Truncatula Seedlings Research Articles

Jarin-1, an inhibitor of JA-Ile biosynthesis in Arabidopsis thaliana, acts differently in other plant species

ABSTRACT Jasmonates (JAs), including jasmonic acid (JA) and its biologically active derivative JA-Ile, are lipid-derived plant signaling molecules. They govern plant responses to stresses, such as wounding and insect herbivory. Wounding elicits a rapid increase of JA and JA-Ile levels as well as the expression of JAR1, coding for the enzyme involved in JA-Ile biosynthesis. Endogenous increase and application of JAs, such as MeJA, a JA methylester, result in increased defense levels, often accompanied by diminished growth. A JA-Ile biosynthesis inhibitor, jarin-1, was shown to exclusively inhibit the JA-conjugating enzyme JAR1 in Arabidopsis thaliana. To investigate whether jarin-1 does function similarly in other plants, we tested this in Medicago truncatula, Solanum lycopersicum, and Brassica nigra seedlings in a root growth inhibition assay. Application of jarin-1 alleviated the inhibition of root growth after MeJA application in M. truncatula seedlings, proving that jarin-1 is biologically active in M. truncatula. Jarin-1 did not show, however, a similar effect in S. lycopersicum and B. nigra seedlings treated with MeJA. Even JA-Ile levels were not affected by application of jarin-1 in wounded leaf disks from S. lycopersicum. Based on these results, we conclude that the effect of jarin-1 is highly species-specific. Researchers intending to use jarin-1 for studying the function of JAR1 or JA-Ile in their model plants, must test its functionality before use.

Translational Landscape of Medicago truncatula Seedlings under Salt Stress.

The expression of plant genes under salt stress at the transcriptional level has been extensively studied. However, less attention has been paid to gene translation regulation under salt stress. In this study, Ribo-seq and RNA-seq analyses were conducted in Medicago truncatula seedlings grown under normal and salt stress conditions. The results showed that salt stress significantly altered the gene expression at the transcriptional and translational levels, with 2755 genes showing significant changes only at the translational level. Salt stress significantly inhibited the gene translation efficiency. Small ORFs (including uORFs in the 5'UTR, dORFs in 3'UTRs, and sORFs in lncRNAs) were identified throughout the genome of M. truncatula. The efficiency of gene translation was simultaneously regulated by the uORFs, dORFs, and miRNAs. In summary, our results provide valuable information about translatomic resources and new insights into plant responses to salt stress.

Oxidative Status of Medicago truncatula Seedlings after Inoculation with Rhizobacteria of the Genus Pseudomonas, Paenibacillus and Sinorhizobium

An increasing number of scientists working to raise agricultural productivity see the potential in the roots and the soil adjacent to them, together with a wealth of micro-organisms. The first mechanisms activated in the plant during any abiotic or biotic stress concern changes in the oxidative status of the plant. With this in mind, for the first time, an attempt was made to check whether the inoculation of seedlings of the model plant Medicago truncatula with rhizobacteria belonging to the genus Pseudomonas (P. brassicacearum KK5, P. corrugata KK7), Paenibacillus borealis KK4 and a symbiotic strain Sinorhizobium meliloti KK13 would change the oxidative status in the days following inoculation. Initially, an increase in H2O2 synthesis was observed, which led to an increase in the activity of antioxidant enzymes responsible for regulating hydrogen peroxide levels. The main enzyme involved in the reduction of H2O2 content in the roots was catalase. The observed changes indicate the possibility of using the applied rhizobacteria to induce processes related to plant resistance and thus to ensure protection against environmental stress factors. In the next stages, it seems reasonable to check whether the initial changes in the oxidative state affect the activation of other pathways related to plant immunity.

Effects of autotoxicity and allelopathy on seed germination and seedling growth in Medicago truncatula.

