HAL

During the past ten years, emerging data revealed the role of E2F factors in various plant physiology aspects. E2F involvement in cell cycle and plant development was particularly investigated and highlighted some plant specificities, espacially in postembryonic development. A major role of E2F is its transcriptional activity governing specific gene induction throughout the cell cycle and also strong induction of DNA repair genes in response to DNA damage, notably in the context of DNA double strand break (DSB) response. More challenging is the implication of E2F in nuclear foci with gamma-H2AX, a marker of DSB, probably independently of its transcriptional activity, in the DNA damage response.

Members of the phylum Actinomycetota are widely distributed across diverse environments and are well known for their metabolic versatility and capacity to produce bioactive compounds. In this study, strain ZE1316R2Aᵀ was isolated from the saline water collected from Lake Zima (Morocco) and subjected to comprehensive polyphasic taxonomic characterisation. Phylogenetic analysis based on the 16 S rRNA gene placed strain ZE1316R2Aᵀ within the genus Streptomyces , showing highest sequence similarity with S. albidoflavus DSM 40,455 T (99.71%). However, genome-based indices, including average nucleotide identity (ANIb = 94.84%, ANIm = 96.09%) and digital DNA-DNA hybridization (dDDH = 64.9%), supported its distinction as a separate species. The draft genome (7.41 Mb; G + C = 73.26 mol%) comprises 6,464 coding sequences and reveals the presence of strain-specific genomic regions and biosynthetic gene clusters. Comparative analyses highlighted both a conserved core genome and a substantial accessory genome component, reflecting genomic differentiation relative to closely related taxa. Phenotypic and chemotaxonomic characteristics were consistent with assignment to the genus Streptomyces , while supporting its differentiation at the species level. Based on the combined genomic, phenotypic, and chemotaxonomic evidence, strain ZE1316R2Aᵀ represents a novel species of the genus Streptomyces , for which the name Streptomyces zimensis sp. nov., is proposed. This study expands current knowledge of Streptomyces diversity associated with saline environments and highlights the genomic diversity present within closely related taxa. The type strain is ZE1316R2Aᵀ (= CCMM B1331 T = DSM 120541 T ).

Transfer RNAs (tRNAs) are essential components of the translation machinery. Their abundance and diversity shape decoding capacity as well as the efficiency and accuracy of protein synthesis. Because tRNA abundance is encoded in the genome through tDNA copy number, chromosomal organization, and cisregulatory sequences controlling transcription, these features are expected to influence the translational system. However, the principles governing nuclear tDNA organization remain poorly understood. Here, we analyzed nuclear tDNA repertoires across 53 photosynthetic eukaryotes spanning major Archaeplastida lineages and secondary endosymbionts, along with seven non-plant eukaryotic outgroups, using comparative genomic approaches at sequence, chromosomal, and genome-wide scales. To standardize these analyses and enable interactive exploration of tDNA organization, we developed ShinytRNA (https://nebula.ibmp. unistra.fr/shinytRNA/), a web application for genome-scale analysis of chromosomal tDNA organization. Nuclear tDNA copy numbers vary by more than two orders of magnitude across species, yet the relative representation of tRNA families corresponding to each amino acid remains strikingly conserved across lineages, revealing strong evolutionary constraints on tDNA dosage. Angiosperm tDNAs exhibit coordinated enrichment of cis-regulatory elements involved in RNA polymerase III transcription, including expanded AT-rich upstream regions, positional enrichment of CAA motifs, and extended poly(T) termination stretches. At the chromosomal scale, tDNAs are predominantly dispersed along chromosome arms, with homogeneous spacing that scales with genome size, while also showing non-random chromosomal distribution, exclusion from centromeric regions, and occasional clustering. Together, these patterns reveal conserved yet lineage-specific principles governing nuclear tDNA organization in plants and highlight how multiple genomic constraints shape the evolution of nuclear tDNA repertoires.

Saline and arid ecosystems are recognized as promising reservoirs of novel Actinomycetota with unique adaptations and secondary metabolic potential. In this study, a halotolerant Actinomycetota strain, designated ZS3416R2A, was isolated from saline soil in the hypersaline wetland of Lake Zima, Morocco. Phylogenetic analysis based on the 16S rRNA gene sequence showed 99.03% similarity to Nocardiopsis terrae DSM 45157 T , indicating close affiliation with the genus Nocardiopsis. Whole-genome sequencing revealed a 6.1 Mb circular chromosome with a G + C content of 70.48% and 5,855 coding sequences. Genome-based metrics (ANI 95.07%, dDDH 66.4%) supported its classification as a novel species distinct from N. terrae, while AAI (95.87%) and POCP (82.4%) confirmed its placement within the genus. Comparative analyses revealed unique gene families, biosynthetic gene clusters, and regions of genomic plasticity in strain ZS3416R2A. Phenotypically, strain ZS3416R2A grew over a broad range of salinity (0-10%), temperature (16-37 °C), and pH (7.0-8.0), and formed denser aerial mycelia than strain DSM 45157 T , reflecting adaptation to the arid saline environment of Lake Zima. Chemotaxonomic characterization identified MK-10(H 6 ), MK-10(H 4 ) and MK-9(H 4 ) as predominant menaquinones, iso-C 16:0 and 10-methyl-C 18:0 as major fatty acids, and diphosphatidylglycerol, phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, and phosphatidylinositol as major polar lipids. Based on this polyphasic evidence, strain ZS3416R2A represents a novel species within the genus Nocardiopsis, for which the name Nocardiopsis moroccensis sp. nov. is proposed.

