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Sample records for da alfafa medicago

  1. Lucerne transient streak virus; a Recently Detected Virus Infecting Alfafa (Medicago sativa) in Central Saudi Arabia

    PubMed Central

    Raza, Ahmed; Al-Shahwan, Ibrahim M.; Abdalla, Omer A.; Al-Saleh, Mohammed A.; Amer, Mahmoud A.

    2017-01-01

    A survey was conducted to determine the status of Lucerne transient streak virus (LTSV) in three high-yielding alfalfa regions in central Saudi Arabia (Riyadh, Qassim, and Hail) during 2014. Three hundred and eight symptomatic alfalfa, and seven Sonchus oleraceus samples were collected. DAS-ELISA indicated that 59 of these samples were positive to LTSV. Two isolates of LTSV from each region were selected for molecular studies. RT-PCR confirmed the presence of LTSV in the selected samples using a specific primer pair. Percentage identity and homology tree comparisons revealed that all Saudi isolates were more closely related to each other but also closely related to the Canadian isolate-JQ782213 (97.1–97.6%) and the New Zealand isolate-U31286 (95.8–97.1%). Comparing Saudi isolates of LTSV with ten other sobemoviruses based on the coat protein gene sequences confirmed the distant relationship between them. Eleven out of fourteen plant species used in host range study were positive to LTSV. This is the first time to document that Trifolium alexandrinum, Nicotiana occidentalis, Chenopodium glaucum, and Lathyrus sativus are new host plant species for LTSV and that N. occidentalis being a good propagative host for it. PMID:28167887

  2. Lucerne transient streak virus; a Recently Detected Virus Infecting Alfafa (Medicago sativa) in Central Saudi Arabia.

    PubMed

    Raza, Ahmed; Al-Shahwan, Ibrahim M; Abdalla, Omer A; Al-Saleh, Mohammed A; Amer, Mahmoud A

    2017-02-01

    A survey was conducted to determine the status of Lucerne transient streak virus (LTSV) in three high-yielding alfalfa regions in central Saudi Arabia (Riyadh, Qassim, and Hail) during 2014. Three hundred and eight symptomatic alfalfa, and seven Sonchus oleraceus samples were collected. DAS-ELISA indicated that 59 of these samples were positive to LTSV. Two isolates of LTSV from each region were selected for molecular studies. RT-PCR confirmed the presence of LTSV in the selected samples using a specific primer pair. Percentage identity and homology tree comparisons revealed that all Saudi isolates were more closely related to each other but also closely related to the Canadian isolate-JQ782213 (97.1-97.6%) and the New Zealand isolate-U31286 (95.8-97.1%). Comparing Saudi isolates of LTSV with ten other sobemoviruses based on the coat protein gene sequences confirmed the distant relationship between them. Eleven out of fourteen plant species used in host range study were positive to LTSV. This is the first time to document that Trifolium alexandrinum, Nicotiana occidentalis, Chenopodium glaucum, and Lathyrus sativus are new host plant species for LTSV and that N. occidentalis being a good propagative host for it.

  3. CPSF30 in Medicago

    USDA-ARS?s Scientific Manuscript database

    CPSF30 is a component in 3’ RNA processing that has been well characterized in the model species Arabidopsis thaliana. In this study the gene for CPSF30 has been cloned out of Medicago truncatula and Medicago sativa. A large region within the gene exhibits significant conservation between A. thalian...

  4. CPSF30 in medicago

    USDA-ARS?s Scientific Manuscript database

    CPSF30 is a component in 3’ RNA processing that has been well characterized in the model species Arabidopsis thaliana. In this study the CPSF30 gene has been cloned out of Medicago truncatula and Medicago sativa. A large region within the gene exhibits significant conservation between A. thaliana an...

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

    PubMed

    Tian, D; Niu, C; Rose, R J

    2002-01-01

    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.

  6. Alfalfa (Medicago sativa L.).

    PubMed

    Fu, Chunxiang; Hernandez, Timothy; Zhou, Chuanen; Wang, Zeng-Yu

    2015-01-01

    Alfalfa (Medicago sativa L.) is a high-quality forage crop widely grown throughout the world. This chapter describes an efficient protocol that allows for the generation of large number of transgenic alfalfa plants by sonication-assisted Agrobacterium-mediated transformation. Binary vectors carrying different selectable marker genes that confer resistance to phosphinothricin (bar), kanamycin (npt II), or hygromycin (hph) were used to generate transgenic alfalfa plants. Intact trifoliates collected from clonally propagated plants in the greenhouse were sterilized with bleach and then inoculated with Agrobacterium strain EHA105. More than 80 % of infected leaf pieces could produce rooted transgenic plants in 4-5 months after Agrobacterium-mediated transformation.

  7. Inhibition of the catalase activity from Phaseolus vulgaris and Medicago sativa by sodium chloride.

    PubMed

    Tejera García, Noel A; Iribarne, Carmen; Palma, Francisco; Lluch, Carmen

    2007-08-01

    Changes in catalase activity during the development of the Rhizobium-legume symbiosis as well as its response in salinized plants of Phaseolus vulgaris and Medicago sativa, was studied. Besides, it was examined the behavior of the enzyme, isolated from leaves and root nodules, during in vitro incubation with NaCl doses. Nodule catalase activities of both legumes were assayed with several enzyme inhibitors and also purified. Leaf catalase activity of Phaseolus vulgaris and Medicago sativa decreased and increased respectively throughout the ontogeny, but root nodule catalase kept a high and stable value. This last result suggests that both legumes require the maintenance of high nodule catalase in nitrogen-fixing nodules. Under salt stress conditions leaf and nodule catalase activity decreased in both, grain and pasture legumes. Because catalase from leaf of Medicago sativa and nodules of Phaseolus vulgaris were relatively sensitive to NaCl during in vitro experiments, the detoxifying role of this enzyme for H(2)O(2) should be limited in such conditions. Both catalases, from determinate and indeterminate nodules, were affected neither by oxygen nor superoxide radicals but showed a strong (Phaseolus vulgaris) or partial (Medicago sativa) inhibition with dithiothreitol, dithionite and beta-mercaptoethanol. Besides, cyanide was the most potent inhibitor of nodule catalases. Finally, catalases partially purified by immobilized metal ion affinity chromatography migrated at 42 (Phaseolus vulgaris) and 46kDa (Medicago sativa) on SDS-PAGE, whereas native forms on sephacryl S-300 columns exhibited a molecular mass of 59 and 48kDa (Phaseolus vulgaris) and 88 and 53kDa (Medicago sativa).

  8. G-protein from Medicago sativa: functional association to photoreceptors.

    PubMed Central

    Muschietti, J P; Martinetto, H E; Coso, O A; Farber, M D; Torres, H N; Flawia, M M

    1993-01-01

    G-protein subunits were characterized from Medicago sativa (alfalfa) seedlings. Crude membranes and GTP-Sepharose-purified fractions were electrophoresed on SDS/polyacrylamide gels and analysed by Western blotting with 9193 (anti-alpha common) and AS/7 (anti-alpha t, anti-alpha i1 and anti-alpha i2) polyclonal antibodies. These procedures led to the identification of a specific polypeptide band of about 43 kDa. Another polypeptide reacting with the SW/1 (anti-beta) antibody, of about 37 kDa, was also detected. The 43 kDa polypeptide bound specifically [alpha-32P]GTP by a photoaffinity reaction and was ADP-ribosylated by activated cholera toxin, but not by pertussis toxin. Irradiation of etiolated Medicago sativa protoplast preparations at 660 nm for 1 min produced a maximal increase in the guanosine 5'-[gamma-thio]triphosphate (GTP[35S])-binding rate. After this period of irradiation, the binding rate tended to decrease. The effect of a red-light (660 nm) pulse on the binding rate was reversed when it was immediately followed by a period of far-red (> 730 nm) illumination. These results may suggest that activation of GTP[S]-binding rate was a consequence of conversion of phytochrome Pr into the Ptr form. Images Figure 1 Figure 2 Figure 3 PMID:8484719

  9. MTGD: The Medicago truncatula genome database.

    PubMed

    Krishnakumar, Vivek; Kim, Maria; Rosen, Benjamin D; Karamycheva, Svetlana; Bidwell, Shelby L; Tang, Haibao; Town, Christopher D

    2015-01-01

    Medicago truncatula, a close relative of alfalfa (Medicago sativa), is a model legume used for studying symbiotic nitrogen fixation, mycorrhizal interactions and legume genomics. J. Craig Venter Institute (JCVI; formerly TIGR) has been involved in M. truncatula genome sequencing and annotation since 2002 and has maintained a web-based resource providing data to the community for this entire period. The website (http://www.MedicagoGenome.org) has seen major updates in the past year, where it currently hosts the latest version of the genome (Mt4.0), associated data and legacy project information, presented to users via a rich set of open-source tools. A JBrowse-based genome browser interface exposes tracks for visualization. Mutant gene symbols originally assembled and curated by the Frugoli lab are now hosted at JCVI and tie into our community annotation interface, Medicago EuCAP (to be integrated soon with our implementation of WebApollo). Literature pertinent to M. truncatula is indexed and made searchable via the Textpresso search engine. The site also implements MedicMine, an instance of InterMine that offers interconnectivity with other plant 'mines' such as ThaleMine and PhytoMine, and other model organism databases (MODs). In addition to these new features, we continue to provide keyword- and locus identifier-based searches served via a Chado-backed Tripal Instance, a BLAST search interface and bulk downloads of data sets from the iPlant Data Store (iDS). Finally, we maintain an E-mail helpdesk, facilitated by a JIRA issue tracking system, where we receive and respond to questions about the website and requests for specific data sets from the community. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  10. Genomic relationships between Medicago murex Willd. and Medicago lesinsii E. Small. investigated by in situ hybridization.

    PubMed

    Falistocco, E.; Torricelli, R.; Falcinelli, M.

    2002-11-01

    Medicago murex Willd. is an annual species (2n = 14) widespread in the wild and of remarkable interest for pastures in regions with a mediterranean climate. It is considered closely related to Medicago lesinsii E. Small (2n = 16) but, up to now, there is no evidence demonstrating their genetic affinity. This research was undertaken to investigate the genomic relationships between M. murex and M. lesinsii by using genomic in situ hybridization (GISH). In this study GISH experiments were performed using both species as sources of chromosomes and genomic probes. To better evaluate the results of the hybridization, the labelled DNA of each species was hybridized to chromosomes of the same species and to chromosomes of the diploid Medicago littoralis (2n = 16). Strong hybridization signals were found on chromosomes of M. murex and M. lesinsii after GISH. Differences in the hybridization strength were not observed when slides from interspecific hybridization were compared with the control preparations. These results suggest that consistent divergences of the DNA sequences did not occur after the separation of the two species. Instead very reduced cross hybridization was found on chromosome spreads of M. littoralis hybridized with the DNA of M. lesinsii or M. murex. The distribution of the ribosomal genes (rDNA) investigated by fluorescent in situ hybridization (FISH) appeared similar in both M. murex and M. lesinsii. The GISH technique may be a valuable approach to obtain information on evolution of the 2n = 14 species and on the origin of the polyploids Medicago rugosa (2n = 30) and Medicago scutellata (2n = 30). The first attempt to investigate the genomic composition of M. scutellata using a genomic probe is reported in this paper.

  11. Fate map of Medicago truncatula root nodules.

    PubMed

    Xiao, Ting Ting; Schilderink, Stefan; Moling, Sjef; Deinum, Eva E; Kondorosi, Eva; Franssen, Henk; Kulikova, Olga; Niebel, Andreas; Bisseling, Ton

    2014-09-01

    Legume root nodules are induced by N-fixing rhizobium bacteria that are hosted in an intracellular manner. These nodules are formed by reprogramming differentiated root cells. The model legume Medicago truncatula forms indeterminate nodules with a meristem at their apex. This organ grows by the activity of the meristem that adds cells to the different nodule tissues. In Medicago sativa it has been shown that the nodule meristem is derived from the root middle cortex. During nodule initiation, inner cortical cells and pericycle cells are also mitotically activated. However, whether and how these cells contribute to the mature nodule has not been studied. Here, we produce a nodule fate map that precisely describes the origin of the different nodule tissues based on sequential longitudinal sections and on the use of marker genes that allow the distinction of cells originating from different root tissues. We show that nodule meristem originates from the third cortical layer, while several cell layers of the base of the nodule are directly formed from cells of the inner cortical layers, root endodermis and pericycle. The latter two differentiate into the uninfected tissues that are located at the base of the mature nodule, whereas the cells derived from the inner cortical cell layers form about eight cell layers of infected cells. This nodule fate map has then been used to re-analyse several mutant nodule phenotypes. This showed, among other things, that intracellular release of rhizobia in primordium cells and meristem daughter cells are regulated in a different manner.

  12. Heteroplasmy of chloroplast DNA in Medicago.

    PubMed

    Johnson, L B; Palmer, J D

    1989-01-01

    Two chloroplast DNA (cpDNA) regions exhibiting a high frequency of intra- or inter-species variation were identified in 12 accessions of the genus Medicago. Restriction maps of both regions were prepared for alfalfa, and the probable nature of the events causing the DNA differences was identified. Specific DNA fragments were then cloned for use in identification of variants in each region. Two each of M. sativa ssp. varia and ssp. caerulea and one of six M. sativa ssp. sativa single plants examined possessed cpDNA heterogeneity as identified by screening extracts for fragments generated by the presence and absence of a specific Xba I restriction site. Three plants of M. sativa ssp. sativa, two of each of sspp. varia and caerulea, and three M. scutellata were also examined for single-plant cpDNA heterogeneity at a hypervariable region where differences resulted from small insertion-deletion events. A single M. scutellata plant with mixed cpDNAs was identified. Sorting out was seen when one spp. sativa plant with mixed plastid types identifiable by the Xba I restriction site difference was vegetatively propagated. This indicated that the initial stock plant was heteroplastidic. Controlled crosses will be required in order to test whether heteroplasmy results from chloroplast transmission in the pollen and to examine the dynamic of sorting out. However, heteroplasmy is apparently not a rare situation in Medicago.

  13. Recent Advances in Medicago truncatula Genomics

    PubMed Central

    Ané, Jean-Michel; Zhu, Hongyan; Frugoli, Julia

    2008-01-01

    Legume rotation has allowed a consistent increase in crop yield and consequently in human population since the antiquity. Legumes will also be instrumental in our ability to maintain the sustainability of our agriculture while facing the challenges of increasing food and biofuel demand. Medicago truncatula and Lotus japonicus have emerged during the last decade as two major model systems for legume biology. Initially developed to dissect plant-microbe symbiotic interactions and especially legume nodulation, these two models are now widely used in a variety of biological fields from plant physiology and development to population genetics and structural genomics. This review highlights the genetic and genomic tools available to the M. truncatula community. Comparative genomic approaches to transfer biological information between model systems and legume crops are also discussed. PMID:18288239

  14. Recent Advances in Medicago truncatula Genomics.

    PubMed

    Ané, Jean-Michel; Zhu, Hongyan; Frugoli, Julia

    2008-01-01

    Legume rotation has allowed a consistent increase in crop yield and consequently in human population since the antiquity. Legumes will also be instrumental in our ability to maintain the sustainability of our agriculture while facing the challenges of increasing food and biofuel demand. Medicago truncatula and Lotus japonicus have emerged during the last decade as two major model systems for legume biology. Initially developed to dissect plant-microbe symbiotic interactions and especially legume nodulation, these two models are now widely used in a variety of biological fields from plant physiology and development to population genetics and structural genomics. This review highlights the genetic and genomic tools available to the M. truncatula community. Comparative genomic approaches to transfer biological information between model systems and legume crops are also discussed.

  15. The Medicago truncatula gene expression atlas web server

    PubMed Central

    2009-01-01

    Background Legumes (Leguminosae or Fabaceae) play a major role in agriculture. Transcriptomics studies in the model legume species, Medicago truncatula, are instrumental in helping to formulate hypotheses about the role of legume genes. With the rapid growth of publically available Affymetrix GeneChip Medicago Genome Array GeneChip data from a great range of tissues, cell types, growth conditions, and stress treatments, the legume research community desires an effective bioinformatics system to aid efforts to interpret the Medicago genome through functional genomics. We developed the Medicago truncatula Gene Expression Atlas (MtGEA) web server for this purpose. Description The Medicago truncatula Gene Expression Atlas (MtGEA) web server is a centralized platform for analyzing the Medicago transcriptome. Currently, the web server hosts gene expression data from 156 Affymetrix GeneChip® Medicago genome arrays in 64 different experiments, covering a broad range of developmental and environmental conditions. The server enables flexible, multifaceted analyses of transcript data and provides a range of additional information about genes, including different types of annotation and links to the genome sequence, which help users formulate hypotheses about gene function. Transcript data can be accessed using Affymetrix probe identification number, DNA sequence, gene name, functional description in natural language, GO and KEGG annotation terms, and InterPro domain number. Transcripts can also be discovered through co-expression or differential expression analysis. Flexible tools to select a subset of experiments and to visualize and compare expression profiles of multiple genes have been implemented. Data can be downloaded, in part or full, in a tabular form compatible with common analytical and visualization software. The web server will be updated on a regular basis to incorporate new gene expression data and genome annotation, and is accessible at: http

  16. Stress responses in alfalfa (Medicago sativa L. )

    SciTech Connect

    Kessmann, H.; Edwards, R.; Dixon, R.A. ); Geno, P.W. )

    1990-09-01

    The isoflavonoid conjugates medicarpin-3-O-glucoside-6{double prime}-O-malonate (MGM), afrormosin-7-O-glucoside (AG), and afrormosin-7-O-glucoside-6{double prime}-O-malonate (AGM) were isolated and characterized from cell suspension cultures of alfalfa (Medicago sativa L.), where they were the major constitutive secondary metabolites. They were also found in alfalfa roots but not in other parts of the plant. The phytoalexin medicarpin accumulated rapidly in suspension cultured cells treated with elicitor from Colletotrichum lindemuthianum, and this was subsequently accompanied by an increase in the levels of MGM. In contrast, net accumulation of afrormosin conjugates was not affected by elicitor treatment. Labeling studies with ({sup 14}C)phenylalanine indicated that afrormosin conjugates were the major de novo synthesized isoflavonoid products in unelicited cells. During elicitation, ({sup 14}C)phenylalanine was incorporated predominantly into medicarpin, although a significant proportion of the newly synthesized medicarpin was also conjugated. Treatment of {sup 14}C-labeled, elicited cells with L-{alpha}-aminooxy-{beta}-phenylpropionic acid, a potent inhibitor of PAL activity in vivo, resulted in the initial appearance of labeled medicarpin of very low specific activity, suggesting that the phytoalexin could be released from a preformed conjugate under these conditions. Our data draw attention to the involvement of isoflavone hydroxylases during the constitutive and elicitor-induced accumulation of isoflavonoids and their conjugates in alfalfa cell cultures.

  17. Stress Responses in Alfalfa (Medicago sativa L.)

    PubMed Central

    Jorrin, Jesus; Dixon, Richard A.

    1990-01-01

    l-Phenylalanine ammonia-lyase has been purified from elicitor-treated alfalfa (Medicago sativa L.) cell suspension cultures using two protocols based on different sequences of chromatofocusing and hydrophobic interaction chromatography. Three distinct forms of the intact enzyme were separated on the basis of affinity for Octyl-Sepharose, with isoelectric points in the range pH 5.1 to 5.4. The native enzyme was a tetramer of Mr 311,000; the intact subunit Mr was about 79,000, although polypeptides of Mr 71,000, 67,000 and 56,000, probably arising from degradation of the intact subunit, were observed in all preparations. Two-dimensional gel analysis revealed the presence of several subunit isoforms of differing isoelectric points. The purified isoforms of the native enzyme had different Km values for l-phenylalanine in the range 40 to 110 micromolar, although mixtures of the forms in crude preparations exhibited apparent negative rate cooperativity. The enzyme activity was induced approximately 16-fold within 6 hours of exposure of alfalfa cells to a fungal elicitor or yeast extract. Analysis by hydrophobic interaction chromatography revealed different proportions of the different active enzyme isoforms, depending upon either time after elicitation or the elicitor used. The elicitor-induced increase in enzyme activity was associated with increased translatable phenylalanine ammonia-lyase mRNA activity in the polysomal fraction. Images Figure 4 Figure 7 PMID:16667296

  18. Sub-cellular proteomics of Medicago truncatula

    PubMed Central

    Lee, Jeonghoon; Lei, Zhentian; Watson, Bonnie S.; Sumner, Lloyd W.

    2013-01-01

    Medicago truncatula is a leading model species and substantial molecular, genetic, genomics, proteomics, and metabolomics resources have been developed for this species to facilitate the study of legume biology. Currently, over 60 proteomics studies of M. truncatula have been published. Many of these have focused upon the unique symbiosis formed between legumes and nitrogen fixing rhizobia bacteria, while others have focused on seed development and the specialized proteomes of distinct tissues/organs. These include the characterization of sub-cellular organelle proteomes such as nuclei and mitochondria, as well as proteins distributed in plasma or microsomal membranes from various tissues. The isolation of sub-cellular proteins typically requires a series of steps that are labor-intensive. Thus, efficient protocols for sub-cellular fractionation, purification, and enrichment are necessary for each cellular compartment. In addition, protein extraction, solubilization, separation, and digestion prior to mass spectral identification are important to enhance the detection of low abundance proteins and to increase the overall detectable proportion of the sub-cellular proteome. This review summarizes the sub-cellular proteomics studies in M. truncatula. PMID:23641248

  19. Stress Responses in Alfalfa (Medicago sativa L.)

    PubMed Central

    Gowri, Ganesan; Bugos, Robert C.; Campbell, Wilbur H.; Maxwell, Carl A.; Dixon, Richard A.

    1991-01-01

    S-Adenosyl-l-methionine:caffeic acid 3-O-methyltransferase (COMT, EC 2.1.1.6) catalyzes the conversion of caffeic acid to ferulic acid, a key step in the biosynthesis of lignin monomers. We have isolated a functionally active cDNA clone (pCOMT1) encoding alfalfa (Medicago sativa L.) COMT by immunoscreening a λZAPII cDNA expression library with anti-(aspen COMT) antibodies. The derived amino acid sequence of pCOMT1 is 86% identical to that of COMT from aspen. Southern blot analysis indicates that COMT in alfalfa is encoded by at least two genes. Addition of an elicitor preparation from bakers' yeast to alfalfa cell suspension cultures resulted in a rapid accumulation of COMT transcripts, which reached a maximum level around 19 hours postelicitation. Northern blot analysis of total RNA from different organs of alfalfa plants at various developmental stages showed that COMT transcripts are most abundant in roots and stems. Transcripts encoding ATP: i-methionine-S-adenosyl transferase (AdoMet synthetase, EC 2.5.1.6), the enzyme responsible for the synthesis of the methyl donor for the COMT reaction, were coinduced with COMT transcripts in elicitor-treated cells and exhibited a similar pattern of expression to that of COMT in different organs of alfalfa plants at various stages of development. ImagesFigure 3Figure 4Figure 6 PMID:16668418

  20. An Italian functional genomic resource for Medicago truncatula

    PubMed Central

    Porceddu, Andrea; Panara, Francesco; Calderini, Ornella; Molinari, Lorna; Taviani, Paola; Lanfaloni, Luisa; Scotti, Carla; Carelli, Maria; Scaramelli, Laura; Bruschi, Gianluca; Cosson, Viviane; Ratet, Pascal; de Larembergue, Henri; Duc, Gerard; Piano, Efisio; Arcioni, Sergio

    2008-01-01

    Background Medicago truncatula is a model species for legumes. Its functional genomics have been considerably boosted in recent years due to initiatives based both in Europe and US. Collections of mutants are becoming increasingly available and this will help unravel the genetic control of important traits for many species of legumes. Findings Our report is on the production of three complementary mutant collections of the model species Medicago truncatula produced in Italy in the frame of a national genomic initiative. Well established strategies were used: Tnt1 mutagenesis, TILLING and activation tagging. Both forward and reverse genetics screenings proved the efficiency of the mutagenesis approaches adopted, enabling the isolation of interesting mutants which are in course of characterization. We anticipate that the reported collections will be complementary to the recently established functional genomics tools developed for Medicago truncatula both in Europe and in the United States. PMID:19077311

  1. The medicago genome provides insight into evolution of rhizobial symbiosis

    USDA-ARS?s Scientific Manuscript database

    Medicago truncatula is an excellent model for the study of legume-specific biology, especially endosymbiotic interactions with bacteria and fungi. This paper describes the sequence of the euchromatic portion of the M. truncatula genome based on a recently completed BAC-based assembly supplemented by...

  2. Comparison of the phytoremediation potentials of Medicago falcata L. And Medicago sativa L. in aged oil-sludge-contaminated soil.

    PubMed

    Panchenko, Leonid; Muratova, Anna; Turkovskaya, Olga

    2017-01-01

    Thirteen-year monitoring of the vegetation growing in the industrial and adjacent areas of an oil refinery showed the prevalence of yellow medick (Medicago falcata L.) over other plant species, including alfalfa (Medicago sativa L.). A comparative field study of the two Medicago species established that yellow medick and alfalfa exhibited similar resistance to soil petroleum hydrocarbons and that the pollutant concentration in their rhizosphere was 30% lower than that in the surrounding bulk soil. In laboratory pot experiments, yellow medick reduced the contaminant content by 18% owing to the degradation of the major heavy oil fractions, such as paraffins, naphthenes, and alcohol and benzene tars; and it was more successful than alfalfa. Both species were equally effective in stimulating the total number of soil microorganisms, but the number of hydrocarbon-oxidizing microorganisms, including polycyclic aromatic hydrocarbon degraders, was larger in the root zone of alfalfa. In turn, yellow medick provided a favorable balance of available nitrogen. Both Medicago species equally stimulated the dehydrogenase and peroxidase activities of the soil, and yellow medick increased the activity of soil polyphenol oxidase but reduced the activity of catalase. The root tissue activity of catalase, ascorbate oxidase, and tyrosinase was grater in alfalfa than in yellow medick. The peroxidase activity of plant roots was similar in both species, but nondenaturing polyacrylamide gel electrophoresis showed some differences in the peroxidase profiles of the root extracts of alfalfa and yellow medick. Overall, this study suggests that the phytoremediation potentials of yellow medick and alfalfa are similar, with some differences.

  3. Gene silencing in Medicago truncatula roots using RNAi.

    PubMed

    Floss, Daniela S; Schmitz, Alexa M; Starker, Colby G; Gantt, J Stephen; Harrison, Maria J

    2013-01-01

    Medicago truncatula is used widely as a model system for studies of root symbioses, interactions with parasitic nematodes and fungal pathogens, as well as studies of development and secondary metabolism. In Medicago truncatula as well as other legumes, RNA interference (RNAi) coupled with Agrobacterium rhizogenes-mediated root transformation, has been used very successfully for analyses of gene function in roots. One of the major advantages of this approach is the ease and relative speed with which transgenic roots can be generated. There are several methods, both for the generation of the RNAi constructs and the root transformation. Here we provide details of an RNAi and root transformation protocol that has been used successfully in M. truncatula and which can be scaled up to enable the analysis of several hundred constructs.

  4. Triterpene saponins from aerial parts of Medicago arabica L.

    PubMed

    Bialy, Zbigniew; Jurzysta, Marian; Mella, Mariella; Tava, Aldo

    2004-03-10

    Eight major triterpene saponins have been isolated from the aerial parts of Medicago arabica and their structures elucidated by FAB-MS and NMR analysis. Three of them are new compounds and are identified as 3-O-(alpha-L-arabinopyranoside) bayogenin, 3-O-(alpha-L-arabinopyranosyl), 28-O-(beta-D-glucopyranoside) bayogenin, and 3-O-[alpha-L-arabinopyranosyl(1-->2)-beta-D-glucuronopyranosyl], 28-O-beta-D-glucopyranoside 2-beta-hydroxyoleanolic acid. Two saponins, identified as 3-O-(alpha-L-arabinopyranoside) hederagenin and 3-O-(alpha-L-arabinopyranosyl), 28-O-(beta-D-glucopyranoside) hederagenin are known compounds but not previously reported as saponin constituents of Medicago species, while three other saponins, being mono- and bidesmosides of hederagenin, have been previously isolated from roots of M. sativa.

  5. Adenylate cyclase activity in a higher plant, alfalfa (Medicago sativa).

    PubMed Central

    Carricarte, V C; Bianchini, G M; Muschietti, J P; Téllez-Iñón, M T; Perticari, A; Torres, N; Flawiá, M M

    1988-01-01

    An adenylate cyclase activity in Medicago sativa L. (alfalfa) roots was partially characterized. The enzyme activity remains in the supernatant fluid after centrifugation at 105,000 g and shows in crude extracts an apparent Mr of about 84,000. The enzyme is active with Mg2+ and Ca2+ as bivalent cations, and is inhibited by EGTA and by chlorpromazine. Calmodulin from bovine brain or spinach leaves activates this adenylate cyclase. PMID:3128270

  6. Triterpene saponins from barrel medic (Medicago truncatula) aerial parts.

    PubMed

    Kapusta, Ireneusz; Stochmal, Anna; Perrone, Angela; Piacente, Sonia; Pizza, Cosimo; Oleszek, Wieslaw

    2005-03-23

    Triterpene saponins from Medicago truncatula aerial parts have been separated and their structures determined by the extensive use of 1D- and 2D-NMR experiments including 1H-1H (DQF-COSY, 1D-TOCSY) and 1H-13C (HSQC, HMBC) spectroscopy along with ESIMS. Fifteen individual compounds were isolated that included seven medicagenic acid and eight zanhic acid glycosides. Additionally, two soyasapogenol B and soyasapogenol E glycosides were identified by MS/MS and TLC. Four medicagenic acid glycosides (5, 11, 12, 14) and eight zanhic acid glycosides (1-4, 6-9) are reported here for the first time. The common feature of M. truncatula aerial part saponins is the (1-->3) linkage between the two glucose units at C-3 of medicagenic and zanhic acids, which is different from that found in alfalfa (Medicago sativa), where this linkage was always (1-->2). This may suggest differences in glucosyltransferases between these two Medicago species.

  7. Comparison of rhizobia that nodulate Medicago laciniata and Medicago truncatula present in a single Tunisian arid soil.

    PubMed

    Badri, Y; Zribi, K; Badri, M; Huguet, T; van Berkum, P; Aouani, M E

    2007-02-01

    The rhizobia present in a single arid region Tunisian soil that nodulate Medicago laciniata and Medicago truncatula were compared. All isolates, 40 from each host, were Sinorhizobium meliloti based on 16S rRNA polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) patterns and subsequent confirmation by sequence analysis of the 16S rRNA genes in four representatives from each host species. There was no apparent relationship between Medicago host species of isolation and the nodulating rhizobial genome as determined by repetitive extragenic palandromic PCR. The isolates of M. laciniata were distinguished from those of M. truncatula present in the same soil by variation in PCR-RFLP of nifDK, indicating that this dissimilarity is originally genetic and not geographic. While forming effective symbioses with their own respective isolates, both M. laciniata and M. truncatula formed ineffective true nodules, nodule-like structures, or no nodules at all in cross-inoculation tests, as confirmed by the histological observations.

  8. The role of CPSF30 in 3’ RNA processing of Medicago sativa

    USDA-ARS?s Scientific Manuscript database

    CPSF30 is a component in 3’ RNA processing that has been well characterized in Arabidopsis thaliana. In this study the gene for CPSF30 has been cloned out of Medicago truncatula and Medicago sativa. A large region within the gene exhibits significant conservation between A. thaliana and both Medicag...

  9. Implementing a gap analysis to conserve Caucasus Medicago species for ex situ conservation

    USDA-ARS?s Scientific Manuscript database

    This paper discusses the results of a Medicago gap analysis of the Former Soviet Union, with a focus on the Caucasus Region. A comprehensive database of 2400 accessions of Medicago crop wild relatives that had been collected in the area of the Former Soviet Union (FSU) was developed. Using the data...

  10. Legume Resources: MtDB and Medicago.Org.

    PubMed

    Retzel, Ernest F; Johnson, James E; Crow, John A; Lamblin, Anne F; Paule, Charles E

    2007-01-01

    To identify the genes and gene functions that underlie key aspects of legume biology, researchers have selected the cool season legume Medicago truncatula as a model system for legume research. The mission of the M. truncatula Consortium is to promote unrestricted sharing of data and information that are provided by Medicago research groups worldwide. Through integration of a variety of data and tools, the medicago.org site intends to facilitate progress in the fields of structural, comparative, and functional genomics. To this goal, and as a consortium partner, the Center for Computational Genomics and Bioinformatics (CCGB) at the University of Minnesota has developed MtDB2.0, the M. truncatula database version 2.0. The MtDB2.0 database is the first step toward the global integration of M. truncatula genomic, genetic, and biological information. MtDB2.0 is a relational database that integrates M. truncatula transcriptome data and provides a wide range of user-defined data mining options. The database is interrogated through a series of interfaces, with 58 options grouped into two filters. Sequence identifiers from all public M. truncatula sites [e.g., IDs from GenBank, CCGB, The Institute for Genomic Research (TIGR), National Center for Genome Resources (NCGR), and I'Institut National de la Recherche Agronomique (INRA)] are fully cross-referenced to facilitate comparisons between different sites, and hypertext links to the appropriate database records are provided for all queries' results. MtDB's goal is to provide researchers with the means to quickly and independently identify sequences that match specific research interests based on user-defined criteria. MtDB2.0 offers unrestricted access to advanced and powerful querying tools unmatched by any other public databases. Structurad Query Language (SQL)-encoded queries with a Java-based Web user interface, incorporate different filtering that allow sophisticated data mining of the expressed sequence tag sequencing

  11. Artefact formation during acid hydrolysis of saponins from Medicago spp.

    PubMed

    Tava, Aldo; Biazzi, Elisa; Mella, Mariella; Quadrelli, Paolo; Avato, Pinarosa

    2017-02-28

    Artefact compounds obtained during acid hydrolysis of saponins from Medicago spp. (Fabaceae), have been monitored and evaluated by GC-FID. Their identification has been performed by GC-MS and (1)H and (13)C NMR. Saponins with different substituents on the triterpenic pentacyclic aglycones were considered, and their hydrolysis products were detected and quantified during 10 h of time course reaction. From soyasapogenol B glycoside the well known soyasapogenols B, C, D and F were obtained together with a previously undescribed sapogenol artefact identified as 3β,22β,24-trihydroxyolean-18(19)-en and named soyasapogenol H. From a zanhic acid saponin two major artefact compounds identified as 2β,3β,16α-trihydroxyolean-13(18)-en-23,28-dioic acid and 2β,3β,16α-trihydroxyolean-28,13β-olide-23-oic acid were obtained, together with some zanhic acid. Other compounds, detected in very small amount in the reaction mixture, were also tentatively identified based on their GC-MS and UV spectra. The other most characteristic saponins in Medicago spp., hederagenin, bayogenin and medicagenic acid glycosides, under acidic condition of hydrolysis, released instead the correspondent aglycones and generated a negligible amount of artefacts. Nature of artefacts and mechanism of their formation, involving a stable tertiary carbocation, is here proposed and discussed for the first time.

  12. A new approach to direct somatic embryogenesis in Medicago.

    PubMed

    Denchev, P; Velcheva, M; Atanassov, A

    1991-09-01

    A highly efficient system for direct somatic embryogenesis is described. Leaf sections originating from young trifoliate leaves of Medicago falcata line 47/1-5 and Medicago sativa line No2/9R, directly produced embryos after cultivation in liquid B5IV induction medium. In comparison with indirect somatic embryogenesis the system omits the callus stage and thus allows shortening of the process of somatic embryogenesis in alfalfa by 35-40 days. It permits the avoidance of secondary changes occurring during the process of dedifferentiation. A modified B5/3H medium containing Polyethylene Glycol 6000 promoted embryo development from globular up to torpedo stage. It was clearly shown that 2.5% Polyethylene Glycol stimulated this process for both H. falcata 47/1-5 and M. sativa No 2/9R. Maturation of torpedo stage embryos was carried out on solidified or liquid abscisic acidcontaining medium. A 30μM abscisic acid concentration was optimal in allowing one embryo to yield one plant. Somatic embryo conversion to plants and plant regeneration was performed on Murashige and Skoog medium. Regenerated plants showed a normal morphology.

  13. Analysis of annual Medicago species using RAPD markers.

    PubMed

    Brummer, E C; Bouton, J H; Kochert, G

    1995-04-01

    Annual species of the genus Medicago have attracted interest as green manure and temporary forage crops. This study was conducted to determine if randomly amplified polymorphic DNA (RAPD) markers could be used to assess the variability within and among species. Several accessions of each six species (M. scutellata Mill., M. disciformis DC., M. murex Willd., M. truncatula Gaertn., M. polymorpha L., and M. rugosa Desr.) were studied. A phylogeny reconstructed with the computer program Phylogenetic Analysis Using Parsimony (PAUP) showed the same relationships as traditional taxonomy. Variation was present among accessions of all species. Several accessions were considerably different from others within the species (one of each M. scutellata and M. polymorpha) and four accessions of M. murex were differentiated by both morphology and RAPD banding patterns from the other accessions. These accessions may be useful to include in a core collection. Variation within accessions was present. Although the species are autogamous, the original seed collections may have been made from a number of plants in the same area. Also, some outcrossing or seed mixing may have occurred. Finally, at least 10 RAPD primers appear to be necessary in order to develop reliable estimates of relatedness among annual Medicago accessions.

  14. Isolation and functional characterization of salt-stress induced RCI2-like genes from Medicago sativa and Medicago truncatula.

    PubMed

    Long, Ruicai; Zhang, Fan; Li, Zhenyi; Li, Mingna; Cong, Lili; Kang, Junmei; Zhang, Tiejun; Zhao, Zhongxiang; Sun, Yan; Yang, Qingchuan

    2015-07-01

    Salt stress is one of the most significant adverse abiotic factors, causing crop failure worldwide. So far, a number of salt stress-induced genes, and genes improving salt tolerance have been characterized in a range of plants. Here, we report the isolation and characterization of a salt stress-induced Medicago sativa (alfalfa) gene (MsRCI2A), which showed a high similarity to the yeast plasma membrane protein 3 gene (PMP3) and Arabidopsis RCI2A. The sequence comparisons revealed that five genes of MtRCI2(A-E) showed a high similarity to MsRCI2A in the Medicago truncatula genome. MsRCI2A and MtRCI2(A-E) encode small, highly hydrophobic proteins containing two putative transmembrane domains, predominantly localized in the plasma membrane. The transcript analysis results suggest that MsRCI2A and MtRCI2(A-D) genes are highly induced by salt stress. The expression of MsRCI2A and MtRCI2(A-C) in yeast mutants lacking the PMP3 gene can functionally complement the salt sensitivity phenotype resulting from PMP3 deletion. Overexpression of MsRCI2A in Arabidopsis plants showed improved salt tolerance suggesting the important role of MsRCI2A in salt stress tolerance in alfalfa.

  15. Comparative Proteomic Analysis Reveals Differential Root Proteins in Medicago sativa and Medicago truncatula in Response to Salt Stress

    PubMed Central

    Long, Ruicai; Li, Mingna; Zhang, Tiejun; Kang, Junmei; Sun, Yan; Cong, Lili; Gao, Yanli; Liu, Fengqi; Yang, Qingchuan

    2016-01-01

    Salt stress is an important abiotic stress that causes decreased crop yields. Root growth and plant activities are affected by salt stress through the actions of specific genes that help roots adapt to adverse environmental conditions. For a more comprehensive understanding of proteins affected by salinity, we used two-dimensional gel electrophoresis and mass spectrometry to characterize the proteome-level changes associated with salt stress response in Medicago sativa cv. Zhongmu-1 and Medicago truncatula cv. Jemalong A17 roots. Our physiological and phenotypic observations indicated that Zhongmu-1 was more salt tolerant than Jemalong A17. We identified 93 and 30 proteins whose abundance was significantly affected by salt stress in Zhongmu-1 and Jemalong A17 roots, respectively. The tandem mass spectrometry analysis of the differentially accumulated proteins resulted in the identification of 60 and 26 proteins in Zhongmu-1 and Jemalong A17 roots, respectively. Function analyses indicated molecule binding and catalytic activity were the two primary functional categories. These proteins have known functions in various molecular processes, including defense against oxidative stress, metabolism, photosynthesis, protein synthesis and processing, and signal transduction. The transcript levels of four identified proteins were determined by quantitative reverse transcription polymerase chain reaction. Our results indicate that some of the identified proteins may play key roles in salt stress tolerance. PMID:27066057

  16. Comparative Proteomic Analysis Reveals Differential Root Proteins in Medicago sativa and Medicago truncatula in Response to Salt Stress.

    PubMed

    Long, Ruicai; Li, Mingna; Zhang, Tiejun; Kang, Junmei; Sun, Yan; Cong, Lili; Gao, Yanli; Liu, Fengqi; Yang, Qingchuan

    2016-01-01

    Salt stress is an important abiotic stress that causes decreased crop yields. Root growth and plant activities are affected by salt stress through the actions of specific genes that help roots adapt to adverse environmental conditions. For a more comprehensive understanding of proteins affected by salinity, we used two-dimensional gel electrophoresis and mass spectrometry to characterize the proteome-level changes associated with salt stress response in Medicago sativa cv. Zhongmu-1 and Medicago truncatula cv. Jemalong A17 roots. Our physiological and phenotypic observations indicated that Zhongmu-1 was more salt tolerant than Jemalong A17. We identified 93 and 30 proteins whose abundance was significantly affected by salt stress in Zhongmu-1 and Jemalong A17 roots, respectively. The tandem mass spectrometry analysis of the differentially accumulated proteins resulted in the identification of 60 and 26 proteins in Zhongmu-1 and Jemalong A17 roots, respectively. Function analyses indicated molecule binding and catalytic activity were the two primary functional categories. These proteins have known functions in various molecular processes, including defense against oxidative stress, metabolism, photosynthesis, protein synthesis and processing, and signal transduction. The transcript levels of four identified proteins were determined by quantitative reverse transcription polymerase chain reaction. Our results indicate that some of the identified proteins may play key roles in salt stress tolerance.

  17. Global reprogramming of transcription and metabolism in Medicago truncatula during progressive drought and after rewatering

    PubMed Central

    Zhang, Ji-Yi; Cruz de Carvalho, Maria H; Torres-Jerez, Ivone; Kang, Yun; Allen, Stacy N; Huhman, David V; Tang, Yuhong; Murray, Jeremy; Sumner, Lloyd W; Udvardi, Michael K

    2014-01-01

    Medicago truncatula is a model legume forage crop native to the arid and semi-arid environments of the Mediterranean. Given its drought-adapted nature, it is an ideal candidate to study the molecular and biochemical mechanisms conferring drought resistance in plants. Medicago plants were subjected to a progressive drought stress over 14 d of water withholding followed by rewatering under controlled environmental conditions. Based on physiological measurements of plant water status and changes in morphology, plants experienced mild, moderate and severe water stress before rehydration. Transcriptome analysis of roots and shoots from control, mildly, moderately and severely stressed, and rewatered plants, identified many thousands of genes that were altered in expression in response to drought. Many genes with expression tightly coupled to the plant water potential (i.e. drought intensity) were identified suggesting an involvement in Medicago drought adaptation responses. Metabolite profiling of drought-stressed plants revealed the presence of 135 polar and 165 non-polar compounds in roots and shoots. Combining Medicago metabolomic data with transcriptomic data yielded insight into the regulation of metabolic pathways operating under drought stress. Among the metabolites detected in drought-stressed Medicago plants, myo-inositol and proline had striking regulatory profiles indicating involvement in Medicago drought tolerance. Global transcriptional and metabolic responses to drought and rewatering were investigated in Medicago truncatula, a naturally drought-adapted model legume species. Integration of metabolomic and transcriptomic data yielded insights into the regulation of metabolic pathways underlying drought-stress adaptation. Many genes and metabolites with expression tightly coupled to drought intensity were identified, suggesting active involvement in Medicago drought resistance. These could prove useful targets for future translational approaches to improve

  18. Global reprogramming of transcription and metabolism in Medicago truncatula during progressive drought and after rewatering.

    PubMed

    Zhang, Ji-Yi; Cruz DE Carvalho, Maria H; Torres-Jerez, Ivone; Kang, Yun; Allen, Stacy N; Huhman, David V; Tang, Yuhong; Murray, Jeremy; Sumner, Lloyd W; Udvardi, Michael K

    2014-11-01

    Medicago truncatula is a model legume forage crop native to the arid and semi-arid environments of the Mediterranean. Given its drought-adapted nature, it is an ideal candidate to study the molecular and biochemical mechanisms conferring drought resistance in plants. Medicago plants were subjected to a progressive drought stress over 14 d of water withholding followed by rewatering under controlled environmental conditions. Based on physiological measurements of plant water status and changes in morphology, plants experienced mild, moderate and severe water stress before rehydration. Transcriptome analysis of roots and shoots from control, mildly, moderately and severely stressed, and rewatered plants, identified many thousands of genes that were altered in expression in response to drought. Many genes with expression tightly coupled to the plant water potential (i.e. drought intensity) were identified suggesting an involvement in Medicago drought adaptation responses. Metabolite profiling of drought-stressed plants revealed the presence of 135 polar and 165 non-polar compounds in roots and shoots. Combining Medicago metabolomic data with transcriptomic data yielded insight into the regulation of metabolic pathways operating under drought stress. Among the metabolites detected in drought-stressed Medicago plants, myo-inositol and proline had striking regulatory profiles indicating involvement in Medicago drought tolerance. © 2014 The Authors. Plant, Cell & Environment published by John Wiley & Sons Ltd.

  19. [Quality standard of uygur medicine Medicago sativa seeds].

    PubMed

    Ding, Wen-Huan; Xu, Hai-Yan; Wang, Dong-Dong; Li, Jie; Tian, Shu-Ge

    2013-11-01

    In this paper, microscopic identification method was adopted to observe the microscopic characters of ten batches of Medicago sativa seeds. And M. sativa seeds were identificated by TLC method in contrast to trigonelline and stachydrine hydrochloride. The impurities, moisture, ash, sour insoluble ash were detected based on Chinese Pharmacopoeia 2010 version (Vol I ). An HPLC method was also established for determination of trigonelline in the M. sativa seeds. The contents of impurities, moisture, ash, sour insoluble ash should not exceed 5%, 10%, 6%, and 2%, respectively. The content of trigonelline should be not less than 0.795 6 mg x g(-1). The experimental methods were accurate and reliable, and can be used as the quality control of the seeds of M. sativa.

  20. Immature seeds and embryos of Medicago truncatula cultured in vitro.

    PubMed

    Ochatt, Sergio J

    2011-01-01

    Legumes are an important source of proteins and lipids for food and feed. In addition, they are -environmentally friendly because of their capacity to fix nitrogen through a symbiosis with Rhizobium that permits them to produce abundant proteins even in the absence of nitrogen fertilization. Seed development in plants follows three chronological steps (1) seed coat differentiation, embryo morphogenesis and endosperm development; (2) embryo maturation with storage accumulation and (3) dehydration and the acquisition of desiccation tolerance. Finally, germination occurs when the environmental conditions become favourable. Working with the model legume Medicago truncatula, an in vitro protocol was developed for the culture of immature embryos that permits their development in a way comparable to that observed in plants.In this chapter, the usefulness of this system for investigating embryo development in legumes is outlined.

  1. Root Surface Association in Relation to Nodulation of Medicago sativa

    PubMed Central

    van Rensburg, Henri Jansen; Strijdom, Barend W.

    1982-01-01

    Nine strains of Rhizobium meliloti, ranging in competitive ability on Medicago sativa from excellent to poor in autoclaved soils, were paired in 29 combinations and used to inoculate M. sativa in a liquid rooting medium. A positive correlation (r = 0.545) between strain ratios in nodules after 28 days and root surface cell ratios after 7 days was determined. Two cell fractions from the root surface, representing loosely and firmly adhering cells, were investigated. Infectivity was linked to the more firmly adhering cells. A significant relationship was established between the cell ratios of competing strains in the two fractions. In another experiment, adherence of cells of both infective and noninfective Rhizobium strains to roots of M. sativa and Trifolium repens was demonstrated; the ratios of loosely to firmly adhering cells on the root surface were significantly narrower with the infective combinations than with noninfective strain-legume associations. PMID:16346071

  2. Crystal structure of isoflavone reductase from alfalfa (Medicago sativa L.).

    PubMed

    Wang, Xiaoqiang; He, Xianzhi; Lin, Jianqiao; Shao, Hui; Chang, Zhenzhan; Dixon, Richard A

    2006-05-19

    Isoflavonoids play important roles in plant defense and exhibit a range of mammalian health-promoting activities. Isoflavone reductase (IFR) specifically recognizes isoflavones and catalyzes a stereospecific NADPH-dependent reduction to (3R)-isoflavanone. The crystal structure of Medicago sativa IFR with deletion of residues 39-47 has been determined at 1.6A resolution. Structural analysis, molecular modeling and docking, and comparison with the structures of other NADPH-dependent enzymes, defined the putative binding sites for co-factor and substrate and potential key residues for enzyme activity and substrate specificity. Further mutagenesis has confirmed the role of Lys144 as a catalytic residue. This study provides a structural basis for understanding the enzymatic mechanism and substrate specificity of IFRs as well as the functions of IFR-like proteins.

  3. Insights into symbiotic nitrogen fixation in Medicago truncatula.

    PubMed

    Tesfaye, Mesfin; Samac, Deborah A; Vance, Carroll P

    2006-03-01

    In silico analysis of the Medicago truncatula gene index release 8.0 at The Institute for Genomic Research identified approximately 530 tentative consensus sequences (TC) clustered from 2,700 expressed sequence tags (EST) derived solely from Sinorhizobium meliloti-inoculated root and nodule tissues. A great majority (76%) of these TC were derived exclusively from nitrogen-fixing and senescent nodules. A cDNA filter array was constructed using approximately 58% of the in silico-identified TC as well as cDNAs representing selected carbon and nitrogen metabolic pathways. The purpose of the array was to analyze transcript abundance in M. truncatula roots and nodules following inoculation by a wild-type S. meliloti strain, a mutant strain that forms ineffective nodules, an uninoculated root control, and roots following nitrate or ammonium treatments. In all, 81 cDNAs were upregulated in both effective and ineffective nodules, and 78% of these cDNAs represent in silico-identified TC. One group of in silico-identified TC encodes genes with similarity to putative plant disease resistance (R) genes of the nucleotide binding site-leucine-rich repeat type. Expression of R genes was enhanced in effective nodules, and transcripts also were detected in ineffective nodules at 14 days postinoculation (dpi). Homologous R gene sequences also have been identified in the Medicago genome. However, their functional importance in nodules remains to be established. Genes for enzymes involved in organic acid synthesis along with genes involved in nitrogen metabolism were shown to be coexpressed in nitrate-fed roots and effective nodules of M. truncatula.

  4. The efficiency of nitrogen fixation of the model legume Medicago truncatula (Jemalong A17) is low compared to Medicago sativa.

    PubMed

    Sulieman, Saad; Schulze, Joachim

    2010-06-15

    Medicago truncatula (Gaertn.) (barrel medic) serves as a model legume in plant biology. Numerous studies have addressed molecular aspects of the biology of M. truncatula, while comparatively little is known about the efficiency of N(2) fixation at the whole plant level. The objective of the present study was to compare the efficiency of N(2) fixation of M. truncatula to the genetically closely related Medicago sativa (L.) (alfalfa). The relative growth of both species relying exclusively on N(2) fixation versus nitrate nutrition, H(2) evolution, nitrogen assimilation, the concentration of amino acids and organic acids in nodules, and (15)N(2) uptake and distribution were studied. M. truncatula showed much lower efficiency of N(2) fixation. Nodule-specific activity was several-fold lower when compared to M. sativa, partially as a result of a lower electron allocation to N(2) versus H(+). M. truncatula or M. sativa plants grown solely on N(2) fixation as a nitrogen source reached about 30% or 80% of growth, respectively, when compared to plants supplied with sufficient nitrate. Moreover, M. truncatula had low %N in shoots and a lower allocation of (15)N to shoots during 1h (15)N(2) labeling period. Amino acid concentration was about 20% higher in M. sativa nodules, largely as a result of more asparagine, while the organic acid concentration was about double in M. sativa, coinciding with a six-fold higher concentration of malate. Total soluble protein in nodules was about three times lower in M. truncatula and the pattern of enzyme activity in that fraction was strongly different. Sucrose cleaving enzymes displayed higher activity in M. truncatula nodules, while the activity of phosphoenolpyruvate carboxylase (PEPC) was much lower. It is concluded that the low efficiency of the M. truncatula symbiotic system is related to a low capacity of organic acid formation and limited nitrogen export from nodules.

  5. Kinetics and Strain Specificity of Rhizosphere and Endophytic Colonization by Enteric Bacteria on Seedlings of Medicago sativa and Medicago truncatula

    PubMed Central

    Dong, Yuemei; Iniguez, A. Leonardo; Ahmer, Brian M. M.; Triplett, Eric W.

    2003-01-01

    The presence of human-pathogenic, enteric bacteria on the surface and in the interior of raw produce is a significant health concern. Several aspects of the biology of the interaction between these bacteria and alfalfa (Medicago sativa) seedlings are addressed here. A collection of enteric bacteria associated with alfalfa sprout contaminations, along with Escherichia coli K-12, Salmonella enterica serotype Typhimurium strain ATCC 14028, and an endophyte of maize, Klebsiella pneumoniae 342, were labeled with green fluorescent protein, and their abilities to colonize the rhizosphere and the interior of the plant were compared. These strains differed widely in their endophytic colonization abilities, with K. pneumoniae 342 and E. coli K-12 being the best and worst colonizers, respectively. The abilities of the pathogens were between those of K. pneumoniae 342 and E. coli K-12. All Salmonella bacteria colonized the interiors of the seedlings in high numbers with an inoculum of 102 CFU, although infection characteristics were different for each strain. For most strains, a strong correlation between endophytic colonization and rhizosphere colonization was observed. These results show significant strain specificity for plant entry by these strains. Significant colonization of lateral root cracks was observed, suggesting that this may be the site of entry into the plant for these bacteria. At low inoculum levels, a symbiosis mutant of Medicago truncatula, dmi1, was colonized in higher numbers on the rhizosphere and in the interior by a Salmonella endophyte than was the wild-type host. Endophytic entry of M. truncatula appears to occur by a mechanism independent of the symbiotic infections by Sinorhizobium meliloti or mycorrhizal fungi. PMID:12620870

  6. The Medicago sativa gene index 1.2: a web-accessible gene expression atlas for investigating expression differences between Medicago sativa subspecies

    USDA-ARS?s Scientific Manuscript database

    Alfalfa (Medicago sativa L.) is the primary forage legume crop species in the United States and plays essential economic and ecological roles in agricultural systems across the country. Modern alfalfa is the result of hybridization between tetraploid M. sativa ssp. sativa and M. sativa ssp. falcata....

  7. The Medicago sativa Gene Index 1.2: A web-accessible expression atlas of two Medicago sativa sub-species

    USDA-ARS?s Scientific Manuscript database

    Alfalfa is an important crop, both financially and ecologically, in the agricultural system of the United States. Using Illumina RNA-seq technology we have developed a de-novo transcriptome assembly from two Medicago sativa sub-species, sativa (B47) and falcata (F56). These two genotypes have proven...

  8. Genome sequence of the Medicago-nodulating Ensifer meliloti commercial inoculant strain RRI128

    PubMed Central

    Reeve, Wayne; Ballard, Ross; Drew, Elizabeth; Tian, Rui; Bräu, Lambert; Goodwin, Lynne; Huntemann, Marcel; Han, James; Tatiparthi, Reddy; Chen, Amy; Mavrommatis, Konstantinos; Markowitz, Victor; Palaniappan, Krishna; Ivanova, Natalia; Pati, Amrita; Woyke, Tanja; Kyrpides, Nikos

    2014-01-01

    Ensifer meliloti strain RRI128 is an aerobic, motile, Gram-negative, non-spore-forming rod. RRI128 was isolated from a nodule recovered from the roots of barrel medic (Medicago truncatula) grown in the greenhouse and inoculated with soil collected from Victoria, Australia. The strain is used in commercial inoculants in Australia. RRI128 nodulates and forms an effective symbiosis with a diverse range of lucerne cultivars (Medicago sativa) and several species of annual medic (M. truncatula, Medicago littoralis and Medicago tornata), but forms an ineffective symbiosis with Medicago polymorpha. Here we describe the features of E. meliloti strain RRI128, together with genome sequence information and annotation. The 6,900,273 bp draft genome is arranged into 156 scaffolds of 157 contigs, contains 6,683 protein-coding genes and 87 RNA-only encoding genes, and is one of 100 rhizobial genomes sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project. PMID:25197447

  9. Genome sequence of the Medicago-nodulating Ensifer meliloti commercial inoculant strain RRI128.

    PubMed

    Reeve, Wayne; Ballard, Ross; Drew, Elizabeth; Tian, Rui; Bräu, Lambert; Goodwin, Lynne; Huntemann, Marcel; Han, James; Tatiparthi, Reddy; Chen, Amy; Mavrommatis, Konstantinos; Markowitz, Victor; Palaniappan, Krishna; Ivanova, Natalia; Pati, Amrita; Woyke, Tanja; Kyrpides, Nikos

    2014-06-15

    Ensifer meliloti strain RRI128 is an aerobic, motile, Gram-negative, non-spore-forming rod. RRI128 was isolated from a nodule recovered from the roots of barrel medic (Medicago truncatula) grown in the greenhouse and inoculated with soil collected from Victoria, Australia. The strain is used in commercial inoculants in Australia. RRI128 nodulates and forms an effective symbiosis with a diverse range of lucerne cultivars (Medicago sativa) and several species of annual medic (M. truncatula, Medicago littoralis and Medicago tornata), but forms an ineffective symbiosis with Medicago polymorpha. Here we describe the features of E. meliloti strain RRI128, together with genome sequence information and annotation. The 6,900,273 bp draft genome is arranged into 156 scaffolds of 157 contigs, contains 6,683 protein-coding genes and 87 RNA-only encoding genes, and is one of 100 rhizobial genomes sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project.

  10. Host and nonhost resistance in Medicago-Colletotrichum interactions.

    PubMed

    Jaulneau, Valérie; Cazaux, Marc; Wong Sak Hoi, Joanne; Fournier, Sylvie; Esquerré-Tugayé, Marie-Thérèse; Jacquet, Christophe; Dumas, Bernard

    2010-09-01

    Medicago truncatula lines resistant (A17) or susceptible (F83005.5) to the alfalfa pathogen Colletotrichum trifolii were used to compare defense reactions induced upon inoculation with C. trifolii or with the nonadapted pathogens C. lindemuthianum and C. higginsianum. Nonadapted Colletotrichum spp. induced a hypersensitive response (HR)-like reaction similar to the one induced during the host-incompatible interaction. Molecular analyses indicated an induction of PR10 and chalcone synthase genes in host and nonhost interactions but delayed responses were observed in the F83005.5 line. The clste12 penetration-deficient C. lindemuthianum mutant induced an HR and defense gene expression, showing that perception of nonadapted strains occurs before penetration of epidermal cells. Cytological and transcriptomic analyses performed upon inoculation of near-isogenic M. truncatula lines, differing only at the C. trifolii resistance locus, Ct1, with the nonadapted Colletotrichum strain, showed that nonhost responses are similar in the two lines. These included a localized oxidative burst, accumulation of fluorescent compounds, and transient expression of a small number of genes. Host interactions were characterized by a group of defense and signaling-related genes induced at 3 days postinoculation, associated with an accumulation of salicylic acid. Together, these results show that M. truncatula displays a rapid and transient response to nonadapted Colletotrichum strains and that this response is not linked to the C. trifolii resistance locus.

  11. Embryogenesis and plant regeneration of Medicago spp. in tissue culture.

    PubMed

    Nagarajan, P; McKenzie, J S; Walton, P D

    1986-02-01

    Ten cultivars and breeding lines from two species of alfalfa (Medicago media and M. sativa) were screened for their ability to produce embryos and plantlets from the root and hypocotyl under three different tissue culture protocols. The three protocols differed in basal salt composition, vitamins, hormones and cytokinin additions. That protocol having a high 2-4,D low cytokinin induction step gave the highest percentage of embryogenic calli in some cultivars and lines. M. media cultivars and breeding lines had a high percentage of embryoid formation. M. sativa cultivars gave no embryoid formation. Two M. media breeding lines (Br1 and Le1), which were intermediate in the percentage of embryogenic calli formed from explants, had the highest number of regenerated plants established in soil. The creeping rooted M. media cultivar Heinrichs produced the highest percentage of embryogenic calli from explants but most of these embryoids were abnormal and failed to grow in soil or vermiculite. Accordingly, successful regeneration is directly related to the quality and quantity of the embryoids produced.

  12. Allelopathic effect of alfalfa (Medicago sativa) on bladygrass (Imperata cylindrica).

    PubMed

    Abdul-Rahman, A A; Habib, S A

    1989-09-01

    Greenhouse and laboratory experiments were conducted at the Agricultural and Water Resources Research Center Station, Baghdad, in 1985 and 1986 to investigate the possible allelopathic potential of alfalfa (Medicago saliva L.) and its decomposed residues on bladygrass (Imperata cylin-drica L. Beauv.), a noxious weed in Iraq, and to isolate, characterize, and quantify possible allelopathic agents in alfalfa residues and root exudates. Results indicated that decomposed alfalfa roots and their associated soil produced a 51-56% reduction in bladygrass seed germination. Root and shoot length of bladygrass seedlings were reduced by an average of 88%. Decayed and undecayed mixtures of alfalfa roots and soil at 0.015∶1 (w/w) inhibited bladygrass seedlings reproduced from rhizomes by 30 and 42%. It was found that root exudates of alfalfa seedlings caused significant reduction in shoot and root dry weights of bladygrass seedlings when alfalfa and bladygrass were grown together in nutrient culture. Caffeic, chlorogenic, isochloro-genic,p-coumaric,p-OH-benzoic, and ferulic acids were detected in alfalfa root exudates and residues. The highest amount (126 fig phenolic acids/g soil) of these compounds was found in alfalfa root residues after six months of decomposition in soil.

  13. Root developmental programs shape the Medicago truncatula nodule meristem.

    PubMed

    Franssen, Henk J; Xiao, Ting Ting; Kulikova, Olga; Wan, Xi; Bisseling, Ton; Scheres, Ben; Heidstra, Renze

    2015-09-01

    Nodules on the roots of legume plants host nitrogen-fixing Rhizobium bacteria. Several lines of evidence indicate that nodules are evolutionarily related to roots. We determined whether developmental control of the Medicago truncatula nodule meristem bears resemblance to that in root meristems through analyses of root meristem-expressed PLETHORA genes. In nodules, MtPLETHORA 1 and 2 are preferentially expressed in cells positioned at the periphery of the meristem abutting nodule vascular bundles. Their expression overlaps with an auxin response maximum and MtWOX5, which is a marker for the root quiescent center. Strikingly, the cells in the central part of the nodule meristem have a high level of cytokinin and display MtPLETHORA 3 and 4 gene expression. Nodule-specific knockdown of MtPLETHORA genes results in a reduced number of nodules and/or in nodules in which meristem activity has ceased. Our nodule gene expression map indicates that the nodule meristem is composed of two distinct domains in which different MtPLETHORA gene subsets are expressed. Our mutant studies show that MtPLETHORA genes function redundantly in nodule meristem maintenance. This indicates that Rhizobium has recruited root developmental programs for nodule formation. © 2015. Published by The Company of Biologists Ltd.

  14. A Snapshot of Functional Genetic Studies in Medicago truncatula

    PubMed Central

    Kang, Yun; Li, Minguye; Sinharoy, Senjuti; Verdier, Jerome

    2016-01-01

    In the current context of food security, increase of plant protein production in a sustainable manner represents one of the major challenges of agronomic research, which could be partially resolved by increased cultivation of legume crops. Medicago truncatula is now a well-established model for legume genomic and genetic studies. With the establishment of genomics tools and mutant populations in M. truncatula, it has become an important resource to answer some of the basic biological questions related to plant development and stress tolerance. This review has an objective to overview a decade of genetic studies in this model plant from generation of mutant populations to nowadays. To date, the three biological fields, which have been extensively studied in M. truncatula, are the symbiotic nitrogen fixation, the seed development, and the abiotic stress tolerance, due to their significant agronomic impacts. In this review, we summarize functional genetic studies related to these three major biological fields. We integrated analyses of a nearly exhaustive list of genes into their biological contexts in order to provide an overview of the forefront research advances in this important legume model plant. PMID:27555857

  15. Optimizing TILLING populations for reverse genetics in Medicago truncatula.

    PubMed

    Le Signor, Christine; Savois, Vincent; Aubert, Grégoire; Verdier, Jérôme; Nicolas, Marie; Pagny, Gaelle; Moussy, Françoise; Sanchez, Myriam; Baker, Dave; Clarke, Jonathan; Thompson, Richard

    2009-06-01

    Medicago truncatula has been widely adopted as a model plant for crop legume species of the Vicieae. Despite the availability of transformation and regeneration protocols, there are currently limited tools available in this species for the systematic investigation of gene function. Within the framework of the European Grain Legumes Integrated Project (http://www.eugrainlegumes.org), chemical mutagenesis was applied to M. truncatula to create two mutant populations that were used to establish a TILLING (targeting induced local lesions in genomes) platform and a phenotypic database, allowing both reverse and forward genetics screens. Both populations had the same M2 line number, but differed in their M1 population size: population 1 was derived from a small M1 population (one-tenth the size of the M2 generation), whereas population 2 was generated by single seed descent and therefore has M1 and M2 generations of equal size. Fifty-six targets were screened, 10 on both populations, and 546 point mutations were identified. Population 2 had a mutation frequency of 1/485 kb, twice that of population 1. The strategy used to generate population 2 is more efficient than that used to generate population 1, with regard to mutagenesis density and mutation recovery. However, the design of population 1 allowed us to estimate the genetically effective cell number to be three in M. truncatula. Phenotyping data to help forward screenings are publicly available, as well as a web tool for ordering seeds at http://www.inra.fr/legumbase.

  16. Transcriptional profiling of Medicago truncatula during Erysiphe pisi infection

    PubMed Central

    Curto, Miguel; Krajinski, Franziska; Schlereth, Armin; Rubiales, Diego

    2015-01-01

    Resistance to powdery mildew has been studied in a number of plant species, yet the molecular mechanisms remain largely unknown. Transcription factors (TFs) play a critical role in the plant defense response by regulating the transcriptional machinery which coordinates the expression of a large group of genes involved in plant defense. Using high-throughput quantitative real-time PCR (qPCR) technology more than 1000 Medicago truncatula TFs were screened in a pair of susceptible and resistant genotypes of M. truncatula after 4 h of Erysiphe pisi infection. Seventy nine TF genes, belonging to 33 families showed a significant transcriptional change in response to E. pisi infection. Forty eight TF genes were differentially expressed in the resistant genotypes compared to the susceptible one in response to E. pisi infection, including pathogenesis-related transcriptional factors, AP2/EREBP (APETALA2/ETHYLENE-RESPONSIVE ELEMENT BINDING FACTORS), WRKY (highly conserved WRKYGQK amino-acid sequence), MYB (Myeloblastoma), homeodomain (HD) and zinc finger C2C2 (CYS2-CYS2), C2H2, (CYS2-HIS2), LIM (Lin-11, Isl-1, Mec-3) gene families, which are involved in known defense responses. Our results suggest that these TF genes are among the E. pisi responsive genes in resistant M. truncatula that may constitute a regulatory network which controls the transcriptional changes in defense genes involved in resistance to E. pisi. PMID:26217367

  17. Bioaccumulation and degradation of pentachloronitrobenzene in Medicago sativa.

    PubMed

    Li, Ying Ying; Yang, Hong

    2013-04-15

    Pentachloronitrobenzene (PCNB) is a fungicide belonging to the organochlorine family and used extensively in agriculture for crop production. Many studies have implied that PCNB has become an environmental concern due to its widespread contamination in eco-systems. However, whether PCNB is bioaccumulated, degraded and phytotoxic in plants is poorly understood. In this study, several alfalfa (Medicago sativa) cultivars were grown in soil with PCNB to investigate their absorption and catabolism, including PCNB residues in the soil and PCNB-induced toxic responses in plants. Alfalfa plants varied widely in their ability to accumulate and degrade PCNB. The degradation rate of PCNB was 66.26-77.68% after alfalfa growth in the soils for 20 d, while the rates in the control (soil without alfalfa) were only 48.42%. Moreover, concentrations of PCNB residues in the rhizosphere soil were significantly higher than those in the non-rhizosphere soils. Alfalfa exposed to 10 mg kg(-1) PCNB showed inhibited growth and oxidative damage, but the effects of PCNB on the cultivars differed significantly, indicating that the alfalfa cultivars have different tolerance to PCNB. Activities of invertase (INV), urease (URE), polyphenol oxidase (PPO), alkaline phosphatase (ALP) and acid phosphatase (ACP) were assayed in the treated soils and showed that the enzyme activities were altered after PCNB exposure. The URE, PPO, ALP and ACP activities were increased in soil following the planting of alfalfa. The objective of the study was to analyze the potential of different cultivars of alfalfa to accumulate and degrade PCNB from the contaminated soil.

  18. CLE peptides control Medicago truncatula nodulation locally and systemically.

    PubMed

    Mortier, Virginie; Den Herder, Griet; Whitford, Ryan; Van de Velde, Willem; Rombauts, Stephane; D'Haeseleer, Katrien; Holsters, Marcelle; Goormachtig, Sofie

    2010-05-01

    The CLAVATA3/embryo-surrounding region (CLE) peptides control the fine balance between proliferation and differentiation in plant development. We studied the role of CLE peptides during indeterminate nodule development and identified 25 MtCLE peptide genes in the Medicago truncatula genome, of which two genes, MtCLE12 and MtCLE13, had nodulation-related expression patterns that were linked to proliferation and differentiation. MtCLE13 expression was up-regulated early in nodule development. A high-to-low expression gradient radiated from the inner toward the outer cortical cell layers in a region defining the incipient nodule. At later stages, MtCLE12 and MtCLE13 were expressed in differentiating nodules and in the apical part of mature, elongated nodules. Functional analysis revealed a putative role for MtCLE12 and MtCLE13 in autoregulation of nodulation, a mechanism that controls the number of nodules and involves systemic signals mediated by a leucine-rich repeat receptor-like kinase, SUNN, which is active in the shoot. When MtCLE12 and MtCLE13 were ectopically expressed in transgenic roots, nodulation was abolished at the level of the nodulation factor signal transduction, and this inhibition involved long-distance signaling. In addition, composite plants with roots ectopically expressing MtCLE12 or MtCLE13 had elongated petioles. This systemic effect was not observed in transgenic roots ectopically expressing MtCLE12 and MtCLE13 in a sunn-1 mutant background, although nodulation was still strongly reduced. These results suggest multiple roles for CLE signaling in nodulation.

  19. Flavonoids from barrel medic (Medicago truncatula) aerial parts.

    PubMed

    Kowalska, Iwona; Stochmal, Anna; Kapusta, Ireneusz; Janda, Bogdan; Pizza, Cosimo; Piacente, Sonia; Oleszek, Wieslaw

    2007-04-04

    Twenty-three flavonoids have been identified in the aerial parts of barrel medic, and their structures were established by spectrometric and spectroscopic (ESI-MS/MS and NMR) techniques. Eight of the identified compounds, including apigenin 7-O-beta-D-glucuronopyranosyl-(1-->3)-O-beta-D-glucuronopyranosyl-(1-->2)-O-beta-D-glucuronopyranoside, apigenin 7-O-[2'-O-sinapoyl-beta-D-glucuronopyranosyl-(1-->2)-O-beta-D-glucuronopyranoside], apigenin 7-O-{2-O-feruloyl-[beta-D-glucuronopyranosyl-(1-->3)]-O-beta-D-glucuronopyranosyl-(1-->2)-O-beta-D-glucopyranoside}, chrysoeriol 7-O-[beta-D-glucuronopyranosyl-(1-->2)-O-beta-D-glucuronopyranoside, chrysoeriol 7-O-{2'-O-p-coumaroyl-[beta-D-glucuronopyranosyl-(1-->3)]-O-beta-D-glucuronopyranosyl(1-->2)-O-beta-D-glucuronopyranoside}, tricin 7-O-beta-D-glucuronopyranosyl-4'-O-glucopyranoside, tricin 7-O-[2'-O-feruloyl-beta-D-glucuronopyranosyl-(1-->2)-O-beta-D-glucopyranoside], and tricin 7-O-{2'-O-p-coumaroyl-[beta-D-glucuronopyranosyl-(1-->3)]-O-beta-D-glucuronopyranosyl(1-->2)-O-beta-D-glucuronopyranoside}, have not been reported before in the plant kingdom. Additionally, the presence of two luteolin, three apigenin, one chrysoeriol, and six tricin glycosides, previously identified in alfalfa (Medicago sativa), was confirmed in M. truncatula. Moreover, besides the above flavones, the aerial parts of this species contained three flavonols including rutin, laricitrin 3,7,5'-triglucoside, and laricitrin 3,5'-diglucoside.

  20. Salt stress alters DNA methylation levels in alfalfa (Medicago spp).

    PubMed

    Al-Lawati, A; Al-Bahry, S; Victor, R; Al-Lawati, A H; Yaish, M W

    2016-02-26

    Modification of DNA methylation status is one of the mechanisms used by plants to adjust gene expression at both the transcriptional and posttranscriptional levels when plants are exposed to suboptimal conditions. Under abiotic stress, different cultivars often show heritable phenotypic variation accompanied by epigenetic polymorphisms at the DNA methylation level. This variation may provide the raw materials for plant breeding programs that aim to enhance abiotic stress tolerance, including salt tolerance. In this study, methylation-sensitive amplified polymorphism (MSAP) analysis was used to assess cytosine methylation levels in alfalfa (Medicago spp) roots exposed to increasing NaCl concentrations (0.0, 8.0, 12.0, and 20.0 dS/m). Eleven indigenous landraces were analyzed, in addition to a salt-tolerant cultivar that was used as a control. There was a slight increase in DNA methylation upon exposure to high levels of soil salinity. Phylogenetic analysis using MSAP showed epigenetic variation within and between the alfalfa landraces when exposed to saline conditions. Based on MSAP and enzyme-linked immunosorbent assay results, we found that salinity increased global DNA methylation status, particularly in plants exposed to the highest level of salinity (20 dS/m). Quantitative reverse transcription-polymerase chain reaction indicated that this might be mediated by the overexpression of methyltransferase homolog genes after exposure to saline conditions. DNA demethylation using 5-azacytidine reduced seedling lengths and dry and fresh weights, indicating a possible decrease in salinity tolerance. These results suggest that salinity affects DNA methylation flexibility.

  1. Small RNA deep sequencing identifies novel and salt-stress-regulated microRNAs from roots of Medicago sativa and Medicago truncatula.

    PubMed

    Long, Rui-Cai; Li, Ming-Na; Kang, Jun-Mei; Zhang, Tie-Jun; Sun, Yan; Yang, Qing-Chuan

    2015-05-01

    Small 21- to 24-nucleotide (nt) ribonucleic acids (RNAs), notably the microRNA (miRNA), are emerging as a posttranscriptional regulation mechanism. Salt stress is one of the primary abiotic stresses that cause the crop losses worldwide. In saline lands, root growth and function of plant are determined by the action of environmental salt stress through specific genes that adapt root development to the restrictive condition. To elucidate the role of miRNAs in salt stress regulation in Medicago, we used a high-throughput sequencing approach to analyze four small RNA libraries from roots of Zhongmu-1 (Medicago sativa) and Jemalong A17 (Medicago truncatula), which were treated with 300 mM NaCl for 0 and 8 h. Each library generated about 20 million short sequences and contained predominantly small RNAs of 24-nt length, followed by 21-nt and 22-nt small RNAs. Using sequence analysis, we identified 385 conserved miRNAs from 96 families, along with 68 novel candidate miRNAs. Of all the 68 predicted novel miRNAs, 15 miRNAs were identified to have miRNA*. Statistical analysis on abundance of sequencing read revealed specific miRNA showing contrasting expression patterns between M. sativa and M. truncatula roots, as well as between roots treated for 0 and 8 h. The expression of 10 conserved and novel miRNAs was also quantified by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). The miRNA precursor and target genes were predicted by bioinformatics analysis. We concluded that the salt stress related conserved and novel miRNAs may have a large variety of target mRNAs, some of which might play key roles in salt stress regulation of Medicago.

  2. Physical characteristics of Medicago truncatula calcium oxalate crystals determine their effectiveness in insect defense

    USDA-ARS?s Scientific Manuscript database

    Plant structural traits can act as defense against herbivorous insects, causing them to avoid feeding on a given plant or tissue. Mineral crystals of calcium oxalate in leaves of Medicago truncatula Gaertn. have previously been shown to be effective deterrents of lepidopteran insect feeding. They ar...

  3. Structural and chemical insect defenses in calcium oxalate defective mutants of Medicago truncatula

    USDA-ARS?s Scientific Manuscript database

    Plant structures can act as defense against herbivorous insects, causing them to avoid feeding on a given plant or tissue. Mineral crystals of calcium oxalate in leaves of Medicago truncatula Gaertn. are effective deterrents of lepidopteran feeding, and they inhibit conversion of leaves into insect ...

  4. Ractopamine uptake by alfalfa (Medicago sativa) and wheat (Triticum aestivum) from soil

    USDA-ARS?s Scientific Manuscript database

    Ractopamine is a beta adrenergic agonist used as a growth promoter in swine, cattle and turkeys. To test whether ractopamine has the potential to accumulate in plants grown in contaminated soil, a greenhouse study was conducted with alfalfa (Medicago sativa) and wheat (Triticum aestivum) grown in t...

  5. A set of GFP organelle marker lines for intracellular localization studies in Medicago truncatula

    USDA-ARS?s Scientific Manuscript database

    Genomics advances in the model legume Medicago truncatula have led to an increase in the number of identified genes encoding proteins with unknown biological function. Determining the intracellular location of uncharacterized proteins often aids in the elucidation of biological function. To expedite...

  6. First report of race 2 of Colletotrichum trifolii causing anthracnose on alfalfa (Medicago sativa) in Wisconsin

    USDA-ARS?s Scientific Manuscript database

    Anthracnose of alfalfa (Medicago sativa), caused by Colletotrichum trifolii, is widespread in the United States. Three physiological races have been described. Race 1 is reported to be the dominant race that is present wherever alfalfa is grown, while race 2 was reported in a limited area in the Mid...

  7. Crop wild relatives of Medicago in Russia and neighboring countries: gap analysis for effective conservation

    USDA-ARS?s Scientific Manuscript database

    The genus Medicago includes important species used for forage, fodder and land improvement. The countries of the Former Soviet Union have a wide diversity of alfalfa crop wild relatives and annual medic species. The N.I. Vavilov Institute of Plant Industry (VIR) has a long history of collecting and ...

  8. Identification of novel RNA viruses in alfalfa (Medicago sativa): an Alphapartitivirus, a Deltapartitivirus and a Marafivirus.

    PubMed

    Kim, Hyein; Park, Dongbin; Hahn, Yoonsoo

    2017-09-30

    Genomic RNA molecules of plant RNA viruses are often co-isolated with the host RNAs, and their sequences can be detected in plant transcriptome datasets. Here, an alfalfa (Medicago sativa) transcriptome dataset was analyzed and three new RNA viruses were identified, which were named Medicago sativa alphapartitivirus 1 (MsAPV1), Medicago sativa deltapartitivirus 1 (MsDPV1), and Medicago sativa marafivirus 1 (MsMV1). The RNA-dependent RNA polymerases of MsAPV1, MsDPV1, and MsMV1 showed about 68%, 58%, and 46% amino acid sequence identity, respectively, with their closest virus species. Sequence similarity and phylogenetic analyses indicated that MsAPV1, MsDPV1, and MsMV1 were novel RNA virus species that belong to the genus Alphapartitivirus of the family Partitiviridae, the genus Deltapartitivirus of the family Partitiviridae, and the genus Marafivirus of the family Tymoviridae, respectively. The bioinformatics procedure applied in this study may facilitate the identification of novel RNA viruses from plant transcriptome data. Copyright © 2017. Published by Elsevier B.V.

  9. Sequence divergence of microsatellites for phylogeographic assessment of Moroccan Medicago species.

    PubMed

    Zitouna, N; Marghali, S; Gharbi, M; Haddioui, A; Trifi-Farah, N

    2014-03-12

    Six Medicago species were investigated to characterize and valorize plant genetic resources of pastoral interest in Morocco. Samples were obtained from the core collection of the South Australian Research and Development Institute (SARDI). The transferability of single sequence repeat markers of Medicago truncatula was successful with 97.6% efficiency across the five species. A total of 283 alleles and 243 genotypes were generated using seven SSR markers, confirming the high level of polymorphism that is characteristic of the Medicago genus, despite a heterozygosity deficit (HO = 0.378; HE = 0.705). In addition, a high level of gene flow was revealed among the species analyzed with significant intra-specific variation. The unweighted pair group method with arithmetic mean dendrogram generated by the dissimilarity matrix revealed that M. polymorpha and M. orbicularis are closely related, and that M. truncatula is likely the ancestral species. The Pearson correlation index revealed no significant correlations between the geographic distribution of the Moroccan species and genetic similarities, indicating local adaptation of these species to different ecological environments independent of their topographical proximities. The substantial genetic variation observed was likely due to the predominance of selfing species, the relative proximity of prospected sites, human impacts, and the nature of the SARDI core collections, which are selected for their high genetic diversity. The results of this first report on Moroccan Medicago species will be of great interest for establishing strategies aiming at reasonable management and selection programs for local and Mediterranean germplasm in the face of increasing environmental change.

  10. Characterization of a bi-pistil mutant in Medicago truncatula Gaertn

    USDA-ARS?s Scientific Manuscript database

    We propose the name bi-pistil, bip, for a floral organ mutant observed in transgenic Medicago truncatula plants. The mutant has two separate stigmas borne on two separate styles that emerge from a single superior carpel primordium. The bip plant was crossed to a previously reported male sterile mtap...

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

    USDA-ARS?s Scientific Manuscript database

    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 m...

