Genome-wide association mapping uncovers sex-associated copy number variation markers and female hemizygous regions on the W chromosome in Salix viminalis.

BACKGROUND: Sex chromosomes are in some species largely undifferentiated (homomorphic) with restricted sex determination regions. Homomorphic but different sex chromosomes are found in the closely related genera Populus and Salix indicating flexible sex determination systems, ideal for studies of processes involved in sex chromosome evolution. We have performed genome-wide association studies of sex and analysed sex chromosomes in a population of 265 wild collected Salix viminalis accessions and studied the sex determining locus. RESULTS: A total of 19,592 markers were used in association analyses using both Fisher's exact tests and a single-marker mixed linear model, which resulted in 48 and 41 sex-associated (SA) markers respectively. Across all 48 SA markers, females were much more often heterozygous than males, which is expected if females were the heterogametic sex. The majority of the SA markers were, based on positions in the S. purpurea genome, located on chromosome 15, previously demonstrated to be the sex chromosome. Interestingly, when mapping the genotyping-by-sequencing sequence tag harbouring the two SA markers with the highest significance to the S. viminalis genomic scaffolds, five regions of very high similarity were found: three on a scaffold that represents a part of chromosome 15, one on a scaffold that represents a part of chromosome 9 and one on a scaffold not anchored to the genome. Based on segregation differences of the alleles at the two marker positions and on differences in PCR amplification between females and males we conclude that females had multiple copies of this DNA fragment (chromosome 9 and 15), whereas males only had one (chromosome 9). We therefore postulate that the female specific sequences have been copied from chromosome 9 and inserted on chromosome 15, subsequently developing into a hemizygous W chromosome linked region. CONCLUSIONS: Our results support that sex determination in S. viminalis is controlled by one locus on chromosome 15. The segregation patterns observed at the SA markers furthermore confirm that S. viminalis females are the heterogametic sex. We also identified a translocation from chromosome 9 to the W chromosome.

Antioxidant Activity and Chemical Characteristics of Supercritical CO2 and Water Extracts from Willow and Poplar.

Renewable feedstock from perennial industrial crops, including those cultivated on marginal land in a short-rotation coppice system, could be an important contribution to the bioeconomy. The majority of data available on the topic are limited to the production of bioenergy from this type of biomass. According to the concept of bioeconomy, biomass-based bioproducts have priority over energy production. This paper characterizes the chemical composition and antioxidant activity of extracts from bark (b), wood (w) or a mixture of bark and wood (b + w) from Salix purpurea, Salix viminalis and Populus nigra obtained using supercritical carbon dioxide (scCO2), scCO2 and water (1%, w/w) or only water. Generally, a high concentration of polyphenols was obtained after extraction with scCO2 and water, while the lowest concentration was found in extracts obtained with scCO2. The highest concentration of polyphenols (p < 0.05) was obtained in an extract from P. nigra (b) (502.62 ± 9.86 mg GAE/g dry matter (d.m.)) after extraction with scCO2 and water, whereas the lowest polyphenol concentration was observed in an scCO2 extract from S. purpurea (b) (6.02 ± 0.13 mg GAE/g d.m.). The flavonoids were effectively separated by extraction with scCO2 (0.88-18.37 mg QE/g d.m.). A positive linear relationship between the antioxidant activity determined by DPPH and ABTS assays and the concentration of polyphenols was demonstrated, R2 = 0.8377 and R2 = 0.9568, respectively. It is most probable that the concentration of flavonoids, rather than the concentration of polyphenols, determines the chelating activity of Fe2+. The Fe2+-chelating activity of scCO2 extracts ranged from 75.11% (EC50 = 5.41 mg/cm3, S. purpurea, b + w) to 99.43% (EC50 = 0.85 mg/cm3, P. nigra, b + w). The lowest chelating activity was demonstrated by the extracts obtained with scCO2 and water (maximum 26.36%, S. purpurea, b + w). In extracts obtained with scCO2 and water, p-hydroxybenzoic acid (210-428 µg/g), p-coumaric acid (56-281 µg/g), saligenin (142-300 µg/g) and salicortin (16-164 µg/g) were the dominant polyphenols. All of these chemical compounds occurred mainly in the free form. The S. purpurea, S. viminalis and P. nigra biomass proved to be an attractive source of biologically active compounds for various possible applications in food, drugs or cosmetics. These compounds could be extracted using an environmentally friendly method with scCO2 and water as a co-solvent.

