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Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers: Medtr4g107930: CP3; Medtr4g079770: CP4; Medtr5g022560: CP2; Medtr4g079470: CP5; TC106667: Actine; Medtr1g099310.1: PR8; Medtr4g120970.1/Medtr6g033450.1: PR10; Medtr5g010640.1: PR5.3; Medtr8g096910.1: PR5.6; Medtr5g088770.1: PHYTOCYSTATIN32; Medtr2g026040.1: PHYTOCYSTATIN5; Medtr4g085800: DNF2; Medtr3g079850: SymCRK; MtrunA17_Chr7g0239441: RSD. Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers: Medtr4g107930: CP3; Medtr4g079770: CP4; Medtr5g022560: CP2; Medtr4g079470: CP5; TC106667: Actine; Medtr1g099310.1: PR8; Medtr4g120970.1/Medtr6g033450.1: PR10; Medtr5g010640.1: PR5.3; Medtr8g096910.1: PR5.6; Medtr5g088770.1: PHYTOCYSTATIN32; Medtr2g026040.1: PHYTOCYSTATIN5; Medtr4g085800: DNF2; Medtr3g079850: SymCRK; MtrunA17_Chr7g0239441: RSD.; International audience; Medicago (Medicago truncatula) establishes a symbiosis with the rhizobia Sinorhizobium sp, resulting in the formation of nodules where the bacteria fix atmospheric nitrogen. The loss of immunity repression or early senescence activation compromises symbiont survival and leads to the formation of nonfunctional nodules (fix−). Despite many studies exploring an overlap between immunity and senescence responses outside the nodule context, the relationship between these processes in the nodule remains poorly understood. To investigate this phenomenon, we selected and characterized three Medicago mutants developing fix− nodules and showing senescence responses. Analysis of specific defense (PATHOGENESIS-RELATED PROTEIN) or senescence (CYSTEINE PROTEASE) marker expression demonstrated that senescence and immunity seem to be antagonistic in fix− nodules. The growth of senescence mutants on non-sterile (sand/perlite) substrate instead of sterile in vitro conditions decreased nodule senescence and enhanced defense, indicating that environment can affect the immunity/senescence balance. The application of wounding stress on wild-type (WT) fix+ nodules led to the death of intracellular rhizobia and associated with co-stimulation of defense and senescence markers, indicating that in fix+ nodules the relationship between the two processes switches from opposite to synergistic to control symbiont survival during response to the stress. Our data show that the immune response in stressed WT nodules is linked to the repression of DEFECTIVE IN NITROGEN FIXATION 2 (DNF2), Symbiotic CYSTEINE-RICH RECEPTOR-LIKE KINASE (SymCRK), and REGULATOR OF SYMBIOSOME DIFFERENTIATION (RSD), key genes involved in symbiotic immunity suppression. This study provides insight to understand the links between senescence and immunity in Medicago nodules. Analyses of Medicago mutants with non-functional nodules highlight the relationship and mechanisms controlling the establishment of the immune and senescence programs during nodule organogenesis.

Leghemoglobins enable the endosymbiotic fixation of molecular nitrogen (N2) in legume nodules by channeling O2 for bacterial respiration while maintaining a micro-oxic environment to protect O2-sensitive nitrogenase. We found that the NIN-like protein (NLP) transcription factors NLP2 and NIN directly activate the expression of leghemoglobins through a promoter motif, resembling a “double” version of the nitrate-responsive elements (NREs) targeted by other NLPs, that has conserved orientation and position across legumes. CRISPR knockout of the NRE-like element resulted in strongly decreased expression of the associated leghemoglobin. Our findings indicate that the origins of the NLP-leghemoglobin module for O2 buffering in nodules can be traced to an ancient pairing of NLPs with nonsymbiotic hemoglobins that function in hypoxia.

Most legumes can establish a symbiotic association with soil rhizobia that trigger the development of root nodules. These nodules host the rhizobia and allow them to fix nitrogen efficiently. The perception of bacterial lipo-chitooligosaccharides (LCOs) in the epidermis initiates a signaling cascade that allows rhizobial intracellular infection in the root and de-differentiation and activation of cell division that gives rise to the nodule. Thus, nodule organogenesis and rhizobial infection need to be coupled in space and time for successful nodulation. The plant hormone cytokinin (CK) contributes to the coordination of this process, acting as an essential positive regulator of nodule organogenesis. However, the temporal regulation of tissue-specific CK signaling and biosynthesis in response to LCOs or Sinorhizobium meliloti inoculation in Medicago truncatula remains poorly understood. In this study, using a fluorescence-based CK sensor (pTCSn::nls:tGFP), we performed a high-resolution tissue-specific temporal characterization of the sequential activation of CK response during root infection and nodule development in M. truncatula after inoculation with S. meliloti. Loss-of-function mutants of the CK-biosynthetic gene ISOPENTENYLTRANSFERASE 3 (IPT3) showed impairment of nodulation, suggesting that IPT3 is required for nodule development in M. truncatula. Simultaneous live imaging of pIPT3::nls:tdTOMATO and the CK sensor showed that IPT3 induction in the pericycle at the base of nodule primordium contributes to CK biosynthesis, which in turn promotes expression of positive regulators of nodule organogenesis in M. truncatula., Precise spatial and temporal characterization of cytokinin (CK) responses reveals the function of the CK biosynthesis gene ISOPENTENYLTRANSFERASE 3 during nodule development in Medicago truncatula.

Flowering of the reference legume Medicago truncatula is promoted by winter cold (vernalization) followed by long-day photoperiods (VLD) similar to winter annual Arabidopsis. However, Medicago lacks FLC and CO, key regulators of Arabidopsis VLD flowering. Most plants have two INHIBITOR OF GROWTH (ING) genes (ING1 and ING2), encoding proteins with an ING domain with two anti-parallel alpha-helices and a plant homeodomain (PHD) finger, but their genetic role has not been previously described. In Medicago, Mting1 gene-edited mutants developed and flowered normally, but an Mting2-1 Tnt1 insertion mutant and gene-edited Mting2 mutants had developmental abnormalities including delayed flowering particularly in VLD, compact architecture, abnormal leaves with extra leaflets but no trichomes, and smaller seeds and barrels. Mting2 mutants had reduced expression of activators of flowering, including the FT-like gene MtFTa1, and increased expression of the candidate repressor MtTFL1c, consistent with the delayed flowering of the mutant. MtING2 overexpression complemented Mting2-1, but did not accelerate flowering in wild type. The MtING2 PHD finger bound H3K4me2/3 peptides weakly in vitro, but analysis of gene-edited mutants indicated that it was dispensable to MtING2 function in wild-type plants. RNA sequencing experiments indicated that7000 genes are mis-expressed in the Mting2-1 mutant, consistent with its strong mutant phenotypes. Interestingly, ChIP-seq analysis identified5000 novel H3K4me3 locations in the genome of Mting2-1 mutants compared to wild type R108. Overall, our mutant study has uncovered an important physiological role of a plant ING2 gene in development, flowering, and gene expression, which likely involves an epigenetic mechanism.

Yellow Stripe-Like (YSL) proteins are a family of plant transporters that are typically involved in transition metal homeostasis. Three of the four YSL clades (I, II and IV) transport metals complexed with the non-proteinogenic amino acid nicotianamine or its derivatives. No such capability has been shown for any member of clade III, but the link between these YSLs and metal homeostasis could be masked by functional redundancy. We studied the role of the clade III YSL protein MtSYL7 in Medicago truncatula nodules. MtYSL7, which encodes a plasma membrane-bound protein, is mainly expressed in the pericycle and cortex cells of the root nodules. Yeast complementation assays revealed that MtSYL7 can transport short peptides. M. truncatula transposon insertion mutants with decreased expression of MtYSL7 had lower nitrogen fixation rates and showed reduced plant growth whether grown in symbiosis with rhizobia or not. YSL7 mutants accumulated more copper and iron in the nodules, which is likely to result from the increased expression of iron uptake and delivery genes in roots. Taken together, these data suggest that MtYSL7 plays an important role in the transition metal homeostasis of nodules and symbiotic nitrogen fixation.

