FaRIF as a key regulator of strawberry fruit ripening: deciphering its targets and interaction networks

Abstract

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.

Acknowledgements

This work was mainly supported by a grant from the Spanish Ministry of Science and Innovation and Universities (MICIU, PID2021-123677OB-I00 and PID2024-155863OB-I00 to D.P.) and funding from the Junta de Andalucía (UMA20-FEDERJA-093 and Postdoctoral program, POSTDOC_21_00893 to C.M.-P) and EMBO (EMBO Scientific Exchange Grant, ref 10186 to C.M.-P.). Further funding support was provided by the Juan de la Cierva Program and from the Junta de Andalucía (JDC2022-048407-I and DGP_POST_2024_00657 to M.F.-P.), from the MICIU (PID2023-149400NB-I00 to J.M.F.-Z.), and by the Excellence Strategy of the German Federal and State Governments (to R.L.-D.). We thank Dr. José F. Sánchez Sevilla for facilitating growing the transgenic plants at the Andalusian Institute for Research and Training in Agriculture, Fishery, Food and Ecological Production (IFAPA) facilities in Churriana, Málaga, Spain, and Francisco Durán for its maintenance, respectively. We thank GeminiTeam (ZMBP, University of Tübingen) for their support with the TurboID experiments, and Irina Droste-Borel (Proteome Center Tübingen (PCR), University of Tübingen), for the NanoLC-MS/MS analysis and MS data processing. Finally, we, C.M.-P. and D.P., would like to express our deepest gratitude to Dr. Victoriano Valpuesta, a true mentor and an inspiring scientific guide. You named FaRIF and introduced us to the fascinating world of strawberry research. Your vision and generosity shaped who we are as scientists, and we will always be grateful. Open access funding was provided by the University of Málaga/CBUA

Author contributions

C.M.-P., G.Q., and D.P. conceived and designed the research. C.M.-P. performed and analyzed most experiments and data. M.F.P. performed the transactivation and FRET-FLIM assays in Nicotiana. J.M.F.-Z. analyzed the ChIP-seq data. R.L.-D. contributed to the TurboID-based proximity labeling experiment. C.M.-P. and D.P. secured funds, designed and supervised the experiments, and wrote the article. All authors commented on the article.

Data availability

The data that support the findings of this study are available in Figs S1–S12, Supplementary Dataset S1, and Tables S1–S8. RNA-seq data and ChIP-seq data are available at GEO under the accession numbers GSE167107 and GSE304403, respectively.

Conflicts of interest statement

The authors declare no competing interests.

Supplementary material

Supplementary material is available at Horticulture Research online.

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