It has been widely reported that biochar can be used as a cost-effective amendment to immobilize of heavy metal contaminants in soil. While less research has been conducted on effect of biochar long-term field aging on its properties and the adsorption capability. In this study, the characteristics of aged biochar were investigated by comprehensive characterization to elucidate its mechanism transformation for heavy metal immobilization. Our results showed that, compared to fresh biochar, the relative content of C of aged biochar was reduced by 34.12%, while O was increased by 8.79%. Additionally, the specific surface area, pore volume, pore size and oxygen-containing functional groups of aged biochar were significantly increased compared to the fresh biochar. Batch adsorption experiment indicated that the maximum adsorption for Cd²⁺ (Q_m = 32.157 mg/g) and Pb²⁺ (Q_m = 39.216 mg/g) on aged biochar surface was much larger than that of Cd²⁺ (Q_m = 7.573 mg/g) and Pb²⁺ (Q_m = 8.134 mg/g) on fresh biochar. The underlying adsorption mechanisms for Cd²⁺ and Pb²⁺ on fresh biochar were dominated by coprecipitation, cation exchange and cation-π interaction, whereas surface complexation and cation exchange appeared to be more vital for aged biochar, as more active adsorption sites and Oxygen-containing functional groups were formed on its surface during aging, which was well explained by BET, XPS, FTIR and Elemental Analysis. Our study found that the physicochemical properties of biochar changed significantly during field aging. Although these changes increased the adsorption of heavy metals by biochar, the reduced stability of biochar to passivated heavy metal ions.

Biochar, a cost-effective amendment, has been reported to play pivotal roles in improving soil fertility and immobilizing soil pollutants due to its well-developed porous structure and tunable functionality. However, the properties of biochar and soils can vary inconsistently after field application. This may affect the remediation of biochar on heavy metal (HM)-contaminated soil being altered. Therefore, we selected lettuce as a model crop to determine the effects of short-term, long-term, and reapplication of biochar on soil physicochemical properties, microbial community, HM bioavailability, and plant toxicity. Our investigation revealed that the long-term application of biochar remarkably improved soil fertility, increased the relative abundance of the phylum Proteobacteria which was highly resistant to HMs, and reduced the abundance of phylum Acidobacteria. These changes in soil properties decreased the accumulation of Cd and Pb in lettuce tissues. The short- and long-term applications of biochar had no substantial effects on biomass, quality, and photosynthesis of lettuce. Moreover, the short-term and reapplication of biochar had no significant effects on soil bacterial communities but decreased the accumulation of Cd and Pb in lettuce tissues. It showed that the changes in the physical, chemical, and biological properties of soil after long-term application of biochar promoted the remediation of HM-contaminated soil. Furthermore, microbial community compositions varied with metal stress and biochar application, while the relative abundance of the phylum Actinobacteria in HM-contaminated soil with long-term biochar application was markedly higher than in HM-contaminated soil without biochar application.

Cadmium (Cd) responses in metal-accumulating plants involve coordinated stress programs and specialized metal transporters, but the relationship between these levels remains poorly resolved in Cardamine violifolia. Here, seeds were germinated for seven days under 0, 10, 25, or 100 mg L⁻¹ CdCl₂, followed by root phenotyping and dose-resolved transcriptome analysis. Root growth showed a biphasic pattern: root length increased at 10 mg L⁻¹, whereas growth at 100 mg L⁻¹ was strongly reduced relative to the low-dose treatment. RNA-seq of 12 libraries identified 5665 genes that responded in at least one comparison and resolved six expression trajectories. Pathway analysis indicated low-dose metabolic adjustment, strong activation of phenylpropanoid, signaling, and xenobiotic-response pathways at 25 mg L⁻¹, and broader transcriptional repression at 100 mg L⁻¹. Cross-referencing the transcriptome with 16 reference-identified CvHMA loci prioritized CvHMA15 and CvHMA16 as a 99.87 identical protein pair with divergent regulation: CvHMA15 was moderately down-regulated across the Cd gradient, whereas CvHMA16 was induced and met the combined differential-expression threshold at 100 mg L⁻¹. In a pYES2.0-based yeast spot assay, expression of either CvHMA15 or CvHMA16 maintained colony growth across five cell densities on medium containing 50 μM CdCl₂, whereas the empty-vector control was strongly inhibited; growth was comparable among transformants in the absence of Cd. These results establish a transcriptome-guided framework for candidate prioritization while leaving transport direction and in planta mechanism to direct functional tests.