RNAi suppression of DNA methylation affects the drought stress response and genome integrity in transgenic poplar.

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 downregulation of the chromatin remodeler DECREASED IN DNA METHYLATION 1 (DDM1) in poplar (Populus tremula × 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.

Winter-dormant shoot apical meristem in poplar trees shows environmental epigenetic memory.

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.

Changes in the epigenome and transcriptome of the poplar shoot apical meristem in response to water availability affect preferentially hormone pathways.

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.

Insights into B-type RR members as signaling partners acting downstream of HPt partners of HK1 in the osmotic stress response in Populus.

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.

Genetic variation for leaf morphology, leaf structure and leaf carbon isotope discrimination in European populations of black poplar (Populus nigra L.).

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 (Δ(13)C) in natural populations of black poplar. Twenty-four to 62 genotypes were sampled in nine metapopulations covering a latitudinal range from 48 °N to 42 °N in France and in Italy and grown in two common gardens at Orléans (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 × environment (G × E) interaction variance being comparable to or even greater than genetic effects. For LA, G × 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 Δ(13)C. The nine P. nigra metapopulations were highly differentiated for LA, as attested by the high coefficient of genetic differentiation (QST = 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 Δ(13)C (QST = 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.

Stem xylem resistance to cavitation is related to xylem structure but not to growth and water-use efficiency at the within-population level in Populus nigra L.

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.

Vulnerability to drought-induced cavitation in poplars: synthesis and future opportunities.

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.

Vapour pressure deficit during growth has little impact on genotypic differences of transpiration efficiency at leaf and whole-plant level: an example from Populus nigra†L.

Poplar genotypes differ in transpiration efficiency (TE) at leaf and whole-plant level under similar conditions. We tested whether atmospheric vapour pressure deficit (VPD) affected TE to the same extent across genotypes. Six Populus nigra genotypes were grown under two VPD. We recorded (1) (13)C content in soluble sugars; (2) (18)O enrichment in leaf water; (3) leaf-level gas exchange; and (4) whole-plant biomass accumulation and water use. Whole-plant and intrinsic leaf TE and (13)C content in soluble sugars differed significantly among genotypes. Stomatal conductance contributed more to these differences than net CO2 assimilation rate. VPD increased water use and reduced whole-plant TE. It increased intrinsic leaf-level TE due to a decline in stomatal conductance. It also promoted higher (18)O enrichment in leaf water. VPD had no genotype-specific effect. We detected a deviation in the relationship between (13)C in leaf sugars and (13)C predicted from gas exchange and the standard discrimination model. This may be partly due to genotypic differences in mesophyll conductance, and to its lack of sensitivity to VPD. Leaf-level (13)C discrimination was a powerful predictor of the genetic variability of whole-plant TE irrespective of VPD during growth.

Characterization of histidine-aspartate kinase HK1 and identification of histidine phosphotransfer proteins as potential partners in a Populus multistep phosphorelay.

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.

Genotype differences in 13C discrimination between atmosphere and leaf matter match differences in transpiration efficiency at leaf and whole-plant levels in hybrid Populus deltoides x nigra.

(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.

Methylome of DNase I sensitive chromatin in Populus trichocarpa shoot apical meristematic cells: a simplified approach revealing characteristics of gene-body DNA methylation in open chromatin state.

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.

Identification of five B-type response regulators as members of a multistep phosphorelay system interacting with histidine-containing phosphotransfer partners of Populus osmosensor.

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.

Integrating genome annotation and QTL position to identify candidate genes for productivity, architecture and water-use efficiency in Populus spp.

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.

Variations in bulk leaf carbon isotope discrimination, growth and related leaf traits among three Populus nigra L. populations.

The ongoing global change could be an additional threat to the establishment and the long-term survival of Populus nigra L., an emblematic European riparian species. With the general aim of gaining insights into the adaptive potential of this species, we (i) quantified variations within and among three French P. nigra populations for key physiological attributes, i.e., water-use efficiency (assessed from bulk leaf carbon isotope discrimination, Δ(13)C), growth performance and related leaf traits, (ii) examined genotype and population by environment interactions, and (iii) explored the relationship between Δ(13)C and growth. Thirty genotypes were sampled in each of three naturally established populations and grown in two different sites, Orléans (ORL) and Guémené-Penfao (GMN). In ORL, two similar plots were established and different watering regimes were applied in order to test for the drought response. Significant variations were observed for all traits within and among populations irrespective of site and watering. Trait variation was larger within than among populations. The effect of drought was neither genotype- nor population-dependent, contrary to the effect of site. The population ranking was maintained in all sites and watering regimes for the two most complex traits: Δ(13)C and growth. Moreover, these two traits were unrelated, which indicates that (i) water-use efficiency and growth are largely uncoupled in this species, and (ii) the environmental factors driving genetic structuration for Δ(13)C and growth act independently. The large variations found within populations combined with the consistent differences among populations suggest a large adaptive potential for P. nigra.

Time course of δ1¹³C in poplar wood: genotype ranking remains stable over the life cycle in plantations despite some differences between cellulose and bulk wood.

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.

Time course and amplitude of DNA methylation in the shoot apical meristem are critical points for bolting induction in sugar beet and bolting tolerance between genotypes.

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.

Hydraulic efficiency and coordination with xylem resistance to cavitation, leaf function, and growth performance among eight unrelated Populus deltoidesxPopulus nigra hybrids.

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.

Common trade-offs between xylem resistance to cavitation and other physiological traits do not hold among unrelated Populus deltoides x Populus nigra hybrids.

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.

Xylem anatomy correlates with gas exchange, water-use efficiency and growth performance under contrasting water regimes: evidence from Populus deltoides x Populus nigra hybrids.

Six Populus deltoides Bartr. ex Marsh. x 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 microm and from 7.5 to 10.5 microm, 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.

Leaf proteome analysis of eight Populus xeuramericana genotypes: genetic variation in drought response and in water-use efficiency involves photosynthesis-related proteins.

Genetic variation of leaf proteome in drought response was investigated among eight Populus xeuramericana genotypes contrasting for their leaf carbon isotope discrimination (Delta), an estimate of intrinsic water-use efficiency. Plants were grown in open field on two similar plots. Drought was induced by an 86-day irrigation cessation on one plot, whereas a second plot remained regularly irrigated. Using 2-DE, 863 reproducible spots were detected; about 60% presented at least one significant effect i.e. treatment, genotype and/or genotype by treatment interaction effect. A significant genotype by treatment interaction was detected for 62 reliably identified proteins among which, about 65% consisted in chloroplast-associated proteins either involved in the Calvin cycle or in the electron-transport chains. The other proteins were involved in oxidative stress, amino acid or protein metabolisms. Correlations between protein abundance and Delta variations were found for 45 reliably identified proteins. The abundance of ribulose-1,5-bisphosphate carboxylase/oxygenase activase isoforms scaled negatively with Delta regardless of the treatment, suggesting that a large intrinsic water-use efficiency could be due to higher abundance of ribulose-1,5-bisphosphate carboxylase/oxygenase activase. Under control condition, abundance of enzymes involved in carbon fixation was also negatively correlated with Delta, whereas abundance of enzymes involved in photorespiration or respiration was positively correlated with Delta.