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Seed priming is routinely applied to improve germination rates and seedling establishment, but the decrease in longevity observed in primed seeds constitutes a major drawback that compromises long-term storability. The optimization of priming protocols able to preserve primed seeds from aging processes represents a promising route to expand the scope of seed priming. The present work explores this possibility in the model legume Medicago truncatula by testing the effectiveness of quercetin- and rutin-supplemented seed priming at improving the response to subsequent artificial aging. In comparison with a non-supplemented hydropriming protocol, supplementation with quercetin or rutin was able to mitigate the effects of post-priming aging by increasing germination percentage and speed, improving seed viability and seedling phenotype, with consistent correlations with a decrease in the levels of reactive oxygen species and an increase in antioxidant potential. The results suggest that quercetin and rutin can reduce the effects of post-priming aging by improving the seed antioxidant profiles. The present work provides novel information to explore the physiological changes associated with seed priming and aging, with possible outcomes for the development of tailored vigorization protocols able to overcome the storability constrains associated with post-priming aging processes. [ABSTRACT FROM AUTHOR]

Seed priming is routinely applied to improve germination rates and seedling establishment, but the decrease in longevity observed in primed seeds constitutes a major drawback that compromises long-term storability. The optimization of priming protocols able to preserve primed seeds from aging processes represents a promising route to expand the scope of seed priming. The present work explores this possibility in the model legume Medicago truncatula by testing the effectiveness of quercetin- and rutin-supplemented seed priming at improving the response to subsequent artificial aging. In comparison with a non-supplemented hydropriming protocol, supplementation with quercetin or rutin was able to mitigate the effects of post-priming aging by increasing germination percentage and speed, improving seed viability and seedling phenotype, with consistent correlations with a decrease in the levels of reactive oxygen species and an increase in antioxidant potential. The results suggest that quercetin and rutin can reduce the effects of post-priming aging by improving the seed antioxidant profiles. The present work provides novel information to explore the physiological changes associated with seed priming and aging, with possible outcomes for the development of tailored vigorization protocols able to overcome the storability constrains associated with post-priming aging processes.

Plants simultaneously interact with belowground symbionts such as arbuscular mycorrhizal (AM) fungi and aboveground antagonists such as aphids. Generally, plants gain access to valuable resources including nutrients and water through the AM symbiosis and are more resistant to pests. Nevertheless, aphids' performance improves on mycorrhizal plants, and it remains unclear whether a more nutritious food source and/or attenuated defenses are the contributing factors. This study examined the shoot and root transcriptome of barrel medic (Medicago truncatula Gaertn.) plants highly colonized by the AM fungus Rhizophagus irregularis (Blaszk. Wubet, Renker, and Buscot) C. Walker and A. Schüßler (Glomerales: Glomeraceae) and exposed to 7 days of mixed age pea aphid (Acyrthosiphon pisum (Harris)) herbivory. The RNA-seq samples chosen for this study showed that aphids were heavier when fed mycorrhizal plants compared to nonmycorrhizal plants. We hypothesized that (i) insect-related plant defense pathways will be downregulated in shoots of mycorrhizal plants with aphids compared to nonmycorrhizal plants with aphids; (ii) pathways involved in nutrient acquisition, carbohydrate-related and amino acid transport will be upregulated in shoots of mycorrhizal plants with aphids compared to nonmycorrhizal plants with aphids; and (iii) roots of mycorrhizal plants with aphids will exhibit mycorrhiza-induced resistance. The transcriptome data revealed that the gene repertoire related to defenses, nutrient transport, and carbohydrates differs between nonmycorrhizal and mycorrhizal plants with aphids, which could explain the weight gain in aphids. We also identified novel candidate genes that are differentially expressed in nonmycorrhizal plants with aphids, thus setting the stage for future functional studies. [ABSTRACT FROM AUTHOR]

Late embryogenesis abundant (LEA) proteins are important for abiotic stress tolerance in diverse organisms. Within the LEA protein superfamily, group 4 members are characterized by a conserved N-terminal region and a structurally disordered C-terminal region that varies regarding length and amino acid content. Previous in vitro assays have suggested that the conserved N-terminal region shared by group 4 LEA proteins is critical for forming an amphipathic α-helix and protecting enzymatic activities from the adverse effects of desiccation or freezing. However, the cellular roles of the varying C-terminal region remain largely to be characterized. Medicago truncatula contains five subgroup LEA4B proteins encoding loci of which three are tandemly arranged on chromosome 7 due to local gene duplication events. In this study, abiotic stresses and addition of abscisic acid (ABA) induced the transcription of the four LEA4B genes. Escherichia coli cells overexpressing the three tandemly aligned LEA genes indicated significantly increased tolerance to salt, osmotic, heat, and freezing stresses. However, the extent of the protective effects on the survival and growth of bacterial cells differed among the LEA proteins, potentially because of variations in the C-terminal region. This possibility was further supported by the observation that the protective effects of the native truncated MtLEA3140, which only contains a conserved N-terminal region, were inferior to the effects of the full-length mutant version. The results suggest that the structurally flexible C-terminal region of group 4 LEA proteins plays roles in protecting cells from damages caused by various abiotic stresses, and provide clues for elucidating the mechanisms underlying the intracellular functions of these proteins.

