A BpbZIP4 Transcription Factor Enhances Drought Resistance and Root Development in <i>Betula platyphylla</i> : Insights from a Gene Regulatory Network

Abstract Drought is a major abiotic stress that poses a significant threat to plants. Basic leucine zipper (bZIP) transcription factors (TFs) are important for plant stress signal transduction. However, the specific functions and molecular mechanisms of bZIP TFs under drought stress are still unclear. In this study, a BpbZIP4 TF of Betula platyphylla (birch) that responds strongly to drought stress was identified. Transgenic birch plants with BpbZIP4 overexpression and RNA interference were developed for gain- and loss-of-function assays. Results from phenotypic, staining, and physiological analyses showed that BpbZIP4 significantly enhances drought resistance and promotes root growth in birch. A four-layer drought-responsive gene regulatory network (GRN) was constructed based on BpbZIP4 transgenic lines. ChIP-PCR and qRT-PCR assays verified the putative interactions among genes at different hierarchical levels, confirming the reliability of the GRN. TF-Centered Y1H, ChIP, and GUS assays revealed that BpbZIP4 regulates the expression of second-layer TFs in the GRN by binding to two novel elements and one photosynthesis-responsive element. Furthermore, six randomly selected second-layer GRN TFs (BpMYB61, BpBEL1, BpWOX4, BpbHLH100, BpZAT11 and BpHB17), when transformed into birch plants, significantly influence birch’s drought tolerance. These results indicate that BpbZIP4 regulates second-layer TFs, thereby hierarchically relaying signals to bottom-layer functional genes, engaging multiple biological pathways, and ultimately enhancing drought resistance in birch. Collectively, these findings clarify the drought regulatory mechanism of BpbZIP4 and identify additional key genes for breeding drought-resistant birch varieties.