This study aimed at determining the cadmium phytoextraction potential of three Populus alba L. clones cultivated in the presence of increasing sodium chloride concentrations. Plantlets of a commercial and two autochthonous poplar clones were grown in perlite with nutrient solution enriched in CdSO (50 and 100 ?M) and NaCl (25 and 50 mM), administered either alone or in combination. The three clones showed significant variation not only in cadmium and salt tolerance, accumulation and content, but also in the effect of the interaction between the two elements on these parameters. The toxic effect of Cd and salt excess on plants was mutually exacerbated by the presence of both. Even though the outcome of the joint treatment was always a decrease in shoot Cd or Na accumulation, the three clones showed variation in the extent of such reduction. Evaluating the total element content per plant shoot, the fast-growing commercial clone displayed the highest phytoextraction potential for Cd and Na, either alone or in mixture. Our results demonstrated for the first time that the Cd response in presence of salt can vary in the different clones.

In drought-stressed plants a coordinated cascade of chemical and transcriptional adjustments occurs at the same time as embolism formation. While these processes do not affect embolism formation during stress, they may prime stems for recovery during rehydration by modifying apoplast pH and increasing sugar concentration in the xylem sap. Here we show thatin vivotreatments modifying apoplastic pH (stem infiltration with a pH buffer) or reducing stem metabolic activity (infiltration with sodium vanadate and sodium cyanide; plant exposure to carbon monoxide) can reduce sugar accumulation, thus disrupting or delaying the recovery process. Application of the vanadate treatment (NaVO(3,)an inhibitor of many ATPases) completely halted recovery from drought-induced embolism for up to 24 h after re-irrigation, while partial recovery was observedin vivoin control plants using X-ray microcomputed tomography. Our results suggest that stem hydraulic recovery in poplar is a biological, energy-dependent process that coincides with accumulation of sugars in the apoplast during stress. Recovery and damage are spatially coordinated, with embolism formation occurring from the inside out and refilling from the outside in. The outside-in pattern highlights the importance of xylem proximity to the sugars within the phloem to the embolism recovery process.

Next-generation sequencing (NGS) approaches are attractive alternatives to the PCR-based characterisation of genetically modified plants for safety assessment and labelling since NGS is highly sensitive to the detection of T-DNA inserts as well as vector backbone sequences in transgenic plants. In this study, two independent transgenic male Populus tremula lines, T193-2 and T195-1, both carrying the FLOWERING LOCUS T gene from Arabidopsis thaliana under control of a heat-inducible promoter (pHSP::AtFT) and the non-transgenic control clone W52, were further characterised by NGS and third-generation sequencing. The results support previous findings that the T-DNA was hemizygously inserted in one genomic locus of each line. However, the T-DNA insertions consist of conglomerations of one or two T-DNA copies together with a small T-DNA fragment without AtFT parts. Based on NGS data, no additional T-DNA splinters or vector backbone sequences could be identified in the genome of the two transgenic lines. Seedlings derived from crosses between the pHSP::AtFT transgenic male parents and female wild type plants are therefore expected to be T-DNA splinter or vector backbone free. Thus, PCR analyses amplifying a partial T-DNA fragment with AtFT-specific primers are sufficient to determine whether the seedlings are transgenic or not. An analysis of 72 second generation-seedlings clearly showed that about 50% of them still reveal the presence of the T-DNA, confirming data already published. To prove if unanticipated genomic changes were induced by T-DNA integration, extended future studies using long-range sequencing technologies are required once a suitable chromosome-level P. tremula reference genome sequence is available.

Some plant species are capable of significant reduction of xylem embolism during recovery from drought despite stem water potential remains negative. However, the functional biology underlying this process is elusive. We subjected poplar trees to drought stress followed by a period of recovery. Water potential, hydraulic conductivity, gas exchange, xylem sap pH, and carbohydrate content in sap and woody stems were monitored in combination with an analysis of carbohydrate metabolism, enzyme activity, and expression of genes involved in sugar metabolic and transport pathways. Drought resulted in an alteration of differential partitioning between starch and soluble sugars. Upon stress, an increase in the starch degradation rate and the overexpression of sugar symporter genes promoted the efflux of disaccharides (mostly maltose and sucrose) to the apoplast. In turn, the efflux activity of the sugar-proton cotransporters caused a drop in xylem pH. The newly acidic environment induced the activity of apoplastic invertases leading to the accumulation of monosaccharides in the apoplast, thus providing the main osmoticum necessary for recovery. During drought and recovery, a complex network of coordinated molecular and biochemical signals was activated at the interface between xylem and parenchyma cells that appeared to prime the xylem for hydraulic recovery.