Autotoxicity is a form of intraspecific allelopathy, in which a plant species inhibits the establishment or growth of the same species through the release of toxic chemical compounds into the environment. The phenomenon of autotoxicity in crops is best traced in alfalfa (Medicago sativa). A close relative of alfalfa, M. truncatula, has been developed into an excellent model species for leguminous plants. However, it is not known whether M. truncatula has autotoxicity. In this study, M. truncatula root exudates showed a negative impact on the growth of M. truncatula seedlings, indicating autotoxicity. Detailed analyses with plant extracts from M. truncatula and alfalfa revealed varying degrees of suppression effects in the two species. The extracts negatively affected seed germination potential, germination rate, radicle length, hypocotyl length, synthetic allelopathic effect index, plant height, root growth, fresh weight, dry weight, net photosynthetic rate, transpiration rate, and stomatal conductance in both M. truncatula and alfalfa. The results demonstrated that autotoxicity and allelopathic effects exist in M. truncatula. This opens up a new way to use M. truncatula as a model species to carry out in-depth studies of autotoxicity and allelopathy to elucidate biochemical pathways of allelochemicals and molecular networks controlling biosynthesis of the chemicals.

Genome-Wide Association Study Reveals Complex Genetic Architecture of Cadmium and Mercury Accumulation and Tolerance Traits in Medicago truncatula.

Heavy metals are an increasing problem due to contamination from human sources that and can enter the food chain by being taken up by plants. Understanding the genetic basis of accumulation and tolerance in plants is important for reducing the uptake of toxic metals in crops and crop relatives, as well as for removing heavy metals from soils by means of phytoremediation. Following exposure of Medicago truncatula seedlings to cadmium (Cd) and mercury (Hg), we conducted a genome-wide association study using relative root growth (RRG) and leaf accumulation measurements. Cd and Hg accumulation and RRG had heritability ranging 0.44 – 0.72 indicating high genetic diversity for these traits. The Cd and Hg trait associations were broadly distributed throughout the genome, indicated the traits are polygenic and involve several quantitative loci. For all traits, candidate genes included several membrane associated ATP-binding cassette transporters, P-type ATPase transporters, oxidative stress response genes, and stress related UDP-glycosyltransferases. The P-type ATPase transporters and ATP-binding cassette protein-families have roles in vacuole transport of heavy metals, and our findings support their wide use in physiological plant responses to heavy metals and abiotic stresses. We also found associations between Cd RRG with the genes CAX3 and PDR3, two linked adjacent genes, and leaf accumulation of Hg associated with the genes NRAMP6 and CAX9. When plant genotypes with the most extreme phenotypes were compared, we found significant divergence in genomic regions using population genomics methods that contained metal transport and stress response gene ontologies. Several of these genomic regions show high linkage disequilibrium (LD) among candidate genes suggesting they have evolved together. Minor allele frequency (MAF) and effect size of the most significant SNPs was negatively correlated with large effect alleles being most rare. This is consistent with purifying selection against alleles that increase toxicity and abiotic stress. Conversely, the alleles with large affect that had higher frequencies that were associated with the exclusion of Cd and Hg. Overall, macroevolutionary conservation of heavy metal and stress response genes is important for improvement of forage crops by harnessing wild genetic variants in gene banks such as the Medicago HapMap collection.

Bacteria-derived N,N-dimethylhexadecylamine modulates the endophytic microbiome of Medicago truncatula in vitro

Beneficial rhizobacteria such as Arthrobacter agilis and Pseudomonas fluorescens produce N,N-dimethylhexadecylamine (DMHDA), a volatile organic compound that acts as a plant-microorganism signal, inducing bacterial motility and plant defense mechanisms. In the present study, Medicago truncatula seedlings were cultured in vitro and inoculated with the microbial fraction of soil with or without DMHDA, and DNA from Actinobacteria, Firmicutes, and α, β, and γ-proteobacteria were quantified using quantitative polymerase chain reaction to obtain a bacterial community profile of root and shoot endophytic compartments. In seedlings cultured without DMHDA, β-proteobacteria and α-proteobacteria were more abundant in root and shoot endophytic compartments, respectively. However, when seedlings were cultured with DMHDA, the proportion of Actinobacteria and Firmicutes increased in both compartments. These changes were dependent on the dose of DMHDA. We propose that DMHDA modulates the endophytic microbiome by inducing plant immune responses.

