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Plant hydraulic traits related to leaf drought tolerance, like the water potential at turgor loss point (TLP) and the water potential inducing 50% loss of hydraulic conductance (P 50), are extremely useful to predict the potential impacts of drought on plants. While novel techniques have allowed the inclusion of TLP in studies targeting a large group of species, fast and reliable protocols to measure leaf P 50 are still lacking. Recently, the optical method coupled with the gas injection (GI) technique has been proposed as a possibility to speed up the P 50 estimation. Here, we present a comparison of leaf optical vulnerability curves (OVcs) measured in three woody species, namely Acer campestre (Ac), Ostrya carpinifolia (Oc) and Populus nigra (Pn), based on bench dehydration (BD) or GI of detached branches. For Pn, we also compared optical data with direct micro-computed tomography (micro-CT) imaging in both intact saplings and cut shoots subjected to BD. Based on the BD procedure, Ac, Oc and Pn had P 50 values of −2.87, −2.47 and −2.11 MPa, respectively, while the GI procedure overestimated the leaf vulnerability (−2.68, −2.04 and −1.54 MPa for Ac, Oc and Pn, respectively). The overestimation was higher for Oc and Pn than for Ac, likely reflecting the species-specific vessel lengths. According to micro-CT observations performed on Pn, the leaf midrib showed none or very few embolized conduits at −1.2 MPa, consistent with the OVcs obtained with the BD procedure but at odds with that derived on the basis of GI. Overall, our data suggest that coupling the optical method with GI might not be a reliable technique to quantify leaf hydraulic vulnerability since it could be affected by the 'open-vessel' artifact. Accurate detection of xylem embolism in the leaf vein network should be based on BD, preferably of intact up-rooted plants. [ABSTRACT FROM AUTHOR]

To investigate the pivotal physiological processes modulating lead (Pb) tolerance capacities of poplars, the saplings of two contrasting poplar species, Populus × canescens with high Pb sensitivity and Populus nigra with relatively low Pb sensitivity, were treated with either 0 or 8 mM Pb for 6 weeks. Lead was absorbed by the roots and accumulated massively in the roots and leaves, leading to overproduction of reactive oxygen species, reduced photosynthesis and biomass in both poplar species. Particularly, the tolerance index of P. × canescens was significantly lower than that of P. nigra. Moreover, the physiological responses including the concentrations of nutrient elements, thiols, organic acids, phytohormones and nonenzymatic antioxidants, and the activities of antioxidative enzymes in the roots and leaves were different between the two poplar species. Notably, the differences in concentrations of nutrient elements, organic acids and phytohormones were remarkable between the two poplar species. A further evaluation of the Pb tolerance-related physiological processes showed that the change of 'sulfur (S) metabolism' in the roots was greater, and that of 'organic acid accumulation' in the roots and 'phytohormone regulation' in the leaves were markedly smaller in P. × canescens than those in P. nigra. These results suggest that there are differences in Pb tolerance capacities between P. × canescens and P. nigra , which is probably associated with their contrasting physiological responses to Pb stress, and that S metabolism, organic acid accumulation and phytohormone regulation are probably the key physiological processes modulating the different Pb tolerance capacities between the two poplar species. [ABSTRACT FROM AUTHOR]

Many terrestrial biosphere models depend on an understanding of the relationship between stomatal conductance and photosynthesis. However, unlike the measurement of photosynthetic parameters, such as the maximum carboxylation capacity, where standard methods (e.g. CO2 response or AC i curves) are widely accepted, a consensus method for empirically measuring parameters representing stomatal response has not yet emerged. Most models of stomatal response to environment represent stomatal conductance as being bounded by a lower intercept parameter (g 0), and linearly scaled based on a multivariate term described by the stomatal slope parameter (g 1). Here we employ the widely used Unified Stomatal Optimization model, to test whether g 1 and g 0 parameters are impacted by the choice of measurement method, either on an intact branch or a cut branch segment stored in water. We measured paired stomatal response curves on intact and excised branches of a hybrid poplar clone (Populus deltoides Bartr. ×  Populus nigra L. OP367), measured twice over a diurnal period. We found that predawn branch excision did not significantly affect measured g 0 and g 1 when measured within 4 h of excision. Measurement in the afternoon resulted in significantly higher values of g 1 and lower values of g 0, with values changing by 55% and 56%, respectively. Excision combined with afternoon measurement resulted in a marked effect on parameter estimates, with g 1 increasing 89% from morning to afternoon and a 25% lower g 1 for cut branches than those measured in situ. We also show that in hybrid poplar the differences in parameter estimates obtained from plants measured under different conditions can directly impact models of canopy function, reducing modeled transpiration by 18% over a simulated 12.5-h period. Although these results are only for a single isohydric woody species, our findings suggest that stomatal optimality parameters may not remain constant throughout the day. [ABSTRACT FROM AUTHOR]

Low temperature is a major stress that severely affects plant growth and development. Inducer of CBF expression 1 (ICE1) plays a key role in plant cold tolerance by regulating the expression of cold stress-responsive genes. In the present study, we characterized the function and underlying regulatory mechanism of PsnICE1 from Xiaohei poplar (Populus simonii × Populus nigra). PsnICE1 was significantly induced in response to cold stress in the roots, stems and leaves. PsnICE1 proteins were found to localize to the nucleus and exert transactivation activity via their N-terminal transactivation domain. Compared with non-transgenic poplar, transgenic poplar overexpressing PsnICE1 showed substantially enhanced tolerance to cold stress, with higher survival rates and antioxidant enzyme activity levels and reduced reactive oxygen species (ROS) accumulation. In contrast, plants with RNA inhibition-mediated silencing of PsnICE1 showed the opposite phenotype. PsnICE1 can bind to H-box element and abscisic acid-responsive element (ABRE), and more importantly, it mainly binds to IBS1 (a newly discovered cis-acting element) and E-box elements to regulate stress-related genes involved in ROS scavenging. Overall, these results indicated that PsnICE1 functions as a positive regulator of cold tolerance and serves as a potential candidate gene for plant cold tolerance improvement via molecular breeding. [ABSTRACT FROM AUTHOR]

