miR169y-PtrNFYA6-PtrNCED3a/3b/6 module cascade regulates ABA synthesis and poplar drought tolerance

Drought is a major abiotic stress factor that limits tree growth. In this study, we determined the role and underlying mechanism of miR169y during the drought response. Expression analysis revealed that miR169y is notably downregulated under abscisic acid (ABA) and drought treatments. Stable transgenic lines expressing miR169y were generated using Agrobacterium-mediated transformation techniques. Suppression of miR169y expression increased drought tolerance in poplar, whereas its overexpression led to a decrease in tolerance. Nuclear factor Y subunit A6 gene (PtrNFYA6) was identified as the primary target of miR169y, exhibiting considerable upregulation in response to drought and ABA treatments. PtrNFYA6-overexpressing lines exhibited higher relative water content, lower malondialdehyde levels, increased proline accumulation and antioxidant enzyme activities, reduced reactive oxygen species levels, and enhanced stomatal closure. Molecular experiments confirmed that PtrNFYA6 directly binds to the CCAAT-box and ABRE elements in the promoters of 9-cis-epoxycarotenoid dioxygenase genes (PtrNCED3a/3b/6), thereby activating their transcription and promoting ABA synthesis. The overexpression of PtrNCED3b further substantiated the importance of ABA in enhancing drought tolerance. This research introduces a molecular framework in which the miR169y-PtrNFYA6-PtrNCED3a/3b/6 cascade modulates ABA production and drought resilience in poplar, thereby offering crucial targets for the molecular breeding of drought-resistant forest trees.