The bZIP transcription factor plays a very important role in abiotic stresses for instance drought, salt, and low-temperature stress, but the mechanism of action at low temperature is still unclear. In this study, overexpression of DgbZIP3 led to increased whereas antisense suppression of DgbZIP3 resulted in decreased tolerance of chrysanthemum (Chrysanthemum morifolium Ramat.) to cold stress. Electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP), luciferase complementary imaging analysis (LCI), and dual luciferase reporter gene detection (DLA) experiments indicated that DgbZIP3 directly bound to the promoter of DgPOD and activated it expression. DgbZIP2 was identified as a DgbZIP3-interacting protein using yeast two-hybrid (Y2H), co-immunoprecipitation (Co-IP), LCI, and bimolecular fluorescence complementation (BIFC) assays. Overexpression of DgbZIP2 led to increased whereas antisense suppression of DgbZIP2 resulted in decreased tolerance of chrysanthemum to cold stress. A ChIP–qPCR experiment showed that DgbZIP2 was highly enriched in the promoter of DgPOD, while DLA, EMSA, and LCI experiments further showed that DgbZIP2 could not directly regulate the expression of DgPOD. The above results show that DgbZIP3 interacts with DgbZIP2 to regulate the expression of DgPOD to promote an increase in peroxidase activity, thereby regulating the balance of ROS and improving the tolerance of chrysanthemum to low-temperature stress.

中文翻译：

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DgbZIP3 与 DgbZIP2 相互作用增加 DgPOD 在菊花耐寒胁迫下的表达

bZIP转录因子在干旱、盐分和低温胁迫等非生物胁迫中发挥着非常重要的作用，但在低温下的作用机制尚不清楚。在这项研究中，DgbZIP3 的过表达导致增加，而 DgbZIP3 的反义抑制导致菊花 (Chrysanthemum morifolium Ramat.) 对冷胁迫的耐受性降低。电泳迁移率变动分析 (EMSA)、染色质免疫沉淀 (ChIP)、荧光素酶互补成像分析 (LCI) 和双荧光素酶报告基因检测 (DLA) 实验表明 DgbZIP3 直接与 DgPOD 的启动子结合并激活它的表达。使用酵母双杂交 (Y2H)、免疫共沉淀 (Co-IP)、LCI、和双分子荧光互补 (BIFC) 测定。DgbZIP2 的过度表达导致增加，而 DgbZIP2 的反义抑制导致菊花对冷胁迫的耐受性降低。ChIP-qPCR 实验表明 DgbZIP2 在 DgPOD 的启动子中高度富集，而 DLA、EMSA 和 LCI 实验进一步表明 DgbZIP2 不能直接调节 DgPOD 的表达。上述结果表明，DgbZIP3与DgbZIP2相互作用，调控DgPOD的表达，促进过氧化物酶活性的增加，从而调节ROS的平衡，提高菊花对低温胁迫的耐受性。ChIP-qPCR 实验表明 DgbZIP2 在 DgPOD 的启动子中高度富集，而 DLA、EMSA 和 LCI 实验进一步表明 DgbZIP2 不能直接调节 DgPOD 的表达。上述结果表明，DgbZIP3与DgbZIP2相互作用，调控DgPOD的表达，促进过氧化物酶活性的增加，从而调节ROS的平衡，提高菊花对低温胁迫的耐受性。ChIP-qPCR 实验表明 DgbZIP2 在 DgPOD 的启动子中高度富集，而 DLA、EMSA 和 LCI 实验进一步表明 DgbZIP2 不能直接调节 DgPOD 的表达。上述结果表明，DgbZIP3与DgbZIP2相互作用，调控DgPOD的表达，促进过氧化物酶活性的增加，从而调节ROS的平衡，提高菊花对低温胁迫的耐受性。