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

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Abstract

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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.

Abstract

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.

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Abbreviations

2,4-D:

2,4-Dichlorophenoxyacetic acid

6-BA:

6-Benzylaminopurine

ABA:

Abscisic acid

CCD:

Carotenoid cleavage dioxygenase

CRISPR:

Clustered regularly interspaced short palindromic repeats

Cas9:

CRISPR-associated protein 9

crRNA:

CRISPR RNA

DNA:

Deoxyribonucleic acid

EB:

Ethidium bromide

EDTA:

Ethylene diamine tetraacetic acid

HPTII:

Hygromycin phosphotransferase II gene

NAA:

1-Naphthaleneacetic acid

NCED:

Nine-cis-epoxy carotenoid dioxygenase

NLS:

Nulcear localization signal

PAM:

Protospacer-adjacent motif

PCR:

Polymerase chain reaction

PSY:

Phytoene synthase

RNA:

Ribonucleic acid

RT-PCR:

Reverse transcription PCR

SDS:

Sodium dodecyl sulfate

sgRNA:

Single guide RNA

SSR:

Site-specific recombination

TALEN:

Transcription activator-like effector nuclease

tracrRNA:

Trans-activating crRNA

WT:

Wild type

ZFN:

Zinc finger nuclease

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Acknowledgements

This work was supported in part by the National Natural Science Foundation of China (Project No. 31271422) to WY, Shenzhen Peacock Innovation Team Project (No. KQTD201101) to WY and JH, Guangdong Innovation Research Team Fund (2014ZT05S078) to WY, Natural Science Foundation of Guangdong Province (Project No. 2015A030310421) to LC, and Hong Kong RGC AoE Project AoE/M-05/12 to JH. We thank Chao Sun, Meiting Wu, Linlin Luo and Ye Jin for assistance in analyzing the off-target cleavage activity.

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Author notes

  1. Xiaoyu Yang and Lei Chen contributed equally to this work.

Authors and Affiliations

  1. Guangdong Provincial Key Laboratory for Plant Epigenetics, College of Life Sciences, Shenzhen University, Shenzhen, 518060, China

    Xiaoyu Yang & Weichang Yu

  2. Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, College of Optoelectronic Engineering, Shenzhen University, Shenzhen, China

    Xiaoyu Yang

  3. Shenzhen Research Institute, State Key Laboratory of Agrobiotechnology, The Chinese University of Hong Kong, Shenzhen, China

    Lei Chen

  4. School of Life Sciences, State Key Laboratory of Agrobiotechnology, The Chinese University of Hong Kong, Sha Tin, New Territories, Hong Kong

    Junxian He

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  1. Xiaoyu Yang
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Correspondence to Junxian He or Weichang Yu.

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Communicated by Kan Wang.

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Yang, X., Chen, L., He, J. et al. Knocking out of carotenoid catabolic genes in rice fails to boost carotenoid accumulation, but reveals a mutation in strigolactone biosynthesis. Plant Cell Rep 36, 1533–1545 (2017). https://doi.org/10.1007/s00299-017-2172-6

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  • DOI: https://doi.org/10.1007/s00299-017-2172-6

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