The PbNAC56-PbMYB31 regulatory module mediates volatile ester biosynthesis in pear

Volatile aroma compounds in fruits are key determinants of fruit quality and consumer acceptance. Research has increasingly clarified the pathways of biosynthesis of key volatile compounds, yet their transcriptional regulatory mechanisms remain poorly understood. In this study, 'Wubu green pear', a variety with a rich aroma, was used to investigate the molecular mechanism underlying ester synthesis, which is the main source of aroma in pear fruits. By integrating gas chromatography-mass spectrometry (GC-MS) metabolomics and RNA sequencing transcriptomics data during fruit development, it was found that the lipoxygenase gene PbLOX2-1 is significantly associated with ester synthesis during post-harvest ripening. Functional verification confirmed that PbLOX2-1 plays a key role in ester formation. Further analysis revealed that PbNAC56 is a major upstream activator of PbLOX2-1 , validated by dual-luciferase reporter assay, yeast one-hybrid assay (Y1H), and electrophoretic mobility shift assay (EMSA). Notably, physical interactions between PbNAC56 and PbMYB31 were verified using yeast two-hybrid (Y2H), luciferase complementation assay (LCA), and GST pull-down assays, with the two forming a functional complex. Dual-luciferase and transient transformation assays demonstrated that the PbNAC56-PbMYB31 complex significantly enhanced PbLOX2-1 promoter activity through synergistic transactivation. Our findings clarify a novel transcriptional regulatory module, PbNAC56-PbMYB31, which synergistically and significantly enhances the expression of PbLOX2-1 , thereby affecting ester biosynthesis in pear. This work provides key molecular targets and a theoretical basis for understanding the regulatory network of fruit aroma biosynthesis and for molecular breeding aimed at improving the flavor quality of fruit crops.