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At least 19 recordsLinked to original sources

PubMed2002

DNA transfer by highly asymmetric somatic hybridisation in Medicago truncatula (+) Medicago rugosaand Medicago truncatula(+) Medicago scutellata.

A regenerable line of Medicago truncatula(Jemalong 2HA) as a recipient species, was fused with the sexually incompatible species Medicago scutellataor Medicago rugosa.The treatments described maintain the chromosome number of the recipient but enable the transfer of small amounts of DNA of the donor species, probably by intergenomic recombination. Without a chromosome number-change fusion products can readily regenerate to produce fertile plants; and potentially a library with a diverse array of new genetic material. The selection of fused cells is based on treatment of the recipient cells with iodoacetamide (IOA), a non-regenerable donor, gamma-irradiation of the donor, and regeneration on a medium favouring the recipient. DNA transfer was demonstrated by amplified fragment length polymorphism (AFLP), Southern hybridisation and changed morphology.

Journal Article↗

PubMed2006

Annotating the genome of Medicago truncatula.

Medicago truncatula will be among the first plant species to benefit from the completion of a whole-genome sequencing project. For each of these species, Arabidopsis, rice and now poplar and Medicago, annotation, the process of identifying gene structures and defining their functions, is essential for the research community to benefit from the sequence data generated. Annotation of the Arabidopsis genome involved gene-by-gene curation of the entire genome, but the larger genomes of rice, Medicago and other species necessitate the automation of the annotation process. Profiting from the experience gained from previous whole-genome efforts, a uniform set of Medicago gene annotations has been generated by coordinated international effort and, along with other views of the genome data, has been provided to the research community at several websites.

Automation↗

PubMed2006

A CDPK isoform participates in the regulation of nodule number in Medicago truncatula.

Medicago spp. are able to develop root nodules via symbiotic interaction with Sinorhizobium meliloti. Calcium-dependent protein kinases (CDPKs) are involved in various signalling pathways in plants, and we found that expression of MtCPK3, a CDPK isoform present in roots of the model legume Medicago truncatula, is regulated during the nodulation process. Early inductions were detected 15 min and 3-4 days post-inoculation (dpi). The very early induction of CPK3 messengers was also present in inoculated M. truncatula dmi mutants and in wild-type roots subjected to salt stress, indicating that this rapid response is probably stress-related. In contrast, the later response was concomitant with cortical cell division and the formation of nodule primordia, and was not observed in wild-type roots inoculated with nod (-) strains. This late induction correlated with a change in the subcellular distribution of CDPK activities. Accordingly, an anti-MtCPK3 antibody detected two bands in soluble root extracts and one in the particulate fraction. CPK3::GFP fusions are targeted to the plasma membrane in epidermal onion cells, a localization that depends on myristoylation and palmitoylation sites of the protein, suggesting a dual subcellular localization. MtCPK3 mRNA and protein were also up-regulated by cytokinin treatment, a hormone linked to the regulation of cortical cell division and other nodulation-related responses. An RNAi-CDPK construction was used to silence CPK3 in Agrobacterium rhizogenes-transformed roots. Although no major phenotype was detected in these roots, when infected with rhizobia, the total number of nodules was, on average, twofold higher than in controls. This correlates with the lack of MtCPK3 induction in the inoculated super-nodulator sunn mutant. Our results suggest that CPK3 participates in the regulation of the symbiotic interaction.

Calcium-Calmodulin-Dependent Protein Kinases↗

PubMed2003

Genomic organization of rDNA loci in natural populations of Medicago truncatula Gaertn.

Medicago truncatula Gaertn. is an annual self-pollinating species characterized by a diploid complement 2n = 16 and low DNA content. It responds very well to transformation methods so it is used as a model species for Leguminosae. In contrast with the advanced studies in molecular biology, cytogenetic research has remained limited even though it is an extremely valuable approach to the analysis of the genome structure. In the present study we examined the chromosomal distribution of rDNA sequences in five natural populations of M. truncatula, explored the genomic diversity of this species and found markers for chromosome identification. FISH experiments revealed three distribution patterns of rDNA sequences, distinguished by one, two and three loci of 5S genes; 18S-5.8S-25S genes were always localized at a single locus. The results add information to the genome structure of M. truncatula, revealing a pattern of distribution of rDNA genes unobserved previously, which consists of 5S genes clustered at a single locus. The physical mapping of rDNA sequences is a first contribution towards the construction of a detailed molecular karyotype of M. truncatula.

