Responses of soil and rhizosphere microbial communities to Cd-hyperaccumulating willows and Cd contamination.

BACKGROUND: The pollution of soil by heavy metals, particularly Cd, is constitutes a critical international environmental concern. Willow species are renowned for their efficacy in the phytoremediation of heavy metals owing to their high Cd absorption rate and rapid growth. However, the mechanisms underlying microbial regulation for high- and low-accumulating willow species remain poorly understood. Therefore, we investigated the responses of soil and rhizosphere microbial communities to high- and low-Cd-accumulating willows and Cd contamination. We analyzed soil properties were analyzed in bulk soil (SM) and rhizosphere soil (RM) planted with high-accumulating (H) and low-accumulating (L) willow species. RESULTS: Rhizosphere soil for different willow species had more NH4+ than that of bulk soil, and RM-H soil had more than RM-L had. The available phosphorus content was greater in hyper-accumulated species than it was in lower-accumulated species, especially in RM-H. Genome sequencing of bacterial and fungal communities showed that RM-L exhibited the highest bacterial diversity, whereas RM-H displayed the greatest richness than the other groups. SM-L exhibited the highest diversity and richness of fungal communities. Ralstonia emerged as the predominant bacterium in RM-H, whereas Basidiomycota and Cercozoa were the most enriched fungi in SM-H. Annotation of the N and C metabolism pathways revealed differential patterns: expression levels of NRT2, NarB, nirA, nirD, nrfA, and nosZ were highest in RM-H, demonstrating the effects of NO3-and N on the high accumulation of Cd in RM-H. The annotated genes associated with C metabolism indicated a preference for the tricarboxylic pathway in RM-H, whereas the hydroxypropionate-hydroxybutyrate cycle was implicated in C sequestration in SM-L. CONCLUSIONS: These contribute to elucidation of the mechanism underlying high Cd accumulation in willows, particularly in respect of the roles of microbes and N and C utilization. This will provide valuable insights for repairing polluted soil using N and employing organic acids to improve heavy metal remediation efficiency.

Phosphorus deficiency-induced cell wall pectin demethylesterification enhances cadmium accumulation in roots of Salix caprea.

Regions affected by heavy metal contamination frequently encounter phosphorus (P) deficiency. Numerous studies highlight crucial role of P in facilitating cadmium (Cd) accumulation in woody plants. However, the regulatory mechanism by which P affects Cd accumulation in roots remains ambiguous. This study aims to investigate the effects of phosphorus (P) deficiency on Cd accumulation, Cd subcellular distribution, and cell wall components in the roots of Salix caprea under Cd stress. The results revealed that under P deficiency conditions, there was a 35.4% elevation in Cd content in roots, coupled with a 60.1% reduction in Cd content in shoots, compared to the P sufficiency conditions. Under deficient P conditions, the predominant response of roots to Cd exposure was the increased sequestration of Cd in root cell walls. The sequestration of Cd in root cell walls increased from 37.1% under sufficient P conditions to 66.7% under P deficiency, with pectin identified as the primary Cd binding site under both P conditions. Among cell wall components, P deficiency led to a significant 31.7% increase in Cd content within pectin compared to P sufficiency conditions, but did not change the pectin content. Notably, P deficiency significantly increased pectin methylesterase (PME) activity by regulating the expression of PME and PMEI genes, leading to a 10.4% reduction in the degree of pectin methylesterification. This may elucidate the absence of significant changes in pectin content under P deficiency conditions and the concurrent increase in Cd accumulation in pectin. Fourier transform infrared spectroscopy (FTIR) results indicated an increase in carboxyl groups in the root cell walls under P deficiency compared to sufficient P treatment. The results provide deep insights into the mechanisms of higher Cd accumulation in root mediated by P deficiency.

Impact of Heavy Metals on Cold Acclimation of Salix viminalis Roots.