Drought dramatically affects wood production by adversely impacting cambial cells and their derivatives. Photosynthesis and assimilate transport are also affected by drought conditions. Two poplar genotypes, Populus deltoides 'Dvina' and Populus alba 'Marte', demonstrated contrasting growth performance and water-carbon balance strategies; a mechanistic understanding of the water deficit response was provided by these poplar species. 'Marte' was found to be more anisohydric than 'Dvina'. This characteristic was associated with the capacity to reallocate carbohydrates during water deficits. In contrast, 'Dvina' displayed more conservative water management; carbohydrates were preferably stored or used for cellulose production rather than to achieve an osmotic balance between the phloem and the xylem. Data confirmed that the more 'risk-taking' characteristic of 'Marte' allowed a rapid recovery following water deficit and was connected to a different carbohydrate metabolism.

We calibrated and evaluated the agricultural model AquaCrop for the simulation of water use and yield of a short-rotation coppice (SRC) plantation with poplar (Populus) in East Flanders (Belgium) during the second and the third rotation (first 2 years only). Differences in crop development and growth during the course of the rotations were taken into account during the model calibration. Overall, the AquaCrop model showed good performance for the daily simulation of soil water content (R of 0.57-0.85), of green canopy cover (R > 0.87), of evapotranspiration (ET; R > 0.76), and of potential yield. The simulated, total yearly water use of the SRC ranged between 55% and 85% of the water use of a reference grass ecosystem calculated under the same environmental conditions. Crop transpiration was between 67% and 93% of total ET, with lower percentages in the first than in the second year of each rotation. The observed (dry mass) yield ranged from 6.61 to 14.76 Mg ha yr. A yield gap of around 30% was observed between the second and the third rotation, as well as between simulated and observed yield during the third rotation. This could possibly be explained by the expansion of the understory (weed) layer; the relative cover of understory weeds was 22% in the third year of the third rotation. The agricultural AquaCrop model simulated total water use and potential yield of the operational SRC in a reliable way. As the plantation was extensively managed, potential effects of irrigation and/or fertilization on ET and on yield were not considered in this study.

The involvement of auxin, abscisic acid (ABA), polyamines (PAs), and proline in adaptation to long-term exposure of woody plants to high levels of heavy metals in soil has received scant attention, even in poplar which is a good candidate for phytoremediation of metal-polluted soils and is regarded as a model for basic research in tree species. Three poplar clones (M1, PE19/66, and B229) were comparatively analyzed in a pot experiment for their responses to 300 mg kg-1 Cu(NO3)2 at morphological, physiological, and biochemical levels. After 4 months, despite the prevalent accumulation of Cu in roots, where the metal reached potentially toxic concentrations, the three clones showed distinct Cu accumulation and translocation capacities, whereas they did not display evident toxicity symptoms or growth inhibition. Several protective mechanisms, namely decreased photosynthetic functionality, enhanced guaiacol peroxidase (GPOD) activity, and accumulation of proline and PAs, were differentially activated in Cu-treated plants in an organ- and clone-specific manner. Overall, a positive relationship between root Cu concentration with GPOD, proline, and PAs was observed. In M1, higher Cu accumulation in roots and leaves compared with other clones was reflected in stimulation of GPOD activity in both organs and in enhanced proline, and PA levels. In PE19/66, these responses were observed only in roots concomitant with high Cu accumulation. Clone B229 accumulated very low amounts of Cu, therefore, these defense responses were attenuated compared with other clones. Enhanced ABA concentrations in response to Cu were observed in PE19/66 and B229; this was likely responsible for stomatal limitation of photosynthesis in PE19/66, whereas in B229 this effect may have been counteracted by increased IAA. Essentially unchanged leaf auxin levels under Cu stress may account for the lack of shoot growth inhibition observed in all three clones; B229 was the only clone that displayed Cu-induced IAA accumulation in roots. Results are discussed in terms of clone-specific adaptive mechanisms to Cu stress in tolerant poplars.