Plant growth-promoting rhizobacteria (PGPR) enhance plant growth and development through diverse mechanisms, including phytohormone production, nutrient acquisition, and stress mitigation. This study describes the isolation and characterization of two bacterial strains, DT1 and S10, from the rhizospheres of Diplotaxis tenuifolia and Cynodon dactylon , respectively capable of solubilizing phosphate and zinc, fix nitrogen and produce indole acetic acid (IAA) and siderophores. Using whole genome sequencing and taxonomic analyses, these two strains were identified as Acinetobacter calcoaceticus (DT1) and Citrobacter braakii (S10). Functional genomic annotation revealed numerous genes associated with key plant growth-promoting traits, including those involved in indole-3-acetic acid (IAA) ( trpABCDE , ipdC ), cytokinin ( miaABE ), and riboflavin biosynthesis, confirmed by targeted metabolomics. In addition, genes associated with nitrogen metabolism ( nirB , narGHI ) and phosphate solubilization ( gcd , phoARP , pstABCS , pqqEFG ) were identified and supported by phenotypic assays. Interestingly, biosynthetic gene clusters for the secondary metabolites enterobactin, bacillibactin, and staphyloferrin B, known to contribute to plant growth promotion, were identified in both genomes. Both strains also harbored genes encoding ACC deaminase, an enzyme known to enhance plant tolerance to abiotic stress. Furthermore, non-targeted metabolomic analysis revealed that DT1 and S10 produced a range of intracellular and extracellular metabolites associated with plant growth promotion and stress resilience, including cadaverine, biotin, arginine, and GABA. Collectively, these findings position DT1 and S10 as promising bioinoculant candidates, offering an integrative genomic and metabolic foundation for their application in next-generation sustainable agricultural strategies.

Background Plant microbiota has received increasing attention in recent years. In particular, the microbiota associated with cereals is being extensively studied to identify bacterial strains that can promote plant health and growth. Barley is the fourth most important cereal worldwide in terms of agricultural production. Intensive barley agriculture requires the use of chemical fertilizers to compensate for nutrient deficiencies in soils and limit pathogen development. The isolation and use of bacteria that can enhance the bioavailability of soil nutrients and inhibit the development of plant pathogens could ultimately limit the use of these chemicals. In this study, we have isolated from a barley microbiota three bacterial strains belonging to the genus Streptomyces. These strains were characterized and named GPA1, GPAT2, and GPN2. <div>Results<p>These three closely related isolates were from the same bacterial genus Streptomyces. Based on a phylogenetic analysis, the strains GPAT2 and GPN2 were classified as Streptomyces murinus, while GPA1 was identified as a new species. All strains showed antagonistic activity against two microorganisms that inhibit barley germination: Pseudomonas sp. MRN1 and Fusarium sp. CK. In addition, these strains exhibited different effects on the growth of barley cultivated under hydroponic and axenic conditions. In fact, GPN2 appeared to have no effect whereas the inoculation of barley seedlings with GPAT2 and GPA1 resulted in a reduction and an increase in root length after two weeks of growth, respectively. GPA1 had various Plant Growth-Promoting (PGP) abilities, including phosphate and zinc solubilization and siderophore production. A metabolite profiling of the GPA1 bacterial culture also showed its production and excretion of indole-3-acetic acid (IAA).</p></div> <div>Conclusion<p>In this study, we have characterized three closely related bacteria, which display different effects on barley seedlings growth. These results revealed that the type of interactions of Streptomyces with barley is straindependent, suggesting that these interactions may arise from specific molecular mechanisms acquired through coevolutionary processes.</p></div>

Over the course of evolution, land plant mitochondrial genomes have lost many transfer RNA (tRNA) genes and the import of nucleus-encoded tRNAs is essential for mitochondrial protein synthesis. By contrast, plastidial genomes of photosynthetic land plants generally possess a complete set of tRNA genes and the existence of plastidial tRNA import remains a long-standing question. The early vascular plants of the Selaginella genus show an extensive loss of plastidial tRNA genes while retaining photosynthetic capacity, and represent an ideal model for answering this question. Using purification, northern blot hybridization, and high-throughput tRNA sequencing, a global analysis of total and plastidial tRNA populations was undertaken in Selaginella kraussiana. We confirmed the expression of all plastidial tRNA genes and, conversely, observed that nucleus-encoded tRNAs corresponding to these plastidial tRNAs were generally excluded from the chloroplasts. We then demonstrated a selective and differential plastidial import of around forty nucleus-encoded tRNA species, likely compensating for the insufficient coding capacity of plastidial-encoded tRNAs. In-depth analysis revealed differential import of tRNA isodecoders, leading to the identification of specific situations. This includes the expression and import of nucleus-encoded tRNAs expressed from plastidial or bacterial-like genes inserted into the nuclear genome. Overall, our results confirm the existence of molecular processes that enable tRNAs to be selectively imported not only into mitochondria, as previously described, but also into chloroplasts, when necessary.