In nature, plants are exposed to a range of climatic conditions. Those negatively impacting plant growth and survival are called abiotic stresses. Although abiotic stresses have been extensively studied separately, little is known about their interactions. Here, we investigate the impact of long-term mild metal exposure on the cold acclimation of Salix viminalis roots using physiological, transcriptomic, and proteomic approaches. We found that, while metal exposure significantly affected plant morphology and physiology, it did not impede cold acclimation. Cold acclimation alone increased glutathione content and glutathione reductase activity. It also resulted in the increase in transcripts and proteins belonging to the heat-shock proteins and related to the energy metabolism. Exposure to metals decreased antioxidant capacity but increased catalase and superoxide dismutase activity. It also resulted in the overexpression of transcripts and proteins related to metal homeostasis, protein folding, and the antioxidant machinery. The simultaneous exposure to both stressors resulted in effects that were not the simple addition of the effects of both stressors taken separately. At the antioxidant level, the response to both stressors was like the response to metals alone. While this should have led to a reduction of frost tolerance, this was not observed. The impact of the simultaneous exposure to metals and cold acclimation on the transcriptome was unique, while at the proteomic level the cold acclimation component seemed to be dominant. Some genes and proteins displayed positive interaction patterns. These genes and proteins were related to the mitigation and reparation of oxidative damage, sugar catabolism, and the production of lignans, trehalose, and raffinose. Interestingly, none of these genes and proteins belonged to the traditional ROS homeostasis system. These results highlight the importance of the under-studied role of lignans and the ROS damage repair and removal system in plants simultaneously exposed to multiple stressors.

Impacts of willow (Salix babylonica L.) leaf extract on growth, cecal microbial population, and blood biochemical parameters of broilers.

The investigation examined the use of willow leaf extract (WLE) on broiler chickens, examining carcass characteristics, cecal microbiota, antioxidants, and blood parameters. In 4 groups of 300 chicks, a basal diet was given for 5 wk, and the first treatment was basal diet (C). The diets for the remaining 3 treatments (WLE150, WLE300, and WLE450) contained 150, 300, and 450 mg of willow leaf extract /kg, respectively. The study found that birds fed willow leaf extract supplements had significantly greater body weight (BW), body weight gain (BWG), and enhanced feed conversion ratio (FCR) vs. the control group. Birds fed at 450 mg/kg food showed the greatest growth features, carcass weight, liver weight, lower abdominal fat, better low-density lipoprotein (LDL), and high-density lipoprotein (HDL) concentrations, and highest hematological characteristics. Chickens fed diets supplemented with varied doses of willow leaf extract showed significantly increased antioxidant enzyme activity, with higher amounts of glutathione peroxidase (GPx) activity, superoxide dismutase (SOD), total antioxidant capacity (TAC), and lower malondialdehyde (MDA). However, in the study, birds fed a diet supplemented with 450 mg of willow leaf extract per kg meal showed a significant drop of 13.02%, which found no significant variations in hazardous bacteria (Escherichia coli) across 2 treatments (WLE150 and WLE300). In addition, the study discovered that birds fed with varied doses of willow leaf extract had fewer cecum infections (Staphylococci aureus). We conclude that using willow at a level of 450 mg/kg diet can significantly enhance the BWG, FCR, antioxidant levels and beneficial bacteria activity besides the condition of broiler chicken's general health.

Cell number regulator 8 from Salix linearistipularis enhances cadmium tolerance in poplar by reducing cadmium uptake and accumulation.

Trace metals have relatively high density and high toxicity at low concentrations. Willow (Salix genus) is an excellent phytoremediation species for soil contaminated by trace metal ions. This study identified a cell number regulator (CNR) gene family member in Salix linearistipularis exhibiting strong metal ion resistance: SlCNR8. SlCNR8 expression was affected by various metal ions, including cadmium (Cd), zinc (Zn), copper (Cu), iron (Fe), and manganese (Mn). SlCNR8 overexpression enhanced Cd, Zn, Cu, and Fe resistance in transgenic poplar seedlings (84K) compared with the wild-type (WT). Moreover, transgenic poplar seedlings showed lower root Cd uptake and less Cd accumulation than WT under Cd stress. SlCNR8 was primarily localized to the nucleus and the plasma membrane-like cell periphery. Furthermore, SlCNR8 had transcriptional activation activity in yeast. The transcript levels of multiple metal ion transporters were altered in the roots of transgenic poplar seedlings compared to WT roots under Cd stress. These results suggest that SlCNR8 may enhance Cd resistance in transgenic poplar by reducing Cd uptake and accumulation. This may be related to altered transcription levels of other transporters or to itself. Our study suggests that SlCNR8 can be used as a candidate gene for genetic improvement of phytostabilisation of trace metals by genetic engineering.

