A cargo sorting receptor mediates chloroplast protein trafficking through the secretory pathway.

Nucleus-encoded chloroplast proteins can be transported via the secretory pathway. The molecular mechanisms underlying the trafficking of chloroplast proteins between the intracellular compartments are largely unclear, and a cargo sorting receptor has not previously been identified in the secretory pathway. Here we report a cargo sorting receptor that is specifically present in Viridiplantae and mediates the transport of cargo proteins to the chloroplast. Using a forward genetic analysis, we identified a gene encoding a transmembrane protein (MtTP930) in barrel medic (Medicago truncatula). Mutation of MtTP930 resulted in impaired chloroplast function and a dwarf phenotype. MtTP930 is highly expressed in the aerial parts of the plant and is localized to the ER exit sites (ERESs) and Golgi. MtTP930 contains typical cargo sorting receptor motifs, interacts with Sar1, Sec12 and Sec24, and participates in coat protein II (COPII) vesicular transport. Importantly, MtTP930 can recognize the cargo proteins plastidial N-glycosylated nucleotide pyrophosphatase/ phosphodiesterase (MtNPP) and α-carbonic anhydrase (MtCAH) in the ER, and then transport them to the chloroplast via the secretory pathway. Mutation of a homolog of MtTP930 in Arabidopsis (Arabidopsis thaliana) resulted in a similar dwarf phenotype. Furthermore, MtNPP-GFP failed to localize to chloroplasts when transgenically expressed in Attp930 protoplasts, implying that these cargo sorting receptors are conserved in plants. These findings fill a gap in our understanding of the mechanism by which chloroplast proteins are sorted and transported via the secretory pathway.

The small peptide CEP1 and the NIN-like protein NLP1 regulate NRT2.1 to mediate root nodule formation across nitrate concentrations.

Legumes acquire fixed nitrogen (N) from the soil and through endosymbiotic association with diazotrophic bacteria. However, establishing and maintaining N2-fixing nodules are expensive for the host plant, relative to taking up N from the soil. Therefore, plants suppress symbiosis when N is plentiful and enhance symbiosis when N is sparse. Here, we show that the nitrate transporter MtNRT2.1 is required for optimal nodule establishment in Medicago truncatula under low-nitrate conditions and the repression of nodulation under high-nitrate conditions. The NIN-like protein (NLP) MtNLP1 is required for MtNRT2.1 expression and regulation of nitrate uptake/transport under low- and high-nitrate conditions. Under low nitrate, the gene encoding the C-terminally encoded peptide (CEP) MtCEP1 was more highly expressed, and the exogenous application of MtCEP1 systemically promoted MtNRT2.1 expression in a compact root architecture 2 (MtCRA2)-dependent manner. The enhancement of nodulation by MtCEP1 and nitrate uptake were both impaired in the Mtnrt2.1 mutant under low nitrate. Our study demonstrates that nitrate uptake by MtNRT2.1 differentially affects nodulation at low- and high-nitrate conditions through the actions of MtCEP1 and MtNLP1.

MtNPF6.5 mediates chloride uptake and nitrate preference in Medicago roots.

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.

VPT-like genes modulate Rhizobium-legume symbiosis and phosphorus adaptation.

Although vacuolar phosphate transporters (VPTs) are essential for plant phosphorus adaptation, their role in Rhizobium-legume symbiosis is unclear. In this study, homologous genes of VPT1 (MtVPTs) were identified in Medicago truncatula to assess their roles in Rhizobium-legume symbiosis and phosphorus adaptation. MtVPT2 and MtVPT3 mainly positively responded to low and high phosphate, respectively. However, both mtvpt2 and mtvpt3 mutants displayed shoot phenotypes with high phosphate sensitivity and low phosphate tolerance. The root-to-shoot phosphate transfer efficiency was significantly enhanced in mtvpt3 but weakened in mtvpt2, accompanied by lower and higher root cytosolic inorganic phosphate (Pi) concentration, respectively. Low phosphate induced MtVPT2 and MtVPT3 expressions in nodules. MtVPT2 and MtVPT3 mutations markedly reduced the nodule number and nitrogenase activity under different phosphate conditions. Cytosolic Pi concentration in nodules was significantly lower in mtvpt2 and mtvpt3 than in the wildtype, especially in tissues near the base of nodules, probably due to inhibition of long-distance Pi transport and cytosolic Pi supply. Also, mtvpt2 and mtvpt3 could not maintain a stable cytosolic Pi level in the nodule fixation zone as the wildtype under low phosphate stress. These findings show that MtVPT2 and MtVPT3 modulate phosphorus adaptation and rhizobia-legume symbiosis, possibly by regulating long-distance Pi transport.

