Exploring phytoremediation potential of willow NJU513 for cadmium-contaminated soil with and without epibrassinolide treatment.

There has been a growing concern over soil cadmium (Cd) pollution, underscoring the importance of finding effective remediation strategies. Willow trees have emerged as promising candidates for phytoremediation of Cd-contaminated soils. Nevertheless, the specific potential of a novel willow genotype, NJU513, in remediating Cd-polluted soil remains unexplored. Hence, the primary objectives of this study were twofold: firstly, to ascertain the suitability of the willow genotype NJU513 for remediating Cd-contaminated soil; and secondly, to elevate its remediation efficciency with the application of epibrassinolide (Brs). In the pot-culture experiment without Brs, its leaf and stem Cd concentrations were 203 mg kg-1 and 65.1 mg kg-1, with a bioaccumulation factor (BCF) of 20.8 and 6.68, respectively. In the pot-culture experiment with Brs, the corresponding Cd concentrations were 226 mg kg-1 and 59.2 mg kg-1, with a BCF of 23.1 and 6.06, respectively. In addition, the extracted Cd contents were higher in the Brs treatments (1.11-1.37 mg plant-1) than in the no-Brs treatments (0.78-0.96 mg plant-1) because Brs increased the plant biomass and leaf BCF. The mechanism underlying the Cd accumulation of NJU513 leaves with and without Brs was revealed by a transcriptome analysis. The expression levels of genes related to metal ion binding, channel activity, and transporters in leaves were up-regulated, which contributed to the high Cd accumulation and stress tolerance. Analyses of soil metabolites and bacteria in the presence and absence of Brs spraying on willow leaves indicated that soil organic compounds with carboxyl and amino groups may induce Cd activation and passivation, respectively. This study provides valuable insights for developing woody plant varieties that can be used for remediating Cd-contaminated soil.

High cadmium-accumulating Salix ecotype shapes rhizosphere microbiome to facilitate cadmium extraction.

Cadmium (Cd) contamination poses a significant threat to agricultural soils and food safety, necessitating effective remediation strategies. Salix species, with their high coverage and Cd accumulating capacity, hold promise for remediation efforts. The rhizosphere microbiome is crucial for enhancing Cd accumulating capacity for Salix. However, the mechanisms by how Salix interacts with its rhizosphere microbiome to enhance Cd extraction remains poorly understood. In this study, we compared the remediation performance of two Salix ecotypes: 51-3 (High Cd-accumulating Ecotype, HAE) and P646 (Low Cd-accumulating Ecotype, LAE). HAE exhibited notable advantages over LAE, with 10.80 % higher plant height, 43.80 % higher biomass, 20.26 % higher Cd accumulation in aboveground tissues (93.09 µg on average), and a superior Cd translocation factor (1.97 on average). Analysis of the rhizosphere bacterial community via 16S rRNA amplicon sequencing revealed that HAE harbored a more diverse bacterial community with a distinct composition compared to LAE. Indicator analysis identified 84 genera specifically enriched in HAE, predominantly belonging to Proteobacteria, Actinobacteria, and Firmicutes, including beneficial microbes such as Streptomyces, Bacillus, and Pseudomonas. Network analysis further elucidated three taxa groups specifically recruited by HAE, which were highly correlated with functional genes that associated with biosynthesis of secondary metabolites, glycan biosynthesis and metabolism, and metabolism of cofactors and vitamins. These functions contribute to enhancing plant growth, Cd uptake, and resistance to Cd in Salix. Overall, our findings highlight the importance of the rhizosphere microbiome in facilitating Cd extraction and provide insights into microbiome-based strategies for sustainable agricultural practices.

Integrated physiological, transcriptomic and metabolomic analyses provide insights into phosphorus-mediated cadmium detoxification in Salix caprea roots.

