Whole-genome landscape of Medicago truncatula symbiotic genes.

Advances in deciphering the functional architecture of eukaryotic genomes have been facilitated by recent breakthroughs in sequencing technologies, enabling a more comprehensive representation of genes and repeat elements in genome sequence assemblies, as well as more sensitive and tissue-specific analyses of gene expression. Here we show that PacBio sequencing has led to a substantially improved genome assembly of Medicago truncatula A17, a legume model species notable for endosymbiosis studies1, and has enabled the identification of genome rearrangements between genotypes at a near-base-pair resolution. Annotation of the new M. truncatula genome sequence has allowed for a thorough analysis of transposable elements and their dynamics, as well as the identification of new players involved in symbiotic nodule development, in particular 1,037 upregulated long non-coding RNAs (lncRNAs). We have also discovered that a substantial proportion (~35% and 38%, respectively) of the genes upregulated in nodules or expressed in the nodule differentiation zone colocalize in genomic clusters (270 and 211, respectively), here termed symbiotic islands. These islands contain numerous expressed lncRNA genes and display differentially both DNA methylation and histone marks. Epigenetic regulations and lncRNAs are therefore attractive candidate elements for the orchestration of symbiotic gene expression in the M. truncatula genome.

Stable Inactivation of MicroRNAs in Medicago truncatula Roots.

MicroRNAs are key regulators in the development processes or stress responses in plants. In the last decade, several conserved or non-conserved microRNAs have been identified in Medicago truncatula. Different strategies leading to the inactivation of microRNAs in plants have been described. Here, we propose a protocol for an effective inactivation of microRNAs using a STTM strategy in M. truncatula transgenic roots.

Root Development in Medicago truncatula: Lessons from Genetics to Functional Genomics.

This decade introduced "omics" approaches, such as genomics, transcriptomics, proteomics, and metabolomics in association with reverse and forward genetic approaches, developed earlier, to try to identify molecular pathways involved in the development or in the response to environmental conditions as well as in animals and plants. This review summarizes studies that utilized "omics" strategies to unravel the root development in the model legume Medicago truncatula and how external factors such as soil mineral status or the presence of bacteria and fungi affect root system architecture in this species. We also compare these "omics" data to the knowledges concerning the Arabidopsis thaliana root development, nowadays considered as the model of allorhiz root systems. However, unlike legumes, this species is unable to interact with soil nitrogen-fixing rhizobia and arbuscular-mycorrhizal (AM) fungi to develop novel root-derived symbiotic structures. Differences in root organization, development, and regulatory pathways between these two model species have been highlighted.

The sunflower genome provides insights into oil metabolism, flowering and Asterid evolution.

The domesticated sunflower, Helianthus annuus L., is a global oil crop that has promise for climate change adaptation, because it can maintain stable yields across a wide variety of environmental conditions, including drought. Even greater resilience is achievable through the mining of resistance alleles from compatible wild sunflower relatives, including numerous extremophile species. Here we report a high-quality reference for the sunflower genome (3.6 gigabases), together with extensive transcriptomic data from vegetative and floral organs. The genome mostly consists of highly similar, related sequences and required single-molecule real-time sequencing technologies for successful assembly. Genome analyses enabled the reconstruction of the evolutionary history of the Asterids, further establishing the existence of a whole-genome triplication at the base of the Asterids II clade and a sunflower-specific whole-genome duplication around 29 million years ago. An integrative approach combining quantitative genetics, expression and diversity data permitted development of comprehensive gene networks for two major breeding traits, flowering time and oil metabolism, and revealed new candidate genes in these networks. We found that the genomic architecture of flowering time has been shaped by the most recent whole-genome duplication, which suggests that ancient paralogues can remain in the same regulatory networks for dozens of millions of years. This genome represents a cornerstone for future research programs aiming to exploit genetic diversity to improve biotic and abiotic stress resistance and oil production, while also considering agricultural constraints and human nutritional needs.

Noncoding RNAs, Emerging Regulators in Root Endosymbioses.

