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SummarySpecialized or secondary metabolites (SMs) play a key role in plant resistance against abiotic stresses and defences against bioaggressors. For example, in sessile oaksQuercus petraea, phenolics contribute to reduce herbivore damage and improve drought resistance. Here, we explored the natural variation of SMs in nine European provenances of sessile oaks and aimed to detect its underlying genetic bases.We sampled mature leaves from high and low branches on 225 sessile oak trees located in a common garden and used untargeted metabolomics to characterise the variation of 219 specialized metabolites. In addition, we used whole genome low-depth sequencing to genotype individuals for 1.4M genetic markers. We then performed genome-wide association analyses to identify the genetic bases underlying the variation of leaf SMs.We found that leaf SMs displayed extensive within-provenance variation, but very little differentiation between provenances. For ∼10% of the metabolites we detected, most of this variation could be explained by a single genetic marker.Our results suggest that genetic variation for most leaf SMs is unlikely to be locally adaptive, and that selective pressures may act locally to maintain diversity at loci associated with leaf SM variation within oak populations.

Whole genome characterizations of crop plants based on ancient DNA have provided unique keys for a better understanding of the evolutionary origins of modern cultivars, the pace and mode of selection underlying their adaptation to new environments and the production of phenotypes of interest. Although forests are among the most biologically rich ecosystems on earth and represent a fundamental resource for human societies, no ancient genome sequences have been generated for trees. This contrasts with the generation of multiple ancient reference genomes for important crops. Here, we sequenced the first ancient tree genomes using two white oak wood remains from Germany dating to the Last Little Ice Age (15th century CE, 7.3× and 4.0×) and one from France dating to the Bronze Age (1700 BCE, 3.4×). We assessed the underlying species and identified one medieval remains as a hybrid between two common oak species (Quercus robur and Q. petraea) and the other two remains as Q. robur. We found that diversity at the global genome level had not changed over time. However, exploratory analyses suggested that a reduction of diversity took place at different time periods. Finally, we determined the timing of leaf unfolding for ancient trees for the first time. The study extends the application of ancient wood beyond the classical proxies of dendroclimatology, dendrochronology, dendroarchaeology and dendroecology, thereby enhancing resolution of inferences on the responses of forest ecosystems to past environmental changes, epidemics and silvicultural practices.

[Motivation] Trait variation within species can reveal plastic and/or genetic responses to environmental gradients, and may indicate where local adaptation has occurred. Here, we present a dataset of rangewide variation in leaf traits from seven of the most ecologically and economically important tree species in Europe. Sample collection and trait assessment are embedded in the GenTree project (EU-Horizon 2020), which aims at characterizing the genetic and phenotypic variability of forest tree species to optimize the management and sustainable use of forest genetic resources. Our dataset captures substantial intra- and interspecific leaf phenotypic variability, and provides valuable information for studying the relationship between ecosystem functioning and trait variability of individuals, and the response and resilience of species to environmental changes., [Main types of variable contained] We chose morphological and chemical characters linked to trade-offs between acquisition and conservation of resources and water use, namely specific leaf area, leaf size, carbon and nitrogen content and their ratio, and the isotopic signature of stable isotope 13C and 15N in leaves., [Spatial location and grain] We surveyed between 18 and 22 populations per species, 141 in total, across Europe., [Time period] Leaf sampling took place between 2016 and 2017., [Major taxa and level of measurement] We sampled at least 25 individuals in each population, 3,569 trees in total, and measured traits in 35,755 leaves from seven European tree species, i.e. the conifers Picea abies, Pinus pinaster and Pinus sylvestris, and the broadleaves Betula pendula, Fagus sylvatica, Populus nigra and Quercus petraea., [Software format] The data files are in ASCII text, tab delimited, not compressed., All authors acknowledge support from the European Union’s Horizon 2020 Research and Innovation Programme under grant agreement no. 676876 (Project GenTree). Laboratory work was also supported by other projects: COMEDIAS (CGL2017- 83170-R, Spanish Ministry of Science, Innovation and Universities), REMEDINAL IV (TE-CM S2018/EMT-4338, Project of the Autonomous Community of Madrid, Spain), SPONFOREST (BIODIVERSA-3, PCIN-2016-055), and International Laboratory of Global Change (LINCGlobal 4540-143AP). The Swiss contribution was supported by the Swiss Secretariat for Education, Research and Innovation (SERI) under contract no. 6.0032. BC was funded by a Scholarship from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES (Brazil).

Drought and waterlogging impede tree growth and may even lead to tree death. Oaks, an emblematic group of tree species, have evolved a range of adaptations to cope with these constraints. The two most widely distributed European species, pedunculate (PO; Quercus robur L.) and sessile oak (SO; Quercus petraea Matt. Lieb), have overlapping ranges, but their respective distribution are highly constrained by local soil conditions. These contrasting ecological preferences between two closely related and frequently hybridizing species constitute a powerful model to explore the functional bases of the adaptive responses in oak. We exposed oak seedlings to waterlogging and drought, conditions typically encountered by the two species in their respective habitats, and studied changes in gene expression in roots using RNA-seq. We identified genes that change in expression between treatments differentially depending on species. These “species × environment”-responsive genes revealed adaptive molecular strategies involving adventitious and lateral root formation, aerenchyma formation in PO, and osmoregulation and ABA regulation in SO. With this experimental design, we also identified genes with different expression between species independently of water conditions imposed. Surprisingly, this category included genes with functions consistent with a role in intrinsic reproductive barriers. Finally, we compared our findings with those for a genome scan of species divergence and found that the expressional candidate genes included numerous highly differentiated genetic markers between the two species. By combining transcriptomic analysis, gene annotation, pathway analyses, as well as genome scan for genetic differentiation among species, we were able to highlight loci likely involved in adaptation of the two species to their respective ecological niches.