  12. Identification and Expression Analysis of BURP Domain-Containing Genes in Medicago truncatula

    PubMed Central

    Li, Yuan; Chen, Xue; Chen, Zhu; Cai, Ronghao; Zhang, Hongmei; Xiang, Yan

    2016-01-01

    BURP domain-containing proteins belong to a newly identified protein class that is unique to plants and plays an important role in plant development and metabolism. Although systematic characterization of BURP domain-containing proteins have been carried out in many species, such as rice, poplar and maize, little is known about BURP domain-containing proteins in Medicago. In this study, multiple bioinformatics approaches were employed to identify all the members of BURP family genes in Medicago. A complete set of 39 BURP family genes were identified. These genes have diverse structures and were distributed on chromosome 1–8 except 7. According to phylogenetic analysis, these BURP family genes could be classified into eight classes. Motif and exon-intron organization, stress-related cis-elements in promoter regions and microarray analysis of MtBURPs were also performed. Furthermore, transcript level analysis of MtBURP genes in response to drought stress revealed that all of the 39 BURP genes were regulated by drought stress. The results of this study reveal a comprehensive overview of the Medicago BURP gene family and provide the first step toward the selection of MtBURP genes for cloning and functional analysis of the BURP gene family in Medicago truncatula. PMID:27148311

  13. Identification of Novel Defense Response Genes in Medicago truncatula for Improving Disease Resistance in Alfalfa

    USDA-ARS?s Scientific Manuscript database

    Infection of plants by pathogens initiates a cascade of defense responses that halt or limit pathogen growth. However, the role of many of the genes induced by pathogens is unknown. Transcript profiling was used to identify genes associated with defense responses in the model legume Medicago truncat...

  14. Induction of peroxidases and superoxide dismutases in transformed embryogenic calli of alfalfa (Medicago sativa L.)

    USDA-ARS?s Scientific Manuscript database

    Activities of peroxidase (POD) and superoxide dismutase (SOD) enzymes were analyzed in non-regenerative transformed embryogenic lines of alfalfa (Medicago sativa L.) carrying wound-inducible oryzacystatin I (OC-I), wound-inducible oryzacystatin I antisense (OC-Ias) or hygromycin phosphotransferase (...

  15. The TFIIS and TFIIS-like genes from Medicago truncatula are involved in oxidative stress response.

    PubMed

    Macovei, Anca; Balestrazzi, Alma; Confalonieri, Massimo; Buttafava, Armando; Carbonera, Daniela

    2011-01-01

    The cDNA sequence coding for a novel putative TFIIS (transcription elongation factor II-S), hereby named MtTFIIS-like, was isolated from barrel medic (Medicago truncatula Gaertn.) by reverse transcriptase-polymerase chain reaction. The nucleotide sequence contains an open reading frame of 1074 bp, predicting a 40.0 kDa protein, conserved among plant species. The N-terminal region of the MtTFIIS-like protein includes a LW motif, characterized by highly conserved leucine (L) and tryptophan (W) residues, also found in the canonical TFIIS protein, elongin A (transcription elongation factor S-III) and CRSP70 (cofactor required for Sp1 activation), while a proline-rich region is present in the C-terminal domain. The expression profiles of the MtTFIIS-like gene were evaluated by quantitative real-time PCR (QRT-PCR) in barrel medic plantlets grown in vitro under oxidative stress conditions induced by copper (CuCl(2) 0.05, 0.1 and 0.2mM) and polyethylene glycol (PEG6000 50, 100 and 150 g/L), respectively. Both stress agents caused ROS (reactive oxygen species) accumulation. Moreover, EPR spectra of leaves from plantlets exposed to toxic copper doses confirmed that the heavy metal is translocated from roots to the aerial parts, where it is found predominantly in the Cu(2+) redox state. The MtTFIIS-like gene expression was significantly enhanced (up to 2.9-fold) in aerial parts of copper-treated plants, and in roots (up to 4.4-fold) in response to PEG treatments. The expression profiles of the MtTFIIS-like gene were compared to those of the MtTFIIS gene, encoding the canonical TFIIS protein, which was similarly up-regulated in response to both stresses. Interestingly, the MtTFIIS-like and MtTFIIS genes were significantly up-regulated (up to 3.2- and 4.3-fold, respectively) during seed imbibition, a physiological process which requires active DNA repair. Based on the reported data, the possible roles played in planta by the novel MtTFIIS-like gene are discussed.

  16. Starch Grain Distribution in Taproots of Defoliated Medicago sativa L.

    PubMed

    Habben, J E; Volenec, J J

    1990-11-01

    Defoliation of alfalfa (Medicago sativa L.) results in a cyclic pattern of starch degradation followed by reaccumulation in taproots. Characterization of changes in anatomical distribution of starch grains in taproots will aid our understanding of biochemical and physiological mechanisms involved in starch metabolism in taproots of this species. Our objectives were to determine the influence of defoliation on starch grain distribution and size variation in taproots of two alfalfa lines selected for contrasting concentrations of taproot starch. In addition, we used electron microscopy to examine the cellular environment of starch grains, and computer-based image optical analysis to determine how cross-sectional area of tissues influenced starch accumulation. Taproots of field-grown plants were sampled at defoliation and weekly thereafter over a 28-day period. Taproot segments were fixed in glutaraldehyde and prepared for either light or electron microscopy. Transverse sections were examined for number and size of starch grains and tissue areas were measured. Starch grains were located throughout bark tissues, but were confined primarily to ray parenchyma cells in wood tissues. During the first week of foliar regrowth after defoliation, starch grains in ray cells near the cambium disappeared first, while degradation of those near the center of the taproot was delayed. During the third and fourth weeks of regrowth, there was a uniform increase in number of starch grains per cell profile across the rays, but by 28 days after defoliation there were more starch grains in ray cells near the cambium than in cells near the center of the taproot (low starch line only). Bark tissues from both lines showed synchronous degradation and synthesis of starch grains that was not influenced greatly by cell location. Diameter of starch grains varied with cell location in medullary rays during rapid starch degradation, but was not influenced by cell position in bark tissues. Therefore

  17. Physiological and molecular characterization of aluminum resistance in Medicago truncatula

    PubMed Central

    Chandran, Divya; Sharopova, Natasha; VandenBosch, Kathryn A; Garvin, David F; Samac, Deborah A

    2008-01-01

    Background Aluminum (Al) toxicity is an important factor limiting crop production on acid soils. However, little is known about the mechanisms by which legumes respond to and resist Al stress. To explore the mechanisms of Al toxicity and resistance in legumes, we compared the impact of Al stress in Al-resistant and Al-sensitive lines of the model legume, Medicago truncatula Gaertn. Results A screen for Al resistance in 54 M. truncatula accessions identified eight Al-resistant and eight Al-sensitive lines. Comparisons of hydroponic root growth and root tip hematoxylin staining in an Al-resistant line, T32, and an Al-sensitive line, S70, provided evidence that an inducible Al exclusion mechanism occurs in T32. Transcriptional events associated with the Al resistance response were analyzed in T32 and S70 after 12 and 48 h Al treatment using oligonucleotide microarrays. Fewer genes were differentially regulated in response to Al in T32 compared to S70. Expression patterns of oxidative stress-related genes, stress-response genes and microscopic examination of Al-treated root tips suggested a lower degree of Al-induced oxidative damage to T32 root tips compared to S70. Furthermore, genes associated with cell death, senescence, and cell wall degradation were induced in both lines after 12 h of Al treatment but preferentially in S70 after 48 h of Al treatment. A multidrug and toxin efflux (MATE) transporter, previously shown to exude citrate in Arabidopsis, showed differential expression patterns in T32 and S70. Conclusion Our results identified novel genes induced by Al in Al-resistant and sensitive M. truncatula lines. In T32, transcription levels of genes related to oxidative stress were consistent with reactive oxygen species production, which would be sufficient to initiate cell death of Al-accumulating cells thereby contributing to Al exclusion and root growth recovery. In contrast, transcriptional levels of oxidative stress-related genes were consistent with

  18. Expression of coordinately regulated defense response genes and analysis of their role in disease resistance in Medicago truncatula

    USDA-ARS?s Scientific Manuscript database

    Microarray technology was used to identify genes associated with disease defense responses in the model legume Medicago truncatula. Transcript profiles from leaves inoculated with Colletotrichum trifolii and Erysiphe pisi and roots infected with Phytophthora medicaginis were compared to identify gen...

  19. Amplification, contraction and genomic spread of a satellite DNA family (E180) in Medicago (Fabaceae) and allied genera

    PubMed Central

    Rosato, Marcela; Galián, José A.; Rosselló, Josep A.

    2012-01-01

    Background and Aims Satellite DNA is a genomic component present in virtually all eukaryotic organisms. The turnover of highly repetitive satellite DNA is an important element in genome organization and evolution in plants. Here we assess the presence and physical distribution of the repetitive DNA E180 family in Medicago and allied genera. Our goals were to gain insight into the karyotype evolution of Medicago using satellite DNA markers, and to evaluate the taxonomic and phylogenetic signal of a satellite DNA family in a genus hypothesized to have a complex evolutionary history. Methods Seventy accessions from Medicago, Trigonella, Melilotus and Trifolium were analysed by PCR to assess the presence of the repetitive E180 family, and fluorescence in situ hybridization (FISH) was used for physical mapping in somatic chromosomes. Key Results The E180 repeat unit was PCR-amplified in 37 of 40 taxa in Medicago, eight of 12 species of Trigonella, six of seven species of Melilotus and in two of 11 Trifolium species. Examination of the mitotic chromosomes revealed that only 13 Medicago and two Trigonella species showed FISH signals using the E180 probe. Stronger hybridization signals were observed in subtelomeric and interstitial loci than in the pericentromeric loci, suggesting this satellite family has a preferential genomic location. Not all 13 Medicago species that showed FISH localization of the E180 repeat were phylogenetically related. However, nine of these species belong to the phylogenetically derived clade including the M. sativa and M. arborea complexes. Conclusions The use of the E180 family as a phylogenetic marker in Medicago should be viewed with caution. Its amplification appears to have been produced through recurrent and independent evolutionary episodes in both annual and perennial Medicago species as well as in basal and derived clades. PMID:22186276

  20. Molecular cloning of a bifunctional beta-xylosidase/alpha-L-arabinosidase from alfalfa roots: heterologous expression in Medicago truncatula and substrate specificity of the purified enzyme.

    PubMed

    Xiong, Jin-Song; Balland-Vanney, Maud; Xie, Zhi-Ping; Schultze, Michael; Kondorosi, Adam; Kondorosi, Eva; Staehelin, Christian

    2007-01-01

    Glycoside hydrolases are often members of a multigene family, suggesting individual roles for each isoenzyme. Various extracellular glycoside hydrolases have an important but poorly understood function in remodelling the cell wall during plant growth. Here, MsXyl1, a concanavalin A-binding protein from alfalfa (Medicago sativa L.) belonging to the glycoside hydrolase family 3 (beta-D-xylosidase branch) is characterized. Transcripts of MsXyl1 were detected in roots (particularly root tips), root nodules, and flowers. MsXyl1 under the control of the CaMV 35S promoter was expressed in the model legume Medicago truncatula (Gaertner). Concanavalin A-binding proteins from the transgenic plants exhibited 5-8-fold increased activities towards three p-nitrophenyl (PNP) glycosides, namely PNP-beta-D-xyloside, PNP-alpha-L-arabinofuranoside, and PNP-alpha-L-arabinopyranoside. An antiserum raised against a synthetic peptide recognized MsXyl1, which was processed to a 65 kDa form. To characterize the substrate specificity of MsXyl1, the recombinant protein was purified from transgenic M. truncatula leaves by concanavalin A and anion chromatography. MsXyl1cleaved beta-1,4-linked D-xylo-oligosaccharides and alpha-1,5-linked L-arabino-oligosaccharides. Arabinoxylan (from wheat) and arabinan (from sugar beet) were substrates for MsXyl1, whereas xylan (from oat spelts) was resistant to degradation. Furthermore, MsXyl1 released xylose and arabinose from cell wall polysaccharides isolated from alfalfa roots. These data suggest that MsXyl1 is a multifunctional beta-xylosidase/alpha-L-arabinofuranosidase/alpha-L-arabinopyranosidase implicated in cell wall turnover of arabinose and xylose, particularly in rapidly growing root tips. Moreover, the findings of this study demonstrate that stable transgenic M. truncatula plants serve as an excellent expression system for purification and characterization of proteins.

  1. The Medicago sativa gene index 1.2: a web-accessible gene expression atlas for investigating expression differences between Medicago sativa subspecies.

    PubMed

    O'Rourke, Jamie A; Fu, Fengli; Bucciarelli, Bruna; Yang, S Sam; Samac, Deborah A; Lamb, JoAnn F S; Monteros, Maria J; Graham, Michelle A; Gronwald, John W; Krom, Nick; Li, Jun; Dai, Xinbin; Zhao, Patrick X; Vance, Carroll P

    2015-07-07

    Alfalfa (Medicago sativa L.) is the primary forage legume crop species in the United States and plays essential economic and ecological roles in agricultural systems across the country. Modern alfalfa is the result of hybridization between tetraploid M. sativa ssp. sativa and M. sativa ssp. falcata. Due to its large and complex genome, there are few genomic resources available for alfalfa improvement. A de novo transcriptome assembly from two alfalfa subspecies, M. sativa ssp. sativa (B47) and M. sativa ssp. falcata (F56) was developed using Illumina RNA-seq technology. Transcripts from roots, nitrogen-fixing root nodules, leaves, flowers, elongating stem internodes, and post-elongation stem internodes were assembled into the Medicago sativa Gene Index 1.2 (MSGI 1.2) representing 112,626 unique transcript sequences. Nodule-specific and transcripts involved in cell wall biosynthesis were identified. Statistical analyses identified 20,447 transcripts differentially expressed between the two subspecies. Pair-wise comparisons of each tissue combination identified 58,932 sequences differentially expressed in B47 and 69,143 sequences differentially expressed in F56. Comparing transcript abundance in floral tissues of B47 and F56 identified expression differences in sequences involved in anthocyanin and carotenoid synthesis, which determine flower pigmentation. Single nucleotide polymorphisms (SNPs) unique to each M. sativa subspecies (110,241) were identified. The Medicago sativa Gene Index 1.2 increases the expressed sequence data available for alfalfa by ninefold and can be expanded as additional experiments are performed. The MSGI 1.2 transcriptome sequences, annotations, expression profiles, and SNPs were assembled into the Alfalfa Gene Index and Expression Database (AGED) at http://plantgrn.noble.org/AGED/ , a publicly available genomic resource for alfalfa improvement and legume research.

  2. Legume genome evolution viewed through the Medicago truncatula and Lotus japonicus genomes

    PubMed Central

    Cannon, Steven B.; Sterck, Lieven; Rombauts, Stephane; Sato, Shusei; Cheung, Foo; Gouzy, Jérôme; Wang, Xiaohong; Mudge, Joann; Vasdewani, Jayprakash; Schiex, Thomas; Spannagl, Manuel; Monaghan, Erin; Nicholson, Christine; Humphray, Sean J.; Schoof, Heiko; Mayer, Klaus F. X.; Rogers, Jane; Quétier, Francis; Oldroyd, Giles E.; Debellé, Frédéric; Cook, Douglas R.; Retzel, Ernest F.; Roe, Bruce A.; Town, Christopher D.; Tabata, Satoshi; Van de Peer, Yves; Young, Nevin D.

    2006-01-01

    Genome sequencing of the model legumes, Medicago truncatula and Lotus japonicus, provides an opportunity for large-scale sequence-based comparison of two genomes in the same plant family. Here we report synteny comparisons between these species, including details about chromosome relationships, large-scale synteny blocks, microsynteny within blocks, and genome regions lacking clear correspondence. The Lotus and Medicago genomes share a minimum of 10 large-scale synteny blocks, each with substantial collinearity and frequently extending the length of whole chromosome arms. The proportion of genes syntenic and collinear within each synteny block is relatively homogeneous. Medicago–Lotus comparisons also indicate similar and largely homogeneous gene densities, although gene-containing regions in Mt occupy 20–30% more space than Lj counterparts, primarily because of larger numbers of Mt retrotransposons. Because the interpretation of genome comparisons is complicated by large-scale genome duplications, we describe synteny, synonymous substitutions and phylogenetic analyses to identify and date a probable whole-genome duplication event. There is no direct evidence for any recent large-scale genome duplication in either Medicago or Lotus but instead a duplication predating speciation. Phylogenetic comparisons place this duplication within the Rosid I clade, clearly after the split between legumes and Salicaceae (poplar). PMID:17003129

  3. A functional genomics approach to (iso)flavonoid glycosylation in the model legume Medicago truncatula.

    PubMed

    Modolo, Luzia V; Blount, Jack W; Achnine, Lahoucine; Naoumkina, Marina A; Wang, Xiaoqiang; Dixon, Richard A

    2007-07-01

    Analysis of over 200,000 expressed sequence tags from a range of Medicago truncatula cDNA libraries resulted in the identification of over 150 different family 1 glycosyltransferase (UGT) genes. Of these, 63 were represented by full length clones in an EST library collection. Among these, 19 gave soluble proteins when expressed in E. coli, and these were screened for catalytic activity against a range of flavonoid and isoflavonoid substrates using a high-throughput HPLC assay method. Eight UGTs were identified with activity against isoflavones, flavones, flavonols or anthocyanidins, and several showed high catalytic specificity for more than one class of (iso)flavonoid substrate. All tested UGTs preferred UDP-glucose as sugar donor. Phylogenetic analysis indicated that the Medicago (iso)flavonoid glycosyltransferase gene sequences fell into a number of different clades, and several clustered with UGTs annotated as glycosylating non-flavonoid substrates. Quantitative RT-PCR and DNA microarray analysis revealed unique transcript expression patterns for each of the eight UGTs in Medicago organs and cell suspension cultures, and comparison of these patterns with known phytochemical profiles suggested in vivo functions for several of the enzymes.

  4. Cell- and Tissue-Specific Transcriptome Analyses of Medicago truncatula Root Nodules

    PubMed Central

    Limpens, Erik; Moling, Sjef; Hooiveld, Guido; Pereira, Patrícia A.; Bisseling, Ton; Becker, Jörg D.; Küster, Helge

    2013-01-01

    Legumes have the unique ability to host nitrogen-fixing Rhizobium bacteria as symbiosomes inside root nodule cells. To get insight into this key process, which forms the heart of the endosymbiosis, we isolated specific cells/tissues at different stages of symbiosome formation from nodules of the model legume Medicago truncatula using laser-capture microdissection. Next, we determined their associated expression profiles using Affymetrix Medicago GeneChips. Cells were collected from the nodule infection zone divided into a distal (where symbiosome formation and division occur) and proximal region (where symbiosomes are mainly differentiating), as well as infected cells from the fixation zone containing mature nitrogen fixing symbiosomes. As non-infected cells/tissue we included nodule meristem cells and uninfected cells from the fixation zone. Here, we present a comprehensive gene expression map of an indeterminate Medicago nodule and selected genes that show specific enriched expression in the different cells or tissues. Validation of the obtained expression profiles, by comparison to published gene expression profiles and experimental verification, indicates that the data can be used as digital “in situ”. This digital “in situ” offers a genome-wide insight into genes specifically associated with subsequent stages of symbiosome and nodule cell development, and can serve to guide future functional studies. PMID:23734198

  5. Databases and information integration for the Medicago truncatula genome and transcriptome.

    PubMed

    Cannon, Steven B; Crow, John A; Heuer, Michael L; Wang, Xiaohong; Cannon, Ethalinda K S; Dwan, Christopher; Lamblin, Anne-Francoise; Vasdewani, Jayprakash; Mudge, Joann; Cook, Andrew; Gish, John; Cheung, Foo; Kenton, Steve; Kunau, Timothy M; Brown, Douglas; May, Gregory D; Kim, Dongjin; Cook, Douglas R; Roe, Bruce A; Town, Chris D; Young, Nevin D; Retzel, Ernest F

    2005-05-01

    An international consortium is sequencing the euchromatic genespace of Medicago truncatula. Extensive bioinformatic and database resources support the marker-anchored bacterial artificial chromosome (BAC) sequencing strategy. Existing physical and genetic maps and deep BAC-end sequencing help to guide the sequencing effort, while EST databases provide essential resources for genome annotation as well as transcriptome characterization and microarray design. Finished BAC sequences are joined into overlapping sequence assemblies and undergo an automated annotation process that integrates ab initio predictions with EST, protein, and other recognizable features. Because of the sequencing project's international and collaborative nature, data production, storage, and visualization tools are broadly distributed. This paper describes databases and Web resources for the project, which provide support for physical and genetic maps, genome sequence assembly, gene prediction, and integration of EST data. A central project Web site at medicago.org/genome provides access to genome viewers and other resources project-wide, including an Ensembl implementation at medicago.org, physical map and marker resources at mtgenome.ucdavis.edu, and genome viewers at the University of Oklahoma (www.genome.ou.edu), the Institute for Genomic Research (www.tigr.org), and Munich Information for Protein Sequences Center (mips.gsf.de).

  6. Acacia nilotica and Medicago sativa, suitable plants for agro-forestry in southern coasts of Iran.

    PubMed

    Emtehani, Mohammad Hassan; Tabari, Masoud

    2007-05-15

    Habitats of the multipurpose tree, Acacia nilotica, were identified along the coastline of the Persian Gulf and Oman Sea, south of Iran. Four sites were randomly chosen and in each one, vegetation as well as climatic and soil characteristics were studied. Likewise, biometry of Acacia trees was conducted and compared in the sites. The fodder values in leaf and fruit of Acacia were determined and compared with those of in foliage of Medicago sativa being managed under an agro-forestry system. By analyzing data it was revealed that the biggest trees were found in Dashteyari region and the smallest in Bamani region (both in the Oman Sea coast). Values in most of nutritional elements were higher in foliage of Medicago than in leaf and fruit of Acacia, respectively. From this investigation it is concluded that in south of Iran where the site is favorable for Acacia plantation, cultivation of Medicago or other adaptable crops together with Acacia can be developed as agroforestry systems (such as undercropping and intercropping) if water is available.

  7. Isolation and characterization of a gene from Medicago sativa L., encoding a bZIP transcription factor.

    PubMed

    Li, Yan; Sun, Yan; Yang, Qingchuan; Fang, Feng; Kang, Junmei; Zhang, Tiejun

    2013-02-01

    A full-length cDNA of 1,537 nucleotides was cloned from Medicago sativa L. cv. "Zhongmu No. 1" by rapid amplification of cDNA ends. It was designated as MsZIP, encoding a protein of 340 amino acids. The protein molecular weight was 36.43 kDa, and the theoretical isoelectric point was 5.72. The MsZIP preferentially localized in nucleus and have signal peptide. Blast analysis revealed that MsZIP shared the highest homology with some bZIP proteins of M. truncatula. The transcript of MsZIP was strongly enriched in leaf compared with root and stem of mature alfalfa plants. MsZIP was strongly induced by 15 % PEG6000 (polyethylene glycol), 50 μM abscisic acid, 200 mM NaCl, 70 μM gibberellic acid, 5 mM salicylic acid and 200 μM methyl jasmonate. Physiological resistance parameters were measured in the transgenic tobacco. Malondialdehyde content, relative water content, soluble sugar content, soluble protein content and proline content in transgenic tobacco increased compared with non-transgenic tobacco under salt stress or drought stress. The results showed that accumulation of the MsZIP protein in the vegetative tissues of transgenic plants enhanced their tolerance to osmotic pressure stress. These results demonstrate a role for the MsZIP protein in stress protection and suggest the potential of the MsZIP gene for genetic engineering of salt tolerance and drought tolerance.

  8. Peroxidases from root exudates of Medicago sativa and Sorghum bicolor: Catalytic properties and involvement in PAH degradation.

    PubMed

    Dubrovskaya, Ekaterina; Pozdnyakova, Natalia; Golubev, Sergey; Muratova, Anna; Grinev, Vyacheslav; Bondarenkova, Anastasiya; Turkovskaya, Olga

    2017-02-01

    Peroxidases from root exudates of sorghum (Sorghum bicolor L. Moench) and alfalfa (Medicago sativa L.) were purified and characterized, and their ability to oxidize native PAHs and PAH-derivatives was evaluated. The obtained data confirm that peroxidases are involved in the rhizosphere degradation of PAHs. Nondenaturing PAGE showed that the peroxidases of both plants were represented by a range of isoforms/isoenzymes (five to eight). Minor forms were lost during further purification, and as a result, the major anionic form from alfalfa root exudates and the major cationic form from those of sorghum were obtained. Both electrophoretically homogeneous peroxidases were monomeric proteins with a molecular weight of about 46-48 kDa. The pH optima and the main catalytic constants for the test substrates were determined. On the basis of their molecular and catalytic properties, the obtained enzymes were found to be typical plant peroxidases. Derivatives of PAHs and potential products of their microbial degradation (9-phenanthrol and 9,10-phenanthrenequinone), unlike the parent PAH (phenanthrene), inhibited the catalytic activity of the peroxidases, possibly indicating greater availability of the enzymes' active centers to these substances. Peroxidase-catalyzed decreases in the concentrations of a number of PAHs and their derivatives were observed. Sorghum peroxidase oxidized anthracene and phenanthrene, while alfalfa peroxidase oxidized only phenanthrene. 1-Hydroxy-2-naphthoic acid was best oxidized by peroxidase of alfalfa. However, quinone derivatives of PAHs were unavailable to sorghum peroxidase, but were oxidized by alfalfa peroxidase. These results indicate that the major peroxidases from root exudates of alfalfa and sorghum can have a role in the rhizosphere degradation of PAHs.

  9. An improved genome release (version Mt4.0) for the model legume Medicago truncatula

    PubMed Central

    2014-01-01

    Background Medicago truncatula, a close relative of alfalfa, is a preeminent model for studying nitrogen fixation, symbiosis, and legume genomics. The Medicago sequencing project began in 2003 with the goal to decipher sequences originated from the euchromatic portion of the genome. The initial sequencing approach was based on a BAC tiling path, culminating in a BAC-based assembly (Mt3.5) as well as an in-depth analysis of the genome published in 2011. Results Here we describe a further improved and refined version of the M. truncatula genome (Mt4.0) based on de novo whole genome shotgun assembly of a majority of Illumina and 454 reads using ALLPATHS-LG. The ALLPATHS-LG scaffolds were anchored onto the pseudomolecules on the basis of alignments to both the optical map and the genotyping-by-sequencing (GBS) map. The Mt4.0 pseudomolecules encompass ~360 Mb of actual sequences spanning 390 Mb of which ~330 Mb align perfectly with the optical map, presenting a drastic improvement over the BAC-based Mt3.5 which only contained 70% sequences (~250 Mb) of the current version. Most of the sequences and genes that previously resided on the unanchored portion of Mt3.5 have now been incorporated into the Mt4.0 pseudomolecules, with the exception of ~28 Mb of unplaced sequences. With regard to gene annotation, the genome has been re-annotated through our gene prediction pipeline, which integrates EST, RNA-seq, protein and gene prediction evidences. A total of 50,894 genes (31,661 high confidence and 19,233 low confidence) are included in Mt4.0 which overlapped with ~82% of the gene loci annotated in Mt3.5. Of the remaining genes, 14% of the Mt3.5 genes have been deprecated to an “unsupported” status and 4% are absent from the Mt4.0 predictions. Conclusions Mt4.0 and its associated resources, such as genome browsers, BLAST-able datasets and gene information pages, can be found on the JCVI Medicago web site (http://www.jcvi.org/medicago). The assembly and annotation has

  10. Molecular docking of Glycine max and Medicago truncatula ureases with urea; bioinformatics approaches.

    PubMed

    Filiz, Ertugrul; Vatansever, Recep; Ozyigit, Ibrahim Ilker

    2016-03-01

    Urease (EC 3.5.1.5) is a nickel-dependent metalloenzyme catalyzing the hydrolysis of urea into ammonia and carbon dioxide. It is present in many bacteria, fungi, yeasts and plants. Most species, with few exceptions, use nickel metalloenzyme urease to hydrolyze urea, which is one of the commonly used nitrogen fertilizer in plant growth thus its enzymatic hydrolysis possesses vital importance in agricultural practices. Considering the essentiality and importance of urea and urease activity in most plants, this study aimed to comparatively investigate the ureases of two important legume species such as Glycine max (soybean) and Medicago truncatula (barrel medic) from Fabaceae family. With additional plant species, primary and secondary structures of 37 plant ureases were comparatively analyzed using various bioinformatics tools. A structure based phylogeny was constructed using predicted 3D models of G. max and M. truncatula, whose crystallographic structures are not available, along with three additional solved urease structures from Canavalia ensiformis (PDB: 4GY7), Bacillus pasteurii (PDB: 4UBP) and Klebsiella aerogenes (PDB: 1FWJ). In addition, urease structures of these species were docked with urea to analyze the binding affinities, interacting amino acids and atom distances in urease-urea complexes. Furthermore, mutable amino acids which could potentially affect the protein active site, stability and flexibility as well as overall protein stability were analyzed in urease structures of G. max and M. truncatula. Plant ureases demonstrated similar physico-chemical properties with 833-878 amino acid residues and 89.39-90.91 kDa molecular weight with mainly acidic (5.15-6.10 pI) nature. Four protein domain structures such as urease gamma, urease beta, urease alpha and amidohydro 1 characterized the plant ureases. Secondary structure of plant ureases also demonstrated conserved protein architecture, with predominantly α-helix and random coil structures. In

  11. MATE transporters facilitate vacuolar uptake of epicatechin 3'-O-glucoside for proanthocyanidin biosynthesis in Medicago truncatula and Arabidopsis.

    PubMed

    Zhao, Jian; Dixon, Richard A

    2009-08-01

    Expression of the Arabidopsis thaliana MYB transcription factor TRANSPARENT TESTA 2 (TT2) in Medicago trunculata hairy roots induces both proanthocyanidin accumulation and the ATP-dependent vacuolar/vesicular uptake of epicatechin 3'-O-glucoside; neither process is active in control roots that do, however, possess anthocyanidin 3-O-glucoside vacuolar uptake activity. A vacuolar membrane-localized multidrug and toxic compound extrusion (MATE) transporter, Medicago MATE1, was identified at the molecular level and shown to preferentially transport epicatechin 3'-O-glucoside. Genetic evidence has implicated TT12, a tonoplastic MATE transporter from Arabidopsis, in the transport of precursors for proanthocyanidin biosynthesis in the seed coat. However, although Arabidopsis TT12 facilitates the transport of cyanidin 3-O-glucoside into membrane vesicles when expressed in yeast, there is no evidence that cyanidin 3-O-glucoside is converted to proanthocyanidins after transport into the vacuole. Here, we show that Arabidopsis TT12, like Medicago MATE1, functions to transport epicatechin 3'-O-glucoside as a precursor for proanthocyanidin biosynthesis, and Medicago MATE1 complements the seed proanthocyanidin phenotype of the Arabidopsis tt12 mutant both quantitatively and qualitatively. On the basis of biochemical properties, tissue-specific expression pattern, and genetic loss-of-function analysis, we conclude that MATE1 is an essential membrane transporter for proanthocyanidin biosynthesis in the Medicago seed coat. Implications of these findings for the assembly of oligomeric proanthocyanidins are discussed.

  12. The succinoglycan endoglycanase encoded by exoK is required for efficient symbiosis of Sinorhizobium meliloti 1021 with the host plants Medicago truncatula and Medicago sativa (Alfalfa).

    PubMed

    Mendis, Hajeewaka C; Queiroux, Clothilde; Brewer, Tess E; Davis, Olivia M; Washburn, Brian K; Jones, Kathryn M

    2013-09-01

    The acidic polysaccharide succinoglycan produced by the nitrogen-fixing rhizobial symbiont Sinorhizobium meliloti 1021 is required for this bacterium to invade the host plant Medicago truncatula and to efficiently invade the host plant M. sativa (alfalfa). The β-glucanase enzyme encoded by exoK has previously been demonstrated to cleave succinoglycan and participate in producing the low molecular weight form of this polysaccharide. Here, we show that exoK is required for efficient S. meliloti invasion of both M. truncatula and alfalfa. Deletion mutants of exoK have a substantial reduction in symbiotic productivity on both of these plant hosts. Insertion mutants of exoK have an even less productive symbiosis than the deletion mutants with the host M. truncatula that is caused by a secondary effect of the insertion itself, and may be due to a polar effect on the expression of the downstream exoLAMON genes.

  13. [Natural nucleotide polymorphism of the Srlk gene that determines salt stress tolerance in alfalfa (Medicago sativa L)].

    PubMed

    Vishnevskaia, M S; Pavlov, A V; Dziubenko, E A; Dziubenko, N I; Potokina, E K

    2014-04-01

    Based on legume genome syntheny, the nucleotide sequence of Srlk gene, key role of which in response to salt stress was demonstrated for the model species Medicago truncatula, was identified in the major forage and siderate crop alfalfa (Medicago sativa). In twelve alfalfa samples originating from regions with contrasting growing conditions, 19 SNPs were revealed in the Srlk gene. For two nonsynonymous SNPs, molecular markers were designed that could be further used to analyze the association between Srlk gene nucleotide polymorphism and the variability in salt stress tolerance among alfalfa cultivars.

  14. Border sequences of Medicago truncatula CLE36 are specifically cleaved by endoproteases common to the extracellular fluids of Medicago and soybean.

    PubMed

    Djordjevic, Michael A; Oakes, Marie; Wong, Chui E; Singh, Mohan; Bhalla, Prem; Kusumawati, Lucia; Imin, Nijat

    2011-08-01

    CLE (CLAVATA3/ESR-related) peptides are developmental regulators that are secreted into the apoplast. Little is known about the role of the sequences that flank CLE peptides in terms of their biological activity or how they are targeted by proteases that are known to liberate the final active CLE peptides from their precursor sequences. The biological activity of Medicago truncatula CLE36, which possesses broadly conserved border sequences flanking the putative final active CLE36 peptide product, was assessed. Using in vitro root growth assays and an in vitro root and callus formation assay it is shown that CLE36 peptides of different lengths possess differential biological activities. Using mass spectrometry, Glycine max and Medicago extracellular fluids were each shown to possess an endoproteolytic activity that recognizes and cleaves at border sequences in a synthetic 31 amino acid CLE36 'propeptide bait' to liberate biologically active peptide products. Inhibitor studies suggest that a subtilisin, in combination with a carboxypeptidase, liberated and trimmed CLE36, respectively, to form biologically relevant 11-15 amino acid cleavage products. The 15 amino acid cleavage product is more biologically potent on Arabidopsis than shorter or longer CLE peptides. In situ hybridization shows that the soybean orthologue of CLE36 (GmCLE34) is expressed in the provascular tissue. The results suggest that secreted subtilisins can specifically recognize the border sequences of CLE36 propeptides and liberate biologically active cleavage products. These secreted proteases may affect the stability and biological activity of CLE peptides in the apoplast or be involved in CLE36 processing. © 2011 The Author(s).

  15. Roles of poly-3-hydroxybutyrate (PHB) and glycogen in symbiosis of Sinorhizobium meliloti with Medicago sp.

    PubMed

    Wang, Chunxia; Saldanha, Marsha; Sheng, Xiaoyan; Shelswell, Kristopher J; Walsh, Keith T; Sobral, Bruno W S; Charles, Trevor C

    2007-02-01

    Poly-3-hydroxybutyrate (PHB) and glycogen are major carbon storage compounds in Sinorhizobium meliloti. The roles of PHB and glycogen in rhizobia-legume symbiosis are not fully understood. Glycogen synthase mutations were constructed by in-frame deletion (glgA1) or insertion (glgA2). These mutations were combined with a phbC mutation to make all combinations of double and triple mutants. PHB was not detectable in any of the mutants containing the phbC mutation; glycogen was not detectable in any of the mutants containing the glgA1 mutation. PHB levels were significantly lower in the glgA1 mutant, while glycogen levels were increased in the phbC mutant. Exopolysaccharide (EPS) was not detected in any of the phbC mutants, while the glgA1 and glgA2 mutants produced levels of EPS similar to the wild-type. Symbiotic properties of these strains were investigated on Medicago truncatula and Medicago sativa. The results indicated that the strains unable to synthesize PHB, or glycogen, were still able to form nodules and fix nitrogen. However, phbC mutations caused greater nodule formation delay on M. truncatula than on M. sativa. Time-course studies showed that (1) the ability to synthesize PHB is important for N(2) fixation in M. truncatula nodules and younger M. sativa nodules, and (2) the blocking of glycogen synthesis resulted in lower levels of N(2) fixation on M. truncatula and older nodules on M. sativa. These data have important implications for understanding how PHB and glycogen function in the interactions of S. meliloti with Medicago spp.

  16. Genomic characterization of the LEED..PEEDs, a gene family unique to the medicago lineage.

    PubMed

    Trujillo, Diana I; Silverstein, Kevin A T; Young, Nevin D

    2014-08-25

    The LEED..PEED (LP) gene family in Medicago truncatula (A17) is composed of 13 genes coding small putatively secreted peptides with one to two conserved domains of negatively charged residues. This family is not present in the genomes of Glycine max, Lotus japonicus, or the IRLC species Cicer arietinum. LP genes were also not detected in a Trifolium pratense draft genome or Pisum sativum nodule transcriptome, which were sequenced de novo in this study, suggesting that the LP gene family arose within the past 25 million years. M. truncatula accession HM056 has 13 LP genes with high similarity to those in A17, whereas M. truncatula ssp. tricycla (R108) and M. sativa have 11 and 10 LP gene copies, respectively. In M. truncatula A17, 12 LP genes are located on chromosome 7 within a 93-kb window, whereas one LP gene copy is located on chromosome 4. A phylogenetic analysis of the gene family is consistent with most gene duplications occurring prior to Medicago speciation events, mainly through local tandem duplications and one distant duplication across chromosomes. Synteny comparisons between R108 and A17 confirm that gene order is conserved between the two subspecies, although a further duplication occurred solely in A17. In M. truncatula A17, all 13 LPs are exclusively transcribed in nodules and absent from other plant tissues, including roots, leaves, flowers, seeds, seed shells, and pods. The recent expansion of LP genes in Medicago spp. and their timing and location of expression suggest a novel function in nodulation, possibly as an aftermath of the evolution of bacteroid terminal differentiation or potentially associated with rhizobial-host specificity. Copyright © 2014 Trujillo et al.

  17. Genomic Characterization of the LEED..PEEDs, a Gene Family Unique to the Medicago Lineage

    PubMed Central

    Trujillo, Diana I.; Silverstein, Kevin A. T.; Young, Nevin D.

    2014-01-01

    The LEED..PEED (LP) gene family in Medicago truncatula (A17) is composed of 13 genes coding small putatively secreted peptides with one to two conserved domains of negatively charged residues. This family is not present in the genomes of Glycine max, Lotus japonicus, or the IRLC species Cicer arietinum. LP genes were also not detected in a Trifolium pratense draft genome or Pisum sativum nodule transcriptome, which were sequenced de novo in this study, suggesting that the LP gene family arose within the past 25 million years. M. truncatula accession HM056 has 13 LP genes with high similarity to those in A17, whereas M. truncatula ssp. tricycla (R108) and M. sativa have 11 and 10 LP gene copies, respectively. In M. truncatula A17, 12 LP genes are located on chromosome 7 within a 93-kb window, whereas one LP gene copy is located on chromosome 4. A phylogenetic analysis of the gene family is consistent with most gene duplications occurring prior to Medicago speciation events, mainly through local tandem duplications and one distant duplication across chromosomes. Synteny comparisons between R108 and A17 confirm that gene order is conserved between the two subspecies, although a further duplication occurred solely in A17. In M. truncatula A17, all 13 LPs are exclusively transcribed in nodules and absent from other plant tissues, including roots, leaves, flowers, seeds, seed shells, and pods. The recent expansion of LP genes in Medicago spp. and their timing and location of expression suggest a novel function in nodulation, possibly as an aftermath of the evolution of bacteroid terminal differentiation or potentially associated with rhizobial–host specificity. PMID:25155275

  18. Chemical characterisation of bioactive compounds in Medicago sativa growing in the desert of Oman.

    PubMed

    Hanif, Muhammad Asif; Al-Maskari, Ahmed Yahya; Al-Sabahi, Jamal Nasser; Al-Hdhrami, Ibtisam; Khan, Muhammad Mumtaz; Al-Azkawi, Ahlam; Hussain, Abdullah Ijaz

    2015-01-01

    Medicago sativa Linn growing in Omani desert were chemically characterised using flame photometry, inductively coupled plasma, gas chromatography-mass spectrometry and high performance liquid chromatographic (HPLC) analysis. HPLC analyses were performed to determine the phenolics and flavonoids present in M. sativa. The major compounds detected in M. sativa leaves were protchaechenic acid (3.22%), hydroxyl benzoic acid (1.05%), β-Phenyl caffate (0.97%) and kaempherol (0.89%). Pterostilbene, a cholesterol-lowering compound, was detected in M. sativa.

  19. MtDB: a database for personalized data mining of the model legume Medicago truncatula transcriptome.

    PubMed

    Lamblin, Anne-Françoise J; Crow, John A; Johnson, James E; Silverstein, Kevin A T; Kunau, Timothy M; Kilian, Alan; Benz, Diane; Stromvik, Martina; Endré, Gabriella; VandenBosch, Kathryn A; Cook, Douglas R; Young, Nevin D; Retzel, Ernest F

    2003-01-01

    In order to identify the genes and gene functions that underlie key aspects of legume biology, researchers have selected the cool season legume Medicago truncatula (Mt) as a model system for legume research. A set of >170 000 Mt ESTs has been assembled based on in-depth sampling from various developmental stages and pathogen-challenged tissues. MtDB is a relational database that integrates Mt transcriptome data and provides a wide range of user-defined data mining options. The database is interrogated through a series of interfaces with 58 options grouped into two filters. In addition, the user can select and compare unigene sets generated by different assemblers: Phrap, Cap3 and Cap4. Sequence identifiers from all public Mt sites (e.g. IDs from GenBank, CCGB, TIGR, NCGR, INRA) are fully cross-referenced to facilitate comparisons between different sites, and hypertext links to the appropriate database records are provided for all queries' results. MtDB's goal is to provide researchers with the means to quickly and independently identify sequences that match specific research interests based on user-defined criteria. The underlying database and query software have been designed for ease of updates and portability to other model organisms. Public access to the database is at http://www.medicago.org/MtDB.

  20. Modulation of endogenous indole-3-acetic acid biosynthesis in bacteroids within Medicago sativa nodules.

    PubMed

    Bianco, C; Senatore, B; Arbucci, S; Pieraccini, G; Defez, R

    2014-07-01

    To evaluate the dose-response effects of endogenous indole-3-acetic acid (IAA) on Medicago plant growth and dry weight production, we increased the synthesis of IAA in both free-living and symbiosis-stage rhizobial bacteroids during Rhizobium-legume symbiosis. For this purpose, site-directed mutagenesis was applied to modify an 85-bp promoter sequence, driving the expression of iaaM and tms2 genes for IAA biosynthesis. A positive correlation was found between the higher expression of IAA biosynthetic genes in free-living bacteria and the increased production of IAA under both free-living and symbiotic conditions. Plants nodulated by RD65 and RD66 strains, synthetizing the highest IAA concentration, showed a significant (up to 73%) increase in the shoot fresh weight and upregulation of nitrogenase gene, nifH, compared to plants nodulated by the wild-type strain. When these plants were analyzed by confocal microscopy, using an anti-IAA antibody, the strongest signal was observed in bacteroids of Medicago sativa RD66 (Ms-RD66) plants, even when they were located in the senescent nodule zone. We show here a simple system to modulate endogenous IAA biosynthesis in bacteria nodulating legumes suitable to investigate which is the maximum level of IAA biosynthesis, resulting in the maximal increase of plant growth.

  1. Mycorrhiza Symbiosis Increases the Surface for Sunlight Capture in Medicago truncatula for Better Photosynthetic Production

    PubMed Central

    Adolfsson, Lisa; Keresztes, Áron; Uddling, Johan; Schoefs, Benoît; Spetea, Cornelia

    2015-01-01

    Arbuscular mycorrhizal (AM) fungi play a prominent role in plant nutrition by supplying mineral nutrients, particularly inorganic phosphate (Pi), and also constitute an important carbon sink. AM stimulates plant growth and development, but the underlying mechanisms are not well understood. In this study, Medicago truncatula plants were grown with Rhizophagus irregularis BEG141 inoculum (AM), mock inoculum (control) or with Pi fertilization. We hypothesized that AM stimulates plant growth through either modifications of leaf anatomy or photosynthetic activity per leaf area. We investigated whether these effects are shared with Pi fertilization, and also assessed the relationship between levels of AM colonization and these effects. We found that increased Pi supply by either mycorrhization or fertilization led to improved shoot growth associated with increased nitrogen uptake and carbon assimilation. Both mycorrhized and Pi-fertilized plants had more and longer branches with larger and thicker leaves than the control plants, resulting in an increased photosynthetically active area. AM-specific effects were earlier appearance of the first growth axes and increased number of chloroplasts per cell section, since they were not induced by Pi fertilization. Photosynthetic activity per leaf area remained the same regardless of type of treatment. In conclusion, the increase in growth of mycorrhized and Pi-fertilized Medicago truncatula plants is linked to an increase in the surface for sunlight capture, hence increasing their photosynthetic production, rather than to an increase in the photosynthetic activity per leaf area. PMID:25615871

  2. Nitric oxide is formed in Medicago truncatula-Sinorhizobium meliloti functional nodules.

    PubMed

    Baudouin, Emmanuel; Pieuchot, Laurent; Engler, Gilbert; Pauly, Nicolas; Puppo, Alain

    2006-09-01

    Nitric oxide (NO) has recently gained interest as a major signaling molecule during plant development and response to environmental cues. Its role is particularly crucial for plant-pathogen interactions, during which it participates in the control of plant defense response and resistance. Indication for the presence of NO during symbiotic interactions has also been reported. Here, we defined when and where NO is produced during Medicago truncatula-Sinorhizobium meliloti symbiosis. Using the NO-specific fluorescent probe 4,5-diaminofluorescein diacetate, NO production was detected by confocal microscopy in functional nodules. NO production was localized in the bacteroid-containing cells of the nodule fixation zone. The infection of Medicago roots with bacterial strains impaired in nitrogenase or nitrite reductase activities lead to the formation of nodules with an unaffected NO level, indicating that neither nitrogen fixation nor denitrification pathways are required for NO production. On the other hand, the NO synthase inhibitor N-methyl-L-arginine impaired NO detection, suggesting that a NO synthase may participate to NO production in nodules. These data indicate that a NO production occurs in functional nodules. The location of such a production in fully metabolically active cells raises the hypothesis of a new function for NO during this interaction unrelated to defense and cell-death activation.

  3. Identification of a molecular dialogue between developing seeds of Medicago truncatula and seedborne xanthomonads.

    PubMed

    Terrasson, Emmanuel; Darrasse, Armelle; Righetti, Karima; Buitink, Julia; Lalanne, David; Ly Vu, Benoit; Pelletier, Sandra; Bolingue, William; Jacques, Marie-Agnès; Leprince, Olivier

    2015-07-01

    Plant pathogenic bacteria disseminate and survive mainly in association with seeds. This study addresses whether seeds are passive carriers or engage a molecular dialogue with pathogens during their development. We developed two pathosystems using Medicago truncatula with Xanthomonas alfalfae subsp. alfalfae (Xaa), the natural Medicago sp. pathogen and Xanthomonas campestris pv. campestris (Xcc), a Brassicaceae pathogen. Three days after flower inoculation, the transcriptome of Xcc-infected pods showed activation of an innate immune response that was strongly limited in Xcc mutated in the type three secretion system, demonstrating an incompatible interaction of Xcc with the reproductive structures. In contrast, the presence of Xaa did not result in an activation of defence genes. Transcriptome profiling during development of infected seeds exhibited time-dependent and differential responses to Xcc and Xaa. Gene network analysis revealed that the transcriptome of Xcc-infected seeds was mainly affected during seed filling whereas that of Xaa-infected seeds responded during late maturation. The Xcc-infected seed transcriptome exhibited an activation of defence response and a repression of targeted seed maturation pathways. Fifty-one percent of putative ABSCISIC ACID INSENSITIVE3 targets were deregulated by Xcc, including oleosin, cupin, legumin and chlorophyll degradation genes. At maturity, these seeds displayed decreased weight and increased chlorophyll content. In contrast, these traits were not affected by Xaa infection. These findings demonstrate the existence of a complex molecular dialogue between xanthomonads and developing seeds and provides insights into a previously unexplored trade-off between seed development and pathogen defence.

  4. Characterization and genetic dissection of resistance to spotted alfalfa aphid (Therioaphis trifolii) in Medicago truncatula

    PubMed Central

    Kamphuis, Lars G.; Lichtenzveig, Judith; Peng, Kefan; Guo, Su-Min; Klingler, John P.

    2013-01-01

    Aphids cause significant yield losses in agricultural crops worldwide. Medicago truncatula, a model legume, cultivated pasture species in Australia and close relative of alfalfa (Medicago sativa), was used to study the defence response against Therioaphis trifolii f. maculate [spotted alfalfa aphid (SAA)]. Aphid performance and plant damage were compared among three accessions. A20 is highly susceptible, A17 has moderate resistance, and Jester is strongly resistant. Subsequent analyses using A17 and A20, reciprocal F1s and an A17×A20 recombinant inbred line (RIL) population revealed that this moderate resistance is phloem mediated and involves antibiosis and tolerance but not antixenosis. Electrical penetration graph analysis also identified a novel waveform termed extended potential drop, which occurred following SAA infestation of M. truncatula. Genetic dissection using the RIL population revealed three quantitative trait loci on chromosomes 3, 6, and 7 involved in distinct modes of aphid defence including antibiosis and tolerance. An antibiosis locus resides on linkage group 3 (LG3) and is derived from A17, whereas a plant tolerance and antibiosis locus resides on LG6 and is derived from A20, which exhibits strong temporary tolerance. The loci identified reside in regions harbouring classical resistance genes, and introgression of these loci in current medic cultivars may help provide durable resistance to SAA, while elucidation of their molecular mechanisms may provide valuable insight into other aphid–plant interactions. PMID:24058162

  5. Complex patterns of autopolyploid evolution in alfalfa and allies (Medicago sativa; Leguminosae).

    PubMed

    Havananda, Tee; Brummer, E Charles; Doyle, Jeff J

    2011-10-01

    Although there is growing evidence that autopolyploidy is a widespread and important evolutionary phenomenon, it has received less attention than allopolyploidy. Medicago sativa comprises several diploid and autopolyploid taxa, including autotetraploid cultivated alfalfa, and affords an opportunity to elucidate the evolutionary history of a morphologically and genetically complex autopolyploid system. Phylogenies and haplotype networks were constructed from two chloroplast noncoding regions (rpl20-rps12 and trnS-trnG spacers) across seven diploid and polyploid infraspecific taxa of M. sativa and five additional closely related Medicago species, and genetic differentiation was estimated. The two most prominent M. sativa autopolyploids have contrasting evolutionary histories. Chloroplast data support a simple autopolyploid origin of subsp. sativa (alfalfa) from diploid subsp. caerulea, from which it is distinguishable in several quantitative characters. In contrast, morphologically identical diploid and autopolyploid cytotypes of subsp. falcata were found to possess very different chloroplast haplotypes, suggesting past introgression from M. prostrata into the polyploid. Despite the presence of hybrids between tetraploid subspecies falcata and sativa, there was little evidence of introgression of chloroplast genomes from either subspecies into the other. Autopolyploid evolution in M. sativa is complicated and has followed very different paths in different subspecific taxa. The potential exists for gene flow in virtually all combinations of subspecies both within and between ploidies, yet despite the existence of hybrids, morphologically and genetically distinctive subspecies persist.

  6. Regeneration of Medicago truncatula from protoplasts isolated from kanamycin-sensitive and kanamycin-resistant plants.

    PubMed

    Rose, R J; Nolan, K E

    1995-03-01

    Medicago truncatula (barrel medic) is an annual legume of agricultural and biological interest. In this report regeneration from isolated mesophyll protoplasts is described. A specifically developed, highly regenerable seed line is essential for regeneration. Other critical requirements for regeneration are the starting plant material, the use of agarose droplets incubated in a shallow layer of liquid medium, and protoplast density. Plants are grown in controlled environment conditions. Protoplasts are purified using a Percoll-based flotation procedure, then embedded in 100 μl agarose droplets containing a basal medium plus 25 μM NAA and 4 μM BAP (the same medium as in the surrounding shallow liquid layer) to induce protoplast division. A protoplast density of 6-8×10(5) ml(-1) is required for maximum colony formation. M. truncatula plants previously transformed for kanamycin resistance yielded embryogenic callus and also regenerated plants. Protoplasts from other annual Medicago (M.intertexta and M.scutellata) species readily form calli by the procedure we have described.

  7. Novel SINEs families in Medicago truncatula and Lotus japonicus: bioinformatic analysis.

    PubMed

    Gadzalski, Marek; Sakowicz, Tomasz

    2011-07-01

    Although short interspersed elements (SINEs) were discovered nearly 30 years ago, the studies of these genomic repeats were mostly limited to animal genomes. Very little is known about SINEs in legumes--one of the most important plant families. Here we report identification, genomic distribution and molecular features of six novel SINE elements in Lotus japonicus (named LJ_SINE-1, -2, -3) and Medicago truncatula (MT_SINE-1, -2, -3), model species of legume. They possess all the structural features commonly found in short interspersed elements including RNA polymerase III promoter, polyA tail and flanking repeats. SINEs described here are present in low to moderate copy numbers from 150 to 3000. Bioinformatic analyses were used to searched public databases, we have shown that three of new SINE elements from M. truncatula seem to be characteristic of Medicago and Trifolium genera. Two SINE families have been found in L. japonicus and one is present in both M. truncatula and L. japonicus. In addition, we are discussing potential activities of the described elements. Copyright © 2011 Elsevier B.V. All rights reserved.

  8. Transcriptional response of Medicago truncatula sulphate transporters to arbuscular mycorrhizal symbiosis with and without sulphur stress.

    PubMed

    Casieri, Leonardo; Gallardo, Karine; Wipf, Daniel

    2012-06-01

    Sulphur is an essential macronutrient for plant growth, development and response to various abiotic and biotic stresses due to its key role in the biosynthesis of many S-containing compounds. Sulphate represents a very small portion of soil S pull and it is the only form that plant roots can uptake and mobilize through H(+)-dependent co-transport processes implying sulphate transporters. Unlike the other organically bound forms of S, sulphate is normally leached from soils due to its solubility in water, thus reducing its availability to plants. Although our knowledge of plant sulphate transporters has been growing significantly in the past decades, little is still known about the effect of the arbuscular mycorrhiza interaction on sulphur uptake. Carbon, nitrogen and sulphur measurements in plant parts and expression analysis of genes encoding putative Medicago sulphate transporters (MtSULTRs) were performed to better understand the beneficial effects of mycorrhizal interaction on Medicago truncatula plants colonized by Glomus intraradices at different sulphate concentrations. Mycorrhization significantly promoted plant growth and sulphur content, suggesting increased sulphate absorption. In silico analyses allowed identifying eight putative MtSULTRs phylogenetically distributed over the four sulphate transporter groups. Some putative MtSULTRs were transcribed differentially in roots and leaves and affected by sulphate concentration, while others were more constitutively transcribed. Mycorrhizal-inducible and -repressed MtSULTRs transcripts were identified allowing to shed light on the role of mycorrhizal interaction in sulphate uptake.

  9. Medicago truncatula-derived calcium oxalate crystals have a negative impact on chewing insect performance via their physical properties

    USDA-ARS?s Scientific Manuscript database

    Plant structural traits often act as defenses against herbivorous insects, causing them to avoid feeding on a given plant or tissue. Mineral crystals of calcium oxalate in Medicago truncatula Gaertn. (Fabaceae) leaves have previously been shown to be effective deterrents of lepidopteran insect feedi...

  10. Genetically modified Medicago truncatula lacking calcium oxalate has increased calcium bioavailability and partially rescues vitamin D receptor knockout mice phenotypes

    USDA-ARS?s Scientific Manuscript database

    How the distribution and sequestered form of plant macro/micro-nutrients influence their bioavailability, and ultimately impact human health, is poorly understood. The legume Medicago truncatula has a portion of its tissue calcium sequestered in the form of the calcium oxalate crystal, which reduces...

  11. Contrasting calcium localization and speciation in leaves of Medicago trunculata mutant COD5 analyzed via synchrotron X-ray techniques

    USDA-ARS?s Scientific Manuscript database

    Oxalate-producing plants accumulate calcium oxalate crystals (CaOx(C)) in the range of 3-80%(w/w) of their dry weight, reducing calcium (Ca) bioavailability. The calcium oxalate deficient 5 (cod5) mutant of Medicago truncatula has been previously shown to contain similar Ca, but lower oxalate and Ca...

  12. Phylogenetic properties of 50 nuclear loci in Medicago (Leguminosae) generated using multiplexed sequence capture and next-generation sequencing.

    PubMed

    de Sousa, Filipe; Bertrand, Yann J K; Nylinder, Stephan; Oxelman, Bengt; Eriksson, Jonna S; Pfeil, Bernard E

    2014-01-01

    Next-generation sequencing technology has increased the capacity to generate molecular data for plant biological research, including phylogenetics, and can potentially contribute to resolving complex phylogenetic problems. The evolutionary history of Medicago L. (Leguminosae: Trifoliae) remains unresolved due to incongruence between published phylogenies. Identification of the processes causing this genealogical incongruence is essential for the inference of a correct species phylogeny of the genus and requires that more molecular data, preferably from low-copy nuclear genes, are obtained across different species. Here we report the development of 50 novel LCN markers in Medicago and assess the phylogenetic properties of each marker. We used the genomic resources available for Medicago truncatula Gaertn., hybridisation-based gene enrichment (sequence capture) techniques and Next-Generation Sequencing to generate sequences. This alternative proves to be a cost-effective approach to amplicon sequencing in phylogenetic studies at the genus or tribe level and allows for an increase in number and size of targeted loci. Substitution rate estimates for each of the 50 loci are provided, and an overview of the variation in substitution rates among a large number of low-copy nuclear genes in plants is presented for the first time. Aligned sequences of major species lineages of Medicago and its sister genus are made available and can be used in further probe development for sequence-capture of the same markers.

  13. Phylogenetic Properties of 50 Nuclear Loci in Medicago (Leguminosae) Generated Using Multiplexed Sequence Capture and Next-Generation Sequencing

    PubMed Central

    de Sousa, Filipe; Bertrand, Yann J. K.; Nylinder, Stephan; Oxelman, Bengt; Eriksson, Jonna S.; Pfeil, Bernard E.

    2014-01-01

    Next-generation sequencing technology has increased the capacity to generate molecular data for plant biological research, including phylogenetics, and can potentially contribute to resolving complex phylogenetic problems. The evolutionary history of Medicago L. (Leguminosae: Trifoliae) remains unresolved due to incongruence between published phylogenies. Identification of the processes causing this genealogical incongruence is essential for the inference of a correct species phylogeny of the genus and requires that more molecular data, preferably from low-copy nuclear genes, are obtained across different species. Here we report the development of 50 novel LCN markers in Medicago and assess the phylogenetic properties of each marker. We used the genomic resources available for Medicago truncatula Gaertn., hybridisation-based gene enrichment (sequence capture) techniques and Next-Generation Sequencing to generate sequences. This alternative proves to be a cost-effective approach to amplicon sequencing in phylogenetic studies at the genus or tribe level and allows for an increase in number and size of targeted loci. Substitution rate estimates for each of the 50 loci are provided, and an overview of the variation in substitution rates among a large number of low-copy nuclear genes in plants is presented for the first time. Aligned sequences of major species lineages of Medicago and its sister genus are made available and can be used in further probe development for sequence-capture of the same markers. PMID:25329401

  14. Identification of molecular markers associated with verticillium wilt resistance in alfalfa (medicago sativa l.) using high-resolution melting

    USDA-ARS?s Scientific Manuscript database

    Verticillium wilt (VW), caused by the soilborne fungus, Verticillium alfalfae, is one of the most serious diseases of alfalfa (Medicago sativa L.) worldwide. To identify loci associated with resistance to VW, an association study was conducted using autotetraploid alfalfa populations composed of 352...

  15. ECOLOGICAL RISK ASSESSMENT OF ALFALFA (MEDICAGO VARIA L.) GENETICLALY ENGINEERED TO EXPRESS A HUMAN METALLOTHIONEIN (HMT) GENE

    EPA Science Inventory

    The objectives of these studies were two-fold: (1) to determine efficacy of low and high expression hMT gene constructs by assessing accumulation of Cu in shoots of parental and transgenic plants of alfalfa (Medicago varia L.) exposed to different concentrations of CuSO4 by addit...

  16. Application of Multilocus Sequence Typing To Study the Genetic Structure of Megaplasmids in Medicago-Nodulating Rhizobia

    USDA-ARS?s Scientific Manuscript database

    A multilocus sequence typing (MLST) analysis was used to examine the relatedness and distribution of genotypic variants of the two large extrachromosomal replicons in Medicago-nodulating rhizobia (Sinorhizobium meliloti and S. medicae). One goal was to develop a strategy for the characterization of...

  17. Genetic evidence for differences in the pathways of druse and prismatic calcium oxalate crystal formation in Medicago truncatula

    USDA-ARS?s Scientific Manuscript database

    Current evidence supports a single pathway of oxalate biosynthesis utilising ascorbic acid as the precursor. In this study, we begin to address the possibility that more than one pathway of oxalate biosynthesis and calcium oxalate formation occurs in Medicago truncatula Gaertn. (cv. Jemalong genotyp...

  18. Leaf calcium oxalate crystal structure and its role in defense against a chewing insect in Medicago truncatula

    USDA-ARS?s Scientific Manuscript database

    Crystals of calcium oxalate are common in plants and widely distributed among many plant families. These hard and largely insoluble crystals take on many shapes and sizes depending on the tissue and species. In Medicago truncatula, calcium oxalate crystals are abundant in leaves and accumulate in sh...

  19. Effective conservation of Medicago Crop Wild Relatives in Russia and neighbouring countries: a gap analysis points the way forward

    USDA-ARS?s Scientific Manuscript database

    Gap analysis is an effective way to review and refine conservation strategies for crop wild relatives. We developed a comprehensive database containing over 2400 accessions of Medicago crop wild relatives that had been collected in the area of the Former Soviet Union (FSU). Using the data we develop...

  20. Characterization of zinc transport by divalent metal transporters of the ZIP family from the model legume medicago truncatula

    USDA-ARS?s Scientific Manuscript database

    To understand how plants from the Fabaceae family maintain zinc (Zn) homeostasis, we have characterized the kinetics of the Zn transporting proteins from the ZIP family of divalent metal transporters in the model legume Medicago truncatula. MtZIP1, MtZIP5, and MtZIP6 were the only members from this ...

  1. Effect of alfalfa (medicago sativa) on fermentation profile and nutritive value of switchgrass (panicum virgatum) and bermudagrass (cynodon dactylon) silages

    USDA-ARS?s Scientific Manuscript database

    An experiment was conducted at the University of Kentucky Spindletop Farm in Lexington, Kentucky between October and November, 2009 to evaluate the effect of different percentages of alfalfa (Medicago sativa) as mixtures in switchgrass (Panicum virgatus) and bermudagrass (Cynodon dactylon) silages. ...

  2. Effect of wheel traffic and green manure treatments on forage yield and crown rot in alfalfa (Medicago sativa)

    USDA-ARS?s Scientific Manuscript database

    Harvesting alfalfa (Medicago sativa) results in mechanical wheel traffic on plants, which may damage crowns and increase the opportunity for entry of pathogens causing crown rot. Developing resistance to crown rot is problematic due to the large number of pathogens involved. Incorporation of plant b...

  3. Expression analysis of Medicago stem internodes identifies genes associated with regulatory control and biogenesis of plant cell walls

    USDA-ARS?s Scientific Manuscript database

    Legumes have many traits that make them attractive bioenergy crops, especially as components of mixed grass stands or in crop rotations with maize. Alfalfa (Medicago sativa) is a legume with an advantage as a biomass crop because of its ability to fix atmospheric nitrogen and the easy separation of ...

  4. Occurrence of transgenic feral alfalfa (Medicago sativa subsp. sativa L.) in alfalfa seed production areas in the United States

    USDA-ARS?s Scientific Manuscript database

    Genetically-engineered glyphosate-resistant alfalfa (Medicago sativa subsp. sativa) was commercialized in 2011. The potential risk of transgene dispersal into the environment is not clearly understood for alfalfa, a perennial crop that is cross-pollinated by insects. We gathered data on feral and tr...

  5. ECOLOGICAL RISK ASSESSMENT OF ALFALFA (MEDICAGO VARIA L.) GENETICLALY ENGINEERED TO EXPRESS A HUMAN METALLOTHIONEIN (HMT) GENE

    EPA Science Inventory

    The objectives of these studies were two-fold: (1) to determine efficacy of low and high expression hMT gene constructs by assessing accumulation of Cu in shoots of parental and transgenic plants of alfalfa (Medicago varia L.) exposed to different concentrations of CuSO4 by addit...

  6. Fractionation of Synteny in a Genomic Region Containing Tandemly Duplicated Genes Across Glycine max, Medicago truncatula and Arabidopsis thaliana

    USDA-ARS?s Scientific Manuscript database

    Extended comparison of gene sequences found on homeologous soybean BACs to Medicago truncatula and Arabidopsis thaliana genomic sequences demonstrated a network of synteny within conserved regions interrupted by gene addition and/or deletions. Consolidation of gene order among all three species prov...

  7. Relationships of the Woody Medicago Species (Section Dendrotelis) Assessed by Molecular Cytogenetic Analyses

    PubMed Central

    Rosato, Marcela; Castro, Mercedes; Rosselló, Josep A.

    2008-01-01

    Background and Aims The organization of rDNA genes in the woody medic species from the agronomically important Medicago section Dendrotelis was analysed to gain insight into their taxonomic relationships, to assess the levels of infraspecific variation concerning ribosomal loci in a restricted and fragmented insular species (M. citrina) and to assess the nature of its polyploidy. Methods Fluorescence in situ hybridization (FISH) was used for physical mapping of 5S and 45S ribosomal DNA genes in the three species of section Dendrotelis (M. arborea, M. citrina, M. strasseri) and the related M. marina from section Medicago. Genomic in situ hybridization (GISH) was used to assess the genomic relationships of the polyploid M. citrina with the putatively related species from section Dendrotelis. Key Results The diploid (2n = 16) M. marina has a single 45S and two 5S rDNA loci, a pattern usually detected in previous studies of Medicago diploid species. However, polyploid species from section Dendrotelis depart from expectations. The tetraploid species (2n = 32) M. arborea and M. strasseri have one 45S rDNA locus and two 5S rDNA loci, whereas in the hexaploid (2n = 48) M. citrina four 45S rDNA and five 5S rDNA loci have been detected. No single chromosome of M. citrina was uniformly labelled after using genomic probes from M. arborea and M. strasseri. Instead, cross-hybridization signals in M. citrina were restricted to terminal chromosome arms and NOR regions. Conclusions FISH results support the close taxonomic interrelationship between M. arborea and M. strasseri. In these tetraploid species, NOR loci have experienced a diploidization event through physical loss of sequences, a cytogenetic feature so far not reported in other species of the genus. The high number of rDNA loci and GISH results support the specific status for the hexaploid M. citrina, and it is suggested that this species is not an autopolyploid derivative of M. arborea or M. strasseri. Further, molecular

  8. A Saturated Genetic Linkage Map of Autotetraploid Alfalfa (Medicago sativa L.) Developed Using Genotyping-by-Sequencing Is Highly Syntenous with the Medicago truncatula Genome

    PubMed Central

    Li, Xuehui; Wei, Yanling; Acharya, Ananta; Jiang, Qingzhen; Kang, Junmei; Brummer, E. Charles

    2014-01-01

    A genetic linkage map is a valuable tool for quantitative trait locus mapping, map-based gene cloning, comparative mapping, and whole-genome assembly. Alfalfa, one of the most important forage crops in the world, is autotetraploid, allogamous, and highly heterozygous, characteristics that have impeded the construction of a high-density linkage map using traditional genetic marker systems. Using genotyping-by-sequencing (GBS), we constructed low-cost, reasonably high-density linkage maps for both maternal and paternal parental genomes of an autotetraploid alfalfa F1 population. The resulting maps contain 3591 single-nucleotide polymorphism markers on 64 linkage groups across both parents, with an average density of one marker per 1.5 and 1.0 cM for the maternal and paternal haplotype maps, respectively. Chromosome assignments were made based on homology of markers to the M. truncatula genome. Four linkage groups representing the four haplotypes of each alfalfa chromosome were assigned to each of the eight Medicago chromosomes in both the maternal and paternal parents. The alfalfa linkage groups were highly syntenous with M. truncatula, and clearly identified the known translocation between Chromosomes 4 and 8. In addition, a small inversion on Chromosome 1 was identified between M. truncatula and M. sativa. GBS enabled us to develop a saturated linkage map for alfalfa that greatly improved genome coverage relative to previous maps and that will facilitate investigation of genome structure. GBS could be used in breeding populations to accelerate molecular breeding in alfalfa. PMID:25147192

  9. A saturated genetic linkage map of autotetraploid alfalfa (Medicago sativa L.) developed using genotyping-by-sequencing is highly syntenous with the Medicago truncatula genome.

    PubMed

    Li, Xuehui; Wei, Yanling; Acharya, Ananta; Jiang, Qingzhen; Kang, Junmei; Brummer, E Charles

    2014-08-21

    A genetic linkage map is a valuable tool for quantitative trait locus mapping, map-based gene cloning, comparative mapping, and whole-genome assembly. Alfalfa, one of the most important forage crops in the world, is autotetraploid, allogamous, and highly heterozygous, characteristics that have impeded the construction of a high-density linkage map using traditional genetic marker systems. Using genotyping-by-sequencing (GBS), we constructed low-cost, reasonably high-density linkage maps for both maternal and paternal parental genomes of an autotetraploid alfalfa F1 population. The resulting maps contain 3591 single-nucleotide polymorphism markers on 64 linkage groups across both parents, with an average density of one marker per 1.5 and 1.0 cM for the maternal and paternal haplotype maps, respectively. Chromosome assignments were made based on homology of markers to the M. truncatula genome. Four linkage groups representing the four haplotypes of each alfalfa chromosome were assigned to each of the eight Medicago chromosomes in both the maternal and paternal parents. The alfalfa linkage groups were highly syntenous with M. truncatula, and clearly identified the known translocation between Chromosomes 4 and 8. In addition, a small inversion on Chromosome 1 was identified between M. truncatula and M. sativa. GBS enabled us to develop a saturated linkage map for alfalfa that greatly improved genome coverage relative to previous maps and that will facilitate investigation of genome structure. GBS could be used in breeding populations to accelerate molecular breeding in alfalfa.

  10. 3D imaging and mechanical modeling of helical buckling in Medicago truncatula plant roots

    PubMed Central

    Silverberg, Jesse L.; Noar, Roslyn D.; Packer, Michael S.; Harrison, Maria J.; Henley, Christopher L.; Cohen, Itai; Gerbode, Sharon J.

    2012-01-01

    We study the primary root growth of wild-type Medicago truncatula plants in heterogeneous environments using 3D time-lapse imaging. The growth medium is a transparent hydrogel consisting of a stiff lower layer and a compliant upper layer. We find that the roots deform into a helical shape just above the gel layer interface before penetrating into the lower layer. This geometry is interpreted as a combination of growth-induced mechanical buckling modulated by the growth medium and a simultaneous twisting near the root tip. We study the helical morphology as the modulus of the upper gel layer is varied and demonstrate that the size of the deformation varies with gel stiffness as expected by a mathematical model based on the theory of buckled rods. Moreover, we show that plant-to-plant variations can be accounted for by biomechanically plausible values of the model parameters. PMID:23010923

  11. Genetic Markers and Quantitative Genetic Variation in Medicago Truncatula (Leguminosae): A Comparative Analysis of Population Structure

    PubMed Central

    Bonnin, I.; Prosperi, J. M.; Olivieri, I.

    1996-01-01

    Two populations of the selfing annual Medicago truncatula Gaertn. (Leguminoseae), each subdivided into three subpopulations, were studied for both metric traits (quantitative characters) and genetic markers (random amplified polymorphic DNA and one morphological, single-locus marker). Hierarchical analyses of variance components show that (1) populations are more differentiated for quantitative characters than for marker loci, (2) the contribution of both within and among subpopulations components of variance to overall genetic variance of these characters is reduced as compared to markers, and (3) at the population level, within population structure is slightly but not significantly larger for markers than for quantitative traits. Under the hypothesis that most markers are neutral, such comparisons may be used to make hypotheses about the strength and heterogeneity of natural selection in the face of genetic drift and gene flow. We thus suggest that in these populations, quantitative characters are under strong divergent selection among populations, and that gene flow is restricted among populations and subpopulations. PMID:8844165

  12. [The plant growth-promoting rhizobacterium Arthrobacter agilis UMCV2 endophytically colonizes Medicago truncatula].

    PubMed

    Aviles-Garcia, Maria Elizabeth; Flores-Cortez, Idolina; Hernández-Soberano, Christian; Santoyo, Gustavo; Valencia-Cantero, Eduardo

    Arthrobacter agilis UMCV2 is a rhizosphere bacterium that promotes legume growth by solubilization of iron, which is supplied to the plant. A second growth promotion mechanism produces volatile compounds that stimulate iron uptake activities. Additionally, A. agilis UMCV2 is capable of inhibiting the growth of phytopathogens. A combination of quantitative polymerase chain reaction and fluorescence in situ hybridization techniques were used here to detect and quantify the presence of the bacterium in the internal tissues of the legume Medicago truncatula. Our results demonstrate that A. agilis UMCV2 behaves as an endophytic bacterium of M. truncatula, particularly in environments where iron is available. Copyright © 2016 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

  13. [Effects of copper stress on Medicago sativa seedlings leaf antioxidative system].

    PubMed

    Wang, Song-hua; Zhang, Hua; He, Qing-yuan

    2011-09-01

    This paper studied the effects of different concentration (0, 10, 30, 50, and 100 micromol x L(-1)) CuSO4 on the leaf physiological and biochemical characteristics of Medicago sativa seedlings cultured with 1/4-strength Hoagland nutrient solution. In treatments 30, 50, and 100 micromol x L(-1) of CuSO4, the leaf H2O2, OH., and MDA contents and Fe-SOD and EST activities increased, and GSH and AsA contents increased significantly. With increasing concentration Cu, the POD, GR, and APX activities increased gradually, and the CAT and G6PDH activities decreased after an initial increase. In treatments >10 micromol x L(-1) of Cu, the capacity of leaf antioxidative system in reactive oxygen species scavenging increased to prevent the injury from copper-induced oxidative stress.

  14. Triterpenoid glycosides from Medicago sativa as antifungal agents against Pyricularia oryzae.

    PubMed

    Abbruscato, Pamela; Tosi, Solveig; Crispino, Laura; Biazzi, Elisa; Menin, Barbara; Picco, Anna M; Pecetti, Luciano; Avato, Pinarosa; Tava, Aldo

    2014-11-19

    The antifungal properties of saponin mixtures from alfalfa (Medicago sativa L.) tops and roots, the corresponding mixtures of prosapogenins from tops, and purified saponins and sapogenins against the causal agent of rice blast Pyricularia oryzae isolates are presented. In vitro experiments highlighted a range of activities, depending upon the assayed metabolite. The antifungal effects of the most promising prosapogenin mixture from alfalfa tops were confirmed by means of in planta tests using three different Italian cultivars of rice (Oryza sativa L. ssp. japonica), known to possess high, medium, and low blast resistance. The evidenced antifungal properties of the tested metabolites allowed some considerations on their structure-activity relationship. Results indicate that prosapogenins are active compounds to prevent the fungal attack of P. oryzae on different rice cultivars. Therefore, if properly formulated, these substances could represent a promising and environmentally friendly treatment to control rice blast.

  15. Transcriptomic Analysis of Sinorhizobium meliloti and Medicago truncatula Symbiosis Using Nitrogen Fixation-Deficient Nodules.

    PubMed

    Lang, Claus; Long, Sharon R

    2015-08-01

    The bacterium Sinorhizobium meliloti interacts symbiotically with legume plant hosts such as Medicago truncatula to form nitrogen-fixing root nodules. During symbiosis, plant and bacterial cells differentiate in a coordinated manner, resulting in specialized plant cells that contain nitrogen-fixing bacteroids. Both plant and bacterial genes are required at each developmental stage of symbiosis. We analyzed gene expression in nodules formed by wild-type bacteria on six plant mutants with defects in nitrogen fixation. We observed differential expression of 482 S. meliloti genes with functions in cell envelope homeostasis, cell division, stress response, energy metabolism, and nitrogen fixation. We simultaneously analyzed gene expression in M. truncatula and observed differential regulation of host processes that may trigger bacteroid differentiation and control bacterial infection. Our analyses of developmentally arrested plant mutants indicate that plants use distinct means to control bacterial infection during early and late symbiotic stages.

  16. Photoperiodic Effects on the Emanation of Volatiles from Alfalfa (Medicago sativa L.) Florets 1

    PubMed Central

    Loper, G. M.; Lapioli, A. M.

    1972-01-01

    Alfalfa (Medicago sativa L.) plants acclimated to photoperiods of 18 hours light, 6 hour dark in plant growth chambers exhibited a daily cyclic pattern of floret volatile emanation with a maximum emanation of about 6.5 nanograms of hydrocarbons/floret·30 minutes. This maximum was reached about 6 to 8 hours into the light period. After 8 hours of light, emanation of volatiles decreased rapidly to less than 0.1 ng/floret·30 min even though light and temperature remained constant. Under continuous illumination, only a small increase of volatile emanation occurred during the following 24 hours. It appeared that a dark period was necessary to promote floret volatile emanation. Floret volatile emanation was drastically affected for at least 7 days following a photoperiod change. A photoperiod change caused 6-fold concentration oscillations every 2 hours. The results are interpreted on the basis of a very active floral metabolism controlled by photoperiodically induced rhythms. PMID:16658038

  17. [Intergeneric somatic hybridization between Astragalus adsurgens Pall and Medicago sativa L].

    PubMed

    Jin, Hong; Jia, Jing Fen; Hao, Jian Guo

    2004-06-01

    The protoplast fusion was induced between Astragalus adsurgens Pall and Agrobacterium rhizogenes-transformed cell line of Medicago Sativa L. by use of PEG method. Putative somatic hybrid cells were selected based on the observation that only intergeneric hybrid cells continued to divide on the DPD medium without any phytohormone while the cells and clusters derived from parental cells did not survive further. After being cultured, the somatic hybrid calli were obtained. Although both of the parental calli were not capable of regenerating , one of the hydrid cell lines recovered the differentiation ability and produced small shoots. Characterizations of the hybrid calli were carried out by examination of chromsome number, opine synthetase assay, isoenzyme and RAPD analyses.

  18. 3D imaging and mechanical modeling of helical buckling in Medicago truncatula plant roots.

    PubMed

    Silverberg, Jesse L; Noar, Roslyn D; Packer, Michael S; Harrison, Maria J; Henley, Christopher L; Cohen, Itai; Gerbode, Sharon J

    2012-10-16

    We study the primary root growth of wild-type Medicago truncatula plants in heterogeneous environments using 3D time-lapse imaging. The growth medium is a transparent hydrogel consisting of a stiff lower layer and a compliant upper layer. We find that the roots deform into a helical shape just above the gel layer interface before penetrating into the lower layer. This geometry is interpreted as a combination of growth-induced mechanical buckling modulated by the growth medium and a simultaneous twisting near the root tip. We study the helical morphology as the modulus of the upper gel layer is varied and demonstrate that the size of the deformation varies with gel stiffness as expected by a mathematical model based on the theory of buckled rods. Moreover, we show that plant-to-plant variations can be accounted for by biomechanically plausible values of the model parameters.

  19. A method for the isolation of root hairs from the model legume Medicago truncatula.

    PubMed

    Ramos, Javier; Bisseling, Ton

    2003-10-01

    A new method for the isolation of root hairs from the model legume, Medicago truncatula, was developed. The procedure involves the propagation of detached roots on agar plates and the collection of root hairs by immersion in liquid nitrogen. Yields of up to 40 micro g of root hair protein were obtained from 50-100 root tips grown for 3 weeks on a single plate. The high purity of the root hair fraction was monitored by western blot analysis using an antibody to the pea epidermis specific protein PsRH2. Sequence analyses revealed that the protein homologous to PsRH2 in M. truncatula, MtRH2, is identical to the root protein MtPR10-1. The MtRH2 protein proved to be a useful endogenous marker to monitor root hair isolation since it is also specifically expressed in the root epidermis.

  20. QTL mapping of physiological traits associated with salt tolerance in Medicago truncatula Recombinant Inbred Lines.

    PubMed

    Arraouadi, Soumaya; Badri, Mounawer; Abdelly, Chedly; Huguet, Thierry; Aouani, Mohamed Elarbi

    2012-02-01

    In this study, QTL mapping of physiological traits in the model Legume (Medicago truncatula) was performed using a set of RILs derived from LR5. Twelve parameters associated with Na+ and K+ content in leaves, stems and roots were measured. Broad-sense heritability of these traits was ranged from 0.15 to 0.83 in control and from 0.14 to 0.61 in salt stress. Variation among RILs was dependent on line, treatment and line by treatment effect. We mapped 6 QTLs in control, 2 in salt stress and 5 for sensitivity index. No major QTL was identified indicating that tolerance to salt stress is governed by several genes with low effects. Detected QTL for leaf, stem and root traits did not share the same map locations, suggesting that genes controlling transport of Na+ and K+ may be different. The maximum of QTL was observed on chromosome 1, no QTL was detected on chromosomes 5 and 6.

