The Medicago genome provides insight into the evolution of rhizobial symbioses

BackgroundLegumes can establish on nitrogen-deprived soils a symbiotic interaction with Rhizobia bacteria, leading to the formation of nitrogen-fixing root nodules. Cytokinin phytohormones are critical for triggering root cortical cell divisions at the onset of nodule initiation. Cytokinin signaling is based on a Two-Component System (TCS) phosphorelay cascade, involving successively Cytokinin-binding Histidine Kinase receptors, phosphorelay proteins shuttling between the cytoplasm and the nucleus, and Type-B Response Regulator (RRB) transcription factors activating the expression of cytokinin primary response genes. Among those, Type-A Response Regulators (RRA) exert a negative feedback on the TCS signaling. To determine whether the legume plant nodulation capacity is linked to specific features of TCS proteins, a genome-wide identification was performed in six legume genomes (Cajanus cajan, pigeonpea; Cicer arietinum, chickpea; Glycine max, soybean; Phaseolus vulgaris, common bean; Lotus japonicus; Medicago truncatula). The diversity of legume TCS proteins was compared to the one found in two non-nodulating species, Arabidopsis thaliana and Vitis vinifera, which are references for functional analyses of TCS components and phylogenetic analyses, respectively.ResultsA striking expansion of non-canonical RRBs was identified, notably leading to the emergence of proteins where the conserved phosphor-accepting aspartate residue is replaced by a glutamate or an asparagine. M. truncatula genome-wide expression datasets additionally revealed that only a limited subset of cytokinin-related TCS genes is highly expressed in different organs, namely MtCHK1/MtCRE1, MtHPT1, and MtRRB3, suggesting that this “core” module potentially acts in most plant organs including nodules.ConclusionsFurther functional analyses are required to determine the relevance of these numerous non-canonical TCS RRBs in symbiotic nodulation, as well as of canonical MtHPT1 and MtRRB3 core signaling elements.

Whole-genome duplication events (polyploidy events) and gene loss events have played important roles in the evolution of legumes. Here we show that the vast majority of Hsf gene duplications resulted from whole genome duplication events rather than tandem duplication, and significant differences in gene retention exist between species. By searching for intraspecies gene colinearity (microsynteny) and dating the age distributions of duplicated genes, we found that genome duplications accounted for 42 of 46 Hsf-containing segments in Glycine max, while paired segments were rarely identified in Lotus japonicas, Medicago truncatula and Cajanus cajan. However, by comparing interspecies microsynteny, we determined that the great majority of Hsf-containing segments in Lotus japonicas, Medicago truncatula and Cajanus cajan show extensive conservation with the duplicated regions of Glycine max. These segments formed 17 groups of orthologous segments. These results suggest that these regions shared ancient genome duplication with Hsf genes in Glycine max, but more than half of the copies of these genes were lost. On the other hand, the Glycine max Hsf gene family retained approximately 75% and 84% of duplicated genes produced from the ancient genome duplication and recent Glycine-specific genome duplication, respectively. Continuous purifying selection has played a key role in the maintenance of Hsf genes in Glycine max. Expression analysis of the Hsf genes in Lotus japonicus revealed their putative involvement in multiple tissue-/developmental stages and responses to various abiotic stimuli. This study traces the evolution of Hsf genes in legume species and demonstrates that the rates of gene gain and loss are far from equilibrium in different species.