Nodule-specific Cu+ -chaperone NCC1 is required for symbiotic nitrogen fixation in Medicago truncatula root nodules.

Cu+ -chaperones are a diverse group of proteins that allocate Cu+ ions to specific copper proteins, creating different copper pools targeted to specific physiological processes. Symbiotic nitrogen fixation carried out in legume root nodules indirectly requires relatively large amounts of copper, for example for energy delivery via respiration, for which targeted copper deliver systems would be required. MtNCC1 is a nodule-specific Cu+ -chaperone encoded in the Medicago truncatula genome, with a N-terminus Atx1-like domain that can bind Cu+ with picomolar affinities. MtNCC1 is able to interact with nodule-specific Cu+ -importer MtCOPT1. MtNCC1 is expressed primarily from the late infection zone to the early fixation zone and is located in the cytosol, associated with plasma and symbiosome membranes, and within nuclei. Consistent with its key role in nitrogen fixation, ncc1 mutants have a severe reduction in nitrogenase activity and a 50% reduction in copper-dependent cytochrome c oxidase activity. A subset of the copper proteome is also affected in the ncc1 mutant nodules. Many of these proteins can be pulled down when using a Cu+ -loaded N-terminal MtNCC1 moiety as a bait, indicating a role in nodule copper homeostasis and in copper-dependent physiological processes. Overall, these data suggest a pleiotropic role of MtNCC1 in copper delivery for symbiotic nitrogen fixation.

Methylated chalcones are required for rhizobial nod gene induction in the Medicago truncatula rhizosphere.

Legume nodulation requires the detection of flavonoids in the rhizosphere by rhizobia to activate their production of Nod factor countersignals. Here we investigated the flavonoids involved in nodulation of Medicago truncatula. We biochemically characterized five flavonoid-O-methyltransferases (OMTs) and a lux-based nod gene reporter was used to investigate the response of Sinorhizobium medicae NodD1 to various flavonoids. We found that chalcone-OMT 1 (ChOMT1) and ChOMT3, but not OMT2, 4, and 5, were able to produce 4,4'-dihydroxy-2'-methoxychalcone (DHMC). The bioreporter responded most strongly to DHMC, while isoflavones important for nodulation of soybean (Glycine max) showed no activity. Mutant analysis revealed that loss of ChOMT1 strongly reduced DHMC levels. Furthermore, chomt1 and omt2 showed strongly reduced bioreporter luminescence in their rhizospheres. In addition, loss of both ChOMT1 and ChOMT3 reduced nodulation, and this phenotype was strengthened by the further loss of OMT2. We conclude that: the loss of ChOMT1 greatly reduces root DHMC levels; ChOMT1 or OMT2 are important for nod gene activation in the rhizosphere; and ChOMT1/3 and OMT2 promote nodulation. Our findings suggest a degree of exclusivity in the flavonoids used for nodulation in M. truncatula compared to soybean, supporting a role for flavonoids in rhizobial host range.

Roles of very long-chain fatty acids in compound leaf patterning in Medicago truncatula.

Plant cuticles are composed of hydrophobic cuticular waxes and cutin. Very long-chain fatty acids (VLCFAs) are components of epidermal waxes and the plasma membrane and are involved in organ morphogenesis. By screening a barrelclover (Medicago truncatula) mutant population tagged by the transposable element of tobacco (Nicotiana tabacum) cell type1 (Tnt1), we identified two types of mutants with unopened flower phenotypes, named unopened flower1 (uof1) and uof2. Both UOF1 and UOF2 encode enzymes that are involved in the biosynthesis of VLCFAs and cuticular wax. Comparative analysis of the mutants indicated that the mutation in UOF1, but not UOF2, leads to the increased number of leaflets in M. truncatula. UOF1 was specifically expressed in the outermost cell layer (L1) of the shoot apical meristem (SAM) and leaf primordia. The uof1 mutants displayed defects in VLCFA-mediated plasma membrane integrity, resulting in the disordered localization of the PIN-FORMED1 (PIN1) ortholog SMOOTH LEAF MARGIN1 (SLM1) in M. truncatula. Our work demonstrates that the UOF1-mediated biosynthesis of VLCFAs in L1 is critical for compound leaf patterning, which is associated with the polarization of the auxin efflux carrier in M. truncatula.

