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Transgenic Research

, Volume 27, Issue 1, pp 25–38 | Cite as

Alteration of flower colour in Ipomoea nil through CRISPR/Cas9-mediated mutagenesis of carotenoid cleavage dioxygenase 4

  • Kenta Watanabe
  • Chihiro Oda-Yamamizo
  • Kimiyo Sage-Ono
  • Akemi Ohmiya
  • Michiyuki Ono
Original Paper

Abstract

Japanese morning glory, Ipomoea nil, exhibits a variety of flower colours, except yellow, reflecting the accumulation of only trace amounts of carotenoids in the petals. In a previous study, we attributed this effect to the low expression levels of carotenogenic genes in the petals, but there may be other contributing factors. In the present study, we investigated the possible involvement of carotenoid cleavage dioxygenase (CCD), which cleaves specific double bonds of the polyene chains of carotenoids, in the regulation of carotenoid accumulation in the petals of I. nil. Using bioinformatics analysis, seven InCCD genes were identified in the I. nil genome. Sequencing and expression analyses indicated potential involvement of InCCD4 in carotenoid degradation in the petals. Successful knockout of InCCD4 using the CRISPR/Cas9 system in the white-flowered cultivar I. nil cv. AK77 caused the white petals to turn pale yellow. The total amount of carotenoids in the petals of ccd4 plants was increased 20-fold relative to non-transgenic plants. This result indicates that in the petals of I. nil, not only low carotenogenic gene expression but also carotenoid degradation leads to extremely low levels of carotenoids.

Keywords

Carotenoid cleavage dioxygenase Carotenoid metabolic engineering CRISPR/Cas9 Flower colour alteration Ipomoea nil Targeted mutagenesis 

Notes

Acknowledgements

We are grateful to the National BioResource Project (NBRP) “Morning Glory,” which is supported by the Agency for Medical Research and Development (AMED) of Japan, for supplying information on the DNA sequence of I. nil. We also thank Dr. Masaki Endo and Dr. Seiichi Toki (Plant Genome Engineering Research Unit, Institute of Agrobiological Sciences, National Agriculture and Food Research Organization) for providing the vectors pDeCas9-Kan, pMR203, pMR204 and pMR205. This work was partially supported by a Cooperative Research Grant of the Plant Transgenic Design Initiative (PTraD), the Gene Research Center, T-PIRC, and the University of Tsukuba.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

11248_2017_51_MOESM1_ESM.pptx (685 kb)
Supplementary material 1 (PPTX 684 kb)

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Copyright information

© Springer International Publishing AG, part of Springer Nature 2017

Authors and Affiliations

  1. 1.Graduate School of Life and Environmental SciencesUniversity of TsukubaTsukubaJapan
  2. 2.Institute of Vegetable and Floriculture ScienceNational Agriculture and Food Research OrganizationTsukubaJapan
  3. 3.Faculty of Life and Environmental Sciences, Gene Research Center, Tsukuba Plant Innovation Research Center (T-PIRC)University of TsukubaTsukubaJapan
  4. 4.Biological Resources and Post-Harvest DivisionJapan International Research Center for Agricultural SciencesTsukubaJapan

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