Research conductedWith the ongoing global warming, there are serious concerns about the persistence of locally adapted populations. Indeed, with the raising of temperature, the phenological cycle of tree species may be strongly affected since higher winter temperatures may have a negative impact on endodormancy release if chilling requirements are not fulfilled during winter and late frost in spring may expose trees if buds flush too early. Thus, Environmental gradients (showing continuous variations of environmental conditions) constitute a design of choice to analyze the effect of winter dormancy in locally adapted population.MethodsIn the present study, we used an elevation gradient in the Pyrenees to explore the gene expression network involved in dormancy regulation in natural populations of sessile oak locally adapted to temperature. Terminal buds were harvested during dormancy induction and release at different elevations. Then, gene expression was quantified using RNAseq and we used a likelihood ratio test to identify genes displaying significant dormancy, elevation or dormancy-by-elevation interaction effects.Key resultsOur results highlight molecular processes in locally adapted populations along this elevation cline, and made it possible to identify key dormancy-by-elevation responsive genes revealing that locally adapted populations have evolved distinct molecular strategies to adapt their bud phenology in response to environmental variation (i.e. temperature).

The pace of tree microevolution during Anthropocene warming is largely unknown. We used a retrospective approach to monitor genomic changes in oak trees since the Little Ice Age (LIA). Allelic frequency changes were assessed from whole-genome pooled sequences for four age-structured cohorts of sessile oak (Quercus petraea) dating back to 1680, in each of three different oak forests in France. The genetic covariances of allelic frequency changes increased between successive time periods, highlighting genome-wide effects of linked selection. We found imprints of convergent linked selection in the three forests during the late LIA, and a shift of selection during more recent time periods. The changes in allelic covariances within and between forests mirrored the documented changes in the occurrence of extreme events (droughts and frosts) over the last three hundred years. The genomic regions with the highest covariances were enriched in genes involved in plant responses to pathogens and abiotic stresses (temperature and drought). These responses are consistent with the reported sequence of frost (or drought) and disease damage ultimately leading to the oak dieback after extreme events. Our results therefore provide evidence of selection operating on long-lived species during recent climatic changes.

Oaks are dominant forest tree species widely distributed across the northern hemisphere, where they constitute natural resources of economic, ecological, social and historical value. Hybridization and adaptive introgression have long been thought to be major evolutionary drivers of the ecological success of oaks. However, the maintenance of species barriers remains a key question, given the extent of interspecific gene flow. We used a demographically-explicit genome scan approach combining approximate Bayesian computation and backward simulations to scan the genomes of four European white oak species for reproductive barriers. We first confirmed earlier findings of interspecific contact since the last glacial maximum. We then made use of the tremendous genetic variation among these four species (31 million SNPs) to identify genomic regions enriched in SNPs deviating from demographic expectations under a hypothesis of recent secondary contact. The outlier-enriched regions detected were of restricted size and widely distributed, over all the chromosomes. We identified 215 regions, containing 227 oak genes. A Literature-based functional annotation of these genes highlighted important functions driving the reproductive isolation of the four species. These functions included tolerance to biotic and abiotic constraints, and intrinsic mating barriers. The functions of the annotated genes were highly consistent with the ecological preferences of the four species. These genes may, therefore, be considered candidate genes in studies of species responses to past and ongoing environmental changes.

The European Beech is the dominant climax tree in most regions of Central Europe and valued for its ecological versatility and hardwood timber. Even though a draft genome has been published recently, higher resolution is required for studying aspects of genome architecture and recombination. Here, we present a chromosome-level assembly of the more than 300 year-old reference individual, Bhaga, from the Kellerwald-Edersee National Park (Germany). Its nuclear genome of 541 Mb was resolved into 12 chromosomes varying in length between 28 and 73 Mb. Multiple nuclear insertions of parts of the chloroplast genome were observed, with one region on chromosome 11 spanning more than 2 Mb which fragments up to 54,784 bp long and covering the whole chloroplast genome were inserted randomly. Unlike in

The European Beech is the dominant climax tree in most regions of Central Europe and valued for its ecological versatility and hardwood timber. Even though a draft genome has been published recently, higher resolution is required for studying aspects of genome architecture and recombination. Here we present a chromosome-level assembly of the more than 300 year-old reference individual, Bhaga, from the Kellerwald-Edersee National Park (Germany). Its nuclear genome of 541 Mb was resolved into 12 chromosomes varying in length between 28 Mb and 73 Mb. Multiple nuclear insertions of parts of the chloroplast genome were observed, with one region on chromosome 11 spanning more than 2 Mb of the genome in which fragments up to 54,784 bp long and covering the whole chloroplast genome were inserted randomly. Unlike in Arabidopsis thaliana, ribosomal cistrons are present in Fagus sylvatica only in four major regions, in line with FISH studies. On most assembled chromosomes, telomeric repeats were found at both ends, while centromeric repeats were found to be scattered throughout the genome apart from their main occurrence per chromosome. The genome- wide distribution of SNPs was evaluated using a second individual from Jamy Nature Reserve (Poland). SNPs, repeat elements and duplicated genes were unevenly distributed in the genomes, with one major anomaly on chromosome 4. The genome presented here adds to the available highly resolved plant genomes and we hope it will serve as a valuable basis for future research on genome architecture and for understanding the past and future of European Beech populations in a changing climate.

