Symbiotic Nitrogen Fixation In Legumes Research Articles

The Potential of Cold Plasma-Based Seed Treatments in Legume–Rhizobia Symbiotic Nitrogen Fixation: A Review

The use of cold plasma (CP) seed treatment is an emerging agricultural technology that exhibits the potential to enhance nodulation and symbiotic nitrogen fixation (SNF) in legumes. CP is composed of a diverse mixture of excited atoms, molecules, ions, and radicals that have the potential to affect the physical properties of the seed and influence gene expressions that could have a lasting impact on the nodulation, SNF, growth, and yield of legumes. The direct participation of the CP in the nodulation process and its correlation with the escalation of nodules and SNF is still not fully understood. This review discussed four areas in the nodulation and SNF process that can directly or indirectly affect CP seed treatments: root–rhizobia signal exchange pathways, root/shoot growth and development, phytohormone production, and the nitrogen fixation process. We also discuss the potential challenges and future research requirements associated with plasma technology to enhance SNF in legumes.

Characterization of drought stress-mitigating Rhizobium from faba bean (Vicia faba L.) in the Chinese Qinghai-Tibet Plateau.

Rhizobium-driven symbiotic nitrogen-fixation in legumes not only benefits the growth but also enhances the stress tolerance of plants. Isolating and characterizing efficient, drought-tolerant rhizobia is a central goal for improving crop yields in arid regions. Here, we phylogenetically and biochemically characterized a novel strain of Rhizobium ('QHCD11') sampled from the root nodules of faba beans growing in an arid agricultural area in Qinghai-Tibet. We further tested the drought tolerance of the strain as well as of 'Qingcan 14' faba bean seedlings inoculated with it. Biochemical characterization involved bromothymol blue (BTB) tests, carbon metabolic profiling (Biolog GENIII), DNA-DNA hybridization (dDDH) assays, average nucleotide identity (ANI) analyses, and 16S rRNA sequencing. The result indicated that strain 'QHCD11' likely belongs to the Rhizobium indicum species. Drought stress tolerance was assessed by exposure to polyethylene glycol (PEG-6000) at concentrations of 0, 10, 15, and 20%. Increasing concentrations of PEG-6000 tended to result in decreased growth of 'QHCD11', although the strain performed better at 20% PEG 6000 than at 15%. Inoculation of drought-stressed faba bean seedlings with strain 'QHCD11' improved root vitality, chlorophyll content, antioxidant enzyme activities, and plant height. We suggest that inoculation of faba beans with 'QHCD11' is an environmentally sound strategy for mitigating crop drought stress in arid and semi-arid regions. In addition, the results presents here will benefit future studies into faba bean-rhizobia symbioses under drought stress.

The interaction between drought stress and nodule formation under multiple environments in chickpea.

Environmental stresses, particularly drought, limit symbiotic nitrogen fixation in legumes, resulting in decreased yielding capacity. Drought is one of the most important constraints limiting yield potential in crops and it is the major abiotic stress that can cause more than 70% yield loss in chickpea. In this study, a total of two hundred four chickpea (Cicer arietinum L.) genotypes were selected to study the interaction between drought stress and nodule formation. This interaction was assessed by using morphological, yield and yield components. The field experiments were laid out in two locations (Terbol and Kfardan stations, Bekaa valley, Lebanon) using Alpha lattice design with two replications and two watering treatments (irrigation and rainfed) during 2016 and 2017 seasons. Parameters that were measured include days to 50% flowering (DFL), day to maturity (DM), plant height (PLH), nodule biomass (NB), nodule fresh weight (NFW), nodule dry weight (NDW), grain yield (GY), Biological yield (BY), 100 seed weight (100SW) and drought tolerance stress (DTS). The results indicated a significant variation between genotypes, environments and other morphological, yield and yield components traits. Drought stress reduced significantly the yield and the nodule’s characteristics, biological and grain yield. The genotypes with the highest levels of drought tolerance, such as IG70399, IG8256, IG71832, IG70270, and IG70272, showed a minimal decrease in yield and nodule biomass. Nodule observations significantly and positively correlated with GY (0.36-0.38) under drought stress treatment. The correlation values for nodule characteristics with DFL and DM were higher under drought stress compared to irrigated conditions. This is a comparative study between drought stress and nodule formation traits associated with morphological, yield and yield components traits.

