces a similar EPS structure. This suggests that EPS contributes to host-plant specificity of nodulation. Here, a comparison was made of i) coinoculation with heterologous strains with different EPS structures, and ii) introduction of the pRL1JI Sym plasmid or a nod gene-encoding fragment in the same heterologous strains. Most strains not complementing in coinoculation experiments were able to nodulate V. sativa roots as transconjugants. Apparently, coinoculation is a delicate approach in which differences in root colonization ability or bacterial growth rate easily affect successful infection-thread formation. Obviously, lack of infection-thread formation in coinoculation studies is not solely determined by EPS structure. Transconjugation data show that different EPS structures can allow infection-thread formation and subsequent nodulation of V. sativa roots.

Carbohydrate Sequence↗
PubMed1993

Regulation and function of rhizobial nodulation genes.

This review focuses on the functions of nodulation (nod) genes in the interaction between rhizobia and legumes. The nod genes are the key bacterial determinants of the signal exchange between the two symbiotic partners. The product of the nodD gene is a transcriptional activator protein that functions as receptor for a flavonoid plant compound. This signaling induces the expression of a set of nod genes that produces several related Nod factors, substituted lipooligosaccharides. The Nod factors are then excreted and serve as signals sent from the bacterium to the plant. The plant responds with the development of a root nodule. The plant-derived flavonoid, as well as the rhizobial signal, must have distinct chemical structures which guarantee that only matching partners are brought together.

Amino Acid Sequence↗
PubMed2005

Lotus japonicus metabolic profiling. Development of gas chromatography-mass spectrometry resources for the study of plant-microbe interactions.

Symbiotic nitrogen fixation (SNF) in legume root nodules requires differentiation and integration of both plant and bacterial metabolism. Classical approaches of biochemistry, molecular biology, and genetics have revealed many aspects of primary metabolism in legume nodules that underpin SNF. Functional genomics approaches, especially transcriptomics and proteomics, are beginning to provide a more holistic picture of the metabolic potential of nodules in model legumes like Medicago truncatula and Lotus japonicus. To extend these approaches, we have established protocols for nonbiased measurement and analysis of hundreds of metabolites from L. japonicus, using gas chromatography coupled with mass spectrometry. Following creation of mass spectral tag libraries, which represent both known and unknown metabolites, we measured and compared relative metabolite levels in nodules, roots, leaves, and flowers of symbiotic plants. Principal component analysis of the data revealed distinct metabolic phenotypes for the different organs and led to the identification of marker metabolites for each. Metabolites that were enriched in nodules included: octadecanoic acid, asparagine, glutamate, homoserine, cysteine, putrescine, mannitol, threonic acid, gluconic acid, glyceric acid-3-P, and glycerol-3-P. Hierarchical cluster analysis enabled discrimination of 10 groups of metabolites, based on distribution patterns in diverse Lotus organs. The resources and tools described here, together with ongoing efforts in the areas of genome sequencing, and transcriptome and proteome analysis of L. japonicus and Mesorhizobium loti, should lead to a better understanding of nodule metabolism that underpins SNF.

Gas Chromatography-Mass Spectrometry↗
PubMed2003

Overexpression of alfalfa cytosolic glutamine synthetase in nodules and flowers of transgenic Lotus japonicus plants.

Legumes can obtain nitrogen from symbiotic nitrogen fixation in root nodules. The glutamine synthetase/glutamate synthase cycle is responsible for the initial nitrogen assimilation. This work reports the analysis of transgenic Lotus japonicus plants with the chimeric gene containing the alfalfa cytosolic glutamine synthetase (GS1) (EC 6.3.1.2) gene controlled by the Sesbania rostrata leghemoglobin gene promoter (Srglb3p). Surprisingly, all of the transgenic primary transformants analysed were sterile. Two transformants designated GS39 and GS44 were further analysed. GS in nodules of GS39 and GS44 plants was upregulated, at the level of transcript and protein. The transgenic plants had 2-fold higher nodule GS activity and similar root GS activity compared to control plants. The GS39 and GS44 sterile plants showed morphological alterations in pollen grains and in ovules. An increase in GS transcript abundance and enzyme activity was measured during early and late stages of flower development of GS plants. Flowers of GS plants showed higher glutamine content, resulting in an increased glutamine/glutamate ratio. The GS transcript and protein were detected in ovules. These data indicate that overexpression of GS1 in reproductive organs critically affects their development and might be a reason for sterility of L. japonicus plants.

Journal Article↗
PubMed1991

Glutamate Oxaloacetate Transaminase in Pea Root Nodules : Participation in a Malate/Aspartate Shuttle between Plant and Bacteroid.