The success of virus transmission by vectors relies on intricate trophic interactions between three partners, the host plant, the virus, and the vector. Despite numerous studies that showed the capacity of plant viruses to manipulate their host plant to their benefit, and potentially of their transmission, the molecular mechanisms sustaining this phenomenon has not yet been extensively analyzed at the molecular level. In this study, we focused on the deregulations induced in Arabidopsis thaliana by an aphid vector that were alleviated when the plants were infected with turnip yellows virus (TuYV), a polerovirus strictly transmitted by aphids in a circulative and nonpropagative mode. By setting up an experimental design mimicking the natural conditions of virus transmission, we analyzed the deregulations in plants infected with TuYV and infested with aphids by a dual transcriptomic and metabolomic approach. We observed that the virus infection alleviated most of the gene deregulations induced by the aphids in a noninfected plant at both time points analyzed (6 and 72 h) with a more pronounced effect at the later time point of infestation. The metabolic composition of the infected and infested plants was altered in a way that could be beneficial for the vector and the virus transmission. Importantly, these substantial modifications observed in infected and infested plants correlated with a higher TuYV transmission efficiency. This study revealed the capacity of TuYV to alter the plant nutritive content and the defense reaction against the aphid vector to promote the viral transmission.

Streptomycetaceae are found ubiquitously within plant microbiota. Several species belonging to this family are plant growth-promoting bacteria or may inhibit phytopathogens. Such bacteria therefore exert crucial functions in host development and resistance to stresses. Recent studies have shown that plants select beneficial bacteria into their microbiota. However, the selection process and the molecular mechanisms by which selected bacteria modulate the physiology of their host are not yet fully understood. Previous work revealed that the metabolic status of Arabidopsis thaliana was crucial for the selection of Streptomycetaceae into the microbiota, in particular bacteria phylogenetically related to Streptomyces cocklensis or Actinacidiphila bryophytorum (previously named Streptomyces bryophytorum). Here, the Arabidopsis-Streptomycetaceae interaction was further depicted by inoculating axenic A. thaliana with S. cocklensis DSM 42063 or A. bryophytorum DSM 42138. We showed that these two bacteria colonize A. thaliana ecotype Columbia-0 plants, but the colonization efficiency is reduced in a chs5 mutant of the same ecotype, being altered in isoprenoid, phenylpropanoid, and lipid profiles. We observed that A. bryophytorum inhibits growth of the chs5 mutant but not of the wild type, suggesting that the Arabidopsis-Actinacidiphila interaction depends on the metabolic status of the host. Using a mass spectrometry-based proteomic approach, we showed that S. cocklensis and A. bryophytorum modulate the A. thaliana proteome, in particular components involved in photosynthesis or phytohormone homeostasis. This study unveils specific aspects of the Arabidopsis-Streptomycetaceae interaction and highlights its complexity and diversity. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Plant responses to salt exposure involve large reconfiguration of hormonal pathways that orchestrate physiological changes towards tolerance. Jasmonate (JA) hormones are essential to withstand biotic and abiotic assaults, but their roles in salt tolerance remain unclear. Here we describe the dynamics of JA metabolism and signaling in root and leaf of rice, a plant species that is highly exposed and sensitive to salt. Roots activate the JA pathway in an early pulse, while 2nd leaf displays a biphasic JA response with peaks at 1 hour and 3 days post-exposure. Based on higher salt tolerance of a rice JA-deficient mutant (aoc), we examined through kinetic transcriptome and physiological analysis the salt-triggered processes that are under JA control. Profound genotype-differential features emerged that could underlie observed phenotypes. ABA content and ABA-dependent water deprivation responses were impaired in aoc shoots. Moreover, aoc accumulated more Na+ in roots, and less in leaf, with reduced ion translocation correlating with root derepression of the HAK4 Na+ transporter. Distinct reactive oxygen species scavengers were also stronger in aoc leaf, along with reduced senescence and chlorophyll catabolism markers. Collectively, the data identify contrasted contributions of JA signaling to different sectors of the salt stress response in rice.

