Rhizobium symbiotic efficiency meets CEP signaling peptides.

C-terminally encoded peptides (CEP) signaling peptides are drivers of systemic pathways regulating nitrogen (N) acquisition in different plants, from Arabidopsis to legumes, depending on mineral N availability (e.g. nitrate) and on the whole plant N demand. Recent studies in the Medicago truncatula model legume revealed how root-produced CEP peptides control the root competence for endosymbiosis with N fixing rhizobia soil bacteria through the activity of the Compact Root Architecture 2 (CRA2) CEP receptor in shoots. Among CEP genes, MtCEP7 was shown to be tightly linked to nodulation, and the dynamic temporal regulation of its expression reflects the plant ability to maintain a different symbiotic root competence window depending on the symbiotic efficiency of the rhizobium strain, as well as to reinitiate a new window of root competence for nodulation.

The Medicago SymCEP7 hormone increases nodule number via shoots without compromising lateral root number.

Legumes acquire soil nutrients through nitrogen-fixing root nodules and lateral roots. To balance the costs and benefits of nodulation, legumes negatively control root nodule number by autoregulatory and hormonal pathways. How legumes simultaneously coordinate root nodule and lateral root development to procure nutrients remains poorly understood. In Medicago (Medicago truncatula), a subset of mature C-TERMINALLY ENCODED PEPTIDE (CEP) hormones can systemically promote nodule number, but all CEP hormones tested to date negatively regulate lateral root number. Here we showed that Medicago CEP7 produces a mature peptide, SymCEP7, that promotes nodulation from the shoot without compromising lateral root number. Rhizobial inoculation induced CEP7 in the susceptible root nodulation zone in a Nod factor-dependent manner, and, in contrast to other CEP genes, its transcription level was elevated in the ethylene signaling mutant sickle. Using mass spectrometry, fluorescence microscopy and expression analysis, we demonstrated that SymCEP7 activity requires the COMPACT ROOT ARCHITECTURE 2 receptor and activates the shoot-to-root systemic effector, miR2111. Shoot-applied SymCEP7 rapidly promoted nodule number in the pM to nM range at concentrations up to five orders of magnitude lower than effects mediated by root-applied SymCEP7. Shoot-applied SymCEP7 also promoted nodule number in White Clover (Trifolium repens) and Lotus (Lotus japonicus), which suggests that this biological function may be evolutionarily conserved. We propose that SymCEP7 acts in the Medicago shoot to counter balance the autoregulation pathways induced rapidly by rhizobia to enable nodulation without compromising lateral root growth, thus promoting the acquisition of nutrients other than nitrogen to support their growth.

The Compact Root Architecture 2 systemic pathway is required for the induction of cytokinins and of the miR399 in Medicago truncatula N-satisfied plants.

Plants use a combination of sophisticated local and systemic pathways to optimize growth depending on heterogeneous nutrient availability in the soil. Legume plants can acquire mineral nitrogen (N) either through their roots or via a symbiotic interaction with N-fixing rhizobia bacteria housed in so-called root nodules. To identify shoot-to-root systemic signals acting in Medicago truncatula plants at N-deficit or N-satiety, plants were grown in a split-root experimental design, in which either high or low N was provided to a half of the root system, allowing the analysis of systemic pathways independently of any local N response. Among the plant hormone families analyzed, the cytokinin trans-Zeatin accumulated in plants at N-satiety. Cytokinin application by petiole feeding led to an inhibition of both root growth and nodulation. In addition, an exhaustive analysis of miRNAs revealed that miR2111 accumulates systemically under N-deficit in both shoots and non-treated distant roots, whereas a miRNA related to inorganic Phosphate (Pi)-acquisition, the miR399, does so in plants grown at N-satiety. These two accumulation patterns are dependent on CRA2 (Compact Root Architecture 2), a receptor required for CEP (C-terminally Encoded Peptide) signaling. Constitutive ectopic expression of the miR399 reduced nodule numbers and root biomass depending on Pi availability, suggesting that the miR399-dependent Pi-acquisition regulatory module controlled by N-availability affects the development of the whole legume plant root system.

