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.

Localized osmotic stress activates systemic responses to N limitation in Medicago truncatula-Sinorhizobium symbiotic plants.

In mature symbiotic root nodules, differentiated rhizobia fix atmospheric dinitrogen and provide ammonium to fulfill the plant nitrogen (N) demand. The plant enables this process by providing photosynthates to the nodules. The symbiosis is adjusted to the whole plant N demand thanks to systemic N signaling controlling nodule development. Symbiotic plants under N deficit stimulate nodule expansion and activate nodule senescence under N satiety. Besides, nodules are highly sensitive to drought. Here, we used split-root systems to characterize the systemic responses of symbiotic plants to a localized osmotic stress. We showed that polyéthylène glycol (PEG) application rapidly inhibited the symbiotic dinitrogen fixation activity of nodules locally exposed to the treatment, resulting to the N limitation of the plant supplied exclusively by symbiotic dinitrogen fixation. The localized PEG treatment triggered systemic signaling stimulating nodule development in the distant untreated roots. This response was associated with an enhancement of the sucrose allocation. Our analyses showed that transcriptomic reprogramming associated with PEG and N deficit systemic signaling(s) shared many targets transcripts. Altogether, our study suggests that systemic N signaling is a component of the adaptation of the symbiotic plant to the local variations of its edaphic environment.

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.

HSFA1a modulates plant heat stress responses and alters the 3D chromatin organization of enhancer-promoter interactions.

The complex and dynamic three-dimensional organization of chromatin within the nucleus makes understanding the control of gene expression challenging, but also opens up possible ways to epigenetically modulate gene expression. Because plants are sessile, they evolved sophisticated ways to rapidly modulate gene expression in response to environmental stress, that are thought to be coordinated by changes in chromatin conformation to mediate specific cellular and physiological responses. However, to what extent and how stress induces dynamic changes in chromatin reorganization remains poorly understood. Here, we comprehensively investigated genome-wide chromatin changes associated with transcriptional reprogramming response to heat stress in tomato. Our data show that heat stress induces rapid changes in chromatin architecture, leading to the transient formation of promoter-enhancer contacts, likely driving the expression of heat-stress responsive genes. Furthermore, we demonstrate that chromatin spatial reorganization requires HSFA1a, a transcription factor (TF) essential for heat stress tolerance in tomato. In light of our findings, we propose that TFs play a key role in controlling dynamic transcriptional responses through 3D reconfiguration of promoter-enhancer contacts.

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.

A dual legume-rhizobium transcriptome of symbiotic nodule senescence reveals coordinated plant and bacterial responses.

Senescence determines plant organ lifespan depending on aging and environmental cues. During the endosymbiotic interaction with rhizobia, legume plants develop a specific organ, the root nodule, which houses nitrogen (N)-fixing bacteria. Unlike earlier processes of the legume-rhizobium interaction (nodule formation, N fixation), mechanisms controlling nodule senescence remain poorly understood. To identify nodule senescence-associated genes, we performed a dual plant-bacteria RNA sequencing approach on Medicago truncatula-Sinorhizobium meliloti nodules having initiated senescence either naturally (aging) or following an environmental trigger (nitrate treatment or salt stress). The resulting data allowed the identification of hundreds of plant and bacterial genes differentially regulated during nodule senescence, thus providing an unprecedented comprehensive resource of new candidate genes associated with this process. Remarkably, several plant and bacterial genes related to the cell cycle and stress responses were regulated in senescent nodules, including the rhizobial RpoE2-dependent general stress response. Analysis of selected core nodule senescence plant genes allowed showing that MtNAC969 and MtS40, both homologous to leaf senescence-associated genes, negatively regulate the transition between N fixation and senescence. In contrast, overexpression of a gene involved in the biosynthesis of cytokinins, well-known negative regulators of leaf senescence, may promote the transition from N fixation to senescence in nodules.

Nitrate-induced CLE35 signaling peptides inhibit nodulation through the SUNN receptor and miR2111 repression.

Legume plants form nitrogen (N)-fixing symbiotic nodules when mineral N is limiting in soils. As N fixation is energetically costly compared to mineral N acquisition, these N sources, and in particular nitrate, inhibit nodule formation and N fixation. Here, in the model legume Medicago truncatula, we characterized a CLAVATA3-like (CLE) signaling peptide, MtCLE35, the expression of which is upregulated locally by high-N environments and relies on the Nodule Inception-Like Protein (NLP) MtNLP1. MtCLE35 inhibits nodule formation by affecting rhizobial infections, depending on the Super Numeric Nodules (MtSUNN) receptor. In addition, high N or the ectopic expression of MtCLE35 represses the expression and accumulation of the miR2111 shoot-to-root systemic effector, thus inhibiting its positive effect on nodulation. Conversely, ectopic expression of miR2111 or downregulation of MtCLE35 by RNA interference increased miR2111 accumulation independently of the N environment, and thus partially bypasses the nodulation inhibitory action of nitrate. Overall, these results demonstrate that the MtNLP1-dependent, N-induced MtCLE35 signaling peptide acts through the MtSUNN receptor and the miR2111 systemic effector to inhibit nodulation.