New Insights into the Taxonomy of Bacteria in the Genomic Era and a Case Study with Rhizobia.

Since early studies, the history of prokaryotes taxonomy has dealt with many changes driven by the development of new and more robust technologies. As a result, the number of new taxa descriptions is exponentially increasing, while an increasing number of others has been subject of reclassification, demanding from the taxonomists more effort to maintain an organized hierarchical system. However, expectations are that the taxonomy of prokaryotes will acquire a more stable status with the genomic era. Other analyses may continue to be necessary to determine microbial features, but the use of genomic data might be sufficient to provide reliable taxa delineation, helping taxonomy to reach the goal of correct classification and identification. Here we describe the evolution of prokaryotes' taxonomy until the genomic era, emphasizing bacteria and taking as an example the history of rhizobia taxonomy. This example was chosen because of the importance of the symbiotic nitrogen fixation of legumes with rhizobia to the nitrogen input to both natural ecosystems and agricultural crops. This case study reports the technological advances and the methodologies used to classify and identify bacterial species and indicates the actual rules required for an accurate description of new taxa.

A proteomic approach to understand the impact of nodulation on salinity stress response in alfalfa (Medicago sativa L.).

Symbiotic nitrogen fixation in legumes is an important source of nitrogen supply in sustainable agriculture. Salinity is a key abiotic stress that negatively affects host plant growth, rhizobium-legume symbiosis and nitrogen fixation. This work investigates how the symbiotic relationship impacts plant response to salinity stress. We assayed the physiological changes and the proteome profile of alfalfa plants with active nodules (NA), inactive nodules (NI) or without nodules (NN) when plants were subjected to salinity stress. Our data suggest that NA plants respond to salinity stress through some unique signalling regulations. NA plants showed upregulation of proteins related to cell wall remodelling and reactive oxygen species scavenging, and downregulation of proteins involved in protein synthesis and degradation. The data also show that NA plants, together with NI plants, upregulated proteins involved in photosynthesis, carbon fixation and respiration, anion transport and plant defence against pathogens. The study suggests that the symbiotic relationship gave the host plant a better capacity to adjust key processes, probably to more efficiently use energy and resources, deal with oxidative stress, and maintain ion homeostasis and health during salinity stress.

Pedospheric Microbial Nitric Oxide Production Challenges Root Symbioses.

Recent studies indicate that a multitude of microbial processes are involved in nitric oxide production and consumptions in the pedosphere. Due to its dual function as a toxic metabolite and signaling compound, we speculate that this pedospheric nitric oxide of microbial origin can significantly interact with mycorrhizal symbioses and symbiotic nitrogen fixation of legumes.

Folates in legume root nodules.

Folates are multifunctional metabolites in plants that are essential for cell division, nucleic acids and amino acid synthesis. During symbiotic nitrogen fixation in legumes, these cofactors are needed for de novo purine biosynthesis, meaning that changes in the folate pools could directly affect the flow of fixed nitrogen to the plant. Its role related to symbiotic nitrogen fixation has not been yet explored, but recent data suggest a relevant role during the first steps. Transcriptomic, metabolomic and proteomic analyses indicate that folates are accumulated in symbiotic plant tissue, as they are involved, not only in de novo purines biosynthesis, but in nitrogen translocation, endoreduplication and phytohormones biosynthesis. Understanding the possible implication of folate pools during the nitrogen fixation and assimilation, might aid for new engineering targets, in relation to the two transformylations or the production of glycine by serine hydroxymethyltransferase during the de novo purine biosynthetic pathway. In this review, we intend to deliver and discuss the available evidence that support a relevant role of folates during the symbiotic nitrogen fixation.

