Unraveling Cadmium Tolerance Mechanisms in Betula platyphylla through a Hierarchical Gene Regulatory Network in Hormone Signaling

Cadmium (Cd), a toxic heavy metal, is a significant pollutant that impacts plant productivity. While some studies have been conducted, the underlying mechanisms by which plants respond to Cd stress remain largely unclear. Here, we performed RNA-seq analysis of Betula platyphylla (birch) under CdCl₂ treatment. The findings revealed a substantial enrichment of differentially expressed genes (DEGs) in pathways associated with plant hormones. A gene regulatory network (GRN) was constructed, and the regulatory relationships between genes were determined using a partial correlation coefficient algorithm. The GRN comprises 2,151 regulatory interactions, including 7 transcription factors (TFs) from the first layer, 25 TFs from the second layer, and 168 structural genes from the third layer, all of which are linked to ten enriched biological processes. ChIP-PCR and qRT-PCR assays validated approximately 85.2% of the predicted interactions between the first and second layers, along with 88.3% of the interactions between the second and third layers, supporting the validity of the GRN. Eighteen genes were selected from the third layer of multiple biological pathways to analyze their functions, and the results indicated that these genes can enhance Cd tolerance in birch plants. Additionally, two TFs in the first layer, BpHD-zip7 and BpRAV1, were successfully introduced into birch plants, confirming their role in improving Cd tolerance. Our findings elucidate the regulatory mechanisms and key determinants that function in the adaptation of B. platyphylla to Cd stress.