Ripening inducing factor (RIF) is a key NAC transcription factor (TF) regulating strawberry fruit ripening. Previous studies using RIF-RNAi and overexpression lines in Fragaria × ananassa and CRISPR knock-out lines in F. vesca have established the role of RIF in controlling ABA biosynthesis and signaling, cell wall remodeling, and secondary metabolism. In this study, we deciphered FaRIF’s transcriptional regulatory network by combining ChIP-seq-based identification of its direct targets with an analysis of FaRIF-RNAi transcriptome data. These analyses revealed FaRIF’s direct role in multiple aspects of strawberry fruit ripening, including the regulation of ripening-related TFs, phytohormone content and signaling, primary and secondary metabolism, and cell wall degradation. Additionally, using the TurboID-based proximity labeling approach, we have identified FaRIF interactors, including proteins involved in mRNA and protein homeostasis, as well as several NAC TFs. Among these, FaNAC021 and FaNAC034 were found to potentially cooperate with FaRIF to enhance the transcription of shared target genes. This integrative analysis, combining transcriptome analysis, in vivo ChIP-seq, and proximity labeling, broadens our understanding of FaRIF-mediated transcriptional networks and interaction partners, providing valuable insights into the molecular regulation of strawberry fruit ripening by this TF.

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