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Zinc (Zn) is an essential micronutrient for plants, but it is toxic beyond a certain threshold. Populus alba (L.) 'Villafranca' clone is known for its good tolerance to high Zn concentration compared to other poplar species. A line of this species overexpressing the tonoplast intrinsic aquaporin AQUA1 gene has showed an improved tolerance to Zn excess in comparison to the wild-type (wt) line. The aims of this work were to: 1) verify if AQUA1 plants can uptake Zn more efficiently after a longer period of exposure; 2) evaluate if a higher Zn uptake in transgenic lines can have negative effects; 3) assess Zn competing elements (iron and manganese), soluble sugars, osmolytes, and potassium to investigate differences in water and osmotic homeostasis between lines. Under Zn excess, AQUA1 plants showed a twofold Zn translocation factor and a higher xylem sap Zn concentration than the wt plants. Transgenic plants preferentially allocated Zn in aerial biomass and this different behaviour matched with modified manganese and iron balances suggesting that the increased Zn uptake might be related to a decrease in iron transport in the transgenic line. Moreover, a higher instantaneous water use efficiency in control conditions and an increase in bark soluble sugars under Zn excess could allow a higher resistance of AQUA1 plants to the water and osmotic perturbations caused by Zn. Indeed, the Zn excess increased the xylem osmolyte content only in wt plants. Further investigations are required to understand the role of AQUA1 in osmotic regulation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

The plant association of Populus alba L. 'Villafranca', Brassica oleracea var. acephala sebellica (kale), and B. oleracea var. capitata 'sonsma' (cabbage) was exposed to Zn, Cd, and exogenous caffeine ( 13 CFN)-contaminated water under growth chamber conditions. In the short term of treatment (15 days), poplar increased the root dry biomass (+ 25%) and decreased the chlorophyll content in new leaves (- 32%), compared to control. On the contrary, cabbage decreased the root dry biomass, enhancing the shoot dry biomass (+ 50%). Heavy metals were mainly concentrated in plant roots and in poplar reached the highest concentrations of 705 ± 232.6 and 338 ± 85.5 μg g -1 DW for Zn and Cd, respectively. The ability of poplar to accumulate more Zn and Cd than kale and cabbage in plant biomass was confirmed by heavy metal contents, following the order: poplar > kale = cabbage. However, poplar and Brassica sp. association was very useful for Zn and Cd decontaminations as reported by the bioconcentration factors (> 1). The concentration of 13 CFN was below 2.4 ng g -1 FW in poplar and 7.4 ng g -1 FW in Brassica species, suggesting the caffeine uptake and degradation by plant association. Under our experimental conditions, the removal efficiency of the system was upper to 79%, indicating the capability of Populus-Brassica association to efficiently remove Zn, Cd, and 13 CFN from mixed inorganic-organic-contaminated water in short term.

Key Message: Poplar callus maintained a specific difference in osmotic potential with respect to media when supplemented with different carbohydrate concentrations. This balance in osmotic potential guaranteed the growth capacity. Osmotic stress is caused by several abiotic factors such as drought, salinity, or freezing. However, the threshold of osmotic potential that allows the growth under stress conditions has not been thoroughly studied. In this study, different levels of osmotic stress in Populus alba (L.) callus have been induced with the addition of mannitol or sorbitol in the medium (from 0 to 500 mM). The key factor for preserving the growth was observed to be the restoration of a constant difference in osmotic potential between callus and medium for all the tested conditions. The osmotic adjustments were primarily achieved with the uptake of mannitol or sorbitol from the media considering their chemical properties instead of their biological functions. The decrease in water content (from - 1 to - 10% after 21 days) and mineral elements, such as potassium, calcium, and magnesium, together with the alterations in cell morphology, did not show negative effects on growth. The activity of sorbitol dehydrogenase was detected for the first time in poplar (+ 4.7 U l -1 in callus treated with sorbitol compared to control callus). This finding suggested the importance of choosing carefully the molecules used to exert osmotic stress for separating the dual function of carbohydrates in osmotic adjustments and cell metabolism.

