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Lead (Pb) contamination is a widespread environmental problem due to its toxicity to living organisms. Hirschfeldia incana L., a member of the Brassicaceae family, commonly found in the Mediterranean regions, is characterized by its ability to tolerate and accumulate Pb in soils and hydroponic cultures. This plant has been reported as an excellent model to assess the response of plants to Pb. However, the lack of genomic data for H. incana hinders research at the molecular level. In the present study, we carried out RNA deep transcriptome sequencing (RNA-seq) of H. incana under two conditions, control without Pb(NO3)2 and treatment with 100 µM of Pb(NO3)2 for 15 days. A total of 797.83 million reads were generated using Illumina sequencing technology. We assembled 77,491 transcript sequences with an average length of 959 bp and N50 of 1330 bp. Sequence similarity analyses and annotation of these transcripts were performed against the Arabidopsis thaliana nr protein database, Gene Ontology (GO), and KEGG databases. As a result, 13,046 GO terms and 138 KEGG maps were created. Under Pb stress, 577 and 270 genes were differentially expressed in roots and aboveground parts, respectively. Detailed elucidation of regulation of metal transporters, transcription factors (TFs), and plant hormone genes described the role of actors that allow the plant to fine-tune Pb stress responses. Our study revealed that several genes related to jasmonic acid biosynthesis and alpha-linoleic acid were upregulated, suggesting these components’ implication in Hirschfeldia incana L responses to Pb stress. This study provides data for further genomic analyses of the biological and molecular mechanisms leading to Pb tolerance and accumulation in Hirschfeldia incana L.

International audience; Tomato is a widely cultivated crop, which can grow in many environments. However, temperature above 30 degrees C impairs its reproduction, subsequently impacting fruit yield. We assessed the impact of high-temperature stress (HS) in two tomato experimental populations, a multi-parental advanced generation intercross (MAGIC) population and a core-collection (CC) of small-fruited tomato accessions. Both populations were evaluated for 11 traits related to yield components, phenology and fruit quality in optimal and HS conditions. HS significantly impacted all traits in both populations, but a few genotypes with stable yield under HS were identified. A plasticity index was computed for each individual to measure the extent of the heat impact for each trait. Quantitative trait loci (QTL) were detected in control and HS conditions as well as for plasticity index. Linkage and genome-wide association analyses in the MAGIC and CC populations identified a total of 98 and 166 QTLs, respectively. Taking the two populations together, 69 plasticity QTLs (pQTLs) were involved in tomato heat response for 11 traits. The transcriptome changes in the ovary of six genotypes with contrasted responses to HS were studied, and 837 genes differentially expressed according to the conditions were detected. Combined with previous transcriptome studies, these results were used to propose candidate genes for HS response QTLs.

Root-knot nematodes (RKN) from genus Meloidogyne induce the dedifferentiation of root vascular cells into giant multinucleate feeding cells. These feeding cells result from an extensive reprogramming of gene expression in targeted root cells, as shown by transcriptomic analyses of galls or giant cells from various plant species.Small non-coding RNAs, and messenger RNAs from tomato (Solanum lycopersicum) galls and uninfected roots were sequenced. De novo microRNA prediction in the tomato genome identified microRNAs expressed in galls and uninfected roots. Statistical analyses identified 174 miRNA genes differentially expressed in galls at 7 and/or 14 days post infection (dpi).Integrative analyses combining small non-coding RNA and transcriptome datasets with the specific sequencing of cleaved transcripts identified miRNA targets in tomato galls. Functional analyses of promoter-GUS fusions and CRISPR-Cas9 mutants highlighted the role of the miR167-regulated transcription factor AUXIN RESPONSE FACTOR 8 (ARF8) in giant cell formation.

Understanding the mechanisms underlying differentiation of inflorescence and flower meristems is essential towards enlarging our knowledge of reproductive organ formation and to open new prospects for improving yield traits. Here, we show that SlDOF9 is a new modulator of floral differentiation in tomato. CRISPR/Cas9 knockout strategy uncovered the role of SlDOF9 in controlling inflorescence meristem and floral meristem differentiation via the regulation of cell division genes and inflorescence architecture regulator LIN. Tomato dof9-KO lines have more flowers in both determinate and indeterminate cultivars and produce more fruit upon vibration-assisted fertilization. SlDOF9 regulates inflorescence development through an auxin-dependent ARF5-DOF9 module that seems to operate, at least in part, differently in Arabidopsis and tomato. Our findings add a new actor to the complex mechanisms underlying reproductive organ differentiation in flowering plants and provide leads towards addressing the diversity of factors controlling the transition to reproductive organs.

Auxin controls multiple aspects of plant growth and development. However, its role in stress responses remains poorly understood. Auxin acts on the transcriptional regulation of target genes, mainly through Auxin Response Factors (ARF). This study focuses on the involvement of SlARF4 in tomato tolerance to salinity and osmotic stress. Using a reverse genetic approach, we found that the antisense down-regulation of SlARF4 promotes root development and density, increases soluble sugars content and maintains chlorophyll content at high levels under stress conditions. Furthermore, ARF4-as displayed higher tolerance to salt and osmotic stress through reduced stomatal conductance coupled with increased leaf relative water content and Abscisic acid (ABA) content under normal and stressful conditions. This increase in ABA content was correlated with the activation of ABA biosynthesis genes and the repression of ABA catabolism genes. Cu/ZnSOD and mdhar genes were up-regulated in ARF4-as plants which can result in a better tolerance to salt and osmotic stress. A CRISPR/Cas9 induced SlARF4 mutant showed similar growth and stomatal responses as ARF4-as plants, which suggest that arf4-cr can tolerate salt and osmotic stresses. Our data support the involvement of ARF4 as a key factor in tomato tolerance to salt and osmotic stresses and confirm the use of CRISPR technology as an efficient tool for functional reverse genetics studies.

