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Trees are long-lived organisms that continuously adapt to their environments, a process in which epigenetic mechanisms are likely to play a key role. Via down regulation of the chromatin remodeler DECREASED IN DNA METHYLATION 1 (DDM1) in poplar (Populus tremula 9 Populus alba) RNAi lines, we examined how DNA methylation coordinates genomic and physiological responses to moderate water deficit.We compared the growth and drought response of two RNAi-ddm1 lines to wild-type (WT) trees under well-watered and water deficit/rewatering conditions, and analyzed their methylomes, transcriptomes, mobilomes and phytohormone contents in the shoot apical meristem. The RNAi-ddm1 lines were more tolerant to drought-induced cavitation but did not differ in height or stem diameter growth. About 5000 differentially methylated regions were consistently detected in both RNAi-ddm1 lines, colocalizing with 910 genes and 89 active transposable elements. Under water deficit conditions, 136 differentially expressed genes were found, including many involved in phytohormone pathways ; changes in phytohormone concentrations were also detected. Finally, the combination of hypomethylation and drought led to the mobility of two transposable elements. Our findings suggest major roles for DNA methylation in regulation of genes involved in hormone-related stress responses, and the maintenance of genome integrity through repression of transposable elements.

As a renewable energy resource, woody biomass has a role to play in reducing worldwide fossil fuel consumption. Species in the genera Salix and Populus are widely used for this purpose through short rotation tree plantations under temperate latitudes. In warmer climates, common short rotation species include genera such as eucalyptus or black locust (Robinia) although this latter is also cultivated in temperate climates. The objective of our study was to compare the potential of poplar (Populus deltoides × P. nigra, clone 'Dorskamp') and black locust (Robinia pseudoacacia, provenances 'Nagybudmerii' and 'Nyirseg') for biomass production in the Northwest of France. We estimated biomass production and efficiency of water use of poplar compared to those of the two provenances of black locust, (1) at two planting densities (short rotation forestry: SRF, 1428 trees per ha vs. short rotation coppice: SRC, 7272 trees per ha), and (2) 2 and 4 years after planting. Only few effects of the initial planting density on plant growth and physiology were visible two years after planting. Stem height and circumference became higher in SRF than in SRC for both species after four years. Yield at plantation level of the first SRC rotation was almost twice as high for black locust than for poplar, while both species exhibited similar stem height or stem increase rate likely due to the development of more sylleptic branches in black locust. At the end of the second rotation, plantation yield was significantly higher for poplar due to black locust mortality. The combination of higher leaf N and greater WUE probably enhanced black locust performance under the dry conditions of our study site in 2010. During the fourth year (2012), the gap between the two species in terms of Δ 13 C, used as a surrogate of water-use efficiency, was less marked than during the first years due to the fact that poplar Δ 13 C decreased (increase in WUE), while black locust Δ 13 C slightly increased (decrease in WUE). To conclude, black locust appears as an interesting option for biomass production at sites prone to water shortage. However, at high planting density, poplar exhibited a lower mortality after harvest leading to an improved biomass production at the end of the second SRC rotation compared to black locust. The choice of species/cultivar must be adapted to the production system (SRC vs. SRF) and to the specific site conditions.

As fixed and long living organisms subjected to repeated environmental stresses, trees have developed mechanisms such as phenotypic plasticity that help them to cope with fluctuating environmental conditions. Here, we tested the role DNA methylation as a hub of integration, linking plasticity and physiological response to water deficit in the shoot apical meristem of the model tree poplar (Populus). Using a reverse genetic approach, we compared hypomethylated RNAi-ddm1lines to wild-type trees for drought tolerance. An integrative analysis was realized with phytohormone balance, methylomes, transcriptomes and mobilomes.Hypomethylated lines were more tolerant when subjected to moderate water deficit and were intrinsically more tolerant to drought-induced cavitation. The alteration of theDDM1machinery induced variation in DNA methylation in a cytosine context dependent manner, both in genes and transposable elements. Hypomethylated lines subjected to water deficit showed altered expression of genes involved in phytohormone pathways, such as salicylic acid and modified hormonal balance. Several transposable elements showed stress- and/or line-specific patterns of reactivation, and we could detect copy number variations for two of them in stressedddm1lines.Overall, our data highlight two major roles for DNA methylation in the shoot apical meristem: control of stress response and plasticity through transduction of hormone signaling and maintenance of genome integrity through the control of transposable elements.

International audience; To make intensive bioenergy poplar plantations attractive on an economic and environmental point of view, their sustainable resource management is mandatory. These plantations are indeed often installed on marginal lands to avoid competition with food agriculture and large amounts of nutrients are repeatedly exported in the young harvested biomass. Besides the application of organic residues to reclaim impoverished soils, the use of species or genotypes efficiently using nutrients, and especially nitrogen, is also a way to slow down fertility losses in short-rotation poplar plantations. Our goal in the present study was to document the genotypic variability available in terms of yield, nitrogen-use efficiency (NUE), and nitrogen export rate, the links between these traits, and their plasticity in response to contrasting site conditions and during two rotations in a wide set of Populus deltoides × Populus nigra genotypes. A wide range of variation has been highlighted for biomass production, tree dimensions, and nitrogen use and export at both sites, and particularly at the most productive one. Environmental and genotype by environment effects were very strong and the ranking of genotypes was not maintained between the two sites for most studied traits. Because of climatic conditions less contrasted between sites during the second rotation, the differences were less marked than during the first rotation. The most productive and efficient genotypes to use nitrogen were also those responsible for the highest nitrogen exports during harvest. Indeed, although the most productive genotypes had high NUE, their high biomass production resulted in a more important nitrogen export at harvest than the less productive genotypes.

International audience; In a context of climate change and forest decline, a better understanding of the sources of tree flexibility involved in phenotypic plasticity and adaptation is needed. These last years, the role of epigenetics in the response to environmental variations has been established in several model plants at the genotype level but little is known at the level of natural populations grown in pedoclimatic sites. Here, we focused on three French natural populations of black poplar, a key pioneer tree from watersheds, planted in common garden and subjected to controlled variations of water availability. We estimated common genetic parameters such as narrow-sense heritability (h(2)), phenotypic differentiation index (P-ST), and the overall genetic differentiation index (F-ST) from genome-wide SNPs to evaluate the extent of epigenetic variations. Indeed, global DNA methylation levels from individuals exposed to drought or irrigated in a common garden were used. We found that the three populations were not distinguished by their levels of DNA methylation. However, a moderate drought was associated to a significant decrease in DNA methylation in the populations. Narrow-sense heritability and P-ST estimates of DNA methylation were similar to those found for biomass productivity. Heritability and P-ST were higher when trees were subjected to drought than in control condition. Negative genetic correlations between global DNA methylation and height or biomass were detected in drought condition only. Altogether, our data highlight that global DNA methylation acts as a genetic marker of natural population differentiation under drought stress in a pedoclimatic context.

