Novel antimicrobial peptides identified in legume plant, Medicago truncatula.

One of the major issues in healthcare today is antibiotic resistance. Antimicrobial peptides (AMPs), a subclass of host defense peptides, have been suggested as a viable solution for the multidrug resistance problem. Legume plants express more than 700 nodule-specific cysteine-rich (NCR) peptides. Three NCR peptides (NCR094, NCR888, and NCR992) were predicted to have antimicrobial activity using in silico AMP prediction programs. This study focused on investigating the roles of the NCRs in antimicrobial activity and antibiofilm activity, followed by in vitro toxicity profiling. Different variants were synthesized, i.e., mutated and truncated derivatives. The effect on the growth of Klebsiella pneumoniae and methicillin-resistant Staphylococcus aureus (MRSA) was monitored post-treatment, and survived cells were counted using an in vitro and ex vivo killing assay. The antibiofilm assay was conducted using subinhibitory concentrations of the NCRs and monitoring K. pneumoniae biomass, followed by crystal violet staining. The cytotoxicity profile was evaluated using erythrocyte hemolysis and leukemia (K562) cell line toxicity assays. Out of the NCRs, NCR094 and NCR992 displayed mainly in vitro and ex vivo bactericidal activity on K. pneumoniae. NCR094 wild type (WT) and NCR992 eradicated K. pneumoniae at different potency; NCR094 and NCR992 killed K. pneumoniae completely at 25 and 50 µM, respectively. However, both peptides in the wild type showed negligible bactericidal effect on MRSA in vitro and ex vivo. NCR094 and its derivatives relatively retained the antimicrobial activity on K. pneumoniae in vitro and ex vivo. NCR992 WT lost its antimicrobial activity on K. pneumoniae ex vivo, yet the different truncated and mutated variants retained some of the antimicrobial role ex vivo. All the different variants of NCR094 had no effect on MRSA in vitro and ex vivo. Similarly, NCR992's variants had a negligible bactericidal role on MRSA in vitro, yet the truncated variants had a significantly high bactericidal effect on MRSA ex vivo. NCR094.3 (cystine replacement variant) and NCR992.1 displayed significant antibiofilm activity more than 90%. NCR992.3 and NCR992.2 displayed more than 50% of antibiofilm activity. All the NCR094 forms had no toxicity, except NCR094.1 (49.38%, SD ± 3.46) and all NCR992 forms (63%-93%), which were above the cutoff (20%). Only NCR992.2 showed low toxicity on K562 (24.8%, SD ± 3.40), yet above the 20% cutoff. This study provided preliminary antimicrobial and safety data for the potential use of these peptides for therapeutical applications.IMPORTANCEThe discovery of new antibiotics is urgently needed, given the global expansion of antibiotic-resistant bacteria and the rising mortality rate. One of the initial lines of defense against microbial infections is antimicrobial peptides (AMPs). Plants can express hundreds of such AMPs as defensins and defensin-like peptides. The nodule-specific cysteine-rich (NCR) peptides are a class of defensin-like peptides that have evolved in rhizobial-legume symbioses. This study screened the antimicrobial activity of a subset of NCR sequences using online computational AMP prediction algorithms. Two novel NCRs, NCR094 and NCR992, with different variants were identified to exhibit antimicrobial activity with various potency on two problematic pathogens, K. pneumoniae and MRSA, using in vitro and ex vivo killing assays. Yet, one variant, NCR094.3, had no toxicity toward human cells and displayed antibiofilm activity, which make it a promising lead for antimicrobial drug development.

Sinorhizobium meliloti Functions Required for Resistance to Antimicrobial NCR Peptides and Bacteroid Differentiation.