Many plant species open their leaves during the daytime and close them at night as if sleeping. This leaf movement is known as nyctinasty, a unique and intriguing phenomenon that been of great interest to scientists for centuries. Nyctinastic leaf movement occurs widely in leguminous plants, and is generated by a specialized motor organ, the pulvinus. Although a key determinant of pulvinus development, PETIOLULE-LIKE PULVINUS (PLP), has been identified, the molecular genetic basis for pulvinus function is largely unknown. Here, through an analysis of knockout mutants in barrelclover (Medicago truncatula), we showed that neither altering brassinosteroid (BR) content nor blocking BR signal perception affected pulvinus determination. However, BR homeostasis did influence nyctinastic leaf movement. BR activity in the pulvinus is regulated by a BR-inactivating gene PHYB ACTIVATION TAGGED SUPPRESSOR1 (BAS1), which is directly activated by PLP. A comparative analysis between M. truncatula and the non-pulvinus forming species Arabidopsis and tomato (Solanum lycopersicum) revealed that PLP may act as a factor that associates with unknown regulators in pulvinus determination in M. truncatula. Apart from exposing the involvement of BR in the functionality of the pulvinus, these results have provided insights into whether gene functions among species are general or specialized.

Stipule morphology is a classical botanical key character used in plant identification. Stipules are considerably diverse in size, function and architecture, such as leaf-like stipules, spines or tendrils. However, the molecular mechanism that regulates stipule identity remains largely unknown. We isolated mutants with abnormal stipules. The mutated gene encodes the NODULE ROOT1 (MtNOOT1), which is the ortholog of BLADE-ON-PETIOLE (BOP) in Medicago truncatula. We also obtained mutants of MtNOOT2, the homolog of MtNOOT1, but they do not show obvious defects in stipules. The mtnoot1 mtnoot2 double mutant shows a higher proportion of transformation from stipules to leaflet-like stipules than the single mutants, suggesting that they redundantly determine stipule identity. Further investigations show that MtNOOTs control stipule initiation together with SINGLE LEAFLET1 (SGL1), which functions in development of lateral leaflets. Increasing SGL1 activity in mtnoot1 mtnoot2 is sufficient for the transformation of stipules to leaves. Moreover, MtNOOTs inhibit SGL1 expression during stipule development, which is probably conserved in legume species. Our study proposes a genetic regulatory model for stipule development, specifically with regard to the MtNOOTs-SGL1 module, which functions in two phases of stipule development, first in the control of stipule initiation and second in stipule patterning.

A 3-ketoacyl-CoA synthase involved in biosynthesis of very long chain fatty acids and cuticular wax plays a vital role in aerial organ development in M. truncatula. Cuticular wax is composed of very long chain fatty acids and their derivatives. Defects in cuticular wax often result in organ fusion, but little is known about the role of cuticular wax in compound leaf and flower development in Medicago truncatula. In this study, through an extensive screen of a Tnt1 retrotransposon insertion population in M. truncatula, we identified four mutant lines, named wrinkled flower and leaf (wfl) for their phenotype. The phenotype of the wfl mutants is caused by a Tnt1 insertion in Medtr3g105550, encoding 3-ketoacyl-CoA synthase (KCS), which functions as a rate-limiting enzyme in very long chain fatty acid elongation. Reverse transcription-quantitative PCR showed that WFL was broadly expressed in aerial organs of the wild type, such as leaves, floral organs, and the shoot apical meristem, but was expressed at lower levels in roots. In situ hybridization showed a similar expression pattern, mainly detecting the WFL transcript in epidermal cells of the shoot apical meristem, leaf primordia, and floral organs. The wfl mutant leaves showed sparser epicuticular wax crystals on the surface and increased water permeability compared with wild type. Further analysis showed that in wfl leaves, the percentage of C20:0, C22:0, and C24:0 fatty acids was significantly increased, the amount of cuticular wax was markedly reduced, and wax constituents were altered compared to the wild type. The reduced formation of cuticular wax and wax composition changes on the leaf surface might lead to the developmental defects observed in the wfl mutants. These findings suggest that WFL plays a key role in cuticular wax formation and in the late stage of leaf and flower development in M. truncatula.

As sessile organisms, plants perceive, respond, and adapt to the environmental changes for optimal growth and survival. The plant growth and fitness are enhanced by circadian clocks through coordination of numerous biological events. In legume species, nitrogen-fixing root nodules were developed as the plant organs specialized for symbiotic transfer of nitrogen between microsymbiont and host. Here, we report that the endogenous circadian rhythm in nodules is regulated by MtLHY in legume species Medicago truncatula. Loss of function of MtLHY leads to a reduction in the number of nodules formed, resulting in a diminished ability to assimilate nitrogen. The operation of the 24-h rhythm in shoot is further influenced by the availability of nitrogen produced by the nodules, leading to the irregulated nyctinastic leaf movement and reduced biomass in mtlhy mutants. These data shed new light on the roles of MtLHY in the orchestration of circadian oscillator in nodules and shoots, which provides a mechanistic link between nodulation, nitrogen assimilation, and clock function.

Necrotrophic fungi constitute the largest group of plant fungal pathogens that cause heavy crop losses worldwide. Phymatotrichopsis omnivora is a broad host, soil‐borne necrotrophic fungal pathogen that infects over 2,000 dicotyledonous plants. The molecular basis of such broad host range is unknown. We conducted cell biology and transcriptomic studies in Medicago truncatula (susceptible), Brachypodium distachyon (resistant/nonhost), and Arabidopsis thaliana (partially resistant) to understand P. omnivora virulence mechanisms. We performed defence gene analysis, gene enrichments, and correlational network studies during key infection stages. We identified that P. omnivora infects the susceptible plant as a traditional necrotroph. However, it infects the partially resistant plant as a hemi‐biotroph triggering salicylic acid‐mediated defence pathways in the plant. Further, the infection strategy in partially resistant plants is determined by the host responses during early infection stages. Mutant analyses in A. thaliana established the role of small peptides PEP1 and PEP2 in defence against P. omnivora. The resistant/nonhost B. distachyon triggered stress responses involving sugars and aromatic acids. Bdwat1 mutant analysis identified the role of cell walls in defence. This is the first report that describes the plasticity in infection strategies of P. omnivora providing insights into broad host range., Phymatotrichopsis omnivora can amend its infection strategy between necrotrophy and hemi‐biotrophy in dicotyledonous plants based on host defences, which indicates the basis of its broad host range.

Arbuscular mycorrhizal fungi help their host plant in the acquisition of nutrients, and this association is itself impacted by soil nutrient levels. High phosphorus levels inhibit the symbiosis, whereas high nitrogen levels enhance it. The genetic mechanisms regulating the symbiosis in response to soil nutrients are poorly understood. Here, we characterised the symbiotic phenotypes in four Medicago truncatula Tnt1-insertion mutants affected in arbuscular mycorrhizal colonisation. We located their Tnt1 insertions and identified alleles for two genes known to be involved in mycorrhization, RAM1 and KIN3. We compared the effects of the kin3-2 and ram1-4 mutations on gene expression, revealing that the two genes alter the expression of overlapping but not identical gene sets, suggesting that RAM1 acts upstream of KIN3. Additionally, KIN3 appears to be involved in the suppression of plant defences in response to the fungal symbiont. KIN3 is located on the endoplasmic reticulum of arbuscule-containing cortical cells, and kin3-2 mutants plants hosted significantly fewer arbuscules than the wild type. KIN3 plays an essential role in the symbiotic response to soil nitrogen levels, as, contrary to wild-type plants, the kin3-2 mutant did not exhibit increased root colonisation under high nitrogen.