Latitudinal and elevational gradients provide valuable experimental settings for studies of the potential impact of global warming on forest tree species. The availability of long-term phenological surveys in common garden experiments for traits associated with climate, such as bud flushing, for sessile oaks (Quercus petraea), provides an ideal opportunity to investigate this impact.We sequenced 18 sessile oak populations, and used available sequencing data for three other closely related European white oak species (Q. pyrenaica, Q. pubescens, Q. robur), to explore the evolutionary processes responsible for shaping the genetic variation across latitudinal and elevational gradients in extant sessile oaks. We used phenotypic surveys in common garden experiments and climatic data for the population of origin, to perform genome-wide scans for population differentiation, genotype-environment (GEA) and genotype-phenotype associations (GPA).The inferred historical relationships betweenQ. petraeapopulations suggest that interspecific gene flow occurred betweenQ. roburandQ. petraeapopulations from cooler or wetter areas. A genome-wide scan of differentiation betweenQ. petraeapopulations identified SNPs displaying strong interspecific relative divergence between these two species. These SNPs followed genetic clines along climatic or phenotypic gradients, providing further support for the likely contribution of introgression to the adaptive divergence ofQ. petraeapopulations.Overall, the results of this study indicate that adaptive introgression ofQ. roburalleles has occurred. We discuss the results of this study in the framework of the post-glacial colonization scenario, in which introgression and diversifying selection have been proposed as essential drivers ofQ. petraeamicroevolution.

Changes in leaf soluble proteome were explored in 3-month-old plants of metallicolous (M) and nonmetallicolous (NM) Agrostis capillaris L. populations exposed to increasing Cu concentrations (1-50 μM) to investigate molecular mechanisms underlying plant responses to Cu excess and tolerance of M plants. Plants were cultivated on perlite (CuSO4 spiked-nutrient solution). Soluble proteins, extracted by the trichloroacetic acid/acetone procedure, were separated with 2-DE (linear 4-7 pH gradient). Analysis of CCB-stained gels (PDQuest) reproducibly detected 214 spots, and 64 proteins differentially expressed were identified using LC-MS/MS. In both populations, Cu excess impacted both light-dependent (OEE, cytochrome b6-f complex, and chlorophyll a-b binding protein), and -independent (RuBisCO) photosynthesis reactions, more intensively in NM leaves (ferredoxin-NADP reductase and metalloprotease FTSH2). In both populations, upregulation of isocitrate dehydrogenase and cysteine/methionine synthases respectively suggested increased isocitrate oxidation and enhanced need for S-containing amino-acids, likely for chelation and detoxification. In NM leaves, an increasing need for energetic compounds was indicated by the stimulation of ATPases, glycolysis, pentose phosphate pathway, and Calvin cycle enzymes; impacts on protein metabolism and oxidative stress increase were respectively suggested by the rise of chaperones and redox enzymes. Overexpression of a HSP70 may be pivotal for M Cu tolerance by protecting protein metabolism. All MS data have been deposited in the ProteomeXchange with the dataset identifier PXD001930 (http//proteomecentral.proteomexchange.org/dataset/PXD001930).

International audience; Secondary xylem (wood) is formed through an intricate biological process that results in a highly variable final product. Studies have focused on understanding the molecular events for wood formation in conifers. In this process environmental, ontogenic and genetic factors influence variation in wood characteristics, including anatomical, chemical and physical properties. The main objective of this study was to analyse the ageing (ontogenic) effect on protein accumulation in wood-forming tissues along a cambial age (CA) gradient, ranging from juvenile wood (JW) sampled at the top of the tree, to mature wood (MW) sampled at the bottom of the tree. A total of 62 proteins whose accumulation varied by at least 1.5-fold according to CA were selected and identified by ESI-MS/MS; 30 of these were more abundant in MW and 32 were more abundant in JW. Consistent with earlier findings, our results show that JW is a tissue characterized by a high energy demand with the accumulation of gene products involved in energy, protein fate and cellular transport, while proteins identified in MW (heat shock response, oxygen and radical detoxification, and the S-adenosyl methionine cycle) support the idea that this tissue undergoes extended cell-wall thickening and a delay of programmed cell death.

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In species with high gene flow and consequent low interpopulation differentiation over wide geographic ranges, differential gene expression along ecological gradients often reveals adaptive significance. We investigated potential differences in protein expression between Picea abies ecotypes adapted to contrasting altitude conditions. Protein expression patterns were compared between needles and roots of 2-month-old P. abies seedlings by means of 2-dimensional electrophoresis. Proteins exhibiting differential expression between the 2 ecotypes were analyzed by tandem mass spectrometry. A total of 19 proteins exhibited qualitative or quantitative polymorphism between the 2 populations. These proteins exhibited organ-specific expression, and the level of interpopulation protein polymorphism was organ dependent. Among differentially expressed proteins, we identified proteins involved in photosynthesis, photorespiration, root tracheary element differentiation, and transmitochondrial membrane transport. Our results show that P. abies seedlings from locally adapted ecotypes exhibit consistent differences in protein expression. The expression polymorphism of some of these proteins has potential adaptive significance.