The "Specific Tissue" (ST) are proteins of unknown function present only in some plant families, mainly Fabaceae and Asteraceae. They are included in the PF10950 protein family and characterized by the presence of at least one domain of unknown function (DUF)2775. In this work we studied the involvement of the six members of the Medicago truncatula ST family (ST1 to ST6) in the development of flowers, fruits, and seeds by analysing the activity of their promoters (pST) after the construction of M. truncatula transgenic plants expressing the b-glucuronidase (GUS) reporter gene under the control of the six pSTs. The GUS activity was analysed in whole flowers and fruits and also in histological sections of these organs. The pST expression in the reproductive organs was mainly associated with the vascular bundles, especially throughout fruit development. These results pointed to an important role of ST proteins during the reproductive development stage, related to nutrient mobilization during the fruit and seed formation, that could be facilitated by their presence in the pod vascular bundles, as well as in the connective tissue of the anthers (ST3, ST4, ST6), in the placenta, the funiculus, and the outer parts of the developing seed (ST2, ST3, ST6). The observations made in this study were in agreement with the functions previously established for the three groups of M. truncatula ST proteins, as in the proposed function for ST1 in the transport and assimilation of nutrients, or the involvement of ST4, ST5, and ST6 in floral defence.

Late embryogenesis abundant (LEA) proteins are important for abiotic stress tolerance in diverse organisms. Within the LEA protein superfamily, group 4 members are characterized by a conserved N-terminal region and a structurally disordered C-terminal region that varies regarding length and amino acid content. Previous in vitro assays have suggested that the conserved N-terminal region shared by group 4 LEA proteins is critical for forming an amphipathic α-helix and protecting enzymatic activities from the adverse effects of desiccation or freezing. However, the cellular roles of the varying C-terminal region remain largely to be characterized. Medicago truncatula contains five subgroup LEA4B proteins encoding loci of which three are tandemly arranged on chromosome 7 due to local gene duplication events. In this study, abiotic stresses and addition of abscisic acid (ABA) induced the transcription of the four LEA4B genes. Escherichia coli cells overexpressing the three tandemly aligned LEA genes indicated significantly increased tolerance to salt, osmotic, heat, and freezing stresses. However, the extent of the protective effects on the survival and growth of bacterial cells differed among the LEA proteins, potentially because of variations in the C-terminal region. This possibility was further supported by the observation that the protective effects of the native truncated MtLEA3140, which only contains a conserved N-terminal region, were inferior to the effects of the full-length mutant version. The results suggest that the structurally flexible C-terminal region of group 4 LEA proteins plays roles in protecting cells from damages caused by various abiotic stresses, and provide clues for elucidating the mechanisms underlying the intracellular functions of these proteins. [ABSTRACT FROM AUTHOR]

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Aphids, including the bluegreen aphid (BGA; Acyrthosiphon kondoi), are important pests in agriculture. Two BGA resistance genes have been identified in the model legume Medicago truncatula , namely AKR (Acyrthosiphon kondoi resistance) and AIN (Acyrthosiphon induced necrosis). In this study, progeny derived from a cross between a resistant accession named Jester and a highly susceptible accession named A20 were used to study the interaction between the AKR and AIN loci with respect to BGA performance and plant response to BGA infestation. These studies demonstrated that AKR and AIN have additive effects on the BGA resistance phenotype. However, AKR exerts dominant suppression epistasis on AIN -controlled macroscopic necrotic lesions. Nevertheless, both AKR and AIN condition production of H2O2 at the BGA feeding site. Electrical penetration graph analysis demonstrated that AKR prevents phloem sap ingestion, irrespective of the presence of AIN. Similarly, the jasmonic acid defense signaling pathway is recruited by AKR , irrespective of AIN. This research identifies an enhancement of aphid resistance through gene stacking, and insights into the interaction of distinct resistance genes against insect pests. [ABSTRACT FROM AUTHOR]