Phytosulfokine-α (PSK-α), a tyrosine-sulfated pentapeptide with the sequence YSO3IYSO3TQ, is widely distributed across the plant kingdom and plays multiple roles in plant growth, development, and immune response. Here, we report a novel type of phytosulfokine, PSK-δ, and its precursor proteins (MtPSKδ, LjPSKδ, and GmPSKδ1), specifically from legume species. The sequence YSO3IYSO3TN of sulfated PSK-δ peptide is different from PSK-α at the last amino acid. Expression pattern analysis revealed PSK-δ-encoding precursor genes to be expressed primarily in legume root nodules. Specifically, in Medicago truncatula, MtPSKδ expression was detected in root cortical cells undergoing nodule organogenesis, in nodule primordia and young nodules, and in the apical region of mature nodules. Accumulation of sulfated PSK-δ peptide in M. truncatula nodules was detected by LC/MS. Application of synthetic PSK-δ peptide significantly increased nodule number in legumes. Similarly, overexpression of MtPSKδ in transgenic M. truncatula markedly promoted symbiotic nodulation. This increase in nodule number was attributed to enhanced nodule organogenesis induced by PSK-δ. Additional genetic evidence from the MtPSKδ mutant and RNA interference assays suggested that the PSK-δ and PSK-α peptides function redundantly in regulating nodule organogenesis. These results suggest that PSK-δ, a legume-specific novel type of phytosulfokine, promotes symbiotic nodulation by enhancing nodule organogenesis.

The preference for nitrate over chloride through regulation of transporters is a fundamental feature of plant ion homeostasis. We show that Medicago truncatula MtNPF6.5, an ortholog of Arabidopsis thaliana AtNPF6.3/NRT1.1, can mediate nitrate and chloride uptake in Xenopus oocytes but is chloride selective and that its close homologue, MtNPF6.7, can transport nitrate and chloride but is nitrate selective. The MtNPF6.5 mutant showed greatly reduced chloride content relative to wild type, and MtNPF6.5 expression was repressed by high chloride, indicating a primary role for MtNPF6.5 in root chloride uptake. MtNPF6.5 and MtNPF6.7 were repressed and induced by nitrate, respectively, and these responses required the transcription factor MtNLP1. Moreover, loss of MtNLP1 prevented the rapid switch from chloride to nitrate as the main anion in nitrate-starved plants after nitrate provision, providing insight into the underlying mechanism for nitrate preference. Sequence analysis revealed three sub-types of AtNPF6.3 orthologs based on their predicted substrate-binding residues: A (chloride selective), B (nitrate selective), and C (legume specific). The absence of B-type AtNPF6.3 homologues in early diverged plant lineages suggests that they evolved from a chloride-selective MtNPF6.5-like protein.

In almost all symbiotic interactions between rhizobia and leguminous plants, host flavonoid–induced synthesis of Nod factors in rhizobia is required to initiate symbiotic response in plants. In this study, we found that Lotus japonicus Nod factor receptor 5 (LjNFR5) might directly regulate flavonoid biosynthesis during symbiotic interaction with rhizobia. A yeast two-hybrid analysis revealed that a dihydroflavonol-4-reductase-like protein (LjDFL1) interacts with LjNFR5. The interaction between MtDFL1 and MtNFP, two Medicago truncatula proteins with homology to LjDFL1 and LjNFR5, respectively, was also shown, suggesting that interaction between these two proteins might be conserved in different legumes. LjDFL1 was highly expressed in root hairs and epidermal cells of root tips. Lotus ljdfl1 mutants and Medicago mtdfl1 mutants produced significantly fewer infection threads (ITs) than the wild-type control plants following rhizobial treatment. Furthermore, the roots of stable transgenic L. japonicus plants overexpressing LjDFL1 formed more ITs than control roots after exposure to rhizobia. These data indicated that LjDFL1 is a positive regulator of symbiotic signaling. However, the expression of LjDFL1 was suppressed by rhizobial treatment, suggesting that a negative feedback loop might be involved in regulation of the symbiotic response in L. japonicus.

From a single transgenic line harboring five Tnt1 transposon insertions, we generated a near-saturated insertion population in Medicago truncatula. Using thermal asymmetric interlaced-polymerase chain reaction followed by sequencing, we recovered 388 888 flanking sequence tags (FSTs) from 21 741 insertion lines in this population. FST recovery from 14 Tnt1 lines using the whole-genome sequencing (WGS) and/or Tnt1-capture sequencing approaches suggests an average of 80 insertions per line, which is more than the previous estimation of 25 insertions. Analysis of the distribution pattern and preference of Tnt1 insertions showed that Tnt1 is overall randomly distributed throughout the M. truncatula genome. At the chromosomal level, Tnt1 insertions occurred on both arms of all chromosomes, with insertion frequency negatively correlated with the GC content. Based on 174 546 filtered FSTs that show exact insertion locations in the M. truncatula genome version 4.0 (Mt4.0), 0.44 Tnt1 insertions occurred per kb, and 19 583 genes contained Tnt1 with an average of 3.43 insertions per gene. Pathway and gene ontology analyses revealed that Tnt1-inserted genes are significantly enriched in processes associated with 'stress', 'transport', 'signaling' and 'stimulus response'. Surprisingly, gene groups with higher methylation frequency were more frequently targeted for insertion. Analysis of 19 583 Tnt1-inserted genes revealed that 59% (1265) of 2144 transcription factors, 63% (765) of 1216 receptor kinases and 56% (343) of 616 nucleotide-binding site-leucine-rich repeat genes harbored at least one Tnt1 insertion, compared with the overall 38% of Tnt1-inserted genes out of 50 894 annotated genes in the genome.

Floral development is one of the model systems for investigating the mechanisms underlying organogenesis in plants. Floral organ identity is controlled by the well-known ABC model, which has been generalized to many flowering plants. Here, we report a previously uncharacterized MYB-like gene, AGAMOUS-LIKE FLOWER (AGLF), involved in flower development in the model legume Medicago truncatula. Loss-of-function of AGLF results in flowers with stamens and carpel transformed into extra whorls of petals and sepals. Compared with the loss-of-function mutant of the class C gene AGAMOUS (MtAG) in M. truncatula, the defects in floral organ identity are similar between aglf and mtag, but the floral indeterminacy is enhanced in the aglf mutant. Knockout of AGLF in the mutants of the class A gene MtAP1 or the class B gene MtPI leads to an addition of a loss-of-C-function phenotype, reflecting a conventional relationship of AGLF with the canonical A and B genes. Furthermore, we demonstrate that AGLF activates MtAG in transcriptional levels in control of floral organ identity. These data shed light on the conserved and diverged molecular mechanisms that control flower development and morphology among plant species.

Medicago truncatula is a model system for legume plants, which has substantially expanded the genome relative to the prototypical model dicot plant, Arabidopsis thaliana. An essential transcriptional regulator, FCP1 (transcription factor IIF-interacting RNA polymerase II carboxyl-terminal phosphatase 1) ortholog, is encoded by a single essential gene CPL4 (CTD-phosphatase-like 4), whereas M. truncatula genome contains four genes homologous to FCP1/AtCPL4, and splicing variants of MtCPL4 are observed. Functional diversification of MtCPL4 family proteins was analyzed using recombinant proteins (MtCPL4a1, MtCPL4a2, and MtCPL4b) produced in Arabidopsis cell culture system developed for plant protein overexpression. In vitro CTD phosphatase assay using highly purified MtCPL4 preparations revealed a potent CTD phosphatase activity in MtCPL4b, but not two splicing variants of MtCPL4a. On the other hand, in planta binding assay to RNA polymerase II (pol II) revealed a greater pol II-binding activity of both MtCPL4a variants. Our results indicate functional diversification of MtCPL4 isoforms and suggest the presence of a large number of functionally specialized CTD-phosphatase-like proteins in plants.