Fast-growing willows significantly reduce invasive knotweed spread.

Competitive interactions seem to play a major role in invasive plant success. However, they have mostly been addressed through the invader impacts on other species of the plant community and rarely through the way plant communities can contain alien species. Understanding such mechanisms would help in designing restoration projects using plant community competitive properties to control invasive populations. In this study, we looked at the role of competitive interactions in the success of Fallopia japonica (Houtt.) Ronse Decraene using a native willow frequently used in bioengineering techniques: Salix viminalis L. S. viminalis has a high growth rate and is, as such, a potential candidate to compete with F. japonica in restoration projects of invaded areas. Both species were grown in semi-controlled conditions in mesocosms (truck dumpsters), alone or in competition. Morphological traits (plant height, specific leaf area) as well as biomass (aboveground and underground) were measured on each species during two growing seasons. We also quantified spatial expansion of F. japonica. Even under a dense canopy of S. viminalis, F. japonica was able to survive and grow. However, its performance was significantly reduced compared to monocultures and its spatial colonization was less extended. Although S. viminalis biomass was affected by F. japonica, F. japonica expressed competitive stress through a modification of ramet density and height. There was no significant effect of F. japonica on S. viminalis height, enabling this species to dominate. Synthesis and applications: We conclude that S. viminalis succeeded in reducing F. japonica growth by developing a competitive canopy. Bioengineering techniques aiming at restoring a competitive neighborhood can control F. japonica. However, F. japonica's broad underground extension should be taken into account in any management strategy in order to successfully limit its development and spatial spread.

Growth, physiology, and phytoextraction potential of poplar and willow established in soils amended with heavy-metal contaminated, dredged river sediments.

Phytotechnologies have been used worldwide to remediate and restore damaged ecosystems, especially those caused by industrial byproducts leaching into rivers and other waterways. The objective of this study was to test the growth, physiology, and phytoextraction potential of poplar and willow established in soils amended with heavy-metal contaminated, dredged river sediments from the Great Backa Canal near Vrbas City, Serbia. The sediments were applied to greenhouse-grown trees of Populus deltoides Bartr. ex Marsh. clone 'Bora' and Salix viminalis L. clone 'SV068'. Individual pots with trees previously grown for two months were amended with 0, 0.5 and 1.0 kg of sediment containing 400 mg Cr kg-1, 295 mg Cu kg-1, 465 mg Zn kg-1, 124 mg Ni kg-1, 1.87 mg Cd kg-1, and 61 mg Pb kg-1. Following amendment, trees were grown for two seasons (i.e., 2014, 2015), with coppicing after the first season. In addition to growth parameters, physiological traits related to the photosynthesis and nitrogen metabolism were assessed during both growing seasons. At the end of the study, trees were harvested for biomass analysis and accumulation of heavy metals in tree tissues and soils. Application of sediment decreased aboveground biomass by 37.3% in 2014, but increased height (16.4%) and leaf area (19.2%) in 2015. Sediment application negatively impacted the content of pigments and nitrate reductase activity, causing them to decrease over time. Generally, the effect of treatments on growth was more pronounced in poplars, while willows had more pronounced physiological activity. Accumulation patterns were similar to previously-published results. In particular, Zn and Cd were mostly accumulated in leaves of both poplar and willow, which indicated successful phytoextraction. In contrast, other metals (e.g., Cr, Ni, Pb, Cu) were mostly phytostabilized in the roots. Differences in metal allocation between poplar and willow were recorded only for Cu, while other metals followed similar distribution patterns in both genera. Results of this study indicated that the composition of heavy metals in the sediments determined the mechanisms of the applied phytoremediation technique.

Genetics of phenotypic plasticity and biomass traits in hybrid willows across contrasting environments and years.

