Molecular Characterization and Evolutionary Relationships of WRKYs in Five Rubus Species and Functional Analysis of RiWRKY48 in Rubus idaeus

Abstract

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.