Importance of Legumes and Role of Sulphur Oxidizing Bacteria for Their Production: A Review

Background: Legumes are relatively cheap, non-animal good source of valuable proteins, micro-nutrients and vitamins in human and animal nutrition for many years. Recognizing the potential of legumes in achieving the sustainable solution to the global food security, protein access, eradicate hunger and malnutrition, FAO of the United Nations (The Food and Agriculture Organization), facilitated 2016 as the International Year of Pulses (grain legumes) under the banner ‘nutritious seeds for a sustainable future’. The nutrient, nitrogen and biological nitrogen fixation is very crucial for legume’s growth, besides sulphur deficiency is very sensitive to the nodulation and nitrogen fixation. Despite the amazing beneficial properties, legumes are neglected by most of us due to having tough competition with low price and high yielding cereal varieties. Methods: Therefore keeping in mind the above points, this review discusses the importance and application of legumes in different perspectives, legume cultivation patterns, importance of sulphur nutrition to legumes, role of sulphur oxidizing bacteria in sulphur nutrition, improving soil and environment, challenges and future of legumes. Conclusion: Legumes have variety of applications including food, health, environment and many other sectors but we are not able to produce enough amount according to their genetic potential due to inefficient breeding programs. Sulphur is an important nutrient along with N effecting its growth and yield. Sulphur oxidizing bacteria (SOB) have been proved as an important tool for improving yield and symbiotic nitrogen fixation in legumes. Therefor application of biofertilizers along with SOB and improved genetic breeding programmes may prove leading steps to enhance their production.

Evaluation of beneficial and inhibitory effects of nitrate on nodulation and nitrogen fixation in common bean (Phaseolus vulgaris)

AbstractThe effects of applied nitrate on symbiotic nitrogen fixation in legumes are complex. Both inhibition and promotion of nodulation by nitrate have been observed in a dose‐dependent manner. The objectives of this study were to determine the effects of nitrate at different concentrations on root nodulation in different genotypes in common bean (Phaseolus vulgaris). Six genotypes were inoculated with the same rhizobial strain and grown hydroponically in growth pouches in a growth chamber and exposed to six nitrate concentrations, including 0, 2.5, 5, 10, 15, and 20 mM for 4 weeks. The tested genotypes included three recombinant inbred lines (RILs, 25, 46, and 70) that differed in their responses to nitrogen (based on observations of one field growing season), their parents (Mist and Sanilac—registered varieties), which are different in N‐fixing abilities, and one nonnodulating mutant (R99). Our results showed that small amounts of nitrate (2.5 and 5 mM) promoted nodule formation and increased nodule biomass, compared with plants in the 0 nitrate control treatment. In contrast, nitrate concentrations over 10 mM inhibited nodulation, resulting in reductions in nodule number and nodule biomass. Nodulation was completely inhibited by 15‐mM nitrate in all the genotypes. Regression analyses indicated that 5‐mM nitrate is the optimum concentration for promoting nodulation as measured by the total number of nodules formed, the number of effective nodules formed, and the nodule biomass formed. In contrast, nitrogen fixation was inhibited by all levels of nitrate. No genotypic differences were observed in nodulation among the three RILs and their parental cultivars, but all were significantly different than R99, a nonnodulating mutant.

Advances in the mechanism of cereal/legume intercropping promotion of symbiotic nitrogen fixation