Glutamate oxaloacetate transaminase (l-glutamate: oxaloacetate aminotransferase, EC 2.6.1.1 [GOT]), a key enzyme in the flow of carbon between the organic acid and amino acid pools in pea (Pisum sativum L.) root nodules, was studied. By ion exchange chromatography, the presence of two forms of GOT in the cytoplasm of pea root nodule cells was established. The major root nodule form was present in only a small quantity in the cytoplasm of root cells. Fractionation of root nodule cell extracts demonstrated that the increase in the GOT activity during nodule development was due to the increase of the activity in the cytoplasm of the plant cells, and not to an increase in activity in the plastids or in the mitochondria. The kinetic properties of the different cytoplasmic forms of GOT were studied. Some of the K(m) values differed, but calculations indicated that not the kinetic properties but a high concentration of the major root nodule form caused the observed increase in GOT activity in the pea root nodules. It was found that the reactions of the malate/aspartate shuttle are catalyzed by intact bacteroids, and that these reactions can support nitrogen fixation. It is proposed that the main function of the nodule-stimulated cytoplasmic form of GOT is participation in this shuttle.

Journal Article↗
PubMed1964

ROOT NODULE SYMBIOSIS. I. ENDOPHYTE OF MYRICA CERIFERA L.

Silver, Warren S. (University of Florida, Gainesville). Root nodule symbiosis. I. Endophyte of Myrica cerifera. J. Bacteriol. 87:416-421. 1964.-Electron microscopy of 0.1-mu thick sections of root nodules, fixed with permanganate and embedded with methacrylate, showed that infected plant cells were filled with a mycelial endophyte. The endophyte was filamentous, 1 mu in diameter, septate, and had an enlarged, club-shaped terminus. Although structurally the endophyte strongly resembles an actinomycete, it was not isolated in pure culture on a variety of appropriate media.

Actinomycetales↗
PubMed1994

The molecular basis of the host specificity of the Rhizobium bacteria.

The interaction between soil bacteria belonging to the genera Rhizobium, Bradyrhizobium and Azorhizobium and leguminous plants results in the induction of a new plant organ, the root nodule. After invading these root nodules via infection threads the bacteria start to fix atmospheric nitrogen into ammonia which is beneficial for the host plant. This symbiotic interaction is highly host-specific in that each rhizobial strain is able to associate with only a limited number of host plant species. The subject of this presentation is the molecular mechanism by which the bacterium determines its host-specific characteristics. This mechanism appears to be based on at least two stages of molecular signaling between the bacterium and the plant host. In the first stage, flavonoids secreted by the plant root induce, in a host specific way, the transcription of bacterial genes which are involved in nodulation, the so-called nod genes. This leads to the second step of the signaling system: the production and secretion of lipo-oligosaccharide molecules by the Rhizobium bacteria. These signal molecules, which are acylated forms of small fragments of chitin, have various discernable effects on the roots of the host plants. One of these effects is the dedifferentiation of groups of cells located in the cortex which leads to the formation of nodule meristems. In their mitogenic activity the bacterial signals resemble several well-known plant hormones like auxins and cytokinins. However, there are two major differences: (i) the bacterial signals lead to the induction of a specific organ and (ii) they are host-specific in that only the signals produced by compatible bacteria are able to induce meristems. The nod genes determine this stage of host specificity by their essential role in the biosynthesis of the signal molecules. They appear to encode enzymes which are involved in the processes of fatty acid biosynthesis, fatty acid transfer, chitin synthesis and chitin modification. I will illustrate the statement that the nod gene products are ideal model enzymes for the study of these important processes because they are not needed in the free-living state of the bacteria.

Base Sequence↗
PubMed1998

Ethylene-mediated phenotypic plasticity in root nodule development on Sesbania rostrata.

Leguminous plants in symbiosis with rhizobia form either indeterminate nodules with a persistent meristem or determinate nodules with a transient meristematic region. Sesbania rostrata was thought to possess determinate stem and root nodules. However, the nature of nodule development is hybrid, and the early stages resemble those of indeterminate nodules. Here we show that, depending on the environmental conditions, mature root nodules can be of the indeterminate type. In situ hybridizations with molecular markers for plant cell division, as well as the patterns of bacterial nod and nif gene expression, confirmed the indeterminate nature of 30-day-old functional root nodules. Experimental data provide evidence that the switch in nodule type is mediated by the plant hormone ethylene.

Journal Article↗
PubMed2006

Recruitment of novel calcium-binding proteins for root nodule symbiosis in Medicago truncatula.

