Enhancing bread wheat resilience to cadmium and drought stress: insights from physiological, morphological, and transcriptomic responses to biochar and 24-epibrassinolide application

Abstract Drought and cadmium (Cd) stress threaten sustainable crop production, highlighting the need for resilent agricultural practices. Individual application of biochar (BC) and brassinosteroids (24-epibrassinolide; EBL) can mitigate drought and Cd stress. However, their synergistic effects on alleviating drought and Cd stress at transcriptomic level in wheat ( Triticum aestivum L.) remain underexplored. This study investigated the combined impact of BC (B 0 = 0% w/w and B 1 = 5% w/w) and EBL (H 0 = control and H 1 = 10 –6 M) on wheat physiology, biomass, and digital gene expression under Cd (30 ppm) and drought stress (D 0 = 75% water holding capacity (WHC) and D 1 = 35% WHC). Drought and Cd stress significantly reduced biomass and photosynthetic activity while increasing oxidative stress and Cd uptake. However, the combined application of BC and EBL treatments showed notable improvements: root fresh biomass, leaf area, and shoot fresh biomass were increased by 39.41%, 66.49%, and 78.25% under D 0 and by 48.24%, 63.76%, and 73.49% under D 1 , respectively, compared to the control. Moreover, Cd uptake by wheat leaves decreased by 71.42% under D 0 and 184.10% under D 1 with BC and EBL combined application. Transcriptome analysis identified 6,174 differentially expressed genes linked to detoxification, carbon and nitrogen metabolism, and stress responses. Gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses highlighted metabolic processes and catalytic functions. Weighted gene co-expression network analysis revealed key modules for stress adaptation, including secondary metabolite biosynthesis and signaling pathways. Transcription factor profiling showed upregulation of AP2/ERF , MYB , and WRKY families in the combined BC and EBL treatments. qPCR validation of RNA-seq data confirmed significant changes in gene expression, with the nitrate transporter and photosystem II CP47 exhibiting increased expression levels by 53.60% and 29.66%, respectively, under BC + EBL treatment at optimal moisture, and 53.38% and 48.82% under drought stress. In contrast, heavy metal transporter genes PMPCB and YCF1 were downregulated, which correlated with a reduction in Cd uptake. Interestingly, the regression analysis demonstrated that Cd concentration in leaves negatively correlated with ( dehydrin-/LEA group ) and ( cadmium tolerance factor ). Overall, this study confirms that combining BC and EBL effectively mitigates Cd stress in drought-affected wheat, enhancing growth and resilience. Graphical abstract