A linkage disequilibrium-based statistical test for Genome-Wide Epistatic Selection Scans in structured populations.

The quest for signatures of selection using single nucleotide polymorphism (SNP) data has proven efficient to uncover genes involved in conserved and/or adaptive molecular functions, but none of the statistical methods were designed to identify interacting alleles as targets of selective processes. Here, we propose a statistical test aimed at detecting epistatic selection, based on a linkage disequilibrium (LD) measure accounting for population structure and heterogeneous relatedness between individuals. SNP-based ([Formula: see text]) and window-based ([Formula: see text]) statistics fit a Student distribution, allowing to test the significance of correlation coefficients. As a proof of concept, we use SNP data from the Medicago truncatula symbiotic legume plant and uncover a previously unknown gene coadaptation between the MtSUNN (Super Numeric Nodule) receptor and the MtCLE02 (CLAVATA3-Like) signaling peptide. We also provide experimental evidence supporting a MtSUNN-dependent negative role of MtCLE02 in symbiotic root nodulation. Using human HGDP-CEPH SNP data, our new statistical test uncovers strong LD between SLC24A5 (skin pigmentation) and EDAR (hairs, teeth, sweat glands development) world-wide, which persists after correction for population structure and relatedness in Central South Asian populations. This result suggests that epistatic selection or coselection could have contributed to the phenotypic make-up in some human populations. Applying this approach to genome-wide SNP data will facilitate the identification of coadapted gene networks in model or non-model organisms.

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.

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.

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.

The NIN transcription factor coordinates CEP and CLE signaling peptides that regulate nodulation antagonistically.

Legumes tightly regulate nodule number to balance the cost of supporting symbiotic rhizobia with the benefits of nitrogen fixation. C-terminally Encoded Peptides (CEPs) and CLAVATA3-like (CLE) peptides positively and negatively regulate nodulation, respectively, through independent systemic pathways, but how these regulations are coordinated remains unknown. Here, we show that rhizobia, Nod Factors, and cytokinins induce a symbiosis-specific CEP gene, MtCEP7, which positively regulates rhizobial infection. Via grafting and split root studies, we reveal that MtCEP7 increases nodule number systemically through the MtCRA2 receptor. MtCEP7 and MtCLE13 expression in rhizobia-inoculated roots rely on the MtCRE1 cytokinin receptor and on the MtNIN transcription factor. MtNIN binds and transactivates MtCEP7 and MtCLE13, and a NIN Binding Site (NBS) identified within the proximal MtCEP7 promoter is required for its symbiotic activation. Overall, these results demonstrate that a cytokinin-MtCRE1-MtNIN regulatory module coordinates the expression of two antagonistic, symbiosis-related, peptide hormones from different families to fine-tune nodule number.

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.

Compact Root Architecture 2 Promotes Root Competence for Nodulation through the miR2111 Systemic Effector.

Nitrogen-deprived legume plants form new root organs, the nodules, following a symbiosis with nitrogen-fixing rhizobial bacteria [1]. Because this interaction is beneficial for the plant but has a high energetic cost, nodulation is tightly controlled by host plants through systemic pathways (acting at long distance) to promote or limit rhizobial infections and nodulation depending on earlier infections and on nitrogen availability [2]. In the Medicago truncatula model legume, CLE12 (Clavata3/Embryo surrounding region 12) and CLE13 signaling peptides produced in nodulated roots act in shoots through the SUNN (Super Numeric Nodule) receptor to negatively regulate nodulation and therefore autoregulate nodule number [3-5]. Conversely, CEP (C-terminally Encoded Peptide) signaling peptides produced in nitrogen-starved roots act in shoots through the CRA2 (Compact Root Architecture 2) receptor to promote nodulation already in the absence of rhizobia [6-9]. We show in this study that a downstream shoot-to-root signaling effector of these systemic pathways is the shoot-produced miR2111 microRNA [10] that negatively regulates TML1 (Too Much Love 1) and TML2 [11] transcripts accumulation in roots, ultimately promoting nodulation. Low nitrogen conditions and CEP1 signaling peptides induce in the absence of rhizobia the production of miR2111 depending on CRA2 activity in shoots, thus favoring root competence for nodulation. Together with the SUNN pathway negatively regulating the same miR2111 systemic effector when roots are nodulated, this allows a dynamic fine-tuning of the nodulation capacity of legume roots by nitrogen availability and rhizobial cues.