Plant leaves have evolved into diverse shapes and LATE MERISTEM IDENTITY1 (LMI1) and its putative paralogous genes encode homeodomain leucine zipper transcription factors that are proposed evolutionary hotspots for the regulation of leaf development in plants. However, the LMI1-mediated regulatory mechanism underlying leaf shape formation is largely unknown. MtLMI1a and MtLMI1b are putative orthologs of LMI1 in the model legume barrelclover (Medicago truncatula). Here, we investigated the role of MtLMI1a and MtLMI1b in leaf margin morphogenesis by characterizing loss-of-function mutants. MtLMI1a and MtLMI1b are expressed along leaf margin in a near-complementary pattern, and they redundantly promote development of leaf margin serrations, as revealed by the relatively smooth leaf margin in their double mutants. Moreover, MtLMI1s directly activate expression of SMOOTH LEAF MARGIN1 (SLM1), which encodes an auxin efflux carrier, thereby regulating auxin distribution along the leaf margin. Further analysis indicates that MtLMI1s genetically interact with NO APICAL MERISTEM (MtNAM) and the ARGONAUTE7 (MtAGO7)-mediated trans-acting short interfering RNA3 (TAS3 ta-siRNA) pathway to develop the final leaf margin shape. The participation of MtLMI1s in auxin-dependent leaf margin formation is interesting in the context of functional conservation. Furthermore, the diverse expression patterns of LMI1s and their putative paralogs within key domains are important drivers for functional specialization, despite their functional equivalency among species.

Most legumes can establish a symbiotic association with soil rhizobia that triggers the development of root nodules. These nodules host the rhizobia and allow them to fix nitrogen efficiently. The perception of bacterial lipo-chitooligosaccharide (LCO) signal in the epidermis initiates a signaling cascade that allows rhizobial intracellular infection in the root and de-differentiation and activation of cell division that gives rise to the nodule. Nodule organogenesis and rhizobial infection need to be coupled in space and time for successful nodulation. The plant hormone cytokinin (CK) acts as an essential positive regulator of nodule organogenesis, and specific CK receptors are required for nodule formation. Temporal regulation of tissue-specific CK signaling and biosynthesis in response to LCOs or Sinorhizobium meliloti inoculation in Medicago truncatula remains poorly understood. In the present study, using a fluorescence-based CK sensor (TCSn::nls:tGFP), we performed a high-resolution tissue-specific temporal characterization of the CK response’s sequential activation during root infection and nodule development in M. truncatula after inoculation with S. meliloti. Loss-of-function mutants of the CK-biosynthetic gene ISOPENTENYL TRANSFERASE 3 (IPT3) showed impairment of nodulation, suggesting that IPT3 is required for nodule development in M. truncatula. Simultaneous live imaging of pIPT3::tdTOMATO and the CK sensor showed that IPT3 induction in the root stele at the base of nodule primordium contributes to CK biosynthesis, which in turn promotes expression of positive regulators of nodule organogenesis in M. truncatula.One-sentence summaryHigh-resolution spatiotemporal imaging of cytokinin signaling reveals IPT3 function during indeterminate nodule development in Medicago truncatula

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.

Flowering time influences the yield and productivity of legume crops. Medicago truncatula is a reference temperate legume that, like the winter annual Arabidopsis thaliana, shows accelerated flowering in response to vernalization (extended cold) and long-day (LD) photoperiods (VLD). However, unlike A. thaliana, M. truncatula appears to lack functional homologs of core flowering time regulators CONSTANS (CO) and FLOWERING LOCUS C (FLC) which act upstream of the mobile florigen FLOWERING LOCUS T (FT). Medicago truncatula has three LD-induced FT-like genes (MtFTa1, MtFTb1, and MtFTb2) with MtFTa1 promoting M. truncatula flowering in response to VLD. Another photoperiodic regulator in A. thaliana, FE, acts to induce FT expression. It also regulates the FT transport pathway and is required for phloem development. Our study identifies a M. truncatula FE homolog Medtr6g444980 (MtFE) which complements the late flowering fe-1 mutant when expressed from the phloem-specific SUCROSE-PROTON SYMPORTER 2 (SUC2) promoter. Analysis of two M. truncatula Tnt1 insertional mutants indicate that MtFE promotes flowering in LD and VLD and growth in all conditions tested. Expression of MtFTa1, MtFTb1, and MtFTb2 are reduced in Mtfe mutant (NF5076), correlating with its delayed flowering. The NF5076 mutant plants are much smaller than wild type indicating that MtFE is important for normal plant growth. The second mutant (NF18291) displays seedling lethality, like strong fe mutants. We searched for mutants in MtFTb1 and MtFTb2 identifying a Mtftb2 knock out Tnt1 mutant (NF20803). However, it did not flower significantly later than wild type. Previously, yeast-two-hybrid assays (Y2H) suggested that Arabidopsis FE interacted with CO and NUCLEAR FACTOR-Y (NF-Y)-like proteins to regulate FT. We found that MtFE interacts with CO and also M. truncatula NF-Y-like proteins in Y2H experiments. Our study indicates that despite the apparent absence of a functional MtCO-like gene, M. truncatula FE likely influences photoperiodic FT expression and flowering time in M. truncatula via a partially conserved mechanism with A. thaliana.

Legumes house nitrogen-fixing endosymbiotic rhizobia in specialized polyploid cells within root nodules, which are factories of metabolic activity. We discovered that the circadian clock-associated transcriptional factor LATE ELONGATED HYPOCOTYL (LHY) affects nodulation in Medicago truncatula. By carrying out expression analysis of transcripts over time in nodules we found that the clock enables coordinated control of metabolic and regulatory processes linked to nitrogen fixation. Rhythmic transcripts in root nodules include a subset of Nodule-specific Cysteine Rich peptides (NCRs) that have the LHY-bound conserved Evening Element in their promoters. Until now, studies have suggested that NCRs act to regulate bacteroid differentiation and keep the rhizobial population in check. However, these conclusions came from the study of a few members of this very large gene family that has complex diversified spatio-temporal expression. We suggest that rhythmic expression of NCRs may be important for temporal coordination of bacterial activity with the rhythms of the plant host, in order to ensure optimal symbiosis.HighlightsThe circadian clock-associated transcriptional factor LATE ELONGATED HYPOCOTYL (LHY) impacts on successful Medicago truncatula-rhizobial symbiosisThe plant clock coordinates rhythmic patterns of metabolic and regulatory activity in nodules and drives rhythmic expression of a subset of Nodule-specific Cysteine Rich (NCR) genes.Rhythmic expression of NCRs may be important for temporal coordination of bacterial activity with plant host rhythms to ensure optimal symbiosis.

Flowering plants display a vast diversity of flowering time and inflorescence architecture, which plays an important role in determining seed yield and fruit production. However, the molecular mechanism underlying the flowering control and compound inflorescence development, especially in legumes, remain to be elucidated. Here, we reported the identification of MtFDa, an essential regulator of flowering in the model legume Medicago truncatula. Mutation of MtFDa, led to the late flowering, abnormal secondary inflorescences as well as severe floral organ defects. Biochemical and molecular analyses revealed that MtFDa physically interacts with M. truncaula FLOWERING LOCUS T homolog, MtFTa1, a key regulator of Medicago flowering time, and this interaction facilitates MtFDa's function in activating the expression of MtSOC1a. Moreover, we demonstrated that MtFDa may affect secondary inflorescence development via regulating MtFULc expression in M. truncatula. Our findings help elucidate the mechanism of MtFDa-mediated regulation of flowering time and inflorescence development and provide insights into understanding the genetic regulatory network underlying complex productive development in legumes.