Drought events impair the carbon and water balances in plants. Climate changes highlight the importance to understand the limits of woody species to reallocate carbon in different processes and the mechanisms driving the osmotic adjustments during the day under stress. In this frame, the aim of this work was to investigate the plant capability to shift energy among competing sinks and preserve the osmotic balance during the day under severe short periods of water deficit. The role of carbohydrates as osmolytes as well as energy sources was investigated in poplar plants. Results highlighted that during water deficit soluble sugars, derived both from the new synthetised carbon and starch degradation, were principally convoyed in the bark. This increase in carbohydrates allowed the maintenance of a water reserve used during the day to prevent a water decrease within the xylem. The decrease of xylem sap osmotic potential during the night, driven by an increase of K, Ca, and fructose (+0.46, 0.52, and 0.26 mg ml -1 in water limited plants after 8 days of withholding water, respectively), probably further attracted water into the xylem. This response mechanism increased at higher water deficit intensity. The little variations in carbohydrates and mineral elements within the leaves highlighted the main role of sinks rather than sources in the early response to water deficit., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

Phthalates are micro-pollutants of great concern due to their negative effects on ecosystem functioning and human health. Thanks to its capability in uptake and accumulation of organic pollutants, Populus alba L. "Villafranca" clone could be a good candidate for reducing the impacts derived by the persistence of such compounds in the environment. We investigated plant response and uptake of dioctyl phthalate (DOP) by poplar, grown in hydroponics condition, for 21 days with 0, 40, and 400 μg L -1 of d 4 -DOP. Treated plants, after 21 days of 400 μg L -1 d 4 -DOP, showed an increase in root dry biomass (+ 29%) at the expense of aerial parts (- 8%) compared with control. The root development could be sustained by the increase of Mg uptake by poplar. LC-MS/MS analysis demonstrated the uptake and accumulation in roots of d 4 -DOP starting from day one (3.5 ± 3.29 and 7.1 ± 3.28 in 40 and 400 μg L -1 d 4 -DOP respectively), despite volatilization of d 4 -DOP was observed from nutritive solution. The chemical interaction between d 4 -DOP and Zn occurred in roots of plants treated with the high d 4 -DOP concentration, without limiting the Zn concentration in leaves. Results confirm the high tolerance of "Villafranca" clone to xenobiotic and suggest the poplar capability in d 4 -DOP uptake and accumulation at root level.

Poplar ( Populus spp.) is a tree species considered for the remediation of soil contaminated by metals, including zinc (Zn). To improve poplar's capacity for Zn assimilation and compartmentalization, it is necessary to understand the physiological and biochemical mechanisms that enable these features as well as their regulation at the molecular level. We observed that the molecular response of poplar roots to Zn excess overlapped with that activated by hypoxia. Therefore, we tested the effect of Zn excess on hypoxia-sensing components and investigated the consequence of root hypoxia on poplar fitness and Zn accumulation capacity. Our results suggest that high intracellular Zn concentrations mimic iron deficiency and inhibit the activity of the oxygen sensors Plant Cysteine Oxidases, leading to the stabilization and activation of ERF-VII transcription factors, which are key regulators of the molecular response to hypoxia. Remarkably, excess Zn and waterlogging similarly decreased poplar growth and development. Simultaneous excess Zn and waterlogging did not exacerbate these parameters, although Zn uptake was limited. This study unveils the contribution of the oxygen-sensing machinery to the Zn excess response in poplar, which may be exploited to improve Zn tolerance and increase Zn accumulation capacity in plants., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