Exploring microRNA Signatures of DNA Damage Response Using an Innovative System of Genotoxic Stress in Medicago truncatula Seedlings.

One of the challenges that living organisms face is to promptly respond to genotoxic stress to avoid DNA damage. To this purpose, all organisms, including plants, developed complex DNA damage response (DDR) mechanisms. These mechanisms are highly conserved among organisms and need to be finely regulated. In this scenario, microRNAs (miRNAs) are emerging as active players, thus attracting the attention of the research community. The involvement of miRNAs in DDR has been investigated prominently in human cells whereas studies in plants are still scarce. To experimentally investigate the involvement of plant miRNAs in the regulation of DDR-associated pathways, an ad hoc system was developed, using the model legume Medicago truncatula. Specific treatments with camptothecin (CPT) and/or NSC120686 (NSC), targeting distinct components of DDR, namely topoisomerase I (TopI) and tyrosyl-DNA phosphodiesterase 1 (TDP1), were used. Phenotypic (germination percentage and speed, seedling growth) and molecular (cell death, DNA damage, and gene expression profiles) analyses demonstrated that the imposed treatments impact DDR. Our results show that these treatments do not influence the germination process but rather inhibit seedling development, causing an increase in cell death and accumulation of DNA damage. Moreover, treatment-specific changes in the expression of suppressor of gamma response 1 (SOG1), master-regulator of plant DDR, were observed. Additionally, the expression of multiple genes playing important roles in different DNA repair pathways and cell cycle regulation were differentially expressed in a treatment-specific manner. Subsequently, specific miRNAs identified from our previous bioinformatics approaches as putatively targeting genes involved in DDR processes were investigated alongside their targets. The obtained results indicate that under most conditions when a miRNA is upregulated the corresponding candidate target gene is downregulated, providing an indirect evidence of miRNAs action over these targets. Hence, the present study extends the present knowledge on the information available regarding the roles played by miRNAs in the post-transcriptional regulation of DDR in plants.

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.

Brassinosteroids Inhibit Autotropic Root Straightening by Modifying Filamentous-Actin Organization and Dynamics.

When positioned horizontally, roots grow down toward the direction of gravity. This phenomenon, called gravitropism, is influenced by most of the major plant hormones including brassinosteroids. Epi-brassinolide (eBL) was previously shown to enhance root gravitropism, a phenomenon similar to the response of roots exposed to the actin inhibitor, latrunculin B (LatB). This led us to hypothesize that eBL might enhance root gravitropism through its effects on filamentous-actin (F-actin). This hypothesis was tested by comparing gravitropic responses of maize (Zea mays) roots treated with eBL or LatB. LatB- and eBL-treated roots displayed similar enhanced downward growth compared with controls when vertical roots were oriented horizontally. Moreover, the effects of the two compounds on root growth directionality were more striking on a slowly-rotating two-dimensional clinostat. Both compounds inhibited autotropism, a process in which the root straightened after the initial gravistimulus was withdrawn by clinorotation. Although eBL reduced F-actin density in chemically-fixed Z. mays roots, the impact was not as strong as that of LatB. Modification of F-actin organization after treatment with both compounds was also observed in living roots of barrel medic (Medicago truncatula) seedlings expressing genetically encoded F-actin reporters. Like in fixed Z. mays roots, eBL effects on F-actin in living M. truncatula roots were modest compared with those of LatB. Furthermore, live cell imaging revealed a decrease in global F-actin dynamics in hypocotyls of etiolated M. truncatula seedlings treated with eBL compared to controls. Collectively, our data indicate that eBL-and LatB-induced enhancement of root gravitropism can be explained by inhibited autotropic root straightening, and that eBL affects this process, in part, by modifying F-actin organization and dynamics.