Background and Aims: Phenotypic plasticity can affect the geographical distribution of taxa and greatly impact the productivity of crops across contrasting and variable environments. The main objectives of this study were to identify genotype-phenotype associations in key biomass and phenology traits and the strength of phenotypic plasticity of these traits in a short-rotation coppice willow population across multiple years and contrasting environments to facilitate marker-assisted selection for these traits. Methods: A hybrid Salix viminalis × ( S. viminalis × Salix schwerinii ) population with 463 individuals was clonally propagated and planted in three common garden experiments comprising one climatic contrast between Sweden and Italy and one water availability contrast in Italy. Several key phenotypic traits were measured and phenotypic plasticity was estimated as the trait value difference between experiments. Quantitative trait locus (QTL) mapping analyses were conducted using a dense linkage map and phenotypic effects of S. schwerinii haplotypes derived from detected QTL were assessed. Key Results: Across the climatic contrast, clone predictor correlations for biomass traits were low and few common biomass QTL were detected. This indicates that the genetic regulation of biomass traits was sensitive to environmental variation. Biomass QTL were, however, frequently shared across years and across the water availability contrast. Phenology QTL were generally shared between all experiments. Substantial phenotypic plasticity was found among the hybrid offspring, that to a large extent had a genetic origin. Individuals carrying influential S. schwerinii haplotypes generally performed well in Sweden but less well in Italy in terms of biomass production. Conclusions: The results indicate that specific genetic elements of S. schwerinii are more suited to Swedish conditions than to those of Italy. Therefore, selection should preferably be conducted separately for such environments in order to maximize biomass production in admixed S. viminalis × S. schwerinii populations.

Transpiration and metabolisation of TCE by willow plants - a pot experiment.

Willows were grown in glass cylinders filled with compost above water-saturated quartz sand, to trace the fate of TCE in water and plant biomass. The experiment was repeated once with the same plants in two consecutive years. TCE was added in nominal concentrations of 0, 144, 288, and 721 mg l(-1). Unplanted cylinders were set-up and spiked with nominal concentrations of 721 mg l(-1) TCE in the second year. Additionally, (13)C-enriched TCE solution (δ(13)C = 110.3 ‰) was used. Periodically, TCE content and metabolites were analyzed in water and plant biomass. The presence of TCE-degrading microorganisms was monitored via the measurement of the isotopic ratio of carbon ((13)C/(12)C) in TCE, and the abundance of (13)C-labeled microbial PLFAs (phospholipid fatty acids). More than 98% of TCE was lost via evapotranspiration from the planted pots within one month after adding TCE. Transpiration accounted to 94 to 78% of the total evapotranspiration loss. Almost 1% of TCE was metabolized in the shoots, whereby trichloroacetic acid (TCAA) and dichloroacetic acid (DCAA) were dominant metabolites; less trichloroethanol (TCOH) and TCE accumulated in plant tissues. Microbial degradation was ruled out by δ(13)C measurements of water and PLFAs. TCE had no detected influence on plant stress status as determined by chlorophyll-fluorescence and gas exchange.

Antitoxic Activity of Extract from Salix Viminalis Leaves under Conditions of 5-Fluorouracil Treatment.

Injection of 5-fluorouracil to animals caused a pronounced toxic effect. Therapeutic and preventive treatment with Salix viminalis leaf extract significantly reduced the negative effects of the antitumor drug: promoted recovery of the bone marrow, peripheral blood, and visceral parameters and prevented ulceration. Combined use of the cytostatic and Salix viminalis extract increased the efficiency of antitumor therapy.

Cloning and Functional Identification of Gibberellin Receptor SvGID1s Gene of Salix viminalis.

Gibberellins (GAs) play a key role in the transition from vegetative growth to flowering and the GA receptor GID1 (GIBBERELLIN INSENSITIVE DWARF1) is the central part of GA-signaling. The differential expression of SvGID1 was found in the transcriptome sequencing in our previous study, which was further verified at different stages of flowering of Salix viminalis. In order to reveal the function GID1 of S. viminalis, two genes of SvGID1b and SvGID1c were cloned and transformed into Arabidopsis thaliana, respectively. The results showed that the full ORF length of SvGID1b and SvGID1c genes were both 1035 bp, encoding 344 amino acids, which were typical globular proteins. The peptide chain contained more α-helix structure, and had 99% similarity with GID1b and GID1c amino acid sequences of Salix suchowensis. Phylogenetic analysis showed that SvGID1s had close genetic relationship with woody plants such as Populus alba and Populus tomentosa, and had far genetic relationship with rice. After overexpression in A. thaliana, the total gibberellin, active gibberellin content and the expression level of GA3ox1, the key gene for GA4 synthesis, were not significantly different from those in the wild-type, while the expression levels of FUL, SOC1 and FT, the key genes for flowering in plants, were increased, and the expression levels of FLC and GAI were decreased. The ectopic expression of SvGID1s increased the sensitivity of plants to gibberellin and enhanced gibberellin effect, caused early bolting, budding and flowering, led to higher plant, longer hypocotyl and other phenomena. The results provide a theoretical basis for clarifying the regulation of gibberellin on flower bud differentiation of flowering plants.