Auxin controls multiple aspects of plant growth and development. However, its role in stress responses remains poorly understood. Auxin acts on the transcriptional regulation of target genes, mainly through Auxin Response Factors (ARF). This study focuses on the involvement of SlARF4 in tomato tolerance to salinity and osmotic stress. Using a reverse genetic approach, we found that the antisense down-regulation of SlARF4 promotes root development and density, increases soluble sugars content and maintains chlorophyll content at high levels under stress conditions. Furthermore, ARF4-as displayed higher tolerance to salt and osmotic stress through reduced stomatal conductance coupled with increased leaf relative water content and ABA content under normal and stressful conditions. This increase in ABA content was correlated with the activation of ABA biosynthesis genes and the repression of ABA catabolism genes. cat1, Cu/ZnSOD and mdhar genes were up-regulated in ARF4-as plants which can result in a better tolerance to salt and osmotic stress. A CRISPR/Cas9 induced SlARF4 mutant showed similar growth and stomatal responses as ARF4-as plants, which suggest that arf4-cr can tolerate salt and osmotic stresses. Our data support the involvement of ARF4 as a key factor in tomato tolerance to salt and osmotic stresses and confirm the use of CRISPR technology as an efficient tool for functional reverse genetics studies.

Although the reference genome of Solanum tuberosum Group Phureja double-monoploid (DM) clone is available, knowledge on the genetic diversity of the highly heterozygous tetraploid Group Tuberosum, representing most cultivated varieties, remains largely unexplored. This lack of knowledge hinders further progress in potato research. In conducted investigation, we first merged and manually curated the two existing partially-overlapping DM genome-based gene models, creating a union of genes in Phureja scaffold. Next, we compiled available and newly generated RNA-Seq datasets (cca. 1.5 billion reads) for three tetraploid potato genotypes (cultivar Désirée, cultivar Rywal, and breeding clone PW363) with diverse breeding pedigrees. Short-read transcriptomes were assembled using several de novo assemblers under different settings to test for optimal outcome. For cultivar Rywal, PacBio Iso-Seq full-length transcriptome sequencing was also performed. EvidentialGene redundancy-reducing pipeline complemented with in-house developed scripts was employed to produce accurate and complete cultivar-specific transcriptomes, as well as to attain the pan-transcriptome. The generated transcriptomes and pan-transcriptome represent a valuable resource for potato gene variability exploration, high-throughput omics analyses, and breeding programmes., Measurement(s) genome • RNA • sequence_assembly • transcriptome Technology Type(s) digital curation • RNA sequencing • sequence assembly process Factor Type(s) cultivar Sample Characteristic - Organism Solanum tuberosum Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.12562733

Nanotechnology is currently undergoing rapid development partly due to the increasing use of carbon-based nanoparticles, such as Few Layer Graphene (FLG). Owing to its numerous applications, its industrial production is likely to lead to environmental release, including into aquatic ecosystems. In this study, a transcriptomic approach was used to assess the effect of FLG at low (0.1 mg L-1) and high (50 mg L-1) concentrations on the benthic freshwater diatom Nitzschia palea after 48 h of exposure. Direct contact with FLG and induced shading were distinguished to compare the transcriptomic responses. Genes that were differentially expressed after exposure compared with control conditions were identified, and their functional descriptions are discussed. A slight transcriptomic response related to cell wall repair was observed in diatoms exposed to the low FLG concentration. Exposure to the high FLG concentration induced a strong response involving 1907 transcripts. Notably, 16 transcripts involved in the chlorophyll biosynthesis process were under-expressed (log2 fold change between -3 and -1.2), suggesting a down-regulation of the photosynthetic metabolism. Diatoms exposed to the high FLG concentration over-expressed about 13 transcripts encoding for extracellular proteins that play a role in cellular adhesion, and two transcripts involved in cell wall repair were highly up-regulated. Light deprivation caused by shading induced a downregulation of genes involved in the energetic metabolism of N. palea. Overall, these results revealed that metabolic pathways impacted by FLG exposure are concentration and contact dependent. Moreover, this study suggests that a low FLG concentration, close to that in environmental conditions, will have a minor impact on diatom biofilms whereas a high FLG concentration, mimicking accidental release, might be critical for ecosystems.

This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession PSQE00000000.; 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.

[Background]: “Omics” approaches may provide useful information for a deeper understanding of speciation events, diversification and function innovation. This can be achieved by investigating the molecular similarities at sequence level between species, allowing the definition of ortholog and paralog genes. However, the spreading of sequenced genome, often endowed with still preliminary annotations, requires suitable bioinformatics to be appropriately exploited in this framework., [Results]: We presented here a multilevel comparative approach to investigate on genome evolutionary relationships and peculiarities of two fleshy fruit species of relevant agronomic interest, Solanum lycopersicum (tomato) and Vitis vinifera (grapevine). We defined 17,823 orthology relationships between tomato and grapevine reference gene annotations. The resulting orthologs are associated with the detected paralogs in each species, permitting the definition of gene networks, useful to investigate the different relationships. The reconciliation of the compared collections in terms of an updating of the functional descriptions was also exploited. All the results were made accessible in ComParaLogs, a dedicated bioinformatics platform available at http://biosrv.cab.unina.it/comparalogs/gene/search., [Conclusions]: The aim of the work was to suggest a reliable approach to detect all similarities of gene loci between two species based on the integration of results from different levels of information, such as the gene, the transcript and the protein sequences, overcoming possible limits due to exclusive protein versus protein comparisons. This to define reliable ortholog and paralog genes, as well as species specific gene loci in the two species, overcoming limits due to the possible draft nature of preliminary gene annotations. Moreover, reconciled functional descriptions, as well as common or peculiar enzymatic classes and protein domains from tomato and grapevine, together with the definition of species-specific gene sets after the pairwise comparisons, contributed a comprehensive set of information useful to comparatively exploit the two species gene annotations and investigate on differences between species with climacteric and non-climacteric fruits. In addition, the definition of networks of ortholog genes and of associated paralogs, and the organization of web-based interfaces for the exploration of the results, defined a friendly computational bench-work in support of comparative analyses between two species., Publication costs for this manuscript were sponsored by the Genopom Pro (PON02_00395_3082360) and HORT (PON02_00395_3215002) Projects (Ministero dell’Istruzione, dell’Università e della Ricerca (MIUR), Italy) and from the Solanaceae Pollen Thermotolerance - Marie Curie Initial Training Network project (Grant Agreement No. 289220). This work is the frame of the Cost Action FA1106.

