Jemalong A17 Research Articles

Responses of flavonoids to solar UV radiation and gradual soil drying in two Medicago truncatula accessions

Ground level UV-B (290–315 nm) and UV-A (315–400 nm) radiation regulates multiple aspects of plant growth and development. In a natural environment, UV radiation interacts in a complex manner with other environmental factors (e.g., drought) to regulate plants’ morphology, physiology, and growth. To assess the interactive effects of UV radiation and soil drying on plants’ secondary metabolites and transcript abundance, we performed a field experiment using two different accessions of Medicago truncatula (F83005-5 French origin and Jemalong A17 Australian origin). Plants were grown for 37 days under long-pass filters to assess the effects of UV short wavelength (290–350 nm, UVsw) and UV-A long wavelength (350–400 nm, UV-Alw). Soil–water deficit was induced by not watering half of the plants during the last seven days of the experiment. The two accessions differed in the concentration of flavonoids in the leaf epidermis and in the whole leaf: F83005-5 had higher concentration than Jemalong A17. They also differed in the composition of the flavonoids: a greater number of apigenin derivatives than tricin derivatives in Jemalong A17 and the opposite in F83005-5. Furthermore, UVsw and soil drying interacted positively to regulate the biosynthesis of flavonoids in Jemalong A17 through an increase in transcript abundance of CHALCONE SYNTHASE (CHS). However, in F83005-5, this enhanced CHS transcript abundance was not detected. Taken together the observed metabolite and gene transcript responses suggest differences in mechanisms for acclimation and stress tolerance between the accessions.Graphical abstract

Medicago truncatula resources to study legume biology and symbiotic nitrogen fixation.

Medicago truncatula is a chosen model for legumes towards deciphering fundamental legume biology, especially symbiotic nitrogen fixation. Current genomic resources for M. truncatula include a completed whole genome sequence information for R108 and Jemalong A17 accessions along with the sparse draft genome sequences for other 226 M. truncatula accessions. These genomic resources are complemented by the availability of mutant resources such as retrotransposon (Tnt1) insertion mutants in R108 and fast neutron bombardment (FNB) mutants in A17. In addition, several M. truncatula databases such as small secreted peptides (SSPs) database, transporter protein database, gene expression atlas, proteomic atlas, and metabolite atlas are available to the research community. This review describes these resources and provide information regarding how to access these resources.

A newly evolved chimeric lysin motif receptor-like kinase in Medicago truncatula spp. tricycla R108 extends its Rhizobia symbiotic partnership.

Rhizobial lipochitooligosaccharidic Nod factors (NFs), specified by nod genes, are the primary determinants of host specificity in the legume-Rhizobia symbiosis. We examined the nodulation ability of Medicago truncatula cv Jemalong A17 and M. truncatula ssp. tricycla R108 with the Sinorhizobium meliloti nodF/nodL mutant, which produces modified NFs. We then applied genetic and functional approaches to study the genetic basis and mechanism of nodulation of R108 by this mutant. We show that the nodF/nodL mutant can nodulate R108 but not A17. Using genomics and reverse genetics, we identified a newly evolved, chimeric LysM receptor-like kinase gene in R108, LYK2bis, which is responsible for the phenotype and can allow A17 to gain nodulation with the nodF/nodL mutant. We found that LYK2bis is involved in nodulation by mutants producing nonO-acetylated NFs and interacts with the key receptor protein NFP. Many, but not all, natural S. meliloti and S. medicae strains tested require LYK2bis for efficient nodulation of R108. Our findings reveal that a newly evolved gene in R108, LYK2bis, extends nodulation specificity to mutants producing nonO-acetylated NFs and is important for nodulation by many natural Sinorhizobia. Evolution of this gene may present an adaptive advantage to allow nodulation by a greater variety of strains.