Characterization of the complete Salix viminalis var. gmelinii Turcz 1854 chloroplast genome from the northeast of China.

The Salix viminalis var. gmelinii Turcz 1854 is a variant of the Salix genus, from the Salicaceae family, and possesses an extremely high economic value. In this study the complete chloroplast genome of the woody plant S. viminalis var. gmelinii was characterized for the first time using a high-throughput approach in conjunction with de novo assembly technology. The S.viminalis var. gmelinii chloroplast genome is 155,405 base pairs (bp) in length and contains 36.71% GC content. It incorporates a large single-copy region (LSC, 84,287bp) alongside one small-copy region (SSC, 16,198bp), and two inverted repeat regions (IRA and IRB, 27,460bp). Moreover, this chloroplast genome encodes 128 genes, which comprises 83 protein-coding genes, 37 transfer RNA (tRNA) genes, and eight ribosomal RNA (rRNA) genes. Furthermore, the phylogenetic analysis revealed that S.viminalis var. gmelinii is closely related to S. cupularis and S.gordejevii.

The contribution of G-layer glucose in Salix clones for biofuels: comparative enzymatic and HPLC analysis of stem cross sections.

BACKGROUND: Interest on the use of short rotation willow as a lignocellulose resource for liquid transport fuels has increased greatly over the last 10 years. Investigations have shown the advantages and potential of using Salix spp. for such fuels but have also emphasized the wide variations existing in the compositional structure between different species and genotypes in addition to their effects on overall yield. The present work studied the importance of tension wood (TW) as a readily available source of glucose in 2-year-old stems of four Salix clones (Tora, Björn, Jorr, Loden). Studies involved application of a novel approach whereby TW-glucose and residual sugars and lignin were quantified using stem cross sections with results correlated with HPLC analyses of milled wood. Compositional analyses were made for four points along stems and glucose derived from enzyme saccharification of TW gelatinous (G) layers (G-glucose), structural cell wall glucose (CW-glucose) remaining after saccharification and total glucose (T-glucose) determined both theoretically and from HPLC analyses. Comparisons were also made between presence of other characteristic sugars as well as acid-soluble and -insoluble lignin. RESULTS: Preliminary studies showed good agreement between using stem serial sections and milled powder from Salix stems for determining total sugar and lignin. Therefore, sections were used throughout the work. HPLC determination of T-glucose in Salix clones varied between 47.1 and 52.8%, showing a trend for higher T-glucose with increasing height (Björn, Tora and Jorr). Using histochemical/microscopy and image analysis, Tora (24.2%) and Björn (28.2%) showed greater volumes of % TW than Jorr (15.5%) and Loden (14.0%). Total G-glucose with enzyme saccharification of TW G-layers varied between 3.7 and 14.7% increasing as the total TW volume increased. CW-glucose measured after enzyme saccharification showed mean values of 41.9-49.1%. Total lignin between and within clones showed small differences with mean variations of 22.4-22.8% before and 22.4-24.3% after enzyme saccharification. Calculated theoretical and quantified values for CW-glucose at different heights for clones were similar with strong correlation: T-glucose = G-glucose + CW-glucose. Pearson's correlation displayed a strong and positive correlation between T-glucose and G-glucose, % TW and stem height, and between G-glucose with % TW and stem height. CONCLUSIONS: The use of stem cross sections to estimate TW together with enzyme saccharification represents a viable approach for determining freely available G-glucose from TW allowing comparisons between Salix clones. Using stem sections provides for discrete morphological/compositional tissue comparisons between clones with results consistent with traditional wet chemical analysis approaches where entire stems are milled and analyzed. The four clones showed variable TW and presence of total % G-glucose in the order Björn > Tora > Jorr > Loden. Calculated in terms of 1 m3, Salix stems Tora and Björn would contain ca. 0.24 and 0.28 m3 of tension wood representing a significant amount of freely available glucose.

