Alfalfa Root Nodules Research Articles

Sinorhizobium meliloti lsrB is involved in alfalfa root nodule development and nitrogen-fixing bacteroid differentiation

Rhizobia interact with host legumes to induce the formation of nitrogen-fixing nodules, which is very important in agriculture and ecology. The development of nitrogen-fixing nodules is stringently regulated by host plants and rhizobial symbionts. In our previous work, a new Sinorhizobium meliloti LysR regulator gene (lsrB) was identified to be essential for alfalfa nodulation. However, how this gene is involved in alfalfa nodulation was not yet understood. Here, we found that this gene was associated with prevention of premature nodule senescence and abortive bacteroid formation. Heterogeneous deficient alfalfa root nodules were induced by the in-frame deletion mutant of lsrB (lsrB1-2), which was similar to the plasmid-insertion mutant, lsrB1. Irregular senescence zones earlier appeared in these nodules where bacteroid differentiation was blocked at different stages from microscopy observations. Interestingly, oxidative bursts were observed in these nodules by DAB staining. The decreased expression of lipopolysaccharide core genes (lpsCDE) was correspondingly determined in these nodules. S. meliloti lipopolysaccharide is required for suppression of oxidative bursts or host cell defense. These findings demonstrate that the S. meliloti lsrB gene is involved in alfalfa root nodule development and bacteroid differentiation by suppressing oxidative bursts or defense responses in host cells.

Salicylic acid improves the salinity tolerance of Medicago sativa in symbiosis with Sinorhizobium meliloti by preventing nitrogen fixation inhibition

In this work we have investigated the contribution of pretreatment with 0.1 and 0.5mM salicylic acid (SA) to the protection against salt stress in root nodules of Medicago sativa in symbiosis with Sinorhizobium meliloti. SA alleviated the inhibition induced by salinity in the plant growth and photosynthetic capacity of M. sativa–S. meliloti symbiosis. In addition, SA prevented the inhibition of the nitrogen fixation capacity under salt stress since nodule biomass was not affected by salinity in SA pretreated plants. Antioxidant enzymes peroxidase (POX), superoxide dismutase (SOD), ascorbate peroxidase (APX), dehidroascorbate reductase (DHAR) and glutathione reductase (GR), key in the main pathway that scavenges H2O2 in plants, were induced by SA pretreatments which suggest that SA may participate in the redox balance in root nodules under salt stress. Catalase activity (CAT) was inhibited around 40% by SA which could be behind the increase of H2O2 detected in nodules of plants pretreated with SA. The accumulation of polyamines (PAs) synthesized in response to salinity was prevented by SA which together with the induction of 1-aminocyclopropane-l-carboxylic acid (ACC) content suggest the prevalence of the ethylene signaling pathway induced by SA in detriment of the synthesis of PAs. In conclusion, SA alleviated the negative effect of salt stress in the M. sativa–S. meliloti symbiosis through the increased level of nodule biomass and the induction of the nodular antioxidant metabolism under salt stress. The H2O2 accumulation and the PAs inhibition induced by SA in nodules of M. sativa suggest that SA activates a hypersensitive response dependent on ethylene.

Deciphering the Role of Sucrose Phosphate Synthase in Root Nodules of Medicago sativa (alfalfa)

Leguminous plants, like alfalfa, can form a symbiotic relationship with a N fixing bacteria that can result in the formation root nodules. They have the ability to sustain the N fixing bacteria by providing optimal conditions for the bacterially encoded enzyme, nitrogenase, to reduce N to ammonia. The ammonia is then assimilated and exported to the rest of the plant. The plant provides the bacteria in the nodules with a source of C and energy in the form of sucrose in exchange for the fixed N. Sucrose Phosphate Synthase (SPS) catalyzes the penultimate step in sucrose biosynthesis and plays a key regulatory role in C partitioning in the pathway. Preliminary work form our lab has shown one isoform to have high SPS activity in the nodules, SPSA. To address the issue of what might be role of SPSA gene product in the nodules, we are using both immunolocalization and site of promoter function to localize the site of expression of SPSA in the nodules in context of N fixation, ammonia assimilation, and C metabolism. We are also using a genetic engineering approach to modulate the expression of SPS genes in a cell specific manner to monitor any changes in the function of the nodules.

