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INTRODUCTION: Alterations in the expression of the Angiotensin II type 1 receptors (AT1R) have been demonstrated in the development of several heart and renal diseases. The aim of this study was to evaluate the novel compound [18F]fluoropyridine-candesartan as a PET imaging tracer of AT1R in rat kidneys. METHODS: Competition binding assays were carried out with membranes from CHO-K1 cells expressing human AT1R. Binding to plasma proteins was assessed by ultrafiltration. Radiolabeled metabolites in rat plasma and kidneys of control and pretreated animals (candesartan 10 mg/kg or losartan 30 mg/kg) were analyzed by column-switch HPLC. Dynamic PET/CT images of [18F]fluoropyridine-candesartan in male Sprague-Dawley rats were acquired for 60 min at baseline, pre-treatment with the AT1R antagonist losartan (30 mg/kg) or the AT2R antagonist PD123,319 (5 mg/kg). RESULTS: Fluoropyridine-candesartan bound with a high affinity for AT1R (Ki = 5.9 ± 1.1 nM), comparable to fluoropyridine-losartan but lower than the parent compound candesartan (Ki = 0.4 ± 0.1 nM). [18F]Fluoropyridine-candesartan bound strongly to plasma proteins (99.3%) and was mainly metabolized to radiolabeled hydrophilic compounds, displaying minimal interference on renal AT1R binding with 82% of unchanged tracer in the kidneys at 20 min post-injection. PET imaging displayed high renal and liver accumulations and slow clearances, with maximum tissue-to-blood ratios of 14 ± 3 and 54 ± 12 in kidney cortex and liver, respectively, at 10 min post-injection. Binding specificity for AT1R was demonstrated with marked reductions in kidney cortex (-84%) and liver (-93%) tissue-to-blood ratios at 20 min post-injection, when blocking with AT1R antagonist losartan (30 mg/kg). No change was observed in kidney cortex of rats pre-treated with AT2R antagonist PD 123,319 (5 mg/kg), confirming binding selectivity for AT1 over AT2 receptors. CONCLUSION: High kidney-to-blood ratios and binding selectivity to renal AT1R combined with tracer in vivo stability displaying minimal interference from labeled metabolites support further PET imaging studies with [18F]fluoropyridine-candesartan.

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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.

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Alternative splicing (AS) of pre-mRNA represents a major mechanism underlying increased transcriptome and proteome complexity. Here, we show that the nuclear speckle RNA-binding protein (NSR) and the AS competitor long noncoding RNA (or ASCO-lncRNA) constitute an AS regulatory module. AtNSR-GFP translational fusions are expressed in primary and lateral root (LR) meristems. Double Atnsr mutants and ASCO overexpressors exhibit an altered ability to form LRs after auxin treatment. Interestingly, auxin induces a major change in AS patterns of many genes, a response largely dependent on NSRs. RNA immunoprecipitation assays demonstrate that AtNSRs interact not only with their alternatively spliced mRNA targets but also with the ASCO-RNA in vivo. The ASCO-RNA displaces an AS target from an NSR-containing complex in vitro. Expression of ASCO-RNA in Arabidopsis affects the splicing patterns of several NSR-regulated mRNA targets. Hence, lncRNA can hijack nuclear AS regulators to modulate AS patterns during development.

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Symbiosis between legume plants and soil rhizobia culminates in the formation of a novel root organ, the 'nodule', containing bacteria differentiated as facultative nitrogen-fixing organelles. MtNF-YA1 is a Medicago truncatula CCAAT box-binding transcription factor (TF), formerly called HAP2-1, highly expressed in mature nodules and required for nodule meristem function and persistence. Here a role for MtNF-YA1 during early nodule development is demonstrated. Detailed expression analysis based on RNA sequencing, quantitiative real-time PCR (qRT-PCR), as well as promoter-ß-glucuronidase (GUS) fusions reveal that MtNF-YA1 is first induced at the onset of symbiotic development during preparation for, and initiation and progression of, symbiotic infection. Moreover, using a new knock-out mutant, Mtnf-ya1-1, it is shown that MtNF-YA1 controls infection thread (IT) progression from initial root infection through colonization of nodule tissues. Extensive confocal and electronic microscopic observations suggest that the bulbous and erratic IT growth phenotypes observed in Mtnf-ya1-1 could be a consequence of the fact that walls of ITs in this mutant are thinner and less coherent than in the wild type. It is proposed that MtNF-YA1 controls rhizobial infection progression by regulating the formation and the wall of ITs.

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Some bacterial group II introns are widely used for genetic engineering in bacteria, because they can be reprogrammed to insert into the desired DNA target sites. There is considerable interest in developing this group II intron gene targeting technology for use in eukaryotes, but nuclear genomes present several obstacles to the use of this approach. The nuclear genomes of eukaryotes do not contain group II introns, but these introns are thought to have been the progenitors of nuclear spliceosomal introns. We investigated the expression and subcellular localization of the bacterial RmInt1 group II intron-encoded protein (IEP) in Arabidopsis thaliana protoplasts. Following the expression of translational fusions of the wild-type protein and several mutant variants with EGFP, the full-length IEP was found exclusively in the nucleolus, whereas the maturase domain alone targeted EGFP to nuclear speckles. The distribution of the bacterial RmInt1 IEP in plant cell protoplasts suggests that the compartmentalization of eukaryotic cells into nucleus and cytoplasm does not prevent group II introns from invading the host genome. Furthermore, the trafficking of the IEP between the nucleolus and the speckles upon maturase inactivation is consistent with the hypothesis that the spliceosomal machinery evolved from group II introns.

