Morphological characterisation and genetic analysis of a bi-pistil mutant (bip) in Medicago truncatula Gaertn.

A floral organ mutant was observed in transgenic Medicago truncatula Gaertn. plants that had two separate stigmas borne on two separate styles that emerged from a single superior carpel primordium. We propose the name bi-pistil, bip for the mutation. We believe this is the first report of such a mutation in this species. Genetic and molecular analyses of the mutant were conducted. The mutant plant was crossed to a mtapetala plant with a wild-type pistil. Expression of the mutant trait in the F₁ and F₂ generations indicates that the bi-pistil trait is under the control of a single recessive gene. Other modifying genes may influence its expression. The mutation was associated with the presence of a T-DNA insert consisting of the Alfalfa mosaic virus (AMV) coat protein gene in antisense orientation and the nptII selectable marker gene. It is suggested that the mutation is due to gene disruption because multiple copies of the T-DNA were observed in the mutant. The bi-pistil gene was found to be independent of the male-sterile gene, tap. This novel mutant may assist in understanding pistil development in legumes.     

Medicago truncatula Mtha1-2 mutants loose metabolic responses to mycorrhizal colonization

Bidirectional nutrient transfer is one of the key features of the arbuscular mycorrhizal symbiosis. Recently we were able to identify a Medicago truncatula mutant (mtha1-2) that is defective in the uptake of phosphate from the periarbuscular space due to a lack of the energy providing proton gradient provided by the symbiosis specific proton ATPase MtHA1¹ In order to further characterize the impact of fungal colonization on the plant metabolic status, without the beneficial aspect of improved mineral nutrition, we performed leaf ion analyses in mutant and wildtype plants with and without fungal colonization. Although frequency of fungal colonization was unaltered, the mutant did not show a positive growth response to mycorrhizal colonization. This indicates that nutrient transfer into the plant cell fails in the truncated arbuscules due to lacking expression of a functional MtHA1 protein. The leaves of wildtype plants showed clear metabolic responses to root mycorrhizal colonization, whereas no changes of leaf metabolite levels of mycorrhizal mtha1-2 plants were detected, even though they were colonized. These results show that MtHa1 is indispensable for a functional mycorrhizal symbiosis and, moreover, suggest that fungal root colonization per se does not depend on nutrient transfer to the plant host.     

Impact of sewage sludges on Medicago truncatula symbiotic proteome

The effects of sewage sludges were investigated on the symbiotic interactions between the model plant Medicago truncatula and the arbuscular mycorrhizal fungus Glomus mosseae or the rhizobial bacteria Sinorhizobium meliloti. By comparison to a control sludge showing positive effects on plant growth and root symbioses, sludges enriched with polycylic aromatic hydrocarbons or heavy metals were deleterious. Symbiosis-related proteins were detected and identified by two-dimensional electrophoresis and matrix-assisted laser desorption ionization mass spectrometry, and image analysis was used to study the effects of sewage sludges on M. truncatula symbiotic proteome.     

Two Distinct Plastid Genome Configurations and Unprecedented Intraspecies Length Variation in the accD Coding Region in Medicago truncatula

We fully sequenced four and partially sequenced six additional plastid genomes of the model legume Medicago truncatula. Three accessions, Jemalong 2HA, Borung and Paraggio, belong to ssp. truncatula, and R108 to ssp. tricycla. We report here that the R108 ptDNA has a ∼45-kb inversion compared with the ptDNA in ssp. truncatula, mediated by a short, imperfect repeat. DNA gel blot analyses of seven additional ssp. tricycla accessions detected only one of the two alternative genome arrangements, represented by three and four accessions each. Furthermore, we found a variable number of repeats in the essential accD and ycf1 coding regions. The repeats within accD are recombinationally active, yielding variable-length insertions and deletions in the central part of the coding region. The length of ACCD was distinct in each of the 10 sequenced ecotypes, ranging between 650 and 796 amino acids. The repeats in the ycf1 coding region are also recombinationally active, yielding short indels in 10 regions of the reading frames. Thus, the plastid genome variability we report here could be linked to repeat-mediated genome rearrangements. However, the rate of recombination was sufficiently low, so that no heterogeneity of ptDNA could be observed in populations maintained by single-seed descent.     