Drought compromises plant's ability to replace transpired water vapor with water absorbed from the soil, leading to extensive xylem dysfunction and causing plant desiccation and death. Short-term plant responses to drought rely on stomatal closure, and on the plant's ability to recover hydraulic functioning after drought relief. We hypothesize a key role for abscisic acid (ABA) not only in the control of stomatal aperture, but also in hydraulic recovery. Young plants of Populus nigra L. were used to investigate possible relationships among ABA, non-structural carbohydrates (NSC) and xylem hydraulic function under drought and after re-watering. In Populus nigra L. plants subjected to drought, water transport efficiency and hydraulic recovery after re-watering were monitored by measuring the percentage loss of hydraulic conductivity (PLC) and stem specific hydraulic conductivity (K stem). In the same plants ABA and NSC were quantified in wood and bark. Drought severely reduced stomatal conductance (g L) and markedly increased the PLC. Leaf and stem water potential, and stem hydraulic efficiency fully recovered within 24 h after re-watering, but g L values remained low. After re-watering, we found significant correlations between changes in ABA content and hexoses concentration both in wood and bark. Our findings suggest a role for ABA in the regulation of stem carbohydrate metabolism and starch mobilization upon drought relief, possibly promoting the restoration of xylem transport capacity. [ABSTRACT FROM AUTHOR]

Soil moisture heterogeneity in the root zone is common both during the establishment of tree seedlings and in experiments aiming to impose semi-constant soil moisture deficits, but its effects on regulating plant water use compared with homogenous soil drying are not well known in trees. Pronounced vertical soil moisture heterogeneity was imposed on black poplar (Populus nigra L.) grown in soil columns by altering irrigation frequency, to test whether plant water use, hydraulic responses, root phytohormone concentrations and root xylem sap chemical composition differed between wet (well-watered, WW), and homogeneously (infrequent deficit irrigation, IDI) and heterogeneously dry soil (frequent deficit irrigation, FDI). At the same bulk soil water content, FDI plants had greater water use than IDI plants, probably because root abscisic acid (ABA) concentration was low in the upper wetter layer of FDI plants, which maintained root xylem sap ABA concentration at basal levels in contrast with IDI. Soil drying did not increase root xylem concentration of any other hormone. Nevertheless, plant-to-plant variation in xylem jasmonic acid (JA) concentration was negatively related to leaf stomatal conductance within WW and FDI plants. However, feeding detached leaves with high (1200 nM) JA concentrations via the transpiration stream decreased transpiration only marginally. Xylem pH and sulphate concentration decreased in FDI plants compared with well-watered plants. Frequent deficit irrigation increased root accumulation of the cytokinin trans -zeatin (t Z), especially in the dry lower layer, and of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), in the wet upper soil layer. Root hormone accumulation might explain the maintenance of high root hydraulic conductance and water use in FDI plants (similar to well-watered plants) compared with IDI plants. In irrigated tree crops, growers could vary irrigation scheduling to control water use by altering the hormone balance. [ABSTRACT FROM AUTHOR]

Background and Aims The advent of molecular breeding is advocated to improve the productivity and sustainability of second-generation bioenergy crops. Advanced molecular breeding in bioenergy crops relies on the ability to massively sample the genetic diversity. Genotyping-by-sequencing has become a widely adopted method for cost-effective genotyping. It basically requires no initial investment for design as compared with array-based platforms which have been shown to offer very robust assays. The latter, however, has the drawback of being limited to analyse only the genetic diversity accounted during selection of a set of polymorphisms and design of the assay. In contrast, genotyping-by-sequencing with random sampling of genomic loci via restriction enzymes or random priming has been shown to be fast and convenient but lacks the ability to target specific regions of the genome and to maintain high reproducibility across laboratories. Methods Here we present a first adoption of single-primer enrichment technology (SPET) which provides a highly efficient and scalable system to obtain targeted sequence-based large genotyping data sets, bridging the gaps between array-based systems and traditional sequencing-based protocols. To fully explore SPET performance, we conducted a benchmark study in ten Zea mays lines and a large-scale study of a natural black poplar population of 540 individuals with the aim of discovering polymorphisms associated with biomass-related traits. Key Results Our results showed the ability of this technology to provide dense genotype information on a customized panel of selected polymorphisms, while yielding hundreds of thousands of untargeted variable sites. This provided an ideal resource for association analysis of natural populations harbouring unexplored allelic diversities and structure such as in black poplar. Conclusion The improvement of sequencing throughput and the development of efficient library preparation methods has made it feasible to carry out targeted genotyping-by-sequencing experiments cost-competitively with either random complexity reduction systems or traditional array-based platforms, while maintaining the key advantages of both technologies. [ABSTRACT FROM AUTHOR]