Poplar short rotation coppice represents a very good opportunity for the production of bioenergy as a strategy against global changes. In recent years, numerous plantations in central Italy showed worrying stump mortality of uncertain cause. Investigations have therefore been conducted to determine the etiology and symptomatology of this phenomenon. The external symptoms begin with leaf yellowing and sometimes necrosis, microphyllia, and reduced shoots growth, followed by withering and death of the stump. Upon sectioning of the stump, we detected browning of internal tissues of roots and crown, gradually deepening in. In severe cases, we observed wood decay in the central part of the stump, eventually deepening into the root system. Cultures from the edge of necrotic tissue on PDA yielded isolates of the same fungus. Sequence analysis of the Internal Transcriber Spacer region (ITS1-5.8S-ITS2) and of the ?-tubulin gene showed that the isolates belong to the genus Cryptosphaeria. Species identification is still ongoing. Eventually, wood decay organisms belonging to the genera Pholiota and Collibia colonized the tissues as secondary invaders. Future goals will be to identify the species of the pathogen, define the disease predisposing factors and identify possible control measures.

Poplar species are economically important sources of timber and bioenergy and they also have a valuableapplication in phytoremediation and in forest restoration programmes. The characterization of theirresponses in the face of environmental constraints is essential to ascertain their adaptation capacity.Quantification of chloroplast pigments and antioxidant compounds, nutrients and carbon isotope dis-crimination (D13C) of a large set of samples in ecophysiological studies is important for determiningthe plant's physiological responses.D13C and nitrogen are parameters of special interest for providinginformation on photosynthesis, water relations and nutritional characteristics in forest stands.Photoprotective and antioxidant compounds participate in the physiological defence of plants subjectedto stressful environmental conditions. The measurement of these leaf components by traditional tech-niques is laborious and expensive. To evaluate them, a calibration model was obtained using Near-Infrared Reflectance Spectroscopy (NIRS) and chemical analyses ofPopulusspp.leaves sampled from dif-ferent clones in different years under different physiological conditions. Predictive calibration equationsfor the concentration of chloroplast pigments (Chlorophyllsaandb, lutein, neoxanthin,b-carotene(b-Car), VAZ (violaxanthin + antheraxanthin + zeaxanthin), epoxidation index (EI = (0.5antheraxanthin+ violaxanthin)/VAZ)), antioxidants (total phenolics (TPhe), ascorbate anda-tocopherol),D13C and N con-tent were established using a partial least squares regression algorithm. Results showed a remarkableaccuracy of the calibration equations for quantifying several antioxidative and photoprotective defencecompounds such as neoxanthin (R2= 0.844),b-Car (R2= 0.827), VAZ (R2= 0.846), EI (R2= 0.831) andexcellent accuracy for TPhe (R2= 0.957), N (R2= 0.963) andD13C(R2= 0.922). As shown, results can beobtained on multiple physiological attributes with a single measurement, saving time and money anddemonstrating the suitability of NIRS for ecophysiological purposes and bioremediation programs.

A cDNA coding for a plastidic P2-type G6PDH isoform from poplar (Populus tremula x tremuloides) has been used to express and purify to homogeneity the mature recombinant protein with a N-terminus His-tag. The study of the kinetic properties of the recombinant enzyme showed an in vitro redox sensing modulation exerted by reduced DTT. The interaction with thioredoxins (TRX5) was then investigated.

The present paper investigates the influence of thermo-vacuum treatment at 170, 190 and 210 °C on the mechanical and physical properties of poplar wood rotary-cut veneers obtained from two different poplar clones ('I-214' and 'Lena'). The modulus of rupture in bending was determined according to a method derived from European Standard EN-310 and previously validated by the authors, while the density was determined on the basis of EN-323. With both clones no significant decrease was recorded either in bending strength or in density with treatment temperatures up to 190 °C. On the contrary, at 210 °C a highly significant decrease in modulus of rupture ('I-214': -18%; 'Lena': -15%) was recorded; the density showed a similar, though lower, trend ('I- 214': -5 %; 'Lena': -8.5 %).

