PcTCP9-PcANK1 module regulates ester biosynthesis during post-harvest ripening of pear (Pyrus communis L.)

Fruit aroma is a key trait determining the comprehensive quality and consumer acceptance. Although progress has been achieved in the characterization of biosynthetic pathways of volatile compounds, the regulatory mechanisms of these pathways have not been fully elucidated. In this study, the high-aroma European pear cultivar 'Starkrimson' ( Pyrus communis L.) was used as the experimental material. By integrating gas chromatography-mass spectrometry (GC-MS) and RNA-sequencing analysis, the association network between volatile metabolite accumulation and gene expression during fruit development was systematically characterized. The study identified the lipoxygenase gene PcLOX5 as a key enzyme-encoding gene for ester biosynthesis in pear fruits, and its function was verified through experimental systems. Further validation using dual-luciferase reporter assays, yeast one-hybrid (Y1H) assays, and electrophoretic mobility shift assays (EMSA) confirmed that the transcription factor PcTCP9 acts as a key upstream activator of PcLOX5 . Using yeast two-hybrid (Y2H) assays, luciferase complementation analysis (LCA), GST pull-down assays, dual-luciferase reporter assays, and transient transformation in pear fruits, results demonstrated that PcTCP9 interacts with the ankyrin-repeat-containing protein PcANK1 to form a functional protein complex. This complex synergistically promotes the transcriptional activation of PcLOX5 . This study reveals the molecular mechanism by which the PcTCP9-PcANK1 protein complex synergistically regulates PcLOX5 expression to modulate ester biosynthesis. The findings offer crucial molecular targets for genetic manipulation, along with a theoretical framework to deepen our comprehension of the regulatory network in fruit aroma biosynthesis. This framework also serves as a basis for improving fruit aroma quality through molecular breeding approaches. • PcTCP9 transcriptionally activates PcLOX5 to promote ester biosynthesis. • An ankyrin repeat protein affects the synthesis of esters via protein interactions. • A TCP-ANK module boosting ester biosynthesis in pears.