Although many genes and biological processes involved in abiotic stress responses have been identified, how they are regulated remains largely unclear. Here, to study the regulatory mechanism of birch (Betula platyphylla) responding to drought induced by polyethylene glycol 6000 (20%, w/v), a partial correlation coefficient-based algorithm for constructing a gene regulatory network (GRN) was proposed, and a three-layer hierarchical GRN was constructed, including 68 transcription factors and 252 structural genes. A total of 1448 predicted regulatory relationships are included, and most of them are novel. The reliability of the GRN was verified by chromatin immunoprecipitation (ChIP)–PCR and qRT–PCR based on transient transformation. About 55% of genes in the bottom layer of the GRN could confer drought tolerance. We selected two TFs, BpMADS11 and BpNAC090, from the top layer and characterized their function in drought tolerance. Overexpression of BpMADS11 and BpNAC090 reduces electrolyte leakage, reactive oxygen species (ROS) and malondialdehyde (MDA) contents, giving greater drought tolerance than wild-type birch. According to this GRN, the important biological processes involved in drought were identified, including ‘signaling hormone pathways’, ‘water transport’, ‘regulation of stomatal movement’, and ‘response to oxidative stress’. This work indicated that BpERF017, BpAGL61, and BpNAC090 are the key upstream regulators of birch drought tolerance. Our data clearly revealed that upstream regulators and transcription factor–DNA interaction regulate different biological processes to adapt to drought stress.

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