Aquaporins are water channel proteins that regulate plant development, growth, and response to environmental stresses. Populus trichocarpa is one of the plants with the highest number of aquaporins in its genome, but only few of them have been characterized at the whole plant functional level. Here we analyzed a putative aquaporin gene, aqua1, a gene that encodes for a protein of 257 amino acid with the typical NPA (Asp-Pro-Ala) signature motif of the aquaporin gene family. aqua1 was down-regulated of ∼10 fold under excess Zn in both leaves and roots, and conferred Zn tolerance when expressed in yeast Zn hypersensitive strain. In vivo localization of AQUA1-GFP in Arabidopsis protoplast showed a heterogeneous distribution of this protein on different membranes destined to form aggregates related to autophagic multivesicular bodies. Zn-dependent AQUA1-GFP re-localization was perturbed by phosphatases' and kinases' inhibitors that could affect both intracellular trafficking and aquaporins' activity. Exposed to high concentration of Zn, AQUA1 also co-localized with AtTIP1;1, a well-known Arabidopsis vacuolar marker, probably in pro-vacuolar multivesicular bodies. These findings suggest that high concentration of Zn down-regulates aqua1 and causes its re-localization in new forming pro-vacuoles. This Zn-dependent re-localization appears to be mediated by mechanisms regulating intracellular trafficking and aquaporins' post-translational modifications. This functional characterization of a poplar aquaporin in response to excess Zn will be a useful reference for understanding aquaporins' roles and regulation in response to high concentration of Zn in poplar., (Copyright © 2018 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Poplar plants may have an important role in the removal of pharmaceuticals from contaminated waters. In this context, plant uptake of the non-steroidal anti-inflammatory drug diclofenac, as well as physiological response in terms of growth traits and stress enzymes activity was assessed in Populus alba Villafranca clone, in order to establish the effectiveness of this species against pharmaceutical active compounds pollution. This evaluation was conducted in mesocosms with 1 mg L -1 of this pharmaceutical for a maximum period of 28 days. Root appears to be the organ with clear uptake of diclofenac (14.76 ± 2.42 ng g -1 fresh weight after 1 day of treatment), and presence of products derived from its metabolism. Indeed, 4-OH-diclofenac metabolite was detected in root tissues, indicating diclofenac uptake and metabolism inside the plants, already after 1 day of treatment. Regarding enzyme activities, glutathione-S-transferases increased in roots after long-term exposure to diclofenac, while an increase in activity of ascorbate peroxidase and glutathione reductase was detected in short and medium-term exposure, as a result of abiotic stress caused by diclofenac. Results suggest the ability of poplar to actively participate in the removal of diclofenac from water when used for phytoremediation purpose., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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.

Surfactants are widely used detergent ingredients and, thanks to their chemical properties, they are applied for remediation of sites polluted by heavy metals and organic contaminants, both in soil flushing and in phytoremediation. However, their direct effects on tree physiology especially in consociation with heavy metal pollution, as well as their possible absorption by plants, have not been appropriately investigated. In order to evaluate plant uptake/translocation of the surfactant sodium dodecyl sulfate (SDS) and the heavy metal zinc (Zn) in Populus alba L. Villafranca clone, SDS was applied alone (0.5 mM) or in combination with Zn (1 mM). Physiological effects on plant growth and photosynthetic performance were investigated. An increasing trend of Zn translocation towards basal leaves as a consequence of SDS co-treatment (1 mM Zn + 0.5 mM SDS; P = 0.03) was observed, proving the ability of SDS to improve heavy metals translocation. However, SDS exposure (both in 0.5 mM SDS and 1 mM Zn + 0.5 mM SDS treated plants) resulted in the appearance of foliar necrosis that expanded with an acropetal trend and finally led to leaf abscission. This phenotype may be caused by the emergence of an additional stress during the experimental trial, which could be related to the dissociation of sodium (Na) ions from the dodecyl sulfate molecules in the hydroponic system. In fact, while liquid chromatography-tandem mass spectrometry measurements revealed that dodecyl sulfate is mainly retained at root levels, Na is translocated to the aerial parts of the plant., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Populus alba Villafranca clone was chosen for a proof of concept study to determine the potential uptake and accumulation of antibiotics by trees. Plants were grown hydroponically and irrigated with a recirculating Hoagland's nutrient solution (control) and Hoagland's nutrient solution fortified with erythromycin at 0.01, 0.1 and 1mgL(-1). After 3 and 28days of treatment, poplar plants were separated into roots, stem, and leaves. Plants showed good health all over the period of treatment, and no differences in poplar growth for all the concentrations of erythromycin tested were observed. Quantification of erythromycin was performed using liquid chromatography electrospray ionization tandem mass spectrometry (LC-MS/MS) in positive ion mode using multiple reaction ion monitoring. Erythromycin was detected in all organs analyzed. Roots showed an erythromycin concentration tenfold higher than leaves. The photochemical efficiency of photosystem II did not show a dose-dependant trend. From the quenching analysis of chlorophyll fluorescence, low nonphotochemical quenching (NPQ) and high photochemical quenching (qP) for the first week of erythromycin exposure was observed, depending on leaves position along the stem. Results suggest a short term adaptation of the photosynthetic apparatus of Populus alba in response to environmental realistic erythromycin concentrations., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Cadmium (Cd) is a heavy metal toxic to humans and its occurrence in soils represents a significant environmental problem. Poplar trees may provide one possible option to help remove Cd contamination from soil. However, before this is practicable, the ability of poplar to accumulate Cd needs to be enhanced. A better understanding of the genes involved in Cd accumulation in poplar would help to achieve this goal. Here, we monitored the expression of genes known to be involved in Cd uptake, accumulation and translocation from other species, in order to provide information on their potential role in Cd accumulation in poplar. Cd concentration in poplar was significantly higher in roots than in stem and leaves in Cd treated plants. Expression of the poplar homologues of IRT1, NRAMP and OPT3 was initially increased after exposure to Cd but reduced after longer term Cd exposure. Exposure to Cd also influenced the accumulation of Fe, Ca, Cu, Mg and Mn in poplar. In particular, Cd treated plants had a higher concentration of Fe, Ca, Cu, and Mg in leaves and stem compared to control plants after one day and one week of experiment; while in roots after one month Cd treated plants had a lower concentration of Mn, Fe, Cu, Co, and Mg., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

