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The roots of plants play multiple functions that are essential for growth and development, including anchoring to the soil as well as water and nutrient acquisition. These underground organs exhibit the plasticity to modify their root system architecture in response to environmental cues, allowing adaptation to change in water and nutrient availability. In addition, roots enter in mutualistic interactions with soil microorganisms, for example, the root nodule symbiosis (RNS) established between a limited group of plants and nitrogen-fixing soil bacteria and the arbuscular mycorrhiza symbiosis involving most land plants and fungi of the Glomeromycetes phylum. In the past 20 years, genetic approaches allowed the identification and functional characterization of genes required for the specific programs of root development, root nodule, and arbuscular mycorrhiza symbioses. These genetic studies provided evidence that the program of the RNS recruited components of the arbuscular mycorrhiza symbiosis and the root developmental programs. The execution of these programs is strongly influenced by epigenetic changes-DNA methylation and histone post-translational modifications-that alter chromatin conformation modifying the expression of key genes. In this review, we summarize recent advances that highlight how DNA methylation and histone post-translational modifications, as well as chromatin remodeling factors and long noncoding RNAs, shape the root system architecture and allow the successful establishment of both root nodule and arbuscular mycorrhiza symbioses. We anticipate that the analysis of dynamic epigenetic changes and chromatin 3D structure in specific single cells or tissue types of root organs will illuminate our understanding of how root developmental and symbiotic programs are orchestrated, opening exciting questions and new perspectives to modulate agronomical and ecological traits linked to nutrient acquisition., Competing Interests: Conflict of interest statement: None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

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.)

Background: Legume roots show a remarkable plasticity to adapt their architecture to biotic and abiotic constraints, including symbiotic interactions. However, global analysis of miRNA regulation in roots is limited, and a global view of the evolution of miRNA-mediated diversification in different ecotypes is lacking., Results: In the model legume Medicago truncatula, we analyze the small RNA transcriptome of roots submitted to symbiotic and pathogenic interactions. Genome mapping and a computational pipeline identify 416 miRNA candidates, including known and novel variants of 78 miRNA families present in miRBase. Stringent criteria of pre-miRNA prediction yield 52 new mtr-miRNAs, including 27 miRtrons. Analyzing miRNA precursor polymorphisms in 26 M. truncatula ecotypes identifies higher sequence polymorphism in conserved rather than Medicago-specific miRNA precursors. An average of 19 targets, mainly involved in environmental responses and signalling, is predicted per novel miRNA. We identify miRNAs responsive to bacterial and fungal pathogens or symbionts as well as their related Nod and Myc-LCO symbiotic signals. Network analyses reveal modules of new and conserved co-expressed miRNAs that regulate distinct sets of targets, highlighting potential miRNA-regulated biological pathways relevant to pathogenic and symbiotic interactions., Conclusions: We identify 52 novel genuine miRNAs and large plasticity of the root miRNAome in response to the environment, and also in response to purified Myc/Nod signaling molecules. The new miRNAs identified and their sequence variation across M. truncatula ecotypes may be crucial to understand the adaptation of root growth to the soil environment, notably in the agriculturally important legume crops.

A C subunit of the heterotrimeric nuclear factor Y (NF-YC1) was shown to play a key role in nodule organogenesis and bacterial infection during the nitrogen fixing symbiosis established between common bean (Phaseolus vulgaris) and Rhizobium etli. To identify other proteins involved in this process, we used the yeast (Saccharomyces cerevisiae) two-hybrid system to screen for NF-YC1-interacting proteins. One of the positive clones encodes a member of the Phytochrome A Signal Transduction1 subfamily of GRAS (for Gibberellic Acid-Insensitive (GAI), Repressor of GAI, and Scarecrow) transcription factors. The protein, named Scarecrow-like13 Involved in Nodulation (SIN1), localizes both to the nucleus and the cytoplasm, but in transgenic Nicotiana benthamiana cells, bimolecular fluorescence complementation suggested that the interaction with NF-YC1 takes place predominantly in the nucleus. SIN1 is expressed in aerial and root tissues, with higher levels in roots and nodules. Posttranscriptional gene silencing of SIN1 using RNA interference (RNAi) showed that the product of this gene is involved in lateral root elongation. However, root cell organization, density of lateral roots, and the length of root hairs were not affected by SIN1 RNAi. In addition, the expression of the RNAi of SIN1 led to a marked reduction in the number and size of nodules formed upon inoculation with R. etli and affected the progression of infection threads toward the nodule primordia. Expression of NF-YA1 and the G2/M transition cell cycle genes Cyclin B and Cell Division Cycle2 was reduced in SIN1 RNAi roots. These data suggest that SIN1 plays a role in lateral root elongation and the establishment of root symbiosis in common bean.

