Integrative transcriptomics nominates a 1R-MYB candidate associated with epidermal wax-related traits and osmotic-stress responses in Kentucky bluegrass

Cuticular wax-associated epidermal traits may contribute to water-loss regulation, yet their regulatory basis in Kentucky bluegrass ( Poa pratensis L.) remains poorly defined. Here, we combined comparative phenotyping and de novo transcriptomics across three cultivars exhibiting contrasting wax-associated epidermal structures. Scanning electron microscopy revealed significant cultivar differences in wax crystal density (quantified as crystals per 100 μm 2 ), and a pot water-withholding assay showed cultivar-dependent injury phenotypes accompanied by differential ROS staining, antioxidant enzyme activities, relative water content, and lipid peroxidation levels. De novo transcriptome assembly (BUSCO completeness 76.8%) of pooled reads from nine libraries identified 228310 unigenes, among which pairwise comparisons revealed 7257–29713 differentially expressed genes with recurrent enrichment of hormone signal transduction pathways. A multi-criteria filtering strategy highlighted a 1R-MYB gene ( PpMYBS3 ) displaying the strongest expression concordance with the cultivar wax-density gradient and progressive stress inducibility by qRT-PCR. Stable overexpression of PpMYBS3 in two independent transgenic lines increased wax-associated epidermal structures, upregulated canonical wax biosynthesis genes ( CER1 , KCS6 , ABCG11 ), reduced stomatal aperture area, enhanced root architecture traits, and mitigated ROS accumulation under PEG-induced osmotic stress. These results support PpMYBS3 as a candidate regulator sufficient to promote epidermal and root traits linked to stress adaptation, while emphasizing that direct quantification of wax composition (e.g., GC-MS wax profiling) and cuticular water-loss assays will be required to establish mechanistic causality.