Transcription factors PaWRKY57 and PaNAC29 regulate fruit color and growth during sweet cherry development

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 physiological, biochemical and transcriptomic analysis of pedicel and fruit tissues across 8 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 widespread transcriptomic and metabolomic reprogramming, particularly in sugar metabolism within the pedicel, suggesting that cherry ripening may be partially regulated by pedicel-derived signals. Through integrative analysis, we identified key transcription factors (TFs), most notably PaWRKY57 and PaNAC29, as putative regulators of fruit development. Silencing the genes encoding these TFs at the color breaking stage in both the "Regina" and early-maturing "Carmen" cultivars resulted in delayed pigmentation and reduced fruit size. Subsequent transcriptomic and proteomic analysis of silenced fruit revealed several candidate downstream targets and regulatory networks specifically 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) levels following PaWRKY57 and PaNAC29 silencing. Furthermore, we demonstrate that PaWRKY57 and PaNAC29 interact with the promoter regions of dihydroflavonol4-reductase (PaDFR) and leucoanthocyanidin dioxygenase (PaLDOX), regulating flavonoid biosynthesis. Notably, PaNAC29 also binds to the promoters of PACLOBUTRAZOL RESISTANCE 6 (PaPRE6) and linoleate 9S-lipoxygenase 5 (PaLOX5), influencing the biosynthesis of ABA and aroma-related volatile compounds. This work provides insights into tissue-specific regulatory dynamics in sweet cherry, establishes a framework for non-climacteric fruit ripening, and identifies promising targets for improving cherry yield and fruit quality.