背景回顾：Plant ethylene-responsive factors (ERFs) play essential roles in cold stress response, but the molecular mechanisms underlying this process remain poorly understood. 

主要研究：In this study, we characterized PtrERF9from trifoliate orange (Poncirus trifoliata (L.) Raf.), a cold-hardy plant.

结果1-低温诱导表达：PtrERF9 was up-regulated bycold in an ethylene-dependent manner. 

结果2-过表达和敲低表型：Overexpression of PtrERF9 conferred prominently enhanced freezing tolerance, which was drastically impaired when PtrERF9 was knocked down by virus-induced gene silencing. 

结果3-转录组分析：Global transcriptome profiling indicated that silencing of PtrERF9 resulted in substantial transcriptional reprogramming of stress-responsive genes involved in different biological processes. 

结果4-PtrERF9下游基因：PtrERF9 was further verified to directly and specifically bind with the promoters of glutathione S-transferase U17 (PtrGSTU17) and ACC synthase1 (PtrACS1). Consistently,PtrERF9-overexpressing plants had higher levels of PtrGSTU17 transcript and GST activity, but accumulated less ROS, whereas the silenced plants showed the opposite changes. Meanwhile, knockdown of PtrERF9 decreased PtrACS1 expression, ACS activity and ACC content. 

结果5-异源表达：However, overexpression of PtrERF9 in lemon, a cold-sensitive species, caused negligible alterations of ethylene biosynthesis, which was attributed to perturbed interaction between PtrERF9, along with lemon homologue ClERF9, and the promoter of lemon ACS1 gene (ClACS1) due to mutation of the cis-acting element. 

结论1-乙烯-PtrERF9-PtrFSTU17-ROS-耐寒性：Taken together, these results indicate that PtrERF9 acts downstream of ethylene signaling and functions positively in cold tolerance via modulation of ROS homeostasis by regulating PtrGSTU17. 

结论2-乙烯-PtrERF9-ACS1-乙烯合成：In addition, PtrERF9 regulates ethylene biosynthesis by activating PtrACS1 gene, forming a feedback regulation loop to reinforce the transcriptional regulation of its target genes, which may contribute to the elite cold tolerance of Poncirus trifoliata.

 摘 要 

植物乙烯响应因子（ERFs）在低温胁迫反应中发挥重要作用，但其分子机制尚不清楚。本文中，作者对耐寒植物三叶橙（Poncirus trifoliata（L.）Raf.）的PtrERF9基因进行功能鉴定。低温能够诱导PtrERF9基因的上调表达，但需要依赖于乙烯。PtrERF9的过表达能够显著增强植株的抗冻性，而通过病毒诱导基因沉默的方法敲低PtrERF9的表达则会降低植株的抗冻性。转录组分析表明，PtrERF9的沉默导致了参与不同生物学过程的胁迫响应基因的转录重编程。进一步的试验显示，PtrERF9能够直接特异性结合到谷胱甘肽S-转移酶编码基因PtrGSTU17和ACC合成酶编码基因PtrACS1的启动子区域。与此一致，PtrERF9过表达的植物具有较高水平的PtrGSTU17转录本丰度和GST活性，但会积累更少的ROS，而PtrERF9基因沉默的植株则表现出相反的变化。同时，PtrERF9基因敲除降低了PtrACS1的表达、ACS活性和ACC含量。然而，在低温敏感植物柠檬中过表达PtrERF9基因，乙烯生物合成的变化十分微小，这可能是由于PtrERF9与ACS1基因之间的互作被干扰，一是存在ERF9的同源物ClERF9，二是ClACS1基因的启动子上顺式作用元件的突变。综上所述，本文的研究结果表明PtrERF9在乙烯信号下游发挥作用，并通过调节PtrGSTU17来调节ROS稳态，从而在三叶橙的耐寒性中发挥正向调控作用。此外，PtrERF9通过激活PtrACS1基因来调节乙烯生物合成，形成一个反馈调节环以加强其靶标基因的转录调节，这可能作用于三叶橙的优良耐寒性。

 通讯作者 

** 刘继红 **