Polar auxin transport mediated by PIN-FORMED (PIN) proteins is critical for plant growth and development. As an environmental cue, shade stimulates hypocotyls, petiole, and stem elongation by inducing auxin synthesis and asymmetric distributions, which is modulated by PIN3,4,7 in Arabidopsis. Here, we characterize the MtPIN1 and MtPIN3, which are the orthologs of PIN3,4,7, in model legume species Medicago truncatula. Under the low Red:Far-Red (R:FR) ratio light, the expression of MtPIN1 and MtPIN3 is induced, and shadeavoidance response is disrupted in mtpin1 mtpin3 double mutant, indicating that MtPIN1 and MtPIN3 have a conserved function in shade response. Surprisingly, under the normal growth condition, mtpin1 mtpin3 displayed the constitutive shade avoidance responses, such as the elongated petiole, smaller leaf, and increased auxin and chlorophyll content. Therefore, MtPIN1 and MtPIN3 play dual roles in regulation of shadeavoidance response under different environments. Furthermore, these data suggest that PIN3,4,7 and its orthologs have evolved conserved and specific functions among species.

Patterned leaf coloration in plants generates remarkable diversity in nature, but the underlying mechanisms remain largely unclear. Here, using Medicago truncatula leaf marking as a model, we show that the classic M. truncatula leaf anthocyanin spot trait depends on two R2R3 MYB paralogous regulators, RED HEART1 (RH1) and RH2. RH1 mainly functions as an anthocyanin biosynthesis activator that specifically determines leaf marking formation depending on its C‐terminal activation motif. RH1 physically interacts with the M. truncatula bHLH protein MtTT8 and the WDR family member MtWD40‐1, and this interaction facilitates RH1 function in leaf anthocyanin marking formation. RH2 has lost transcriptional activation activity, due to a divergent C‐terminal domain, but retains the ability to interact with the same partners, MtTT8 and MtWD40‐1, as RH1, thereby acting as a competitor in the regulatory complex and exerting opposite effects. Moreover, our results demonstrate that RH1 can activate its own expression and that RH2‐mediated competition can repress RH1 expression. Our findings reveal the molecular mechanism of the antagonistic gene paralogs RH1 and RH2 in determining anthocyanin leaf markings in M. truncatula, providing a multidimensional paralogous–antagonistic regulatory paradigm for fine‐tuning patterned pigmentation.

13 Pags.- 5 Figs. © The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology., Symbiotic nitrogen fixation carried out in legume root nodules requires transition metals. These nutrients are delivered by the host plant to the endosymbiotic nitrogen-fixing bacteria living within the nodule cells, a process in which vascular transport is essential. As members of the Yellow Stripe-Like (YSL) family of metal transporters are involved in root to shoot transport, they should also be required for root to nodule metal delivery. The genome of the model legume Medicago truncatula encodes eight YSL proteins, four of them with a high degree of similarity to Arabidopsis thaliana YSLs involved in long-distance metal trafficking. Among them, MtYSL3 is a plasma membrane protein expressed by vascular cells in roots and nodules and by cortical nodule cells. Reducing the expression level of this gene had no major effect on plant physiology when assimilable nitrogen was provided in the nutrient solution. However, nodule functioning was severely impaired, with a significant reduction of nitrogen fixation capabilities. Further, iron and zinc accumulation and distribution changed. Iron was retained in the apical region of the nodule, while zinc became strongly accumulated in the nodule veins in the ysl3 mutant. These data suggest a role for MtYSL3 in vascular delivery of iron and zinc to symbiotic nitrogen fixation., This research was funded by a European Research Council Starting Grant (ERC-2013-StG-335284) and the Spanish State Research Agency (AEI) grant (AGL2015-65866-P) to MGG, and a AEI grant (AGL2016-75226-R) to JA and AA-F co-financed by the European Regional Development Fund (FEDER). RC-R and FJJ-P were supported by a Formación del Personal Investigador fellowship (BES2013-062674 and BES-2017-082913, respectively). IA was the recipient of a Juan de la Cierva- Formación postdoctoral fellowship from Ministerio de Ciencia, Innovación y Universidades (FJCI-2017- 33222). VE was partially funded by the Severo Ochoa Programme for Centres of Excellence in R&D from the AEI (grant SEV-2016- 0672) received by the Centro de Biotecnología y Genómica de Plantas (UPM-INIA). Development of the M. truncatula Tnt1 mutant population was, in part, funded by the National Science Foundation, USA (DBI-0703285) to KSM. AM and HK and the µXRF measurements were supported by the Ministry of Education, Youth and Sports of the Czech Republic with co-financing from the European Union (grant ‘KOROLID’, CZ.02.1.01/0.0/0.0/15_003/0000336) and the Czech Academy of Sciences (RVO: 60077344).

l‐Tyrosine (Tyr) is an aromatic amino acid synthesized de novo in plants and microbes downstream of the shikimate pathway. In plants, Tyr and a Tyr pathway intermediate, 4‐hydroxyphenylpyruvate (HPP), are precursors to numerous specialized metabolites, which are crucial for plant and human health. Tyr is synthesized in the plastids by a TyrA family enzyme, arogenate dehydrogenase (ADH/TyrAa), which is feedback inhibited by Tyr. Additionally, many legumes possess prephenate dehydrogenases (PDH/TyrAp), which are insensitive to Tyr and localized to the cytosol. Yet the role of PDH enzymes in legumes is currently unknown. This study isolated and characterized Tnt1‐transposon mutants of MtPDH1 (pdh1) in Medicago truncatula to investigate PDH function. The pdh1 mutants lacked PDH transcript and PDH activity, and displayed little aberrant morphological phenotypes under standard growth conditions, providing genetic evidence that MtPDH1 is responsible for the PDH activity detected in M. truncatula. Though plant PDH enzymes and activity have been specifically found in legumes, nodule number and nitrogenase activity of pdh1 mutants were not significantly reduced compared with wild‐type (Wt) during symbiosis with nitrogen‐fixing bacteria. Although Tyr levels were not significantly different between Wt and mutants under standard conditions, when carbon flux was increased by shikimate precursor feeding, mutants accumulated significantly less Tyr than Wt. These data suggest that MtPDH1 is involved in Tyr biosynthesis when the shikimate pathway is stimulated and possibly linked to unidentified legume‐specific specialized metabolism.

International audience; The formation of nitrogen-fixing nodules on legume hosts is a finely tuned process involving many components of both symbiotic partners. Production of the exopolysaccharide succinoglycan by the nitrogen-fixing bacterium Sinorhizobium meliloti 1021 is needed for an effective symbiosis with Medicago spp., and the succinyl modification to this polysaccharide is critical. However, it is not known when succinoglycan intervenes in the symbiotic process, and it is not known whether the plant lysin-motif receptor-like kinase MtLYK10 intervenes in recognition of succinoglycan, as might be inferred from work on the Lotus japonicus MtLYK10 ortholog, LjEPR3. We studied the symbiotic infection phenotypes of S. meliloti mutants deficient in succinoglycan production or producing modified succinoglycan, in wild-type Medicago truncatula plants and in Mtlyk10 mutant plants. On wild-type plants, S. meliloti strains producing no succinoglycan or only unsuccinylated succinoglycan still induced nodule primordia and epidermal infections, but further progression of the symbiotic process was blocked. These S. meliloti mutants induced a more severe infection phenotype on Mtlyk10 mutant plants. Nodulation by succinoglycan-defective strains was achieved by in trans rescue with a Nod factor-deficient S. meliloti mutant. While the Nod factor-deficient strain was always more abundant inside nodules, the succinoglycan-deficient strain was more efficient than the strain producing only unsuccinylated succinoglycan. Together, these data show that succinylated succinoglycan is essential for infection thread formation in M. truncatula, and that MtLYK10 plays an important, but different role in this symbiotic process. These data also suggest that succinoglycan is more important than Nod factors for bacterial survival inside nodules.