Complete Populus genome sequences are available for the nucleus 25 (P. trichocarpa; section Tacamahaca) and for chloroplasts (seven species), but not for mitochondria. Here, we provide the complete genome sequences of the chloroplast and the mitochondrion for the clones P. tremula W52 and P. tremula x P. alba 717-1B4 (section Populus). The organization of the chloroplast genomes of both Populus clones is described. A phylogenetic tree constructed from all available complete chloroplast DNA sequences of Populus was not congruent with the assignment of the related species to different Populus sections. In total, 3,024 variable nucleotide positions were identified among all compared Populus chloroplast DNA sequences. The 5-prime part of the LSC from trnH to atpA showed the highest frequency of variations. The variable positions included 163 positions with SNPs allowing for differentiating the two clones with P. tremula chloroplast genomes (W52, 717-1B4) from the other seven Populus individuals. These potential P. tremula-specific SNPs were displayed as a whole-plastome barcode on the P. tremula W52 chloroplast DNA sequence. Three of these SNPs and one InDel in the trnH-psbA linker were successfully validated by Sanger sequencing in an extended set of Populus individuals. The complete mitochondrial genome sequence of P. tremula is the first in the family of Salicaceae. The mitochondrial genomes of the two clones are 783,442 bp (W52) and 783,513 bp (717-1B4) in size, structurally very similar and organized as single circles. DNA sequence regions with high similarity to the W52 chloroplast sequence account for about 2% of the W52 mitochondrial genome. The mean SNP frequency was found to be nearly six fold higher in the chloroplast than in the mitochondrial genome when comparing 717-1B4 with W52. The availability of the genomic information of all three DNA-containing cell organelles will allow a holistic approach in poplar molecular breeding in the future.

The synergistic activity between plants and microorganisms may contribute to the implementation of proactive management strategies in the stabilization of contaminated sites, although heavy metals, such as cadmium (Cd), are potentially toxic to them. The aim of this study was to evaluate the degree of tolerance to Cd contamination (supplying twice 40 mg kg(-1) of Cd) in poplar cuttings [clone I-214, P. x euramericana (Dode) Guinier] inoculated or not with two concentrations of Serratia marcescens strain (1 x 10(7) CFU/g and 2 x 10(7) CFU/g of potting mix). The response of the plant-bacteria system to excess Cd was investigated with special reference to the structural traits of plants and the functional efficiency of bacteria. Bacterial colonization and substrate components were previously assessed in order to define the best solution for formulating the experimental plant growth media. The tested plant-bacteria association, especially when bacteria were provided in double concentration, stimulated specific tolerance mechanisms to Cd through the promotion of the poplar growth. Inoculated plants produced larger leaves and increased stem diameter, while roots grew longer and wider in Cd-treated plants. The effect of bacterial inoculum on plant growth traits and metal partitioning in plant organs was assessed in order to define the potential of this poplar clone to be a suitable candidate for phytostabilization of Cd-contaminated soil. The final effect of the inoculation with bacteria, which alleviated the metal load and Cd phytotoxicity due to their bioaccumulation ability, suggests promising phytostabilization potential of these plant-bacteria associations.

The plant-microorganism combinations may contribute to the success of phytoextraction of heavy metal-polluted soil. The purpose of this study was to investigate the effects of cadmium (Cd) soil concentration on selected physiological parameters of the poplar clone "I-214" inoculated at root level with a strain (BT4) of Pseudomonas fluorescens and a commercial product based on microbial consortia (Micosat F FitoA (R)). Plants were subjected to Cd treatment of 40 mg kg(-1) in greenhouse. The effects of plant-microbe interactions, plant growth, leaf physiology, and microbial activity were periodically monitored. Metal concentration and translocation factors in plant tissues proved enhanced Cd uptake in roots of plants inoculated with P. fluorescens and transfer to shoots in plants inoculated with Micosat F FitoA (R), suggesting a promising strategy for using microbes in support of Cd uptake. Plant-microbe integration increased total removal of Cd, without interfering with plant growth, while improving the photosynthetic capacity. Two major mechanisms of metal phytoextraction inducted by microbial inoculation may be suggested: improved Cd accumulation in roots inoculated with P. fluorescens, implying phytostabilization prospective and high Cd transfer to shoots of inoculated plants, outlining enhanced metal translocation.

