RsKNAT3 Interacts Antagonistically With RsKNAT1 to Confer Thermotolerance by Regulating RsDREB2A Transcription in Radish

Knotted1-like homeobox (KNOX) transcription factors (TFs) are widely involved in plant growth and development processes, including shoot apical meristem division and leaf and root organ development. However, the critical roles of KNOXs in response to abiotic stress, especially heat stress (HS), remain largely unexplored in plants. In this study, both the transcriptome and RT-qPCR analysis revealed that two KNOX TFs, RsKNAT1 and RsKNAT3, were significantly responsive to HS in radish (Raphanus sativus L.). RsKNAT3, highly expressed in heat-tolerant genotypes, enhances thermotolerance and ROS scavenging, acting as a transcriptional activator. Conversely, RsKNAT1, elevated in heat-susceptible genotypes, negatively regulates thermotolerance and increases ROS accumulation, functioning as a repressor. Interestingly, the RsKNAT1 interacts with RsKNAT3 by forming a heterodimer. The identification of regulatory elements showed that RsKNAT3 bound to the Knotted I binding site and activate RsDREB2A expression, while RsKNAT1 acts as the specific repressor of RsKNAT3 and inhibits the regulation of RsKNAT3-targeted RsDREB2A under HS. These findings provided insights into the regulatory mechanism underlying thermotolerance mediated by RsKNAT3 interacting antagonistically with RsKNAT1, facilitating the genetically improvement of the heat tolerance in radish breeding programs.