Yellow Stripe-Like (YSL) proteins are a family of plant transporters typically involved in transition metal homeostasis. The substrate of three of the four YSL clades (clades I, II, and IV) are metal complexes with non-proteinogenic amino acid nicotianamine or its derivatives. No such transport capabilities have been shown for any member of the remaining clade (clade III), which is able to translocate short peptides across the membranes instead. The connection between clade III YSL members and metal homeostasis might have been masked by the functional redundancy characteristic of this family. This might have been circumvented in legumes through neofunctionalization of YSLs to ensure a steady supply of transition metals for symbiotic nitrogen fixation in root nodules. To test this possibility,Medicago truncatulaclade III transporter MtYSL7 has been studied both when the plant was fertilized with ammonium nitrate or when nitrogen had to be provided by endosymbiotic rhizobia within the root nodules. MtYSL7 is a plasma membrane protein expressed in the vasculature and in the nodule cortex. This protein is able to transport short peptides into the cytosol, although none with known metal homeostasis roles. ReducingMtYSL7expression levels resulted in diminished nitrogen fixation rates. In addition, nodules of mutant lines lackingYSL7accumulated more copper and iron, the later the likely result of increased expression in roots of iron uptake and delivery genes. The available data is indicative of a role of MtYSL7, and likely other clade III YSLs, in transition metal homeostasis.ONE SENTENCE SUMMARYMedicago truncatulaYSL7 is a peptide transporter required for symbiotic nitrogen fixation in legume nodules, likely controlling transition metal allocation to these organs.

CLE53 negatively regulates root colonization with arbuscular mycorrhizal fungi in a SUNN- and RDN1-dependent manner., Plants and arbuscular mycorrhizal fungi (AMF) engage in mutually beneficial symbioses based on a reciprocal exchange of nutrients. The beneficial character of the symbiosis is maintained through a mechanism called autoregulation of mycorrhization (AOM). AOM includes root-to-shoot-to-root signaling; however, the molecular details of AOM are poorly understood. AOM shares many features of autoregulation of nodulation (AON) where several genes are known, including the receptor-like kinase SUPER NUMERIC NODULES (SUNN), root-to-shoot mobile CLAVATA3/ENDOSPERM SURROUNDING REGION (ESR)-RELATED (CLE) peptides, and the hydroxyproline O-arabinosyltransferase ROOT DETERMINED NODULATION1 (RDN1) required for post-translational peptide modification. In this work, CLE53 was identified to negatively regulate AMF symbiosis in a SUNN- and RDN1-dependent manner. CLE53 expression was repressed at low phosphorus, while it was induced by AMF colonization and high phosphorus. CLE53 overexpression reduced AMF colonization in a SUNN- and RDN1 dependent manner, while cle53, rdn1, and sunn mutants were more colonized than the wild type. RNA-sequencing identified 700 genes with SUNN-dependent regulation in AMF-colonized plants, providing a resource for future identification of additional AOM genes. Disruption of AOM genes in crops potentially constitutes a novel route for improving AMF-derived phosphorus uptake in agricultural systems with high phosphorus levels.

Iron is an essential cofactor for symbiotic nitrogen fixation. It is required by many of the enzymes facilitating the conversion of N2into NH4+by endosymbiotic bacteria living within root nodule cells, including signal transduction proteins, O2homeostasis systems, and nitrogenase itself. Consequently, host plants have developed a transport network to deliver essential iron to nitrogen-fixing nodule cells. Model legumeMedicago truncatula Ferroportin2(MtFPN2) is a nodule-specific gene that encodes an iron-efflux protein. MtFPN2 is located in intracellular membranes in the nodule vasculature, and in the symbiosome membranes that contain the nitrogen-fixing bacteria in the differentiation and early-fixation zones of the nodules. Loss-of-function ofMtFPN2leads to altered iron distribution and speciation in nodules, which causes a reduction in nitrogenase activity and in biomass production. Using promoters with different tissular activity to driveMtFPN2expression inMtFPN2mutants, we determined that MtFPN2-facilitated iron delivery across symbiosomes is essential for symbiotic nitrogen fixation, while its presence in the vasculature does not seem to play a major role in in the conditions tested.

[EN] Legumes have unique features, such as compound inflorescences and a complex floral ontogeny. Thus, the study of regulatory genes in these species during inflorescence and floral development is essential to understand their role in the evolutionary origin of developmental novelties. The SUPERMAN (SUP) gene encodes a C2H2 zinc-finger transcriptional repressor that regulates the floral organ number in the third and fourth floral whorls of Arabidopsis thaliana. In this work, we present the functional characterization of the Medicago truncatula SUPERMAN (MtSUP) gene based on gene expression analysis, complementation and overexpression assays, and reverse genetic approaches. Our findings provide evidence that MtSUP is the orthologous gene of SUP in M. truncatula. We have unveiled novel functions for a SUP-like gene in eudicots. MtSUP controls not only the number of floral organs in the inner two whorls, but also in the second whorl of the flower. Furthermore, MtSUP regulates the activity of the secondary inflorescence meristem, thus controlling the number of flowers produced. Our work provides insight into the regulatory network behind the compound inflorescence and flower development in this angiosperm family., This work was supported by a grant from the Spanish Ministry of Economy and Competitiveness (MINECO; BIO2016-75485-R). A.L.R. acknowledges a Santiago Grisolia fellowship (GRISOLIA 2017/168) from the Generalitat Valenciana. We thank Dr Beatriz Sabater (IBMCP, Valencia) for valuable suggestions and comments regarding evolutionary biology and Dr Javier Forment, head of the Bioinformatics Core Service at the IBMCP, for providing data from the OrthoVenn web platform. We thank Dr Lynne Yenush (IBMCP, Valencia) for English correction, and Dr Miguel A. Blazquez (IBMCP, Valencia) and Dr Javier Paz-Ares (CNB, Madrid) for critical reading of the manuscript. We would also like to acknowledge the technical assistance of Maria Victoria Palau in the glasshouse and Marisol Gascon in microscopy. Document

Legume plants can assimilate inorganic nitrogen and have access to fixed nitrogen through symbiotic interaction with diazotrophic bacteria called rhizobia. Symbiotic nitrogen fixation is an energy-consuming process and is strongly inhibited when sufficient levels of fixed nitrogen are available, but the molecular mechanisms governing this regulation are largely unknown. The transcription factor nodule inception (NIN) is strictly required for nodulation and belongs to a family of NIN-like proteins (NLPs), which have been implicated in the regulation of nitrogen homeostasis in Arabidopsis. Here, we show that mutation or downregulation of NLP genes prevents nitrate inhibition of infection, nodule formation and nitrogen fixation. We find that NIN and NLPs physically interact through their carboxy-terminal PB1 domains. Furthermore, we find that NLP1 is required for the expression of nitrate-responsive genes and that nitrate triggers NLP1 re-localization from the cytosol to the nucleus. Finally, we show that NLP1 can suppress NIN activation of CRE1 expression in Nicotiana benthamiana and Medicago truncatula. Our findings highlight a central role for NLPs in the suppression of nodulation by nitrate.