Contrasting effects of the inoculation time with passenger endophytic Agrobacterium sp.10C2 on the nodule functioning and growth of Medicago truncatula

Agrobacterium species are one of the most frequently isolated endophytes from root nodules of a wide range of wild and cultivated legumes. Many studies have investigated the role of endophytes in phytoprotection and phytoremediation enhancement. However, their biological significance and their implication in nodule functioning have been scarcely studied, especially, the effect of the inoculation time on nodule activity and plant growth which still unknown. Accordingly, in this study, nodule-endophytic Agrobacterium sp.10C2 was inoculated before and after nodule formation, to determine the effect of the inoculation time on the plant growth and nodule functioning in the reference line Jemalong A17 of the model legume Medicago truncatula in symbiotic association with Sinorhizobium meliloti TII7. The results revealed that nodule functioning was strongly influenced by the inoculation time of the endophytic Agrobacterium sp.10C2. In fact, the early inoculation with Agrobacterium sp.10C2 induced an increase of the highest nodules nitrogenase activity, recorded at the day 66 after the inoculation, by 66%, the leghemoglobin concentration by 26% and the total sugar content by 67%, in addition to a higher photosynthesis activity and biomass plant production compared to the control. Interestingly, when inoculation occurred after nodule formation, nodules have induced a defense response against Agrobacterium sp. 10C2 evidenced by an overproduction of guaicol peroxidase which increased from 1.04 μmol H2O2 min-1 mg-1 proteins in the control to 2.36 μmol H2O2 min-1 mg-1 proteins in the late inoculated plants, leading to a loss of the beneficial impact of the endophytic Agrobacterium on both biomass production and nodule functioning parameters. We showed that nodule response to endophytic agrobacteria change according to the time of inoculation. When inoculated at early stage, 10C2 plays the role of a Plant Growth Promoting Rhizobacteria (PGPR), however, after nodule formation, 10C2 could act as a pathogen agent. Thus, the inoculation time could be considered as a crucial parameter for the nodule functioning and plant growth.

Comparison of structural variants in the whole genome sequences of two Medicago truncatula ecotypes: Jemalong A17 and R108

BackgroundStructural variants (SVs) constitute a large proportion of the genomic variation that results in phenotypic variation in plants. However, they are still a largely unexplored feature in most plant genomes. Here, we present the whole-genome landscape of SVs between two model legume Medicago truncatula ecotypes–Jemalong A17 and R108– that have been extensively used in various legume biology studies.ResultsTo catalogue SVs, we first resolved the previously published R108 genome assembly (R108 v1.0) to chromosome-scale using 124 × Hi-C data, resulting in a high-quality genome assembly. The inter-chromosomal reciprocal translocations between chromosomes 4 and 8 were confirmed by performing syntenic analysis between the two genomes. Combined with the Hi-C data, it appears that these translocation events had a significant effect on chromatin organization. Using both whole-genome and short-read alignments, we identified the genomic landscape of SVs between the two genomes, some of which may account for several phenotypic differences, including their differential responses to aluminum toxicity and iron deficiency, and the development of different anthocyanin leaf markings. We also found extensive SVs within the nodule-specific cysteine-rich gene family which encodes antimicrobial peptides essential for terminal bacteroid differentiation during nitrogen-fixing symbiosis.ConclusionsOur results provide a near-complete R108 genome assembly and the first genomic landscape of SVs obtained by comparing two M. truncatula ecotypes. This may provide valuable genomic resources for the functional and molecular research of legume biology in the future.

Sinorhizobium medicae WSM419 Genes That Improve Symbiosis between Sinorhizobium meliloti Rm1021 and Medicago truncatula Jemalong A17 and in Other Symbiosis Systems.