International audience; The 1.5 Gbp/2C genome of pedunculate oak (Quercus robur) has been sequenced. A strategy was established for dealing with the challenges imposed by the sequencing of such a large, complex and highly heterozygous genome by a whole-genome shotgun (WGS) approach, without the use of costly and time-consuming methods, such as fosmid or BAC clone-based hierarchical sequencing methods. The sequencing strategy combined short and long reads. Over 49 million reads provided by Roche 454 GS-FLX technology were assembled into contigs and combined with shorter Illumina sequence reads from paired-end and mate-pair libraries of different insert sizes, to build scaffolds. Errors were corrected and gaps filled with Illumina paired-end reads and contaminants detected, resulting in a total of 17,910 scaffolds (>2 kb) corresponding to 1.34 Gb. Fifty per cent of the assembly was accounted for by 1468 scaffolds (N50 of 260 kb). Initial comparison with the phylogenetically related Prunus persica gene model indicated that genes for 84.6% of the proteins present in peach (mean protein coverage of 90.5%) were present in our assembly. The second and third steps in this project are genome annotation and the assignment of scaffolds to the oak genetic linkage map. In accordance with the Bermuda and Fort Lauderdale agreements and the more recent Toronto Statement, the oak genome data have been released into public sequence repositories in advance of publication. In this presubmission paper, the oak genome consortium describes its principal lines of work and future directions for analyses of the nature, function and evolution of the oak genome.

Background Many northern-hemisphere forests are dominated by oaks. These species extend over diverse environmental conditions and are thus interesting models for studies of plant adaptation and speciation. The genomic toolbox is an important asset for exploring the functional variation associated with natural selection. Results The assembly of previously available and newly developed long and short sequence reads for two sympatric oak species, Quercus robur and Quercus petraea, generated a comprehensive catalog of transcripts for oak. The functional annotation of 91 k contigs demonstrated the presence of a large proportion of plant genes in this unigene set. Comparisons with SwissProt accessions and five plant gene models revealed orthologous relationships, making it possible to decipher the evolution of the oak genome. In particular, it was possible to align 9.5 thousand oak coding sequences with the equivalent sequences on peach chromosomes. Finally, RNA-seq data shed new light on the gene networks underlying vegetative bud dormancy release, a key stage in development allowing plants to adapt their phenology to the environment. Conclusion In addition to providing a vast array of expressed genes, this study generated essential information about oak genome evolution and the regulation of genes associated with vegetative bud phenology, an important adaptive traits in trees. This resource contributes to the annotation of the oak genome sequence and will provide support for forward genetics approaches aiming to link genotypes with adaptive phenotypes. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1331-9) contains supplementary material, which is available to authorized users.

Genetic markers developed from expressed sequence tags (ESTs) were used as orthologous loci for comparative genome studies in the genus Pinus. A total of 309 ESTs derived from conifer gene sequences were tested for amplification and polymorphism in maritime pine (Pinus pinaster Ait.). Electrophoresis-based techniques made it possible to map 50 expressed sequence tag polymorphisms (ESTPs). The map positions of 32 markers were compared to putative orthologous loci on the loblolly pine (Pinus taeda L.) linkage map, which is the reference map of the conifer genetic mapping community. Overall, synteny was maintained between the two species. This report agrees with other pairwise genome comparisons in pine and supports the cytogenetic evidence that chromosome evolution in the genus is conservative. The alignment of homologous linkage groups allowed, for the first time in conifers, the comparison of QTL location. The position of two QTLs controlling wood density and cell wall components were found to be conserved between the two species.

International audience; We constructed a high-density linkage map of maritime pine (Pinus pinaster Ait.) based on AFLP (Amplified Fragment Length Polymorphism) markers using a three-generation outbred pedigree. In a first step, male and female maps were established independently with test-cross markers segregating 1:1 (presence:absence of the amplified fragment in the full-sib progeny). In a second step, both maps were merged using intercross markers segregating 3:1 in the progeny. A combination of MAPMAKER and JOINMAP softwares was used for the mapping process. A consensus map was obtained and is available at URL http://www.pierroton.inra.fr/genetics/pinus/Map3/index.html. It covers 1441 cM and comprises a total of 620 AFLP markers on 12 linkage groups. The physical size of the maritime pine genome (51.5 pg/2C) was measured by flow cytometry, providing a physical/genetic size ratio of 13.78 Mb/cM. This map will be used to dissect the genetic architecture of economically (growth, wood quality) and ecologically (water-use efficiency) important traits into mendelian inherited components (QTLs: Quantitative Trait Loci). It will also provide a framework to localize more informative markers (ESTs: Expressed Sequence Tags) to be used as candidate genes in QTL detection experiments. The location of orthologous markers (ESTs and SSRs: Simple Sequence Repeats) will also allow the study of the genome structure of closely related conifer species using a comparative genome mapping approach.; Établissement d'une carte génétique à haute densité du pin maritime à partir de marqueurs AFLP. Nous avons construit une carte génétique du pin maritime (Pinus pinaster Ait.) en génotypant une famille de plein-frères appartenant à la troisième génération du programme d'amélioration, avec des marqueurs AFLP. Dans un premier temps, les cartes des parents mâle et femelle ont été établies indépendamment avec des marqueurs de type " test-cross " ségréguant dans les proportions 1:1 (présence:absence du fragment amplifié dans la famille de plein-frères). Dans un second temps ces deux cartes ont été fusionnées à l'aide de marqueurs de type " intercross ", ségréguant dans les proportions 3:1. La construction des cartes a été réalisée à l'aide des logiciels de cartographie génétique JOINMAP et MAPMAKER. Une carte génétique consensus des deux parents comprenant 12 groupes de liaison a finalement été obtenue et est accessible à l'URL suivante : http://www.pierroton.inra.fr/genetics/pinus/Map3/index.html. Elle couvre 1441 cM et comprend 620 marqueurs. Par ailleurs, la taille physique du génome du pin maritime a été estimée par cytométrie de flux à 51.5 pg/2C, donnant un rapport taille physique/taille génétique de 13.78 Mb/cM. Cette carte sera maintenant utilisée pour étudier l'architecture génétique de caractères d'intérêt économique (croissance, qualité du bois) et écologique (efficience d'utilisation de l'eau). Il s'agira de localiser les zones du génome (QTL, Quantitative Trait Loci) impliquées dans le contrôle génétique de ces caractères complexes. La carte génétique fournira aussi un support pour localiser d'autres types de marqueurs, tels que des gènes (EST, Expressed Sequence Tags) qui seront utilisés comme marqueurs candidats pouvant correspondre aux QTL. La localisation de marqueurs orthologues (EST et SSR, Simple Sequence Repeats) permettra d'étudier en outre la structure du génome des conifères en utilisant une approche par cartographie comparée.

