ZjICE2 works as a positive regulator in abiotic stress responses and ZjICE2 is a valuable genetic resource to improve abiotic stress tolerance in the molecular breeding program of Zoysia japonica. The basic helix-loop-helix (bHLH) family transcription factors (TFs) play an important role in response to biotic or abiotic stresses in plants. However, the functions of bHLH TFs in Zoysia japonica, one of the warm-season turfgrasses, remain poorly understood. Here, we identified ZjICE2 from Z. japonica, a novel MYC-type bHLH transcription factor that was closely related to ICE homologs in the phylogenetic tree, and its expression was regulated by various abiotic stresses. Transient expression of ZjICE2-GFP in onion epidermal cells revealed that ZjICE2 was a nuclear-localized protein. Also, ZjICE2 bound the MYC cis-element in the promoter of dehydration responsive element binding 1 of Z. japonica (ZjDREB1) using yeast one-hybrid assay. A phenotypic analysis showed that overexpression of the ZjICE2 in Arabidopsis enhanced tolerance to cold, drought, and salt stresses. The transgenic Arabidopsis and Z. japonica accumulated more transcripts of cold-responsive DREB/CBFs and their downstream genes than the wild type (WT) after cold treatment. Furthermore, the transgenic plants exhibited an enhanced Reactive oxygen species (ROS) scavenging ability, which resulted in an efficient maintenance of oxidant-antioxidant homeostasis. In addition, overexpression of the ZjICE2 in Z. japonica displayed intensive cold tolerance with increases in chlorophyll contents and photosynthetic efficiency. Our study suggests that ZjICE2 works as a positive regulator in abiotic stress responses and the ICE-DREB/CBFs response pathway involved in cold stress tolerance is also conserved in Z. japonica. These results provide a valuable genetic resource for the molecular breeding program especially for warm-season grasses as well as other leaf crop plants.

中文翻译：

---

来自结缕草的新型碱性螺旋-环-螺旋转录因子 ZjICE2 通过激活 DREB/CBF 调节子和增强 ROS 清除来赋予转基因植物非生物胁迫耐受性。

ZjICE2作为非生物胁迫响应的正调节因子，ZjICE2是提高结缕草分子育种计划中非生物胁迫耐受性的宝贵遗传资源。基本螺旋-环-螺旋 (bHLH) 家族转录因子 (TFs) 在植物应对生物或非生物胁迫方面发挥着重要作用。然而，对于暖季型草坪草之一的结缕草（Zoysia japonica）中 bHLH TFs 的功能仍知之甚少。在这里，我们从 Z. japonica 中鉴定出 ZjICE2，这是一种新的 MYC 型 bHLH 转录因子，与系统发育树中的 ICE 同源物密切相关，其表达受各种非生物胁迫的调节。ZjICE2-GFP 在洋葱表皮细胞中的瞬时表达表明 ZjICE2 是一种核定位蛋白。还，ZjICE2 使用酵母单杂交试验与日本粳稻 (ZjDREB1) 脱水反应元件结合 1 的启动子中的 MYC 顺式元件结合。表型分析表明，ZjICE2 在拟南芥中的过表达增强了对寒冷、干旱和盐胁迫的耐受性。转基因拟南芥和日本粳稻在冷处理后比野生型（WT）积累了更多的冷响应DREB / CBFs及其下游基因的转录本。此外，转基因植物表现出增强的活性氧（ROS）清除能力，从而有效地维持了氧化剂-抗氧化剂的稳态。此外，ZjICE2 在 Z. japonica 中的过表达表现出强烈的耐寒性，叶绿素含量和光合效率增加。我们的研究表明，ZjICE2 在非生物胁迫响应中作为正调节因子起作用，并且参与冷胁迫耐受的 ICE-DREB/CBFs 响应途径在 Z. japonica 中也是保守的。这些结果为分子育种计划提供了宝贵的遗传资源，特别是对于暖季草以及其他叶作物植物。