Medicago truncatula root developmental changes by growth-promoting microbes isolated from Fabaceae, growing on organic farms, involve cell cycle changes and WOX5 gene expression.

Both root nodules and the rhizosphere of Fabaceae plants grown on organic farms are a rich source of bacteria, mainly from the families Enterobacteriaceae and Pseudomonadaceae. The enhanced root system growth in M. truncatula after inoculation with selected bacteria includes an increase of nuclei in the cell cycle S phase and a reduction in phase G2 as well as an enhanced expression of the WOX5 gene. Synthetic fertilizers and pesticides are commonly used to improve plant quality and health. However, it is necessary to look for other efficient and also environmentally safe methods. One such method involves the use of bacteria known as plant growth-promoting bacteria (PGPB). Seventy-two bacterial isolates from the rhizospheric soil and root nodule samples of legumes, including bean, alfalfa, lupine and barrel medic, grown on an organic farm in Western Pomerania (Poland) were screened for their growth-promoting capacities and 38 selected isolates were identified based on 16S rRNA gene sequencing. The analysis showed the isolates to represent 17 strains assigned to 6 families: Enterobacteriaceae, Pseudomonadaceae, Xanthomonadaceae, Rhizobiaceae, Bacillaceae and Alcaligenaceae. Pot experiments showed that 13 strains, capable of producing indole compounds from tryptophan in vitro, could significantly enhance the root and shoot weight of 10-week-old Medicago truncatula seedlings. Compared to non-inoculated seedlings, the root system of inoculated ones was more branched; in addition, the root length, surface area and, especially, the root volume were higher. The 24-h root inoculation with the three selected strains increased the nuclei population in the G1 and S phases, decreased it in the G2 phase and enhanced the WUSCHEL-related Homeobox5 (WOX5) gene expression in root tips and lateral zones. The "arrest" of nuclei in the S phase and the enhancement of the WOX5 gene expression were observed to gradually disappear once the bacterial suspension was rinsed off the seedling roots and the roots were transferred to water for further growth. This study shows that the nodules and rhizosphere of legumes grown on organic farms are a rich source of different PGPB species and provides new data on the ability of these bacteria to interfere with cell cycle and gene expression during the root development.

Involvement of Medicago truncatula glutamate receptor-like channels in nitric oxide production under short-term water deficit stress

Early stages of plant development are highly susceptible to environmental cues, and seedlings have to develop sophisticated mechanisms to sense and respond to abiotic stresses. We have previously identified that abscisic acid (ABA), nitric oxide (NO) and modulation of nitrogen metabolism are involved in adaptive responses in Medicago truncatula seedlings under water deficit stress. Here, we investigated whether glutamate receptor-like channels (GLRs) played a role in the developmental physiological processes of Medicago seedlings during post-germination after a short-term water deficit stress. Twenty-nine independent MtGLR genes have been identified and then divided into four clades following a phylogenetic analysis; seventeen of them exhibited specific domains which are characteristic of animal ionotropic glutamate receptors. Under drought stress, ABA-induced NO accumulation was significantly reduced in presence of a GLR competitive antagonist, suggesting that this water deficit-induced endogenous NO production was mediated through a MtGLR-dependent pathway. Water deficit-induced inhibition of embryo axis elongation was strongly reduced whereas loss of water content was alleviated when MtGLRs were inhibited. These results suggest that glutamate receptors-like channels are required, through their involvement in NO production, in adaptive responses under short-term water-deficit stress during Medicago seedling establishment.

Genome-Wide Identification of microRNAs in Response to Salt/Alkali Stress in Medicago truncatula through High-Throughput Sequencing.