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International audience; Trees have a long lifespan and must continually adapt to environmental pressures, notably in the context of climate change. Epigenetic mechanisms are doubtless involved in phenotypic plasticity and in stress memory; however, little evidence of the role of epigenetic processes is available for trees growing in fields. Here, we analyzed the possible involvement of epigenetic mechanisms in the winter-dormant shoot apical meristem of Populus × euramericana clones in memory of the growing conditions faced during the vegetative period. We aimed to estimate the range of genetic and environmentally induced variations in global DNA methylation and to evaluate their correlation with changes in biomass production, identify differentially methylated regions (DMRs), and characterize common DMRs between experiments. We showed that the variations in global DNA methylation between conditions were genotype dependent and correlated with biomass production capacity. Microarray chip analysis allowed detection of DMRs 6 months after the stressful summer period. The 161 DMRs identified as common to three independent experiments most notably targeted abiotic stress and developmental response genes. Results are consistent with a winter-dormant shoot apical meristem epigenetic memory of stressful environmental conditions that occurred during the preceding summer period. This memory may facilitate tree acclimation.

International audience; The adaptive capacity of long-lived organisms such as trees to the predicted climate changes, including severe and successive drought episodes, will depend on the presence of genetic diversity and phenotypic plasticity. Here, the involvement of epigenetic mechanisms in phenotypic plasticity toward soil water availability was examined in Populus×euramericana. This work aimed at characterizing (i) the transcriptome plasticity, (ii) the genome-wide plasticity of DNA methylation, and (iii) the function of genes affected by a drought-rewatering cycle in the shoot apical meristem. Using microarray chips, differentially expressed genes (DEGs) and differentially methylated regions (DMRs) were identified for each water regime. The rewatering condition was associated with the highest variations of both gene expression and DNA methylation. Changes in methylation were observed particularly in the body of expressed genes and to a lesser extent in transposable elements. Together, DEGs and DMRs were significantly enriched in genes related to phytohormone metabolism or signaling pathways. Altogether, shoot apical meristem responses to changes in water availability involved coordinated variations in DNA methylation, as well as in gene expression, with a specific targeting of genes involved in hormone pathways, a factor that may enable phenotypic plasticity.

International audience; Successful plant manipulation by herbivores requires a reconfiguration of the primary and secondary metabolisms of the host-plant. Water deficit is generally predicted to negatively affect the development of gall-inducing insects, by impairing their ability to remodel the primary metabolism of their host. We assessed whether host genotype could modulate water deficit effects on a gall-inducing aphid, Phloeomyzus passerinii, developing on poplars, which are known to be among the most susceptible trees to water deficit. Stem-cuttings of two poplar genotypes, with a different resistance level to P. passerinii, were grown under three irrigation treatments, and subsequently infested with the same aphid clone. Plant growth parameters and aphid colonies development were recorded, and the development and organization of the pseudogall induced by-the aphid was observed using histological investigations. The three irrigation treatments resulted in three levels of water deficit. Mean predawn leaf water potential reached -0.10, -0.49 and -0.75 MPa in the optimal, intermediate and low irrigation treatments respectively. In both genotypes, growth, architecture and photosynthesis were similarly affected. The severe water deficit reduced the development of aphids on both genotypes, which is in agreement with the general prediction for gall-inducing insects. Nonetheless, while the intermediate water deficit did not affect the development of aphids on the susceptible genotype, it enhanced their development on the resistant one. This latter observation contradicts the prediction for gall-inducing insects, but is in agreement with the plant stress hypothesis for sucking insects. The size of the pseudogall induced by the aphids in the bark of their host progressively diminished when water deficit increased. Lignification, believed to be an important component of poplar resistance to P. passerinii, also progressively decreased with water deficit. The involvement of physiological and architecture modifications due to water deficit on the development of aphids is discussed. The results demonstrate that host genotype can modulate water deficit effects on the development of a galling aphid, thereby compromising predictions on the outcome of droughts on stand resistance to plant-manipulating herbivores.

1. Phytophagous insects frequently manipulate their host-plant to improve their immediate environment. This generally implies substantial modifications of host metabolism, and sometimes an alteration of nitrogen allocation within the host-plant. However, the outcome of plant manipulation on amino acid or protein content can be modulated by environmental factors and host-plant traits. 2. It was investigated whether the pseudogall induced by the aphid Phloeomyzus passerinii (Signoret) (Aphididae: Phloeomyzinae) in the bark of its host-plant affects the amino acid content in bark tissues, and whether the strength of the modification is modulated by the fertilisation regime and/or the resistance level of the host-plant. The development of aphid colonies on a resistant and a susceptible poplar genotype, under three fertilisation regimes, was studied. After the development of colonies, the free and protein-bound amino acid content of the infested bark were quantified. 3. Fertilisation enhanced poplar growth and increased the free amino acid content of bark tissues. Infestation also triggered accumulations of both free and protein-bound amino acids in the feeding sites, but in the susceptible genotype only. The increase in amino acid content was more pronounced when fertilisation was low, and fertilisation did not enhance aphid development. 4. In conclusion, infestation by P. passerinii triggers an accumulation of amino acids, but the effect is influenced by both the fertilisation regime and the resistance level of the host-plant. This suggests that P. passerinii could affect the allocation of nutrients within trees during outbreaks.

International audience; To buffer against the high spatial and temporal heterogeneity of the riparian habitat, riparian tree species, such as black poplar (Populus nigra L.), may display a high level of genetic variation and phenotypic plasticity for functional traits. Using a multisite common garden experiment, we estimated the relative contribution of genetic and environmental effects on the phenotypic variation expressed for individual leaf area, leaf shape, leaf structure and leaf carbon isotope discrimination (Delta C-13) in natural populations of black poplar. Twenty-four to 62 genotypes were sampled in nine metapopulations covering a latitudinal range from 48A degrees N to 42A degrees N in France and in Italy and grown in two common gardens at Orl,ans (ORL) and at Savigliano (SAV). In the two common gardens, substantial genetic variation was expressed for leaf traits within all metapopulations, but its expression was modulated by the environment, as attested by the genotype x environment (G x E) interaction variance being comparable to or even greater than genetic effects. For LA, G x E interactions were explained by both changes in genotype ranking between common gardens and increased variation in SAV, while these interactions were mainly attributed to changes in genotype ranking for Delta C-13. The nine P. nigra metapopulations were highly differentiated for LA, as attested by the high coefficient of genetic differentiation (Q(ST) = 0.50 at ORL and 0.51 at SAV), and the pattern of metapopulation differentiation was highly conserved between the two common gardens. In contrast, they were moderately differentiated for Delta C-13 (Q(ST) = 0.24 at ORL and 0.25 at SAV) and the metapopulation clustering changed significantly between common gardens. Our results evidenced that the nine P. nigra metapopulations present substantial genetic variation and phenotypic plasticity for leaf traits, which both represent potentially significant determinants of populations' capacities to respond, on a short-term basis and over generations, to environmental variations.

Vulnerability to drought-induced cavitation is a key trait of plant water relations. Here, we summarize the available literature on vulnerability to drought-induced cavitation in poplars (Populus spp.), a genus of agronomic, ecological and scientific importance. Vulnerability curves and vulnerability parameters (including the water potential inducing 50% loss in hydraulic conductivity, P50) were collected from 37 studies published between 1991 and 2014, covering a range of 10 species and 12 interspecific hybrid crosses. Results of our meta-analysis confirm that poplars are among the most vulnerable woody species to drought-induced cavitation (mean P50 = -1.44 and -1.55 MPa across pure species and hybrids, respectively). Yet, significant variation occurs among species (P50 range: 1.43 MPa) and among hybrid crosses (P50 range: 1.12 MPa), within species and hybrid crosses (max. P50 range reported: 0.8 MPa) as well as in response to environmental factors including nitrogen fertilization, irradiance, temperature and drought (max. P50 range reported: 0.75 MPa). Potential implications and gaps in knowledge are discussed in the context of poplar cultivation, species adaptation and climate modifications. We suggest that poplars represent a valuable model for studies on drought-induced cavitation, especially to elucidate the genetic and molecular basis of cavitation resistance in Angiosperms.