Genetic diversity of rhizobia associated with alfalfa in Serbian soils

We have evaluated the genetic diversity and phylogeny of alfalfa rhizobia, originating from different types of soils in Serbia and their ability to establish an effective symbiosis with alfalfa (Medicago sativa L.). A collection of 65 strains isolated from root nodules of alfalfa were characterized by rep-PCR analysis, partial and complete 16S rDNA gene and recA gene sequencing, as well as atpD gene sequencing and DNA-DNA hybridizations. The results of the sequence analyses revealed that Sinorhizobium meliloti is the dominant species in alfalfa nodules. Only one strain was identified as Sinorhizobium medicae, two strains as Rhizobium tibeticum and one strain as Rhizobium sp. Despite the fact that the majority of strains were identified as S. meliloti, a high genetic diversity at strain level was detected. Almost all isolates shared the ability to nodulate and fix nitrogen with M. sativa, except 11 of them, which were incapable of fixing nitrogen with this species. About 50% of the isolates showed values of symbiotic effectiveness (SE) above 50%, while 10% of the strains were highly effective with SE values above 70%. Some of the strains which were highly effective in nitrogen fixation at the same time could intensively solubilize phosphates, offering a possibility for multipurpose inoculum development. This was the first genetic study of rhizobia isolated from this region and also the first report of natural presence of R. tibeticum in root nodules of M. sativa.

Phenotypic and genetic characterization of rhizobia associated with alfalfa in the Hokkaido and Ishigaki regions of Japan

Twenty five rhizobial isolates were obtained from root nodules of Medicago sativa inoculated with soil samples collected from the Sapporo region and Ishigaki Island in Japan. To study their diversity and characterize them in relation to the climatic conditions of their soils of origin, a polyphasic approach analyzing stress tolerance, symbiotic and genetic properties was used. Stress tolerance assays revealed marked variations in salinity, pH and temperature tolerance. Isolates originating from a sub-tropical climate in alkaline soil (Ishigaki Island) tolerated high temperature, salinity and pH levels. Moreover, isolates recovered from a temperate climate in acidic soil (Sapporo) were sensitive to high temperature and salinity, and tolerated acidic pH. Phylogenetic analysis of conserved 16S rRNA and recA genes, and symbiotic nodA and nifDK revealed 25 isolates to be closely related to Ensifer meliloti. Furthermore, the branch patterns of phylogenetic trees constructed from different genes revealed the existence of at least two E. meliloti types in the soils studied. These results may be relevant to programs directed towards improving crop productivity through biofertilization with locally adapted and genetically defined strains.

Phenotypic and genetic diversity in Sinorhizobium meliloti and S. medicae from drought and salt affected regions of Morocco

BackgroundSinorhizobium meliloti and S. medicae are symbiotic nitrogen fixing bacteria in root nodules of forage legume alfalfa (Medicago sativa L.). In Morocco, alfalfa is usually grown in marginal soils of arid and semi-arid regions frequently affected by drought, extremes of temperature and soil pH, soil salinity and heavy metals, which affect biological nitrogen fixing ability of rhizobia and productivity of the host. This study examines phenotypic diversity for tolerance to the above stresses and genotypic diversity at Repetitive Extragenic Pallindromic DNA regions of Sinorhizobium nodulating alfalfa, sampled from marginal soils of arid and semi-arid regions of Morocco.ResultsRsaI digestion of PCR amplified 16S rDNA of the 157 sampled isolates, assigned 136 isolates as S. meliloti and the rest as S. medicae. Further phenotyping of these alfalfa rhizobia for tolerance to the environmental stresses revealed a large degree of variation: 55.41%, 82.16%, 57.96% and 3.18% of the total isolates were tolerant to NaCl (>513 mM), water stress (-1.5 MPa), high temperature (40°C) and low pH (3.5), respectively. Sixty-seven isolates of S. meliloti and thirteen isolates of S. medicae that were tolerant to salinity were also tolerant to water stress. Most of the isolates of the two species showed tolerance to heavy metals (Cd, Mn and Zn) and antibiotics (chloramphenicol, spectinomycin, streptomycin and tetracycline). The phenotypic clusters observed by the cluster analysis clearly showed adaptations of the S. meliloti and S. medicae strains to the multiple stresses. Genotyping with rep-PCR revealed higher genetic diversity within these phenotypic clusters and classified all the 157 isolates into 148 genotypes. No relationship between genotypic profiles and the phenotypes was observed. The Analysis of Molecular Variance revealed that largest proportion of significant (P < 0.01) genetic variation was distributed within regions (89%) than among regions (11%).ConclusionHigh degree of phenotypic and genotypic diversity is present in S. meliloti and S. medicae populations from marginal soils affected by salt and drought, in arid and semi-arid regions of Morocco. Some of the tolerant strains have a potential for exploitation in salt and drought affected areas for biological nitrogen fixation in alfalfa.