The Acyl-Activating Enzyme (AAE) 3 gene encodes an oxalyl-CoA synthetase that catalyzes the conversion of oxalate to oxalyl-CoA in a CoA and ATP-dependent manner. Although the biochemical activity of AAE3 has been established, its biological role in plant growth and development remains unclear. To advance our understanding of the role of AAE3 in plant growth and development, we report here the characterization of two Medicago truncatula AAE3 (Mtaae3) mutants. Characterization of a Mtaae3 RNAi mutant revealed an accumulation of calcium oxalate crystals and increased seed permeability. These phenotypes were also exhibited in the Arabidopsis aae3 (Ataae3) mutants. Unlike the Ataae3 mutants, the Mtaae3 RNAi mutant did not show a reduction in vegetative growth, decreased seed germination, or increased seed calcium concentration. In an effort to clarify these phenotypic differences, a Mtaae3 Tnt1 mutant was identified and characterized. This Mtaae3 Tnt1 mutant displayed reduced vegetative growth, decreased seed germination, and increased seed calcium concentration as well as an accumulation of calcium oxalate crystals and increased seed permeability as found in Ataae3. Overall, the results presented here show the importance of AAE3 in the growth and development of plants. In addition, this study highlights the ability to separate specific growth and development phenotypes based on the level of AAE3 gene expression.

The WOX family transcription factor HEADLESS exhibits a repressive activity and performs conserved and novel functions in the regulation of shoot meristems and leaf shape in Medicago truncatula., The formation and maintenance of the shoot apical meristem (SAM) are critical for plant development. However, the underlying molecular mechanism of regulating meristematic cell activity is poorly understood in the model legume Medicago truncatula. Using forward genetic approaches, we identified HEADLESS (HDL), a homolog of Arabidopsis WUSCHEL, required for SAM maintenance and leaf development in M. truncatula. Disruption of HDL led to disorganized specification and arrest of the SAM and axillary meristems, resulting in the hdl mutant being locked in the vegetative phase without apparent stem elongation. hdl mutant leaves are shorter in the proximal–distal axis due to reduced leaf length elongation, which resulted in a higher blade width/length ratio and altered leaf shape, uncovering novel phenotypes undescribed in the Arabidopsis wus mutant. HDL functions as a transcriptional repressor by recruiting MtTPL through its conserved WUS-box and EAR-like motif. Further genetic analysis revealed that HDL and STENOFOLIA (STF), a key regulator of M. truncatula lamina outgrowth, act independently in leaf development although HDL could recruit MtTPL in the same manner as STF does. Our results indicate that HDL has conserved and novel functions in regulating shoot meristems and leaf shape in M. truncatula, providing new avenues for understanding meristem biology and plant development.

27 Symbiotic nitrogen fixation in legume root nodules requires a steady supply of 28 molybdenum for synthesis of the iron-molybdenum cofactor of nitrogenase. This nutrient 29 has to be provided by the host plant from the soil, crossing several symplastically 30 disconnected compartments through molybdate transporters, including members of the 31 MOT1 family. MtMOT1.2 is a Medicago truncatula MOT1 family member located in 32 the endodermal cells in roots and nodules. Immunolocalization of a tagged MtMOT1.2 33 indicates that it is associated to the plasma membrane and to intracellular membrane 34 systems, where it would be transporting molybdate towards the cytosol, as indicated in 35 yeast transport assays. Loss-of-function mot1.2-1 mutant showed reduced growth 36 compared to wild-type plants when nitrogen fixation was required, but not when nitrogen 37 was provided as nitrate. While no effect on molybdenum-dependent nitrate reductase 38 activity was observed, nitrogenase activity was severely affected, explaining the observed 39 difference of growth depending on nitrogen source. This phenotype was the result of 40 molybdate not reaching the nitrogen-fixing nodules, since genetic complementation with 41 a wild-type MtMOT1.2 gene or molybdate-fortification of the nutrient solution, both 42 restored wild-type levels of growth and nitrogenase activity. These results support a 43 model in which MtMOT1.2 would mediate molybdate delivery by the vasculature into 44 the nodules. 45 46, 19 molybdate delivery for nitrogen fixation is occurring at the 450 level of nodule vessels and 451 not in loading the root vasculature with Mo. Otherwise, an accumulation of Mo in mot1.2 452 roots would be expected as well as a decrease in shoots, and none was detected in either 453 (in this case, even slightly higher levels were detected, which might correspond to surplus 454 Mo being delivered to the shoots). 455 In summary, MtMOT1.2 would position itself between molybdate root uptake 456 transporter, likely MtMOT1.1 as the closest LjMOT1 orthologue, and the nodule apoplast 457 molybdate uptake protein MtMOT1.3 (Fig. 6). MtMOT1.2 would facilitate the transfer 458 of this nutrient into endodermal cells mediating the sink-to-source molybdate trafficking, 459 which would be controlled by mass-effects to ensure that it reaches its destination. 460 However, a critical point remains to be solved, which is the identity of the proteins 461 mediating molybdate efflux from the cytosol to the symbiosome. Whether these are 462 sulfate transporters, or whether a novel family of Mo transporters with a direction of 463 transport opposite to MOT1 proteins, remains to be unveiled. 464 465 ACKNOWLEDGEMENTS 466 This research was funded by a European Research Council Starting Grant (ERC- 467 2013-StG-335284) and a Spanish Ministry of Economy and Competitiveness grant 468 (AGL2015-65866-P) to M.G-G. Development of the M. truncatula Tnt1 mutant 469 population was, in part, funded by the National Science Foundation, USA (DBI-0703285 470 & IOS-1127155) to K.S.M. Yeast transport assays were partially funded by the Plan 471 Propio de la Universidad de Córdoba (to M.T-J) and MINECO (BFU2015-70649-P). The 472 authors would like to thank Dr. Emilio Fernández and Dr. Aurora Galván (Universidad 473 de Córdoba) for their help with the yeast transport assays, as well as to members of 20 Laboratory 281 at Centro de Biotecnología y Genómica de 474 Plantas (UPM-INIA) for their 475 support and feed-back in preparing this manuscript

Plant cell infection is tightly controlled by cell surface receptor-like kinases (RLKs) Alike other RLKs the Medicago truncatula entry receptor LYK3 laterally segregates into membrane nanodomains in a stimulus-dependent manner. Although nanodomain localization arises as a generic feature of plant membrane proteins, molecular mechanisms underlying such dynamic transitions and their functional relevance remained poorly understood. Here, we demonstrate that actin and the flotillin protein FLOT4 form the primary and indispensable core of a specific nanodomain. Infection-dependent induction of the remorin protein and secondary molecular scaffold SYMREM1 results in subsequent recruitment of ligand-activated LYK3 and its stabilization within these membrane subcompartments. Reciprocally, the majority of this LYK3 receptor pool is destabilized at the plasma membrane and undergoes rapid endocytosis in symrem1 mutants upon rhizobial inoculation resulting in premature abortion of host cell infections. These data reveal that receptor recruitment into nanodomains is indispensable for their function during host cell infection.SIGNIFICANCE STATEMENTPattern recognition receptors control the cellular entry of pathogenic as well as symbiotic microbes. While ligand-induced changes in receptor mobility at the plasma membrane and their localization in membrane nanodomains appears as a general feature, the molecular mechanism and the biological relevance of this phenomenon remained unknown. Here, we show that immobilization of the symbiotic cell entry receptor LYK3 in nanodomains requires the presence of actin and the two molecular scaffold proteins FLOT4 and SYMREM1. While FLOT4 forms the initial core structure, infection-induced expression and subsequent physical interaction of SYMREM1 with LYK3 stabilizes the activated receptors in membrane nanodomains. This recruitment prevents its stimulus-dependent endocytosis and ensures progression of the primary infection thread into root cortical cells.

