Genetic foundations of copper stress tolerance in safflower: bridging heavy metal contamination and global warming challenges

Copper (Cu) contamination threatens agriculture, yet safflower's genetic basis for Cu tolerance remains unexplored—especially as global warming intensifies risks through nighttime warming and soil acidification. We conducted a pot experiment using 94 safflower genotypes under four copper chloride (CuCl₂) concentrations, revealing substantial variation among genotypes and treatments with trait reductions ranging from 1.75% (root-shoot ratio) (RSR) to 57.14% (dry root weight) (DRW). Correlation analysis showed positive trait relationships, while principal component analysis (PCA) grouped genotypes into three clusters. Ten genotypes consistently performed better under Cu stress, indicating strong tolerance potential for climate-resilient breeding. Genome-wide association study (GWAS) detected 180 significant marker-trait associations (MTAs) across eight traits, and gene annotation linked candidate genes to brassinosteroid signaling, ion homeostasis, metal response, and transport. Co-expression analysis revealed a tightly connected tolerance module in which AT5G01610 and AT5G14640 shared the same top co-expressed partners ( r > 0.8), including HMA4 , CAX1 , and SOS1 , suggesting coordinated roles in Cu stress tolerance. Convergent protein-protein interaction (PPI) validation confirmed these candidates as distinct hubs that are physically interacting and transcriptionally co-regulated, supporting their function as Cu tolerance modules. In silico heatmap-based expression profiling showed distinct, stress-specific transcriptional responses in Arabidopsis roots, with Cu inducing, cadmium (Cd) eliciting mixed, and (Al) aluminum repressing expression. RNA-seq analysis identified seven Cu stress tolerance genes, validated by qRT-PCR. These findings highlight promising genotypes for developing Cu-resilient safflower cultivars, while further investigation of the highly expressed genes is needed to clarify their roles under Cu stress.