International audience; As part of normal development most eukaryotic organisms, ranging from insects and mammals to plants, display variations in nuclear ploidy levels resulting from somatic endopolyploidy. Endoreduplication is the major source of endopolyploidy in higher plants. Endoreduplication is a remarkable characteristic of the fleshy pericarp tissue of developing tomato fruits, where it establishes a highly integrated cellular system that acts as a morphogenetic factor supporting cell growth. However, the functional significance of endoreduplication is not fully understood. Although endoreduplication is thought to increase metabolic activity due to a global increase in transcription, the issue of gene-specific ploidy-regulated transcription remains open. To investigate the influence of endoreduplication on transcription in tomato fruit, we tested the feasibility of a RNA sequencing (RNA-Seq) approach using total nuclear RNA extracted from purified populations of flow cytometry-sorted nuclei based on their DNA content. Here we show that cell-based approaches to the study of RNA-Seq profiles need to take into account the putative global shift in expression between samples for correct analysis and interpretation of the data. From ploidy-specific expression profiles we found that the activity of cells inside the pericarp is related both to the ploidy level and their tissue location.

International audience; RNA-Seq is a widely used technology that allows an efficient genome-wide quantification of gene expressions for, for example, differential expression (DE) analysis. After a brief review of the main issues, methods and tools related to the DE analysis of RNA-Seq data, this article focuses on the impact of both the replicate number and library size in such analyses. While the main drawback of previous relevant studies is the lack of generality, we conducted both an analysis of a two-condition experiment (with eight biological replicates per condition) to compare the results with previous benchmark studies, and a meta-analysis of 17 experiments with up to 18 biological conditions, eight biological replicates and 100 million (M) reads per sample. As a global trend, we concluded that the replicate number has a larger impact than the library size on the power of the DE analysis, except for low-expressed genes, for which both parameters seem to have the same impact. Our study also provides new insights for practitioners aiming to enhance their experimental designs. For instance, by analyzing both the sensitivity and specificity of the DE analysis, we showed that the optimal threshold to control the false discovery rate (FDR) is approximately 2 −r , where r is the replicate number. Furthermore, we showed that the false positive rate (FPR) is rather well controlled by all three studied R packages: DESeq, DESeq2, and edgeR. We also analyzed the impact of both the replicate number and library size on gene ontology (GO) enrichment analysis. Interestingly, we concluded that increases in the replicate number and library size tend to enhance the sensitivity and specificity, respectively, of the GO analysis. Finally, we recommend to RNA-Seq practitioners the production of a pilot data set to strictly analyze the power of their experimental design, or the use of a public data set, which should be similar to the data set they will obtain. For individuals working on tomato research, on the basis of the meta-analysis, we recommend at least four biological replicates per condition and 20 M reads per sample to be almost sure of obtaining about 1000 DE genes if they exist.

International audience; Survival biomass production and crop yield are heavily constrained by a wide range of environmental stresses. Several phytohormones among which abscisic acid (ABA), ethylene and salicylic acid (SA) are known to mediate plant responses to these stresses. By contrast, the role of the plant hormone auxin in stress responses remains so far poorly studied. Auxin controls many aspects of plant growth and development, and Auxin Response Factors play a key role in the transcriptional activation or repression of auxin-responsive genes through direct binding to their promoters. As a mean to gain more insight on auxin involvement in a set of biotic and abiotic stress responses in tomato, the present study uncovers the expression pattern of SlARF genes in tomato plants subjected to biotic and abiotic stresses. In silico mining of the RNAseq data available through the public TomExpress web platform, identified several SlARFs as responsive to various pathogen infections induced by bacteria and viruses. Accordingly, sequence analysis revealed that 5’ regulatory regions of these SlARFs are enriched in biotic and abiotic stress-responsive cis-elements. Moreover, quantitative qPCR expression analysis revealed that many SlARFs were differentially expressed in tomato leaves and roots under salt, drought and flooding stress conditions. Further pointing to the putative role of SlARFs in stress responses, quantitative qPCR expression studies identified some miRNA precursors as potentially involved in the regulation of their SlARF target genes in roots exposed to salt and drought stresses. These data suggest an active regulation of SlARFs at the post-transcriptional level under stress conditions. Based on the substantial change in the transcript accumulation of several SlARF genes, the data presented in this work strongly support the involvement of auxin in stress responses thus enabling to identify a set of candidate SlARFs as potential mediators of biotic and abiotic stress responses.

