Siyue Qi, Tongtong Yao, Zhanqi Ren, Jiechen Wang, Zheyuan Wang, Hongzhen Liu, Jiaqi Song, Huijiao Li, Tingting Liu, Changjun Ding, Yanbo Hu, Huihui Zhang
Drought stress severely limits agricultural and forestry productivity, and the identification of key regulatory genes or modules-and elucidation of their molecular mechanisms-is essential for developing drought-resistant crops. In this study, we selected three Populus cultivars from the “Zhongxiong” series with contrasting drought tolerance (ZX3, ZX4, and ZX5) and performed integrated microRNA and transcriptome sequencing analyses. A total of 266 miRNA-mRNA interactions were predicted, of which 66 miRNAs and 86 target genes exhibited significant drought responsiveness; most targets encoded transcription factors (TFs). Notably, 36 miRNAs targeting 55 TFs showed pronounced differential expression, implicating modules such as miR156–SPL and miR169-NF-Y in the drought response. In the highly tolerant ZX4, miR156g was significantly upregulated under drought, concomitant with suppression of its target, PagSPL1B , a nucleus-localized TF, whereas miR156g levels remained unchanged in the sensitive ZX3 and ZX5. Dual-luciferase assays confirmed that miR156g directly cleaves PagSPL1B mRNA. Transgenic 84 K poplar overexpressing PagSPL1B exhibited enhanced drought sensitivity relative to wild type, manifested by exacerbated leaf wilting, reduced relative water content, increased reactive oxygen species accumulation, decreased osmolyte (proline and soluble sugar) levels, and pronounced impairment of PSII and PSI activities. These results demonstrate that PagSPL1B negatively regulates drought tolerance by compromising osmotic adjustment, antioxidant capacity, and photoprotection. This work is the first to show that miR156g positively modulates poplar drought adaptation through targeted repression of PagSPL1B , offering a novel molecular target for the genetic improvement of drought resistance in poplar. • Identification of key drought-related miRNA-target pairs through differential miRNA profiling. • The miR156- SPL module is involved in the regulatory network of drought response in poplar. • PagSPL1B negatively regulates drought tolerance by suppressing osmotic adjustment and antioxidant capacity.