Saline-alkaline stress is a universal abiotic stress that adversely affects plant growth and productivity. Saline-alkaline conditions results in plant abnormal transcriptome expression finally manifesting as defective phenotypes. Considerable research has revealed the active role of microRNA in various stress conditions. This study was aimed to identify novel miRNAs and the miRNA expression patterns in the leguminous model plant R108 (Medicago truncatula). The miRNA contained in the total RNA extracted from Medicago truncatula seedlings (72 h) that had been treated with solutions mimicking saline and alkaline soils was subjected to miRNA deep sequencing. The Illumina HiSeq sequencing platform was used to analyze nine small RNA libraries of three treatment groups: distilled water, 20 mM NaCl + Na2SO4 and 5 mM Na2CO3 + NaHCO3. Sequencing revealed that 876 miRNAs including 664 known miRNAs and 212 potential novel miRNAs were present in all the libraries. The miR159 family, miR156 family, miR2086-3p, miR396, miR166, miR319, miR167, miR5213-5p, miR1510 and miR2643 were among the most expressed miRNAs in all libraries. The results of miRNAs expression under treatments were validated by reverse-transcription quantitative PCR (RT-qPCR). Target gene prediction through computational analysis and pathway annotation analysis revealed that the primary pathways affected by stress were related to plant development, including metabolic processes, single-organism processes and response to the stimulus. Our results provide valuable information towards elucidating the molecular mechanisms of salt/alkali tolerance in Medicago truncatula and provide insight into the putative role of miRNAs in plant stress resistance.

Pathway-specific metabolome analysis with 18O2-labeled Medicago truncatula via a mass spectrometry-based approach

IntroductionOxygen from carbon dioxide, water or molecular oxygen, depending on the responsible enzyme, can lead to a large variety of metabolites through chemical modification.ObjectivesPathway-specific labeling using isotopic molecular oxygen (18O2) makes it possible to determine the origin of oxygen atoms in metabolites and the presence of biosynthetic enzymes (e.g., oxygenases). In this study, we established the basis of 18O2-metabolome analysis.Methods18O2 labeled whole Medicago truncatula seedlings were prepared using 18O2-air and an economical sealed-glass bottle system. Metabolites were analyzed using high-accuracy and high-resolution mass spectrometry. Identification of the metabolite was confirmed by NMR following UHPLC–solid-phase extraction (SPE).ResultsA total of 511 peaks labeled by 18O2 from shoot and 343 peaks from root were annotated by untargeted metabolome analysis. Additionally, we identified a new flavonoid, apigenin 4′-O-[2′-O-coumaroyl-glucuronopyranosyl-(1–2)-O-glucuronopyranoside], that was labeled by 18O2. To the best of our knowledge, this is the first report of apigenin 4′-glucuronide in M. truncatula. Using MSn analysis, we estimated that 18O atoms were specifically incorporated in apigenin, the coumaroyl group, and glucuronic acid. For apigenin, an 18O atom was incorporated in the 4′-hydroxy group. Thus, non-specific incorporation of an 18O atom by recycling during one month of labeling is unlikely compared with the more specific oxygenase-catalyzing reaction.ConclusionOur finding indicated that 18O2 labeling was effective not only for the mining of unknown metabolites which were biosynthesized by oxygenase-related pathway but also for the identification of metabolites whose oxygen atoms were derived from oxygenase activity.

Effect of short-term aluminum stress and mycorrhizal inoculation on nitric oxide metabolism in Medicago truncatula roots

Aluminum (Al) toxicity can induce oxidative and nitrosative stress, which limits growth and yield of crop plants. Nevertheless, plant tolerance to stress may be improved by symbiotic associations including arbuscular mycorrhiza (AM). Nitric oxide (NO) is a signaling molecule involved in physiological processes and plant responses to abiotic and biotic stresses. However, almost no information about the NO metabolism has been gathered about AM. In the present work, Medicago truncatula seedlings were inoculated with Rhizophagus irregularis, and 7-week-old plants were treated with 50μM AlCl3 for 3h. Cytochemical and molecular techniques were used to measure the components of the NO metabolism, including NO content and localization, expression of genes encoding NO-synthesis (MtNR1, MtNR2 and MtNIR1) and NO-scavenging (MtGSNOR1, MtGSNOR2, MtHB1 and MtHB2) enzymes and the profile of protein tyrosine nitration (NO2-Tyr) in Medicago roots.For the first time, NO and NO2-Tyr accumulation was connected with fungal structures (arbuscules, vesicles and intercellular hyphae). Expression analysis of genes encoding NO-synthesis enzymes indicated that AM symbiosis results in lower production of NO in Al-treated roots in comparison to non-mycorrhizal roots. Elevated levels of transcription of genes encoding NO-scavenging enzymes indicated more active NO scavenging in AMF-inoculated Al-treated roots compared to non-inoculated roots. These results were confirmed by less NO accumulation and lower protein nitration in Al-stressed mycorrhizal roots in comparison to non-mycorrhizal roots.This study provides a new insight in NO metabolism in response to arbuscular mycorrhiza under normal and metal stress conditions. Our results suggest that mycorrhizal fungi decrease NO and tyrosine nitrated proteins content in Al-treated Medicago roots, probably via active NO scavenging system.