International audience; MADS-box transcription factors are key elements of the genetic networks controlling flower and fruit development. Among these, the class D clade gathers AGAMOUS-like genes which are involved in seed, ovule, and funiculus development. The tomato genome comprises two class D genes, Sl-AGL11 and Sl-MBP3 , both displaying high expression levels in seeds and in central tissues of young fruits. The potential effects of Sl-AGL11 on fruit development were addressed through RNAi silencing and ectopic expression strategies. Sl-AGL11-down-regulated tomato lines failed to show obvious phenotypes except a slight reduction in seed size. In contrast, Sl-AGL11 overexpression triggered dramatic modifications of flower and fruit structure that include: the conversion of sepals into fleshy organs undergoing ethylene-dependent ripening, a placenta hypertrophy to the detriment of locular space, starch and sugar accumulation, and an extreme softening that occurs well before the onset of ripening. RNA-Seq transcriptomic profiling high-lighted substantial metabolic reprogramming occurring in sepals and fruits, with major impacts on cell wall-related genes. While several Sl-AGL11-related phenotypes are reminiscent of class C MADS-box genes (TAG1 and TAGL1), the modifications observed on the placenta and cell wall and the Sl-AGL11 expression pattern suggest an action of this class D MADS-box factor on early fleshy fruit development.

A gene encoding an elongation factor LeEF-Tsmt that participates in the protein synthesis process in mitochondria shows strong expression in ripening fruit as compared to other organs. It is strongly up-regulated during the first stages of the ripening process in parallel with the climacteric rise in respiration. LeEF-Tsmt expression is stimulated by ethylene, wounding and high temperature but ethylene-insensitive mutants exhibit normal expression. Transgenic fruit have been generated in which LeEF-Tsmt has been constitutively up- and down-regulated. Surprisingly, altering the expression of the gene by genetic transformation with antisense and sense LeEF-Tsmt constructs did not affect the pattern of respiration and ethylene production during ripening and upon wounding. In addition, expression of the alternative oxidase gene which is known to play an important role in respiratory climacteric was not affected. Possible reasons for the absence of effect on respiration of variations of LeEF-Tsmt gene expression are discussed. Made available in DSpace on 2016-10-25T21:37:34Z (GMT). No. of bitstreams: 1 GIRARDIOstharvestBiolTechv42n1p12006.pdf: 466398 bytes, checksum: 9306c559caab243abde7e824345aea9c (MD5) Previous issue date: 2012-08-06

The involvement of ethylene in fruit ripening is well documented, though knowledge regarding the crosstalk between ethylene and other hormones in ripening is lacking. We discovered that AUXIN RESPONSE FACTOR 2A (ARF2A), a recognized auxin signaling component, functions in the control of ripening. ARF2A expression is ripening regulated and reduced in the rin, nor and nr ripening mutants. It is also responsive to exogenous application of ethylene, auxin and abscisic acid (ABA). Over-expressing ARF2A in tomato resulted in blotchy ripening in which certain fruit regions turn red and possess accelerated ripening. ARF2A over-expressing fruit displayed early ethylene emission and ethylene signaling inhibition delayed their ripening phenotype, suggesting ethylene dependency. Both green and red fruit regions showed the induction of ethylene signaling components and master regulators of ripening. Comprehensive hormone profiling revealed that altered ARF2A expression in fruit significantly modified abscisates, cytokinins and salicylic acid while gibberellic acid and auxin metabolites were unaffected. Silencing of ARF2A further validated these observations as reducing ARF2A expression let to retarded fruit ripening, parthenocarpy and a disturbed hormonal profile. Finally, we show that ARF2A both homodimerizes and interacts with the ABA STRESS RIPENING (ASR1) protein, suggesting that ASR1 might be linking ABA and ethylene-dependent ripening. These results revealed that ARF2A interconnects signals of ethylene and additional hormones to co-ordinate the capacity of fruit tissue to initiate the complex ripening process., Author Summary The hormone ethylene is known to be involved in fleshy fruit ripening, although the role of other hormones is less well studied. Here we investigated the role of AUXIN RESPONSE FACTOR 2A (ARF2A) in tomato fruit ripening and suggest that it may be involved in the crosstalk between ethylene and other hormones. We show that over-expression of ARF2A (ARF2-OX) causes the fruit to ripen in an uneven, blotchy manner. The timing of ripening in ARF2-OX fruit is affected by applying exogenous ethylene, but the variegated appearance of ripening regions is independent of ethylene. In agreement with findings in ARF2-OX fruit, silencing of both ARF2 paralogs, ARF2A and ARF2B (ARF2as), delayed the ripening process. Comprehensive hormone profiling revealed that altered ARF2 expression in fruit significantly impacted abscisates, cytokinins and salicylic acid while gibberellic acid and auxin metabolites were unaffected. Transcriptome analysis of ARF2-OX fruit patches revealed that normal ripening does occur, however, the timing and co-ordination is affected. These observations were reinforced in ARF2as fruit that displayed the opposite gene expression and metabolic phenotypes. Finally, we show that ARF2A homodimerizes as well as interacts with the known ABA STRESS RIPENING (ASR1) protein, suggesting that ASR1 might be linking ABA and ethylene-dependent ripening. Our results reveal that ARF2A may interconnect signals of ethylene and additional hormones to co-ordinate the capacity of fruit tissue to initiate the complex ripening process.