Background and Aims Hydraulic and chemical signals operate in tandem to regulate systemic plant responses to drought. Transport of abscisic acid (ABA) through the xylem and phloem from the root to shoot has been suggested to serve as the main signal of water deficit. There is evidence that ABA and its ABA-glycosyl-ester (ABA-GE) are also formed in leaves and stems through the chloroplastic 2-C-methylerythritol-5-phosphate (MEP) pathway. This study aimed to evaluate how hormonal and hydraulic signals contribute to optimize stomatal (g s ), mesophyll (g m) and leaf hydraulic (K leaf) conductance under well-watered and water-stressed conditions in Populus nigra (black poplar) plants. In addition, we assessed possible relationships between ABA and soluble carbohydrates within the leaf and stem. Methods Plants were subjected to three water treatments: well-watered (WW), moderate stress (WS1) and severe stress (WS2). This experimental set-up enabled a time-course analysis of the response to water deficit at the physiological [leaf gas exchange, plant water relations, (K leaf)], biochemical (ABA and its metabolite/catabolite quantification in xylem sap, leaves, wood, bark and roots) and molecular (gene expression of ABA biosynthesis) levels. Key Results Our results showed strong coordination between g s , g m and K leaf under water stress, which reduced transpiration and increased intrinsic water use efficiency (WUEint). Analysis of gene expression of 9- cis -epoxycarotenoid dioxygenase (NCED) and ABA content in different tissues showed a general up-regulation of the biosynthesis of this hormone and its finely-tuned catabolism in response to water stress. Significant linear relationships were found between soluble carbohydrates and ABA contents in both leaves and stems, suggesting a putative function for this hormone in carbohydrate mobilization under severe water stress. Conclusions This study demonstrates the tight regulation of the photosynthetic machinery by levels of ABA in different plants organs on a daily basis in both well-watered and water stress conditions to optimize WUEint and coordinate whole plant acclimation responses to drought. [ABSTRACT FROM AUTHOR]

A major environmental pollution problem is the release into the atmosphere of particulate matter, including nanoparticles (NPs), which causes serious hazards to human and ecosystem health, particularly in urban areas. However, knowledge about the uptake, translocation and accumulation of NPs in plant tissues is almost completely lacking. The uptake of silver nanoparticles (Ag-NPs) and their transport and accumulation in the leaves, stems and roots of three different tree species, downy oak (Quercus pubescens Willd.), Scots pine (Pinus sylvestris L.) and black poplar (Populus nigra L.), were assessed. In the experiment, Ag-NPs were supplied separately to the leaves (via spraying, the foliar treatment) and roots (via watering, the root treatment) of the three species. Uptake, transport and accumulation of Ag were investigated through spectroscopy. The concentration of Ag in the stem was higher in the foliar than in the root treatment, and in poplar more than in oak and pine. Foliar treatment with Ag-NPs reduced aboveground biomass and stem length in poplars, but not in oaks or pines. Species-specific signals of oxidative stress were observed; foliar treatment of oak caused the accumulation of H2O2 in leaves, and both foliar and root treatments of poplar led to increased O2− in leaves. Ag-NPs affected leaf and root bacteria and fungi; in the case of leaves, foliar treatment reduced bacterial populations in oak and poplar and fungi populations in pine, and in the case of roots, root treatment reduced bacteria and increased fungi in poplar. Species-specific mechanisms of interaction, transport, allocation and storage of NPs in trees were found. We demonstrated definitively that NPs enter into the tree stem through leaves faster than through roots in all of the investigated tree species. [ABSTRACT FROM AUTHOR]

Wood is a renewable resource that can be employed for the production of second generation biofuels by enzymatic saccharification and subsequent fermentation. Knowledge on how the saccharification potential is affected by genotype-related variation of wood traits and drought is scarce. Here, we used three Populus nigra L. genotypes from habitats differing in water availability to (i) investigate the relationships between wood anatomy, lignin content and saccharification and (ii) identify genes and coexpressed gene clusters related to genotype and drought-induced variation in wood traits and saccharification potential. The three poplar genotypes differed in wood anatomy, lignin content and saccharification potential. Drought resulted in reduced cambial activity, decreased vessel and fiber lumina, and increased the saccharification potential. The saccharification potential was unrelated to lignin content as well as to most wood anatomical traits. RNA sequencing of the developing xylem revealed that 1.5% of the analyzed genes were differentially expressed in response to drought, while 67% differed among the genotypes. Weighted gene correlation network analysis identified modules of co-expressed genes correlated with saccharification potential. These modules were enriched in gene ontology terms related to cell wall polysaccharide biosynthesis and modification and vesicle transport, but not to lignin biosynthesis. Among the most strongly saccharification-correlated genes, those with regulatory functions, especially kinases, were prominent. We further identified transcription factors whose transcript abundances differed among genotypes, and which were co-regulated with genes for biosynthesis and modifications of hemicelluloses and pectin. Overall, our study suggests that the regulation of pectin and hemicellulose metabolism is a promising target for improving wood quality of second generation bioenergy crops. The causal relationship of the identified genes and pathways with saccharification potential needs to be validated in further experiments. [ABSTRACT FROM AUTHOR]