Acceptability of interventional materials: public participation in public health interventional research / Acceptabilité d'une intervention : exemple de participation des publics en recherche interventionnelle en santé publique

Introduction: The uptake rate of colorectal cancer screening remains insufficient in France and decreases as the level of deprivation increases. Participants' health literacy appears to be an important determinant of screening uptake. Aim of the study: The aim of this study, nested in our multicenter-randomized controlled trial, was to present the development and acceptability of interventional material (training and a pictorial brochure) for general practitioners and healthcare users in disadvantaged geographical areas using a participatory involvement approach. Methods: The development of the brochure and the training was carried out in three stages, two for the development, usability, and acceptability testing and a third for its evaluation with the target audience. We used a qualitative approach based on focus groups and cognitive interviews. The qualitative analysis was based on Morville's "Honeycomb" conceptual model and the COREQ checklist. Results: The development and test of the acceptability of the material enabled us to adjust the content of the training by proposing examples that were more rooted in professional reality, and to produce a brochure that was easy to read, understand, acceptable and adapted to the intervention's targeted audience. Conclusions: This experience illustrates in a concrete way the feasibility of public participation and its value in the context of interventional research, and more generally in the creation of interventional material.

Introduction: Le taux de participation au dépistage du cancer colorectal reste insuffisant en France et diminue à mesure que le niveau de précarité augmente. La littératie en santé est un déterminant important du recours au dépistage. But de l'étude: Cette étude, nichée dans notre essai randomisé multicentrique, a pour but de présenter la procédure d'élaboration (procédé itératif de test d'utilisabilité et d'acceptabilité) et de vérification de l'acceptabilité de l'intervention (formation et brochure imagée) ciblant les médecins généralistes et usagers du soin dans des zones géographiques défavorisées, selon une approche participative. Méthodes: Le développement de la brochure et de la formation a été réalisé en trois étapes : deux pour l'élaboration et tests itératifs d'utilisabilité et acceptabilité et une troisième pour vérifier l'acceptabilité auprès des publics cibles. Nous avons utilisé une approche qualitative par focus group et entretiens individuels cognitifs dont l'analyse repose sur le « nid d'abeille ¼ de Morville et la grille COREQ. Résultats: Le développement itératif et la vérification de l'acceptabilité du matériel nous ont permis, d'une part, de réaliser des ajustements quant au contenu de la formation, en proposant des exemples plus ancrés dans la réalité professionnelle et, d'autre part, de produire une brochure imagée facile à lire et à comprendre, acceptable et adaptée au public ciblé par l'intervention. Conclusions: Cette expérience illustre, de manière concrète, la faisabilité de cette modalité de participation des publics concernés et son intérêt dans le cadre de la recherche interventionnelle et, plus généralement, dans le matériel interventionnel.

A Cytokinin Signaling Type-B Response Regulator Transcription Factor Acting in Early Nodulation.

Nitrogen-fixing root nodulation in legumes challenged with nitrogen-limiting conditions requires infection of the root hairs by soil symbiotic bacteria, collectively referred to as rhizobia, and the initiation of cell divisions in the root cortex. Cytokinin hormones are critical for early nodulation to coordinate root nodule organogenesis and the progression of bacterial infections. Cytokinin signaling involves regulation of the expression of cytokinin primary response genes by type-B response regulator (RRB) transcription factors. RNA interference or mutation of MtRRB3, the RRB-encoding gene most strongly expressed in Medicago truncatula roots and nodules, significantly decreased the number of nodules formed, indicating a function of this RRB in nodulation initiation. Fewer infection events were also observed in rrb3 mutant roots associated with a reduced Nod factor induction of the Early Nodulin 11 (MtENOD11) infection marker, and of the cytokinin-regulated Nodulation Signaling Pathway 2 (Mt NSP2) gene. Rhizobial infections correlate with an expansion of the nuclear area, suggesting the activation of endoreduplication cycles linked to the cytokinin-regulated Cell Cycle Switch 52A (Mt CCS52A) gene. Although no significant difference in nucleus size and endoreduplication were detected in rhizobia-infected rrb3 mutant roots, expression of the MtCCS52A endoreduplication marker was reduced. As the MtRRB3 expression pattern overlaps with those of MtNSP2 and MtCCS52A in roots and nodule primordia, chromatin immunoprecipitation-quantitative PCR and protoplast trans-activation assays were used to show that MtRRB3 can interact with and trans-activate MtNSP2 and MtCCS52A promoters. Overall, we highlight that the MtRRB3 cytokinin signaling transcription factor coordinates the expression of key early nodulation genes.

