Dehydrins are intrinsically disordered proteins that play central roles in cryoprotection and osmotic stress tolerance in plants. In this study, we investigated orthologous KS dehydrin promoters from the arctic Oxytropis arctobia (Fabaceae) and the temperate O. splendens using reporter gene constructs to evaluate transcriptional responses under abiotic stress conditions in transgenic Lotus japonicus. Reporter gene assays in freeze- and heat-exposed L. japonicus plantlets showed that a specific region of the O. splendens promoter functions as a repressor, while both promoters contain temperature-responsive cis-elements. In parallel, we analyzed the nuclear genome sequences of O. arctobia and O. splendens, focusing on identifying KS dehydrin orthologs and their regulatory regions. This revealed that the intron in the 3′ UTR is highly conserved between three Oxytropis species and in close relatives, which may contribute to post-transcriptional regulation and adaptation to harsh environments. Together, these results demonstrate cis-regulatory divergence in dehydrin promoters and provide new insights into the molecular mechanisms and evolutionary trajectories of cold adaptation in Oxytropis.

Structural annotation was performed using BRAKER v3.0.3 (Gabriel et al. 2024) with homology-based predictions. Fabales protein sequences from OrthoDB and protein sequences from O. ochrocephala, were combined to create a protein database for BRAKER. The annotations were curated using gFACs v1.1.2 (Caballero and Wegrzyn 2019) with --rem-all-incompletes --rem-genes-without-start-codon --rem-genes-without-stop-codon --allow-alternate-starts --unique-genes-only parameters.

The functional annotation of protein-coding sequences was performed using AHRD v3.3.3 (Sato et al., 2012) against Trembl, swiss-prot, and Araprot databases and using Interproscan v5.73-104 against the PFAM database (Blum et al., 2025).