Isoprene is synthesized through the 2-C-methylerythritol-5-phosphate (MEP) pathway that also produces abscisic acid (ABA). Increases in foliar free ABA concentration during drought induce stomatal closure and may also alter ethylene biosynthesis. We hypothesized a role of isoprene biosynthesis in protecting plants challenged by increasing water deficit, by influencing ABA production and ethylene evolution. We performed a split-root experiment on Populus nigra L. subjected to three water treatments: well-watered (WW) plants with both root sectors kept at pot capacity, plants with both root compartments allowed to dry for 5 days (DD) and plants with one-half of the roots irrigated to pot capacity, while the other half did not receive water (WD). WD and WW plants were similar in photosynthesis, water relations, foliar ABA concentration and isoprene emission, whereas these parameters were significantly affected in DD plants: leaf isoprene emission increased despite the fact that photosynthesis declined by 85% and the ABA-glucoside/free ABA ratio decreased significantly. Enhanced isoprene biosynthesis in water-stressed poplars may have contributed to sustaining leaf ABA biosynthesis by keeping the MEP pathway active. However, this enhancement in ABA was accompanied by no change in ethylene biosynthesis, likely confirming the antagonistic role between ABA and ethylene. These results may indicate a potential cross-talk among isoprene, ABA and ethylene under drought. [ABSTRACT FROM AUTHOR]

Molecular regulation of growth must include spatial and temporal coupling of cell production and cell expansion. The underlying mechanisms, especially under environmental challenge, remain obscure. Spatial patterns of cell processes make the root apex well suited to deciphering stress signaling pathways, and to investigating both processes. Kinematics and RNA-sequencing were used to analyze the immediate growth response of hydroponically grown Populus nigra cuttings submitted to osmotic stress. About 7400 genes and unannotated transcriptionally active regions were differentially expressed between the division and elongation zones. Following the onset of stress, growth decreased sharply, probably due to mechanical effects, before recovering partially. Stress impaired cell expansion over the apex, progressively shortened the elongation zone, and reduced the cell production rate. Changes in gene expression revealed that growth reduction was mediated by a shift in hormone homeostasis. Osmotic stress rapidly elicited auxin, ethylene, and abscisic acid. When growth restabilized, transcriptome remodeling became complex and zone specific, with the deployment of hormone signaling cascades, transcriptional regulators, and stress-responsive genes. Most transcriptional regulations fit growth reduction, but stress also promoted expression of some growth effectors, including aquaporins and expansins. Together, osmotic stress interfered with growth by activating regulatory proteins rather than by repressing the machinery of expansive growth. [ABSTRACT FROM AUTHOR]

Summer droughts are likely to increase in frequency and intensity across Europe, yet long-lived trees may have a limited ability to tolerate drought. It is therefore critical that we improve our understanding of phenotypic plasticity to drought in natural populations for ecologically and economically important trees such as Populus nigra L. A common garden experiment was conducted using ~500 wild P. nigra trees, collected from 11 river populations across Europe. Phenotypic variation was found across the collection, with southern genotypes from Spain and France characterized by small leaves and limited biomass production. To examine the relationship between phenotypic variation and drought tolerance, six genotypes with contrasting leaf morphologies were subjected to a water deficit experiment. 'North eastern' genotypes were collected at wet sites and responded to water deficit with reduced biomass growth, slow stomatal closure and reduced water use efficiency (WUE) assessed by Δ13C. In contrast, 'southern' genotypes originating from arid sites showed rapid stomatal closure, improved WUE and limited leaf loss. Transcriptome analyses of a genotype from Spain (Sp2, originating from an arid site) and another from northern Italy (Ita, originating from a wet site) revealed dramatic differences in gene expression response to water deficit. Transcripts controlling leaf development and stomatal patterning, including SPCH, ANT, ER, AS1, AS2, PHB, CLV1, ERL1-3 and TMM, were down-regulated in Ita but not in Sp2 in response to drought. [ABSTRACT FROM AUTHOR]

Altering gene dosage through variation in gene copy number is a powerful approach to addressing questions regarding gene regulation, quantitative trait loci, and heterosis, but one that is not easily applied to sexually transmitted species. Elite poplar (Populus spp) varieties are created through interspecific hybridization, followed by clonal propagation. Altered gene dosage relationships are believed to contribute to hybrid performance. Clonal propagation allows for replication and maintenance of meiotically unstable ploidy or structural variants and provides an alternative approach to investigating gene dosage effects not possible in sexually propagated species. Here, we built a genome-wide structural variation system for dosage-based functional genomics and breeding of poplar. We pollinated Populus deltoides with gamma-irradiated Populus nigra pollen to produce >500 F1 seedlings containing dosage lesions in the form of deletions and insertions of chromosomal segments (indel mutations). Using high-precision dosage analysis, we detected indel mutations in ∼55% of the progeny. These indels varied in length, position, and number per individual, cumulatively tiling >99% of the genome, with an average of 10 indels per gene. Combined with future phenotype and transcriptome data, this population will provide an excellent resource for creating and characterizing dosage-based variation in poplar, including the contribution of dosage to quantitative traits and heterosis. [ABSTRACT FROM AUTHOR]

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 (H²ind = 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. [ABSTRACT FROM AUTHOR]

