Landscape of the Noncoding Transcriptome Response of Two Arabidopsis Ecotypes to Phosphate Starvation.

Root architecture varies widely between species; it even varies between ecotypes of the same species, despite strong conservation of the coding portion of their genomes. By contrast, noncoding RNAs evolve rapidly between ecotypes and may control their differential responses to the environment, since several long noncoding RNAs (lncRNAs) are known to quantitatively regulate gene expression. Roots from ecotypes Columbia and Landsberg erecta of Arabidopsis (Arabidopsis thaliana) respond differently to phosphate starvation. Here, we compared transcriptomes (mRNAs, lncRNAs, and small RNAs) of root tips from these two ecotypes during early phosphate starvation. We identified thousands of lncRNAs that were largely conserved at the DNA level in these ecotypes. In contrast to coding genes, many lncRNAs were specifically transcribed in one ecotype and/or differentially expressed between ecotypes independent of phosphate availability. We further characterized these ecotype-related lncRNAs and studied their link with small interfering RNAs. Our analysis identified 675 lncRNAs differentially expressed between the two ecotypes, including antisense RNAs targeting key regulators of root-growth responses. Misregulation of several lincRNAs showed that at least two ecotype-related lncRNAs regulate primary root growth in ecotype Columbia. RNA-sequencing analysis following deregulation of lncRNA NPC48 revealed a potential link with root growth and transport functions. This exploration of the noncoding transcriptome identified ecotype-specific lncRNA-mediated regulation in root apexes. The noncoding genome may harbor further mechanisms involved in ecotype adaptation of roots to different soil environments.

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.

Arabidopsis CLAVATA1 and CLAVATA2 receptors contribute to Ralstonia solanacearum pathogenicity through a miR169-dependent pathway.

Bacterial wilt caused by Ralstonia solanacearum is one of the most destructive bacterial plant diseases. Although many molecular determinants involved in R. solanacearum adaptation to hosts and pathogenesis have been described, host components required for disease establishment remain poorly characterized. Phenotypical analysis of Arabidopsis mutants for leucine-rich repeat (LRR)-receptor-like proteins revealed that mutations in the CLAVATA1 (CLV1) and CLAVATA2 (CLV2) genes confer enhanced disease resistance to bacterial wilt. We further investigated the underlying mechanisms using genetic, transcriptomic and molecular approaches. The enhanced resistance of both clv1 and clv2 mutants to the bacteria did not require the well characterized CLV signalling modules involved in shoot meristem homeostasis, and was conditioned by neither salicylic acid nor ethylene defence-related hormones. Gene expression microarray analysis performed on clv1 and clv2 revealed deregulation of genes encoding nuclear transcription factor Y subunit alpha (NF-YA) transcription factors whose post-transcriptional regulation is known to involve microRNAs from the miR169 family. Both clv mutants showed a defect in miR169 accumulation. Conversely, overexpression of miR169 abrogated the resistance phenotype of clv mutants. We propose that CLV1 and CLV2, two receptors involved in CLV3 perception during plant development, contribute to bacterial wilt through a signalling pathway involving the miR169/NF-YA module.

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.

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.

Genome-wide Medicago truncatula small RNA analysis revealed novel microRNAs and isoforms differentially regulated in roots and nodules.

Posttranscriptional regulation of a variety of mRNAs by small 21- to 24-nucleotide RNAs, notably the microRNAs (miRNAs), is emerging as a novel developmental mechanism. In legumes like the model Medicago truncatula, roots are able to develop a de novo meristem through the symbiotic interaction with nitrogen-fixing rhizobia. We used deep sequencing of small RNAs from root apexes and nodules of M. truncatula to identify 100 novel candidate miRNAs encoded by 265 hairpin precursors. New atypical precursor classes producing only specific 21- and 24-nucleotide small RNAs were found. Statistical analysis on sequencing reads abundance revealed specific miRNA isoforms in a same family showing contrasting expression patterns between nodules and root apexes. The differentially expressed conserved and nonconserved miRNAs may target a large variety of mRNAs. In root nodules, which show diverse cell types ranging from a persistent meristem to a fully differentiated central region, we discovered miRNAs spatially enriched in nodule meristematic tissues, vascular bundles, and bacterial infection zones using in situ hybridization. Spatial regulation of miRNAs may determine specialization of regulatory RNA networks in plant differentiation processes, such as root nodule formation.