  1. Analysis and modeling of the integrative response of Medicago truncatula to nitrogen constraints.

    PubMed

    Salon, Christophe; Lepetit, Marc; Gamas, Pascal; Jeudy, Christian; Moreau, Sandra; Moreau, Delphine; Voisin, Anne-Sophie; Duc, Gérard; Bourion, Virginie; Munier-Jolain, Nathalie

    2009-11-01

    An integrative biology approach was conducted in Medicago truncatula for: (i) unraveling the coordinated regulation of NO3-, NH4+ and N(2) acquisition by legumes to fulfill the plant N demand; and (ii) modeling the emerging properties occurring at the whole plant level. Upon localized addition of a high level of mineral N, the three N acquisition pathways displayed similar systemic feedback repression to adjust N acquisition capacities to the plant N status. Genes associated to these responses were in contrast rather specific to the N source. Following an N deficit, NO3- fed plants maintained efficiently their N status through rapid functional and developmental up regulations while N(2) fed plants responded by long term plasticity of nodule development. Regulatory genes associated with various symbiotic stages were further identified. An ecophysiological model simulating relations between leaf area and roots N retrieval was developed and now furnishes an analysis grid to characterize a spontaneous or induced genetic variability for plant N nutrition.

  2. Characterization of a copper-resistant symbiotic bacterium isolated from Medicago lupulina growing in mine tailings.

    PubMed

    Fan, Lian-Mei; Ma, Zhan-Qiang; Liang, Jian-Qiang; Li, Hui-Fen; Wang, En-Tao; Wei, Ge-Hong

    2011-01-01

    A root nodule bacterium, Sinorhizobium meliloti CCNWSX0020, resistant to 1.4 mM Cu2+ was isolated from Medicago lupulina growing in mine tailings. In medium supplied with copper, this bacterium showed cell deformation and aggregation due to precipitation of copper on the cell surface. Genes similar to the copper-resistant genes, pcoR and pcoA from Escherichia coli, were amplified by PCR from a 1.4-Mb megaplasmid. Inoculation with S. meliloti CCNWSX0020 increased the biomass of M. lupulina grown in medium added 0 and 100 mg Cu2+ kg(-1) by 45.8% and 78.2%, respectively, and increased the copper concentration inside the plant tissues grown in medium supplied with 100 μM Cu2+ by 39.3%, demonstrating that it is a prospective symbiotic system for bioremediation purposes.

  3. Natural occurrence of entomophthoroid fungi of aphid pests on Medicago sativa L. in Argentina.

    PubMed

    Manfrino, Romina G; Zumoffen, Leticia; Salto, César E; Lastra, Claudia C López

    2014-01-01

    Four species of entomophthoroid fungi, Pandora neoaphidis (Entomophthorales: Entomophthoraceae), Zoophthora radicans (Entomophthorales: Entomophthoraceae), Entomophthora planchoniana (Entomophthorales: Entomophthoraceae) and Neozygites fresenii (Neozygitales: Neozygitaceae) were found to infect Aphis craccivora, Therioaphis trifolii, and Acyrthosiphon pisum and unidentified species of Acyrthosiphon on lucerne in Argentina. Samples were collected from five sites (Ceres, Rafaela, Sarmiento, Monte Vera and Bernardo de Irigoyen) in the province of Santa Fe. In this study, Zoophthora radicans was the most important pathogen and was recorded mainly on Acyrthosiphon sp. Zoophthora radicans was successfully isolated and maintained in pure cultures. This study is the first report of entomophthoroid fungi infecting lucerne (Medicago sativa L.) aphids in Argentina. Copyright © 2014 Asociación Colombiana de Psiquiatría. Publicado por Elsevier España. All rights reserved.

  4. [Control of the fermentation activity during ensilage of Lolium italicum A. Br. and Medicago sativa L].

    PubMed

    Ozino Marletto, O I; Ambrosoli, R; Piccone, G; Biasiol, B

    1982-01-01

    Samples with different dry matter contents of Lolium italicum A. Br., Medicago sativa L., have been ensiled in hermetically sealed containers, in order to study the evolution of microflora and its activity. The principal microbial groups (coliforms, proteolytics , lactic acid bacteria, clostridia, yeasts) have been detected and enumerated, in anaerobic atmosphere, after ensiling periods of 3, 6, 10, 13, 20, 100 days. At the same time, the samples were chemically analyzed for the detection of: pH, moisture, ashes, total and ammoniacal nitrogen, total reducing sugars, lactic acid, volatile fatty acids, short chain alcohols. The relations found between chemical and microbiological data, show that a strong lactic fermentation is not enough for the inhibition of silage spoiling microorganisms, such as coliforms, yeasts, and (less) proteolytics . This phenomenon may be related to the "quality" of the lactic acid microflora, more than to the characteristics of the environment.

  5. Possible Role of Nutritional Priming for Early Salt and Drought Stress Responses in Medicago truncatula

    PubMed Central

    Staudinger, Christiana; Mehmeti, Vlora; Turetschek, Reinhard; Lyon, David; Egelhofer, Volker; Wienkoop, Stefanie

    2012-01-01

    Most legume species establish a symbiotic association with soil bacteria. The plant accommodates the differentiated rhizobia in specialized organs, the root nodules. In this environment, the microsymbiont reduces atmospheric nitrogen (N) making it available for plant metabolism. Symbiotic N-fixation is driven by the respiration of the host photosynthates and thus constitutes an additional carbon sink for the plant. Molecular phenotypes of symbiotic and non-symbiotic Medicago truncatula are identified. The implication of nodule symbiosis on plant abiotic stress response mechanisms is not well understood. In this study, we exposed nodulated and non-symbiotic N-fertilized plants to salt and drought conditions. We assessed the stress effects with proteomic and metabolomic methods and found a nutritionally regulated phenotypic plasticity pivotal for a differential stress adjustment strategy. PMID:23267362

  6. In vitro culture of pods from annual and perennial Medicago species.

    PubMed

    Wang, J W; Sorensen, E L; Liang, G H

    1984-08-01

    Because most interspecific Medicago embryos abort before they can be excised and cultured, our objective was to grow young pods in vitro. Various media were used to grow three-day-old pods of annuals [diploids, M. blancheana Boiss., M. disciformis DC., tetraploid M. scutellata (L.) Mill.] and perennials (diploid M. falcata L., tetraploid M. sativa L.).Few pods of perennial species grew to maturity on media containing modified Hoagland's plus 1% glucose or sucrose with or without 5% potato extract. Increasing sucrose to 6% increased the percentage of M. sativa pods that produced mature seeds. On DM (differentiation medium), the best medium, the percentage of pods producing viable seeds was: M. blancheana (82), M. disciformis (81), M. scutellata (48), M. sativa (63), M. falcata (15). DM plus 1 ppm indoleacetic or gibberellic acid did not enhance seed production.

  7. [Restorative morphogenesis in vitro of the annual Medicago species in presence of benzylaminopurine].

    PubMed

    Smolenskaia, S E; Ibragimova, S S

    2002-01-01

    Four annual medick species (Medicago orbicularis (L.) All., M. rigidula (L.) Desr., M. scutellata (L.) Miller, and M. rugosa Desr.) were used as model objects for studying the spectrum of morphogenetic reactions in vitro. The seeds were incubated on nutrient media with benzylaminopurine at different concentrations until germination and, thereafter, the explants of seedlings were cultivated in order to obtain primary calluses and morphogenetic structures. Normal and abnormal (with reduced root and/or apex) seedlings were cultivated in the presence of benzylaminopurine. Further cultivation of explants from the seedlings of both types showed a considerable intra- and interspecific polymorphism by the capacity for callusogenesis, frequency of primary restorative reactions, and pattern of microreproduction in vitro. In the control (the seeds were incubated on a hormone-free medium), no cases of microreproduction by way of organogenesis or somatic embryogenesis were observed. In all experimental variants, the restorative reactions preceded microreproduction in vitro.

  8. Contrasted patterns of selective pressure in three recent paralogous gene pairs in the Medicago genus (L.)

    PubMed Central

    2012-01-01

    Background Gene duplications are a molecular mechanism potentially mediating generation of functional novelty. However, the probabilities of maintenance and functional divergence of duplicated genes are shaped by selective pressures acting on gene copies immediately after the duplication event. The ratio of non-synonymous to synonymous substitution rates in protein-coding sequences provides a means to investigate selective pressures based on genic sequences. Three molecular signatures can reveal early stages of functional divergence between gene copies: change in the level of purifying selection between paralogous genes, occurrence of positive selection, and transient relaxed purifying selection following gene duplication. We studied three pairs of genes that are known to be involved in an interaction with symbiotic bacteria and were recently duplicated in the history of the Medicago genus (Fabaceae). We sequenced two pairs of polygalacturonase genes (Pg11-Pg3 and Pg11a-Pg11c) and one pair of auxine transporter-like genes (Lax2-Lax4) in 17 species belonging to the Medicago genus, and sought for molecular signatures of differentiation between copies. Results Selective histories revealed by these three signatures of molecular differentiation were found to be markedly different between each pair of paralogs. We found sites under positive selection in the Pg11 paralogs while Pg3 has mainly evolved under purifying selection. The most recent paralogs examined Pg11a and Pg11c, are both undergoing positive selection and might be acquiring new functions. Lax2 and Lax4 paralogs are both under strong purifying selection, but still underwent a temporary relaxation of purifying selection immediately after duplication. Conclusions This study illustrates the variety of selective pressures undergone by duplicated genes and the effect of age of the duplication. We found that relaxation of selective constraints immediately after duplication might promote adaptive divergence. PMID

  9. The identification of novel loci required for appropriate nodule development in Medicago truncatula.

    PubMed

    Domonkos, Agota; Horvath, Beatrix; Marsh, John F; Halasz, Gabor; Ayaydin, Ferhan; Oldroyd, Giles E D; Kalo, Peter

    2013-10-11

    The formation of functional symbiotic nodules is the result of a coordinated developmental program between legumes and rhizobial bacteria. Genetic analyses in legumes have been used to dissect the signaling processes required for establishing the legume-rhizobial endosymbiotic association. Compared to the early events of the symbiotic interaction, less attention has been paid to plant loci required for rhizobial colonization and the functioning of the nodule. Here we describe the identification and characterization of a number of new genetic loci in Medicago truncatula that are required for the development of effective nitrogen fixing nodules. Approximately 38,000 EMS and fast neutron mutagenized Medicago truncatula seedlings were screened for defects in symbiotic nitrogen fixation. Mutant plants impaired in nodule development and efficient nitrogen fixation were selected for further genetic and phenotypic analysis. Nine mutants completely lacking in nodule formation (Nod-) represented six complementation groups of which two novel loci have been identified. Eight mutants with ineffective nodules (Fix-) represented seven complementation groups, out of which five were new monogenic loci. The Fix- M. truncatula mutants showed symptoms of nitrogen deficiency and developed small white nodules. Microscopic analysis of Fix- nodules revealed that the mutants have defects in the release of rhizobia from infection threads, differentiation of rhizobia and maintenance of persistence of bacteria in nodule cells. Additionally, we monitored the transcriptional activity of symbiosis specific genes to define what transcriptional stage of the symbiotic process is blocked in each of the Fix- mutants. Based on the phenotypic and gene expression analysis a functional hierarchy of the FIX genes is proposed. The new symbiotic loci of M. truncatula isolated in this study provide the foundation for further characterization of the mechanisms underpinning nodulation, in particular the later

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

    PubMed

    Neupane, Achal; Nepal, Madhav P; Benson, Benjamin V; Macarthur, Kenton J; Piya, Sarbottam

    2013-11-01

    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.

  11. Definition and evolution of a new symbiovar, sv. rigiduloides, among Ensifer meliloti efficiently nodulating Medicago species.

    PubMed

    Gubry-Rangin, Cécile; Béna, Gilles; Cleyet-Marel, Jean-Claude; Brunel, Brigitte

    2013-10-01

    Understanding functional diversity is one of the main goals of microbial ecology, and definition of new bacterial ecotypes contributes significantly to this objective. Nitrogen-fixing bacteria provide a good system for investigation of ecotypes/biovars/symbiovars, as they present different specific associations with several host plants. This specific symbiosis is reflected both in the nodulation and fixation efficiency and in genetic characters of the bacteria, and several biovars have already been described in the bacterial species Ensifer meliloti. In the present study, the species affiliation of E. meliloti strains trapped from nodules sampled from Medicago rigiduloïdes roots was analyzed using housekeeping recA genes and DNA-DNA hybridization. The genetic diversity of these isolates was also investigated using several symbiotic markers: nodulation (nodA, nodB, nodC) and nitrogen fixation (nifH) genes, as well as the performance of phenotypic tests of nodulation capacity and nitrogen fixation efficiency. These analyses led to the proposal of a new bacterial symbiovar, E. meliloti sv. rigiduloides, that fixed nitrogen efficiently on M. rigiduloïdes, but not on Medicago truncatula. Using phylogenetic reconstructions, including the different described symbiovars, several hypotheses of lateral gene transfer and gene loss are proposed to explain the emergence of symbiovars within this species. The widespread geographical distribution of this symbiovar around the Mediterranean Basin, in contrast to restriction of M. rigiduloïdes to Eastern European countries, suggests that these isolates might also be associated with other plant species. The description of a new symbiovar within E. meliloti confirms the need for accurate bacterial ecological classification, especially for analysis of bacterial populations.

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

    PubMed

    Gargantini, Pablo R; Gonzalez-Rizzo, Silvina; Chinchilla, Delphine; Raices, Marcela; Giammaria, Verónica; Ulloa, Rita M; Frugier, Florian; Crespi, Martin D

    2006-12-01

    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.

  13. Response of Medicago truncatula seedlings to colonization by Salmonella enterica and Escherichia coli O157:H7.

    PubMed

    Jayaraman, Dhileepkumar; Valdés-López, Oswaldo; Kaspar, Charles W; Ané, Jean-Michel

    2014-01-01

    Disease outbreaks due to the consumption of legume seedlings contaminated with human enteric bacterial pathogens like Escherichia coli O157:H7 and Salmonella enterica are reported every year. Besides contaminations occurring during food processing, pathogens present on the surface or interior of plant tissues are also responsible for such outbreaks. In the present study, surface and internal colonization of Medicago truncatula, a close relative of alfalfa, by Salmonella enterica and Escherichia coli O157:H7 were observed even with inoculum levels as low as two bacteria per plant. Furthermore, expression analyses revealed that approximately 30% of Medicago truncatula genes were commonly regulated in response to both of these enteric pathogens. This study highlights that very low inoculum doses trigger responses from the host plant and that both of these human enteric pathogens may in part use similar mechanisms to colonize legume seedlings.

  14. Response of Medicago truncatula Seedlings to Colonization by Salmonella enterica and Escherichia coli O157:H7

    PubMed Central

    Jayaraman, Dhileepkumar; Valdés-López, Oswaldo; Kaspar, Charles W.; Ané, Jean-Michel

    2014-01-01

    Disease outbreaks due to the consumption of legume seedlings contaminated with human enteric bacterial pathogens like Escherichia coli O157:H7 and Salmonella enterica are reported every year. Besides contaminations occurring during food processing, pathogens present on the surface or interior of plant tissues are also responsible for such outbreaks. In the present study, surface and internal colonization of Medicago truncatula, a close relative of alfalfa, by Salmonella enterica and Escherichia coli O157:H7 were observed even with inoculum levels as low as two bacteria per plant. Furthermore, expression analyses revealed that approximately 30% of Medicago truncatula genes were commonly regulated in response to both of these enteric pathogens. This study highlights that very low inoculum doses trigger responses from the host plant and that both of these human enteric pathogens may in part use similar mechanisms to colonize legume seedlings. PMID:24551073

  15. Genomic and Coexpression Analyses Predict Multiple Genes Involved in Triterpene Saponin Biosynthesis in Medicago truncatula[C][W

    PubMed Central

    Naoumkina, Marina A.; Modolo, Luzia V.; Huhman, David V.; Urbanczyk-Wochniak, Ewa; Tang, Yuhong; Sumner, Lloyd W.; Dixon, Richard A.

    2010-01-01

    Saponins, an important group of bioactive plant natural products, are glycosides of triterpenoid or steroidal aglycones (sapogenins). Saponins possess many biological activities, including conferring potential health benefits for humans. However, most of the steps specific for the biosynthesis of triterpene saponins remain uncharacterized at the molecular level. Here, we use comprehensive gene expression clustering analysis to identify candidate genes involved in the elaboration, hydroxylation, and glycosylation of the triterpene skeleton in the model legume Medicago truncatula. Four candidate uridine diphosphate glycosyltransferases were expressed in Escherichia coli, one of which (UGT73F3) showed specificity for multiple sapogenins and was confirmed to glucosylate hederagenin at the C28 position. Genetic loss-of-function studies in M. truncatula confirmed the in vivo function of UGT73F3 in saponin biosynthesis. This report provides a basis for future studies to define genetically the roles of multiple cytochromes P450 and glycosyltransferases in triterpene saponin biosynthesis in Medicago. PMID:20348429

  16. Genome sequence of Ensifer medicae strain WSM1369; an effective microsymbiont of the annual legume Medicago sphaerocarpos.

    PubMed

    Terpolilli, Jason; Garau, Giovanni; Hill, Yvette; Tian, Rui; Howieson, John; Bräu, Lambert; Goodwin, Lynne; Han, James; Liolios, Konstantinos; Huntemann, Marcel; Pati, Amrita; Woyke, Tanja; Mavromatis, Konstantinos; Markowitz, Victor; Ivanova, Natalia; Kyrpides, Nikos; Reeve, Wayne

    2013-12-20

    Ensifer medicae WSM1369 is an aerobic, motile, Gram-negative, non-spore-forming rod that can exist as a soil saprophyte or as a legume microsymbiont of Medicago. WSM1369 was isolated in 1993 from a nodule recovered from the roots of Medicago sphaerocarpos growing at San Pietro di Rudas, near Aggius in Sardinia (Italy). WSM1369 is an effective microsymbiont of the annual forage legumes M. polymorpha and M. sphaerocarpos. Here we describe the features of E. medicae WSM1369, together with genome sequence information and its annotation. The 6,402,557 bp standard draft genome is arranged into 307 scaffolds of 307 contigs containing 6,656 protein-coding genes and 79 RNA-only encoding genes. This rhizobial genome is one of 100 sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project.

  17. Genome sequence of Ensifer medicae strain WSM1369; an effective microsymbiont of the annual legume Medicago sphaerocarpos

    PubMed Central

    Terpolilli, Jason; Garau, Giovanni; Hill, Yvette; Tian, Rui; Howieson, John; Bräu, Lambert; Goodwin, Lynne; Han, James; Liolios, Konstantinos; Huntemann, Marcel; Pati, Amrita; Woyke, Tanja; Mavromatis, Konstantinos; Markowitz, Victor; Ivanova, Natalia; Kyrpides, Nikos; Reeve, Wayne

    2013-01-01

    Ensifer medicae WSM1369 is an aerobic, motile, Gram-negative, non-spore-forming rod that can exist as a soil saprophyte or as a legume microsymbiont of Medicago. WSM1369 was isolated in 1993 from a nodule recovered from the roots of Medicago sphaerocarpos growing at San Pietro di Rudas, near Aggius in Sardinia (Italy). WSM1369 is an effective microsymbiont of the annual forage legumes M. polymorpha and M. sphaerocarpos. Here we describe the features of E. medicae WSM1369, together with genome sequence information and its annotation. The 6,402,557 bp standard draft genome is arranged into 307 scaffolds of 307 contigs containing 6,656 protein-coding genes and 79 RNA-only encoding genes. This rhizobial genome is one of 100 sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project. PMID:24976897

  18. Microgravity Effects on the Early Events of Biological Nitrogen Fixation in Medicago Truncatula: Results from the SyNRGE Experiment

    NASA Technical Reports Server (NTRS)

    Stutte, Gary W.; Roberts, Michael

    2012-01-01

    SyNRGE (Symbiotic Nodulation in a Reduced Gravity Environment) was a sortie mission on STS-135 in the Biological Research in Canisters (BRIC) hardware to study the effect of microgravity on a plant-microbe symbiosis resulting in biological nitrogen fixation. Medicago truncatula, a model species for th legume family, was inoculated with its bacterial symbiont, Sinorhizobium meliloti, to observe early biomolecular events associated with infection and nodulation in Petri Dish Fixation Units (PDFU's).

  19. Adaptive evolution of the symbiotic gene NORK is not correlated with shifts of rhizobial specificity in the genus Medicago

    PubMed Central

    De Mita, Stéphane; Santoni, Sylvain; Ronfort, Joëlle; Bataillon, Thomas

    2007-01-01

    Background The NODULATION RECEPTOR KINASE (NORK) gene encodes a Leucine-Rich Repeat (LRR)-containing receptor-like protein and controls the infection by symbiotic rhizobia and endomycorrhizal fungi in Legumes. The occurrence of numerous amino acid changes driven by directional selection has been reported in this gene, using a limited number of messenger RNA sequences, but the functional reason of these changes remains obscure. The Medicago genus, where changes in rhizobial associations have been previously examined, is a good model to test whether the evolution of NORK is influenced by rhizobial interactions. Results We sequenced a region of 3610 nucleotides (encoding a 392 amino acid-long region of the NORK protein) in 32 Medicago species. We confirm that positive selection in NORK has occurred within the Medicago genus and find that the amino acid positions targeted by selection occur in sites outside of solvent-exposed regions in LRRs, and other sites in the N-terminal region of the protein. We tested if branches of the Medicago phylogeny where changes of rhizobial symbionts occurred displayed accelerated rates of amino acid substitutions. Only one branch out of five tested, leading to M. noeana, displays such a pattern. Among other branches, the most likely for having undergone positive selection is not associated with documented shift of rhizobial specificity. Conclusion Adaptive changes in the sequence of the NORK receptor have involved the LRRs, but targeted different sites than in most previous studies of LRR proteins evolution. The fact that positive selection in NORK tends not to be associated to changes in rhizobial specificity indicates that this gene was probably not involved in evolving rhizobial preferences. Other explanations (e.g. coevolutionary arms race) must be tested to explain the adaptive evolution of NORK. PMID:17986323

  20. Effects of simulated acidic rain on yields of Raphanus sativus, Lactuca sativa, Triticum aestivum and Medicago sativa

    SciTech Connect

    Evans, L.S.; Gmur, N.F.; Mancini, D.

    1982-01-01

    Experiments were performed to determine effects of simulated acidic rain on radishes (Raphanus sativus), wheat(Triticum aestivum) and alfalfa (Medicago sativa) grown under greenhouse conditions. Experimental designs allowed the detection of statistically significant differences among means that differed by less than 10%. These results suggest that the efficiency of radish foliage in increasing; root mass decreases with increased rainfall acidity since only foliage was exposed to the treatments.

  1. A sequence-based genetic map of Medicago truncatula and comparison of marker colinearity with M. sativa.

    PubMed Central

    Choi, Hong-Kyu; Kim, Dongjin; Uhm, Taesik; Limpens, Eric; Lim, Hyunju; Mun, Jeong-Hwan; Kalo, Peter; Penmetsa, R Varma; Seres, Andrea; Kulikova, Olga; Roe, Bruce A; Bisseling, Ton; Kiss, Gyorgy B; Cook, Douglas R

    2004-01-01

    A core genetic map of the legume Medicago truncatula has been established by analyzing the segregation of 288 sequence-characterized genetic markers in an F(2) population composed of 93 individuals. These molecular markers correspond to 141 ESTs, 80 BAC end sequence tags, and 67 resistance gene analogs, covering 513 cM. In the case of EST-based markers we used an intron-targeted marker strategy with primers designed to anneal in conserved exon regions and to amplify across intron regions. Polymorphisms were significantly more frequent in intron vs. exon regions, thus providing an efficient mechanism to map transcribed genes. Genetic and cytogenetic analysis produced eight well-resolved linkage groups, which have been previously correlated with eight chromosomes by means of FISH with mapped BAC clones. We anticipated that mapping of conserved coding regions would have utility for comparative mapping among legumes; thus 60 of the EST-based primer pairs were designed to amplify orthologous sequences across a range of legume species. As an initial test of this strategy, we used primers designed against M. truncatula exon sequences to rapidly map genes in M. sativa. The resulting comparative map, which includes 68 bridging markers, indicates that the two Medicago genomes are highly similar and establishes the basis for a Medicago composite map. PMID:15082563

  2. Genome sequence of Ensifer medicae strain WSM1115; an acid-tolerant Medicago-nodulating microsymbiont from Samothraki, Greece.

    PubMed

    Reeve, Wayne; Ballard, Ross; Howieson, John; Drew, Elizabeth; Tian, Rui; Bräu, Lambert; Munk, Christine; Davenport, Karen; Chain, Patrick; Goodwin, Lynne; Pagani, Ioanna; Huntemann, Marcel; Mavrommatis, Konstantinos; Pati, Amrita; Markowitz, Victor; Ivanova, Natalia; Woyke, Tanja; Kyrpides, Nikos

    2014-06-15

    Ensifer medicae strain WSM1115 forms effective nitrogen fixing symbioses with a range of annual Medicago species and is used in commercial inoculants in Australia. WSM1115 is an aerobic, motile, Gram-negative, non-spore-forming rod. It was isolated from a nodule recovered from the root of burr medic (Medicago polymorpha) collected on the Greek Island of Samothraki. WSM1115 has a broad host range for nodulation and N2 fixation capacity within the genus Medicago, although this does not extend to all medic species. WSM1115 is considered saprophytically competent in moderately acid soils (pH(CaCl2) 5.0), but it has failed to persist at field sites where soil salinity exceeded 10 ECe (dS/m). Here we describe the features of E. medicae strain WSM1115, together with genome sequence information and its annotation. The 6,861,065 bp high-quality-draft genome is arranged into 7 scaffolds of 28 contigs, contains 6,789 protein-coding genes and 83 RNA-only encoding genes, and is one of 100 rhizobial genomes sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project.

  3. Genome sequence of Ensifer medicae strain WSM1115; an acid-tolerant Medicago-nodulating microsymbiont from Samothraki, Greece

    PubMed Central

    Reeve, Wayne; Ballard, Ross; Howieson, John; Drew, Elizabeth; Tian, Rui; Bräu, Lambert; Munk, Christine; Davenport, Karen; Chain, Patrick; Goodwin, Lynne; Pagani, Ioanna; Huntemann, Marcel; Mavrommatis, Konstantinos; Pati, Amrita; Markowitz, Victor; Ivanova, Natalia; Woyke, Tanja; Kyrpides, Nikos

    2013-01-01

    Ensifer medicae strain WSM1115 forms effective nitrogen fixing symbioses with a range of annual Medicago species and is used in commercial inoculants in Australia. WSM1115 is an aerobic, motile, Gram-negative, non-spore-forming rod. It was isolated from a nodule recovered from the root of burr medic (Medicago polymorpha) collected on the Greek Island of Samothraki. WSM1115 has a broad host range for nodulation and N2 fixation capacity within the genus Medicago, although this does not extend to all medic species. WSM1115 is considered saprophytically competent in moderately acid soils (pH(CaCl2) 5.0), but it has failed to persist at field sites where soil salinity exceeded 10 ECe (dS/m). Here we describe the features of E. medicae strain WSM1115, together with genome sequence information and its annotation. The 6,861,065 bp high-quality-draft genome is arranged into 7 scaffolds of 28 contigs, contains 6,789 protein-coding genes and 83 RNA-only encoding genes, and is one of 100 rhizobial genomes sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project. PMID:25197437

  4. Sapogenin content variation in Medicago inter-specific hybrid derivatives highlights some aspects of saponin synthesis and control.

    PubMed

    Carelli, Maria; Biazzi, Elisa; Tava, Aldo; Losini, Ilaria; Abbruscato, Pamela; Depedro, Claudia; Scotti, Carla

    2015-04-01

    In the Medicago genus, saponins are a complex mixture of triterpene glycosides showing a broad spectrum of biological properties. Here we analyzed the variation in the sapogenin content and composition of inter-specific hybrid Medicago sativa × Medicago arborea derivatives to highlight the pattern of this variation in plant organs (leaves/roots) and the possible mechanisms underlying it. In Sativa Arborea Cross (SAC) leaves and roots, saponins and sapogenins were evaluated using chromatographic methods. Phenotypic correlations between sapogenin content and bio-agronomic traits were examined. Expression studies on β-amyrin synthase and four cytochromes P450 (CYPs) involved in sapogenin biosynthesis and sequence analysis of the key gene of the hemolytic sapogenin pathway (CYP716A12) were performed. Chromatographic analyses revealed a different pattern of among-family variation for hemolytic and nonhemolytic sapogenins and saponins and for the two organs/tissues. Different correlation patterns of gene expression in roots and leaves were found. Diachronic analysis revealed a relationship between sapogenin content and gene transcriptional levels in the early stages of the productive cycle. The results suggest that there are different control mechanisms acting on sapogenin biosynthesis for leaves and roots, which are discussed. A key role for medicagenic acid in the control of sapogenin content in both the tissues is proposed and discussed.

  5. Arbuscular Mycorrhiza Stimulates Biological Nitrogen Fixation in Two Medicago spp. through Improved Phosphorus Acquisition.

    PubMed

    Püschel, David; Janoušková, Martina; Voříšková, Alena; Gryndlerová, Hana; Vosátka, Miroslav; Jansa, Jan

    2017-01-01

    Legumes establish root symbioses with rhizobia that provide plants with nitrogen (N) through biological N fixation (BNF), as well as with arbuscular mycorrhizal (AM) fungi that mediate improved plant phosphorus (P) uptake. Such complex relationships complicate our understanding of nutrient acquisition by legumes and how they reward their symbiotic partners with carbon along gradients of environmental conditions. In order to disentangle the interplay between BNF and AM symbioses in two Medicago species (Medicago truncatula and M. sativa) along a P-fertilization gradient, we conducted a pot experiment where the rhizobia-treated plants were either inoculated or not inoculated with AM fungus Rhizophagus irregularis 'PH5' and grown in two nutrient-poor substrates subjected to one of three different P-supply levels. Throughout the experiment, all plants were fertilized with (15)N-enriched liquid N-fertilizer to allow for assessment of BNF efficiency in terms of the fraction of N in the plants derived from the BNF (%NBNF). We hypothesized (1) higher %NBNF coinciding with higher P supply, and (2) higher %NBNF in mycorrhizal as compared to non-mycorrhizal plants under P deficiency due to mycorrhiza-mediated improvement in P nutrition. We found a strongly positive correlation between total plant P content and %NBNF, clearly documenting the importance of plant P nutrition for BNF efficiency. The AM symbiosis generally improved P uptake by plants and considerably stimulated the efficiency of BNF under low P availability (below 10 mg kg(-1) water extractable P). Under high P availability (above 10 mg kg(-1) water extractable P), the AM symbiosis brought no further benefits to the plants with respect to P nutrition even as the effects of P availability on N acquisition via BNF were further modulated by the environmental context (plant and substrate combinations). As a response to elevated P availability in the substrate, the extent of root length colonization by AM fungi was

  6. An integrated genetic linkage map for white clover (Trifolium repens L.) with alignment to Medicago

    PubMed Central

    2013-01-01

    Background White clover (Trifolium repens L.) is a temperate forage legume with an allotetraploid genome (2n=4×=32) estimated at 1093 Mb. Several linkage maps of various sizes, marker sources and completeness are available, however, no integrated map and marker set has explored consistency of linkage analysis among unrelated mapping populations. Such integrative analysis requires tools for homoeologue matching among populations. Development of these tools provides for a consistent framework map of the white clover genome, and facilitates in silico alignment with the model forage legume, Medicago truncatula. Results This is the first report of integration of independent linkage maps in white clover, and adds to the literature on methyl filtered GeneThresher®-derived microsatellite (simple sequence repeat; SSR) markers for linkage mapping. Gene-targeted SSR markers were discovered in a GeneThresher® (TrGT) methyl-filtered database of 364,539 sequences, which yielded 15,647 SSR arrays. Primers were designed for 4,038 arrays and of these, 465 TrGT-SSR markers were used for parental consensus genetic linkage analysis in an F1 mapping population (MP2). This was merged with an EST-SSR consensus genetic map of an independent population (MP1), using markers to match homoeologues and develop a multi-population integrated map of the white clover genome. This integrated map (IM) includes 1109 loci based on 804 SSRs over 1274 cM, covering 97% of the genome at a moderate density of one locus per 1.2 cM. Eighteen candidate genes and one morphological marker were also placed on the IM. Despite being derived from disparate populations and marker sources, the component maps and the derived IM had consistent representations of the white clover genome for marker order and genetic length. In silico analysis at an E-value threshold of 1e-20 revealed substantial co-linearity with the Medicago truncatula genome, and indicates a translocation between T. repens groups 2 and 6 relative to

  7. Arbuscular Mycorrhiza Stimulates Biological Nitrogen Fixation in Two Medicago spp. through Improved Phosphorus Acquisition

    PubMed Central

    Püschel, David; Janoušková, Martina; Voříšková, Alena; Gryndlerová, Hana; Vosátka, Miroslav; Jansa, Jan

    2017-01-01

    Legumes establish root symbioses with rhizobia that provide plants with nitrogen (N) through biological N fixation (BNF), as well as with arbuscular mycorrhizal (AM) fungi that mediate improved plant phosphorus (P) uptake. Such complex relationships complicate our understanding of nutrient acquisition by legumes and how they reward their symbiotic partners with carbon along gradients of environmental conditions. In order to disentangle the interplay between BNF and AM symbioses in two Medicago species (Medicago truncatula and M. sativa) along a P-fertilization gradient, we conducted a pot experiment where the rhizobia-treated plants were either inoculated or not inoculated with AM fungus Rhizophagus irregularis ‘PH5’ and grown in two nutrient-poor substrates subjected to one of three different P-supply levels. Throughout the experiment, all plants were fertilized with 15N-enriched liquid N-fertilizer to allow for assessment of BNF efficiency in terms of the fraction of N in the plants derived from the BNF (%NBNF). We hypothesized (1) higher %NBNF coinciding with higher P supply, and (2) higher %NBNF in mycorrhizal as compared to non-mycorrhizal plants under P deficiency due to mycorrhiza-mediated improvement in P nutrition. We found a strongly positive correlation between total plant P content and %NBNF, clearly documenting the importance of plant P nutrition for BNF efficiency. The AM symbiosis generally improved P uptake by plants and considerably stimulated the efficiency of BNF under low P availability (below 10 mg kg-1 water extractable P). Under high P availability (above 10 mg kg-1 water extractable P), the AM symbiosis brought no further benefits to the plants with respect to P nutrition even as the effects of P availability on N acquisition via BNF were further modulated by the environmental context (plant and substrate combinations). As a response to elevated P availability in the substrate, the extent of root length colonization by AM fungi was

  8. MATE Transporters Facilitate Vacuolar Uptake of Epicatechin 3′-O-Glucoside for Proanthocyanidin Biosynthesis in Medicago truncatula and Arabidopsis[C][W

    PubMed Central

    Zhao, Jian; Dixon, Richard A.

    2009-01-01

    Expression of the Arabidopsis thaliana MYB transcription factor TRANSPARENT TESTA 2 (TT2) in Medicago trunculata hairy roots induces both proanthocyanidin accumulation and the ATP-dependent vacuolar/vesicular uptake of epicatechin 3′-O-glucoside; neither process is active in control roots that do, however, possess anthocyanidin 3-O-glucoside vacuolar uptake activity. A vacuolar membrane-localized multidrug and toxic compound extrusion (MATE) transporter, Medicago MATE1, was identified at the molecular level and shown to preferentially transport epicatechin 3′-O-glucoside. Genetic evidence has implicated TT12, a tonoplastic MATE transporter from Arabidopsis, in the transport of precursors for proanthocyanidin biosynthesis in the seed coat. However, although Arabidopsis TT12 facilitates the transport of cyanidin 3-O-glucoside into membrane vesicles when expressed in yeast, there is no evidence that cyanidin 3-O-glucoside is converted to proanthocyanidins after transport into the vacuole. Here, we show that Arabidopsis TT12, like Medicago MATE1, functions to transport epicatechin 3′-O-glucoside as a precursor for proanthocyanidin biosynthesis, and Medicago MATE1 complements the seed proanthocyanidin phenotype of the Arabidopsis tt12 mutant both quantitatively and qualitatively. On the basis of biochemical properties, tissue-specific expression pattern, and genetic loss-of-function analysis, we conclude that MATE1 is an essential membrane transporter for proanthocyanidin biosynthesis in the Medicago seed coat. Implications of these findings for the assembly of oligomeric proanthocyanidins are discussed. PMID:19684242

  9. Medicago sativa--Sinorhizobium meliloti Symbiosis Promotes the Bioaccumulation of Zinc in Nodulated Roots.

    PubMed

    Zribi, Kais; Nouairi, Issam; Slama, Ines; Talbi-Zribi, Ons; Mhadhbi, Haythem

    2015-01-01

    In this study we investigated effects of Zn supply on germination, growth, inorganic solutes (Zn, Ca, Fe, and Mg) partitioning and nodulation of Medicago sativa This plant was cultivated with and without Zn (2 mM). Treatments were plants without (control) and with Zn tolerant strain (S532), Zn intolerant strain (S112) and 2 mM urea nitrogen fertilisation. Results showed that M. sativa germinates at rates of 50% at 2 mM Zn. For plants given nitrogen fertilisation, Zn increased plant biomass production. When grown with symbionts, Zn supply had no effect on nodulation. Moreover, plants with S112 showed a decrease of shoot and roots biomasses. However, in symbiosis with S532, an increase of roots biomass was observed. Plants in symbiosis with S. meliloti accumulated more Zn in their roots than nitrogen fertilised plants. Zn supply results in an increase of Ca concentration in roots of fertilised nitrogen plants. However, under Zn supply, Fe concentration decreased in roots and increased in nodules of plants with S112. Zn supply showed contrasting effects on Mg concentrations for plants with nitrogen fertilisation (increase) and plants with S112 (decrease). The capacity of M. sativa to accumulate Zn in their nodulated roots encouraged its use in phytostabilisation processes.

  10. Glutamine synthetase in Medicago truncatula, unveiling new secrets of a very old enzyme.

    PubMed

    Seabra, Ana R; Carvalho, Helena G

    2015-01-01

    Glutamine synthetase (GS) catalyzes the first step at which nitrogen is brought into cellular metabolism and is also involved in the reassimilation of ammonium released by a number of metabolic pathways. Due to its unique position in plant nitrogen metabolism, GS plays essential roles in all aspects of plant development, from germination to senescence, and is a key component of nitrogen use efficiency (NUE) and plant yield. Understanding the mechanisms regulating GS activity is therefore of utmost importance and a great effort has been dedicated to understand how GS is regulated in different plant species. The present review summarizes exciting recent developments concerning the structure and regulation of GS isoenzymes, using the model legume Medicago truncatula. These include the understanding of the structural determinants of both the cytosolic and plastid located isoenzymes, the existence of a seed-specific GS gene unique to M. truncatula and closely related species and the discovery that GS isoenzymes are regulated by nitric oxide at the post-translational level. The data is discussed and integrated with the potential roles of the distinct GS isoenzymes within the whole plant context.

  11. Transcriptome analysis of Glomus mosseae/Medicago sativa mycorrhiza on atrazine stress

    PubMed Central

    Song, Fuqiang; Li, Jize; Fan, Xiaoxu; Zhang, Quan; Chang, Wei; Yang, Fengshan; Geng, Gui

    2016-01-01

    Arbuscular mycorrhizal fungi (AMF) protect host plants against diverse biotic and abiotic stresses, and promote biodegradation of various contaminants. In this study effect of Glomus mosseae/Medicago sativa mycorrhiza on atrazine degradation was investigated. It was observed that the atrazine degradation rates with any addition level in mycorrhizal treatments were all significantly higher than those in non- mycorrhizal treatments. When atrazine was applied at 20 mg kg−1, the removal efficiency was up to 74.65%. Therefore, G. mosseae can be considered as ideal inhabitants of technical installations to facilitate phytoremediation. Furthermore, a total of 10.4 Gb was used for de novo transcriptome assembly, resulting in a comprehensive data set for the identification of genes corresponding to atrazine stress in the AM association. After comparative analysis with edgeR, a total of 2,060 differential expressed genes were identified, including 570 up-regulated genes and 1490 down-regulated genes. After excluding ‘function unknown’ and ‘general function predictions only’ genes, 172 up-regulated genes were obtained. The differentially expressed genes in AM association with and without atrazine stress were associated with molecular processes/other proteins, zinc finger protein, intracellular/extracellular enzymes, structural proteins, anti-stress/anti-disease protein, electron transport-related protein, and plant growth associated protein. Our results not only prove AMF has important ecological significance on atrazine degradation but also provide evidence for the molecular mechanisms of atrazine degradation by AMF. PMID:26833403

  12. Nitric oxide is required for an optimal establishment of the Medicago truncatula–Sinorhizobium meliloti symbiosis

    PubMed Central

    del Giudice, Jennifer; Cam, Yvan; Damiani, Isabelle; Fung-Chat, Franck; Meilhoc, Eliane; Bruand, Claude; Brouquisse, Renaud; Puppo, Alain; Boscari, Alexandre

    2011-01-01

    Nitric oxide (NO) is a gaseous molecule that participates in numerous plant signalling pathways. It is involved in plant responses to pathogens and development processes such as seed germination, flowering and stomatal closure. Using a permeable NO-specific fluorescent probe and a bacterial reporter strain expressing the lacZ gene under the control of a NO-responsive promoter, we detected NO production in the first steps, during infection threads growth, of the Medicago truncatula–Sinorhizobium meliloti symbiotic interaction. Nitric oxide was also detected, by confocal microscopy, in nodule primordia. Depletion of NO caused by cPTIO (2-(4-carboxyphenyl)-4,4,5,5-tetramethyl imidazoline-1-oxyl-3-oxide), an NO scavenger, resulted in a significant delay in nodule appearance. The overexpression of a bacterial hmp gene, encoding a flavohaemoglobin able to scavenge NO, under the control of a nodule-specific promoter (pENOD20) in transgenic roots, led to the same phenotype. The NO scavenging resulting from these approaches provoked the downregulation of plant genes involved in nodule development, such as MtCRE1 and MtCCS52A. Furthermore, an Hmp-overexpressing S. meliloti mutant strain was found to be less competitive than the wild type in the nodulation process. Taken together, these results indicate that NO is required for an optimal establishment of the M. truncatula–S. meliloti symbiotic interaction. PMID:21457261

  13. Hydrogen peroxide-regulated genes in the Medicago truncatula-Sinorhizobium meliloti symbiosis.

    PubMed

    Andrio, Emilie; Marino, Daniel; Marmeys, Anthony; de Segonzac, Marion Dunoyer; Damiani, Isabelle; Genre, Andrea; Huguet, Stéphanie; Frendo, Pierre; Puppo, Alain; Pauly, Nicolas

    2013-04-01

    Reactive oxygen species (ROS), particularly hydrogen peroxide (H(2)O(2)), play an important role in signalling in various cellular processes. The involvement of H(2)O(2) in the Medicago truncatula-Sinorhizobium meliloti symbiotic interaction raises questions about its effect on gene expression. A transcriptome analysis was performed on inoculated roots of M. truncatula in which ROS production was inhibited with diphenylene iodonium (DPI). In total, 301 genes potentially regulated by ROS content were identified 2 d after inoculation. These genes included MtSpk1, which encodes a putative protein kinase and is induced by exogenous H(2)O(2) treatment. MtSpk1 gene expression was also induced by nodulation factor treatment. MtSpk1 transcription was observed in infected root hair cells, nodule primordia and the infection zone of mature nodules. Analysis with a fluorescent protein probe specific for H(2)O(2) showed that MtSpk1 expression and H(2)O(2) were similarly distributed in the nodule infection zone. Finally, the establishment of symbiosis was impaired by MtSpk1 downregulation with an artificial micro-RNA. Several genes regulated by H(2)O(2) during the establishment of rhizobial symbiosis were identified. The involvement of MtSpk1 in the establishment of the symbiosis is proposed. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

  14. Molecular Signals Controlling the Inhibition of Nodulation by Nitrate in Medicago truncatula

    PubMed Central

    van Noorden, Giel E.; Verbeek, Rob; Dinh, Quy Dung; Jin, Jian; Green, Alexandra; Ng, Jason Liang Pin; Mathesius, Ulrike

    2016-01-01

    The presence of nitrogen inhibits legume nodule formation, but the mechanism of this inhibition is poorly understood. We found that 2.5 mM nitrate and above significantly inhibited nodule initiation but not root hair curling in Medicago trunatula. We analyzed protein abundance in M. truncatula roots after treatment with either 0 or 2.5 mM nitrate in the presence or absence of its symbiont Sinorhizobium meliloti after 1, 2 and 5 days following inoculation. Two-dimensional gel electrophoresis combined with mass spectrometry was used to identify 106 differentially accumulated proteins responding to nitrate addition, inoculation or time point. While flavonoid-related proteins were less abundant in the presence of nitrate, addition of Nod gene-inducing flavonoids to the Sinorhizobium culture did not rescue nodulation. Accumulation of auxin in response to rhizobia, which is also controlled by flavonoids, still occurred in the presence of nitrate, but did not localize to a nodule initiation site. Several of the changes included defense- and redox-related proteins, and visualization of reactive oxygen species indicated that their induction in root hairs following Sinorhizobium inoculation was inhibited by nitrate. In summary, the presence of nitrate appears to inhibit nodulation via multiple pathways, including changes to flavonoid metabolism, defense responses and redox changes. PMID:27384556

  15. Genome-wide identification and characterization of the Dof gene family in Medicago truncatula.

    PubMed

    Shu, Y J; Song, L L; Zhang, J; Liu, Y; Guo, C H

    2015-09-09

    The DNA-binding one zinc finger (Dof) family is a classic plant-specific zinc-finger transcription factor family, which is involved in many important processes, including seed maturation and germination, plant growth and development, and light responses. Investigation of the Medicago truncatula genome revealed 42 putative Dof genes, each of which holds one Dof domain. These genes were classified into four groups based on phylogenetic analysis, which are similar to the groups reported for Arabidopsis and rice. Based on genome duplication analysis, it was found that the MtDof genes were distributed on all chromosomes and had expanded through tandem gene duplication and segmental duplication events. Two main duplication regions were identified, one from tandem duplication and another from segmental duplication. By analyzing high-throughput sequencing data from M. truncatula, we found that most of the MtDof genes showed specific expression patterns in different tissues. According to cis-regulatory element analysis, these MtDof genes are regulated by different cis-acting motifs, which are important for the functional divergence of the MtDof genes in different processes. Thus, using genome-wide identification, evolution, and expression pattern analysis of the Dof genes in M. truncatula, our study provides valuable information for understanding the potential function of the Dof genes in regulating the growth and development of M. truncatula.

  16. Complete genome sequence of the Medicago microsymbiont Ensifer (Sinorhizobium) medicae strain WSM419

    PubMed Central

    Reeve, Wayne; Chain, Patrick; O’Hara, Graham; Ardley, Julie; Nandesena, Kemanthi; Bräu, Lambert; Tiwari, Ravi; Malfatti, Stephanie; Kiss, Hajnalka; Lapidus, Alla; Copeland, Alex; Nolan, Matt; Land, Miriam; Hauser, Loren; Chang, Yun-Juan; Ivanova, Natalia; Mavromatis, Konstantinos; Markowitz, Victor; Kyrpides, Nikos; Gollagher, Margaret; Yates, Ron; Dilworth, Michael; Howieson, John

    2010-01-01

    Ensifer (Sinorhizobium) medicae is an effective nitrogen fixing microsymbiont of a diverse range of annual Medicago (medic) species. Strain WSM419 is an aerobic, motile, non-spore forming, Gram-negative rod isolated from a M. murex root nodule collected in Sardinia, Italy in 1981. WSM419 was manufactured commercially in Australia as an inoculant for annual medics during 1985 to 1993 due to its nitrogen fixation, saprophytic competence and acid tolerance properties. Here we describe the basic features of this organism, together with the complete genome sequence, and annotation. This is the first report of a complete genome sequence for a microsymbiont of the group of annual medic species adapted to acid soils. We reveal that its genome size is 6,817,576 bp encoding 6,518 protein-coding genes and 81 RNA only encoding genes. The genome contains a chromosome of size 3,781,904 bp and 3 plasmids of size 1,570,951 bp, 1,245,408 bp and 219,313 bp. The smallest plasmid is a feature unique to this medic microsymbiont. PMID:21304680

  17. Detection of polycyclic aromatic hydrocarbons (PAHs) in Medicago sativa L. by fluorescence microscopy.

    PubMed

    Alves, Wilber S; Manoel, Evelin A; Santos, Noemi S; Nunes, Rosane O; Domiciano, Giselli C; Soares, Marcia R

    2017-04-01

    Green technologies, such as phytoremediation, are effective for removing organic pollutants derived from oil and oil products, including polycyclic aromatic hydrocarbons (PAHs). Given the increasing popularity of these sustainable remediation techniques, methods based on fluorescence microscopy and multiphoton microscopy for the environmental monitoring of such pollutants have emerged in recent decades as effective tools for phytoremediation studies aimed at understanding the fate of these contaminants in plants. However, little is known about the cellular and molecular mechanisms involved in PAH uptake, responses and degradation by plants. Thus, the present study aimed to detect the location of pyrene, anthracene and phenanthrene using fluorescence microscopy techniques in shoots and roots of Medicago sativa L. (alfalfa) plants grown in artificially contaminated soil (150ppm PAHs) for 40days. Leaflet and root samples were then collected and observed under a fluorescence microscope to detect the presence of PAHs in various tissues. One important finding of the present study was intense fluorescence in the glandular secreting trichomes (GSTs) of plants grown in contaminated soil. These trichomes, with a previously unknown function, may be sites of PAH conjugation and degradation.

  18. A Medicago truncatula NADPH oxidase is involved in symbiotic nodule functioning

    PubMed Central

    Marino, Daniel; Andrio, Emilie; Danchin, Etienne G J; Oger, Elodie; Gucciardo, Sébastien; Lambert, Annie; Puppo, Alain; Pauly, Nicolas

    2011-01-01

    Summary The plant plasma membrane-localized NADPH oxidases, known as respiratory burst oxidase homologues (RBOHs), appear to play crucial roles in plant growth and development. They are involved in important processes, such as root hair growth, plant defence reactions and abscisic acid signalling.Using sequence similarity searches, we identified seven putative RBOH-encoding genes in the Medicago truncatula genome. A phylogenetic reconstruction showed that Rboh gene duplications occurred in legume species. We analysed the expression of these MtRboh genes in different M. truncatula tissues: one of them, MtRbohA, was significantly up-regulated in Sinorhizobium meliloti-induced symbiotic nodules.MtRbohA expression appeared to be restricted to the nitrogen-fixing zone of the functional nodule. Moreover, using S. meliloti bacA and nifH mutants unable to form efficient nodules, a strong link between nodule nitrogen fixation and MtRbohA up-regulation was shown. MtRbohA expression was largely enhanced under hypoxic conditions. Specific RNA interference for MtRbohA provoked a decrease in the nodule nitrogen fixation activity and the modulation of genes encoding the microsymbiont nitrogenase.These results suggest that hypoxia, prevailing in the nodule-fixing zone, may drive the stimulation of MtRbohA expression, which would, in turn, lead to the regulation of nodule functioning. PMID:21155825

  19. Stoichiometric Characteristics of Carbon, Nitrogen, and Phosphorus in Leaves of Differently Aged Lucerne (Medicago sativa) Stands

    PubMed Central

    Wang, Zhennan; Lu, Jiaoyun; Yang, Mei; Yang, Huimin; Zhang, Qingping

    2015-01-01

    Element concentration within a plant which is vital to function maintenance and adaptation to environment, may change with plant growth. However, how carbon (C), nitrogen (N), and phosphorus (P) vary stoichiometrically with stand growth, i.e., ages or cuts, was still untouched in perennial species. This study tested the hypothesis that lucerne (Medicago sativa) C:N, C:P, and N:P should change with stand age and cut. Leaf C:N, C:P, and N:P changed with stand age, showing various trends in different cuts of lucerne. Generally the greatest stoichiometric ratios were measured in 8 year stand and in the second cut. They were affected significantly and negatively by total N and P concentrations of leaf, but not by organic C concentration. There were significantly positive correlations among leaf C:N, C:P, and N:P. However, leaf C:N, C:P, and N:P were hardly affected by soil features. Conclusively, lucerne C, N, and P stoichiometry are age- and cut-specific, and regulated mainly by leaf N, P concentrations and stoichiometry. There are few correlations with soil fertility. To our knowledge, it is the first try to elucidate the stoichiometry in the viewpoint of age and cut with a perennial herbaceous legume. PMID:26697029

  20. Evolution by gene duplication of Medicago truncatula PISTILLATA-like transcription factors.

    PubMed

    Roque, Edelín; Fares, Mario A; Yenush, Lynne; Rochina, Mari Cruz; Wen, Jiangqi; Mysore, Kirankumar S; Gómez-Mena, Concepción; Beltrán, José Pío; Cañas, Luis A

    2016-03-01

    PISTILLATA (PI) is a member of the B-function MADS-box gene family, which controls the identity of both petals and stamens in Arabidopsis thaliana. In Medicago truncatula (Mt), there are two PI-like paralogs, known as MtPI and MtNGL9. These genes differ in their expression patterns, but it is not known whether their functions have also diverged. Describing the evolution of certain duplicated genes, such as transcription factors, remains a challenge owing to the complex expression patterns and functional divergence between the gene copies. Here, we report a number of functional studies, including analyses of gene expression, protein-protein interactions, and reverse genetic approaches designed to demonstrate the respective contributions of each M. truncatula PI-like paralog to the B-function in this species. Also, we have integrated molecular evolution approaches to determine the mode of evolution of Mt PI-like genes after duplication. Our results demonstrate that MtPI functions as a master regulator of B-function in M. truncatula, maintaining the overall ancestral function, while MtNGL9 does not seem to have a role in this regard, suggesting that the pseudogenization could be the functional evolutionary fate for this gene. However, we provide evidence that purifying selection is the primary evolutionary force acting on this paralog, pinpointing the conservation of its biochemical function and, alternatively, the acquisition of a new role for this gene.

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

    PubMed

    Hubberten, Hans-Michael; Sieh, Daniela; Zöller, Daniela; Hoefgen, Rainer; Krajinski, Franziska

    2015-01-01

    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 MtHA1 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.

  2. Characterization of a novel Medicago sativa NAC transcription factor gene involved in response to drought stress.

    PubMed

    Wang, Yong Xin

    2013-11-01

    Relying on the regulation of transcription factors, plants resist to various abiotic and biotic stresses. NAC (NAM, ATAF1/2, CUC2) are one of the largest families of plant-specific transcription factors and known to play important roles in plant development and response to environmental stresses. A new NAC gene was cloned on the basis of 503 bp EST fragment from the SSH cDNA library of Medicago sativa. It was 1,115 bp including an 816 bp ORF and encodes 271 amino acids. A highly conserved region is located from the 7th amino acid to the 315th amino acid in its N-terminal domain. The NAC protein is subcellularly localized in the nucleus of onion epidemical cells and possible functions as a transcription factor. The relative quantitative real-time RT-PCR was performed at different stress time. The results revealed that the transcription expression of NAC gene could be induced by drought, high salinity and ABA. The transgenic Arabidopsis with NAC gene has the drought tolerance better than the wild-type.

  3. Allelopatic effects of cyanobacteria extracts containing microcystins on Medicago sativa-Rhizobia symbiosis.

    PubMed

    El Khalloufi, Fatima; Oufdou, Khalid; Lahrouni, Majida; El Ghazali, Issam; Saqrane, Sanaa; Vasconcelos, Vitor; Oudra, Brahim

    2011-03-01

    The eutrophication of water leads to massive blooms of cyanobacteria potentially producers of highly toxic substances: cyanotoxins, especially microcystins (MC). The contamination of water used for irrigation by these toxins, can cause several adverse effects on plants and microorganisms. In this work, we report the phytotoxic effects of microcystins on the development of symbiosis between the leguminous plant Medicago sativa (Alfalfa) and rhizobia strains. The exposure of rhizobial strains to three different concentrations 0.01, 0.05 and 0.1 μg MC ml(-1) led to decrease on the bacteria growth. The strains of rhizobia Rh L1, Rh L2, Rh L3 and Rh L4 reduced their growth to, respectively, 20.85%, 20.80%, 33.19% and 25.65%. The chronic exposure of alfalfa seeds and seedlings to different MC concentrations affects the whole stages of plant development. The germination process has also been disrupted with an inhibition, which reaches 68.34% for a 22.24 μg MC ml(-1). Further, seedlings growth and photosynthetic process were also disrupted. The toxins reduced significantly the roots length and nodule formation and leads to an oxidative stress. Thus, the MCs contained in lake water and used for irrigation affect the development of symbiosis between M. sativa and Rhizobia.

  4. Evaluation of Medicago sativa L. sprouts as antihyperlipidemic and antihyperglycemic agent.

    PubMed

    Seida, Ahmed; El-Hefnawy, Hala; Abou-Hussein, Dina; Mokhtar, Fatma Alzahraa; Abdel-Naim, Ashraf

    2015-11-01

    Medicago sativa L. (Alfalfa) is traditionally used to treat diabetes. This study was designed to investigate the potential antihyperlipidemic and antihyperglycemic activity of M. sativa sprouts in streptozotocin (STZ) induced diabetes via i.p. injection of 55 mg/kg of STZ. Experimental animals were divided into the following groups: GP1 (normal), GP2 (STZ-hyperlipidemic), GP3 (rouvastatin), GP4 (metformin), GP 5-9 (diabetic treated with methanolic, petroleum ether, chloroform, ethyl acetate and butanol extracts). The administration of the total methanolic extract (500 mg/kg), the petroleum ether (32.5mg) and butanol fractions (60 mg) for 4 weeks significantly decreased (p<0.05) triglycerides (TG), total cholesterol (TC), low-density lipoproteins (LDL) and very low density lipoproteins (VLDL) in comparison to rouvastatin. Petroleum ether fraction proved to exhibit the best activity as antihyperlipidemic agent (12.23%). On the other hand, ethyl acetate fraction retained the best activity (vs. metformin) as antihyperglycemic agent. Histopathological evidences on liver, pancreas and spleen were in agreement with the above mentioned results. Purification, characterization, and identification of isolated compounds from the active fractions afforded 9 compounds: β-sitosterol and stigmasterol from the petroleum ether fraction; 10-hydroxy-coumestrol, apigenin, genistein, p-hydroxy-benzoic-acid, 7, 4'- dihydroxyflavone, quercetin-3-glucoside and sissotrin from the ethyl acetate fraction.

  5. Ractopamine up take by alfalfa (Medicago sativa) and wheat (Triticum aestivum) from soil.

    PubMed

    Shelver, Weilin L; DeSutter, Thomas M

    2015-08-01

    Ractopamine is a beta adrenergic agonist used as a growth promoter in swine, cattle and turkeys. To test whether ractopamine has the potential to accumulate in plants grown in contaminated soil, a greenhouse study was conducted with alfalfa (Medicago sativa) and wheat (Triticum aestivum) grown in two soils having different concentrations of organic matter (1.3% and 2.1%), amended with 0, 0.5, and 10 μg/g of ractopamine. Plant growth ranged from 2.7 to 8.8 g dry weight (dw) for alfalfa, and 8.7 to 40 g dw for wheat and was generally greater in the higher organic matter content soil. The uptake of ractopamine in plant tissues ranged from non-detectable to 897 ng/g and was strongly dependent on soil ractopamine concentration across soil and plant tissue. When adjusted to the total fortified quantities, the amount of ractopamine taken up by the plant tissue was low, <0.01% for either soil.

  6. Selective lignin downregulation leads to constitutive defense response expression in alfalfa (Medicago sativa L.).

    PubMed

    Gallego-Giraldo, Lina; Jikumaru, Yusuke; Kamiya, Yuji; Tang, Yuhong; Dixon, Richard A

    2011-05-01

    • Downregulation of hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase (HCT) in alfalfa (Medicago sativa) reduces lignin levels and improves forage quality and saccharification efficiency for bioethanol production. However, the plants have reduced stature. It was previously reported that HCT-down-regulated Arabidopsis have impaired auxin transport, but this has recently been disproved. • To address the basis for the phenotypes of lignin-modified alfalfa, we measured auxin transport, profiled a range of metabolites including flavonoids and hormones, and performed in depth transcriptome analyses. • Auxin transport is unaffected in HCT antisense alfalfa despite increased flavonoid biosynthesis. The plants show increased cytokinin and reduced auxin levels, and gibberellin levels and sensitivity are both reduced. Levels of salicylic, jasmonic and abscisic acids are elevated, associated with massive upregulation of pathogenesis and abiotic stress-related genes and enhanced tolerance to fungal infection and drought. • We suggest that HCT downregulated alfalfa plants exhibit constitutive activation of defense responses, triggered by release of bioactive cell wall fragments and production of hydrogen peroxide as a result of impaired secondary cell wall integrity.

  7. Accumulation and residue of napropamide in alfalfa (Medicago sativa) and soil involved in toxic response.

    PubMed

    Cui, Li E; Yang, Hong

    2011-06-15

    Napropamide belongs to the amide herbicide family and widely used to control weeds in farmland. Intensive use of the herbicide has resulted in widespread contamination to ecosystems. The present study demonstrated an analysis on accumulation of the toxic pesticide napropamide in six genotypes of alfalfa (Medicago sativa), along with biological parameters and its residues in soils. Soil was treated with napropamide at 3 mg kg(-1) dry soil and alfalfa plants were cultured for 10 or 30 d, respectively. The maximum value for napropamide accumulation is 0.426 mg kg(-1) in shoots and 2.444 mg kg(-1) in roots. The napropamide-contaminated soil with alfalfa cultivation had much lower napropamide concentrations than the control (soil without alfalfa cultivation). Also, the content of napropamide residue in the rhizosphere was significantly lower than that in the non-rhizosphere soil. M. sativa exposed to 3 mg kg(-1) napropamide showed inhibited growth. Further analysis revealed that plants treated with napropamide accumulated more reactive oxygen species (O(2)(-) and H(2)O(2)) and less amounts of chlorophyll. However, not all cultivars showed oxidative injury, suggesting that the alfalfa cultivars display different tolerance to napropamide.

  8. Micromonospora from nitrogen fixing nodules of alfalfa (Medicago sativa L.). A new promising Plant Probiotic Bacteria.

    PubMed

    Martínez-Hidalgo, Pilar; Galindo-Villardón, Purificación; Trujillo, Martha E; Igual, José M; Martínez-Molina, Eustoquio

    2014-09-17

    Biotic interactions can improve agricultural productivity without costly and environmentally challenging inputs. Micromonospora strains have recently been reported as natural endophytes of legume nodules but their significance for plant development and productivity has not yet been established. The aim of this study was to determine the diversity and function of Micromonospora isolated from Medicago sativa root nodules. Micromonospora-like strains from field alfalfa nodules were characterized by BOX-PCR fingerprinting and 16S rRNA gene sequencing. The ecological role of the interaction of the 15 selected representative Micromonospora strains was tested in M. sativa. Nodulation, plant growth and nutrition parameters were analyzed. Alfalfa nodules naturally contain abundant and highly diverse populations of Micromonospora, both at the intra- and at interspecific level. Selected Micromonospora isolates significantly increase the nodulation of alfalfa by Ensifer meliloti 1021 and also the efficiency of the plant for nitrogen nutrition. Moreover, they promote aerial growth, the shoot-to-root ratio, and raise the level of essential nutrients. Our results indicate that Micromonospora acts as a Rhizobia Helper Bacteria (RHB) agent and has probiotic effects, promoting plant growth and increasing nutrition efficiency. Its ecological role, biotechnological potential and advantages as a plant probiotic bacterium (PPB) are also discussed.

  9. Chemical modification and degradation of atrazine in Medicago sativa through multiple pathways.

    PubMed

    Zhang, Jing Jing; Lu, Yi Chen; Yang, Hong

    2014-10-08

    Atrazine is a member of the triazine herbicide family intensively used to control weeds for crop production. In this study, atrazine residues and its degraded products in alfalfa (Medicago sativa) were characterized using UPLC-TOF-MS/MS. Most of atrazine absorbed in plants was found as chemically modified derivatives like deisopropylated atrazine (DIA), dehydrogenated atrazine (DHA), or methylated atrazine (MEA), and some atrazine derivatives were conjugated through different functional groups such as sugar, glutathione, and amino acids. Interestingly, the specific conjugates DHA+hGSH (homoglutathione) and MEA-HCl+hGSH in alfalfa were detected. These results suggest that atrazine in alfalfa can be degraded through different pathways. The increased activities of glycosyltransferase and glutathione S-transferase were determined to support the atrazine degradation models. The outcome of the work uncovered the detailed mechanism for the residual atrazine accumulation and degradation in alfalfa and will help to evaluate whether the crop is suitable to be cultivated in the atrazine-polluted soil.

  10. Transcriptome analysis of Glomus mosseae/Medicago sativa mycorrhiza on atrazine stress.

    PubMed

    Song, Fuqiang; Li, Jize; Fan, Xiaoxu; Zhang, Quan; Chang, Wei; Yang, Fengshan; Geng, Gui

    2016-02-02

    Arbuscular mycorrhizal fungi (AMF) protect host plants against diverse biotic and abiotic stresses, and promote biodegradation of various contaminants. In this study effect of Glomus mosseae/Medicago sativa mycorrhiza on atrazine degradation was investigated. It was observed that the atrazine degradation rates with any addition level in mycorrhizal treatments were all significantly higher than those in non-mycorrhizal treatments. When atrazine was applied at 20 mg kg(-1), the removal efficiency was up to 74.65%. Therefore, G. mosseae can be considered as ideal inhabitants of technical installations to facilitate phytoremediation. Furthermore, a total of 10.4 Gb was used for de novo transcriptome assembly, resulting in a comprehensive data set for the identification of genes corresponding to atrazine stress in the AM association. After comparative analysis with edgeR, a total of 2,060 differential expressed genes were identified, including 570 up-regulated genes and 1490 down-regulated genes. After excluding 'function unknown' and 'general function predictions only' genes, 172 up-regulated genes were obtained. The differentially expressed genes in AM association with and without atrazine stress were associated with molecular processes/other proteins, zinc finger protein, intracellular/extracellular enzymes, structural proteins, anti-stress/anti-disease protein, electron transport-related protein, and plant growth associated protein. Our results not only prove AMF has important ecological significance on atrazine degradation but also provide evidence for the molecular mechanisms of atrazine degradation by AMF.

  11. Microbiote shift in the Medicago sativa rhizosphere in response to cyanotoxins extract exposure.

    PubMed

    El Khalloufi, Fatima; Oufdou, Khalid; Bertrand, Marie; Lahrouni, Majida; Oudra, Brahim; Ortet, Philippe; Barakat, Mohamed; Heulin, Thierry; Achouak, Wafa

    2016-01-01

    The bloom-containing water bodies may have an impact due to cyanotoxins production on other microorganisms and aquatic plants. Where such water is being used for crops irrigation, the presence of cyanotoxins may also have a toxic impact on terrestrial plants and their rhizosphere microbiota. For that purpose, PCR-based 454 pyrosequencing was applied to phylogenetically characterize the bacterial community of Medicago sativa rhizosphere in response to cyanotoxins extract. This analysis revealed a wide diversity at species level, which decreased from unplanted soil to root tissues indicating that only some populations were able to compete for nutrients and niches in this selective habitat. Gemmatimonas, Actinobacteria, Deltaproteobacteria and Opitutae mainly inhabited the bulk soil, whereas, the root-adhering soil and the root tissues were inhabited by Gammaproteobacteria and Alphaproteobacteria. The proportion of these populations fluctuated in response to cyanotoxins extract exposure. Betaproteobacteria proportion increased in the three studied compartments, whereas Gammaproteobacteria proportion decreased except in the bulk soil. This study revealed the potential toxicity of cyanotoxins extract towards Actinobacteria, Gemmatimonas, Deltaproteobacteria, and Gammaproteobacteria, however Clostridia, Opitutae and bacteria related with Betaproteobacteria, were stimulated denoting their tolerance. Altogether, these data indicate that crop irrigation using cyanotoxins containing water might alter the rhizosphere functioning.

  12. Germination of Medicago sativa is inhibited by soluble compounds in cement dust.

    PubMed

    Lafragüeta, Cristina; García-Criado, Balbino; Arranz, Angel; Vázquez-de-Aldana, Beatriz R

    2014-01-01

    Deposition of cement dust on soils and plant surfaces is known to affect plant growth and the species composition of plant communities, but little is known about its effects (and those of its pH and constituents) on germination. Therefore, the aim of this study was to assess the toxicity of an aqueous cement extract, constituents of the extract and pH on the germination of seeds of a selected species, Medicago sativa. First, the effects of the extract were tested in assays with concentrations and exposure durations ranging from 0 to 1.0 g/mL and 4 to 96 h, respectively. At 0.8 g/mL, the extract strongly inhibited germination; a 4-h exposure reduced the germination rate, from 77 ± 1.8 to 50 ± 2.6% (mean ± SE), while 8-h exposure completely inhibited it. Further, treatment at this concentration killed the non-germinating seeds, thus the inhibition was due to toxic effects. Neither the pH of the extract nor the concentration of its main soluble elements separately (K, Ca, S, Na, or Cr) caused the toxicity since germination rates were not significantly reduced when these variables were tested individually. However, a mixture of the elements in solution reduced germination rates, suggesting that they have adverse synergistic effects.

  13. In silico identification of transcription factors in Medicago sativa using available transcriptomic resources.

    PubMed

    Postnikova, Olga A; Shao, Jonathan; Nemchinov, Lev G

    2014-06-01

    Transcription factors (TFs) are proteins that govern organismal development and response to the environment by regulating gene expression. Information on the amount and diversity of TFs within individual plant species is critical for understanding of their biological roles and evolutionary history across the plant kingdom. Currently, only scattered information on separate TFs is available for alfalfa, the most extensively cultivated forage legume in the world. In the meantime, several large transcriptomic resources that can be used to identify and characterize alfalfa TF genes are freely accessible online. In this study, we have performed an in silico analysis of transcriptome data generated in our laboratory and publicly acquirable from other sources to reveal and systematize alfalfa transcription factors. Transcriptome-wide mining enabled prediction of 983 TFs along with their sequence features and putative phylogenies of the largest families. All data were assembled into a simple open-access database named AlfalfaTFDB ( http://plantpathology.ba.ars.usda.gov/alfalfatfdb.html ). Transcriptomic analysis used in this work represents an effective approach for the identification of TF genes in plants with incomplete genomes, such as alfalfa. Integrated TF repertoires of Medicago sativa will provide an important tool for studying regulation of gene expression in other complex non-model species of agricultural significance.

  14. Spectinomycin resistance mutations in the rrn16 gene are new plastid markers in Medicago sativa.

    PubMed

    Dudas, Brigitta; Jenes, Barnabas; Kiss, Gyorgy Botond; Maliga, Pal

    2012-11-01

    We report here the isolation of spectinomycin-resistant mutants in cultured cells of Medicago sativa line RegenSY-T2. Spectinomycin induces bleaching of cultured alfalfa cells due to inhibition of protein synthesis on the prokaryotic type 70S plastid ribosomes. Spontaneous mutants resistant to spectinomycin bleaching were identified by their ability to form green shoots on plant regeneration medium containing selective spectinomycin concentrations in the range of 25-50 mg/l. Sequencing of the plastid rrn16 gene revealed that spectinomycin resistance is due to mutations in a conserved stem structure of the 16S rRNA. Resistant plants transferred to the greenhouse developed normally and produced spectinomycin-resistant seed progeny. In light of their absence in soybean, a related leguminous plant, the isolation of spectinomycin-resistant mutants in M. sativa was unexpected. The new mutations are useful for the study of plastid inheritance, as demonstrated by detection of predominantly paternal plastid inheritance in the RegenSY-T2 × Szapko57 cross, and can be used as selective markers in plastid transformation vectors to obtain cisgenic plants.

  15. Enhanced salt tolerance of alfalfa (Medicago sativa) by rstB gene transformation.

    PubMed

    Zhang, Wan-Jun; Wang, Tao

    2015-05-01

    Generating salt tolerance forage plant is essential for use of the land affected by high salinity. A salt tolerance gene rstB was used as a selectable marker gene in Agrobacterium-mediated transformation of tobacco under a selective regime of 170mM NaCl. The transgenic plants showed clear improvement in salt tolerance. To improve salt tolerance of alfalfa (Medicago sativa L.), rstB gene was introduced into alfalfa genome by Agrobacterium-mediated transformation. No abnormal phenotype was observed among the transgenic plants when compared with wild type (wt) plants. Significant enhancement of resistance to salt-shock treatment was noted on the rstB transgenic (T0) plants. Transgenic second-generation (T1) seeds showed improved germination rate and seedling growth under salt-stress condition. Hindered Na(+) accumulation, but enhanced Ca(2+) accumulation was observed on the rstB T1 plants when subjected to salt-stresses. Enhanced calcium accumulation in transgenic plants was also verified by cytohistochemical localization of calcium. Under salt-stress of 50mM NaCl, about 15% of the transgenic plants finished their life-cycle but the wt plants had no flower formation. The results demonstrated that the expression of rstB gene improved salt tolerance in transgenic alfalfa.

  16. Exploring root symbiotic programs in the model legume Medicago truncatula using EST analysis.

    PubMed

    Journet, Etienne-Pascal; van Tuinen, Diederik; Gouzy, Jérome; Crespeau, Hervé; Carreau, Véronique; Farmer, Mary-Jo; Niebel, Andreas; Schiex, Thomas; Jaillon, Olivier; Chatagnier, Odile; Godiard, Laurence; Micheli, Fabienne; Kahn, Daniel; Gianinazzi-Pearson, Vivienne; Gamas, Pascal

    2002-12-15

    We report on a large-scale expressed sequence tag (EST) sequencing and analysis program aimed at characterizing the sets of genes expressed in roots of the model legume Medicago truncatula during interactions with either of two microsymbionts, the nitrogen-fixing bacterium Sinorhizobium meliloti or the arbuscular mycorrhizal fungus Glomus intraradices. We have designed specific tools for in silico analysis of EST data, in relation to chimeric cDNA detection, EST clustering, encoded protein prediction, and detection of differential expression. Our 21 473 5'- and 3'-ESTs could be grouped into 6359 EST clusters, corresponding to distinct virtual genes, along with 52 498 other M.truncatula ESTs available in the dbEST (NCBI) database that were recruited in the process. These clusters were manually annotated, using a specifically developed annotation interface. Analysis of EST cluster distribution in various M.truncatula cDNA libraries, supported by a refined R test to evaluate statistical significance and by 'electronic northern' representation, enabled us to identify a large number of novel genes predicted to be up- or down-regulated during either symbiotic root interaction. These in silico analyses provide a first global view of the genetic programs for root symbioses in M.truncatula. A searchable database has been built and can be accessed through a public interface.

  17. Adaptation to climate through flowering phenology: a case study in Medicago truncatula.

    PubMed

    Burgarella, Concetta; Chantret, Nathalie; Gay, Laurène; Prosperi, Jean-Marie; Bonhomme, Maxime; Tiffin, Peter; Young, Nevin D; Ronfort, Joelle

    2016-07-01

    Local climatic conditions likely constitute an important selective pressure on genes underlying important fitness-related traits such as flowering time, and in many species, flowering phenology and climatic gradients strongly covary. To test whether climate shapes the genetic variation on flowering time genes and to identify candidate flowering genes involved in the adaptation to environmental heterogeneity, we used a large Medicago truncatula core collection to examine the association between nucleotide polymorphisms at 224 candidate genes and both climate variables and flowering phenotypes. Unlike genome-wide studies, candidate gene approaches are expected to enrich for the number of meaningful trait associations because they specifically target genes that are known to affect the trait of interest. We found that flowering time mediates adaptation to climatic conditions mainly by variation at genes located upstream in the flowering pathways, close to the environmental stimuli. Variables related to the annual precipitation regime reflected selective constraints on flowering time genes better than the other variables tested (temperature, altitude, latitude or longitude). By comparing phenotype and climate associations, we identified 12 flowering genes as the most promising candidates responsible for phenological adaptation to climate. Four of these genes were located in the known flowering time QTL region on chromosome 7. However, climate and flowering associations also highlighted largely distinct gene sets, suggesting different genetic architectures for adaptation to climate and flowering onset. © 2016 John Wiley & Sons Ltd.

  18. [Influence of salt stress on the genetically polymorphic system of Sinorhizobium meliloti-Medicago truncatula].

    PubMed

    Kurchak, O N; Provorov, N A; Onishchuk, O P; Vorobyov, N I; Roumiantseva, M L; Simarov, B V

    2014-07-01

    The impacts of salt stress (75 mM NaC1) on the ecological efficiency of the genetically polymorphic Sinorhizobium meliloti-Medicago truncatula system were studied. Its impact on a symbiotic system results in an increase of the partners' variability for symbiotic traits and of the symbiosis integrity as indicated by: a) the specificity of the partners' interactions--the nonadditive inputs of their genotypes into the variation of symbiotic parameters; and b) the correlative links between these parameters. The structure of the nodDI locus and the content correlates to the efficiency of the symbiosis between S. meliloti and M. truncatula genotypes under stress conditions more sufficiently than in the absence of stress. Correlations between the symbiotic efficiency of rhizobia strains and their growth rate outside symbiosis are expressed under stress conditions, not in the absence of stress. Under salt stress symbiotic effectiveness was decreased for M. truncatula line F83005.5, which was salt sensitive for mineral nutrition. The inhibition of symbiotic activity for this line is linked with decreased nodule formation, whereas for Jemalong 6 and DZA315.16 lines it is associated with repressed N2-fixation. It was demonstrated for the first time that salt stress impairs the M. truncatula habitus (the mass : height ratio increased 2- to 6-fold), which in the salt-resistant cultivar Jemalong 6 is normalized as the result of rhizobia inoculation.

  19. Overlap of proteome changes in Medicago truncatula in response to auxin and Sinorhizobium meliloti.

    PubMed

    van Noorden, Giel E; Kerim, Tursun; Goffard, Nicolas; Wiblin, Robert; Pellerone, Flavia I; Rolfe, Barry G; Mathesius, Ulrike

    2007-06-01

    We used proteome analysis to identify proteins induced during nodule initiation and in response to auxin in Medicago truncatula. From previous experiments, which found a positive correlation between auxin levels and nodule numbers in the M. truncatula supernodulation mutant sunn (supernumerary nodules), we hypothesized (1) that auxin mediates protein changes during nodulation and (2) that auxin responses might differ between the wild type and the supernodulating sunn mutant during nodule initiation. Increased expression of the auxin response gene GH3:beta-glucuronidase was found during nodule initiation in M. truncatula, similar to treatment of roots with auxin. We then used difference gel electrophoresis and tandem mass spectrometry to compare proteomes of wild-type and sunn mutant roots after 24 h of treatment with Sinorhizobium meliloti, auxin, or a control. We identified 131 of 270 proteins responding to treatment with S. meliloti and/or auxin, and 39 of 89 proteins differentially displayed between the wild type and sunn. The majority of proteins changed similarly in response to auxin and S. meliloti after 24 h in both genotypes, supporting hypothesis 1. Proteins differentially accumulated between untreated wild-type and sunn roots also showed changes in auxin response, consistent with altered auxin levels in sunn. However, differences between the genotypes after S. meliloti inoculation were largely not due to differential auxin responses. The role of the identified candidate proteins in nodule initiation and the requirement for their induction by auxin could be tested in future functional studies.

  20. Nitric oxide (NO): a key player in the senescence of Medicago truncatula root nodules.

    PubMed

    Cam, Yvan; Pierre, Olivier; Boncompagni, Eric; Hérouart, Didier; Meilhoc, Eliane; Bruand, Claude

    2012-10-01

    Nitric oxide (NO) is a signalling and defence molecule involved in diverse plant developmental processes, as well as in the plant response to pathogens. NO has also been detected at different steps of the symbiosis between legumes and rhizobia. NO is required for an optimal establishment of the Medicago truncatula-Sinorhizobium meliloti symbiotic interaction, but little is known about the role of NO in mature nodules. Here, we investigate the role of NO in the late steps of symbiosis. Genetic and pharmacological approaches were conducted to modulate the NO level inside root nodules, and their effects on nitrogen fixation and root nodule senescence were monitored. An increase in endogenous NO levels led to a decrease in nitrogen fixation and early nodule senescence, characterized by cytological modifications of the nodule structure and the early expression of a specific senescence marker. By contrast, a decrease in NO levels led to a delay in nodule senescence. Together, our results strongly suggest that NO is a signal in developmental as well as stress-induced nodule senescence. In addition, this work demonstrates the pivotal role of the bacterial NO detoxification response in the prevention of early nodule senescence, and hence the maintenance of efficient symbiosis.

  1. Medicago truncatula root nodule proteome analysis reveals differential plant and bacteroid responses to drought stress.

    PubMed

    Larrainzar, Estíbaliz; Wienkoop, Stefanie; Weckwerth, Wolfram; Ladrera, Rubén; Arrese-Igor, Cesar; González, Esther M

    2007-07-01

    Drought is one of the environmental factors most affecting crop production. Under drought, symbiotic nitrogen fixation is one of the physiological processes to first show stress responses in nodulated legumes. This inhibition process involves a number of factors whose interactions are not yet understood. This work aims to further understand changes occurring in nodules under drought stress from a proteomic perspective. Drought was imposed on Medicago truncatula 'Jemalong A17' plants grown in symbiosis with Sinorhizobium meliloti strain 2011. Changes at the protein level were analyzed using a nongel approach based on liquid chromatography coupled to tandem mass spectrometry. Due to the complexity of nodule tissue, the separation of plant and bacteroid fractions in M. truncatula root nodules was first checked with the aim of minimizing cross contamination between the fractions. Second, the protein plant fraction of M. truncatula nodules was profiled, leading to the identification of 377 plant proteins, the largest description of the plant nodule proteome so far. Third, both symbiotic partners were independently analyzed for quantitative differences at the protein level during drought stress. Multivariate data mining allowed for the classification of proteins sets that were involved in drought stress responses. The isolation of the nodule plant and bacteroid protein fractions enabled the independent analysis of the response of both counterparts, gaining further understanding of how each symbiotic member is distinctly affected at the protein level under a water-deficit situation.