Background: A total of 62,591 cowpea expressed sequence tags (ESTs)were BLAST aligned to the whole-genome sequence of barrel medic (Medicago truncatula) to develop conserved intron scanning primers (CISPs). The efficacy of the primers was tested across 10 different legumes and on different varieties of cowpea, chickpea, and pigeon pea. Genetic diversity was assessed using the same primers on different cowpea genotypes. Singlenucleotide polymorphisms (SNPs) were detected, which were later converted to length polymorphism markers for easy genotyping. CISPs developed in this study were used in tagging resistance to bacterial leaf blight disease in cowpea. Results: A total of 1262 CISPs were designed. The single-copy amplification success rates using these primers on 10 different legumes and on different varieties of cowpea, chickpea, and pigeon pea were approximately 60% inmost of the legumes except soybean (47%) and peanut (37%). Genetic diversity analysis of 35 cowpea genotypes using 179 CISPs revealed 123 polymorphic markers with PIC values ranging from 0.05 to 0.59. Potential SNPs identified in cowpea, chickpea, and pigeon pea were converted to PCR primers of various sizes for easy genotyping. Using the markers developed in this study, a genetic linkage map was constructed with 11 linkage groups in cowpea. QTL mapping with 194 F3 progeny families derived from the cross C-152 × V-16 resulted in the identification of three QTLs for resistance to bacterial leaf blight disease. Conclusions: CISPs were proved to be efficient markers to identify various other marker classes like SNPs through comparative genomic studies in lesser studied crops and to aid in systematic sampling of the entire genome for well-distributed markers at low cost. [ABSTRACT FROM AUTHOR]

Abstract: The infection of the model legume Medicago truncatula with Ralstonia solanacearum GMI1000 gives rise to bacterial wilt disease via colonisation of roots. The root and leaf responses to early infection (1 and 3 days post infection) were characterised to investigate the molecular mechanisms of plant resistance or susceptibility. A proteomics approach based on pools of susceptible and resistant recombinant inbred lines was used to specifically target the mechanisms for tolerance. Differential abundances were evidenced for proteins involved in defence (e.g., PR5, PR10, or Kunitz protease inhibitors) and signalling pathways (such as cyclophilin). R. solanacearum inoculation modifies expression levels of those genes, either in both genotypes (AOS1, LOX4, and proteinase inhibitors) or specifically in the resistant line (PR proteins). Exogenous application of salicylic acid (SA) enhanced tolerance to the bacteria, whereas methyl jasmonate (MeJA) enhanced short‐term tolerance then promoted disease in the susceptible ecotype, suggesting that they may mediate defence responses. Conversely, proteomics‐identified genes were also shown to be SA or MeJA responsive. This is the first description of differential response to R. solanacearum in M. truncatula. Our results suggest that root basal defence is activated at 1 dpi, together with the JA pathway. Specific resistance is then evidenced at three dpi, with the up‐regulation of SA‐dependent PR proteins. [ABSTRACT FROM AUTHOR]

The mainstream pasture legume species such as Trifolium subterraneum, T. repens and annual Medicago spp. used in the temperate pasture systems of southern Australia have high critical external requirements for phosphorus (P) (i.e. P required to achieve 90% of maximum yield). This work aimed to identify alternative pasture legume species that could be used in systems with lower P input. Shoot and root biomass of 12 species of pasture legume was measured in response to seven rates of P applied to the top 48 mm of soil in a pot experiment. Most species had maximum yields similar to T. subterraneum, but some required only one-third of the applied P to achieve this. The critical external P requirement of the species, ranked from lowest to highest, was as follows: Ornithopus compressus = O. sativus < Biserrula pelecinus < T. michelianum = T. vesiculosum = T. glanduliferum < T. hirtum = Medicago truncatula = T. purpureum = T. incarnatum < T. spumosum = T. subterraneum. An ability to maximise soil exploration through a combination of high root-length density, high specific root length and long root hairs (i.e. a large specific root-hair-cylinder volume) was associated with a low critical external P requirement. The results indicate that Ornithopus spp. could be used to achieve productive, low P-input pasture systems. [ABSTRACT FROM AUTHOR]

In this article, we present the response of Medicago truncatula Gaert. cv. Jemalong plants expressing constitutively the Dsp22 gene from Craterostigma plantagineum to water stress and rehydration. The Dsp22 gene encodes an ELIP-like protein thought to protect the chloroplast against photooxidative damage during the dehydration and rehydration. The Dsp22 transgenic homozygous M. truncatula plants showed higher amount of chlorophyll (Chl), lower Chl a/Chl b ratio and higher actual efficiency of energy conversion in photosystem 2 (ΦPSII) after rehydration, when compared to the wild type. The combined data from the Chl a fluorescence analysis, pigment quantification and biomass accumulation showed that transgenic M. truncatula plants are able to recover from water deprivation better than wild type plants.