Orthologous markers transferable between distantly related legume species allow for the rapid generation of genetic maps in species where there is little pre-existing genomic or EST information. We are using the model legume Medicago truncatula Gaertn. to develop such markers in legumes of importance to Australian agriculture. This will enable the construction of comparative genetic maps, help to determine patterns of chromosomal evolution in the legume family, and characterise syntenic relationships between M. truncatula and cultivated legumes. This information can then be used to identify markers that are tightly linked to the genes of interest, candidate gene(s) for a trait, and expedite the isolation of such genes. Among the Papilionoideae, we compared ESTs from the phylogenetically distant species, M. truncatula, Lupinus albus and Glycine max, to produce 500 intron-targeted amplified polymorphic markers (ITAPs). In addition to 126 M. truncatula cross-species markers from Department of Plant Pathology, University of California (USA), these markers were used to generate comparative genetic maps of lentil (Lens culinaris Medik.) and white lupin (Lupinus albus Linn.). Our results showed that 90% of the ITAPs markers amplified genomic DNA in M. truncatula, 80% in Lupinus albus, and 70% in Lens culinaris. The comparative map of Lens culinaris was constructed based on 79 ITAP markers. The Lupinus albus comparative map was developed from 105 gene-based markers together with 223 AFLP markers. Although a direct and simple syntenic relationship was observed between M. truncatula and Lens culinaris genomes, there is evidence of moderate chromosomal rearrangement. This may account for the different chromosome numbers in the two species. A more complicated pattern among homologous blocks was apparent between the Lupinus albus and M. truncatula genomes.

F-box proteins constitute a large gene family that regulates processes from hormone signaling to stress response. F-box proteins are the substrate recognition modules of SCF E3 ubiquitin ligases. Here we report very distinct trends in family size, duplication, synteny and transcription of F-box genes in two nitrogen-fixing legumes, Glycine max (soybean) and Medicago truncatula (alfafa). While the soybean FBX genes emerged mainly through segmental duplications (including whole-genome duplications), M. truncatula genome is dominated by locally-duplicated (tandem) F-box genes. Many of these young FBX genes evolved complex transcriptional patterns, including preferential transcription in different tissues, suggesting that they have probably been recruited to important biochemical pathways (e.g. nodulation and seed development).

Peptide sequencing by computational assignment of tandem mass spectra to a database of putative protein sequences provides an independent approach to confirming or refuting protein predictions based on large-scale DNA and RNA sequencing efforts. This use of mass spectrometrically-derived sequence data for testing and refining predicted gene models has been termed proteogenomics. We report herein the application of proteogenomic methodology to a database of 10.9 million tandem mass spectra collected over a period of two years from proteolytically generated peptides isolated from the model legume Medicago truncatula. These spectra were searched against a database of predicted M. truncatula protein sequences generated from public databases, in silico gene model predictions, and a whole-genome six-frame translation. This search identified 78,647 distinct peptide sequences, and a comparison with the publicly available proteome from the recently published M. truncatula genome supported translation of 9,843 existing gene models and identified 1,568 novel peptides suggesting corrections or additions to the current annotations. Each supporting and novel peptide was independently validated using mRNA-derived deep sequencing coverage and an overall correlation of 93% between the two data types was observed. We have additionally highlighted examples of several aspects of structural annotation for which tandem MS provides unique evidence not easily obtainable through typical DNA or RNA sequencing. Proteogenomic analysis is a valuable and unique source of information for the structural annotation of genomes and should be included in such efforts to ensure that the genome models used by biologists mirror as accurately as possible what is present in the cell.

BackgroundAlternative polyadenylation (APA) plays an important role in the post-transcriptional regulation of gene expression. Little is known about how APA sites may evolve in homologous genes in different plant species. To this end, comparative studies of APA sites in different organisms are needed. In this study, a collection of poly(A) sites in Medicago truncatula, a model system for legume plants, has been generated and compared with APA sites in Arabidopsis thaliana.ResultsThe poly(A) tags from a deep-sequencing protocol were mapped to the annotated M. truncatula genome, and the identified poly(A) sites used to update the annotations of 14,203 genes. The results show that 64% of M. truncatula genes possess more than one poly(A) site, comparable to the percentages reported for Arabidopsis and rice. In addition, the poly(A) signals associated with M. truncatula genes were similar to those seen in Arabidopsis and other plants. The 3′-UTR lengths are correlated in pairs of orthologous genes between M. truncatula and Arabidopsis. Very little conservation of intronic poly(A) sites was found between Arabidopsis and M. truncatula, which suggests that such sites are likely to be species-specific in plants. In contrast, there is a greater conservation of CDS-localized poly(A) sites in these two species. A sizeable number of M. truncatula antisense poly(A) sites were found. A high percentage of the associated target genes possess Arabidopsis orthologs that are also associated with antisense sites. This is suggestive of important roles for antisense regulation of these target genes.ConclusionsOur results reveal some distinct patterns of sense and antisense poly(A) sites in Arabidopsis and M. truncatula. In so doing, this study lends insight into general evolutionary trends of alternative polyadenylation in plants.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-615) contains supplementary material, which is available to authorized users.