Correspondance: chervin@ensat.fr; International audience; Grape is considered as a non-climacteric fruit, the maturation of which is independent of ethylene. However, previous work had shown that ethylene is capable of affecting the physiological processes during maturation of grape berries. Experiments were designed to screen the gene pool affected by ethylene at the ripening inception in Cabernet Sauvignon berries. The results showed that only 73 of 14 562 genes of microarray slides were significantly modulated by a 24-h ethylene treatment (4 mu l l(-1)), performed 8 weeks after flowering. The study then focused on accumulation of several mRNAs affected by ethylene in relation to the berry size. Indeed, we observed that ethylene application at veraison led to a berry diameter increase. This increase is mainly because of sap intake and cell wall modifications, enabling cell elongation. This was related to changes in the expression pattern of many genes, classified in two groups: (1) 'water exchange' genes: various aquaporins (AQUA) and (2) 'cell wall structure' genes: polygalacturonases, xyloglucan endotransglucosylases (XTH), pectin methyl esterases, cellulose synthases and expansins. The expression patterns were followed either along berry development or in three berry tissues (peel, pulp and seeds). Ethylene stimulates the accumulation of most gene transcripts in 1 h, and in several parts of the berry, this stimulation may last for 24 h in some cases. One XTH and one AQUA seem to be good candidates to explain the ethylene-induced berry expansion. This work brings more clues about the ethylene involvement in the development and ripening of grape berries

Gene amplifications have been detected as a transitory phenomenon in bacterial cultures. They are predicted to contribute to rapid adaptation by simultaneously increasing the expression of genes clustered on the chromosome. However, genome amplifications have rarely been described in natural isolates. Through DNA array analysis, we have identified two Streptococcus agalactiae strains carrying tandem genome amplifications: a fourfold amplification of 13.5 kb and a duplication of 92 kb. Both amplifications were located close to the terminus of replication and originated independently from any long repeated sequence. They probably arose in the human host and showed different stabilities, the 13.5-kb amplification being lost at a frequency of 0.003 per generation and the 92-kb tandem duplication at a frequency of 0.035 per generation. The 13.5-kb tandem amplification carried the five genes required for dihydrofolate biosynthesis and led to both trimethoprim (TMP) and sulfonamide (SU) resistance. Resistance to SU probably resulted from the increased synthesis of dihydropteroate synthase, the target of this antibiotic, whereas the amplification of the whole pathway was responsible for TMP resistance. This revealed a new mechanism of resistance to TMP involving an increased dihydrofolate biosynthesis. This is, to our knowledge, the first reported case of naturally occurring antibiotic resistance resulting from genome amplification in bacteria. The low stability of DNA segment amplifications suggests that their role in antibiotic resistance might have been underestimated.

International audience; Our knowledge of the factors mediating ethylene-dependent ripening of climacteric fruit remains limited. The transcription of ethylene-regulated genes is mediated by ethylene response factors (ERFs), but mutants providing information on the specific role of the ERFs in fruit ripening are still lacking, likely due to functional redundancy among this large multigene family of transcription factors. We present here a comprehensive expression profiling of tomato (Solanum lycopersicum) ERFs in wild-type and tomato ripening-impaired tomato mutants (Never-ripe [Nr], ripening-inhibitor [rin], and non-ripening [nor]), indicating that out of the 77 ERFs present in the tomato genome, 27 show enhanced expression at the onset of ripening while 28 display a ripeningassociated decrease in expression, suggesting that different ERFs may have contrasting roles in fruit ripening. Among the 19 ERFs exhibiting the most consistent up-regulation during ripening, the expression of 11 ERFs is strongly down-regulated in rin, nor, and Nr tomato ripening mutants, while only three are consistently up-regulated. Members of subclass E, SlERF.E1, SlERF.E2, and SlERF.E4, show dramatic down-regulation in the ripening mutants, suggesting that their expression might be instrumental in fruit ripening. This study illustrates the high complexity of the regulatory network connecting RIN and ERFs and identifies subclass E members as the most active ERFs in ethylene- and RIN/NOR-dependent ripening.

International audience; Parthenocarpy is a desired trait in fruit crops; it enables fruit set under environmental conditions suboptimal for pollination, and seedless fruits represent a valuable consumer product. We employed TILLING-based screening of a mutant tomato population to find genetic lesions in Aux/IAA9, a negative regulator of the auxin response involved in the control of fruit set. We identified three mutations located in the coding region of this gene, including two singlebase substitutions and one single-base deletion, which leads to a frame shift and premature stop codon. The transcription of IAA9 was strongly reduced in the frame-shift mutant, and partial loss of mutated protein activity was evidenced by an in vitro transactivation assay. Whereas missense mutations were predicted to be tolerated and did not cause mutant phenotypes, the frame-shift mutation-induced phenotypes expected for a loss of IAA9 function, including altered axillary shoot growth, reduced leaf compoundness and a strong tendency to produce parthenocarpic fruits. Mutant flowers showed pleiotropic anther cone defects, a phenotype frequently associated with parthenocarpy in tomato and other species. Mutant fruits were larger than those of the seeded control, with higher brix

Streptococcus agalactiae is a commensal bacterium colonizing the intestinal tract of a significant proportion of the human population. However, it is also a pathogen which is the leading cause of invasive infections in neonates and causes septicaemia, meningitis and pneumonia. We sequenced the genome of the serogroup III strain NEM316, responsible for a fatal case of septicaemia. The genome is 2 211 485 base pairs long and contains 2118 protein coding genes. Fifty-five per cent of the predicted genes have an ortholog in the Streptococcus pyogenes genome, representing a conserved backbone between these two streptococci. Among the genes in S. agalactiae that lack an ortholog in S. pyogenes, 50% are clustered within 14 islands. These islands contain known and putative virulence genes, mostly encoding surface proteins as well as a number of genes related to mobile elements. Some of these islands could therefore be considered as pathogenicity islands. Compared with other pathogenic streptococci, S. agalactiae shows the unique feature that pathogenicity islands may have an important role in virulence acquisition and in genetic diversity.