The Seed Repair Response during Germination: Disclosing Correlations between DNA Repair, Antioxidant Response, and Chromatin Remodeling in Medicago truncatula.

This work provides novel insights into the effects caused by the histone deacetylase inhibitor trichostatin A (TSA) during Medicago truncatula seed germination, with emphasis on the seed repair response. Seeds treated with H2O and TSA (10 and 20 μM) were collected during imbibition (8 h) and at the radicle protrusion phase. Biometric data showed delayed germination and impaired seedling growth in TSA-treated samples. Comet assay, performed on radicles at the protrusion phase and 4-days old M. truncatula seedlings, revealed accumulation of DNA strand breaks upon exposure to TSA. Activation of DNA repair toward TSA-mediated genotoxic damage was evidenced by the up-regulation of MtOGG1(8-OXOGUANINE GLYCOSYLASE/LYASE) gene involved in the removal of oxidative DNA lesions, MtLIGIV(LIGASE IV) gene, a key determinant of seed quality, required for the rejoining of DNA double strand breaks and TDP(TYROSYL-DNA PHOSPHODIESTERASE) genes encoding the multipurpose DNA repair enzymes tyrosyl-DNA phosphodiesterases. Since radical scavenging can prevent DNA damage, the specific antioxidant activity (SAA) was measured by DPPH (1,1-diphenyl-2-picrylhydrazyl) and Folin-Ciocalteu reagent assays. Fluctuations of SAA were observed in TSA-treated seeds/seedlings concomitant with the up-regulation of antioxidant genes MtSOD(SUPEROXIDE DISMUTASE, MtAPX(ASCORBATE PEROXIDASE) and MtMT2(TYPE 2 METALLOTHIONEIN). Chromatin remodeling, required to facilitate the access of DNA repair enzymes at the damaged sites, is also part of the multifaceted seed repair response. To address this aspect, still poorly explored in plants, the MtTRRAP(TRANSFORMATION/TRANSACTIVATION DOMAIN-ASSOCIATED PROTEIN) gene was analyzed. TRRAP is a transcriptional adaptor, so far characterized only in human cells where it is needed for the recruitment of histone acetyltransferase complexes to chromatin during DNA repair. The MtTRRAP gene and the predicted interacting partners MtHAM2 (HISTONE ACETYLTRANSFERASE OF THE MYST FAMILY) and MtADA2A (TRANSCRIPTIONAL ADAPTOR) showed tissue- and dose-dependent fluctuations in transcript levels. PCA (Principal Component Analysis) and correlation analyses suggest for a new putative link between DNA repair and chromatin remodeling that involves MtOGG1 and MtTRRAP genes, in the context of seed germination. Interesting correlations also connect DNA repair and chromatin remodeling with antioxidant players and proliferation markers.

The U-box family genes in Medicago truncatula: Key elements in response to salt, cold, and drought stresses.