International audience; Systematic sequencing is the method of choice for generating genomic resources for molecular marker development and candidate gene identification in non-model species. We generated 47,357 Sanger ESTs and 2.2M Roche-454 reads from five cDNA libraries for European beech (Fagus sylvatica L.). This tree species of high ecological and economic value in Europe is among the most representative trees of deciduous broadleaf forests. The sequences generated were assembled into 21,057 contigs with MIRA software. Functional annotations were obtained for 85% of these contigs, from the proteomes of four plant species, Swissprot accessions and the Gene Ontology database. We were able to identify 28,079 in silico SNPs for future marker development. Moreover, RNAseq and qPCR approaches identified genes and gene networks regulated differentially between two critical phenological stages preceding vegetative bud burst (the quiescent and swelling buds stages). According to climatic model based projection, some European beech populations may be endangered, particularly at the southern and eastern edges of the European distribution range, which are strongly affected by current climate change. This first genomic resource for the genus Fagus should facilitate the identification of key genes for beech adaptation and management strategies for preserving beech adaptability.

International audience; Differential expression of soluble proteins was explored in roots of metallicolous (M) and non-M (NM) plants of Agrostis capillaris L. exposed to increasing Cu to partially identify molecular mechanisms underlying higher Cu tolerance in M plants. Plants were cultivated for 2 months on perlite with a CuSO4 (1–30 μM) spiked-nutrient solution. Soluble proteins extracted by the trichloroacetic acid/acetone procedure were separated with 2DE (linear 4–7 pH gradient). After Coomassie Blue staining and image analysis, 19 proteins differentially expressed were identified using LC-MS/MS and Expressed Sequence Tag (ESTs) databases. At supra-optimal Cu exposure (15–30 μM), glycolysis was likely altered in NM roots with increased production of glycerone-P and methylglyoxal based on overexpression of triosephosphate isomerase and fructose bisphosphate aldolase. Changes in tubulins and higher expressions of 5-methyltetrahydropteroyltriglutamatehomocysteine methyltransferase and S-adenosylmethionine synthase underpinned impacts on the cytoskeleton and stimulation of ethylene metabolism. Increased l-methionine and S-adenosylmethionine amounts may also facilitate production of nicotianamine, which complexes Cu, and of l-cysteine, needed for metallothioneins and GSH. In M roots, the increase of [Cu/Zn] superoxide dismutase suggested a better detoxification of superoxide, when Cu exposure rose. Higher Cu-tolerance of M plants would rather result from simultaneous cooperation of various processes than from a specific mechanism.

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Article fédérateur entre toutes les équipes Européennes travaillant sur le pin maritime. Assemblage du transcriptome des données venant de France (INRA Orléans + INRA Pierroton) + Espagne + Portugal = projet Sustainpine; International audience; Maritime pine (Pinus pinasterAit.) is a widely distributed conifer species in Southwestern Europe and one of the most advanced models for conifer research. In the current work, comprehensive characterization of the maritime pine transcriptome was performed using a combination of two different next-generation sequencing platforms, 454 and Illumina. De novo assembly of the transcriptome provided a catalogue of 26 020 unique transcripts in maritime pine trees and a collection of 9641 full-length cDNAs. Quality of the transcriptome assembly was validated by RT-PCR amplification of selected transcripts for structural and regulatory genes. Transcription factors and enzyme-encoding transcripts were annotated. Furthermore, the available sequencing data permitted the identification of polymorphisms and the establishment of robust single nucleotide polymorphism (SNP) and simple-sequence repeat (SSR) databases for genotyping applications and integration of translational genomics in maritime pine breeding programmes. All our data are freely available at SustainpineDB, the P. pinaster expressional database. Results reported here on the maritime pine transcriptome represent a valuable resource for future basic and applied studies on this ecological and economically important pine species.