In plants and some animal lineages, RNA silencing is an efficient and adaptable defense mechanism against viruses. To counter it, viruses encode suppressor proteins that interfere with RNA silencing. Phloem-restricted viruses are spreading at an alarming rate and cause substantial reduction of crop yield, but how they interact with their hosts at the molecular level is still insufficiently understood. Here, we investigate the antiviral response against phloem-restricted turnip yellows virus (TuYV) in the model plant Arabidopsis thaliana. Using a combination of genetics, deep sequencing, and mechanical vasculature enrichment, we show that the main axis of silencing active against TuYV involves 22-nt vsiRNA production by DCL2, and their preferential loading into AGO1. Moreover, we identify vascular secondary siRNA produced from plant transcripts and initiated by DCL2-processed AGO1-loaded vsiRNA. Unexpectedly, and despite the viral encoded VSR P0 previously shown to mediate degradation of AGO proteins, vascular AGO1 undergoes specific post-translational stabilization during TuYV infection. Collectively, our work uncovers the complexity of antiviral RNA silencing against phloem-restricted TuYV and prompts a re-assessment of the role of its suppressor of silencing P0 during genuine infection

Beyond their key role in translation, cytosolic transfer RNAs (tRNAs) are involved in a wide range of other biological processes. Nuclear tRNA genes (tD-NAs) are transcribed by the RNA polymerase III (RNAP III) and cis-elements, trans-factors as well as genomic features are known to influence their expression. In Arabidopsis, besides a predominant population of dispersed tDNAs spread along the 5 chromosomes, some clustered tDNAs have been identified. Here, we demonstrate that these tDNA clusters are transcriptionally silent and that pathways involved in the maintenance of DNA methy-lation play a predominant role in their repression. Moreover, we show that clustered tDNAs exhibit repressive chromatin features whilst their dispersed counterparts contain permissive euchromatic marks. This work demonstrates that both genomic and epigenomic contexts are key players in the regulation of tDNAs transcription. The conservation of most of these regulatory processes suggests that this pioneering work in Arabidopsis can provide new insights into the regulation of RNA Pol III transcription in other organisms, including vertebrates.

Rhodococcus strain IGTS8 is the most extensively studied model bacterium for biodesulfurization of fossil fuels via the non-destructive sulfur-specific 4S pathway. This strain was initially assigned to Rhodococcus rhodochrous and later to Rhodococcus erythropolis thus making its taxonomic status debatable and reflecting the limited resolution of methods available at the time. In this study, phylogenomic analyses of the whole genome sequences of strain IGTS8 and closely related rhodococci showed that R. erythropolis and Rhodococcus qingshengii are very closely related species, that Rhodococcus strain IGTS8 is a R. qingshengii strain and that several strains identified as R. erythropolis should be reclassified as R. qingshengii. The genomes of strains assigned to these species contain potentially novel biosynthetic gene clusters showing that members of these taxa should be given greater importance in the search for new antimicrobials and other industrially important biomolecules. The plasmid-borne dsz operon encoding fossil fuel desulfurization enzymes was present in R. qingshengii IGTS8 and R. erythropolis XP suggesting that it might be transferable between members of these species.

Genome packaging by nucleosomes is a hallmark of eukaryotes. Histones and the pathways that deposit, remove, and read histone modifications are deeply conserved. Yet, we lack information regarding chromatin landscapes in extant representatives of ancestors of the main groups of eukaryotes, and our knowledge of the evolution of chromatin-related processes is limited. We used the bryophyte Marchantia polymorpha, which diverged from vascular plants circa 400 mya, to obtain a whole chromosome genome assembly and explore the chromatin landscape and three-dimensional genome organization in an early diverging land plant lineage. Based on genomic profiles of ten chromatin marks, we conclude that the relationship between active marks and gene expression is conserved across land plants. In contrast, we observed distinctive features of transposons and other repetitive sequences in Marchantia compared with flowering plants. Silenced transposons and repeats did not accumulate around centromeres. Although a large fraction of constitutive heterochromatin was marked by H3K9 methylation as in flowering plants, a significant proportion of transposons were marked by H3K27me3, which is otherwise dedicated to the transcriptional repression of protein-coding genes in flowering plants. Chromatin compartmentalization analyses of Hi-C data revealed that repressed B compartments were densely decorated with H3K27me3 but not H3K9 or DNA methylation as reported in flowering plants. We conclude that, in early plants, H3K27me3 played an essential role in heterochromatin function, suggesting an ancestral role of this mark in transposon silencing.

Plants are exposed to the damaging effect of sunlight that induces DNA photolesions. In order to maintain genome integrity, specific DNA repair pathways are mobilized. Upon removal of UV-induced DNA lesions, the accurate re-establishment of epigenome landscape is expected to be a prominent step of these DNA repair pathways. However, it remains poorly documented whether DNA methylation is accurately maintained at photoda-maged sites and how photodamage repair pathways contribute to the maintenance of genome/methylome integrities. Using genome wide approaches, we report that UV-C irradiation leads to CHH DNA methylation changes. We identified that the specific DNA repair pathways involved in the repair of UV-induced DNA lesions, Direct Repair (DR), Global Genome Repair (GGR) and small RNA-mediated GGR prevent the excessive alterations of DNA methylation landscape. Moreover, we identified that UV-C irradiation induced chromo-center reorganization and that photodamage repair factors control this dynamics. The methylome changes rely on misregulation of maintenance, de novo and active DNA demethylation pathways highlighting that molecular processes related to genome and methylome integrities are closely interconnected. Importantly, we identified that photole-sions are sources of DNA methylation changes in repressive chromatin. This study unveils that DNA repair factors, together with small RNA, act to accurately maintain both genome and methylome integrities at photodamaged silent genomic regions, strengthening the idea that plants have evolved sophisticated interplays between DNA methylation dynamics and DNA repair.