Insight into the control of nodule immunity and senescence during Medicago truncatula symbiosis.

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.

Enabling Medicago truncatula forward genetics: identification of genetic crossing partner for R108 and development of mapping resources for Tnt1 mutants.

Though Medicago truncatula Tnt1 mutants are widely used by researchers in the legume community, they are mainly used for reverse genetics because of the availability of the BLAST-searchable large-scale flanking sequence tags database. However, these mutants should have also been used extensively for forward genetic screens, an effort that has been hindered due to the lack of a compatible genetic crossing partner for the M. truncatula genotype R108, from which Tnt1 mutants were generated. In this study, we selected three Medicago HapMap lines (HM017, HM018 and HM022) and performed reciprocal genetic crosses with R108. After phenotypic analyses in F1 and F2 progenies, HM017 was identified as a compatible crossing partner with R108. By comparing the assembled genomic sequences of HM017 and R108, we developed and confirmed 318 Indel markers evenly distributed across the eight chromosomes of the M. truncatula genome. To validate the effectiveness of these markers, by employing the map-based cloning approach, we cloned the causative gene in the dwarf mutant crs isolated from the Tnt1 mutant population, identifying it as gibberellin 3-ß-dioxygenase 1, using some of the confirmed Indel markers. The primer sequences and the size difference of each marker were made available for users in the web-based database. The identification of the crossing partner for R108 and the generation of Indel markers will enhance the forward genetics and the overall usage of the Tnt1 mutants.

MtING2 encodes an ING domain PHD finger protein which affects Medicago growth, flowering, global patterns of H3K4me3, and gene expression.

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 that >7000 genes are mis-expressed in the Mting2-1 mutant, consistent with its strong mutant phenotypes. Interestingly, ChIP-seq analysis identified >5000 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.

Multiplex CRISPR/Cas9-mediated mutagenesis of alfalfa FLOWERING LOCUS Ta1 (MsFTa1) leads to delayed flowering time with improved forage biomass yield and quality.

Alfalfa (Medicago sativa L.) is a perennial flowering plant in the legume family that is widely cultivated as a forage crop for its high yield, forage quality and related agricultural and economic benefits. Alfalfa is a photoperiod sensitive long-day (LD) plant that can accomplish its vegetative and reproductive phases in a short period of time. However, rapid flowering can compromise forage biomass yield and quality. Here, we attempted to delay flowering in alfalfa using multiplex CRISPR/Cas9-mediated mutagenesis of FLOWERING LOCUS Ta1 (MsFTa1), a key floral integrator and activator gene. Four guide RNAs (gRNAs) were designed and clustered in a polycistronic tRNA-gRNA system and introduced into alfalfa by Agrobacterium-mediated transformation. Ninety-six putative mutant lines were identified by gene sequencing and characterized for delayed flowering time and related desirable agronomic traits. Phenotype assessment of flowering time under LD conditions identified 22 independent mutant lines with delayed flowering compared to the control. Six independent Msfta1 lines containing mutations in all four copies of MsFTa1 accumulated significantly higher forage biomass yield, with increases of up to 78% in fresh weight and 76% in dry weight compared to controls. Depending on the harvesting schemes, many of these lines also had reduced lignin, acid detergent fibre (ADF) and neutral detergent fibre (NDF) content and significantly higher crude protein (CP) and mineral contents compared to control plants, especially in the stems. These CRISPR/Cas9-edited Msfta1 mutants could be introduced in alfalfa breeding programmes to generate elite transgene-free alfalfa cultivars with improved forage biomass yield and quality.

An extracellular ß-N-acetylhexosaminidase of Medicago truncatula hydrolyzes chitooligosaccharides and is involved in arbuscular mycorrhizal symbiosis but not required for nodulation.