A Comparative Analysis of Phytochemicals, Metal Ions, Volatile Metabolites in Heart Wood, Stem Bark and Leaves of Salix alba L. along with in Vitro Antioxidant, Antacid, Antimicrobial Activities for Sake of Environment Conservation by Substitution of Stem Bark With Leaf.

The genus of Salix is used in food, medicine and nutraceuticals, and standardized by using the single marker compound Salicin only. Stem bark is the official part used for the preparation of various drugs, nutraceuticals and food products, which may lead to overexploitation and damage of tree. There is need to search substitution of the stem bark with leaf of Salix alba L. (SA), which is yet not reported. Comparative phytochemicals viz. Salicin, Procyanidin B1 and Catechin were quantified in the various parts of SA viz. heart wood (SA-HW), stem bark (SA-SB) and leaves (SA-L) of Salix alba L.by using newly developed HPLC method. It was observed that SA-HW and SA-L contained far better amount of Salicin, Procyanidin B and Catechin as compared to SA-SB (SA-HW~SA-L≫SA-SB). Essential and toxic metal ions of all three parts were analysed using newly developed ICP-OES method, where SA-L were founded as a rich source of micronutrients and essential metal ions as compared to SA-SB and SA-HW. GC-MS analysis has shown the presence of fatty acids and volatile compounds. The observed TPC and TFC values for all three parts were ranged from 2.69 to 32.30 mg GAE/g of wt. and 37.57 to 220.76 mg QCE/g of wt. respectively. In DPPH assay the IC50 values of SA-SB, SA-HW, and SA-L were 1.09 (±0.02), 5.42 (±0.08), and 8.82 (±0.10) mg/mL, respectively. The order of antibacterial activities against E. coli, S. aureus, P. aeruginosa, and B. subtilis strains was SA-L>SA-HW>SA-SB with strong antibacterial activities against S. aureus, and B. subtilis strains. The antacid activities order was SA-L>SA-SB>SA-HW. The leaves of SA have shown significant source of nutrients, phytochemicals and medicinal properties than SA-HW and SA-SB. The leaves of SA may be considered as substitute of stem bark to save the environment or to avoid over exploitation, but after the complete pharmacological and toxicological studies.

Perfluoroalkyl substances exposure alters stomatal opening and xylem hydraulics in willow plants.

Climate change and pollution are increasingly important stress factors for life on Earth. Dispersal of poly- and perfluoroalkyl substances (PFAS) are causing worldwide contamination of soils and water tables. PFAS are partially hydrophobic and can easily bioaccumulate in living organisms, causing metabolic alterations. Different plant species can uptake large amounts of PFAS, but little is known about its consequences for the plant water relation and other physiological processes, especially in woody plants. In this study, we investigated the fractionation of PFAS bioaccumulation from roots to leaves and its effects on the conductive elements of willow plants. Additionally, we focused on the stomal opening and the phytohormonal content. For this purpose, willow cuttings were exposed to a mixture of 11 PFAS compounds and the uptake was evaluated by LC-MS/MS. Stomatal conductance was measured and the xylem vulnerability to air embolism was tested and further, the abscisic acid and salicylic acid contents were quantified using LC-MS/MS. PFAS accumulated from roots to leaves based on their chemical structure. PFAS-exposed plants showed reduced stomatal conductance, while no differences were observed in abscisic acid and salicylic acid contents. Interestingly, PFAS exposure caused a higher vulnerability to drought-induced xylem embolism in treated plants. Our study provides novel information about the PFAS effects on the xylem hydraulics, suggesting that the plant water balance may be affected by PFAS exposure. In this perspective, drought events may be more stressful for PFAS-exposed plants, thus reducing their potential for phytoremediation.

Immunochemical Characterization of Salix alba (S. alba) Pollen Allergens and Evaluation of the Cross-Reactivity Pattern with Common Allergenic Pollen Grains.