Legumes of the Medicago genus have a symbiotic relationship with the bacterium Sinorhizobium meliloti and develop root nodules housing large numbers of intracellular symbionts. Members of the nodule-specific cysteine-rich peptide (NCR) family induce the endosymbionts into a terminal differentiated state. Individual cationic NCRs are antimicrobial peptides that have the capacity to kill the symbiont, but the nodule cell environment prevents killing. Moreover, the bacterial broad-specificity peptide uptake transporter BacA and exopolysaccharides contribute to protect the endosymbionts against the toxic activity of NCRs. Here, we show that other S. meliloti functions participate in the protection of the endosymbionts; these include an additional broad-specificity peptide uptake transporter encoded by the yejABEF genes and lipopolysaccharide modifications mediated by lpsB and lpxXL, as well as rpoH1, encoding a stress sigma factor. Strains with mutations in these genes show a strain-specific increased sensitivity profile against a panel of NCRs and form nodules in which bacteroid differentiation is affected. The lpsB mutant nodule bacteria do not differentiate, the lpxXL and rpoH1 mutants form some seemingly fully differentiated bacteroids, although most of the nodule bacteria are undifferentiated, while the yejABEF mutants form hypertrophied but nitrogen-fixing bacteroids. The nodule bacteria of all the mutants have a strongly enhanced membrane permeability, which is dependent on the transport of NCRs to the endosymbionts. Our results suggest that S. meliloti relies on a suite of functions, including peptide transporters, the bacterial envelope structures, and stress response regulators, to resist the aggressive assault of NCR peptides in the nodule cells. IMPORTANCE The nitrogen-fixing symbiosis of legumes with rhizobium bacteria has a predominant ecological role in the nitrogen cycle and has the potential to provide the nitrogen required for plant growth in agriculture. The host plants allow the rhizobia to colonize specific symbiotic organs, the nodules, in large numbers in order to produce sufficient reduced nitrogen for the plants' needs. Some legumes, including Medicago spp., produce massively antimicrobial peptides to keep this large bacterial population in check. These peptides, known as NCRs, have the potential to kill the rhizobia, but in nodules, they rather inhibit the division of the bacteria, which maintain a high nitrogen-fixing activity. In this study, we show that the tempering of the antimicrobial activity of the NCR peptides in the Medicago symbiont Sinorhizobium meliloti is multifactorial and requires the YejABEF peptide transporter, the lipopolysaccharide outer membrane, and the stress response regulator RpoH1.

Unusually Fast bis-Histidyl Coordination in a Plant Hemoglobin.

The recently identified nonsymbiotic hemoglobin gene MtGlb1-2 of the legume Medicago truncatula possesses unique properties as it generates four alternative splice forms encoding proteins with one or two heme domains. Here we investigate the ligand binding kinetics of MtGlb1-2.1 and MtGlb1-2.4, bearing two hemes and one heme, respectively. Unexpectedly, the overall time-course of ligand rebinding was unusually fast. Thus, we complemented nanosecond laser flash photolysis kinetics with data collected with a hybrid femtosecond-nanosecond pump-probe setup. Most photodissociated ligands are rebound geminately within a few nanoseconds, which leads to rates of the bimolecular rebinding to pentacoordinate species in the 108 M-1s-1 range. Binding of the distal histidine to the heme competes with CO rebinding with extremely high rates (kh ~ 105 s-1). Histidine dissociation from the heme occurs with comparable rates, thus resulting in moderate equilibrium binding constants (KH ~ 1). The rate constants for ligation and deligation of distal histidine to the heme are the highest reported for any plant or vertebrate globin. The combination of microscopic rates results in unusually high overall ligand binding rate constants, a fact that contributes to explaining at the mechanistic level the extremely high reactivity of these proteins toward the physiological ligands oxygen, nitric oxide and nitrite.

Symbiotic NCR Peptide Fragments Affect the Viability, Morphology and Biofilm Formation of Candida Species.

The increasing rate of fungal infections causes global problems not only in human healthcare but agriculture as well. To combat fungal pathogens limited numbers of antifungal agents are available therefore alternative drugs are needed. Antimicrobial peptides are potent candidates because of their broad activity spectrum and their diverse mode of actions. The model legume Medicago truncatula produces >700 nodule specific cysteine-rich (NCR) peptides in symbiosis and many of them have in vitro antimicrobial activities without considerable toxicity on human cells. In this work we demonstrate the anticandidal activity of the NCR335 and NCR169 peptide derivatives against five Candida species by using the micro-dilution method, measuring inhibition of biofilm formation with the XTT (2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide) assay, and assessing the morphological change of dimorphic Candida species by microscopy. We show that both the N- and C-terminal regions of NCR335 possess anticandidal activity as well as the C-terminal sequence of NCR169. The active peptides inhibit biofilm formation and the yeast-hypha transformation. Combined treatment of C. auris with peptides and fluconazole revealed synergistic interactions and reduced 2-8-fold the minimal inhibitory concentrations. Our results demonstrate that shortening NCR peptides can even enhance and broaden their anticandidal activity and therapeutic potential.