Understanding drought tolerance mechanisms requires knowledge about the induced weakness that leads to tree death. Bud survival is vital to sustain tree growth across seasons. We hypothesized that the hydraulic connection of the bud to stem xylem structures was critical for its survival. During an artificial drastic water stress, we carried out a census of bud metabolic activity of young Populus nigra L. trees by microcalorimetry. We monitored transcript expression of aquaporins (AQPs; plasma membrane intrinsic proteins (PIPs), X intrinsic proteins (XIPs) and tonoplast membrane intrinsic proteins (TIPs)) and measured local water status within the bud and tissues in the bearer shoot node by nuclear magnetic resonance (NMR) imaging. We found that the bud respiration rate was closely correlated with its water content and decreased concomitantly in buds and their surrounding bearer tissues. At the molecular level, we observed a modulation of AQP pattern expressions (PIP, TIP and XIP subfamilies) linked to water movements in living cells. However, AQP functions remain to be investigated. Both the bud and tree died beyond a threshold water content and respiration rate. Nuclear magnetic resonance images provided relevant local information about the various water reservoirs of the stem, their dynamics and their interconnections. Comparison of pith, xylem and cambium tissues revealed that the hydraulic connection between the bud and saturated parenchyma cells around the pith allowed bud desiccation to be delayed. At the tree death date, NMR images showed that the cambium tissues remained largely hydrated. Overall, the respiration rate (Rco2) and a few AQP isoforms were found to be two suitable, complementary criteria to assess the bud metabolic activity and the ability to survive a severe drought spell. Bud moisture content could be a key factor in determining the capacity of poplar to recover from water stress. [ABSTRACT FROM AUTHOR]

It is assumed that the refilling of drought-induced embolism requires the creation of an osmotic gradient between xylem parenchyma cells and vessel lumens to generate the water efflux needed to fill the void. To assess the mechanism of embolism repair, it is crucial to determine if plants can up-regulate the efflux of osmotically active substances into embolized vessels and identify the major components of the released osmoticum. Here, we introduce a new approach of sap collection designed to separate water from nonembolized (functional) and embolized (nonfunctional) vessels. This new approach made possible the chemical analysis of liquid collected from both types of vessels in plants subjected to different levels of water stress. The technique also allowed us to determine the water volumes in nonfunctional vessels as a function of stress level. Overall, with the increase of water stress in plants, the osmotic potential of liquid collected from nonfunctional vessels increased while its volume decreased. These results revealed the presence of both sugars and ions in nonfunctional vessels at elevated levels in comparison with liquid collected from functional vessels, in which only traces of sugars were found. The increased sugar concentration was accompanied by decreased xylem sap pH. These results provide new insight into the biology of refilling, underlining the role of sugar and sugar transporters, and imply that a large degree of hydraulic compartmentalization must exist in the xylem during the refilling process. [ABSTRACT FROM AUTHOR]

Background and Aims Morphological and biomechanical alterations occurring in woody roots of many plant species in response to mechanical stresses are well documented; however, little is known about the molecular mechanisms regulating these important alterations. The first forest tree genome to be decoded is that of Populus, thereby providing a tool with which to investigate the mechanisms controlling adaptation of woody roots to changing environments. The aim of this study was to use a proteomic approach to investigate the response of Populus nigra woody taproot to mechanical stress. Methods To simulate mechanical perturbations, the taproots of 30 one-year-old seedlings were bent to an angle of 90 ° using a steel net. A spatial and temporal two-dimensional proteome map of the taproot axis was obtained. We compared the events occurring in the above-bending, central bending and below-bending sectors of the taproot. Key Results The first poplar woody taproot proteome map is reported here; a total of 207 proteins were identified. Spatial and temporal proteomic analysis revealed that factors involved in plant defence, metabolism, reaction wood formation and lateral root development were differentially expressed in the various sectors of bent vs. control roots, seemingly in relation to the distribution of mechanical forces along the stressed woody taproots. A complex interplay among different signal transduction pathways involving reactive oxygen species appears to modulate these responses. Conclusions Poplar woody root uses different temporal and spatial mechanisms to respond to mechanical stress. Long-term bending treatment seem to reinforce the defence machinery, thereby enabling the taproot to better overcome winter and to be ready to resume growth earlier than controls. [ABSTRACT FROM AUTHOR]

Little is known about the variability of response to heavy metal stress within tree species, although it could be a key for a better understanding of tolerance mechanisms and for breeding. The aim of the present study was to characterize the natural variation of response to cadmium (Cd) in Populus nigra L. in order to understand the mechanisms of Cd tolerance. For that, two P. nigra genotypes, originating from contrasting environments in northern (genotype 58-861) and southern (genotype Poli) Italy, were exposed to Cd stress in hydroponics for 3 weeks. The effect of stress was estimated by measuring biomass production, photosynthetic performance and accumulation and translocation of Cd at the end of the experiment. To better understand the mechanisms of Cd tolerance, the expression of some candidate genes involved in the ascorbate–glutathione cycle (ascorbate peroxidase, glutathione reductase, glutathione S-transferase) and in metal sequestration (metallothioneins) was analyzed in leaves. Biomass production and photosynthesis were affected by the treatment in both clones but the southern clone was markedly more tolerant to Cd stress than the other. Nevertheless, the Cd content in leaves was not significantly different between the two clones and was quite low compared to other species. The content of thiols and phytochelatins (PCs), associated with the transcription profile of the glutathione S-transferase gene, indicated relevant differences in the use of the PCs pathway under Cd stress, which could explain the different tolerance to Cd. The northern clone accumulated thiols but down-regulated the GST gene, whereas the southern clone accumulated PCs and up-regulated the GST gene, which can be useful to complex and detoxify Cd. These results suggest that the glutathione pathway is involved in the differential Cd tolerance of the two genotypes. The natural germplasm of P. nigra represents a valuable resource for understanding tolerance to Cd and for selection of plant material for phytoremediation. [ABSTRACT FROM PUBLISHER]

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, Δ13C), growth performance and related leaf traits, (ii) examined genotype and population by environment interactions, and (iii) explored the relationship between Δ13C 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: Δ13C 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 Δ13C 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. [ABSTRACT FROM PUBLISHER]

