The PbWRKY7-PbMADS2 transcriptional module coordinates volatile ester biosynthesis in pear fruit

Fruit aroma significantly influences fruit quality and shapes consumer preferences. Despite significant advancements in deciphering the metabolic pathways related to volatile compound biosynthesis, the regulatory mechanisms governing these pathways remain relatively underexplored. 'Fragrant pear' (Pyrus sinkiangensis Yu) and some pear varieties with Pyrus sinkiangensis lineage possess intense aromas, providing materials for studying the metabolic regulatory pathways of aroma compound biosynthesis. In this study, we used the 'Chongyanghong' pear (Pyrus sinkiangensis × Pyrus bretschneideri) as the experimental material. Gas chromatography-mass spectrometry (GC-MS) and RNA sequencing (RNA-Seq) were used to systematically analyze the relationship between metabolites and gene regulation. PbAAT1, an alcohol acyltransferase, was identified as the key enzyme responsible for ester biosynthesis in pears. Multiple experiments collectively confirmed that PbAAT1 was involved in ester synthesis. PbWRKY7 was identified as an important upstream transcriptional activator of PbAAT1 using dual-luciferase, yeast one-hybrid (Y1H), and electrophoretic mobility shift assays (EMSA). This transcriptional activation effect was further enhanced by the complex formed between PbWRKY7 and the MADS-box protein PbMADS2, as revealed using yeast 2-hybrid (Y2H), luciferase complementation assays, GST pull-down, dual-luciferase, and transient transformation assays in pears. This study revealed a molecular network in which the PbWRKY7-PbMADS2 interaction synergistically regulates PbAAT1 expression, providing insights into ester biosynthesis and pinpointing regulatory targets for enhancing fruit aroma quality.