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Background The availability of a large expressed sequence tags (EST) resource and recent advances in high-throughput genotyping technology have made it possible to develop highly multiplexed SNP arrays for multi-objective genetic applications, including the construction of meiotic maps. Such approaches are particularly useful in species with a large genome size, precluding the use of whole-genome shotgun assembly with current technologies. Results In this study, a 12 k-SNP genotyping array was developed for maritime pine from an extensive EST resource assembled into a unigene set. The offspring of three-generation outbred and inbred mapping pedigrees were then genotyped. The inbred pedigree consisted of a classical F2 population resulting from the selfing of a single inter-provenance (Landes x Corsica) hybrid tree, whereas the outbred pedigree (G2) resulted from a controlled cross of two intra-provenance (Landes x Landes) hybrid trees. This resulted in the generation of three linkage maps based on SNP markers: one from the parental genotype of the F2 population (1,131 markers in 1,708 centimorgan (cM)), and one for each parent of the G2 population (1,015 and 1,110 markers in 1,447 and 1,425 cM for the female and male parents, respectively). A comparison of segregation patterns in the progeny obtained from the two types of mating (inbreeding and outbreeding) led to the identification of a chromosomal region carrying an embryo viability locus with a semi-lethal allele. Following selfing and segregation, zygote mortality resulted in a deficit of Corsican homozygous genotypes in the F2 population. This dataset was also used to study the extent and distribution of meiotic recombination along the length of the chromosomes and the effect of sex and/or genetic background on recombination. The genetic background of trees in which meiotic recombination occurred was found to have a significant effect on the frequency of recombination. Furthermore, only a small proportion of the recombination hot- and cold-spots were common to all three genotypes, suggesting that the spatial pattern of recombination was genetically variable. Conclusion This study led to the development of classical genomic tools for this ecologically and economically important species. It also identified a chromosomal region bearing a semi-lethal recessive allele and demonstrated the genetic variability of recombination rate over the genome.

Waterlogging causes stressful conditions for perennial species. The temporary overabundance of water in waterlogged soil can induce hypoxia in the rhizosphere, leading to root death, tree decline and even dieback. Two closely related members of the European white oak complex, pedunculate (Quercus robur L.) and sessile (Quercus petraea Matt. Liebl.) oaks, have different ecological characteristics, especially regarding their adaptation to soil waterlogging. The tolerance of waterlogging observed in pedunculate oak is driven principally by its ability to produce adaptive structures, hypertrophied lenticels and adventitious roots, and to switch rapidly its metabolism to the fermentative pathway. This study had two objectives: (i) to identify genes important for adaptation to waterlogging and (ii) to gain insight into the molecular mechanisms involved in hypertrophied lenticel formation in pedunculate oak. We subjected seedlings of the two species to hypoxia by maintaining the water level 2 cm above the collar. The immersed part of the stem (i.e., containing hypertrophied lenticels in pedunculate oak) was sampled after 9 days of waterlogging stress and its gene expression was investigated by RNA-seq. Genes displaying differential expression between the two species were identified with the DESeq R package and a false discovery rate of 0.001. We found that 3705 contigs were differentially regulated between the two species. Twenty-two differentially expressed genes were validated by real-time quantitative polymerase chain reaction. The suberin biosynthesis pathway was found to be upregulated in pedunculate oak, consistent with molecular mechanisms analogous to those operating in the radial oxygen loss barrier in waterlogging-tolerant species.

Résumé de la présentation oraleRésumé de la présentation orale; absent

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Background Plants are subjected to continuous stimuli from the environment and have evolved an ability to respond through various growth and development processes. Phototropism and gravitropism responses enable the plant to reorient with regard to light and gravity. Results We quantified the speed of maritime pine seedlings to reorient with regard to light and gravity over 22 days. Seedlings were inclined at 15, 30 and 45 degrees with vertical plants as controls. A lateral light source illuminated the plants and stem movement over time was recorded. Depending on the initial angle of stem lean, the apical response to the lateral light source differed. In control and 15° inclined plants, the apex turned directly towards the light source after only 2 h. In plants inclined at 30° and 45°, the apex first reoriented in the vertical plane after 2 h, then turned towards the light source after 24 h. Two-dimensional gel electrophoresis coupled with mass spectrometry was then used to describe the molecular response of stem bending involved in photo- and gravi-tropism after 22 hr and 8 days of treatment. A total of 486 spots were quantitatively analyzed using image analysis software. Significant changes were determined in the protein accumulation of 68 protein spots. Early response gravitropic associated proteins were identified, which are known to function in energy related and primary metabolism. A group of thirty eight proteins were found to be involved in primary metabolism and energy related metabolic pathways. Degradation of Rubisco was implicated in some protein shifts. Conclusions Our study demonstrates a rapid gravitropic response in apices of maritime pine seedlings inclined >30°. Little or no response was observed at the stem bases of the same plants. The primary gravitropic response is concomitant with a modification of the proteome, consisting of an over accumulation of energy and metabolism associated proteins, which may allow the stem to reorient rapidly after bending.

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International audience; Although tree species typically exhibit low genetic differentiation between populations, ecotypes adapted to different environmental conditions can vary in their capacity to withstand and recover from environmental stresses like heat stress. Two month old seedlings of a Picea abies ecotype adapted to high elevation showed lower level of thermotolerance and higher level of tolerance to oxidative stress relative to a low elevation ecotype. Protein expression patterns following exposure to severe heat stress of the two ecotypes were compared by means of 2-DE. Several proteins exhibiting ecotype and tissue specific expression were identified by MS/MS. Among them, small heat shock proteins of the HSP 20 family and proteins involved in protection from oxidative stress displayed qualitative and quantitative differences in expression between the ecotypes correlated with the observed phenotypic differences. On the basis of these results, it can be speculated that the observed interpopulation polymorphism of protein regulation in response to heat stress could underlie their different capacities to withstand and recover from heat stress. These local adaptations are potentially relevant for the species adaptation to the conditions predicted by the current models for climate change.

In this chapter we present a protocol for total protein extraction optimized for wood-forming tissue (differentiating secondary xylem). The protocol is then used for a series of other organs (root, leaf, pollen, bud, flower, cambium, and phloem) in broadleaf (oak and poplar) and conifer (pine) species. Proteins are first extracted from tissue powdered in liquid nitrogen using the TCA-acetone method and then solubilized in an optimized buffer. The resulting 2D gels can be viewed at http://cbi.labri.fr/outils/protic/index.php.