Biological nitrogen fixation by rhizobia in symbiotic association with legumes is a valuable N source for agroecosystems, and thus it is crucial for increased sustainability of agricultural production. Previous studies found that legume and cereal intercropping can promote symbiotic nitrogen fixation mainly due to rhizosphere nitrogen depletion by the cereal and that soil mineral nitrogen is absorbed more by cereals than legumes, which forces the legume to increase its reliance on symbiotic nitrogen fixation. However, in addition to the nitrogen depletion theory in the rhizosphere, stoichiometry (such as nitrogen and phosphorus coupling) and molecular regulation mechanisms are also important in understanding legume nitrogen fixation. The legume usually maintains a stable stoichiometric relationship between nitrogen and phosphorus. Symbiotic nitrogen fixation of legumes tends to have a high demand for phosphorus, mainly because nitrogen fixation by legumes is a high energy-consuming process, and the required energy is directly provided by ATP. Therefore, when the phosphorus obtained by the intercropped legume is insufficient to meet the increase in symbiotic nitrogen fixation, legume growth is limited by phosphorus. Under phosphorus limitation, the phosphorus uptake strategy of the legume can be stimulated, i.e. by increasing the infection of arbuscular mycorrhizal fungi (AMF) and secretion of root exudates. The AMF mycelia of legumes can extend beyond the rhizosphere, linking the roots to the surrounding soil microhabitats and expanding the area of phosphorus absorbed by the roots. On the other hand, mobilizing rhizosphere insoluble phosphorus through root exudates is also an important pathway for improving phosphorus utilization. Increased soil phosphorus availability promotes plant growth and increased allocation of carbon (C) sources to roots and nodules, resulting in a larger root system or greater nodule formation, finally higher symbiotic nitrogen fixation. In addition, the formation of root nodules is influenced by rhizosphere talk between the cereal and legume, which are directly related to the level of nitrogen. A recent study found that under low N concentration conditions, cereal root exudates stimulated flavonoid biosynthesis and secretion partly through upregulating expression of the Chalcone-flavanone isomerase gene ( CFI ) in legumes. Meanwhile, an auxin-responsive GH3 family gene ( GH3.1 ) and the nodulation gene ENODL2 displayed significant increases, suggesting that nodulation continues under the regulation of auxin signaling and the nodulin-like protein. At nodule maturity, cereal root exudates continue to up-regulate key nodulation genes, such as NODL4 , ENOD93 and the N fixation gene FixI3 , finally promoting symbiotic nitrogen fixation. Moreover, a high concentration of soil mineral N significantly suppressed expression of the early nodulin gene NIN in Lotus corniculatus , which inhibited nodulation and symbiotic nitrogen fixation. In the cereal/legume intercropping system, rhizosphere nitrogen depletion can relieve the inhibition of nitrate on the expression of key nodulation and N fixation genes, thus promoting nodulation and symbiotic nitrogen fixation. In conclusion, intercropping can promote nitrogen uptake and optimize nitrogen use efficiency through the coupling effects of rhizosphere nitrogen depletion, phosphorus mobilization and the regulation of nitrogen fixation genes. This review will help improve our understanding of the mechanism of symbiotic nitrogen fixation in the cereal/legume intercropping system.

Nodule-specific PLAT domain proteins are expanded in the Medicago lineage and required for nodulation.

Symbiotic nitrogen fixation in legumes is mediated by an interplay of signaling processes between plant hosts and rhizobial symbionts. In legumes, several secreted protein families have undergone expansions and play key roles in nodulation. Thus, identifying lineage-specific expansions (LSEs) of nodulation-associated genes can be a strategy to discover candidate gene families. Using bioinformatic tools, we identified 13 LSEs of nodulation-related secreted protein families, each unique to either Glycine, Arachis or Medicago lineages. In the Medicago lineage, nodule-specific Polycystin-1, Lipoxygenase, Alpha Toxin (PLAT) domain proteins (NPDs) expanded to five members. We examined NPD function using CRISPR/Cas9 multiplex genome editing to create Medicago truncatula NPD knockout lines, targeting one to five NPD genes. Mutant lines with differing combinations of NPD gene inactivations had progressively smaller nodules, earlier onset of nodule senescence, or ineffective nodules compared to the wild-type control. Double- and triple-knockout lines showed dissimilar nodulation phenotypes but coincided in upregulation of a DHHC-type zinc finger and an aspartyl protease gene, possible candidates for the observed disturbance of proper nodule function. By postulating that gene family expansions can be used to detect candidate genes, we identified a family of nodule-specific PLAT domain proteins and confirmed that they play a role in successful nodule formation.