Some soil bacteria, called rhizobia, can interact symbiotically with legumes, in which they form nodules on the plant roots, where they can reduce atmospheric dinitrogen to ammonia, a form of nitrogen that can be used by growing plants. Rhizobium-plant combinations can differ in how successful this symbiosis is: for example, Sinorhizobium meliloti Rm1021 forms a relatively ineffective symbiosis with Medicago truncatula Jemalong A17, but Sinorhizobium medicae WSM419 is able to support more vigorous plant growth. Using proteomic data from free-living and symbiotic S. medicae WSM419, we previously identified a subset of proteins that were not closely related to any S. meliloti Rm1021 proteins and speculated that adding one or more of these proteins to S. meliloti Rm1021 would increase its effectiveness on M. truncatula A17. Three genes, Smed_3503, Smed_5985, and Smed_6456, were cloned into S. meliloti Rm1021 downstream of the E. coli lacZ promoter. Strains with these genes increased nodulation and improved plant growth, individually and in combination with one another. Smed_3503, renamed iseA (increased symbiotic effectiveness), had the largest impact, increasing M. truncatula biomass by 61%. iseA homologs were present in all currently sequenced S. medicae strains but were infrequent in other Sinorhizobium isolates. Rhizobium leguminosarum bv. viciae 3841 containing iseA led to more nodules on pea and lentil. Split-root experiments with M. truncatula A17 indicated that S. meliloti Rm1021 carrying the S. medicae iseA is less sensitive to plant-induced resistance to rhizobial infection, suggesting an interaction with the plant's regulation of nodule formation. IMPORTANCE Legume symbiosis with rhizobia is highly specific. Rhizobia that can nodulate and fix nitrogen on one legume species are often unable to associate with a different species. The interaction can be more subtle. Symbiotically enhanced growth of the host plant can differ substantially when nodules are formed by different rhizobial isolates of a species, much like disease severity can differ when conspecific isolates of pathogenic bacteria infect different cultivars. Much is known about bacterial genes essential for a productive symbiosis, but less is understood about genes that marginally improve performance. We used a proteomic strategy to identify Sinorhizobium genes that contribute to plant growth differences that are seen when two different strains nodulate M. truncatula A17. These genes could also alter the symbiosis between R. leguminosarum bv. viciae 3841 and pea or lentil, suggesting that this approach identifies new genes that may more generally contribute to symbiotic productivity.

Delineating the Tnt1 Insertion Landscape of the Model Legume Medicago truncatula cv. R108 at the Hi-C Resolution Using a Chromosome-Length Genome Assembly.

Legumes are of great interest for sustainable agricultural production as they fix atmospheric nitrogen to improve the soil. Medicago truncatula is a well-established model legume, and extensive studies in fundamental molecular, physiological, and developmental biology have been undertaken to translate into trait improvements in economically important legume crops worldwide. However, M. truncatula reference genome was generated in the accession Jemalong A17, which is highly recalcitrant to transformation. M. truncatula R108 is more attractive for genetic studies due to its high transformation efficiency and Tnt1-insertion population resource for functional genomics. The need to perform accurate synteny analysis and comprehensive genome-scale comparisons necessitates a chromosome-length genome assembly for M. truncatula cv. R108. Here, we performed in situ Hi-C (48×) to anchor, order, orient scaffolds, and correct misjoins of contigs in a previously published genome assembly (R108 v1.0), resulting in an improved genome assembly containing eight chromosome-length scaffolds that span 97.62% of the sequenced bases in the input assembly. The long-range physical information data generated using Hi-C allowed us to obtain a chromosome-length ordering of the genome assembly, better validate previous draft misjoins, and provide further insights accurately predicting synteny between A17 and R108 regions corresponding to the known chromosome 4/8 translocation. Furthermore, mapping the Tnt1 insertion landscape on this reference assembly presents an important resource for M. truncatula functional genomics by supporting efficient mutant gene identification in Tnt1 insertion lines. Our data provide a much-needed foundational resource that supports functional and molecular research into the Leguminosae for sustainable agriculture and feeding the future.

Involvement of Polyamine Metabolism in the Response of Medicago truncatula Genotypes to Salt Stress.

Salinity constitutes one of the most important causes leading to severe reduction in plant yield. Several reports correlate the accumulation of polyamines in plants with tolerance to abiotic stress cues. The present study examined three Medicago truncatula genotypes with differing sensitivities to salinity (TN1.11, tolerant; Jemalong A17, moderately sensitive; TN6.18, sensitive), with the aim of examining the genotype-specific involvement of the polyamine metabolic pathway in plant response to salinity. The study was carried out with leaves harvested 48 h after watering plants with 200 mM NaCl. A comprehensive profile of free polyamines was determined using high performance liquid chromatography. All genotypes showed spermidine and spermine as the most abundant polyamines under control conditions. In salinity conditions, spermine levels increased at the expense of putrescine and spermidine, indicating a drift of polyamine metabolism towards the synthesis of increasing polycationic forms as a stress response. The increasing balance between high and low polycationic forms was clearly diminished in the salt-sensitive genotype TN6.18, showing a clear correlation with its sensitive phenotype. The polyamine metabolic profile was then supported by molecular evidence through the examination of polyamine metabolism transcript levels by RT-qPCR. General suppression of genes that are involved upstream in the PA biosynthetic pathway was determined. Contrarily, an induction in the expression of genes involved in the biosynthesis of spermine and spermidine was observed, in agreement with the metabolic analysis. A significant induction in diamino oxidase expression, involved in the catabolism of putrescine, was specifically found in the sensitive genotype ΤΝ6.18, indicating a distinct metabolic response to stress. Present findings highlight the involvement of polyamines in the defense response of Medicago genotypes showing sensitivity to salt stress.

