Integrated transcriptomic, metagenomic, and physiological analyses reveal the mechanisms by which biochar regulates tolerance to Cd-Pb-As combined stress in cotton at the bud stage

The impact of heavy metals on crop development is mitigated by biochar. Uncertainty surrounds biochar's role in controlling cotton bud stage reactions to Cd-Pb-As combined stress and the alterations in soil microbes that accompany it. Here, we showed that biochar (10 g·kg⁻¹) successfully reduced the harmful effects of cotton under Cd-Pb-As combined stress. Biochar accelerated bud initiation by two days, increased biomass, and decreased the amounts of Cd (8.58 %) and Pb (15.62 %) the roots absorbed, Pb (32.46 %) and As (26.44 %) by the stems, and Pb (36.19 %) by the leaves. Biochar influenced metal transporter ( ABC and ZIP ) gene expression, contributing to ion homeostasis. To counteract the adverse effects of Cd-Pb-As combined stress, biochar enhanced photosynthetic efficiency and downregulated genes involved in photosynthesis. Biochar reduces oxidative damage induced by heavy metals by decreasing MDA levels. It also helped cotton maintain cellular and redox balance. This was achieved by regulating antioxidant enzyme activities and HSPs . Additionally, it controlled genes related to lignin synthesis and terpene metabolism through signal transduction pathways. Under Cd-Pb-As combined stress, biochar changed the composition of the rhizosphere bacterial community at the bud stage. It increased the abundance of heavy metal stress-tolerant Proteobacteria , Actinobacteria , Gemmatimonadetes , Nitrospirae , and Chloroflexi while decreasing the abundance of Acidobacteria , Bacteroidetes , Sphingomonas , and Acidobacterium . The findings elucidate the molecular mechanisms by which biochar mitigates Cd-Pb-As combined toxicity in bud-stage cotton and highlight associated changes in the rhizosphere microbial community. • Biochar mitigated the detrimental impacts of Cd-Pb-As combined stress on cotton development and expedited bud initiation by two days. • Biochar enhances photosynthetic performance despite downregulating photosynthesis-related gene expression. • Biochar decreases MDA, boosts antioxidant enzymes, and modulates HSPs expression • Biochar modulated the expression of metal transport genes, thereby reducing the absorption and translocation of Cd, Pb, and As in cotton. • Biochar reshapes rhizosphere microbial communities, enriching metal-tolerant beneficial bacteria.