The bp-miR156c-BpSPL2 module positive regulates drought tolerance by mediating lateral root development and reactive oxygen species scavenging in Betula platyphylla

Drought is one of the major abiotic stress factors affecting plant growth and productivity. The miR156/SPL module plays a crucial role in plant growth, development, and responses to abiotic stress; however, its regulatory mechanism in mediating drought adaptation in woody plants such as birch remains incompletely understood. In this study, we used transgenic plants overexpressing bp-miR156c and BpSPL2 as experimental materials and employed GUS staining, RNA-seq, yeast one-hybrid assay, ChIP-PCR and dual-luciferase reporter assays to investigate the mechanism by which the miR156/SPL module regulates drought tolerance in birch. GUS staining results indicated that BpSPL2 is a target gene of bp-miR156c and subject to its cleavage. Compared with wild-type plants, bp-miR156c -overexpressing transgenic plants exhibited reduced drought tolerance under drought stress, whereas plants overexpressing its target gene BpSPL2 showed enhanced drought resistance. Specifically, BpSPL2-OE lines under drought stress displayed alleviated photodamage in both PSII and PSI, along with reduced oxidative damage. Moreover, overexpression of BpSPL2 significantly promoted root system development, particularly lateral root growth, in birch. RNA-seq analysis revealed that, compared with the wild type, differentially expressed genes (DEGs) in BpSPL2-OE plants under drought stress were significantly enriched not only in photosynthesis-related pathways but also in tryptophan metabolism, redox processes, and glutathione metabolism. We speculate that the alleviation of photosynthetic inhibition and oxidative damage in birch leaves under drought stress by BpSPL2 may be related to its regulation of ROS metabolism, while the promotion of lateral root development may be associated with activation of the tryptophan metabolic pathway and subsequent accumulation of IAA. Further studies demonstrated that the BpSPL2 transcription factor recognizes the GTAC motif and binds to the promoters of glutathione-S-transferase BpGSTF3 and the key rate-limiting enzyme in tryptophan synthesis gene BpASA1 , thereby enhancing their transcription. On one hand, this upregulates GST and antioxidant enzyme activities, mitigating drought-induced photodamage and oxidative injury; on the other hand, it promotes IAA accumulation, stimulating lateral root formation and ultimately improving drought tolerance in birch. In summary, our findings demonstrate that the miR156/SPL module enhances drought tolerance in birch by modulating ROS homeostasis and lateral root development, providing novel molecular insights and a theoretical foundation for drought resistance research in birch trees.