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

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

Unraveling new molecular players involved in the autoregulation of nodulation in Medicago truncatula.

The number of legume root nodules resulting from a symbiosis with rhizobia is tightly controlled by the plant. Certain members of the CLAVATA3/Embryo Surrounding Region (CLE) peptide family, specifically MtCLE12 and MtCLE13 in Medicago truncatula, act in the systemic autoregulation of nodulation (AON) pathway that negatively regulates the number of nodules. Little is known about the molecular pathways that operate downstream of the AON-related CLE peptides. Here, by means of a transcriptome analysis, we show that roots ectopically expressing MtCLE13 deregulate only a limited number of genes, including three down-regulated genes encoding lysin motif receptor-like kinases (LysM-RLKs), among which are the nodulation factor (NF) receptor NF Perception gene (NFP) and two up-regulated genes, MtTML1 and MtTML2, encoding Too Much Love (TML)-related Kelch-repeat containing F-box proteins. The observed deregulation was specific for the ectopic expression of nodulation-related MtCLE genes and depended on the Super Numeric Nodules (SUNN) AON RLK. Moreover, overexpression and silencing of these two MtTML genes demonstrated that they play a role in the negative regulation of nodule numbers. Hence, the identified MtTML genes are the functional counterpart of the Lotus japonicus TML gene shown to be central in the AON pathway. Additionally, we propose that the down-regulation of a subset of LysM-RLK-encoding genes, among which is NFP, might contribute to the restriction of further nodulation once the first nodules have been formed.

Flavonoids and Auxin Transport Inhibitors Rescue Symbiotic Nodulation in the Medicago truncatula Cytokinin Perception Mutant cre1.

Initiation of symbiotic nodules in legumes requires cytokinin signaling, but its mechanism of action is largely unknown. Here, we tested whether the failure to initiate nodules in the Medicago truncatula cytokinin perception mutant cre1 (cytokinin response1) is due to its altered ability to regulate auxin transport, auxin accumulation, and induction of flavonoids. We found that in the cre1 mutant, symbiotic rhizobia cannot locally alter acro- and basipetal auxin transport during nodule initiation and that these mutants show reduced auxin (indole-3-acetic acid) accumulation and auxin responses compared with the wild type. Quantification of flavonoids, which can act as endogenous auxin transport inhibitors, showed a deficiency in the induction of free naringenin, isoliquiritigenin, quercetin, and hesperetin in cre1 roots compared with wild-type roots 24 h after inoculation with rhizobia. Coinoculation of roots with rhizobia and the flavonoids naringenin, isoliquiritigenin, and kaempferol, or with the synthetic auxin transport inhibitor 2,3,5,-triiodobenzoic acid, rescued nodulation efficiency in cre1 mutants and allowed auxin transport control in response to rhizobia. Our results suggest that CRE1-dependent cytokinin signaling leads to nodule initiation through the regulation of flavonoid accumulation required for local alteration of polar auxin transport and subsequent auxin accumulation in cortical cells during the early stages of nodulation.

KNAT3/4/5-like class 2 KNOX transcription factors are involved in Medicago truncatula symbiotic nodule organ development.