Chromosomes, Plant↗

PubMed2002

The Nod factor-elicited annexin MtAnn1 is preferentially localised at the nuclear periphery in symbiotically activated root tissues of Medicago truncatula.

The Medicago truncatula MtAnn1 gene, encoding a putative annexin, is transcriptionally activated in root tissues in response to rhizobial Nod factors. To gain further insight into MtAnn1 function during the early stages of nodulation, we have examined in detail both spatio-temporal gene expression patterns and MtAnn1 activity and localisation in root tissues. Analysis of transgenic Medicago plants expressing a pMtAnn1-GUS fusion has revealed a novel pattern of transcription in both outer and inner cell layers of the root following either Nod factor-treatment or rhizobial inoculation. The highest gene expression levels were observed in the endodermis and outer cortex. These transgenic plants also revealed that MtAnn1 expression is associated with lateral root development and cell differentiation in the root apex independent of nodulation. By purifying recombinant MtAnn1 we were able to demonstrate that this plant annexin indeed possesses the calcium-dependent binding to acidic phospholipids typical of the annexin family. Antisera against recombinant MtAnn1 were then used to show that tissue-specific localisation of the MtAnn1 protein in Medicago roots matches the pMtAnn1-GUS expression pattern. Finally, both immunolabelling and in vivo studies using MtAnn1-GFP reporter fusions have revealed that MtAnn1 is cytosolic and in particular localises to the nuclear periphery in cortical cells activated during the early stages of nodulation. In the light of our findings, we discuss the possible role of this annexin in root tissues responding to symbiotic rhizobial signals.

Annexin A1↗

PubMed2002

Genome-wide identification of nodule-specific transcripts in the model legume Medicago truncatula.

The Medicago truncatula expressed sequence tag (EST) database (Gene Index) contains over 140,000 sequences from 30 cDNA libraries. This resource offers the possibility of identifying previously uncharacterized genes and assessing the frequency and tissue specificity of their expression in silico. Because M. truncatula forms symbiotic root nodules, unlike Arabidopsis, this is a particularly important approach in investigating genes specific to nodule development and function in legumes. Our analyses have revealed 340 putative gene products, or tentative consensus sequences (TCs), expressed solely in root nodules. These TCs were represented by two to 379 ESTs. Of these TCs, 3% appear to encode novel proteins, 57% encode proteins with a weak similarity to the GenBank accessions, and 40% encode proteins with strong similarity to the known proteins. Nodule-specific TCs were grouped into nine categories based on the predicted function of their protein products. Besides previously characterized nodulins, other examples of highly abundant nodule-specific transcripts include plantacyanin, agglutinin, embryo-specific protein, and purine permease. Six nodule-specific TCs encode calmodulin-like proteins that possess a unique cleavable transit sequence potentially targeting the protein into the peribacteroid space. Surprisingly, 114 nodule-specific TCs encode small Cys cluster proteins with a cleavable transit peptide. To determine the validity of the in silico analysis, expression of 91 putative nodule-specific TCs was analyzed by macroarray and RNA-blot hybridizations. Nodule-enhanced expression was confirmed experimentally for the TCs composed of five or more ESTs, whereas the results for those TCs containing fewer ESTs were variable.

Agglutinins↗

PubMed2006

A standardized method for analysis of Medicago truncatula phenotypic development.

Medicago truncatula has become a model system to study legume biology. It is imperative that detailed growth characteristics of the most commonly used cultivar, line A17 cv Jemalong, be documented. Such analysis creates a basis to analyze phenotypic alterations due to genetic lesions or environmental stress and is essential to characterize gene function and its relationship to morphological development. We have documented morphological development of M. truncatula to characterize its temporal developmental growth pattern; developed a numerical nomenclature coding system that identifies stages in morphological development; tested the coding system to identify phenotypic differences under phosphorus (P) and nitrogen (N) deprivation; and created visual models using the L-system formalism. The numerical nomenclature coding system, based on a series of defined growth units, represents incremental steps in morphological development. Included is a decimal component dividing growth units into nine substages. A measurement component helps distinguish alterations that may be missed by the coding system. Growth under N and P deprivation produced morphological alterations that were distinguishable using the coding system and its measurement component. N and P deprivation resulted in delayed leaf development and expansion, delayed axillary shoot emergence and elongation, decreased leaf and shoot size, and altered root growth. Timing and frequency of flower emergence in P-deprived plants was affected. This numerical coding system may be used as a standardized method to analyze phenotypic variation in M. truncatula due to nutrient stress, genetic lesions, or other factors and should allow valid growth comparisons across geographically distant laboratories.