CEP receptor signalling controls root system architecture in Arabidopsis and Medicago.

Root system architecture (RSA) influences the effectiveness of resources acquisition from soils but the genetic networks that control RSA remain largely unclear. We used rhizoboxes, X-ray computed tomography, grafting, auxin transport measurements and hormone quantification to demonstrate that Arabidopsis and Medicago CEP (C-TERMINALLY ENCODED PEPTIDE)-CEP RECEPTOR signalling controls RSA, the gravitropic set-point angle (GSA) of lateral roots (LRs), auxin levels and auxin transport. We showed that soil-grown Arabidopsis and Medicago CEP receptor mutants have a narrower RSA, which results from a steeper LR GSA. Grafting showed that CEPR1 in the shoot controls GSA. CEP receptor mutants exhibited an increase in rootward auxin transport and elevated shoot auxin levels. Consistently, the application of auxin to wild-type shoots induced a steeper GSA and auxin transport inhibitors counteracted the CEP receptor mutant's steep GSA phenotype. Concordantly, CEP peptides increased GSA and inhibited rootward auxin transport in wild-type but not in CEP receptor mutants. The results indicated that CEP-CEP receptor-dependent signalling outputs in Arabidopsis and Medicago control overall RSA, LR GSA, shoot auxin levels and rootward auxin transport. We propose that manipulating CEP signalling strength or CEP receptor downstream targets may provide means to alter RSA.

Diversification of cytokinin phosphotransfer signaling genes in Medicago truncatula and other legume genomes.

BACKGROUND: Legumes can establish on nitrogen-deprived soils a symbiotic interaction with Rhizobia bacteria, leading to the formation of nitrogen-fixing root nodules. Cytokinin phytohormones are critical for triggering root cortical cell divisions at the onset of nodule initiation. Cytokinin signaling is based on a Two-Component System (TCS) phosphorelay cascade, involving successively Cytokinin-binding Histidine Kinase receptors, phosphorelay proteins shuttling between the cytoplasm and the nucleus, and Type-B Response Regulator (RRB) transcription factors activating the expression of cytokinin primary response genes. Among those, Type-A Response Regulators (RRA) exert a negative feedback on the TCS signaling. To determine whether the legume plant nodulation capacity is linked to specific features of TCS proteins, a genome-wide identification was performed in six legume genomes (Cajanus cajan, pigeonpea; Cicer arietinum, chickpea; Glycine max, soybean; Phaseolus vulgaris, common bean; Lotus japonicus; Medicago truncatula). The diversity of legume TCS proteins was compared to the one found in two non-nodulating species, Arabidopsis thaliana and Vitis vinifera, which are references for functional analyses of TCS components and phylogenetic analyses, respectively. RESULTS: A striking expansion of non-canonical RRBs was identified, notably leading to the emergence of proteins where the conserved phosphor-accepting aspartate residue is replaced by a glutamate or an asparagine. M. truncatula genome-wide expression datasets additionally revealed that only a limited subset of cytokinin-related TCS genes is highly expressed in different organs, namely MtCHK1/MtCRE1, MtHPT1, and MtRRB3, suggesting that this "core" module potentially acts in most plant organs including nodules. CONCLUSIONS: Further functional analyses are required to determine the relevance of these numerous non-canonical TCS RRBs in symbiotic nodulation, as well as of canonical MtHPT1 and MtRRB3 core signaling elements.

Gibberellins negatively regulate the development of Medicago truncatula root system.

The root system displays a remarkable plasticity that enables plants to adapt to changing environmental conditions. This plasticity is tightly linked to the activity of root apical meristems (RAMs) and to the formation of lateral roots, both controlled by related hormonal crosstalks. In Arabidopsis thaliana, gibberellins (GAs) were shown to positively control RAM growth and the formation of lateral roots. However, we showed in Medicago truncatula that GAs negatively regulate root growth and RAM size as well as the number of lateral roots depending at least on the MtDELLA1 protein. By using confocal microscopy and molecular analyses, we showed that GAs primarily regulate RAM size by affecting cortical cell expansion and additionally negatively regulate a subset of cytokinin-induced root expansin encoding genes. Moreover, GAs reduce the number of cortical cell layers, resulting in the formation of both shorter and thinner roots. These results suggest contrasting effects of GA regulations on the root system architecture depending on plant species.