Systems biology reveals key tissue-specific metabolic and transcriptional signatures involved in the response of Medicago truncatula plant genotypes to salt stress

Salt stress is an important factor limiting plant productivity by affecting plant physiology and metabolism. To explore salt tolerance adaptive mechanisms in the model legume Medicago truncatula, we used three genotypes with differential salt-sensitivity: TN6.18 (highly sensitive), Jemalong A17 (moderately sensitive), and TN1.11 (tolerant). Cellular damage was monitored in roots and leaves 48 h after 200 mM NaCl treatment by measuring lipid peroxidation, nitric oxide, and hydrogen peroxide contents, further supported by leaf stomatal conductance and chlorophyll readings. The salt-tolerant genotype TN1.11 displayed the lowest level of oxidative damage, in contrast to the salt sensitive TN6.18, which showed the highest responses. Metabolite profiling was employed to explore the differential genotype-related responses to stress at the molecular level. The metabolic data in the salt tolerant TN1.11 roots revealed an accumulation of metabolites related to the raffinose pathway. To further investigate the sensitivity to salinity, global transcriptomic profiling using microarray analysis was carried out on the salt-stressed sensitive genotypes. In TN6.18, the transcriptomic analysis identified a lower expression of many genes related to stress signalling, not previously linked to salinity, and corresponding to the TIR-NBS-LRR gene class. Overall, this global approach contributes to gaining significant new insights into the complexity of stress adaptive mechanisms and to the identification of potential targets for crop improvement.

The alternative oxidase pathway is involved in optimizing photosynthesis inMedicago truncatulainfected byFusarium oxysporumandRhizoctonia solani

Phytopathogen infection alters primary metabolism status and plant development. The alternative oxidase (AOX) has been hypothesized to increase under pathogen attack preventing reductions, thus optimizing photosynthesis and growth. In this study, two genotypes of Medicago truncatula, one relatively resistant (Jemalong A17) and one susceptible (TN1.11), were infected with Fusarium oxysporum and Rhizoctonia solani. The in vivo foliar respiratory activities of the cytochrome oxidase pathway (COP) and the alternative oxidase pathway (AOP) were measured using the oxygen isotope fractionation. Gas exchange and photosynthesis-related parameters were measured and calculated together with antioxidant enzymes activities and organic acids contents. Our results show that the in vivo activity of AOX (valt ) plays a role under fungal infection. When infected with R. solani, the increase of valt in A17 was concomitant to an increase in net assimilation, in mesophyll conductance, to an improvement in the maximum velocity of Rubisco carboxylation and to unchanged malate content. However, under F. oxysporum infection, the induced valt was accompanied by an enhancement in the antioxidant enzymes, superoxide dismutase (SOD; EC1.15.1.1), catalase (CAT; EC1.11.1.6) and guaiacol peroxidase (GPX; EC1.11.1.7), activities and to an unchanged tricarboxylic acid cycle intermediates. These results provide new insight into the role of the in vivo activity of AOX in coordinating primary metabolism interactions that, partly, modulate the relative resistance of M. truncatula to diseases caused by soil-borne pathogenic fungi.

Identification of tissue-specific and cold-responsive lncRNAs in Medicago truncatula by high-throughput RNA sequencing