This is the first study reporting the presence of endogenous caffeine, theobromine, and theophylline in all organs of poplar plants. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was used in order to evaluate the uptake, translocation, and metabolism of caffeine-(trimethyl-(13)C) in Populus alba L. Villafranca clone grown in hydroponic conditions. We investigated the remediation of caffeine since it is one of the most widely consumed drugs and it is frequently detected in wastewater treatment plant effluents, surface water, and groundwater worldwide. Our results demonstrated that poplar can absorb and degrade exogenous caffeine without negative effects on plant health. Data showed that concentrations of all endogenous compounds varied depending on caffeine-(trimethyl-(13)C) treatments. In particular, in control conditions, endogenous caffeine, theobromine, and theophylline were mainly distributed in roots. On the other hand, once caffeine-(trimethyl-(13)C) was provided, this compound and its dimethy-(13)C metabolites are mainly localized at leaf level. In conclusion, our results support the possible use of Villafranca clone in association with other water treatment systems in order to complete the process of caffeine remediation.

Key Message: Transgenic Populus alba over-expressing a TIP aquaporin ( aqua1) showed a higher growth rate under Zn excess, suggesting that aqua1 could be involved in water homeostasis, rather than in Zn homeostasis. Populus is the internationally accepted model for physiological and developmental studies of tree traits under stress. In plants, aquaporins facilitate and regulate the diffusion of water, however, few poplar aquaporins have been characterized to date. In this study, we reported for the first time an in vivo characterization of Populus alba clone Villafranca transgenic plants over-expressing a TIP aquaporin (aqua1) of P. x euramericana clone I-214. An AQUA1:GFP chimeric construct, over-expressed in P. alba Villafranca clones, shows a cytoplasmic localization in roots, and it localizes in guard cells in leaves. When over-expressed in transgenic plants, aqua1 confers a higher growth rate compared to wild-type (wt) plants, without affecting chlorophyll accumulation, relative water content (RWC), and fluorescence performances, but increasing the intrinsic Transpiration Efficiency. In response to Zn (1 mM), transgenic lines did not show a significant increase in Zn accumulation as compared to wt plants, even though the over-expression of this gene confers higher tolerance in root tissues. These results suggest that, in poplar plants, this gene could be principally involved in regulation of water homeostasis and biomass production, rather than in Zn homeostasis.

Populus x canadensis clone I-214 exhibits a general indicator phenotype in response to excess Zn, and a higher metal uptake in roots than in shoots with a reduced translocation to aerial parts under hydroponic conditions. This physiological adaptation seems mainly regulated by roots, although the molecular mechanisms that underlie these processes are still poorly understood. Here, differential expression analysis using RNA-sequencing technology was used to identify the molecular mechanisms involved in the response to excess Zn in root. In order to maximize specificity of detection of differentially expressed (DE) genes, we consider the intersection of genes identified by three distinct statistical approaches (61 up- and 19 down-regulated) and validate them by RT-qPCR, yielding an agreement of 93% between the two experimental techniques. Gene Ontology (GO) terms related to oxidation-reduction processes, transport and cellular iron ion homeostasis were enriched among DE genes, highlighting the importance of metal homeostasis in adaptation to excess Zn by P. x canadensis clone I-214. We identified the up-regulation of two Populus metal transporters (ZIP2 and NRAMP1) probably involved in metal uptake, and the down-regulation of a NAS4 gene involved in metal translocation. We identified also four Fe-homeostasis transcription factors (two bHLH38 genes, FIT and BTS) that were differentially expressed, probably for reducing Zn-induced Fe-deficiency. In particular, we suggest that the down-regulation of FIT transcription factor could be a mechanism to cope with Zn-induced Fe-deficiency in Populus. These results provide insight into the molecular mechanisms involved in adaption to excess Zn in Populus spp., but could also constitute a starting point for the identification and characterization of molecular markers or biotechnological targets for possible improvement of phytoremediation performances of poplar trees.