BACKGROUND: Pollens, particularly tree and plant pollens, are one of the major causes of allergic respiratory diseases worldwide. Allergy to pollens of different species of Salix trees has been reported in various regions of the world. The most common type of Salix tree in Iran is white willow (Salix alba). OBJECTIVES: This study aimed to identify and determine the immunochemical characteristics of allergenic proteins in S. alba tree pollen extract using SDS-PAGE and IgE- immunoblotting methods. Moreover, the cross-reaction pattern of the specific IgE antibody of S. alba tree pollen proteins with pollen allergens of common allergenic trees, i.e., Populus nigra (P. nigra), Cupressus sempervirens (C. sempervirens), Pinus brutia (P. brutia) and Platanus orientalis (P. orientalis) in the region was investigated. METHODS: The reaction of allergenic proteins in S. alba pollen extract with specific IgE antibodies in patients' sera was investigated using SDS-PAGE and IgE-immunoblotting methods. The cross-reaction of specific IgE antibodies of the proteins present in S. alba pollen extract with pollen allergens of common allergenic trees in the region was investigated using ELISA and immunoblotting inhibition methods. In silico methods such as phylogenetic tree drawing and alignment of amino acid sequences were used to examine the evolutionary relationship and homology structure of common allergenic proteins (Panallergens) responsible for cross reactions. RESULTS: More than 11 protein bands binding to specific IgE antibodies in patients' sera with a molecular weight between 13 and 95 kDa were identified in the S. alba tree pollen extract. ELISA and immunoblotting inhibition results showed that P. nigra extract could inhibit the binding of IgE antibodies to S. alba pollen extract proteins to a greater extent than C. sempervirens, P. brutia, and P. orientalis tree extracts. In silico methods investigated the results of ELISA and immunoblotting inhibition methods. Moreover, a high structural homology and evolutionary relationship were observed between S. alba and P. nigra tree pollen panallergens. CONCLUSION: In this study, it was found that more than 80 % of the sensitive patients who were examined had specific IgE antibodies reacting with the approximately a 15 kDa-protein present in the S. alba pollen extract. Furthermore, the specific IgE-binding proteins found in the pollens of S. alba and P. nigra trees had relative structural homology, and it is likely that if recombinant forms are produced, they can be used for diagnostic and therapeutic purposes for both of the trees.

Nutritional, biological, and structural properties of bioactive peptides from bellflower, Persian-willow, and bitter-orange pollens.

In this study, the composition of amino acids, nutritional characteristics, degree of hydrolysis (DH), antioxidant properties, and antibacterial activity of proteins and hydrolysates of bellflower (Campanula latifolia), Persian willow (Salix aegyptiaca), and bitter orange (Citrus aurantium L.) were investigated under the influence of different proteases (Alcalase: Al, trypsin: Tr, pancreatin: Pa, and pepsin: Pe). Evaluation of the structural features of the proteins showed amide regions (amide A, B, I-III) and secondary structures. Hydrophobic amino acids (∼38%), antioxidants (∼21%), and essential types (∼46%) form a significant part of the structure of flower pollen. The digestibility and nutritional quality (PER) of the hydrolyzed samples (CP: 1.67; CA: 1.89, and PW: 1.93) were more than the original protein. Among proteins and peptides, the highest degree of hydrolysis (34.6%: Al-PWH), inhibition of free radicals DPPH (84.2%: Al-CPH), ABTS (95.2%: Pa-CPH), OH (86.7%: Tr-CAH), NO (57.8%: Al-CPH), reducing power (1.31: Pa-CPH), total antioxidant activity (1.46: Pa-CPH), and chelation of iron ions (80%: Al-CPH and Al-CAH) and copper (50.3%: Pa-CAH) were affected by protein type, enzyme type, and amino acid composition. Also, the highest inhibition of the growth of Escherichia coli (25 mm) and Bacillus cereus (24 mm) were related to CP and PW hydrolysates, respectively. The results of this research showed that hydrolyzed flower pollens can be used as a rich source of essential amino acids as well as natural antioxidants and antibacterial in food and dietary products. PRACTICAL APPLICATION: Enzymatic hydrolysis of Campanula latifolia, Persian willow, and Citrus aurantium pollen proteins was performed. The hydrolyzed ones had high nutritional quality and digestibility (essential amino acids and PER index). Antioxidant properties and chelation of metal ions of peptides were affected by the type of protein and enzyme. The hydrolysates showed inhibitory activity against the growth of Esherichia coli and Bacillus cereus.