A small-plot trial with eight Australian barrel medic (Medicago truncatula Gaertn) cultivars was carried out in 2010 at Rimski Šančevi. The average green forage and forage dry matter yields were highest in Jemalong (30.7 t ha-1 and 7.3 t ha-1) and Parabinga (30.7 t ha-1 and 8.0 t ha-1). Forage dry matter crude protein content ranged between 150.8 g kg-1 in Parabinga and 179.4 g kg-1 in Jester. Forage neutral detergent fibre content varied from 305.2 g kg-1 in Sephi to 458.8 g kg-1 in Caliph, while the average forage acid detergent fibre content was 312.8 g kg-1. The average seed yield for all cultivars was 281 kg ha-1 and may be considered satisfying, as it was obtained in a very rainy and warm growing season. Jemalong and Parabinga had the highest aboveground nitrogen yield (190 kg ha-1 and 193 kg ha-1) and thus the greatest potential for green manure.

Resistance to the Australian pea aphid (PA; Acyrthosiphon pisum) biotype in cultivar Jester of the model legume Medicago truncatula is mediated by a single dominant gene and is phloem-mediated. The genetic map position for this resistance gene, APR (Acyrthosiphon pisum resistance), is provided and shows that APR maps 39 centiMorgans (cM) distal of the A. kondoi resistance (AKR) locus, which mediates resistance to a closely related species of the same genus bluegreen aphid (A. kondoi). The APR region on chromosome 3 is dense in classical nucleotide binding site leucine-rich repeats (NLRs) and overlaps with the region harbouring the RAP1 gene which confers resistance to a European PA biotype in the accession Jemalong A17. Further screening of a core collection of M. truncatula accessions identified seven lines with strong resistance to PA. Allelism experiments showed that the single dominant resistance to PA in M. truncatula accessions SA10481 and SA1516 are allelic to SA10733, the donor of the APR locus in cultivar Jester. While it remains unclear whether there are multiple PA resistance genes in an R-gene cluster or the resistance loci identified in the other M. truncatula accessions are allelic to APR, the introgression of APR into current M. truncatula cultivars will provide more durable resistance to PA. [ABSTRACT FROM AUTHOR]

Transposable elements (TEs) are mobile DNA segments, abundant and dynamic in plant genomes. Because their mobility can be potentially deleterious to the host, a variety of mechanisms evolved limiting that negative impact, one of them being preference for a specific target insertion site. Here, we describe a family of Mutator-like DNA transposons in Medicago truncatula targeting TA microsatellites. We identified 218 copies of MuTAnTs and an element carrying a complete ORF encoding a mudrA-like transposase. Most insertion sites are flanked by a variable number of TA tandem repeats, indicating that MuTAnTs are specifically targeting TA microsatellites. Other TE families flanked by TA repeats (e.g. TAFT elements in maize) were described previously, however we identified the first putative autonomous element sharing that characteristics with a related group of short non-autonomous transposons. [ABSTRACT FROM AUTHOR]

A major strigolactone produced by the model legume Medicago truncatula (barrel medic) has been tentatively identified as a didehydro-orobanchol isomer. In this study, a putative didehydro-orobanchol isomer was isolated from root exudates collected from barrel medic grown hydroponically under phosphate-starved conditions. The structure and absolute configurations of this strigolactone, named medicaol, were determined by a combination of spectroscopic analysis and spectral comparison with 4-deoxymedicaol which was synthesized using solvolysis and rearrangement of hydroxymethylhexahydroindenone to tetrahydroazulenone as a key step. Medicaol has a seven-membered cycloheptadiene in the A ring instead of a typical six-membered cyclohexene. Medicaol and 4-deoxymedicaol showed activity comparable to their corresponding six-membered A ring relatives, orobanchol and 4-deoxyorobanchol ( ent -2′- epi -5-deoxystrigol), in inducing hyphal branching of germinating spores of an arbuscular mycorrhizal fungus Gigaspora margarita . Plausible biosynthetic pathways from 4-deoxyorobanchol to medicaol are also proposed. [ABSTRACT FROM AUTHOR]

Resistance to the Australian pea aphid (PA; Acyrthosiphon pisum) biotype in cultivar Jester of the model legume Medicago truncatula is mediated by a single dominant gene and is phloem-mediated. The genetic map position for this resistance gene, APR (Acyrthosiphon pisum resistance), is provided and shows that APR maps 39 centiMorgans (cM) distal of the A. kondoi resistance (AKR) locus, which mediates resistance to a closely related species of the same genus bluegreen aphid (A. kondoi). The APR region on chromosome 3 is dense in classical nucleotide binding site leucine-rich repeats (NLRs) and overlaps with the region harbouring the RAP1 gene which confers resistance to a European PA biotype in the accession Jemalong A17. Further screening of a core collection of M. truncatula accessions identified seven lines with strong resistance to PA. Allelism experiments showed that the single dominant resistance to PA in M. truncatula accessions SA10481 and SA1516 are allelic to SA10733, the donor of the APR locus in cultivar Jester. While it remains unclear whether there are multiple PA resistance genes in an R-gene cluster or the resistance loci identified in the other M. truncatula accessions are allelic to APR, the introgression of APR into current M. truncatula cultivars will provide more durable resistance to PA.