Endosymbiosis interactions allow plants to grow in nutrient-deficient soil environments. The arbuscular mycorrhizal (AM) symbiosis is an ancestral interaction between land plants and fungi, whereas nitrogen-fixing symbioses are highly specific for certain plants, notably major crop legumes. The signaling pathways triggered by specific lipochitooligosaccharide molecules involved in these interactions have common components that also overlap with plant root development. These pathways include receptor-like kinases, transcription factors (TFs), and various intermediate signaling effectors, including noncoding (nc)RNAs. These latter molecules have emerged as major regulators of gene expression and small ncRNAs, composed of micro (mi)RNAs and small interfering (si)RNAs, are known to control gene expression at transcriptional (chromatin) or posttranscriptional levels. In this review, we describe exciting recent data connecting variants of conserved si/miRNAs with the regulation of TFs, such as NSP2, NFY-A1, auxin-response factors, and AP2-like proteins, known to be involved in symbiosis. The link between hormonal regulations and these si- and miRNA-TF nodes is proposed in a model in which different feedback loops or regulations controlling endosymbiosis signaling are integrated. The diversity and emerging regulatory networks of young legume miRNAs are also highlighted.

Small RNA profiles in soybean primary root tips under water deficit.

BACKGROUND: Soybean (Glycine max) production is significantly hampered by frequent droughts in many regions of the world including the United States. Identifying microRNA (miRNA)-controlled posttranscriptional gene regulation under drought will enhance our understanding of molecular basis of drought tolerance in this important cash crop. Indeed, miRNA profiles in soybean exposed to drought were studied but not from the primary root tips, which is not only a main zone of water uptake but also critical for water stress sensing and signaling. METHODS: Here we report miRNA profiles specifically from well-watered and water-stressed primary root tips (0 to 8 mm from the root apex) of soybean. Small RNA sequencing confirmed the expression of vastly diverse miRNA (303 individual miRNAs) population, and, importantly several conserved miRNAs were abundantly expressed in primary root tips. RESULTS: Notably, 12 highly conserved miRNA families were differentially regulated in response to water-deficit; six were upregulated while six others were downregulated at least by one fold (log2) change. Differentially regulated soybean miRNAs are targeting genes include auxin response factors, Cu/Zn Superoxide dismutases, laccases and plantacyanin and several others. CONCLUSIONS: These results highlighted the importance of miRNAs in primary root tips both under control and water-deficit conditions; under control conditions, miRNAs could be important for cell division, cell elongation and maintenance of the root apical meristem activity including quiescent centre whereas under water stress differentially regulated miRNAs could decrease auxin signaling and oxidative stress as well as other metabolic processes that save energy and water.

The small RNA diversity from Medicago truncatula roots under biotic interactions evidences the environmental plasticity of the miRNAome.

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 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.

A miR169 isoform regulates specific NF-YA targets and root architecture in Arabidopsis.

In plants, roots are essential for water and nutrient acquisition. MicroRNAs (miRNAs) regulate their target mRNAs by transcript cleavage and/or inhibition of protein translation and are known as major post-transcriptional regulators of various developmental pathways and stress responses. In Arabidopsis thaliana, four isoforms of miR169 are encoded by 14 different genes and target diverse mRNAs, encoding subunits A of the NF-Y transcription factor complex. These miRNA isoforms and their targets have previously been linked to nutrient signalling in plants. By using mimicry constructs against different isoforms of miR169 and miR-resistant versions of NF-YA genes we analysed the role of specific miR169 isoforms in root growth and branching. We identified a regulatory node involving the particular miR169defg isoform and NF-YA2 and NF-YA10 genes that acts in the control of primary root growth. The specific expression of MIM169defg constructs altered specific cell type numbers and dimensions in the root meristem. Preventing miR169defg-regulation of NF-YA2 indirectly affected laterial root initiation. We also showed that the miR169defg isoform affects NF-YA2 transcripts both at mRNA stability and translation levels. We propose that a specific miR169 isoform and the NF-YA2 target control root architecture in Arabidopsis.