BackgroundLong non-coding RNAs (lncRNAs) play important roles in the regulation of plant responses to environmental stress by acting as essential regulators of gene expression. However, whether and how lncRNAs are involved in cold acclimation-dependent freezing tolerance in plants remains largely unknown. Medicago truncatula is a prominent model for studies of legume genomics, and distinguished by its cold-acclimation characteristics. To determine the roles of lncRNAs in plant cold stress response, we conducted genome-wide high-throughput sequencing in the legume model plant M. truncatula.ResultsRNA-seq data were generated from twelve samples for the four treatments, i.e., non-cold treated leaves and roots, cold-treated leaves and roots of M. truncatula Jemalong A17 seedlings. A total of 1204 million raw reads were generated. Of them, 1150 million filtered reads after quality control (QC) were subjected to downstream analysis. A large number of 24,368 unique lncRNAs were identified from the twelve samples. Among these lncRNAs, 983 and 1288 were responsive to cold treatment in the leaves and roots, respectively. We further found that the intronic-lncRNAs were most sensitive to the cold treatment. The cold-responsive lncRNAs were unevenly distributed across the eight chromosomes in M. truncatula seedlings with obvious preferences for locations. Further analyses revealed that the cold-responsive lncRNAs differed between leaves and roots. The putative target genes of the lncRNAs were predicted to mainly involve the processes of protein translation, transport, metabolism and nucleic acid transcription. Furthermore, the networks of a tandem array of CBF/DREB1 genes that were reported to be located in a major freezing tolerance QTL region on chromosome 6 and their related lncRNAs were dissected based on their gene expression and chromosome location.ConclusionsWe identified a comprehensive set of lncRNAs that were responsive to cold treatment in M. truncatula seedlings, and discovered tissue-specific cold-responsive lncRNAs in leaves and roots. We further dissected potential regulatory networks of CBF Intergenic RNA (MtCIR1) and MtCBFs that play critical roles in response and adaptation of M. truncatula to cold stress.

Sinorhizobium spp inoculation alleviates the effect of Fusarium oxysporum on Medicago truncatula plants by increasing antioxidant capacity and sucrose accumulation

In this study, the effect of the phyto-pathogenic fungus Fusarium oxysporum on Medicago truncatula-Sinorhizobium symbiosis performance was investigated. Fusarium oxysporum (KLR13) was inoculated on two genotypes of M. truncatula, one tolerant (Jemalong A17) and one susceptible (TN1.11), in symbiosis with Sinorhizobium meliloti (TII7), Sinorhizobium medicae (SII4) or fertilized with KNO3. Fungus infection reduced significantly biomass production and photosynthetic related parameters of non-symbiotic M. truncatula A17 and TN1.11. Reduction was less pronounced in A17, where a lower accumulation of H2O2 and lipid peroxidation (MDA) in concomitance to high activities of antioxidant enzymes, catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD) was detected. In symbiosis, the fungus affected the nodule number and weight and inhibited the nitrogen fixing capacity (NFC), as well as the fructose and glucose contents. Nevertheless, inoculation with S. meliloti and S. medicae alleviated the effect of F. oxysporum on plant biomass production compared to the KNO3 fertilized plants. Moreover, the Sinorhizobium inoculation decreased H2O2 and MDA over-production caused by the fungal infection in leaves of both genotypes. This tolerance enhancing effect was more pronounced in S. medicae-TN1.11 symbiosis, where the relative maintenance of photosynthesis related parameters, growth capacity and NFC performance was concomitant to the induction of SOD and CAT activities and an increase of the sucrose content in leaves. For A17 genotype, both Sinorhizobium strains induced an increase of the proline and sucrose accumulation. In conclusion, F. oxysporum effect on M. truncatula was mitigated when plants were in symbiosis with Sinorhizobium species, mainly Sinorhizobium medicae that alleviated the oxidative stress and enhanced the accumulation of sucrose as energy source in TN1.11 genotype, which attenuates its susceptibility to F. oxysporum infection.

Drought stress stimulates endocytosis and modifies membrane lipid order of rhizodermal cells of Medicago truncatula in a genotype-dependent manner

BackgroundDrought stress negatively affects plant growth and productivity. Plants sense soil drought at the root level but the underlying mechanisms remain unclear. At the cell level, we aim to reveal the short-term root perception of drought stress through membrane dynamics.ResultsIn our study, 15 Medicago truncatula accessions were exposed to a polyethylene glycol (PEG)-induced drought stress, leading to contrasted ecophysiological responses, in particular related to root architecture plasticity. In the reference accession Jemalong A17, identified as drought susceptible, we analyzed lateral roots by imaging of membrane-localized fluorescent probes using confocal microscopy. We found that PEG stimulated endocytosis especially in cells belonging to the growth differentiation zone (GDZ). The mapping of membrane lipid order in cells along the root apex showed that membranes of root cap cells were more ordered than those of more differentiated cells. Moreover, PEG triggered a significant increase in membrane lipid order of rhizodermal cells from the GDZ. We initiated the membrane analysis in the drought resistant accession HM298, which did not reveal such membrane modifications in response to PEG.ConclusionsOur data demonstrated that the plasma membranes of root cells from a susceptible genotype perceived drought stress by modulating their physical state both via a stimulation of endocytosis and a modification of the degree of lipid order, which could be proposed as mechanisms required for signal transduction.

