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Nissolia brasiliensis as a nonnodulating model legume.

The nitrogen-fixing root nodule symbiosis is specifically formed by 4 orders of angiosperms. The largest of these 4 orders includes the legume family, the Fabaceae. Among legumes, historical model species have emerged, such as the root nodule symbiosis-forming Medicago truncatula and Lotus japonicus or, more recently, Aeschynomene evenia. By contrast, legume species that have lost root nodule symbiosis have been largely ignored. Here, we describe the first near chromosome-level assembly for a non-root nodule symbiosis-forming legume, the tropical Papilionoideae Nissolia brasiliensis. We compared its genome to closely related legumes and identified genes associated with root nodule symbiosis. Finally, we developed a stable transformation protocol that can be deployed in the future to reevolve root nodule symbiosis in legumes, a first step toward the goal of engineering root nodule symbiosis in nonlegume crops.

Fabaceae

Beyond the CO-FT regulatory module: E1 and PHYA emerge as players in photoperiodic regulation of flowering in legumes.

The legume family (Fabaceae) is the third largest in plants and includes several crop species that are able to fix nitrogen, promote soil health, and contribute to food security worldwide. Recent progress in legume genetics and genomics allowed the identification of photoperiod-dependent flowering loci, which were incorporated into specific signalling networks. Functional characterization of these regulators revealed new roles for known photoreceptors such as phytochrome A, and it also identified legume-specific B3 domain transcriptional factors (E1 and E1-like proteins). This suggests some diversification from the traditional CONSTANS-FLOWERING LOCUS T module present in other angiosperms. Although most of the findings discussed herein pertain to species from the two main legume clades, the Galegoids (e.g. alfalfa, clover, and pea) and the Phaseoloids (e.g. common bean, soybean, cowpea, and pigeon pea), research on flowering regulation in the basal Genistoid clade (e.g. lupins) will also be addressed. We propose that functional diversification of photoperiod-dependent flowering strategies in the different legume species could have contributed to their environmental adaptation and allowed their geographical expansion and success worldwide.

Photoperiod

Integrating genomics, multi-omics, CRISPR and speed breeding for stress-resilient vegetable legume improvement.

Vegetable legumes are nutritionally and ecologically important crops. However, their genetic improvement has not kept pace with the increasing challenges posed by climate change due to the polygenic nature of stress tolerance, narrow genetic diversity, and the persistent gap between molecular discoveries and field-level cultivar development. Although recent reviews have examined individual genomic tools or specific stress responses, a comprehensive synthesis integrating genomics-assisted breeding, multi-omics technologies, genome editing, and speed breeding within a unified crop improvement framework has been lacking. This review addresses that gap by critically evaluating how these complementary approaches can accelerate the development of stress-resilient vegetable legumes, including pea, common bean, cowpea, faba bean, cluster bean, yard-long bean, and hyacinth bean. This review synthesizes advances in QTL mapping, genome-wide association studies, transcriptomics, metabolomics, and CRISPR-based functional genomics that have identified key regulators and pathways underlying resistance to major biotic and abiotic stresses. Rather than considering these technologies independently, the review emphasizes their convergence into a systems-level breeding framework integrating genomic discovery, functional validation, predictive breeding, and accelerated generation advancement to improve breeding efficiency. Speed breeding, enabling up to seven to eight generations annually under optimized controlled-environment experimental conditions in cowpea, is discussed as a complementary strategy with genomic selection and genome editing. The review further identifies major translational bottlenecks, including transformation recalcitrance, limited genomic resources for underutilized vegetable legumes, inadequate multi-environment validation, and fragmented omics integration, and presents an integrated systems-breeding framework to bridge the gap between gene discovery and cultivar development.

Fabaceae

Estimating Rhizobial Fitness During Legume Symbiosis: Enriching Viable Undifferentiated Bacteria from Root Nodules.

