Pan-genomic analysis of Cation/H+ exchanger (CAX) family variation in soybean reveals structural and functional insights into heavy metal stress response

Cadmium (Cd), a pervasive environmental toxicant, jeopardizes ecosystem integrity and human health. The cation/H + exchanger (CAX) family, a key class of metal transporters, mediates Cd uptake, translocation, and detoxification in plants. Despite extensive soybean genomic studies, systematic pan-genomic exploration of CAX genes remains limited. Here, we conducted a comprehensive pan-genome analysis across 35 high-quality soybean genomes, identifying 30 CAX genes implicated in stress response, including 2 core genes conserved in all accessions. Genomic structural variation (SV) analysis revealed 299 SVs overlapping with 22 CAX loci and their flanking regions (±2 kb). Notably, SVs significantly modulated NCL2 expression (P < 0.05). Structural dissection of NCL2 showed that 53 % of soybean genomes retain domains identical to the reference (Wm82.a4), while atypical variants exhibit expanded exons and motifs. Expression profiling demonstrated that CAX5 , NCL2 , CCX2 , and CCX8 transcripts were significantly elevated in typical vs. atypical structural genomes (P < 0.01). Furthermore, CCX7 , CAX1 , CAX5 , and CAX6 displayed dynamic responses to dehydration/salt stress. Our findings propose a TF-CAX regulatory module fine-tuning heavy metal stress adaptation, providing a foundational resource for soybean functional genomics and stress-resilient crop design. Soybean (Glycine max), a vital source of oil and protein, was domesticated in China from the wild soybean (Glycine soja) around 5000 years ago. However, the genetic diversity within the species may not be fully represented by a single or limited reference genomes, hindering the identification of key genetic variations, particularly structural variations crucial for agronomic traits. The cation/H + exchanger (CAX) family, known for their role in intracellular ion transport, is essential for maintaining ion balance, pH regulation, and responding to environmental stresses, including heavy metals.This study delves into the pan-genomic analysis of the CAX gene family in soybean, examining evolutionary trends, structural variations, and gene expression patterns, providing insights into how soybean responds to heavy metal stress. The CAX gene family's widespread presence in the soybean genome underscores its pivotal role in plant growth, development, and resistance to various stresses, emphasizing the importance of further research into this family for soybean improvement.