International audience; Poplar is the first forest tree genome to be decoded. As an initial step to the comprehensive analysis of poplar proteome, we described reference 2-D-maps for eight tissues/organs of the plant, and the functional characterization of some proteins. A total of 398 proteins were excised from the gels. About 91.2% were identified by nanospray LC-MS/MS, based on comparison with 260 000 Populus sp. ESTs. In comparison, reliable PMFs were obtained for only 51% of the spots by MALDI-TOF-MS, from which 43% (83 spots) positively matched gene models of the Populus trichocarpa genome sequence. Among these 83 spots, 58% matched with the same proteins as identified by LC-MS/MS, 21.7% with unknown function proteins and 19.3% with completely different functions. In the second phase, we studied the effect of drought stress on poplar root and leaf proteomes. The function of up- and down-regulated proteins is discussed with respect to the physiological response of the plants and compared with transcriptomic data. Some important clues regarding the way poplar copes with water deficit were revealed.

PROTICdb is a web-based application, mainly designed to store and analyze plant proteome data obtained by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and mass spectrometry (MS). The purposes of PROTICdb are (i) to store, track, and query information related to proteomic experiments, i.e., from tissue sampling to protein identification and quantitative measurements, and (ii) to integrate information from the user's own expertise and other sources into a knowledge base, used to support data interpretation (e.g., for the determination of allelic variants or products of post-translational modifications). Data insertion into the relational database of PROTICdb is achieved either by uploading outputs of image analysis and MS identification software, or by filling web forms. 2-D PAGE annotated maps can be displayed, queried, and compared through a graphical interface. Links to external databases are also available. Quantitative data can be easily exported in a tabulated format for statistical analyses. PROTICdb is based on the Oracle or the PostgreSQL Database Management System and is freely available upon request at the following URL: http://moulon.inra.fr/ bioinfo/PROTICdb.

International audience; Wood is one of our most important natural resources. Surprisingly, we know hardly anything about the details of the process of wood formation. The aim of this work was to describe the main proteins expressed in wood forming tissue of a conifer species (Pinus pinaster Ait.). Using high resolution 2-DE with linear pH gradient ranging from 4 to 7, a total of 1039 spots were detected. Out of the 240 spots analyzed by MS/MS, 67.9% were identified, 16.7% presented no homology in the databases, and 15.4% corresponded to protein mixtures. Out of the 57 spots analyzed by MALDI-MS, only 15.8% were identified. Most of the 175 identified proteins play a role in either defense (19.4%), carbohydrates (16.6%) and amino acid (14.9%) metabolisms, genes and proteins expression (13.1%), cytoskeleton (8%), cell wall biosynthesis (5.7%), secondary (5.1%) and primary (4%) metabolisms. A summary of the identified proteins, their putative functions, and behavior in different types of wood are presented. This information was introduced into the PROTICdb database and is accessible at http://cbib1.cbib.u-bordeaux2.fr/Protic/Protic/home/index.php. Finally, the average protein amount was compared with their respective transcript abundance as quantified through EST counting in a cDNA-library constructed with mRNA extracted from wood forming tissue

International audience; Hepatocellular carcinoma (HCC) is a major complication of chronic viral hepatitis C. Therapy for HCC is still disappointing. It is thus of great importance to identify novel HCC markers for early detection of the disease, and tumor-specific proteins as potential therapeutic targets. We have used a proteomic approach to identify new proteins involved in HCC development. Four cases of HCC developing from chronic viral hepatitis C were analyzed by two-dimensional electrophoresis (2-DE), and results were compared to those of paired adjacent non-tumorous liver tissues. For MS fingerprinting, protein spots with differential intensity between HCC and non-tumorous liver were directly cut out of gels and processed for MALDI-MS and nano-LC-ESI-MS/MS analysis. Approximately 850 spots were visualized in each gel. The comparative analysis of paired samples indicated that 345 protein spots showed significant differences in expression level between non-tumor and tumor tissue. Among the 345 protein spots analyzed, 238 spots corresponding to 155 different proteins were identified; 49 proteins were up-regulated, whereas 106 proteins were down-regulated. Among these 155 proteins, 91 proteins were regulated in at least three cases. Although 52 out of these 91 proteins have been already described by previous proteomic or transcriptomic studies, or are already known to be involved in hepatocarcinogenesis, this experiment revealed 39 new proteins differentially expressed in HCC developing from viral hepatitis C. Variations in protein accumulation were confirmed for two selected proteins (apolipoprotein E, chloride intracellular channel 1) by Western blotting in ten additional cases of HCC developing in patients with viral hepatitis C

In order to analyze the large-scale structure of the genome of Norway spruce (Picea abies Karst.), a pseudo-testcross genetic linkage map was built using markers of six different types, belonging to the low (amplified fragment length polymorphisms, simple sequence repeats) or high (sequence-specific amplified polymorphisms, inter-retrotransposon amplified polymorphisms) copy-number fraction of the genome, and including expressed region-derived markers (expressed sequence tag polymorphisms). Twenty seven and 23 linkage groups of at least four markers were obtained for the female and the male parent maps, respectively. A subset of these linkage groups coalesced into 13 bi-parental linkage groups through markers shared between the two maps. This map was used to investigate the frequency of each marker type over chromosomes and the distribution of marker types relative to each other, using autocorrelation techniques. Our results show that, while the composition of chromosomes is homogeneous, low- and high-copy-number markers tend to occupy separate regions of the linkage groups, and that expressed sequences are preferentially associated with microsatellites and separated from retrotransposons. These results indicate that the spatial structure of Norway spruce chromosomes is not homogeneous.