Plant uridine 5′-diphosphate glycosyltransferases (UGTs) influence the physiochemical properties of several classes of specialized metabolites including triterpenoids via glycosylation. To uncover the evolutionary past of UGTs of soyasaponins (a group of beneficial triterpene glycosides widespread among Leguminosae), the UGT gene superfamily in Medicago truncatula, Glycine max, Phaseolus vulgaris, Lotus japonicus, and Trifolium pratense genomes were systematically mined. A total of 834 nonredundant UGTs were identified and categorized into 98 putative orthologous loci (POLs) using tree-based and graph-based methods. Major key findings in this study were of, (i) 17 POLs represent potential catalysts for triterpene glycosylation in legumes, (ii) UGTs responsible for the addition of second (UGT73P2: galactosyltransferase and UGT73P10: arabinosyltransferase) and third (UGT91H4: rhamnosyltransferase and UGT91H9: glucosyltransferase) sugars of the C-3 sugar chain of soyasaponins were resulted from duplication events occurred before and after the hologalegina–millettoid split, respectively, and followed neofunctionalization in species-/ lineage-specific manner, and (iii) UGTs responsible for the C-22-O glycosylation of group A (arabinosyltransferase) and DDMP saponins (DDMPtransferase) and the second sugar of C-22 sugar chain of group A saponins (UGT73F2: glucosyltransferase) may all share a common ancestor. Our findings showed a way to trace the evolutionary history of UGTs involved in specialized metabolism.

Under nitrogen starvation, most legume plants form a nitrogen-fixing symbiosis with Rhizobium bacteria. The bacteria induce the formation of a novel organ called the nodule in which rhizobia reside as intracellular symbionts and convert atmospheric nitrogen into ammonia. During this symbiosis, miRNAs are essential for coordinating the various plant processes required for nodule formation and function. miRNAs are non-coding, endogenous RNA molecules, typically 20-24 nucleotides long, that negatively regulate the expression of their target mRNAs. Some miRNAs can move systemically within plant tissues through the vascular system, which mediates, for example, communication between the stem/leaf tissues and the roots. In this review, we summarize the growing number of miRNAs that function during legume nodulation focusing on two model legumes, Lotus japonicus and Medicago truncatula, and two important legume crops, soybean (Glycine max) and common bean (Phaseolus vulgaris). This regulation impacts a variety of physiological processes including hormone signaling and spatial regulation of gene expression. The role of mobile miRNAs in regulating legume nodule number is also highlighted.

Legumes are protein-rich grains of Fabaceae family that enrich soil through symbiotic nitrogen fixation (SNF). Genome editing employing clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) system allows a precise modification of genome using either endogenous non-homologous end-joining (NHEJ), homology-dependent repair (HDR) or microhomology-mediated end-joining (MMEJ) pathways. This facile RNA-guided tool is extensively used to characterize gene functions and traits of agricultural importance. Here, we discuss the reports of genome editing using CRISPR/Cas system in five important legume species, viz. birdsfoot trefoil (Lotus japonicus), barrel medic (Medicago truncatula), alfalfa (Medicago sativa), soya bean (Glycine max) and cowpea (Vigna unguiculata), and some common web-based tools for designing reagents for editing. In birdsfoot trefoil, an efficient editing of representative symbiotic nitrogen-fixing genes via. Agrobacterium-mediated stable or hairy root transformation has been reported. Notably, this tool was used to decipher the synergistic roles of leghaemoglobin genes in SNF. In barrel medic, the amenability of CRISPR/Cas system was demonstrated for genes/loci using phytoene desaturase (PDS); both homoallelic and biallelic mutants were reported with albino phenotype. Subsequently, editing genes for small RNA processing were also demonstrated to be heritable in nature. In polyploid alfalfa, successful CRISPR/Cas-mediated genome editing was reported for traits like hyperaccumulation of phosphate and regulators of plant developmental pathway. In cowpea, the disruption of symbiotic nitrogen fixation gene, symbiosis receptor-like kinase (SYMRK), in hairy root-transformed plant exhibits a complete blockage of nodule formation. The extensively worked-out legume is soya bean, where endogenous and exogenous genes/loci were amenable to genome editing via. hairy root composite plants, protoplast and whole-plant transformation employing genetic engineering and RNP delivery routes. Besides several endogenous loci, genes for traits like plant architecture, SNF, early flowering, oil composition etc. were reported to be edited. Possibilities of HDR, multiplexing and base editing were also demonstrated. Gene-edited high-oleic soya bean oil (Calyno™) developed by Calyxt, Inc is the first gene-edited food released for consumers in the USA. Current research efforts should be focused to translate the findings in model legumes to legumes of local importance, so that the benefit of the technology can be harnessed.

