CRISPR/Cas9-mediated SNAC9 mutants reveal the positive regulation of tomato ripening by SNAC9 and the mechanism of carotenoid metabolism regulation

Abstract

NAC transcriptional regulators are crucial for tomato ripening. Virus-induced gene silencing (VIGS) of SNAC9 (SlNAC19, Gene ID: 101248665) affects tomato ripening, and SNAC9 is involved in ethylene and abscisic acid (ABA) metabolic pathways. However, the function of SNAC9 in pigment metabolism in tomatoes remains unclear. This work seeks to discover the mechanism of SNAC9 involvement in pigment metabolism during tomato ripening by establishing a SNAC9 knockout model using CRISPR/Cas9 technology. The results indicated that fruit ripening was delayed in knockout (KO) mutants, and SNAC9 mutation significantly affected carotenoid metabolism. The chlorophyll (Chl) degradation rate, total carotenoid content, and lycopene content decreased significantly in the mutants. The transformation rate of chloroplasts to chromoplasts in mutants was slower, which was related to the carotenoid content. Furthermore, SNAC9 changed the expression of critical genes (PSY1, PDS, CRTISO, Z-ISO, SGR1, DXS2, LCYE, LCYB, and CrtR-b2) involved in pigment metabolism in tomato ripening. SNAC9 knockout also altered the expression levels of critical genes involved in the biosynthesis of ethylene and ABA. Accordingly, SNAC9 regulated carotenoid metabolism by directly regulating PSY1, DXS2, SGR1, and CrtR-b2. This research provides a foundation for developing the tomato ripening network and precise tomato ripening regulation.

Acknowledgements

This work was supported by the National Natural Science Foundation of China, China [Grant No. 32072274 and 31871848]. We thank Yaoguang Liu (South China Agricultural University) and Hongliang Zhu (China Agricultural University) for helping with pYLgRNA and pYLCRISPR/Cas9Pubi-H vector.

Author contributions

All the authors contributed to the discussion. Y.F. and H.X.K. conceived and designed the experiment; Y.F. performed experiments and data analyses and wrote the manuscript; H.X.K., H.Z.X., and E.C.W. were involved in revising the manuscript; S.Y. and Y.X.Z. collected the literature and revised the manuscript; X.Q.L. measured ethylene release; Y.Z.H. and J.Y.S. checked the language. All authors read and approved the final manuscript.

Data availability

All data related to this manuscript are available in this paper and its supporting materials.

Conflict of interest

None declared.

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