Plant defensin antibacterial mode of action against Pseudomonas species.

BACKGROUND: Though many plant defensins exhibit antibacterial activity, little is known about their antibacterial mode of action (MOA). Antimicrobial peptides with a characterized MOA induce the expression of multiple bacterial outer membrane modifications, which are required for resistance to these membrane-targeting peptides. Mini-Tn5-lux mutant strains of Pseudomonas aeruginosa with Tn insertions disrupting outer membrane protective modifications were assessed for sensitivity against plant defensin peptides. These transcriptional lux reporter strains were also evaluated for lux gene expression in response to sublethal plant defensin exposure. Also, a plant pathogen, Pseudomonas syringae pv. syringae was modified through transposon mutagenesis to create mutants that are resistant to in vitro MtDef4 treatments. RESULTS: Plant defensins displayed specific and potent antibacterial activity against strains of P. aeruginosa. A defensin from Medicago truncatula, MtDef4, induced dose-dependent gene expression of the aminoarabinose modification of LPS and surface polycation spermidine production operons. The ability for MtDef4 to damage bacterial outer membranes was also verified visually through fluorescent microscopy. Another defensin from M. truncatula, MtDef5, failed to induce lux gene expression and limited outer membrane damage was detected with fluorescent microscopy. The transposon insertion site on MtDef4 resistant P. syringae pv. syringae mutants was sequenced, and modifications of ribosomal genes were identified to contribute to enhanced resistance to plant defensin treatments. CONCLUSIONS: MtDef4 damages the outer membrane similar to polymyxin B, which stimulates antimicrobial peptide resistance mechanisms to plant defensins. MtDef5, appears to have a different antibacterial MOA. Additionally, the MtDef4 antibacterial mode of action may also involve inhibition of translation.

Plant phenolics mediated bottom-up effects of elevated CO2 on Acyrthosiphon pisum and its parasitoid Aphidius avenae.

Elevated concentrations of atmospheric CO2 can alter plant secondary metabolites, which play important roles in the interactions among plants, herbivorous insects and natural enemies. However, few studies have examined the cascading effects of host plant secondary metabolites on tri-trophic interactions under elevated CO2 (eCO2 ). In this study, we determined the effects of eCO2 on the growth and foliar phenolics of Medicago truncatula and the cascading effects on two color genotypes of Acyrthosiphon pisum (pink vs. green) and their parasitoid Aphidius avenae in the field open-top chambers. Our results showed that eCO2 increased photosynthetic rate, nodule number, yield and the total phenolic content of M. truncatula. eCO2 had contrasting effects on two genotypes of A. pisum; the green genotype demonstrated increased population abundance, fecundity, growth and feeding efficiency, while the pink genotype showed decreased fitness and these were closely associated with the foliar genstein content. Furthermore, eCO2 decreased the parasitic rate of A. avenae independent of aphid genotypes. eCO2 prolonged the emergence time and reduced the emergence rate and percentage of females when associated with the green genotype, but little difference, except for increased percentage of females, was observed in A. avenae under eCO2 when associated with the pink genotype, indicating that parasitoids can perceive and discriminate the qualities of aphid hosts. We concluded that eCO2 altered plant phenolics and thus the performance of aphids and parasitoids. Our results indicate that plant phenolics vary by different abiotic and biotic stimuli and could potentially deliver the cascading effects of eCO2 to the higher trophic levels. Our results also suggest that the green genotype is expected to perform better in future eCO2 because of decreased plant resistance after its infestation and decreased parasitic rate.

A comparison of the effects of apigenin and seven of its derivatives on selected biomarkers of oxidative stress and coagulation in vitro.

Apigenin is a phenolic compound widely present in many fruits, vegetables and herbs. Its name originates from Apium: a genus of the Apiaceae. The aim of the present study was to determine the antioxidant or pro-oxidant properties of apigenin and seven of its derivatives, isolated from the aerial parts of barrel medic (Medicago truncatula) and common wheat (Triticum aestivum), in human plasma treated with a hydroxyl radical donor (OH•) in vitro. It also examines their influence on the parameters of coagulation. The compounds were found to demonstrate different effects on oxidative stress and coagulation which may be related to differences in their structure. In particular, apigenin 7-O-{2'-O-feruloyl-[ß-D-glucuronopyranosyl(1 â†’ 3)]-ß-D- glucuronopyranosyl(1 â†’ 2)-O-ß-D-glucopyranoside} demonstrates both antioxidant and anticoagulant activities, and may offer the most promise for the prevention and treatment of cardiovascular disorders of all the phenolic compounds tested so far.