Evaluating the Effectiveness of Rhizobium Inoculants and Micronutrients as Technologies for Nepalese Common Bean Smallholder Farmers in the Real-World Context of Highly Variable Hillside Environments and Indigenous Farming Practices

Studies have shown the potential of rhizobia and associated micronutrients to enhance symbiotic nitrogen fixation in legumes. Tens of millions of smallholder farmers, however, farm on mountain hillsides in highly variable soil and microenvironments, with different crop rotations, inputs and cultural practices. Here, on the terraces of the Nepalese Himalayas, we evaluated rhizobium inoculants (local, exotic), micronutrients (molybdenum, boron) and their combinations as technologies for smallholder farmers under highly variable microenvironments and traditional practices. The study was conducted as a series of participatory on-farm trials with 39 terrace farmers in two mid-hill districts of Nepal (Dhading, Kaski) from 2015 to 2017. Plots were measured for relevant agronomic traits. As expected, when comparing treatment plots with adjacent control plots within each farm, the results demonstrated tremendous farm-to-farm variability for nodulation, vegetative biomass, shoot nitrogen content, grain yield, and grain N content. Despite the variation observed, the data showed that the number of farms that showed yield increases from the rhizobium interventions, compared to those that suffered yield losses, was generally 2:1. We discuss potential experimental and socio-agronomic reasons for the variable results, including rainfall, which appeared critical. The results demonstrate the promise of rhizobium interventions for hillside smallholder farmers, even in a highly variable context.

Development of RR soybean in function of glyphosate doses and Bradyrhizobium inoculation

ABSTRACT Soybean has traditionally been produced in systems that include the use of herbicides, often in higher than recommended doses. The process of symbiotic nitrogen fixation in legumes can be hampered by these herbicides, both by direct effects on rhizobia and indirect effects on the host plant. An outdoor experiment was performed to evaluate the effects of different doses of a glyphosate herbicide on Bradyrhizobium strains and biological nitrogen fixation in soybean BMX Potência RR plants. Soybean seeds were inoculated with Bradyrhizobium elkanii (SEMIA 5019) and Bradyrhizobium japonicum (SEMIA 5079) strains in a commercial liquid inoculant. The treatments consisted of the absence and presence of Bradyrhizobium genotypes inoculated via seed and four doses of the herbicide glyphosate applied on the leaves (0, 1.0, 2.0, and 4.0 L ha-1 of the commercial product) at the V3 stage. The leaf chlorophyll index of inoculated RR soybean plants did not change on the application of glyphosate and, regardless of inoculation, plants had the capacity to recover from the effects of glyphosate application, without impaired development.

Challenges in Using Precision Agriculture to Optimize Symbiotic Nitrogen Fixation in Legumes: Progress, Limitations, and Future Improvements Needed in Diagnostic Testing

Precision agriculture (PA) has been used for ≥25 years to optimize inputs, maximize profit, and minimize negative environmental impacts. Legumes play an important role in cropping systems, by associating with rhizobia microbes that convert plant-unavailable atmospheric nitrogen into usable nitrogen through symbiotic nitrogen fixation (SNF). However, there can be field-level spatial variability for SNF activity, as well as underlying soil factors that influence SNF (e.g., macro/micronutrients, pH, and rhizobia). There is a need for PA tools that can diagnose spatial variability in SNF activity, as well as the relevant environmental factors that influence SNF. Little information is available in the literature concerning the potential of PA to diagnose/optimize SNF. Here, we critically analyze SNF/soil diagnostic methods that hold promise as PA tools in the short–medium term. We also review the challenges facing additional diagnostics currently used for research, and describe the innovations needed to move them forward as PA tools. Our analysis suggests that the nitrogen difference method, isotope methods, and proximal and remote sensing techniques hold promise for diagnosing field-level variability in SNF. With respect to soil diagnostics, soil sensors and remote sensing techniques for nitrogen, phosphorus, pH, and salinity have short–medium term potential to optimize legume SNF under field conditions.

Epidermal auxin biosynthesis facilitates rhizobial infection in Lotus japonicus.

Symbiotic nitrogen fixation in legumes requires nodule organogenesis to be coordinated with infection by rhizobia. The plant hormone auxin influences symbiotic infection, but the precise timing of auxin accumulation and the genetic network governing it remain unclear. We used a Lotus japonicus optimised variant of the DII-based auxin accumulation sensor and identified a rapid accumulation of auxin in the epidermis, specifically in the root hair cells. This auxin accumulation occurs in the infected root hairs during rhizobia invasion, while Nod factor application induces this response across a broader range of root hairs. Using the DR5 auxin responsive promoter, we demonstrate that activation of auxin signalling also occurs specifically in infected root hairs. Analysis of root hair transcriptome data identified induction of an auxin biosynthesis gene of the Tryptophan Amino-transferase Related (LjTar1) family following both bacteria inoculation and Nod factor treatment. Genetic analysis showed that both expression of the LjTar1 biosynthesis gene and the auxin response requires Nod factor perception, while common symbiotic pathway transcription factors are only partially required or act redundantly to initiate auxin accumulation. Using a chemical genetics approach, we confirmed that auxin biosynthesis has a functional role in promoting symbiotic infection events in the epidermis.