Mapping the sex determination region in the Salix F1 hybrid common parent population confirms a ZW system in six diverse species.

Within the genus Salix, there are approximately 350 species native primarily to the northern hemisphere and adapted to a wide range of habitats. This diversity can be exploited to mine novel alleles conferring variation important for production as a bioenergy crop, but also to identify evolutionarily important genes, such as those involved in sex determination. To leverage this diversity, we created a mapping population by crossing 6 Salix species (Salix viminalis, Salix suchowensis, Salix integra, Salix koriyanagi, Salix udensis, and Salix alberti) to common male and female Salix purpurea parents. Each family was genotyped via genotyping-by-sequencing and assessed for kinship and population structure as well as the construction of 16 backcross linkage maps to be used as a genetic resource for breeding and selection. Analyses of population structure resolved both the parents and F1 progeny to their respective phylogenetic section and indicated that the S. alberti parent was misidentified and was most likely S.suchowensis. Sex determining regions were identified on Salix chromosome 15 in the female-informative maps for seven of the eight families indicating that these species share a common female heterogametic ZW sex system. The eighth family, S. integra × S. purpurea, was entirely female and had a truncated chromosome 15. Beyond sex determination, the Salix F1 hybrid common parent population (Salix F1 HCP) introduced here will be useful in characterizing genetic factors underlying complex traits, aid in marker-assisted selection, and support genome assemblies for this promising bioenergy crop.

Effect of Supercritical Extract from Black Poplar and Basket Willow on the Quality of Natural and Probiotic Drinkable Yogurt.

Yogurt is a fermented milk drink produced by Streptococcus thermophilus, Lactobacillus delbrüeckii ssp. bulgaricus, or Lactobacillus rhamnosus, which can be enriched with polyphenolic compounds to enhance its antioxidant properties. Supercritical (scCO2/H2O) extracts obtained from the mixture of bark and wood of black poplar (Populus nigra) and basket willow (Salix viminalis) are the source of bioactive compounds. The aim of the study was to assess the effect of supercritical extracts from the P. nigra and S. viminalis on the fermentation, quality, and bioactive properties of drinkable natural and probiotic yogurts. The incorporation of scCO2/H2O extracts at a dose of 0.01% (w/v) into milk for the production of natural and probiotic yogurts increases their functional properties by enhancing the antioxidant activity without causing negative effects on the physicochemical and organoleptic properties of products. The antioxidant activity of yogurt with scCO2/H2O extract from P. nigra and S. viminalis was higher than control yogurts by 1.3-13.2% and 4.4-37.5%, respectively. The addition of a supercritical S. viminalis extract reduced the time of natural and probiotic yogurt fermentation. Natural and probiotic yogurt with scCO2/H2O extracts added was characterised by a bacterial population size of over 7 log cfu/g, and the microflora was active throughout the cold storage period. FTIR analysis confirmed the presence of scCO2/H2O extracts from P. nigra or S. viminalis in both types of yogurt. A secondary structure analysis confirmed interactions between compounds of scCO2/H2O extract from P. nigra and S. viminalis extract with milk proteins. These interactions affect the compounds' structural and functional properties by changing, e.g., their digestibility and antioxidant properties.

Transcriptomic and metabolomic insights into the adaptive response of Salix viminalis to phenanthrene.