Advances in understanding the evolutionary ecology of the rhizobia-legume mutualism have been constrained by methodological limitations in efficiently measuring relative strain frequencies alongside measurements of absolute population sizes of rhizobia living in nodules. To examine strain competition in natural and agricultural ecosystems that harbor multiple strains of rhizobia, an increasing number of manipulative and observational studies have recently begun to examine dozens or hundreds of strains simultaneously. Assessing the competitive fitness of multiple strains in legume nodules requires, first, processing pools of dozens to hundreds of nodules to overcome the stochasticity of nodule formation; second, focusing on the reproductively viable rhizobial population, since this trait represents rhizobia's reproductive success in nodules and is pivotal for evolutionary interpretations. Our approach has been optimized in the Medicago truncatula-Sinorhizobium meliloti system, where rhizobia induce the formation of indeterminate nodules that harbor two subpopulations: terminally-differentiated bacteroids and undifferentiated rhizobia that retain reproductive viability. This protocol has also been used for other legumes with terminally-differentiated bacteroids, such as pea and vetch, as well as for those with non-terminally differentiated bacteroids, such as soybean and cowpeas. The protocol we present enables rapid and reproducible homogenization of pools containing hundreds of nodules using a tissue homogenizer. We also enrich for undifferentiated rhizobia using two centrifugation steps: first, a low-speed centrifugation to deplete nodule debris and large, endoreduplicated, terminally-differentiated bacteroids, followed by a high-speed centrifugation to pellet the remaining undifferentiated rhizobia. The pellet can later be used for DNA extraction, followed by whole-genome or amplicon sequencing, and then downstream analysis to estimate strain fitness. Finally, we include an optional step for a reliable, reproducible system for nodule imaging, which is especially useful for quantifying nodule abundance and studying morphological variation. ​.

Symbiosis

Molecular characterization of vitellogenin and its receptor with CRISPR-based sgRNA validation in the legume pod borer, Maruca vitrata (Geyer) (Lepidoptera: Crambidae).

Maruca vitrata, the legume pod borer, causes yield losses of up to 80% in grain legumes. Increasing insecticide resistance and environmental concerns necessitate sustainable pest management alternatives. In the present study, the complete vitellogenin (Vg) coding sequence (CDS), a key reproductive gene involved in oogenesis and embryonic development, was cloned and molecularly characterised from M. vitrata. The assembled Vg CDS (∼5.3 kb) shared 99.04% sequence identity with the reported M. vitrata Vg sequence (MG799570.1). Phylogenetic analysis demonstrated close evolutionary association with related Lepidopteran species, while protein domain analysis identified three conserved domains, namely LPD_N, DUF1943, and VWD. Among these, the single exon-encoded LPD_N domain was selected as the target region for CRISPR/Cas9-mediated editing. Homology models of Vg and vitellogenin receptor (VgR) (Global Model Quality Estimation (GMQE): 0.58 and 0.51) showed a favourable interaction by protein-protein docking (score: -295.66). Three single-guide RNAs (sgRNAs) were designed, synthesised through in-vitro transcription, and evaluated using in vitro cleavage assays. sgRNA1 targeting the LPD_N domain and sgRNA2 targeting the signal peptide region exhibited efficient site-specific cleavage activity, whereas sgRNA3 failed to induce cleavage because of an unfavourable secondary structure that likely impaired Cas9-sgRNA complex formation. Overall, this study provides the first CRISPR-oriented functional characterisation and sgRNA validation of the M. vitrata Vg gene, together with structural characterisation of VgR and Vg-VgR interaction analysis, providing preliminary molecular resources for future CRISPR/Cas9 studies and supporting future embryo microinjection and heritable genome editing for sustainable management of M. vitrata.

CRISPR/Cas9

Evolution and Expression Divergence of Legume PAL Genes Suggest Associations with Drought Response and Root Nodule Development.