Independent Regulation of Symbiotic Nodulation by the SUNN Negative and CRA2 Positive Systemic Pathways

Plant systemic signaling pathways allow the integration and coordination of shoot and root organ metabolism and development at the whole-plant level depending on nutrient availability. In legumes, two systemic pathways have been reported in the Medicago truncatula model to regulate root nitrogen-fixing symbiotic nodulation. Both pathways involve leucine-rich repeat receptor-like kinases acting in shoots and proposed to perceive signaling peptides produced in roots depending on soil nutrient availability. In this study, we characterized in the M. truncatula Jemalong A17 genotype a mutant allelic series affecting the Compact Root Architecture2 (CRA2) receptor. These analyses revealed that this pathway acts systemically from shoots to positively regulate nodulation and is required for the activity of carboxyl-terminally encoded peptides (CEPs). In addition, we generated a double mutant to test genetic interactions of the CRA2 systemic pathway with the CLAVATA3/EMBRYO SURROUNDING REGION peptide (CLE)/Super Numeric Nodule (SUNN) receptor systemic pathway negatively regulating nodule number from shoots, which revealed an intermediate nodule number phenotype close to the wild type. Finally, we showed that the nitrate inhibition of nodule numbers was observed in cra2 mutants but not in sunn and cra2 sunn mutants. Overall, these results suggest that CEP/CRA2 and CLE/SUNN systemic pathways act independently from shoots to regulate nodule numbers.

Physical Mutagenesis in Medicago truncatula Using Fast Neutron Bombardment (FNB) for Symbiosis and Developmental Biology Studies.

Medicago truncatula has been selected as a model species for legume molecular genetics and functional genomics studies. With the completion of the Medicago truncatula cv. Jemalong A17 genome sequencing, a major challenge is to determine the function of the large number of genes in the genome. Development of diverse mutant resources is crucial for gene functional studies. In the past years, M2 seeds from over 150,000 Medicago truncatula mutant lines in the Jemalong A17 background have been generated coordinately at the Noble Research Institute, USA, and the John Innes Centre, UK, using fast neutron bombardment (FNB) mutagenesis. These mutant resources have been used in screening and characterization of different categories of mutants including symbiotic nitrogen fixation, nodule development, and growth and patterning of leaf, stem, and root system architecture in the legume system. Here, we describe the detail procedure that has been used for screening of mutants derived from fast neutron bombardment mutagenesis in Medicago truncatula.

Nodule-Specific Cysteine-Rich Peptides Negatively Regulate Nitrogen-Fixing Symbiosis in a Strain-Specific Manner inMedicago truncatula

Medicago truncatula shows a high level of specificity when interacting with its symbiotic partner Sinorhizobium meliloti. This specificity is mainly manifested at the nitrogen-fixing stage of nodule development, such that a particular bacterial strain forms nitrogen-fixing nodules (Nod+/Fix+) on one plant genotype but ineffective nodules (Nod+/Fix-) on another. Recent studies have just begun to reveal the underlying molecular mechanisms that control this specificity. The S. meliloti strain A145 induces the formation of Fix+ nodules on the accession DZA315.16 but Fix- nodules on Jemalong A17. A previous study reported that the formation of Fix- nodules on Jemalong A17 by S. meliloti A145 was conditioned by a single recessive allele named Mtsym6. Here we demonstrate that the specificity associated with S. meliloti A145 is controlled by multiple genes in M. truncatula, including NFS1 and NFS2 that encode nodule-specific cysteine-rich (NCR) peptides. The two NCR peptides acted dominantly to block rather than promote nitrogen fixation by S. meliloti A145. These two NCR peptides are the same ones that negatively regulate nitrogen-fixing symbiosis associated with S. meliloti Rm41.

Is there genetic variation in mycorrhization of Medicago truncatula?