Multistep phosphorelays involve a phosphate transfer from sensor histidine-aspartate kinases (HKs) to response regulators (RRs), via histidine-containing phosphotransfer proteins (HPts). In Arabidopsis, some AHK receptors are organized as homodimers and able to interact with HPts (AHPs). However, there are no data available concerning the dimerization of the Arabidopsis osmosensor AHK1. Although only AHP2 is able to interact with AHK1 in yeast, validation of this interaction remains to be clarified in planta. The ability of poplar HK1 osmosensor, homologous to AHK1, to homodimerize and interact with three HPts (HPt2, 7 and 9) as preferential partners has been previously shown by yeast two-hybrid assay. However, protein interaction studies need to use complementary approaches to avoid interaction artifacts. Here, we confirmed in planta homodimerization of the cytoplasmic part of HK1 (HK1-CP) and the functional relevance of HK1-CP/HPt interactions by bimolecular fluorescence complementation assays. This wor...

DNA methylation is involved in the control of plant development and adaptation to the environment through modifications of chromatin compaction and gene expression. In poplar (Populus trichocarpa), a perennial plant, variations in DNA methylation have been reported between genotypes and tissues or in response to drought. Nevertheless, the relationships between gene-body DNA methylation, gene expression and chromatin compaction still need clarification. Here, DNA methylation was mapped in the noncondensed chromatin fraction from P. trichocarpa shoot apical meristematic cells, the center of plant morphogenesis, where DNA methylation variations could influence the developmental trajectory. DNase I was used to isolate the noncondensed chromatin fraction. Methylated sequences were immunoprecipitated, sequenced using Illumina/Solexa technology and mapped on the v2.0 poplar genome. Bisulfite sequencing of candidate sequences was used to confirm mapping data and to assess cytosine contexts and methylation levels. While the methylated DNase I hypersensitive site fraction covered 1.9% of the poplar genome, it contained sequences corresponding to 74% of poplar gene models, mostly exons. The level and cytosine context of gene-body DNA methylation varied with the structural characteristics of the genes. Taken together, our data show that DNA methylation is widespread and variable among genes in open chromatin of meristematic cells, in agreement with a role in their developmental trajectory.

National audience; Global climate changes in progress will impact tree productivity in the northern hemisphere. The understanding of physiological as well as genetic/molecular processes controlling plants response to abiotic stress will help to improve plant breeding. Recently, epigenetic mechanisms such as DNA methylation have been shown to participate to the control of plant development and their adaptation to environment through modifications of chromatin compaction and gene expression profiles. Phenotypic plasticity defines as the different phenotypes for a given genotype in distinct environments is a key process for plant to adapt to their changing environment. This is particularly relevant for perennialplants such as trees that are exposed to repeated fluctuations of their living conditions. In this context, drought is a significant threat to forest health and agro-ecosystem productivity. With the availability of its genome and its important natural genetic and phenotypic variations, Populus became a model tree.The aim of our project is to develop an integrative approach to identify ecophysiological and molecular bases, particularly DNA methylation, involved in phenotypic plasticity. Contrary to animals, plants exhibit a continuous development and their germline arise from very specific somatic tissue: the shoot apical meristem. We focus our effort to the study the role of DNA methylation in shoot apical meristem using various epigenomic approaches (MeDIP-SEQ, MeDIP-CHIP, WGBS) in parallel to transcriptomics and phenotyping. Our experimental designs include collection of genotypes and are established in controlled conditions such as in greenhouse and nursery as well as in field plantations. We have also characterized the first hypomethylated tree (DDM1-RNAi poplars) in different environments.Altogether, our results demonstrate the role of DNA methylation in memorizing environmental influence and identify the corresponding genes network in relation to phenotypic plasticity.

(13) C discrimination between atmosphere and bulk leaf matter (Δ(13) C(lb) ) is frequently used as a proxy for transpiration efficiency (TE). Nevertheless, its relevance is challenged due to: (1) potential deviations from the theoretical discrimination model, and (2) complex time integration and upscaling from leaf to whole plant. Six hybrid genotypes of Populus deltoides×nigra genotypes were grown in climate chambers and tested for whole-plant TE (i.e. accumulated biomass/water transpired). Net CO(2) assimilation rates (A) and stomatal conductance (g(s) ) were recorded in parallel to: (1) (13) C in leaf bulk material (δ(13) C(lb) ) and in soluble sugars (δ(13) C(ss) ) and (2) (18) O in leaf water and bulk leaf material. Genotypic means of δ(13) C(lb) and δ(13) C(ss) were tightly correlated. Discrimination between atmosphere and soluble sugars was correlated with daily intrinsic TE at leaf level (daily mean A/g(s) ), and with whole-plant TE. Finally, g(s) was positively correlated to (18) O enrichment of bulk matter or water of leaves at individual level, but not at genotype level. We conclude that Δ(13) C(lb) captures efficiently the genetic variability of whole-plant TE in poplar. Nevertheless, scaling from leaf level to whole-plant TE requires to take into account water losses and respiration independent of photosynthesis, which remain poorly documented.

International audience; Six Populus deltoides Bartr. ex Marsh. × P. nigra L. genotypes were selected to investigate whether stem xylem anatomy correlated with gas exchange rates, water-use efficiency (WUE) and growth performance. Clonal copies of the genotypes were grown in a two-plot common garden test under contrasting water regimes, with one plot maintained irrigated and the other one subjected to moderate summer water deficit. The six genotypes displayed a large range of xylem anatomy, mean vessel and fibre diameter varying from about 40 to 60 μm and from 7.5 to 10.5 μm, respectively. Decreased water availability resulted in a reduced cell size and an important rise in vessel density, but the extent of xylem plasticity was both genotype and trait dependent. Vessel diameter and theoretical xylem-specific hydraulic conductivity correlated positively with stomatal conductance, carbon isotope discrimination and growth performance-related traits and negatively with intrinsic WUE, especially under water deficit conditions. Vessel diameter and vessel density measured under water deficit conditions correlated with the relative losses in biomass production in response to water deprivation; this resulted from the fact that a more plastic xylem structure was generally accompanied by a larger loss in biomass production.

National audience; Recent findings suggest that epigenetic processes such as DNA methylation are involved in water-stress acclimation. Poplars are increasingly used for tree research as model organisms and poplar RNAi lines represent powerful tools to address physiological questions. Here, three RNAi poplar lines inhibited for the DECREASED DNA METHYLATION 1 gene (DDM1, involved in the maintenance of DNA methylation profile) were compared to the control genotype (Populus tremula × Populus alba). A drought-rewatering experiment was conducted to evaluate the role of DNA methylation in phenotypic plasticity. In vitro plantlets of the four genotypes were propagated and transferred to a greenhouse where they were grown under controlled conditions of temperature, light and photoperiod. At the onset of the drought experiment, half of the plants were subjected to a controlled and moderate water deficit while the other half was maintained at field capacity. After three weeks of water depletion, waterstressed plants were re-watered for one week before being harvested. Results related to growth behaviour, photosynthetic capacities, water-use efficiency, xylem vulnerability to cavitation and xylem anatomy will be presented. This phenotypic characterization will drive future work in epigenomic such as the identification of ‘Differentially Methylated Regions’ and their relationships with corresponding gene expression profiles.