Ectopic Defense Gene Expression Is Associated with Growth Defects in Medicago truncatula Lignin Pathway Mutants.

Lignin provides essential mechanical support for plant cell walls but decreases the digestibility of forage crops and increases the recalcitrance of biofuel crops. Attempts to modify lignin content and/or composition by genetic modification often result in negative growth effects. Although several studies have attempted to address the basis for such effects in individual transgenic lines, no common mechanism linking lignin modification with perturbations in plant growth and development has yet been identified. To address whether a common mechanism exists, we have analyzed transposon insertion mutants resulting in independent loss of function of five enzymes of the monolignol pathway, as well as one double mutant, in the model legume Medicago truncatula These plants exhibit growth phenotypes from essentially wild type to severely retarded. Extensive phenotypic, transcriptomic, and metabolomics analyses, including structural characterization of differentially expressed compounds, revealed diverse phenotypic consequences of lignin pathway perturbation that were perceived early in plant development but were not predicted by lignin content or composition alone. Notable phenotypes among the mutants with severe growth impairment were increased trichome numbers, accumulation of a variety of triterpene saponins, and extensive but differential ectopic expression of defense response genes. No currently proposed model explains the observed phenotypes across all lines. We propose that reallocation of resources into defense pathways is linked to the severity of the final growth phenotype in monolignol pathway mutants of M. truncatula, although it remains unclear whether this is a cause or an effect of the growth impairment.

Crystallographic identification of spontaneous oxidation intermediates and products of protein sulfhydryl groups.

In the absence of protective reducing agents, Cys residues in purified proteins can be oxidized spontaneously by oxygen in the air, as frequently observed in protein crystal structures. However, the formation of an O-bridge via dehydration mechanism between a peroxidized Cys side chain and a primary amine of Lys side chain in proteins has not yet been reported. When an electron density feature was observed for an extra group or an extra atom between side chains of Cys-245 and Lys-158 in the crystal structure of histidinol phosphate phosphatase, mass spectrometric analysis was carried out for its chemical identification. That analysis led to a conclusion that this extra density corresponded to a methylene group. It was then proposed that these two residues were able to absorb CO2 and reduced it to CH2 spontaneously. Further examination of other protein structures in the PDB showed that the formation of this cross-linking species was a widespread phenomenon. This claim is examined in this study using methods recently developed for quantification of electrons around nucleus as the means for direct chemical identification. It is found that an O-bridge is actually formed between Cys and Lys side chains, instead of a CH2 -bridge.

Differential accumulation of leucine and methionine in red and green pea aphids leads to different fecundity in response to nitrogen fertilization.

BACKGROUND: Nitrogen fertilization affects plants directly and herbivorous insects indirectly. Although insect species and even genotypes are known to differ in their responses to nitrogen fertilization, the physiological and molecular mechanisms remain unclear. This study assessed the fecundity and related regulatory signaling pathways in the green and red morphs of pea aphid (Acyrthosiphon pisum) feeding on Medicago truncatula with and without nitrogen fertilization. RESULTS: Nitrogen fertilization significantly increased foliar amino acid concentrations and consequently increased the concentrations of several individual essential amino acids in body tissue of the green morph. The increased concentration of Leu, Ile, Met and Val was consistent with enhanced biosynthesis of these amino acids in the endosymbiont Buchnera. Under nitrogen fertilization, Leu and Met accumulated in the green morph enhanced the target of rapamycin (TOR) signaling pathway, which consequently increased fecundity by promoting vitellogenin synthesis. In the red morph, however, nitrogen fertilization did not change the concentration of essential amino acids, TOR signaling or fecundity. CONCLUSION: Specific amino acids accumulation and the nutrient transduction pathway in pea aphids are responsible for genotype-specific fecundity in response to nitrogen fertilization, which could be used as potential target for pest control. © 2018 Society of Chemical Industry.

Synthesis of lipo-chitooligosaccharide analogues and their interaction with LYR3, a high affinity binding protein for Nod factors and Myc-LCOs.