Jasmonate (JA)-mediated defences play important roles in host responses to pathogen attack, in particular to necrotrophic fungal pathogens that kill host cells in order to extract nutrients and live off the dead plant tissue. The root-infecting fungal pathogen Fusarium oxysporum initiates a necrotrophic growth phase towards the later stages of its lifecycle and is responsible for devastating Fusarium wilt disease on numerous legume crops worldwide. Here we describe the use of the model legume Medicago truncatula to study legume–F. oxysporum interactions and compare and contrast this against knowledge from other model pathosystems, in particular Arabidopsis thaliana–F. oxysporum interactions. We describe publically-available genomic, transcriptomic and genetic (mutant) resources developed in M. truncatula that enable dissection of host jasmonate responses and apply aspects of these herein during the M. truncatula-–F. oxysporum interaction. Our initial results suggest not all components of JA-responses observed in M. truncatula are shared with Arabidopsis in response to F. oxysporum infection.

Background Genetic crossing is an essential tool in both forward and reverse genetic approaches to understand the biological functions of genes. For Medicago truncatula (barrel medic) various crossing techniques have been used which differ in the methods used to dissect the female parent's unopened flower bud to remove immature anthers for prevention of self-pollination. Previously described methods including front, side or back incision methods may damage the flower bud, impeding successful fertilization and/or seed development because they may allow pollen to dislodge and floral organs to desiccate after crossing, all of which diminish the success rates of crossing. Results We report the keel petal incision method for genetic crossing in M. truncatula ecotype R108 and demonstrate successful crosses with two other M. truncatula ecotypes, A17 and A20. In the method presented here, an incision is made along the central line of the keel petal from the bottom 1/3rd of the female parent's flower bud to its distal end. This allows easy removal of anthers from the flower bud and access for cross-pollination. After pollination, the stigma and the deposited pollen from the male donor are covered by the keel petal, wing petals and standard petal, forming a natural pouch. The pouch prevents dislodging of deposited pollen from the stigma and protects the internal floral organs from drying out, without using clingfilm or water-containing chambers to maintain a humid environment. The keel petal incision method showed an approximate 80% success rate in the M. truncatula R108 ecotype and also in other ecotypes including Jemalong A17 and A20. Conclusions Our keel petal incision protocol shows marked improvement over existing methods with respect to the ease of crossing and the percentage of successful crosses. Developed for the M. truncatula R108 ecotype, the protocol has been demonstrated with A17 and A20 ecotypes and is expected to work with other ecotypes. Investigators of varying experience have achieved genetic crosses in M. truncatula using this method. [ABSTRACT FROM AUTHOR]

Ascochyta blight caused by Didymella pinodes (formerly Mycosphaerella pinodes) is one of the most important fungal diseases of pea ( Pisum sativum) worldwide that can also infect the model legume Medicago truncatula. The objective of this study was to identify quantitative trait loci (QTLs) controlling resistance to D. pinodes in M. truncatula. Response to D. pinodes was studied under controlled conditions in seedlings of a population derived from the cross J6 × F83005.5, two M. truncatula lines that are, respectively, resistant and susceptible to D. pinodes. A combined map using two different recombinant inbred line populations was then used to identify the genomic regions bearing putative QTLs and to improve the position of the QTLs. A single QTL associated with resistance to D. pinodes was detected on linkage group 2, explaining up to 13 % of the total phenotypic variation for relative disease severity against the pathogen. Two simple sequence repeat markers, MTE80 and mtic890 (3 cM apart) were the ones most significantly associated with the QTL. These markers are located in bacterial artifical chromosomes AC119409 and AC125474, respectively, both of them overlapping on M. truncatula chromosome 2. The integration of QTL analysis and genomics in M. truncatula will contribute to the development of new markers and facilitate the identification of candidate genes for Ascochyta blight resistance. [ABSTRACT FROM AUTHOR]

