State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University, Harbin 150040, PR China.
Beijing Advanced Innovation Center for Tree Breeding by Molecular Design, Beijing Forestry University, Beijing 100083, PR China.
Plant Sci. 2020 Mar;292:110375. doi: 10.1016/j.plantsci.2019.110375. Epub 2019 Dec 12.
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 °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.
AP2/ERF（APETALA2/ethylene-responsive factor）转录因子家族参与调控植物的生物和非生物胁迫反应。为了探索 AP2/ERFs 在木本植物耐寒性中的作用，克隆并鉴定了山梨醇桦（Betula platyphylla）中的 BpERF13，该物种主要分布在亚洲的温带和寒带气候区。基于系统发育分析，BpERF13 是 ERF 的 IXb 亚家族的成员。通过 qRT-PCR 分析，发现 BpERF13 在不同组织中差异表达，并且其表达可以被冷处理（4°C）诱导。融合 GFP 的 BpERF13 蛋白仅定位于细胞核。为了进一步评估 BpERF13 在耐寒性中的作用，在山梨醇桦中生成了 BpERF13 过表达（OE）转基因株系，并用于冷胁迫处理和生化/生理研究。BpERF13 过表达株系在亚致死温度处理下表现出显著的耐受性，并减少了活性氧的产生。利用以 TF 为中心的酵母单杂交（Y1H）实验系统，我们表明 BpERF13 可以与 LTRECOREATCOR15 和 MYBCORE 顺式元件结合，激活报告基因。ChIP-seq 和 ChIP-PCR 实验进一步表明，BpERF13 在超氧化物歧化酶（SOD）、过氧化物酶（POD）和 C-重复结合因子（CBF）基因的 5'近端区域存在时，与这些顺式元件结合。通过 qRT-PCR 检测这些基因在冷胁迫下的表达水平；与野生型植物相比，SOD、POD 和 CBF 基因在 BpERF13 转基因株系中对冷胁迫的响应显著上调。这些结果表明，转录因子 BpERF13 调节木本植物耐寒性相关的生理过程。