Phosphorus (P) plays a crucial role in facilitating plant adaptation to cadmium (Cd) stress. However, the molecular mechanisms underlying P-mediated responses to Cd stress in roots remain elusive. This study investigates the effects of P on the growth, physiology, transcriptome, and metabolome of Salix caprea under Cd stress. The results indicate that Cd significantly inhibits plant growth, while sufficient P alleviates this inhibition. Under Cd exposure, P sufficiency resulted in increased Cd accumulation in roots, along with reduced oxidative stress levels (superoxide anion and hydrogen peroxide contents were reduced by 16.8% and 30.1%, respectively). This phenomenon can be attributed to the enhanced activities of antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT), as well as increased levels of antioxidants including ascorbic acid (AsA) and flavonoids under sufficient P conditions. A total of 4208 differentially expressed genes (DEGs) and 552 differentially accumulated metabolites (DAMs) were identified in the transcriptomic and metabolomic analyses, with 2596 DEGs and 113 DAMs identified among treatments with different P levels under Cd stress, respectively. Further combined analyses reveal the potential roles of several pathways in P-mediated Cd detoxification, including flavonoid biosynthesis, ascorbate biosynthesis, and plant hormone signal transduction pathways. Notably, sufficient P upregulates the expression of genes including HMA, ZIP, NRAMP and CAX, all predicted to localize to the cell membrane. This may elucidate the heightened Cd accumulation under sufficient P conditions. These findings provide insights into the roles of P in enhancing plant resistance to Cd stress and improving of phytoremediation.

Exploring the combined effect of heavy metals on accumulation efficiency of Salix alba raised on lead and cadmium contaminated soils.

The present study illustrated that Salix alba can accumulate high level of Pb and Cd in different plant parts, with maximum accumulation in roots followed by stem and leaves in the order Cd > Pb > Cd + Pb. The phytoremediation evaluation factors such as bioconcentration factor (BCF) and translocation factor (TF) was higher for Cd over Pb in all plant parts, further the BCF for both Pb and Cd was maximum in root (BCF > 1) followed by stem and leaves. Higher accumulation of Cd over the Pb was observed inside the plant tissues due to Cd mimics with other elements and gets transported through respective transporters. The combined treatment of Pb and Cd affected the bioaccumulation at every treatment level suggesting the negative effect among both elements. Higher survival rate (>85%) was recorded up to 200mgPb/kg and 15mgCd/kg, while further increase in metal concentration reduced the plant efficiency to remediate contaminated soils, hence results in declined survival rate. The FTIR analysis revealed that Pb and Cd accumulation in plants induced changes in carboxy, amino, hydroxyl and phosphate groups that ultimately caused alteration in physiological and biochemical processes of plant and thus provided an insight to the interaction, binding and accumulation of heavy metals.

The present study conferred that Salix alba is a heavy metal (Pb and Cd) excluder plant on the basis of phytoremediation efficiency evaluation factors such BCF >1 (root) and TF <1. The correlation studies suggested the negative correlation among Pb and Cd accumulation and morphological traits. Physiological studies indicated that Pb and Cd accumulation negatively affect chlorophyll concentration and the antioxidant mechanism of plants gets activated, further these results are confirmed with FTIR studies, which reported the alteration in functional groups and associated compounds in plant tissues under Pb and Cd stress.

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.

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.

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.

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.

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.

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.

"Willow Branch" DNA Self-Assembly for Cancer Dual-Target and Proliferation Inhibition.

DNA nanotechnology is beginning to yield unique advantages in the area of drug delivery. For the dual-targeting and proliferation suppression of cancer cells, a "willow branch" DNA assembly based on rolling circle amplification (RCA) was built. Three single-stranded DNAs, including antibody modified cDNAs, aptamer cDNAs, and simple cDNAs, were employed in the DNA self-assembly, along with the RCA scaffolds (every 63 bases is a repeat unit). "Willow branch" DNA (WB DNA) assembly successfully linked multiple antibodies and aptamers together to achieve dual targeting of cancer cells. Binding of CD44 antibodies and S2.2 aptamers to receptors on the cell membrane inhibits both pathways, ß-catenin signaling and nuclear factor-kappa B-specific transcription activity, through feedback regulation. Results demonstrated that WB DNA assembly could effectively exert multivalency clustering cell-surface receptors, modulating signal pathways and inhibiting proliferation. This study proposes a new approach for cancer dual-target and proliferation inhibition by clustering multivalent receptors.

Effects of salt stress on the photosynthetic physiology and mineral ion absorption and distribution in white willow (Salix alba L.).