  2. Local and systemic proteomic changes in medicago truncatula at an early phase of Sinorhizobium meliloti infection.

    PubMed

    Molesini, Barbara; Cecconi, Daniela; Pii, Youry; Pandolfini, Tiziana

    2014-02-07

    A symbiotic association with N-fixing bacteria facilitates the growth of leguminous plants under nitrogen-limiting conditions. The establishment of the symbiosis requires signal exchange between the host and the bacterium, which leads to the formation of root nodules, inside which bacteria are hosted. The formation of nodules is controlled through local and systemic mechanisms, which involves root-shoot communication. Our study was aimed at investigating the proteomic changes occurring in shoots and concomitantly in roots of Medicago truncatula at an early stage of Sinorhizobium meliloti infection. The principal systemic effects consisted in alteration of chloroplast proteins, induction of proteins responsive to biotic stress, and changes in proteins involved in hormonal signaling and metabolism. The most relevant local effect was the induction of proteins involved in the utilization of photosynthates and C-consuming processes (such as sucrose synthase and fructose-bisphosphate aldolase). In addition, some redox enzymes such as peroxiredoxin and ascorbate peroxidase showed an altered abundance. The analysis of local and systemic proteome changes suggests the occurrence of a stress response in the shoots and the precocious alteration of energy metabolism in roots and shoots. Furthermore, our data indicate the possibility that ABA and ethylene participate in the communicative network between root and shoot in the control of rhizobial infection.

  3. Enhancement of lead uptake by alfalfa (Medicago sativa) using EDTA and a plant growth promoter.

    PubMed

    López, Martha L; Peralta-Videa, Jose R; Benitez, Tenoch; Gardea-Torresdey, Jorge L

    2005-10-01

    Phytoremediation is a novel cleanup technology for the removal of contaminants from polluted waters and soils. In phytoremediation, the plant uptake capability and the availability of the pollutant in the media are important. Here we show the results of a study on the combined effects of ethylenediaminetetraacetic acid (EDTA) and the phytohormone indole-3-acetic acid (IAA) on Pb uptake by Medicago sativa (alfalfa). Plants were grown in hydroponics media containing a nutrient solution amended with Pb at 0.2mM and different combinations of EDTA, and IAA. After 10d of treatment, the Pb content in plant tissues was quantified using an Inductively Coupled Plasma Optical Emission Spectrometer (ICP/OES). The results showed that the combination of 100 microM IAA/0.2 mM EDTA increased the Pb accumulation in leaves by about 2800% and by about 600%, as compared to Pb content in leaves of plants exposed to Pb alone and those cultivated with Pb/EDTA, respectively. These results indicate that non-metal hyperaccumulator plants could increase their hyperaccumulating potential without genetic manipulation.

  4. The Medicago Genome Provides Insight into the Evolution of Rhizobial Symbioses

    PubMed Central

    Young, Nevin D.; Debellé, Frédéric; Oldroyd, Giles E. D.; Geurts, Rene; Cannon, Steven B.; Udvardi, Michael K.; Benedito, Vagner A.; Mayer, Klaus F. X.; Gouzy, Jérôme; Schoof, Heiko; Van de Peer, Yves; Proost, Sebastian; Cook, Douglas R.; Meyers, Blake C.; Spannagl, Manuel; Cheung, Foo; De Mita, Stéphane; Krishnakumar, Vivek; Gundlach, Heidrun; Zhou, Shiguo; Mudge, Joann; Bharti, Arvind K.; Murray, Jeremy D.; Naoumkina, Marina A.; Rosen, Benjamin; Silverstein, Kevin A. T.; Tang, Haibao; Rombauts, Stephane; Zhao, Patrick X.; Zhou, Peng; Barbe, Valérie; Bardou, Philippe; Bechner, Michael; Bellec, Arnaud; Berger, Anne; Bergès, Hélène; Bidwell, Shelby; Bisseling, Ton; Choisne, Nathalie; Couloux, Arnaud; Denny, Roxanne; Deshpande, Shweta; Dai, Xinbin; Doyle, Jeff; Dudez, Anne-Marie; Farmer, Andrew D.; Fouteau, Stéphanie; Franken, Carolien; Gibelin, Chrystel; Gish, John; Goldstein, Steven; González, Alvaro J.; Green, Pamela J.; Hallab, Asis; Hartog, Marijke; Hua, Axin; Humphray, Sean; Jeong, Dong-Hoon; Jing, Yi; Jöcker, Anika; Kenton, Steve M.; Kim, Dong-Jin; Klee, Kathrin; Lai, Hongshing; Lang, Chunting; Lin, Shaoping; Macmil, Simone L; Magdelenat, Ghislaine; Matthews, Lucy; McCorrison, Jamison; Monaghan, Erin L.; Mun, Jeong-Hwan; Najar, Fares Z.; Nicholson, Christine; Noirot, Céline; O’Bleness, Majesta; Paule, Charles R.; Poulain, Julie; Prion, Florent; Qin, Baifang; Qu, Chunmei; Retzel, Ernest F.; Riddle, Claire; Sallet, Erika; Samain, Sylvie; Samson, Nicolas; Sanders, Iryna; Saurat, Olivier; Scarpelli, Claude; Schiex, Thomas; Segurens, Béatrice; Severin, Andrew J.; Sherrier, D. Janine; Shi, Ruihua; Sims, Sarah; Singer, Susan R.; Sinharoy, Senjuti; Sterck, Lieven; Viollet, Agnès; Wang, Bing-Bing; Wang, Keqin; Wang, Mingyi; Wang, Xiaohong; Warfsmann, Jens; Weissenbach, Jean; White, Doug D.; White, Jim D.; Wiley, Graham B.; Wincker, Patrick; Xing, Yanbo; Yang, Limei; Yao, Ziyun; Ying, Fu; Zhai, Jixian; Zhou, Liping; Zuber, Antoine; Dénarié, Jean; Dixon, Richard A.; May, Gregory D.; Schwartz, David C.; Rogers, Jane; Quétier, Francis; Town, Christopher D.; Roe, Bruce A.

    2011-01-01

    Legumes (Fabaceae or Leguminosae) are unique among cultivated plants for their ability to carry out endosymbiotic nitrogen fixation with rhizobial bacteria, a process that takes place in a specialized structure known as the nodule. Legumes belong to one of the two main groups of eurosids, the Fabidae, which includes most species capable of endosymbiotic nitrogen fixation 1. Legumes comprise several evolutionary lineages derived from a common ancestor 60 million years ago (Mya). Papilionoids are the largest clade, dating nearly to the origin of legumes and containing most cultivated species 2. Medicago truncatula (Mt) is a long-established model for the study of legume biology. Here we describe the draft sequence of the Mt euchromatin based on a recently completed BAC-assembly supplemented with Illumina-shotgun sequence, together capturing ~94% of all Mt genes. A whole-genome duplication (WGD) approximately 58 Mya played a major role in shaping the Mt genome and thereby contributed to the evolution of endosymbiotic nitrogen fixation. Subsequent to the WGD, the Mt genome experienced higher levels of rearrangement than two other sequenced legumes, Glycine max (Gm) and Lotus japonicus (Lj). Mt is a close relative of alfalfa (M. sativa), a widely cultivated crop with limited genomics tools and complex autotetraploid genetics. As such, the Mt genome sequence provides significant opportunities to expand alfalfa’s genomic toolbox. PMID:22089132

  5. Identification and Network-Enabled Characterization of Auxin Response Factor Genes in Medicago truncatula

    PubMed Central

    Burks, David J.; Azad, Rajeev K.

    2016-01-01

    The Auxin Response Factor (ARF) family of transcription factors is an important regulator of environmental response and symbiotic nodulation in the legume Medicago truncatula. While previous studies have identified members of this family, a recent spurt in gene expression data coupled with genome update and reannotation calls for a reassessment of the prevalence of ARF genes and their interaction networks in M. truncatula. We performed a comprehensive analysis of the M. truncatula genome and transcriptome that entailed search for novel ARF genes and the co-expression networks. Our investigation revealed 8 novel M. truncatula ARF (MtARF) genes, of the total 22 identified, and uncovered novel gene co-expression networks as well. Furthermore, the topological clustering and single enrichment analysis of several network models revealed the roles of individual members of the MtARF family in nitrogen regulation, nodule initiation, and post-embryonic development through a specialized protein packaging and secretory pathway. In summary, this study not just shines new light on an important gene family, but also provides a guideline for identification of new members of gene families and their functional characterization through network analyses. PMID:28018393

  6. Regulation of Compound Leaf Development by PHANTASTICA in Medicago truncatula1[C][W][OPEN

    PubMed Central

    Ge, Liangfa; Peng, Jianling; Berbel, Ana; Madueño, Francisco; Chen, Rujin

    2014-01-01

    Plant leaves, simple or compound, initiate as peg-like structures from the peripheral zone of the shoot apical meristem, which requires class I KNOTTED-LIKE HOMEOBOXI (KNOXI) transcription factors to maintain its activity. The MYB domain protein encoded by the ASYMMETRIC LEAVES1/ROUGH SHEATH2/PHANTASTICA (ARP) gene, together with other factors, excludes KNOXI gene expression from incipient leaf primordia to initiate leaves and specify leaf adaxial identity. However, the regulatory relationship between ARP and KNOXI is more complex in compound-leafed species. Here, we investigated the role of ARP and KNOXI genes in compound leaf development in Medicago truncatula. We show that the M. truncatula phantastica mutant exhibited severe compound leaf defects, including curling and deep serration of leaf margins, shortened petioles, increased rachises, petioles acquiring motor organ characteristics, and ectopic development of petiolules. On the other hand, the M. truncatula brevipedicellus mutant did not exhibit visible compound leaf defects. Our analyses show that the altered petiole development requires ectopic expression of ELONGATED PETIOLULE1, which encodes a lateral organ boundary domain protein, and that the distal margin serration requires the auxin efflux protein M. truncatula PIN-FORMED10 in the M. truncatula phantastica mutant. PMID:24218492

  7. Mapping the genetic basis of symbiotic variation in legume-rhizobium interactions in Medicago truncatula.

    PubMed

    Gorton, Amanda J; Heath, Katy D; Pilet-Nayel, Marie-Laure; Baranger, Alain; Stinchcombe, John R

    2012-11-01

    Mutualisms are known to be genetically variable, where the genotypes differ in the fitness benefits they gain from the interaction. To date, little is known about the loci that underlie such genetic variation in fitness or whether the loci influencing fitness are partner specific, and depend on the genotype of the interaction partner. In the legume-rhizobium mutualism, one set of potential candidate genes that may influence the fitness benefits of the symbiosis are the plant genes involved in the initiation of the signaling pathway between the two partners. Here we performed quantitative trait loci (QTL) mapping in Medicago truncatula in two different rhizobium strain treatments to locate regions of the genome influencing plant traits, assess whether such regions are dependent on the genotype of the rhizobial mutualist (QTL × rhizobium strain), and evaluate the contribution of sequence variation at known symbiosis signaling genes. Two of the symbiotic signaling genes, NFP and DMI3, colocalized with two QTL affecting average fruit weight and leaf number, suggesting that natural variation in nodulation genes may potentially influence plant fitness. In both rhizobium strain treatments, there were QTL that influenced multiple traits, indicative of either tight linkage between loci or pleiotropy, including one QTL with opposing effects on growth and reproduction. There was no evidence for QTL × rhizobium strain or genotype × genotype interactions, suggesting either that such interactions are due to small-effect loci or that more genotype-genotype combinations need to be tested in future mapping studies.

  8. The RPG gene of Medicago truncatula controls Rhizobium-directed polar growth during infection

    PubMed Central

    Arrighi, Jean-François; Godfroy, Olivier; de Billy, Françoise; Saurat, Olivier; Jauneau, Alain; Gough, Clare

    2008-01-01

    Rhizobia can infect roots of host legume plants and induce new organs called nodules, in which they fix atmospheric nitrogen. Infection generally starts with root hair curling, then proceeds inside newly formed, intracellular tubular structures called infection threads. A successful symbiotic interaction relies on infection threads advancing rapidly at their tips by polar growth through successive cell layers of the root toward developing nodule primordia. To identify a plant component that controls this tip growth process, we characterized a symbiotic mutant of Medicago truncatula, called rpg for rhizobium-directed polar growth. In this mutant, nitrogen-fixing nodules were rarely formed due to abnormally thick and slowly progressing infection threads. Root hair curling was also abnormal, indicating that the RPG gene fulfils an essential function in the process whereby rhizobia manage to dominate the process of induced tip growth for root hair infection. Map-based cloning of RPG revealed a member of a previously unknown plant-specific gene family encoding putative long coiled-coil proteins we have called RRPs (RPG-related proteins) and characterized by an “RRP domain” specific to this family. RPG expression was strongly associated with rhizobial infection, and the RPG protein showed a nuclear localization, indicating that this symbiotic gene constitutes an important component of symbiotic signaling. PMID:18621693

  9. The RPG gene of Medicago truncatula controls Rhizobium-directed polar growth during infection.

    PubMed

    Arrighi, Jean-François; Godfroy, Olivier; de Billy, Françoise; Saurat, Olivier; Jauneau, Alain; Gough, Clare

    2008-07-15

    Rhizobia can infect roots of host legume plants and induce new organs called nodules, in which they fix atmospheric nitrogen. Infection generally starts with root hair curling, then proceeds inside newly formed, intracellular tubular structures called infection threads. A successful symbiotic interaction relies on infection threads advancing rapidly at their tips by polar growth through successive cell layers of the root toward developing nodule primordia. To identify a plant component that controls this tip growth process, we characterized a symbiotic mutant of Medicago truncatula, called rpg for rhizobium-directed polar growth. In this mutant, nitrogen-fixing nodules were rarely formed due to abnormally thick and slowly progressing infection threads. Root hair curling was also abnormal, indicating that the RPG gene fulfils an essential function in the process whereby rhizobia manage to dominate the process of induced tip growth for root hair infection. Map-based cloning of RPG revealed a member of a previously unknown plant-specific gene family encoding putative long coiled-coil proteins we have called RRPs (RPG-related proteins) and characterized by an "RRP domain" specific to this family. RPG expression was strongly associated with rhizobial infection, and the RPG protein showed a nuclear localization, indicating that this symbiotic gene constitutes an important component of symbiotic signaling.

  10. Concerted changes in N and C primary metabolism in alfalfa (Medicago sativa) under water restriction

    PubMed Central

    Aranjuelo, Iker

    2013-01-01

    Although the mechanisms of nodule N2 fixation in legumes are now well documented, some uncertainty remains on the metabolic consequences of water deficit. In most cases, little consideration is given to other organs and, therefore, the coordinated changes in metabolism in leaves, roots, and nodules are not well known. Here, the effect of water restriction on exclusively N2-fixing alfalfa (Medicago sativa L.) plants was investigated, and proteomic, metabolomic, and physiological analyses were carried out. It is shown that the inhibition of nitrogenase activity caused by water restriction was accompanied by concerted alterations in metabolic pathways in nodules, leaves, and roots. The data suggest that nodule metabolism and metabolic exchange between plant organs nearly reached homeostasis in asparagine synthesis and partitioning, as well as the N demand from leaves. Typically, there was (i) a stimulation of the anaplerotic pathway to sustain the provision of C skeletons for amino acid (e.g. glutamate and proline) synthesis; (ii) re-allocation of glycolytic products to alanine and serine/glycine; and (iii) subtle changes in redox metabolites suggesting the implication of a slight oxidative stress. Furthermore, water restriction caused little change in both photosynthetic efficiency and respiratory cost of N2 fixation by nodules. In other words, the results suggest that under water stress, nodule metabolism follows a compromise between physiological imperatives (N demand, oxidative stress) and the lower input to sustain catabolism. PMID:23440170

  11. Medicago truncatula Zinc-Iron Permease6 provides zinc to rhizobia-infected nodule cells.

    PubMed

    Abreu, Isidro; Saéz, Ángela; Castro-Rodríguez, Rosario; Escudero, Viviana; Rodríguez-Haas, Benjamín; Senovilla, Marta; Larue, Camille; Grolimund, Daniel; Tejada-Jiménez, Manuel; Imperial, Juan; González-Guerrero, Manuel

    2017-07-21

    Zinc is a micronutrient required for symbiotic nitrogen fixation. It has been proposed that in model legume Medicago truncatula, zinc is delivered by the root vasculature into the nodule and released in the infection/differentiation zone. There, transporters must introduce this element into rhizobia-infected cells to metallate the apoproteins that use zinc as a cofactor. MtZIP6 (Medtr4g083570) is a M. truncatula Zinc-Iron Permease (ZIP) that is expressed only in roots and nodules, with the highest expression levels in the infection/differentiation zone. Immunolocalization studies indicate that it is located in the plasma membrane of nodule rhizobia-infected cells. Down-regulating MtZIP6 expression levels with RNAi does not result in any strong phenotype when plants are fed mineral nitrogen. However, these plants displayed severe growth defects when they depended on nitrogen fixed by their nodules, losing of 80% of their nitrogenase activity. The reduction of this activity was likely an indirect effect of zinc being retained in the infection/differentiation zone and not reaching the cytosol of rhizobia-infected cells. These data are consistent with a model in which MtZIP6 would be responsible for zinc uptake by rhizobia-infected nodule cells in the infection/differentiation zone. This article is protected by copyright. All rights reserved.

  12. Exploring root symbiotic programs in the model legume Medicago truncatula using EST analysis

    PubMed Central

    Journet, Etienne-Pascal; van Tuinen, Diederik; Gouzy, Jérome; Crespeau, Hervé; Carreau, Véronique; Farmer, Mary-Jo; Niebel, Andreas; Schiex, Thomas; Jaillon, Olivier; Chatagnier, Odile; Godiard, Laurence; Micheli, Fabienne; Kahn, Daniel; Gianinazzi-Pearson, Vivienne; Gamas, Pascal

    2002-01-01

    We report on a large-scale expressed sequence tag (EST) sequencing and analysis program aimed at characterizing the sets of genes expressed in roots of the model legume Medicago truncatula during interactions with either of two microsymbionts, the nitrogen-fixing bacterium Sinorhizobium meliloti or the arbuscular mycorrhizal fungus Glomus intraradices. We have designed specific tools for in silico analysis of EST data, in relation to chimeric cDNA detection, EST clustering, encoded protein prediction, and detection of differential expression. Our 21 473 5′- and 3′-ESTs could be grouped into 6359 EST clusters, corresponding to distinct virtual genes, along with 52 498 other M.truncatula ESTs available in the dbEST (NCBI) database that were recruited in the process. These clusters were manually annotated, using a specifically developed annotation interface. Analysis of EST cluster distribution in various M.truncatula cDNA libraries, supported by a refined R test to evaluate statistical significance and by ‘electronic northern’ representation, enabled us to identify a large number of novel genes predicted to be up- or down-regulated during either symbiotic root interaction. These in silico analyses provide a first global view of the genetic programs for root symbioses in M.truncatula. A searchable database has been built and can be accessed through a public interface. PMID:12490726

  13. A Medicago truncatula phosphate transporter indispensable for the arbuscular mycorrhizal symbiosis.

    PubMed

    Javot, Hélène; Penmetsa, R Varma; Terzaghi, Nadia; Cook, Douglas R; Harrison, Maria J

    2007-01-30

    The arbuscular mycorrhizal (AM) symbiosis is a mutualistic endosymbiosis formed by plant roots and AM fungi. Most vascular flowering plants have the ability to form these associations, which have a significant impact on plant health and consequently on ecosystem function. Nutrient exchange is a central feature of the AM symbiosis, and AM fungi obtain carbon from their plant host while assisting the plant with the acquisition of phosphorus (as phosphate) from the soil. In the AM symbiosis, the fungus delivers P(i) to the root through specialized hyphae called arbuscules. The molecular mechanisms of P(i) and carbon transfer in the symbiosis are largely unknown, as are the mechanisms by which the plant regulates the symbiosis in response to its nutrient status. Plants possess many classes of P(i) transport proteins, including a unique clade (Pht1, subfamily I), members of which are expressed only in the AM symbiosis. Here, we show that MtPT4, a Medicago truncatula member of subfamily I, is essential for the acquisition of P(i) delivered by the AM fungus. However, more significantly, MtPT4 function is critical for AM symbiosis. Loss of MtPT4 function leads to premature death of the arbuscules; the fungus is unable to proliferate within the root, and symbiosis is terminated. Thus, P(i) transport is not only a benefit for the plant but is also a requirement for the AM symbiosis.

  14. Comparative physical mapping reveals features of microsynteny between Glycine max, Medicago truncatula, and Arabidopsis thaliana.

    PubMed

    Yan, H H; Mudge, J; Kim, D-J; Shoemaker, R C; Cook, D R; Young, N D

    2004-02-01

    To gain insight into genomic relationships between soybean (Glycine max) and Medicago truncatula, eight groups of bacterial artificial chromosome (BAC) contigs, together spanning 2.60 million base pairs (Mb) in G. max and 1.56 Mb in M. truncatula, were compared through high-resolution physical mapping combined with sequence and hybridization analysis of low-copy BAC ends. Cross-hybridization among G. max and M. truncatula contigs uncovered microsynteny in six of the contig groups and extensive microsynteny in three. Between G. max homoeologous (within genome duplicate) contigs, 85% of coding and 75% of noncoding sequences were conserved at the level of cross-hybridization. By contrast, only 29% of sequences were conserved between G. max and M. truncatula, and some kilobase-scale rearrangements were also observed. Detailed restriction maps were constructed for 11 contigs from the three highly microsyntenic groups, and these maps suggested that sequence order was highly conserved between G. max duplicates and generally conserved between G. max and M. truncatula. One instance of homoeologous BAC contigs in M. truncatula was also observed and examined in detail. A sequence similarity search against the Arabidopsis thaliana genome sequence identified up to three microsyntenic regions in A. thaliana for each of two of the legume BAC contig groups. Together, these results confirm previous predictions of one recent genome-wide duplication in G. max and suggest that M. truncatula also experienced ancient large-scale genome duplications.

  15. Identification and Network-Enabled Characterization of Auxin Response Factor Genes in Medicago truncatula.

    PubMed

    Burks, David J; Azad, Rajeev K

    2016-01-01

    The Auxin Response Factor (ARF) family of transcription factors is an important regulator of environmental response and symbiotic nodulation in the legume Medicago truncatula. While previous studies have identified members of this family, a recent spurt in gene expression data coupled with genome update and reannotation calls for a reassessment of the prevalence of ARF genes and their interaction networks in M. truncatula. We performed a comprehensive analysis of the M. truncatula genome and transcriptome that entailed search for novel ARF genes and the co-expression networks. Our investigation revealed 8 novel M. truncatula ARF (MtARF) genes, of the total 22 identified, and uncovered novel gene co-expression networks as well. Furthermore, the topological clustering and single enrichment analysis of several network models revealed the roles of individual members of the MtARF family in nitrogen regulation, nodule initiation, and post-embryonic development through a specialized protein packaging and secretory pathway. In summary, this study not just shines new light on an important gene family, but also provides a guideline for identification of new members of gene families and their functional characterization through network analyses.

  16. Nitrogen Fixation, Nodule Development, and Vegetative Regrowth of Alfalfa (Medicago sativa L.) following Harvest 1

    PubMed Central

    Vance, Carroll P.; Heichel, Gary H.; Barnes, Donald K.; Bryan, Jeff W.; Johnson, Lois E.

    1979-01-01

    Nitrogenase-dependent acetylene reduction, nodule function, and nodule regrowth were studied during vegetative regrowth of harvested (detopped) alfalfa (Medicago sativa L.) seedlings grown in the glasshouse. Compared with controls, harvesting caused an 88% decline in acetylene reduction capacity of detached root systems within 24 hours. Acetylene reduction in harvested plants remained low for 13 days, then increased to a level comparable to the controls by day 18. Protease activity increased in nodules from harvested plants, reached a maximum at day 7 after harvest, and then declined to a level almost equal to the control by day 22 after harvest. Soluble protein and leghemoglobin decreased in nodules from harvested plants in an inverse relationship to protease activity. Nitrate reductase activity of nodules from harvested plants increased significantly within 24 hours and was inversely associated with acetylene reduction. The difference in nitrate reductase between nodules from harvested plants and control plants became less evident as shoot regrowth occurred and as acetylene reduction increased in the harvested plants. No massive loss of nodules occurred after harvest as evidenced by little net change in nodule fresh weight. There was, however, a rapid localized senescence which occurred in nodules of harvested plants. Histology of nodules from harvested plants showed that they degenerated at the proximal end after harvest. Starch in the nodule was depleted by 10 days after harvest. The meristem and vascular bundles of nodules from harvested plants remained intact. The senescent nodules began to regrow and fix nitrogen after shoot growth resumed. Images PMID:16660893

  17. Legume adaptation to sulfur deficiency revealed by comparing nutrient allocation and seed traits in Medicago truncatula.

    PubMed

    Zuber, Hélène; Poignavent, Germain; Le Signor, Christine; Aimé, Delphine; Vieren, Eric; Tadla, Charlène; Lugan, Raphaël; Belghazi, Maya; Labas, Valérie; Santoni, Anne-Lise; Wipf, Daniel; Buitink, Julia; Avice, Jean-Christophe; Salon, Christophe; Gallardo, Karine

    2013-12-01

    Reductions in sulfur dioxide emissions and the use of sulfur-free mineral fertilizers are decreasing soil sulfur levels and threaten the adequate fertilization of most crops. To provide knowledge regarding legume adaptation to sulfur restriction, we subjected Medicago truncatula, a model legume species, to sulfur deficiency at various developmental stages, and compared the yield, nutrient allocation and seed traits. This comparative analysis revealed that sulfur deficiency at the mid-vegetative stage decreased yield and altered the allocation of nitrogen and carbon to seeds, leading to reduced levels of major oligosaccharides in mature seeds, whose germination was dramatically affected. In contrast, during the reproductive period, sulfur deficiency had little influence on yield and nutrient allocation, but the seeds germinated slowly and were characterized by low levels of a biotinylated protein, a putative indicator of germination vigor that has not been previously related to sulfur nutrition. Significantly, plants deprived of sulfur at an intermediary stage (flowering) adapted well by remobilizing nutrients from source organs to seeds, ensuring adequate quantities of carbon and nitrogen in seeds. This efficient remobilization of photosynthates may be explained by vacuolar sulfate efflux to maintain leaf metabolism throughout reproductive growth, as suggested by transcript and metabolite profiling. The seeds from these plants, deprived of sulfur at the floral transition, contained normal levels of major oligosaccharides but their germination was delayed, consistent with low levels of sucrose and the glycolytic enzymes required to restart seed metabolism during imbibition. Overall, our findings provide an integrative view of the legume response to sulfur deficiency.

  18. NODULES WITH ACTIVATED DEFENSE 1 is required for maintenance of rhizobial endosymbiosis in Medicago truncatula.

    PubMed

    Wang, Chao; Yu, Haixiang; Luo, Li; Duan, Liujian; Cai, Liuyang; He, Xinxing; Wen, Jiangqi; Mysore, Kirankumar S; Li, Guoliang; Xiao, Aifang; Duanmu, Deqiang; Cao, Yangrong; Hong, Zonglie; Zhang, Zhongming

    2016-10-01

    The symbiotic interaction between legume plants and rhizobia results in the formation of root nodules, in which symbiotic plant cells host and harbor thousands of nitrogen-fixing rhizobia. Here, a Medicago truncatula nodules with activated defense 1 (nad1) mutant was identified using reverse genetics methods. The mutant phenotype was characterized using cell and molecular biology approaches. An RNA-sequencing technique was used to analyze the transcriptomic reprogramming of nad1 mutant nodules. In the nad1 mutant plants, rhizobial infection and propagation in infection threads are normal, whereas rhizobia and their symbiotic plant cells become necrotic immediately after rhizobia are released from infection threads into symbiotic cells of nodules. Defense-associated responses were detected in nad1 nodules. NAD1 is specifically present in root nodule symbiosis plants with the exception of Morus notabilis, and the transcript is highly induced in nodules. NAD1 encodes a small uncharacterized protein with two predicted transmembrane helices and is localized at the endoplasmic reticulum. Our data demonstrate a positive role for NAD1 in the maintenance of rhizobial endosymbiosis during nodulation.

  19. Two Direct Targets of Cytokinin Signaling Regulate Symbiotic Nodulation in Medicago truncatula[W][OA

    PubMed Central

    Ariel, Federico; Brault-Hernandez, Marianne; Laffont, Carole; Huault, Emeline; Brault, Mathias; Plet, Julie; Moison, Michael; Blanchet, Sandrine; Ichanté, Jean Laurent; Chabaud, Mireille; Carrere, Sébastien; Crespi, Martin; Chan, Raquel L.; Frugier, Florian

    2012-01-01

    Cytokinin regulates many aspects of plant development, and in legume crops, this phytohormone is necessary and sufficient for symbiotic nodule organogenesis, allowing them to fix atmospheric nitrogen. To identify direct links between cytokinins and nodule organogenesis, we determined a consensus sequence bound in vitro by a transcription factor (TF) acting in cytokinin signaling, the nodule-enhanced Medicago truncatula Mt RR1 response regulator (RR). Among genes rapidly regulated by cytokinins and containing this so-called RR binding site (RRBS) in their promoters, we found the nodulation-related Type-A RR Mt RR4 and the Nodulation Signaling Pathway 2 (NSP2) TF. Site-directed mutagenesis revealed that RRBS cis-elements in the RR4 and NSP2 promoters are essential for expression during nodule development and for cytokinin induction. Furthermore, a microRNA targeting NSP2 (miR171 h) is also rapidly induced by cytokinins and then shows an expression pattern anticorrelated with NSP2. Other primary targets regulated by cytokinins depending on the Cytokinin Response1 (CRE1) receptor were a cytokinin oxidase/dehydrogenase (CKX1) and a basic Helix-Loop-Helix TF (bHLH476). RNA interference constructs as well as insertion of a Tnt1 retrotransposon in the bHLH gene led to reduced nodulation. Hence, we identified two TFs, NSP2 and bHLH476, as direct cytokinin targets acting at the convergence of phytohormonal and symbiotic cues. PMID:23023168

  20. Molecular Signals Controlling the Inhibition of Nodulation by Nitrate in Medicago truncatula.

    PubMed

    van Noorden, Giel E; Verbeek, Rob; Dinh, Quy Dung; Jin, Jian; Green, Alexandra; Ng, Jason Liang Pin; Mathesius, Ulrike

    2016-07-02

    The presence of nitrogen inhibits legume nodule formation, but the mechanism of this inhibition is poorly understood. We found that 2.5 mM nitrate and above significantly inhibited nodule initiation but not root hair curling in Medicago trunatula. We analyzed protein abundance in M. truncatula roots after treatment with either 0 or 2.5 mM nitrate in the presence or absence of its symbiont Sinorhizobium meliloti after 1, 2 and 5 days following inoculation. Two-dimensional gel electrophoresis combined with mass spectrometry was used to identify 106 differentially accumulated proteins responding to nitrate addition, inoculation or time point. While flavonoid-related proteins were less abundant in the presence of nitrate, addition of Nod gene-inducing flavonoids to the Sinorhizobium culture did not rescue nodulation. Accumulation of auxin in response to rhizobia, which is also controlled by flavonoids, still occurred in the presence of nitrate, but did not localize to a nodule initiation site. Several of the changes included defense- and redox-related proteins, and visualization of reactive oxygen species indicated that their induction in root hairs following Sinorhizobium inoculation was inhibited by nitrate. In summary, the presence of nitrate appears to inhibit nodulation via multiple pathways, including changes to flavonoid metabolism, defense responses and redox changes.

  1. Role of LONELY GUY genes in indeterminate nodulation on Medicago truncatula.

    PubMed

    Mortier, Virginie; Wasson, Anton; Jaworek, Pavel; De Keyser, Annick; Decroos, Martijn; Holsters, Marcelle; Tarkowski, Petr; Mathesius, Ulrike; Goormachtig, Sofie

    2014-04-01

    LONELY GUY (LOG) genes encode cytokinin riboside 5'-monophosphate phosphoribohydrolases and are directly involved in the activation of cytokinins. To assess whether LOG proteins affect the influence of cytokinin on nodulation, we studied two LOG genes of Medicago truncatula. Expression analysis showed that MtLOG1 and MtLOG2 were upregulated during nodulation in a CRE1-dependent manner. Expression was mainly localized in the dividing cells of the nodule primordium. In addition, RNA interference revealed that MtLOG1 is involved in nodule development and that the gene plays a negative role in lateral root development. Ectopic expression of MtLOG1 resulted in a change in cytokinin homeostasis, triggered cytokinin-inducible genes and produced roots with enlarged vascular tissues and shortened primary roots. In addition, those 35S:LOG1 roots also displayed fewer nodules than the wild-type. This inhibition in nodule formation was local, independent of the SUPER NUMERIC NODULES gene, but coincided with an upregulation of the MtCLE13 gene, encoding a CLAVATA3/EMBRYO SURROUNDING REGION peptide. In conclusion, we demonstrate that in M. truncatula LOG proteins might be implicated in nodule primordium development and lateral root formation. © 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.

  2. Abscisic acid coordinates nod factor and cytokinin signaling during the regulation of nodulation in Medicago truncatula.

    PubMed

    Ding, Yiliang; Kalo, Peter; Yendrek, Craig; Sun, Jongho; Liang, Yan; Marsh, John F; Harris, Jeanne M; Oldroyd, Giles E D

    2008-10-01

    Nodulation is tightly regulated in legumes to ensure appropriate levels of nitrogen fixation without excessive depletion of carbon reserves. This balance is maintained by intimately linking nodulation and its regulation with plant hormones. It has previously been shown that ethylene and jasmonic acid (JA) are able to regulate nodulation and Nod factor signal transduction. Here, we characterize the nature of abscisic acid (ABA) regulation of nodulation. We show that application of ABA inhibits nodulation, bacterial infection, and nodulin gene expression in Medicago truncatula. ABA acts in a similar manner as JA and ethylene, regulating Nod factor signaling and affecting the nature of Nod factor-induced calcium spiking. However, this action is independent of the ethylene signal transduction pathway. We show that genetic inhibition of ABA signaling through the use of a dominant-negative allele of ABSCISIC ACID INSENSITIVE1 leads to a hypernodulation phenotype. In addition, we characterize a novel locus of M. truncatula, SENSITIVITY TO ABA, that dictates the sensitivity of the plant to ABA and, as such, impacts the regulation of nodulation. We show that ABA can suppress Nod factor signal transduction in the epidermis and can regulate cytokinin induction of the nodule primordium in the root cortex. Therefore, ABA is capable of coordinately regulating the diverse developmental pathways associated with nodule formation and can intimately dictate the nature of the plants' response to the symbiotic bacteria.

  3. Concerted changes in N and C primary metabolism in alfalfa (Medicago sativa) under water restriction.

    PubMed

    Aranjuelo, Iker; Tcherkez, Guillaume; Molero, Gemma; Gilard, Françoise; Avice, Jean-Christophe; Nogués, Salvador

    2013-02-01

    Although the mechanisms of nodule N(2) fixation in legumes are now well documented, some uncertainty remains on the metabolic consequences of water deficit. In most cases, little consideration is given to other organs and, therefore, the coordinated changes in metabolism in leaves, roots, and nodules are not well known. Here, the effect of water restriction on exclusively N(2)-fixing alfalfa (Medicago sativa L.) plants was investigated, and proteomic, metabolomic, and physiological analyses were carried out. It is shown that the inhibition of nitrogenase activity caused by water restriction was accompanied by concerted alterations in metabolic pathways in nodules, leaves, and roots. The data suggest that nodule metabolism and metabolic exchange between plant organs nearly reached homeostasis in asparagine synthesis and partitioning, as well as the N demand from leaves. Typically, there was (i) a stimulation of the anaplerotic pathway to sustain the provision of C skeletons for amino acid (e.g. glutamate and proline) synthesis; (ii) re-allocation of glycolytic products to alanine and serine/glycine; and (iii) subtle changes in redox metabolites suggesting the implication of a slight oxidative stress. Furthermore, water restriction caused little change in both photosynthetic efficiency and respiratory cost of N(2) fixation by nodules. In other words, the results suggest that under water stress, nodule metabolism follows a compromise between physiological imperatives (N demand, oxidative stress) and the lower input to sustain catabolism.

  4. Examination of Endogenous Peptides in Medicago truncatula Using Mass Spectrometry Imaging.

    PubMed

    Gemperline, Erin; Keller, Caitlin; Jayaraman, Dhileepkumar; Maeda, Junko; Sussman, Michael R; Ané, Jean-Michel; Li, Lingjun

    2016-12-02

    Plant science is an important, rapidly developing area of study. Within plant science, one area of study that has grown tremendously with recent technological advances, such as mass spectrometry, is the field of plant-omics; however, plant peptidomics is relatively underdeveloped in comparison with proteomics and metabolomics. Endogenous plant peptides can act as signaling molecules and have been shown to affect cell division, development, nodulation, reproduction, symbiotic associations, and defense reactions. There is a growing need to uncover the role of endogenous peptides on a molecular level. Mass spectrometric imaging (MSI) is a valuable tool for biological analyses as it allows for the detection of thousands of analytes in a single experiment and also displays spatial information for the detected analytes. Despite the prediction of a large number of plant peptides, their detection and imaging with spatial localization and chemical specificity is currently lacking. Here we analyzed the endogenous peptides and proteins in Medicago truncatula using matrix-assisted laser desorption/ionization (MALDI)-MSI. Hundreds of endogenous peptides and protein fragments were imaged, with interesting peptide spatial distribution changes observed between plants in different developmental stages.

  5. MAP Kinase-Mediated Negative Regulation of Symbiotic Nodule Formation in Medicago truncatula

    PubMed Central

    Ryu, Hojin; Laffont, Carole; Frugier, Florian; Hwang, Ildoo

    2017-01-01

    Mitogen-activated protein kinase (MAPK) signaling cascades play critical roles in various cellular events in plants, including stress responses, innate immunity, hormone signaling, and cell specificity. MAPK-mediated stress signaling is also known to negatively regulate nitrogen-fixing symbiotic interactions, but the molecular mechanism of the MAPK signaling cascades underlying the symbiotic nodule development remains largely unknown. We show that the MtMKK5-MtMPK3/6 signaling module negatively regulates the early symbiotic nodule formation, probably upstream of ERN1 (ERF Required for Nodulation 1) and NSP1 (Nod factor Signaling Pathway 1) in Medicago truncatula. The overexpression of MtMKK5 stimulated stress and defense signaling pathways but also reduced nodule formation in M. truncatula roots. Conversely, a MAPK specific inhibitor, U0126, enhanced nodule formation and the expression of an early nodulation marker gene, MtNIN. We found that MtMKK5 directly activates MtMPK3/6 by phosphorylating the TEY motif within the activation loop and that the MtMPK3/6 proteins physically interact with the early nodulation-related transcription factors ERN1 and NSP1. These data suggest that the stress signaling-mediated MtMKK5/MtMPK3/6 module suppresses symbiotic nodule development via the action of early nodulation transcription factors. PMID:28152300

  6. Expression Analysis of PIN Genes in Root Tips and Nodules of Medicago truncatula.

    PubMed

    Sańko-Sawczenko, Izabela; Łotocka, Barbara; Czarnocka, Weronika

    2016-07-25

    Polar auxin transport is dependent on the family of PIN-formed proteins (PINs), which are membrane transporters of anionic indole-3-acetic acid (IAA(-)). It is assumed that polar auxin transport may be essential in the development and meristematic activity maintenance of Medicago truncatula (M. truncatula) root nodules. However, little is known about the involvement of specific PIN proteins in M. truncatula nodulation. Using real-time quantitative PCR, we analyzed the expression patterns of all previously identified MtPIN genes and compared them between root nodules and root tips of M. truncatula. Our results demonstrated significant differences in the expression level of all 11 genes (MtPIN1-MtPIN11) between examined organs. Interestingly, MtPIN9 was the only PIN gene with higher expression level in root nodules compared to root tips. This result is the first indication of PIN9 transporter potential involvement in M. truncatula nodulation. Moreover, relatively high expression level in root nodules was attributed to MtPINs encoding orthologs of Arabidopsis thaliana PIN5 subclade. PIN proteins from this subclade have been found to localize in the endoplasmic reticulum, which may indicate that the development and meristematic activity maintenance of M. truncatula root nodules is associated with intracellular homeostasis of auxins level and their metabolism in the endoplasmic reticulum.

  7. A combined histology and transcriptome analysis unravels novel questions on Medicago truncatula seed coat

    PubMed Central

    Abirached-Darmency, Mona

    2013-01-01

    The seed coat is involved in the determination of seed quality traits such as seed size, seed composition, seed permeability, and hormonal regulation. Understanding seed coat structure is therefore a prerequisite to deciphering the genetic mechanisms that govern seed coat functions. By combining histological and transcriptomic data analyses, cellular and molecular events occurring during Medicago truncatula seed coat development were dissected in order to relate structure to function and pinpoint target genes potentially involved in seed coat traits controlling final seed quality traits. The analyses revealed the complexity of the seed coat transcriptome, which contains >30 000 genes. In parallel, a set of genes showing a preferential expression in seed coat that may be involved in more specific functions was identified. The study describes how seed coat anatomy and morphological changes affect final seed quality such as seed size, seed composition, seed permeability, and hormonal regulation. Putative regulator genes of different processes have been identified as potential candidates for further functional genomic studies to improve agronomical seed traits. The study also raises new questions concerning the implication of seed coat endopolyploidy in cell expansion and the participation of the seed coat in de novo abscisic acid biosynthesis at early seed filling. PMID:23125357

  8. Complete genome sequence of the Medicago microsymbiont Ensifer (Sinorhizobium) medicae strain WSM419

    SciTech Connect

    Reeve, Wayne; Chain, Patrick S. G.; O'Hara, Graham; Ardley, Julie; Nandesena, Kemanthi; Brau, Lambert; Tiwari, Ravi; Malfatti, Stephanie; Kiss, Hajnalka; Lapidus, Alla L.; Copeland, A; Nolan, Matt; Land, Miriam L; Hauser, Loren John; Chang, Yun-Juan; Ivanova, N; Mavromatis, K; Markowitz, Victor; Kyrpides, Nikos C; Gollagher, Margaret; Yates, Ron; Dilworth, Michael; Howieson, John

    2010-01-01

    Ensifer (Sinorhizobium) medicae is an effective nitrogen fixing microsymbiont of a diverse range of annual Medicago (medic) species. Strain WSM419 is an aerobic, motile, non-spore forming, Gram-negative rod isolated from a M. murex root nodule collected in Sardinia, Italy in 1981. WSM419 was manufactured commercially in Australia as an inoculant for annual medics during 1985 to 1993 due to its nitrogen fixation, saprophytic competence and acid tolerance properties. Here we describe the basic features of this organism, together with the complete genome sequence, and annotation. This is the first report of a complete genome sequence for a microsymbiont of the group of annual medic species adapted to acid soils. We reveal that its genome size is 6,817,576 bp encoding 6,518 protein-coding genes and 81 RNA only encoding genes. The genome contains a chromosome of size 3,781,904 bp and 3 plasmids of size 1,570,951 bp, 1,245,408 bp and 219,313 bp. The smallest plasmid is a feature unique to this medic microsymbiont.

  9. A Standardized Method for Analysis of Medicago truncatula Phenotypic Development1[W][OA

    PubMed Central

    Bucciarelli, Bruna; Hanan, Jim; Palmquist, Debra; Vance, Carroll P.

    2006-01-01

    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. PMID:16877701

  10. A Medicago truncatula mutant hyper-responsive to mycorrhiza and defective for nodulation.

    PubMed

    Morandi, Dominique; le Signor, Christine; Gianinazzi-Pearson, Vivienne; Duc, Gérard

    2009-08-01

    One key strategy for the identification of plant genes required for mycorrhizal development is the use of plant mutants affected in mycorrhizal colonisation. In this paper, we report a new Medicago truncatula mutant defective for nodulation but hypermycorrhizal for symbiosis development and response. This mutant, called B9, presents a poor shoot and, especially, root development with short laterals. Inoculation with Glomus intraradices results in significantly higher root colonisation of the mutant than the wild-type genotype A17 (+20% for total root length, +16% for arbuscule frequency in the colonised part of the root, +39% for arbuscule frequency in the total root system). Mycorrhizal effects on shoot and root biomass of B9 plants are about twofold greater than in the wild-type genotype. The B9 mutant of M. truncatula is characterised by considerably higher root concentrations of the phytoestrogen coumestrol and by the novel synthesis of the coumestrol conjugate malonyl glycoside, absent from roots of wild-type plants. In conclusion, this is the first time that a hypermycorrhizal plant mutant affected negatively for nodulation (Myc(++), Nod (-/+) phenotype) is reported. This mutant represents a new tool for the study of plant genes differentially regulating mycorrhiza and nodulation symbioses, in particular, those related to autoregulation mechanisms.

  11. Medicago truncatula gene responses specific to arbuscular mycorrhiza interactions with different species and genera of Glomeromycota.

    PubMed

    Massoumou, M; van Tuinen, D; Chatagnier, O; Arnould, C; Brechenmacher, L; Sanchez, L; Selim, S; Gianinazzi, S; Gianinazzi-Pearson, V

    2007-05-01

    Plant genes exhibiting common responses to different arbuscular mycorrhizal (AM) fungi and not induced under other biological conditions have been sought for to identify specific markers for monitoring the AM symbiosis. A subset of 14 candidate Medicago truncatula genes was identified as being potentially mycorrhiza responsive in previous cDNA microarray analyses and exclusive to cDNA libraries derived from mycorrhizal root tissues. Transcriptional activity of the selected plant genes was compared during root interactions with seven AM fungi belonging to different species of Glomus, Acaulospora, Gigaspora, or Scutellospora, and under widely different biological conditions (mycorrhiza, phosphate fertilization, pathogenic/beneficial microbe interactions, incompatible plant genotype). Ten of the M. truncatula genes were commonly induced by all the tested AM fungal species, and all were activated by at least two fungi. Most of the plant genes were transcribed uniquely in mycorrhizal roots, and several were already active at the appressorium stage of fungal development. Novel data provide evidence that common recognition responses to phylogenetically different Glomeromycota exist in plants during events that are unique to mycorrhiza interactions. They indicate that plants should possess a mycorrhiza-specific genetic program which is comodulated by a broad spectrum of AM fungi.

  12. Early somatic embryo induction events in alfalfa callus cultures. [Medicago sativa

    SciTech Connect

    El-Bakry, A.A.; Hildebrand, D.F.

    1987-04-01

    High and low regenerating alfalfa Medicago sativa L. cv Regen S full sibs were isolated from a callus culture screen on modified Blaydes medium. The average number of embryos per ovary were thirty and zero for the high and low genotypes respectively after six weeks in culture. Proembryonic cell masses (4-8 celled) were observed after 4-5 days in culture and maximum meristematic activity was at 6-7 days in culture, for the high regenerating genotypes. Well formed globular embryos, both epidermal and subepidermal in origin, were observed after 2 weeks is culture. Samples in culture for 3, 6 and 14 days from the high and low regenerating genotypes were radiolabeled in vivo with /sup 35/S-methionine and run both on one and two dimension gels. The results will be discussed in relation to differences in proteins between the high and low regenerating genotypes at the stage of maximum meristematic activity (day 6) and differences occurring relative to the appearance of globular stage embryos (day 14) will be presented.

  13. The Mechanics of Surface Expansion Anisotropy in Medicago truncatula Root Hairs1

    PubMed Central

    Dumais, Jacques; Long, Sharon R.; Shaw, Sidney L.

    2004-01-01

    Wall expansion in tip-growing cells shows variations according to position and direction. In Medicago truncatula root hairs, wall expansion exhibits a strong meridional gradient with a maximum near the pole of the cell. Root hair cells also show a striking expansion anisotropy, i.e. over most of the dome surface the rate of circumferential wall expansion exceeds the rate of meridional expansion. Concomitant measurements of expansion rates and wall stresses reveal that the extensibility of the cell wall must vary abruptly along the meridian of the cell to maintain the gradient of wall expansion. To determine the mechanical basis of expansion anisotropy, we compared measurements of wall expansion with expansion patterns predicted from wall structural models that were either fully isotropic, transversely isotropic, or fully anisotropic. Our results indicate that a model based on a transversely isotropic wall structure can provide a good fit of the data although a fully anisotropic model offers the best fit overall. We discuss how such mechanical properties could be controlled at the microstructural level. PMID:15448192

  14. Comparative transcriptomic analysis of salt adaptation in roots of contrasting Medicago truncatula genotypes.

    PubMed

    Zahaf, Ons; Blanchet, Sandrine; de Zélicourt, Axel; Alunni, Benoît; Plet, Julie; Laffont, Carole; de Lorenzo, Laura; Imbeaud, Sandrine; Ichanté, Jean-Laurent; Diet, Anouck; Badri, Mounawer; Zabalza, Ana; González, Esther M; Delacroix, Hervé; Gruber, Véronique; Frugier, Florian; Crespi, Martin

    2012-09-01

    Evolutionary diversity can be driven by the interaction of plants with different environments. Molecular bases involved in ecological adaptations to abiotic constraints can be explored using genomic tools. Legumes are major crops worldwide and soil salinity is a main stress affecting yield in these plants. We analyzed in the Medicago truncatula legume the root transcriptome of two genotypes having contrasting responses to salt stress: TN1.11, sampled in a salty Tunisian soil, and the reference Jemalong A17 genotype. TN1.11 plants show increased root growth under salt stress as well as a differential accumulation of sodium ions when compared to A17. Transcriptomic analysis revealed specific gene clusters preferentially regulated by salt in root apices of TN1.11, notably those related to the auxin pathway and to changes in histone variant isoforms. Many genes encoding transcription factors (TFs) were also differentially regulated between the two genotypes in response to salt. Among those selected for functional studies, overexpression in roots of the A17 genotype of the bHLH-type TF most differentially regulated between genotypes improved significantly root growth under salt stress. Despite the global complexity of the differential transcriptional responses, we propose that an increase in this bHLH TF expression may be linked to the adaptation of M. truncatula to saline soil environments.

  15. Stem nematode counteracts plant resistance of aphids in alfalfa, Medicago sativa.

    PubMed

    Ramirez, Ricardo A; Spears, Lori R

    2014-10-01

    Plants are exploited by a diverse community of insect herbivores and phytopathogens that interact indirectly through plant-mediated interactions. Generally, plants are thought to respond to insects and pathogens through different defensive signaling pathways. As plants are selected for resistance to one phytophagous organism type (insect vs. pathogen) in managed systems, it is not clear how this selection may affect community interactions. This study examined the effect of nematode-resistant varieties on aphid (Acyrthosiphon pisum) suppression, and then determined how infection by the stem nematode, Ditylenchus dipsaci, mediated ecological effects on aphids and on plant defense proteins. Four alfalfa (Medicago sativa) varieties were selected with resistance to nematodes only (+,-), aphids only (-,+), nematodes and aphids (+,+), and susceptibility to nematodes and aphids (-,-). Field and greenhouse experiments were conducted to isolate the effect of nematode infection and aphid abundance on each variety. We found that varieties resistant to nematode, regardless of aphid resistance, had the lowest aphid counts, suggesting possible cross-resistance. Aphid abundance, however, increased when plants were exposed to nematodes. Resistant varieties were associated with elevated saponins but these compounds were not affected by insect or pathogen feeding. Concentrations of peroxidases and trypsin inhibitors, however, were increased in nematode resistant varieties when exposed to nematodes and aphids, respectively. The patterns of plant defense were variable, and a combination of resistance traits and changes in nutrient availability may drive positive interactions between nematodes and aphids aboveground.

  16. Effect of drought and rewatering on the cellular status and antioxidant response of Medicago truncatula plants

    PubMed Central

    Filippou, Panagiota; Antoniou, Chrystalla

    2011-01-01

    Effects of water stress on plants have been well-documented. However, the combined responses to drought and rewatering and their underlying mechanisms are relatively unknown. The present study attempts to describe spatiotemporal alterations in the physiology and cellular status of Medicago truncatula tissues that result from and subsequently follow a period of moderate water deficit. Physiological processes and cellular damage levels were monitored in roots and leaves by determining lipid peroxidation levels, as well as nitric oxide and hydrogen peroxide content, further supported by stomatal conductance and chlorophyll fluorescence measurements in leaves. During water stress, cells in both organs displayed increased damage levels and reactive oxygen and nitrogen species content, while leaves showed reduced stomatal conductance. Furthermore, both tissues demonstrated increased proline content. Upon rewatering, plants recovered displaying readings similar to pre-stress control conditions. Furthermore, molecular analysis of antioxidant gene expression by quantitative real-time RT-PCR revealed differential spatiotemporal regulation in a number of genes examined (including catalase, cytosolic ascorbate peroxidase, copper/zinc and iron superoxide dismutase and alternative oxidase). Overall, M. truncatula plants demonstrated increased sensitivity to drought-induced oxidative damage; however, this was reversed following rewatering indicating a great elasticity in the plant's capacity to cope with free oxygen and nitrogen radicals. PMID:21330785

  17. Transcriptional responses of Medicago truncatula upon sulfur deficiency stress and arbuscular mycorrhizal symbiosis

    PubMed Central

    Wipf, Daniel; Mongelard, Gaëlle; van Tuinen, Diederik; Gutierrez, Laurent; Casieri, Leonardo

    2014-01-01

    Sulfur plays an essential role in plants' growth and development and in their response to various abiotic and biotic stresses despite its leachability and its very low abundance in the only form that plant roots can uptake (sulfate). It is part of amino acids, glutathione (GSH), thiols of proteins and peptides, membrane sulfolipids, cell walls and secondary products, so reduced availability can drastically alter plant growth and development. The nutritional benefits of symbiotic interactions can help the plant in case of S deficiency. In particular the arbuscular mycorrhizal (AM) interaction improves N, P, and S plant nutrition, but the mechanisms behind these exchanges are not fully known yet. Although the transcriptional changes in the leguminous model plant Medicago truncatula have been already assessed in several biotic and/or abiotic conditions, S deficiency has not been considered so far. The aim of this work is to get a first overview on S-deficiency responses in the leaf and root tissues of plants interacting with the AM fungus Rhizophagus irregularis. Several hundred genes displayed significantly different transcript accumulation levels. Annotation and GO ID association were used to identify biological processes and molecular functions affected by sulfur starvation. Beside the beneficial effects of AM interaction, plants were greatly affected by the nutritional status, showing various differences in their transcriptomic footprints. Several pathways in which S plays an important role appeared to be differentially affected according to mycorrhizal status, with a generally reduced responsiveness to S deficiency in mycorrhized plants. PMID:25520732

  18. Soil decontamination of 2,4,6- trinitrotoluene by alfalfa (Medicago sativa).

    PubMed

    Fallahi, S; Habibi-Rezaei, M; Khayami, M; Heydari, R

    2007-12-15

    Present study investigate the toxicity effect of 2,4,6-trinitrotoluene (TNT) on a terrestrial plant, alfalfa (Medicago sativa) in artificial soils. In this study, TNT toxicity assessment was performed on spiked silica with this nitroaromatic compound by determination of the percent of emergence and shoots and roots biomasses at the concentration range of 3.2-10000 mg kg(-1) Dry Weight (DW). The emergence was reduced by 22-32% after 5 days of exposure at TNT concentrations up to 100 mg kg(-1) DW; shoot and root biomasses were reduced by 48-50 and 63-74%, respectively after 30 days exposure at TNT concentrations < or = 32 mg kg(-1) DW. Concentrations higher than 100 mg kg(-1) DW can not be tolerated at all. Concentrations of TNT and its metabolites in silica, root and shoot were measured by High-Performance Liquid Chromatography (HPLC). Analyses of TNT spiked soil extracts reveal hat during alfalfa cultivation for 30 days, TNT was partially transformed at the extent of 15-27%. This transformation decreased at higher TNT soil concentrations. TNT is taken up and metabolized by plants to its downstream derivatives.

  19. Nitrogen modulation of Medicago truncatula resistance to Aphanomyces euteiches depends on plant genotype.

    PubMed

    Thalineau, Elise; Fournier, Carine; Gravot, Antoine; Wendehenne, David; Jeandroz, Sylvain; Truong, Hoai-Nam

    2017-03-15

    Nitrogen (N) availability can impact plant resistance to pathogens by regulating plant immunity. To better understand the links between N nutrition and plant defence, we analysed the impact of N availability on Medicago truncatula resistance to the root pathogen Aphanomyces euteiches. This oomycete is considered as the most limiting factor for legume production. Ten plant genotypes were tested in vitro for their resistance to A. euteiches in either complete or N-deficient medium. N-deficiency led to enhanced or reduced susceptibility depending on plant genotype. Focusing on four genotypes displaying contrasted responses we determined the impact of N-deficiency on plant growth and shoot N concentration and performed expression analyses on N- and defence-related genes as well as quantification of soluble phenolics and of root contents in different amino-acids. Our analyses suggest that N modulation of plant resistance is not linked to plant response to N deprivation nor to mechanisms previously identified to be involved in plant resistance. Furthermore our studies highlight a role of glutamine in mediating susceptibility to A. euteiches in M. truncatula. This article is protected by copyright. All rights reserved.

  20. api, A novel Medicago truncatula symbiotic mutant impaired in nodule primordium invasion.

    PubMed

    Teillet, Alice; Garcia, Joseph; de Billy, Françoise; Gherardi, Michèle; Huguet, Thierry; Barker, David G; de Carvalho-Niebel, Fernanda; Journet, Etienne-Pascal

    2008-05-01

    Genetic approaches have proved to be extremely useful in dissecting the complex nitrogen-fixing Rhizobium-legume endosymbiotic association. Here we describe a novel Medicago truncatula mutant called api, whose primary phenotype is the blockage of rhizobial infection just prior to nodule primordium invasion, leading to the formation of large infection pockets within the cortex of noninvaded root outgrowths. The mutant api originally was identified as a double symbiotic mutant associated with a new allele (nip-3) of the NIP/LATD gene, following the screening of an ethylmethane sulphonate-mutagenized population. Detailed characterization of the segregating single api mutant showed that rhizobial infection is also defective at the earlier stage of infection thread (IT) initiation in root hairs, as well as later during IT growth in the small percentage of nodules which overcome the primordium invasion block. Neither modulating ethylene biosynthesis (with L-alpha-(2-aminoethoxyvinylglycine or 1-aminocyclopropane-1-carboxylic acid) nor reducing ethylene sensitivity in a skl genetic background alters the basic api phenotype, suggesting that API function is not closely linked to ethylene metabolism or signaling. Genetic mapping places the API gene on the upper arm of the M. truncatula linkage group 4, and epistasis analyses show that API functions downstream of BIT1/ERN1 and LIN and upstream of NIP/LATD and the DNF genes.

  1. Adaptation of Medicago truncatula to nitrogen limitation is modulated via local and systemic nodule developmental responses.

    PubMed

    Jeudy, Christian; Ruffel, Sandrine; Freixes, Sandra; Tillard, Pascal; Santoni, Anne Lise; Morel, Sylvain; Journet, Etienne-Pascal; Duc, Gérard; Gojon, Alain; Lepetit, Marc; Salon, Christophe

    2010-02-01

    Adaptation of Medicago truncatula to local nitrogen (N) limitation was investigated to provide new insights into local and systemic N signaling. The split-root technique allowed a characterization of the local and systemic responses of NO(3)(-) or N(2)-fed plants to localized N limitation. (15)N and (13)C labeling were used to monitor plant nutrition. Plants expressing pMtENOD11-GUS and the sunn-2 hypernodulating mutant were used to unravel mechanisms involved in these responses. Unlike NO(3)(-)-fed plants, N(2)-fixing plants lacked the ability to compensate rapidly for a localized N limitation by up-regulating the N(2)-fixation activity of roots supplied elsewhere with N. However they displayed a long-term response via a growth stimulation of pre-existing nodules, and the generation of new nodules, likely through a decreased abortion rate of early nodulation events. Both these responses involve systemic signaling. The latter response is abolished in the sunn mutant, but the mutation does not prevent the first response. Local but also systemic regulatory mechanisms related to plant N status regulate de novo nodule development in Mt, and SUNN is required for this systemic regulation. By contrast, the stimulation of nodule growth triggered by systemic N signaling does not involve SUNN, indicating SUNN-independent signaling.

  2. A model-based framework for the phenotypic characterization of the flowering of Medicago truncatula.

    PubMed

    Moreau, Delphine; Salon, Christophe; Munier-Jolain, Nathalie

    2007-02-01

    To facilitate the phenotypic characterization of Medicago truncatula, our aim was to provide a framework of analysis of flowering in response to environmental factors. The flowering of the line A17 was analysed in different conditions of temperature, duration of vernalization and photoperiod. Flowering was characterized using three descriptors at the axis level: the position of the first reproductive node (1RN), the date of beginning of flowering (DBF) and the florochron (RFa-1) corresponding to the reciprocal of the rate of progression of flowering along each axis. As for vegetative development, it was found that flowering could be analysed as a function of thermal time using a base temperature (Tb) of 5 degrees C. Vernalization displayed a sound impact on the flowering. For all the studied axes, increasing the duration of vernalization lowered the 1RN and hastened the DBF. By contrast, for most of the studied axes, RFa-1 was only slightly affected by vernalization. For the branch B0, RFa-1 was a genotypic constant when thermal time was used. Considering B0 as a reference axis, an ecophysiological model was developed to simulate the impact of environmental factors on the three components of flowering. Concrete practical applications of the model-based framework presented herein are proposed for helping the genetic and genomic studies of M. truncatula.

  3. Morphological and microsatellite diversity associated with ecological factors in natural populations of Medicago laciniata Mill. (Fabaceae).

    PubMed

    Badri, Mounawer; Zitoun, Adel; Ilahi, Houcine; Huguet, Thierry; Aouani, Mohamed Elarbi

    2008-12-01

    Genetic variability in 10 natural Tunisian populations of Medicago laciniata were analysed using 19 quantitative traits and 12 polymorphic microsatellite loci. A large degree of genetic variability within-populations and among-populations was detected for both quantitative characters and molecular markers. High genetic differentiation among populations for quantitative traits was seen, with Q(ST) = 0.47, and F(ST) = 0.47 for microsatellite markers. Several quantitative traits displayed no statistical difference in the levels of Q(ST) and F(ST). Further, significant correlations between quantitative traits and eco-geographical factors suggest that divergence in the traits among populations may track environmental differences. There was no significant correlation between genetic variability at quantitative traits and microsatellite markers within populations. The site-of-origin of eco-geographical factors explain between 18.13% and 23.40% of genetic variance among populations at quantitative traits and microsatellite markers, respectively. The environmental factors that most influence variation in measured traits among populations are assimilated phosphorus (P(2)0(5)) and mean annual rainfall, followed by climate and soil texture, altitude and organic matter. Significant associations between eco-geographical factors and gene diversity, He, were established in five microsatellite loci suggesting that these simple sequence repeats (SSRs) are not necessarily biologically neutral.

  4. From embryo sac to oil and protein bodies: embryo development in the model legume Medicago truncatula.

    PubMed

    Wang, Xin-Ding; Song, Youhong; Sheahan, Michael B; Garg, Manohar L; Rose, Ray J

    2012-01-01

    • The cell and developmental biology of zygotic embryogenesis in the model legume Medicago truncatula has received little attention. We studied M. truncatula embryogenesis from embryo sac until cotyledon maturation, including oil and protein body biogenesis. • We characterized embryo development using light and electron microscopy, measurement of protein and lipid fatty acid accumulation and by profiling the expression of key seed storage genes. • Embryo sac development in M. truncatula is of the Polygonum type. A distinctive multicellular hypophysis and suspensor develops before the globular stage and by the early cotyledon stage, the procambium connects the developing apical meristems. In the storage parenchyma of cotyledons, ovoid oil bodies surround protein bodies and the plasma membrane. Four major lipid fatty acids accumulate as cotyledons develop, paralleling the expression of OLEOSIN and the storage protein genes, VICILIN and LEGUMIN. • Zygotic embryogenesis in M. truncatula features the development of a distinctive multicellular hypophysis and an endopolyploid suspensor with basal transfer cell. A clear procambial connection between the apical meristems is evident and there is a characteristic arrangement of oil bodies in the cotyledons and radicle. Our data help link embryogenesis to the genetic regulation of oil and protein body biogenesis in legume seed.

  5. Influence of calcium oxalate crystal accumulation on the calcium content of seeds from Medicago truncatula.

    PubMed

    Nakata, Paul A

    2012-04-01

    Crystals of calcium oxalate often form in cells adjacent to the vascular bundles in the tissues along the xylem stream. This spatial crystal pattern suggests a role for calcium oxalate formation in regulating calcium transport and partitioning to edible organs such as seeds. To investigate this potential role, microscopic and biochemical comparisons were conducted on the different tissues of Medicago truncatula wild-type and the calcium oxalate defective (cod) 5 which lacks the ability to accumulate prismatic crystals in the cells adjacent to the vascular bundles. Calcium measurements showed that cod5 seeds had more calcium and cod5 pods contained less calcium than the corresponding wild-type tissues. Roots, stems, and leaves from cod5 and wild-type had similar calcium content. Although cod5 was devoid of prismatic crystals, cod5 pods were observed to form druse crystals of calcium oxalate not found in wild-type pods. Taken together these findings suggest a functional role for calcium oxalate formation in regulating calcium transport to the seeds. Regulating calcium uptake at the roots also appeared to be another point of control in determining seed calcium content. Overall, regulating the long distance transport and partitioning of calcium to the seeds appears to be a complex process with multiple points of control.

  6. MicroRNA156 improves drought stress tolerance in alfalfa (Medicago sativa) by silencing SPL13.

    PubMed

    Arshad, Muhammad; Feyissa, Biruk A; Amyot, Lisa; Aung, Banyar; Hannoufa, Abdelali

    2017-05-01

    Alfalfa (Medicago sativa) is an important forage crop that is often grown in areas that frequently experience drought and water shortage. MicroRNA156 (miR156) is an emerging tool for improving various traits in plants. We tested the role of miR156d in drought response of alfalfa, and observed a significant improvement in drought tolerance of miR156 overexpression (miR156OE) alfalfa genotypes compared to the wild type control (WT). In addition to higher survival and reduced water loss, miR156OE genotypes also maintained higher stomatal conductance compared to WT during drought stress. Furthermore, we observed an enhanced accumulation of compatible solute (proline) and increased levels of abscisic acid (ABA) and antioxidants in miR156OE genotypes. Similarly, alfalfa plants with reduced expression of miR156-targeted SPL13 showed reduced water loss and enhanced stomatal conductance, chlorophyll content and photosynthetic assimilation. Several genes known to be involved in drought tolerance were differentially expressed in leaf and root of miR156 overexpression plants. Taken together, our findings reveal that miR156 improves drought tolerance in alfalfa at least partially by silencing SPL13. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

  7. Micromonospora from nitrogen fixing nodules of alfalfa (Medicago sativa L.). A new promising Plant Probiotic Bacteria.

    PubMed Central

    Martínez-Hidalgo, Pilar; Galindo-Villardón, Purificación; Igual, José M.; Martínez-Molina, Eustoquio

    2014-01-01

    Biotic interactions can improve agricultural productivity without costly and environmentally challenging inputs. Micromonospora strains have recently been reported as natural endophytes of legume nodules but their significance for plant development and productivity has not yet been established. The aim of this study was to determine the diversity and function of Micromonospora isolated from Medicago sativa root nodules. Micromonospora-like strains from field alfalfa nodules were characterized by BOX-PCR fingerprinting and 16S rRNA gene sequencing. The ecological role of the interaction of the 15 selected representative Micromonospora strains was tested in M. sativa. Nodulation, plant growth and nutrition parameters were analyzed. Alfalfa nodules naturally contain abundant and highly diverse populations of Micromonospora, both at the intra- and at interspecific level. Selected Micromonospora isolates significantly increase the nodulation of alfalfa by Ensifer meliloti 1021 and also the efficiency of the plant for nitrogen nutrition. Moreover, they promote aerial growth, the shoot-to-root ratio, and raise the level of essential nutrients. Our results indicate that Micromonospora acts as a Rhizobia Helper Bacteria (RHB) agent and has probiotic effects, promoting plant growth and increasing nutrition efficiency. Its ecological role, biotechnological potential and advantages as a plant probiotic bacterium (PPB) are also discussed. PMID:25227415

  8. Nutraceutical Potential of New Alfalfa (Medicago sativa) Ingredients for Beverage Preparations.

    PubMed

    Soto-Zarazúa, Maria Guadalupe; Bah, Moustapha; Costa, Anabela Silvia Gomes; Rodrigues, Francisca; Pimentel, Filipa Botelho; Rojas-Molina, Isela; Rojas, Alejandra; Pinto Oliveira, Maria Beatriz Prior

    2017-07-27

    Alfalfa (Medicago sativa) has been extensively used as animal feed, due to its fiber, protein, minerals, and vitamins, being also a useful source of phenolic compounds with potential therapeutic benefits. Nevertheless, its potential use as human ingredient is scarce. The aim of this work was to assess the nutritional composition, amino acid profile, and antioxidant capacity (AOC) of freeze-dried juice (FDJ) and fibrous residual material (RM), two new alfalfa-derived products (Adps) recently launched as ingredients for beverage preparations. Results demonstrated a high content of proteins (23-30 g/100 g FDJ and 13-17 g/100 g RM), crude fiber (29 g/100 g RM), and minerals (such as sodium, calcium, iron, and zinc). No significant difference was found in caloric content (4 kcal/g). Essential and nonessential amino acids were quantified in both Adps being leucine and lysine the most abundant. Total phenolic and total flavonoid contents (TPC and TFC, respectively) and their changes along the different harvesting periods of the year were also examined. FDJ presented the highest TPC in May (19 mg gallic acid equivalents/g dry weight [dw]), while in October TFC had the maximum value (4 mg catechin equivalents/g dw). Both products exhibited an interesting AOC by 1,1-diphenyl-2-picrylhydrazyl (DPPH) and ferric reducing antioxidant power assays. This study reports the nutraceutical potential of two new types of Adps.

  9. Metabolomic Analysis of Alfalfa (Medicago sativa L.) Root-Symbiotic Rhizobia Responses under Alkali Stress

    PubMed Central

    Song, Tingting; Xu, Huihui; Sun, Na; Jiang, Liu; Tian, Pu; Yong, Yueyuan; Yang, Weiwei; Cai, Hua; Cui, Guowen

    2017-01-01

    Alkaline salts (e.g., NaHCO3 and Na2CO3) causes more severe morphological and physiological damage to plants than neutral salts (e.g., NaCl and Na2SO4) due to differences in pH. The mechanism by which plants respond to alkali stress is not fully understood, especially in plants having symbotic relationships such as alfalfa (Medicago sativa L.). Therefore, a study was designed to evaluate the metabolic response of the root-nodule symbiosis in alfalfa under alkali stress using comparative metabolomics. Rhizobium-nodulized (RI group) and non-nodulized (NI group) alfalfa roots were treated with 200 mmol/L NaHCO3 and, roots samples were analyzed for malondialdehydyde (MDA), proline, glutathione (GSH), superoxide dismutase (SOD), and peroxidase (POD) content. Additionally, metabolite profiling was conducted using gas chromatography combined with time-of-flight mass spectrometry (GC/TOF-MS). Phenotypically, the RI alfalfa exhibited a greater resistance to alkali stress than the NI plants examined. Physiological analysis and metabolic profiling revealed that RI plants accumulated more antioxidants (SOD, POD, GSH), osmolytes (sugar, glycols, proline), organic acids (succinic acid, fumaric acid, and alpha-ketoglutaric acid), and metabolites that are involved in nitrogen fixation. Our pairwise metabolomics comparisons revealed that RI alfalfa plants exhibited a distinct metabolic profile associated with alkali putative tolerance relative to NI alfalfa plants. Data provide new information about the relationship between non-nodulized, rhizobium-nodulized alfalfa and alkali resistance. PMID:28744296

  10. [Determination of Hard Rate of Alfalfa (Medicago sativa L.) Seeds with Near Infrared Spectroscopy].

    PubMed

    Wang, Xin-xun; Chen, Ling-ling; Zhang, Yun-wei; Mao, Pei-sheng

    2016-03-01

    Alfalfa (Medicago sativa L.) is the most commonly grown forage crop due to its better quality characteristics and high adaptability in China. However, there was 20%-80% hard seeds in alfalfa which could not be identified easily from non hard seeds which would cause the loss of seed utilization value and plant production. This experiment was designed for 121 samples of alfalfa. Seeds were collected according to different regions, harvested year and varieties. 31 samples were artificial matched as hard rates ranging from 20% to 80% to establish a model for hard seed rate by near infrared spectroscopy (NIRS) with Partial Least Square (PLS). The objective of this study was to establish a model and to estimate the efficiency of NIRS for determining hard rate of alfalfa seeds. The results showed that the correlation coefficient (R2(cal)) of calibration model was 0.981 6, root mean square error of cross validation (RMSECV) was 5.32, and the ratio of prediction to deviation (RPD) was 3.58. The forecast model in this experiment presented the satisfied precision. The proposed method using NIRS technology is feasible for identification and classification of hard seed in alfalfa. A new method, as nondestructive testing of hard seed rate, was provided to theoretical basis for fast nondestructive detection of hard seed rates in alfalfa.

  11. Allelopathic interference of alfalfa (Medicago sativa L.) genotypes to annual ryegrass (Lolium rigidum).

    PubMed

    Zubair, Hasan Muhammad; Pratley, James E; Sandral, G A; Humphries, A

    2017-07-01

    Alfalfa (Medicago sativa L.) genotypes at varying densities were investigated for allelopathic impact using annual ryegrass (Lolium rigidum) as the target species in a laboratory bioassay. Three densities (15, 30, and 50 seedlings/beaker) and 40 alfalfa genotypes were evaluated by the equal compartment agar method (ECAM). Alfalfa genotypes displayed a range of allelopathic interference in ryegrass seedlings, reducing root length from 5 to 65%. The growth of ryegrass decreased in response to increasing density of alfalfa seedlings. At the lowest density, Q75 and Titan9 were the least allelopathic genotypes. An overall inhibition index was calculated to rank each alfalfa genotype. Reduction in seed germination of annual ryegrass occurred in the presence of several alfalfa genotypes including Force 10, Haymaster7 and SARDI Five. A comprehensive metabolomic analysis using Quadruple Time of Flight (Q-TOF), was conducted to compare six alfalfa genotypes. Variation in chemical compounds was found between alfalfa root extracts and exudates and also between genotypes. Further individual compound assessments and quantitative study at greater chemical concentrations are needed to clarify the allelopathic activity. Considerable genetic variation exists among alfalfa genotypes for allelopathic activity creating the opportunity for its use in weed suppression through selection.

  12. Metabolomic Analysis of Alfalfa (Medicago sativa L.) Root-Symbiotic Rhizobia Responses under Alkali Stress.

    PubMed

    Song, Tingting; Xu, Huihui; Sun, Na; Jiang, Liu; Tian, Pu; Yong, Yueyuan; Yang, Weiwei; Cai, Hua; Cui, Guowen

    2017-01-01

    Alkaline salts (e.g., NaHCO3 and Na2CO3) causes more severe morphological and physiological damage to plants than neutral salts (e.g., NaCl and Na2SO4) due to differences in pH. The mechanism by which plants respond to alkali stress is not fully understood, especially in plants having symbotic relationships such as alfalfa (Medicago sativa L.). Therefore, a study was designed to evaluate the metabolic response of the root-nodule symbiosis in alfalfa under alkali stress using comparative metabolomics. Rhizobium-nodulized (RI group) and non-nodulized (NI group) alfalfa roots were treated with 200 mmol/L NaHCO3 and, roots samples were analyzed for malondialdehydyde (MDA), proline, glutathione (GSH), superoxide dismutase (SOD), and peroxidase (POD) content. Additionally, metabolite profiling was conducted using gas chromatography combined with time-of-flight mass spectrometry (GC/TOF-MS). Phenotypically, the RI alfalfa exhibited a greater resistance to alkali stress than the NI plants examined. Physiological analysis and metabolic profiling revealed that RI plants accumulated more antioxidants (SOD, POD, GSH), osmolytes (sugar, glycols, proline), organic acids (succinic acid, fumaric acid, and alpha-ketoglutaric acid), and metabolites that are involved in nitrogen fixation. Our pairwise metabolomics comparisons revealed that RI alfalfa plants exhibited a distinct metabolic profile associated with alkali putative tolerance relative to NI alfalfa plants. Data provide new information about the relationship between non-nodulized, rhizobium-nodulized alfalfa and alkali resistance.

  13. Mapping the Genetic Basis of Symbiotic Variation in Legume-Rhizobium Interactions in Medicago truncatula

    PubMed Central

    Gorton, Amanda J.; Heath, Katy D.; Pilet-Nayel, Marie-Laure; Baranger, Alain

    2012-01-01

    Mutualisms are known to be genetically variable, where the genotypes differ in the fitness benefits they gain from the interaction. To date, little is known about the loci that underlie such genetic variation in fitness or whether the loci influencing fitness are partner specific, and depend on the genotype of the interaction partner. In the legume-rhizobium mutualism, one set of potential candidate genes that may influence the fitness benefits of the symbiosis are the plant genes involved in the initiation of the signaling pathway between the two partners. Here we performed quantitative trait loci (QTL) mapping in Medicago truncatula in two different rhizobium strain treatments to locate regions of the genome influencing plant traits, assess whether such regions are dependent on the genotype of the rhizobial mutualist (QTL × rhizobium strain), and evaluate the contribution of sequence variation at known symbiosis signaling genes. Two of the symbiotic signaling genes, NFP and DMI3, colocalized with two QTL affecting average fruit weight and leaf number, suggesting that natural variation in nodulation genes may potentially influence plant fitness. In both rhizobium strain treatments, there were QTL that influenced multiple traits, indicative of either tight linkage between loci or pleiotropy, including one QTL with opposing effects on growth and reproduction. There was no evidence for QTL × rhizobium strain or genotype × genotype interactions, suggesting either that such interactions are due to small-effect loci or that more genotype-genotype combinations need to be tested in future mapping studies. PMID:23173081

  14. Nodule and Leaf Nitrate Reductases and Nitrogen Fixation in Medicago sativa L. under Water Stress

    PubMed Central

    Aparicio-Tejo, P.; Sánchez-Díaz, Manuel

    1982-01-01

    The effect of water stress on patterns of nitrate reductase activity in the leaves and nodules and on nitrogen fixation were investigated in Medicago sativa L. plants watered 1 week before drought with or without NO3−. Nitrogen fixation was decreased by water stress and also inhibited strongly by the presence of NO3−. During drought, leaf nitrate reductase activity (NRA) decreased significantly particularly in plants watered with NO3−, while with rewatering, leaf NRA recovery was quite important especially in the NO3−-watered plants. As water stress progressed, the nodular NRA increased both in plants watered with NO3− and in those without NO3− contrary to the behavior of the leaves. Beyond −15.105 pascal, nodular NRA began to decrease in plants watered with NO3−. This phenomenon was not observed in nodules of plants given water only. Upon rewatering, it was observed that in plants watered with NO3− the nodular NRA increased again, while in plants watered but not given NO3−, such activity began to decrease. Nitrogen fixation increased only in plants without NO3−. PMID:16662233

  15. Unveiling common responses of Medicago truncatula to appropriate and inappropriate rust species

    PubMed Central

    Vaz Patto, Maria Carlota; Rubiales, Diego

    2014-01-01

    Little is known about the nature of effective defense mechanisms in legumes to pathogens of remotely related plant species. Some rust species are among pathogens with broad host range causing dramatic losses in various crop plants. To understand and compare the different host and nonhost resistance (NHR) responses of legume species against rusts, we characterized the reaction of the model legume Medicago truncatula to one appropriate (Uromyces striatus) and two inappropriate (U. viciae-fabae and U. lupinicolus) rusts. We found that similar pre and post-haustorial mechanisms of resistance appear to be operative in M. truncatula against appropriate and inappropriate rust fungus. The appropriate U. striatus germinated better on M. truncatula accessions then the inappropriate U. viciae-fabae and U. lupinicolus, but once germinated, germ tubes of the three rusts had a similar level of success in finding stomata and forming an appressoria over a stoma. However, responses to different inappropriate rust species also showed some specificity, suggesting a combination of non-specific and specific responses underlying this legume NHR to rust fungi. Further genetic and expression analysis studies will contribute to the development of the necessary molecular tools to use the present information on host and NHR mechanisms to breed for broad-spectrum resistance to rust in legume species. PMID:25426128

  16. Uptake of phosphorus and lead by Brassica juncea and Medicago sativa from chloropyromorphite.

    PubMed

    Abbaspour, A; Arocena, J M; Kalbasi, M

    2012-07-01

    In situ remediation of lead (Pb)-contaminated soils via phosphate amendments has been extensively used to immobilize Pb as pyromorphite. However, in phosphorus (P) deficient soils, plants may develop extensive root systems to access P in any P-containing minerals, thereby affecting the stability of Pb5 (PO4)3Cl (Chloropyromorphite; CP). We grew Brassica juncea and Medicago sativa in sand culture to evaluate the stability of CP in the presence or absence of hydroxyapatite (HA) as P source. Treatments (per kilogram of sand) watered with P-nutrient solution were control [PC0, (without CP)], 1, and 5 g Pb as CP [PC1, and PC5] and 0.45 g P as HA (PA), and those of watered with P-free nutrient solution were 1 and 5 g Pb as CP [NC1 and NC5], 5 g Pb as CP plus 0.45 g P as HA [NAC5], and 0.45 g P as HA [NA]. Plants in NC1 and NC5 treatments showed stunted growth and reductions in shoot elongation and leaf size. Among CP treated pots, the highest shoot Pb uptake was observed in NAC5 treatment. The results suggested that Pb accumulation and translocation in the plants was markedly higher in P-sufficient conditions than in P-deficient conditions.

  17. Molecular cloning and characterization of a gene regulating flowering time from Alfalfa (Medicago sativa L.).

    PubMed

    Zhang, Tiejun; Chao, Yuehui; Kang, Junmei; Ding, Wang; Yang, Qingchuan

    2013-07-01

    Genes that regulate flowering time play crucial roles in plant development and biomass formation. Based on the cDNA sequence of Medicago truncatula (accession no. AY690425), the LFY gene of alfalfa was cloned. Sequence similarity analysis revealed high homology with FLO/LFY family genes of other plants. When fused to the green fluorescent protein, MsLFY protein was localized in the nucleus of onion (Allium cepa L.) epidermal cells. The RT-qPCR analysis of MsLFY expression patterns showed that the expression of MsLFY gene was at a low level in roots, stems, leaves and pods, and the expression level in floral buds was the highest. The expression of MsLFY was induced by GA3 and long photoperiod. Plant expression vector was constructed and transformed into Arabidopsis by the agrobacterium-mediated methods. PCR amplification with the transgenic Arabidopsis genome DNA indicated that MsLFY gene had integrated in Arabidopsis genome. Overexpression of MsLFY specifically caused early flowering under long day conditions compared with non-transgenic plants. These results indicated MsLFY played roles in promoting flowering time.