Differences in the plant’s response among ecotypes or accessions are often used to identify molecular markers for the respective process. In order to analyze genetic diversity of Medicago truncatula in respect to interaction with the arbuscular mycorrhizal (AM) fungus Rhizophagus irregularis, mycorrhizal colonization was evaluated in 32 lines of the nested core collection representing the genetic diversity of the SARDI collection. All studied lines and the reference line Jemalong A17 were inoculated with R. irregularis and the mycorrhization rate was determined at three time points after inoculation. There were, however, no reliable and consistent differences in mycorrhization rates among all lines. To circumvent possible overlay of potential differences by use of the highly effective inoculum, native sandy soil was used in an independent experiment. Here, significant differences in mycorrhization rates among few of the lines were detectable, but the overall high variability in the mycorrhization rate hindered clear conclusions. To narrow down the number of lines to be tested in more detail, root system architecture (RSA) of in vitro-grown seedlings of all lines under two different phosphate (Pi) supply condition was determined in terms of primary root length and number of lateral roots. Under high Pi supply (100 µM), only minor differences were observed, whereas in response to Pi-limitation (3 µM) several lines exhibited a drastically changed number of lateral roots. Five lines showing the highest alterations or deviations in RSA were selected and inoculated with R. irregularis using two different Pi-fertilization regimes with either 13 mM or 3 mM Pi. Mycorrhization rate of these lines was checked in detail by molecular markers, such as transcript levels of RiTubulin and MtPT4. Under high phosphate supply, the ecotypes L000368 and L000555 exhibited slightly increased fungal colonization and more functional arbuscules, respectively. To address the question, whether capability for mycorrhizal colonization might be correlated to general invasion by microorganisms, selected lines were checked for infection by the root rot causing pathogen, Aphanoymces euteiches. The mycorrhizal colonization phenotype, however, did not correlate with the resistance phenotype upon infection with two strains of A. euteiches as L000368 showed partial resistance and L000555 exhibited high susceptibility as determined by quantification of A. euteiches rRNA within infected roots. Although there is genetic diversity in respect to pathogen infection, genetic diversity in mycorrhizal colonization of M. truncatula is rather low and it will be rather difficult to use it as a trait to access genetic markers.

Polymorphic responses of Medicago truncatula accessions to potassium deprivation

ABSTRACTPotassium (K+) is an essential macronutrient for plants and the most abundant cation in cells. Due to variable K+ availability in the environment, plants must be able to adjust their developmental, physiological and transcriptional responses. The plant development to K+ deprivation was not well studied in legumes thus far. We recently described the first adaptation mechanisms of the model legume Medicago truncatula Jemalong A17 to long-term K+ deprivation and analyzed these responses in the context of arbuscular mycorrhizal symbiosis. Here we report polymorphic growth variations of two genetically very different accessions of M. truncatula to K+-limiting conditions, Jemalong A17, and the Tunisian accession Tn11.1. The faster adaptation of Tn11.1 than A17 to K+ shortage might be due to its greater adaptation to saline soils. Examining in a more systematic way the developmental adaptation of various M. truncatula accessions to K+ deprivation will provide a better understanding of how legume evolved to cope with this stressful condition.

Impact of Hypoxia on Nodulation and Growth of the Legume Plant Medicago truncatula in Symbiosis with Sinorhizobium meliloti

Legumes form a symbiotic interaction with bacteria of the Rhizobiaceae family to develop nitrogen-fixing root nodules under nitrogen-limiting conditions. The present study aimed at analysing the impact of two- and four-week long periods of hypoxia on the nodulation and the growth of the legume plant Medicago truncatula (var Jemalong A17) in symbiosis with Sinorhizobium meliloti. Results showed that the nodulation was not affected by moderate hypoxia (4.5% O2), but the number of nodules decreases for high hypoxia (0.1% O2) and anoxia. The analysis of growth parameters (fresh and dry weight, leaf area and thickness, chlorophyll contents) and of various metabolite contents (total sugars, chlorophylls, inorganic nitrogen compounds) in the leaves, roots and nodules showed that nodulated Medicago truncatula tolerates periods of 4 weeks hypoxia and may be considered as a hypoxia-tolerant legume plant.

Medicago truncatula genotypes Jemalong A17 and R108 show contrasting variations under drought stress

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.