Abstract: Flavonoids are the most common antioxidant compounds produced in plants. In this study, two wild types and two independent mutants of Medicago truncatula with altered anthocyanin content in leaves were characterized at the phenotype, metabolite profile, gene structure and transcript levels. Flavonoid profiles showed conserved levels of dihydroflavonols, leucoanthocyanidins and flavonols, while anthocyanidin, anthocyanin and isoflavone levels were lower in the mutants (up to 90% less) compared with the wild types. Genes encoding key enzymes of the anthocyanin pathway and transcriptional factors were analyzed by RT-PCR. Genes involved in the later steps of the anthocyanin pathway (dihydrokaempferol reductase 2, UDP-glucose:anthocyanin 3-O-glucosyltransferase and glutathione S-transferase) were found under-expressed in both mutants. Dihydrokaempferol reductase 1 was downregulated two-fold in the anthocyanin-less mutant while the UDP-glucose:anthocyanin 5-O-glucosyltransferase was strongly repressed only in the mutant with low pigmentation, suggesting a different regulation in the two genotypes. The common feature was that the first enzymes of the flavonoid biosynthesis pathway were not altered in rate of expression. A very high reduction in transcript accumulation was also found for two homologous R2R3 MYB genes, namely MtMYBA and AN2, suggesting that these genes have a role in anthocyanin accumulation in leaves. More evidence was found on analyzing their nucleotide sequence: several SNPs, insertions and deletions in the coding and non-coding regions of both MYB genes were found between mutants and wild types that could influence anthocyanin biosynthesis. Moreover, a subfamily of eight MYB genes with a high homology to MtMYBA was discovered in tandem on chromosome 5 of M. truncatula. [Copyright &y& Elsevier]

Leaf explants from leaflets collected from either in vivo grown or in vitro grown seedlings of Medicago truncatula genotype R108-1 were co-cultivated with bacterial cells of Agrobacterium tumefaciens strains EHA105 or C58pMP90. Each of these strains was carrying the pCambia 1390 plasmid harbouring a hygromycin resistance gene cassette. Explants were then incubated on a medium containing 10 mg/l hygromycin and 800 mg/l augmentin to suppress Agrobacterium growth, and subcultured 4-5 times every 2 weeks for the proliferation of calli. After 8-10 weeks, callusing explants were transferred to hormone-free medium with 10 mg/l hygromycin and 400 mg/l augmentin for shoot regeneration. After rooting, a total of about 300 putative transformants were grown into plantlets, transferred to soil, acclimatized, and then moved to the greenhouse. Of these, a total of 43 independent PCR positive primary transformants and their T1 and T2 progeny were subjected to flow cytometric analysis, to assessing their trueness-to-type, as well as to southern blot analysis. [ABSTRACT FROM AUTHOR]

In this article, we present the response of Medicago truncatula Gaert. cv. Jemalong plants expressing constitutively the Dsp22 gene from Craterostigma plantagineum to water stress and rehydration. The Dsp22 gene encodes an ELIP-like protein thought to protect the chloroplast against photooxidative damage during the dehydration and rehydration. The Dsp22 transgenic homozygous M. truncatula plants showed higher amount of chlorophyll (Chl), lower Chl a/Chl b ratio and higher actual efficiency of energy conversion in photosystem 2 (Φ) after rehydration, when compared to the wild type. The combined data from the Chl a fluorescence analysis, pigment quantification and biomass accumulation showed that transgenic M. truncatula plants are able to recover from water deprivation better than wild type plants. [ABSTRACT FROM AUTHOR]

Abstract: The expression profiles of genes involved in DNA repair, namely MtTdp1 (tyrosyl-DNA phosphodiesterase), top1 (DNA topoisomerase I), MtTFIIS (transcription elongation factor II-S) and MtTFIIS-like, were evaluated in Medicago truncatula Gaertn. during seed imbibition carried out with the osmotic agent polyethylene glycol (PEG6000, 100g/L). The use of PEG6000 resulted in delayed water up-take by seeds, and reduced levels of oxidative DNA damage, measured in terms of 7,8-dihydro-8-oxoguanine (8-oxo-dG) were observed compared to seeds imbibed with water. The prolonged exposure to PEG6000 caused an increase in DNA oxidative damage; after 24h of treatment with the osmotic agent, the estimated amount of 8-oxo-dG was 1.25-fold higher compared to the value detected in seeds imbibed with water. Three days after imbibition, consistent cell damage and reactive oxygen species (ROS) production were also detected in radicles emerging from the PEG-treated seeds. All of the tested genes were known to be up-regulated during seed imbibition, with the highest transcript levels accumulating at approximately 8–12h of rehydration. Exposure to PEG6000 caused a delayed up-regulation of MtTdp1α and MtTdp1β genes, with transcript peaks occurring at 12–24h, when the highest levels of DNA damage were also recorded. For the top1, MtTFIIS and MtTFIIS-like genes, different expression profiles were observed in response to PEG6000. The possible roles of these genes in the repair response activated during seed imbibition are discussed. [Copyright &y& Elsevier]

Abstract: 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 1074bp, predicting a 40.0kDa 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 (CuCl2 0.05, 0.1 and 0.2mM) and polyethylene glycol (PEG6000 50, 100 and 150g/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 Cu2+ 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. [Copyright &y& Elsevier]