We investigated the role of KNOX genes in legume root nodule organogenesis. Class 1 KNOX homeodomain transcription factors (TFs) are involved in plant shoot development and leaf shape diversity. Class 2 KNOX genes are less characterized, even though an antagonistic function relative to class 1 KNOXs was recently proposed. In silico expression data and further experimental validation identified in the Medicago truncatula model legume three class 2 KNOX genes, belonging to the KNAT3/4/5-like subclass (Mt KNAT3/4/5-like), as expressed during nodulation from early stages. RNA interference (RNAi)-mediated silencing and overexpression studies were used to unravel a function for KNOX TFs in nodule development. Mt KNAT3/4/5-like genes encoded four highly homologous proteins showing overlapping expression patterns during nodule organogenesis, suggesting functional redundancy. Simultaneous reduction of Mt KNAT3/4/5-like genes indeed led to an increased formation of fused nodule organs, and decreased the expression of the MtEFD (Ethylene response Factor required for nodule Differentiation) TF and its direct target MtRR4, a cytokinin response gene. Class 2 KNOX TFs therefore regulate legume nodule development, potentially through the MtEFD/MtRR4 cytokinin-related regulatory module, and may control nodule organ boundaries and shape like class 2 KNOX function in leaf development.

Different Pathways Act Downstream of the CEP Peptide Receptor CRA2 to Regulate Lateral Root and Nodule Development.

C-TERMINALLY ENCODED PEPTIDEs (CEPs) control root system architecture in a non-cell-autonomous manner. In Medicago truncatula, MtCEP1 affects root development by increasing nodule formation and inhibiting lateral root emergence by unknown pathways. Here, we show that the MtCEP1 peptide-dependent increase in nodulation requires the symbiotic signaling pathway and ETHYLENE INSENSITIVE2 (EIN2)/SICKLE (SKL), but acts independently of SUPER NUMERIC NODULES. MtCEP1-dependent inhibition of lateral root development acts through an EIN2-independent mechanism. MtCEP1 increases nodulation by promoting rhizobial infections, the developmental competency of roots for nodulation, the formation of fused nodules, and an increase in frequency of nodule development that initiates at proto-phloem poles. These phenotypes are similar to those of the ein2/skl mutant and support that MtCEP1 modulates EIN2-dependent symbiotic responses. Accordingly, MtCEP1 counteracts the reduction in nodulation induced by increasing ethylene precursor concentrations, and an ethylene synthesis inhibitor treatment antagonizes MtCEP1 root phenotypes. MtCEP1 also inhibits the development of EIN2-dependent pseudonodule formation. Finally, mutants affecting the COMPACT ROOT ARCHITECTURE2 (CRA2) receptor, which is closely related to the Arabidopsis CEP Receptor1, are unresponsive to MtCEP1 effects on lateral root and nodule formation, suggesting that CRA2 is a CEP peptide receptor mediating both organogenesis programs. In addition, an ethylene inhibitor treatment counteracts the cra2 nodulation phenotype. These results indicate that MtCEP1 and its likely receptor, CRA2, mediate nodulation and lateral root development through different pathways.

Local and systemic regulation of plant root system architecture and symbiotic nodulation by a receptor-like kinase.

In plants, root system architecture is determined by the activity of root apical meristems, which control the root growth rate, and by the formation of lateral roots. In legumes, an additional root lateral organ can develop: the symbiotic nitrogen-fixing nodule. We identified in Medicago truncatula ten allelic mutants showing a compact root architecture phenotype (cra2) independent of any major shoot phenotype, and that consisted of shorter roots, an increased number of lateral roots, and a reduced number of nodules. The CRA2 gene encodes a Leucine-Rich Repeat Receptor-Like Kinase (LRR-RLK) that primarily negatively regulates lateral root formation and positively regulates symbiotic nodulation. Grafting experiments revealed that CRA2 acts through different pathways to regulate these lateral organs originating from the roots, locally controlling the lateral root development and nodule formation systemically from the shoots. The CRA2 LRR-RLK therefore integrates short- and long-distance regulations to control root system architecture under non-symbiotic and symbiotic conditions.

Two direct targets of cytokinin signaling regulate symbiotic nodulation in Medicago truncatula.