Simulating the impact of genetic diversity of Medicago truncatula on germination and emergence using a crop emergence model for ideotype breeding

Background and Aims

Germination and heterotrophic growth are crucial steps for stand establishment. Numerical experiments based on the modelling of these early stages in relation to major environmental factors at sowing were used as a powerful tool to browse the effects of the genetic diversity of Medicago truncatula, one of the model legume species, under a range of agronomic scenarios, and to highlight the most important plant parameters for emergence. To this end, the emergence of several genotypes of M. truncatula was simulated under a range of sowing conditions with a germination and emergence simulation model.

Methods

After testing the predictive quality of the model by comparing simulations to field observations of several genotypes of M. truncatula, numerical experiments were performed under a wide range of environmental conditions (sowing dates × years × seedbed structure). Germination and emergence was simulated for a set of five genotypes previously parameterized and for two virtual genotypes engineered to maximize the potential effects of genetic diversity.

Key Results

The simulation results gave an average value of 5–10 % difference in final emergence between genotypes, which was low, but the analysis underlined considerable inter-annual variation. The effects of parameters describing germination and emergence processes were quantified and ranked according to their contribution to the variation in emergence. Seedling non-emergence was mainly related to mechanical obstacles (40–50 %). More generally, plant parameters that accelerated the emergence time course significantly contributed to limiting the risk of soil surface crusting occurring before seedling emergence.

Conclusions

The model-assisted analysis of the effects of genetic diversity demonstrated its usefulness in helping to identify the parameters which have most influence that could be improved by breeding programmes. These results should also enable a deeper analysis of the genetic determinism of the main plant parameters influencing emergence, using the genomic tools available for this model plant.     

Recent Progress in Development of Tnt1 Functional Genomics Platform for Medicago truncatula and Lotus japonicus in Bulgaria

Legumes, as protein-rich crops, are widely used for human food, animal feed and vegetable oil production. Over the past decade, two legume species, Medicago truncatula and Lotus japonicus, have been adopted as model legumes for genomics and physiological studies. The tobacco transposable element, Tnt1, is a powerful tool for insertional mutagenesis and gene inactivation in plants. A large collection of Tnt1-tagged lines of M. truncatula cv. Jemalong was generated during the course of the project 'GLIP': Grain Legumes Integrated Project, funded by the European Union (www.eugrainlegumes.org). In the project 'IFCOSMO': Integrated Functional and COmparative genomics Studies on the MOdel Legumes Medicago truncatula and Lotus japonicus, supported by a grant from the Ministry of Education, Youth and Science, Bulgaria, these lines are used for development of functional genomics platform of legumes in Bulgaria. This review presents recent advances in the evaluation of the M. truncatula Tnt1 mutant collection and outlines the steps that are taken in using the Tnt1-tagging for generation of a mutant collection of the second model legume L. japonicus. Both collections will provide a number of legume-specific mutants and serve as a resource for functional and comparative genomics research on legumes. Genomics technologies are expected to advance genetics and breeding of important legume crops (pea, faba bean, alfalfa and clover) in Bulgaria and worldwide.     

Evolutionary history of mitogen-activated protein kinase (MAPK) genes in Lotus,Medicago, and Phaseolus

