Ethylene‐Mediated RsCBF2 and RsERF18 Enhance Salt Tolerance by Directly Regulating Aquaporin Gene <i>RsPIP2‐1</i> in Radish (<i>Raphanus sativus</i> L.)

Salt stress is a major environmental factor limiting the production and quality of plants worldwide. Radish (Raphanus sativus L.), one of the most important root crops, is susceptible to salt stress worldwide. Plasma membrane intrinsic proteins (PIPs) have been identified to play a crucial role in regulating plants' salt tolerance. However, the underlying molecular regulatory mechanisms involved in salt stress tolerance are largely unknown. Here, a salt-induced water transport gene RsPIP2-1 associated with the regulatory mechanisms in response to salt stress was clarified in radish. Overexpression of RsPIP2-1 had high-water channel and H₂O₂ transport activity in Xenopus laevis oocytes and yeast, and it also conferred prominently salt tolerance through promoting reactive oxygen species (ROS) scavenging and enhancing antioxidant enzyme activity in transgenic radish. Moreover, yeast one-hybrid (Y1H) was used to screen the upstream regulators of RsPIP2-1, and two ethylene-responsive transcription factors including RsCBF2 and RsERF18 were identified. Y1H, dual-luciferase assay (DLA) and electrophoretic mobility shift assays (EMSA) showed that these two genes could active the transcription of RsPIP2-1 by directly binding to the DRE/CRT element and GCC-box element in its promoter. In addition, the salt tolerance and the expression levels of these two transcription factors could be significantly upregulated when treated with exogenous application of an ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), while the plants' resistance as well as the expression patterns could be reduced when exposure to the inhibitor of ethylene action (AgNO₃), suggesting that RsCBF2 and RsERF18 positively regulated the salt tolerance in a manner of dependent on ethylene synthesis pathway. Taken together, these findings uncover a novel transcriptional regulatory module based on the RsCBF2/RsERF18-RsPIP2-1 underlying salt tolerance in radish and could provide new insights into the salt-tolerant vegetable crop breeding programs.