Identification of a Hydroxyproline Transport System in the Legume Endosymbiont Sinorhizobium meliloti

Hydroxyproline-rich proteins in plants offer a source of carbon and nitrogen to soil-dwelling microorganisms in the form of root exudates and decaying organic matter. This report describes an ABC-type transport system dedicated to the uptake of hydroxyproline in the legume endosymbiont Sinorhizobium meliloti. We have designated genes involved in hydroxyproline metabolism as hyp genes and show that an S. meliloti strain lacking putative transport genes (DeltahypMNPQ) is unable to grow with or transport trans-4-hydroxy-l-proline when this compound is available as a sole source of carbon. Expression of hypM is upregulated in the presence of trans-4-hydroxy-l-proline and cis-4-hydroxy-d-proline, as modulated by a repressor (HypR) of the GntR/FadR subfamily. Although alfalfa root nodules contain hydroxyproline-rich proteins, we demonstrate that the transport system is not highly expressed in nodules, suggesting that bacteroids are not exposed to high levels of free hydroxyproline in planta. In addition to hypMNPQ, we report that S. meliloti encodes a second independent mechanism that enables transport of trans-4-hydroxy-l-proline. This secondary transport mechanism is induced in proline-grown cells and likely entails a system involved in l-proline uptake. This study represents the first genetic description of a prokaryotic hydroxyproline transport system, and the ability to metabolize hydroxyproline may contribute significantly toward the ecological success of plant-associated bacteria such as the rhizobia.

Flavodoxin overexpression reduces cadmium-induced damage in alfalfa root nodules

Flavodoxins are electron carrier flavoproteins that are involved in the response to oxidative stress in bacteria and cyanobacteria. Recently, we obtained Sinorhizobium meliloti bacteria that overexpressed a flavodoxin from the cyanobacterium Anabaena variabilis [Redondo et al. (2009) Plant Physiology 149:1166–1178]. In the present work, tolerance to cadmium was evaluated in free-living transformed S. meliloti and in alfalfa plants nodulated by the flavodoxin-overexpressing rhizobia, in comparison with plants nodulated by wild-type bacteria. Overexpression of flavodoxin protected free-living S. meliloti from cadmium toxicity and had a positive effect on nitrogen fixation of alfalfa plants subjected to cadmium stress. Flavodoxin notably reduced cadmium-induced structural and ultrastructural alterations in alfalfa nodules. Putative protection mechanisms in flavodoxin-overexpressing nodules are discussed. Flavodoxin could have applications as a biotechnological tool to improve the symbiotic performance of alfalfa and other legumes in cadmium polluted soils.

Genotypic characterization of indigenous Sinorhizobium meliloti and Rhizobium sullae by rep- PCR, RAPD and ARDRA analyses

The rhizobia, Sinorhizobium meliloti and Rhizobium sullae, which fix nitrogen in root nodules of alfalfa (Medicago sativa L.) and sulla (Hedysarum sp.) forage legumes, respectively, were isolated from root nodules and soils from Morocco. We used three PCR-based techniques namely, rep-PCR, RAPD and ARDRA techniques for genotypic characterization of 10 isolates of S. meliloti and R. sullae, in order to identify rapid and reliable techniques for applications in population genetic analysis of these species. The analysis revealed characteristic banding patterns for S. meliloti and R. sullae isolates by all the three techniques, even though the isolates are from a narrow geographic region in Morocco. Furthermore, the results showed that the rep-PCR with REP and ERIC primers was more efficient than RAPD and ARDRA technique for genotyping S. meliloti isolates; and rep-PCR with REP primers and the ARDRA technique with restriction enzyme HinfI, were more efficient than the other rep-PCR and RAPD-PCR techniques for genotyping R. sullae isolates.

Genotypic characterization of indigenous Sinorhizobium meliloti and Rhizobium sullae by rep- PCR, RAPD and ARDRA analyses

The rhizobia, Sinorhizobium meliloti and Rhizobium sullae, which fix nitrogen in root nodules of alfalfa (Medicago sativa L.) and sulla (Hedysarum sp.) forage legumes, respectively, were isolated from root nodules and soils from Morocco. We used three PCR-based techniques namely, rep-PCR, RAPD and ARDRA techniques for genotypic characterization of 10 isolates of S. meliloti and R. sullae, in order to identify rapid and reliable techniques for applications in population genetic analysis of these species. The analysis revealed characteristic banding patterns for S. meliloti and R. sullae isolates by all the three techniques, even though the isolates are from a narrow geographic region in Morocco. Furthermore, the results showed that the rep-PCR with REP and ERIC primers was more efficient than RAPD and ARDRA technique for genotyping S. meliloti isolates; and rep-PCR with REP primers and the ARDRA technique with restriction enzyme HinfI, were more efficient than the other rep-PCR and RAPD-PCR techniques for genotyping R. sullae isolates.