The Fabaceae (legume family) is the third largest and the second of agricultural importance among flowering plant groups. In this study, we report the reconstruction of a composite comparative map composed of ten legume genomes, including seven species from the galegoid clade (Medicago truncatula, Medicago sativa, Lens culinaris, Pisum sativum, Lotus japonicus, Cicer arietinum, Vicia faba) and three species from the phaseoloid clade (Vigna radiata, Phaseolus vulgaris, Glycine max). To accomplish this comparison, a total of 209 cross-species gene-derived markers were employed. The comparative analysis resulted in a single extensive genetic/genomic network composed of 93 chromosomes or linkage groups, from which 110 synteny blocks and other evolutionary events (e.g., 13 inversions) were identified. This comparative map also allowed us to deduce several large scale evolutionary events, such as chromosome fusion/fission, with which might explain differences in chromosome numbers among compared species or between the two clades. As a result, useful properties of cross-species genic markers were re-verified as an efficient tool for cross-species translation of genomic information, and similar approaches, combined with a high throughput bioinformatic marker design program, should be effective for applying the knowledge of trait-associated genes to other important crop species for breeding purposes. Here, we provide a basic comparative framework for the ten legume species, and expect to be usefully applied towards the crop improvement in legume breeding.

The early response to dehydration (ERD) gene family plays a central role in abiotic stress regulation, plant development and defense mechanisms, particularly under dehydration conditions. However, the genomic characterization and functional roles of ERD genes in alfalfa (Medicago sativa) remain largely unexplored. This study aims to identify and analyze ERD genes in alfalfa to understand their evolutionary conservation and involvement in stress responses. A total of 27 ERD genes were identified and categorized into five distinct phylogenetic groups (Group-1 to Group-5) based on comparative analysis with homologs from Glycine max, Medicago truncatula, Lotus japonicus and Arabidopsis thaliana. Comprehensive analyses of gene structure, chromosomal localization, conserved protein motifs, cis-elements, synteny and transcriptomic profiles were conducted to uncover the structural and functional attributes of the MsERD genes. Notably, the results indicated that MsERD2, MsERD15 and MsERD27 were significantly overexpressed in response to low selenium concentrations and salt stress mediated by melatonin pathways. Evidence of segmental duplication and motif conservation implies evolutionary conservation and potential functional redundancy. Differential expression patterns in leaf tissues emphasize their roles in adaptive stress responses. This comprehensive genomic and expression study provides valuable insights into the molecular regulation of ERD genes in alfalfa. The findings underscore the potential roles of specific MsERD genes in abiotic stress tolerance and mineral homeostasis, highlighting potential targets for the genetic improvement of stress-resilient alfalfa cultivars.