Polycyclic aromatic hydrocarbons (PAHs) are widespread, persistent environmental pollutants. They exert toxic effects at different developmental stages of plants. Plant defense mechanisms against PAHs are poorly understood. To this end, transcriptomics and widely targeted metabolomic sequencing were used to study the changes in gene expression and metabolites that occur in the roots of Salix viminalis subjected to phenanthrene stress. Significant variations in genes and metabolites were observed between treatment groups and the control group. Thirteen amino acids and key genes involved in their biosynthesis were upregulated exposed to phenanthrene. Cysteine biosynthesis was upregulated. Sucrose, inositol galactoside, and mellidiose were the main carbohydrates that were largely accumulated. Glutathione biosynthesis was enhanced in order to scavenge reactive oxygen species and detoxify the phenanthrene. Glucosinolate and flavonoid biosynthesis were upregulated. The production of pinocembrin, apigenin, and epigallocatechin increased, which may play a role in antioxidation to resist phenanthrene stress. In addition, levels of six amino acids and N,N'-(p-coumaroyl)-cinnamoyl-caffeoyl-spermidine were significantly increased, which may have helped protect the plant against phenanthrene stress. These results demonstrated that S. viminalis had a positive defense strategy in response to phenanthrene challenge. Subsequent defense-related reactions may have also occurred within 24 h of phenanthrene exposure. The findings of the present study would be useful in elucidating the molecular mechanisms regulating plant responses to PAH challenges and would help guide crop and plant breeders in enhancing PAH resistance.

Enzymatic hydrolysis of the gelatinous layer in tension wood of Salix varieties as a measure of accessible cellulose for biofuels.

BACKGROUND: Salix (willow) species represent an important source of bioenergy and offer great potential for producing biofuels. Salix spp. like many hardwoods, produce tension wood (TW) characterized by special fibres (G-fibres) that produce a cellulose-rich lignin-free gelatinous (G) layer on the inner fibre cell wall. Presence of increased amounts of TW and G-fibres represents an increased source of cellulose. In the present study, the presence of TW in whole stems of different Salix varieties was characterized (i.e., physical measurements, histochemistry, image analysis, and microscopy) as a possible marker for the availability of freely available cellulose and potential for releasing D-glucose. Stem cross sections from different Salix varieties (Tora, Björn) were characterized for TW, and subjected to cellulase hydrolysis with the free D-glucose produced determined using a glucose oxidase/peroxidase (GOPOD) assay. Effect of cellulase on the cross sections and progressive hydrolysis of the G-layer was followed using light microscopy after staining and scanning electron microscopy (SEM). RESULTS: Tension wood fibres with G-layers were developed multilaterally in all stems studied. Salix TW from varieties Tora and Björn showed fibre G-layers were non-lignified with variable thickness. Results showed: (i) Differences in total % TW at different stem heights; (ii) that using a 3-day incubation period at 50 °C, the G-layers could be hydrolyzed with no apparent ultrastructural effects on lignified secondary cell wall layers and middle lamellae of other cell elements; and (iii) that by correlating the amount of D-glucose produced from cross sections at different stem heights together with total % TW and density, an estimate of the total free D-glucose in stems can be derived and compared between varieties. These values were used together with a literature value (45%) for estimating the contribution played by G-layer cellulose to the total cellulose content. CONCLUSIONS: The stem section-enzyme method developed provides a viable approach to compare different Salix varieties ability to produce TW and thus freely available D-glucose for fermentation and biofuel production. The use of Salix stem cross sections rather than comminuted biomass allows direct correlation between tissue- and cell types with D-glucose release. Results allowed correlation between % TW in cross sections and entire Salix stems with D-glucose production from digested G-layers. Results further emphasize the importance of TW and G-fibre cellulose as an important marker for enhanced D-glucose release in Salix varieties.

Soil microbial community succession and interactions during combined plant/white-rot fungus remediation of polycyclic aromatic hydrocarbons.