In the model plant Arabidopsis thaliana, four Dicer-like proteins (DCL1-4) mediate the production of various classes of small RNAs (sRNAs). Among these four proteins, DCL4 is by far the most versatile RNaseIII-like enzyme, and previously identified dcl4 missense alleles were shown to uncouple the production of the various classes of DCL4-dependent sRNAs. Yet little is known about the molecular mechanism behind this uncoupling. Here, by studying the subcellular localization, interactome and binding to the sRNA precursors of three distinct dcl4 missense alleles, we simultaneously highlight the absolute requirement of a specific residue in the helicase domain for the efficient production of all DCL4-dependent sRNAs, and identify, within the PAZ domain, an important determinant of DCL4 versatility that is mandatory for the efficient processing of intramolecular fold-back double-stranded RNA (dsRNA) precursors, but that is dispensable for the production of small interfering RNAs (siRNAs) from RDR-dependent dsRNA susbtrates. This study not only provides insights into the DCL4 mode of action, but also delineates interesting tools to further study the complexity of RNA silencing pathways in plants, and possibly other organisms.

In the expanding repertoire of small noncoding RNAs (ncRNAs), tRNA-derived RNA fragments (tRFs) have been identified in all domains of life. Their existence in plants has been already proven but no detailed analysis has been performed. Here, short tRFs of 19-26 nucleotides were retrieved from Arabidopsis thaliana small RNA libraries obtained from various tissues, plants submitted to abiotic stress or fractions immunoprecipitated with ARGONAUTE 1 (AGO1). Large differences in the tRF populations of each extract were observed. Depending on the tRNA, either tRF-5D (due to a cleavage in the D region) or tRF-3T (via a cleavage in the T region) were found and hot spots of tRNA cleavages have been identified. Interestingly, up to 25% of the tRFs originate from plastid tRNAs and we provide evidence that mitochondrial tRNAs can also be a source of tRFs. Very specific tRF-5D deriving not only from nucleus-encoded but also from plastid-encoded tRNAs are strongly enriched in AGO1 immunoprecipitates. We demonstrate that the organellar tRFs are not found within chloroplasts or mitochondria but rather accumulate outside the organelles. These observations suggest that some organellar tRFs could play regulatory functions within the plant cell and may be part of a signaling pathway.

Summary Intracellular sorting of mRNA s is an essential process for regulating gene expression and protein localization. Most mitochondrial proteins are nuclear‐encoded and imported into the mitochondria through post‐translational or co‐translational processes. In the latter case, mRNA s are found to be enriched in the vicinity of mitochondria. A genome‐scale analysis of mRNA s associated with mitochondria has been performed to determine plant cytosolic mRNA s targeted to the mitochondrial surface. Many messengers encoding mitochondrial proteins were found associated with mitochondria. These mRNA s correspond to particular functions and complexes, such as respiration or mitoribosomes, which indicates a coordinated control of mRNA localization within metabolic pathways. In addition, upstream AUG s in 5' untranslated regions ( UTR s), which modulate the translation efficiency of downstream sequences, were found to negatively affect the association of mRNA s with mitochondria. A mutational approach coupled with in vivo mRNA visualization confirmed this observation. Moreover, this technique allowed the identification of 3'‐ UTR s as another essential element for mRNA localization at the mitochondrial surface. Therefore, this work offers new insights into the mechanism, function and regulation of the association of cytosolic mRNA s with plant mitochondria.

In plants, several dsRNA-binding proteins (DRBs) have been shown to play important roles in various RNA silencing pathways, mostly by promoting the efficiency and/or accuracy of Dicer-like proteins (DCL)mediated small RNA production. Among the DRBs encoded by the Arabidopsis genome, we recently identified DRB7.2 whose function in RNA silencing was unknown. Here, we show that DRB7.2 is specifically involved in siRNA production from endogenous inverted-repeat (endoIR) loci. This function requires its interacting partner DRB4, the main cofactor of DCL4 and is achieved through specific sequestration of endoIR dsRNA precursors, thereby repressing their access and processing by the siRNA-generating DCLs. The present study also provides multiple lines of evidence showing that DRB4 is partitioned into, at least, two distinct cellular pools fulfilling different functions, through mutually exclusive binding with either DCL4 or DRB7.2. Collectively, these findings revealed that plants have evolved a specific DRB complex that modulates selectively the production of endoIR-siRNAs. The existence of such a complex and its implication regarding the still elusive biological function of plant endoIR-siRNA will be discussed.

In eukaryotes, DNA repair pathways help to maintain genome integrity and epigenomic patterns. However, the factors at the nexus of DNA repair and chromatin modification/remodeling remain poorly characterized. Here, we uncover a previously unrecognized interplay between the DNA repair factor DNA DAMAGE BINDING PROTEIN2 (DDB2) and the DNA methylation machinery in Arabidopsis thaliana. Loss-of-function mutation in DDB2 leads to genome-wide DNA methylation alterations. Genetic and biochemical evidence indicate that at many repeat loci, DDB2 influences de novo DNA methylation by interacting with ARGONAUTE4 and by controlling the local abundance of 24-nucleotide short interfering RNAs (siRNAs). We also show that DDB2 regulates active DNA demethylation mediated by REPRESSOR OF SILENCING1 and DEMETER LIKE3. Together, these findings reveal a role for the DNA repair factor DDB2 in shaping the Arabidopsis DNA methylation landscape in the absence of applied genotoxic stress.