Pertinent evolutionary studies are based on a correct use of homology terms such as paralogues, metalogues and orthologues. Such crucial concepts have been applied to intragenomic and intergenomic analyses. A further requisite is a proper definition of what is a structural segment of homology. Such segments are called modules to reflect that they play a role in the mechanism of combinational construction of a gene from ready-made basic components. Since identifying a module is operationally equivalent to determining the ancestor to this gene segment, it becomes possible to track back protein history and genome evolution. Such studies underline the importance of two fundamental processes, gene duplication and gene fusion. Moreover, grouping the closest orthologues in families is a pertinent way to reconstruct a genomic tree for all available prokaryotes.

Most land plant species live in symbiosis with arbuscular mycorrhizal fungi. These fungi differentiate essential functional structures called arbuscules in root cortical cells from which mineral nutrients are released to the plant. We investigated the role of microRNA393 (miR393), an miRNA that targets several auxin receptors, in arbuscular mycorrhizal root colonization. Expression of the precursors of the miR393 was down-regulated during mycorrhization in three different plant species: Solanum lycopersicum, Medicago truncatula, and Oryza sativa. Treatment of S. lycopersicum, M. truncatula, and O. sativa roots with concentrations of synthetic auxin analogs that did not affect root development stimulated mycorrhization, particularly arbuscule formation. DR5-GUS, a reporter for auxin response, was preferentially expressed in root cells containing arbuscules. Finally, overexpression of miR393 in root tissues resulted in down-regulation of auxin receptor genes (transport inhibitor response1 and auxin-related F box) and underdeveloped arbuscules in all three plant species. These results support the conclusion that miR393 is a negative regulator of arbuscule formation by hampering auxin perception in arbuscule-containing cells.

International audience; Members of the TOPLESS gene family emerged recently as key players in gene repression in several mechanisms, especially in auxin perception. The TOPLESS genes constitute, in higher-plant genomes, a small multigenic family comprising four to 11 members. In this study, this family was investigated in tomato, a model plant for Solanaceae species and fleshy fruits. Six open reading frames predicted to encode topless-like proteins (SlTPLs) containing the canonical domains (LisH, CTLH, and two WD40 repeats) were identified in the tomato genome. Nuclear localization was confirmed for all members of the SlTPL family with the exception SlTPL6, which localized at the cytoplasm and was excluded from the nucleus. SlTPL genes displayed distinctive expression patterns in different tomato organs, with SlTPL1 showing the highest levels of transcript accumulation in all tissues tested except in ripening fruit where SlTPL3 and SlTPL4 were the most prominently expressed. To gain insight into the specificity of the different TOPLESS paralogues, a proteinprotein interaction map between TOPLESS and auxin/indole-3-acetic acid (Aux/IAA) proteins was built using a yeast two-hybrid approach. The PPI map enabled the distinction of two patterns: TOPLESS isoforms interacting with the majority of Aux/IAA, and isoforms with limited capacity for interaction with these protein partners. Interestingly, evolutionary analyses of the TOPLESS gene family revealed that the highly expressed isoforms (SlTPL1, SlTPL3, and SlTPL4) corresponded to the three TPL-related genes undergoing the strongest purifying selection, while the selection was much weaker for SlTPL6, which was expressed at a low level and encoded a protein lacking the capacity to interact with Aux/IAAs.

International audience; Auxin is one of the most prominent phytohormones regulating many aspects of fleshy fruit development including fruit set, fruit size through the control of cell division and cell expansion, and fruit ripening. To shed light on the role of auxin fruit ripening, we have previously shown that Sl-ARF4 is a major player in mediating the auxin control of sugar metabolism in tomato fruit (cv MicroTom). Further extending this study, we show here that down-regulation of Sl-ARF4 in tomato alters some ripening-related fruit quality traits including enhanced fruit density at mature stage, increased firmness, prolonged shelf-life and reduced water (weight) loss at red ripe stage. These findings suggest that Sl-ARF4 plays a role in determining fruit cell wall architecture and thus providing a potential genetic marker for improving post-harvest handling and shelf life of tomato fruits.

International audience; In recent years, RNA-Seq technologies became a powerful tool for transcriptome studies. However, computational methods dedicated to the analysis of high-throughput sequencing data are yet to be standardized. In particular, it is known that the choice of a normalization procedure leads to a great variability in results of differential gene expression analysis. The present study compares the most widespread normalization procedures and proposes a novel one aiming at removing an inherent bias of studied transcriptomes related to their relative size. Comparisons of the normalization procedures are performed on real and simulated data sets. Real RNA-Seq data sets analyses, performed with all the different normalization methods, show that only 50% of significantly differentially expressed genes are common. This result highlights the influence of the normalization step on the differential expression analysis. Real and simulated data sets analyses give similar results showing 3 different groups of procedures having the same behavior. The group including the novel method named "Median Ratio Normalization" (MR N) gives the lower number of false discoveries. Within this group the MR N method is less sensitive to the modification of parameters related to the relative size of transcriptomes such as the number of down- and upregulated genes and the gene expression levels. The newly proposed MR N method efficiently deals with intrinsic bias resulting from relative size of studied transcriptomes. Validation with real and simulated data sets confirmed that MR N is more consistent and robust than existing methods.