  18. Variation in Preference for Rhizobium meliloti Within and Between Medicago sativa Cultivars Grown in Soil †

    PubMed Central

    Bromfield, E. S. P.

    1984-01-01

    Variation in nodulation preferences for Rhizobium strains within and between Medicago sativa cultivars was assessed in the greenhouse with plants grown in Leonard jars and two soils of diverse origin (Lanark and Ottawa), using inocula consisting of effective individual or paired strains of R. meliloti which could be recognized by high-concentration antibiotic resistance. The results indicated considerable variability in host preferences for R. meliloti among plants within cultivars but not between cultivars. The implications of this variation are discussed from the point of view of possible improvement of symbiotic nitrogen fixation. With one exception, the differences in nodulation success between inoculant R. meliloti strains were consistent in Leonard jars and both soils. All introduced strains formed significantly more nodules in Renfrew soil containing few native rhizobia than in Ottawa soil with a large resident R. meliloti population. Plants grown in Lanark soil without inoculation were ineffectively nodulated by native rhizobia and yielded significantly less growth than those receiving inoculation. In contrast, the yield of inoculated plants in Ottawa soil did not significantly differ from those without inoculation due to effective nodulation by native R. meliloti. The data indicated synergistic effects on yield by certain paired strain inocula relative to the same strains inoculated individually in Lanark but not in Ottawa soil or Leonard jars. PMID:16346682

  19. Genetic diversity of an Italian Rhizobium meliloti population from different Medicago sativa varieties.

    PubMed Central

    Paffetti, D; Scotti, C; Gnocchi, S; Fancelli, S; Bazzicalupo, M

    1996-01-01

    We investigated the genetic diversity of 96 Rhizobium meliloti strains isolated from nodules of four Medicago sativa varieties from distinct geographic areas and planted in two different northern Italian soils. The 96 isolates, which were phenotypically indistinguishable, were analyzed for DNA polymorphism with the following three methods: (i) a randomly amplified polymorphic DNA (RAPD) method, (ii) a restriction fragment length polymorphism (RFLP) analysis of the 16S-23S ribosomal operon spacer region, and (iii) an RFLP analysis of a 25-kb region of the pSym plasmid containing nod genes. Although the bacteria which were studied constituted a unique genetic population, a considerable level of genetic diversity was found. The new analysis of molecular variance (AMOVA) method was used to estimate the variance among the RAPD patterns. The results indicated that there was significant genetic diversity among strains nodulating different varieties. The AMOVA method was confirmed to be a useful tool for investigating the genetic variation in an intraspecific population. Moreover, the data obtained with the two RFLP methods were consistent with the RAPD results. The genetic diversity of the population was found to reside on the whole bacterial genome, as suggested by the RAPD analysis results, and seemed to be distributed on both the chromosome and plasmid pSym. PMID:8779566

  20. Effect of salt stress on glycine betaine biosynthesis and catabolism by Medicago sativa bacteroids

    SciTech Connect

    Fougere, F.; Poggi, M.-C.; Le Rudulier, D. )

    1990-05-01

    Previous works have shown that glycine betaine (GB) and choline (Cho) are actively taken up by Medicago sativa bacteroids isolated from 4-week-old nodules. Here, we have investigated the effects of NaCl on the fte of Cho and GB. Bacteroids were incubated in low- or high-salt-medium (0.4 M NaCl) and supplemented with {sup 14}C 1,2-Cho or {sup 14}C 1,2-GB. After 3 hours, radioactivity was measured in CO{sub 2} released, in ethanol-soluble and insoluble fractions. In absence of salt, a low proportion of the labelling was found in soluble fraction: 47 and 19% after Cho or GB supply, respectively. On the contrary, in high-salt-medium, the soluble fraction still contained 85% of the radioactivity with GB corresponding to 92-98%. Both enzymes involved in GB biosynthesis from Cho were studied. Choline oxidase activity was enhanced by 59%, while betainal dehydrogenase activity remained unchanged after bacteroid incubation in high-salt-medium. Thus, GB accumulation in salt-stressed bacteroids would be likely a consequence of a decrease of its catabolism rather than an increase of its biosynthesis.

  1. The Effects of Clinorotation on the Host Plant, Medicago truncatula, and Its Microbial Symbionts

    NASA Astrophysics Data System (ADS)

    Dauzart, Ariel; Vandenbrink, Joshua; Kiss, John

    2016-02-01

    Understanding the outcome of the plant-microbe symbiosis in altered gravity is vital to developing life support systems for long-distance space travel and colonization of other planets. Thus, the aim of this research was to understand mutualistic relationships between plants and endophytic microbes under the influence of altered gravity. This project utilized the model tripartite relationship among Medicago truncatula ¬- Sinorhizobium meliloti - Rhizophagus irregularis. Plants were inoculated with rhizobial bacteria (S. meliloti), arbuscular mycorrhizal fungi (R. irregularis), or both microbes, and placed on a rotating clinostat. Vertical and horizontal static controls were also performed. Clinorotation significantly reduced M. truncatula dry mass and fresh mass compared to the static controls. The addition of rhizobia treatments under clinorotation also altered total root length and root-to-shoot fresh mass ratio. Nodule size decreased under rhizobia + clinorotation treatment, and nodule density was significantly decreased compared to the vertical treatment. However, inoculation with arbuscular mycorrhizal fungi was shown to increase biomass accumulation and nodule size. Thus, clinorotation significantly affected M. truncatula and its symbiotic relationships with S. meliloti and R. irregularis. In the long term, the results observed in this clinostat study on the changes of plant-microbe mutualism need to be investigated in spaceflight experiments. Thus, careful consideration of the symbiotic microbes of plants should be included in the design of bioregenerative life support systems needed for space travel.

  2. Cotyledon damage affects seed number through final plant size in the annual grassland species Medicago lupulina.

    PubMed

    Zhang, Shiting; Zhao, Chuan; Lamb, Eric G

    2011-03-01

    The effects of cotyledon damage on seedling growth and survival are relatively well established, but little is known about the effects on aspects of plant fitness such as seed number and size. Here the direct and indirect mechanisms linking cotyledon damage and plant fitness in the annual species Medicago lupulina are examined. Growth and reproductive traits, including mature plant size, time to first flowering, flower number, seed number and individual seed mass were monitored in M. lupulina plants when zero, one or two cotyledons were removed at 7 d old. Structural equation modelling (SEM) was used to examine the mechanisms linking cotyledon damage to seed number and seed mass. Cotyledon damage reduced seed number but not individual seed mass. The primary mechanism was a reduction in plant biomass with cotyledon damage that in turn reduced seed number primarily through a reduction in flower numbers. Although cotyledon damage delayed flower initiation, it had little effect on seed number. Individual seed mass was not affected by cotyledon removal, but there was a trade-off between seed number and seed mass. It is shown how a network of indirect mechanisms link damage to cotyledons and fitness in M. lupulina. Cotyledon damage had strong direct effects on both plant size and flowering phenology, but an analysis of the causal relationships among plant traits and fitness components showed that a reduction in plant size associated with cotyledon damage was an important mechanism influencing fitness.

  3. RNA interference identifies a calcium-dependent protein kinase involved in Medicago truncatula root development.

    PubMed

    Ivashuta, Sergey; Liu, Jinyuan; Liu, Junqi; Lohar, Dasharath P; Haridas, Sajeet; Bucciarelli, Bruna; VandenBosch, Kathryn A; Vance, Carroll P; Harrison, Maria J; Gantt, J Stephen

    2005-11-01

    Changes in cellular or subcellular Ca2+ concentrations play essential roles in plant development and in the responses of plants to their environment. However, the mechanisms through which Ca2+ acts, the downstream signaling components, as well as the relationships among the various Ca2+-dependent processes remain largely unknown. Using an RNA interference-based screen for gene function in Medicago truncatula, we identified a gene that is involved in root development. Silencing Ca2+-dependent protein kinase1 (CDPK1), which is predicted to encode a Ca2+-dependent protein kinase, resulted in significantly reduced root hair and root cell lengths. Inactivation of CDPK1 is also associated with significant diminution of both rhizobial and mycorrhizal symbiotic colonization. Additionally, microarray analysis revealed that silencing CDPK1 alters cell wall and defense-related gene expression. We propose that M. truncatula CDPK1 is a key component of one or more signaling pathways that directly or indirectly modulates cell expansion or cell wall synthesis, possibly altering defense gene expression and symbiotic interactions.

  4. Role of motility and chemotaxis in efficiency of nodulation by Rhizobium meliloti. [Medicago sation

    SciTech Connect

    Caetano-Anolles, G.; Wall, L.G.; De Micheli, A.T.; Macchi, E.M.; Bauer, W.D.; Favelukes, G. )

    1988-04-01

    Spontaneous mutants of Rhizobium meliloti L5-30 defective in motility or chemotaxis were isolated and compared against the parent with respect to symbiotic competence. Each of the mutants were able to generate normal nodules on the host plant alfalfa (Medicago sativa), but had slightly delayed nodule formation, diminished nodulation int he initially susceptible region of the host root, and relatively low representation in nodules following co-inoculation with equal numbers of the parent. When inoculated in growth pouches with increasing dosages of the parental strain, the number of nodules formed in the initially susceptible region of the root increased sigmoidally, with an optimum concentration of about 10{sup 5} to 10{sup 6} bacteria/plant. The dose-response behavior of the nonmotile and nonchemotactic mutants was similar, but they required 10- to 30-fold higher concentrations of bacteria to generate the same number of nodules. The distribution frequencies of nodules at different positions along the primary root were very similar for the mutants and parent, indicating that reduced nodulation by the mutants in dose-response experiments probably reflects reduced efficiency of nodule initiation rather than developmentally delayed nodule initiation. The number of bacteria that firmly adsorbed to the host root surface during several hours of incubation was 5- to 20-fold greater for the parent than the mutants.

  5. The thiol compounds glutathione and homoglutathione differentially affect cell development in alfalfa (Medicago sativa L.).

    PubMed

    Pasternak, Taras; Asard, Han; Potters, Geert; Jansen, Marcel A K

    2014-01-01

    Glutathione (GSH) is an important scavenger of Reactive Oxygen Species (ROS), precursor of metal chelating phytochelatins, xenobiotic defence compound and regulator of cell proliferation. Homoglutathione (hGSH) is a GSH homologue that is present in several taxa in the family of Fabaceae. It is thought that hGSH performs many of the stress-defence roles typically ascribed to GSH, yet little is known about the potential involvement of hGSH in controlling cell proliferation. Here we show that hGSH/GSH ratios vary across organs and cells and that these changes in hGSH/GSH ratio occur during dedifferentiation and/or cell cycle activation events. The use of a GSH/hGSH biosynthesis inhibitor resulted in impaired cytokinesis in isolated protoplasts, showing the critical importance of these thiol-compounds for cell division. However, exposure of isolated protoplasts to exogenous GSH accelerated cytokinesis, while exogenous hGSH was found to inhibit the same process. We conclude that GSH and hGSH have distinct functional roles in cell cycle regulation in Medicago sativa L. GSH is associated with meristemic cells, and promotes cell cycle activation and induction of somatic embryogenesis, while hGSH is associated with differentiated cells and embryo proliferation.

  6. Development of simple sequence repeat markers and diversity analysis in alfalfa (Medicago sativa L.).

    PubMed

    Wang, Zan; Yan, Hongwei; Fu, Xinnian; Li, Xuehui; Gao, Hongwen

    2013-04-01

    Efficient and robust molecular markers are essential for molecular breeding in plant. Compared to dominant and bi-allelic markers, multiple alleles of simple sequence repeat (SSR) markers are particularly informative and superior in genetic linkage map and QTL mapping in autotetraploid species like alfalfa. The objective of this study was to enrich SSR markers directly from alfalfa expressed sequence tags (ESTs). A total of 12,371 alfalfa ESTs were retrieved from the National Center for Biotechnology Information. Total 774 SSR-containing ESTs were identified from 716 ESTs. On average, one SSR was found per 7.7 kb of EST sequences. Tri-nucleotide repeats (48.8 %) was the most abundant motif type, followed by di-(26.1 %), tetra-(11.5 %), penta-(9.7 %), and hexanucleotide (3.9 %). One hundred EST-SSR primer pairs were successfully designed and 29 exhibited polymorphism among 28 alfalfa accessions. The allele number per marker ranged from two to 21 with an average of 6.8. The PIC values ranged from 0.195 to 0.896 with an average of 0.608, indicating a high level of polymorphism of the EST-SSR markers. Based on the 29 EST-SSR markers, assessment of genetic diversity was conducted and found that Medicago sativa ssp. sativa was clearly different from the other subspecies. The high transferability of those EST-SSR markers was also found for relative species.

  7. Screening of Cd tolerant genotypes and isolation of metallothionein genes in alfalfa (Medicago sativa L.).

    PubMed

    Wang, Xiaojuan; Song, Yu; Ma, Yanhua; Zhuo, Renying; Jin, Liang

    2011-12-01

    In order to evaluate Cd tolerance in wide-ranging sources of alfalfa (Medicago sativa) and to identify Cd tolerant genotypes which may potentially be useful for restoring Cd-contaminated environments, thirty-six accessions of alfalfa were screened under hydroponic culture. Our results showed that the relative root growth rate varied from 0.48 to 1.0, which indicated that different alfalfa accessions had various responses to Cd stress. The candidate fragments derived from differentially expressed metallothionein (MT) genes were cloned from leaves of two Cd tolerant genotypes, YE and LZ. DNA sequence and the deduced protein sequence showed that MsMT2a and MsMT2b had high similarity to those in leguminous plants. DDRT-PCR analysis showed that MsMT2a expressed in both YE and LZ plants under control and Cd stress treatment, but MsMT2b only expressed under Cd stress treatment. This suggested that MsMT2a was universally expressed in leaves of alfalfa but expression of MsMT2b was Cadmium (Cd) inducible.

  8. Influence of arbuscular mycorrhizal colonisation on cadmium induced Medicago truncatula root isoflavonoid accumulation.

    PubMed

    Aloui, Achref; Dumas-Gaudot, Eliane; Daher, Zeina; van Tuinen, Diederik; Aschi-Smit, Samira; Morandi, Dominique

    2012-11-01

    Cadmium is a serious environmental pollution threats to the planet. Its accumulation in plants affects many cellular functions, resulting in growth and development inhibition, whose mechanisms are not fully understood. However, some fungi forming arbuscular mycorrhizal symbiosis with the majority of plant species have the capacity to buffer the deleterious effect of this heavy metal. In the present work we investigated the capacity of Rhizophagus irregularis (syn. Glomus irregularis) to alleviate cadmium stress in Medicago truncatula. In spite of a reduction in all mycorrhizal parameters, plants colonized for 21 days by R. irregularis and treated by 2 mg kg⁻¹ cadmium displayed less growth inhibition in comparison to plants grown without cadmium. Cadmium strongly increased the accumulation of some isoflavonoids and their derivates: formononetin, malonylononin, medicarpin 3-O-β-(6'-malonylglucoside), medicarpin and coumestrol. Interestingly, in plants colonized by R. irregularis we noticed a strong reduction of the cadmium-induced accumulation of root isoflavonoids, a part for medicarpin and coumestrol. Moreover, transcripts of chalcone reductase, a protein that we reported previously as being down-regulated in R. irregularis-colonized M. truncatula roots, revealed a similar expression pattern with a strong increase in response to cadmium and a reduced expression in cadmium-treated mycorrhizal roots.

  9. MtVRN2 is a Polycomb VRN2-like gene which represses the transition to flowering in the model legume Medicago truncatula.

    PubMed

    Jaudal, Mauren; Zhang, Lulu; Che, Chong; Hurley, Daniel G; Thomson, Geoffrey; Wen, Jiangqi; Mysore, Kirankumar S; Putterill, Joanna

    2016-04-01

    Optimising the timing of flowering contributes to successful sexual reproduction and yield in agricultural plants. FLOWERING LOCUS T (FT) genes, first identified in Arabidopsis thaliana (Arabidopsis), promote flowering universally, but the upstream flowering regulatory pathways can differ markedly among plants. Flowering in the model legume, Medicago truncatula (Medicago) is accelerated by winter cold (vernalisation) followed by long day (LD) photoperiods leading to elevated expression of the floral activator, FT-like gene FTa1. However, Medicago, like some other plants, lacks the activator CONSTANS (CO) and the repressor FLOWERING LOCUS C (FLC) genes which directly regulate FT and are key to LD and vernalisation responses in Arabidopsis. Conversely, Medicago has a VERNALISATION2-LIKE VEFS-box gene (MtVRN2). In Arabidopsis AtVRN2 is a key member of a Polycomb complex involved in stable repression of Arabidopsis FLC after vernalisation. VRN2-like genes have been identified in other eudicot plants, but their function has never been reported. We show that Mtvrn2 mutants bypass the need for vernalisation for early flowering in LD conditions in Medicago. Investigation of the underlying mechanism by transcriptome analysis reveals that Mtvrn2 mutants precociously express FTa1 and other suites of genes including floral homeotic genes. Double-mutant analysis indicates that early flowering is dependent on functional FTa1. The broad significance of our study is that we have demonstrated a function for a VRN2-like VEFS gene beyond the Brassicaceae. In particular, MtVRN2 represses the transition to flowering in Medicago by regulating the onset of expression of the potent floral activator, FTa1.

  10. Flavone Synthases from Medicago truncatula Are Flavanone-2-Hydroxylases and Are Important for Nodulation1[W][OA

    PubMed Central

    Zhang, Juan; Subramanian, Senthil; Zhang, Yansheng; Yu, Oliver

    2007-01-01

    Flavones are important copigments found in the flowers of many higher plants and play a variety of roles in plant adaptation to stress. In Medicago species, flavones also act as signal molecules during symbiotic interaction with the diazotropic bacterium Sinorhizobium meliloti. They are the most potent nod gene inducers found in root exudates. However, flavone synthase II (FNS II), the key enzyme responsible for flavone biosynthesis, has not been characterized in Medicago species. We cloned two FNS II genes from Medicago truncatula using known FNS II sequences from other species and named them MtFNSII-1 and MtFNSII-2. Functional assays in yeast (Saccharomyces cerevisiae) suggested that the catalytic mechanisms of both cytochrome P450 monooxygenases were similar to the other known legume FNS II from licorice (Glycyrrhiza echinata). MtFNSII converted flavanones to 2-hydroxyflavanones instead of flavones whereas FNS II from the nonlegume Gerbera hybrida, converted flavanones to flavones directly. The two MtFNSII genes had distinct tissue-specific expression patterns. MtFNSII-1 was highly expressed in roots and seeds whereas MtFNSII-2 was highly expressed in flowers and siliques. In addition, MtFNSII-2 was inducible by S. meliloti and methyl jasmonate treatment, whereas MtFNSII-1 was not. Histochemical staining of transgenic hairy roots carrying the promoter-reporter constructs indicated that the MtFNSII-2 induction was tissue specific, mostly localized to vascular tissues and root hairs. RNA interference-mediated suppression of MtFNSII genes resulted in flavone depleted roots and led to significantly reduced nodulation when inoculated with S. meliloti. Our results provide genetic evidence supporting that flavones are important for nodulation in M. truncatula. PMID:17434990

  11. MsPG3, a Medicago sativa polygalacturonase gene expressed during the alfalfa–Rhizobium meliloti interaction

    PubMed Central

    Muñoz, Jose A.; Coronado, Carmen; Pérez-Hormaeche, Javier; Kondorosi, Adam; Ratet, Pascal; Palomares, Antonio J.

    1998-01-01

    Polygalacturonase (PG) is one of the most important enzymes associated with plant cell wall degradation. It has been proposed to participate in the early steps of the Rhizobium–legume interaction. We have identified two classes of cDNA fragments corresponding to two classes of PG genes in the Medicago genome. One of this class, represented by E2 in M. truncatula and Pl1 in M. sativa, seems to be related to previously characterized plant PG genes expressed in pollen. We have isolated the genomic clone containing the entire gene corresponding to the second class (E3). We showed that MsPG3 is a single gene in the Medicago genome coding for PG. By reverse transcription-PCR, MsPG3 expression was detected in roots 1 day after Rhizobium inoculation. The early induction of the MsPG3, as also seen by in situ hybridization experiments, supports its involvement in the early stages of the Rhizobium-legume infection process. In addition, by analyzing the expression of a MsPG3 promoter-gus construct in Vicia hirsuta-transgenic root nodules, we showed that MsPG3 was expressed in all cells of nodule primordia and in the cells of the invasion zone. By Northern blot, MsPG3 transcripts are not detected in various Medicago tissues, indicating that the function of this gene is related closely to symbiosis. Thus, our results strongly suggest the involvement of MsPG3 gene during meristem formation and/or in the infection process, probably by facilitating cell wall rearrangement, penetration of the bacteria through the root hair wall, or infection thread formation and release of bacteria in plant cells. MsPG3 represents a class of PG genes, distinct from the pollen-specific genes, and it is the first pectic encoded enzyme demonstrated to be involved in Rhizobium-legume symbiosis. PMID:9689142

  12. Comparative genomic sequence and expression analyses of Medicago truncatula and alfalfa subspecies falcata COLD-ACCLIMATION-SPECIFIC genes.

    PubMed

    Pennycooke, Joyce C; Cheng, Hongmei; Stockinger, Eric J

    2008-03-01

    In Arabidopsis (Arabidopsis thaliana) the low-temperature induction of genes encoding the C-REPEAT BINDING FACTOR (CBF) transcriptional activators is a key step in cold acclimation. CBFs in turn activate a battery of downstream genes known as the CBF regulon, which collectively act to increase tolerance to low temperatures. Fundamental questions are: What determines the size and scope of the CBF regulon, and is this is a major determinant of the low-temperature tolerance capacity of individual plant species? Here we have begun to address these questions through comparative analyses of Medicago truncatula and Medicago sativa subsp. falcata. M. truncatula survived to -4 degrees C but did not cold acclimate, whereas Medicago falcata cold acclimated and survived -14 degrees C. Both species possessed low-temperature-induced CBFs but differed in the expression of the COLD-ACCLIMATION-SPECIFIC (CAS) genes, which are candidate CBF targets. M. falcata CAS30 was robustly cold-responsive whereas the MtCAS31 homolog was not. M. falcata also possessed additional CAS30 homologs in comparison to the single CAS31 gene in M. truncatula. MfCAS30 possessed multiple pairs of closely spaced C-REPEAT/DEHYDRATION RESPONSIVE ELEMENT (CRT/DRE) motifs, the cognate CBF binding site in its upstream region whereas MtCAS31 lacked one CRT/DRE partner of the two proximal partner pairs. CAS genes also shared a promoter structure comprising modules proximal and distal to the coding sequence. CAS15, highly cold-responsive in both species, harbored numerous CRT/DRE motifs, but only in the distal module. However, fusion of the MtCAS15 promoter, including the distal module, to a reporter gene did not result in low-temperature responsiveness in stably transformed Arabidopsis. In contrast, both MtCAS31 and MfCAS30 promoter fusions were low-temperature responsive, although the MfCAS31 fusion was less robust than the MfCAS30 fusion. From these studies we conclude that CAS genes harbor CRT/DRE motifs, their

  13. Fine Mapping Links the FTa1 Flowering Time Regulator to the Dominant Spring1 Locus in Medicago

    PubMed Central

    Yeoh, Chin Chin; Balcerowicz, Martin; Zhang, Lulu; Jaudal, Mauren; Brocard, Lysiane; Ratet, Pascal; Putterill, Joanna

    2013-01-01

    To extend our understanding of flowering time control in eudicots, we screened for mutants in the model legume Medicago truncatula (Medicago). We identified an early flowering mutant, spring1, in a T-DNA mutant screen, but spring1 was not tagged and was deemed a somaclonal mutant. We backcrossed the mutant to wild type R108. The F1 plants and the majority of F2 plants were early flowering like spring1, strongly indicating that spring1 conferred monogenic, dominant early flowering. We hypothesized that the spring1 phenotype resulted from over expression of an activator of flowering. Previously, a major QTL for flowering time in different Medicago accessions was located to an interval on chromosome 7 with six candidate flowering- time activators, including a CONSTANS gene, MtCO, and three FLOWERING LOCUS T (FT) genes. Hence we embarked upon linkage mapping using 29 markers from the MtCO/FT region on chromosome 7 on two populations developed by crossing spring1 with Jester. Spring1 mapped to an interval of ∼0.5 Mb on chromosome 7 that excluded MtCO, but contained 78 genes, including the three FT genes. Of these FT genes, only FTa1 was up-regulated in spring1 plants. We then investigated global gene expression in spring1 and R108 by microarray analysis. Overall, they had highly similar gene expression and apart from FTa1, no genes in the mapping interval were differentially expressed. Two MADS transcription factor genes, FRUITFULLb (FULb) and SUPPRESSOR OF OVER EXPRESSION OF CONSTANS1a (SOC1a), that were up-regulated in spring1, were also up-regulated in transgenic Medicago over-expressing FTa1. This suggested that their differential expression in spring1 resulted from the increased abundance of FTa1. A 6255 bp genomic FTa1 fragment, including the complete 5′ region, was sequenced, but no changes were observed indicating that the spring1 mutation is not a DNA sequence difference in the FTa1 promoter or introns. PMID:23308229

  14. Mineral accumulation in vegetative and reproductive tissues during seed development in Medicago truncatula

    PubMed Central

    Garcia, Christina B.; Grusak, Michael A.

    2015-01-01

    Enhancing nutrient density in legume seeds is one of several strategies being explored to improve the nutritional quality of the food supply. In order to develop crop varieties with increased seed mineral concentration, a more detailed understanding of mineral translocation within the plant is required. By studying mineral accumulation in different organs within genetically diverse members of the same species, it may be possible to identify variable traits that modulate seed mineral concentration. We utilized two ecotypes (A17 and DZA315.16) of the model legume, Medicago truncatula, to study dry mass and mineral accumulation in the leaves, pod walls, and seeds during reproductive development. The pod wall dry mass was significantly different between the two ecotypes beginning at 12 days after pollination, whereas there was no significant difference in the average dry mass of individual seeds between the two ecotypes at any time point. There were also no significant differences in leaf dry mass between ecotypes; however, we observed expansion of A17 leaves during the first 21 days of pod development, while DZA315.16 leaves did not display a significant increase in leaf area. Mineral profiling of the leaves, pod walls, and seeds highlighted differences in accumulation patterns among minerals within each tissue as well as genotypic differences with respect to individual minerals. Because there were differences in the average seed number per pod, the total seed mineral content per pod was generally higher in A17 than DZA315.16. In addition, mineral partitioning to the seeds tended to be higher in A17 pods. These data revealed that mineral retention within leaves and/or pod walls might attenuate mineral accumulation within the seeds. As a result, strategies to increase seed mineral content should include approaches that will enhance export from these tissues. PMID:26322063

  15. Physiological and antioxidant responses of Medicago sativa-rhizobia symbiosis to cyanobacterial toxins (Microcystins) exposure.

    PubMed

    El Khalloufi, Fatima; Oufdou, Khalid; Lahrouni, Majida; Faghire, Mustapha; Peix, Alvaro; Ramírez-Bahena, Martha Helena; Vasconcelos, Vitor; Oudra, Brahim

    2013-12-15

    Toxic cyanobacteria in freshwaters can induce potent harmful effects on growth and development of plants irrigated with contaminated water. In this study, the effect of cyanobacteria extract containing Microcystins (MC) on Medicago sativa-rhizobia symbiosis was investigated in order to explore plants response through biomass production, photosynthetic pigment and antioxidant enzymes analysis: Peroxidase (POD), Polyphenoloxidase (PPO) and Catalase (CAT). Alfalfa plants were inoculated with two endosymbiotic rhizobial strains: RhOL1 (MC less sensitive strain) and RhOL3 (MC more sensitive strain), to evaluate the rhizobial contribution on the plant response cultured under cyanobacterial toxins stress. The two rhizobia strains were identified as Ensifer meliloti by sequence analysis of their rrs and atpD genes. The chronic exposure to MC extract showed shoot, root and nodules dry weight decrease, in both symbiosis cultures. The rate of decline in plants inoculated with RhOL3 was higher than that in symbiosis with RhOL1 mainly at 20 μg L(-1) of MC. Cyanotoxins also reduced photosynthetic pigment content and generated an oxidative stress observed at cellular level. POD, PPO and CAT activities were significantly increased in leaves, roots and nodules of alfalfa plants exposed to MC. These enzyme activities were higher in plants inoculated with RhOL3 especially when alfalfa plants were exposed to 20 μg L(-1) of MC. The present paper reports new scientific finding related to the behavior of rhizobia-M. sativa associations to MC (Microcystins) for later recommendation concerning the possible use of these symbiosis face to crops exposure to MC contaminated water irrigation.

  16. Interplay of Pathogen-Induced Defense Responses and Symbiotic Establishment in Medicago truncatula

    PubMed Central

    Chen, Tao; Duan, Liujian; Zhou, Bo; Yu, Haixiang; Zhu, Hui; Cao, Yangrong; Zhang, Zhongming

    2017-01-01

    Suppression of host innate immunity appears to be required for the establishment of symbiosis between rhizobia and host plants. In this study, we established a system that included a host plant, a bacterial pathogen and a symbiotic rhizobium to study the role of innate immunity during symbiotic interactions. A pathogenic bacterium, Pseudomonas syringae pv. tomato strain DC3000 (Pst DC3000), was shown to cause chlorosis in Medicago truncatula A17. Sinorhizobium meliloti strain Sm2011 (Sm2011) and Pst DC3000 strain alone induced similar defense responses in M. truncatula. However, when co-inoculated, Sm2011 specifically suppressed the defense responses induced by Pst DC3000, such as MAPK activation and ROS production. Inoculation with Sm2011 suppressed the transcription of defense-related genes triggered by Pst DC3000 infection, including the receptor of bacterial flagellin (FLS2), pathogenesis-related protein 10 (PR10), and the transcription factor WRKY33. Interestingly, inoculation with Pst DC3000 specifically inhibited the expression of the symbiosis marker genes nodule inception and nodulation pectate lyase and reduced the numbers of infection threads and nodules on M. truncatula A17 roots, indicating that Pst DC3000 inhibits the establishment of symbiosis in M. truncatula. In addition, defense-related genes, such as MAPK3/6, RbohC, and WRKY33, exhibited a transient increase in their expression in the early stage of symbiosis with Sm2011, but the expression dropped down to normal levels at later symbiotic stages. Our results suggest that plant innate immunity plays an antagonistic role in symbiosis by directly reducing the numbers of infection threads and nodules. PMID:28611764

  17. Interplay of Pathogen-Induced Defense Responses and Symbiotic Establishment in Medicago truncatula.

    PubMed

    Chen, Tao; Duan, Liujian; Zhou, Bo; Yu, Haixiang; Zhu, Hui; Cao, Yangrong; Zhang, Zhongming

    2017-01-01

    Suppression of host innate immunity appears to be required for the establishment of symbiosis between rhizobia and host plants. In this study, we established a system that included a host plant, a bacterial pathogen and a symbiotic rhizobium to study the role of innate immunity during symbiotic interactions. A pathogenic bacterium, Pseudomonas syringae pv. tomato strain DC3000 (Pst DC3000), was shown to cause chlorosis in Medicago truncatula A17. Sinorhizobium meliloti strain Sm2011 (Sm2011) and Pst DC3000 strain alone induced similar defense responses in M. truncatula. However, when co-inoculated, Sm2011 specifically suppressed the defense responses induced by Pst DC3000, such as MAPK activation and ROS production. Inoculation with Sm2011 suppressed the transcription of defense-related genes triggered by Pst DC3000 infection, including the receptor of bacterial flagellin (FLS2), pathogenesis-related protein 10 (PR10), and the transcription factor WRKY33. Interestingly, inoculation with Pst DC3000 specifically inhibited the expression of the symbiosis marker genes nodule inception and nodulation pectate lyase and reduced the numbers of infection threads and nodules on M. truncatula A17 roots, indicating that Pst DC3000 inhibits the establishment of symbiosis in M. truncatula. In addition, defense-related genes, such as MAPK3/6, RbohC, and WRKY33, exhibited a transient increase in their expression in the early stage of symbiosis with Sm2011, but the expression dropped down to normal levels at later symbiotic stages. Our results suggest that plant innate immunity plays an antagonistic role in symbiosis by directly reducing the numbers of infection threads and nodules.

  18. The alternative Medicago truncatula defense proteome of ROS—defective transgenic roots during early microbial infection

    PubMed Central

    Kiirika, Leonard M.; Schmitz, Udo; Colditz, Frank

    2014-01-01

    ROP-type GTPases of plants function as molecular switches within elementary signal transduction pathways such as the regulation of ROS synthesis via activation of NADPH oxidases (RBOH-respiratory burst oxidase homolog in plants). Previously, we reported that silencing of the Medicago truncatula GTPase MtROP9 led to reduced ROS production and suppressed induction of ROS-related enzymes in transgenic roots (MtROP9i) infected with pathogenic (Aphanomyces euteiches) and symbiotic microorganisms (Glomus intraradices, Sinorhizobium meliloti). While fungal infections were enhanced, S. meliloti infection was drastically impaired. In this study, we investigate the temporal proteome response of M. truncatula MtROP9i transgenic roots during the same microbial interactions under conditions of deprived potential to synthesize ROS. In comparison with control roots (Mtvector), we present a comprehensive proteomic analysis using sensitive MS protein identification. For four early infection time-points (1, 3, 5, 24 hpi), 733 spots were found to be different in abundance: 213 spots comprising 984 proteins (607 unique) were identified after S. meliloti infection, 230 spots comprising 796 proteins (580 unique) after G. intraradices infection, and 290 spots comprising 1240 proteins (828 unique) after A. euteiches infection. Data evaluation by GelMap in combination with a heatmap tool allowed recognition of key proteome changes during microbial interactions under conditions of hampered ROS synthesis. Overall, the number of induced proteins in MtROP9i was low as compared with controls, indicating a dual function of ROS in defense signaling as well as alternative response patterns activated during microbial infection. Qualitative analysis of induced proteins showed that enzymes linked to ROS production and scavenging were highly induced in control roots, while in MtROP9i the majority of proteins were involved in alternative defense pathways such as cell wall and protein degradation. PMID

  19. Transcriptome analysis of a bacterially induced basal and hypersensitive response of Medicago truncatula.

    PubMed

    Bozsó, Zoltán; Maunoury, Nicolas; Szatmari, Agnes; Mergaert, Peter; Ott, Péter G; Zsíros, László R; Szabó, Erika; Kondorosi, Eva; Klement, Zoltán

    2009-08-01

    Research using the well-studied model legume Medicago truncatula has largely focused on rhizobium symbiosis, while little information is currently available for this species on pathogen-induced transcriptome changes. We have performed a transcriptome analysis of this species with the objective of studying the basal (BR, no visible symptoms) and hypersensitive response (HR, plant cell death) in its leaves at 6 and at 24 h after infection by HR-negative (hrcC mutant) and HR-inducing Pseudomonas syringae pv. syringae strains, respectively. Although there were no visible symptoms at the BR, the alterations in gene expression were comparable to those found with the HR. Both responses resulted in the transcriptional alteration of hundreds of plant genes; however, the responses in the HR were usually more intense. The reactions to HR-inducing and HR-negative bacterial strains were significantly overlapping. Parallel up- or down-regulation of genes with the same function occurred frequently. However, some plant processes were regulated in one direction; for example, most of the protein synthesis-related genes were activated and all of the photosynthetic/chloroplast genes were suppressed during BR. The possible roles of several functional classes (e.g., cell rescue, signaling, defense, cell death, etc.) of transcriptionally altered genes are discussed. The results of the comparison with available mycorrhizal and nodule expression data show that there is a significant overlap between nodulation and the leaf defense response and that during the early stage of the nodulation in roots, Sinorhizobium meliloti induces a fluctuation in the transcription of BR- and HR-responsive genes.

  20. The association of homeobox gene expression with stem cell formation and morphogenesis in cultured Medicago truncatula.

    PubMed

    Chen, S-K; Kurdyukov, S; Kereszt, A; Wang, X-D; Gresshoff, P M; Rose, R J

    2009-09-01

    Somatic embryogenesis (SE) is induced in vitro in Medicago truncatula 2HA by auxin and cytokinin but rarely in wild type Jemalong. The putative WUSCHEL (MtWUS), CLAVATA3 (MtCLV3) and the WUSCHEL-related homeobox gene WOX5 (MtWOX5) were investigated in M. truncatula (Mt) and identified by the similarity to Arabidopsis WUS, CLV3 and WOX5 in amino acid sequence, phylogeny and in planta and in vitro expression patterns. MtWUS was induced throughout embryogenic cultures by cytokinin after 24-48 h and maximum expression occurred after 1 week, which coincides with the induction of totipotent stem cells. During this period there was no MtCLV3 expression to suppress MtWUS. MtWUS expression, as illustrated by promoter-GUS studies, subsequently localised to the embryo, and there was then the onset of MtCLV3 expression. This suggests that the expression of the putative MtCLV3 coincides with the WUS-CLAVATA feedback loop becoming operational. RNAi studies showed that MtWUS expression is essential for callus and somatic embryo production. Based on the presence of MtWUS promoter binding sites, MtWUS may be required for the induction of MtSERF1, postulated to have a key role in the signalling required for SE induced in 2HA. MtWOX5 expressed in auxin-induced root primordia and root meristems and appears to be involved in pluripotent stem cell induction. The evidence is discussed that the homeobox genes MtWUS and MtWOX5 are "hijacked" for stem cell induction, which is key to somatic embryo and de novo root induction. In relation to SE, a role for WUS in the signalling involved in induction is discussed.

  1. Salinity Adaptation and the Contribution of Parental Environmental Effects in Medicago truncatula

    PubMed Central

    Moriuchi, Ken S.; Friesen, Maren L.; Cordeiro, Matilde A.; Badri, Mounawer; Vu, Wendy T.; Main, Bradley J.; Aouani, Mohamed Elarbi; Nuzhdin, Sergey V.; Strauss, Sharon Y.; von Wettberg, Eric J. B.

    2016-01-01

    High soil salinity negatively influences plant growth and yield. Some taxa have evolved mechanisms for avoiding or tolerating elevated soil salinity, which can be modulated by the environment experienced by parents or offspring. We tested the contribution of the parental and offspring environments on salinity adaptation and their potential underlying mechanisms. In a two-generation greenhouse experiment, we factorially manipulated salinity concentrations for genotypes of Medicago truncatula that were originally collected from natural populations that differed in soil salinity. To compare population level adaptation to soil salinity and to test the potential mechanisms involved we measured two aspects of plant performance, reproduction and vegetative biomass, and phenological and physiological traits associated with salinity avoidance and tolerance. Saline-origin populations had greater biomass and reproduction under saline conditions than non-saline populations, consistent with local adaptation to saline soils. Additionally, parental environmental exposure to salt increased this difference in performance. In terms of environmental effects on mechanisms of salinity adaptation, parental exposure to salt spurred phenological differences that facilitated salt avoidance, while offspring exposure to salt resulted in traits associated with greater salt tolerance. Non-saline origin populations expressed traits associated with greater growth in the absence of salt while, for saline adapted populations, the ability to maintain greater performance in saline environments was also associated with lower growth potential in the absence of salt. Plastic responses induced by parental and offspring environments in phenology, leaf traits, and gas exchange contribute to salinity adaptation in M. truncatula. The ability of plants to tolerate environmental stress, such as high soil salinity, is likely modulated by a combination of parental effects and within-generation phenotypic

  2. Plant growth-promoting endophyte Piriformospora indica alleviates salinity stress in Medicago truncatula.

    PubMed

    Li, Liang; Li, Lei; Wang, Xiaoyang; Zhu, Pengyue; Wu, Hongqing; Qi, Shuting

    2017-10-01

    Piriformospora indica, a cultivable root endophytic fungus, induces growth promotion as well as biotic stress resistance and tolerance to abiotic stress in a broad range of host plants. In this study, the potential protection for M Medicago truncatula plants from salinity stress by P. indica was explored. The improved plant growth under severe saline condition was exhibited in P. indica-colonized lines. Moreover, the antioxidant enzymes activities and hyphae density in roots were increased by the endophyte under high salt concentration. Conversely, reduced malondialdehyde (MDA) activity, Na(+) content and relative electrolyte conductivity (REC) were observed in P. indica colonized plants. Especially, osmoprotectant proline accumulated and the expression of Delta 1-Pyrroline-5-carboxylate synthetase gene (P5CS2) was induced. The defense related genes PR1 and PR10 and the transcription factors MtAlfin1-like and C2H2-type zinc finger protein MtZfp-c2h2 were induced by P. indica colonization as well. Further work indicated that salinity resistance was increased in overexpressing P5CS2, MtAlfin1-like and MtZfp-c2h2 transgenic M. truncatula plants. Interestingly, our data showed that the transcription factors MtAlfin1-like and MtZfp-c2h2 were positively contributed to P. indica colonization. These results demonstrate that tolerance to salinity stress was conferred by P. indica in M. truncatula via accumulation of osmoprotectant, stimulating antioxidant enzymes and the expression of defense-related genes. This work revealed the potential application of P. indica's as a plant growth-promoting fungus for the target improvement either in crop protection or in the salinized soil improvement indirectly. Copyright © 2017. Published by Elsevier Masson SAS.

  3. Identification of a Chemically Induced Point Mutation Mediating Herbicide Tolerance in Annual Medics (Medicago spp.)

    PubMed Central

    Oldach, Klaus H.; Peck, David M.; Cheong, Judy; Williams, Kevin J.; Nair, Ramakrishnan M.

    2008-01-01

    Background and Aims Sulfonylurea (SU) herbicides are used extensively in cereal–livestock farming zones as effective and cheap herbicides with useful levels of residual activity. These residues can persist beyond the cropping year, severely affecting legumes in general, and annual medics in particular, resulting in reduced dry matter production, lower seed yields and decreased nitrogen fixation. A strand medic cultivar, Medicago littoralis ‘Angel’, has been developed via chemical mutagenesis with tolerance to SU soil residues. Identifying the molecular basis of the observed tolerance was the aim of this study. Methods Two F2 populations were generated from crosses between ‘Angel’ and varieties of intolerant M. truncatula, the male-sterile mutant tap and the cultivar ‘Caliph’. Genetic mapping with SSR (single sequence repeat) and gene-based markers allowed identification of the trait-defining gene. Quantitative gene expression studies showed the activity of the respective alleles. Key Results Segregation ratios indicated the control of SU-herbicide tolerance by a single dominant gene. SU herbicides inhibit the biosynthesis of the branched-chain amino acids by targeting the acetolactate synthase enzyme, allowing the choice of a mapping approach using acetolactate synthase (ALS) gene homologues as candidates. SSR-marker analysis suggested the ALS-gene homologue on chromosome 3 in M. truncatula. The ALS-gene sequences from ‘Angel’ and intolerant genotypes were sequenced. In ‘Angel’, a single point mutation from C to T translating into an amino acid change from proline to leucine was identified. The polymorphism was used to develop a diagnostic marker for the tolerance trait. Expression of the mutant ALS allele was confirmed by quantitative RT-PCR and showed no differences at various seedling stages and treatments to the corresponding wild-type allele. Conclusions The identification of the trait-defining gene and the development of a diagnostic

  4. Complete nucleotide sequence of Alfalfa mosaic virus isolated from alfalfa (Medicago sativa L.) in Argentina.

    PubMed

    Trucco, Verónica; de Breuil, Soledad; Bejerman, Nicolás; Lenardon, Sergio; Giolitti, Fabián

    2014-06-01

    The complete nucleotide sequence of an Alfalfa mosaic virus (AMV) isolate infecting alfalfa (Medicago sativa L.) in Argentina, AMV-Arg, was determined. The virus genome has the typical organization described for AMV, and comprises 3,643, 2,593, and 2,038 nucleotides for RNA1, 2 and 3, respectively. The whole genome sequence and each encoding region were compared with those of other four isolates that have been completely sequenced from China, Italy, Spain and USA. The nucleotide identity percentages ranged from 95.9 to 99.1 % for the three RNAs and from 93.7 to 99 % for the protein 1 (P1), protein 2 (P2), movement protein and coat protein (CP) encoding regions, whereas the amino acid identity percentages of these proteins ranged from 93.4 to 99.5 %, the lowest value corresponding to P2. CP sequences of AMV-Arg were compared with those of other 25 available isolates, and the phylogenetic analysis based on the CP gene was carried out. The highest percentage of nucleotide sequence identity of the CP gene was 98.3 % with a Chinese isolate and 98.6 % at the amino acid level with four isolates, two from Italy, one from Brazil and the remaining one from China. The phylogenetic analysis showed that AMV-Arg is closely related to subgroup I of AMV isolates. To our knowledge, this is the first report of a complete nucleotide sequence of AMV from South America and the first worldwide report of complete nucleotide sequence of AMV isolated from alfalfa as natural host.

  5. Biogenic Pt uptake and nanoparticle formation in Medicago sativa and Brassica juncea

    NASA Astrophysics Data System (ADS)

    Bali, Roza; Siegele, Rainer; Harris, Andrew T.

    2010-10-01

    The ability of the facultative metallophyte plants, Medicago sativa ( M. sativa) and Brassica juncea ( B. juncea) to accumulate and translocate platinum (Pt) from aqueous substrates is reported. The influence of Pt concentration in the substrate (5, 10, 20, 40 and 80 ppm), exposure time (24, 48 and 72 h) and substrate pH (2, 3, 5, 7 and 9) was determined. In both plants the concentration of Pt increased with substrate concentration and exposure time. Greater accumulation was detected in the roots of M. sativa than B. juncea, up to a maximum of 94.19 mg Pt g-1 (dry biomass) compared with 38.5 mg Pt g-1 (dry biomass) following exposure to 80 ppm Pt after 72 h exposure, respectively. However, at lower substrate concentrations (5 and 20 ppm) greater quantities of Pt were detected in the shoots of B. juncea, ranging between 0.02 and 0.32 mg Pt g-1 (dry biomass) at 5 ppm across the different time intervals studied, compared with 0.02-0.14 mg Pt g-1 (dry biomass) for M. sativa, suggesting B. juncea to be a better translocator of Pt under idealised conditions at low concentrations. Higher Pt uptake was also observed in acidic media, with a maximum at pH 2 for M. sativa and pH 3 for B. juncea, indicating the role of net surface charge on the bioaccumulation of Pt. Once sequestered Pt(II) was reduced to Pt(0) due to the action of local metabolites. TEM images of M. sativa root samples showed the in vivo formation of Pt nanoparticles between 3 and 100 nm in size and of varying morphologies in the epidermal root cells. In vivo Pt distribution profiles were assessed using proton induced X-ray emission (μ-PIXE) spectroscopy, which showed even distribution across all tissue systems (epidermal, cortical and vascular) within the roots of both M. sativa and B. juncea.

  6. Nonphotosynthetic CO/sub 2/ fixation by alfalfa (Medicago sativa L. ) roots and nodules

    SciTech Connect

    Anderson, M.P.; Heichel, G.H.; Vance, C.P.

    1987-09-01

    The dependence of alfalfa (Medicago sativa L.) root and nodule nonphotosynthetic CO/sub 2/ fixation on the supply of currently produced photosynthate and nodule nitrogenase activity was examined a various times after phloem-girdling and exposure of nodules to Ar:O/sub 2/. Phloem-girdling was effected 20 hours and exposure to Ar:O/sub 2/ was effected 2 to 3 hours before initiation of experiments. Nodule and root CO/sub 2/ fixation rates of phloem-girdled plants were reduced to 38 and 50%, respectively, of those of control plants. Exposure to Ar:O/sub 2/ decreased nodule CO/sub 2/ fixation rates to 45%, respiration rates to 55%, and nitrogenase activities to 51% of those of the controls. The products of nodule CO/sub 2/ fixation were exported through the xylem to the shoot mainly as amino acids within 30 to 60 minutes after exposure to /sup 14/CO/sub 2/. In contrast to nodules, roots exported very little radioactivity, and most of the /sup 14/C was exported as organic acids. The nonphotosynthetic CO/sub 2/ fixation rate of roots and nodules averaged 26% of the gross respiration rate, i.e. the sum of net respiration and nonphotosynthetic CO/sub 2/ assimilation. Nodules fixed CO/sub 2/ at a rate 5.6 times that of roots, but since nodules comprised a small portion of root system mass, roots accounted for 76% of the nodulated roots system CO/sub 2/ fixation. The results indicate that nodule CO/sub 2/ fixation in alfalfa is associated with N assimilation.

  7. Superoxide dismutase enhances tolerance of freezing stress in transgenic alfalfa (Medicago sativa L.).

    PubMed Central

    McKersie, B D; Chen, Y; de Beus, M; Bowley, S R; Bowler, C; Inzé, D; D'Halluin, K; Botterman, J

    1993-01-01

    Activated oxygen or oxygen free radicals have been implicated in a number of physiological disorders in plants including freezing injury. Superoxide dismutase (SOD) catalyzes the dismutation of superoxide into O2 and H2O2 and thereby reduces the titer of activated oxygen molecules in the cell. To further examine the relationship between oxidative and freezing stresses, the expression of SOD was modified in transgenic alfalfa (Medicago sativa L.). The Mn-SOD cDNA from Nicotiana plumbaginifolia under the control of the cauliflower mosaic virus 35S promoter was introduced into alfalfa using Agrobacterium tumefaciens-mediated transformation. Two plasmid vectors, pMitSOD and pChlSOD, contained a chimeric Mn-SOD construct with a transit peptide for targeting to the mitochondria or one for targeting to the chloroplast, respectively. The putatively transgenic plants were selected for resistance to kanamycin and screened for neomycin phosphotransferase activity and the presence of an additional Mn-SOD isozyme. Detailed analysis of a set of four selected transformants indicated that some had enhanced SOD activity, increased tolerance to the diphenyl ether herbicide, acifluorfen, and increased regrowth after freezing stress. The F1 progeny of one line, RA3-ChlSOD-30, were analyzed by SOD isozyme activity, by polymerase chain reaction for the Mn-SOD gene, and by polymerase chain reaction for the neo gene. RA3-ChlSOD-30 had three sites of insertion of pChlSOD, but only one gave a functional Mn-SOD isozyme; the other two were apparently partial insertions. The progeny with a functional Mn-SOD transgene had more rapid regrowth following freezing stress than those progeny lacking the functional Mn-SOD transgene, suggesting that Mn-SOD serves a protective role by minimizing oxygen free radical production after freezing stress. PMID:8290627

  8. Speciation Matters: Bioavailability of Silver and Silver Sulfide Nanoparticles to Alfalfa (Medicago sativa).

    PubMed

    Stegemeier, John P; Schwab, Fabienne; Colman, Benjamin P; Webb, Samuel M; Newville, Matthew; Lanzirotti, Antonio; Winkler, Christopher; Wiesner, Mark R; Lowry, Gregory V

    2015-07-21

    Terrestrial crops are directly exposed to silver nanoparticles (Ag-NPs) and their environmentally transformed analog silver sulfide nanoparticles (Ag2S-NPs) when wastewater treatment biosolids are applied as fertilizer to agricultural soils. This leads to a need to understand their bioavailability to plants. In the present study, the mechanisms of uptake and distribution of silver in alfalfa (Medicago sativa) were quantified and visualized upon hydroponic exposure to Ag-NPs, Ag2S-NPs, and AgNO3 at 3 mg total Ag/L. Total silver uptake was measured in dried roots and shoots, and the spatial distribution of elements was investigated using transmission electron microscopy (TEM) and synchrotron-based X-ray imaging techniques. Despite large differences in release of Ag(+) ions from the particles, Ag-NPs, Ag2S-NPs, and Ag(+) became associated with plant roots to a similar degree, and exhibited similarly limited (<1%) amounts of translocation of silver into the shoot system. X-ray fluorescence (XRF) mapping revealed differences in the distribution of Ag into roots for each treatment. Silver nanoparticles mainly accumulated in the (columella) border cells and elongation zone, whereas Ag(+) accumulated more uniformly throughout the root. In contrast, Ag2S-NPs remained largely adhered to the root exterior, and the presence of cytoplasmic nano-SixOy aggregates was observed. Exclusively in roots exposed to particulate silver, NPs smaller than the originally dosed NPs were identified by TEM in the cell walls. The apparent accumulation of Ag in the root apoplast determined by XRF, and the presence of small NPs in root cell walls suggests uptake of partially dissolved NPs and translocation along the apoplast.

  9. Concerted modulation of alanine and glutamate metabolism in young Medicago truncatula seedlings under hypoxic stress.

    PubMed

    Limami, Anis M; Glévarec, Gaëlle; Ricoult, Claudie; Cliquet, Jean-Bernard; Planchet, Elisabeth

    2008-01-01

    The modulation of primary nitrogen metabolism by hypoxic stress was studied in young Medicago truncatula seedlings. Hypoxic seedlings were characterized by the up-regulation of glutamate dehydrogenase 1 (GDH1) and mitochondrial alanine aminotransferase (mAlaAT), and down-regulation of glutamine synthetase 1b (GS1b), NADH-glutamate synthase (NADH-GOGAT), glutamate dehydrogenase 3 (GDH3), and isocitrate dehydrogenase (ICDH) gene expression. Hypoxic stress severely inhibited GS activity and stimulated NADH-GOGAT activity. GDH activity was lower in hypoxic seedlings than in the control, however, under either normoxia or hypoxia, the in vivo activity was directed towards glutamate deamination. (15)NH(4) labelling showed for the first time that the adaptive reaction of the plant to hypoxia consisted of a concerted modulation of nitrogen flux through the pathways of both alanine and glutamate synthesis. In hypoxic seedlings, newly synthesized (15)N-alanine increased and accumulated as the major amino acid, asparagine synthesis was inhibited, while (15)N-glutamate was synthesized at a similar rate to that in the control. A discrepancy between the up-regulation of GDH1 expression and the down-regulation of GDH activity by hypoxic stress highlighted for the first time the complex regulation of this enzyme by hypoxia. Higher rates of glycolysis and ethanol fermentation are known to cause the fast depletion of sugar stores and carbon stress. It is proposed that the expression of GDH1 was stimulated by hypoxia-induced carbon stress, while the enzyme protein might be involved during post-hypoxic stress contributing to the regeneration of 2-oxoglutarate via the GDH shunt.

  10. Influence of pCO2 on carbon allocation in nodulated Medicago sativa L.

    NASA Astrophysics Data System (ADS)

    Pereyra, Gabriela; Hartmann, Henrik; Ziegler, Waldemar; Michalzik, Beate; Gonzalez-Meler, Miquel; Trumbore, Susan

    2016-04-01

    Atmospheric CO2 concentrations (pCO_2) have been related to changes in plant carbon (C) availability and photosynthetic capacity, yet there is no clear consensus as to the effect of pCO2 on the plant C balance and on nitrogen fixation in symbiotic systems. We investigated how different pCO2 (Pleistocene: 170 ppm, ambient: 400 ppm and projected future: 700 ppm) influence C allocation in nodulated Medicago sativa L. We labeled 17 week old plants with depleted 13C (-34.7±1.2‰) and traced the label over a 9-day period, to assess the redistribution of newly assimilated C across different sinks, including nodules. We analyzed N concentrations in plant tissues and found no significant differences in leaves and roots across treatments. However, growth and C fixation rates increased with pCO_2, and differences were greatest between 170 ppm and 700 ppm. Across pCO2 treatments we observed a 13C-enrichment in roots compared to leaves. We further observed the highest 13C depletion of non-structural carbohydrates (NSCs) and respired CO2 in tissues of plants grown at 700 ppm, especially in leaves and nodules. Our preliminary results suggest that sink organs like roots and nodules are fed with newly-assimilated NSCs from leaves to support respiration, and especially in 170 ppm plants represented a major respiratory loss of newly assimilated C (≈ 35{%} of the total plant respiration). Our results suggest that although plant metabolic processes like photosynthesis and respiration are affected by changes in pCO_2, nitrogen acquisition in such a symbiotic system is not.

  11. Mineral accumulation in vegetative and reproductive tissues during seed development in Medicago truncatula.

    PubMed

    Garcia, Christina B; Grusak, Michael A

    2015-01-01

    Enhancing nutrient density in legume seeds is one of several strategies being explored to improve the nutritional quality of the food supply. In order to develop crop varieties with increased seed mineral concentration, a more detailed understanding of mineral translocation within the plant is required. By studying mineral accumulation in different organs within genetically diverse members of the same species, it may be possible to identify variable traits that modulate seed mineral concentration. We utilized two ecotypes (A17 and DZA315.16) of the model legume, Medicago truncatula, to study dry mass and mineral accumulation in the leaves, pod walls, and seeds during reproductive development. The pod wall dry mass was significantly different between the two ecotypes beginning at 12 days after pollination, whereas there was no significant difference in the average dry mass of individual seeds between the two ecotypes at any time point. There were also no significant differences in leaf dry mass between ecotypes; however, we observed expansion of A17 leaves during the first 21 days of pod development, while DZA315.16 leaves did not display a significant increase in leaf area. Mineral profiling of the leaves, pod walls, and seeds highlighted differences in accumulation patterns among minerals within each tissue as well as genotypic differences with respect to individual minerals. Because there were differences in the average seed number per pod, the total seed mineral content per pod was generally higher in A17 than DZA315.16. In addition, mineral partitioning to the seeds tended to be higher in A17 pods. These data revealed that mineral retention within leaves and/or pod walls might attenuate mineral accumulation within the seeds. As a result, strategies to increase seed mineral content should include approaches that will enhance export from these tissues.

  12. The coupling of the plant and microbial catabolisms of phenanthrene in the rhizosphere of Medicago sativa.

    PubMed

    Muratova, Anna; Dubrovskaya, Ekaterina; Golubev, Sergey; Grinev, Vyacheslav; Chernyshova, Marina; Turkovskaya, Olga

    2015-09-01

    We studied the catabolism of the polycyclic aromatic hydrocarbon phenanthrene by four rhizobacterial strains and the possibility of enzymatic oxidation of this compound and its microbial metabolites by the root exudates of alfalfa (Medicago sativa L.) in order to detect the possible coupling of the plant and microbial metabolisms under the rhizospheric degradation of the organic pollutant. A comparative study of phenanthrene degradation pathways in the PAH-degrading rhizobacteria Ensifer meliloti, Pseudomonas kunmingensis, Rhizobium petrolearium, and Stenotrophomonas sp. allowed us to identify the key metabolites from the microbial transformation of phenanthrene, including 9,10-phenanthrenequinone, 2-carboxybenzaldehyde, and 1-hydroxy-2-naphthoic, salicylic, and o-phthalic acids. Sterile alfalfa plants were grown in the presence and absence of phenanthrene (0.03 g kg(-1)) in quartz sand under controlled environmental conditions to obtain plant root exudates. The root exudates were collected, concentrated by ultrafiltration, and the activity of oxidoreductases was detected spectrophotometrically by the oxidation rate for various substrates. The most marked activity was that of peroxidase, whereas the presence of oxidase and tyrosinase was detected on the verge of the assay sensitivity. Using alfalfa root exudates as a crude enzyme preparation, we found that in the presence of the synthetic mediator, the plant peroxidase could oxidize phenanthrene and its microbial metabolites. The results indicate the possibility of active participation of plants in the rhizospheric degradation of polycyclic aromatic hydrocarbons and their microbial metabolites, which makes it possible to speak about the coupling of the plant and microbial catabolisms of these contaminants in the rhizosphere.

  13. Flavones and flavonols play distinct critical roles during nodulation of Medicago truncatula by Sinorhizobium meliloti.

    PubMed

    Zhang, Juan; Subramanian, Senthil; Stacey, Gary; Yu, Oliver

    2009-01-01

    Flavonoids play critical roles in legume-rhizobium symbiosis. However, the role of individual flavonoid compounds in this process has not yet been clearly established. We silenced different flavonoid-biosynthesis enzymes to generate transgenic Medicago truncatula roots with different flavonoid profiles. Silencing of chalcone synthase, the key entry-point enzyme for flavonoid biosynthesis led to flavonoid-deficient roots. Silencing of isoflavone synthase and flavone synthase led to roots deficient for a subset of flavonoids, isoflavonoids (formononetin and biochanin A) and flavones (7,4'-dihydroxyflavone), respectively. When tested for nodulation by Sinorhizobium meliloti, flavonoid-deficient roots had a near complete loss of nodulation, whereas flavone-deficient roots had reduced nodulation. Isoflavone-deficient roots nodulated normally, suggesting that isoflavones might not play a critical role in M. truncatula nodulation, even though they are the most abundant root flavonoids. Supplementation of flavone-deficient roots with 7, 4'-dihydroxyflavone, a major inducer of S. meliloti nod genes, completely restored nodulation. However, the same treatment did not restore nodulation in flavonoid-deficient roots, suggesting that other non-nod gene-inducing flavonoid compounds are also critical to nodulation. Supplementation of roots with the flavonol kaempferol (an inhibitor of auxin transport), in combination with the use of flavone pre-treated S. meliloti cells, completely restored nodulation in flavonoid-deficient roots. In addition, S. meliloti cells constitutively producing Nod factors were able to nodulate flavone-deficient roots, but not flavonoid-deficient roots. These observations indicated that flavones might act as internal inducers of rhizobial nod genes, and that flavonols might act as auxin transport regulators during nodulation. Both these roles of flavonoids appear critical for symbiosis in M. truncatula.

  14. Crosstalk between the nodulation signaling pathway and the autoregulation of nodulation in Medicago truncatula.

    PubMed

    Saur, Isabel M L; Oakes, Marie; Djordjevic, Michael A; Imin, Nijat

    2011-06-01

    A subset of CLAVATA3/endosperm-surrounding region-related (CLE) peptides are involved in autoregulation of nodulation (AON) in Medicago truncatula (e.g. MtCLE12 and MtCLE13). However, their linkage to other components of the AON pathways downstream of the shoot-derived inhibitor (SDI) is not understood. We have ectopically expressed the putative peptide ligand encoding genes MtCLE12 and MtCLE13 in M. truncatula which abolished nodulation completely in wild-type roots but not in the supernodulating null mutant sunn-4. Further, root growth inhibition was detected when MtCLE12 was ectopically expressed in wild-type roots or synthetic CLE12 peptide was applied exogenously. To identify downstream genes, roots of wild-type and sunn-4 mutant overexpressing MtCLE12 were used for quantitative gene expression analysis. We found that, in 35S:MtCLE12 roots, NODULE INCEPTION (NIN, a central regulator of nodulation) was down-regulated, whereas MtEFD (ethylene response factor required for nodule differentiation) and MtRR8 (a type-A response regulator thought to be involved in the negative regulation of cytokinin signaling), were up-regulated. Moreover, we found that the up-regulation of MtEFD and MtRR8 caused by overexpressing MtCLE12 is SUNN-dependent. Hence, our data link for the first time the pathways for Nod factor signaling, cytokinin perception and AON. © 2011 The Authors. New Phytologist © 2011 New Phytologist Trust.

  15. CLE Peptides Control Medicago truncatula Nodulation Locally and Systemically1[C][W][OA

    PubMed Central

    Mortier, Virginie; Den Herder, Griet; Whitford, Ryan; Van de Velde, Willem; Rombauts, Stephane; D'haeseleer, Katrien; Holsters, Marcelle; Goormachtig, Sofie

    2010-01-01

    The CLAVATA3/embryo-surrounding region (CLE) peptides control the fine balance between proliferation and differentiation in plant development. We studied the role of CLE peptides during indeterminate nodule development and identified 25 MtCLE peptide genes in the Medicago truncatula genome, of which two genes, MtCLE12 and MtCLE13, had nodulation-related expression patterns that were linked to proliferation and differentiation. MtCLE13 expression was up-regulated early in nodule development. A high-to-low expression gradient radiated from the inner toward the outer cortical cell layers in a region defining the incipient nodule. At later stages, MtCLE12 and MtCLE13 were expressed in differentiating nodules and in the apical part of mature, elongated nodules. Functional analysis revealed a putative role for MtCLE12 and MtCLE13 in autoregulation of nodulation, a mechanism that controls the number of nodules and involves systemic signals mediated by a leucine-rich repeat receptor-like kinase, SUNN, which is active in the shoot. When MtCLE12 and MtCLE13 were ectopically expressed in transgenic roots, nodulation was abolished at the level of the nodulation factor signal transduction, and this inhibition involved long-distance signaling. In addition, composite plants with roots ectopically expressing MtCLE12 or MtCLE13 had elongated petioles. This systemic effect was not observed in transgenic roots ectopically expressing MtCLE12 and MtCLE13 in a sunn-1 mutant background, although nodulation was still strongly reduced. These results suggest multiple roles for CLE signaling in nodulation. PMID:20348212

  16. Genomic Signature of Selective Sweeps Illuminates Adaptation of Medicago truncatula to Root-Associated Microorganisms

    PubMed Central

    Bonhomme, Maxime; Boitard, Simon; San Clemente, Hélène; Dumas, Bernard; Young, Nevin; Jacquet, Christophe

    2015-01-01

    Medicago truncatula is a model legume species used to investigate plant–microorganism interactions, notably root symbioses. Massive population genomic and transcriptomic data now available for this species open the way for a comprehensive investigation of genomic variations associated with adaptation of M. truncatula to its environment. Here we performed a fine-scale genome scan of selective sweep signatures in M. truncatula using more than 15 million single nucleotide polymorphisms identified on 283 accessions from two populations (Circum and Far West), and exploited annotation and published transcriptomic data to identify biological processes associated with molecular adaptation. We identified 58 swept genomic regions with a 15 kb average length and comprising 3.3 gene models on average. The unimodal sweep state probability distribution in these regions enabled us to focus on the best single candidate gene per region. We detected two unambiguous species-wide selective sweeps, one of which appears to underlie morphological adaptation. Population genomic analyses of the remaining 56 sweep signatures indicate that sweeps identified in the Far West population are less population-specific and probably more ancient than those identified in the Circum population. Functional annotation revealed a predominance of immunity-related adaptations in the Circum population. Transcriptomic data from accessions of the Far West population allowed inference of four clusters of coregulated genes putatively involved in the adaptive control of symbiotic carbon flow and nodule senescence, as well as in other root adaptations upon infection with soil microorganisms. We demonstrate that molecular adaptations in M. truncatula were primarily triggered by selective pressures from root-associated microorganisms. PMID:25901015

  17. Fine-Scale Population Recombination Rates, Hotspots, and Correlates of Recombination in the Medicago truncatula Genome

    PubMed Central

    Paape, Timothy; Zhou, Peng; Branca, Antoine; Briskine, Roman; Young, Nevin; Tiffin, Peter

    2012-01-01

    Recombination rates vary across the genome and in many species show significant relationships with several genomic features, including distance to the centromere, gene density, and GC content. Studies of fine-scale recombination rates have also revealed that in several species, there are recombination hotspots, that is, short regions with recombination rates 10–100 greater than those in surrounding regions. In this study, we analyzed whole-genome resequence data from 26 accessions of the model legume Medicago truncatula to gain insight into the genomic features that are related to high- and low-recombination rates and recombination hotspots at 1 kb scales. We found that high-recombination regions (1-kb windows among those in the highest 5% of the distribution) on all three chromosomes were significantly closer to the centromere, had higher gene density, and lower GC content than low-recombination windows. High-recombination windows are also significantly overrepresented among some gene functional categories—most strongly NB–ARC and LRR genes, both of which are important in plant defense against pathogens. Similar to high-recombination windows, recombination hotspots (1-kb windows with significantly higher recombination than the surrounding region) are significantly nearer to the centromere than nonhotspot windows. By contrast, we detected no difference in gene density or GC content between hotspot and nonhotspot windows. Using linear model wavelet analysis to examine the relationship between recombination and genomic features across multiple spatial scales, we find a significant negative correlation with distance to the centromere across scales up to 512 kb, whereas gene density and GC content show significantly positive and negative correlations, respectively, only up to 64 kb. Correlations between recombination and genomic features, particularly gene density and polymorphism, suggest that they are scale dependent and need to be assessed at scales relevant

  18. Karyotypic evolution of the Medicago complex: sativa-caerulea-falcata inferred from comparative cytogenetic analysis.

    PubMed

    Yu, Feng; Wang, Haiqing; Zhao, Yanyan; Liu, Ruijuan; Dou, Quanwen; Dong, Jiangli; Wang, Tao

    2017-04-21

    Polyploidy plays an important role in the adaptation and speciation of plants. The alteration of karyotype is a significant event during polyploidy formation. The Medicago sativa complex includes both diploid (2n = 2× = 16) and tetraploid (2n = 2× = 32) subspecies. The tetraploid M. ssp. sativa was regarded as having a simple autopolyploid origin from diploid ssp. caerulea, whereas the autopolyploid origin of tetraploid ssp. falcata from diploid form ssp. falcata is still in doubt. In this study, detailed comparative cytogenetic analysis between diploid to tetraploid species, as well as genomic affinity across different species in the M. sativa complex, were conducted based on comparative mapping of 11 repeated DNA sequences and two rDNA sequences by a fluorescence in situ hybridization (FISH) technique. FISH patterns of the repeats in diploid subspecies caerulea were highly similar to those in tetraploid subspecies sativa. Distinctly different FISH patterns were first observed in diploid ssp. falcata, with only centromeric hybridizations using centromeric and multiple region repeats and a few subtelomeric hybridizations using subtelomeric repeats. Tetraploid subspecies falcata was unexpectedly found to possess a highly variable karyotype, which agreed with neither diploid ssp. falcata nor ssp. sativa. Reconstruction of chromosome-doubling process of diploid ssp. caerulea showed that chromosome changes have occurred during polyploidization process. The comparative cytogenetic results provide reliable evidence that diploid subspecies caerulea is the direct progenitor of tetraploid subspecies sativa. And autotetraploid ssp. sativa has been suggested to undergo a partial diploidization by the progressive accumulation of chromosome structural rearrangements during evolution. However, the tetraploid subspecies falcata is far from a simple autopolyploid from diploid subspecies falcata although no obvious morphological change was observed between these two

  19. Comparative gene expression profiles between heterotic and non-heterotic hybrids of tetraploid Medicago sativa

    PubMed Central

    Li, Xuehui; Wei, Yanling; Nettleton, Dan; Brummer, E Charles

    2009-01-01

    Background Heterosis, the superior performance of hybrids relative to parents, has clear agricultural value, but its genetic control is unknown. Our objective was to test the hypotheses that hybrids expressing heterosis for biomass yield would show more gene expression levels that were different from midparental values and outside the range of parental values than hybrids that do not exhibit heterosis. Results We tested these hypotheses in three Medicago sativa (alfalfa) genotypes and their three hybrids, two of which expressed heterosis for biomass yield and a third that did not, using Affymetrix M. truncatula GeneChip arrays. Alfalfa hybridized to approximately 47% of the M. truncatula probe sets. Probe set signal intensities were analyzed using MicroArray Suite v.5.0 (MAS) and robust multi-array average (RMA) algorithms. Based on MAS analysis, the two heterotic hybrids performed similarly, with about 27% of genes showing differential expression among the parents and their hybrid compared to 12.5% for the non-heterotic hybrid. At a false discovery rate of 0.15, 4.7% of differentially expressed genes in hybrids (~300 genes) showed nonadditive expression compared to only 0.5% (16 genes) in the non-heterotic hybrid. Of the nonadditively expressed genes, approximately 50% showed expression levels that fell outside the parental range in heterotic hybrids, but only one of 16 showed a similar profile in the non-heterotic hybrid. Genes whose expression differed in the parents were three times more likely to show nonadditive expression than genes whose parental transcript levels were equal. Conclusion The higher proportions of probe sets with expression level that differed from the parental midparent value and that were more extreme than either parental value in the heterotic hybrids compared to a non-heterotic hybrid were also found using RMA. We conclude that nonadditive expression of transcript levels may contribute to heterosis for biomass yield in alfalfa. PMID

  20. Sexual Polyploidization in Medicago sativa L.: Impact on the Phenotype, Gene Transcription, and Genome Methylation.

    PubMed

    Rosellini, Daniele; Ferradini, Nicoletta; Allegrucci, Stefano; Capomaccio, Stefano; Zago, Elisa Debora; Leonetti, Paola; Balech, Bachir; Aversano, Riccardo; Carputo, Domenico; Reale, Lara; Veronesi, Fabio

    2016-04-07

    Polyploidization as the consequence of 2n gamete formation is a prominent mechanism in plant evolution. Studying its effects on the genome, and on genome expression, has both basic and applied interest. We crossed two diploid (2n = 2x = 16) Medicago sativa plants, a subsp. falcata seed parent, and a coerulea × falcata pollen parent that form a mixture of n and 2n eggs and pollen, respectively. Such a cross produced full-sib diploid and tetraploid (2n = 4x = 32) hybrids, the latter being the result of bilateral sexual polyploidization (BSP). These unique materials allowed us to investigate the effects of BSP, and to separate the effect of intraspecific hybridization from those of polyploidization by comparing 2x with 4x full sib progeny plants. Simple sequence repeat marker segregation demonstrated tetrasomic inheritance for all chromosomes but one, demonstrating that these neotetraploids are true autotetraploids. BSP brought about increased biomass, earlier flowering, higher seed set and weight, and larger leaves with larger cells. Microarray analyses with M. truncatula gene chips showed that several hundred genes, related to diverse metabolic functions, changed their expression level as a consequence of polyploidization. In addition, cytosine methylation increased in 2x, but not in 4x, hybrids. Our results indicate that sexual polyploidization induces significant transcriptional novelty, possibly mediated in part by DNA methylation, and phenotypic novelty that could underpin improved adaptation and reproductive success of tetraploid M. sativa with respect to its diploid progenitor. These polyploidy-induced changes may have promoted the adoption of tetraploid alfalfa in agriculture. Copyright © 2016 Rosellini et al.

  1. Sexual Polyploidization in Medicago sativa L.: Impact on the Phenotype, Gene Transcription, and Genome Methylation

    PubMed Central

    Rosellini, Daniele; Ferradini, Nicoletta; Allegrucci, Stefano; Capomaccio, Stefano; Zago, Elisa Debora; Leonetti, Paola; Balech, Bachir; Aversano, Riccardo; Carputo, Domenico; Reale, Lara; Veronesi, Fabio

    2016-01-01

    Polyploidization as the consequence of 2n gamete formation is a prominent mechanism in plant evolution. Studying its effects on the genome, and on genome expression, has both basic and applied interest. We crossed two diploid (2n = 2x = 16) Medicago sativa plants, a subsp. falcata seed parent, and a coerulea × falcata pollen parent that form a mixture of n and 2n eggs and pollen, respectively. Such a cross produced full-sib diploid and tetraploid (2n = 4x = 32) hybrids, the latter being the result of bilateral sexual polyploidization (BSP). These unique materials allowed us to investigate the effects of BSP, and to separate the effect of intraspecific hybridization from those of polyploidization by comparing 2x with 4x full sib progeny plants. Simple sequence repeat marker segregation demonstrated tetrasomic inheritance for all chromosomes but one, demonstrating that these neotetraploids are true autotetraploids. BSP brought about increased biomass, earlier flowering, higher seed set and weight, and larger leaves with larger cells. Microarray analyses with M. truncatula gene chips showed that several hundred genes, related to diverse metabolic functions, changed their expression level as a consequence of polyploidization. In addition, cytosine methylation increased in 2x, but not in 4x, hybrids. Our results indicate that sexual polyploidization induces significant transcriptional novelty, possibly mediated in part by DNA methylation, and phenotypic novelty that could underpin improved adaptation and reproductive success of tetraploid M. sativa with respect to its diploid progenitor. These polyploidy-induced changes may have promoted the adoption of tetraploid alfalfa in agriculture. PMID:26858330

  2. Medicago truncatula genotypes Jemalong A17 and R108 show contrasting variations under drought stress.

    PubMed

    Luo, Shi-Shuai; Sun, Yan-Ni; Zhou, Xue; Zhu, Tong; Zhu, Li-Sha; Arfan, Muhammad; Zou, Li-Juan; Lin, Hong-Hui

    2016-12-01

    Drought is one of the most significant abiotic stresses that restrict crop productivity. Medicago truncatula is a model legume species with a wide genetic diversity. We compared the differential physiological and molecular changes of two genotypes of M. truncatula (Jemalong A17 and R108) in response to progressive drought stress and rewatering. The MtNCED and MtZEP activation and higher abscisic acid (ABA) content was observed in Jemalong A17 plants under normal conditions. Additionally, a greater increase in ABA content and expression of MtNCED and MtZEP in Jemalong A17 plants than that of R108 plants were observed under drought conditions. A more ABA-sensitive stomatal closure and a slower water loss was found in excised leaves of Jemalong A17 plants. Meanwhile, Jemalong A17 plants alleviated leaf wilting and maintained higher relative water content under drought conditions. Exposed to drought stress, Jemalong A17 plants exhibited milder oxidative damage which has less H2O2 and MDA accumulation, lower electrolyte leakage and higher chlorophyll content and PSII activity. Furthermore, Jemalong A17 plants enhanced expression of stress-upregulated genes under drought conditions. These results suggest that genotypes Jemalong A17 and R108 differed in their response and adaptation to drought stress. Given the relationship between ABA and these physiological responses, the MtNCED and MtZEP activation under normal conditions may play an important role in regulation of greater tolerance of Jemalong A17 plants to drought stress. The activation of MtNCED and MtZEP may lead to the increase of ABA content which may activate expression of drought-stress-regulated genes and cause a series of physiological resistant responses. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  3. Enhanced Secondary- and Hormone Metabolism in Leaves of Arbuscular Mycorrhizal Medicago truncatula1[OPEN

    PubMed Central

    Adolfsson, Lisa; Šimura, Jan; Beebo, Azeez; Aboalizadeh, Jila; Široká, Jitka

    2017-01-01

    Arbuscular mycorrhizas (AM) are the most common symbiotic associations between a plant’s root compartment and fungi. They provide nutritional benefit (mostly inorganic phosphate [Pi]), leading to improved growth, and nonnutritional benefits, including defense responses to environmental cues throughout the host plant, which, in return, delivers carbohydrates to the symbiont. However, how transcriptional and metabolic changes occurring in leaves of AM plants differ from those induced by Pi fertilization is poorly understood. We investigated systemic changes in the leaves of mycorrhized Medicago truncatula in conditions with no improved Pi status and compared them with those induced by high-Pi treatment in nonmycorrhized plants. Microarray-based genome-wide profiling indicated up-regulation by mycorrhization of genes involved in flavonoid, terpenoid, jasmonic acid (JA), and abscisic acid (ABA) biosynthesis as well as enhanced expression of MYC2, the master regulator of JA-dependent responses. Accordingly, total anthocyanins and flavonoids increased, and most flavonoid species were enriched in AM leaves. Both the AM and Pi treatments corepressed iron homeostasis genes, resulting in lower levels of available iron in leaves. In addition, higher levels of cytokinins were found in leaves of AM- and Pi-treated plants, whereas the level of ABA was increased specifically in AM leaves. Foliar treatment of nonmycorrhized plants with either ABA or JA induced the up-regulation of MYC2, but only JA also induced the up-regulation of flavonoid and terpenoid biosynthetic genes. Based on these results, we propose that mycorrhization and Pi fertilization share cytokinin-mediated improved shoot growth, whereas enhanced ABA biosynthesis and JA-regulated flavonoid and terpenoid biosynthesis in leaves are specific to mycorrhization. PMID:28698354

  4. Exploring the nuclear proteome of Medicago truncatula at the switch towards seed filling.

    PubMed

    Repetto, Ombretta; Rogniaux, Hélène; Firnhaber, Christian; Zuber, Hélène; Küster, Helge; Larré, Colette; Thompson, Richard; Gallardo, Karine

    2008-11-01

    Despite its importance in determining seed composition, and hence quality, regulation of the development of legume seeds is incompletely understood. Because of the cardinal role played by the nucleus in gene expression and regulation, we have characterized the nuclear proteome of Medicago truncatula at the 12 days after pollination (dap) stage that marks the switch towards seed filling. Nano-liquid chromatography-tandem mass spectrometry analysis of nuclear protein bands excised from one-dimensional SDS-PAGE identified 179 polypeptides (143 different proteins), providing an insight into the complexity and distinctive feature of the seed nuclear proteome and highlighting new plant nuclear proteins with possible roles in the biogenesis of ribosomal subunits (PESCADILLO-like) or nucleocytoplasmic trafficking (dynamin-like GTPase). The results revealed that nuclei of 12-dap seeds store a pool of ribosomal proteins in preparation for intense protein synthesis activity, occurring subsequently during seed filling. Diverse proteins of the molecular machinery leading to the synthesis of ribosomal subunits were identified along with proteins involved in transcriptional regulation, RNA processing or transport. Some had already been shown to play a role during the early stages of seed formation whereas for others the findings are novel (e.g. the DIP2 and ES43 transcriptional regulators or the RNA silencing-related ARGONAUTE proteins). This study also revealed the presence of chromatin-modifying enzymes and RNA interference proteins that have roles in RNA-directed DNA methylation and may be involved in modifying genome architecture and accessibility during seed filling and maturation.

  5. Catalase and superoxide dismutase in alfalfa root nodules. [Medicago sativa L

    SciTech Connect

    Becana, M.; Aparicio-Tejo, P.M.; Sanchez-Diaz, M.

    1986-04-01

    Catalase and superoxide dismutase (SOD), in scavenging H/sub 2/ O/sub 2/ and O/sub 2/, respectively, have been recently proposed to play a role in leghemoglobin protection. The occurrence of catalase and SOD activities in alfalfa (Medicago sativa L.) nodule cytosol is reported here. Enzymes were extracted at 0-4/sup 0/C from 0.5 g fresh nodules with 12 ml of a medium containing K-phosphate buffer 50 mM, pH 7.8 and Na/sub 2/EDTA 0.1 mM. The homogenate was filtered and centrifuged at 18,000 xg for 10 min, and the resulting supernatant was used for catalase assay. A further precipitation of leghemoglobin was required to avoid interferences with SOD determination. Catalase was determined by back-titration with KMnO/sub 4/. SOD was assayed by measuring the inhibition of nitro blue tetrazolium reduction. The sensitivity of SOD activity to CN/sup -/ was tested by including 1 mM KCN in the reaction mixture. Catalase activity of alfalfa nodule cytosol was 237 +/- 1 units/mg protein, decreasing very significantly (P < 0.01, Duncan's multiple range test) at 20 mM NO/sub 3//sup -/. Typical specific SOD activities were 94 +/- 5 and 65 +/- 4 units/mg protein, without CN/sup -/ and with CN/sup -/, respectively. Both activities increased very significantly at 20 mM NO/sub 3//sup -/. SOD activities with CN/sup -/ were 70-80% those without CN/sup -/ within the range of NO/sub 3//sup -/ investigated (0-20 mM).