PlantRNA database (http://plantrna.ibmp.cnrs.fr/) compiles transfer RNA (tRNA) gene sequences retrieved from fully annotated plant nuclear, plastidial and mitochondrial genomes. The set of annotated tRNA gene sequences has been manually curated for maximum quality and confidence. The novelty of this database resides in the inclusion of biological information relevant to the function of all the tRNAs entered in the library. This includes 5'- and 3'-flanking sequences, A and B box sequences, region of transcription initiation and poly(T) transcription termination stretches, tRNA intron sequences, aminoacyl-tRNA synthetases and enzymes responsible for tRNA maturation and modification. Finally, data on mitochondrial import of nuclear-encoded tRNAs as well as the bibliome for the respective tRNAs and tRNA-binding proteins are also included. The current annotation concerns complete genomes from 11 organisms: five flowering plants (Arabidopsis thaliana, Oryza sativa, Populus trichocarpa, Medicago truncatula and Brachypodium distachyon), a moss (Physcomitrella patens), two green algae (Chlamydomonas reinhardtii and Ostreococcus tauri), one glaucophyte (Cyanophora paradoxa), one brown alga (Ectocarpus siliculosus) and a pennate diatom (Phaeodactylum tricornutum). The database will be regularly updated and implemented with new plant genome annotations so as to provide extensive information on tRNA biology to the research community.

Legumes overcome nitrogen shortage by developing root nodules in which symbiotic bacteria fix atmospheric nitrogen in exchange for host-derived carbohydrates and mineral nutrients. Nodule development involves the distinct processes of nodule organogenesis, bacterial infection, and the onset of nitrogen fixation. These entail profound, dynamic gene expression changes, notably contributed to by microRNAs (miRNAs). Here, we used deep-sequencing, candidate-based expression studies and a selection of Lotus japonicus mutants uncoupling different symbiosis stages to identify miRNAs involved in symbiotic nitrogen fixation. Induction of a noncanonical miR171 isoform, which targets the key nodulation transcription factor Nodulation Signaling Pathway2, correlates with bacterial infection in nodules. A second candidate, miR397, is systemically induced in the presence of active, nitrogen-fixing nodules but not in that of noninfected or inactive nodule organs. It is involved in nitrogen fixation-related copper homeostasis and targets a member of the laccase copper protein family. These findings thus identify two miRNAs specifically responding to symbiotic infection and nodule function in legumes.

Although transfer RNA (tRNA) has a fundamental role in cell life, little is known about tRNA gene organization and expression on a genome-wide scale in eukaryotes, particularly plants. Here, we analyse the content and distribution of tRNA genes in five flowering plants and one green alga. The tRNA gene content is homogenous in plants, and is mostly correlated with genome size. The number of tRNA pseudogenes and organellar-like tRNA genes present in nuclear genomes varies greatly from one plant species to another. These pseudogenes or organellar-like genes appear to be generated or inserted randomly during evolution. Interestingly, we identified a new family of tRNA-related short interspersed nuclear elements (SINEs) in the Populus trichocarpa nuclear genome. In higher plants, intron-containing tRNA genes are rare, and correspond to genes coding for tRNATyr and tRNAMete. By contrast, in green algae, more than half of the tRNA genes contain an intron. This suggests divergent means of intron acquisition and the splicing process between green algae and land plants. Numerous tRNAs are co-transcribed in Chlamydomonas, but they are mostly transcribed as a single unit in flowering plants. The only exceptions are tRNAGly–snoRNA and tRNAMete–snoRNA cotranscripts in dicots and monocots, respectively. The internal or external motifs required for efficient transcription of tRNA genes by RNA polymerase III are well conserved among angiosperms. A brief analysis of the mitochondrial and plastidial tRNA gene populations is also provided.

In mammals, microRNAs (miRNAs) can play diverse roles in viral infection through their capacity to regulate both host and viral genes. Recent reports have demonstrated that specific miRNAs change in expression level upon infection and can impact viral production and infectivity. It is clear that miRNAs are an integral component of viral-host interactions, and it is likely that both host and virus contain mechanisms to regulate miRNA expression and/or activity. To date, little is known about the mechanisms by which miRNAs are regulated in viral infection. Here we report the rapid down-regulation of miR-27a in multiple mouse cell lines as well as primary macrophages upon infection with the murine cytomegalovirus. Down-regulation of miR-27a occurs independently from two other miRNAs, miR-23a and miR-24, located within the same genomic cluster, and analysis of pri-miRNA levels suggest that regulation occurs post-transcriptionally. miR-27b, a close homolog of miR-27a (20/21 nucleotide identity), also decreases upon infection, and we demonstrate that both miR-27a and miR-27b exert an antiviral function upon over-expression. Drug sensitivity experiments suggest that virus entry is not sufficient to induce the down-regulation of miR-27 and that the mechanism requires synthesis of RNA. Altogether, our findings indicate that miR-27a and miR-27b have antiviral activity against MCMV, and that either the virus or the host encodes molecule(s) for regulating miR-27 accumulation, most likely by inducing the rapid decay of the mature species.