BuOH fraction of Salix Babylonica L. extract increases pancreatic beta-cell tumor death at lower doses without harming their function.

Salix babylonica L. is a species of the willow tree. Insulinoma is a tumor originating from pancreatic beta cells. This study aims to research the effect of different fractions of Salix babylonica L. leaf extract on INS-1 cells for treating pancreatic tumors. Cell death occurred at lower doses in the EtOAc fraction. The cells are functional in the BuOH fraction but not in EtOAc and H2O fractions. The EtOAc fraction has a higher percentage of necrosis and ROS. INS1, INS2, and AKT gene expressions in the H2O fraction, GLUT2, IR, HSP70 gene expressions, and WNT4 protein levels increased in the BuOH fraction. HSP90 gene expression, Beta-actin, GAPDH, insulin, HSP70, HSP90, HSF1, Beta-Catenin, and WNT7A protein levels were decreased, while IR immunolabelling intensity increased in both fractions. Ca+2, K+, Na+, and CA-19-9 in the cell, Ca+2 and K+ in secretion increased. The secondary metabolites in the EtOAc fraction cause more damage in INS-1 cells. Since the water fraction also causes the cells to die in high doses, cell function is damaged. The secondary metabolites in the BuOH fraction kill INS-1 cells with less damage. This makes the BuOH fraction of Salix babylonica L. more valuable.

Biodegradation of willow sawdust by novel cellulase-producing bacterial consortium from wood-feeding termites for enhancing methane production.

This study was designed to develop a cellulase-producing bacterial consortium (CBC) from wood-feeding termites that could effectively degrade willow sawdust (WSD) and consequently enhance methane production. The bacterial strains Shewanella sp. SSA-1557, Bacillus cereus SSA-1558, and Pseudomonas mosselii SSA-1568 exhibited significant cellulolytic activity. Their CBC consortium showed positive effects on cellulose bioconversion, resulting in accelerated WSD degradation. After nine days of pretreatment, the WSD had lost 63%, 50%, and 28% of its cellulose, hemicellulose, and lignin, respectively. The hydrolysis rate of treated WSD (352 mg/g) was much higher than that of untreated WSD (15.2 mg/g). The highest biogas production (66.1 NL/kg VS) with 66% methane was observed in the anaerobic digester M-2, which contained a combination of pretreated WSD and cattle dung in a 50/50 ratio. The findings will enrich knowledge for the development of cellulolytic bacterial consortia from termite guts for biological wood pretreatment in lignocellulosic anaerobic digestion biorefineries.

Salix babylonica L. mitigates pancreatic damage by regulating the Beclin-P62/SQSTM1 autophagy pathway in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Salix babylonica L. belongs to the genus Salix, family Salicaceae. It is traditionally used as an antipyretic, antirheumatic, antidiabetic and for the treatment of ulcers and parasite skin diseases. It also has a range of pharmacological effects, such as anti-inflammatory, anti-tumor, antioxidant, and antibacterial effects. However, there are no reports on the phytochemical profile and efficacy of its leaves extract to modulate dexamethasone induced pancreatic damage. AIM OF THE STUDY: The present study was performed to annotate the phytoconstituents of Salix babylonica leaf extract and explore whether and how it could modulate dexamethasone-induced pancreatic damage and the role of oxidative stress and autophagy in mediating its protective effects. MATERIALS AND METHODS: Wistar rats were used for this study. Salix babylonica in two dose levels (100 and 200 mg/kg) or metformin (50 mg/kg) was given by oral gavage concurrently with dexamethasone which was injected SC in a dose of 10 mg/kg for 4 consecutive days. RESULTS: LC-MS analysis furnished 84 secondary metabolites belonging to phenolic acids, salicinoids, proanthocyanidins, flavonoids, cyclohexanediol glycosides, and hydroxy fatty acids. S. babylonica at both dose levels and metformin decreased the elevated pancreatic beclin while elevated the decreased pancreatic P62/SQSTM1 content compared to dexamethasone. These effects were associated with improved histopathological changes, glycemic and lipid parameters indicating that there might be a connection between autophagy and dexamethasone-induced pancreatic damage. Given that the level of GSH was negatively correlated with the levels of beclin and positively correlated with P62/SQSTM1, while both MDA and NO levels were positively correlated with beclin and negatively correlated with P62/SQSTM1, it seems that dexamethasone induced autophagy may be attributed to dexamethasone induced pancreatic oxidative stress. CONCLUSION: Our results indicate that S. babylonica protects pancreatic tissues against dexamethasone-induced damage by decreasing oxidative stress and its associated autophagy. Our study reveals a new mechanism for dexamethasone effects on pancreas and shows the potential therapeutic role of S. babylonica in mitigating dexamethasone adverse effects on pancreas and establishes the groundwork for future clinical applications.