The Tdp1 gene encoding tyrosyl-DNA phosphodiesterase has been extensively investigated in animal cells, due to the role of this enzyme in the repair of topoisomerase I-DNA covalent lesions. In contrast, information in this regard is totally missing in plants. We report for the first time in plants on the Tdp1 gene family from barrel medic ( Medicago truncatula Gaertn.), composed of two members, hereby named MtTdp1α and MtTdp1β. The expression profiles of MtTdp1α and MtTdp1β genes were evaluated in plantlets grown in vitro using copper and polyethylene glycol (PEG 6000) as stress agents. In situ detection of reactive oxygen species (ROS) was carried out by histochemical staining, while the level of oxidative DNA damage, quantified in terms of 7,8-dihydro-8-oxoguanine (8-oxo-dG), increased up to 7.4- and 6.7-fold in response to copper and PEG 6000 treatments, respectively. Quantitative real-time polymerase chain reaction revealed that both Tdp1 genes were significantly up-regulated in response to copper and PEG. The Tdp1 genes were also significantly up-regulated during seed rehydration, an aspect of seed physiology in which DNA repair is a key component. Thus, the Tdp1 genes might be used as novel tools for improving stress tolerance in crops. The expression patterns of the barrel medic top1α and top1β genes, encoding distinct isoforms of DNA topoisomerase I, were also analyzed and discussed to acquire additional information on their specific functions, closely related to that of the Tdp1 gene in animal cells. [ABSTRACT FROM AUTHOR]

Abstract: Miniature inverted-repeat transposable elements (MITEs) are small and high copy number transposons, related to and mobilized by some class II autonomous elements. New MITE families can be identified by computer-based mining of sequenced genomes. We describe four MITE families related to MtPH transposons mined de novo in the genome of Medicago truncatula, together with one previously described family MITRAV. Different levels of their intra-family sequence diversity and insertion polymorphism indicate that they were active at different evolutionary periods. MetMIT1 and MITRAV families were uniform in sequence and produced highly polymorphic insertion sites in 26 ecotypes representing a M. truncatula core collection. A subset of insertions was present only in the reference genome of A17 ‘Jemalong’, suggesting that the two families might have been active in the course of domestication. In contrast, all investigated insertions of the MetMIT2 family were fixed, showing that it was not active after M. truncatula speciation. MetMIT1 elements were divided into three clusters, i.e. (I) relatively heterogenous copies fixed in the genome of M. truncatula, (II) uniform but also mostly fixed, and (III) uniform and polymorphic among the investigated accessions. It might reflect the evolutionary history of the MetMIT1 family, showing multiple bursts of activity. A number of MetMIT1 and MITRAV insertions were present within 1 kb upstream or downstream the ORF. A high proportion of insertions proximal to coding regions was unique to A17 ‘Jemalong’. [Copyright &y& Elsevier]

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 feeding. They are also inhibitors of conversion of plant material into insect body mass during or after consumption. Growth of beet armyworm, Spodoptera exigua Hübner (Lepidoptera: Noctuidae), larvae was correspondingly greater on calcium oxalate-defective ( cod) mutants of M. truncatula with lower levels of crystal accumulation. Data presented here show that insects feeding on M. truncatula leaves with calcium oxalate crystals experience greater negative effects on growth and mandible wear than those feeding on artificial diet amended with smaller amorphous crystals from commercial preparations. Commercial calcium oxalate can be added to insect artificial diet at levels up to 7.5-fold higher than levels found in wild-type M. truncatula leaves with minimal effect on insect growth or lepidopteran mandibles. These data suggest that negative impacts of calcium oxalate in the diet of larvae are due to physical factors, and not toxicity of the compound, as high levels of the commercial crystals are readily tolerated. In contrast to the dramatic physical effects that M. truncatula-derived crystals have on insect mandibles, we could detect no damage to insect peritrophic gut membranes due to consumption of these crystals. Taken together, the data indicate that the size and shape of prismatic M. truncatula oxalate crystals are important factors in determining effects on insect growth. If manipulation of calcium oxalate is to be used in developing improved insect resistance in plants, then our findings suggest that controlling not only the overall amount, but also the size and shape of crystals, could be valuable traits in selecting desirable plant lines. [ABSTRACT FROM AUTHOR]