Mitogen-Activated Protein Kinase (MAPK) genes encode proteins that mediate various signaling pathways associated with biotic and abiotic stress responses in eukaryotes. The MAPK genes form a 3-tier signal transduction cascade between cellular stimuli and physiological responses. Recent identification of soybean MAPKs and availability of genome sequences from other legume species allowed us to identify their MAPK genes. The main objectives of this study were to identify MAPKs in 3 legume species, Lotus japonicus, Medicago truncatula, and Phaseolus vulgaris, and to assess their phylogenetic relationships. We used approaches in comparative genomics for MAPK gene identification and named the newly identified genes following Arabidopsis MAPK nomenclature model. We identified 19, 18, and 15 MAPKs and 7, 4, and 9 MAPKKs in the genome of Lotus japonicus, Medicago truncatula, and Phaseolus vulgaris, respectively. Within clade placement of MAPKs and MAPKKs in the 3 legume species were consistent with those in soybean and Arabidopsis. Among 5 clades of MAPKs, 4 founder clades were consistent to MAPKs of other plant species and orthologs of MAPK genes in the fifth clade-"Clade E" were consistent with those in soybean. Our results also indicated that some gene duplication events might have occurred prior to eudicot-monocot divergence. Highly diversified MAPKs in soybean relative to those in 3 other legume species are attributable to the polyploidization events in soybean. The identification of the MAPK genes in the legume species is important for the legume crop improvement; and evolutionary relationships and functional divergence of these gene members provide insights into plant genome evolution.     

Sub-cellular proteomic analysis of a Medicago truncatula root microsomal fraction

Since the last decade, Medicago truncatula has emerged as one of the model plants particularly investigated in the field of plant-microbe interactions. Several genetic and molecular approaches including proteomics have been developed to increase knowledge about this plant species. To complement the proteomic data, which have mainly focused on the total root proteins from M. truncatula, we carried out a sub-cellular approach to gain access to the total membrane-associated proteins. Following the setting up of the purification process, microsomal proteins were separated on 2-DE. Ninety-six out of the 440 well-resolved proteins were identified by MALDI-TOF peptide mass fingerprinting. A high percent (83%) of successful protein identification was obtained when using M. truncatula clustered EST database for queries. During the purification process, the enrichment in membrane-associated proteins was monitored on 2-D gels. The membrane location of microsomal proteins was further confirmed using PMF identification. This study reports a fractionation process for characterizing microsomal root proteins of M. truncatula, which could be an interesting tool for investigating the molecular mechanisms involved in root symbioses.     

Abscisic acid promotes pre-emergence stages of lateral root development in Medicago truncatula

The plant root system is important for plant anchorage and nutrition. Among the different characteristics of the root system, root branching is a major factor of plasticity and adaptation to changing environments. Indeed, many biotic and abiotic stresses, such as drought or symbiotic interactions, influence root branching. Many studies concerning root development and root branching were performed on the model plant Arabidopsis thaliana, but this model plant has a very simplified root structure and is not able to establish any symbiotic interactions. We have recently described 7 stages for lateral root development in the model legume Medicago truncatula and found significant differences in the tissular contribution of root cell layers to the formation of new lateral roots (LR). We have also described 2 transgenic lines expressing the DR5:GUS and DR5:VENUS-N7 reporter genes that are useful to follow LR formation at early developmental stages. Here, we describe the use of these transgenic lines to monitor LR developmental responses of M. truncatula to the phytohormone abscisic acid (ABA) which is a major actor of stress and symbiotic interactions. We show that ABA promotes the formation of new lateral root primordia and their development, mostly at the late, pre-emergence stage.     

Microtubule dynamics in root hairs of Medicago truncatula

The microtubular cytoskeleton plays an important role in the development of tip-growing plant cells, but knowledge about its dynamics is incomplete. In this study, root hairs of the legume Medicago truncatula have been chosen for a detailed analysis of microtubular cytoskeleton dynamics using GFP-MBD and EB1-YFP as markers and 4D imaging. The microtubular cytoskeleton appears mainly to be composed of bundles which form tracks along which new microtubules polymerise. Polymerisation rates of microtubules are highest in the tip of growing root hairs. Treatment of root hairs with Nod factor and latrunculin B result in a twofold decrease in polymerisation rate. Nonetheless, no direct, physical interaction between the actin filament cytoskeleton and microtubules could be observed. A new picture of how the plant cytoskeleton is organised in apically growing root hairs emerges from these observations, revealing similarities with the organisation in other, non-plant, tip-growing cells.     

MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules

Most techniques used to study small molecules, such as pharmaceutical drugs or endogenous metabolites, employ tissue extracts which require the homogenization of the tissue of interest that could potentially cause changes in the metabolic pathways being studied¹. Mass spectrometric imaging (MSI) is a powerful analytical tool that can provide spatial information of analytes within intact slices of biological tissue samples^1-5. This technique has been used extensively to study various types of compounds including proteins, peptides, lipids, and small molecules such as endogenous metabolites. With matrix-assisted laser desorption/ionization (MALDI)-MSI, spatial distributions of multiple metabolites can be simultaneously detected. Herein, a method developed specifically for conducting untargeted metabolomics MSI experiments on legume roots and root nodules is presented which could reveal insights into the biological processes taking place. The method presented here shows a typical MSI workflow, from sample preparation to image acquisition, and focuses on the matrix application step, demonstrating several matrix application techniques that are useful for detecting small molecules. Once the MS images are generated, the analysis and identification of metabolites of interest is discussed and demonstrated. The standard workflow presented here can be easily modified for different tissue types, molecular species, and instrumentation.     

The involvement of Medicago truncatula non-specific lipid transfer protein N5 in the control of rhizobial infection

Cysteine-rich proteins seem to play important regulatory roles in Medicago truncatula/Sinorhizobium meliloti symbiosis. In particular, a large family of nodule-specific cysteine-rich (NCR) peptides is crucial for the differentiation of nitrogen-fixing bacteroids. The Medicago truncatula N5 protein (MtN5) is currently the only reported non-specific lipid transfer protein necessary for successful rhizobial symbiosis; in addition, MtN5 shares several characteristics with NCR peptides: a small size, a conserved cysteine-rich motif, an N-terminal signal peptide for secretion and antimicrobial activity. Unlike NCR peptides, MtN5 expression is not restricted to the root nodules and is induced during the early phases of symbiosis in root hairs and nodule primordia. Recently, MtN5 was determined to be involved in the regulation of root tissue invasion; while, it was dispensable for nodule primordia formation. Here, we discuss the hypothesis that MtN5 participates in linking the progression of bacterial invasion with restricting the competence of root hairs for infection.     

Abscisic acid rescues the root meristem defects of the Medicago truncatula latd mutant

The LATD gene of the model legume, Medicago truncatula, is required for the normal function of three meristems, i.e. the primary root, lateral roots and nitrogen-fixing nodules. In latd mutants, primary root growth eventually arrests, resulting in a disorganized root tip lacking a presumptive meristem and root cap columella cells. Lateral root organs are more severely affected; latd lateral roots and nodules arrest immediately after emerging from the primary root, and reveal a lack of organization. Here we show that the plant hormone, abscisic acid (ABA), can rescue the latd root, but not nodule, meristem defects. Growth on ABA is sufficient to restore formation of small, cytoplasm-rich cells in the presumptive meristem region, rescue meristem organization and root growth and formation of root cap columella cells. In contrast, inhibition of ethylene synthesis or signaling fails to restore latd primary root growth. We find that latd mutants have normal levels of ABA, but exhibit reduced sensitivity to the hormone in two other ABA-dependent processes: seed germination and stomatal closure. Together, these observations demonstrate that the latd mutant is defective in the ABA response and indicate a role for LATD-dependent ABA signaling in M. truncatula root meristem function.     

Haplometra cylindracea (Zeder, 1800) (Trematoda:Plagiorchiidae): variation in the dates of cercarial shedding for overwintering Galba truncatula

Natural infections of Galba truncatula with Haplometra cylindracea were followed from 2001 to 2009 to determine if their characteristics were similar when snails came from water collections frequented by Bufo bufo or by frogs and newts for their egg-laying. Snail samples were collected from both types of sites to count shed cercariae for three days and also free cercariae when snails were dissected. In sites only frequented by B. bufo, cercarial shedding occurred earlier than in those colonized by frogs and newts (March instead of April-May). In contrast, the number of cercariae shed during three successive days was significantly higher in May. This variation in the dates of cercarial shedding might be due, either to a synchronism between cercaria-releasing snails and the presence of the definitive host (tadpoles) in water collections, or to an earlier infection of overwintering snails in autumn by H. cylindracea miracidia in the case of toad-frequented sites.