Acetic acid enhances drought tolerance more in female than in male willows.

Drought is an important stress factor that limits plant growth and development. Female willows generally display stronger drought tolerance than males. The application of exogenous acetic acid (AA) has emerged as an efficient and eco-friendly approach to facilitate drought tolerance in willows. However, whether AA exerts sexually different effects on willows remains undefined. In this study, we comprehensively performed morphological and physiological analyses on three willow species, Salix rehderiana, Salix babylonica, and Salix matsudana, to investigate the sexually different responses to drought and AA. The results indicated that willow females were more drought-tolerant than males. AA application effectively enhanced willows' drought tolerance, and females applied with AA displayed greater root distribution and activity, stronger osmotic and antioxidant capacity and photosynthetic rate but less reactive oxygen species, or abscisic acid-mediated stomatal closure than males. In addition, AA application enhanced the jasmonic acid signaling pathway in females but inhibited it in males, conferring stronger drought defense capacity in female willows than in males. Overall, AA application improves drought tolerance more in female than in male willows, further enlarging the sexual differences in willows under drought-stressed conditions.

Exogenous hydrogen sulfide and methylglyoxal alleviate cadmium-induced oxidative stress in Salix matsudana Koidz by regulating glutathione metabolism.

BACKGROUND: Cadmium (Cd) is a highly toxic element for plant growth. In plants, hydrogen sulfide (H2S) and methylglyoxal (MG) have emerged as vital signaling molecules that regulate plant growth processes under Cd stress. However, the effects of sodium hydrosulfide (NaHS, a donor of H2S) and MG on Cd uptake, physiological responses, and gene expression patterns of Salix to Cd toxicity have been poorly understood. Here, Salix matsudana Koidz. seedlings were planted in plastic pot with applications of MG (108 mg kg- 1) and NaHS (50 mg kg- 1) under Cd (150 mg kg- 1) stress. RESULTS: Cd treatment significantly increased the reactive oxygen species (ROS) levels and malondialdehyde (MDA) content, but decreased the growth parameters in S. matsudana. However, NaHS and MG supplementation significantly decreased Cd concentration, ROS levels, and MDA content, and finally enhanced the growth parameters. Cd stress accelerated the activities of antioxidative enzymes and the relative expression levels of stress-related genes, which were further improved by NaHS and MG supplementation. However, the activities of monodehydroascorbate reductase (MDHAR), and dehydroascorbate reductase (DHAR) were sharply decreased under Cd stress. Conversely, NaHS and MG applications restored the MDHAR and DHAR activities compared with Cd-treated seedlings. Furthermore, Cd stress decreased the ratios of GSH/GSSG and AsA/DHA but considerably increased the H2S and MG levels and glyoxalase I-II system in S. matsudana, while the applications of MG and NaHS restored the redox status of AsA and GSH and further improved glyoxalase II activity. In addition, compared with AsA, GSH showed a more sensitive response to exogenous applications of MG and NaHS and plays more important role in the detoxification of Cd. CONCLUSIONS: The present study illustrated the crucial roles of H2S and MG in reducing ROS-mediated oxidative damage to S. matsudana and revealed the vital role of GSH metabolism in regulating Cd-induced stress.

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.

A 24-epibrassinolide treatment and intercropping willow with alfalfa increase the efficiency of the phytoremediation of cadmium-contaminated soil.