Overexpression of flavodoxin in bacteroids induces changes in antioxidant metabolism leading to delayed senescence and starch accumulation in alfalfa root nodules.

Sinorhizobium meliloti cells were engineered to overexpress Anabaena variabilis flavodoxin, a protein that is involved in the response to oxidative stress. Nodule natural senescence was characterized in alfalfa (Medicago sativa) plants nodulated by the flavodoxin-overexpressing rhizobia or the corresponding control bacteria. The decline of nitrogenase activity and the nodule structural and ultrastructural alterations that are associated with nodule senescence were significantly delayed in flavodoxin-expressing nodules. Substantial changes in nodule antioxidant metabolism, involving antioxidant enzymes and ascorbate-glutathione cycle enzymes and metabolites, were detected in flavodoxin-containing nodules. Lipid peroxidation was also significantly lower in flavodoxin-expressing nodules than in control nodules. The observed amelioration of the oxidative balance suggests that the delay in nodule senescence was most likely due to a role of the protein in reactive oxygen species detoxification. Flavodoxin overexpression also led to high starch accumulation in nodules, without reduction of the nitrogen-fixing activity.

Mutational Analysis of the Sinorhizobium meliloti Short-Chain Dehydrogenase/Reductase Family Reveals Substantial Contribution to Symbiosis and Catabolic Diversity

The short-chain dehydrogenase/reductase (SDR) family is one of the largest and most ubiquitous protein families in bacterial genomes. Despite there being a few well-characterized examples, the substrate specificities or functions of most members of the family are unknown. In this study, we carried out a large-scale mutagenesis of the SDR gene family in the alfalfa root nodule symbiont Sinorhizobium meliloti. Subsequent phenotypic analysis revealed phenotypes for mutants of 21 of the SDR-encoding genes. This brings the total number of S. meliloti SDR-encoding genes with known function or associated phenotype to 25. Several of the mutants were deficient in the utilization of specific carbon sources, while others exhibited symbiotic deficiencies on alfalfa (Medicago sativa), ranging from partial ineffectiveness to complete inability to form root nodules. Five of the mutants had both symbiotic and carbon utilization phenotypes. These results clearly demonstrate the importance of the SDR family in both symbiosis and saprotrophy, and reinforce the complex nature of the interaction of S. meliloti with its plant hosts. Further analysis of the genes identified in this study will contribute to the overall understanding of the biology and metabolism of S. meliloti in relation to its interaction with alfalfa.

Low levels of ferredoxin, ATP and leghemoglobin contribute to limited N2 fixation of peas (Pisum sativum L.) and alfalfa (Medicago sativa L.) under S deficiency conditions

Sulphur (S) has become a major limiting factor for plant production in industrial as well as in remote industrial rural areas. Limitation of S can reduce legume N2 fixation by affecting nodule development and function. In pot experiments with pea (Pisum sativum L.) and alfalfa (Medicago sativa L.), we investigated the influence of S on growth, ferredoxin, ATP and leghemoglobin concentrations. Addition of 200 mg S pot−1 increased yield of shoots, roots and nodules of both plant species significantly. However, the influence of S on nodule yield formation was most pronounced. Pea and alfalfa roots were found to have higher S concentrations than shoots and being up to 2.9 times the S concentration in the shoots of peas under S-sufficient conditions. Sulphur addition also increased N2 fixation significantly. The ferredoxin concentration in bacteroids of root nodules of pea was increased significantly by S only 10 weeks after planting and in bacteroids of root nodules of alfalfa 10 and 17 weeks after planting, while on per pot base the amounts of ferredoxin were higher throughout the experimental period of time. The ATP concentration of bacteroids of root nodules of both plant species as well as of mitochondria of root nodules of pea were significantly higher with optimum S supply. The effects of S deficiency on N2 fixation are likely to be caused by the shortage of ferredoxin and ATP. The amount of leghemoglobin was reduced in comparison to nodules of the S-sufficient plants.