Chronology as Topic↗

PubMed2005

Development and composition of the seeds of nine genotypes of the Medicago truncatula species complex.

The seed development and composition of Medicago truncatula Gaertn., the new model plant for grain legumes, was studied using nine genotypes of the species complex: M. truncatula-Medicago littoralis (M. truncatula). The seed development of M. truncatula was very similar to that of other legumes, the only notable exception being the presence, in the mature seed, of an endosperm layer that is absent in grain legumes. During early embryogenesis and until mid-maturation, transient storage of starch occurred in the seed coat and embryo. This temporary storage probably contributed to the early development of the embryo and reserve synthesis. During maturation the synthesis and accumulation of proteins and oil took place at quasi-constant rates. Conversely oligosaccharides, mainly stachyose, were synthesised only during late maturation and at the beginning of desiccation. Proteins represented the major class of storage compounds and their average amino acid composition was found to be very close to that of pea and robust in various environmental conditions. Similar compositions between the two species and other grain legumes were also found for the fatty acids and the soluble sugars; most of these characters varied depending on the various environmental conditions used for seed production. All these similarities fully justify the use of M. truncatula as a model plant for genomic approaches to grain legume improvement. The major difference between M. truncatula seeds and European grain legume seeds resides in the nature of their carbon storage namely triacylglycerides for M. truncatula and starch for pea and faba bean.

Amino Acids↗

PubMed2003

Regiospecific hydroxylation of isoflavones by cytochrome p450 81E enzymes from Medicago truncatula.

Mining of Medicago truncatula EST databases and screening of a root cDNA library led to the identification of three cytochrome p450 81E subfamily members. Two were functionally characterized by expression in yeast. The recombinant enzymes in yeast microsomes utilized the same isoflavone substrates, but produced different products hydroxylated at the 2' and/or 3' positions of the B-ring. When transiently expressed in alfalfa leaves, green fluorescent protein (GFP) fusions of the isoflavone 2'- and 3'-hydroxylases localized to the endoplasmic reticulum. The isoflavone 2'-hydroxylase was functional when expressed in Arabidopsis. Differential tissue-specific and biotic/abiotic stress-dependent expression patterns were observed for the isoflavone 2'-hydroxylase and 3'-hydroxylase genes, suggesting differential involvement of 2'- and 3'-hydroxylated isoflavonoids in pathogen defense and insect-induced responses, respectively, in Medicago.

Amino Acid Sequence↗

PubMed2001

Guidelines for genetic nomenclature and community governance for the model legume Medicago truncatula.

At the 2nd Medicago meeting (a satellite of the 1999 IS-MPMI meeting in Amsterdam), investigators perceived a need for standardization of genetic nomenclature in Medicago truncatula, due to the rapid growth of research on this species in the past few years. Establishment of such standards grew out of discussions begun at this meeting and continued electronically throughout the M. truncatula community. The proposed standards presented here are the consensus results of those discussions. In addition to standards for gene nomenclature, a method for community governance and a website for cataloging gene names and submitting new ones are presented. The purpose of implementing these guidelines is to help maintain consistency in the literature, to avoid redundancy, to contribute to the accuracy of databases, and, in general, to aid the international collaborations that have made M. truncatula a model system for legume biology.

Genes, Bacterial↗

PubMed2004

Satellite repeats in the functional centromere and pericentromeric heterochromatin of Medicago truncatula.