Arbuscular mycorrhiza (AM) is a root endosymbiosis between plants and glomeromycete fungi. It is the most widespread terrestrial plant symbiosis, improving plant uptake of water and mineral nutrients. Yet, despite its crucial role in land ecosystems, molecular mechanisms leading to its formation are just beginning to be unravelled. Recent evidence suggests that AM fungi produce diffusible symbiotic signals. Here we show that Glomus intraradices secretes symbiotic signals that are a mixture of sulphated and non-sulphated simple lipochitooligosaccharides (LCOs), which stimulate formation of AM in plant species of diverse families (Fabaceae, Asteraceae and Umbelliferae). In the legume Medicago truncatula these signals stimulate root growth and branching by the symbiotic DMI signalling pathway. These findings provide a better understanding of the evolution of signalling mechanisms involved in plant root endosymbioses and will greatly facilitate their molecular dissection. They also open the way to using these natural and very active molecules in agriculture.

Legume root architecture is characterized by the development of two de novo meristems, leading to the formation of lateral roots or symbiotic nitrogen-fixing nodules. Organogenesis involves networks of transcription factors, the encoding mRNAs of which are frequently targets of microRNA (miRNA) regulation. Most plant miRNAs, in contrast with animal miRNAs, are encoded as single entities in an miRNA precursor. In the model legume Medicago truncatula, we have identified the MtMIR166a precursor containing tandem copies of MIR166 in a single transcriptional unit. These miRNAs post-transcriptionally regulate a new family of transcription factors associated with nodule development, the class-III homeodomain-leucine zipper (HD-ZIP III) genes. In situ expression analysis revealed that these target genes are spatially co-expressed with MIR166 in vascular bundles, and in apical regions of roots and nodules. Overexpression of the tandem miRNA precursor correlated with MIR166 accumulation and the downregulation of several class-III HD-ZIP genes, indicating its functionality. MIR166 overexpression reduced the number of symbiotic nodules and lateral roots, and induced ectopic development of vascular bundles in these transgenic roots. Hence, plant polycistronic miRNA precursors, although rare, can be processed, and MIR166-mediated post-transcriptional regulation is a new regulatory pathway involved in the regulation of legume root architecture.

Root growth and function are determined by the action of environmental stresses through specific genes that adapt root development to these restrictive conditions. We have defined in vitro conditions affecting the growth and recovery of Medicago truncatula roots after a salt stress. A dedicated macroarray containing 384 genes, based on a large-scale subtractive hybridization approach, was constructed and used to analyze gene expression during salt stress and recovery of root growth from this stress. Several potential regulatory genes were identified as being linked to this recovery process: a novel RNA-binding protein, a small G-protein homologous to ROP9, a receptor-like kinase, two TF IIIA-like and an AP2-like transcription factors (TF), MtZpt2-1, MtZpt2-2 and MtAp2, and a histidine kinase associated with cytokinin transduction pathways. The two ZPT2-type TFs were also rapidly induced by cold stress in roots. By analyzing transgenic M. truncatula plants showing reduced expression levels of both TFs and affected in their capacity to recover root growth after a salt stress, we identified potential target genes that were either activated or repressed in these plants. Overexpression of MtZpt2-1 in roots conferred salt tolerance and affected the expression of three putative targets in the predicted manner: a cold-regulated A (CORA) homolog, a flower-promoting factor (FPF1) homolog and an auxin-induced proline-rich protein (PRP) gene. Hence, regulatory networks depending on TFIIIA-like transcription factors are involved in the control of root adaptation to salt stress.

This article describes an efficient procedure to study Nod factor-induced gene expression in root hairs of the model legume Medicago truncatula. By developing an improved method of fracturing frozen root hairs, it has been possible to obtain a highly purified root hair fraction from M. truncatula seedlings yielding sufficient RNA for real-time quantitative RT-PCR expression analysis. After Nod factor treatment it was possible to detect up to 100-fold increases of MtENOD11 and pMtENOD11-gus transcript levels in root hair RNA. This corresponds to 5-7-fold higher induction levels than for entire root tissue preparations. Furthermore, the use of these enriched RNA samples has revealed for the first time a very significant induction (30-fold) of the MtENOD40 gene in root hairs in response to Nod factors. It is concluded that the rapid and convenient procedure described here will be particularly useful for detecting tissue-specific low-level gene expression in root hairs responding to Rhizobium Nod factors or other exogenous signals.

To study the role of LecRK (lectin-like receptor kinase) genes in the legumerhizobia symbiosis, we have characterized the four Medicago truncatula Gaernt. LecRK genes that are most highly expressed in roots. Three of these genes, MtLecRK7;1, MtLecRK7;2, and MtLecRK7;3, encode proteins most closely related to the Class A LecRKs of Arabidopsis, whereas the protein encoded by the fourth gene, MtLecRK1;1, is most similar to a Class B Arabidopsis LecRK. All four genes show a strongly enhanced root expression, and detailed studies on MtLecRK1;1 and MtLecRK7;2 revealed that the levels of their mRNAs are increased by nitrogen starvation and transiently repressed after either rhizobial inoculation or addition of lipochitooligosaccharidic Nod factors. Studies of the MtLecRK1;1 and MtLecRK7;2 proteins, using green fluorescent protein fusions in transgenic M. truncatula roots, revealed that they are located in the plasma membrane and that their central transmembrane-spanning helix is required for correct sorting. Moreover, their lectin-like domains appear to be highly glycosylated. Of the four proteins, only MtLecRK1;1 shows a high conservation of key residues implicated in monosaccharide binding, and molecular modeling revealed that this protein may be capable of interacting with Nod factors. However, no increase in Nod factor binding was found in roots overexpressing a fusion in which the kinase domain of this protein had been replaced with green fluorescent protein. Roots expressing this fusion protein however showed an increase in nodule number, suggesting that expression of MtLecRK1;1 influences nodulation. The potential role of LecRKs in the legume-rhizobia symbiosis is discussed.