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of the mammalian blastocyst. Cellular differentiation entails loss of pluripotency and gain of lineage-specific characteristics. However, the molecular controls that govern the differentiation process remain poorly understood. We have characterized small RNA expression profiles in differentiating ES cells as a model for early mammalian development. High-throughput 454 pyro-sequencing was performed on 19-30 nt RNAs isolated from undifferentiated male and female ES cells, as well as day 2 and 5 differentiating derivatives. A discrete subset of microRNAs (miRNAs) largely dominated the small RNA repertoire, and the dynamics of their accumulation could be readily used to discriminate pluripotency from early differentiation events. Unsupervised partitioning around meloids (PAM) analysis revealed that differentiating ES cell miRNAs can be divided into three expression clusters with highly contrasted accumulation patterns. PAM analysis afforded an unprecedented level of definition in the temporal fluctuations of individual members of several miRNA genomic clusters. Notably, this unravelled highly complex post-transcriptional regulations of the key pluripotency miR-290 locus, and helped identify miR-293 as a clear outlier within this cluster. Accordingly, the miR-293 seed sequence and its predicted cellular targets differed drastically from those of the other abundant cluster members, suggesting that previous conclusions drawn from whole miR-290 over-expression need to be reconsidered. Our analysis in ES cells also uncovered a striking male-specific enrichment of the miR-302 family, which share the same seed sequence with most miR-290 family members. Accordingly, a miR-302 representative was strongly enriched in embryonic germ cells derived from primordial germ cells of male but not female mouse embryos. Identifying the chromatin remodelling and E2F-dependent transcription repressors Ari4a and Arid4b as additional targets of miR-302 and miR-290 supports and possibly expands a model integrating possible overlapping functions of the two miRNA families in mouse cell totipotency during early development. This study demonstrates that small RNA sampling throughout early ES cell differentiation enables the definition of statistically significant expression patterns for most cellular miRNAs. We have further shown that the transience of some of these miRNA patterns provides highly discriminative markers of particular ES cell states during their differentiation, an approach that might be broadly applicable to the study of early mammalian development.

Ribonucleotide reductase (RNR) is an essential enzyme that provides dNTPs for DNA replication and repair. Arabidopsis (Arabidopsis thaliana) encodes three AtRNR2-like catalytic subunit genes (AtTSO2, AtRNR2A, and AtRNR2B). However, it is currently unclear what role, if any, each gene contributes to the DNA damage response, and in particular how each gene is transcriptionally regulated in response to replication blocks and DNA damage. To address this, we investigated transcriptional changes of 17-d-old Arabidopsis plants (which are enriched in S-phase cells over younger seedlings) in response to the replication-blocking agent hydroxyurea (HU) and to the DNA double-strand break inducer bleomycin (BLM). Here we show that AtRNR2A and AtRNR2B are specifically induced by HU but not by BLM. Early AtRNR2A induction is decreased in an atr mutant, and this induction is likely required for the replicative stress checkpoint since rnr2a mutants are hypersensitive to HU, whereas AtRNR2B induction is abolished in the rad9-rad17 double mutant. In contrast, AtTSO2 transcription is only activated in response to double-strand breaks (BLM), and this activation is dependent upon AtE2Fa. Both TSO2 and E2Fa are likely required for the DNA damage response since tso2 and e2fa mutants are hypersensitive to BLM. Interestingly, TSO2 gene expression is increased in atr versus wild type, possibly due to higher ATM expression in atr. On the other hand, a transient ATR-dependent H4 up-regulation was observed in wild type in response to HU and BLM, perhaps linked to a transient S-phase arrest. Our results therefore suggest that individual RNR2-like catalytic subunit genes participate in unique aspects of the cellular response to DNA damage in Arabidopsis.

Abstract In Chlamydomonas reinhardtii, 259 tRNA genes were identified and classified into 49 tRNA isoaccepting families. By constructing phylogenetic trees, we determined the evolutionary history for each tRNA gene family. The majority of the tRNA sequences are more closely related to their plant counterparts than to animals ones. Northern experiments also permitted us to show that at least one member of each tRNA isoacceptor family is transcribed and correctly processed in vivo. A short stretch of T residues known to be a signal for termination of polymerase III transcription was found downstream of most tRNA genes. It allowed us to propose that the vast majority of the tRNA genes are expressed and to confirm that numerous tRNA genes separated by short spacers are indeed cotranscribed. Interestingly, in silico analyses and hybridization experiments show that the cellular tRNA abundance is correlated with the number of tRNA genes and is adjusted to the codon usage to optimize translation efficiency. Finally, we studied the origin of SINEs, short interspersed elements related to tRNAs, whose presence in Chlamydomonas is exceptional. Phylogenetic analysis strongly suggests that tRNAAsp-related SINEs originate from a prokaryotic-type tRNA either horizontally transferred from a bacterium or originally present in mitochondria or chloroplasts.