Cadmium contamination in agricultural soils threatens food security and human health, and that has caused widespread concern worldwide. Willow and alfalfa are widely used for the phytoremediation of cadmium (Cd)-contaminated soil, and willow NJU513 is the promising plant for remediating Cd-contaminated soil. In order to discuss the effect of intercropping willow NJU513 with alfalfa on the phytoremediation of Cd-contaminated soil, a pot-culture experiment was conducted in the greenhouse. The result showed that the phytoremediation of Cd-contaminated soil was enhanced by this intercropping because of the 25.90 % increase in the available Cd content. In order to increase the phytoremediation efficiency of Cd in the intercropping treatment, a 24-epibrassinolide (Brs) treatment was designed in the current study. The results showed that the phytoremediation of Cd-contaminated soil by willow and alfalfa improved following a Brs treatment because of the 16.32-74.15 % and 16.91-44.48 % increases in the plant biomass and available Cd content, respectively. Additionally, the extracted Cd by plants in the intercropping treatments with and without Brs was 0.56 and 0.31 mg pot-1, respectively. Transcriptome analyses of willow leaves revealed that Brs up-regulated the expression of genes related to calcium channel activity, calcium and zinc transmembrane transport, photosynthesis, catalase/antioxidant activity, glutathione metabolic processes and detoxification, phagosomes, and vacuoles, and that these upregulated genes promoted plant remediation efficiency and resistance to Cd stress. Brs promoted the phosphate ion transporter activity in willow leaves, which may have enhanced the solubilization of insoluble phosphate minerals by bacterial species (e.g., Vicinamibacterales, Bacillus, and Gaiella) to release Cd, ultimately leading to increased phytoremediation efficiency. In addition, plants with and without Brs treatments induced the bacteria-mediated transformation of available Cd to stable Cd. The study findings may be useful for improving the phytoremediation of Cd-contaminated paddy soil.

Allochthonous arbuscular mycorrhizal fungi promote Salix viminalis L.-mediated phytoremediation of polycyclic aromatic hydrocarbons characterized by increasing the release of organic acids and enzymes in soils.

Polycyclic aromatic hydrocarbons (PAHs) are well known persistent organic pollutants that have carcinogenic, teratogenic, and mutagenic effects on humans and animals. Arbuscular mycorrhizal fungi (AMF) that can infest plant hosts and form symbioses may help plants to enhance potential rhizosphere effects, thus contributing to the rhizodegradation of PAH-contaminated soils. The present study aimed to assess the effectiveness of AMF on enhancing Salix viminalis-mediated phytoremediation of PAH-polluted soil and clarify the plant enzymatic and organic acid mechanisms induced by AMF. Natural attenuation (NA), phytoremediation (P, Salix viminalis), S. viminalis-AMF combined remediation using willow inoculated with Funneliformis mosseae (PM), Laroideoglomus etunicatum (PE), and Rhizophagus intraradices (PI) were used as strategies for the remediation of PAH-polluted soils. The results showed that AMF inoculation contributed to the dissipation of the high-molecular-weight PAH benzo (α) pyrene that had concentrations in PM, PE, and PI treatments of 40.1 %, 24.49 %, and 36.28 % of the level in the NA treatment, and 62.32 %, 38.05 %, and 56.38 % of the level in the P treatment after 90 days. The mycorrhizal treatment also improved the removal efficiency of phenanthrene and pyrene, as their concentrations were sharply decreased after 30 days compared to the NA and P treatments. The research further clarified the changes in rhizosphere substances induced by AMF. Organic acids including arachidonic acid, octadecanedioic acid, α-linolenic acid, 10,12,14-octadecarachidonic acid and 5-methoxysalicylic acid that can act as co-metabolic substrates for certain microbial species to metabolize PAHs were significantly increased in AMF-inoculated treatments. AMF inoculation also elevated the levels of polyphenol oxidase, laccase, and dehydrogenase, that played crucial roles in PAHs biodegradation. These findings provide an effective strategy for using AMF-assisted S. viminalis to remediate PAH-polluted soils, and the results have confirmed the key roles of organic acids and soil enzymes in plant-AMF combined remediation of PAHs.

Pulsed Light Mutagenesis of the Willow Bracket Mushroom, Phellinus igniarius (Agaricomycetes), for Enhanced Production of Flavonoids, Laccase, and Fermentation Biomass.