Comparative genomic analyses provide insight into the mechanisms underlying gene-family evolution and crop adaptation. Here, we used the legume phenylalanine ammonia-lyase (PAL) gene family as a model and integrated pan-genomic, phylogenetic, molecular evolutionary, duplication-mode, and transcriptomic analyses, while developing GFtool for gene family identification. Across 45 genomes, we identified 302 PAL genes and classified them into five Groups. Groups 1-3 represented ancient lineages shared with outgroups, whereas Groups 4 and 5 were legume-specific. Molecular-clock analyses placed the divergence of Group 2 near the Paleocene-Eocene transition, while Groups 4 and 5 diversified from the middle Eocene to the early Oligocene. WGD/segmental duplication broadly contributed to PAL copy-number expansion, whereas tandem duplication was enriched in Group 5 of Papilionoideae. Group 2 genes showed drought-induced expression, whereas Group 5 genes were associated with early root nodule development. GFtool provides a scalable framework for gene-family studies.

Fabaceae

Structure and development of P-protein in phloem parenchyma and companion cells of legumes.

Phloem parenchyma and companion cells from four species of legumes, Phaseolus vulgaris L., Melilotus alba Desr., Desmodium canadense, L. and Dolichos lablab L., were examined electron microscopically to estabish the presence, structure and development of P-protein. P-protein components consisting of granular, fibrillar, tubular or crystalline structures were found in parenchyma cells of all species and in companion cells of M. alba. The earliest stages of P-protein formation were closely associated with dictyosome cisternae, dictyosome vesicles and/or spiny vesicles. After their formation, the P-protein bodies were frequently transformed into one or more structurally different components. Although these components appeared to be develop-mentally related, their specific associations and transformations differed in each species examined.

Inclusion Bodies

Role of complement in the toxicity of dietary legumes.

On the basis of in vivo data Jayne-Williams (1) has proposed that the toxicity of dietary legumes is due to their content of lectins which are immunosuppressive. On the basis of in vitro data with cultured lymphocytes (2), it is now proposed that ingested lectins bind to cell surfaces and cause autologous complement components to bind to and destroy immunologically competent cells. The hypothesis throws a possible light on the aetiology of favism and Whipple's disease.

Complement System Proteins

Concurrent ecological and evolutionary processes contribute to mutualism breakdown between legumes and rhizobia.

Though they jointly shape community responses to environmental perturbations, ecology and evolution are often examined separately, even in microorganisms where both occur over short timescales. Here we examine ecological and evolutionary responses to 33 years of nitrogen fertilization using the legume-rhizobium mutualism. Pairing a manipulative inoculation study with full-length 16S rRNA gene amplicon sequencing and structural equation modeling allows us to synthesize across biological scales: whole bacterial community, genus Rhizobium, Rhizobium ASVs, and symbiosis plasmids. Clover's preferred partner decreases in N-addition soils, limiting host growth, while a diverse and largely uncharacterized Rhizobium community increases. This ecological change is compounded by a concurrent evolutionary degradation of symbiont partner quality via changing frequencies of symbiotic plasmids. Ecological (rarer symbionts) and evolutionary (inferior symbionts) processes each accounted for roughly half of this loss of host benefit, revealing that ecology and evolution jointly shape mutualism breakdown over the short timescales typical of microbial systems.

ecology

The Rhizobium--legume symbiosis.

The rhizobia are soil microorganisms that can interact with leguminous plants to form root nodules within which conditions are favourable for bacterial nitrogen fixation. Legumes allow the development of very large rhizobial populations in the vicinity of their roots. Infections and nodule formation require the specific recognition of host and Rhizobium, probably mediated by plant lectins. Penetration of the host by a compatible Rhizobium species usually provokes host root cell division to form the nodule, and a process of differentiation by both partners then ensues. In most cases the rhizobia alter morphologically to form bacteroids, which are usually larger than the free-living bacteria and have altered cell walls. At all stages during infection, the bacteria are bounded by host cell plasmalemma. The enzyme nitrogenase is synthesized by the bacteria and, if leghaemoglobin is present, nitrogen fixation will occur. Leghaemoglobin is a product of the symbiotic interaction, since the globin is produced by the plant while the haem is synthesized by the bacteria. In the intracellular habitat the bacteria are dependent upon the plant for supplies of energy and the bacteroids, in particular, appear to differentiate so that they are no longer able to utilize the nitrogen that they fix. Regulation of the supply of carbohydrate and the use of the fixed nitrogen thus appear to be largely governed by the host.