The ubiquitination pathway regulates growth, development, and stress responses in plants, and the U-box protein family of ubiquitin ligases has important roles in this pathway. Here, 64 putative U-box proteins were identified in the Medicago truncatula genome. In addition to the conserved U-box motif, other functional domains, such as the ARM, kinase, KAP, and WD40 domains, were also detected. Phylogenetic analysis of the M. truncatula U-box proteins grouped them into six subfamilies, and chromosomal mapping and synteny analyses indicated that tandem and segmental duplications may have contributed to the expansion and evolution of the U-box gene family in this species. Using RNA-seq data from M. truncatula seedlings subjected to three different abiotic stresses, we identified 33 stress-inducible plant U-box genes (MtPUBs). Specifically, 25 salinity-, 15 drought-, and 16 cold-regulated MtPUBs were detected. Among them, MtPUB10, MtPUB17, MtPUB18, MtPUB35, MtPUB42, and MtPUB44 responded to all three stress conditions. Expression profiling by qRT-PCR was consistent with the RNA-seq data, and stress-related elements were identified in the promoter regions. The present findings strongly indicate that U-box proteins play critical roles in abiotic stress response in M. truncatula.

Transcriptome analysis of the fungal pathogen Fusarium oxysporum f. sp. medicaginis during colonisation of resistant and susceptible Medicago truncatula hosts identifies differential pathogenicity profiles and novel candidate effectors.

BackgroundPathogenic members of the Fusarium oxysporum species complex are responsible for vascular wilt disease on many important crops including legumes, where they can be one of the most destructive disease causing necrotrophic fungi. We previously developed a model legume-infecting pathosystem based on the reference legume Medicago truncatula and a pathogenic F. oxysporum forma specialis (f. sp.) medicaginis (Fom). To dissect the molecular pathogenicity arsenal used by this root-infecting pathogen, we sequenced its transcriptome during infection of a susceptible and resistant host accession.ResultsHigh coverage RNA-Seq of Fom infected root samples harvested from susceptible (DZA315) or resistant (A17) M. truncatula seedlings at early or later stages of infection (2 or 7 days post infection (dpi)) and from vegetative (in vitro) samples facilitated the identification of unique and overlapping sets of in planta differentially expressed genes. This included enrichment, particularly in DZA315 in planta up-regulated datasets, for proteins associated with sugar, protein and plant cell wall metabolism, membrane transport, nutrient uptake and oxidative processes. Genes encoding effector-like proteins were identified, including homologues of the F. oxysporum f. sp. lycopersici Secreted In Xylem (SIX) proteins, and several novel candidate effectors based on predicted secretion, small protein size and high in-planta induced expression. The majority of the effector candidates contain no known protein domains but do share high similarity to predicted proteins predominantly from other F. oxysporum ff. spp. as well as other Fusaria (F. solani, F. fujikori, F. verticilloides, F. graminearum and F. pseudograminearum), and from another wilt pathogen of the same class, a Verticillium species. Overall, this suggests these novel effector candidates may play important roles in Fusaria and wilt pathogen virulence.ConclusionCombining high coverage in planta RNA-Seq with knowledge of fungal pathogenicity protein features facilitated the identification of differentially expressed pathogenicity associated genes and novel effector candidates expressed during infection of a resistant or susceptible M. truncatula host. The knowledge from this first in depth in planta transcriptome sequencing of any F. oxysporum ff. spp. pathogenic on legumes will facilitate the dissection of Fusarium wilt pathogenicity mechanisms on many important legume crops.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3192-2) contains supplementary material, which is available to authorized users.

Cross-Regulation between N Metabolism and Nitric Oxide (NO) Signaling during Plant Immunity.