Cytokinin regulates many aspects of plant development, and in legume crops, this phytohormone is necessary and sufficient for symbiotic nodule organogenesis, allowing them to fix atmospheric nitrogen. To identify direct links between cytokinins and nodule organogenesis, we determined a consensus sequence bound in vitro by a transcription factor (TF) acting in cytokinin signaling, the nodule-enhanced Medicago truncatula Mt RR1 response regulator (RR). Among genes rapidly regulated by cytokinins and containing this so-called RR binding site (RRBS) in their promoters, we found the nodulation-related Type-A RR Mt RR4 and the Nodulation Signaling Pathway 2 (NSP2) TF. Site-directed mutagenesis revealed that RRBS cis-elements in the RR4 and NSP2 promoters are essential for expression during nodule development and for cytokinin induction. Furthermore, a microRNA targeting NSP2 (miR171 h) is also rapidly induced by cytokinins and then shows an expression pattern anticorrelated with NSP2. Other primary targets regulated by cytokinins depending on the Cytokinin Response1 (CRE1) receptor were a cytokinin oxidase/dehydrogenase (CKX1) and a basic Helix-Loop-Helix TF (bHLH476). RNA interference constructs as well as insertion of a Tnt1 retrotransposon in the bHLH gene led to reduced nodulation. Hence, we identified two TFs, NSP2 and bHLH476, as direct cytokinin targets acting at the convergence of phytohormonal and symbiotic cues.

A Medicago truncatula rdr6 allele impairs transgene silencing and endogenous phased siRNA production but not development.

RNA-dependent RNA polymerase 6 (RDR6) and suppressor of gene silencing 3 (SGS3) act together in post-transcriptional transgene silencing mediated by small interfering RNAs (siRNAs) and in biogenesis of various endogenous siRNAs including the tasiARFs, known regulators of auxin responses and plant development. Legumes, the third major crop family worldwide, has been widely improved through transgenic approaches. Here, we isolated rdr6 and sgs3 mutants in the model legume Medicago truncatula. Two sgs3 and one rdr6 alleles led to strong developmental defects and impaired biogenesis of tasiARFs. In contrast, the rdr6.1 homozygous plants produced sufficient amounts of tasiARFs to ensure proper development. High throughput sequencing of small RNAs from this specific mutant identified 354 potential MtRDR6 substrates, for which siRNA production was significantly reduced in the mutant. Among them, we found a large variety of novel phased loci corresponding to protein-encoding genes or transposable elements. Interestingly, measurement of GFP expression revealed that post-transcriptional transgene silencing was reduced in rdr6.1 roots. Hence, this novel mis-sense mutation, affecting a highly conserved amino acid residue in plant RDR6s, may be an interesting tool both to analyse endogenous pha-siRNA functions and to improve transgene expression, at least in legume species.

Dual involvement of a Medicago truncatula NAC transcription factor in root abiotic stress response and symbiotic nodule senescence.

Legume crops related to the model plant Medicago truncatula can adapt their root architecture to environmental conditions, both by branching and by establishing a symbiosis with rhizobial bacteria to form nitrogen-fixing nodules. Soil salinity is a major abiotic stress affecting plant yield and root growth. Previous transcriptomic analyses identified several transcription factors linked to the M. truncatula response to salt stress in roots, including NAC (NAM/ATAF/CUC)-encoding genes. Over-expression of one of these transcription factors, MtNAC969, induced formation of a shorter and less-branched root system, whereas RNAi-mediated MtNAC969 inactivation promoted lateral root formation. The altered root system of over-expressing plants was able to maintain its growth under high salinity, and roots in which MtNAC969 was down-regulated showed improved growth under salt stress. Accordingly, expression of salt stress markers was decreased or induced in MtNAC969 over-expressing or RNAi roots, respectively, suggesting a repressive function for this transcription factor in the salt-stress response. Expression of MtNAC969 in central symbiotic nodule tissues was induced by nitrate treatment, and antagonistically affected by salt in roots and nodules, similarly to senescence markers. MtNAC969 RNAi nodules accumulated amyloplasts in the nitrogen-fixing zone, and were prematurely senescent. Therefore, the MtNAC969 transcription factor, which is differentially affected by environmental cues in root and nodules, participates in several pathways controlling adaptation of the M. truncatula root system to the environment.