  6. Ensifer meliloti is the preferred symbiont of Medicago arborea in eastern Morocco soils.

    PubMed

    Guerrouj, Kamal; Pérez-Valera, Eduardo; Abdelmoumen, Hanaa; Bedmar, Eulogio J; Missbah El Idrissi, Mustapha

    2013-08-01

    Polyphasic characterization of 61 bacteria isolated from root nodules of Medicago arborea (Medic tree) plants growing in 4 arid soils of the arid eastern area of Morocco was studied. All the isolates characterized were fast growers. The phenotypic, symbiotic, and cultural characteristics analyzed allowed the description of a broad physiological diversity among the isolates. The results obtained suggest that the phenotype of these rhizobia might have evolved to adapt to the local conditions. The genetic characterization consisted of an analysis of the rep-PCR (repetitive extragenic palindromic polymerase chain reaction) fingerprints and a PCR-based RFLP (restriction fragment length polymorphism) of the 16S rDNA patterns. The diversity of the isolates was investigated by rep-PCR, giving a similarity of 62%, delineated into 3 clusters, 4 groups, and 6 subclusters. This wide diversity was also observed by a phenotypic approach, where the carbohydrate assimilation test was the most discriminating. The results show a relationship between rep-PCR fingerprinting and sugar assimilation, which are complementary in diversity investigation. The nearly complete 16S rRNA gene sequence from representative strains of each soil showed they are closely related to members of the genus Ensifer of the family Rhizobiaceae within the Alphaproteobacteria and shows the highest similitude values (99.93%/100%) with Ensifer meliloti LMG 6133(T) (X67222). Sequencing of the symbiotic nodC gene from 7 representative strains revealed they had 94.89% identity with the nodC sequence of the type strain E. meliloti LMG 6133(T) (EF428922). Therefore, the 61 M. arborea isolates from the 4 different soils have the same phylogenetic affiliation, which proves the restricted host specificity among M. arborea species.

  7. Proteomic Identification of Differentially Expressed Proteins during Alfalfa (Medicago sativa L.) Flower Development.

    PubMed

    Chen, Lingling; Chen, Quanzhu; Zhu, Yanqiao; Hou, Longyu; Mao, Peisheng

    2016-01-01

    Flower development, pollination, and fertilization are important stages in the sexual reproduction process of plants; they are also critical steps in the control of seed formation and development. During alfalfa (Medicago sativa L.) seed production, some distinct phenomena such as a low seed setting ratio, serious flower falling, and seed abortion commonly occur. However, the causes of these phenomena are complicated and largely unknown. An understanding of the mechanisms that regulate alfalfa flowering is important in order to increase seed yield. Hence, proteomic technology was used to analyze changes in protein expression during the stages of alfalfa flower development. Flower samples were collected at pre-pollination (S1), pollination (S2), and the post-pollination senescence period (S3). Twenty-four differentially expressed proteins were successfully identified, including 17 down-regulated in pollinated flowers, one up-regulated in pollinated and senesced flowers, and six up-regulated in senesced flowers. The largest proportions of the identified proteins were involved in metabolism, signal transduction, defense response, oxidation reduction, cell death, and programmed cell death (PCD). Their expression profiles demonstrated that energy metabolism, carbohydrate metabolism, and amino acid metabolism provided the nutrient foundation for pollination in alfalfa. Furthermore, there were three proteins involved in multiple metabolic pathways: dual specificity kinase splA-like protein (kinase splALs), carbonic anhydrase, and NADPH: quinone oxidoreductase-like protein. Expression patterns of these proteins indicated that MAPK cascades regulated multiple processes, such as signal transduction, stress response, and cell death. PCD also played an important role in the alfalfa flower developmental process, and regulated both pollination and flower senescence. The current study sheds some light on protein expression profiles during alfalfa flower development and

  8. Genome-Wide Association Mapping and Genomic Selection for Alfalfa (Medicago sativa) Forage Quality Traits.

    PubMed

    Biazzi, Elisa; Nazzicari, Nelson; Pecetti, Luciano; Brummer, E Charles; Palmonari, Alberto; Tava, Aldo; Annicchiarico, Paolo

    2017-01-01

    Genetic progress for forage quality has been poor in alfalfa (Medicago sativa L.), the most-grown forage legume worldwide. This study aimed at exploring opportunities for marker-assisted selection (MAS) and genomic selection of forage quality traits based on breeding values of parent plants. Some 154 genotypes from a broadly-based reference population were genotyped by genotyping-by-sequencing (GBS), and phenotyped for leaf-to-stem ratio, leaf and stem contents of protein, neutral detergent fiber (NDF) and acid detergent lignin (ADL), and leaf and stem NDF digestibility after 24 hours (NDFD), of their dense-planted half-sib progenies in three growing conditions (summer harvest, full irrigation; summer harvest, suspended irrigation; autumn harvest). Trait-marker analyses were performed on progeny values averaged over conditions, owing to modest germplasm × condition interaction. Genomic selection exploited 11,450 polymorphic SNP markers, whereas a subset of 8,494 M. truncatula-aligned markers were used for a genome-wide association study (GWAS). GWAS confirmed the polygenic control of quality traits and, in agreement with phenotypic correlations, indicated substantially different genetic control of a given trait in stems and leaves. It detected several SNPs in different annotated genes that were highly linked to stem protein content. Also, it identified a small genomic region on chromosome 8 with high concentration of annotated genes associated with leaf ADL, including one gene probably involved in the lignin pathway. Three genomic selection models, i.e., Ridge-regression BLUP, Bayes B and Bayesian Lasso, displayed similar prediction accuracy, whereas SVR-lin was less accurate. Accuracy values were moderate (0.3-0.4) for stem NDFD and leaf protein content, modest for leaf ADL and NDFD, and low to very low for the other traits. Along with previous results for the same germplasm set, this study indicates that GBS data can be exploited to improve both quality traits

  9. miR396 affects mycorrhization and root meristem activity in the legume Medicago truncatula.

    PubMed

    Bazin, Jérémie; Khan, Ghazanfar Abbas; Combier, Jean-Philippe; Bustos-Sanmamed, Pilar; Debernardi, Juan Manuel; Rodriguez, Ramiro; Sorin, Céline; Palatnik, Javier; Hartmann, Caroline; Crespi, Martin; Lelandais-Brière, Christine

    2013-06-01

    The root system is crucial for acquisition of resources from the soil. In legumes, the efficiency of mineral and water uptake by the roots may be reinforced due to establishment of symbiotic relationships with mycorrhizal fungi and interactions with soil rhizobia. Here, we investigated the role of miR396 in regulating the architecture of the root system and in symbiotic interactions in the model legume Medicago truncatula. Analyses with promoter-GUS fusions suggested that the mtr-miR396a and miR396b genes are highly expressed in root tips, preferentially in the transition zone, and display distinct expression profiles during lateral root and nodule development. Transgenic roots of composite plants that over-express the miR396b precursor showed lower expression of six growth-regulating factor genes (MtGRF) and two bHLH79-like target genes, as well as reduced growth and mycorrhizal associations. miR396 inactivation by mimicry caused contrasting tendencies, with increased target expression, higher root biomass and more efficient colonization by arbuscular mycorrhizal fungi. In contrast to MtbHLH79, repression of three GRF targets by RNA interference severely impaired root growth. Early activation of mtr-miR396b, concomitant with post-transcriptional repression of MtGRF5 expression, was also observed in response to exogenous brassinosteroids. Growth limitation in miR396 over-expressing roots correlated with a reduction in cell-cycle gene expression and the number of dividing cells in the root apical meristem. These results link the miR396 network to the regulation of root growth and mycorrhizal associations in plants. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

  10. Concerted modulation of alanine and glutamate metabolism in young Medicago truncatula seedlings under hypoxic stress

    PubMed Central

    Limami, Anis M.; Glévarec, Gaëlle; Ricoult, Claudie; Cliquet, Jean-Bernard; Planchet, Elisabeth

    2008-01-01

    The modulation of primary nitrogen metabolism by hypoxic stress was studied in young Medicago truncatula seedlings. Hypoxic seedlings were characterized by the up-regulation of glutamate dehydrogenase 1 (GDH1) and mitochondrial alanine aminotransferase (mAlaAT), and down-regulation of glutamine synthetase 1b (GS1b), NADH-glutamate synthase (NADH-GOGAT), glutamate dehydrogenase 3 (GDH3), and isocitrate dehydrogenase (ICDH) gene expression. Hypoxic stress severely inhibited GS activity and stimulated NADH-GOGAT activity. GDH activity was lower in hypoxic seedlings than in the control, however, under either normoxia or hypoxia, the in vivo activity was directed towards glutamate deamination. 15NH4 labelling showed for the first time that the adaptive reaction of the plant to hypoxia consisted of a concerted modulation of nitrogen flux through the pathways of both alanine and glutamate synthesis. In hypoxic seedlings, newly synthesized 15N-alanine increased and accumulated as the major amino acid, asparagine synthesis was inhibited, while 15N-glutamate was synthesized at a similar rate to that in the control. A discrepancy between the up-regulation of GDH1 expression and the down-regulation of GDH activity by hypoxic stress highlighted for the first time the complex regulation of this enzyme by hypoxia. Higher rates of glycolysis and ethanol fermentation are known to cause the fast depletion of sugar stores and carbon stress. It is proposed that the expression of GDH1 was stimulated by hypoxia-induced carbon stress, while the enzyme protein might be involved during post-hypoxic stress contributing to the regeneration of 2-oxoglutarate via the GDH shunt. PMID:18508812

  11. Identification of QTL affecting seed mineral concentrations and content in the model legume Medicago truncatula.

    PubMed

    Sankaran, Renuka P; Huguet, Thierry; Grusak, Michael A

    2009-07-01

    Increasing the amount of bioavailable micronutrients such as iron and zinc in plant foods for human consumption is an international goal, intended especially for developing countries where micronutrient deficiencies are an ongoing health risk. Legume seeds have the potential to provide the essential nutrients required by humans, but concentrations of several minerals are low when compared to other foods. In order to increase seed mineral concentrations, it is important to understand the genes and processes involved in mineral distribution within the plant. The main objectives of this study were to use a Medicago truncatula recombinant inbred population (Jemalong-6 x DZA 315.16) to determine loci governing seed mineral concentrations, seed mineral content, and average seed weight, and to use these loci to propose candidate genes whose expression might contribute to these traits. Ninety-three lines in 2004 and 169 lines in 2006 were grown for seed harvest and subsequent analysis of seed Ca, Cu, Fe, K, Mg, Mn, P, and Zn concentrations and content. Quantitative trait loci (QTL) cartographer was used to identify QTL using composite interval mapping (CIM). CIM identified 46 QTL for seed mineral concentration, 26 for seed mineral content, and 3 for average seed weight. At least one QTL was detected for each mineral trait, and colocation of QTL for several minerals was found in both years. Results comparing seed weight with seed mineral concentration and content QTL demonstrate that seed size can be an important determinant of seed mineral concentration. The identification, in this model legume, of transgressive segregation for nearly all the minerals suggests that allelic recombination of relevant mineral-related genes in agronomic legumes could be a successful strategy to increase seed mineral concentrations above current levels.

  12. Ontogeny of embryogenic callus in Medicago truncatula: the fate of the pluripotent and totipotent stem cells.

    PubMed

    Wang, Xin-Ding; Nolan, Kim E; Irwanto, Rina R; Sheahan, Michael B; Rose, Ray J

    2011-04-01

    Understanding the fate and dynamics of cells during callus formation is essential to understanding totipotency and the mechanisms of somatic embryogenesis. Here, the fate of leaf explant cells during the development of embryogenic callus was investigated in the model legume Medicago truncatula. Callus development was examined from cultured leaf explants of the highly regenerable genotype Jemalong 2HA (2HA) and from mesophyll protoplasts of 2HA and wild-type Jemalong. Callus development was studied by histology, manipulation of the culture system, detection of early production of reactive oxygen species and visualization of SERK1 (SOMATIC EMBRYO RECEPTOR KINASE1) gene expression. Callus formation in leaf explants initiates at the cut surface and within veins of the explant. The ontogeny of callus development is dominated by the division and differentiation of cells derived from pluripotent procambial cells and from dedifferentiated mesophyll cells. Procambium-derived cells differentiated into vascular tissue and rarely formed somatic embryos, whereas dedifferentiated mesophyll cells were competent to form somatic embryos. Interestingly, explants incubated adaxial-side down had substantially less cell proliferation associated with veins yet produced similar numbers of somatic embryos to explants incubated abaxial-side down. Somatic embryos mostly formed on the explant surface originally in contact with the medium, while in protoplast microcalli, somatic embryos only fully developed once at the surface of the callus. Mesophyll protoplasts of 2HA formed embryogenic callus while Jemalong mesophyll protoplasts produced callus rich in vasculature. The ontogeny of embryogenic callus in M. truncatula relates to explant orientation and is driven by the dynamics of pluripotent procambial cells, which proliferate and differentiate into vasculature. The ontogeny is also related to de-differentiated mesophyll cells that acquire totipotency and form the majority of embryos. This

  13. Proteomic Identification of Differentially Expressed Proteins during Alfalfa (Medicago sativa L.) Flower Development

    PubMed Central

    Chen, Lingling; Chen, Quanzhu; Zhu, Yanqiao; Hou, Longyu; Mao, Peisheng

    2016-01-01

    Flower development, pollination, and fertilization are important stages in the sexual reproduction process of plants; they are also critical steps in the control of seed formation and development. During alfalfa (Medicago sativa L.) seed production, some distinct phenomena such as a low seed setting ratio, serious flower falling, and seed abortion commonly occur. However, the causes of these phenomena are complicated and largely unknown. An understanding of the mechanisms that regulate alfalfa flowering is important in order to increase seed yield. Hence, proteomic technology was used to analyze changes in protein expression during the stages of alfalfa flower development. Flower samples were collected at pre-pollination (S1), pollination (S2), and the post-pollination senescence period (S3). Twenty-four differentially expressed proteins were successfully identified, including 17 down-regulated in pollinated flowers, one up-regulated in pollinated and senesced flowers, and six up-regulated in senesced flowers. The largest proportions of the identified proteins were involved in metabolism, signal transduction, defense response, oxidation reduction, cell death, and programmed cell death (PCD). Their expression profiles demonstrated that energy metabolism, carbohydrate metabolism, and amino acid metabolism provided the nutrient foundation for pollination in alfalfa. Furthermore, there were three proteins involved in multiple metabolic pathways: dual specificity kinase splA-like protein (kinase splALs), carbonic anhydrase, and NADPH: quinone oxidoreductase-like protein. Expression patterns of these proteins indicated that MAPK cascades regulated multiple processes, such as signal transduction, stress response, and cell death. PCD also played an important role in the alfalfa flower developmental process, and regulated both pollination and flower senescence. The current study sheds some light on protein expression profiles during alfalfa flower development and

  14. Salinity Adaptation and the Contribution of Parental Environmental Effects in Medicago truncatula.

    PubMed

    Moriuchi, Ken S; Friesen, Maren L; Cordeiro, Matilde A; Badri, Mounawer; Vu, Wendy T; Main, Bradley J; Aouani, Mohamed Elarbi; Nuzhdin, Sergey V; Strauss, Sharon Y; von Wettberg, Eric J B

    2016-01-01

    High soil salinity negatively influences plant growth and yield. Some taxa have evolved mechanisms for avoiding or tolerating elevated soil salinity, which can be modulated by the environment experienced by parents or offspring. We tested the contribution of the parental and offspring environments on salinity adaptation and their potential underlying mechanisms. In a two-generation greenhouse experiment, we factorially manipulated salinity concentrations for genotypes of Medicago truncatula that were originally collected from natural populations that differed in soil salinity. To compare population level adaptation to soil salinity and to test the potential mechanisms involved we measured two aspects of plant performance, reproduction and vegetative biomass, and phenological and physiological traits associated with salinity avoidance and tolerance. Saline-origin populations had greater biomass and reproduction under saline conditions than non-saline populations, consistent with local adaptation to saline soils. Additionally, parental environmental exposure to salt increased this difference in performance. In terms of environmental effects on mechanisms of salinity adaptation, parental exposure to salt spurred phenological differences that facilitated salt avoidance, while offspring exposure to salt resulted in traits associated with greater salt tolerance. Non-saline origin populations expressed traits associated with greater growth in the absence of salt while, for saline adapted populations, the ability to maintain greater performance in saline environments was also associated with lower growth potential in the absence of salt. Plastic responses induced by parental and offspring environments in phenology, leaf traits, and gas exchange contribute to salinity adaptation in M. truncatula. The ability of plants to tolerate environmental stress, such as high soil salinity, is likely modulated by a combination of parental effects and within-generation phenotypic

  15. The plasma membrane proteome of Medicago truncatula roots as modified by arbuscular mycorrhizal symbiosis.

    PubMed

    Aloui, Achref; Recorbet, Ghislaine; Lemaître-Guillier, Christelle; Mounier, Arnaud; Balliau, Thierry; Zivy, Michel; Wipf, Daniel; Dumas-Gaudot, Eliane

    2017-07-19

    In arbuscular mycorrhizal (AM) roots, the plasma membrane (PM) of the host plant is involved in all developmental stages of the symbiotic interaction, from initial recognition to intracellular accommodation of intra-radical hyphae and arbuscules. Although the role of the PM as the agent for cellular morphogenesis and nutrient exchange is especially accentuated in endosymbiosis, very little is known regarding the PM protein composition of mycorrhizal roots. To obtain a global overview at the proteome level of the host PM proteins as modified by symbiosis, we performed a comparative protein profiling of PM fractions from Medicago truncatula roots either inoculated or not with the AM fungus Rhizophagus irregularis. PM proteins were isolated from root microsomes using an optimized discontinuous sucrose gradient; their subsequent analysis by liquid chromatography followed by mass spectrometry (MS) identified 674 proteins. Cross-species sequence homology searches combined with MS-based quantification clearly confirmed enrichment in PM-associated proteins and depletion of major microsomal contaminants. Changes in protein amounts between the PM proteomes of mycorrhizal and non-mycorrhizal roots were monitored further by spectral counting. This workflow identified a set of 82 mycorrhiza-responsive proteins that provided insights into the plant PM response to mycorrhizal symbiosis. Among them, the association of one third of the mycorrhiza-responsive proteins with detergent-resistant membranes pointed at partitioning to PM microdomains. The PM-associated proteins responsive to mycorrhization also supported host plant control of sugar uptake to limit fungal colonization, and lipid turnover events in the PM fraction of symbiotic roots. Because of the depletion upon symbiosis of proteins mediating the replacement of phospholipids by phosphorus-free lipids in the plasmalemma, we propose a role of phosphate nutrition in the PM composition of mycorrhizal roots.

  16. Integrated Metabolomics and Transcriptomics Reveal Enhanced Specialized Metabolism in Medicago truncatula Root Border Cells1[OPEN

    PubMed Central

    Watson, Bonnie S.; Bedair, Mohamed F.; Urbanczyk-Wochniak, Ewa; Huhman, David V.; Yang, Dong Sik; Allen, Stacy N.; Li, Wensheng; Tang, Yuhong; Sumner, Lloyd W.

    2015-01-01

    Integrated metabolomics and transcriptomics of Medicago truncatula seedling border cells and root tips revealed substantial metabolic differences between these distinct and spatially segregated root regions. Large differential increases in oxylipin-pathway lipoxygenases and auxin-responsive transcript levels in border cells corresponded to differences in phytohormone and volatile levels compared with adjacent root tips. Morphological examinations of border cells revealed the presence of significant starch deposits that serve as critical energy and carbon reserves, as documented through increased β-amylase transcript levels and associated starch hydrolysis metabolites. A substantial proportion of primary metabolism transcripts were decreased in border cells, while many flavonoid- and triterpenoid-related metabolite and transcript levels were increased dramatically. The cumulative data provide compounding evidence that primary and secondary metabolism are differentially programmed in border cells relative to root tips. Metabolic resources normally destined for growth and development are redirected toward elevated accumulation of specialized metabolites in border cells, resulting in constitutively elevated defense and signaling compounds needed to protect the delicate root cap and signal motile rhizobia required for symbiotic nitrogen fixation. Elevated levels of 7,4′-dihydroxyflavone were further increased in border cells of roots exposed to cotton root rot (Phymatotrichopsis omnivora), and the value of 7,4′-dihydroxyflavone as an antimicrobial compound was demonstrated using in vitro growth inhibition assays. The cumulative and pathway-specific data provide key insights into the metabolic programming of border cells that strongly implicate a more prominent mechanistic role for border cells in plant-microbe signaling, defense, and interactions than envisioned previously. PMID:25667316

  17. Medicago truncatula symbiotic peptide NCR247 contributes to bacteroid differentiation through multiple mechanisms.

    PubMed

    Farkas, Attila; Maróti, Gergely; Durgő, Hajnalka; Györgypál, Zoltán; Lima, Rui M; Medzihradszky, Katalin F; Kereszt, Attila; Mergaert, Peter; Kondorosi, Éva

    2014-04-08

    Symbiosis between rhizobia soil bacteria and legume plants results in the formation of root nodules where plant cells are fully packed with nitrogen fixing bacteria. In the host cells, the bacteria adapt to the intracellular environment and gain the ability for nitrogen fixation. Depending on the host plants, the symbiotic fate of bacteria can be either reversible or irreversible. In Medicago and related legume species, the bacteria undergo a host-directed multistep differentiation process culminating in the formation of elongated and branched polyploid bacteria with definitive loss of cell division ability. The plant factors are nodule-specific symbiotic peptides. Approximately 600 of them are nodule-specific cysteine-rich (NCR) peptides produced in the rhizobium-infected plant cells. NCRs are targeted to the endosymbionts, and concerted action of different sets of peptides governs different stages of endosymbiont maturation, whereas the symbiotic function of individual NCRs is unknown. This study focused on NCR247, a cationic peptide exhibiting in vitro antimicrobial activities. We show that NCR247 acts in those nodule cells where bacterial cell division is arrested and cell elongation begins. NCR247 penetrates the bacteria and forms complexes with many bacterial proteins. Interaction with FtsZ required for septum formation is one of the host interventions for inhibiting bacterial cell division. Complex formation with the ribosomal proteins affects translation and contributes to altered proteome and physiology of the endosymbiont. Binding to the chaperone GroEL amplifies the NCR247-modulated biological processes. We show that GroEL1 of Sinorhizobium meliloti is required for efficient infection, terminal differentiation, and nitrogen fixation.

  18. Comparative gene expression profiles between heterotic and non-heterotic hybrids of tetraploid Medicago sativa.

    PubMed

    Li, Xuehui; Wei, Yanling; Nettleton, Dan; Brummer, E Charles

    2009-08-13

    Heterosis, the superior performance of hybrids relative to parents, has clear agricultural value, but its genetic control is unknown. Our objective was to test the hypotheses that hybrids expressing heterosis for biomass yield would show more gene expression levels that were different from midparental values and outside the range of parental values than hybrids that do not exhibit heterosis. We tested these hypotheses in three Medicago sativa (alfalfa) genotypes and their three hybrids, two of which expressed heterosis for biomass yield and a third that did not, using Affymetrix M. truncatula GeneChip arrays. Alfalfa hybridized to approximately 47% of the M. truncatula probe sets. Probe set signal intensities were analyzed using MicroArray Suite v.5.0 (MAS) and robust multi-array average (RMA) algorithms. Based on MAS analysis, the two heterotic hybrids performed similarly, with about 27% of genes showing differential expression among the parents and their hybrid compared to 12.5% for the non-heterotic hybrid. At a false discovery rate of 0.15, 4.7% of differentially expressed genes in hybrids (approximately 300 genes) showed nonadditive expression compared to only 0.5% (16 genes) in the non-heterotic hybrid. Of the nonadditively expressed genes, approximately 50% showed expression levels that fell outside the parental range in heterotic hybrids, but only one of 16 showed a similar profile in the non-heterotic hybrid. Genes whose expression differed in the parents were three times more likely to show nonadditive expression than genes whose parental transcript levels were equal. The higher proportions of probe sets with expression level that differed from the parental midparent value and that were more extreme than either parental value in the heterotic hybrids compared to a non-heterotic hybrid were also found using RMA. We conclude that nonadditive expression of transcript levels may contribute to heterosis for biomass yield in alfalfa.

  19. Cloning and characterization of chromosomal markers in alfalfa (Medicago sativa L.).

    PubMed

    Yu, Feng; Lei, Yunting; Li, Yuan; Dou, Quanwen; Wang, Haiqing; Chen, Zhiguo

    2013-07-01

    Eleven tandemly repetitive sequences were identified from a Cot-1 library by FISH and sequence analysis of alfalfa (Medicago sativa). Five repetitive sequences (MsCR-1, MsCR-2, MsCR-3, MsCR-4, and MsCR-5) were centromeric or pericentromeric, of which three were satellite DNAs and two were minisatellite DNAs. Monomers of 144, 148, and 168 bp were identified in MsCR-1, MsCR-2, and MsCR-3, respectively, while 15 and 39 bp monomers were identified in MsCR-4 and MsCR-5, respectively. Three repetitive sequences were characterized as subtelomeric; one repetitive sequence, MsTR-1, had a 184 bp monomer, and two repetitive sequences had fragments of 204 and 327 bp. Sequence analysis revealed homology (70-80 %) between MsTR-1 and a highly repeated sequence (C300) isolated from M. ssp. caerulea. Three identified repetitive sequences produced hybridization signals at multiple sites in a few of the chromosomes; one repetitive sequence was identified as the E180 satellite DNA previously isolated from M. sativa, while the other 163 and 227 bp fragments had distinct sequences. Physical mapping of the repetitive sequences with double-target FISH revealed different patterns. Thus, nine novel tandemly repetitive sequences that can be adopted as distinct chromosome markers in alfalfa were identified in this study. Furthermore, the chromosome distribution of each sequence was well described. Though significant chromosome variations were detected within and between cultivars, a molecular karyotype of alfalfa was suggested with the chromosome markers we identified. Therefore, these novel chromosome markers will still be a powerful tool for genome composition analysis, phylogenetic studies, and breeding applications.

  20. Composite Medicago truncatula plants harbouring Agrobacterium rhizogenes-transformed roots reveal normal mycorrhization by Glomus intraradices

    PubMed Central

    Mrosk, Cornelia; Forner, Susanne; Hause, Gerd; Küster, Helge; Kopka, Joachim; Hause, Bettina

    2009-01-01

    Composite plants consisting of a wild-type shoot and a transgenic root are frequently used for functional genomics in legume research. Although transformation of roots using Agrobacterium rhizogenes leads to morphologically normal roots, the question arises as to whether such roots interact with arbuscular mycorrhizal (AM) fungi in the same way as wild-type roots. To address this question, roots transformed with a vector containing the fluorescence marker DsRed were used to analyse AM in terms of mycorrhization rate, morphology of fungal and plant subcellular structures, as well as transcript and secondary metabolite accumulations. Mycorrhization rate, appearance, and developmental stages of arbuscules were identical in both types of roots. Using Mt16kOLI1Plus microarrays, transcript profiling of mycorrhizal roots showed that 222 and 73 genes exhibited at least a 2-fold induction and less than half of the expression, respectively, most of them described as AM regulated in the same direction in wild-type roots. To verify this, typical AM marker genes were analysed by quantitative reverse transcription-PCR and revealed equal transcript accumulation in transgenic and wild-type roots. Regarding secondary metabolites, several isoflavonoids and apocarotenoids, all known to accumulate in mycorrhizal wild-type roots, have been found to be up-regulated in mycorrhizal in comparison with non-mycorrhizal transgenic roots. This set of data revealed a substantial similarity in mycorrhization of transgenic and wild-type roots of Medicago truncatula, validating the use of composite plants for studying AM-related effects. PMID:19574251

  1. Natural diversity in the model legume Medicago truncatula allows identifying distinct genetic mechanisms conferring partial resistance to Verticillium wilt.

    PubMed

    Ben, Cécile; Toueni, Maoulida; Montanari, Sara; Tardin, Marie-Claire; Fervel, Magalie; Negahi, Azam; Saint-Pierre, Laure; Mathieu, Guillaume; Gras, Marie-Christine; Noël, Dominique; Prospéri, Jean-Marie; Pilet-Nayel, Marie-Laure; Baranger, Alain; Huguet, Thierry; Julier, Bernadette; Rickauer, Martina; Gentzbittel, Laurent

    2013-01-01

    Verticillium wilt is a major threat to alfalfa (Medicago sativa) and many other crops. The model legume Medicago truncatula was used as a host for studying resistance and susceptibility to Verticillium albo-atrum. In addition to presenting well-established genetic resources, this wild plant species enables to investigate biodiversity of the response to the pathogen and putative crosstalk between disease and symbiosis. Symptom scoring after root inoculation and modelling of disease curves allowed assessing susceptibility levels in recombinant lines of three crosses between susceptible and resistant lines, in a core collection of 32 lines, and in mutants affected in symbiosis with rhizobia. A GFP-expressing V. albo-atrum strain was used to study colonization of susceptible plants. Symptoms and colonization pattern in infected M. truncatula plants were typical of Verticillium wilt. Three distinct major quantitative trait loci were identified using a multicross, multisite design, suggesting that simple genetic mechanisms appear to control Verticillium wilt resistance in M. truncatula lines A17 and DZA45.5. The disease functional parameters varied largely in lines of the core collection. This biodiversity with regard to disease response encourages the development of association genetics and ecological approaches. Several mutants of the resistant line, impaired in different steps of rhizobial symbiosis, were affected in their response to V. albo-atrum, which suggests that mechanisms involved in the establishment of symbiosis or disease might have some common regulatory control points.

  2. The lipopolysaccharide of Sinorhizobium meliloti suppresses defense-associated gene expression in cell cultures of the host plant Medicago truncatula.

    PubMed

    Tellström, Verena; Usadel, Björn; Thimm, Oliver; Stitt, Mark; Küster, Helge; Niehaus, Karsten

    2007-02-01

    In the establishment of symbiosis between Medicago truncatula and the nitrogen-fixing bacterium Sinorhizobium meliloti, the lipopolysaccharide (LPS) of the microsymbiont plays an important role as a signal molecule. It has been shown in cell cultures that the LPS is able to suppress an elicitor-induced oxidative burst. To investigate the effect of S. meliloti LPS on defense-associated gene expression, a microarray experiment was performed. For evaluation of the M. truncatula microarray datasets, the software tool MapMan, which was initially developed for the visualization of Arabidopsis (Arabidopsis thaliana) datasets, was adapted by assigning Medicago genes to the ontology originally created for Arabidopsis. This allowed functional visualization of gene expression of M. truncatula suspension-cultured cells treated with invertase as an elicitor. A gene expression pattern characteristic of a defense response was observed. Concomitant treatment of M. truncatula suspension-cultured cells with invertase and S. meliloti LPS leads to a lower level of induction of defense-associated genes compared to induction rates in cells treated with invertase alone. This suppression of defense-associated transcriptional rearrangement affects genes induced as well as repressed by elicitation and acts on transcripts connected to virtually all kinds of cellular processes. This indicates that LPS of the symbiont not only suppresses fast defense responses as the oxidative burst, but also exerts long-term influences, including transcriptional adjustment to pathogen attack. These data indicate a role for LPS during infection of the plant by its symbiotic partner.

  3. Multifaceted Investigation of Metabolites During Nitrogen Fixation in Medicago via High Resolution MALDI-MS Imaging and ESI-MS

    PubMed Central

    Gemperline, Erin; Jayaraman, Dhileepkumar; Maeda, Junko; Ané, Jean-Michel; Li, Lingjun

    2014-01-01

    Legumes have developed the unique ability to establish a symbiotic relationship with soil bacteria known as rhizobia. This interaction results in the formation of root nodules in which rhizobia thrive and reduce atmospheric dinitrogen into plant-usable ammonium through biological nitrogen fixation (BNF). Due to the availability of genetic information for both of the symbiotic partners, the Medicago truncatula–Sinorhizobium meliloti association is an excellent model for examining the BNF process. Although metabolites are important in this symbiotic association, few studies have investigated the array of metabolites that influence this process. Of these studies, most target only a few specific metabolites, the roles of which are either well known or are part of a well-characterized metabolic pathway. Here, we used a multifaceted mass spectrometric (MS) approach to detect and identify the key metabolites that are present during BNF using the Medicago truncatula–Sinorhizobium meliloti association as the model system. High mass accuracy and high resolution matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) Orbitrap instruments were used in this study and provide complementary results for more in-depth characterization of the nitrogen-fixation process. We used well-characterized plant and bacterial mutants to highlight differences between the metabolites that are present in functional vs. non-functional nodules. Our study highlights the benefits of using a combination of mass spectrometric techniques to detect differences in metabolite composition and the distributions of these metabolites in plant biology. PMID:25323862

  4. Enhanced nodulation and nodule development by nolR mutants of Sinorhizobium medicae on specific Medicago host genotypes.

    PubMed

    Sugawara, Masayuki; Sadowsky, Michael J

    2014-04-01

    The nolR gene encodes a negatively acting, transcriptional regulatory protein of core Nod-factor biosynthetic genes in the sinorhizobia. Although previous reports showed that nolR modulates Nod-factor production and enhances nodulation speed of Sinorhizobium meliloti on alfalfa, there have been no reports for the symbiotic function of this gene in the S. medicae-Medicago truncatula symbiosis. Here, we constructed an nolR mutant of S. medicae WSM419 and evaluated mutant and wild-type strains for their nodulation ability, competitiveness, host specificity, and density-dependent nodulation phenotypes. When the mutant was inoculated at low and medium population densities, it showed enhanced nodule formation during the initial stages of nodulation. Results of quantitative competitive nodulation assays indicated that an nolR mutant had 2.3-fold greater competitiveness for nodulation on M. truncatula 'A17' than did the wild-type strain. Moreover, the nodulation phenotype of the nolR mutant differed among Medicago genotypes and showed significantly enhanced nodule development on M. tricycla. Taken together, these results indicated that mutation of nolR in S. medicae positively influenced nodule initiation, competitive nodulation, and nodule development at later nodulation stages. This may allow nolR mutants of S. medicae to have a selective advantage under field conditions.

  5. Medicago truncatula CYP716A12 Is a Multifunctional Oxidase Involved in the Biosynthesis of Hemolytic Saponins[W

    PubMed Central

    Carelli, Maria; Biazzi, Elisa; Panara, Francesco; Tava, Aldo; Scaramelli, Laura; Porceddu, Andrea; Graham, Neil; Odoardi, Miriam; Piano, Efisio; Arcioni, Sergio; May, Sean; Scotti, Carla; Calderini, Ornella

    2011-01-01

    Saponins, a group of glycosidic compounds present in several plant species, have aglycone moieties that are formed using triterpenoid or steroidal skeletons. In spite of their importance as antimicrobial compounds and their possible benefits for human health, knowledge of the genetic control of saponin biosynthesis is still poorly understood. In the Medicago genus, the hemolytic activity of saponins is related to the nature of their aglycone moieties. We have identified a cytochrome P450 gene (CYP716A12) involved in saponin synthesis in Medicago truncatula using a combined genetic and biochemical approach. Genetic loss-of-function analysis and complementation studies showed that CYP716A12 is responsible for an early step in the saponin biosynthetic pathway. Mutants in CYP716A12 were unable to produce hemolytic saponins and only synthetized soyasaponins, and were thus named lacking hemolytic activity (lha). In vitro enzymatic activity assays indicate that CYP716A12 catalyzes the oxidation of β-amyrin and erythrodiol at the C-28 position, yielding oleanolic acid. Transcriptome changes in the lha mutant showed a modulation in the main steps of triterpenic saponin biosynthetic pathway: squalene cyclization, β-amyrin oxidation, and glycosylation. The analysis of CYP716A12 expression in planta is reported together with the sapogenin content in different tissues and stages. This article provides evidence for CYP716A12 being a key gene in hemolytic saponin biosynthesis. PMID:21821776

  6. The Hybridization Barrier between Herbaceous Medicago sativa and Woody M. arborea Is Weakened by Selection of Seed Parents

    PubMed Central

    Bingham, Edwin; Armour, David; Irwin, John

    2013-01-01

    Medicago sativa, alfalfa or lucerne, and M. arborea were considered reproductively isolated until recently. Then, in 2003, an alfalfa genotype was identified that produced a few seeds and progeny with hybrid traits after a large number of pollinations by M. arborea. A derivative of this alfalfa genotype also produced a low frequency of progeny with hybrid traits. Thus, the hybridization barrier was weakened by selection of seed parents. Hybrids from both events expressed traits from M. arborea and M. arborea-specific DNA bands, although more of the M. sativa genome was retained, based on the DNA results. Thus, there was chromatin elimination during embryogenesis, resulting in partial hybrids (hereafter hybrids). However, more than 30 hybrids with an array of M. arborea traits have been obtained thus far, and research continues on the nature of the hybrids. Traits have been genetically transmitted in crosses, and selected traits are in use for alfalfa breeding. This paper reviews the first hybrids and then focuses on further weakening of the hybridization barrier with the discovery of a more efficient hybridizer derived from crossing Medicago sativa subspecies, sativa, coerulea and falcata. This genotype was found to have reproductive abnormalities associated with its complex subspecies origin that are best described as hybrid breakdown. In effect, this subspecies derivative is a bridge-cross parent that consistently produces hybrids. Reproductive abnormalities in the bridge-cross parent are reported and discussed. PMID:27137379

  7. How grow-and-switch gravitropism generates root coiling and root waving growth responses in Medicago truncatula.

    PubMed

    Tan, Tzer Han; Silverberg, Jesse L; Floss, Daniela S; Harrison, Maria J; Henley, Christopher L; Cohen, Itai

    2015-10-20

    Experimental studies show that plant root morphologies can vary widely from straight gravity-aligned primary roots to fractal-like root architectures. However, the opaqueness of soil makes it difficult to observe how environmental factors modulate these patterns. Here, we combine a transparent hydrogel growth medium with a custom built 3D laser scanner to directly image the morphology of Medicago truncatula primary roots. In our experiments, root growth is obstructed by an inclined plane in the growth medium. As the tilt of this rigid barrier is varied, we find Medicago transitions between randomly directed root coiling, sinusoidal root waving, and normal gravity-aligned morphologies. Although these root phenotypes appear morphologically distinct, our analysis demonstrates the divisions are less well defined, and instead, can be viewed as a 2D biased random walk that seeks the path of steepest decent along the inclined plane. Features of this growth response are remarkably similar to the widely known run-and-tumble chemotactic behavior of Escherichia coli bacteria, where biased random walks are used as optimal strategies for nutrient uptake.

  8. How grow-and-switch gravitropism generates root coiling and root waving growth responses in Medicago truncatula

    PubMed Central

    Tan, Tzer Han; Silverberg, Jesse L.; Floss, Daniela S.; Harrison, Maria J.; Henley, Christopher L.; Cohen, Itai

    2015-01-01

    Experimental studies show that plant root morphologies can vary widely from straight gravity-aligned primary roots to fractal-like root architectures. However, the opaqueness of soil makes it difficult to observe how environmental factors modulate these patterns. Here, we combine a transparent hydrogel growth medium with a custom built 3D laser scanner to directly image the morphology of Medicago truncatula primary roots. In our experiments, root growth is obstructed by an inclined plane in the growth medium. As the tilt of this rigid barrier is varied, we find Medicago transitions between randomly directed root coiling, sinusoidal root waving, and normal gravity-aligned morphologies. Although these root phenotypes appear morphologically distinct, our analysis demonstrates the divisions are less well defined, and instead, can be viewed as a 2D biased random walk that seeks the path of steepest decent along the inclined plane. Features of this growth response are remarkably similar to the widely known run-and-tumble chemotactic behavior of Escherichia coli bacteria, where biased random walks are used as optimal strategies for nutrient uptake. PMID:26432881

  9. Analysis of Cell Wall-Related Genes in Organs of Medicago sativa L. under Different Abiotic Stresses

    PubMed Central

    Behr, Marc; Legay, Sylvain; Hausman, Jean-Francois; Guerriero, Gea

    2015-01-01

    Abiotic constraints are a source of concern in agriculture, because they can have a strong impact on plant growth and development, thereby affecting crop yield. The response of plants to abiotic constraints varies depending on the type of stress, on the species and on the organs. Although many studies have addressed different aspects of the plant response to abiotic stresses, only a handful has focused on the role of the cell wall. A targeted approach has been used here to study the expression of cell wall-related genes in different organs of alfalfa plants subjected for four days to three different abiotic stress treatments, namely salt, cold and heat stress. Genes involved in different steps of cell wall formation (cellulose biosynthesis, monolignol biosynthesis and polymerization) have been analyzed in different organs of Medicago sativa L. Prior to this analysis, an in silico classification of dirigent/dirigent-like proteins and class III peroxidases has been performed in Medicago truncatula and M. sativa. The final goal of this study is to infer and compare the expression patterns of cell wall-related genes in response to different abiotic stressors in the organs of an important legume crop. PMID:26193255

  10. Analysis of Cell Wall-Related Genes in Organs of Medicago sativa L. under Different Abiotic Stresses.

    PubMed

    Behr, Marc; Legay, Sylvain; Hausman, Jean-Francois; Guerriero, Gea

    2015-07-16

    Abiotic constraints are a source of concern in agriculture, because they can have a strong impact on plant growth and development, thereby affecting crop yield. The response of plants to abiotic constraints varies depending on the type of stress, on the species and on the organs. Although many studies have addressed different aspects of the plant response to abiotic stresses, only a handful has focused on the role of the cell wall. A targeted approach has been used here to study the expression of cell wall-related genes in different organs of alfalfa plants subjected for four days to three different abiotic stress treatments, namely salt, cold and heat stress. Genes involved in different steps of cell wall formation (cellulose biosynthesis, monolignol biosynthesis and polymerization) have been analyzed in different organs of Medicago sativa L. Prior to this analysis, an in silico classification of dirigent/dirigent-like proteins and class III peroxidases has been performed in Medicago truncatula and M. sativa. The final goal of this study is to infer and compare the expression patterns of cell wall-related genes in response to different abiotic stressors in the organs of an important legume crop.

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

    PubMed

    Revalska, Miglena; Vassileva, Valya; Goormachtig, Sofie; Van Hautegem, Tom; Ratet, Pascal; Iantcheva, Anelia

    2011-04-01

    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.

  12. Natural diversity in the model legume Medicago truncatula allows identifying distinct genetic mechanisms conferring partial resistance to Verticillium wilt

    PubMed Central

    Gentzbittel, Laurent

    2013-01-01

    Verticillium wilt is a major threat to alfalfa (Medicago sativa) and many other crops. The model legume Medicago truncatula was used as a host for studying resistance and susceptibility to Verticillium albo-atrum. In addition to presenting well-established genetic resources, this wild plant species enables to investigate biodiversity of the response to the pathogen and putative crosstalk between disease and symbiosis. Symptom scoring after root inoculation and modelling of disease curves allowed assessing susceptibility levels in recombinant lines of three crosses between susceptible and resistant lines, in a core collection of 32 lines, and in mutants affected in symbiosis with rhizobia. A GFP-expressing V. albo-atrum strain was used to study colonization of susceptible plants. Symptoms and colonization pattern in infected M. truncatula plants were typical of Verticillium wilt. Three distinct major quantitative trait loci were identified using a multicross, multisite design, suggesting that simple genetic mechanisms appear to control Verticillium wilt resistance in M. truncatula lines A17 and DZA45.5. The disease functional parameters varied largely in lines of the core collection. This biodiversity with regard to disease response encourages the development of association genetics and ecological approaches. Several mutants of the resistant line, impaired in different steps of rhizobial symbiosis, were affected in their response to V. albo-atrum, which suggests that mechanisms involved in the establishment of symbiosis or disease might have some common regulatory control points. PMID:23213135

  13. Genome sequence of Ensifer meliloti strain WSM1022; a highly effective microsymbiont of the model legume Medicago truncatula A17

    PubMed Central

    Terpolilli, Jason; Hill, Yvette; Tian, Rui; Howieson, John; Bräu, Lambert; Goodwin, Lynne; Han, James; Liolios, Konstantinos; Huntemann, Marcel; Pati, Amrita; Woyke, Tanja; Mavromatis, Konstantinos; Markowitz, Victor; Ivanova, Natalia; Kyrpides, Nikos; Reeve, Wayne

    2013-01-01

    Ensifer meliloti WSM1022 is an aerobic, motile, Gram-negative, non-spore-forming rod that can exist as a soil saprophyte or as a legume microsymbiont of Medicago. WSM1022 was isolated in 1987 from a nodule recovered from the roots of the annual Medicago orbicularis growing on the Cyclades Island of Naxos in Greece. WSM1022 is highly effective at fixing nitrogen with M. truncatula and other annual species such as M. tornata and M. littoralis and is also highly effective with the perennial M. sativa (alfalfa or lucerne). In common with other characterized E. meliloti strains, WSM1022 will nodulate but fixes poorly with M. polymorpha and M. sphaerocarpos and does not nodulate M. murex. Here we describe the features of E. meliloti WSM1022, together with genome sequence information and its annotation. The 6,649,661 bp high-quality-draft genome is arranged into 121 scaffolds of 125 contigs containing 6,323 protein-coding genes and 75 RNA-only encoding genes, and is one of 100 rhizobial genomes sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project. PMID:24976888

  14. Genome sequence of Ensifer meliloti strain WSM1022; a highly effective microsymbiont of the model legume Medicago truncatula A17.

    PubMed

    Terpolilli, Jason; Hill, Yvette; Tian, Rui; Howieson, John; Bräu, Lambert; Goodwin, Lynne; Han, James; Liolios, Konstantinos; Huntemann, Marcel; Pati, Amrita; Woyke, Tanja; Mavromatis, Konstantinos; Markowitz, Victor; Ivanova, Natalia; Kyrpides, Nikos; Reeve, Wayne

    2013-12-20

    Ensifer meliloti WSM1022 is an aerobic, motile, Gram-negative, non-spore-forming rod that can exist as a soil saprophyte or as a legume microsymbiont of Medicago. WSM1022 was isolated in 1987 from a nodule recovered from the roots of the annual Medicago orbicularis growing on the Cyclades Island of Naxos in Greece. WSM1022 is highly effective at fixing nitrogen with M. truncatula and other annual species such as M. tornata and M. littoralis and is also highly effective with the perennial M. sativa (alfalfa or lucerne). In common with other characterized E. meliloti strains, WSM1022 will nodulate but fixes poorly with M. polymorpha and M. sphaerocarpos and does not nodulate M. murex. Here we describe the features of E. meliloti WSM1022, together with genome sequence information and its annotation. The 6,649,661 bp high-quality-draft genome is arranged into 121 scaffolds of 125 contigs containing 6,323 protein-coding genes and 75 RNA-only encoding genes, and is one of 100 rhizobial genomes sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project.

  15. Chemical investigation of saponins from twelve annual Medicago species and their bioassay with the brine shrimp Artemia salina.

    PubMed

    Tava, Aldo; Pecetti, Luciano

    2012-07-01

    The saponin and sapogenin composition of the aerial growth of 12 annual Medicago species sampled at full senescence were investigated. Saponins were extracted from the plant material and obtained in a highly pure grade by reverse-phase chromatography, with a yield ranging from 0.38 +/- 0.04% to 1.35 +/- 0.08% dry matter, depending on the species. Sapogenins were then obtained after acid hydrolysis of saponins, and evaluated by GC/FID and GC/MS methods. Different compositions of the aglycone moieties were observed in the 12 Medicago species. Medicagenic acid was the dominant aglycone in M. x blancheana, M. doliata, M. littoralis, M. rotata, M. rugosa, M. scutellata, M. tornata and M. truncatula, bayogenin and hederagenin in M. arabica and M. rigidula, echinocystic acid in M. polymorpha, and soyasapogenol B in M. aculeata. The purified saponin mixtures, characterized by different chemical compositions, were then used in a toxicity test using the brine shrimp Artemia salina. The most active compounds were the saponins from M. arabica and M. rigidula with LD50 values of 10.1 and 4.6 microg/mL, respectively. A structure-activity relationship for the tested saponin mixtures was observed.

  16. Multifaceted Investigation of Metabolites During Nitrogen Fixation in Medicago via High Resolution MALDI-MS Imaging and ESI-MS

    NASA Astrophysics Data System (ADS)

    Gemperline, Erin; Jayaraman, Dhileepkumar; Maeda, Junko; Ané, Jean-Michel; Li, Lingjun

    2015-01-01

    Legumes have developed the unique ability to establish a symbiotic relationship with soil bacteria known as rhizobia. This interaction results in the formation of root nodules in which rhizobia thrive and reduce atmospheric dinitrogen into plant-usable ammonium through biological nitrogen fixation (BNF). Owing to the availability of genetic information for both of the symbiotic partners, the Medicago truncatula- Sinorhizobium meliloti association is an excellent model for examining the BNF process. Although metabolites are important in this symbiotic association, few studies have investigated the array of metabolites that influence this process. Of these studies, most target only a few specific metabolites, the roles of which are either well known or are part of a well-characterized metabolic pathway. Here, we used a multifaceted mass spectrometric (MS) approach to detect and identify the key metabolites that are present during BNF using the Medicago truncatula- Sinorhizobium meliloti association as the model system. High mass accuracy and high resolution matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) Orbitrap instruments were used in this study and provide complementary results for more in-depth characterization of the nitrogen-fixation process. We used well-characterized plant and bacterial mutants to highlight differences between the metabolites that are present in functional versus nonfunctional nodules. Our study highlights the benefits of using a combination of mass spectrometric techniques to detect differences in metabolite composition and the distributions of these metabolites in plant biology.

  17. Molecular cloning and functional analysis of the drought tolerance gene MsHSP70 from alfalfa (Medicago sativa L.).

    PubMed

    Li, Zhenyi; Long, Ruicai; Zhang, Tiejun; Wang, Zhen; Zhang, Fan; Yang, Qingchuan; Kang, Junmei; Sun, Yan

    2017-03-01

    Heat shock proteins (HSPs) are a ubiquitously expressed class of protective proteins that play a key role in plant response to stressful conditions. This study aimed to characterize and investigate the function of an HSP gene in alfalfa (Medicago sativa). MsHSP70, which contains a 2028-bp open reading frame, was identified through homology cloning. MsHSP70 shares high sequence identity (94.47%) with HSP70 from Medicago truncatula. Expression analysis of MsHSP70 in alfalfa organs revealed a relatively higher expression level in aerial organs such as flowers, stems and leaves than in roots. MsHSP70 was induced by heat shock, abscisic acid (ABA) and hydrogen peroxide. Transgenic Arabidopsis seedlings overexpressing MsHSP70 were hyposensitive to polyethylene glycol (PEG) and ABA treatments, suggesting that exogenous expression of MsHSP70 enhanced Arabidopsis tolerance to these stresses. Examination of physiological indexes related to drought and ABA stress demonstrated that in comparison with non-transgenic plants, T3 transgenic Arabidopsis plants had an increased proline content, higher superoxide dismutase (SOD) activity, and decreased malondialdehyde (MDA) content. Furthermore, higher relative water content (RWC) was detected in transgenic plants compared with non-transgenic plants under drought stress. These findings clearly indicate that molecular manipulation of MsHSP70 in plants can have substantial effects on stress tolerance.

  18. Control of Dissected Leaf Morphology by a Cys(2)His(2) Zinc Finger Transcription Factor in the Model Legume Medicago Truncatula

    USDA-ARS?s Scientific Manuscript database

    Different plants may have different leaf types. Diversity in leaf types contributes to a large degree of plant diversity in the natural environment. How different leaf morphology is determined is not yet understood. The leguminous plant Medicago truncatula exhibits dissected leaves with three leafl...

  19. Effects of Fe deficiency on the protein profiles and lignin composition of stem tissues of Medicago truncatula in absence and presence of calcium carbonate

    USDA-ARS?s Scientific Manuscript database

    Iron deficiency is a yield-limiting factor with major implications for crop production, especially in soils with high CaCO3. Because stems are essential for the delivery of nutrients to the shoots, the aim of this work was to study the effects of Fe deficiency on the stem proteome of Medicago trunca...

  20. How planting configuration influences plant secondary metabolites and total N in tall fescue (Festuca arundinacea Schreb.), alfalfa (Medicago sativa L.) and birdsfoot trefoil (Lotus corniculatus L.)

    USDA-ARS?s Scientific Manuscript database

    Theories suggest that incorporating alfalfa (Medicago sativa L.; Alf) or birdsfoot trefoil (Lotus corniculatus L.; BFT) into endophyte-infected tall fescue (Festuca arundinaceas Schreb.; E+TF) pasturelands may improve livestock production. We investigated how planting configuration might influence p...

  1. Annual Medicago: From a Model Crop Challenged by a Spectrum of Necrotrophic Pathogens to a Model Plant to Explore the Nature of Disease Resistance

    PubMed Central

    TIVOLI, B.; BARANGER, A.; SIVASITHAMPARAM, K.; BARBETTI, M. J.

    2006-01-01

    • Background Annual Medicago spp., including M. truncatula, play an important agronomic role in dryland farming regions of the world where they are often an integral component of cropping systems, particularly in regions with a Mediterranean or Mediterranean-type climate where they grow as winter annuals that provide both nitrogen and disease breaks for rotational crops. Necrotrophic foliar and soil-borne pathogens dominate these regions and challenge the productivity of annual Medicago and crop legume species. • Scope This review outlines some of the major and/or widespread diseases these necrotrophic pathogens cause on Medicago spp. It then explores the potential for using the spectrum of necrotrophic pathogen–host interactions, with annual Medicago as the host plant, to better understand and model pathosystems within the diseases caused by nectrotrophic pathogens across forage and grain legume crops. • Conclusions Host resistance clearly offers the best strategy for cost-effective, long-term control of necrotrophic foliar and soil-borne pathogens, particularly as useful resistance to a number of these diseases has been identified. Recently and initially, the annual M. truncatula has emerged as a more appropriate and agronomically relevant substitute to Arabidopsis thaliana as a model plant for legumes, and is proving an excellent model to understand the mechanisms of resistance both to individual pathogens and more generally to most forage and grain legume necrotrophic pathogens. PMID:16803846

  2. Transgene silencing of sucrose synthase in alfalfa (Medicago sativa L.) stem vascular tissue suggests a role for invertase in cell wall cellulose synthesis

    USDA-ARS?s Scientific Manuscript database

    Alfalfa (Medicago sativa L.) plants were transformed with two constructs: (1) a truncated phosphoenolpyruvate carboxylase promoter isolated from alfalfa nodules (PEPC-4) fused to GUS; and (2) PEPC-4 fused with sucrose synthase (SUS) isolated from alfalfa nodules. Histochemical staining for GUS in st...

  3. Stress tolerance and symbiotic and phylogenic features of root nodule bacteria associated with Medicago species in different bioclimatic regions of Tunisia.

    PubMed

    Djedidi, Salem; Yokoyama, Tadashi; Ohkama-Ohtsu, Naoko; Risal, Chandra Prasad; Abdelly, Chedly; Sekimoto, Hitoshi

    2011-01-01

    Thirty two rhizobial isolates were obtained from different bioclimatic regions of Tunisia using as trap plants, Medicago sativa, Medicago ciliaris, Medicago polymorpha and Medicago minima. To study their diversity and characterize them in relation to Mediterranean conditions, abiotic stress resistance, symbiotic properties and genetic diversity in terms of 16S rRNA and nodA sequences were assessed. Five isolates from M. sativa, three from M. ciliaris and three from M. minima could grow at 45°C. Only two isolates from M. sativa grew at 4% NaCl. The most stress tolerant isolates were obtained from arid soils. A phylogenetic analysis of 16S rRNA genes revealed 29 isolates to be closely related to Ensifer including one (Pl.3-9) that showed a 16S rRNA sequence similar to that of Ensifer meliloti and nodA sequence similar to that of Ensifer medicae. However, three isolates were categorized into Agrobacterium containing the nodA of Ensifer. Furthermore, these isolates developed nodules on original hosts. The results for the four isolates suggest horizontal gene transfer between the species.

  4. The Medicago FLOWERING LOCUS T Homolog, MtFTa1, Is a Key Regulator of Flowering Time1[C][W][OA

    PubMed Central

    Laurie, Rebecca E.; Diwadkar, Payal; Jaudal, Mauren; Zhang, Lulu; Hecht, Valérie; Wen, Jiangqi; Tadege, Million; Mysore, Kirankumar S.; Putterill, Joanna; Weller, James L.; Macknight, Richard C.

    2011-01-01

    FLOWERING LOCUS T (FT) genes encode proteins that function as the mobile floral signal, florigen. In this study, we characterized five FT-like genes from the model legume, Medicago (Medicago truncatula). The different FT genes showed distinct patterns of expression and responses to environmental cues. Three of the FT genes (MtFTa1, MtFTb1, and MtFTc) were able to complement the Arabidopsis (Arabidopsis thaliana) ft-1 mutant, suggesting that they are capable of functioning as florigen. MtFTa1 is the only one of the FT genes that is up-regulated by both long days (LDs) and vernalization, conditions that promote Medicago flowering, and transgenic Medicago plants overexpressing the MtFTa1 gene flowered very rapidly. The key role MtFTa1 plays in regulating flowering was demonstrated by the identification of fta1 mutants that flowered significantly later in all conditions examined. fta1 mutants do not respond to vernalization but are still responsive to LDs, indicating that the induction of flowering by prolonged cold acts solely through MtFTa1, whereas photoperiodic induction of flowering involves other genes, possibly MtFTb1, which is only expressed in leaves under LD conditions and therefore might contribute to the photoperiodic regulation of flowering. The role of the MtFTc gene is unclear, as the ftc mutants did not have any obvious flowering-time or other phenotypes. Overall, this work reveals the diversity of the regulation and function of the Medicago FT family. PMID:21685176

  5. Diversity and structure of PIF/Harbinger-like elements in the genome of Medicago truncatula

    PubMed Central

    Grzebelus, Dariusz; Lasota, Slawomir; Gambin, Tomasz; Kucherov, Gregory; Gambin, Anna

    2007-01-01

    Background Transposable elements constitute a significant fraction of plant genomes. The PIF/Harbinger superfamily includes DNA transposons (class II elements) carrying terminal inverted repeats and producing a 3 bp target site duplication upon insertion. The presence of an ORF coding for the DDE/DDD transposase, required for transposition, is characteristic for the autonomous PIF/Harbinger-like elements. Based on the above features, PIF/Harbinger-like elements were identified in several plant genomes and divided into several evolutionary lineages. Availability of a significant portion of Medicago truncatula genomic sequence allowed for mining PIF/Harbinger-like elements, starting from a single previously described element MtMaster. Results Twenty two putative autonomous, i.e. carrying an ORF coding for TPase and complete terminal inverted repeats, and 67 non-autonomous PIF/Harbinger-like elements were found in the genome of M. truncatula. They were divided into five families, MtPH-A5, MtPH-A6, MtPH-D,MtPH-E, and MtPH-M, corresponding to three previously identified and two new lineages. The largest families, MtPH-A6 and MtPH-M were further divided into four and three subfamilies, respectively. Non-autonomous elements were usually direct deletion derivatives of the putative autonomous element, however other types of rearrangements, including inversions and nested insertions were also observed. An interesting structural characteristic – the presence of 60 bp tandem repeats – was observed in a group of elements of subfamily MtPH-A6-4. Some families could be related to miniature inverted repeat elements (MITEs). The presence of empty loci (RESites), paralogous to those flanking the identified transposable elements, both autonomous and non-autonomous, as well as the presence of transposon insertion related size polymorphisms, confirmed that some of the mined elements were capable for transposition. Conclusion The population of PIF/Harbinger-like elements in the

  6. In Medicago truncatula, water deficit modulates the transcript accumulation of components of small RNA pathways

    PubMed Central

    2011-01-01

    Background Small RNAs (sRNAs) are 20-24 nucleotide (nt) RNAs and are involved in plant development and response to abiotic stresses. Plants have several sRNA pathways implicated in the transcriptional and post-transcriptional silencing of gene expression. Two key enzyme families common to all pathways are the Dicer-like (DCL) proteins involved in sRNAs maturation and the Argonautes (AGOs) involved in the targeting and functional action of sRNAs. Post-transcriptional silencing mediated by AGOs may occur by cleavage or translational repression of target mRNA's, while transcriptional silencing may be controlled by DNA methylation and chromatin remodeling. Thus far, these gene families have not been characterized in legumes, nor has their involvement in adaptation to water deficit been studied. Results A bioinformatic search in Medicago truncatula genome databases, using Arabidopsis thaliana AGO and DCL cDNA and protein sequences, identified three sequences encoding for putative Dicer-like genes and twelve sequences encoding for putative Argonaute genes. Under water deficit conditions and mainly in roots, MtDCL1 and MtAGO1, two enzymes probably involved in the processing and activation of microRNAs (miRNAs), increased their transcript levels. mir162 which target DCL1 mRNA and mir168 which target AGO1 mRNA reduced their expression in the roots of plants subjected to water deficit. Three putative genes, MtDCL3, MtAGO4b and MtAGO4c probably involved in DNA methylation mechanisms, increased their mRNA levels. However, the mRNA levels of MtAGO6 reduced, which probably encodes a protein with functions similar to MtAGO4. MtAGO7 mRNA levels increased and possibly encodes a protein involved in the production of trans-acting small interfering RNAs. The transcript abundance of MtAGO12a, MtAGO12b and MtAGO12c reduced under water deprivation. Plants recovered from water deprivation reacquire the mRNA levels of the controls. Conclusions Our work demonstrates that in M. truncatula

  7. QTL analysis of seed germination and pre-emergence growth at extreme temperatures in Medicago truncatula.

    PubMed

    Dias, Paula Menna Barreto; Brunel-Muguet, Sophie; Dürr, Carolyne; Huguet, Thierry; Demilly, Didier; Wagner, Marie-Helene; Teulat-Merah, Béatrice

    2011-02-01

    Enhancing the knowledge on the genetic basis of germination and heterotrophic growth at extreme temperatures is of major importance for improving crop establishment. A quantitative trait loci (QTL) analysis was carried out at sub- and supra-optimal temperatures at these early stages in the model Legume Medicago truncatula. On the basis of an ecophysiological model framework, two populations of recombinant inbred lines were chosen for the contrasting behaviours of parental lines: LR5 at sub-optimal temperatures (5 or 10°C) and LR4 at a supra-optimal temperature (20°C). Seed masses were measured in all lines. For LR5, germination rates and hypocotyl growth were measured by hand, whereas for LR4, imbibition and germination rates as well as early embryonic axis growth were measured using an automated image capture and analysis device. QTLs were found for all traits. The phenotyping framework we defined for measuring variables, distinguished stages and enabled identification of distinct QTLs for seed mass (chromosomes 1, 5, 7 and 8), imbibition (chromosome 4), germination (chromosomes 3, 5, 7 and 8) and heterotrophic growth (chromosomes 1, 2, 3 and 8). The three QTL identified for hypocotyl length at sub-optimal temperature explained the largest part of the phenotypic variation (60% together). One digenic interaction was found for hypocotyl width at sub-optimal temperature and the loci involved were linked to additive QTLs for hypocotyl elongation at low temperature. Together with working on a model plant, this approach facilitated the identification of genes specific to each stage that could provide reliable markers for assisting selection and improving crop establishment. With this aim in view, an initial set of putative candidate genes was identified in the light of the role of abscissic acid/gibberellin balance in regulating germination at high temperatures (e.g. ABI4, ABI5), the molecular cascade in response to cold stress (e.g. CBF1, ICE1) and hypotheses on

  8. QTL analysis of seed germination and pre-emergence growth at extreme temperatures in Medicago truncatula

    PubMed Central

    Dias, Paula Menna Barreto; Brunel-Muguet, Sophie; Dürr, Carolyne; Huguet, Thierry; Demilly, Didier; Wagner, Marie-Helene

    2010-01-01

    Enhancing the knowledge on the genetic basis of germination and heterotrophic growth at extreme temperatures is of major importance for improving crop establishment. A quantitative trait loci (QTL) analysis was carried out at sub- and supra-optimal temperatures at these early stages in the model Legume Medicago truncatula. On the basis of an ecophysiological model framework, two populations of recombinant inbred lines were chosen for the contrasting behaviours of parental lines: LR5 at sub-optimal temperatures (5 or 10°C) and LR4 at a supra-optimal temperature (20°C). Seed masses were measured in all lines. For LR5, germination rates and hypocotyl growth were measured by hand, whereas for LR4, imbibition and germination rates as well as early embryonic axis growth were measured using an automated image capture and analysis device. QTLs were found for all traits. The phenotyping framework we defined for measuring variables, distinguished stages and enabled identification of distinct QTLs for seed mass (chromosomes 1, 5, 7 and 8), imbibition (chromosome 4), germination (chromosomes 3, 5, 7 and 8) and heterotrophic growth (chromosomes 1, 2, 3 and 8). The three QTL identified for hypocotyl length at sub-optimal temperature explained the largest part of the phenotypic variation (60% together). One digenic interaction was found for hypocotyl width at sub-optimal temperature and the loci involved were linked to additive QTLs for hypocotyl elongation at low temperature. Together with working on a model plant, this approach facilitated the identification of genes specific to each stage that could provide reliable markers for assisting selection and improving crop establishment. With this aim in view, an initial set of putative candidate genes was identified in the light of the role of abscissic acid/gibberellin balance in regulating germination at high temperatures (e.g. ABI4, ABI5), the molecular cascade in response to cold stress (e.g. CBF1, ICE1) and hypotheses on

  9. Genetic transformation and analysis of rice OsAPx2 gene in Medicago sativa.

    PubMed

    Guan, Qingjie; Takano, Tetsuo; Liu, Shenkui

    2012-01-01

    The OsAPx2 gene from rice was cloned to produce PBI121::OsAPx2 dual-expression plants, of which expression level would be increasing under stressful conditions. The enzyme ascorbate peroxidase (APX) in the leaves and roots of the plants increased with increasing exposure time to different sodium chloride (NaCl) and hydrogen peroxide (H(2)O(2))concentrations, as indicated by protein gel blot analysis. The increased enzyme yield improved the ability of the plants to resist the stress treatments. The OsAPx2 gene was localized in the cytoplasm of epidermal onion cells as indicated by the instantaneous expression of green fluorescence. An 80% regeneration rate was observed in Medicago sativa L. plants transformed with the OsAPx2 gene using Agrobacterium tumefaciens, as indicated by specific primer PCR. The OsAPx2 gene was expressed at the mRNA level and the individual M. sativa (T#1,T#2,T#5) were obtained through assaying the generation of positive T2 using RNA gel blot analysis. When the seeds of the wild type (WT) and the T2 (T#1,T#5) were incubated in culture containing MS with NaCl for 7 days, the results as shown of following: the root length of transgenic plant was longer than WT plants, the H(2)O(2) content in roots of WT was more than of transgenic plants, the APX activity under stresses increased by 2.89 times compared with the WT, the malondialdehyde (MDA) content of the WT was higher than the transgenic plants, the leaves of the WT turned yellow, but those of the transgenic plants remained green and remained healthy. The chlorophyll content in the WT leaves was less than in the transgenic plants, after soaking in solutions of H(2)O(2), sodium sulfite (Na(2)SO(3)), and sodium bicarbonate (NaHCO(3)). Therefore, the OsAPx2 gene overexpression in transgenic M. sativa improves the removal of H(2)O(2) and the salt-resistance compared with WT plants. A novel strain of M. sativa carrying a salt-resistance gene was obtained.

  10. Exploring the plant-associated bacterial communities in Medicago sativa L.

    PubMed

    Pini, Francesco; Frascella, Arcangela; Santopolo, Luisa; Bazzicalupo, Marco; Biondi, Emanuele G; Scotti, Carla; Mengoni, Alessio

    2012-05-20

    Plant-associated bacterial communities caught the attention of several investigators which study the relationships between plants and soil and the potential application of selected bacterial species in crop improvement and protection. Medicago sativa L. is a legume crop of high economic importance as forage in temperate areas and one of the most popular model plants for investigations on the symbiosis with nitrogen fixing rhizobia (mainly belonging to the alphaproteobacterial species Sinorhizobium meliloti). However, despite its importance, no studies have been carried out looking at the total bacterial community associated with the plant. In this work we explored for the first time the total bacterial community associated with M. sativa plants grown in mesocosms conditions, looking at a wide taxonomic spectrum, from the class to the single species (S. meliloti) level. Results, obtained by using Terminal-Restriction Fragment Length Polymorphism (T-RFLP) analysis, quantitative PCR and sequencing of 16 S rRNA gene libraries, showed a high taxonomic diversity as well as a dominance by members of the class Alphaproteobacteria in plant tissues. Within Alphaproteobacteria the families Sphingomonadaceae and Methylobacteriaceae were abundant inside plant tissues, while soil Alphaproteobacteria were represented by the families of Hyphomicrobiaceae, Methylocystaceae, Bradyirhizobiaceae and Caulobacteraceae. At the single species level, we were able to detect the presence of S. meliloti populations in aerial tissues, nodules and soil. An analysis of population diversity on nodules and soil showed a relatively low sharing of haplotypes (30-40%) between the two environments and between replicate mesocosms, suggesting drift as main force shaping S. meliloti population at least in this system. In this work we shed some light on the bacterial communities associated with M. sativa plants, showing that Alphaproteobacteria may constitute an important part of biodiversity in this

  11. Exploring the plant-associated bacterial communities in Medicago sativa L

    PubMed Central

    2012-01-01

    Background Plant-associated bacterial communities caught the attention of several investigators which study the relationships between plants and soil and the potential application of selected bacterial species in crop improvement and protection. Medicago sativa L. is a legume crop of high economic importance as forage in temperate areas and one of the most popular model plants for investigations on the symbiosis with nitrogen fixing rhizobia (mainly belonging to the alphaproteobacterial species Sinorhizobium meliloti). However, despite its importance, no studies have been carried out looking at the total bacterial community associated with the plant. In this work we explored for the first time the total bacterial community associated with M. sativa plants grown in mesocosms conditions, looking at a wide taxonomic spectrum, from the class to the single species (S. meliloti) level. Results Results, obtained by using Terminal-Restriction Fragment Length Polymorphism (T-RFLP) analysis, quantitative PCR and sequencing of 16 S rRNA gene libraries, showed a high taxonomic diversity as well as a dominance by members of the class Alphaproteobacteria in plant tissues. Within Alphaproteobacteria the families Sphingomonadaceae and Methylobacteriaceae were abundant inside plant tissues, while soil Alphaproteobacteria were represented by the families of Hyphomicrobiaceae, Methylocystaceae, Bradyirhizobiaceae and Caulobacteraceae. At the single species level, we were able to detect the presence of S. meliloti populations in aerial tissues, nodules and soil. An analysis of population diversity on nodules and soil showed a relatively low sharing of haplotypes (30-40%) between the two environments and between replicate mesocosms, suggesting drift as main force shaping S. meliloti population at least in this system. Conclusions In this work we shed some light on the bacterial communities associated with M. sativa plants, showing that Alphaproteobacteria may constitute an important

  12. Genome-wide transcriptional analysis of super-embryogenic Medicago truncatula explant cultures

    PubMed Central

    Imin, Nijat; Goffard, Nicolas; Nizamidin, Mahira; Rolfe, Barry G

    2008-01-01

    Background The Medicago truncatula (M. truncatula) line 2HA has a 500-fold greater capacity to regenerate plants in culture by somatic embryogenesis than its wild type progenitor Jemalong. To understand the molecular basis for the regeneration capacity of this super-embryogenic line 2HA, using Affymetrix GeneChip®, we have compared transcriptomes of explant leaf cultures of these two lines that were grown on media containing the auxin NAA (1-naphthaleneacetic acid) and the cytokinin BAP (6-benzylaminopurine) for two weeks, an early time point for tissue culture proliferation. Results Using Affymetrix GeneChip®, GCRMA normalisation and statistical analysis, we have shown that more than 196 and 49 probe sets were significantly (p < 0.05) up- or down-regulated respectively more than 2 fold in expression. We have utilised GeneBins, a database for classifying gene expression data to distinguish differentially displayed pathways among these two cultures which showed changes in number of biochemical pathways including carbon and flavonoid biosynthesis, phytohormone biosynthesis and signalling. The up-regulated genes in the embryogenic 2HA culture included nodulins, transporters, regulatory genes, embryogenesis related arabinogalactans and genes involved in redox homeostasis, the transition from vegetative growth to reproductive growth and cytokinin signalling. Down-regulated genes included protease inhibitors, wound-induced proteins, and genes involved in biosynthesis and signalling of phytohormones auxin, gibberellin and ethylene. These changes indicate essential differences between the super-embryogenic line 2HA and Jemalong not only in many aspects of biochemical pathways but also in their response to auxin and cytokinin. To validate the GeneChip results, we used quantitative real-time RT-PCR to examine the expression of the genes up-regulated in 2HA such as transposase, RNA-directed DNA polymerase, glycoside hydrolase, RESPONSE REGULATOR 10, AGAMOUS-LIKE 20, flower

  13. Genome-Wide Association Mapping and Genomic Selection for Alfalfa (Medicago sativa) Forage Quality Traits

    PubMed Central

    Pecetti, Luciano; Brummer, E. Charles; Palmonari, Alberto; Tava, Aldo

    2017-01-01

    Genetic progress for forage quality has been poor in alfalfa (Medicago sativa L.), the most-grown forage legume worldwide. This study aimed at exploring opportunities for marker-assisted selection (MAS) and genomic selection of forage quality traits based on breeding values of parent plants. Some 154 genotypes from a broadly-based reference population were genotyped by genotyping-by-sequencing (GBS), and phenotyped for leaf-to-stem ratio, leaf and stem contents of protein, neutral detergent fiber (NDF) and acid detergent lignin (ADL), and leaf and stem NDF digestibility after 24 hours (NDFD), of their dense-planted half-sib progenies in three growing conditions (summer harvest, full irrigation; summer harvest, suspended irrigation; autumn harvest). Trait-marker analyses were performed on progeny values averaged over conditions, owing to modest germplasm × condition interaction. Genomic selection exploited 11,450 polymorphic SNP markers, whereas a subset of 8,494 M. truncatula-aligned markers were used for a genome-wide association study (GWAS). GWAS confirmed the polygenic control of quality traits and, in agreement with phenotypic correlations, indicated substantially different genetic control of a given trait in stems and leaves. It detected several SNPs in different annotated genes that were highly linked to stem protein content. Also, it identified a small genomic region on chromosome 8 with high concentration of annotated genes associated with leaf ADL, including one gene probably involved in the lignin pathway. Three genomic selection models, i.e., Ridge-regression BLUP, Bayes B and Bayesian Lasso, displayed similar prediction accuracy, whereas SVR-lin was less accurate. Accuracy values were moderate (0.3–0.4) for stem NDFD and leaf protein content, modest for leaf ADL and NDFD, and low to very low for the other traits. Along with previous results for the same germplasm set, this study indicates that GBS data can be exploited to improve both quality traits

  14. Genetic Transformation and Analysis of Rice OsAPx2 Gene in Medicago sativa

    PubMed Central

    Guan, Qingjie; Takano, Tetsuo; Liu, Shenkui

    2012-01-01

    The OsAPx2 gene from rice was cloned to produce PBI121::OsAPx2 dual-expression plants, of which expression level would be increasing under stressful conditions. The enzyme ascorbate peroxidase (APX) in the leaves and roots of the plants increased with increasing exposure time to different sodium chloride (NaCl) and hydrogen peroxide (H2O2)concentrations, as indicated by protein gel blot analysis. The increased enzyme yield improved the ability of the plants to resist the stress treatments. The OsAPx2 gene was localized in the cytoplasm of epidermal onion cells as indicated by the instantaneous expression of green fluorescence. An 80% regeneration rate was observed in Medicago sativa L. plants transformed with the OsAPx2 gene using Agrobacterium tumefaciens, as indicated by specific primer PCR. The OsAPx2 gene was expressed at the mRNA level and the individual M. sativa (T#1,T#2,T#5) were obtained through assaying the generation of positive T2 using RNA gel blot analysis. When the seeds of the wild type (WT) and the T2 (T#1,T#5) were incubated in culture containing MS with NaCl for 7 days, the results as shown of following: the root length of transgenic plant was longer than WT plants, the H2O2 content in roots of WT was more than of transgenic plants, the APX activity under stresses increased by 2.89 times compared with the WT, the malondialdehyde (MDA) content of the WT was higher than the transgenic plants, the leaves of the WT turned yellow, but those of the transgenic plants remained green and remained healthy. The chlorophyll content in the WT leaves was less than in the transgenic plants, after soaking in solutions of H2O2, sodium sulfite (Na2SO3), and sodium bicarbonate (NaHCO3). Therefore, the OsAPx2 gene overexpression in transgenic M. sativa improves the removal of H2O2 and the salt-resistance compared with WT plants. A novel strain of M. sativa carrying a salt-resistance gene was obtained. PMID:22848448

  15. LTR retrotransposon landscape in Medicago truncatula: more rapid removal than in rice

    PubMed Central

    Wang, Hao; Liu, Jin-Song

    2008-01-01

    Background Long terminal repeat retrotransposons (LTR elements) are ubiquitous Eukaryotic TEs that transpose through RNA intermediates. Accounting for significant proportion of many plant genomes, LTR elements have been well established as one of the major forces underlying the evolution of plant genome size, structure and function. The accessibility of more than 40% of genomic sequences of the model legume Medicago truncatula (Mt) has made the comprehensive study of its LTR elements possible. Results We use a newly developed tool LTR_FINDER to identify LTR retrotransposons in the Mt genome and detect 526 full-length elements as well as a great number of copies related to them. These elements constitute about 9.6% of currently available genomic sequences. They are classified into 85 families of which 64 are reported for the first time. The majority of the LTR retrotransposons belong to either Copia or Gypsy superfamily and the others are categorized as TRIMs or LARDs by their length. We find that the copy-number of Copia-like families is 3 times more than that of Gypsy-like ones but the latter contribute more to the genome. The analysis of PBS and protein-coding domain structure of the LTR families reveals that they tend to use only 4–5 types of tRNAs and many families have quite conservative ORFs besides known TE domains. For several important families, we describe in detail their abundance, conservation, insertion time and structure. We investigate the amplification-deletion pattern of the elements and find that the detectable full-length elements are relatively young and most of them were inserted within the last 0.52 MY. We also estimate that more than ten million bp of the Mt genomic sequences have been removed by the deletion of LTR elements and the removal of the full-length structures in Mt has been more rapid than in rice. Conclusion This report is the first comprehensive description and analysis of LTR retrotransposons in the Mt genome. Many important

  16. Molecular phylogenetic approach for studying life-history evolution: the ambiguous example of the genus Medicago L.

    PubMed Central

    Bena, G; Lejeune, B; Prosperi, J M; Olivieri, I

    1998-01-01

    We present a molecular phylogeny including most species of the genus Medicago L. (Fabaceae). Based on the consensus of the 48 most parsimonious trees, life-history and mating-system characters are mapped, and a putative history of the genus is suggested. The most parsimonious reconstruction suggests an ancestral annual and selfing state, and recurrent evolution towards perenniality and outcrossing. Based on theoretical predictions and classical hypotheses of the history of the genus, different assumptions about the ancestral state and different weighting schemes of evolution between the character states are made. Assuming an outcrossing, perennial ancestral state (partly supported by morphological features) does not fundamentally change the reconstruction. To meet theoretical expectations, various weighting schemes favouring evolution towards annuality and selfing are applied. Influence and validity of such weighting schemes are discussed with regard to other studies. PMID:9684377

  17. [Physical localization of ribosomal genes and chromosome DAPI banding by in situ hybridization in Medicago sativa L].

    PubMed

    Chen, Jian-Min; Hong, Yi-Huan; Wang, You-Ping; Bowley, Steve; Wan, Jian-Min

    2006-02-01

    Physical localization of ribosomal genes in diploid and tetraploid alfalfa (Medicago. sativa) was studied using fluorescent in situ hybridization (FISH). It was revealed that 45s gene was only located at nucleolus organizer region (NOR) with a single locus in both diploid and tetraploid alfalfa, while 5s gene had 2-3 loci on chromosomes. Using the genomic DNA from M. coerulea and M. falcata as probe to hybridize with tetraploid species in alfalfa, both diploid species were successfully hybridized with tetraploid chromosomes, only showing the difference in hybridization signals in different numbers of chromosomes. Chromosomes of alfalfa exhibited DAPI banding by FISH analysis. In general, the patterns of distribution of DAPI banding were consistent with C-banding for M. coerulea. The possible origination of tetraploid alfalfa was discussed based on DAPI banding patterns and FISH analysis for ribosomal genes .

  18. Potential regulatory phosphorylation sites in a Medicago truncatula plasma membrane proton pump implicated during early symbiotic signaling in roots.

    PubMed

    Nguyen, Thao T; Volkening, Jeremy D; Rose, Christopher M; Venkateshwaran, Muthusubramanian; Westphall, Michael S; Coon, Joshua J; Ané, Jean-Michel; Sussman, Michael R

    2015-08-04

    In plants and fungi the plasma membrane proton pump generates a large proton-motive force that performs essential functions in many processes, including solute transport and the control of cell elongation. Previous studies in yeast and higher plants have indicated that phosphorylation of an auto-inhibitory domain is involved in regulating pump activity. In this report we examine the Medicago truncatula plasma membrane proton pump gene family, and in particular MtAHA5. Yeast complementation assays with phosphomimetic mutations at six candidate sites support a phosphoregulatory role for two residues, suggesting a molecular model to explain early Nod factor-induced changes in the plasma membrane proton-motive force of legume root cells.

  19. An analysis of synteny of Arachis with Lotus and Medicago sheds new light on the structure, stability and evolution of legume genomes.