The lncRNA MARS modulates the epigenetic reprogramming of the marneral cluster in response to ABA.

Clustered organization of biosynthetic non-homologous genes is emerging as a characteristic feature of plant genomes. The co-regulation of clustered genes seems to largely depend on epigenetic reprogramming and three-dimensional chromatin conformation. In this study, we identified the long non-coding RNA (lncRNA) MARneral Silencing (MARS), localized inside the Arabidopsis marneral cluster, which controls the local epigenetic activation of its surrounding region in response to abscisic acid (ABA). MARS modulates the POLYCOMB REPRESSIVE COMPLEX 1 (PRC1) component LIKE HETEROCHROMATIN PROTEIN 1 (LHP1) binding throughout the cluster in a dose-dependent manner, determining H3K27me3 deposition and chromatin condensation. In response to ABA, MARS decoys LHP1 away from the cluster and promotes the formation of a chromatin loop bringing together the MARNERAL SYNTHASE 1 (MRN1) locus and a distal ABA-responsive enhancer. The enrichment of co-regulated lncRNAs in clustered metabolic genes in Arabidopsis suggests that the acquisition of novel non-coding transcriptional units may constitute an additional regulatory layer driving the evolution of biosynthetic pathways.

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.

Synthesis of methoxyfumimycin with 1,2-addition to ketimines.

The synthesis of (+/-)-methoxyfumimycin, a potential new bacterial peptide deformylase (PDF) inhibitor, is reported. To generate the stereogenic fully substituted carbon, the key step is a 1,2-addition of a methyl Grignard reagent to a ketimine. The overall synthetic strategy involves a Dakin oxidation of a vanillin derivative, Friedel-Crafts acylation, Claisen rearrangement, lactonization, and rhodium-catalyzed olefin isomerization.

Steric modulation of chiral biaryl diamines via Pd-catalyzed directed C-H arylation.

Palladium-catalyzed directed arylation of 2,2'-diacetamidobiaryls with aryl iodides provides efficient access to chiral ortho-substituted biaryl diamines. Aryl iodides with para- and meta-substituents are tolerated. Deprotection of the acetyl groups under basic conditions furnishes the free diamines, which should find broad utility in asymmetric catalysis.

Towards an asymmetric synthesis of the bacterial peptide deformylase (PDF) inhibitor fumimycin.

Studies towards the synthesis of the bacterial peptide deformylase (PDF) inhibitor fumimycin are reported. The synthetic approach features an organocatalytic access to the alpha,alpha-disubstituted amino acid unit and results in the synthesis of an advanced intermediate which already contains all functionalities of fumimycin.

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.

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.

Low proportion of whole exon deletions causing phenylketonuria in Denmark and Germany.

Phenylketonuria (PKU) is an autosomal recessive genetic disorder caused by mutations of the gene encoding phenylalanine hydroxylase (PAH). More than 500 different PAH mutations have been identified and about 90% of these are single base mutations. Although the identification rate of the PAH mutations is generally very high, some variants remain unidentified. A fraction of these mutations are the result of genomic deletions or duplications, which are not recognized with standard PCR-based methods. Here we present the results of exon deletion or duplication analysis in a total of 34 families, in which two mutations had not been identified using conventional diagnostic screening techniques. Using multiplex ligation-dependent probe amplification (MLPA), we found a deletion covering exon 1 and exon 2 (c.1-?_168+?del) in one patient, a deletion of exon 3 (c.169-?_352+?del) in four patients, and a deletion of exon 5 (c.442-?_509+?del) in two patients. A deletion was thus detected in about 20% (7/34) of the families tested. Out of a combined cohort of 570 independent PKU patients from Denmark and Germany, exon deletions were identified in a total of four patients. The estimated allelic frequency of exon deletions in PKU patients in these two populations is therefore below 0.5%.

Identification of novel drought-related mRNAs in common bean roots by differential display RT-PCR.

