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Legume plants develop two types of root postembryonic organs, lateral roots and symbiotic nodules, using shared regulatory components. The module composed by the microRNA390, the Trans-Acting SIRNA3 (TAS3) RNA and the Auxin Response Factors (ARF)2, ARF3, and ARF4 (miR390/TAS3/ARFs) mediates the control of both lateral roots and symbiotic nodules in legumes. Here, a transcriptomic approach identified a member of the Lateral Organ Boundaries Domain (LBD) family of transcription factors in Medicago truncatula, designated MtLBD17/29a, which is regulated by the miR390/TAS3/ARFs module. ChIP-PCR experiments evidenced that MtARF2 binds to an Auxin Response Element present in the MtLBD17/29a promoter. MtLBD17/29a is expressed in root meristems, lateral root primordia, and noninfected cells of symbiotic nodules. Knockdown of MtLBD17/29a reduced the length of primary and lateral roots and enhanced lateral root formation, whereas overexpression of MtLBD17/29a produced the opposite phenotype. Interestingly, both knockdown and overexpression of MtLBD17/29a reduced nodule number and infection events and impaired the induction of the symbiotic genes Nodulation Signaling Pathway (NSP) 1 and 2. Our results demonstrate that MtLBD17/29a is regulated by the miR390/TAS3/ARFs module and a direct target of MtARF2, revealing a new lateral root regulatory hub recruited by legumes to act in the root nodule symbiotic program., (© 2024 The Authors New Phytologist © 2024 New Phytologist Foundation.)

The formation of symbiotic nodule cells in Medicago truncatula is driven by successive endoreduplication cycles and transcriptional reprogramming in different temporal waves including the activation of more than 600 cysteine-rich NCR genes expressed only in nodules. We show here that the transcriptional waves correlate with growing ploidy levels and have investigated how the epigenome changes during endoreduplication cycles. Differential DNA methylation was found in only a small subset of symbiotic nodule-specific genes, including more than half of the NCR genes, whereas in most genes DNA methylation was unaffected by the ploidy levels and was independent of the genes' active or repressed state. On the other hand, expression of nodule-specific genes correlated with ploidy-dependent opening of the chromatin as well as, in a subset of tested genes, with reduced H3K27me3 levels combined with enhanced H3K9ac levels. Our results suggest that endoreduplication-dependent epigenetic changes contribute to transcriptional reprogramming in the differentiation of symbiotic cells., Competing Interests: The authors declare no conflict of interest.

During endosymbiotic interactions between legume plants and nitrogen-fixing rhizobia, successful root infection by bacteria and nodule organogenesis requires the perception and transduction of bacterial lipo-chitooligosaccharidic signal called Nod factor (NF). NF perception in legume roots leads to the activation of an early signaling pathway and of a set of symbiotic genes which is controlled by specific early transcription factors (TFs) including CYCLOPS/IPD3, NSP1, NSP2, ERN1 and NIN. In this study, we bring convincing evidence that the Medicago truncatula CCAAT-box-binding NF-YA1 TF, previously associated with later stages of rhizobial infection and nodule meristem formation is, together with its closest homolog NF-YA2, also an essential positive regulator of the NF-signaling pathway. Here we show that NF-YA1 and NF-YA2 are both expressed in epidermal cells responding to NFs and their knock-down by reverse genetic approaches severely affects the NF-induced expression of symbiotic genes and rhizobial infection. Further over-expression, transactivation and ChIP-PCR approaches indicate that NF-YA1 and NF-YA2 function, at least in part, via the direct activation of ERN1. We thus propose a model in which NF-YA1 and NF-YA2 appear as early symbiotic regulators acting downstream of DMI3 and NIN and possibly within the same regulatory complexes as NSP1/2 to directly activate the expression of ERN1., (© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.)

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.

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., (© 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.)

