Abstract: Drought stress severely impairs plant growth and development, and lignin plays a critical role in plant resistance. In this study, we investigated the physiological and molecular mechanisms underlying drought adaptation regulated by BpSPL2 in birch (Betula platyphylla Suk), using transgenic overexpressing lines (OE3 and OE5) and SRDX repressor lines suppressed BpSPL2 expression (S2 and S4). The results showed that, compared with the wild type, the drought tolerance of two-year-old OE3 and OE5 birch lines was significantly enhanced under natural drought conditions, with reduced accumulation of malondialdehyde (MDA) and H₂O₂ content, increased proline content, and elevated antioxidant enzyme activities. In contrast, S2 and S4 lines exhibited greater drought sensitivity. RNA-seq analysis revealed that, under both normal and drought conditions, differentially expressed genes (DEGs) between genotypes were consistently enriched in pathways related to phenylpropanoid biosynthesis, cell wall formation, and lignin synthesis. Overexpression of BpSPL2 significantly increased the expression of lignin synthesis-related genes in birch and promoted lignin accumulation, indicating that BpSPL2 enhances birch drought tolerance by promoting lignin synthesis. To uncover potential molecular mechanisms, we performed a multi-omics correlation analysis integrating transcriptome and DAP-seq data, identifying 12 DEGs in the phenylpropanoid biosynthesis pathway as potential target genes of BpSPL2. DAP-seq results revealed that both the BpHCT25 and BpCSE5 promoter regions contain conserved BpSPL2 binding motifs. RT-qPCR analysis demonstrated that BpHCT25 and BpCSE5 expression levels were significantly elevated in the OE5 line, whereas they were downregulated in the S4 line. Further validation using biochemical approaches such as yeast one hybrid (Y1H) and dual luciferase reporter (DLR) assays confirmed that BpSPL2 binds to the promoters of its downstream genes BpHCT25 and BpCSE5, thereby promoting their expression. In summary, we report a novel molecular mechanism in birch where BpSPL2 mediates lignin biosynthesis to regulate drought resistance. BpSPL2 serves as a potential target for drought-tolerant breeding in birch, and these findings may hold significant application value for developing molecular breeding strategies for drought-resistant forest trees.

Key words: Betula platyphylla, Drought stress, BpSPL2, Lignin