Bermudagrass (Cynodon dactylon) is one of the most widely cultivated warm-season turfgrass species around the world. Cold stress has been a key environmental factor that adversely affects the growth, development, and geographical distribution of bermudagrass; however, the underlying mechanism of bermudagrass responsive to cold stress remains largely unexplored. Here, we identified a cold-induced WRKY transcription factor CdWRKY2 from bermudagrass and demonstrated its function in cold stress response. Overexpression of CdWRKY2 enhanced cold tolerance in transgenic Arabidopsis and bermudagrass hairy roots, while knocking down CdWRKY2 expression via virus-induced gene silencing increased cold susceptibility. RNA sequencing showed that overexpression of CdWRKY2 in Arabidopsis activated the expression of genes involved in sucrose synthesis and metabolism, including sucrose synthase 1 (AtSUS1) and sucrose phosphate synthase 2F (AtSPS2F). CdSPS1, the homology gene of AtSPS2F in bermudagrass, was subsequently proven to be the direct target of CdWRKY2 by yeast one-hybrid, electrophoretic mobility shift assay, and transient expression analysis. As expected, overexpression of CdSPS1 conferred cold tolerance in transgenic Arabidopsis plants, whereas silencing CdSPS1 expression enhanced cold sensitivity in bermudagrass. Besides, CdCBF1 whose expression was dramatically up-regulated in CdWRKY2-overexpressing bermudagrass hairy roots but down-regulated in CdWRKY2-silencing bermudagrass both under normal and cold stress conditions was confirmed as another target of CdWRKY2. Collectively, this study reveals that CdWRKY2 is a positive regulator in cold stress by targeting CdSPS1 and CdCBF1 promoters and activating their expression to coordinately mediate sucrose biosynthesis and CBF-signalling pathway, which provides valuable information for breeding cold-resistant bermudagrass through gene manipulation.

中文翻译：

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CdWRKY2介导的蔗糖生物合成和CBF信号通路协同促进百慕大草的耐寒性

狗牙根（Cynodon dactylon）是世界上种植最广泛的暖季型草坪草之一。冷胁迫是影响狗牙根生长发育和地理分布的关键环境因素；然而，狗牙根对冷应激反应的潜在机制在很大程度上仍未被探索。在这里，我们从狗牙根中鉴定出一种冷诱导的 WRKY 转录因子 CdWRKY2，并证明了它在冷应激反应中的作用。CdWRKY2的过表达增强了转基因拟南芥和狗牙根毛状根的耐寒性，同时敲低了CdWRKY2通过病毒诱导的基因沉默表达增加了对寒冷的敏感性。RNA测序显示，CdWRKY2在拟南芥中的过表达激活了参与蔗糖合成和代谢的基因的表达，包括蔗糖合酶1（AtSUS1）和蔗糖磷酸合酶2F（AtSPS2F）。CdSPS1是犬牙草中AtSPS2F的同源基因，随后通过酵母单杂交、电泳迁移率变动分析和瞬时表达分析证明是 CdWRKY2 的直接靶标。正如预期的那样，CdSPS1的过表达赋予转基因抗冷性拟南芥植物，而沉默CdSPS1表达增强了狗牙根的冷敏感性。此外，在正常和冷胁迫条件下，CdCBF1 在 CdWRKY2 过表达的狗牙根毛状根中的表达显着上调，但在 CdWRKY2 沉默的狗牙根中下调，被证实是 CdWRKY2的另一个靶标。总的来说，这项研究表明 CdWRKY2 通过靶向CdSPS1和CdCBF1是冷应激的正调节因子启动子并激活其表达以协同介导蔗糖生物合成和CBF信号通路，为通过基因操作培育抗寒狗牙根提供有价值的信息。