Most eukaryotic centromeres contain long arrays of tandem repeats, with unit lengths of 150-300 bp. We searched for such repeats in the functional centromeres of the model legume Medicago truncatula (Medicago) accession Jemalong A17. To this end three repeats, MtR1, MtR2 and MtR3, were identified in 20 Mb of a low-pass, whole genome sequencing data set generated by a random shotgun approach. The nucleotide sequence composition, genomic organization and abundance of these repeats were characterized. Fluorescent in situ hybridization of these repeats on chromosomes at meiosis I showed that only the MtR3 repeat, encompassing stretches of 450 kb to more than 1.0 Mb, is located in the functional portion of all eight centromeres. MtR1 and MtR2 occupy distinct regions in pericentromeric heterochromatin. We also studied the presence and distribution of MtRs in Medicago accession R108-1, a genotype with a genome that is 20% smaller than that of Jemalong A17. We determined that while MtR3 is also centromeric on all pachytene bivalents in R108-1, MtR1 and MtR2 are not present in the R108 genome.

Base Sequence↗

PubMed2006

Medicago truncatula transformation using root explants.

Medicago truncatula is a forage crop that has been developed into a model legume. As a model plant, M. truncatula is particularly useful for the study of root endosymbiotic associations, including nodulation and mycorrhizal colonization. The development of different transformation methods is an important aspect for functional genomic studies in the species. This protocol describes an efficient system for generating transgenic plants from M. truncatula roots based on Agrobacterium tumefaciens-mediated transformation. Furthermore, the protocol can be easily adapted for recovering fertile transgenic plants from transformed hairy roots.

Agrobacterium tumefaciens↗

PubMed2005

A two-dimensional electrophoresis proteomic reference map and systematic identification of 1367 proteins from a cell suspension culture of the model legume Medicago truncatula.

The proteome of a Medicago truncatula cell suspension culture was analyzed using two-dimensional electrophoresis and nanoscale HPLC coupled to a tandem Q-TOF mass spectrometer (QSTAR Pulsar i) to yield an extensive protein reference map. Coomassie Brilliant Blue R-250 was used to visualize more than 1661 proteins, which were excised, subjected to in-gel trypsin digestion, and analyzed using nanoscale HPLC/MS/MS. The resulting spectral data were queried against a custom legume protein database using the MASCOT search engine. A total of 1367 of the 1661 proteins were identified with high rigor, yielding an identification success rate of 83% and 907 unique protein accession numbers. Functional annotation of the M. truncatula suspension cell proteins revealed a complete tricarboxylic acid cycle, a nearly complete glycolytic pathway, a significant portion of the ubiquitin pathway with the associated proteolytic and regulatory complexes, and many enzymes involved in secondary metabolism such as flavonoid/isoflavonoid, chalcone, and lignin biosynthesis. Proteins were also identified from most other functional classes including primary metabolism, energy production, disease/defense, protein destination/storage, protein synthesis, transcription, cell growth/division, and signal transduction. This work represents the most extensive proteomic description of M. truncatula suspension cells to date and provides a reference map for future comparative proteomic and functional genomic studies of the response of these cells to biotic and abiotic stress.

Amino Acid Sequence↗

PubMed2006

Medicago truncatula transformation using cotyledon explants.

Medicago truncatula has been developed into a model species for legumes. Large numbers of ESTs have been sequenced, and sequencing of the complete gene-rich space of the genotype Jemalong A17 is in progress. By using cotyledons as explants for Agrobacterium infection and direct shoot formation, this protocol allows for rapid production of transgenic plants from A17 and other genotypes. Transgenic plants can be regenerated and established in the greenhouse in only 3-4 mo after Agrobacterium-mediated transformation. Transformation frequency is in the range of 5-12%.

Agrobacterium tumefaciens↗

PubMed2006

MtHAP2-1 is a key transcriptional regulator of symbiotic nodule development regulated by microRNA169 in Medicago truncatula.

In the model legume Medicago truncatula, we identified a new transcription factor of the CCAAT-binding family, MtHAP2-1, for which RNA interference (RNAi) and in situ hybridization experiments indicate a key role during nodule development, possibly by controlling nodule meristem function. We could also show that MtHAP2-1 is regulated by microRNA169, whose overexpression leads to the same nodule developmental block as MtHAP2-1 RNAi constructs. The complementary expression pattern of miR169 and MtHAP2-1 and the phenotype of miR169-resistant MtHAP2-1 nodules strongly suggest, in addition, that the miR169-mediated restriction of MtHAP2-1 expression to the nodule meristematic zone is essential for the differentiation of nodule cells.

Base Sequence↗

PubMed2006

Characterization of alanine aminotransferase (AlaAT) multigene family and hypoxic response in young seedlings of the model legume Medicago truncatula.