The model legume Medicago truncatula contains at least six apyrase-like genes, five of which (MtAPY1;1, MtAPY1;2, MtAPY1;3, MtAPY1;4, and MtAPY1;5) are members of a legume-specific family, whereas a single gene (MtAPY2) has closer homologs in Arabidopsis. Phylogenetic analysis has revealed that the proteins encoded by these two plant gene families are more similar to yeast (Saccharomyces cerevisiae) GDA1 and to two proteins encoded by newly described mammalian genes (ENP5 and 6) than they are to mammalian CD39- and CD39-like proteins. Northern analyses and analyses of the frequencies of expressed sequence tags (ESTs) in different cDNA libraries suggest that in roots, leaves, and flowers, the more highly expressed genes are MtAPY1;3/MtAPY2, MtAPY1;3/MtAPY1;5 and MtAPY1;2/MtAPY1;3 respectively. In roots, at least four of the MtAPY1 genes are induced transiently within 3 to 6 h by a stress response that seems to be ethylene independent because it occurs after treatment with an ethylene synthesis inhibitor and also in the skl ethylene-insensitive mutant. This response also occurs in roots of the following symbiotic mutants: dmi1, dmi2, dmi3, nsp, hcl, pdl, lin, and skl. No evidence was obtained for a rapid, transient, and specific induction of the MtAPY genes in roots in response to rhizobia or rhizobial lipochitooligosaccharidic Nod factors. Thus, our data suggest that the apyrase-like genes, which in several legumes have been implicated to play a role in the legume-rhizobia symbiosis (with some members being described as early nodulin genes), are not regulated symbiotically by rhizobia in M. truncatula.

We report on a large-scale expressed sequence tag (EST) sequencing and analysis program aimed at characterizing the sets of genes expressed in roots of the model legume Medicago truncatula during interactions with either of two microsymbionts, the nitrogen-fixing bacterium Sinorhizobium meliloti or the arbuscular mycorrhizal fungus Glomus intraradices. We have designed specific tools for in silico analysis of EST data, in relation to chimeric cDNA detection, EST clustering, encoded protein prediction, and detection of differential expression. Our 21 473 5'- and 3'-ESTs could be grouped into 6359 EST clusters, corresponding to distinct virtual genes, along with 52 498 other M.truncatula ESTs available in the dbEST (NCBI) database that were recruited in the process. These clusters were manually annotated, using a specifically developed annotation interface. Analysis of EST cluster distribution in various M.truncatula cDNA libraries, supported by a refined R test to evaluate statistical significance and by 'electronic northern' representation, enabled us to identify a large number of novel genes predicted to be up- or down-regulated during either symbiotic root interaction. These in silico analyses provide a first global view of the genetic programs for root symbioses in M.truncatula. A searchable database has been built and can be accessed through a public interface.

Plant NF-Y transcription factors control a wide array of biological functions enabling appropriate reproductive and developmental processes as well as adaptation to various abiotic and biotic environments. In Medicago truncatula, MtNF-YA1 was previously identified as a key determinant for nodule development and establishment of rhizobial symbiosis. Here, we highlight a new role for this protein in compatibility to Aphanomyces euteiches, a root pathogenic oomycete. The Mtnf-ya1-1 mutant plants showed better survival rate, reduced symptoms, and increased development of their root apparatus as compared to their wild-type (WT) background A17. MtNF-YA-1 was specifically up-regulated by A. euteiches in F83005.5, a highly susceptible natural accession of M. truncatula while transcript level remained stable in A17, which is partially resistant. The role of MtNF-YA1 in F83005.5 susceptibility was further documented by reducing MtNF-YA1 expression either by overexpression of the miR169q, a microRNA targeting MtNF-YA1, or by RNAi approaches leading to a strong enhancement in the resistance of this susceptible line. Comparative analysis of the transcriptome of WT and Mtnf-ya1-1 led to the identification of 1509 differentially expressed genes. Among those, almost 36 defense-related genes were constitutively expressed in Mtnf-ya1-1, while 20 genes linked to hormonal pathways were repressed. In summary, we revealed an unexpected dual role for this symbiotic transcription factor as a key player in the compatibility mechanisms to a pathogen. [ABSTRACT FROM AUTHOR]