Information on plant responses to combined stresses such as ozone (O3) and cadmium (Cd) is scarce in tree species. On the other hand, high O3 concentrations in the atmosphere and heavy metal contaminations in water and soil simultaneously affect forest ecosystems. Toxic metals may exacerbate the consequences of air pollutants. In this research, two poplar clones, differently sensitive to O3 ("I-214" O3-tolerant and "Eridano" O3-sensitive), were grown for 5 weeks in pots supplied with 0 and 150 mg Cd kg(-1) soil and then exposed to a 15-day O3 fumigation (60 nl l(-1), 5 h a day) or supplied with charcoal-filtered air under the same conditions (referred to as control samples). The effects of the two stressors, alone or in combination, on Cd accumulation, photosynthetic capacity, ethylene emission and oxidative state were investigated in fully expanded leaves. Cadmium accumulation in leaves caused a reduction, but not complete failure, of photosynthesis in Eridano and I-214 poplar clones. The reduction in assimilation rate was more important following O3 fumigation. Stomatal aperture after O3 treatment, instead, increased in I-214 and decreased in Eridano. Overall, Cd treatment was effective in decreasing ethylene emission, whereas O3 fumigation increased it in both clones, although interacting with the metal treatment. Again, O3 fumigation induced a significant increase in ascorbate (ASA) + dehydroascorbate (DHA) content, which was strongly oxidised by O3, thus decreasing the redox state. On the other hand, Cd treatment had a positive effect on ASA content and redox state in I-214, but not in Eridano. Although Cd and O3 are known to share some common toxicity pathways, the combined effects induced distinct clone-specific responses, underlying the complexity of plant reactions to multiple stresses.