Lipo-chitotetrasaccharide analogues where one central GlcNAc residue was replaced by a triazole unit have been synthesized from a derivative obtained by chitin depolymerization and a functionalized N-acetyl-glucosamine via the copper-catalyzed azide-alkyne cycloaddition. Their evaluation in a binding assay using LYR3, a putative lipo-chitooligosaccharide receptor in Medicago truncatula, shows a complete loss of binding.

Receptor-mediated chitin perception in legume roots is functionally separable from Nod factor perception.

The ability of root cells to distinguish mutualistic microbes from pathogens is crucial for plants that allow symbiotic microorganisms to infect and colonize their internal root tissues. Here we show that Lotus japonicus and Medicago truncatula possess very similar LysM pattern-recognition receptors, LjLYS6/MtLYK9 and MtLYR4, enabling root cells to separate the perception of chitin oligomeric microbe-associated molecular patterns from the perception of lipochitin oligosaccharide by the LjNFR1/MtLYK3 and LjNFR5/MtNFP receptors triggering symbiosis. Inactivation of chitin-receptor genes in Ljlys6, Mtlyk9, and Mtlyr4 mutants eliminates early reactive oxygen species responses and induction of defense-response genes in roots. Ljlys6, Mtlyk9, and Mtlyr4 mutants were also more susceptible to fungal and bacterial pathogens, while infection and colonization by rhizobia and arbuscular mycorrhizal fungi was maintained. Biochemical binding studies with purified LjLYS6 ectodomains further showed that at least six GlcNAc moieties (CO6) are required for optimal binding efficiency. The 2.3-Å crystal structure of the LjLYS6 ectodomain reveals three LysM ßααß motifs similar to other LysM proteins and a conserved chitin-binding site. These results show that distinct receptor sets in legume roots respond to chitin and lipochitin oligosaccharides found in the heterogeneous mixture of chitinaceous compounds originating from soil microbes. This establishes a foundation for genetic and biochemical dissection of the perception and the downstream responses separating defense from symbiosis in the roots of the 80-90% of land plants able to develop rhizobial and/or mycorrhizal endosymbiosis.

Genome-Wide Sensitivity Analysis of the Microsymbiont Sinorhizobium meliloti to Symbiotically Important, Defensin-Like Host Peptides.

The model legume species Medicago truncatula expresses more than 700 nodule-specific cysteine-rich (NCR) signaling peptides that mediate the differentiation of Sinorhizobium meliloti bacteria into nitrogen-fixing bacteroids. NCR peptides are essential for a successful symbiosis in legume plants of the inverted-repeat-lacking clade (IRLC) and show similarity to mammalian defensins. In addition to signaling functions, many NCR peptides exhibit antimicrobial activity in vitro and in vivo Bacterial resistance to these antimicrobial activities is likely to be important for symbiosis. However, the mechanisms used by S. meliloti to resist antimicrobial activity of plant peptides are poorly understood. To address this, we applied a global genetic approach using transposon mutagenesis followed by high-throughput sequencing (Tn-seq) to identify S. meliloti genes and pathways that increase or decrease bacterial competitiveness during exposure to the well-studied cationic NCR247 peptide and also to the unrelated model antimicrobial peptide polymyxin B. We identified 78 genes and several diverse pathways whose interruption alters S. meliloti resistance to NCR247. These genes encode the following: (i) cell envelope polysaccharide biosynthesis and modification proteins, (ii) inner and outer membrane proteins, (iii) peptidoglycan (PG) effector proteins, and (iv) non-membrane-associated factors such as transcriptional regulators and ribosome-associated factors. We describe a previously uncharacterized yet highly conserved peptidase, which protects S. meliloti from NCR247 and increases competitiveness during symbiosis. Additionally, we highlight a considerable number of uncharacterized genes that provide the basis for future studies to investigate the molecular basis of symbiotic development as well as chronic pathogenic interactions.IMPORTANCE Soil rhizobial bacteria enter into an ecologically and economically important symbiotic interaction with legumes, in which they differentiate into physiologically distinct bacteroids that provide essential ammonia to the plant in return for carbon sources. Plant signal peptides are essential and specific to achieve these physiological changes. These peptides show similarity to mammalian defensin peptides which are part of the first line of defense to control invading bacterial populations. A number of these legume peptides are indeed known to possess antimicrobial activity, and so far, only the bacterial BacA protein is known to protect rhizobial bacteria against their antimicrobial action. This study identified numerous additional bacterial factors that mediate protection and belong to diverse biological pathways. Our results significantly contribute to our understanding of the molecular roles of bacterial factors during legume symbioses and, second, provide insights into the mechanisms that pathogenic bacteria may use to resist the antimicrobial effects of defensins during infections.