Four alanine aminotransferases (AlaATs) are expressed in Medicago truncatula. In adult plants, two genes encoding mitochondrial isoforms m-AlaAT and alanine-glyoxylate aminotransferase (AGT), catalysing, respectively, reversible reactions of alanine/oxoglutarate<==>glutamate/pyruvate and alanine/glyoxylate<==>glycine/pyruvate, were expressed in roots, stems, and leaves. A gene encoding a cytosolic (c-AlaAT) isoform, catalysing the same reaction as m-AlaAT, was expressed specifically in leaves, while a gene encoding an isoform involved in branched chain amino acid metabolism was expressed in stems and roots. In young seedlings, only m-AlaAT and AGT were expressed in embryo axes. In hypoxic embryo axes, the amounts of transcript and putative protein of m-AlaAT (EC 2.6.1.2) increased while those of AGT (EC 2.6.1.44) decreased and in vivo enzyme activities changed as revealed by [(15)N]alanine and [(15)N]glutamate labelling. Under hypoxia, m-AlaAT catalysed only alanine synthesis while glutamate synthesis using alanine as amino donor was inhibited. As a result, alanine accumulated as the major amino acid in hypoxic seedlings instead of asparagine, in agreement with the involvement of the fermentative AlaAT pathway in hypoxia tolerance. Regulation of m-AlaAT at both the transcriptional and post-translational levels allowed for an increase in gene expression and orientation of the activity of the product of its transcription towards alanine synthesis under hypoxia. Labelling experiments showed that glycine synthesis occurred at the expense of either alanine or glutamate as amino donor, indicating that a glutamate-glyoxylate aminotransferase was operating together with AGT in Medicago truncatula seedlings. Both enzymes seemed to be inhibited by hypoxia, resulting in a very low amount of glycine in hypoxic seedlings.

Alanine↗

PubMed2005

Variation in rDNA locus number and position among legume species and detection of 2 linked rDNA loci in the model Medicago truncatula by FISH.

Within Fabaceae, legume species have a variable genome size, chromosome number, and ploidy level. The genome distribution of ribosomal genes, easily detectable by fluorescent in situ hybridization (FISH), is a good tool for anchoring physical and genetic comparative maps. The organisation of 45S rDNA and 5S loci was analysed by FISH in the 4 closely related species: Pisum sativum, Medicago truncatula, Medicago sativa (2 diploid taxa), and Lathyrus sativus. The 2 types of rDNA arrays displayed interspecific variation in locus number and location, but little intraspecific variation was detected. In the model legume, M. truncatula, the presence of 2 adjacent 45S rDNA loci was demonstrated, and the location of the rDNA loci was independent of the general evolution of the genome DNA. The different parameters relative to clustering of the rDNA loci in specific chromosome regions and the possible basis of rDNA instability are discussed.

DNA, Ribosomal↗

PubMed2000

Genetic analysis of calcium spiking responses in nodulation mutants of Medicago truncatula.

The symbiotic interaction between Medicago truncatula and Sinorhizobium meliloti results in the formation of nitrogen-fixing nodules on the roots of the host plant. The early stages of nodule formation are induced by bacteria via lipochitooligosaccharide signals known as Nod factors (NFs). These NFs are structurally specific for bacterium-host pairs and are sufficient to cause a range of early responses involved in the host developmental program. Early events in the signal transduction of NFs are not well defined. We have previously reported that Medicago sativa root hairs exposed to NF display sharp oscillations of cytoplasmic calcium ion concentration (calcium spiking). To assess the possible role of calcium spiking in the nodulation response, we analyzed M. truncatula mutants in five complementation groups. Each of the plant mutants is completely Nod- and is blocked at early stages of the symbiosis. We defined two genes, DMI1 and DMI2, required in common for early steps of infection and nodulation and for calcium spiking. Another mutant, altered in the DMI3 gene, has a similar mutant phenotype to dmi1 and dmi2 mutants but displays normal calcium spiking. The calcium behavior thus implies that the DMI3 gene acts either downstream of calcium spiking or downstream of a common branch point for the calcium response and the later nodulation responses. Two additional mutants, altered in the NSP and HCL genes, which show root hair branching in response to NF, are normal for calcium spiking. This system provides an opportunity to use genetics to study ligand-stimulated calcium spiking as a signal transduction event.

Calcium↗