The effects of the interaction between high growth temperatures and water stress on gas-exchange properties of Populus nigra saplings were investigated. Water stress was expressed as a function of soil water content (SWC) or fraction of transpirable soil water (FTSW). Isoprene emission and photosynthesis (A) did not acclimate in response to elevated temperature, whereas dark (Rn) and light (Rd) respiration underwent thermal acclimation. Rd was ∼30% lower than Rn irrespective of growth temperature and water stress level. Water stress induced a sharp decline, but not a complete inhibition, of both Rn and Rd. There was no significant effect of high growth temperature on the responses of A, stomatal conductance (gs), isoprene emission, Rn or Rd to FTSW. High growth temperature resulted in a significant increase in the SWC endpoint. Photosynthesis was limited mainly by CO2 acquisition in water-stressed plants. Impaired carbon metabolism became apparent only at the FTSW endpoint. Photosynthesis was restored in about a week following rewatering, indicating transient biochemical limitations. The kinetics of isoprene emission in response to FTSW confirmed that water stress uncouples the emission of isoprene from A, isoprene emission being unaffected by decreasing gs. The different kinetics of A, respiration and isoprene emission in response to the interaction between high temperature and water stress led to rising Rd/A ratio and amount of carbon lost as isoprene. Since respiration and isoprene sensitivity are much lower than A sensitivity to water stress, temperature interactions with water stress may dominate poplar acclimatory capability and maintenance of carbon homeostasis under climate change scenarios. Furthermore, predicted temperature increases in arid environments may reduce the amount of soil water that can be extracted before plant gas exchange decreases, exacerbating the effects of water stress even if soil water availability is not directly affected. [ABSTRACT FROM AUTHOR]

Background and Aims Cambium reactivation after dormancy and budbreak in deciduous trees requires a supply of mobilized reserve materials. The pathway and mode of transfer of these materials are poorly understood. Methods Transport of reserve materials during cambium reactivation in Populus nigra was investigated by conventional and immunocytochemical TEM analyses, SDS-PAGE, western blotting and intracellular microinjection of fluorescent dyes. Key Results Proteinaceous compounds stored in vacuoles and protein bodies of vascular cells and ray cells disappeared within 3 weeks after cambial reactivation and budbreak. Some of these proteins (32 kDa, 30 kDa and 15 kDa) were labelled by lectin antibodies in SDS-PAGE. The same antibodies were localized to plasmodesmata (PDs) between phloem parenchyma, ray cells and fusiform cambial cells. In addition, proteinaceous particles were localized inside the cytoplasmic sleeves of these PDs during budbreak. During this period, the functional diameter of PDs was about 2.2 nm which corresponds approximately to the Stokes' radius of the detected 15-kDa protein. Conclusions Lectin-like reserve proteins or their degradation products seem to be transferred through PDs of phloem parenchyma and rays during cambial reactivation and budbreak. PD transfer of storage proteins is a novelty which supports the concept of symplasmic nutrient supply to the cambial region. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM PUBLISHER]

A portable unilateral nuclear magnetic resonance (NMR) instrument was used to detect in field conditions the water status of leaves of herbaceous crops (Zea mays, Phaseolus vulgaris), mesophyllous trees (Populus nigra), and natural Mediterranean vegetation characterized by water-spending shrubs (Cistus incanus) and water-saving sclerophyllous trees (Quercus ilex). A good relationship was observed between NMR signal, leaf relative water content, and leaf transpiration in herbaceous leaves undergoing fast dehydration or slowly developing a drought stress. A relationship was also observed between NMR signal and water potential of Populus leaves during the development of a water stress and when leaves recovered from the stress. In the natural vegetation, the relationship between NMR signal and water status was found in Cistus, the species characterized by high transpiration rates, when measured during a drought stress period and after a rainfall. In the case of the scierophyllous Quercus, the NMR signal, the relative water content, and the transpiration rate did not change at different leaf water status, possibly because a large amount of water is compartmentalized in cellular structures and macromolecules. The good association between NMR signal and relative water content was lost in leaves exposed for 24 h to dehydration or to an osmotic stress caused by polyethylene glycol feeding. At this time, the transverse relaxation time became longer than in leaves maintained under optimal water conditions, and two indicators of membrane damage, the ion leakage and the emission of products of membrane lipoxygenation [(Z)-3-hexenal, (Z)-3-hexenol, and (E)-2-hexenol], increased. These results taken all together give information on the physiological state of a leaf under a developing stress and show the usefulness of the NMR instrumentation for screening vegetation health and fitness in natural and cultivated conditions. It is concluded that the portable unilateral NMR instrument may be usefully employed in field conditions to monitor nondestructively the water status of plants and to assist agricultural practices, such as irrigation scheduling, to minimize stomatal closure and the consequent limitation to plant production. [ABSTRACT FROM AUTHOR]