Drought is a major constraint for the production of common bean (Phaseolus vulgaris L.). To identify molecular responses to water deficit, we performed a differential display RT-PCR (DDRT) analysis using roots of bean plants grown aeroponically and submitted to dehydration. This allowed us to visualise 1200 DDRT bands, 8.7% of which showed a clear regulation by dehydration, and to clone 42 cDNAs, called PvD1 to PvD42. Among them, 20 early-dehydration-responsive cDNAs were selected by reverse northern that were induced or repressed before detectable water status changes and induction of ABA-regulated genes. Northern analysis for 16 PvD clones confirmed these early regulations and allowed us to identify four late dehydration-responsive genes. Their putative involvement in signalling, protein turn-over and translocation, chaperones as well as root growth modulations in response to water stress is discussed.

Regulation of 7S globulin gene expression in zygotic and somatic embryos of oil palm.

We describe here the characterization and expression analysis of the oil palm GLO7A gene encoding a 7S globulin protein. Previous work carried out in our laboratory showed that 7S globulins accumulate in the oil palm zygotic embryo mostly between the 14 and 17 weeks after pollination. To investigate further the regulation of 7S globulin gene expression in both zygotic and somatic embryos of oil palm, we isolated a cDNA clone, GLO7A, for use as a probe in northern hybridization studies. The nucleotide sequence of the GLO7A cDNA reveals that it encodes a polypeptide of 572 amino acids (66 kDa) sharing significant sequence similarities with various vicilin-like proteins of both dicotyledonous and monocotyledonous plants. Northern hybridization analysis shows that 7S globulin mRNA accumulation in zygotic embryos is temporally regulated with a profile essentially the same as that observed at the protein level. In somatic embryos, 7S globulin proteins were found to occur in amounts approximately 80 times lower than those in zygotic embryos. This lack of 7S globulin protein accumulation in somatic embryos is mirrored by a low accumulation of the GLO7A mRNA. The in vitro production of 7S globulins (and more generally salt-soluble proteins) is improved by the addition to the culture medium of arginine, sucrose and ABA, the effects of these 3 components being additive. To investigate further the action of the 3 molecules of interest, we performed parallel studies on mRNA and protein abundance. Our studies of transcript accumulation suggest that ABA and sucrose act directly on mRNA synthesis or stability; however, it appears that there are also translational or post-translational regulatory factors which act to limit protein accumulation in somatic embryos. The GLO7A gene promoter was cloned and sequenced to assess whether GLO7A gene expression might be modulated by cis-acting promoter elements related to those found in other plants. Two motifs resembling ABREs (ABA-responsive elements) and one motif resembling a seed-specific promoter element were identified within the 5' flanking sequence.

[MicroRNAs: a new class of gene expression regulators]. / Les microARN: une nouvelle classe de régulateurs de l'expression génique.

MicroRNAs (miRs) are small non coding RNA, about 21-25 nucleotides in length, that direct post transcriptional regulation of gene expression through interaction with homologous mRNAs. Hundreds miR genes have been identified in animals and 40 in plants. Many of them are conserved between related species, and in some cases across phyla. Two mechanisms for regulation of gene expression by miRs have been reported. As described for lin-4 and let-7 miR of C.elegans, miRs can inhibit translation, which seems to represent the major mode of regulation in animals, or can direct cleavage of target mRNAs, which seems to represent the major mode of regulation in plants.

Immunohistochemical expression of PDGFR, VEGF-C, and proteins of the mToR pathway before and after androgen deprivation therapy in prostate carcinoma: significant decrease after treatment.

Targeted therapy in hormone refractory prostate cancer (HRPC) is currently under evaluation in many trials. The effect of androgen deprivation therapy (ADT) on many targets in prostate cancer is incompletely known. For the first time, immunohistochemical expression of the platelet-derived growth factor receptor (PDGFR), epidermal growth factor receptor (EGFR), vascular endothelial growth factor C (VEGF-C), mammalian target of rapamycin (mToR), p70 ribosomal protein S6 kinase 1 (PS6K), human epidermal growth factor receptor 2 (c-erbB-2), and carbonic anhydrase IX (CA9) was evaluated in 44 patients with prostate carcinoma treated with or without ADT, at biopsy time and after radical prostatectomy. PDGFR, VEGF-C, mToR, and PS6K expression was significantly reduced (p = 0.002, p = 0.035, p = 0.025, and p = 0.033, respectively) after ADT, whereas expression of EGFR, c-erbB-2, and CA9 was not influenced by ADT. In conclusion, targeting PDGFR, VEGF-C, mToR, or PS6K after ADT should be considered with precaution, as those targets can severely be altered or functionally deregulated by ADT.

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.