Article en open access; International audience; Xylem resistance to drought-induced cavitation is a key trait of plant water relations. This study assesses the genetic variation expressed for stem cavitation resistance within a population of a riparian species, the European black poplar (Populus nigra L.), and explores its relationships with xylem anatomy, water-use efficiency (WUE), and growth. Sixteen structural and physiological traits related to cavitation resistance, xylem anatomy, growth, bud phenology, and WUE were measured on 33 P. nigra genotypes grown under optimal irrigation in a 2-year-old clonal experiment in a nursery. Significant genetic variation was expressed for the xylem tension inducing 50% loss of hydraulic conductivity (Ψ50) within the studied population, as attested by the high value of broad-sense heritability estimated for this trait (H 2 ind = 0.72). Stem cavitation resistance was associated with xylem structure: the more cavitation-resistant genotypes exhibited lower hydraulic efficiency and higher mechanical reinforcement as assessed from stem xylem cross sections. By contrast, Ψ50 was not significantly related to shoot height increment, total above-ground dry mass, or bulk leaf carbon isotope discrimination, a proxy for intrinsic WUE. These findings indicate that the trade-offs between xylem resistance to cavitation, hydraulic efficiency, and mechanical reinforcement can occur at the within-population level. Given that the studied genotypes were exposed to the same environmental conditions and evolutionary drivers in situ, the trade-offs detected at this scale are expected to reflect true functional relationships.

International audience; The B-type response regulators (B-type RRs), final elements of a signaling pathway called "histidine/aspartate phosphorelay system" in plants, are devoted to the regulation of response genes through a transcription factor activity. Signal transduction consists in the transfer of a phosphoryl group from a transmembrane histidine kinase (HK) which recognizes a given stimulus to nuclear RRs via cytosolic shuttle phosphotransfer proteins (HPts). In Arabidopsis, the receptors HK are to date the major characterized candidates to be responsible for initiation of osmotic stress responses. However, little information is available concerning the signaling partners acting downstream of HKs. In Populus, three HPts and five B-type RRs were previously identified as interacting partners of HK1, the Arabidopsis AHK1 homolog. Here, we report the isolation of RR18, a member of the B-type RR family, which shares high sequence similarities with ARR18 characterized to act in the osmosensing signaling pathway in Arabidopsis, from poplar cuttings subjected to osmotic stress conditions. By using yeast and in planta interaction assays, RR18 was further identified as acting downstream of HK1 and its three preferential HPt partners. Besides, our results are in favor of a possible involvement of both RR18 and RR13, the main expressed poplar B-type RR, in the osmotic signaling pathway. Nonetheless, different behaviors of these two B-type RRs in this pathway need to be noted, with one RR, RR13, acting in an early phase, mainly in roots of poplar cuttings, and the other one, RR18, acting in a late phase, mainly in leaves to supply an adequate response. (C) 2015 Elsevier Masson SAS. All rights reserved.

In order to evaluate the permanent chromatin remodeling in plant allowing their high developmental plasticity, three sugarbeet cell lines (Beta vulgaris L. altissima) originating from the same mother plant and exhibiting graduate states of differentiation were analyzed. Cell differentiation has been estimated by the cell redox state characterized by 36 biochemical parameters as reactive oxygen species steady-state levels, peroxidation product contents and enzymatic or non-enzymatic protective systems. Chromatin remodeling has been estimated by the measurement of levels of DNA methylation, histone acetylation and corresponding enzyme activities that were shown to differ between cell lines. Furthermore, distinct loci related to proteins involved in cell cycle, gene expression regulation and cell redox state were shown by restriction landmark genome scanning or bisulfite sequencing to display differential methylation states in relation to the morphogenic capacity of the lines. DNA methylating, demethylating and/or histone acetylating treatments allowed to generate a collection of sugarbeet cell lines differing by their phenotypes (from organogenic to dedifferentiated), methylcytosine percentages (from 15.0 to 43.5%) and acetylated histone ratios (from 0.37 to 0.52). Correlations between methylcytosine or acetylated histone contents and levels of various parameters (23 or 7, respectively, out of 36) of the cell redox state could be established. These data lead to the identification of biomarkers of sugarbeet morphogenesis in vitro under epigenetic regulation and provide evidence for a connection between plant morphogenesis in vitro, cell redox state and epigenetic mechanisms.

L'impact de secheresses moderee et severe sur le contenu en proteines des feuilles et sur les activites de la dismutase de superoxyde (SOD), de la catalase et de la peroxydase a ete etudie chez de jeunes boutures de deux clones de Populus deltoides x nigra, 'Luisa_Avanzo' et 'Dorskamp', connus pour presenter des niveaux differents de tolerance au deficit hydrique. Les feuilles en croissance et matures ont ete analysees separement. La reponse a la secheresse des systemes de protection enzymatiques etait differente selon le clone et le stade de developpement des feuilles etudiees. Pour le clone 'Dorskamp', repute plus tolerant que 'Luisa_Avanzo' a la secheresse,les activites de la SOD et de la catalase augmentaient dans les feuilles matures en reponse a un deficit hydrique severe. Pour le clone 'Luisa_Avanzo', l'activite de la peroxydase augmentât essentiellement dans les feuilles en croissance en reponse a une secheresse moderee. Pour les deux clones, trois isoformes differentes de la SOD, la Mn-SOD, la Fe-SOD et la Cu/Zn-SOD ont ete detectees en quantites variables chez les deux clones en fonction de l'intensite de la contrainte hydrique.

In plants organogenesis, cell differentiation and dedifferentiation are fundamental processes allowing high developmental plasticity. Such plasticity involved epigenetic mechanisms but limited knowledge is available concerning quantitative aspects. Three sugarbeet (Beta vulgaris L. altissima) cell lines originating from the same mother plant and exhibiting graduate states of morphogenesis were used to assess whether these differences could be related or not to changes in DNA methylation levels. Methylcytosine percentages from 18.3 to 28.8% and distinct levels of DNA methyltransferase (EC 2.1.1.37) activities were shown in the three cell lines. The lowest methylcytosine percentage was associated to organogenesis. In order to test the plasticity of these cell lines, various treatments causing DNA hypo or hypermethylation were performed at different times and concentrations. In this collection of treated lines with+/-10% of methylcytosine percentages, loss of organogenic properties and cell dedifferentiation were observed. As cell wall formation fits well with cell differentiation state, the lignification process was further investigated in treated and untreated lines as a biochemical marker of the phenotypic changes. For example, peroxidase specific activities (EC 1.11.1.7) varied from 0.7 to 0.02 pkat mg(-1) of protein in organogenic and dedifferentiated lines, respectively. A negative relationship between peroxidase activities, incorporation of cell wall-bound phenolic compounds as ferulate and sinapate derivatives and methylcytosine percentages was obtained. This is the first biochemical evidence that phenotypic changes in plant cells induced by DNA hypo- or hypermethylating treatments are correlated in a linear relationship to modifications of the cell wall differentiation state.