We assessed the effects of ambient tropospheric ozone on annual tree-ring growth, δ13C in the rings, leaf gas exchange and visible injury in three ozone-sensitive woody plant species in southern Switzerland. Seedlings of Populus nigra L., Viburnum lantana L. and Fraxinus excelsior L. were exposed to charcoal-filtered air (CF) and non-filtered air (NF) in open-top chambers, and to ambient air (AA) in open plots during the 2001 and 2002 growing seasons. Ambient ozone exposures in the region were sufficient to cause visible foliar injury, early leaf senescence and premature leaf loss in all species. Ozone had significant negative effects on net photosynthesis and stomatal conductance in all species in 2002 and in V. lantana and F. excelsior in 2001. Water-use efficiency decreased and intercellular CO2 concentrations increased in all species in response to ozone in 2002 only. The width and δ13C of the 2001 and 2002 growth rings were measured for all species at the end of the 2002 growing season. Compared with CF seedlings, mean ring width in theAAandNFP. nigra seedlings was reduced by 52 and 46%, respectively, in 2002, whereas in V. lantana and F. excelsior, ring width showed no significant reductions in either year. Although δ13C was usually more negative in CF seedlings than in AA and NF seedlings, with the exception of F. excelsior in 2001, ozone effects on δ13C were significant only for V. lantana and P. nigra in 2001. Among species, P. nigra exhibited the greatest response to ozone for the measured parameters as well as the most severe foliar injury andwas the only species to showa significant reduction in ring width in response to ozone exposure, despite significant negative ozone effects on leaf gas exchange and the development of visible foliar injury in V. lantana and F. excelsior. Thus, significant ozone-induced effects at the leaf level did not correspond to reduced tree-ring growth or increased δ13C in all species, indicating that the timing of ozone exposure and severity of leaf-level responses may be important in determining the sensitivity of tree productivity to ozone exposure. [ABSTRACT FROM AUTHOR]

Field-grown poplar trees (Populus nigra L. × P. maximowiczii Henry, clone Kamabuchi) were exposed to severe drought twice during the growing season to evaluate the impact on wood cell development. The drought treatment caused a reduction in leaf water potential, leaf wilting and a decreased concentration of osmotically active solutes in the cambial zone. Drought-induced changes in the anatomy of developing xylem cells were examined in stem sections and macerated wood samples. In early summer, drought significantly reduced the length and cross-sectional area of newly formed fibers, whereas no such effects were observed in late summer. In well-watered trees, fiber cross-sectional area declined between early and late summer. Similarly, drought reduced the cross-sectional area of vessel elements in early summer but not in late summer, whereas in both control and drought-treated trees, the cross-sectional area of vessel elements decreased between early and late summer. The vessel area to xylem area ratio was unaffected by drought because the drought-induced decrease in vessel size was matched by an increase in the number of newly formed vessel cells. In contrast to its effect in early summer, late-summer drought had no significant effect on fiber and vessel cell development, indicating that sensitivity of wood cell development to drought varies seasonally. [ABSTRACT FROM PUBLISHER]

Confocal Raman microscopy was used to illustrate changes of molecular composition in secondary plant cell wall tissues of poplar (Populus nigra × Populus deltoids) wood. Two-dimensional spectral maps were acquired and chemical images calculated by integrating the intensity of characteristic spectral bands. This enabled direct visualization of the spatial variation of the lignin content without any chemical treatment or staining of the cell wall. A small (0.5 μm) lignified border toward the lumen was observed in the gelatinous layer of poplar tension wood. The variable orientation of the cellulose was also characterized, leading to visualization of the S1 layer with dimensions smaller than 0.5 μm. Scanning Raman microscopy was thus shown to be a powerful, nondestructive tool for imaging changes in molecular cell wall organization with high spatial resolution. [ABSTRACT FROM AUTHOR]

Poplar, whose genome is the first to be sequenced among woody plants, is a favorable model for plant biologists to enable them to understand molecular processes of growth, development and responses to environmental stimuli in trees. The sequence will allow the development of a strategy for improving environmental stress tolerance in forest trees. In this study, we have generated a full-length enriched cDNA library from leaves of axenically grown poplar (Populus nigra var. italica) subjected to environmental stress treatments by dehydration, high salinity, chilling, heat, abscisic acid (ABA) and H2O2. We sequenced >30,000 expressed sequence tags (ESTs) from the cDNA library and consequently collected approximately 4,500 non-redundant clones. We further analyzed cDNAs encoding an ERF/AP2-domain transcription factor which is specific in plants and plays an important role under stress. Thirteen candidates containing the ERF/AP2 domain were found within our EST resource. Some of them showed stress-responsive gene expression. We report here the first collection of full-length enriched stress-related ESTs of poplar and discuss environmental stress responses of forest trees in the light of comparative genomics. [ABSTRACT FROM PUBLISHER]

Isoprene emitted from plants is made in chloroplasts from dimethylallyl pyrophosphate (DMAPP). Leaves of Populus nigra and Phragmites australis exposed to 13CO2 for 15 min emitted isoprene that was about 90% 13C, but DMAPP isolated from those leaves was only 28% and 36% 13C, respectively. The labeled DMAPP is likely to represent chloroplastic DMAPP contributing to isoprene emission. A substantial 13C labeling was also found in both emission and DMAPP pool of low-emitting, young leaves of Phragmites. This confirms that low emission of young leaves is not caused by absence of chloroplastic DMAPP but rather by enzyme characteristics. A very low 13C labeling was found in the DMAPP pool and in the residual isoprene emission of leaves previously fed with fosmidomycin to inhibit isoprene formation. This shows that fosmidomycin is a very effective inhibitor of the chloroplastic biosynthetic pathway of isoprene synthesis, that the residual isoprene is formed from extra-chloroplastic sources, and that chloroplastic and extrachloroplastic pathways are not cross-linked, at least following inhibition of the chloroplastic pathway. Refixation of unlabeled respiratory CO2 in the light may explain incomplete labeling of isoprene emission, as we found a good association between these two parameters. [ABSTRACT FROM AUTHOR]

