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Re-evaluation of the rin mutation and the role of RIN in the induction of tomato ripening

Abstract

Tomato (Solanum lycopersicum) rin mutants completely fail to ripen: they do not produce red pigmentation, soften or induce an ethylene burst. Therefore, RIN has long been believed to function as a major regulator that is essential for the induction of ripening. Here, we provide evidence contradicting this concept of RIN function, showing induction of fruit ripening in the absence of RIN. A CRISPR/Cas9-mediated RIN-knockout mutation did not repress initiation of ripening and the mutant fruits showed moderate red colouring. Moreover, inactivation of the rin mutant allele partially restored the induction of ripening. Therefore, RIN is not required for the initiation of ripening and rin is not a null mutation, but rather is a gain-of-function mutation that produces a protein that actively represses ripening. Since the discovery of the rin mutant a half-century ago, many models have depicted RIN as indispensable for the induction of ripening; these models should be reconsidered in light of these results.

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Fig. 1: Molecular properties of the rin mutant protein.
Fig. 2: Induction of ripening in RIN-KO fruits.
Fig. 3: Expression of ripening-associated genes in RIN-KO and rin-inactivated fruits.
Fig. 4: Binding of RIN and rin proteins and RIN-associated transcription factors to ripening-related genes in RIN-KO and rin-inactivated fruits.
Fig. 5: RIN/RIN-KO and RIN/rin fruits exhibit different phenotypes.
Fig. 6: Schematic representation of transcriptional regulation by RIN.

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Acknowledgements

The authors thank A. Koma for technical assistance. This work was supported by the Cross-ministerial Strategic Innovation Promotion Program (SIP) and the Japan Society for Bioscience, Biotechnology, and Agrochemistry.

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Authors

Contributions

Y.I. conceived the idea for the study. Y.I. and S.T. designed the experiments. Y.I., A.N.-Y. and Y.S. produced mutants. Y.I., M.E. and M.M. constructed plasmids. Y.I., N.N. and E.K.-N. did the experiments for mutant characterization. Y.I. and S.K. did assays of protein activities. Y.I. did expression analyses and ChIP assays. Y.I. analysed the data and wrote the manuscript.

Corresponding author

Correspondence to Yasuhiro Ito.

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Supplementary Figures 1–8, Supplementary Tables 1 and 2, Supplementary Methods, Supplementary References.

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Ito, Y., Nishizawa-Yokoi, A., Endo, M. et al. Re-evaluation of the rin mutation and the role of RIN in the induction of tomato ripening. Nature Plants 3, 866–874 (2017). https://doi.org/10.1038/s41477-017-0041-5

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