Small RNA pathways and diversity in model legumes: lessons from genomics.

Small non-coding RNAs (smRNA) participate in the regulation of development, cell differentiation, adaptation to environmental constraints and defense responses in plants. They negatively regulate gene expression by degrading specific mRNA targets, repressing their translation or modifying chromatin conformation through homologous interaction with target loci. MicroRNAs (miRNA) and short-interfering RNAs (siRNA) are generated from long double stranded RNA (dsRNA) that are cleaved into 20-24-nucleotide dsRNAs by RNase III proteins called DICERs (DCL). One strand of the duplex is then loaded onto effective complexes containing different ARGONAUTE (AGO) proteins. In this review, we explored smRNA diversity in model legumes and compiled available data from miRBAse, the miRNA database, and from 22 reports of smRNA deep sequencing or miRNA identification genome-wide in three legumes: Medicago truncatula, soybean (Glycine max) and Lotus japonicus. In addition to conserved miRNAs present in other plant species, 229, 179, and 35 novel miRNA families were identified respectively in these 3 legumes, among which several seems legume-specific. New potential functions of several miRNAs in the legume-specific nodulation process are discussed. Furthermore, a new category of siRNA, the phased siRNAs, which seems to mainly regulate disease-resistance genes, was recently discovered in legumes. Despite that the genome sequence of model legumes are not yet fully completed, further analysis was performed by database mining of gene families and protein characteristics of DCLs and AGOs in these genomes. Although most components of the smRNA pathways are conserved, identifiable homologs of key smRNA players from non-legumes, like AGO10 or DCL4, could not yet be detected in M. truncatula available genomic and expressed sequence (EST) databases. In contrast to Arabidopsis, an important gene diversification was observed in the three legume models (for DCL2, AGO4, AGO2, and AGO10) or specifically in soybean for DCL1 and DCL4. Functional significance of these variant isoforms may reflect peculiarities of smRNA biogenesis and functions in legumes.

miR396 affects mycorrhization and root meristem activity in the legume Medicago truncatula.

The root system is crucial for acquisition of resources from the soil. In legumes, the efficiency of mineral and water uptake by the roots may be reinforced due to establishment of symbiotic relationships with mycorrhizal fungi and interactions with soil rhizobia. Here, we investigated the role of miR396 in regulating the architecture of the root system and in symbiotic interactions in the model legume Medicago truncatula. Analyses with promoter-GUS fusions suggested that the mtr-miR396a and miR396b genes are highly expressed in root tips, preferentially in the transition zone, and display distinct expression profiles during lateral root and nodule development. Transgenic roots of composite plants that over-express the miR396b precursor showed lower expression of six growth-regulating factor genes (MtGRF) and two bHLH79-like target genes, as well as reduced growth and mycorrhizal associations. miR396 inactivation by mimicry caused contrasting tendencies, with increased target expression, higher root biomass and more efficient colonization by arbuscular mycorrhizal fungi. In contrast to MtbHLH79, repression of three GRF targets by RNA interference severely impaired root growth. Early activation of mtr-miR396b, concomitant with post-transcriptional repression of MtGRF5 expression, was also observed in response to exogenous brassinosteroids. Growth limitation in miR396 over-expressing roots correlated with a reduction in cell-cycle gene expression and the number of dividing cells in the root apical meristem. These results link the miR396 network to the regulation of root growth and mycorrhizal associations in plants.

Analyzing small and long RNAs in plant development using non-radioactive in situ hybridization.