L'article original est publié par The American Society of Plant Biologists; International audience; Successful completion of fruit developmental programs depends on the interplay between multiple phytohormones. However, besides ethylene, the impact of other hormones on fruit quality traits remains elusive. A previous study has shown that down-regulation of SlARF4, a member of the tomato (Solanum lycopersicum) auxin response factor (ARF) gene family, results in a dark-green fruit phenotype with increased chloroplasts (Jones et al., 2002). This study further examines the role of this auxin transcriptional regulator during tomato fruit development at the level of transcripts, enzyme activities, and metabolites. It is noteworthy that the dark-green phenotype of antisense SlARF4-suppressed lines is restricted to fruit, suggesting that SlARF4 controls chlorophyll accumulation specifically in this organ. The SlARF4 underexpressing lines accumulate more starch at early stages of fruit development and display enhanced chlorophyll content and photochemical efficiency, which is consistent with the idea that fruit photosynthetic activity accounts for the elevated starch levels. SlARF4 expression is high in pericarp tissues of immature fruit and then undergoes a dramatic decline at the onset of ripening concomitant with the increase in sugar content. The higher starch content in developing fruits of SlARF4 down-regulated lines correlates with the up-regulation of genes and enzyme activities involved in starch biosynthesis, suggesting their negative regulation by SlARF4. Altogether, the data uncover the involvement of ARFs in the control of sugar content, an essential feature of fruit quality, and provide insight into the link between auxin signaling, chloroplastic activity, and sugar metabolism in developing fruit.

International audience; HPB12-L24 was previously described as a bifunctional histone-like and ribosomal protein in Bacillus subtilis. In order to confirm the identity of HPB12 and L24, and to study the properties of this protein, the rplX gene of B. subtilis encoding L24 has been overexpressed in Escherichia coli by an efficient protein overproduction system. A simple and rapid purification scheme using ammonium sulfate precipitation and cation-exchange chromatography is presented. 10 mg of pure L24 per g of Escherichia coli cells were obtained. The purified recombinant protein L24 is heat-stable, acid-soluble and binds preferentially supercoiled DNA like protein HPB12. These results confirm the identity of HPB12 and L24. Overexpression of rplX led to gross alterations of cell morphology and to an abnormal shape of nucleoids.

European Integrated Project EU-SOL [FOOD-CT-2006-016214] Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; International audience; Auxin is a central hormone that exerts pleiotropic effects on plant growth including the development of roots, shoots, flowers and fruit. The perception and signaling of the plant hormone auxin rely on the cooperative action of several components,among which auxin/indole-3-acetic acid (Aux/IAA) proteins play a pivotal role. In this study, we identified and comprehensively analyzed the entire Aux/IAA gene family in tomato (Solanum lycopersicum), a reference species for Solanaceae plants, and the model plant for fleshy fruit development. Functional characterization using a dedicated single cell system revealed that tomato Aux/IAA proteins function as active repressors of auxin-dependent gene transcription, with, however, different Aux/IAA members displaying varying levels of repression. Phylogenetic analysis indicated that the Aux/IAA gene family is slightly contracted in tomato compared with Arabidopsis, with a lower representation of non-canonical proteins. Sl-IAA genes display distinctive expression pattern in different tomato organs and tissues, and some of them display differential responses to auxin and ethylene, suggesting that Aux/IAAs may play a role in linking both hormone signaling pathways. The data presented here shed more light on Sl-IAA genes and provides new leads towards the elucidation of their function during plant development and in mediating hormone cross-talk.

Background The phytohormone ethylene is involved in a wide range of developmental processes and in mediating plant responses to biotic and abiotic stresses. Ethylene signalling acts via a linear transduction pathway leading to the activation of Ethylene Response Factor genes (ERF) which represent one of the largest gene families of plant transcription factors. How an apparently simple signalling pathway can account for the complex and widely diverse plant responses to ethylene remains yet an unanswered question. Building on the recent release of the complete tomato genome sequence, the present study aims at gaining better insight on distinctive features among ERF proteins. Results A set of 28 cDNA clones encoding ERFs in the tomato (Solanum lycopersicon) were isolated and shown to fall into nine distinct subclasses characterised by specific conserved motifs most of which with unknown function. In addition of being able to regulate the transcriptional activity of GCC-box containing promoters, tomato ERFs are also shown to be active on promoters lacking this canonical ethylene-responsive-element. Moreover, the data reveal that ERF affinity to the GCC-box depends on the nucleotide environment surrounding this cis-acting element. Site-directed mutagenesis revealed that the nature of the flanking nucleotides can either enhance or reduce the binding affinity, thus conferring the binding specificity of various ERFs to target promoters. Based on their expression pattern, ERF genes can be clustered in two main clades given their preferential expression in reproductive or vegetative tissues. The regulation of several tomato ERF genes by both ethylene and auxin, suggests their potential contribution to the convergence mechanism between the signalling pathways of the two hormones. Conclusions The data reveal that regions flanking the core GCC-box sequence are part of the discrimination mechanism by which ERFs selectively bind to their target promoters. ERF tissue-specific expression combined to their responsiveness to both ethylene and auxin bring some insight on the complexity and fine regulation mechanisms involving these transcriptional mediators. All together the data support the hypothesis that ERFs are the main component enabling ethylene to regulate a wide range of physiological processes in a highly specific and coordinated manner.

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; International audience; A comparative proteomic approach was performed to identify differentially expressed proteins in plastids at three stages of tomato (Solanum lycopersicum) fruit ripening (mature-green, breaker, red). Stringent curation and processing of the data from three independent replicates identified 1,932 proteins among which 1,529 were quantified by spectral counting. The quantification procedures have been subsequently validated by immunoblot analysis of six proteins representative of distinct metabolic or regulatory pathways. Among the main features of the chloroplast-to-chromoplast transition revealed by the study, chromoplastogenesis appears to be associated with major metabolic shifts: (1) strong decrease in abundance of proteins of light reactions (photosynthesis, Calvin cycle, photorespiration) and carbohydrate metabolism (starch synthesis/degradation), mostly between breaker and red stages and (2) increase in terpenoid biosynthesis (including carotenoids) and stress-response proteins (ascorbate-glutathione cycle, abiotic stress, redox, heat shock). These metabolic shifts are preceded by the accumulation of plastid-encoded acetyl Coenzyme A carboxylase D proteins accounting for the generation of a storage matrix that will accumulate carotenoids. Of particular note is the high abundance of proteins involved in providing energy and in metabolites import. Structural differentiation of the chromoplast is characterized by a sharp and continuous decrease of thylakoid proteins whereas envelope and stroma proteins remain remarkably stable. This is coincident with the disruption of the machinery for thylakoids and photosystem biogenesis (vesicular trafficking, provision of material for thylakoid biosynthesis, photosystems assembly) and the loss of the plastid division machinery. Altogether, the data provide new insights on the chromoplast differentiation process while enriching our knowledge of the plant plastid proteome.

