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.

Salix suchowensis is an ideal model organism for investigating nitrogen (N) transport mechanisms due to its low N-input requirements. Accurate quantification of gene expression is essential for elucidating these processes, with quantitative real-time PCR (RT-qPCR) being the preferred method. However, the identification of stable reference genes for normalization in Salix suchowensis under varying N conditions remains unresolved. In this study, thirteen commonly employed candidate reference genes were evaluated across root, stem, and leaf tissues, under four N treatments (NH₄NO₃, NH₄⁺, NO₃⁻, and N deficiency). Five genes (UBQ1, UBQ3, 18S, H2A2, and H2B2) were excluded due to poor amplification efficiency or irregular melting curves. The remaining eight genes were further assessed for expression stability using the geNorm, NormFinder, and BestKeeper algorithms. Integrated ranking via RefFinder identified EF1α, EFβ, and αTUB as the most stable reference genes. GeNorm analysis suggested that two reference genes were sufficient for reliable normalization. Validation using the N-responsive gene SsAMT1 and SsNRT2 confirmed the stability of EF1α, EFβ, and αTUB as suitable reference genes. Based on comprehensive stability assessments and experimental validation, we recommended EF1α + αTUB as optimal reference gene pairs for RT-qPCR normalization under varying N conditions. Furthermore, the consistent expression of EF1α and αTUB across nine willow genotypes highlighted their broader applicability within Salix species. This study provides valuable methodological guidance for advancing molecular research on N transport in woody perennial plants.

Cadmium (Cd) is a major environmental pollutant due to its high mobility and persistence in soils, facilitating entry into the food chain and threatening ecosystems and human health. However, the mechanisms that enable Salix species, well adapted for Cd remediation, to both tolerate and accumulate Cd remain elusive. Here, two Salix genotypes with contrasting Cd tolerance were examined under control and Cd stress using integrated physiological, transcriptomic, and metabolomic analyses of roots and leaves. The Cd-tolerant genotype (Salix suchowensis P294) maintained biomass under Cd stress, whereas the Cd-sensitive genotype (Salix sinopurpurea × Salix integra P646) showed a ~17% reduction. P294 accumulated more Cd in its stems (132.76 mg kg⁻¹) and leaves (122.25 mg kg⁻¹) than P646 (93.54 and 56.24 mg kg⁻¹). Transcriptomics responses were stronger in roots, with 896 DEGs in P294 and 462 in P646, enriched in nitrogen metabolism, phenylpropanoid biosynthesis, and metal transport, whereas only 167 and 176 DEGs were detected in leaves for P294 and P646, respectively. Metabolomics revealed more altered metabolites in roots (125 in P294, 89 in P646), mainly organic acids, amino acids, and flavonoids, compared with leaves (46 and 66). RT-qPCR validated the root-specific upregulation of key detoxification and transport genes (ABCA7, PRX72, GSTU1, GSTU4, ZIP1). These results reveal a root-centered regulatory network underlying Cd accumulation and tolerance, integrating detoxification, redox homeostasis, and structural reinforcement, as well as providing valuable targets for genetic improvement of phytoremediation efficiency.