Sucrose metabolism plays a critical role in development, stress response, and yield formation of plants. Sucrose phosphate synthase (SPS) is the key rate-limiting enzyme in the sucrose synthesis pathway. To date, genome-wide survey and comprehensive analysis of the SPS gene family in soybean (Glycine max) have yet to be performed. In this study, seven genes encoding SPS were identified in soybean genome. The structural characteristics, phylogenetics, tissue expression patterns, and cold stress response of these GmSPSs were investigated. A comparative phylogenetic analysis of SPS proteins in soybean, Medicago truncatula, Medicago sativa, Lotus japonicus, Arabidopsis, and rice revealed four families. GmSPSs were clustered into three families from A to C, and have undergone five segmental duplication events under purifying selection. All GmSPS genes had various expression patterns in different tissues, and family A members GmSPS13/17 were highly expressed in nodules. Remarkably, all GmSPS promoters contain multiple low-temperature-responsive elements such as potential binding sites of inducer of CBF expression 1 (ICE1), the central regulator in cold response. qRT-PCR proved that these GmSPS genes, especially GmSPS8/18, were induced by cold treatment in soybean leaves, and the expression pattern of GmICE1 under cold treatment was similar to that of GmSPS8/18. Further transient expression analysis in Nicotiana benthamiana and electrophoretic mobility shift assay (EMSA) indicated that GmSPS8 and GmSPS18 transcriptions were directly activated by GmICE1. Taken together, our findings may aid in future efforts to clarify the potential roles of GmSPS genes in response to cold stress in soybean.

A legume specific protein database (LegProt) improves the number of identified peptides, confidence scores and overall protein identification success rates for legume proteomics

Polycomb group (PcG) proteins, which are important epigenetic regulators, play essential roles in the regulatory networks involved in plant growth, development, and environmental stress responses. Currently, as far as we know, no comprehensive and systematic study has been carried out on the PcG family in Medicago truncatula. In the present study, we identified 64 PcG genes with distinct gene structures from the M. truncatula genome. All of the PcG genes were distributed unevenly over eight chromosomes, of which 26 genes underwent gene duplication. The prediction of protein interaction network indicated that 34 M. truncatula PcG proteins exhibited protein–protein interactions, and MtMSI1;4 and MtVRN2 had the largest number of protein–protein interactions. Based on phylogenetic analysis, we divided 375 PcG proteins from 27 species into three groups and nine subgroups. Group I and Group III were composed of five components from the PRC1 complex, and Group II was composed of four components from the PRC2 complex. Additionally, we found that seven PcG proteins in M. truncatula were closely related to the corresponding proteins of Cicer arietinum. Syntenic analysis revealed that PcG proteins had evolved more conservatively in dicots than in monocots. M. truncatula had the most collinearity relationships with Glycine max (36 genes), while collinearity with three monocots was rare (eight genes). The analysis of various types of expression data suggested that PcG genes were involved in the regulation and response process of M. truncatula in multiple developmental stages, in different tissues, and for various environmental stimuli. Meanwhile, many differentially expressed genes (DEGs) were identified in the RNA-seq data, which had potential research value in further studies on gene function verification. These findings provide novel and detailed information on the M. truncatula PcG family, and in the future it would be helpful to carry out related research on the PcG family in other legumes.

Deciphering the mechanisms leading to symbiotic nitrogen-fixing root nodule organogenesis in legumes resulted in the identification of numerous nodule-specific genes and gene families. Among them, NCR and GRP genes encode short secreted peptides with potential antimicrobial activity. These genes appear to form large multigenic families in Medicago truncatula and other closely related legume species, whereas no similar genes were found in databases of Lotus japonicus and Glycine max. We analyzed the genomic organization of these genes as well as their evolutionary dynamics in the M. truncatula genome. A total of 108 NCR and 23 GRP genes have been mapped that were often clustered in the genome. These included 29 new NCR and 17 new GRP genes. Reverse transcription-polymerase chain reaction analyses of the novel genes confirmed their exclusive nodule-specific expression similar to the previously identified members. Protein alignments and phylogenetic analyses revealed traces of several duplication events in the history of GRP and NCR genes. Moreover, microsyntenic evidences between M. truncatula and L. japonicus validated the hypothesis that these genes are specific for the inverted repeat-lacking clade of hologalegoid legumes, which allowed dating the appearance of these two gene families during the evolution of legume plants.