Establishment of symbiosis between plants and arbuscular mycorrhizal (AM) fungi depends on fungal chitooligosaccharides (COs) and lipo-chitooligosaccharides (LCOs). The latter are also produced by nitrogen-fixing rhizobia to induce nodules on leguminous roots. However, host enzymes regulating structure and levels of these signals remain largely unknown. Here, we analyzed the expression of a ß-N-acetylhexosaminidase gene of Medicago truncatula (MtHEXO2) and biochemically characterized the enzyme. Mutant analysis was performed to study the role of MtHEXO2 during symbiosis. We found that expression of MtHEXO2 is associated with AM symbiosis and nodulation. MtHEXO2 expression in the rhizodermis was upregulated in response to applied chitotetraose, chitoheptaose, and LCOs. M. truncatula mutants deficient in symbiotic signaling did not show induction of MtHEXO2. Subcellular localization analysis indicated that MtHEXO2 is an extracellular protein. Biochemical analysis showed that recombinant MtHEXO2 does not cleave LCOs but can degrade COs into N-acetylglucosamine (GlcNAc). Hexo2 mutants exhibited reduced colonization by AM fungi; however, nodulation was not affected in hexo2 mutants. In conclusion, we identified an enzyme, which inactivates COs and promotes the AM symbiosis. We hypothesize that GlcNAc produced by MtHEXO2 may function as a secondary symbiotic signal.

Spatiotemporal cytokinin response imaging and ISOPENTENYLTRANSFERASE 3 function in Medicago nodule development.

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.

A CEP Peptide Receptor-like Kinase Regulates Auxin Biosynthesis and Ethylene Signaling to Coordinate Root Growth and Symbiotic Nodulation in Medicago truncatula.

Because of the high energy consumed during symbiotic nitrogen fixation, legumes must balance growth and symbiotic nodulation. Both lateral roots and nodules form on the root system and the developmental coordination of these organs according to reduced nitrogen (N) availability remains elusive. We show that the Compact Root Architecture 2 (MtCRA2) receptor-like kinase is essential to promote the initiation of early symbiotic nodulation and to inhibit root growth in response to low-N. MtCEP1 peptides can activate MtCRA2 under N-starvation conditions, leading to a repression of MtYUC2 auxin biosynthesis gene expression, and therefore of auxin root responses. Accordingly, the compact root architecture phenotype of cra2 can be mimicked by an auxin treatment or by over-expressing MtYUC2, and conversely, a treatment with YUC inhibitors or a MtYUC2 knock-out rescues the cra2 root phenotype. The MtCEP1-activated CRA2 can additionally interact with and phosphorylate the MtEIN2 ethylene signaling component at Ser643 and Ser924, preventing its cleavage and therefore repressing ethylene responses, thus locally promoting the root susceptibility to rhizobia. In agreement, the cra2 low nodulation phenotype is rescued by an ein2 mutation. Overall, by reducing auxin biosynthesis and inhibiting ethylene signaling, the MtCEP1/MtCRA2 pathway balances root and nodule development under low-N conditions.

A CEP Peptide Receptor-Like Kinase Regulates Auxin Biosynthesis and Ethylene Signaling to Coordinate Root Growth and Symbiotic Nodulation in Medicago truncatula.

Because of the large amount of energy consumed during symbiotic nitrogen fixation, legumes must balance growth and symbiotic nodulation. Both lateral roots and nodules form on the root system, and the developmental coordination of these organs under conditions of reduced nitrogen (N) availability remains elusive. We show that the Medicago truncatula COMPACT ROOT ARCHITECTURE2 (MtCRA2) receptor-like kinase is essential to promote the initiation of early symbiotic nodulation and to inhibit root growth in response to low N. C-TERMINALLY ENCODED PEPTIDE (MtCEP1) peptides can activate MtCRA2 under N-starvation conditions, leading to a repression of YUCCA2 (MtYUC2) auxin biosynthesis gene expression, and therefore of auxin root responses. Accordingly, the compact root architecture phenotype of cra2 can be mimicked by an auxin treatment or by overexpressing MtYUC2, and conversely, a treatment with YUC inhibitors or an MtYUC2 knockout rescues the cra2 root phenotype. The MtCEP1-activated CRA2 can additionally interact with and phosphorylate the MtEIN2 ethylene signaling component at Ser643 and Ser924, preventing its cleavage and thereby repressing ethylene responses, thus locally promoting the root susceptibility to rhizobia. In agreement with this interaction, the cra2 low nodulation phenotype is rescued by an ein2 mutation. Overall, by reducing auxin biosynthesis and inhibiting ethylene signaling, the MtCEP1/MtCRA2 pathway balances root and nodule development under low-N conditions.