Bioremediation is an increasingly popular alternative to conventional methods for treating waste compounds, in line with environmental sustainability, with the possibility to degrade contaminants using natural microbial activity mediated by different consortia of microbial strains. Among the techniques of so-called green remediation, plant-assisted bioremediation seems to be one of the most promising techniques. Plant-assisted bioremediation is a technology that exploits the synergistic actions that are established in the rhizosphere between plant roots and microorganisms in order to remove, transform or immobilize toxic substances. The presence of plant species through the roots promotes the modification of the physico-chemical properties of contaminated soils as well as the release of root exudates. Organic pollutants may be partially co-metabolic degraded by root enzymes and/or completely biodegraded by microorganisms in the rhizosphere. The phytoremediation effectiveness depend on the use of plant species tolerant to the toxic effects of contaminants in the soil and with roots able to promote the development of a microbial community capable of supporting the degradation of the contaminant in the rhizosphere. This technique is used in preliminary experiments, one in the field and in two in greenhouse, using a soil diffusely contaminated by PCBs in which different plant species were tested. In the field experiment, two tree species (Tamarix gallica and clone Monviso of the genus Populus), were used. At the same time, soil microcosms were set up in greenhouse in order to study in detail the possible PCB degradation processes that occur in the rhizosphere. For this purpose, some microcosms were prepared in the presence/absence of Medicago sativa specie and others in the presence of the Monviso clone, the same used in the field experiment. In this work we describe the various experiments and their main results.

Nella presente nota sono riportate alcune recenti esperienze sull'uso delle piantagioni cedue di pioppo da biomassa per il fitori - medio. Le piantagioni cedue da biomassa a corta rotazione (2-3 anni), o Short Rotation Coppice (SRC), hanno interessanti potenzialità di applicazione nel fitorimedio poiché abbinano un'alta capacità di assorbimento e degradazione di contaminanti del suolo con la destinazione ad uso non alimentare della biomassa. L'efficienza di assorbimento è determinata sia dalla rapidità di crescita del pioppo sia da un alta percentuale di tessuti legnosi attivi sulla biomassa totale. Tale biomassa può essere poi desti - nata alla successiva valorizzazione energetica. Le piantagioni SRC possono essere usate per la distribuzione di reflui zootecnici, che hanno significative concentrazioni di fitonutrienti (in primis N) e metalli pesanti, altrimenti dannosi all'ambiente se non smaltiti adeguatamente. La liquamazione in piantagioni SRC di pioppo oltre a costituire un'alternativa per lo smaltimento dei reflui zootecnici, migliora il bilancio energetico della coltura legnosa, di cui la fertilizzazione azotata rappresenta una significa - tiva percentuale. L'esperienza presentata riguarda un caso applicativo in pieno campo di una piantagione SRC di pioppo, costituita nel 2006, con vari cloni, confrontando parcelle non trattate con parcelle nelle quali è stato effettuato uno smaltimento di reflui zootecnici. Il volume di liquamazione distribuito nella SRC di pioppo durante i due cicli vegetativi triennali è stato di 75 t ha-1 per singolo trattamento allo 0.1% di contenuto di N. Sono stati monitorati tutti gli interventi colturali, dall'impianto alla raccolta, con particolare attenzione alle liquamazioni ed irrigazioni. Per verificare gli effetti degli interventi di liquamazione, sono stati rilevati, per i due cicli triennali, le produzioni dei vari cloni posti a confronto. E' stata inoltre determinata la composizione chimica relativamente a nutrienti e metalli pesanti presenti del legno e nel suolo. Infine, è stata condotta un'analisi eco - nomica complessiva dell'operazione al fine di evidenziare possibili benefici relazionati sia ad una riduzione dell'apporto nel terreno di fertilizzanti chimici tradizionali, sia in termini di eliminazione degli oneri di smaltimento dei reflui zootecnici azien - dali.