Phellinus igniarius is a medicinal fungus possessing potent therapeutic activity due to the polysaccharides, polyphenols, flavonoids, and other secondary metabolites they contain. Laccases are crucial enzymes involved in lignin degradation in Ph. igniarius and offer great potential to accomplish several bioprocesses. To generate Ph. igniarius strains with high biomass, flavonoid, and laccase activity, we used pulsed light (PL) technology for mutagenesis of Ph. igniarius protoplasts and screened for mutants with high biomass, flavonoid, and laccase activity. At the irradiation power of 100 J, treated distance 8.5 cm, irradiation frequency was 0.5 s/time, three times treatments, after five generations of selection, three mutants were obtained with higher biomass production. Compared with control, the mycelium biomass and the flavonoid production of the screened mutant strain QB72 were increased 20.87% and 53.51%, respectively. The total amount of the accumulated extracellular laccase of the QB72 in the first 6 and 8 days increased 23.38% and 22.37% respectively, and over the total 16 days it increased 9.62%. In addition, RAPD analysis results indicated that the genetic materials of the mutant QB72 were altered. PL mutagenesis method has great potential for developing strains, especially Phellinus.

Plant cadmium resistance 6 from Salix linearistipularis (SlPCR6) affects cadmium and copper uptake in roots of transgenic Populus.

Phytoextraction in phytoremediation is one of the environmentally friendly methods used for restoring soils contaminated by heavy metals (HMs). The screening and identification of HM-resistant plants and their regulatory genes associated with HM ion transport are the key research aims in this field. In this study, a plant cadmium (Cd) resistance (PCR) gene family member, SlPCR6, was identified in roots of Salix linearistipularis, which exhibits strong HM resistance. The results revealed that SlPCR6 expression was induced in S. linearistipularis roots in response to Cd stress. Furthermore, SlPCR6 was mainly localized on the plasma membrane. Compared with the wild type, SlPCR6 overexpression reduced the Cd and copper (Cu) contents in the transgenic poplar (84 K) and increased its Cd and Cu resistance. The roots of transgenic poplar seedlings had lower net Cd and Cu uptake rates than wild type roots. Further investigation revealed that the transcript levels of multiple HM ion transporters were not significantly different between the roots of the wild type and those of the transgenic poplar. These results suggest that SlPCR6 is directly involved in Cd and Cu transport in S. linearistipularis roots. Therefore, SlPCR6 can serve as a candidate gene to improve the phytoextraction of the HMs Cd and Cu through genetic engineering.

Comparison of two willow genotypes reveals potential roles of iron-regulated transporter 9 and heavy-metal ATPase 1 in cadmium accumulation and resistance in Salix suchowensis.

Willows (Salix spp.) are promising extractors of cadmium (Cd), with fast growth, high biomass production, and high Cd accumulation capacity. However, the molecular mechanisms underlying Cd uptake and detoxification are currently poorly understood. Analysis of the Cd uptake among 30 willow genotypes in hydroponic systems showed that the S. suchowensis and S. integra hybrids, Jw8-26 and Jw9-6, exhibited distinct Cd accumulation and resistance characteristics. Jw8-26 was a high Cd-accumulating and tolerant willow, while Jw9-6 was a low Cd-accumulating and relatively Cd-intolerant willow. Therefore, these two genotypes were ideal specimens for determining the molecular mechanisms of Cd uptake and detoxification. To identify relevant genes in Cd handling, the parent S. suchowensis was treated with Cd and RNA-seq analysis was performed. SsIRT, SsHMA, and SsGST, in addition to the transcription factors SsERF, SsMYB, and SsZAT were identified as being associated with Cd uptake and resistance. Because membrane-localised heavy metal transporters mediate Cd transfer to plant tissues, a total of 17 SsIRT and 12 SsHMA family members in S. suchowensis were identified. Subsequently, a thorough bioinformatics analysis of the SsIRT and SsHMA families was conducted, and their transcript levels were analysed in the roots of the two hybrids. The transcript levels of SsIRT9 in roots were positively correlated with the observed differences in Cd accumulation in Jw8-26 versus Jw9-6. Jw8-26 displayed higher SsIRT9 expression levels and higher Cd accumulation than Jw9-6; therefore, SsIRT9 may be involved in Cd uptake. Gene expression analysis also revealed that SsHMA1 was a candidate gene associated with Cd resistance. These results lay the foundation for understanding the molecular mechanism of Cd transfer and detoxification in willows, and provide guidance for the screening and breeding of high Cd-accumulating and tolerant willow genotypes via genetic engineering.