Constitutive AtRD29A expression reveals divergent BpCBF3 regulatory mechanisms in birch

Abiotic stress resistance in plants is often associated with growth retardation. To address this limitation, stress-inducible promoters can be used to generate plants that exhibit both rapid growth and high stress resistance. RD29A (Response to Desiccation 29A) is a widely used stress-inducible promoter in Arabidopsis thaliana (AtRD29A) that can be activated by multiple factors including NaCl, ABA, and low temperature. Specifically, under cold stress, it is induced through the response to C-repeat/DREB binding factors (CBFs). In this study, we identified five CBF3 homologous genes-BpCBF3e, BpCBF3b, BpCBF3d, BpCBF3c, and BpCBF3a-in birch (Betula platyphylla), but did not detect an RD29A homolog. We proposed BpCBF3d to be the functional CBF because its expression is strongly regulated by circadian rhythm and cold stress, and it activates downstream cold response marker genes. To confirm this hypothesis, we performed Y1H assays and generated BpCBF3d overexpression lines driven by two different promoters-p35S and pRD29A. Notably, we found that BpCBF3d did not bind to the AtRD29A promoter, whereas BpCBF3e, BpCBF3b, BpCBF3c, and BpCBF3a bound strongly, resulting in constitutive expression of heterologous AtRD29A in birch. This study provides new molecular tools for breeding forest trees with enhanced resilience to environmental change.