International audience; ETHYLENE-INSENSITIVE3/ETHYLENE-INSENSITIVE3-like (EIN3/EIL) transcription factors are important downstream components of the ethylene transduction pathway known to regulate the transcription of early ethylene-responsive genes in plants. Previous studies have shown that phosphorylation can repress their transcriptional activity by promoting protein degradation. The present study identifies a new phosphorylation region named EPR1 (EIN3/EIL phosphorylation region 1) in tomato EIL1 proteins. The functional significance of EPR1 was tested by introducing mutations in this region of the Sl-EIL1 gene and by expressing these mutated versions in transgenic tomato plants. Transient expression data and phenotypic analysis of the transgenic lines indicated that EPR1 is essential for the transcriptional activity of Sl-EIL1. Moreover, mutation in the EPR1 site that prevents phosphorylation abolishes ethylene constitutive responses normally displayed by the Sl-EIL1-overexpressing lines. Bimolecular fluorescence complementation (BiFC) studies showed that the presence of a functional phosphorylation site within EPR1 is instrumental in the dimerization of Sl-EIL1 proteins. The results illuminate a new molecular mechanism for the control of EIN3/EIL activity and propose a model where phosphorylation within the EPR1 promotes the dimerization process allowing the initiation of EIL-mediated transcription of early ethylene-regulated genes.

Chromoplasts are nonphotosynthetic plastids that accumulate carotenoids. They derive from other plastid forms, mostly chloroplasts. The biochemical events responsible for the interconversion of one plastid form into another are poorly documented. However, thanks to transcriptomics and proteomics approaches, novel information is now available. Data of proteomic and biochemical analysis revealed the importance of lipid metabolism and carotenoids biosynthetic activities. The loss of photosynthetic activity was associated with the absence of the chlorophyll biosynthesis branch and the presence of proteins involved in chlorophyll degradation. Surprisingly, the entire set of Calvin cycle and of the oxidative pentose phosphate pathway persisted after the transition from chloroplast to chromoplast. The role of plastoglobules in the formation and organisation of carotenoid-containing structures and that of theOrgene in the control of chromoplastogenesis are reviewed. Finally, using transcriptomic data, an overview is given the expression pattern of a number of genes encoding plastid-located proteins during tomato fruit ripening.

International audience; Chromoplasts are non-photosynthetic specialized plastids that are important in ripening tomato fruit (Solanum lycopersicum) since, among other functions, they are the site of accumulation of coloured compounds. Analysis of the proteome of red fruit chromoplasts revealed the presence of 988 proteins corresponding to 802 Arabidopsis unigenes, among which 209 had not been listed so far in plastidial databanks. These data revealed several features of the chromoplast. Proteins of lipid metabolism and trafficking were well represented, including all the proteins of the lipoxygenase pathway required for the synthesis of lipid-derived aroma volatiles. Proteins involved in starch synthesis co-existed with several starch-degrading proteins and starch excess proteins. Chromoplasts lacked proteins of the chlorophyll biosynthesis branch and contained proteins involved in chlorophyll degradation. None of the proteins involved in the thylakoid transport machinery were discovered. Surprisingly, chromoplasts contain the entire set of Calvin cycle proteins including Rubisco, as well as the oxidative pentose phosphate pathway (OxPPP). The present proteomic analysis, combined with available physiological data, provides new insights into the metabolic characteristics of the tomato chromoplast and enriches our knowledge of non-photosynthetic plastids.

Tomato (Solanum lycopersicum) and coffee (Coffea canephora) belong to the sister families Solanaceae and Rubiaceae, respectively. We report herein the mapping of a common set of 257 Conserved Ortholog Set II genes in the genomes of both species. The mapped markers are well distributed across both genomes allowing the first syntenic comparison between species from these two families. The majority (75%) of the synteny blocks are short (

P>Vibrio splendidus is a dominant Vibrio species in seawater presenting a remarkable genetic diversity; several strains have been linked to invertebrate's mortality. We report the complete genome sequence of V. splendidus LGP32, an oyster pathogen, and its comparison with partial genome sequences from related strains. As is typical for the genus, V. splendidus LGP32 contains two chromosomes (3.29 and 1.67 Mb) and most essential cellular processes are encoded by chromosome 1. Comparison with two other V. splendidus partial genome sequences (strains 12B01 and Med222) confirms the previously suggested high genotypic diversity within this species and led to the identification of numerous strain-specific regions that could frequently not be assigned to a specific mechanisms of recombination. Surprisingly, the chromosomal integron, the most variable genetic element in all other Vibrio species analysed to date, is absent from 12B01 and inactivated by a mobile element in Med222, while in LGP32 it only contains a limited number of cassettes. Finally, we found that the LGP32 integron contains a new dfrA cassette, related to those found in resistance integrons of Gram-negative clinical isolates. Those results suggest that marine Vibrio can be a source of antibiotic resistance genes.