Identification and characterization of U-box E3 ubiquitin ligase genes (ChPUBs) in Cardamine hupingshanensis under abiotic stress

Cardamine hupingshanensis is a novel selenium (Se) hyperaccumulator within the Brassicaceae family, yet the genomic basis of its stress adaptation remains poorly understood. Although U-box E3 ubiquitin ligases (PUBs) are known to be key regulators of abiotic stress responses in plants, their composition and functions in C. hupingshanensis have not been determined. We conducted the first genome-wide identification and analysis of the U-box gene family in C. hupingshanensis and identified 114 ChPUB genes. Phylogenetic analysis classified them into five distinct subgroups. These genes were unevenly distributed across the chromosomes and have expanded primarily through segmental duplication. Structural analysis revealed conserved U-box domains and motif organization within subgroups. Promoter analysis uncovered an abundance of cis-elements associated with stress and hormone responses. Expression profiling of representative ChPUBs demonstrated tissue-specific expression patterns and dynamic responses to various abiotic stresses, including selenium, osmotic, salt, temperature, and ABA treatments. Strikingly, most ChPUBs were strongly upregulated by high-concentration selenium in leaves. Importantly, yeast two-hybrid assays revealed that ChPUB12 and ChPUB33 interact with the ABA receptor ChPYL1, while ChPUB33 also interacts with ChPYL6, ChPYL13, and the protein phosphatase ChABI2. Furthermore, ChPUB12, ChPUB33, and ChPUB113 all physically interact with the cold signaling master regulator ChICE1. Our study provides a comprehensive genomic resource for the ChPUB gene family and highlights their potential as integrators of ABA and cold signaling pathways. The strong induction of ChPUBs by selenium stress suggests a novel role in selenium homeostasis. These findings provide key molecular insights into the adaptive mechanisms of this unique selenium hyperaccumulator to environmental challenges.