Transcription factors PaWRKY57 and PaNAC29 regulate color and size in sweet cherry fruit

Although the ripening process of climacteric fruits is well-characterized, the regulatory mechanisms underlying ripening in non-climacteric fruits, such as sweet cherry, remain poorly understood. In this study, we present an extensive transcriptomic analysis of pedicel and fruit tissues across eight developmental stages in the late-maturing sweet cherry cultivar Regina, providing a comprehensive map of tissue-specific gene expression dynamics during fruit ripening. Our data reveal extensive transcriptomic and metabolomic reprogramming, particularly in sugar metabolism pathways within the pedicel during fruit development, suggesting that cherry ripening may be partially regulated by pedicel-derived signals. Through integrative analysis, we identified key transcription factors, most notably PaWRKY57 and PaNAC29, as putative regulators of fruit ripening. Silencing of these genes at the color break stage in both the Regina and early-maturing cultivar Carmen cultivars resulted in delayed pigmentation and reduced fruit size. Subsequent transcriptomic and proteomic analyses of silenced fruit revealed several candidate downstream targets and regulatory networks linked to anthocyanin biosynthesis. Levels of central metabolic components and major anthocyanins, particularly cyanidin glucoside and cyanidin rutinoside were reduced alongside altered abscisic acid (ABA) concentrations. Furhtermore we demonstrate that PaWRKY57 and PaNAC29 directly bind to the promoters of PaDFR and PaLDOX, disrupting flavonoid biosynthesis. Notably, PaNAC29 also binds to PaPRE6 and PaLOX5 promoters, impacting abscisic acid and volatile compound biosynthesis. This work provides a novel insights into tissue-specific regulatory dynamics in sweet cherry, establishes a molecular framework for non-climacteric fruit ripening, and identifies promising targets for improving cherry yield and fruit quality.