We determined: (a) the physiological consequences of overproduction of putrescine in transgenic poplar (Populus nigra × maximoviczii) cells expressing an ornithine decarboxylase transgene; and (b) effects of variation in nitrogen (N) concentration of the medium on cellular polyamine concentration in transgenic and non-transgenic cells. Cells grown in the presence of supplemental (to the normal concentrations of N sources in the growth medium) and reduced amounts of NH4NO3 and KNO3 were used to study effects on membrane permeability, mitochondrial respiratory activity, protein accumulation, growth rates and changes in cellular polyamine concentration. The N concentration of the MS medium was not a limiting factor for continued overproduction of putrescine in transgenic cells. However, continued supplies of NH4 + and NO3 – were required to maintain homeostatic amounts of putrescine in both cell lines. The presence of high amounts of putrescine in transgenic cells had significant effects on the physiological parameters measured. Compared with non-transgenic cells, transgenic cells had greater plasma membrane permeability, less tolerance to NH4NO3, more tolerance to KNO3, and accumulated higher amounts of soluble protein. [ABSTRACT FROM AUTHOR]

Leaf osmotic potential at full turgor (Ψπo) and the major solutes that contribute to osmotic potential were characterized in five hybrid poplar clones of Populus trichocarpa Torr. & Gray × P. deltoides Bartr. (TD) and P. deltoides × P. nigra L. (DN), growing under field conditions at two sites in eastern Washington and Oregon, USA. Trees were drip irrigated with 46, 76 or 137 cm of supplemental irrigation during each growing season. Trees at Wallula, WA, which were in their third growing season in 1994, were sampled twice a year for two years (1994 and 1995), and trees at Boardman, OR, which were in their second growing season in 1994, were sampled once a year for three years (1994–1996). At Wallula, the TD and DN clones exhibited lower predawn leaf water potentials in the 46-cm treatment than in the 137-cm treatment (–1.2 versus –0.7 MPa) during a hot, dry period in July 1994. Clone TD had a lower Ψπo than Clone DN (–1.67 versus –1.56 MPa) during the same period and the difference was also evident in 1995 (–1.81 versus –1.72 MPa) when trees were in their fourth growing season. There was also a significant treatment effect on Ψπo in Clone TD, with trees in the 46-cm treatment having lower Ψπo than trees in the 137-cm treatment in July 1994. At Boardman, Ψπo was generally high with no treatment differences during the 1994–96 samplings. The TD clones had significantly lower Ψπo than the DN clones in 1994 (–1.44 versus –1.36 MPa) and 1996 (–1.72 versus –1.54 MPa), but there was no difference between clones in 1995 (–1.40 versus –1.43 MPa). In 1995, at Wallula, osmotic adjustment in Clone TD was largely accounted for by an increase in sucrose, which constituted 70% of total organic solutes. Although the total concentration of free primary amino acids in this clone was 28% higher in trees in the 46-cm treatment than in trees in the 137-cm treatment, amino acids constituted only a small fraction of the total solute pool. Sixty-two percent of total solutes were inorganic ions in Clone TD compared to 52% in Clone DN, and potassium was the main ion constituting about 30% of total solutes and 50% of total ions. However, the clonal difference in Ψπo was not fully accounted for by the difference in solute concentration. Osmotic potential at full turgor declined over the growing season and with age. We conclude that, because the extent of osmotic adjustment exhibited by these clones was small, other drought resistance mechanisms contributed to the clonal differences in field performance. [ABSTRACT FROM PUBLISHER]

Poplar hybrids were grown with irrigation in a large-scale plantation to investigate the mechanisms underlying clonal differences in drought resistance. Beginning in spring 1992, Populus trichocarpa × P. deltoides (TD) and P. deltoides × P. nigra (DN) cuttings received 46, 76, or 137 cm year−1 of irrigation to supplement the 18–20 cm of annual precipitation, and all trees received the same fertilization regime. Stem volume, assessed as the square of stem diameter at breast height times tree height (D2H), and water relations of the trees were studied from the end of their second growing season until the end of their fifth growing season. [ABSTRACT FROM PUBLISHER]

We assessed the effects of near-lethal heat stress on bud break, heat-shock proteins (HSPs) and ubiquitin in hybrid poplar (Populus nigra (L.) Charkowiensis × P. nigra (L.) incrassata). Shoots, with 10–15 buds each, were collected from September to March and exposed to temperatures between 20 and 60 °C for 2 h. Shoots were then placed in a greenhouse at 18–22 °C with supplemental light and cumulative bud break was recorded over a 4-week period. Samples of bud tissues were collected during and up to 96 h after heat treatment for protein analysis. De novo synthesis of proteins was monitored by exposing excised buds to [35S]-methionine for 3 h before, during, or after heat treatment. Heat treatments of 40–45 °C resulted in both a release from endodormancy and a decrease in thermal units needed for bud break during ecodormancy. The response to near-lethal heat stress was complex and was affected by intrinsic thermal sensitivity. Heat treatments were least effective during August and became progressively more effective as endodormancy progressed. In the later stages of ecodormancy, a heat treatment of 45 °C either inhibited bud break or killed the buds. Although temperatures of 42.5 to 45 °C inhibited incorporation of [35S]-methionine into proteins for at least 48 h, several HSPs were synthesized in response to temperatures of 40–45 °C. Immunoblots indicated that one of the heat-induced proteins was immunologically related to HSP70. Increases in free and conjugated forms of ubiquitin were also observed in response to heat treatment. Production of HSPs and ubiquitin, however, was not consistently associated with the heat treatments that induced the highest percentage of bud break. The roles of heat-induced protein degradation, HSPs, and ubiquitin in overcoming dormancy by near-lethal heat stress are discussed. [ABSTRACT FROM PUBLISHER]