Establishment of symbiosis between legumes and nitrogen-fixing rhizobia depends on bacterial Nod factors (NFs) that trigger symbiosis-related NF signaling in host plants. NFs are modified oligosaccharides of chitin with a fatty acid moiety. NFs can be cleaved and inactivated by host enzymes, such as MtNFH1 (MEDICAGO TRUNCATULA NOD FACTOR HYDROLASE 1). In contrast to related chitinases, MtNFH1 hydrolyzes neither chitin nor chitin fragments, indicating a high cleavage preference for NFs. Here, we provide evidence for a role of MtNFH1 in the symbiosis with Sinorhizobium meliloti. Upon rhizobial inoculation, MtNFH1 accumulated at the curled tip of root hairs, in the so-called infection chamber. Mutant analysis revealed that lack of MtNFH1 delayed rhizobial root hair infection, suggesting that excess amounts of NFs negatively affect the initiation of infection threads. MtNFH1 deficiency resulted in nodule hypertrophy and abnormal nodule branching of young nodules. Nodule branching was also stimulated in plants expressing MtNFH1 driven by a tandem CaMV 35S promoter and plants inoculated by a NF-overproducing S. meliloti strain. We suggest that fine-tuning of NF levels by MtNFH1 is necessary for optimal root hair infection as well as for NF-regulated growth of mature nodules.

Medicago truncatula was chosen by the legume community, along with Lotus japonicus, as a model plant to study legume biology. Since then, numerous resources and tools have been developed for M. truncatula. These include, for example, its genome sequence, core ecotype collections, transformation/regeneration methods, extensive mutant collections, and a gene expression atlas. This review aims to describe the different genetic and genomic tools and resources currently available for M. truncatula. We also describe how these resources were generated and provide all the information necessary to access these resources and use them from a practical point of view. © 2017 by John Wiley & Sons, Inc. Keywords: genomics; genome sequence; genetic diversity; insertion mutagenesis; medicago truncatula; mutagenesis

Phosphate deficiency-responsive lncRNAs in Medicago truncatula are involved in the regulation of Pi deficiency signaling network and Pi transport., Emerging evidence indicates that long non-coding RNAs (lncRNAs) play important roles in the regulation of many biological processes. Inhibition of plant growth due to deficiency in soil inorganic phosphate (Pi) occurs widely across natural and agricultural ecosystems; however, we know little about the function of plant lncRNAs in response to Pi deficiency. To address this issue, we first identified 10 785 lncRNAs in the legume model species Medicago truncatula by sequencing eight strand-specific libraries. Out of these lncRNAs, 358 and 224 were responsive to Pi deficiency in the leaves and roots, respectively. We further predicted and classified the putative targets of those lncRNAs and the results revealed that they may be involved in the processes of signal transduction, energy synthesis, detoxification, and Pi transport. Finally, we functionally characterized three Phosphate Deficiency-Induced LncRNAs (PDILs) using their corresponding Tnt1 mutants. The results showed that PDIL1 suppressed degradation of MtPHO2, which encodes a ubiquitin-conjugating E2 enzyme regulated by miR399, while PDIL2 and PDIL3 directly regulated Pi transport at the transcriptional level. These findings demonstrate that PDILs can regulate Pi-deficiency signaling and Pi transport, highlighting the involvement of lncRNAs in the regulation of responses of plants to Pi deficiency.

Brassinosteroids are phytohormones involved in plant development and physiological processes. Brassinosteroids Insensitive 1 (BRI1) is required for BR perception and initiation of subsequent signal transduction in Arabidopsis. In this study, the orthologue of BRI1 in the model legume species Medicago truncatula, MtBRI1, was identified and characterised. Three allelic Tnt1 insertion mutants, mtbri1-1, mtbri1-2, and mtbri1-3, were obtained from the M. truncatula Tnt1 insertion population. mtbri1 mutants displayed characteristic bri1 mutant phenotypes: extreme dwarfness, dark green curled leaves, short primary roots, less lateral roots, and insensitive to exogenous brassinolide (BL). Moreover, mtbri1 mutants show decreased total nodule number and defects in nitrogen fixation. MtBRI1 is able to complement an Arabidopsis BRI1 mutant, bri1-5. Similar to the interaction of BRI1 and BAK1 in Arabidopsis, MtBRI1 interacts with MtSERK1 in vivo. Global gene expression profiling revealed that the expression of BR biosynthesis genes and SAUR genes are significantly altered in mtbri1 mutants. MapMan analysis indicated that genes involved in signaling, hormone, cell wall, and biotic stress responses are over-represented in differentially expressed genes. Taken together, the results indicate that MtBRI1 is the BR receptor in M. truncatula and that BR signaling may play a conserved role in balancing plant growth and defenses.

Summary The multidrug and toxin extrusion (MATE) transporter family comprises 70 members in the Medicago truncatula genome, and they play seemingly important, yet mostly uncharacterized, physiological functions. Here, we employed bioinformatics and molecular genetics to identify and characterize MATE transporters involved in citric acid export, Al3+ tolerance and Fe translocation. MtMATE69 is a citric acid transporter induced by Fe-deficiency. Overexpression of MtMATE69 in hairy roots altered Fe homeostasis and hormone levels under Fe-deficient or Fe-oversupplied conditions. MtMATE66 is a plasma membrane citric acid transporter primarily expressed in root epidermal cells. The mtmate66 mutant had less root growth than the wild type under Al3+ stress, and seedlings were chlorotic under Fe-deficient conditions. Overexpression of MtMATE66 rendered hairy roots more tolerant to Al3+ toxicity. MtMATE55 is involved in seedling development and iron homeostasis, as well as hormone signaling. The mtmate55 mutant had delayed development and chlorotic leaves in mature plants. Both knock-out and overexpression mutants of MtMATE55 showed altered Fe accumulation and abnormal hormone levels compared with the wild type. We demonstrate that the zinc-finger transcription factor MtSTOP is essentially required for MtMATE66 expression and plant resistance to H+ and Al3+ toxicity. The proper expression of two previously characterized MATE flavonoid transporters MtMATE1 and MtMATE2 also depends on several transcription factors. This study reveals not only functional diversity of MATE transporters and regulatory mechanisms in legumes against H+ and Al3+ stresses, but also casts light on their role in metal nutrition and hormone signaling under various stresses.

Symbiotic nitrogen fixation carried out in legume root nodules requires transition metals. These nutrients are delivered by the host plant to the endosymbiotic nitrogen-fixing bacteria living with the nodule cells, a process in which vascular transport is essential. As occurs in root-to-shoot transport, members of the Yellow Stripe-Like (YSL) family of metal transporters should also be required for root-to-nodule metal delivery. The genome of the model legumeMedicago truncatulaencodes for eight YSL proteins, four of them with a high degree of similarity toArabidopsis thalianaYSLs involved in long-distance metal trafficking. Among them, MtYSL3 is a plasma membrane protein expressed by vascular cells in roots and nodules, and by cortical nodule cells. Reducing expression levels of this gene had no major effect on plant physiology when assimilable nitrogen was provided in the nutrient solution. However, nodule functioning was severely impaired, with a significant reduction of nitrogen fixation capabilities. Further, iron and zinc accumulation and distribution changed. Iron was retained in the apical region of the nodule, while zinc became strongly accumulated in the nodule veins in theysl3mutant. These data suggest a role of MtYSL3 in vascular delivery of iron and zinc to symbiotic nitrogen fixation.HighlightMedicago truncatulaYSL3 transporter is required for optimal nitrogen fixation in root nodules, mediating iron and zinc distribution in these organs.