Overexpression of an AP2/ERF family gene, BpERF13, in birch enhances cold tolerance through upregulating CBF genes and mitigating reactive oxygen species

The AP2/ERF (APETALA2/ethylene-responsive factor) family of transcription factors (TF) is involved in regulating biotic and abiotic stress responses in plants. To explore the role of AP2/ERFs in cold tolerance in woody plants, BpERF13 was cloned and characterized in Betula platyphylla (white birch), a species primarily found in Asia in temperate and boreal climates. Based on phylogenetic analysis, BpERF13 is a member of the IXb subfamily of ERFs. Using qRT-PCR, we found that BpERF13 was differentially expressed in different tissues, and its expression could be induced by cold treatment (4 degrees C). BpERF13 protein, fused with GFP, was exclusively localized to nuclei. To further assess the role of BpERF13 in cold tolerance, BpERF13 overexpression (OE) transgenic lines were generated in B. platyphylla and used for cold stress treatment and biochemical/physiological studies. BpERF13 overexpression lines had significantly increased tolerance to subfreezing treatment and reduced reactive oxygen species. Using a TF-centered yeast one-hybrid (Y1H) experimental system, we showed that BpERF13 could bind to LTRECOREATCOR15 and MYBCORE cis-elements to activate a reporter gene. ChIP-seq and ChIP-PCR experiments further demonstrated that BpERF13 bound to these cis-elements when present in the 5' proximal regions of superoxide dismutase (SOD), peroxidase (POD), and C-repeat-binding factor (CBF) genes. qRT-PCR was employed to examine the expression levels of these genes in response to cold stress; SOD, POD, and CBF genes were significantly upregulated in BpERF13 transgenic lines compared to wild-type plants in response to cold stress. These results indicate that the transcription factor BpERF13 regulates physiological processes underlying cold tolerance in woody plants.