Genetic Characterization and Diversity of Rhizobium Isolated From Root Nodules of Mid-Altitude Climbing Bean (Phaseolus vulgaris L.) Varieties.

The increasing interest in the use of rhizobia as biofertilizers in smallholder agricultural farming systems of the Sub-Saharan Africa has prompted the identification of a large number of tropical rhizobia strains and led to studies on their diversity. Inoculants containing diverse strains of rhizobia have been developed for use as biofertilizers to promote soil fertility and symbiotic nitrogen fixation in legumes. In spite of this success, there is paucity of data on rhizobia diversity and genetic variation associated with the newly released and improved mid-altitude climbing (MAC) bean lines (Phaseolus vulgaris L.). In this study, 41 rhizobia isolates were obtained from the root nodules of MAC 13 and MAC 64 climbing beans grown in upper and lower midland agro-ecological zones of Eastern Kenya. Eastern Kenya was chosen because of its high production potential of diverse common bean cultivars. The rhizobia isolates were characterized phenotypically on the basis of colony morphology, growth and biochemical features. Rhizobia diversity from the different regions of Eastern Kenya was determined based on the amplified ribosomal DNA restriction analysis (ARDRA) of PCR amplified 16S rRNA genes using Msp I, EcoR I, and Hae III restriction enzymes. Notably, native rhizobia isolates were morphologically diverse and grouped into nine different morphotypes. Correspondingly, the analysis of molecular variance based on restriction digestion of 16S rRNA genes showed that the largest proportion of significant (p < 0.05) genetic variation was distributed within the rhizobia population (97.5%) than among rhizobia populations (1.5%) in the four agro-ecological zones. The high degree of morphological and genotypic diversity of rhizobia within Eastern Kenya shows that the region harbors novel rhizobia strains worth exploiting to obtain strains efficient in biological nitrogen fixation with P. vulgaris L. Genetic sequence analysis of the isolates and testing for their symbiotic properties should be carried out to ascertain their identity and functionality in diverse environments.

Dynamics of Ethylene Production in Response to Compatible Nod Factor.

Establishment of symbiotic nitrogen-fixation in legumes is regulated by the plant hormone ethylene, but it has remained unclear whether and how its biosynthesis is regulated by the symbiotic pathway. We established a sensitive ethylene detection system for Lotus japonicus and found that ethylene production increased as early as 6 hours after inoculation with Mesorhizobium loti This ethylene response was dependent on Nod factor production by compatible rhizobia. Analyses of nodulation mutants showed that perception of Nod factor was required for ethylene emission, while downstream transcription factors including CYCLOPS, NIN, and ERN1 were not required for this response. Activation of the nodulation signaling pathway in spontaneously nodulating mutants was also sufficient to elevate ethylene production. Ethylene signaling is controlled by EIN2, which is duplicated in L. japonicus We obtained a L. japonicus Ljein2a Ljein2b double mutant that exhibits complete ethylene insensitivity and confirms that these two genes act redundantly in ethylene signaling. Consistent with this redundancy, both LjEin2a and LjEin2b are required for negative regulation of nodulation and Ljein2a Ljein2b double mutants are hypernodulating and hyperinfected. We also identified an unexpected role for ethylene in the onset of nitrogen fixation, with the Ljein2a Ljein2b double mutant showing severely reduced nitrogen fixation. These results demonstrate that ethylene production is an early and sustained nodulation response that acts at multiple stages to regulate infection, nodule organogenesis, and nitrogen fixation in L. japonicus.

Use of CRISPR/Cas9 for Symbiotic Nitrogen Fixation Research in Legumes.

