LkERF6 enhances drought and salt tolerance in transgenic tobacco by regulating ROS homeostasis

The transcription factor Ethylene Responsive Factor (ERF) is crucial for responding to various environmental stressors. Proteins containing the ERF-associated amphiphilic repression (EAR) motif often inhibit gene expression. However, the functions of LkERF, an EAR motif-containing protein from Larix kaempferi, especially in reactive oxygen species (ROS) homeostasis, are not well understood. In the present research, we introduce a novel transcription factor, LkERF6, which contains an EAR motif and positively regulates gene expression, thereby enhancing drought and salt tolerance in tobacco. LkERF6 is classified within the ERF-B1 subfamily due to its conserved AP2/ERF domain and EAR motif. Subcellular localization assays demonstrated LkERF6 is primarily localized in the nucleus. Further analysis revealed that LkERF6 interacts with GCC and DRE elements and is significantly induced by NaCl and PEG₆₀₀₀. Moreover, LkERF6 transgenic tobacco plants exhibit lower ROS accumulation and higher levels of antioxidant enzyme activities. Additionally, correlation analysis identified a strong association between LkERF6 and three genes: LkSOD, LkCCS, and LkCAT. Y1H, EMAS, and DLR assays confirmed that LkERF6 directly interacts with the promoters of these genes through GCC-box and DRE-box to activate their expression. These findings shed new light on the function of EAR motif-containing transcription factors and highlight LkERF6's crucial role in enhancing abiotic stress resistance by activating multiple ROS clearance genes.