The MIO1-MtKIX8 module regulates the organ size in Medicago truncatula.

Plant organ size is an important agronomic trait tightly related to crop yield. However, the molecular mechanisms underlying organ size regulation remain largely unexplored in legumes. We previously characterized a key regulator F-box protein MINI ORGAN1 (MIO1)/SMALL LEAF AND BUSHY1 (SLB1), which controls plant organ size in the model legume Medicago truncatula. In order to further dissect the molecular mechanism, MIO1 was used as the bait to screen its interacting proteins from a yeast library. Subsequently, a KIX protein, designated MtKIX8, was identified from the candidate list. The interaction between MIO1 and MtKIX8 was confirmed further by Y2H, BiFC, split-luciferase complementation and pull-down assays. Phylogenetic analyses indicated that MtKIX8 is highly homologous to Arabidopsis KIX8, which negatively regulates organ size. Moreover, loss-of-function of MtKIX8 led to enlarged leaves and seeds, while ectopic expression of MtKIX8 in Arabidopsis resulted in decreased cotyledon area and seed weight. Quantitative reverse-transcription PCR and in situ hybridization showed that MtKIX8 is expressed in most developing organs. We also found that MtKIX8 serves as a crucial molecular adaptor, facilitating interactions with BIG SEEDS1 (BS1) and MtTOPLESS (MtTPL) proteins in M. truncatula. Overall, our results suggest that the MIO1-MtKIX8 module plays a significant and conserved role in the regulation of plant organ size. This module could be a good target for molecular breeding in legume crops and forages.

The role of Class â ¡ KNOX family in controlling compound leaf patterning in Medicago truncatula.

Compound leaf development requires the coordination of genetic factors, hormones, and other signals. In this study, we explored the functions of Class Ⅱ KNOTTED-like homeobox (KNOXII) genes in the model leguminous plant Medicago truncatula. Phenotypic and genetic analyses suggest that MtKNOX4, 5 are able to repress leaflet formation, while MtKNOX3, 9, 10 are not involved in this developmental process. Further investigations have shown that MtKNOX4 represses the CK signal transduction, which is downstream of MtKNOXⅠ-mediated CK biosynthesis. Additionally, two boundary genes, FUSED COMPOUND LEAF1 (orthologue of Arabidopsis Class M KNOX) and NO APICAL MERISTEM (orthologue of Arabidopsis CUP-SHAPED COTYLEDON), are necessary for MtKNOX4-mediated compound leaf formation. These findings suggest, that among the members of MtKNOXⅡ, MtKNOX4 plays a crucial role in integrating the CK pathway and boundary regulators, providing new insights into the roles of MtKNOXⅡ in regulating the elaboration of compound leaves in M. truncatula.

MtSUPERMAN plays a key role in compound inflorescence and flower development in Medicago truncatula.

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.

The formation of stipule requires the coordinated actions of the legume orthologs of Arabidopsis BLADE-ON-PETIOLE and LEAFY.

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.

Anthocyanin pigmentation as a quantitative visual marker for arbuscular mycorrhizal fungal colonization of Medicago truncatula roots.

Visualization of root colonization by arbuscular mycorrhizal fungi (AMF) is the most elementary experiment in the field of mycorrhizal symbiosis. The most widely used approach for evaluating levels of AMF colonization is staining with trypan blue or ink, which is scored using the time-consuming grid intersection method. Here we demonstrate the use of an anthocyanin-based visual marker system for visualizing AMF colonization of Medicago truncatula roots. Expression of MtLAP1, a transcription factor which regulates the production of anthocyanins, from the AMF-induced Kunitz Protease Inhibitor 106 promoter, allowed the visualization of arbuscules in live plant tissues without microscopy or staining. This marker system allowed straightforward qualitative evaluation of the ram1, vpy and dmi3 AMF phenotypes using Agrobacterium rhizogenes hairy-root transformation. For the strigolactone biosynthesis mutant carotenoid cleavage dioxygenase 8a and a novel mutant scooby, which show quantitative AMF symbiotic phenotypes, the amount of anthocyanins in the roots estimated by spectrophotometry correlated very well with colonization levels estimated by staining and scoring using the grid intersection method. The LAP1-based marker system therefore provides a highly efficient approach for mutant screening and monitoring of AMF colonization in live tissues by eye, or for quantitative assessment using a simple and quick photometric assay.