The miR169z‐NF‐YA5‐GPDHc1 module improves drought tolerance by increasing NAD+ levels to inhibit ROS production in Populus

Summary The microRNA169 (miR169) family and NF‐YA transcription factors (TFs) are crucial for drought stress responses. However, the mechanisms by which these factors regulate reactive oxygen species (ROS) homeostasis under drought conditions remain inadequately characterized in Populus . Here, we identified an NF‐YA TF, PagNF‐YA5 , from hybrid poplar 84 K ( Populus alba × Populus glandulosa ). Knockout of PagNF‐YA5 reduced drought tolerance in transgenic poplars, while its overexpression enhanced tolerance. Tobacco transient co‐expression, 5′ RACE, and dual‐luciferase reporter assays confirmed that miR169z specifically cleaved PagNF‐YA5 transcripts. Overexpressing miR169z decreased drought tolerance in transgenic poplars, whereas repressing its expression using short tandem target mimics improved tolerance. Transcriptomic and biochemical analyses revealed that NF‐YA5 directly activates glycerol‐3‐phosphate dehydrogenase 1 ( PagGPDHc1 ) expression. PagGPDHc1 upregulation elevates NAD + levels, thereby inhibiting ROS production and enhancing drought tolerance. Conversely, gpdhc1 ‐knockout poplars displayed opposing phenotypic effects. Collectively, this study elucidates a molecular mechanism by which the miR169z‐NF‐YA5‐GPDHc1 module enhances drought tolerance through NAD + ‐mediated inhibit ROS production in Populus . These findings advance our understanding of drought adaptation mechanisms in woody plants and establish a molecular framework for the genetic improvement of forest trees under water deficit conditions.