Knocking out of carotenoid catabolic genes in rice fails to boost carotenoid accumulation, but reveals a mutation in strigolactone biosynthesis.

KEY MESSAGE: Targeted mutations in five carotenoid catabolism genes failed to boost carotenoid accumulation in rice seeds, but produced dwarf and high tillering mutants when OsCCD7 gene was knocked out. Carotenoids play an important role in human diet as a source of vitamin A. Rice is a major staple food in Asia, but does not accumulate carotenoids in the endosperm because of the low carotenoid biosynthesis or the degradation in metabolism. In this study, the CRISPR/Cas9 system was investigated in the targeted knockout of five rice carotenoid catabolic genes (OsCYP97A4, OsDSM2, OsCCD4a, OsCCD4b and OsCCD7) and in an effort to increase β-carotene accumulation in rice endosperm. Transgenic plants that expressed OsNLSCas9 and sgRNAs were generated by Agrobacterium-mediated transformation. Various knockout mutations were identified at the T0 generation of the transgenic rice by TILLING and direct sequencing of the PCR products amplified from the target sites. Carotenoids were not accumulated in both mono-allelic and bi-allelic knockout mutations of the five genes. However, transgenic plants with homozygous or bi-allelic mutations to the OsCCD7 gene were extremely dwarfish with more tillers and lower seed setting than other transgenic or nontransgenic plants. This phenotype was similar to the previously reported ccd7 mutants, which are defective in the biosynthesis of strigolactone, a plant hormone that regulates branching in plants and tiller formation in rice.