Responses to successive drought and re-watering cycles (1-3 cycles) were compared in greenhouse-grown cuttings of Populus x canadensis (Moench) clones, 'Luisa_ Avanzo' and 'Dorskamp.' Total leaf number increment rate, duration of leaf expansion, total and individual leaf area expansion rates and stomatal conductance were recorded periodically during the experiment. Soil water content (SWC) and predawn leaf water potential (Psi(WP)) were measured four times during each drought cycle. In parallel, relative leaf water content (RWC) and specific leaf area (SLA) were estimated on leaves collected from the top to bottom of each cutting. Under well-watered conditions, 'Luisa_Avanzo' and 'Dorskamp' differed in their patterns of leaf area expansion. Although duration of leaf expansion was similar between clones, 'Luisa_ Avanzo' exhibited higher total leaf number increment rates and individual leaf area increases than 'Dorskamp.' As a result, 'Luisa_Avanzo' cuttings reached larger individual and total leaf areas than 'Dorskamp.' 'Dorskamp' leaves had lower SLA than 'Luisa_Avanzo' leaves. In response to successive drought cycles, both clones underwent decreases in total leaf number increment rates and in total leaf area expansion rates, but both whole-plant and individual leaf areas were drastically reduced only in 'Luisa_Avanzo.' 'Dorskamp' maintained a constant leaf area as a result of an increase in the duration of leaf expansion during drought and a significant stimulation of individual leaf area expansion rate and total leaf number increment rate in response to re-watering. Drought caused a greater decrease in SLA in 'Luisa_Avanzo' than in 'Dorskamp.' Expanded leaves of 'Dorskamp' were constitutively dense or thick, or both, whereas leaves of 'Luisa_Avanzo' became dense or thick, or both, only in response to drought. In both clones, re-watering caused partial recovery of SLA to control values. Our data confirm previous field and greenhouse observations that 'Dorskamp' is more tolerant to successive drought and re-watering cycles than 'Luisa_Avanzo.' We conclude that this difference is mainly attributable to the plasticity of 'Dorskamp' after re-watering, rather than to clonal differences in drought responses.

One hundred Norway spruce (Picea abies (L.) Karst.) clones (three ramets per clone) were analyzed for phloem phenol composition and concentration before and 10 days after wound inoculation with sterile malt agar. Fifty clones (Experiment 1) belonged to the same provenance, whereas the remaining clones (Experiment 2) belonged to five provenances from three geographic areas. In Experiment 2, two additional ramets from the same clones were mass inoculated with Ceratocystis polonica (Siem.) C. Moreau to quantify the resistance of each clone. Tree response to wound inoculations was characterized by increased catechin concentration in both experiments, accompanied by increases in astringin and decreases in piceid in Experiment 1. In both experiments, we observed a diverse group of phenolic compounds whose concentrations increased (catechin, astringin) or did not vary (taxifolin glucoside) in response to wound inoculations, whereas concentrations of a homogeneous group of stilbene compounds decreased (piceid) or did not vary (isorhapontin, unidentified stilbene). In Experiment 2, provenances from the alpine and Hercynian-Carpatic areas differed from provenances from the Baltic area with respect to the relative importance of these two groups of compounds, further indicating that the two groupings of phenolic compounds structure the Norway spruce populations. Eighty days after mass inoculation, the percentage of healthy sapwood, which was taken as a measure of tree resistance, indicated that clones from the Baltic area were less resistant to mass inoculations than clones from the alpine and Hercynian-Carpatic areas. We conclude that the degree of resistance of Norway spruce trees to mass inoculations with a bark beetle-associated fungus can be predicted based on the diversity of constitutive phloem phenols and the ability to induce phenol synthesis in response to wounding.

International audience; Because of their impressive growth rates, interspecific hybrid poplars are among the most extensively cultivated trees in temperate latitudes. In line with their general riparian origin, poplar trees are generally highly water-demanding such that the productivity of poplar plantations is tightly dependent on adequate water availability. However, in the current context of rational management of water resources, optimizing the carbon-water trade-off of such plantations is particularly important. Water-use efficiency (WUE, i.e. the amount of plant biomass produced per unit water used) has therefore emerged as a new relevant criterion to be accounted for in poplar selection, in conjunction with other common criteria already used such as disease resistance, productivity and wood quality. We propose here an overview of nearly 15 years of research on WUE in poplar hybrids (Populus deltoides × P. nigra and P. deltoides × P. trichocarpa, the two most commonly interspecific combinations used in Europe). These results were obtained under the framework of seven Ph.D. theses conducted along the 2000-2013 period. Four main questions will be addressed: (1) Is bulk leaf carbon isotope discrimination a good estimator of WUE assessed either at the leaf level (intrinsic WUE, i.e. net CO2 assimilation rate / stomatal conductance to water vapour) or at the whole-plant level (transpiration efficiency, i.e. biomass produced / water loss)? (2) To what extent does WUE vary among and within hybrid crosses and are hybrid rankings for WUE stable with ageing? (3) How do environmental conditions (drought, planting density, soil fertility) affect WUE? and (4) Are WUE and biomass production related and is this relationship modulated by environmental factors? The practical implications opened up by the results and the future research opportunities will then be presented.

Summary The mechanisms by which drought-induced oxidative stress is tolerated in poplar were appreciated by the ability of leaf extracts to scavenge reactive oxygen species (ROS) generated in vitro by a xanthine oxidase-hypoxanthine system. Their effects were detected by the protection towards the denaturation of malate dehydrogenase (MDH) of crude and dialysed extracts of a drought-sensitive ( Populus × euramericana , Luisa Avanzo) and a drought-tolerant ( P × euramericana , Dorskamp) poplar exposed to control and osmotic stress (-0.336 MPa). At the beginning of the incubation period in presence of ROS, MDH from crude extract of control Dorskamp was protected against the denaturation; boiled crude extracts of control Dorskamp also protected MDH from the dialysed extract of control Luisa Avanzo. In control Dorskamp, cysteine, proline, polyamines and ascorbate added in vitro were efficient ROS scavengers. Compared with the control condition, the rate of MDH denaturation in presence of ROS decreased in a dialysed extract of stressed Luisa Avanzo but increased in that of stressed Dorskamp. Under stress conditions, proline in vitro enhanced the rate of MDH denaturation by ROS in Luisa Avanzo, but competed as ascorbate with ROS in Dorskamp. In Dorskamp, the 12h-exposure to osmotic stress resulted in a decrease in catalase and gutathione reductase activities, in an enhancement of superoxide dismutase, ascorbate peroxidase and contents of some putative leaf antioxidants (putrescine and glutathione), but did not affect the contents of spermine, spermidine and ascorbate. The consequences of extract/solute effects are discussed for each clone as regards the drought response.

Changes in phloem phenolic content of Norway spruce (Picea abies (L.) Karst.) clones were followed during the first 12 days of the reaction induced by phloem artificial inoculation with Ceratocystis polonica Siem., a bark beetle (Ips typographus L.) associated fungus. The aim was to confirm our previous results concerning the mechanisms of this reaction and the possible predictors of Norway spruce resistance to bark beetles and their associated fungi. The induced reaction was characterized by a slight decrease of tanning ability and an increase of (+)-catechin concentration, which confirmed our previous observations. The relative resistance of the clones was first predicted using the predictors previously proposed. In addition, the first axis of the principal component analysis describing the phenolic content of all clones was used as a synthetic predictor (resistance axis). Related variables were also tested as predictors. Actual resistance of each clone was then measured, using mass inoculations of C. polonica, and was compared with the predictions. Four predictors were so validated: the resistance axis, tanning ability and isorhapontin concentration in uninoculated phloem, and (+)-catechin concentration in the phloem 6 days after its inoculation. Phloem phenolic composition could thus be used to predict Norway spruce resistance to bark beetles and their associated fungi.