A salt stress-responsive cytokinin receptor homologue isolated from Medicago sativa nodules

A new cytokinin receptor homologue, MsHK1, was isolated from Medicago sativa root nodules. MsHK1 expression was induced in alfalfa seedlings by exogenous application of the cytokinin trans-zeatin. Transcript accumulation was detected in different plant organs. MsHK1 expression was induced by salt stress in alfalfa roots, leaves and nodules, and transcript accumulation in the vascular bundles pointed to a putative role in osmosensing for MsHK1 and/or other close cytokinin receptor homologues. Expression in the meristem and the invasion zone of the nodule suggest a role for cytokinin receptors in cytokinin sensing during nodule cell division and differentiation.

A Sinorhizobium meliloti minE mutant has an altered morphology and exhibits defects in legume symbiosis

Sinorhizobium meliloti differentiates from rod-shaped, free-living cells into pleomorphic, non-dividing, N(2)-fixing bacteroids within alfalfa root nodules. Here, the role of the minCDE genes in bacteroid differentiation and in free-living cell division is examined. Disruption of the minE gene resulted in large, swollen and branched free-living cells, and in symbiosis a minE mutation resulted in a defect in nitrogen fixation with activity reduced by approximately 70 % compared to the wild-type. It has been demonstrated that the minCDE genes form an operon driven by a promoter located 173 bp upstream of minC. The minCDE genes were expressed in free-living cells and in both the infection zone and the symbiotic zone of alfalfa nodules; however, no changes in the free-living cell morphology, growth or symbiotic N(2) fixation were detected as a result of deletion of these genes. Induced production of individual or combinations of Min proteins in S. meliloti altered its rod-shaped cell morphology. Moreover, cell morphologies resulting from the overexpression of the S. meliloti Min proteins in Escherichia coli suggested similar functions for the E. coli and S. meliloti min genes. These data suggest that there is greater redundancy in the roles of cell division genes in S. meliloti compared with E. coli.

Malic Enzyme Cofactor and Domain Requirements for Symbiotic N 2 Fixation by Sinorhizobium meliloti

The NAD(+)-dependent malic enzyme (DME) and the NADP(+)-dependent malic enzyme (TME) of Sinorhizobium meliloti are representatives of a distinct class of malic enzymes that contain a 440-amino-acid N-terminal region homologous to other malic enzymes and a 330-amino-acid C-terminal region with similarity to phosphotransacetylase enzymes (PTA). We have shown previously that dme mutants of S. meliloti fail to fix N(2) (Fix(-)) in alfalfa root nodules, whereas tme mutants are unimpaired in their N(2)-fixing ability (Fix(+)). Here we report that the amount of DME protein in bacteroids is 10 times greater than that of TME. We therefore investigated whether increased TME activity in nodules would allow TME to function in place of DME. The tme gene was placed under the control of the dme promoter, and despite elevated levels of TME within bacteroids, no symbiotic nitrogen fixation occurred in dme mutant strains. Conversely, expression of dme from the tme promoter resulted in a large reduction in DME activity and symbiotic N(2) fixation. Hence, TME cannot replace the symbiotic requirement for DME. In further experiments we investigated the DME PTA-like domain and showed that it is not required for N(2) fixation. Thus, expression of a DME C-terminal deletion derivative or the Escherichia coli NAD(+)-dependent malic enzyme (sfcA), both of which lack the PTA-like region, restored wild-type N(2) fixation to a dme mutant. Our results have defined the symbiotic requirements for malic enzyme and raise the possibility that a constant high ratio of NADPH + H(+) to NADP in nitrogen-fixing bacteroids prevents TME from functioning in N(2)-fixing bacteroids.

Metabolite Profiles of Nodulated Alfalfa Plants Indicate That Distinct Stages of Nodule Organogenesis Are Accompanied by Global Physiological Adaptations

