Flower bud differentiation is crucial to reproductive success in plants. In the present study, RNA-Seq and nutrients quantification were used to identify the stage-specific genes for flower bud differentiation with buds which characterize the marked change during flower bud formation from a widely grown Chinese cherry (Prunus pseudocerasus L.) cultivar ‘Manaohong’. A KEGG enrichment analysis revealed that the sugar metabolism pathways dynamically changed. The gradually decreasing trend in the contents of total sugar, soluble sugar and protein implies that the differentiation was an energy-consuming process. Changes in the contents of D-glucose and sorbitol were conformed with the gene expression trends of bglX and SORD, respectively, which at least partially reflects a key role of the two substances in the transition from physiological to morphological differentiation. Further, the WRKY and SBP families were also significantly differentially expressed during the vegetative-to-reproductive transition. In addition, floral meristem identity genes, e.g., AP1, AP3, PI, AGL6, SEP1, LFY, and UFO demonstrate involvement in the specification of the petal and stamen primordia, and FPF1 might promote the onset of morphological differentiation. Conclusively, the available evidence justifies the involvement of sugar metabolism in the flower bud differentiation of Chinese cherry, and the uncovered candidate genes are beneficial to further elucidate flower bud differentiation in cherries.

Copper amine oxidases (CuAOs) play important roles in PA catabolism, plant growth and development, and abiotic stress response. In order to better understand how PA affects cherry fruit, four potential PavCuAO genes (PavCuAO1–PavCuAO4) that are dispersed over two chromosomes were identified in the sweet cherry genome. Based on phylogenetic analysis, they were classified into three subclasses. RNA-seq analysis showed that the PavCuAO genes were tissue-specific and mostly highly expressed in flowers and young leaves. Many cis-elements associated with phytohormones and stress responses were predicted in the 2 kb upstream region of the promoter. The PavCuAOs transcript levels were increased in response to abscisic acid (ABA) and gibberellin 3 (GA₃) treatments, as well as abiotic stresses (NaCl, PEG, and cold). Quantitative fluorescence analysis and high-performance liquid chromatography confirmed that the Put content fell, and the PavCuAO4 mRNA level rose as the sweet cherry fruit ripened. After genetically transforming Arabidopsis with PavCuAO4, the Put content in transgenic plants decreased significantly, and the expression of the ABA synthesis gene NCED was also significantly increased. At the same time, excessive H₂O₂ was produced in PavCuAO4 transiently expressed tobacco leaves. The above results strongly proved that PavCuAO4 can decompose Put and may promote fruit ripening by increasing the content of ABA and H₂O₂ while suppressing total free PA levels in the fruit.

WRKY transcription factors play crucial roles in plant growth, development, and stress responses. However, their systematic evolution and functions within the genus Rubus remain incompletely understood. In this study, genome-wide identification, evolutionary analysis and functional characterization of the WRKY gene family were conducted in five Rubus species. A total of 37–67 members were identified and classified into three groups by phylogenetic analysis. The collinearity result, gene family expansion and contraction analyses revealed a contraction trend within the Rubus lineage compared with the outgroup sweet cherry. Promoter cis-acting element analysis predicted widespread involvement of these genes in hormone and stress responses. Expression profiling revealed tissue-specific patterns among WRKY genes, with most members down-regulated during fruit development and differentially regulated by biotic and abiotic stresses. Further correlation analysis and experimental validation were focused on RiWRKY48 in red raspberry (Rubus idaeus L.). This protein was localized to the nucleus and lacked transcriptional activation activity. Combined in silico modeling (AlphaFold3) and dual-luciferase reporter assays revealed that RiWRKY48 may repressed the promoter activity of RiUFGT4, a key anthocyanin biosynthetic gene, suggesting a putative interaction with the W-box element. In summary, this study systematically characterized the evolutionary features of the Rubus WRKY family and provided evidence for a potential regulatory role of RiWRKY48 in anthocyanin biosynthesis through modulation of RiUFGT4 promoter activity. These findings provided new insights into the regulatory roles of WRKY transcription factors in fruit quality development in Rubus species.