Despite combined plant/white-rot fungus remediation being effective for remediating polycyclic aromatic hydrocarbon (PAH)-contaminated soil, the complex organismal interactions and their effects on soil PAH degradation remain unclear. Here, we used quantitative PCR, analysis of soil enzyme activities, and sequencing of representative genes to characterize the ecological dynamics of natural attenuation, mycoremediation (MR, using Crucibulum laeve), phytoremediation (PR, using Salix viminalis), and plant-microbial remediation (PMR, using both species) for PAHs in soil for 60 days. On day 60, PMR achieved the highest removal efficiency of all three representative PAHs (65.5%, 47.5%, and 62.4% for phenanthrene, pyrene, and benzo(a)pyrene, respectively) when compared with the other treatments. MR significantly increased the relative abundance of Rhizobium and Bacillus but antagonized the other putative indigenous PAH-degrading bacteria, which were enriched by PR. PR significantly reduced soil nutrients, such as NO3- and NH4+, and available potassium (AK), thereby changing the microbial community composition as reflected by redundancy analysis, significantly reducing the soil bacterial biomass relative to that in other treatments. These disadvantages hampered phenanthrene and pyrene removal. MR provided additional nutrients, which counteracted the nutrient consumption associated with PR, thereby maintaining the microbial community diversity and bacterial biomass of PMR at a level achieved in the NA treatment. Combination remediation therefore overcame the disadvantages of using PR alone. These results indicated that inoculation with the combination of S. viminalis and C. laeve synergistically stimulated the growth of indigenous PAH-degrading microorganisms and maintained bacterial biomass, thus accelerating the dissipation of soil PAHs.

Heterologous Expression of SvMBD5 from Salix viminalis L. Promotes Flowering in Arabidopsis thaliana L.

Methyl-CpG-binding domain (MBD) proteins have diverse molecular and biological functions in plants. Most studies of MBD proteins in plants have focused on the model plant Arabidopsis thaliana L. Here we cloned SvMBD5 from the willow Salix viminalis L. by reverse transcription-polymerase chain reaction (RT-PCR) and analyzed the structure of SvMBD5 and its evolutionary relationships with proteins in other species. The coding sequence of SvMBD5 is 645 bp long, encoding a 214 amino acid protein with a methyl-CpG-binding domain. SvMBD5 belongs to the same subfamily as AtMBD5 and AtMBD6 from Arabidopsis. Subcellular localization analysis showed that SvMBD5 is only expressed in the nucleus. We transformed Arabidopsis plants with a 35S::SvMBD5 expression construct to examine SvMBD5 function. The Arabidopsis SvMBD5-expressing line flowered earlier than the wild type. In the transgenic plants, the expression of FLOWERING LOCUS T and CONSTANS significantly increased, while the expression of FLOWERING LOCUS C greatly decreased. In addition, heterologously expressing SvMBD5 in Arabidopsis significantly inhibited the establishment and maintenance of methylation of CHROMOMETHYLASE 3 and METHYLTRANSFERASE 1, as well as their expression, and significantly increased the expression of the demethylation-related genes REPRESSOR OF SILENCING1 and DEMETER-LIKE PROTEIN3. Our findings suggest that SvMBD5 participates in the flowering process by regulating the methylation levels of flowering genes, laying the foundation for further studying the role of SvMBD5 in regulating DNA demethylation.

Enhancing Salix viminalis L.-mediated phytoremediation of polycyclic aromatic hydrocarbon-contaminated soil by inoculation with Crucibulum laeve (white-rot fungus).

Although plant-white-rot fungi (WRF) remediation is considered efficient in improving polycyclic aromatic hydrocarbon (PAH)-contaminated soil, the prospects for using it remain poorly known. Therefore, we evaluated whether the WRF Crucibulum laeve could improve the phytoremediation of PAH-contaminated soil by Salix viminalis L. A 60-day pot experiment was conducted to investigate the effects of C. laeve inoculation (using two inoculation treatments and a non-inoculated control) on the phytoremediation potential, growth, and antioxidant metabolism of S. viminalis cultivated in PAH-contaminated soil. The S. viminalis-C. laeve association synergistically caused the highest PAH removal rate. Under the S. viminalis-C. laeve treatment, 80% of the biological concentration and translocation factors for all tissues of S. viminalis were > 1, whereas only 20% of these factors were > 1 when S. viminalis was used alone. C. laeve inoculation remarkably enhanced phytoremediation by promoting S. viminalis-based phytoextraction of PAHs from soils. Furthermore, although C. laeve inoculation altered the antioxidant metabolism of S. viminalis by inducing oxidative stress, thereby inhibiting plant growth, the plant's hardiness enabled it to survive and grow normally for 60 days after treatment. Therefore, phytoremediation using S. viminalis inoculated with C. laeve can be considered a feasible approach for the phytoremediation of PAH-contaminated soil.

Urban soil phytomanagement for Zn and Cd in situ removal, greening, and Zn-rich biomass production taking care of snail exposure.