An effective symbiosis between Sinorhizobium meliloti and its host plant Medicago sativa is dependent on a balanced physiological interaction enabling the microsymbiont to fix atmospheric nitrogen. Maintenance of the symbiotic interaction is regulated by still poorly understood control mechanisms. A first step toward a better understanding of nodule metabolism was the determination of characteristic metabolites for alfalfa root nodules. Furthermore, nodules arrested at different developmental stages were analyzed in order to address metabolic changes induced during the progression of nodule formation. Metabolite profiles of bacteroid-free pseudonodule extracts indicated that early nodule developmental processes are accompanied by photosynthate translocation but no massive organic acid formation. To determine metabolic adaptations induced by the presence of nonfixing bacteroids, nodules induced by mutant S. meliloti strains lacking the nitrogenase protein were analyzed. The bacteroids are unable to provide ammonium to the host plant, which is metabolically reflected by reduced levels of characteristic amino acids involved in ammonium fixation. Elevated levels of starch and sugars in Fix(-) nodules provide strong evidence that plant sanctions preventing a transformation from a symbiotic to a potentially parasitic interaction are not strictly realized via photosynthate supply. Instead, metabolic and gene expression data indicate that alfalfa plants react to nitrogen-fixation-deficient bacteroids with a decreased organic acid synthesis and an early induction of senescence. Noneffective symbiotic interactions resulting from plants nodulated by mutant rhizobia also are reflected in characteristic metabolic changes in leaves. These are typical for nitrogen deficiency, but also highlight metabolites potentially involved in sensing the N status.

The Sinorhizobium meliloti chromosomal origin of replication

The predicted chromosomal origin of replication (oriC) from the alfalfa symbiont Sinorhizobium meliloti is shown to allow autonomous replication of a normally non-replicating plasmid within S. meliloti cells. This is the first chromosomal replication origin to be experimentally localized in the Rhizobiaceae and its location, adjacent to hemE, is the same as for oriC in Caulobacter crescentus, the only experimentally characterized alphaproteobacterial oriC. Using an electrophoretic mobility shift assay and purified S. meliloti DnaA replication initiation protein, binding sites for DnaA were mapped in the S. meliloti oriC region. Mutations in these sites eliminated autonomous replication. S. meliloti that expressed DnaA from a plasmid lac promoter was observed to form pleomorphic filamentous cells, suggesting that cell division was perturbed. Interestingly, this cell phenotype is reminiscent of differentiated bacteroids found inside plant cells in alfalfa root nodules.

Regulation and Properties of PstSCAB, a High-Affinity, High-Velocity Phosphate Transport System ofSinorhizobium meliloti

The properties and regulation of the pstSCAB-encoded Pi uptake system from the alfalfa symbiont Sinorhizobium meliloti are reported. We present evidence that the pstSCAB genes and the regulatory phoUB genes are transcribed from a single promoter that contains two PhoB binding sites and that transcription requires PhoB. S. meliloti strain 1021 (Rm1021) and its derivatives were found to carry a C deletion frameshift mutation in the pstC gene (designated pstC1021) that severely impairs activity of the PstSCAB Pi transport system. This mutation is absent in RCR2011, the parent of Rm1021. Correction of the pstC1021 mutation in Rm1021 by site-directed mutagenesis revealed that PstSCAB is a Pi-specific, high-affinity (Km, 0.2 microM), high-velocity (Vmax, 70 nmol/min/mg protein) transport system. The pstC1021 allele was shown to generate a partial pho regulon constitutive phenotype, in which transcription is activated by PhoB even under Pi-excess conditions that render PhoB inactive in a wild-type background. The previously reported symbiotic Fix- phenotype of phoCDET mutants was found to be dependent on the pstC1021 mutation, as Rm1021 phoCDET mutants formed small white nodules on alfalfa that failed to reduce N2, whereas phoCDET mutant strains with a corrected pstC allele (RmP110) formed pink nodules on alfalfa that fixed N2 like the wild type. Alfalfa root nodules formed by the wild-type RCR2011 strain expressed the low-affinity orfA-pit-encoded Pi uptake system and neither the pstSCAB genes nor the phoCDET genes. Thus, metabolism of alfalfa nodule bacteroids is not Pi limited.

A Rab-related small GTP binding protein is predominantly expressed in root nodules of Medicago sativa

Rab-related small GTP-binding proteins are known to be involved in the regulation of the vesicular transport system in eucaryotic cells. In this paper we report the isolation of the cDNA clone MS- rab11f from Medicago sativa (alfalfa) root nodules using a combination of RT-PCR and SSCP analysis. MS- rab11f shows high homology to the Rab-related cDNA clone LJ- rab11f from Lotus japonicus root nodules. The MS-Rab11F protein expressed in Escherichia coli was found to bind GTP, confirming that the isolated cDNA indeed codes for a small GTP-binding protein. Expression analysis by RT-PCR demonstrated that MS- rab11f is preferentially expressed in root nodules of alfalfa. Using the cDNA-sequence of MS-rab11f, a peptide-specific antibody was generated. Western blot analysis with this antibody revealed that two Rab11F isoforms, designated MS-Rab11FA and MS-Rab11FB, are found in M. sativa root nodules.