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The legume plant Medicago truncatula establishes a symbiosis with the nitrogen-fixing bacterium Sinorhizobium meliloti which takes place in root nodules. The formation of nodules employs a complex developmental program involving organogenesis, specific cellular differentiation of the host cells and the endosymbiotic bacteria, called bacteroids, as well as the specific activation of a large number of plant genes. By using a collection of plant and bacterial mutants inducing non-functional, Fix(-) nodules, we studied the differentiation processes of the symbiotic partners together with the nodule transcriptome, with the aim of unravelling links between cell differentiation and transcriptome activation. Two waves of transcriptional reprogramming involving the repression and the massive induction of hundreds of genes were observed during wild-type nodule formation. The dominant features of this "nodule-specific transcriptome" were the repression of plant defense-related genes, the transient activation of cell cycle and protein synthesis genes at the early stage of nodule development and the activation of the secretory pathway along with a large number of transmembrane and secretory proteins or peptides throughout organogenesis. The fifteen plant and bacterial mutants that were analyzed fell into four major categories. Members of the first category of mutants formed non-functional nodules although they had differentiated nodule cells and bacteroids. This group passed the two transcriptome switch-points similarly to the wild type. The second category, which formed nodules in which the plant cells were differentiated and infected but the bacteroids did not differentiate, passed the first transcriptome switch but not the second one. Nodules in the third category contained infection threads but were devoid of differentiated symbiotic cells and displayed a root-like transcriptome. Nodules in the fourth category were free of bacteria, devoid of differentiated symbiotic cells and also displayed a root-like transcriptome. A correlation thus exists between the differentiation of symbiotic nodule cells and the first wave of nodule specific gene activation and between differentiation of rhizobia to bacteroids and the second transcriptome wave in nodules. The differentiation of symbiotic cells and of bacteroids may therefore constitute signals for the execution of these transcriptome-switches.

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Plants use a variety of small peptides for cell to cell communication during growth and development. Leguminous plants are characterized by their ability to develop nitrogen-fixing nodules via an interaction with symbiotic bacteria. During nodule organogenesis, several so-called nodulin genes are induced, including large families that encode small peptides. Using a three-hybrid approach in yeast cells, we identified two new small nodulins, MtSNARP1 and MtSNARP2 (for small nodulin acidic RNA-binding protein), which interact with the RNA of MtENOD40, an early induced nodulin gene showing conserved RNA secondary structures. The SNARPs are acidic peptides showing single-stranded RNA-binding activity in vitro and are encoded by a small gene family in Medicago truncatula. These peptides exhibit two new conserved motifs and a putative signal peptide that redirects a GFP fusion to the endoplasmic reticulum both in protoplasts and during symbiosis, suggesting they are secreted. MtSNARP2 is expressed in the differentiating region of the nodule together with several early nodulin genes. MtSNARP2 RNA interference (RNAi) transgenic roots showed aberrant early senescent nodules where differentiated bacteroids degenerate rapidly. Hence, a functional symbiotic interaction may be regulated by secreted RNA-binding peptides.

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In plants, a diverse group of cell surface receptor-like protein kinases (RLKs) plays a fundamental role in sensing external signals to regulate gene expression. Roots explore the soil environment to optimize their growth via complex signaling cascades, mainly analyzed in Arabidopsis thaliana. However, legume roots have significant physiological differences, notably their capacity to establish symbiotic interactions. These major agricultural crops are affected by environmental stresses such as salinity. Here, we report the identification of a leucine-rich repeat RLK gene, Srlk, from the legume Medicago truncatula. Srlk is rapidly induced by salt stress in roots, and RNA interference (RNAi) assays specifically targeting Srlk yielded transgenic roots whose growth was less inhibited by the presence of salt in the medium. Promoter-beta-glucuronidase fusions indicate that this gene is expressed in epidermal root tissues in response to salt stress. Two Srlk-TILLING mutants also failed to limit root growth in response to salt stress and accumulated fewer sodium ions than controls. Furthermore, early salt-regulated genes are downregulated in Srlk-RNAi roots and in the TILLING mutant lines when submitted to salt stress. We propose a role for Srlk in the regulation of the adaptation of M. truncatula roots to salt stress.

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Long non-protein coding RNAs (npcRNA) represent an emerging class of riboregulators, which either act directly in this long form or are processed to shorter miRNA and siRNA. Genome-wide bioinformatic analysis of full-length cDNA databases identified 76 Arabidopsis npcRNAs. Fourteen npcRNAs were antisense to protein-coding mRNAs, suggesting cis-regulatory roles. Numerous 24-nt siRNA matched to five different npcRNAs, suggesting that these npcRNAs are precursors of this type of siRNA. Expression analyses of the 76 npcRNAs identified a novel npcRNA that accumulates in a dcl1 mutant but does not appear to produce trans-acting siRNA or miRNA. Additionally, another npcRNA was the precursor of miR869 and shown to be up-regulated in dcl4 but not in dcl1 mutants, indicative of a young miRNA gene. Abiotic stress altered the accumulation of 22 npcRNAs among the 76, a fraction significantly higher than that observed for the RNA binding protein-coding fraction of the transcriptome. Overexpression analyses in Arabidopsis identified two npcRNAs as regulators of root growth during salt stress and leaf morphology, respectively. Hence, together with small RNAs, long npcRNAs encompass a sensitive component of the transcriptome that have diverse roles during growth and differentiation.

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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.

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