International audience; In poplar, we identified proteins homologous to yeast proteins involved in osmosensing multistep phosphorelay Sln1p‐Ypd1p‐Ssk1p. This finding led us to speculate that Populus cells could sense osmotic stress by a similar mechanism. This study focuses on first and second protagonists of this possible pathway: a histidine‐aspartate kinase (HK1), putative osmosensor and histidine phosphotransfer proteins (HPt1 to 10), potential partners of this HK. Characterization of HK1 showed its ability to homodimerize in two‐hybrid tests and to act as an osmosensor with a kinase activity in yeast, by functional complementation of sln1Δ sho1Δ strain. Moreover, in plant cells, plasma membrane localization of HK1 is shown. Further analysis on HPts allowed us to isolate seven new cDNAs, leading to a total of 10 different HPts identified in poplar. Interaction tests showed that almost all HPts can interact with HK1, but two of them exhibit stronger interactions, suggesting a preferential partnership in poplar. The importance of the phosphorylation status in these interactions has been investigated with two‐hybrid tests carried out with mutated HK1 forms. Finally, in planta co‐expression analysis of genes encoding these potential partners revealed that only three HPts are co‐expressed with HK1 in different poplar organs. This result reinforces the hypothesis of a partnership between HK1 and these three preferential HPts in planta. Taken together, these results shed some light on proteins partnerships that could be involved in the osmosensing pathway in Populus.

†Background and Aims Extreme water stress episodes induce tree mortality, but the physiological mechanisms causing tree death are still poorly understood. This study tests the hypothesis that a potted tree’s ability to survive extreme monotonic water stress is determined by the cavitation resistance of its xylem tissue. †Methods Two species were selected with contrasting cavitation resistance (beech and poplar), and potted juvenile trees were exposed to a range of water stresses, causing up to 100 % plant death. †KeyResults The lethal dose of water stress, defined as the xylem pressure inducing 50 % mortality, differed sharply across species (1.75 and 4.5 MPa in poplar and beech, respectively). However, the relationships between tree mortality and the degree of cavitation in the stems were similar, with mortality occurring suddenly when .90 % cavitation had occurred. †Conclusions Overall, the results suggest that cavitation resistance is a causal factor of tree mortality under extreme drought conditions.

International audience; Short rotation coppice (SRC) willow plantations may play an important role in the future for biomass production purposes. However, the high planting density schemes combined with the frequent harvests occurring in such plantations could rapidly deplete soil resources. The use of genotypes able to produce greater amounts of biomass by using the least water and nutrients may help mitigating this risk. This study aimed at assessing among six willow genotypes (1) the variability of traits related to productivity (e.g. aboveground dry biomass or stem height), leaf and wood nitrogen (N) contents, N exportation rate and water-use efficiency (WUE) as estimated through bulk leaf carbon isotope discrimination (Δ13C), (2) the relationships among traits, and (3) the plasticity of these traits and of the relationships among them across different sites. The six genotypes were grown under SRC at three sites in northern France differing primarily in pedoclimatic characteristics for two years. A significant genotypic variability was found for all traits, except for the N exportation rate. The pedoclimatic context impacted the values of all traits, and the genotypic ranking for traits related to productivity and for Δ13C. Δ13C was negatively correlated with total shoot dry biomass and/or height irrespective of the site, meaning that the most productive genotypes were also the most efficient to use water. In conclusion, no antagonism was detected between biomass production and WUE. The most productive and most water-use efficient genotypes were the ones responsible for the highest nitrogen removal from the plantation during harvest.

International audience; Poplar coppice plantations for biomass production can be conducted under either short rotation coppice (SRC) or short rotation forestry (SRF) systems, depending on planting density and rotation length. It is likely that differences in planting density affect tree physiology through competition for resource acquisition, including light, water and nutrients. In this paper, we hypothesized that the effects of planting density on growth and water-use efficiency (assessed through bulk leaf carbon isotope discrimination, Delta C-13) in poplar depend on site characteristics in terms of soil fertility and water availability. To test this hypothesis, 56 Populus deltoides x P. nigra genotypes were planted under both SRC and SRF and replicated at two sites differing for pedoclimatic conditions. At the most favorable site for growth, trees grown at the higher density (SRC) displayed higher stem height, lower stem circumference, higher specific leaf area, higher mass-based leaf nitrogen contents and higher Delta C-13, indicating that increased tree density mainly accentuated competition for light. Under less favorable conditions, trees grown under SRC still displayed lower stem circumference, higher specific leaf area and higher mass-based leaf nitrogen contents. However, stem height remained unaffected by increasing planting density while Delta C-13 was lower, likely because of increased competition for water availability. Genotypic rankings across planting densities were overall conserved while they were significantly modified across sites, suggesting that rankings for genotypic performances were much less affected by planting density than by site. Realized growth measured after 2 years (height and circumference) was weakly correlated with Delta C-13, but a negative relationship between Delta C-13 and growing season leaf increment rate was observed in most cases. The absence of trade-off between growth and water-use efficiency combined with the large genotypic variations observed for these traits confirms the potential for selecting genotypes with high water-use efficiency without counter-selecting on biomass production in P. deltoides x P. nigra.

Background Hybrid poplars species are candidates for biomass production but breeding efforts are needed to combine productivity and water use efficiency in improved cultivars. The understanding of the genetic architecture of growth in poplar by a Quantitative Trait Loci (QTL) approach can help us to elucidate the molecular basis of such integrative traits but identifying candidate genes underlying these QTLs remains difficult. Nevertheless, the increase of genomic information together with the accessibility to a reference genome sequence (Populus trichocarpa Nisqually-1) allow to bridge QTL information on genetic maps and physical location of candidate genes on the genome. The objective of the study is to identify QTLs controlling productivity, architecture and leaf traits in a P. deltoides x P. trichocarpa F1 progeny and to identify candidate genes underlying QTLs based on the anchoring of genetic maps on the genome and the gene ontology information linked to genome annotation. The strategy to explore genome annotation was to use Gene Ontology enrichment tools to test if some functional categories are statistically over-represented in QTL regions. Results Four leaf traits and 7 growth traits were measured on 330 F1 P. deltoides x P. trichocarpa progeny. A total of 77 QTLs controlling 11 traits were identified explaining from 1.8 to 17.2% of the variation of traits. For 58 QTLs, confidence intervals could be projected on the genome. An extended functional annotation was built based on data retrieved from the plant genome database Phytozome and from an inference of function using homology between Populus and the model plant Arabidopsis. Genes located within QTL confidence intervals were retrieved and enrichments in gene ontology (GO) terms were determined using different methods. Significant enrichments were found for all traits. Particularly relevant biological processes GO terms were identified for QTLs controlling number of sylleptic branches: intervals were enriched in GO terms of biological process like ‘ripening’ and ‘adventitious roots development’. Conclusion Beyond the simple identification of QTLs, this study is the first to use a global approach of GO terms enrichment analysis to fully explore gene function under QTLs confidence intervals in plants. This global approach may lead to identification of new candidate genes for traits of interest.