Plants are sessile organisms that have evolved a complex immune system which helps them cope with pathogen attacks. However, the capacity of a plant to mobilize different defense responses is strongly affected by its physiological status. Nitrogen (N) is a major nutrient that can play an important role in plant immunity by increasing or decreasing plant resistance to pathogens. Although no general rule can be drawn about the effect of N availability and quality on the fate of plant/pathogen interactions, plants’ capacity to acquire, assimilate, allocate N, and maintain amino acid homeostasis appears to partly mediate the effects of N on plant defense. Nitric oxide (NO), one of the products of N metabolism, plays an important role in plant immunity signaling. NO is generated in part through Nitrate Reductase (NR), a key enzyme involved in nitrate assimilation, and its production depends on levels of nitrate/nitrite, NR substrate/product, as well as on L-arginine and polyamine levels. Cross-regulation between NO signaling and N supply/metabolism has been evidenced. NO production can be affected by N supply, and conversely NO appears to regulate nitrate transport and assimilation. Based on this knowledge, we hypothesized that N availability partly controls plant resistance to pathogens by controlling NO homeostasis. Using the Medicago truncatula/Aphanomyces euteiches pathosystem, we showed that NO homeostasis is important for resistance to this oomycete and that N availability impacts NO homeostasis by affecting S-nitrosothiol (SNO) levels and S-nitrosoglutathione reductase activity in roots. These results could therefore explain the increased resistance we noted in N-deprived as compared to N-replete M. truncatula seedlings. They open onto new perspectives for the studies of N/plant defense interactions.

Cadmium-induced changes in antioxidative systems and differentiation in roots of contrasted Medicago truncatula lines.

Defense pathways and stress responses induced under Cd stress were illustrated in roots of hydroponically grown Medicago truncatula seedlings. Actually, the ascorbate-glutathione and antioxidative system, secondary metabolism events including peroxidases, phenolic compounds, and lignification launching, and developmental modifications were described. Cd (100μM) initially increased reactive oxygen species, enhanced antioxidative (total SOD, CAT, and PRX) and ascorbate-glutathione-related metabolism enzymes (APX and MDAR), except in A17 and TN1.11. In agreement with peroxidase enhancement, physiological measurement and in situ observation illustrated soluble phenolic compound accumulation under Cd treatment. However, lignification was restricted to recently created protoxylem elements established in the root tip area, usually constituting the elongation zone. Cell death was increased. In the absence of necrotic reactions, developmental changes including lignin deposition, increase in cellulose and pectin contents, intercellular meatus, and condensed and deformed hairs were noticed in Cd-treated roots.

The nitrate transporter MtNPF6.8 (MtNRT1.3) transports abscisic acid and mediates nitrate regulation of primary root growth in Medicago truncatula.

Elongation of the primary root during postgermination of Medicago truncatula seedlings is a multigenic trait that is responsive to exogenous nitrate. A quantitative genetic approach suggested the involvement of the nitrate transporter MtNPF6.8 (for Medicago truncatula NITRATE TRANSPORTER1/PEPTIDE TRANSPORTER Family6.8) in the inhibition of primary root elongation by high exogenous nitrate. In this study, the inhibitory effect of nitrate on primary root elongation, via inhibition of elongation of root cortical cells, was abolished in npf6.8 knockdown lines. Accordingly, we propose that MtNPF6.8 mediates nitrate inhibitory effects on primary root growth in M. truncatula. pMtNPF6.8:GUS promoter-reporter gene fusion in Agrobacterium rhizogenes-generated transgenic roots showed the expression of MtNPF6.8 in the pericycle region of primary roots and lateral roots, and in lateral root primordia and tips. MtNPF6.8 expression was insensitive to auxin and was stimulated by abscisic acid (ABA), which restored the inhibitory effect of nitrate in npf6.8 knockdown lines. It is then proposed that ABA acts downstream of MtNPF6.8 in this nitrate signaling pathway. Furthermore, MtNPF6.8 was shown to transport ABA in Xenopus spp. oocytes, suggesting an additional role of MtNPF6.8 in ABA root-to-shoot translocation. (15)NO3(-)-influx experiments showed that only the inducible component of the low-affinity transport system was affected in npf6.8 knockdown lines. This indicates that MtNPF6.8 is a major contributor to the inducible component of the low-affinity transport system. The short-term induction by nitrate of the expression of Nitrate Reductase1 (NR1) and NR2 (genes that encode two nitrate reductase isoforms) was greatly reduced in the npf6.8 knockdown lines, supporting a role of MtNPF6.8 in the primary nitrate response in M. truncatula.