OBJECTIVE: The purpose of this study was to explore the adaptive mechanism underlying the photosynthetic characteristics and the ion absorption and distribution of white willow (Salix alba L.) in a salt stress environment in cutting seedlings. The results lay a foundation for further understanding the distribution of sodium chloride and its effect on the photosynthetic system. METHOD: A salt stress environment was simulated in a hydroponics system with different NaCl concentrations in one-year-old Salix alba L.branches as the test materials. Their growth, ion absorption, transport and distribution in the roots and leaves, and the changes in the photosynthetic fluorescence parameters were studied after 20 days under hydroponics. RESULTS: The results show that The germination and elongation of roots are promoted in the presence of 171mM NaCl, but root growth is comprehensively inhibited under increasing salt stress. Under salt stress, Na+ accumulates significantly in the roots and leaves, and the Na+ content and the Na+/K+ and Na+/Ca2+ root ratios are significantly greater than those in the leaves. When the NaCl concentration is ≤ 342mM, Salix alba can maintain relatively stable K+ and Ca2+ contents in its leaves by improving the selective absorption and accumulation of K+ and Ca2+ and adjusting the transport capacity of mineral ions to aboveground parts, while K+ and Ca2+ levels are clearly decreased under high salt stress. With increasing salt concentrations, the net photosynthetic rate (Pn), transpiration rate (E) and stomatal conductance (gs) of leaves decrease gradually overall, and the intercellular CO2 concentration (Ci) first decreases and then increases. When the NaCl concentration is < 342mM, the decrease in leaf Pn is primarily restricted by the stomata. When the NaCl concentration is > 342mM, the decrease in the Pn is largely inhibited by non-stomatal factors. Due to the salt stress environment, the OJIP curve (Rapid chlorophyll fluorescence) of Salix alba turns into an OKJIP curve. When the NaCl concentration is > 171mM, the fluorescence values of points I and P decrease significantly, which is accompanied by a clear inflection point (K). The quantum yield and energy distribution ratio of the PSⅡ reaction center change significantly (φPo, Ψo and φEo show an overall downward trend while φDo is promoted). The performance index and driving force (PIABS, PICSm and DFCSm) decrease significantly when the NaCl concentration is > 171mM, indicating that salt stress causes a partial inactivation of the PSII reaction center, and the functions of the donor side and the recipient side are damaged. CONCLUSION: The above results indicate that Salix alba can respond to salt stress by intercepting Na+ in the roots, improving the selective absorption of K+ and Ca2+ and the transport capacity to the above ground parts of the plant, and increasing φDo, thus shows an ability to self-regulate and adapt.

Frequent ploidy changes in Salicaceae indicates widespread sharing of the salicoid whole genome duplication by the relatives of Populus L. and Salix L.

BACKGROUNDS: Populus and Salix belong to Salicaceae and are used as models to investigate woody plant physiology. The variation of karyotype and nuclear DNA content can partly reflect the evolutionary history of the whole genome, and can provide critical information for understanding, predicting, and potentially ameliorating the woody plant traits. Therefore, it is essential to study the chromosome number (CN) and genome size in detail to provide information for revealing the evolutionary process of Salicaceae. RESULTS: In this study, we report the somatic CNs of seventeen species from eight genera in Salicaceae. Of these, CNs for twelve species and for five genera are reported for the first time. Among the three subfamilies of Salicaceae, the available data indicate CN in Samydoideae is n = 21, 22, 42. The only two genera, Dianyuea and Scyphostegia, in Scyphostegioideae respectively have n = 9 and 18. In Salicoideae, Populus, Salix and five genera closely related to them (Bennettiodendron, Idesia, Carrierea, Poliothyrsis, Itoa) are based on relatively high CNs from n = 19, 20, 21, 22 to n = 95 in Salix. However, the other genera of Salicoideae are mainly based on relatively low CNs of n = 9, 10, 11. The genome sizes of 35 taxa belonging to 14 genera of Salicaceae were estimated. Of these, the genome sizes of 12 genera and all taxa except Populus euphratica are first reported. Except for Dianyuea, Idesia and Bennettiodendron, all examined species have relatively small genome sizes of less than 1 pg, although polyploidization exists. CONCLUSIONS: The variation of CN and genome size across Salicaceae indicates frequent ploidy changes and a widespread sharing of the salicoid whole genome duplication (WGD) by the relatives of Populus and Salix. The shrinkage of genome size after WGD indicates massive loss of genomic components. The phylogenetic asymmetry in clade of Populus, Salix, and their close relatives suggests that there is a lag-time for the subsequent radiations after the salicoid WGD event. Our results provide useful data for studying the evolutionary events of Salicaceae.

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.