Medicago truncatula Oleanolic-Derived Saponins Are Correlated with Caterpillar Deterrence.

Plant resistance mechanisms to insect herbivory can potentially be bred into crops as an important strategy for integrated pest management. Medicago truncatula ecotypes inoculated with the rhizobium Ensifer medicae (Sinorhizobium medica) WSM419 were screened for resistance to herbivory by caterpillars of the beet armyworm, Spodoptera exigua, through leaf and whole plant choice studies; TN1.11 and F83005.5 are identified as the least and most deterrent ecotypes, respectively. In response to caterpillar herbivory, both ecotypes mount a robust burst of plant defensive jasmonate phytohormones. Restriction of caterpillars to either of these ecotypes does not adversely affect pest performance. This argues for an antixenosis (deterrence) resistance mechanism associated with the F83005.5 ecotype. Unbiased metabolomic profiling identified strong ecotype-specific differences in metabolite profile, particularly in the content of oleanolic-derived saponins that may act as antifeedants. Compared to the more susceptible ecotype, F83005.5 has higher levels of oleanolic-type zanhic acid- and medicagenic acid-derived compounds. Together, these data support saponin-mediated deterrence as a resistance mechanism of the F83005.5 ecotype and implicates these compounds as potential antifeedants that could be used in agricultural sustainable pest management strategies.

PlantMAT: A Metabolomics Tool for Predicting the Specialized Metabolic Potential of a System and for Large-Scale Metabolite Identifications.

Custom software entitled Plant Metabolite Annotation Toolbox (PlantMAT) has been developed to address the number one grand challenge in metabolomics, which is the large-scale and confident identification of metabolites. PlantMAT uses informed phytochemical knowledge for the prediction of plant natural products such as saponins and glycosylated flavonoids through combinatorial enumeration of aglycone, glycosyl, and acyl subunits. Many of the predicted structures have yet to be characterized and are absent from traditional chemical databases, but have a higher probability of being present in planta. PlantMAT allows users to operate an automated and streamlined workflow for metabolite annotation from a user-friendly interface within Microsoft Excel, a familiar, easily accessed program for chemists and biologists. The usefulness of PlantMAT is exemplified using ultrahigh-performance liquid chromatography-electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UHPLC-ESI-QTOF-MS/MS) metabolite profiling data of saponins and glycosylated flavonoids from the model legume Medicago truncatula. The results demonstrate PlantMAT substantially increases the chemical/metabolic space of traditional chemical databases. Ten of the PlantMAT-predicted identifications were validated and confirmed through the isolation of the compounds using ultrahigh-performance liquid chromatography-mass spectrometry-solid-phase extraction (UHPLC-MS-SPE) followed by de novo structural elucidation using 1D/2D nuclear magnetic resonance (NMR). It is further demonstrated that PlantMAT enables the dereplication of previously identified metabolites and is also a powerful tool for the discovery of structurally novel metabolites.

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

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

LYR3, a high-affinity LCO-binding protein of Medicago truncatula, interacts with LYK3, a key symbiotic receptor.

LYR3, LYK3, and NFP are lysin motif-containing receptor-like kinases (LysM-RLKs) from Medicago truncatula, involved in perception of symbiotic lipo-chitooligosaccharide (LCO) signals. Here, we show that LYR3, a high-affinity LCO-binding protein, physically interacts with LYK3, a key player regulating symbiotic interactions. In vitro, LYR3 is phosphorylated by the active kinase domain of LYK3. Fluorescence lifetime imaging/Förster resonance energy transfer (FLIM/FRET) experiments in tobacco protoplasts show that the interaction between LYR3 and LYK3 at the plasma membrane is disrupted or inhibited by addition of LCOs. Moreover, LYR3 attenuates the cell death response, provoked by coexpression of NFP and LYK3 in tobacco leaves.

Genome-wide analysis identifies gain and loss/change of function within the small multigenic insecticidal Albumin 1 family of Medicago truncatula.