Oligonucleotide microarrays corresponding to over 16,000 genes were used to analyze changes in transcript accumulation in root tips of the Al-sensitive Medicago truncatula cultivar Jemalong genotype A17 in response to Al treatment. Out of 2,782 genes with significant changes in transcript accumulation, 324 genes were up-regulated and 267 genes were down-regulated at least twofold by Al. Up-regulated genes were enriched in transcripts involved in cell-wall modification and abiotic and biotic stress responses while down-regulated genes were enriched in transcripts involved in primary metabolism, secondary metabolism, protein synthesis and processing, and the cell cycle. Known markers of Al-induced gene expression including genes associated with oxidative stress and cell wall stiffening were differentially regulated in this study. Transcript profiling identified novel genes associated with processes involved in Al toxicity including cell wall modification, cell cycle arrest and ethylene production. Novel genes potentially associated with Al resistance and tolerance in M. truncatula including organic acid transporters, cell wall loosening enzymes, Ca2+ homeostasis maintaining genes, and Al-binding were also identified. In addition, expression analysis of nine genes in the mature regions of the root revealed that Al-induced gene expression in these regions may play a role in Al tolerance. Finally, interfering RNA-induced silencing of two Al-induced genes, pectin acetylesterase and annexin, in A17 hairy roots slightly increased the sensitivity of A17 to Al suggesting that these genes may play a role in Al resistance. [ABSTRACT FROM AUTHOR]

In this work, we studied the resistance of 277 Medicago truncatula accessions against powdery mildew and further characterized the defense mechanisms of resistant plants. Ten resistant accessions were selected according to macroscopic assessment. Histological studies showed a range of defense mechanisms, acting alone or combined, that impeded fungal development at different stages. Some accessions allowed a reduced spore germination frequency compared with that of the susceptible control. In others, the fungus was arrested at penetration stage due to papilla formation. Epidermal cells of several accessions were penetrated by the fungus but then hypersensitive response (HR) leading to cell death hampered fungal development. In some cases, cell death was very fast and no haustorium could be observed in epidermal cells, whereas in others, haustoria and secondary hyphae indicated a slow HR. Finally, in some accessions in which no HR was observed, colony growth was restricted through posthaustorial defense mechanisms. Characterization of defense mechanisms will be useful for further cellular and molecular studies to unravel the bases of resistance in this species in particular and in legume-powdery mildew interaction in general. [ABSTRACT FROM AUTHOR]

Abstract: An integrated approach utilizing HPLC–UV–ESI–MS and GC–MS was used for the large-scale and systematic identification of polyphenols in Medicago truncatula root and cell culture. Under optimized conditions, we were able to simultaneously quantify and identify 35 polyphenols including 26 isoflavones, 3 flavones, 2 flavanones, 2 aurones and a chalcone. All identifications were based upon UV spectra, mass spectral characteristics of protonated molecules, tandem mass spectral data, mass measurements obtained using a quadrupole time-of-flight mass spectrometer (QtofMS), and confirmed through the co-characterization of authentic compounds. In specific instances where the stereochemistry of sugar conjugates was uncertain, subsequent enzymatic hydrolysis of the conjugate followed by GC–MS was used to assign the sugar stereochemical configuration. Comparative metabolic profiling of Medicago truncatula root and cell cultures was then performed and revealed significant differences in the isoflavonoid composition of these two tissues. [Copyright &y& Elsevier]

Pea albumin 1b (PA1b) is a small sulphur-rich peptide from pea seeds, also called leginsulin due to the binding properties of its soybean orthologue. Its insecticidal properties were discovered more recently. By using a combination of molecular, biochemical and specific insect bioassays, we characterised new genes and their products from the seeds of four legume species. Two species (Glycine max and Phaseolus vulgaris) display most of the characteristics of the Pisum sativum type: homologous genes and predicted toxic hydrophobic peptides with similar post-translational processing. The third species (Medicago truncatula) possesses homologous genes and high insecticidal activity, but no specific biochemical detection of the peptide products was obtained, indicating possible variant post-translational processing. Our combined approach appears to be efficient for a broad study of A1b within legumes. [Copyright &y& Elsevier]

Aphanomyces euteiches causes root rot disease of alfalfa, and two races of the pathogen have been identified based on the reaction of established check cultivars. A majority of alfalfa cultivars grown in regions where A. euteiches is prevalent have resistance to race 1 of the pathogen, but very few cultivars have resistance to race 2. The genetic basis of resistance in alfalfa to A. euteiches is unknown, in part due to characteristics of alfalfa that impede genetic analysis, including its tetraploid genome and susceptibility to inbreeding depression. The barrel medic, Medicago truncatula , has been suggested as a model organism for the study of legume genetics. Our objective was to evaluate plant introduction (PI) accessions of M. truncatula for resistance to A. euteiches race 2. In repeated experiments, employing standardized testing conditions used for evaluating resistance in alfalfa to A. euteiches , we identified seven M. truncatula accessions with levels of resistance similar to the resistant alfalfa check population WAPH-5. Two M. truncatula accessions were identified that were as susceptible as the susceptible alfalfa check cultivar Saranac. The utility of M. truncatula as a model for studying the genetics of resistance in alfalfa to A. euteiches race 2 is discussed. [ABSTRACT FROM AUTHOR]