Soil cadmium (Cd) contamination is becoming a matter of great global concern. The identification of plants differentially sensitive to Cd excess is of interest for the selection of genotype adaptive to grow and develop in polluted areas and capable of ameliorating or reducing the negative environmental effects of this toxic metal. The two poplar clones I-214 (Populus×canadensis) and Eridano (Populus deltoides×maximowiczii) are, respectively, tolerant and sensitive to ozone (O3) exposure. Because stress tolerance is mediated by an array of overlapping defence mechanisms, we tested the hypothesis that these two clones differently sensitive to O3 stress factor also exhibit different tolerance to Cd. With this purpose, an outdoor pot experiment was designed to study the responses of I-214 and Eridano to the distribution of different Cd solutions enriched with CdCl2 (0, 50 and 150μM) for 35 days. Changes in leaf area, biomass allocation and Cd uptake, photosynthesis, chlorophyll fluorescence, leaf concentration of nutrients and pigments, hydrogen peroxide (H2O2) and nitric oxide (NO) production and thiol compounds were investigated. The two poplar clones showed similar sensitivity to excess Cd in terms of biomass production, photosynthesis activity and Cd accumulation, though physiological and biochemical traits revealed different defence strategies. In particular, Eridano maintained in any Cd treatment the number of its constitutively wider blade leaves, while the number of I-214 leaves (with lower size) was reduced. H2O2 increased 4.5- and 13-fold in I-214 leaves after the lowest (L) and highest (H) Cd treatments, respectively, revealing the induction of oxidative burst. NO, constitutively higher in I-214 than Eridano, progressively increased in both clones with the enhancement of Cd concentration in the substrate. I-214 showed a more elevated antioxidative capacity (GSH/GSSG) and higher photochemical efficiency of PSII (Fv/Fm) and de-epoxidation degree of xantophylls-cycle (DEPS). The glutathione pool was not affected by Cd treatment in both clones, while non-protein thiols and phytochelatins were reduced at L Cd treatment in I-214. Overall, these two clones presented high adaptability to Cd stress and are both suitable to develop and growth in environments contaminated with this metal, thus being promising for their potential use in phytoremediation programmes., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Contamination of soil and water by heavy metals has become a widespread problem; environmental pollution by high zinc (Zn) concentration occurs frequently. Although poplar (Populus spp.) has been identified as suitable for phytoremediation approaches, its response to high Zn concentrations are still not clearly understood. For this reason, we investigated the effects of Zn in Populus×euramericana clone I-214 roots by proteomic analysis. Comparative experiments were conducted on rooted woody cuttings grown in nutrient solutions containing 1mM (treatment) or 1μM (control) Zn concentrations. A gel-based proteomic approach coupled with morphological and chemical analysis was used to identify differentially represented proteins in treated roots and to investigate the effect of Zn treatment on the poplar root system. Data shows that Zn was accumulated preferentially in roots, that the antioxidant system, the carbohydrate/energy and amino acid metabolisms were the main pathways modulated by Zn excess, and that mitochondria and vacuoles were the cellular organelles predominately affected by Zn stress. A coordination between cell death and proliferation/growth seems to occur under this condition to counteract the Zn-induced damage., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Zinc (Zn) is an essential element for plant growth and development, but at high levels this metal can become toxic. Hyperaccumulator species are often not suitable for phytoremediation technologies because they need to be fast growing and have high biomass production, such as those of the Populus genus. Comparative genomics studies of poplars subjected to stress conditions such as heavy metal contamination have generated resources useful for improving the annotation of genes and have provided novel insights in the defense/tolerance mechanisms governing adaptation in non-hyperaccumulator plants. Using a microarray-based comparative analysis, we identified functional gene sets that are differentially regulated in the leaves of Populus × euramericana clone I-214 subjected to an excess but sub-lethal dose of Zn (1 mM). Eco-physiological and chemical analyses confirmed the results obtained in previous similar experiments. A total of 3861 expressed sequence tags (ESTs) were differentially expressed and grouped into two distinct libraries of up-regulated (40%) and down-regulated (60%) putative genes. The annotation of genes and gene products according to the Gene Ontology vocabularies was performed using Blast2GO software. The two transcriptome data sets were used to query all known Kyoto Encyclopedia of Genes and Genomes (KEGG) biosynthetic pathways of the genes identified in this study. The most represented molecular functions and biological processes were nucleotide binding and transcription, transport and response to stress and abiotic and biotic stimuli. The chloroplast, mitochondrion and their membrane systems were the cellular components most affected by excess Zn, as well as the photosynthetic, defense, sulfur and glutathione (GSH) metabolic pathways. The most up-regulated genes encoded electron carriers associated with ferrodoxin, the small subunit of ribulose-bisphosphate carboxylase oxygenase, and enzymes involved in GSH metabolism. This study is the most in-depth transcriptome and gene-annotation analysis of a hybrid poplar to date. The results are presented and critically discussed in terms of poplar response/tolerance to Zn stress for the characterization of non-hyperaccumulator phenotypes and the identification of candidate genes in perennial plants. These genetic findings provide useful information on tree species' adaptation to metal stress and provide powerful tools for the selection and/or genetic manipulation of stress-tolerant poplar clones.

Increasing global background concentrations of tropospheric ozone (O(3)) are expected to affect both crops and forest ecosystems negatively. The phytotoxic effects of O(3) are mainly associated with the O(3)-induced production of reactive oxygen species (ROS) in excess of the ability of the plant to maintain ROS below the tolerance threshold. It is the balance between O(3) uptake and cellular antioxidant potential that determines O(3) effects on vegetation. The greater sensitivity to ambient O(3) exposure (60 nl l(-1) O(3), 15 days, 5 h a day) of poplar clone Eridano (Populus deltoides x maximowiczii) compared with clone I-214 (P. x euramericana) was reflected in a lower photosynthetic efficiency, higher stomatal conductance and hydrogen peroxide (H(2)O(2)) accumulation and more pronounced leaf tissue membrane injury in Eridano than in I-214. We checked if the differences in clonal responses to O(3) fumigation were related to differing capacities for antioxidant defense and phenylpropanoid metabolism and found that the increases in foliar ascorbate and phenolic concentrations and phenylpropanoid metabolism in Eridano were insufficient to counteract H(2)O(2) accumulation and the consequent oxidative stress. This was probably because the higher influx of O(3) into Eridano leaves compared with I-214 leaves resulted in a lower potential detoxification capacity per unit of O(3) influx.