BACKGROUND: Albumin 1b peptides (A1b) are small disulfide-knotted insecticidal peptides produced by Fabaceae (also called Leguminosae). To date, their diversity among this plant family has been essentially investigated through biochemical and PCR-based approaches. The availability of high-quality genomic resources for several fabaceae species, among which the model species Medicago truncatula (Mtr), allowed for a genomic analysis of this protein family aimed at i) deciphering the evolutionary history of A1b proteins and their links with A1b-nodulins that are short non-insecticidal disulfide-bonded peptides involved in root nodule signaling and ii) exploring the functional diversity of A1b for novel bioactive molecules. RESULTS: Investigating the Mtr genome revealed a remarkable expansion, mainly through tandem duplications, of albumin1 (A1) genes, retaining nearly all of the same canonical structure at both gene and protein levels. Phylogenetic analysis revealed that the ancestral molecule was most probably insecticidal giving rise to, among others, A1b-nodulins. Expression meta-analysis revealed that many A1b coding genes are silent and a wide tissue distribution of the A1 transcripts/peptides within plant organs. Evolutionary rate analyses highlighted branches and sites with positive selection signatures, including two sites shown to be critical for insecticidal activity. Seven peptides were chemically synthesized and folded in vitro, then assayed for their biological activity. Among these, AG41 (aka MtrA1013 isoform, encoded by the orphan TA24778 contig.), showed an unexpectedly high insecticidal activity. The study highlights the unique burst of diversity of A1 peptides within the Medicago genus compared to the other taxa for which full-genomes are available: no A1 member in Lotus, or in red clover to date, while only a few are present in chick pea, soybean or pigeon pea genomes. CONCLUSION: The expansion of the A1 family in the Medicago genus is reminiscent of the situation described for another disulfide-rich peptide family, the "Nodule-specific Cysteine-Rich" (NCR), discovered within the same species. The oldest insecticidal A1b toxin was described from the Sophorae, dating the birth of this seed-defense function to more than 58 million years, and making this model of plant/insect toxin/receptor (A1b/insect v-ATPase) one of the oldest known.

Inhibition of a multiproduct terpene synthase from Medicago truncatula by 3-bromoprenyl diphosphates.

The multiproduct sesquiterpene synthase MtTPS5 from Medicago truncatula catalyzes the conversion of farnesyl diphosphate (FDP) into a complex mixture of 27 terpenoids. 3-Bromo substrate analogues of geranyl diphosphate (3-BrGDP) and farnesyl diphosphate (3-BrFDP) were evaluated as substrates of MTPS5 enzyme. Kinetic studies demonstrated that these compounds were highly potent competitive inhibitors of the MtTPS5 enzyme with fast binding and slow reversibility. Since there is a lack of knowledge about the crystal structure of multiproduct terpene synthases, these molecules might be ideal candidates for obtaining a co-crystal structure with multiproduct terpene synthases. Due to the structural and mechanistic similarity between various terpene synthases we expect these 3-bromo isoprenoids to be ideal probes for crystal structure studies.

Effect of cadmium pollution on mobilization of embryo reserves in seedlings of six contrasted Medicago truncatula lines.

Six Medicago truncatula genotypes differing in cadmium susceptibility were used to test the effect of this heavy metal on mineral, carbohydrate and amino acid supply in growing radicles. Cadmium treatment caused alteration of macronutrient (Ca and K), microelement (Fe, Zn and Cu), carbohydrate (total soluble sugars (TSS), glucose, fructose and sucrose) and free amino acid (FAAS) accumulations. These mobilization changes differed in the tested genotypes. Carbohydrates were determining to susceptible lines' growth in control condition; free amino acids enabled tolerant lines to counteract cadmium intrusion. Transcriptional changes in response to cadmium treatment were analyzed on MtMST, a gene encoding a monosaccharide transport protein. A significant down-regulation was observed in the most susceptible line TN1.11. Glucose was over-consumed in tolerant lines. Thus, glucose metabolism integrity seems essential to maintain growth under cadmium exposure. Analysis of germination medium showed solute losses at the expense of suitable mobilization to the growing embryonic axis and highlights cadmium-triggered membrane alterations. FAAS and TSS leakages were reduced in tolerant lines while monosaccharide losses were accentuated in susceptible lines. This research work gave an overview of cadmium deleterious effects on biomass mobilization and membrane integrity. Carbon metabolism is shown to be primordial to enhance early embryonic growth and nitrogen metabolism is revealed to be crucial to establish seedling growth under cadmium stress.