    PubMed

    Bertioli, David J; Moretzsohn, Marcio C; Madsen, Lene H; Sandal, Niels; Leal-Bertioli, Soraya C M; Guimarães, Patricia M; Hougaard, Birgit K; Fredslund, Jakob; Schauser, Leif; Nielsen, Anna M; Sato, Shusei; Tabata, Satoshi; Cannon, Steven B; Stougaard, Jens

    2009-01-23

    Most agriculturally important legumes fall within two sub-clades of the Papilionoid legumes: the Phaseoloids and Galegoids, which diverged about 50 Mya. The Phaseoloids are mostly tropical and include crops such as common bean and soybean. The Galegoids are mostly temperate and include clover, fava bean and the model legumes Lotus and Medicago (both with substantially sequenced genomes). In contrast, peanut (Arachis hypogaea) falls in the Dalbergioid clade which is more basal in its divergence within the Papilionoids. The aim of this work was to integrate the genetic map of Arachis with Lotus and Medicago and improve our understanding of the Arachis genome and legume genomes in general. To do this we placed on the Arachis map, comparative anchor markers defined using a previously described bioinformatics pipeline. Also we investigated the possible role of transposons in the patterns of synteny that were observed. The Arachis genetic map was substantially aligned with Lotus and Medicago with most synteny blocks presenting a single main affinity to each genome. This indicates that the last common whole genome duplication within the Papilionoid legumes predated the divergence of Arachis from the Galegoids and Phaseoloids sufficiently that the common ancestral genome was substantially diploidized. The Arachis and model legume genomes comparison made here, together with a previously published comparison of Lotus and Medicago allowed all possible Arachis-Lotus-Medicago species by species comparisons to be made and genome syntenies observed. Distinct conserved synteny blocks and non-conserved regions were present in all genome comparisons, implying that certain legume genomic regions are consistently more stable during evolution than others. We found that in Medicago and possibly also in Lotus, retrotransposons tend to be more frequent in the variable regions. Furthermore, while these variable regions generally have lower densities of single copy genes than the more

  20. An analysis of synteny of Arachis with Lotus and Medicago sheds new light on the structure, stability and evolution of legume genomes

    PubMed Central

    Bertioli, David J; Moretzsohn, Marcio C; Madsen, Lene H; Sandal, Niels; Leal-Bertioli, Soraya CM; Guimarães, Patricia M; Hougaard, Birgit K; Fredslund, Jakob; Schauser, Leif; Nielsen, Anna M; Sato, Shusei; Tabata, Satoshi; Cannon, Steven B; Stougaard, Jens

    2009-01-01

    Background Most agriculturally important legumes fall within two sub-clades of the Papilionoid legumes: the Phaseoloids and Galegoids, which diverged about 50 Mya. The Phaseoloids are mostly tropical and include crops such as common bean and soybean. The Galegoids are mostly temperate and include clover, fava bean and the model legumes Lotus and Medicago (both with substantially sequenced genomes). In contrast, peanut (Arachis hypogaea) falls in the Dalbergioid clade which is more basal in its divergence within the Papilionoids. The aim of this work was to integrate the genetic map of Arachis with Lotus and Medicago and improve our understanding of the Arachis genome and legume genomes in general. To do this we placed on the Arachis map, comparative anchor markers defined using a previously described bioinformatics pipeline. Also we investigated the possible role of transposons in the patterns of synteny that were observed. Results The Arachis genetic map was substantially aligned with Lotus and Medicago with most synteny blocks presenting a single main affinity to each genome. This indicates that the last common whole genome duplication within the Papilionoid legumes predated the divergence of Arachis from the Galegoids and Phaseoloids sufficiently that the common ancestral genome was substantially diploidized. The Arachis and model legume genomes comparison made here, together with a previously published comparison of Lotus and Medicago allowed all possible Arachis-Lotus-Medicago species by species comparisons to be made and genome syntenies observed. Distinct conserved synteny blocks and non-conserved regions were present in all genome comparisons, implying that certain legume genomic regions are consistently more stable during evolution than others. We found that in Medicago and possibly also in Lotus, retrotransposons tend to be more frequent in the variable regions. Furthermore, while these variable regions generally have lower densities of single copy genes

  1. Identification of a dominant gene in Medicago truncatula that restricts nodulation by Sinorhizobium meliloti strain Rm41

    PubMed Central

    2014-01-01

    Background Leguminous plants are able to form a root nodule symbiosis with nitrogen-fixing soil bacteria called rhizobia. This symbiotic association shows a high level of specificity. Beyond the specificity for the legume family, individual legume species/genotypes can only interact with certain restricted group of bacterial species or strains. Specificity in this system is regulated by complex signal exchange between the two symbiotic partners and thus multiple genetic mechanisms could be involved in the recognition process. Knowledge of the molecular mechanisms controlling symbiotic specificity could enable genetic improvement of legume nitrogen fixation, and may also reveal the possible mechanisms that restrict root nodule symbiosis in non-legumes. Results We screened a core collection of Medicago truncatula genotypes with several strains of Sinorhizobium meliloti and identified a naturally occurring dominant gene that restricts nodulation by S. meliloti Rm41. We named this gene as Mt-NS1 (for M.truncatulanodulation specificity 1). We have mapped the Mt-NS1 locus within a small genomic region on M. truncatula chromosome 8. The data reported here will facilitate positional cloning of the Mt-NS1 gene. Conclusions Evolution of symbiosis specificity involves both rhizobial and host genes. From the bacterial side, specificity determinants include Nod factors, surface polysaccharides, and secreted proteins. However, we know relatively less from the host side. We recently demonstrated that a component of this specificity in soybeans is defined by plant NBS-LRR resistance (R) genes that recognize effector proteins delivered by the type III secretion system (T3SS) of the rhizobial symbionts. However, the lack of a T3SS in many sequenced S. meliloti strains raises the question of how the specificity is regulated in the Medicago-Sinorhizobium system beyond Nod-factor perception. Thus, cloning and characterization of Mt-NS1 will add a new dimension to our knowledge about

  2. Transfer of anthracnose resistance and pod coiling traits from Medicago arborea to M. sativa by sexual reproduction.

    PubMed

    Armour, D J; Mackie, J M; Musial, J M; Irwin, J A G

    2008-07-01

    Five asymmetric hybrid plants were obtained between Medicago sativa (2n = 4x = 32) and Medicago arborea (2n = 4x = 32) through sexual reproduction and the use of a cytoplasmically male sterile M. sativa genotype. Over 2,000 pollinations were made to obtain these hybrids. Amplified fragment length polymorphism (AFLP) analysis showed that in the most studied hybrid (WA2273), 4% of the bands unique to the M. arborea parent were present, versus 72% for the unique M. sativa bands. This suggests that only a single M. arborea chromosome or chromosome parts has been transferred. WA2273 had 7% of AFLP bands which were not present in either parent, which is suggestive of chromosome rearrangements as would be expected if only chromosome parts or a single part had been transferred from M. arborea. Phenotypic evidence for hybridity was obtained for pod coiling (1.4 coils in WA2273 versus three coils in the M. sativa parent and its self and testcross populations, and one coil in M. arborea), and Colletotrichum trifolii race 2 resistance (transferred from the resistant M. arborea parent, as the M. sativa parent and the self populations were highly susceptible). The hybrids were self sterile, but were female fertile to a high level when crossed with 4x, but not 2x, M. sativa, indicating they were at or near 4x. Both the pod coiling trait and anthracnose resistance segregated in the progeny of testcrosses between WA2273 and M. sativa. The work demonstrates that agronomically useful traits can be introgressed into M. sativa from M. arborea by use of male sterile M. sativa and sexual reproduction.

  3. Determination of saponins in aerial parts of barrel medic (Medicago truncatula) by liquid chromatography-electrospray ionization/mass spectrometry.

    PubMed

    Kapusta, Ireneusz; Janda, Bogdan; Stochmal, Anna; Oleszek, Wieslaw

    2005-10-05

    Triterpene saponins from aerial parts of Medicago truncatula cv. Jemalong A-17, M. truncatula Gaertn.var. longispina Urb., and M. truncatula Gaertn. var. truncatula were profiled and quantified using reverse-phase liquid chromatography with on-line photodiode array detection and electrospray ionization mass spectrometry (LC-PDA/ESI/MS/MS). The determination was based on standard curves obtained for the 18 available saponin standards, previously isolated from Jemalong A-17. Aerial parts of all three subspecies contained 17 saponins previously identified and also a substantial amount of astragaloside VIII (3-GlcA-Xyl-Rha soyasapogenol B), not previously reported in M. truncatula. The compositions of saponin mixtures were very similar in the three subspecies with three dominant groups, recognized as zanhic acid, medicagenic acid, and soyasapogenol glycosides. Relative proportions of these three groups were also similar in the three subspecies: var. longispina had 49.5, 48.1, and 2.4%; var. truncatula, 41.5, 53.4, and 5.1%; and Jemalong A-17, 42.1, 56.6, and 1.3% of zanhic acid, medicagenic acid, and soyasapogenol glycosides, respectively. Jemalong A-17 had 30% lower total content of saponins as compared to M. truncatula var. longispina and M. truncatula var. truncatula; in relation to the dry matter, var. longispina contained 0.22%, var. truncatula, 0.22%, and Jemalong A-17, 0.15% dry matter of saponins. If one takes into consideration that this determination was performed on spring-collected samples, it can be concluded that the concentration of saponins in M. truncatula is similar to the concentration in alfalfa (Medicago sativa); the proportions of the three groups of saponins in these species are slightly different from those found in alfalfa, having a higher content of zanhic acid glycosides.

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

    PubMed

    Abirached-Darmency, Mona; Prado-Vivant, Emilce; Chelysheva, Liudmila; Pouthier, Thomas

    2005-06-01

    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.

  5. Detection of Changes in the Medicago sativa Retinoblastoma-Related Protein (MsRBR1) Phosphorylation During Cell Cycle Progression in Synchronized Cell Suspension Culture.

    PubMed

    Ayaydin, Ferhan; Kotogány, Edit; Ábrahám, Edit; Horváth, Gábor V

    2017-01-01

    Deepening our knowledge on the regulation of the plant cell division cycle depends on techniques that allow for the enrichment of cell populations in defined cell cycle phases. Synchronization of cell division can be achieved using different plant tissues; however, well-established cell suspension cultures provide large amount of biological sample for further analyses. Here, we describe the methodology of the establishment, propagation, and analysis of a Medicago sativa suspension culture that can be used for efficient synchronization of the cell division. A novel 5-ethynyl-2'-deoxyuridine (EdU)-based method is used for the estimation of cell fraction that enters DNA synthesis phase of the cell cycle and we also demonstrate the changes in the phosphorylation level of Medicago sativa retinoblastoma-related protein (MsRBR1) during cell cycle progression.

  6. Phylogeny and character evolution in Medicago (Leguminosae): Evidence from analyses of plastid trnK/matK and nuclear GA3ox1 sequences.

    PubMed

    Steele, Kelly P; Ickert-Bond, Stefanie M; Zarre, Shahin; Wojciechowski, Martin F

    2010-07-01

    The genus Medicago, with about 87 species, includes the model legume species M. truncatula, and a number of important forage species such as M. sativa (alfalfa), M. scutellata (snail medic), and M. lupulina (black medic). Relationships within the genus are not yet sufficiently resolved, contributing to difficulty in understanding the evolution of a number of distinguishing characteristics such as aneuploidy and polyploidy, life history, structure of cotyledons, and number of seeds per fruit. • Phylogenetic relationships of 70-73 species of Medicago and its sister genus Trigonella (including Melilotus) were reconstructed from nucleotide sequences of the plastid trnK/matK region and the nuclear-encoded GA3ox1 gene (gibberellin 3-β-hydroxylase) using maximum parsimony and Bayesian inference methods. • Our results support certain currently recognized taxonomic groups, e.g., sect. Medicago (with M. sativa) and sect. Buceras. However, other strongly supported clades-the "reduced subsection Leptospireae clade" that includes M. lupulina, the "polymorpha clade" that includes M. murex and M. polymorpha and the "subsection Pachyspireae clade" that includes M. truncatula-each of which includes species presently in different subsections of sect. Spirocarpos, contradict the current classification. • These results support the hypothesis that some characters considered important in existing taxonomies, for example, single-seeded fruits that have arisen more than once in both Medicago and Trigonella, are indeed homoplastic. Others, such as the 2n = 14 chromosome number, have also arisen independently within the genus. In addition, we demonstrate support for the utility of GA3ox1 sequences for phylogenetic analysis among and within closely related genera of legumes.

  7. High-quality permanent draft genome sequence of Ensifer medicae strain WSM244, a microsymbiont isolated from Medicago polymorpha growing in alkaline soil

    DOE PAGES

    Ardley, Julie; Tian, Rui; O’Hara, Graham; ...

    2015-12-01

    We report that Ensifer medicae WSM244 is an aerobic, motile, Gram-negative, non-spore-forming rod that can exist as a soil saprophyte or as a legume microsymbiont of Medicago species. WSM244 was isolated in 1979 from a nodule recovered from the roots of the annual Medicago polymorpha L. growing in alkaline soil (pH 8.0) in Tel Afer, Iraq. WSM244 is the only acid-sensitive E. medicae strain that has been sequenced to date. It is effective at fixing nitrogen with M. polymorpha L., as well as with more alkaline-adapted Medicago spp. such as M. littoralis Loisel., M. scutellata (L.) Mill., M. tornata (L.)more » Mill. and M. truncatula Gaertn. This strain is also effective with the perennial M. sativa L. Here we describe the features of E. medicae WSM244, together with genome sequence information and its annotation. The 6,650,282 bp high-quality permanent draft genome is arranged into 91 scaffolds of 91 contigs containing 6,427 protein-coding genes and 68 RNA-only encoding genes, and is one of the rhizobial genomes sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project proposal.« less

  8. In situ expression of two storage protein genes in relation to histo-differentiation at mid-embryogenesis in Medicago truncatula and Pisum sativum seeds.

    PubMed

    Abirached-Darmency, M; Abdel-gawwad, M R; Conejero, G; Verdeil, J L; Thompson, R

    2005-08-01

    The seed consists of several layers of specialized cell-types that divide and differentiate following a highly regulated programme in time and space. A cytological approach was undertaken in order to study the histo-differentiation at mid-embryogenesis in Medicago truncatula as a model legume, and in Pisum sativum using serial sections of embedded immature seed. Little published information is available about seed development in Medicago species. The observations from this study revealed a number of distinctive features of Medicago seed development and differentiation. Transfer cells, involved in nutrient transfer to the embryo, were clearly identified in the thin-walled parenchyma of the innermost integument. Histological Schiff-naphthol enabled carbohydrate accumulation to be followed in the different seed compartments, and revealed the storage protein bodies. Non-radioactive mRNA in situ hybridization, was carried out using mRNA probes from two highly expressed genes encoding the major vicilin and legumin A storage protein types. The timing of mRNA expression was related to that of the corresponding proteins already identified.

  9. An overall evaluation of the Resistance (R) and Pathogenesis-Related (PR) superfamilies in soybean, as compared with Medicago and Arabidopsis

    PubMed Central

    Wanderley-Nogueira, Ana C.; Belarmino, Luis C.; Soares-Cavalcanti, Nina da M.; Bezerra-Neto, João P.; Kido, Ederson A.; Pandolfi, Valesca; Abdelnoor, Ricardo V.; Binneck, Eliseu; Carazzole, Marcelo F.; Benko-Iseppon, Ana M.

    2012-01-01

    Plants have the ability to recognize and respond to a multitude of pathogens, resulting in a massive reprogramming of the plant to activate defense responses including Resistance (R) and Pathogenesis-Related (PR) genes. Abiotic stresses can also activate PR genes and enhance pathogen resistance, representing valuable genes for breeding purposes. The present work offers an overview of soybean R and PR genes present in the GENOSOJA (Brazilian Soybean Genome Consortium) platform, regarding their structure, abundance, evolution and role in the plant-pathogen metabolic pathway, as compared with Medicago and Arabidopsis. Searches revealed 3,065 R candidates (756 in Soybean, 1,142 in Medicago and 1,167 in Arabidopsis), and PR candidates matching to 1,261 sequences (310, 585 and 366 for the three species, respectively). The identified transcripts were also evaluated regarding their expression pattern in 65 libraries, showing prevalence in seeds and developing tissues. Upon consulting the SuperSAGE libraries, 1,072 R and 481 PR tags were identified in association with the different libraries. Multiple alignments were generated for Xa21 and PR-2 genes, allowing inferences about their evolution. The results revealed interesting insights regarding the variability and complexity of defense genes in soybean, as compared with Medicago and Arabidopsis. PMID:22802711

  10. High-quality permanent draft genome sequence of Ensifer medicae strain WSM244, a microsymbiont isolated from Medicago polymorpha growing in alkaline soil.

    PubMed

    Ardley, Julie; Tian, Rui; O'Hara, Graham; Seshadri, Rekha; Reddy, T B K; Pati, Amrita; Woyke, Tanja; Markowitz, Victor; Ivanova, Natalia; Kyrpides, Nikos; Howieson, John; Reeve, Wayne

    2015-01-01

    Ensifer medicae WSM244 is an aerobic, motile, Gram-negative, non-spore-forming rod that can exist as a soil saprophyte or as a legume microsymbiont of Medicago species. WSM244 was isolated in 1979 from a nodule recovered from the roots of the annual Medicago polymorpha L. growing in alkaline soil (pH 8.0) in Tel Afer, Iraq. WSM244 is the only acid-sensitive E. medicae strain that has been sequenced to date. It is effective at fixing nitrogen with M. polymorpha L., as well as with more alkaline-adapted Medicago spp. such as M. littoralis Loisel., M. scutellata (L.) Mill., M. tornata (L.) Mill. and M. truncatula Gaertn. This strain is also effective with the perennial M. sativa L. Here we describe the features of E. medicae WSM244, together with genome sequence information and its annotation. The 6,650,282 bp high-quality permanent draft genome is arranged into 91 scaffolds of 91 contigs containing 6,427 protein-coding genes and 68 RNA-only encoding genes, and is one of the rhizobial genomes sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project proposal.

  11. High-quality permanent draft genome sequence of Ensifer medicae strain WSM244, a microsymbiont isolated from Medicago polymorpha growing in alkaline soil

    SciTech Connect

    Ardley, Julie; Tian, Rui; O’Hara, Graham; Seshadri, Rekha; Reddy, T. B. K.; Pati, Amrita; Woyke, Tanja; Markowitz, Victor; Ivanova, Natalia; Kyrpides, Nikos; Howieson, John; Reeve, Wayne

    2015-12-01

    We report that Ensifer medicae WSM244 is an aerobic, motile, Gram-negative, non-spore-forming rod that can exist as a soil saprophyte or as a legume microsymbiont of Medicago species. WSM244 was isolated in 1979 from a nodule recovered from the roots of the annual Medicago polymorpha L. growing in alkaline soil (pH 8.0) in Tel Afer, Iraq. WSM244 is the only acid-sensitive E. medicae strain that has been sequenced to date. It is effective at fixing nitrogen with M. polymorpha L., as well as with more alkaline-adapted Medicago spp. such as M. littoralis Loisel., M. scutellata (L.) Mill., M. tornata (L.) Mill. and M. truncatula Gaertn. This strain is also effective with the perennial M. sativa L. Here we describe the features of E. medicae WSM244, together with genome sequence information and its annotation. The 6,650,282 bp high-quality permanent draft genome is arranged into 91 scaffolds of 91 contigs containing 6,427 protein-coding genes and 68 RNA-only encoding genes, and is one of the rhizobial genomes sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project proposal.

  12. The regulatory gamma subunit SNF4b of the sucrose non-fermenting-related kinase complex is involved in longevity and stachyose accumulation during maturation of Medicago truncatula seeds.

    PubMed

    Rosnoblet, Claire; Aubry, Catherine; Leprince, Olivier; Vu, Benoit Ly; Rogniaux, Hélène; Buitink, Julia

    2007-07-01

    The sucrose non-fermenting-related kinase complex (SnRK1) is a heterotrimeric complex that plays a central role in metabolic adaptation to nutritional or environmental stresses. Here we investigate the role of a regulatory gamma-subunit of the complex, MtSNF4b, in Medicago truncatula seeds. Western blot indicated that MtSNF4b accumulated during seed filling, whereas it disappeared during imbibition of mature seeds. Gel filtration chromatography suggested that MtSNF4b assembled into a complex (450-600 kDa) at the onset of maturation drying, and dissociated during subsequent imbibition. Drying of desiccation-tolerant radicles led to a reassembly of the complex, in contrast to sensitive tissues. Silencing of MtSNF4b using a RNA interference (RNAi) approach resulted in a phenotype with reduced seed longevity, evident from the reduction in both germination percentage and seedling vigour in aged RNAi MtSNF4b seeds compared with the wild-type seeds. In parallel to the assembly of the complex, seeds of the RNAi MtSNF4b lines showed impaired accumulation of raffinose family oligosaccharides compared with control seeds. In mature seeds, the amount of stachyose was reduced by 50-80%, whereas the sucrose content was 60% higher. During imbibition, the differences in non-reducing sugar compared with the control disappeared in parallel to the disassembly of the complex. No difference was observed in dry weight or reserve accumulation such as proteins, lipids and starch. These data suggest that the regulatory gamma-subunit MtSNF4b confers a specific and temporal function to SnRK1 complexes in seeds, improving seed longevity and affecting the non-reducing sugar content at later stages of seed maturation.

  13. Genome-Wide Identification of Different Dormant Medicago sativa L. MicroRNAs in Response to Fall Dormancy

    PubMed Central

    Du, Hongqi; Sun, Xiaoge; Shi, Yinghua; Wang, Chengzhang

    2014-01-01

    Background MicroRNAs (miRNAs) are a class of regulatory small RNAs (sRNAs) that regulate gene post-transcriptional expression in plants and animals. High-throughput sequencing technology is capable of identifying small RNAs in plant species. Alfalfa (Medicago sativa L.) is one of the most widely cultivated perennial forage legumes worldwide, and fall dormancy is an adaptive characteristic related to the biomass production and winter survival in alfalfa. Here, we applied high-throughput sRNA sequencing to identify some miRNAs that were responsive to fall dormancy in standard variety (Maverick and CUF101) of alfalfa. Results Four sRNA libraries were generated and sequenced from alfalfa leaves in two typical varieties at distinct seasons. Through integrative analysis, we identified 51 novel miRNA candidates of 206 families. Additionally, we identified 28 miRNAs associated with fall dormancy in standard variety (Maverick and CUF101), including 20 known miRNAs and eight novel miRNAs. Both high-throughput sequencing and RT-qPCR confirmed that eight known miRNA members were up-regulated and six known miRNA members were down-regulated in response to fall dormancy in standard variety (Maverick and CUF101). Among the 51 novel miRNA candidates, five miRNAs were up-regulated and three miRNAs were down-regulated in response to fall dormancy in standard variety (Maverick and CUF101), and five of them were confirmed by Northern blot analysis. Conclusion We identified 20 known miRNAs and eight new miRNA candidates that were responsive to fall dormancy in standard variety (Maverick and CUF101) by high-throughput sequencing of small RNAs from Medicago sativa. Our data provide a useful resource for investigating miRNA-mediated regulatory mechanisms of fall dormancy in alfalfa, and these findings are important for our understanding of the roles played by miRNAs in the response of plants to abiotic stress in general and fall dormancy in alfalfa. PMID:25473944

  14. Contributions of Sinorhizobium meliloti Transcriptional Regulator DksA to Bacterial Growth and Efficient Symbiosis with Medicago sativa.

    PubMed

    Wippel, Kathrin; Long, Sharon R

    2016-05-01

    The stringent response, mediated by the (p)ppGpp synthetase RelA and the RNA polymerase-binding protein DksA, is triggered by limiting nutrient conditions. For some bacteria, it is involved in regulation of virulence. We investigated the role of two DksA-like proteins from the Gram-negative nitrogen-fixing symbiont Sinorhizobium meliloti in free-living culture and in interaction with its host plant Medicago sativa The two paralogs, encoded by the genes SMc00469 and SMc00049, differ in the constitution of two major domains required for function in canonical DksA: the DXXDXA motif at the tip of a coiled-coil domain and a zinc finger domain. Using mutant analyses of single, double, and triple deletions for SMc00469(designated dksA),SMc00049, and relA, we found that the ΔdksA mutant but not the ΔSMc00049 mutant showed impaired growth on minimal medium, reduced nodulation on the host plant, and lower nitrogen fixation activity in early nodules, while its nod gene expression was normal. The ΔrelA mutant showed severe pleiotropic phenotypes under all conditions tested. Only S. meliloti dksA complemented the metabolic defects of an Escherichia coli dksA mutant. Modifications of the DXXDXA motif in SMc00049 failed to establish DksA function. Our results imply a role for transcriptional regulator DksA in the S. meliloti-M. sativa symbiosis. The stringent response is a bacterial transcription regulation process triggered upon nutritional stress.Sinorhizobium meliloti, a soil bacterium establishing agriculturally important root nodule symbioses with legume plants, undergoes constant molecular adjustment during host interaction. Analyzing the components of the stringent response in this alphaproteobacterium helps understand molecular control regarding the development of plant interaction. Using mutant analyses, we describe how the lack of DksA influences symbiosis with Medicago sativa and show that a second paralogous S. meliloti protein cannot substitute for this missing

  15. Contributions of Sinorhizobium meliloti Transcriptional Regulator DksA to Bacterial Growth and Efficient Symbiosis with Medicago sativa

    PubMed Central

    Wippel, Kathrin

    2016-01-01

    ABSTRACT The stringent response, mediated by the (p)ppGpp synthetase RelA and the RNA polymerase-binding protein DksA, is triggered by limiting nutrient conditions. For some bacteria, it is involved in regulation of virulence. We investigated the role of two DksA-like proteins from the Gram-negative nitrogen-fixing symbiont Sinorhizobium meliloti in free-living culture and in interaction with its host plant Medicago sativa. The two paralogs, encoded by the genes SMc00469 and SMc00049, differ in the constitution of two major domains required for function in canonical DksA: the DXXDXA motif at the tip of a coiled-coil domain and a zinc finger domain. Using mutant analyses of single, double, and triple deletions for SMc00469 (designated dksA), SMc00049, and relA, we found that the ΔdksA mutant but not the ΔSMc00049 mutant showed impaired growth on minimal medium, reduced nodulation on the host plant, and lower nitrogen fixation activity in early nodules, while its nod gene expression was normal. The ΔrelA mutant showed severe pleiotropic phenotypes under all conditions tested. Only S. meliloti dksA complemented the metabolic defects of an Escherichia coli dksA mutant. Modifications of the DXXDXA motif in SMc00049 failed to establish DksA function. Our results imply a role for transcriptional regulator DksA in the S. meliloti-M. sativa symbiosis. IMPORTANCE The stringent response is a bacterial transcription regulation process triggered upon nutritional stress. Sinorhizobium meliloti, a soil bacterium establishing agriculturally important root nodule symbioses with legume plants, undergoes constant molecular adjustment during host interaction. Analyzing the components of the stringent response in this alphaproteobacterium helps understand molecular control regarding the development of plant interaction. Using mutant analyses, we describe how the lack of DksA influences symbiosis with Medicago sativa and show that a second paralogous S. meliloti protein cannot

  16. Construction of two genetic linkage maps in cultivated tetraploid alfalfa (Medicago sativa) using microsatellite and AFLP markers

    PubMed Central

    Julier, Bernadette; Flajoulot, Sandrine; Barre, Philippe; Cardinet, Gaëlle; Santoni, Sylvain; Huguet, Thierry; Huyghe, Christian

    2003-01-01

    Background Alfalfa (Medicago sativa) is a major forage crop. The genetic progress is slow in this legume species because of its autotetraploidy and allogamy. The genetic structure of this species makes the construction of genetic maps difficult. To reach this objective, and to be able to detect QTLs in segregating populations, we used the available codominant microsatellite markers (SSRs), most of them identified in the model legume Medicago truncatula from EST database. A genetic map was constructed with AFLP and SSR markers using specific mapping procedures for autotetraploids. The tetrasomic inheritance was analysed in an alfalfa mapping population. Results We have demonstrated that 80% of primer pairs defined on each side of SSR motifs in M. truncatula EST database amplify with the alfalfa DNA. Using a F1 mapping population of 168 individuals produced from the cross of 2 heterozygous parental plants from Magali and Mercedes cultivars, we obtained 599 AFLP markers and 107 SSR loci. All but 3 SSR loci showed a clear tetrasomic inheritance. For most of the SSR loci, the double-reduction was not significant. For the other loci no specific genotypes were produced, so the significant double-reduction could arise from segregation distortion. For each parent, the genetic map contained 8 groups of four homologous chromosomes. The lengths of the maps were 2649 and 3045 cM, with an average distance of 7.6 and 9.0 cM between markers, for Magali and Mercedes parents, respectively. Using only the SSR markers, we built a composite map covering 709 cM. Conclusions Compared to diploid alfalfa genetic maps, our maps cover about 88–100% of the genome and are close to saturation. The inheritance of the codominant markers (SSR) and the pattern of linkage repulsions between markers within each homology group are consistent with the hypothesis of a tetrasomic meiosis in alfalfa. Except for 2 out of 107 SSR markers, we found a similar order of markers on the chromosomes between the

  17. Comparative transcriptome investigation of global gene expression changes caused by miR156 overexpression in Medicago sativa.

    PubMed

    Gao, Ruimin; Austin, Ryan S; Amyot, Lisa; Hannoufa, Abdelali

    2016-08-19

    Medicago sativa (alfalfa) is a low-input forage and potential bioenergy crop, and improving its yield and quality has always been a focus of the alfalfa breeding industry. Transgenic alfalfa plants overexpressing a precursor of alfalfa microRNA156 (MsmiR156) were recently generated by our group. These plants (miR156OE) showed enhanced biomass yield, reduced internodal length, increased shoot branching and trichome density, and a delay in flowering time. Transcripts of three SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) genes (MsSPL6, MsSPL12, and MsSPL13) were found to be targeted for cleavage by MsmiR156 in alfalfa. To further illustrate the molecular mechanisms underlying the effects of miR156 in alfalfa, two miR156OE genotypes (A11a and A17) were subjected to Next Generation RNA Sequencing with Illumina HiSeq. More than 1.11 billion clean reads were obtained from our available sequenced samples. A total of 160,472 transcripts were generated using Trinity de novo assembly and 4,985 significantly differentially expressed genes were detected in miR156OE plants A11a and A17 using the Medicago truncatula genome as reference. A total of 17 genes (including upregulated, downregulated, and unchanged) were selected for quantitative real-time PCR (qRT-PCR) validation, which showed that gene expression levels were largely consistent between qRT-PCR and RNA-Seq data. In addition to the established SPL genes MsSPL6, MsSPL12 and MsSPL13, four new SPLs; MsSPL2, MsSPL3, MsSPL4 and MsSPL9 were also down-regulated significantly in both miR156OE plants. These seven SPL genes belong to genes phylogeny clades VI, IV, VIII, V and VII, which have been reported to be targeted by miR156 in Arabidopsis thaliana. The gene ontology terms characterized electron transporter, starch synthase activity, sucrose transport, sucrose-phosphate synthase activity, chitin binding, sexual reproduction, flavonoid biosynthesis and lignin catabolism correlate well to the phenotypes of miR156OE alfalfa

  18. Genome-wide identification of different dormant Medicago sativa L. MicroRNAs in response to fall dormancy.

    PubMed

    Fan, Wenna; Zhang, Senhao; Du, Hongqi; Sun, Xiaoge; Shi, Yinghua; Wang, Chengzhang

    2014-01-01

    MicroRNAs (miRNAs) are a class of regulatory small RNAs (sRNAs) that regulate gene post-transcriptional expression in plants and animals. High-throughput sequencing technology is capable of identifying small RNAs in plant species. Alfalfa (Medicago sativa L.) is one of the most widely cultivated perennial forage legumes worldwide, and fall dormancy is an adaptive characteristic related to the biomass production and winter survival in alfalfa. Here, we applied high-throughput sRNA sequencing to identify some miRNAs that were responsive to fall dormancy in standard variety (Maverick and CUF101) of alfalfa. Four sRNA libraries were generated and sequenced from alfalfa leaves in two typical varieties at distinct seasons. Through integrative analysis, we identified 51 novel miRNA candidates of 206 families. Additionally, we identified 28 miRNAs associated with fall dormancy in standard variety (Maverick and CUF101), including 20 known miRNAs and eight novel miRNAs. Both high-throughput sequencing and RT-qPCR confirmed that eight known miRNA members were up-regulated and six known miRNA members were down-regulated in response to fall dormancy in standard variety (Maverick and CUF101). Among the 51 novel miRNA candidates, five miRNAs were up-regulated and three miRNAs were down-regulated in response to fall dormancy in standard variety (Maverick and CUF101), and five of them were confirmed by Northern blot analysis. We identified 20 known miRNAs and eight new miRNA candidates that were responsive to fall dormancy in standard variety (Maverick and CUF101) by high-throughput sequencing of small RNAs from Medicago sativa. Our data provide a useful resource for investigating miRNA-mediated regulatory mechanisms of fall dormancy in alfalfa, and these findings are important for our understanding of the roles played by miRNAs in the response of plants to abiotic stress in general and fall dormancy in alfalfa.

  19. Sinorhizobium meliloti-induced chitinase gene expression in Medicago truncatula ecotype R108-1: a comparison between symbiosis-specific class V and defence-related class IV chitinases.

    PubMed

    Salzer, Peter; Feddermann, Nadja; Wiemken, Andres; Boller, Thomas; Staehelin, Christian

    2004-08-01

    The Medicago truncatula (Gaertn.) ecotypes Jemalong A17 and R108-1 differ in Sinorhizobium meliloti-induced chitinase gene expression. The pathogen-inducible class IV chitinase gene, Mtchit 4, was strongly induced during nodule formation of the ecotype Jemalong A17 with the S. meliloti wild-type strain 1021. In the ecotype R108-1, the S. meliloti wild types Sm1021 and Sm41 did not induce Mtchit 4 expression. On the other hand, expression of the putative class V chitinase gene, Mtchit 5, was found in roots of M. truncatula cv. R108-1 nodulated with either of the rhizobial strains. Mtchit 5 expression was specific for interactions with rhizobia. It was not induced in response to fungal pathogen attack, and not induced in roots colonized with arbuscular mycorrhizal (AM) fungi. Elevated Mtchit 5 gene expression was first detectable in roots forming nodule primordia. In contrast to Mtchit 4, expression of Mtchit 5 was stimulated by purified Nod factors. Conversely, Mtchit 4 expression was strongly elevated in nodules formed with the K-antigen-deficient mutant PP699. Expression levels of Mtchit 5 were similarly increased in nodules formed with PP699 and its parental wild-type strain Sm41. Phylogenetic analysis of the deduced amino acid sequences of Mtchit 5 (calculated molecular weight = 41,810 Da, isoelectric point pH 7.7) and Mtchit 4 (calculated molecular weight 30,527 Da, isoelectric point pH 4.9) revealed that the putative Mtchit 5 chitinase forms a separate clade within class V chitinases of plants, whereas the Mtchit 4 chitinase clusters with pathogen-induced class IV chitinases from other plants. These findings demonstrate that: (i) Rhizobium-induced chitinase gene expression in M. truncatula occurs in a plant ecotype-specific manner, (ii) Mtchit 5 is a putative chitinase gene that is specifically induced by rhizobia, and (iii) rhizobia-specific and defence-related chitinase genes are differentially influenced by rhizobial Nod factors and K antigens.

  20. Arbuscular mycorrhizal symbiosis can mitigate the negative effects of night warming on physiological traits of Medicago truncatula L.

    PubMed

    Hu, Yajun; Wu, Songlin; Sun, Yuqing; Li, Tao; Zhang, Xin; Chen, Caiyan; Lin, Ge; Chen, Baodong

    2015-02-01

    Elevated night temperature, one of the main climate warming scenarios, can have profound effects on plant growth and metabolism. However, little attention has been paid to the potential role of mycorrhizal associations in plant responses to night warming, although it is well known that symbiotic fungi can protect host plants against various environmental stresses. In the present study, physiological traits of Medicago truncatula L. in association with the arbuscular mycorrhizal (AM) fungus Rhizophagus irregularis were investigated under simulated night warming. A constant increase in night temperature of 1.53 °C significantly reduced plant shoot and root biomass, flower and seed number, leaf sugar concentration, and shoot Zn and root P concentrations. However, the AM association essentially mitigated these negative effects of night warming by improving plant growth, especially through increased root biomass, root to shoot ratio, and shoot Zn and root P concentrations. A significant interaction was observed between R. irregularis inoculation and night warming in influencing both root sucrose concentration and expression of sucrose synthase (SusS) genes, suggesting that AM symbiosis and increased night temperature jointly regulated plant sugar metabolism. Night warming stimulated AM fungal colonization but did not influence arbuscule abundance, symbiosis-related plant or fungal gene expression, or growth of extraradical mycelium, indicating little effect of night warming on the development or functioning of AM symbiosis. These findings highlight the importance of mycorrhizal symbiosis in assisting plant resilience to climate warming.

  1. Rhizobial symbiosis effect on the growth, metal uptake, and antioxidant responses of Medicago lupulina under copper stress.

    PubMed

    Kong, Zhaoyu; Mohamad, Osama Abdalla; Deng, Zhenshan; Liu, Xiaodong; Glick, Bernard R; Wei, Gehong

    2015-08-01

    The effects of rhizobial symbiosis on the growth, metal uptake, and antioxidant responses of Medicago lupulina in the presence of 200 mg kg(-1) Cu(2+) throughout different stages of symbiosis development were studied. The symbiosis with Sinorhizobium meliloti CCNWSX0020 induced an increase in plant growth and nitrogen content irrespective of the presence of Cu(2+). The total amount of Cu uptake of inoculated plants significantly increased by 34.0 and 120.4% in shoots and roots, respectively, compared with non-inoculated plants. However, although the rhizobial symbiosis promoted Cu accumulation both in shoots and roots, the increase in roots was much higher than in shoots, thus decreasing the translocation factor and helping Cu phytostabilization. The rate of lipid peroxidation was significantly decreased in both shoots and roots of inoculated vs. non-inoculated plants when measured either 8, 13, or 18 days post-inoculation. In comparison with non-inoculated plants, the activities of superoxide dismutase and ascorbate peroxidase of shoots of inoculated plants exposed to excess Cu were significantly elevated at different stages of symbiosis development; similar increases occurred in the activities of superoxide dismutase, catalase, and glutathione reductase of inoculated roots. The symbiosis with S. meliloti CCNWSX0020 also upregulated the corresponding genes involved in antioxidant responses in the plants treated with excess Cu. The results indicated that the rhizobial symbiosis with S. meliloti CCNWSX0020 not only enhanced plant growth and metal uptake but also improved the responses of plant antioxidant defense to excess Cu stress.

  2. The arbuscular mycorrhizal fungus Glomus mosseae gives contradictory effects on phosphorus and arsenic acquisition by Medicago sativa Linn.

    PubMed

    Chen, Baodong; Xiao, Xueyi; Zhu, Yong-Guan; Smith, F Andrew; Xie, Z Miao; Smith, Sally E

    2007-07-01

    Mycorrhizal fungi may play an important role in protecting plants against arsenic (As) contamination. However, little is known about the direct and indirect involvement of arbuscular mycorrhizal fungi (AMF) in detoxification mechanisms. A compartmented pot cultivation system ('cross-pots') is used here to investigate the roles of AMF Glomus mosseae in plant phosphorus (P) and As acquisition by Medicago sativa, and P-As interactions. The results indicate that fungal colonization dramatically increased plant dry weight by a factor of around 6, and also substantially increased both plant P and As contents (i.e. total uptake). Irrespective of P and As addition levels, AM plants had shoot and root P concentrations 2 fold higher, but As concentrations significantly lower, than corresponding uninoculated controls. The decreased shoot As concentrations were largely due to "dilution effects" that resulted from stimulated growth of AM plants and reduced As partitioning to shoots. The study provides further evidence for the protective effects of AMF on host plants against As contamination, and have uncovered key aspects of underlying mechanisms. The possible application of AMF in remediation practices is discussed.

  3. MATE2 Mediates Vacuolar Sequestration of Flavonoid Glycosides and Glycoside Malonates in Medicago truncatula[C][W][OA

    PubMed Central

    Zhao, Jian; Huhman, David; Shadle, Gail; He, Xian-Zhi; Sumner, Lloyd W.; Tang, Yuhong; Dixon, Richard A.

    2011-01-01

    The majority of flavonoids, such as anthocyanins, proanthocyanidins, and isoflavones, are stored in the central vacuole, but the molecular basis of flavonoid transport is still poorly understood. Here, we report the functional characterization of a multidrug and toxin extrusion transporter (MATE2), from Medicago truncatula. MATE 2 is expressed primarily in leaves and flowers. Despite its high similarity to the epicatechin 3′-O-glucoside transporter MATE1, MATE2 cannot efficiently transport proanthocyanidin precursors. In contrast, MATE2 shows higher transport capacity for anthocyanins and lower efficiency for other flavonoid glycosides. Three malonyltransferases that are coexpressed with MATE2 were identified. The malonylated flavonoid glucosides generated by these malonyltransferases are more efficiently taken up into MATE2-containing membrane vesicles than are the parent glycosides. Malonylation increases both the affinity and transport efficiency of flavonoid glucosides for uptake by MATE2. Genetic loss of MATE2 function leads to the disappearance of leaf anthocyanin pigmentation and pale flower color as a result of drastic decreases in the levels of various flavonoids. However, some flavonoid glycoside malonates accumulate to higher levels in MATE2 knockouts than in wild-type controls. Deletion of MATE2 increases seed proanthocyanidin biosynthesis, presumably via redirection of metabolic flux from anthocyanin storage. PMID:21467581

  4. Flavonoids and Auxin Transport Inhibitors Rescue Symbiotic Nodulation in the Medicago truncatula Cytokinin Perception Mutant cre1.

    PubMed

    Ng, Jason Liang Pin; Hassan, Samira; Truong, Thy T; Hocart, Charles H; Laffont, Carole; Frugier, Florian; Mathesius, Ulrike

    2015-08-01

    Initiation of symbiotic nodules in legumes requires cytokinin signaling, but its mechanism of action is largely unknown. Here, we tested whether the failure to initiate nodules in the Medicago truncatula cytokinin perception mutant cre1 (cytokinin response1) is due to its altered ability to regulate auxin transport, auxin accumulation, and induction of flavonoids. We found that in the cre1 mutant, symbiotic rhizobia cannot locally alter acro- and basipetal auxin transport during nodule initiation and that these mutants show reduced auxin (indole-3-acetic acid) accumulation and auxin responses compared with the wild type. Quantification of flavonoids, which can act as endogenous auxin transport inhibitors, showed a deficiency in the induction of free naringenin, isoliquiritigenin, quercetin, and hesperetin in cre1 roots compared with wild-type roots 24 h after inoculation with rhizobia. Coinoculation of roots with rhizobia and the flavonoids naringenin, isoliquiritigenin, and kaempferol, or with the synthetic auxin transport inhibitor 2,3,5,-triiodobenzoic acid, rescued nodulation efficiency in cre1 mutants and allowed auxin transport control in response to rhizobia. Our results suggest that CRE1-dependent cytokinin signaling leads to nodule initiation through the regulation of flavonoid accumulation required for local alteration of polar auxin transport and subsequent auxin accumulation in cortical cells during the early stages of nodulation.

  5. Microgravity Effects on the Early Events of Biological Nitrogen Fixation in Medicago Truncatula: Results from the SyNRGE Experiment

    NASA Astrophysics Data System (ADS)

    Stutte, Gary W.; Roberts, Michael S.

    2013-02-01

    SyNRGE (Symbiotic Nodulation in a Reduced Gravity Environment) was a sortie mission on STS-135 in the Biological Research in Canisters (BRIC) hardware to study the effect of μg on a plant-microbe symbiosis resulting in biological nitrogen fixation. Medicago truncatula, a model species for the legume family, was inoculated with its bacterial symbiont, Sinorhizobium meliloti, to observe early biomolecular events associated with infection and nodulation in Petri Dish Fixation Units (PDFU’s). Two sets of experiments were conducted in orbit and in 24-hour delayed ground controls. Experiments were designed to determine if S. meliloti would infect M. truncatula and initiate biomolecular changes associated with nodule formation and if the μg environment altered the host plant and/or bacteria to induce nodule formation upon return to 1g. Initial analysis results demonstrate that the legumes and bacteria cultivated in μg have potential to develop a symbiotic interaction, but suggest that μg alters their ability to form nodules upon return to 1g. (Research supported by NASA ESMD/ Advance Capabilities Division grant NNX10AR09A)

  6. Characterization of the plant growth promoting bacterium, Enterobacter cloacae MSR1, isolated from roots of non-nodulating Medicago sativa

    PubMed Central

    Khalifa, Ashraf Y.Z.; Alsyeeh, Abdel-Moneium; Almalki, Mohammed A.; Saleh, Farag A.

    2015-01-01

    The aim of the present study was to characterize the endophytic bacterial strain designated MSR1 that was isolated from inside the non-nodulating roots of Medicago sativa after surface-sterilization. MSR1 was identified as Enterobacter cloacae using both 16S rDNA gene sequence analysis and API20E biochemical identification system (Biomerieux, France). Furthermore, this bacterium was characterized using API50CH kit (Biomerieux, France) and tested for antibacterial activities against some food borne pathogens. The results showed that E. cloacae consumed certain carbohydrates such as glycerol, d-xylose, d-maltose and esculin melibiose as a sole carbon source and certain amino acids such as arginine, tryptophan ornithine as nitrogen source. Furthermore, MSR1 possessed multiple plant-growth promoting characteristics; phosphate solubility, production of phytohormones acetoin and bioactive compounds. Inoculation of Pisum sativum with MSR1 significantly improved the growth parameters (the length and dry weight) of this economically important grain legume compared to the non-treated plants. To our knowledge, this is the first report addressing E. cloacae which exist in roots of alfalfa growing in Al-Ahsaa region. The results confirmed that E. cloacae exhibited traits for plant growth promoting and could be developed as an eco-friendly biofertilizer for P. sativum and probably for other important plant species in future. PMID:26858542

  7. Contrasting calcium localization and speciation in leaves of Medicago truncatula mutant COD5 analyzed via synchrotron X-ray techniques

    PubMed Central

    Punshon, Tracy; Tappero, Ryan; Ricachenevsky, Felipe K.; Hirschi, Kendal; Nakata, Paul A.

    2014-01-01

    SUMMARY Oxalate-producing plants accumulate calcium oxalate crystals (CaOx(c)) in the range of 3–80%(w/w) of their dry weight, reducing calcium (Ca) bioavailability. The calcium oxalate deficient 5 (cod5) mutant of Medicago truncatula has been previously shown to contain similar Ca, but lower oxalate and CaOx(c) concentrations than wild type (WT) plants. We imaged the Ca distribution in WT and cod5 leaflets via synchrotron X-ray fluorescence mapping (SXRF). We observed a contrast in the Ca distribution between cod5 and WT leaflets, manifested as an abundance of Ca in the interveinal areas and a lack of Ca along the secondary veins in cod5, the opposite of WT. X-ray microdiffraction (µXRD) of M. truncatula leaves confirmed crystalline CaOx(c) (whewellite; CaC2O4•H2O) was present in WT only, in cells sheathing the secondary veins. Together with µXRD, microbeam Ca K-edge X-ray absorption near-edge structure spectroscopy (µXANES) indicated that among the forms of CaOx – namely crystalline or amorphous – only amorphous CaOx was present in cod5. These results demonstrate that deletion of COD5 changes both Ca localization and the form of CaOx within leaflets. PMID:24033783

  8. An endogenous artificial microRNA system for unraveling the function of root endosymbioses related genes in Medicago truncatula

    PubMed Central

    2013-01-01

    Background Legumes have the unique capacity to undergo two important root endosymbioses: the root nodule symbiosis and the arbuscular mycorrhizal symbiosis. Medicago truncatula is widely used to unravel the functions of genes during these root symbioses. Here we describe the development of an artificial microRNA (amiR)-mediated gene silencing system for M. truncatula roots. Results The endogenous microRNA (miR) mtr-miR159b was selected as a backbone molecule for driving amiR expression. Heterologous expression of mtr-miR159b-amiR constructs in tobacco showed that the backbone is functional and mediates an efficient gene silencing. amiR-mediated silencing of a visible marker was also effective after root transformation of M. truncatula constitutively expressing the visible marker. Most importantly, we applied the novel amiR system to shed light on the function of a putative transcription factor, MtErf1, which was strongly induced in arbuscule-containing cells during mycorrhizal symbiosis. MtPt4 promoter driven amiR-silencing led to strongly decreased transcript levels and deformed, non-fully truncated arbuscules indicating that MtErf1 is required for arbuscule development. Conclusions The endogenous amiR system demonstrated here presents a novel and highly efficient tool to unravel gene functions during root endosymbioses. PMID:23679580

  9. Effect of cadmium pollution on mobilization of embryo reserves in seedlings of six contrasted Medicago truncatula lines.

    PubMed

    Rahoui, Sondès; Chaoui, Abdelilah; Ben, Cécile; Rickauer, Martina; Gentzbittel, Laurent; El Ferjani, Ezzeddine

    2015-03-01

    Six Medicago truncatula genotypes differing in cadmium susceptibility were used to test the effect of this heavy metal on mineral, carbohydrate and amino acid supply in growing radicles. Cadmium treatment caused alteration of macronutrient (Ca and K), microelement (Fe, Zn and Cu), carbohydrate (total soluble sugars (TSS), glucose, fructose and sucrose) and free amino acid (FAAS) accumulations. These mobilization changes differed in the tested genotypes. Carbohydrates were determining to susceptible lines' growth in control condition; free amino acids enabled tolerant lines to counteract cadmium intrusion. Transcriptional changes in response to cadmium treatment were analyzed on MtMST, a gene encoding a monosaccharide transport protein. A significant down-regulation was observed in the most susceptible line TN1.11. Glucose was over-consumed in tolerant lines. Thus, glucose metabolism integrity seems essential to maintain growth under cadmium exposure. Analysis of germination medium showed solute losses at the expense of suitable mobilization to the growing embryonic axis and highlights cadmium-triggered membrane alterations. FAAS and TSS leakages were reduced in tolerant lines while monosaccharide losses were accentuated in susceptible lines. This research work gave an overview of cadmium deleterious effects on biomass mobilization and membrane integrity. Carbon metabolism is shown to be primordial to enhance early embryonic growth and nitrogen metabolism is revealed to be crucial to establish seedling growth under cadmium stress.

  10. The Sinorhizobium meliloti EmrR regulator is required for efficient colonization of Medicago sativa root nodules.

    PubMed

    Santos, Mário R; Marques, Andreia T; Becker, Jörg D; Moreira, Leonilde M

    2014-04-01

    The nitrogen-fixing bacterium Sinorhizobium meliloti must adapt to diverse conditions encountered during its symbiosis with leguminous plants. We characterized a new symbiotically relevant gene, emrR (SMc03169), whose product belongs to the TetR family of repressors and is divergently transcribed from emrAB genes encoding a putative major facilitator superfamily-type efflux pump. An emrR deletion mutant produced more succinoglycan, displayed increased cell-wall permeability, and exhibited higher tolerance to heat shock. It also showed lower tolerance to acidic conditions, a reduced production of siderophores, and lower motility and biofilm formation. The simultaneous deletion of emrA and emrR genes restored the mentioned traits to the wild-type phenotype, except for survival under heat shock, which was lower than that displayed by the wild-type strain. Furthermore, the ΔemrR mutant as well as the double ΔemrAR mutant was impaired in symbiosis with Medicago sativa; it formed fewer nodules and competed poorly with the wild-type strain for nodule colonization. Expression profiling of the ΔemrR mutant showed decreased expression of genes involved in Nod-factor and rhizobactin biosynthesis and in stress responses. Expression of genes directing the biosynthesis of succinoglycan and other polysaccharides were increased. EmrR may therefore be involved in a regulatory network targeting membrane and cell wall modifications in preparation for colonization of root hairs during symbiosis.

  11. Allelopathic Potential of Switchgrass ( Panicum virgatum L.) on Perennial Ryegrass ( Lolium perenne L.) and Alfalfa ( Medicago sativa L.)

    NASA Astrophysics Data System (ADS)

    Shui, Junfeng; An, Yu; Ma, Yongqing; Ichizen, Nobumasa

    2010-10-01

    This study investigated allelopathy and its chemical basis in nine switchgrass ( Panicum virgatum L.) accessions. Perennial ryegrass ( Lolium perenne L.) and alfalfa ( Medicago sativa L.) were used as test species. Undiluted aqueous extracts (5 g plant tissue in 50 ml water) from the shoots and roots of most of the switchgrass accessions inhibited the germination and growth of the test species. However, the allelopathic effect of switchgrass declined when extracts were diluted 5- or 50-fold. Seedling growth was more sensitive than seed germination as an indicator of allelopathic effect. Allelopathic effect was related to switchgrass ecotype but not related to ploidy level. Upland accessions displayed stronger allelopathic potential than lowland accessions. The aqueous extract from one switchgrass accession was separated into phenols, organic acids, neutral chemicals, and alkaloids, and then these fractions were bioassayed to test for allelopathic potential. Alkaloids had the strongest allelopathic effect among the four chemical fractions. In summary, the results indicated that switchgrass has allelopathic potential; however, there is not enough evidence to conclude that allelopathic advantage is the main factor that has contributed to the successful establishment of switchgrass on China’s Loess Plateau.

  12. Identification of molecular markers associated with Verticillium wilt resistance in alfalfa (Medicago sativa L.) using high-resolution melting.

    PubMed

    Zhang, Tiejun; Yu, Long-Xi; McCord, Per; Miller, David; Bhamidimarri, Suresh; Johnson, David; Monteros, Maria J; Ho, Julie; Reisen, Peter; Samac, Deborah A

    2014-01-01

    Verticillium wilt, caused by the soilborne fungus, Verticillium alfalfae, is one of the most serious diseases of alfalfa (Medicago sativa L.) worldwide. To identify loci associated with resistance to Verticillium wilt, a bulk segregant analysis was conducted in susceptible or resistant pools constructed from 13 synthetic alfalfa populations, followed by association mapping in two F1 populations consisted of 352 individuals. Simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers were used for genotyping. Phenotyping was done by manual inoculation of the pathogen to replicated cloned plants of each individual and disease severity was scored using a standard scale. Marker-trait association was analyzed by TASSEL. Seventeen SNP markers significantly associated with Verticillium wilt resistance were identified and they were located on chromosomes 1, 2, 4, 7 and 8. SNP markers identified on chromosomes 2, 4 and 7 co-locate with regions of Verticillium wilt resistance loci reported in M. truncatula. Additional markers identified on chromosomes 1 and 8 located the regions where no Verticillium resistance locus has been reported. This study highlights the value of SNP genotyping by high resolution melting to identify the disease resistance loci in tetraploid alfalfa. With further validation, the markers identified in this study could be used for improving resistance to Verticillium wilt in alfalfa breeding programs.

  13. Involvement of abscisic acid in the response of Medicago sativa plants in symbiosis with Sinorhizobium meliloti to salinity.

    PubMed

    Palma, F; López-Gómez, M; Tejera, N A; Lluch, C

    2014-06-01

    Legumes are classified as salt-sensitive crops with their productivity particularly affected by salinity. Abcisic acid (ABA) plays an important role in the response to environmental stresses as signal molecule which led us to study its role in the response of nitrogen fixation and antioxidant metabolism in root nodules of Medicago sativa under salt stress conditions. Adult plants inoculated with Sinorhizobium meliloti were treated with 1 μM and 10 μM ABA two days before 200 mM salt addition. Exogenous ABA together with the salt treatment provoked a strong induction of the ABA content in the nodular tissue which alleviated the inhibition induced by salinity in the plant growth and nitrogen fixation. Antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR) were induced by ABA pre-treatments under salt stress conditions which together with the reduction of the lipid peroxidation, suggest a role for ABA as signal molecule in the activation of the nodular antioxidant metabolism. Interaction between ABA and polyamines (PAs), described as anti-stress molecules, was studied being detected an induction of the common polyamines spermidine (Spd) and spermine (Spm) levels by ABA under salt stress conditions. In conclusion, ABA pre-treatment improved the nitrogen fixation capacity under salt stress conditions by the induction of the nodular antioxidant defenses which may be mediated by the common PAs Spd and Spm that seems to be involved in the anti-stress response induced by ABA.

  14. New pharmacological properties of Medicago sativa and Saponaria officinalis saponin-rich fractions addressed to Candida albicans.

    PubMed

    Sadowska, Beata; Budzyńska, Aleksandra; Więckowska-Szakiel, Marzena; Paszkiewicz, Małgorzata; Stochmal, Anna; Moniuszko-Szajwaj, Barbara; Kowalczyk, Mariusz; Różalska, Barbara

    2014-08-01

    The antifungal activity of the saponin-rich fractions (SFs) from Medicago sativa (aerial parts and roots) and Saponaria officinalis (used as a well-known source of plant saponins) against Candida albicans reference and clinical strains, their yeast-to-hyphal conversion, adhesion, and biofilm formation was investigated. Direct fungicidal/fungistatic properties of the tested phytochemicals used alone, as well as their synergy with azoles (probably resulting from yeast cell wall instability) were demonstrated. Here, to the best of our knowledge, we report for the first time the ability of saponin-rich extracts of M. sativa and S. officinalis to inhibit C. albicans germ tube formation, limit hyphal growth, reduce yeast adherence and biofilm formation, and eradicate mature (24 h) Candida biofilm. Moreover, M. sativa SFs (mainly obtained from aerial parts), in the range of concentrations which were active modulators of Candida virulence factors, exhibited low cytotoxicity against the mouse fibroblast line L929. These properties seem to be very promising in the context of using plant-derived SFs as potential novel antifungal therapeutics supporting classic drugs or as ingredients of disinfectants.

  15. Identification of transcriptome involved in atrazine detoxification and degradation in alfalfa (Medicago sativa) exposed to realistic environmental contamination.

    PubMed

    Zhang, Jing Jing; Lu, Yi Chen; Zhang, Shu Hao; Lu, Feng Fan; Yang, Hong

    2016-08-01

    Plants are constantly exposed to a variety of toxic compounds (or xenobiotics) such as pesticides (or herbicides). Atrazine (ATZ) as herbicide has become one of the environmental contaminants due to its intensive use during crop production. Plants have evolved strategies to cope with the adverse impact of ATZ. However, the mechanism for ATZ degradation and detoxification in plants is largely unknown. Here we employed a global RNA-sequencing (RNA-Seq) strategy to dissect transcriptome variation in alfalfa (Medicago sativa) exposed to ATZ. Four libraries were constructed including Root-ATZ (root control, ATZ-free), Shoot-ATZ, Root+ATZ (root treated with ATZ) and Shoot+ATZ. Hierarchical clustering was performed to display the expression patterns for all differentially expressed genes (DEGs) under ATZ exposure. Transcripts involved in ATZ detoxification, stress responses (e.g. oxidation and reduction, conjugation and hydrolytic reactions), and regulations of cysteine biosynthesis were identified. Several genes encoding glycosyltransferases, glutathione S-transferases or ABC transporters were up-regulated notably. Also, many other genes involved in oxidation-reduction, conjugation, and hydrolysis for herbicide degradation were differentially expressed. These results suggest that ATZ in alfalfa can be detoxified or degraded through different pathways. The expression patterns of some DEGs by high-throughput sequencing were well confirmed by qRT-PCR. Our results not only highlight the transcriptional complexity in alfalfa exposed to ATZ but represent a major improvement for analyzing transcriptional changes on a large scale as well.

  16. Deep-sequencing transcriptome analysis of field-grown Medicago sativa L. crown buds acclimated to freezing stress.

    PubMed

    Song, Lili; Jiang, Lin; Chen, Yue; Shu, Yongjun; Bai, Yan; Guo, Changhong

    2016-09-01

    Medicago sativa L. (alfalfa) 'Zhaodong' is an important forage legume that can safely survive in northern China where winter temperatures reach as low as -30 °C. Survival of alfalfa following freezing stress depends on the amount and revival ability of crown buds. In order to investigate the molecular mechanisms of frost tolerance in alfalfa, we used transcriptome sequencing technology and bioinformatics strategies to analyze crown buds of field-grown alfalfa during winter. We statistically identified a total of 5605 differentially expressed genes (DEGs) involved in freezing stress including 1900 upregulated and 3705 downregulated DEGs. We validated 36 candidate DEGs using qPCR to confirm the accuracy of the RNA-seq data. Unlike other recent studies, this study employed alfalfa plants grown in the natural environment. Our results indicate that not only the CBF orthologs but also membrane proteins, hormone signal transduction pathways, and ubiquitin-mediated proteolysis pathways indicate the presence of a special freezing adaptation mechanism in alfalfa. The antioxidant defense system may rapidly confer freezing tolerance to alfalfa. Importantly, biosynthesis of secondary metabolites and phenylalanine metabolism, which is of potential importance in coordinating freezing tolerance with growth and development, were downregulated in subzero temperatures. The adaptive mechanism for frost tolerance is a complex multigenic process that is not well understood. This systematic analysis provided an in-depth view of stress tolerance mechanisms in alfalfa.

  17. Salicornia europaea L. Na⁺/H⁺ antiporter gene improves salt tolerance in transgenic alfalfa (Medicago sativa L.).

    PubMed

    Zhang, L Q; Niu, Y D; Huridu, H; Hao, J F; Qi, Z; Hasi, A

    2014-07-24

    In order to obtain a salt-tolerant perennial alfalfa (Medicago sativa L.), we transferred the halophyte Salicornia europaea L. Na(+)/H(+) antiporter gene, SeNHX1, to alfalfa by using the Agrobacterium-mediated transformation method. The transformants were confirmed by both PCR and RT-PCR analyses. Of 197 plants that were obtained after transformation, 36 were positive by PCR analysis using 2 primer pairs for the CaMV35S-SeNHX1 and SeNHX1-Nos fragments; 6 plants survived in a greenhouse. RT-PCR analysis revealed that SeNHX1 was expressed in 5 plants. The resultant transgenic alfalfa had better salt tolerance. After stress treatment for 21 days with 0.6% NaCl, the chlorophyll and MDA contents in transgenic plants were lower, but proline content and SOD, POD, and CAT activities were higher than those in wild-type plants. These results suggest that the salt tolerance of transgenic alfalfa was improved by the overexpression of the SeNHX1 gene.

  18. Sinorhizobium meliloti Controls Nitric Oxide-Mediated Post-Translational Modification of a Medicago truncatula Nodule Protein.

    PubMed

    Blanquet, Pauline; Silva, Liliana; Catrice, Olivier; Bruand, Claude; Carvalho, Helena; Meilhoc, Eliane

    2015-12-01

    Nitric oxide (NO) is involved in various plant-microbe interactions. In the symbiosis between soil bacterium Sinorhizobium meliloti and model legume Medicago truncatula, NO is required for an optimal establishment of the interaction but is also a signal for nodule senescence. Little is known about the molecular mechanisms responsible for NO effects in the legume-rhizobium interaction. Here, we investigate the contribution of the bacterial NO response to the modulation of a plant protein post-translational modification in nitrogen-fixing nodules. We made use of different bacterial mutants to finely modulate NO levels inside M. truncatula root nodules and to examine the consequence on tyrosine nitration of the plant glutamine synthetase, a protein responsible for assimilation of the ammonia released by nitrogen fixation. Our results reveal that S. meliloti possesses several proteins that limit inactivation of plant enzyme activity via NO-mediated post-translational modifications. This is the first demonstration that rhizobia can impact the course of nitrogen fixation by modulating the activity of a plant protein.

  19. (Homo)glutathione depletion modulates host gene expression during the symbiotic interaction between Medicago truncatula and Sinorhizobium meliloti.

    PubMed

    Pucciariello, Chiara; Innocenti, Gilles; Van de Velde, Willem; Lambert, Annie; Hopkins, Julie; Clément, Mathilde; Ponchet, Michel; Pauly, Nicolas; Goormachtig, Sofie; Holsters, Marcelle; Puppo, Alain; Frendo, Pierre

    2009-11-01

    Under nitrogen-limiting conditions, legumes interact with symbiotic rhizobia to produce nitrogen-fixing root nodules. We have previously shown that glutathione and homoglutathione [(h)GSH] deficiencies impaired Medicago truncatula symbiosis efficiency, showing the importance of the low M(r) thiols during the nodulation process in the model legume M. truncatula. In this study, the plant transcriptomic response to Sinorhizobium meliloti infection under (h)GSH depletion was investigated using cDNA-amplified fragment length polymorphism analysis. Among 6,149 expression tags monitored, 181 genes displayed significant differential expression between inoculated control and inoculated (h)GSH depleted roots. Quantitative reverse transcription polymerase chain reaction analysis confirmed the changes in mRNA levels. This transcriptomic analysis shows a down-regulation of genes involved in meristem formation and a modulation of the expression of stress-related genes in (h)GSH-depleted plants. Promoter-beta-glucuronidase histochemical analysis showed that the putative MtPIP2 aquaporin might be up-regulated during nodule meristem formation and that this up-regulation is inhibited under (h)GSH depletion. (h)GSH depletion enhances the expression of salicylic acid (SA)-regulated genes after S. meliloti infection and the expression of SA-regulated genes after exogenous SA treatment. Modification of water transport and SA signaling pathway observed under (h)GSH deficiency contribute to explain how (h)GSH depletion alters the proper development of the symbiotic interaction.

  20. Plant-activated bacterial receptor adenylate cyclases modulate epidermal infection in the Sinorhizobium meliloti-Medicago symbiosis.

    PubMed

    Tian, Chang Fu; Garnerone, Anne-Marie; Mathieu-Demazière, Céline; Masson-Boivin, Catherine; Batut, Jacques

    2012-04-24

    Legumes and soil bacteria called rhizobia have coevolved a facultative nitrogen-fixing symbiosis. Establishment of the symbiosis requires bacterial entry via root hair infection threads and, in parallel, organogenesis of nodules that subsequently are invaded by bacteria. Tight control of nodulation and infection is required to maintain the mutualistic character of the interaction. Available evidence supports a passive bacterial role in nodulation and infection after the microsymbiont has triggered the symbiotic plant developmental program. Here we identify in Sinorhizobium meliloti, the Medicago symbiont, a cAMP-signaling regulatory cascade consisting of three receptor-like adenylate cyclases, a Crp-like regulator, and a target gene of unknown function. The cascade is activated specifically by a plant signal during nodule organogenesis. Cascade inactivation results in a hyperinfection phenotype consisting of abortive epidermal infection events uncoupled from nodulation. These findings show that, in response to a plant signal, rhizobia play an active role in the control of infection. We suggest that rhizobia may modulate the plant's susceptibility to infection. This regulatory loop likely aims at optimizing legume infection.

  1. The Medicago truncatula CRE1 cytokinin receptor regulates lateral root development and early symbiotic interaction with Sinorhizobium meliloti.

    PubMed

    Gonzalez-Rizzo, Silvina; Crespi, Martin; Frugier, Florian

    2006-10-01

    Legumes develop different types of lateral organs from their primary root, lateral roots and nodules, the latter depending on a symbiotic interaction with Sinorhizobium meliloti. Phytohormones have been shown to function in the control of these organogeneses. However, related signaling pathways have not been identified in legumes. We cloned and characterized the expression of Medicago truncatula genes encoding members of cytokinin signaling pathways. RNA interference of the cytokinin receptor homolog Cytokinin Response1 (Mt CRE1) led to cytokinin-insensitive roots, which showed an increased number of lateral roots and a strong reduction in nodulation. Both the progression of S. meliloti infection and nodule primordia formation were affected. We also identified two cytokinin signaling response regulator genes, Mt RR1 and Mt RR4, which are induced early during the symbiotic interaction. Induction of these genes by S. meliloti infection is altered in mutants affected in the Nod factor signaling pathway; conversely, cytokinin regulation of the early nodulin Nodule Inception1 (Mt NIN) depends on Mt CRE1. Hence, cytokinin signaling mediated by a single receptor, Mt CRE1, leads to an opposite control of symbiotic nodule and lateral root organogenesis. Mt NIN, Mt RR1, and Mt RR4 define a common pathway activated during early S. meliloti interaction, allowing crosstalk between plant cytokinins and bacterial Nod factors signals.

  2. Medicago truncatula increases its iron-uptake mechanisms in response to volatile organic compounds produced by Sinorhizobium meliloti.

    PubMed

    Orozco-Mosqueda, Maria del Carmen; Macías-Rodríguez, Lourdes I; Santoyo, Gustavo; Farías-Rodríguez, Rodolfo; Valencia-Cantero, Eduardo

    2013-11-01

    Medicago truncatula represents a model plant species for understanding legume-bacteria interactions. M. truncatula roots form a specific root-nodule symbiosis with the nitrogen-fixing bacterium Sinorhizobium meliloti. Symbiotic nitrogen fixation generates high iron (Fe) demands for bacterial nitrogenase holoenzyme and plant leghemoglobin proteins. Leguminous plants acquire Fe via "Strategy I," which includes mechanisms such as rhizosphere acidification and enhanced ferric reductase activity. In the present work, we analyzed the effect of S. meliloti volatile organic compounds (VOCs) on the Fe-uptake mechanisms of M. truncatula seedlings under Fe-deficient and Fe-rich conditions. Axenic cultures showed that both plant and bacterium modified VOC synthesis in the presence of the respective symbiotic partner. Importantly, in both Fe-rich and -deficient experiments, bacterial VOCs increased the generation of plant biomass, rhizosphere acidification, ferric reductase activity, and chlorophyll content in plants. On the basis of our results, we propose that M. truncatula perceives its symbiont through VOC emissions, and in response, increases Fe-uptake mechanisms to facilitate symbiosis.

  3. The NIN Transcription Factor Coordinates Diverse Nodulation Programs in Different Tissues of the Medicago truncatula Root[OPEN

    PubMed Central

    Kim, Jiyoung; Frances, Lisa; Ding, Yiliang; Sun, Jongho; Guan, Dian; de Carvalho-Niebel, Fernanda; Oldroyd, Giles E.D.

    2015-01-01

    Biological nitrogen fixation in legumes occurs in nodules that are initiated in the root cortex following Nod factor recognition at the root surface, and this requires coordination of diverse developmental programs in these different tissues. We show that while early Nod factor signaling associated with calcium oscillations is limited to the root surface, the resultant activation of Nodule Inception (NIN) in the root epidermis is sufficient to promote cytokinin signaling and nodule organogenesis in the inner root cortex. NIN or a product of its action must be associated with the transmission of a signal between the root surface and the cortical cells where nodule organogenesis is initiated. NIN appears to have distinct functions in the root epidermis and the root cortex. In the epidermis, NIN restricts the extent of Early Nodulin 11 (ENOD11) expression and does so through competitive inhibition of ERF Required for Nodulation (ERN1). In contrast, NIN is sufficient to promote the expression of the cytokinin receptor Cytokinin Response 1 (CRE1), which is restricted to the root cortex. Our work in Medicago truncatula highlights the complexity of NIN action and places NIN as a central player in the coordination of the symbiotic developmental programs occurring in differing tissues of the root that combined are necessary for a nitrogen-fixing symbiosis. PMID:26672071

  4. P-HYDROXYPHENYLPYRUVATE DIOXYGENASE from Medicago sativa is involved in vitamin E biosynthesis and abscisic acid-mediated seed germination

    PubMed Central

    Jiang, Jishan; Chen, Zhihong; Ban, Liping; Wu, Yudi; Huang, Jianping; Chu, Jinfang; Fang, Shuang; Wang, Zan; Gao, Hongwen; Wang, Xuemin

    2017-01-01

    P-HYDROXYPHENYLPYRUVATE DIOXYGENASE (HPPD) is the first committed enzyme involved in the biosynthesis of vitamin E, and is characterized by catalyzing the conversion of p-hydroxyphenyl pyruvate (HPP) to homogentisic acid (HGA). Here, an HPPD gene was cloned from Medicago sativa L. and designated MsHPPD, which was expressed at high levels in alfalfa leaves. PEG 6000 (polyethylene glycol), NaCl, abscisic acid and salicylic acid were shown to significantly induce MsHPPD expression, especially in the cotyledons and root tissues. Overexpression of MsHPPD was found to significantly increase the level of β-tocotrienol and the total vitamin E content in Arabidopsis seeds. Furthermore, these transgenic Arabidopsis seeds exhibited an accelerated germination time, compared with wild-type seeds under normal conditions, as well as under NaCl and ABA treatments. Meanwhile, the expression level of several genes associated with ABA biosynthesis (NCED3, NCED5 and NCED9) and the ABA signaling pathway (RAB18, ABI3 and ABI5) were significantly down-regulated in MsHPPD-overexpressing transgenic lines, as well as the total free ABA content. Taken together, these results demonstrate that MsHPPD functions not only in the vitamin E biosynthetic pathway, but also plays a critical role in seed germination via affecting ABA biosynthesis and signaling. PMID:28084442

  5. Toxic effects of copper-based nanoparticles or compounds to lettuce (Lactuca sativa) and alfalfa (Medicago sativa).

    PubMed

    Hong, Jie; Rico, Cyren M; Zhao, Lijuan; Adeleye, Adeyemi S; Keller, Arturo A; Peralta-Videa, Jose R; Gardea-Torresdey, Jorge L

    2015-01-01

    The increased production and use of nanoparticles (NPs) has generated concerns about their impact on living organisms. In this study, nCu, bulk Cu, nCuO, bulk CuO, Cu(OH)2 (CuPRO 2005, Kocide 3000), and CuCl2 were exposed for 15 days to 10 days-old hydroponically grown lettuce (Lactuca sativa) and alfalfa (Medicago sativa). Each compound was applied at 0, 5, 10, and 20 mg L(-1). At harvest, we measured the size of the plants and determined the concentration of Cu, macro and microelements by using ICP-OES. Catalase and ascorbate peroxidase activity was also determined. Results showed that all Cu NPs/compounds reduced the root length by 49% in both plant species. All Cu NPs/compounds increased Cu, P, and S (>100%, >50%, and >20%, respectively) in alfalfa shoots and decreased P and Fe in lettuce shoot (>50% and >50%, respectively, excluding Fe in CuCl2 treatment). Biochemical assays showed reduced catalase activity in alfalfa (root and shoot) and increased ascorbate peroxidase activity in roots of both plant species. Results suggest that Cu NPs/compounds not only reduced the size of the plants but altered nutrient content and enzyme activity in both plant species.

  6. Medicago N2-fixing symbiosomes acquire the endocytic identity marker Rab7 but delay the acquisition of vacuolar identity.

    PubMed

    Limpens, Erik; Ivanov, Sergey; van Esse, Wilma; Voets, Guido; Fedorova, Elena; Bisseling, Ton

    2009-09-01

    Rhizobium bacteria form N(2)-fixing organelles, called symbiosomes, inside the cells of legume root nodules. The bacteria are generally thought to enter the cells via an endocytosis-like process. To examine this, we studied the identity of symbiosomes in relation to the endocytic pathway. We show that in Medicago truncatula, the small GTPases Rab5 and Rab7 are endosomal membrane identity markers, marking different (partly overlapping) endosome populations. Although symbiosome formation is considered to be an endocytosis-like process, symbiosomes do not acquire Rab5 at any stage during their development, nor do they accept the trans-Golgi network identity marker SYP4, presumed to mark early endosomes in plants. By contrast, the endosomal marker Rab7 does occur on symbiosomes from an early stage of development when they have stopped dividing up to the senescence stage. However, the symbiosomes do not acquire vacuolar SNAREs (SYP22 and VTI11) until the onset of their senescence. By contrast, symbiosomes acquire the plasma membrane SNARE SYP132 from the start of symbiosome formation throughout their development. Therefore, symbiosomes appear to be locked in a unique SYP132- and Rab7-positive endosome stage and the delay in acquiring (lytic) vacuolar identity (e.g., vacuolar SNAREs) most likely ensures their survival and maintenance as individual units.

  7. Relationship between photosynthetic capacity, nitrogen assimilation and nodule metabolism in alfalfa (Medicago sativa) grown with sewage sludge.

    PubMed

    Antolín, M Carmen; Fiasconaro, M Laura; Sánchez-Díaz, Manuel

    2010-10-15

    Sewage sludge has been used as N fertilizer because it contains some of inorganic N, principally as nitrate and ammonium ions. However, sewage sludge addition to legumes could result in impaired nodule metabolism due to the presence of inorganic N from sludge. A greenhouse experiment was conducted to examine the effects of sewage sludge on growth, photosynthesis, nitrogen assimilation and nodule metabolism in alfalfa (Medicago sativa L. cv. Aragón). Plants were grown in pots with a mixture of perlite and vermiculite (2:1, v/v). The experiment included three treatments: (1) plants inoculated with rhizobia and amended with sewage sludge at rate of 10% (w/w) (RS); (2) plants inoculated with rhizobia without any amendment (R); and (3) non-inoculated plants fed with ammonium nitrate (N). N(2)-fixing plants had lower growth and sucrose phosphate synthase activity but higher photosynthesis than nitrate-fed plants because they compensated the carbon cost of the rhizobia. However, sewage sludge-treated plants evidenced a loss of carbon sink strength due to N(2) fixation by means of decreased photosynthetic capacity, leaf chlorophylls and N concentration in comparison to untreated plants. Sewage sludge did no affect nodulation but decreased nodule enzyme activities involved in carbon and N metabolisms that may lead to accumulation of toxic N-compounds.

  8. Concurrent synthesis and release of nod-gene-inducing flavonoids from alfalfa roots. [Medicago sativa L. ; Rhizobium meliloti

    SciTech Connect

    Maxwell, C.A.; Phillips, D.A. )

    1990-08-01

    Flavonoid signals from alfalfa (Medicago sativa L.) induce transcription of nodulation (nod) genes in Rhizobium meliloti. Alfalfa roots release three major nod-gene inducers: 4{prime},7-dihydroxyflavanone, 4{prime},7-dihydroxyflavone, and 4,4{prime}-dihydroxy-2{prime}-methoxychalcone. The objective of the present study was to define temporal relationships between synthesis and exudation for those flavonoids. Requirements for concurrent flavonoid biosynthesis were assessed by treating roots of intact alfalfa seedlings with (U-{sup 14}C)-L-phenylalanine in the presence or absence of the phenylalanine ammonia-lyase inhibitor L-2-aminoxy-3-phenylpropionic acid (AOPP). In the absence of AOPP, each of the three flavonoids in exudates contained {sup 14}C. In the presence of AOPP, {sup 14}C labeling and release of all the exuded nod-gene inducers were reduced significantly. AOPP inhibited labeling and release of the strongest nod-gene inducer, methoxychalcone, by more than 90%. The release process responsible for exudation of nod-gene inducers appears to be specific rather than a general phenomenon such as a sloughing off of cells during root growth.

  9. Allelic differences in Medicago truncatula NIP/LATD mutants correlate with their encoded proteins' transport activities in planta.

    PubMed

    Salehin, Mohammad; Huang, Ying-Sheng; Bagchi, Rammyani; Sherrier, D Janine; Dickstein, Rebecca

    2013-02-01

    Medicago truncatula NIP/LATD gene, required for symbiotic nitrogen fixing nodule and root architecture development, encodes a member of the NRT1(PTR) family that demonstrates high-affinity nitrate transport in Xenopus laevis oocytes. Of three Mtnip/latd mutant proteins, one retains high-affinity nitrate transport in oocytes, while the other two are nitrate-transport defective. To further examine the mutant proteins' transport properties, the missense Mtnip/latd alleles were expressed in Arabidopsis thaliana chl1-5, resistant to the herbicide chlorate because of a deletion spanning the nitrate transporter AtNRT1.1(CHL1) gene. Mtnip-3 expression restored chlorate sensitivity in the Atchl1-5 mutant, similar to wild type MtNIP/LATD, while Mtnip-1 expression did not. The high-affinity nitrate transporter AtNRT2.1 gene was expressed in Mtnip-1 mutant roots; it did not complement, which could be caused by several factors. Together, these findings support the hypothesis that MtNIP/LATD may have another biochemical activity.

  10. Strigolactones contribute to shoot elongation and to the formation of leaf margin serrations in Medicago truncatula R108.

    PubMed

    Lauressergues, Dominique; André, Olivier; Peng, Jianling; Wen, Jiangqi; Chen, Rujin; Ratet, Pascal; Tadege, Million; Mysore, Kirankumar S; Rochange, Soizic F

    2015-03-01

    Strigolactones were recently identified as a new class of plant hormones involved in the control of shoot branching. The characterization of strigolactone mutants in several species has progressively revealed their contribution to several other aspects of development in roots and shoots. In this article, we characterize strigolactone-deficient and strigolactone-insensitive mutants of the model legume Medicago truncatula for aerial developmental traits. The most striking mutant phenotype observed was compact shoot architecture. In contrast with what was reported in other species, this could not be attributed to enhanced shoot branching, but was instead due to reduced shoot elongation. Another notable feature was the modified leaf shape in strigolactone mutants: serrations at the leaf margin were smaller in the mutants than in wild-type plants. This phenotype could be rescued in a dose-dependent manner by exogenous strigolactone treatments of strigolactone-deficient mutants, but not of strigolactone-insensitive mutants. Treatment with the auxin transport inhibitor N-1-naphthylphtalamic acid resulted in smooth leaf margins, opposite to the effect of strigolactone treatment. The contribution of strigolactones to the formation of leaf serrations in M. truncatula R108 line represents a novel function of these hormones, which has not been revealed by the analysis of strigolactone mutants in other species.

  11. EFD Is an ERF transcription factor involved in the control of nodule number and differentiation in Medicago truncatula.

    PubMed

    Vernié, Tatiana; Moreau, Sandra; de Billy, Françoise; Plet, Julie; Combier, Jean-Philippe; Rogers, Christian; Oldroyd, Giles; Frugier, Florian; Niebel, Andreas; Gamas, Pascal

    2008-10-01

    Mechanisms regulating legume root nodule development are still poorly understood, and very few regulatory genes have been cloned and characterized. Here, we describe EFD (for ethylene response factor required for nodule differentiation), a gene that is upregulated during nodulation in Medicago truncatula. The EFD transcription factor belongs to the ethylene response factor (ERF) group V, which contains ERN1, 2, and 3, three ERFs involved in Nod factor signaling. The role of EFD in the regulation of nodulation was examined through the characterization of a null deletion mutant (efd-1), RNA interference, and overexpression studies. These studies revealed that EFD is a negative regulator of root nodulation and infection by Rhizobium and that EFD is required for the formation of functional nitrogen-fixing nodules. EFD appears to be involved in the plant and bacteroid differentiation processes taking place beneath the nodule meristem. We also showed that EFD activated Mt RR4, a cytokinin primary response gene that encodes a type-A response regulator. We propose that EFD induction of Mt RR4 leads to the inhibition of cytokinin signaling, with two consequences: the suppression of new nodule initiation and the activation of differentiation as cells leave the nodule meristem. Our work thus reveals a key regulator linking early and late stages of nodulation and suggests that the regulation of the cytokinin pathway is important both for nodule initiation and development.