Nitrogen-fixing rhizobia have established a symbiotic relationship with the legume family through more than 60 million years of evolution. Hundreds of legume host genes are involved in the SNF (symbiotic nitrogen fixation) process, such as recognition of the bacterial partners, nodulation signaling and nodule development, maintenance of highly efficient nitrogen fixation within nodules, regulation of nodule numbers, and nodule senescence. However, investigations of SNF-related gene functions and dissecting molecular mechanisms of the complicated signaling crosstalk on a genomic scale were significantly restricted by insufficient mutant resources of several representative model legumes. Targeted genome-editing technologies, including ZFNs, TALENs, and CRISPR-Cas systems, have been developed in recent years and rapidly revolutionized biological research in many fields. These technologies were also applied to legume plants, and significant progress has been made in the last several years. Here, we summarize the applications of these genome-editing technologies, especially CRISPR-Cas9, toward the study of SNF in legumes, which should greatly advance our understanding of the basic mechanisms underpinning the legume-rhizobia interactions and guide the engineering of the SNF pathway into nonlegume crops to reduce the dependence on the use of nitrogen fertilizers for sustainable development of modern agriculture.

Molecular Characterization of LjSWEET3, a Sugar Transporter in Nodules of Lotus japonicus.

Symbiotic nitrogen fixation in legumes contributes greatly to the global nitrogen cycle on the earth. In nodules, resident rhizobia supply nitrogen nutrient fixed from atmospheric N2 to the host plant; in turn, the plant provides photosynthetic metabolites to bacteroids as a carbon source. In this process, various transporters are involved at different membrane systems; however, little is known at the molecular level about the flow of carbon from the host cells to the symbiotic bacteria. We have been studying transporters functioning in nodules of Lotus japonicus, and found that out of 13 SWEET genes in the L. japonicus genome LjSWEET3, a member of the SWEET transporter family, is highly expressed in nodules. The SWEET family was first identified in Arabidopsis, where members of the family are involved in phloem loading, nectar secretion, pollen nutrition and seed filling. The expression of LjSWEET3 strongly increased during nodule development and reached the highest level in mature nodules. Histochemical analysis using L. japonicus plants transformed with LjSWEET3 promoter:GUS (β-glucuronidase) showed strong expression in the vascular systems of nodules. Analysis of an LjSWEET3-green fluorescent protein (GFP) fusion expressed in Nicotiana banthamiana and Coptis japonica indicates that LjSWEET3 localizes to the plasma membrane. Together these data are consistent with a role for LjSWEET3 in sugar translocation towards nodules and also suggest the possible existence of multiple routes of carbon supply into nodules.

Symbiotic nitrogen fixation in legumes as a genetic and selection trait

The results of long-term studies on legume symbiogenetics and breeding are summarized by the example of pea Pisum sativum L. A collection of symbiotic mutants was developed by chemical and radiational mutagenesis of pea varieties and genetically characterized. Various sym genes were recognized. From the large set, supernodulating (proved to be recessive) and dominant hypernodulating types of symbiotic mutants were chosen for breeding programs. Varieties differed dramatically in hypernodulation degree. Aiming at nitrogen fixation intensification, accessions bearing recessive genes for supernodulation ( nod4 ) and dominant genes for hypernodulation ( Nod5 ) were selected. The recurrent method of symbiotic mutants utilization in pea breeding for nitrogen fixation intensification was developed. The best results were obtained by combining two sym genes in one pea genotype: the dominant hypernodulation gene Nod5 and the recessive supernodulation gene nod4 . A set of recurrent lines tagged with both these genes was raised to use in breeding programs as donors of intense nitrogen fixation combined with good performance. In addition, they are good preceding crops. After their harvesting, soil accumulates large amounts of nitrogen-rich root and bacterial biomass. The nitrogen is preserved for years, whereas mineral nitrogen is rapidly washed out with precipitation. Endemic pea accessions originated from various regions can be successful starting material in breeding for nitrogen fixation intensification, accessions from Egypt and Syria having provided best results. Nodulation and nitrogen fixation intensities were assessed in seven cultivars derived from three promising pea lines raised at the Siberian Research Institute of Plant Breeding and Selection.