DNA methylation is involved in the control of plant development and adaptation to the environment through modifications of chromatin compaction and gene expression. In poplar (Populus trichocarpa), a perennial plant, variations in DNA methylation have been reported between genotypes and tissues or in response to drought. Nevertheless, the relationships between gene-body DNA methylation, gene expression and chromatin compaction still need clarification. Here, DNA methylation was mapped in the noncondensed chromatin fraction from P. trichocarpa shoot apical meristematic cells, the center of plant morphogenesis, where DNA methylation variations could influence the developmental trajectory. DNase I was used to isolate the noncondensed chromatin fraction. Methylated sequences were immunoprecipitated, sequenced using Illumina/Solexa technology and mapped on the v2.0 poplar genome. Bisulfite sequencing of candidate sequences was used to confirm mapping data and to assess cytosine contexts and methylation levels. While the methylated DNase I hypersensitive site fraction covered 1.9% of the poplar genome, it contained sequences corresponding to 74% of poplar gene models, mostly exons. The level and cytosine context of gene-body DNA methylation varied with the structural characteristics of the genes. Taken together, our data show that DNA methylation is widespread and variable among genes in open chromatin of meristematic cells, in agreement with a role in their developmental trajectory.

Background In plants, the multistep phosphorelay signaling pathway mediates responses to environmental factors and plant hormones. This system is composed of three successive partners: hybrid Histidine-aspartate Kinases (HKs), Histidine-containing Phosphotransfer proteins (HPts), and Response Regulators (RRs). Among the third partners, B-type RR family members are the final output elements of the pathway; they act as transcription factors and clearly play a pivotal role in the early response to cytokinin in Arabidopsis. While interactions studies between partners belonging to the multistep phosphorelay system are mainly focused on protagonists involved in cytokinin or ethylene pathways, very few reports are available concerning partners of osmotic stress signaling pathway. Results In Populus, we identified eight B-type RR proteins, RR12-16, 19, 21 and 22 in the Dorskamp genotype. To assess HPt/B-type RR interactions and consequently determine potential third partners in the osmosensing multistep phosphorelay system, we performed global yeast two-hybrid (Y2H) assays in combination with Bimolecular Fluorescence Complementation (BiFC) assays in plant cells. We found that all B-type RRs are able to interact with HPt predominant partners (HPt2, 7 and 9) of HK1, which is putatively involved in the osmosensing pathway. However, different profiles of interaction are observed depending on the studied HPt. HPt/RR interactions displayed a nuclear localization, while the nuclear and cytosolic localization of HPt and nuclear localization of RR proteins were validated. Although the nuclear localization of HPt/RR interaction was expected, this work constitutes the first evidence of such an interaction in plants. Furthermore, the pertinence of this partnership is reinforced by highlighting a co-expression of B-type RR transcripts and the other partners (HK1 and HPts) belonging to a potential osmosensing pathway. Conclusion Based on the interaction studies between identified B-type RR and HPt proteins, and the co-expression analysis of transcripts of these potential partners in poplar organs, our results favor the model that RR12, 13, 14, 16 and 19 are able to interact with the main partners of HK1, HPt2, 7 and 9, and this HPt/RR interaction occurs within the nucleus. On the whole, the five B-type RRs of interest could be third protagonists putatively involved in the osmosensing signaling pathway in Populus.

Genetic differences in δ¹³C (isotopic composition of dry matter carbon) have been evidenced among poplar genotypes at juvenile stages. To check whether such differences were maintained with age in trees growing in plantations, we investigated the time course of δ¹³C as recorded in annual tree rings from different genotypes growing at three sites in southwestern France and felled at ∼15-17 years. Wood cores were cut from tree discs to record the time course of annual basal area increment (BAI). The isotopic ratio δ¹³C was recorded in bulk wood and in extracted cellulose from the annual rings corresponding to the period 1996-2005. Discrimination against ¹³C between atmosphere and tissues (Δ¹³C) was computed by taking into account the inter-annual time course of δ¹³C in the atmosphere. Annual BAI increased steadily and stabilized at about 8 years. An offset in δ¹³C of ∼1‰ was recorded between extracted cellulose and bulk wood. It was relatively stable among genotypes within sites but varied among sites and increased slightly with age. Site effects as well as genotype differences were detected in Δ¹³C recorded from the cellulose fraction. Absolute values as well as the genotype ranking of Δ¹³C remained stable with age in the three sites. Genotype means of Δ¹³C were not correlated to annual BAI. We conclude that genotypic differences of Δ¹³C occur in older poplar trees in plantations, and that the differences as well as the genotype ranking remain stable while trees age until harvest.

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International audience; - Introduction Because of its vigorous growth, poplar can play an important role for sustainable production of woody biomass to cover renewable energy needs. Hence, the selection of suitable genotypes has to be based on relevant traits, among which intrinsic water use efficiency (Wi, estimated through leaf carbon isotope discrimination, Δ) may be a key trait. Besides a large genetic variation in Δ among the frequently planted poplar hybrids, the use of Δ in deployment or breeding programmes requires insights in the robustness of the genotype ranking for Δ across environments and years. - Methods Two F1 full-sib families of poplar (Populus deltoides × Populus nigra and Populus deltoides × Populus trichocarpa) were grown at two sites in Europe, i.e. northern Italy and central France. For each family, leaf samples from 31 F1 genotypes collected during different field studies were used (1) to assess the effect of genotype, site and year on Δ in leaves, as well as their mutual interactions, and (2) to elucidate the relationships between Δ, leaf morphology and tree dimensions. - Results Under the well-watered conditions of our study, a low to moderate genetic variability was observed in the two poplar families. Within-family broad-sense heritability values ranged from 0 to 0.49. The ranking of genotypes for Δ was more stable between years than between sites. - Conclusions The study confirmed the occurrence of some degree of genetic variability ofΔ in the studied poplar families and the possibility to identify genotypes with low, stable Δ values across years. However, the significant genotype-by-site interactions in our study suggest that selection for larger water use efficiency or lower Δ in these families has to consider specific responses in different environments.

International audience; An epigenetic control of vernalization has been demonstrated in annual plants such as Arabidopsis and cereals, but the situation remains unclear in biennial plants such as sugar beet that has an absolute requirement for vernalization. The role of DNA methylation in flowering induction and the identification of corresponding target loci also need to be clarified. In this context, sugar beet (Beta vulgaris altissima) genotypes differing in bolting tolerance were submitted to various bolting conditions such as different temperatures and/or methylating drugs. DNA hypomethylating treatment was not sufficient to induce bolting while DNA hypermethylation treatment inhibits and delays bolting. Vernalizing and devernalizing temperatures were shown to affect bolting as well as DNA methylation levels in the shoot apical meristem. In addition, a negative correlation was established between bolting and DNA methylation. Genotypes considered as resistant or sensitive to bolting could also be distinguished by their DNA methylation levels. Finally, sugar beet homologues of the Arabidopsis vernalization genes FLC and VIN3 exhibited distinct DNA methylation marks during vernalization independently to the variations of global DNA methylation. These vernalization genes also displayed differences in mRNA accumulation and methylation profiles between genotypes resistant or sensitive to bolting. Taken together, the data suggest that the time course and amplitude of DNA methylation variations are critical points for the induction of sugar beet bolting and represent an epigenetic component of the genotypic bolting tolerance, opening up new perspectives for sugar beet breeding.