  12. Inhibition of nitrogen fixation in symbiotic Medicago truncatula upon Cd exposure is a local process involving leghaemoglobin

    PubMed Central

    Marino, Daniel

    2013-01-01

    Leguminous biological nitrogen fixation (BNF) is very sensitive to environmental fluctuations. It is still contentious how BNF is regulated under stress conditions. The local or systemic control of BNF and the role played by reactive oxygen species (ROS) in such regulation have still not been elucidated completely. Cadmium, which belongs to the so-called heavy metals, is one of the most toxic substances released into the environment. The mechanisms involved in Cd toxicity are still not completely understood but the overproduction of ROS is one of its characteristic symptoms. In this work, we used a split-root system approach to study nodule BNF and the antioxidant machinery’s response to the application of a mild Cd treatment on one side of a nodulated Medicago truncatula root system. Cd induced the majority of nodule antioxidants without generating any oxidative damage. Cd treatment also provoked BNF inhibition exclusively in nodules directly exposed to Cd, without provoking any effect on plant shoot biomass or chlorophyll content. The overall data suggest that the decline in BNF was not due to a generalized breakdown of the plant but to control exerted through leghaemoglobin/oxygen availability, affecting nitrogenase function. PMID:24151304

  13. Transcriptomic and metabolomic analyses identify a role for chlorophyll catabolism and phytoalexin during Medicago nonhost resistance against Asian soybean rust

    PubMed Central

    Ishiga, Yasuhiro; Rao Uppalapati, Srinivasa; Gill, Upinder S.; Huhman, David; Tang, Yuhong; Mysore, Kirankumar S.

    2015-01-01

    Asian soybean rust (ASR) caused by Phakopsora pachyrhizi is a devastating foliar disease affecting soybean production worldwide. Understanding nonhost resistance against ASR may provide an avenue to engineer soybean to confer durable resistance against ASR. We characterized a Medicago truncatula-ASR pathosystem to study molecular mechanisms of nonhost resistance. Although urediniospores formed appressoria and penetrated into epidermal cells of M. truncatula, P. pachyrhizi failed to sporulate. Transcriptomic analysis revealed the induction of phenylpropanoid, flavonoid and isoflavonoid metabolic pathway genes involved in the production of phytoalexin medicarpin in M. truncatula upon infection with P. pachyrhizi. Furthermore, genes involved in chlorophyll catabolism were induced during nonhost resistance. We further characterized one of the chlorophyll catabolism genes, Stay-green (SGR), and demonstrated that the M. truncatula sgr mutant and alfalfa SGR-RNAi lines showed hypersensitive-response-like enhanced cell death upon inoculation with P. pachyrhizi. Consistent with transcriptomic analysis, metabolomic analysis also revealed the accumulation of medicarpin and its intermediate metabolites. In vitro assay showed that medicarpin inhibited urediniospore germination and differentiation. In addition, several triterpenoid saponin glycosides accumulated in M. truncatula upon inoculation with P. pachyrhizi. In summary, using multi-omic approaches, we identified a correlation between phytoalexin production and M. truncatula defense responses against ASR. PMID:26267598

  14. A H+-ATPase That Energizes Nutrient Uptake during Mycorrhizal Symbioses in Rice and Medicago truncatula[C][W][OPEN

    PubMed Central

    Wang, Ertao; Yu, Nan; Bano, S. Asma; Liu, Chengwu; Miller, Anthony J.; Cousins, Donna; Zhang, Xiaowei; Ratet, Pascal; Tadege, Million; Mysore, Kirankumar S.; Downie, J. Allan; Murray, Jeremy D.; Oldroyd, Giles E.D.; Schultze, Michael

    2014-01-01

    Most plant species form symbioses with arbuscular mycorrhizal (AM) fungi, which facilitate the uptake of mineral nutrients such as phosphate from the soil. Several transporters, particularly proton-coupled phosphate transporters, have been identified on both the plant and fungal membranes and contribute to delivering phosphate from fungi to plants. The mechanism of nutrient exchange has been studied in plants during mycorrhizal colonization, but the source of the electrochemical proton gradient that drives nutrient exchange is not known. Here, we show that plasma membrane H+-ATPases that are specifically induced in arbuscule-containing cells are required for enhanced proton pumping activity in membrane vesicles from AM-colonized roots of rice (Oryza sativa) and Medicago truncatula. Mutation of the H+-ATPases reduced arbuscule size and impaired nutrient uptake by the host plant through the mycorrhizal symbiosis. Overexpression of the H+-ATPase Os-HA1 increased both phosphate uptake and the plasma membrane potential, suggesting that this H+-ATPase plays a key role in energizing the periarbuscular membrane, thereby facilitating nutrient exchange in arbusculated plant cells. PMID:24781115

  15. Combined phosphate and nitrogen limitation generates a nutrient stress transcriptome favorable for arbuscular mycorrhizal symbiosis in Medicago truncatula.

    PubMed

    Bonneau, Laurent; Huguet, Stéphanie; Wipf, Daniel; Pauly, Nicolas; Truong, Hoai-Nam

    2013-07-01

    Arbuscular mycorrhizal (AM) symbiosis is stimulated by phosphorus (P) limitation and contributes to P and nitrogen (N) acquisition. However, the effects of combined P and N limitation on AM formation are largely unknown. Medicago truncatula plants were cultivated in the presence or absence of Rhizophagus irregularis (formerly Glomus intraradices) in P-limited (LP), N-limited (LN) or combined P- and N-limited (LPN) conditions, and compared with plants grown in sufficient P and N. The highest AM formation was observed in LPN, linked to systemic signaling by the plant nutrient status. Plant free phosphate concentrations were higher in LPN than in LP, as a result of cross-talk between P and N. Transcriptome analyses suggest that LPN induces the activation of NADPH oxidases in roots, concomitant with an altered profile of plant defense genes and a coordinate increase in the expression of genes involved in the methylerythritol phosphate and isoprenoid-derived pathways, including strigolactone synthesis genes. Taken together, these results suggest that low P and N fertilization systemically induces a physiological state of plants favorable for AM symbiosis despite their higher P status. Our findings highlight the importance of the plant nutrient status in controlling plant-fungus interaction.

  16. GC-MS based metabolite profiling implies three interdependent ways of ammonium assimilation in Medicago truncatula root nodules.

    PubMed

    Barsch, Aiko; Carvalho, Helena G; Cullimore, Julie V; Niehaus, Karsten

    2006-12-15

    In symbiotic interaction with legume plants, bacteria termed Rhizobia can fix massive amounts of atmospheric nitrogen which is primarily provided in the form of ammonium to the host plants. Therefore, legume root nodules that house the symbiotic bacteria are ideally suited to study the process of primary ammonium assimilation. Here, we present a GC-MS based metabolite profiling analysis of Medicago truncatula root nodules (induced by the bacterium Sinorhizobium meliloti) before and after inhibition of glutamine synthetase (GS) by the chemical herbicide phosphinotricine. The primary role of GS in ammonium assimilation was revealed by drastically reduced levels of glutamine in phosphinotricine treated root nodules. In comparison to previous results of increased asparagine synthetase transcript and protein abundances in GS inhibited nodules the metabolic data revealed that decreased amounts of aspartate might preclude taking advantage of this elevated enzymatic activity. A potential role of glutamate dehydrogenase in ammonium assimilation was metabolically indicated 24 and 48 h after GS inhibition. Therefore, nodule ammonium assimilation might in principle involve three interdependent metabolic pathways which are adjusted to control basic nitrogen metabolism.

  17. The Medicago truncatula sucrose transporter family: characterization and implication of key members in carbon partitioning towards arbuscular mycorrhizal fungi.

    PubMed

    Doidy, Joan; van Tuinen, Diederik; Lamotte, Olivier; Corneillat, Marion; Alcaraz, Gérard; Wipf, Daniel

    2012-11-01

    We identified de novo sucrose transporter (SUT) genes involved in long-distance transport of sucrose from photosynthetic source leaves towards sink organs in the model leguminous species Medicago truncatula. The identification and functional analysis of sugar transporters provide key information on mechanisms that underlie carbon partitioning in plant-microorganism interactions. In that way, full-length sequences of the M. truncatula SUT (MtSUT) family were retrieved and biochemical characterization of MtSUT members was performed by heterologous expression in yeast. The MtSUT family now comprises six genes which distribute among Dicotyledonous clades. MtSUT1-1 and MtSUT4-1 are key members in regard to their expression profiles in source leaves and sink roots and were characterized as functional H(+)/sucrose transporters. Physiological and molecular responses to phosphorus supply and inoculation by the arbuscular mycorrhizal fungus (AMF) Glomus intraradices was studied by gene expression and sugar quantification analyses. Sucrose represents the main sugar transport form in M. truncatula and the expression profiles of MtSUT1-1, MtSUT2, and MtSUT4-1 highlight a fine-tuning regulation for beneficial sugar fluxes towards the fungal symbiont. Taken together, these results suggest distinct functions for proteins from the SUT1, SUT2, and SUT4 clades in plant and in biotrophic interactions.

  18. NO loading: Efficiency assessment of five commonly used application methods of sodium nitroprusside in Medicago truncatula plants.

    PubMed

    Filippou, Panagiota; Antoniou, Chrystalla; Yelamanchili, Shirisha; Fotopoulos, Vasileios

    2012-11-01

    Nitric oxide (NO) is a bioactive, diffusible molecule involved in a multitude of physiological and developmental processes in plants, which has been reported to display both antioxidant and pro-oxidant properties in plants. Several reports exist highlighting the protective action of sodium nitroprusside (SNP), an NO donor, which demonstrate its important role as a signal molecule in plants responsible for the expression regulation of antioxidant and other defense enzymes. However, the mode of application of this compound varies greatly between studies. The present study provides a comprehensive efficiency comparison of the most commonly used application methods using 2.5mM SNP on mature (40 day) Medicago truncatula plants. Measurement of NO content in both leaves and roots suggests that vacuum infiltration is the most efficient method for NO donation in leaf tissue, whereas hydroponic application resulted in highest NO content in roots. NO content correlated with activity levels of nitrate reductase (NR; EC 1.7.99.4), a key enzyme involved in the generation of NO in plants and which is known to be regulated by NO itself.

  19. Comparative proteomics of yeast-elicited Medicago truncatula cell suspensions reveals induction of isoflavonoid biosynthesis and cell wall modifications.

    PubMed

    Lei, Zhentian; Chen, Fang; Watson, Bonnie S; Nagaraj, Satish; Elmer, Aaron M; Dixon, Richard A; Sumner, Lloyd W

    2010-12-03

    The temporal proteome response of Medicago truncatula suspension cell cultures to yeast elicitation (which mimics fungal infection) was investigated using two-dimensional polyacrylamide gel electrophoresis (2-DE) and nanoliquid chromatography coupled to tandem mass spectrometry (nano LC-MS/MS). Reproducibility of 2-DE was assessed using the number of the visualized protein spots and spot volume. Average coefficient of variation was determined to be less than 6% for the number of spots and around 50% for spot volume. About 4% of the total visualized proteins, that is, 34 out of 861, were differentially accumulated in the suspension cells 24 h after yeast elicitation, including isoflavononid biosynthetic enzymes and a putative laccase. The induction of the putative laccase was highly correlated with the polymerization of phenolics such as 4-hydroxybenzoic acid, 4-hydroxybenzaldehyde, and ferulic acid into cell walls. In contrast, lignin was only induced at the later stages of the temporal study, indicating that this specific laccase is primarily involved in cell wall modifications and/or fortifications rather than in lignification in response to yeast elicitation.

  20. The Transcriptional Repressor MYB2 Regulates Both Spatial and Temporal Patterns of Proanthocyandin and Anthocyanin Pigmentation in Medicago truncatula.

    PubMed

    Jun, Ji Hyung; Liu, Chenggang; Xiao, Xirong; Dixon, Richard A

    2015-10-01

    Accumulation of anthocyanins and proanthocyanidins (PAs) is limited to specific cell types and developmental stages, but little is known about how antagonistically acting transcriptional regulators work together to determine temporal and spatial patterning of pigmentation at the cellular level, especially for PAs. Here, we characterize MYB2, a transcriptional repressor regulating both anthocyanin and PA biosynthesis in the model legume Medicago truncatula. MYB2 was strongly upregulated by MYB5, a major regulator of PA biosynthesis in M. truncatula and a component of MYB-basic helix loop helix-WD40 (MBW) activator complexes. Overexpression of MYB2 abolished anthocyanin and PA accumulation in M. truncatula hairy roots and Arabidopsis thaliana seeds, respectively. Anthocyanin deposition was expanded in myb2 mutant seedlings and flowers accompanied by increased anthocyanin content. PA mainly accumulated in the epidermal layer derived from the outer integument in the M. truncatula seed coat, starting from the hilum area. The area of PA accumulation and ANTHOCYANIDIN REDUCTASE expression was expanded into the seed body at the early stage of seed development in the myb2 mutant. Genetic, biochemical, and cell biological evidence suggests that MYB2 functions as part of a multidimensional regulatory network to define the temporal and spatial pattern of anthocyanin and PA accumulation linked to developmental processes. © 2015 American Society of Plant Biologists. All rights reserved.

  1. Nod Factor Receptors Form Heteromeric Complexes and Are Essential for Intracellular Infection in Medicago Nodules[W

    PubMed Central

    Moling, Sjef; Pietraszewska-Bogiel, Anna; Postma, Marten; Fedorova, Elena; Hink, Mark A.; Limpens, Erik; Gadella, Theodorus W.J.; Bisseling, Ton

    2014-01-01

    Rhizobial Nod factors are the key signaling molecules in the legume-rhizobium nodule symbiosis. In this study, the role of the Nod factor receptors NOD FACTOR PERCEPTION (NFP) and LYSIN MOTIF RECEPTOR-LIKE KINASE3 (LYK3) in establishing the symbiotic interface in root nodules was investigated. It was found that inside Medicago truncatula nodules, NFP and LYK3 localize at the cell periphery in a narrow zone of about two cell layers at the nodule apex. This restricted accumulation is narrower than the region of promoter activity/mRNA accumulation and might serve to prevent the induction of defense-like responses and/or to restrict the rhizobium release to precise cell layers. The distal cell layer where the receptors accumulate at the cell periphery is part of the meristem, and the proximal layer is part of the infection zone. In these layers, the receptors can most likely perceive the bacterial Nod factors to regulate the formation of symbiotic interface. Furthermore, our Förster resonance energy transfer-fluorescence lifetime imaging microscopy analysis indicates that NFP and LYK3 form heteromeric complexes at the cell periphery in M. truncatula nodules. PMID:25351493

  2. Multiple Autoregulation of Nodulation (AON) Signals Identified through Split Root Analysis of Medicago truncatula sunn and rdn1 Mutants.

    PubMed

    Kassaw, Tessema; Jr, William Bridges; Frugoli, Julia

    2015-04-27

    Nodulation is energetically costly to the host: legumes balance the nitrogen demand with the energy expense by limiting the number of nodules through long-distance signaling. A split root system was used to investigate systemic autoregulation of nodulation (AON) in Medicago truncatula and the role of the AON genes RDN1 and SUNN in the regulatory circuit. Developing nodule primordia did not trigger AON in plants carrying mutations in RDN1 and SUNN genes, while wild type plants had fully induced AON within three days. However, despite lacking an early suppression response, AON mutants suppressed nodulation when roots were inoculated 10 days or more apart, correlated with the maturation of nitrogen fixing nodules. In addition to correlation between nitrogen fixation and suppression of nodulation, suppression by extreme nutrient stress was also observed in all genotypes and may be a component of the observed response due to the conditions of the assay. These results suggest there is more than one systemic regulatory circuit controlling nodulation in M. truncatula. While both signals are present in wild type plants, the second signal can only be observed in plants lacking the early repression (AON mutants). RDN1 and SUNN are not essential for response to the later signal.

  3. Multiple Autoregulation of Nodulation (AON) Signals Identified through Split Root Analysis of Medicago truncatula sunn and rdn1 Mutants

    PubMed Central

    Kassaw, Tessema; Bridges, William; Frugoli, Julia

    2015-01-01

    Nodulation is energetically costly to the host: legumes balance the nitrogen demand with the energy expense by limiting the number of nodules through long-distance signaling. A split root system was used to investigate systemic autoregulation of nodulation (AON) in Medicago truncatula and the role of the AON genes RDN1 and SUNN in the regulatory circuit. Developing nodule primordia did not trigger AON in plants carrying mutations in RDN1 and SUNN genes, while wild type plants had fully induced AON within three days. However, despite lacking an early suppression response, AON mutants suppressed nodulation when roots were inoculated 10 days or more apart, correlated with the maturation of nitrogen fixing nodules. In addition to correlation between nitrogen fixation and suppression of nodulation, suppression by extreme nutrient stress was also observed in all genotypes and may be a component of the observed response due to the conditions of the assay. These results suggest there is more than one systemic regulatory circuit controlling nodulation in M. truncatula. While both signals are present in wild type plants, the second signal can only be observed in plants lacking the early repression (AON mutants). RDN1 and SUNN are not essential for response to the later signal. PMID:27135324

  4. Medicago truncatula natural resistance-associated macrophage Protein1 is required for iron uptake by rhizobia-infected nodule cells.

    PubMed

    Tejada-Jiménez, Manuel; Castro-Rodríguez, Rosario; Kryvoruchko, Igor; Lucas, M Mercedes; Udvardi, Michael; Imperial, Juan; González-Guerrero, Manuel

    2015-05-01

    Iron is critical for symbiotic nitrogen fixation (SNF) as a key component of multiple ferroproteins involved in this biological process. In the model legume Medicago truncatula, iron is delivered by the vasculature to the infection/maturation zone (zone II) of the nodule, where it is released to the apoplast. From there, plasma membrane iron transporters move it into rhizobia-containing cells, where iron is used as the cofactor of multiple plant and rhizobial proteins (e.g. plant leghemoglobin and bacterial nitrogenase). MtNramp1 (Medtr3g088460) is the M. truncatula Natural Resistance-Associated Macrophage Protein family member, with the highest expression levels in roots and nodules. Immunolocalization studies indicate that MtNramp1 is mainly targeted to the plasma membrane. A loss-of-function nramp1 mutant exhibited reduced growth compared with the wild type under symbiotic conditions, but not when fertilized with mineral nitrogen. Nitrogenase activity was low in the mutant, whereas exogenous iron and expression of wild-type MtNramp1 in mutant nodules increased nitrogen fixation to normal levels. These data are consistent with a model in which MtNramp1 is the main transporter responsible for apoplastic iron uptake by rhizobia-infected cells in zone II.

  5. Genome-wide association of drought-related and biomass traits with HapMap SNPs in Medicago truncatula.

    PubMed

    Kang, Yun; Sakiroglu, Muhammet; Krom, Nicholas; Stanton-Geddes, John; Wang, Mingyi; Lee, Yi-Ching; Young, Nevin D; Udvardi, Michael

    2015-10-01

    Improving drought tolerance of crop plants is a major goal of plant breeders. In this study, we characterized biomass and drought-related traits of 220 Medicago truncatula HapMap accessions. Characterized traits included shoot biomass, maximum leaf size, specific leaf weight, stomatal density, trichome density and shoot carbon-13 isotope discrimination (δ(13) C) of well-watered M. truncatula plants, and leaf performance in vitro under dehydration stress. Genome-wide association analyses were carried out using the general linear model (GLM), the standard mixed linear model (MLM) and compressed MLM (CMLM) in TASSEL, which revealed significant overestimation of P-values by CMLM. For each trait, candidate genes and chromosome regions containing SNP markers were found that are in significant association with the trait. For plant biomass, a 0.5 Mbp region on chromosome 2 harbouring a plasma membrane intrinsic protein, PIP2, was discovered that could potentially be targeted to increase dry matter yield. A protein disulfide isomerase-like protein was found to be tightly associated with both shoot biomass and leaf size. A glutamate-cysteine ligase and an aldehyde dehydrogenase family protein with Arabidopsis homologs strongly expressed in the guard cells were two of the top genes identified by stomata density genome-wide association studies analysis.

  6. Genome-wide characterization of GRAS family genes in Medicago truncatula reveals their evolutionary dynamics and functional diversification.

    PubMed

    Zhang, Hailing; Cao, Yingping; Shang, Chen; Li, Jikai; Wang, Jianli; Wu, Zhenying; Ma, Lichao; Qi, Tianxiong; Fu, Chunxiang; Bai, Zetao; Hu, Baozhong

    2017-01-01

    The GRAS gene family is a large plant-specific family of transcription factors that are involved in diverse processes during plant development. Medicago truncatula is an ideal model plant for genetic research in legumes, and specifically for studying nodulation, which is crucial for nitrogen fixation. In this study, 59 MtGRAS genes were identified and classified into eight distinct subgroups based on phylogenetic relationships. Motifs located in the C-termini were conserved across the subgroups, while motifs in the N-termini were subfamily specific. Gene duplication was the main evolutionary force for MtGRAS expansion, especially proliferation of the LISCL subgroup. Seventeen duplicated genes showed strong effects of purifying selection and diverse expression patterns, highlighting their functional importance and diversification after duplication. Thirty MtGRAS genes, including NSP1 and NSP2, were preferentially expressed in nodules, indicating possible roles in the process of nodulation. A transcriptome study, combined with gene expression analysis under different stress conditions, suggested potential functions of MtGRAS genes in various biological pathways and stress responses. Taken together, these comprehensive analyses provide basic information for understanding the potential functions of GRAS genes, and will facilitate further discovery of MtGRAS gene functions.

  7. The Compact Root Architecture1 Gene Regulates Lignification, Flavonoid Production, and Polar Auxin Transport in Medicago truncatula1[W

    PubMed Central

    Laffont, Carole; Blanchet, Sandrine; Lapierre, Catherine; Brocard, Lysiane; Ratet, Pascal; Crespi, Martin; Mathesius, Ulrike; Frugier, Florian

    2010-01-01

    The root system architecture is crucial to adapt plant growth to changing soil environmental conditions and consequently to maintain crop yield. In addition to root branching through lateral roots, legumes can develop another organ, the nitrogen-fixing nodule, upon a symbiotic bacterial interaction. A mutant, cra1, showing compact root architecture was identified in the model legume Medicago truncatula. cra1 roots were short and thick due to defects in cell elongation, whereas densities of lateral roots and symbiotic nodules were similar to the wild type. Grafting experiments showed that a lengthened life cycle in cra1 was due to the smaller root system and not to the pleiotropic shoot phenotypes observed in the mutant. Analysis of the cra1 transcriptome at a similar early developmental stage revealed few significant changes, mainly related to cell wall metabolism. The most down-regulated gene in the cra1 mutant encodes a Caffeic Acid O-Methyl Transferase, an enzyme involved in lignin biosynthesis; accordingly, whole lignin content was decreased in cra1 roots. This correlated with differential accumulation of specific flavonoids and decreased polar auxin transport in cra1 mutants. Exogenous application of the isoflavone formononetin to wild-type plants mimicked the cra1 root phenotype, whereas decreasing flavonoid content through silencing chalcone synthases restored the polar auxin transport capacity of the cra1 mutant. The CRA1 gene, therefore, may control legume root growth through the regulation of lignin and flavonoid profiles, leading to changes in polar auxin transport. PMID:20522723

  8. Medicago truncatula and Glycine max: Different Drought Tolerance and Similar Local Response of the Root Nodule Proteome.

    PubMed

    Gil-Quintana, Erena; Lyon, David; Staudinger, Christiana; Wienkoop, Stefanie; González, Esther M

    2015-12-04

    Legume crops present important agronomical and environmental advantages mainly due to their capacity to reduce atmospheric N2 to ammonium via symbiotic nitrogen fixation (SNF). This process is very sensitive to abiotic stresses such as drought, but the mechanism underlying this response is not fully understood. The goal of the current work is to compare the drought response of two legumes with high economic impact and research importance, Medicago truncatula and Glycine max, by characterizing their root nodule proteomes. Our results show that, although M. truncatula exhibits lower water potential values under drought conditions compared to G. max, SNF declined analogously in the two legumes. Both of their nodule proteomes are very similar, and comparable down-regulation responses in the diverse protein functional groups were identified (mainly proteins related to the metabolism of carbon, nitrogen, and sulfur). We suggest lipoxygenases and protein turnover as newly recognized players in SNF regulation. Partial drought conditions applied to a split-root system resulted in the local down-regulation of the entire proteome of drought-stressed nodules in both legumes. The high degree of similarity between both legume proteomes suggests that the vast amount of research conducted on M. truncatula could be applied to economically important legume crops, such as soybean.

  9. Two Medicago truncatula Half-ABC Transporters Are Essential for Arbuscule Development in Arbuscular Mycorrhizal Symbiosis[W

    PubMed Central

    Zhang, Quan; Blaylock, Laura A.; Harrison, Maria J.

    2010-01-01

    In the symbiotic association of plants and arbuscular mycorrhizal (AM) fungi, the fungal symbiont resides in the root cortical cells where it delivers mineral nutrients to its plant host through branched hyphae called arbuscules. Here, we report a Medicago truncatula mutant, stunted arbuscule (str), in which arbuscule development is impaired and AM symbiosis fails. In contrast with legume symbiosis mutants reported previously, str shows a wild-type nodulation phenotype. STR was identified by positional cloning and encodes a half-size ATP binding cassette (ABC) transporter of a subfamily (ABCG) whose roles in plants are largely unknown. STR is a representative of a novel clade in the ABCG subfamily, and its orthologs are highly conserved throughout the vascular plants but absent from Arabidopsis thaliana. The STR clade is unusual in that it lacks the taxon-specific diversification that is typical of the ABCG gene family. This distinct phylogenetic profile enabled the identification of a second AM symbiosis-induced half-transporter, STR2. Silencing of STR2 by RNA interference results in a stunted arbuscule phenotype identical to that of str. STR and STR2 are coexpressed constitutively in the vascular tissue, and expression is induced in cortical cells containing arbuscules. STR heterodimerizes with STR2, and the resulting transporter is located in the peri-arbuscular membrane where its activity is required for arbuscule development and consequently a functional AM symbiosis. PMID:20453115

  10. Increase in 4-Coumaryl Alcohol Units during Lignification in Alfalfa (Medicago sativa) Alters the Extractability and Molecular Weight of Lignin*

    PubMed Central

    Ziebell, Angela; Gracom, Kristen; Katahira, Rui; Chen, Fang; Pu, Yunqiao; Ragauskas, Art; Dixon, Richard A.; Davis, Mark

    2010-01-01

    The lignin content of biomass can impact the ease and cost of biomass processing. Lignin reduction through breeding and genetic modification therefore has potential to reduce costs in biomass-processing industries (e.g. pulp and paper, forage, and lignocellulosic ethanol). We investigated compositional changes in two low-lignin alfalfa (Medicago sativa) lines with antisense down-regulation of p-coumarate 3-hydroxylase (C3H) or hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyltransferase (HCT). We investigated whether the difference in reactivity during lignification of 4-coumaryl alcohol (H) monomers versus the naturally dominant sinapyl alcohol and coniferyl alcohol lignin monomers alters the lignin structure. Sequential base extraction readily reduced the H monomer content of the transgenic lines, leaving a residual lignin greatly enriched in H subunits; the extraction profile highlighted the difference between the control and transgenic lines. Gel permeation chromatography of isolated ball-milled lignin indicated significant changes in the weight average molecular weight distribution of the control versus transgenic lines (CTR1a, 6000; C3H4a, 5500; C3H9a, 4000; and HCT30a, 4000). PMID:20921228

  11. Increase in 4-coumaryl alcohol units during lignification in alfalfa (Medicago sativa) alters the extractability and molecular weight of lignin.

    PubMed

    Ziebell, Angela; Gracom, Kristen; Katahira, Rui; Chen, Fang; Pu, Yunqiao; Ragauskas, Art; Dixon, Richard A; Davis, Mark

    2010-12-10

    The lignin content of biomass can impact the ease and cost of biomass processing. Lignin reduction through breeding and genetic modification therefore has potential to reduce costs in biomass-processing industries (e.g. pulp and paper, forage, and lignocellulosic ethanol). We investigated compositional changes in two low-lignin alfalfa (Medicago sativa) lines with antisense down-regulation of p-coumarate 3-hydroxylase (C3H) or hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyltransferase (HCT). We investigated whether the difference in reactivity during lignification of 4-coumaryl alcohol (H) monomers versus the naturally dominant sinapyl alcohol and coniferyl alcohol lignin monomers alters the lignin structure. Sequential base extraction readily reduced the H monomer content of the transgenic lines, leaving a residual lignin greatly enriched in H subunits; the extraction profile highlighted the difference between the control and transgenic lines. Gel permeation chromatography of isolated ball-milled lignin indicated significant changes in the weight average molecular weight distribution of the control versus transgenic lines (CTR1a, 6000; C3H4a, 5500; C3H9a, 4000; and HCT30a, 4000).

  12. Calcium and magnesium interference studies for the binding of heavy metal ions in solution by Medicago sativa (alfalfa)

    SciTech Connect

    Gardea-Torresdey, J.L.; Tiemann, K.J.; Gonzalez, J.H.; Henning, J.A.; Townsend, M.S.

    1996-12-31

    Previous batch laboratory experiments performed to determine the potential ability of seven different varieties of Medicago sativa (alfalfa) revealed that the African shoots population was able to efficiently bind copper(II) and nickel(II) from aqueous solutions. Batch laboratory interference studies were performed with various calcium and magnesium concentrations (0.1 mM to 1 M) in order to ascertain the effects of these ions on the heavy metal binding ability of African alfalfa shoots. Results from these studies have shown that calcium and magnesium did not seriously reduce the binding of copper(II) and lead(II) to African alfalfa shoots. However, high concentrations of calcium and magnesium significantly reduced chromium(III), cadmium(II), nickel(II), and zinc(II) binding to African shoots. In addition, all these experiments were repeated maintaining the ionic strength constant, and similar results were obtained. Interference studies were also conducted in order to determine the effects of hard cations under flow conditions with silica-immobilized African alfalfa shoots. The information obtained from these studies will be useful for an innovative method of heavy metal ion removal and recovery from contaminated waters.

  13. Application of sodium nitroprusside results in distinct antioxidant gene expression patterns in leaves of mature and senescing Medicago truncatula plants.

    PubMed

    Fotopoulos, Vasileios; Antoniou, Chrystalla; Filippou, Panagiota; Mylona, Photini; Fasoula, Dionysia; Ioannides, Ioannis; Polidoros, Alexios

    2014-07-01

    Sodium nitroprusside (SNP) represents one of the most commonly used NO donors in biological sciences, which acts as a signal molecule in plants responsible for the regulation of the expression of many defense-related enzymes. This study attempts to provide novel insight into the effect of application of low (100 μΜ) and high (2.5 mM) concentrations of SNP on antioxidant gene expression (cAPX, GST, FeSOD, CAT, and AOX) in mature (40 day) and senescing (65 day) Medicago truncatula plants. Quantitative real-time RT-PCR suggests that low concentration of SNP applied in mature leaves leads to an overall induction of antioxidant gene expression, while increasing concentration results in suppression of these genes. Conversely, older plants demonstrate a much more variable regulation which appears to be time dependent. The observed transcriptional regulation pattern in mature M. truncatula plants comes in support of the previously documented protective or damaging effect of SNP depending on concentration applied, whereas senescing M. truncatula plants demonstrated a general suppression in antioxidant gene expression levels regardless of SNP concentration, indicative of reduced overall plant defense capacity against free radicals.

  14. Is N-feedback involved in the inhibition of nitrogen fixation in drought-stressed Medicago truncatula?

    PubMed Central

    González, Esther M.

    2013-01-01

    Drought stress is a major factor limiting nitrogen fixation (NF) in crop production. However, the regulatory mechanism involved and the origin of the inhibition, whether local or systemic, is still controversial and so far scarcely studied in temperate forage legumes. Medicago truncatula plants were symbiotically grown with a split-root system and exposed to gradual water deprivation. Physiological parameters, NF activity, and amino acid content were measured. The partial drought treatment inhibited NF in the nodules directly exposed to drought stress. Concomitantly, in the droughted below-ground organs, amino acids accumulated prior to any drop in evapotranspiration (ET). It is concluded that drought exerts a local inhibition of NF and drives an overall accumulation of amino acids in diverse plant organs which is independent of the decrease in ET. The general increase in the majority of single amino acids in the whole plant questions the commonly accepted concept of a single amino acid acting as an N-feedback signal. PMID:23175536

  15. In vitro auxin treatment promotes cell division and delays endoreduplication in developing seeds of the model legume species Medicago truncatula.

    PubMed

    Atif, Rana M; Boulisset, Florian; Conreux, Catherine; Thompson, Richard; Ochatt, Sergio J

    2013-08-01

    The role of auxins in the morphogenesis of immature seeds of Medicago truncatula was studied, focusing on the transition from the embryo cell division phase to seed maturation. We analyzed seed development in vitro, by flow cytometry, and through the determination of the kinetics of seed fresh weight and size. Thus, seeds were harvested at 8, 10 and 12 days after pollination and cultured in vitro on a medium either without auxin or supplemented with indole-3-butyric acid (IBA) or naphthalene acetic acid (NAA) at 1 mg l(-1). All parameters studied were determined every 2 days from the start of in vitro culture. The results showed that both auxins increased the weight and size of seeds with NAA having a stronger effect than IBA. We further demonstrated that the auxin treatments modulate the transition between mitotic cycles and endocycles in M. truncatula developing seed by favoring sustained cell divisions while simultaneously prolonging endoreduplication, which is known to be the cytogenetical imprint of the transition from the cell division phase to the storage protein accumulation phase during seed development. Copyright © Physiologia Plantarum 2012.

  16. Unraveling the effect of arsenic on the model Medicago-Ensifer interaction: a transcriptomic meta-analysis.

    PubMed

    Lafuente, Alejandro; Pérez-Palacios, Patricia; Doukkali, Bouchra; Molina-Sánchez, María D; Jiménez-Zurdo, José I; Caviedes, Miguel A; Rodríguez-Llorente, Ignacio D; Pajuelo, Eloísa

    2015-01-01

    The genetic regulation underlying the effect of arsenic (As(III)) on the model symbiosis Medicago-Ensifer was investigated using a combination of physiological (split-roots), microscopy and genetic (microarrays, qRT-PCR and composite plants) tools. Nodulation was very sensitive to As(III) (median inhibitory dose (ID50) = 20 μM). The effect on root elongation and on nodulation was local (nonsystemic). A battery of stress (salt, drought, heat shock, metals, etc.)-related genes were induced. Glutathione played a pivotal role in tolerance/detoxification, together with secondary metabolites ((iso)flavonoids and phenylpropanoids). However, antioxidant enzymes were not activated. Concerning the symbiotic interaction, molecular evidence suggesting that rhizobia alleviate As stress is for the first time provided. Chalcone synthase (which is involved in the first step of the legume-rhizobia cross-talk) was strongly enhanced, suggesting that the plants are biased to establish symbiotic interactions under As(III) stress. In contrast, 13 subsequent nodulation genes (involved in nodulation factors (Nod factors) perception, infection, thread initiation and progression, and nodule morphogenesis) were repressed. Overexpression of the ethylene responsive factor ERN in composite plants reduced root stress and partially restored nodulation, whereas overexpression of the early nodulin ENOD12 enhanced nodulation both in the presence and, particularly, in the absence of As, without affecting root elongation. Several transcription factors were identified, which could be additional targets for genetic engineering aiming to improve nodulation and/or alleviate root stress induced by this toxic.

  17. NFP, a LysM protein controlling Nod factor perception, also intervenes in Medicago truncatula resistance to pathogens.

    PubMed

    Rey, Thomas; Nars, Amaury; Bonhomme, Maxime; Bottin, Arnaud; Huguet, Stéphanie; Balzergue, Sandrine; Jardinaud, Marie-Françoise; Bono, Jean-Jacques; Cullimore, Julie; Dumas, Bernard; Gough, Clare; Jacquet, Christophe

    2013-05-01

    Plant LysM proteins control the perception of microbial-derived N-acetylglucosamine compounds for the establishment of symbiosis or activation of plant immunity. This raises questions about how plants, and notably legumes, can differentiate friends and foes using similar molecular actors and whether any receptors can intervene in both symbiosis and resistance. To study this question, nfp and lyk3 LysM-receptor like kinase mutants of Medicago truncatula that are affected in the early steps of nodulation, were analysed following inoculation with Aphanomyces euteiches, a root oomycete. The role of NFP in this interaction was further analysed by overexpression of NFP and by transcriptome analyses. nfp, but not lyk3, mutants were significantly more susceptible than wildtype plants to A. euteiches, whereas NFP overexpression increased resistance. Transcriptome analyses on A. euteiches inoculation showed that mutation in the NFP gene led to significant changes in the expression of c. 500 genes, notably involved in cell dynamic processes previously associated with resistance to pathogen penetration. nfp mutants also showed an increased susceptibility to the fungus Colletotrichum trifolii. These results demonstrate that NFP intervenes in M. truncatula immunity, suggesting an unsuspected role for NFP in the perception of pathogenic signals. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

  18. N-feedback regulation is synchronized with nodule carbon alteration in Medicago truncatula under excessive nitrate or low phosphorus conditions.

    PubMed

    Sulieman, Saad; Schulze, Joachim; Tran, Lam-Son Phan

    2014-03-15

    The aim of the present study was to test the hypothesis that the higher nodule amino acid content induced under certain treatments may play a role in the N-feedback regulation of nitrogenase (EC 1.18.6.1) activity by restricting the carbon supply to the functioning nodules. Growing Medicago truncatula plants under sub-optimal phosphorus conditions or upon exposure to large supply of nitrate caused significant asparagine accumulation in nodules of the treated plants. In addition, there was a remarkable decline in the nodule succinate content under phosphorus deprivation while malate was tended to increase. Interestingly, the relative share of succinate in the symbiotic tissues was totally inhibited, i.e. reached zero, by excessive nitrate application. These results provide evidence that succinate might be greatly affected by asparagine content of the nodule fraction, thereby restricting cellular carbon supply to the functioning bacteroids which leads to down-regulation of nodule metabolism and nitrogenase activity. Copyright © 2013 Elsevier GmbH. All rights reserved.

  19. Carbon metabolism and bacteroid functioning are involved in the regulation of nitrogen fixation in Medicago truncatula under drought and recovery.

    PubMed

    Larrainzar, Estíbaliz; Wienkoop, Stefanie; Scherling, Christian; Kempa, Stefan; Ladrera, Rubén; Arrese-Igor, Cesar; Weckwerth, Wolfram; González, Esther M

    2009-12-01

    Regulation of symbiotic nitrogen fixation (SNF) during drought stress is complex and not yet fully understood. In the present work, the involvement of nodule C and N metabolism in the regulation of SNF in Medicago truncatula under drought and a subsequent rewatering treatment was analyzed using a combination of metabolomic and proteomic approaches. Drought induced a reduction of SNF rates and major changes in the metabolic profile of nodules, mostly an accumulation of amino acids (Pro, His, and Trp) and carbohydrates (sucrose, galactinol, raffinose, and trehalose). This accumulation was coincidental with a decline in the levels of bacteroid proteins involved in SNF and C metabolism, along with a partial reduction of the levels of plant sucrose synthase 1 (SuSy1). In contrast, the variations in enzymes related to N assimilation were found not to correlate with the reduction in SNF, suggesting that these enzymes do not have a role in the regulation of SNF. Unlike the situation in other legumes such as pea and soybean, the drought-induced inhibition of SNF in M. truncatula appears to be caused by impairment of bacteroid metabolism and N(2)-fixing capacity rather than a limitation of respiratory substrate.

  20. 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase1 Interacts with NORK and Is Crucial for Nodulation in Medicago truncatula

    PubMed Central

    Kevei, Zoltán; Lougnon, Géraldine; Mergaert, Peter; Horváth, Gábor V.; Kereszt, Attila; Jayaraman, Dhileepkumar; Zaman, Najia; Marcel, Fabian; Regulski, Krzysztof; Kiss, György B.; Kondorosi, Adam; Endre, Gabriella; Kondorosi, Eva; Ané, Jean-Michel

    2007-01-01

    NORK in legumes encodes a receptor-like kinase that is required for Nod factor signaling and root nodule development. Using Medicago truncatula NORK as bait in a yeast two-hybrid assay, we identified 3-hydroxy-3-methylglutaryl CoA reductase 1 (Mt HMGR1) as a NORK interacting partner. HMGR1 belongs to a multigene family in M. truncatula, and different HMGR isoforms are key enzymes in the mevalonate biosynthetic pathway leading to the production of a diverse array of isoprenoid compounds. Testing other HMGR members revealed a specific interaction between NORK and HMGR1. Mutagenesis and deletion analysis showed that this interaction requires the cytosolic active kinase domain of NORK and the cytosolic catalytic domain of HMGR1. NORK homologs from Lotus japonicus and Sesbania rostrata also interacted with Mt HMGR1, but homologous nonsymbiotic kinases of M. truncatula did not. Pharmacological inhibition of HMGR activities decreased nodule number and delayed nodulation, supporting the importance of the mevalonate pathway in symbiotic development. Decreasing HMGR1 expression in M. truncatula transgenic roots by RNA interference led to a dramatic decrease in nodulation, confirming that HMGR1 is essential for nodule development. Recruitment of HMGR1 by NORK could be required for production of specific isoprenoid compounds, such as cytokinins, phytosteroids, or isoprenoid moieties involved in modification of signaling proteins. PMID:18156218

  1. Putative use of a Bacillus subtilis L194 strain for biocontrol of Phoma medicaginis in Medicago truncatula seedlings.

    PubMed

    Ben Slimene, Imen; Tabbene, Olfa; Djebali, Naceur; Cosette, Pascal; Schmitter, Jean Marie; Jouenne, Thierry; Urdaci, Maria-Camino; Limam, Ferid

    2012-06-01

    An antagonist L194 strain against Phoma medicaginis pathogenic fungi was isolated from Tunisian soil (vicinity of Tunis) and identified as Bacillus subtilis based on biochemical characteristics and partial 16S rDNA sequence. When cells were grown in a minimal medium for 24 h, spore culture supernatant exhibited 2-fold higher antifungal activity than vegetative cells. MALDI-TOF mass spectrometry analysis showed that L194 spores produced mainly iturins, surfactins and fengycins with long-chain fatty acids, and other not yet identified compounds. Both vegetative cells and spores of L194 efficiently reduced germination of P. medicaginis conidia. As revealed by atomic force microscopy, L194 spores modified conidia morphology from a regular to a deflated shape. Data suggest that lipopeptides interacted with the cytoplasmic membrane, causing pore formation. In vivo, L194 spores were highly protective against P. medicaginis by reducing disease symptoms and alleviating growth disturbance of Medicago truncatula seedlings. As a whole, the lipopeptide-producing L194 strain may be successfully used in biocontrol of plant diseases induced by phytopathogenic fungi such as P. medicaginis.

  2. Characterization of the plant growth promoting bacterium, Enterobacter cloacae MSR1, isolated from roots of non-nodulating Medicago sativa.

    PubMed

    Khalifa, Ashraf Y Z; Alsyeeh, Abdel-Moneium; Almalki, Mohammed A; Saleh, Farag A

    2016-01-01

    The aim of the present study was to characterize the endophytic bacterial strain designated MSR1 that was isolated from inside the non-nodulating roots of Medicago sativa after surface-sterilization. MSR1 was identified as Enterobacter cloacae using both 16S rDNA gene sequence analysis and API20E biochemical identification system (Biomerieux, France). Furthermore, this bacterium was characterized using API50CH kit (Biomerieux, France) and tested for antibacterial activities against some food borne pathogens. The results showed that E. cloacae consumed certain carbohydrates such as glycerol, d-xylose, d-maltose and esculin melibiose as a sole carbon source and certain amino acids such as arginine, tryptophan ornithine as nitrogen source. Furthermore, MSR1 possessed multiple plant-growth promoting characteristics; phosphate solubility, production of phytohormones acetoin and bioactive compounds. Inoculation of Pisum sativum with MSR1 significantly improved the growth parameters (the length and dry weight) of this economically important grain legume compared to the non-treated plants. To our knowledge, this is the first report addressing E. cloacae which exist in roots of alfalfa growing in Al-Ahsaa region. The results confirmed that E. cloacae exhibited traits for plant growth promoting and could be developed as an eco-friendly biofertilizer for P. sativum and probably for other important plant species in future.

  3. Identification of genetic loci associated with crude protein and mineral concentrations in alfalfa (Medicago sativa) using association mapping.

    PubMed

    Jia, Congjun; Wu, Xinming; Chen, Min; Wang, Yunqi; Liu, Xiqiang; Gong, Pan; Xu, Qingfang; Wang, Xuemin; Gao, Hongwen; Wang, Zan

    2017-06-06

    Alfalfa (Medicago sativa) is one of the most important legume forage species in China and many other countries of the world. It provides a quality source of proteins and minerals to animals. Genetic underpinnings for these important traits, however, are elusive. An alfalfa (M. sativa) association mapping study for six traits, namely crude protein (CP), rumen undegraded protein (RUP), and four mineral elements (Ca, K, Mg and P), was conducted in three consecutive years using a large collection encompassing 336 genotypes genotyped with 85 simple sequence repeat (SSR) markers. All the traits were significantly influenced by genotype, environment, and genotype × environment interaction. Eight-five significant associations (P < 0.005) were identified. Among these, five associations with Ca were repeatedly observed and six co-localized associations were identified. The identified marker alleles significantly associated with the traits provided important information for understanding genetic controls of alfalfa quality. The markers could be used in assisting selection for the individual traits in breeding populations for developing new alfalfa cultivars.

  4. Identification of Molecular Markers Associated with Verticillium Wilt Resistance in Alfalfa (Medicago Sativa L.) Using High-Resolution Melting

    PubMed Central

    Zhang, Tiejun; Yu, Long-Xi; McCord, Per; Miller, David; Bhamidimarri, Suresh; Johnson, David; Monteros, Maria J.; Ho, Julie; Reisen, Peter; Samac, Deborah A.

    2014-01-01

    Verticillium wilt, caused by the soilborne fungus, Verticillium alfalfae, is one of the most serious diseases of alfalfa (Medicago sativa L.) worldwide. To identify loci associated with resistance to Verticillium wilt, a bulk segregant analysis was conducted in susceptible or resistant pools constructed from 13 synthetic alfalfa populations, followed by association mapping in two F1 populations consisted of 352 individuals. Simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers were used for genotyping. Phenotyping was done by manual inoculation of the pathogen to replicated cloned plants of each individual and disease severity was scored using a standard scale. Marker-trait association was analyzed by TASSEL. Seventeen SNP markers significantly associated with Verticillium wilt resistance were identified and they were located on chromosomes 1, 2, 4, 7 and 8. SNP markers identified on chromosomes 2, 4 and 7 co-locate with regions of Verticillium wilt resistance loci reported in M. truncatula. Additional markers identified on chromosomes 1 and 8 located the regions where no Verticillium resistance locus has been reported. This study highlights the value of SNP genotyping by high resolution melting to identify the disease resistance loci in tetraploid alfalfa. With further validation, the markers identified in this study could be used for improving resistance to Verticillium wilt in alfalfa breeding programs. PMID:25536106

  5. Genome sequence of Ensifer medicae Di28; an effective N2-fixing microsymbiont of Medicago murex and M. polymorpha

    PubMed Central

    2014-01-01

    Ensifer medicae Di28 is an aerobic, motile, Gram-negative, non-spore-forming rod that can exist as a soil saprophyte or as a legume microsymbiont of Medicago spp. Di28 was isolated in 1998 from a nodule recovered from the roots of M. polymorpha growing in the south east of Sardinia (Italy). Di28 is an effective microsymbiont of the annual forage legumes M. polymorpha and M. murex and is capable of establishing a partially effective symbiotic association with the perennial M. sativa. Here we describe the features of E. medicae Di28, together with genome sequence information and its annotation. The 6,553,624 bp standard draft genome is arranged into 104 scaffolds of 104 contigs containing 6,394 protein-coding genes and 75 RNA-only encoding genes. This rhizobial genome is one of 100 sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project. PMID:25780497

  6. Genome sequence of Ensifer medicae Di28; an effective N2-fixing microsymbiont of Medicago murex and M. polymorpha.

    PubMed

    Garau, Giovanni; Terpolilli, Jason; Hill, Yvette; Tian, Rui; Howieson, John; Bräu, Lambert; Goodwin, Lynne; Han, James; Reddy, Tbk; Huntemann, Marcel; Pati, Amrita; Woyke, Tanja; Mavromatis, Konstantinos; Markowitz, Victor; Ivanova, Natalia; Kyrpides, Nikos; Reeve, Wayne

    2014-01-01

    Ensifer medicae Di28 is an aerobic, motile, Gram-negative, non-spore-forming rod that can exist as a soil saprophyte or as a legume microsymbiont of Medicago spp. Di28 was isolated in 1998 from a nodule recovered from the roots of M. polymorpha growing in the south east of Sardinia (Italy). Di28 is an effective microsymbiont of the annual forage legumes M. polymorpha and M. murex and is capable of establishing a partially effective symbiotic association with the perennial M. sativa. Here we describe the features of E. medicae Di28, together with genome sequence information and its annotation. The 6,553,624 bp standard draft genome is arranged into 104 scaffolds of 104 contigs containing 6,394 protein-coding genes and 75 RNA-only encoding genes. This rhizobial genome is one of 100 sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project.

  7. The Medicago truncatula lysin [corrected] motif-receptor-like kinase gene family includes NFP and new nodule-expressed genes.

    PubMed

    Arrighi, Jean-François; Barre, Annick; Ben Amor, Besma; Bersoult, Anne; Soriano, Lidia Campos; Mirabella, Rossana; de Carvalho-Niebel, Fernanda; Journet, Etienne-Pascal; Ghérardi, Michèle; Huguet, Thierry; Geurts, René; Dénarié, Jean; Rougé, Pierre; Gough, Clare

    2006-09-01

    Rhizobial Nod factors are key symbiotic signals responsible for starting the nodulation process in host legume plants. Of the six Medicago truncatula genes controlling a Nod factor signaling pathway, Nod Factor Perception (NFP) was reported as a candidate Nod factor receptor gene. Here, we provide further evidence for this by showing that NFP is a lysin [corrected] motif (LysM)-receptor-like kinase (RLK). NFP was shown both to be expressed in association with infection thread development and to be involved in the infection process. Consistent with deviations from conserved kinase domain sequences, NFP did not show autophosphorylation activity, suggesting that NFP needs to associate with an active kinase or has unusual functional characteristics different from classical kinases. Identification of nine new M. truncatula LysM-RLK genes revealed a larger family than in the nonlegumes Arabidopsis (Arabidopsis thaliana) or rice (Oryza sativa) of at least 17 members that can be divided into three subfamilies. Three LysM domains could be structurally predicted for all M. truncatula LysM-RLK proteins, whereas one subfamily, which includes NFP, was characterized by deviations from conserved kinase sequences. Most of the newly identified genes were found to be expressed in roots and nodules, suggesting this class of receptors may be more extensively involved in nodulation than was previously known.

  8. Paenibacillus medicaginis sp. nov. a chitinolytic endophyte isolated from the root nodule of alfalfa (Medicago sativa L.).

    PubMed

    Lai, Wei-An; Hameed, Asif; Lin, Shih-Yao; Hung, Mei-Hua; Hsu, Yi-Han; Liu, You-Cheng; Shahina, Mariyan; Shen, Fo-Ting; Young, Chiu-Chung

    2015-08-04

    A Gram-stain-variable, short-rod-shaped, endospore-forming, strictly aerobic, non-motile, chitinolytic and endophytic bacterium, designated strain CC-Alfalfa-19T, exhibiting unusual bipolar appendages was isolated from the root nodule of alfalfa (Medicago sativa L.) in Taiwan and subjected to a polyphasic taxonomy. Based on 16S rRNA gene sequence analysis, strain CC-Alfalfa-19T was found to be most closely related to Paenibacillus puldeungensis CAU 9324T (95.2 %), whereas other Paenibacillus species shared ≤95.0 % sequence similarity. The phylogenetic analysis revealed a distinct phyletic lineage established by strain CC-Alfalfa-19T with respect to other Paenibacillus species. Fatty acids comprised predominantly anteiso-C15:0, C16:0, anteiso-C17:0 and iso-C16. Menaquinone 7 (MK-7) was identified as the sole respiratory quinone and the genomic DNA G+C content was 42.7 mol%. Polar lipids included diphosphatidylglycerol, phosphatidylglycerol, phosphatidylmonomethylethanolamine, phosphatidylethanolamine, an unidentified glycolipid and an unidentified lipid. The diagnostic diamino acid found in the cell-wall peptidoglycan was meso-diaminopimelic acid. Based on the polyphasic taxonomic evidences that were in line with the genus Paenibacillus and additional distinguishing characteristics, strain CC-Alfalfa-19T is considered to represent a novel species, for which the name Paenibacillus medicaginis sp. nov. (type strain CC-Alfalfa-19T = BCRC 80441T = JCM 18446T) is proposed.

  9. Allelopathic potential of switchgrass (Panicum virgatum L.) on perennial ryegrass (Lolium perenne L.) and alfalfa (Medicago sativa L.).

    PubMed

    Shui, Junfeng; An, Yu; Ma, Yongqing; Ichizen, Nobumasa

    2010-10-01

    This study investigated allelopathy and its chemical basis in nine switchgrass (Panicum virgatum L.) accessions. Perennial ryegrass (Lolium perenne L.) and alfalfa (Medicago sativa L.) were used as test species. Undiluted aqueous extracts (5 g plant tissue in 50 ml water) from the shoots and roots of most of the switchgrass accessions inhibited the germination and growth of the test species. However, the allelopathic effect of switchgrass declined when extracts were diluted 5- or 50-fold. Seedling growth was more sensitive than seed germination as an indicator of allelopathic effect. Allelopathic effect was related to switchgrass ecotype but not related to ploidy level. Upland accessions displayed stronger allelopathic potential than lowland accessions. The aqueous extract from one switchgrass accession was separated into phenols, organic acids, neutral chemicals, and alkaloids, and then these fractions were bioassayed to test for allelopathic potential. Alkaloids had the strongest allelopathic effect among the four chemical fractions. In summary, the results indicated that switchgrass has allelopathic potential; however, there is not enough evidence to conclude that allelopathic advantage is the main factor that has contributed to the successful establishment of switchgrass on China's Loess Plateau.

  10. Effects of Endogenous Salicylic Acid on Nodulation in the Model Legumes Lotus japonicus and Medicago truncatula1[W

    PubMed Central

    Stacey, Gary; McAlvin, Crystal Bickley; Kim, Sung-Yong; Olivares, José; Soto, María José

    2006-01-01

    The exogenous addition of salicylic acid (SA) was previously shown to inhibit indeterminate but not determinate-type nodulation. We sought to extend these results by modulating endogenous levels of SA through the transgenic expression of salicylate hydroxylase (NahG) in both stably transformed Lotus japonicus and composite Medicago truncatula plants. NahG expression in L. japonicus resulted in a marked reduction of SA levels. This reduction correlated with an increase in the number of infections and mean nodule number when compared to controls. However, a complicating factor was that NahG-expressing plants had greater root growth. Spot inoculations of NahG-expressing L. japonicus plants confirmed increased nodulation in these plants. Consistent with the reported inhibitory effects of exogenous SA on indeterminate-type nodulation, NahG expression in M. truncatula plants led to enhanced nodulation and infection. These data point to an important role for SA-mediated plant defense pathways in controlling nodule formation on both determinate and indeterminate nodule-forming hosts. PMID:16798946

  11. Crystal structure of the anticarcinogenic Bowman-Birk inhibitor from snail medic (Medicago scutellata) seeds complexed with bovine trypsin.

    PubMed

    Capaldi, Stefano; Perduca, Massimiliano; Faggion, Beniamino; Carrizo, Maria E; Tava, Aldo; Ragona, Laura; Monaco, Hugo L

    2007-04-01

    The structure of the ternary complex of the anticarcinogenic Bowman-Birk protease inhibitor purified from snail medic (Medicago scutellata) seeds (MSTI) and two molecules of bovine trypsin has been solved by X-ray diffraction analysis of single crystals to a resolution of 2.0 A. This is the highest resolution model of a ternary complex of this type currently available. The two binding loops of the MSTI differ in only one amino acid and have in both cases an arginine in position P1. The distances between the residues of the inhibitor at the binding interface and the trypsin side chains that recognize them are almost identical in the two sites. When compared to the NMR model of the uncomplexed MSTI, the inhibitor in the functional assembly with trypsin shows the largest differences in the two P2' residues. Compared with the similar ternary complex of the soybean trypsin inhibitor, this model shows very small differences in the polypeptide chain of the trypsin binding sites and its largest difference in the area between Asp 26 and His 32 of the MSTI which in the soybean inhibitor has an extra Leu inserted in position 29.

  12. Anticarcinogenic Bowman Birk inhibitor isolated from snail medic seeds (Medicago scutellata): solution structure and analysis of self-association behavior.

    PubMed

    Catalano, M; Ragona, L; Molinari, H; Tava, A; Zetta, L

    2003-03-18

    The high-resolution three-dimensional structure of a Bowman Birk inhibitor, purified from snail medic seeds (Medicago scutellata) (MSTI), has been determined in solution by 1H NMR spectroscopy at pH 5.6 and 27 degrees C. The structure of MSTI comprises two distinct symmetric domains each composed of a three-stranded beta-sheet containing a VIb type loop, where the active sites are located. A characteristic geometry of three aromatic residues confers stability to this protein, and we observe that this feature is conserved in all the Bowman Birk inhibitors of known structure. The two active domains exhibit different conformational features: the second domain displays higher flexibility and hydrophobicity with respect to the first one, and these properties have been correlated to a lower trypsin inhibitory specificity, in agreement with titration studies that have shown a stoichiometric ratio MSTI:trypsin of 1:1.5. NMR analysis indicated that MSTI undergoes self-association at concentrations higher than 2 mM, and the residues involved in this mechanism are localized at opposite faces of the molecule, having the highest positive and negative potential, respectively, thus indicating that electrostatic intermolecular interactions are the driving forces for MSTI association. Most of the residues affected by self-association are highly conserved in BBIs from different seeds, suggesting a functional relevance for these charged superficial patches, possibly involved in the interaction with other enzymes or macromolecules, thus triggering anti-carcinogenic activity.

  13. Drought stress provokes the down-regulation of methionine and ethylene biosynthesis pathways in Medicago truncatula roots and nodules.

    PubMed

    Larrainzar, Estíbaliz; Molenaar, Johanna A; Wienkoop, Stefanie; Gil-Quintana, Erena; Alibert, Bénédicte; Limami, Anis M; Arrese-Igor, Cesar; González, Esther M

    2014-09-01

    Symbiotic nitrogen fixation is one of the first physiological processes inhibited in legume plants under water-deficit conditions. Despite the progress made in the last decades, the molecular mechanisms behind this regulation are not fully understood yet. Recent proteomic work carried out in the model legume Medicago truncatula provided the first indications of a possible involvement of nodule methionine (Met) biosynthesis and related pathways in response to water-deficit conditions. To better understand this involvement, the drought-induced changes in expression and content of enzymes involved in the biosynthesis of Met, S-adenosyl-L-methionine (SAM) and ethylene in M. truncatula root and nodules were analyzed using targeted approaches. Nitrogen-fixing plants were subjected to a progressive water deficit and a subsequent recovery period. Besides the physiological characterization of the plants, the content of total sulphur, sulphate and main S-containing metabolites was measured. Results presented here show that S availability is not a limiting factor in the drought-induced decline of nitrogen fixation rates in M. truncatula plants and provide evidences for a down-regulation of the Met and ethylene biosynthesis pathways in roots and nodules in response to water-deficit conditions. © 2014 John Wiley & Sons Ltd.

  14. [MEDICAGO SATIVA L: IMPROVEMENT AND NEW APPROACHES OF ITS NUTRITIONAL AND FUNCTIONAL VALUE BY BACTERIAL CO-INOCULATION].

    PubMed

    Martínez, Rosario; Nebot, Elena; Porres, Jesús María; Kapravelou, Garyfallia; Del Moral, Ana; Talbi, Chouhra; Bedmar, Eulogio Jose; López-Jurado, María

    2015-12-01

    to study the effect of co-inoculation with Ensifer meliloti and Halomonas maura of the leguminous Medicago sativa L., on growth, nutritional and functional value, grown under salinity conditions. plants of M. sativa were grown in a solution with a mixture of salts (CaSO4, MgCl, NaCl and NaHCO 3) and were co-inoculated with its specific rhizobium and the halophilic moderated bacterium H. maura. Different physiologic parameters were determined, as well as, nitrogen and minerals content. Furthermore, an assay of in vitro digestibility was carried out. salinity had a negative effect on the plants; however, co-inoculation increased yield, nitrogen content, total minerals, Ca and Mg. Moreover, physiologic parameters as water potential and leghemoglobin content in fresh nodules were higher compared to those of plants inoculated only with E. meliloti. Both, salinity and bacterial treatment with E. meliloti and H. maura increased the antioxidant capacity of the legume, in dialyzates and retentates collected after an in vitro digestibility assay. co-inoculation of plants with E. meliloti and H. maura could improve the alfalfa yield under specific salinity conditions, increasing the nutritional and functional value of the plants. M. sativa could be considered in the formulations of nutritional supplements for the human diet. Copyright AULA MEDICA EDICIONES 2014. Published by AULA MEDICA. All rights reserved.

  15. Adjustment of host cells for accommodation of symbiotic bacteria: vacuole defunctionalization, HOPS suppression, and TIP1g retargeting in Medicago.

    PubMed

    Gavrin, Aleksandr; Kaiser, Brent N; Geiger, Dietmar; Tyerman, Stephen D; Wen, Zhengyu; Bisseling, Ton; Fedorova, Elena E

    2014-09-01

    In legume-rhizobia symbioses, the bacteria in infected cells are enclosed in a plant membrane, forming organelle-like compartments called symbiosomes. Symbiosomes remain as individual units and avoid fusion with lytic vacuoles of host cells. We observed changes in the vacuole volume of infected cells and thus hypothesized that microsymbionts may cause modifications in vacuole formation or function. To examine this, we quantified the volumes and surface areas of plant cells, vacuoles, and symbiosomes in root nodules of Medicago truncatula and analyzed the expression and localization of VPS11 and VPS39, members of the HOPS vacuole-tethering complex. During the maturation of symbiosomes to become N2-fixing organelles, a developmental switch occurs and changes in vacuole features are induced. For example, we found that expression of VPS11 and VPS39 in infected cells is suppressed and host cell vacuoles contract, permitting the expansion of symbiosomes. Trafficking of tonoplast-targeted proteins in infected symbiotic cells is also altered, as shown by retargeting of the aquaporin TIP1g from the tonoplast membrane to the symbiosome membrane. This retargeting appears to be essential for the maturation of symbiosomes. We propose that these alterations in the function of the vacuole are key events in the adaptation of the plant cell to host intracellular symbiotic bacteria. © 2014 American Society of Plant Biologists. All rights reserved.

  16. Toxic Effects of Copper-based Nanoparticles or Compounds to Lettuce (Lactuca sativa) and Alfalfa (Medicago sativa)

    PubMed Central

    Hong, Jie; Rico, Cyren; Zhao, Lijuan; Adeleye, Adeyemi S.; Keller, Arturo A.; Peralta-Videa, Jose R.; Gardea-Torresdey, Jorge L.

    2014-01-01

    The increased production and use of nanoparticles (NPs) has generated concerns about their impact on living organisms. In this study, nCu, bulk Cu, nCuO, bulk CuO, Cu(OH)2 (CuPRO 2005, Kocide 3000), and CuCl2 were exposed for 15 days to 10 day-old hydroponically grown lettuce (Lactuca sativa) and alfalfa (Medicago sativa). Each compound was applied at 0, 5, 10, and 20 mg/L. At harvest, we measured the size of the plants and determined the concentration of Cu, macro and microelements by using ICP-OES. Catalase and ascorbate peroxidase activity was also determined. Results showed that all Cu NPs/compounds reduced the root length by 49% in both plant species. All Cu NPs/compounds increased Cu, P, and S (>100%, >50%, and >20%, respectively) in alfalfa shoots and decreased P and Fe in lettuce shoot (>50% and >50%, respectively, excluding Fe in CuCl2 treatment). Biochemical assays showed reduced catalase activity in alfalfa (root and shoot) and increased ascorbate peroxidase activity in roots of both plant species. Results suggest that Cu NPs/compounds not only reduced the size of the plants but altered nutrient content and enzyme activity in both plant species. PMID:25474419

  17. Elevated CO2 Modifies N Acquisition of Medicago truncatula by Enhancing N Fixation and Reducing Nitrate Uptake from Soil

    PubMed Central

    Guo, Huijuan; Sun, Yucheng; Li, Yuefei; Liu, Xianghui; Ren, Qin; Zhu-Salzman, Keyan; Ge, Feng

    2013-01-01

    The effects of elevated CO2 (750 ppm vs. 390 ppm) were evaluated on nitrogen (N) acquisition and assimilation by three Medicago truncatula genotypes, including two N-fixing-deficient mutants (dnf1-1 and dnf1-2) and their wild-type (Jemalong). The proportion of N acquisition from atmosphere and soil were quantified by 15N stable isotope, and N transportation and assimilation-related genes and enzymes were determined by qPCR and biochemical analysis. Elevated CO2 decreased nitrate uptake from soil in all three plant genotypes by down-regulating nitrate reductase (NR), nitrate transporter NRT1.1 and NR activity. Jemalong plant, however, produced more nodules, up-regulated N-fixation-related genes and enhanced percentage of N derived from fixation (%Ndf) to increase foliar N concentration and N content in whole plant (Ntotal Yield) to satisfy the requirement of larger biomass under elevated CO2. In contrast, both dnf1 mutants deficient in N fixation consequently decreased activity of glutamine synthetase/glutamate synthase (GS/GOGAT) and N concentration under elevated CO2. Our results suggest that elevated CO2 is likely to modify N acquisition of M. truncatula by simultaneously increasing N fixation and reducing nitrate uptake from soil. We propose that elevated CO2 causes legumes to rely more on N fixation than on N uptake from soil to satisfy N requirements. PMID:24339920

  18. Cytotoxicity and apoptosis induced by alfalfa (Medicago sativa) leaf extracts in sensitive and multidrug-resistant tumor cells.

    PubMed

    Gatouillat, Grégory; Magid, Abdulmagid Alabdul; Bertin, Eric; Okiemy-Akeli, Marie-Genevieve; Morjani, Hamid; Lavaud, Catherine; Madoulet, Claudie

    2014-01-01

    Alfalfa (Medicago sativa) has been used to cure a wide variety of ailments. However, only a few studies have reported its anticancer effects. In this study, extracts were obtained from alfalfa leaves and their cytotoxic effects were assessed on several sensitive and multidrug-resistant tumor cells lines. Using the mouse leukaemia P388 cell line and its doxorubicin-resistant counterpart (P388/DOX), we showed that the inhibition of cell growth induced by alfalfa leaf extracts was mediated through the induction of apoptosis, as evidenced by DNA fragmentation analysis. The execution of programmed cell death was achieved via the activation of caspase-3, leading to PARP cleavage. Fractionation of toluene extract (To-1), the most active extract obtained from crude extract, led to the identification of 3 terpene derivatives and 5 flavonoids. Among them, (-)-medicarpin, (-)-melilotocarpan E, millepurpan, tricin, and chrysoeriol showed cytotoxic effects in P388 as well as P388/DOX cells. These results demonstrate that alfalfa leaf extract may have interesting potential in cancer chemoprevention and therapy.

  19. Alfalfa (Medicago sativa L.) shoot saponins: identification and bio-activity by the assessment of aphid feeding.

    PubMed

    Mazahery-Laghab, H; Yazdi-Samadi, B; Bagheri, M; Bagheri, A R

    2011-01-01

    Biochemical components in alfalfa (Medicago sativa L.), such as saponins, can act as protecting factors against bio-stresses. Saponins are also antifeedants and show oral toxicity towards higher and lower animals. Changes in saponins, such as variation in the carbon skeleton, or hydrolysis of saponin glycosides and other conjugates, may change their biological effects. The aims of this research were to study saponin variation in different growth stages of alfalfa and to investigate the biological role of saponins in the spotted alfalfa aphid, Therioaphis maculata. Saponins from alfalfa shoots in different growth stages were extracted, chemically purified and analysed by TLC. Specific saponins such as soyasaponin1 from root and shoot and two bisdesmosides of medicagenic acid, one from shoot and another from root tissues, were identified using reference compounds allowing changes in saponin composition during plant development in different shoot tissues of alfalfa to be assessed. The response of the alfalfa aphid to feeding on alfalfa in different growth stages was studied. No significant difference in the survival of aphids, from neonate to adult, was observed, but due to the antibiotic effects of saponins, two differences were found in the onset of nymph production and cumulative nymph production. The results show that the saponin composition in alfalfa changes with plant development and this, in turn, can often negatively affect the development of specific insect pests such as the spotted alfalfa aphid, suggesting a possible biological role of alfalfa saponins.

  20. Characterization of two novel cold-inducible K3 dehydrin genes from alfalfa (Medicago sativa spp. sativa L.).

    PubMed

    Dubé, Marie-Pier; Castonguay, Yves; Cloutier, Jean; Michaud, Josée; Bertrand, Annick

    2013-03-01

    Dehydrin defines a complex family of intrinsically disordered proteins with potential adaptive value with regard to freeze-induced cell dehydration. Search within an expressed sequence tags library from cDNAs of cold-acclimated crowns of alfalfa (Medicago sativa spp. sativa L.) identified transcripts putatively encoding K(3)-type dehydrins. Analysis of full-length coding sequences unveiled two highly homologous sequence variants, K(3)-A and K(3)-B. An increase in the frequency of genotypes yielding positive genomic amplification of the K(3)-dehydrin variants in response to selection for superior tolerance to freezing and the induction of their expression at low temperature strongly support a link with cold adaptation. The presence of multiple allelic forms within single genotypes and independent segregation indicate that the two K(3) dehydrin variants are encoded by distinct genes located at unlinked loci. The co-inheritance of the K(3)-A dehydrin with a Y(2)K(4) dehydrin restriction fragment length polymorphism with a demonstrated impact on freezing tolerance suggests the presence of a genome domain where these functionally related genes are located. These results provide additional evidence that dehydrin play important roles with regard to tolerance to subfreezing temperatures. They also underscore the value of recurrent selection to help identify variants within a large multigene family in allopolyploid species like alfalfa.

  1. In vitro and in vivo antioxidant activity of alfalfa (Medicago sativa L.) on carbon tetrachloride intoxicated rats.

    PubMed

    Al-Dosari, Mohammed S

    2012-01-01

    The present study was conducted to determine whether lyophilized aqueous extract of alfalfa, or Medicago sativa L. could exert antioxidant activity against carbon tetrachloride-induced oxidative stress and liver injury in rats. The hepatoprotective activity of alfalfa extract was determined by assessing the levels of serum transaminases, ALP, bilirubin and lipid profile. Further, the effect of the test substance on malondialdehyde (MDA), an end product of lipid peroxidation; antioxidant liver enzyme non-protein sulfhydryl (NP-SH); and total protein (TP) were also studied. Serum transaminase, ALP, bilirubin level, lipid profile and liver MDA were significantly elevated and the antioxidant status in liver NP-SH and TP contents were declined in animals treated with CCl (4) alone. Pretreatment with alfalfa and silymarin for three weeks prior to the administration of CCl (4) significantly prevented the increase in the serum levels of hepatic marker, LDL, VLDL levels enzymes and reduced oxidative stress indicated by elevated NP-SH and TP concentration. The histopathological examination of the livers also showed that the alfalfa extract reduced the incidence of liver lesions induced by CCl (4). The in vitro antioxidant assessment of alfalfa extract on DPPH and carotene-linoleic assays demonstrated a moderate antioxidant potential. Results suggest that the alfalfa extract possesses hepatoprotective and antioxidative stress properties possibly through its antioxidant phytochemical constituents and substantiates its use in various liver disorders as a hepatoprotector.

  2. Architecture of infection thread networks in developing root nodules induced by the symbiotic bacterium Sinorhizobium meliloti on Medicago truncatula.

    PubMed

    Monahan-Giovanelli, Hannah; Pinedo, Catalina Arango; Gage, Daniel J

    2006-02-01

    During the course of the development of nitrogen-fixing root nodules induced by Sinorhizobium meliloti on the model plant Medicago truncatula, tubules called infection threads are cooperatively constructed to deliver the bacterial symbiont from the root surface to cells in the interior of the root and developing nodule. Three-dimensional reconstructions of infection threads inside M. truncatula nodules showed that the threads formed relatively simple, tree-like networks. Some characteristics of thread networks, such as branch length, branch density, and branch surface-to-volume ratios, were remarkably constant across nodules in different stages of development. The overall direction of growth of the networks changed as nodules developed. In 5-d-old nodules, the overall growth of the network was directed inward toward the root. However, well-defined regions of these young networks displayed an outward growth bias, indicating that they were likely in the process of repolarizing their direction of development in response to the formation of the outward-growing nodule meristem. In 10- and 30-d-old nodules, the branches of the network grew outward toward the meristem and away from the roots on which the nodules developed.

  3. Population differentiation for germination and early seedling root growth traits under saline conditions in the annual legume Medicago truncatula (Fabaceae).

    PubMed

    Cordeiro, Matilde A; Moriuchi, Ken S; Fotinos, Tonya D; Miller, Kelsey E; Nuzhdin, Sergey V; von Wettberg, Eric J; Cook, Douglas R

    2014-03-01

    Seedling establishment and survival are highly sensitive to soil salinity and plants that evolved in saline environments are likely to express traits that increase fitness in those environments. Such traits are of ecological interest and they may have practical value for improving salt tolerance in cultivated species. We examined responses to soil salinity and tested potential mechanisms of salt tolerance in Medicago truncatula, using genotypes that originated from natural populations occurring on saline and nonsaline soils. Germination and seedling responses were quantified and compared between saline and nonsaline origin genotypes. Germination treatments included a range of sodium chloride (NaCl) concentrations in both offspring and parental environments. Seedling treatments included NaCl, abscisic acid (ABA), and potassium chloride (KCl). Saline origin genotypes displayed greater salinity tolerance for germination and seedling traits relative to nonsaline origin genotypes. We observed population specific differences for the effects of salinity on time to germination and for the impact of parental environment on germination rates. ABA and NaCl treatments had similar negative effects on root growth, although relative sensitivities differed, with saline population less sensitive to NaCl and more sensitive to ABA compared to their nonsaline counterparts. We report population differentiation for germination and seedling growth traits under saline conditions among populations derived from saline and nonsaline environments. These observations are consistent with a syndrome of adaptations for salinity tolerance during early plant development, including traits that are common among saline environments and those that are idiosyncratic to local populations.

  4. Flavonoids and Auxin Transport Inhibitors Rescue Symbiotic Nodulation in the Medicago truncatula Cytokinin Perception Mutant cre1

    PubMed Central

    Ng, Jason Liang Pin; Hassan, Samira; Truong, Thy T.; Hocart, Charles H.; Laffont, Carole; Frugier, Florian; Mathesius, Ulrike

    2015-01-01

    Initiation of symbiotic nodules in legumes requires cytokinin signaling, but its mechanism of action is largely unknown. Here, we tested whether the failure to initiate nodules in the Medicago truncatula cytokinin perception mutant cre1 (cytokinin response1) is due to its altered ability to regulate auxin transport, auxin accumulation, and induction of flavonoids. We found that in the cre1 mutant, symbiotic rhizobia cannot locally alter acro- and basipetal auxin transport during nodule initiation and that these mutants show reduced auxin (indole-3-acetic acid) accumulation and auxin responses compared with the wild type. Quantification of flavonoids, which can act as endogenous auxin transport inhibitors, showed a deficiency in the induction of free naringenin, isoliquiritigenin, quercetin, and hesperetin in cre1 roots compared with wild-type roots 24 h after inoculation with rhizobia. Coinoculation of roots with rhizobia and the flavonoids naringenin, isoliquiritigenin, and kaempferol, or with the synthetic auxin transport inhibitor 2,3,5,-triiodobenzoic acid, rescued nodulation efficiency in cre1 mutants and allowed auxin transport control in response to rhizobia. Our results suggest that CRE1-dependent cytokinin signaling leads to nodule initiation through the regulation of flavonoid accumulation required for local alteration of polar auxin transport and subsequent auxin accumulation in cortical cells during the early stages of nodulation. PMID:26253705

  5. Three Medicago MtFUL genes have distinct and overlapping expression patterns during vegetative and reproductive development and 35S:MtFULb accelerates flowering and causes a terminal flower phenotype in Arabidopsis.

    PubMed

    Jaudal, Mauren; Zhang, Lulu; Che, Chong; Putterill, Joanna

    2015-01-01

    The timing of the transition to flowering is carefully controlled by plants in order to optimize sexual reproduction and the ensuing production of seeds, grains, and fruits. The genetic networks that regulate floral induction are best characterized in the temperate eudicot Arabidopsis in which the florigen gene FT plays a major role in promoting the transition to flowering. Legumes are an important plant group, but less is known about the regulation of their flowering time. In the model legume Medicago truncatula (Medicago), a temperate annual plant like Arabidopsis, flowering is induced by prolonged cold (vernalization) followed by long day lengths (LD). Recent molecular-genetic experiments have revealed that a FT-like gene, MtFTa1, is a central regulator of flowering time in Medicago. Here, we characterize the three Medicago FRUITFULL (FUL) MADS transcription factors, MtFULa, MtFULb, and MtFULc using phylogenetic analyses, gene expression profiling through developmental time courses, and functional analyses in transgenic plants. MtFULa and MtFULb have similarity in sequence and expression profiles under inductive environmental conditions during both vegetative and reproductive development while MtFULc is only up regulated in the apex after flowering in LD conditions. Sustained up regulation of MtFULs requires functional MtFTa1 but their transcript levels are not affected during cold treatment. Overexpression of MtFULa and MtFULb promotes flowering in transgenic Arabidopsis plants with an additional terminal flower phenotype on some 35S:MtFULb plants. An increase in transcript levels of the MtFULs was also observed in Medicago plants overexpressing MtFTa1. Our results suggest that the MtFULs are targets of MtFTa1. Overall, this work highlights the conserved functions of FUL-like genes in promoting flowering and other roles in plant development and thus contributes to our understanding of the genetic control of the flowering process in Medicago.

  6. Three Medicago MtFUL genes have distinct and overlapping expression patterns during vegetative and reproductive development and 35S:MtFULb accelerates flowering and causes a terminal flower phenotype in Arabidopsis

    PubMed Central

    Jaudal, Mauren; Zhang, Lulu; Che, Chong; Putterill, Joanna

    2015-01-01

    The timing of the transition to flowering is carefully controlled by plants in order to optimize sexual reproduction and the ensuing production of seeds, grains, and fruits. The genetic networks that regulate floral induction are best characterized in the temperate eudicot Arabidopsis in which the florigen gene FT plays a major role in promoting the transition to flowering. Legumes are an important plant group, but less is known about the regulation of their flowering time. In the model legume Medicago truncatula (Medicago), a temperate annual plant like Arabidopsis, flowering is induced by prolonged cold (vernalization) followed by long day lengths (LD). Recent molecular-genetic experiments have revealed that a FT-like gene, MtFTa1, is a central regulator of flowering time in Medicago. Here, we characterize the three Medicago FRUITFULL (FUL) MADS transcription factors, MtFULa, MtFULb, and MtFULc using phylogenetic analyses, gene expression profiling through developmental time courses, and functional analyses in transgenic plants. MtFULa and MtFULb have similarity in sequence and expression profiles under inductive environmental conditions during both vegetative and reproductive development while MtFULc is only up regulated in the apex after flowering in LD conditions. Sustained up regulation of MtFULs requires functional MtFTa1 but their transcript levels are not affected during cold treatment. Overexpression of MtFULa and MtFULb promotes flowering in transgenic Arabidopsis plants with an additional terminal flower phenotype on some 35S:MtFULb plants. An increase in transcript levels of the MtFULs was also observed in Medicago plants overexpressing MtFTa1. Our results suggest that the MtFULs are targets of MtFTa1. Overall, this work highlights the conserved functions of FUL-like genes in promoting flowering and other roles in plant development and thus contributes to our understanding of the genetic control of the flowering process in Medicago. PMID:25745430

  7. Genome-Wide Identification and Expression Profiling Analysis of the Aux/IAA Gene Family in Medicago truncatula during the Early Phase of Sinorhizobium meliloti Infection

    PubMed Central

    Zhang, Lei; Sun, Tao; Xu, Luqin; Tie, Shuanggui; Wang, Huizhong

    2014-01-01

    Background Auxin/indoleacetic acid (Aux/IAA) genes, coding a family of short-lived nuclear proteins, play key roles in wide variety of plant developmental processes, including root system regulation and responses to environmental stimulus. However, how they function in auxin signaling pathway and symbiosis with rhizobial in Medicago truncatula are largely unknown. The present study aims at gaining deeper insight on distinctive expression and function features of Aux/IAA family genes in Medicago truncatula during nodule formation. Principal Findings Using the latest updated draft of the full Medicago truncatula genome, a comprehensive identification and analysis of IAA genes were performed. The data indicated that MtIAA family genes are distributed in all the M. truncatula chromosomes except chromosome 6. Most of MtIAA genes are responsive to exogenous auxin and express in tissues-specific manner. To understand the biological functions of MtIAA genes involved in nodule formation, quantitative real-time polymerase chain reaction (qRT-PCR) was used to test the expression profiling of MtIAA genes during the early phase of Sinorhizobium meliloti (S. meliloti) infection. The expression patterns of most MtIAA genes were down-regulated in roots and up-regulated in shoots by S. meliloti infection. The differences in expression responses between roots and shoots caused by S. meliloti infection were alleviated by 1-NOA application. Conclusion The genome-wide identification, evolution and expression pattern analysis of MtIA