Bioactive cytokinins are selectively secreted by Sinorhizobium meliloti nodulating and nonnodulating strains.

Bacteria present in the rhizosphere of plants often synthesize phytohormones, and these signals can consequently affect root system development. In legumes, plants adapt to nitrogen starvation by forming lateral roots as well as a new organ, the root nodule, following a symbiotic interaction with bacteria collectively referred to as rhizobia. As cytokinin (CK) phytohormones were shown to be necessary and sufficient to induce root nodule organogenesis, the relevance of CK production by symbiotic rhizobia was questioned. In this study, we analyzed quantitatively, by liquid chromatography-tandem mass spectrometry, the production of 25 forms of CK in nine rhizobia strains belonging to four different species. All bacterial strains were able to synthesize a mix of CK, and bioactive forms of CK, such as iP, were notably found to be secreted in bacterial culture supernatants. Use of a mutant affected in extracellular polysaccharide (EPS) production revealed a negative correlation of EPS production with the ability to secrete CK. In addition, analysis of a nonnodulating Sinorhizobium meliloti strain revealed a similar pattern of CK production and secretion when compared with a related nodulating strain. This indicates that bacterially produced CK are not sufficient to induce symbiotic nodulation.

Cell-specific pathways recruited for symbiotic nodulation in the Medicago truncatula legume.

Medicago truncatula is a model legume species that has been studied for decades to understand the symbiotic relationship between legumes and soil bacteria collectively named rhizobia. This symbiosis called nodulation is initiated in roots with the infection of root hair cells by the bacteria, as well as the initiation of nodule primordia from root cortical, endodermal, and pericycle cells, leading to the development of a new root organ, the nodule, where bacteria fix and assimilate the atmospheric dinitrogen for the benefit of the plant. Here, we report the isolation and use of the nuclei from mock and rhizobia-inoculated roots for the single nuclei RNA-seq (sNucRNA-seq) profiling to gain a deeper understanding of early responses to rhizobial infection in Medicago roots. A gene expression map of the Medicago root was generated, comprising 25 clusters, which were annotated as specific cell types using 119 Medicago marker genes and orthologs to Arabidopsis cell-type marker genes. A focus on root hair, cortex, endodermis, and pericycle cell types, showing the strongest differential regulation in response to a short-term (48 h) rhizobium inoculation, revealed not only known genes and functional pathways, validating the sNucRNA-seq approach, but also numerous novel genes and pathways, allowing a comprehensive analysis of early root symbiotic responses at a cell type-specific level.

Transcriptional and post-transcriptional regulation of a NAC1 transcription factor in Medicago truncatula roots.

• Legume roots develop two types of lateral organs, lateral roots and nodules. Nodules develop as a result of a symbiotic interaction with rhizobia and provide a niche for the bacteria to fix atmospheric nitrogen for the plant. • The Arabidopsis NAC1 transcription factor is involved in lateral root formation, and is regulated post-transcriptionally by miRNA164 and by SINAT5-dependent ubiquitination. We analyzed in Medicago truncatula the role of the closest NAC1 homolog in lateral root formation and in nodulation. • MtNAC1 shows a different expression pattern in response to auxin than its Arabidopsis homolog and no changes in lateral root number or nodulation were observed in plants affected in MtNAC1 expression. In addition, no interaction was found with SINA E3 ligases, suggesting that post-translational regulation of MtNAC1 does not occur in M. truncatula. Similar to what was found in Arabidopsis, a conserved miR164 target site was retrieved in MtNAC1, which reduced protein accumulation of a GFP-miR164 sensor. Furthermore, miR164 and MtNAC1 show an overlapping expression pattern in symbiotic nodules, and overexpression of this miRNA led to a reduction in nodule number. • This work suggests that regulatory pathways controlling a conserved transcription factor are complex and divergent between M. truncatula and Arabidopsis.