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International audience; Background: The cuticle is a hydrophobic barrier located at the aerial surface of all terrestrial plants. Recent studies performed on model plants, such as Arabidopsis thaliana, have suggested that the cuticle may be involved in drought stress adaptation, preventing non-stomatal water loss. Although forest trees will face more intense drought stresses (in duration and intensity) with global warming, very few studies on the role of the cuticle in drought stress adaptation in these long-lived organisms have been so far reported. Results: This aspect was investigated in a conifer, maritime pine (Pinus pinaster Ait.), in a factorial design with two genetic units (two half-sib families with different growth rates) and two treatments (irrigated vs non-irrigated), in field conditions. Saplings were grown in an open-sided greenhouse and half were irrigated three times per week for two growing seasons. Needles were sampled three times per year for cuticular wax (composition and content) and transcriptome (of 11 genes involved in cuticle biosynthesis) analysis. Non-irrigated saplings (i) had a higher cuticular wax content than irrigated saplings and (ii) overexpressed most of the genes studied. Both these trends were more marked in the faster growing family. Conclusions: The higher cuticular wax content observed in the non-irrigated treatment associated with strong modifications in products from the decarbonylation pathway suggest that cuticular wax may be involved in drought stress adaptation in maritime pine. This study provides also a set of promising candidate genes for future forward genetic studies in conifers.

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International audience; Simple sequence repeats (SSRs) or microsatellites are valuable tools for genome mapping and population genetic studies for as they are codominant and highly polymorphic markers. Seventy-six SSR primer pairs from four Pinus species were tested to amplify microsatellites in Pinus pinaster. Twenty-six primer pairs were stemmed from a microsatellite library on P. pinaster and the other primer pairs were obtained in other species of the same genus (P. radiata, P. strobus and P. halepensis). Only three out of the 76 SSR primer pairs amplified at a single polymorphic locus in P. pinaster. The Mendelian inheritance of those three primer pairs was studied and their genetic map position was determined. The number of alleles and the level of heterozygosity were assessed in an analysis of a sample of 196 trees. The development of microsatellites in Pinus species has been reported to be a difficult task because of the size and complexity of their genome. The results of this study showed that cross-species amplification was quite unsuccessful.; Marqueurs microsatellites chez Pinus pinaster Ait. Les microsatellites (SSRs) sont des outils de choix pour la cartographie génétique et les études de génétique des populations parce qu'ils sont des marqueurs codominants et très polymorphes. Soixante-seize paires d'amorces de quatre espèces de pin ont été utilisées afin d'amplifier des microsatellites chez Pinus pinaster. Vingt-neuf paires d'amorces étaient issues d'une banque enrichie en microsatellites sur P. pinaster et les autres paires d'amorces avaient été obtenues sur d'autres espèces du même genre (P. radiata, P. strobus et P. halepensis). Sur un total de 76 paires d'amorces, seulement trois ont amplifié un seul locus microsatellite polymorphe chez P. pinaster. Leur ségrégation mendélienne a été étudiée et chaque locus a été localisé sur une carte génétique. Le nombre d'allèles et l'hétérozygotie ont été ensuite évalués en analysant un échantillon de 196 arbres. Le développement de microsatellites chez les espèces du genre Pinus s'est révélée difficile en raison de la taille et de la complexité de leur génome. Les résultats de cette étude ont montré que l'amplification inter-espèces n'a rencontré que peu de succès.

Introduction : notre travail visait à évaluer et comparer les profils psychologiques et psychiatriques de patients atteints de rhumatismes inflammatoires chroniques (RIC) : polyarthrite rhumatoïdes (PR) et spondyloarthrites (SpA), afin de mettre en évidence un profil émotionnel particulier par rapport aux patients sans RIC. La répression émotionnelle, qui décrit une tendance à inhiber l'expression de sentiments négatifs et/ou de cognitions désagréables pour préserver une image positive du soi, a été particulièrement étudiée. Matériel et méthodes : nous avons mené une étude pilote observationnelle prospective monocentrique incluant des patients suivis dans un service universitaire de rhumatologie avec évaluation systématique, par un psychiatre expérimenté, de différents paramètres psychologiques et psychiatriques. Les paramètres cliniques et biologiques de leurs rhumatismes ont été colligés. Les variables qualitatives ont été comparées à l'aide d’un test du Chi-2 ou d'un test Exact de Fisher. Résultats : nNous avons évalué 59 patients dont 47 avec un RIC (27 PR et 20 SpA) et 12 témoins sans RIC (9 arthroses, 1 virose, 1 ostéoporose et 1 ostéomalacie). Une forte répression émotionnelle était retrouvée plus significativement dans le groupe RIC par rapport au groupe témoin (p=0,019). De même, les fortes plaintes psychologiques et somatiques étaient plus significativement retrouvées dans le groupe sans rhumatisme inflammatoire (respectivement p=0,002 et p=0,01). L'intensité forte des évènements de vie précoces était significativement plus élevée dans le groupe RIC (p=0,015). Conclusion : notre étude confirme l'importance de la régulation émotionnelle pour faire face à différents évènements traumatisants de vie, dans le processus étiologique ou évolutif des RIC. Nous soulignons l'intérêt des traitements psychothérapiques en complément de la prise en charge médicale des RIC.