Correlation Research of Corolla Non-Opening Phenotype with Cell Morphology and XTH/EXP-PME Expression Characteristics in Lonicera macranthoides

• • In the conventional (WT) cultivar, adaxial corolla epidermal cells exhibited a spatially sequential expansion pattern: basal cells expanded first, followed by middle cells, with adaxial expansion rates exceeding abaxial rates. In the Xianglei (XL) cultivar, adaxial cell expansion rates were significantly reduced (p < 0.01), and the degree of cell expansion was negligible. This cellular arrest directly accounts for the non-opening phenotype, which extends the bud stage and alters the harvest window for medicinal material, potentially affecting biomass accumulation and secondary metabolite profiles. • • Transcript profiling by qRT-PCR revealed that LmXTH6, LmXTH7, and LmXTH33 were highly expressed in WT during pre-anthesis and anthesis, but their expression was significantly suppressed in XL (p < 0.05). LmEXP7 peaked before corolla opening in WT, whereas its expression remained low in XL at the same stage. These data establish that the down-regulation of cell wall loosening enzymes (XTHs and EXP) is a primary molecular lesion underlying the non-opening trait, providing candidate targets for marker-assisted selection. • • Ruthenium red staining quantified by Image J showed that the overall degree of pectin de-methylesterification in XL corolla cell walls was higher than in WT, consistent with the elevated expression of LmPME28 and LmPME45 (p < 0.05). Increased de-methylesterification can promote calcium-mediated pectin cross-linking, enhancing cell wall rigidity. This biochemical shift likely acts in concert with reduced XTH/EXP activity to lock the corolla in a closed conformation, offering a mechanistic explanation for the prolonged bud stage. • • STRING-based protein-protein interaction network prediction identified potential interactions among LmXTHs, LmEXP7, and LmPMEs, suggesting a coordinated regulatory module. The inverse expression patterns—low XTH/EXP and high PME—indicate that the Xianglei phenotype arises from a coordinated cell wall remodeling program rather than a single gene defect. This multi-gene signature can be exploited for breeding programs aimed at manipulating flower opening time and improving mechanical harvestability or ornamental value.