Phytohormonal interactions are essential to regulate plant organogenesis. In response to the presence of signals from symbiotic bacteria, the Nod factors, legume roots generate a new organ: the nitrogen-fixing nodule. Analysis of mutants in the Medicago truncatula CRE1 cytokinin receptor and of the MtRR4 cytokinin primary response gene expression pattern revealed that cytokinin acts in initial cortical cell divisions and later in the transition between meristematic and differentiation zones of the mature nodule. MtCRE1 signaling is required for activation of the downstream nodulation-related transcription factors MtERN1, MtNSP2 and MtNIN, as well as to regulate expression and accumulation of PIN auxin efflux carriers. Whereas the MtCRE1 pathway is required to allow the inhibition of polar auxin transport in response to rhizobia, nodulation is still negatively regulated by the MtEIN2/SICKLE-dependent ethylene pathway in cre1 mutants. Hence, MtCRE1 signaling acts as a regulatory knob, integrating positive plant and bacterial cues to control legume nodule organogenesis., (© 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.)

Lateral root (LR) formation and emergence are influenced by the environment and determines the architecture of the root system in the soil. Whereas auxins appear as the main hormone controlling LR initiation, patterning and emergence, abscisic acid (ABA) is the key hormone mediating the effect of the environment on root architecture. Hormone signaling act through transcription factors (TFs) and the Medicago truncatula LOB-like TF LBD1 was shown to be auxin-inducible but repressed by the HD-Zip I TF MtHB1 in response to salt stress and ABA during LR formation. Here, we demonstrate that the constitutive expression of Mt LBD1 in Medicago roots alters their global architecture when the plant is subjected to salt stress. Hence, LBD1 may control the final form of the root system in the soil environment.

The adaptation of root architecture to environmental constraints is a major agricultural trait, notably in legumes, the third main crop worldwide. This root developmental plasticity depends on the formation of lateral roots (LRs) emerging from primary roots. In the model legume Medicago truncatula, the HD-Zip I transcription factor HB1 is expressed in primary and lateral root meristems and induced by salt stress. Constitutive expression of HB1 in M. truncatula roots alters their architecture, whereas hb1 TILLING mutants showed increased lateral root emergence. Electrophoretic mobility shift assay, promoter mutagenesis, and chromatin immunoprecipitation-PCR assays revealed that HB1 directly recognizes a CAATAATTG cis-element present in the promoter of a LOB-like (for Lateral Organ Boundaries) gene, LBD1, transcriptionally regulated by auxin. Expression of these genes in response to abscisic acid and auxin and their behavior in hb1 mutants revealed an HB1-mediated repression of LBD1 acting during LR emergence. M. truncatula HB1 regulates an adaptive developmental response to minimize the root surface exposed to adverse environmental stresses.

The AP2/ERF superfamily is a large, plant-specific transcription factor family that is involved in many important processes, including plant growth, development, and stress responses. Using Medicago truncatula genome information, we identified and characterized 123 putative AP2/ERF genes, which were named as MtERF1-123. These genes were classified into four families based on phylogenetic analysis, which is consistent with the results of other plant species. MtERF genes are distributed throughout all chromosomes but are clustered on various chromosomes due to genomic tandem and segmental duplication. Using transcriptome, high-throughput sequencing data, and qRT-PCR analysis, we assessed the expression patterns of the MtERF genes in tissues during development and under abiotic stresses. In total, 87 MtERF genes were expressed in plant tissues, most of which were expressed in specific tissues during development or under specific abiotic stress treatments. These results support the notion that MtERF genes are involved in developmental regulation and environmental responses in M. truncatula. Furthermore, a cluster of DREB subfamily members on chromosome 6 was induced by both cold and freezing stress, representing a positive gene regulatory response under low temperature stress, which suggests that these genes might contribute to freezing tolerance to M. truncatula. In summary, our genome-wide characterization, evolutionary analysis, and expression pattern analysis of MtERF genes in M. truncatula provides valuable information for characterizing the molecular functions of these genes and utilizing them to improve stress tolerance in plants. [ABSTRACT FROM AUTHOR]