The phytoextraction potential of Arabidopsis halleri (L.) O'Kane & Al Shehbaz and Salix viminalis L. to partially remove Zn and Cd in soil was investigated. In an urban field site, a very short rotation coppice of willows was implemented, and growth parameters were monitored for 3 years. A. halleri was cultivated in the same site with or without fertilizer to improve biomass yield and/or Zn and Cd aerial part concentrations. Effects of harvest and co-cultivation on these two parameters were measured. To determine if willows and A. halleri leaves were risky in case of consumption by a herbivorous invertebrate like the landsnail Cantareus aspersus, metal concentrations of snails fed with Zn- and Cd-enriched and low enriched leaves were compared. Willows and A. halleri grew well on the metal-contaminated soil (1.7 and 616 mg kg-1 Cd and Zn, respectively). The A. halleri Zn foliar concentration reached the Zn hyperaccumulation threshold (> 10,000 mg kg-1 DW) in the presence of NPK fertilizer and although the soil was alkaline (pH > 8.2). Cd concentration increased with harvest and fertilizer. Cd and Zn foliar concentrations of willows were far above baseline values. Laboratory snails exposure revealed that willow leaves ingestion caused a moderate increase of Cd, Pb, and Zn bioaccumulation in snails compared to the one caused by A. halleri ingestion. The soil and plant metal concentrations were reflected by field snail biomonitoring. This study confirmed the interest of selecting A. halleri and willows to partially remove Zn and Cd in the soil and emphasized their potential usefulness in greening urban contaminated area and producing raw materials for green chemistry while paying attention to the environmental pollutant transfer.

Assisted phytoremediation of a former mine soil using biochar and iron sulphate: Effects on As soil immobilization and accumulation in three Salicaceae species.

Metal(loid) accumulation in soils, is of increasing concern because of the potential human health risks. Therefore, metal(loid) contaminated sites need rehabilitation. It is becoming increasingly popular to use phytoremediation methods for the reclamation of sites containing metal(loid)s. However, plant establishment and growth on contaminated soils can be difficult due to high metal(loid) concentrations and poor fertility conditions. Consequently, amendments, like biochar and iron sulphate, must be applied. Biochar, obtained from plant biomass or animal wastes pyrolyzed under minimal oxygen supply, showed beneficial effects on soil properties and plant growth. Iron sulphate can effectively immobilize anions, thus mitigating metal(loid) toxicity and hence promoting plant development. This study aimed to assess the effect of two different modalities of biochar amendment application (top third of the tube and all tube height) combined with iron sulphate addition on the physico-chemical properties of a mining polluted soil and the growth and metal(loid) uptake of three Salicaceae species. A 1.5 year mesocosm experiment under field condition was conducted using a former tin mine contaminated by arsenic, amended with biochar and iron sulphate and vegetated with three Salicaceae species. Results showed that the combination of biochar and iron sulphate improved soil characteristics by increasing pH and electrical conductivity and reducing soil pore water metal(loid) concentrations. Between the two biochar application methods, the addition of biochar on the all tube height showed better results. But for such contaminated soil, biochar, in combination with iron sulphate, had no positive effect on plant growth, for all species tested and especially when incorporating on the top third of the tube. Finally, S. purpurea presented high root metal(loid) concentrations associated to the better growth compared to P. euramericana and S. viminalis, making it a better candidate for phytostabilization of the studied soil.

The complete chloroplast genome sequence of Salix viminalis (Salicaceae).

Here, the complete chloroplast (cp) genome of Salix viminalis was reported. The genome is 155,531 bp long, with a GC content of 36.71%, and contains four sub-regions: 84,395 bp of large single copy (LSC) and 16,218 bp of small single copy (SSC) regions, separated by 27,459 bp of inverted repeat (IR) regions. A total of 129 genes were annotated, including 83 protein-coding genes, 38 tRNA genes, and 8 rRNA genes. The Phylogenetic analyses based on the whole cp genome sequence placed S. viminalis into a clade containing Salix rehderiana, Salix taoensis, Salix koriyanagi, and Salix suchowensis. This is the first complete cp genome for S. viminalis that would be useful for phylogenetic and population genetic studies of this species.