In the past decade, hundreds of non-coding RNAs (small and long RNAs) have been identified as crucial elements in developmental processes and stress response in plants. Among small RNAs, the microRNAs or miRNAs control levels of specific mRNA by inhibiting translation or reducing the stability of their mRNA targets through integration into different ribonucleoproteins (RNP). Spatio-temporal expression of small and long RNAs, using reporter genes or in situ hybridization, is essential to understand their functions. We are interested in understanding the role of various non-coding RNAs (including miRNAs) in the regulation of root and nodule development in legumes, which are agriculturally important crops. Here, we present the protocol we are currently using for detection of small and long RNA in model legume plants and tissues, like nodules and roots. The probe selection, as well as the fixation and permeabilization steps allowing to preserve tissues and cell integrity and to increase accessibility to RNA targets, will be specifically discussed.

Analyzing protein distribution in plant tissues using "whole-mount" immunolocalization.

Proteins are distributed in different cellular compartments. Our group studies the role of non-coding RNAs and associated RNPs in the development and stress response in legumes. Ribonucleoproteins (RNPs) are RNA-protein complexes that play different roles in many cellular processes. Long and small non-coding RNAs determine the specificity of action of several RNPs as the RNA Induced Silencing Complex (RISC), or affect mRNA translation, splicing and stability by interacting with other RNPs such as P-bodies, spliceosome or polysomes. Together with small and long RNAs (Chapter 20), the precise localization of the associated RNPs or the translational products regulated by small RNAs (ie target proteins regulated by miRNAs, or translationally-regulated products) by immunocytochemistry could bring novel insights into these regulatory processes. The protocol described is currently used for detection of RNP associated proteins in nodules and roots of Medicago truncatula but could be extended to any other protein. The critical points, as the choice of the antibody and the fixation and permeabilization steps, that allow preservation of tissue and cell integrity and increase the accessibility to epitopes, will be discussed.

The microRNA miR171h modulates arbuscular mycorrhizal colonization of Medicago truncatula by targeting NSP2.

Most land plants live symbiotically with arbuscular mycorrhizal fungi. Establishment of this symbiosis requires signals produced by both partners: strigolactones in root exudates stimulate pre-symbiotic growth of the fungus, which releases lipochito-oligosaccharides (Myc-LCOs) that prepare the plant for symbiosis. Here, we have investigated the events downstream of this early signaling in the roots. We report that expression of miR171h, a microRNA that targets NSP2, is up-regulated in the elongation zone of the root during colonization by Rhizophagus irregularis (formerly Glomus intraradices) and in response to Myc-LCOs. Fungal colonization was much reduced by over-expressing miR171h in roots, mimicking the phenotype of nsp2 mutants. Conversely, in plants expressing an NSP2 mRNA resistant to miR171h cleavage, fungal colonization was much increased and extended into the elongation zone of the roots. Finally, phylogenetic analyses revealed that miR171h regulation of NSP2 is probably conserved among mycotrophic plants. Our findings suggest a regulatory mechanism, triggered by Myc-LCOs, that prevents over-colonization of roots by arbuscular mycorrhizal fungi by a mechanism involving miRNA-mediated negative regulation of NSP2.

Complexity of miRNA-dependent regulation in root symbiosis.

The development of root systems may be strongly affected by the symbiotic interactions that plants establish with soil organisms. Legumes are able to develop symbiotic relationships with both rhizobial bacteria and arbuscular mycorrhizal fungi leading to the formation of nitrogen-fixing nodules and mycorrhizal arbuscules, respectively. Both of these symbiotic interactions involve complex cellular reprogramming and profound morphological and physiological changes in specific root cells. In addition, the repression of pathogenic defence responses seems to be required for successful symbiotic interactions. Apart from typical regulatory genes, such as transcription factors, microRNAs (miRNAs) are emerging as riboregulators that control gene networks in eukaryotic cells through interactions with specific target mRNAs. In recent years, the availability of deep-sequencing technologies and the development of in silico approaches have allowed for the identification of large sets of miRNAs and their targets in legumes. A number of conserved and legume-specific miRNAs were found to be associated with symbiotic interactions as shown by their expression patterns or actions on symbiosis-related targets. In this review, we combine data from recent literature and genomic and deep-sequencing data on miRNAs controlling nodule development or restricting defence reactions to address the diversity and specificity of miRNA-dependent regulation in legume root symbiosis. Phylogenetic analysis of miRNA isoforms and their potential targets suggests a role for miRNAs in the repression of plant defence during symbiosis and revealed the evolution of miRNA-dependent regulation in legumes to allow for the modification of root cell specification, such as the formation of mycorrhized roots and nitrogen-fixing nodules.