The presence of contaminated sites in ever increasing numbers brings several management problems and often leads to a waste of important resources, such as soils and water which cannot be utilised. Remediation with conventional technologies is sometimes unfeasible due to economic reasons or for the opposition of the public opinion, but it is often the only available alternative. Since many years, biological techniques for remediation, bioremediation and phytoremediation, have been studied; they can give acceptable results with a lower environmental impact, even if with longer time periods. In particular, the use of higher plants in decontamination has been limited by legislation constraints, and also for lack of plant species, which can be really effective towards specific contaminants. In this paper the remediation potential of a short rotation coppicing with poplars selected for bioenergy production and for specific remediation needs of two national interest contaminated sites: 1) the agricultur- al soil polluted with lindane and isomers in the River Sacco valley in the roman province, 2) the soil of the industrial area of the ENI Oil Island by Porto Marghera, polluted by multiple heavy metals. It is also shown and evaluated an example of manure spreading on a poplar plantation by a farm of Torre in Pietra in the Agro Romano.

Genetic determination of gender is a fundamental developmental and evolutionary process in plants. Although it appears that dioecy in Populus is genetically controlled, the precise gender-determining systems remain unclear. The recently released second draft assembly and annotated gene set of the Populus genome provided an opportunity to revisit this topic. We hypothesized that over evolutionary time, selective pressure has reformed the genome structure and gene composition in the peritelomeric region of the chromosome XIX, which has resulted in a distinctive genome structure and cluster of genes contributing to gender determination in Populus trichocarpa. Multiple lines of evidence support this working hypothesis. First, the peritelomeric region of the chromosome XIX contains significantly fewer single nucleotide polymorphisms than the rest of Populus genome and has a distinct evolutionary history. Second, the peritelomeric end of chromosome XIX contains the largest cluster of the nucleotide-binding site-leucine-rich repeat (NBS-LRR) class of disease resistance genes in the entire Populus genome. Third, there is a high occurrence of small microRNAs on chromosome XIX, which is coincident to the region containing the putative gender-determining locus and the major cluster of NBS-LRR genes. Further, by analyzing the metabolomic profiles of floral bud in male and female Populus trees using a gas chromatography-mass spectrometry, we found that there are gender-specific accumulations of phenolic glycosides. Taken together, these findings led to the hypothesis that resistance to and regulation of a floral pathogen and gender determination coevolved, and that these events triggered the emergence of a nascent sex chromosome. Further studies of chromosome XIX will provide new insights into the genetic control of gender determination in Populus. © 2012 The Author(s).

The objectives of this work were the characterization of the spatial genetic diversity in poplar populations at the stand level, and the study of the potential breeding between natural and cultivated populations in Mediterranean environment. To this end, two test areas were selected in the Migliarino-San Rossore-Massaciuccoli Regional Park in Tuscany (Italy): A) a mixed forest stand; B) a scattered Populus population in the wetland area of "lake of Massaciuccoli". Test area A is a naturally-originated mixed forest stand near to poplar plantations. In the forest, the prevailing tree species are Populus alba and P. x canescens. In the test area B, single and clustered trees of Populus spp. are scattered along the lake, and close to poplar plantations. In the test area A an experimental plot 2500 m2 large has been designed inside the forest. All Populus trees within the two test area have been identified and their position have been collected by GPS. The analysis of spatial and genetic structure of the two poplar stands was performed using Geneland. Nuclear microsatellites were used to evaluate genetic diversity of poplar populations and level of breeding between natural and cultivated Populus. The results of Geneland clearly showed that four distinct clusters can be identified in the area A, indicating the presence of gene flow barriers. The same analysis indicates two clusters in area B with a gene flow between clusters higher than in area A. In test area A, we found hybridization between the poplar stand and the Populus plantation. Therefore, the possible cultivation of transgenic poplar close to naturally originated stands might influence their biodiversity. The study has been performed within the framework of the European project LIFE08 NAT/IT/342.