Jing Qu, Peng Xiao, Yilei Wang, Fang Tian, Haowei Chen, Chunlong Li, Ji‐Hong Liu
Cold stress adversely affects plant growth and development, significantly limiting fruit yield and quality in citrus. Ichang papeda (Citrus ichangensis), a cold-tolerant citrus species, serves as a valuable genetic resource for studying cold adaptation, yet the key genes and their modes of action underlying the cold stress response remain largely unexplored. In this study, we identified CiWRKY27 as a critical positive regulator of cold tolerance in Ichang papeda. Through DNA affinity purification sequencing (DAP-seq) and RNA-sequencing analyses we uncovered 717 potential target genes of CiWRKY27, many of which are associated with stress adaptation. Further investigation revealed that CiWRKY27 directly activated CiCAD7 and CiGSTF6 by binding to W-box elements in their promoters, thereby enhancing lignin biosynthesis and reactive oxygen species (ROS) detoxification to confer cold tolerance. Functional assays confirmed that both CiCAD7 and CiGSTF6 contributed positively to cold tolerance. Additionally, CiRAP2.7, an RAP2 (AP2/ERF) transcription factor, physically interacted with CiWRKY27 to synergistically amplify the activation of CiCAD7. Intriguingly, CiRAP2.7 was itself regulated by CiWRKY27 and functioned as a transcriptional activator of CiCAD7 for rendering cold tolerance. Taken together, our findings demonstrate that CiWRKY27 works, alone or together with CiRAP2.7, to modulate cold tolerance by regulating CiCAD7-mediated lignin biosynthesis and CiGSTF6-dependent ROS scavenging. This study gains valuable insights into the molecular regulation of lignin biosynthesis and ROS homeostasis under cold stress, and offers a promising molecular module for improving cold tolerance in citrus and other crops.