In this study the effects of different levels of industrial wastes on growth traits and metal accumulation in aerial portions were determined for Populusxeuramericana clone I-214. The experiment started in April 2003. Scions of Populusxeuramericana clone I-214, were grown outdoor near Pisa (Italy), in lisimeters filled with soil naturally present in the land around the experimental site, were daily drip irrigated, hand weeded, monthly fertilized, pruned for a unique shoot and cultivated with four increasing treatments: soil non-amended, soil amended with 4.8 kgm(-2), with 9.6 kgm(-2) and with 19.2 kgm(-2) of fresh tannery waste. The climatic parameters were daily recorded throughout the whole experiment. Growth relieves were performed during the growing season. After six months since the plantation of the scions, aerial portions of every plant were harvested for biomass and metal content analyses. Data demonstrated that the waste exerted beneficial effects on poplars mainly through a general increase of growth traits and that the nutrients relocation is the mechanisms involved in modulating growth rate. The concentration and the amount of the mineral elements analysed (N, P, K, Na, Ca, Mg, S, B, Fe, Mn, Cu, Zn, Cr) changed determinately among treatments, organs and position. We concluded that phytoremediation strategies of tannery wastes might be possible and sustainable for polar plantations in soil amended with non-hazardous levels of industrial waste, which maintain total heavy metals concentration close to background values.

Hydrogen peroxide (H(2)O(2)), ethylene, 1-aminocyclopropane-1-carboxylic acid (ACC) and salicylic acid (SA) concentrations and ACC synthase (ACS) gene expression were measured to establish whether the high sensitivity of the Populus deltoides x maximowiczii clone Eridano to ozone (O(3)) exposure, compared with the O(3)-resistant Populus deltoides x euramericana clone I-214, is attributable to differences in the modulation of signal transduction pathways. In a time-course experiment, Populus deltoides (poplar) clones were exposed to acute fumigation with 150 nl l(-1) O(3) for 5 h. The two poplar clones showed differences in ethylene evolution, I-214 displaying earlier and less pronounced ethylene emission than Eridano. In both clones, ethylene evolution was accompanied by increased ACS transcript levels and enhanced emission of free ACC. I-214 exhibited a greater basal concentration of free SA and a lower concentration of the conjugated pool. However, a slight accumulation of free SA at the end of the 5-h exposure was found only in Eridano, together with an earlier minimal increase in the concentration of conjugated SA. The results show that both clones react to O(3) by producing H(2)O(2), ethylene and SA, but the difference in sensitivity to the pollutant is probably attributable to differences in the kinetics and magnitude of this response.

Transition metals such as zinc (Zn) are essential micronutrients for many physiological processes, but they become toxic at elevated levels. Zinc is one of the most abundant trace heavy metals present in agro-ecosystems. Populus spp. have been suggested as good candidates for using to study the removal and/or immobilization of environmental organic and inorganic pollutants. In order to understand the physiological and biochemical bases of this assumption for Zn, plants of Populus deltoides x P. nigra (P. x euramericana) were grown in hydroponics with different concentrations of Zn [1 microm (control), and 1, 5 and 10 mm] in the nutrient solution.Shoot biomass decreased at 5 and 10 mm Zn, while the Zn content of young leaves increased progressively with increasing Zn concentration (1-10 mm). Total glutathione (GSH+GSSG) content was reduced with increasing Zn concentration, while the contribution of oxidized to total glutathione increased. Despite these observations, semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) revealed that the gene expression of GSH reductase (GR, chloroplastic and cytosolic isoform) and gamma-glutamylcysteine synthetase (gamma-ECS) increased in young leaves of poplars treated with excess Zn. We conclude that GSH synthesis, consumption and redox status play a central role in the response of poplars to high concentrations of Zn.

Plants can remove or immobilize various environmental contaminants; however, little is known about the physiological mechanisms underlying responses to soil amendment with biosolids contaminated with heavy metals. We investigated the responses of cuttings of hybrid poplar clones Eridano and I-214 grown for a season in soil amended with nutrient-rich organic material from tanneries, which contains potentially toxic amounts of heavy elements. Plant growth traits, gas exchange parameters, stomatal density and leaf layer thickness of frozen-hydrated leaves, and foliar concentrations of heavy metals and nitrogen were determined. Overall, soil amendment increased net assimilation rate and growth, but the cuttings accumulated only small quantities of heavy metal soil contaminants.