Legume rhizobia symbiotic nitrogen (N2) fixation plays a critical role in sustainable nitrogen management in agriculture and in the Earth's nitrogen cycle. Signaling between rhizobia and legumes initiates development of a unique plant organ, the root nodule, where bacteria undergo endocytosis and become surrounded by a plant membrane to form a symbiosome. Between this membrane and the encased bacteria exists a matrix-filled space (the symbiosome space) that is thought to contain a mixture of plant- and bacteria-derived proteins. Maintenance of the symbiosis state requires continuous communication between the plant and bacterial partners. Here, we show in the model legume Medicago truncatula that a novel family of six calmodulin-like proteins (CaMLs), expressed specifically in root nodules, are localized within the symbiosome space. All six nodule-specific CaML genes are clustered in the M. truncatula genome, along with two other nodule-specific genes, nodulin-22 and nodulin-25. Sequence comparisons and phylogenetic analysis suggest that an unequal recombination event occurred between nodulin-25 and a nearby calmodulin, which gave rise to the first CaML, and the gene family evolved by tandem duplication and divergence. The data provide striking evidence for the recruitment of a ubiquitous Ca(2+)-binding gene for symbiotic purposes.

Base Sequence↗
PubMed1982

Effect of Atmospheric CO(2) Enrichment on Growth, Nonstructural Carbohydrate Content, and Root Nodule Activity in Soybean.

The objective of this study was to determine whether the supply of current photosynthate was limiting root nodule activity. Both short-term (36 hours) and long-term (16 days) periods of CO(2) enrichment were imposed on vegetative, growth chamber-grown soybean plants (Glycine max. [L.] Merr. cv. ;Clay') to increase the supply of current photosynthate and to observe the effects on photosynthate partitioning in the plants, plant growth, and root nodule activity.Neither total nor specific nodule activities were increased during exposure to short-term (36 hours) CO(2) enrichment. Dry weight of the leaves increased after 12, 24, and 36 hours of CO(2) enrichment and dry weight of the stems plus petioles increased after 36 hours of CO(2) enrichment. Dry weights of the roots and nodules were not altered by short-term CO(2) enrichment. Short-term CO(2) enrichment increased the total nonstructural carbohydrates in the leaves and stems plus petioles, but not in the roots and nodules. Analyses of the separate pools of carbohydrate reserves indicated that the majority of the additional carbohydrate provided by short-term CO(2) enrichment was stored as leaf starch with relatively little being partitioned to the roots and nodules.Long-term CO(2) enrichment (16 days) did not enhance specific nodule activity. Shoot, root, and nodule dry weights were increased 109, 34%, and 56% respectively. Total nodule activity per plant was significantly enhanced only after 16 days of treatment and was related to increased nodule mass. These results indicate that the increased total nodule activity in response to CO(2) enrichment is a consequence of a general growth response of the plant.Results of both studies indicate that nodule activity was not directly limited by current photosynthesis but rather by the partitioning and utilization of photosynthate in the plant.

Journal Article↗
PubMed2005

Stress-induced changes in protease composition are determined by nitrogen supply in non-nodulating white clover.

An inbreeding line of white clover has been identified which remains non-nodulated under appropriate physiological conditions and so the nitrogen concentration of the plant can be manipulated by altering the nitrate supply to the roots. Non-nodulating plants were used to test the hypothesis that acclimation to nitrogen limitation in white clover involves changes in protease activity and composition. These results indicate that acclimation to nitrogen limitation involves the realignment of constituent proteases without necessarily incurring significant changes in total protease activity. Plants grown at 2.5, 5.0, 7.5, and 10 mM nitrate showed a positive correlation between nitrate supply and foliar protein concentration. Protein profiles, revealed by Coomassie-stained SDS-PAGE, were unchanged between treatments for a given amount of protein. Serine, aspartate/metalloprotease, and two cysteine proteases were identified in the leaves. Although total protease activity per gram fresh weight was unchanged between treatments, the relative contributions of these four proteases was determined by nitrate supply. When plants were stressed further by withholding nitrate there was an increase in cysteine protease activity, but a senescence-related aspartate/metalloprotease was not visible. Hence, while protease expression in white clover leaves responded to the current and past nitrogen status of the plant, the proteases involved in remobilization during nutrient limitation were distinct from those involved during the main senescence period. It is suggested that nitrogen limitation induced an early, reversible stage of senescence in which perturbations in protease activity facilitated the degradation of non-essential proteins in order to increase the chances of plant survival or seed set.

Chlorophyll↗