Leghemoglobin

Rhizobium zaerense sp. nov., a novel member of the Rhizobium leguminosarum species complex with a broad geographic distribution and multiple legume hosts.

A novel nitrogen-fixing rhizobial strain, designated Z1P35ᵀ, was isolated from root nodules of Pisum sativum grown in the Zaër region of Morocco. Phylogenetic analysis of the 16S rRNA gene placed strain Z1P35ᵀ within the genus Rhizobium, showing 100% sequence identity with several undescribed genospecies of the Rhizobium leguminosarum species complex (Rlc). Strain Z1P35ᵀ exhibited low average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values with all described Rhizobium species, but high ANI and dDDH values (97.62 and 78.8%, respectively) with Rhizobium sp. SRDI565, representing genospecies M (GsM) of the Rlc, suggesting that Z1P35ᵀ represents a novel species corresponding to GsM within this complex. FastANI screening against all Rhizobium genomes available in GenBank revealed that Z1P35ᵀ shares ANI values above the bacterial species delimitation threshold with 17 unclassified strains, which, together with Z1P35ᵀ and Rhizobium sp. SRDI565 (GsM), form a distinct lineage within the Rlc. These 17 strains originate from root nodules of diverse legume hosts and are distributed across the Mediterranean region and Australia, including representatives of the symbiovars viciae and trifolii. Phylogenomic analysis further confirms the clustering of Z1P35ᵀ with Rhizobium sp. SRDI565 (GsM) and several undescribed Rhizobium strains, forming a unique taxonomic unit clearly distinct from other members of the Rlc. Strain Z1P35ᵀ has a genome of 7.6 Mb with a G+C content of 61 mol% and carries numerous genes associated with chemotaxis, nodulation, nitrogen fixation, phosphate solubilization, iron acquisition and abiotic stress tolerance. Differentiation of Z1P35ᵀ from described Rhizobium species was further supported by phenotypic and chemotaxonomic analyses. Based on these results, we conclude that Z1P35T belongs to a novel species, corresponding to genospecies M within the Rlc, for which we propose the name Rhizobium zaerense sp. nov. The type strain is Z1P35ᵀ (DSM 120601ᵀ=CCMM B1365ᵀ).

Phylogeny

Genomic and Molecular Interaction Analysis of NodD1 in a Novel Bradyrhizobium yuanmingense sp. B64 Isolate for Nodulation and Symbiosis of Legume Plants.

Rhizobial bacteria are known for their ability to fix nitrogen for leguminous plants and their essential function for sustainable agriculture. This study characterizes the taxonomic status and functional potential of the Bradyrhizobium B64 isolate using integrated genomic and molecular approaches. The whole genome of the B64 isolate was sequenced via Illumina paired-end technology. Species delimitation was performed using average nucleotide identity (ANI) and digital DNA-DNA Hybridization (dDDH). The NodD1 protein structure was modeled using AlphaFold3 and validated by Ramachandran plot analysis. Molecular docking was then conducted to evaluate interactions between NodD1 and four signaling flavonoids: Apigenin, Daidzein, Genistein, and Naringenin. Genomic analysis revealed a maximum ANI of 94.4% and dDDH values between 51.4 and 62.4%. Since these values fall below the standard prokaryotic thresholds (ANI&#x2009;<&#x2009;95%; dDDH&#x2009;<&#x2009;70%), the B64 isolate is identified as a novel species. Physiological assays confirmed nitrogen fixation (1.97 ppm), IAA production (3.67 ppm), and phosphate solubilization (26.10 ppm). Structural validation showed 100% of NodD1 residues in allowed regions, ensuring high model reliability. Docking simulations demonstrated strong binding affinities across all flavonoids, with binding free energies ranging from -&#x2009;8.8 to -&#x2009;9.0&#xa0;kcal/mol. Daidzein exhibited the highest thermodynamic stability (-&#x2009;9.0&#xa0;kcal/mol), whereas apigenin showed the most extensive residue interaction network. The B64 isolate is a novel Bradyrhizobium species with a high symbiotic capacity. The stable NodD1-flavonoid interactions provide a molecular basis for efficient nodulation, positioning B64 as a promising candidate for developing lipo-chitooligosaccharide (LCO)-based biofertilizers.