[Non-coding RNAs involved in plant responses to environmental constraints]. / Les ARN non-codants impliqués dans la réponse des plantes aux contraintes environnementales.

In recent years, in addition to mRNAs, the non-protein-coding RNAs (or ncRNAs) have emerged as a major part of the eukaryotic transcriptome. New genomic approaches allowed the discovery of many novel long and small ncRNAs that may be linked to the generation of evolutionary complexity in multicellular organisms. Many long ncRNAs are regulated by abiotic stresses although only very few long ncRNAs have been functionally analyzed. On the other hand, small RNAs act in the regulation of gene expression at transcriptional or post-transcriptional level and several among them have been linked to abiotic stress responses. Here we describe various ncRNAs associated with environmental stress responses such as to salt, cold or nutrient deprivation. The understanding of these RNA networks may reveal novel mechanisms involved in plant adaptation to changing environmental conditions.

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.

MicroRNAs as regulators of root development and architecture.

MicroRNAs (miRNAs) are post-transcriptional regulators of growth and development in both plants and animals. In plants, roots play essential roles in their anchorage to the soil as well as in nutrient and water uptake. In this review, we present recent advances made in the identification of miRNAs involved in embryonic root development, radial patterning, vascular tissue differentiation and formation of lateral organs (i.e., lateral and adventitious roots and symbiotic nitrogen-fixing nodules in legumes). Certain mi/siRNAs target members of the Auxin Response Factors family involved in auxin homeostasis and signalling and participate in complex regulatory loops at several crucial stages of root development. Other miRNAs target and restrict the action of various transcription factors that control root-related processes in several species. Finally, because abiotic stresses, which include nutrient or water deficiencies, generally modulate root growth and branching, we summarise the action of certain miRNAs in response to these stresses that may be involved in the adaptation of the root system architecture to the soil environment.

Small RNA diversity in plants and its impact in development.

MicroRNAs are a class of non-coding RNAs involved in post-transcriptional control of gene expression, either via degradation or translational inhibition of target mRNAs. Both experimental and computational approaches have been used to identify miRNAs and their target genes. In plants, deep sequencing methods have recently allowed the analysis of small RNA diversity in different species and/or mutants. Most sequencing efforts have been concentrated on the identification of miRNAs and their mRNA targets have been predicted based on complementarity criteria. The recent demonstration that certain plant miRNAs could act partly via inhibition of protein translation certainly opens new fields of analysis for plant miRNA function on a broader group of targets. The roles of conserved miRNAs on target mRNA stability have been analysed in different species and defined common mechanisms in development and stress responses. In contrast, much less is known about expression patterns or functions of non-conserved miRNAs. In this review, we focus on the comparative analyses of plant small RNA diversity and the action of si/miRNAs in post-transcriptional regulation of some key genes involved in root development.

Cleavage of a non-conserved target by a specific miR156 isoform in root apexes of Medicago truncatula.

Micro RNAs (miRNAs) have emerged as an important class of gene expression regulators controlling development, growth and metabolism. These short RNA molecules are 20-24 nucleotides in length and act post-transcriptionally to regulate the cleavage or translation of specific mRNA targets. In the model legume Medicago truncatula, we have recently reported identification of 100 novel and 27 conserved miRNAs in root apexes and nodules. Statistical analysis on sequencing results revealed specific miRNA isoforms for the same family (up to 3 mismatches) showing contrasting expression patterns between these tissues. Here, we report the cleavage of a non-conserved target of miR156 in root apexes complementary to a differentially expressed miR156 isoform. This suggests that changes in the abundance of miRNA isoforms may have functional consequences on the post-transcriptional regulation of new mRNA targets in different organs.