The compact root architecture1 gene regulates lignification, flavonoid production, and polar auxin transport in Medicago truncatula.

The root system architecture is crucial to adapt plant growth to changing soil environmental conditions and consequently to maintain crop yield. In addition to root branching through lateral roots, legumes can develop another organ, the nitrogen-fixing nodule, upon a symbiotic bacterial interaction. A mutant, cra1, showing compact root architecture was identified in the model legume Medicago truncatula. cra1 roots were short and thick due to defects in cell elongation, whereas densities of lateral roots and symbiotic nodules were similar to the wild type. Grafting experiments showed that a lengthened life cycle in cra1 was due to the smaller root system and not to the pleiotropic shoot phenotypes observed in the mutant. Analysis of the cra1 transcriptome at a similar early developmental stage revealed few significant changes, mainly related to cell wall metabolism. The most down-regulated gene in the cra1 mutant encodes a Caffeic Acid O-Methyl Transferase, an enzyme involved in lignin biosynthesis; accordingly, whole lignin content was decreased in cra1 roots. This correlated with differential accumulation of specific flavonoids and decreased polar auxin transport in cra1 mutants. Exogenous application of the isoflavone formononetin to wild-type plants mimicked the cra1 root phenotype, whereas decreasing flavonoid content through silencing chalcone synthases restored the polar auxin transport capacity of the cra1 mutant. The CRA1 gene, therefore, may control legume root growth through the regulation of lignin and flavonoid profiles, leading to changes in polar auxin transport.

Characterization of a dual-affinity nitrate transporter MtNRT1.3 in the model legume Medicago truncatula.

Primary root growth in the absence or presence of exogenous NO(3)(-) was studied by a quantitative genetic approach in a recombinant inbred line (RIL) population of Medicago truncatula. A quantitative trait locus (QTL) on chromosome 5 appeared to be particularly relevant because it was seen in both N-free medium (LOD score 5.7; R(2)=13.7) and medium supplied with NO(3)(-) (LOD score, 9.5; R(2)=21.1) which indicates that it would be independent of the general nutritional status. Due to its localization exactly at the peak of this QTL, the putative NRT1-NO(3)(-) transporter (Medtr5g093170.1), closely related to Arabidopsis AtNRT1.3, a putative low-affinity nitrate transporter, appeared to be a significant candidate involved in the control of primary root growth and NO(3)(-) sensing. Functional characterization in Xenopus oocytes using both electrophysiological and (15)NO(3)(-) uptake approaches showed that Medtr5g093170.1, named MtNRT1.3, encodes a dual-affinity NO(3)(-) transporter similar to the AtNRT1.1 'transceptor' in Arabidopsis. MtNRT1.3 expression is developmentally regulated in roots, with increasing expression after completion of germination in N-free medium. In contrast to members of the NRT1 superfamily characterized so far, MtNRT1.3 is environmentally up-regulated by the absence of NO(3)(-) and down-regulated by the addition of the ion to the roots. Split-root experiments showed that the increased expression stimulated by the absence of NO(3)(-) was not the result of a systemic signalling of plant N status. The results suggest that MtNRT1.3 is involved in the response to N limitation, which increases the ability of the plant to acquire NO(3)(-) under N-limiting conditions.

Nitrogen Systemic Signaling: From Symbiotic Nodulation to Root Acquisition.

Plant nutrient acquisition is tightly regulated by resource availability and metabolic needs, implying the existence of communication between roots and shoots to ensure their integration at the whole-plant level. Here, we focus on systemic signaling pathways controlling nitrogen (N) nutrition, achieved both by the root import of mineral N and, in legume plants, through atmospheric N fixation by symbiotic bacteria inside dedicated root nodules. We explore features conserved between systemic pathways repressing or enhancing symbiotic N fixation and the regulation of mineral N acquisition by roots, as well as their integration with other environmental factors, such as phosphate, light, and CO2 availability.