Bradyrhizobium

Effect of adenine nucleotides on NAD-dependent isocitrate dehydrogenases in rhizobia and bacteroids of legume root nodules.

ATP, ADP, AMP and cyclic AMP inhibit NAD-dependent isocitrate dehydrogenase (L-s-isocitrate : NAD-+ oxidoreductase, EC 1.1.1.41) from rhizobia but have no effect on the enzyme from corresponding bacteroids. This was observed using three rhizobial strains two of which are effective, and one ineffective, with Lotus pedunculatus. Using partially purified enzyme from each of the three rhizobial strains it was found that the adenine nucleotides inhibit the enzyme by competing with NAD-+, not with isocritrate. The rate of reaction catalysed by the enzyme (expressed as activity per mg protein) in cell-free extracts of each of the effective rhizobial strains was about three times that of the reaction in extracts of the corresponding bacteroids. No correlation was found between effectiveness and NAD-dependent isocitrate dehydrogenase activity in the rhizobial cells.

Adenine Nucleotides

Kinetic model of a determinate legume root nodule reveals plant metabolic characteristics for more efficient nitrogen fixation symbiosis.

While nitrogen fertilizers are widely used in agricultural production, their application incurs significant environmental and energetic costs. In contrast, some crops are less dependent on these fertilizers because they engage in symbioses with rhizobia, nitrogen-fixing bacteria that provide ammonium to the plant in exchange for carbon. However, the carbon cost associated with nitrogen fixation can negatively impact crop yields. Improving the efficiency of this metabolic process could alleviate this impact on crop productivity. Mathematical models can help us quantitatively explore metabolic behavior and identify potential targets for metabolic engineering. In this work, we developed a kinetic model of determinate root nodule metabolism, where this symbiotic exchange of carbon from the plant and nitrogen from the bacteria occurs. We used this model to evaluate how the predicted metabolic behavior differs between inefficient and efficient nodules, and to identify potential engineering targets for improving nitrogen fixation efficiency and rate. We show that the enzymes phosphoenolpyruvate carboxylase and pyruvate kinase have significant influence on the predicted rate and efficiency of nitrogen fixation, especially when their expression is varied in combination with oxidative Pentose Phosphate Pathway enzymes like glucose-6-phosphate dehydrogenase and 6-phosphogluconolactonase. The model predicts that pairing a 3-fold decrease in glucose-6-phosphate dehydrogenase activity along with either a 3-fold increase in phosphoenolpyruvate carboxylase activity or decrease in pyruvate kinase activity could increase nitrogen fixation rate by 8.82% while improving nitrogen fixation efficiency by 10.99%.

Enzyme kinetics

Enzymes of nitrogen metabolism in legume nodules. Purification and properties of NADH-dependent glutamate synthase from lupin nodules.

An NADH-dependent glutamate synthase has been purified 500-fold from the plant cytoplasm fraction of Lupinus angustifolius nodules. It consists of a single polypeptide chain, Mr 235000. The optimum pH is 8.5, at which Km values for 2-oxoglutarate, glutamine and NADH are 39 micrometer, 400 micrometer and 1.3 micrometer respectively. The catalytic centre activity is of the order of 70 s-1 and is independent of pH between 6.5 and 9.5. Glutamate synthase is inhibited by glutamic acid, oxaloacetic acid, aspartic acid and asparagine, all competitive with 2-oxoglutarate; and by NAD+, which is competitive with NADH. There is evidence of two flavine prosthetic groups per enzyme molecule.

Glutamate Synthase