Tests were carried out to determine whether variations in the hydraulic architecture of eight Populus deltoides×Populus nigra genotypes could be related to variations in leaf function and growth performance. Measurements were performed in a coppice plantation on 1-year-old shoots under optimal irrigation. Hydraulic architecture was characterized through estimates of hydraulic efficiency (the ratio of conducting sapwood area to leaf area, A(X):A(L); leaf- and xylem-specific hydraulic conductance of defoliated shoots, k(SL) and k(SS), respectively; apparent whole-plant leaf-specific hydraulic conductance, k(plant)) and xylem safety (water potential inducing 50% loss in hydraulic conductance). The eight genotypes spanned a significant range of k(SL) from 2.63 kg s(-1) m(-2) MPa(-1) to 4.18 kg s(-1) m(-2) MPa(-1), variations being mostly driven by k(SS) rather than A(X):A(L). There was a strong trade-off between hydraulic efficiency and xylem safety. Values of k(SL) correlated positively with k(plant), indicating that high-pressure flowmeter (HPFM) measurements of stem hydraulic efficiency accurately reflected whole-plant water transport efficiency of field-grown plants at maximum transpiration rate. No clear relationship could be found between hydraulic efficiency and either net CO(2) assimilation rates, water-use efficiency estimates (intrinsic water-use efficiency and carbon isotope discrimination against (13)C), or stomatal characteristics (stomatal density and stomatal pore area index). Estimates of hydraulic efficiency were negatively associated with relative growth rate. This unusual pattern, combined with the trade-off observed between hydraulic efficiency and xylem safety, provides the rationale for the positive link already reported between relative growth rate and xylem safety among the same eight P. deltoides×P. nigra genotypes.

Tests were carried out to determine whether variations in the hydraulic architecture of eight Populus deltoides×Populus nigra genotypes could be related to variations in leaf function and growth performance. Measurements were performed in a coppice plantation on 1-year-old shoots under optimal irrigation. Hydraulic architecture was characterized through estimates of hydraulic efficiency (the ratio of conducting sapwood area to leaf area, AX:AL; leaf- and xylem-specific hydraulic conductance of defoliated shoots, kSL and kSS, respectively; apparent whole-plant leaf-specific hydraulic conductance, kplant) and xylem safety (water potential inducing 50% loss in hydraulic conductance). The eight genotypes spanned a significant range of kSL from 2.63 kg s−1 m−2 MPa−1 to 4.18 kg s−1 m−2 MPa−1, variations being mostly driven by kSS rather than AX:AL. There was a strong trade-off between hydraulic efficiency and xylem safety. Values of kSL correlated positively with kplant, indicating that high-pressure flowmeter (HPFM) measurements of stem hydraulic efficiency accurately reflected whole-plant water transport efficiency of field-grown plants at maximum transpiration rate. No clear relationship could be found between hydraulic efficiency and either net CO2 assimilation rates, water-use efficiency estimates (intrinsic water-use efficiency and carbon isotope discrimination against 13C), or stomatal characteristics (stomatal density and stomatal pore area index). Estimates of hydraulic efficiency were negatively associated with relative growth rate. This unusual pattern, combined with the trade-off observed between hydraulic efficiency and xylem safety, provides the rationale for the positive link already reported between relative growth rate and xylem safety among the same eight P. deltoides×P. nigra genotypes.

International audience; Genetic differences in delta C-13 (isotopic composition of dry matter carbon) have been evidenced among poplar genotypes at juvenile stages. To check whether such differences were maintained with age in trees growing in plantations, we investigated the time course of delta C-13 as recorded in annual tree rings from different genotypes growing at three sites in southwestern France and felled at similar to 15-17 years. Wood cores were cut from tree discs to record the time course of annual basal area increment (BAI). The isotopic ratio delta C-13 was recorded in bulk wood and in extracted cellulose from the annual rings corresponding to the period 1996-2005. Discrimination against C-13 between atmosphere and tissues (delta C-13) was computed by taking into account the inter-annual time course of delta C-13 in the atmosphere. Annual BAI increased steadily and stabilized at about 8 years. An offset in delta C-13 of similar to 1 parts per thousand was recorded between extracted cellulose and bulk wood. It was relatively stable among genotypes within sites but varied among sites and increased slightly with age. Site effects as well as genotype differences were detected in delta C-13 recorded from the cellulose fraction. Absolute values as well as the genotype ranking of delta C-13 remained stable with age in the three sites. Genotype means of delta C-13 were not correlated to annual BAI. We conclude that genotypic differences of delta C-13 occur in older poplar trees in plantations, and that the differences as well as the genotype ranking remain stable while trees age until harvest.

We present results and insights gained from the characterization of the hydraulic architecture of eight field-grown Populus deltoides × Populus nigra genotypes, already known to differ in terms of growth performance, water-use and xylem anatomy (Marron et al. 2005, Monclus et al. 2005, 2006; Fichot et al. 2009). Under well-watered conditions, we provide evidence that (1) there are wide genotypic variations for hydraulic efficiency inferred from the leaf specific hydraulic conductance (kSL) and for resistance to drought-induced cavitation inferred from the water potential inducing 50% loss in hydraulic conductance, (2) a strong trade-off between water transport efficiency and xylem safety occurs at the whole plant level, indicating that genotypes with lower kSL apparently compensate the greater risks of embolism by building a safer xylem, (3) higher relative growth rate is tightly associated with lower whole-plant hydraulic efficiency and higher resistance to cavitation, (4) estimates of leaf water-use efficiency (intrinsic water use efficiency and carbon isotope discrimination against 13C) are weakly associated with whole-plant hydraulics. Under limited water supply, the characterization of xylem resistance to cavitation provides additional evidence that (5) xylem safety acclimates to moderate drought in a genotype-dependent manner, (6) there is no clear relationship between xylem safety and either xylem water transport efficiency or xylem biomechanics, contrary to what is observed at inter-specific scales. Our results provide interesting insights for the possible role of whole plant hydraulic architecture and xylem hydraulics in mediating key aspects of whole-plant physiology in poplar, such as growth and water-use efficiency. Finally, our results also indicate that there may be great opportunity for intra-specific comparisons to unravel specific issues that cannot be fully addressed by interspecific comparisons.

We examined the relationships between xylem resistance to cavitation and 16 structural and functional traits across eight unrelated Populus deltoides x Populus nigra genotypes grown under two contrasting water regimes. The xylem water potential inducing 50% loss of hydraulic conductance (Psi(50)) varied from -1.60 to -2.40 MPa. Drought-acclimated trees displayed a safer xylem, although the extent of the response was largely genotype dependent, with Psi(50) being decreased by as far as 0.60 MPa. At the tissue level, there was no clear relationship between xylem safety and either xylem water transport efficiency or xylem biomechanics; the only structural trait to be strongly associated with Psi(50) was the double vessel wall thickness, genotypes exhibiting a thicker double wall being more resistant. At the leaf level, increased cavitation resistance was associated with decreased stomatal conductance, while no relationship could be identified with traits associated with carbon uptake or bulk leaf carbon isotope discrimination, a surrogate of intrinsic water-use efficiency. At the whole-plant level, increased safety was associated with higher shoot growth potential under well-irrigated regime only. We conclude that common trade-offs between xylem resistance to cavitation and other physiological traits that are observed across species may not necessarily hold true at narrower scales.