MicroRNAs (miRNAs) are small, noncoding RNA molecules that regulate gene expression at the posttranscriptional level. Originally identified in a variety of organisms ranging from plants to mammals, miRNAs have recently been identified in several viruses. Viral miRNAs may play a role in modulating both viral and host gene expression. Here, we report on the identification and characterization of 18 viral miRNAs from mouse fibroblasts lytically infected with the murine cytomegalovirus (MCMV). The MCMV miRNAs are expressed at early times of infection and are scattered in small clusters throughout the genome with up to four distinct miRNAs processed from a single transcript. No significant homologies to human CMV-encoded miRNAs were found. Remarkably, as soon as 24 h after infection, MCMV miRNAs constituted about 35% of the total miRNA pool, and at 72 h postinfection, this proportion was increased to more than 60%. However, despite the abundance of viral miRNAs during the early phase of infection, the expression of some MCMV miRNAs appeared to be regulated. Hence, for three miRNAs we observed polyuridylation of their 3' end, coupled to subsequent degradation. Individual knockout mutants of two of the most abundant MCMV miRNAs, miR-m01-4 and miR-M44-1, or a double knockout mutant of miR-m21-1 and miR-M23-2, incurred no or only a very mild growth deficit in murine embryonic fibroblasts in vitro.

Chlamydomonas reinhardtii is a unicellular green alga whose lineage diverged from land plants over 1 billion years ago. It is a model system for studying chloroplast-based photosynthesis, as well as the structure, assembly, and function of eukaryotic flagella (cilia), which were inherited from the common ancestor of plants and animals, but lost in land plants. We sequenced the approximately 120-megabase nuclear genome of Chlamydomonas and performed comparative phylogenomic analyses, identifying genes encoding uncharacterized proteins that are likely associated with the function and biogenesis of chloroplasts or eukaryotic flagella. Analyses of the Chlamydomonas genome advance our understanding of the ancestral eukaryotic cell, reveal previously unknown genes associated with photosynthetic and flagellar functions, and establish links between ciliopathy and the composition and function of flagella.

A study of molecular diversity was carried out on 136 sugar beets infected with Beet necrotic yellow vein virus (BNYVV, Benyvirus) collected worldwide. The nucleotide sequences of the RNA-2-encoded CP, RNA-3-encoded p25 and RNA-5-encoded p26 proteins were analysed. The resulting phylogenetic trees allowed BNYVV to be classified into groups that show correlations between the virus clusters and geographic origins. The selective constraints on these three sequences were measured by estimating the ratio between synonymous and non-synonymous substitution rates (ω) with maximum-likelihood models. The results suggest that selective constraints are exerted differently on the proteins. CP was the most conserved, with mean ω values ranging from 0·12 to 0·15, while p26 was less constrained, with mean ω values ranging from 0·20 to 0·33. Selection was detected in three amino acid positions of p26, with ω values of about 5·0. The p25 sequences presented the highest mean ω values (0·36–1·10), with strong positive selection (ω=4·7–54·7) acting on 14 amino acids, and particularly on amino acid 68, where the ω value was the highest so far encountered in plant viruses

Abstract Saline and arid ecosystems are recognized as promising reservoirs of novel actinomycetes with unique adaptations and secondary metabolism potential. In this study, a halotolerant actinomycetota strain, designated ZS3416R2A, was isolated from saline soil in the hypersaline wetland of Lake Zima, Morocco. Phylogenetic analysis using the 16S rRNA gene sequence showed 99.03% similarity to Nocardiopsis terrae DSM 45157, indicating a close affiliation with the genus Nocardiopsis . Whole-genome sequencing revealed a 6.1 Mb circular chromosome with a G + C content of 70.48 mol% and 5,855 coding sequences. Genome-based metrics (ANI 95.07%, dDDH 66.4%) supported its classification as a novel species distinct from N. terrae , while AAI (95.87%) and POCP (82.4%) confirmed its placement within the genus. Comparative analyses revealed unique gene families, biosynthetic gene clusters, and regions of genomic plasticity in ZS3416R2A. Phenotypically, ZS3416R2A grew over a broad range of salinity (0–15%), temperature (16–37°C), and pH (7.0–8.0), with denser aerial mycelium formation than DSM 45157, reflecting adaptation to the arid saline environment of Lake Zima. Chemotaxonomic characterization identified MK-10(H 6 ), MK-10(H 4 ) and MK-9(H 4 ) as predominant menaquinones, iso-C16:0 and 10-methyl-C18:0 as major fatty acids, and diphosphatidylglycerol, phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, and phosphatidylinositol as major polar lipids. Based on these polyphasic evidences, strain ZS3416R2A represents a novel species within the genus Nocardiopsis , for which the name Nocardiopsis moroccensis sp. nov. is proposed. The type strain is ZS3416R2A T (= CCMM B1332 T = DSM 120542 T ).