Alteration of flower colour in Ipomoea nil through CRISPR/Cas9-mediated mutagenesis of carotenoid cleavage dioxygenase 4
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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.
KeywordsCarotenoid cleavage dioxygenase Carotenoid metabolic engineering CRISPR/Cas9 Flower colour alteration Ipomoea nil Targeted mutagenesis
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.
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Conflict of interest
The authors declare that they have no conflict of interest.
- Ariizumi T, Kishimoto S, Kakami R et al (2014) Identification of the carotenoid modifying gene PALE YELLOW PETAL 1 as an essential factor in xanthophyll esterification and yellow flower pigmentation in tomato (Solanum lycopersicum). Plant J 79:453–465. https://doi.org/10.1111/tpj.12570 CrossRefPubMedGoogle Scholar
- Bai S, Tuan PA, Tatsuki M et al (2016) Knockdown of Carotenoid Cleavage Dioxygenase 4 (CCD4) via virus-induced gene silencing confers yellow coloration in peach fruit: evaluation of gene function related to fruit traits. Plant Mol Biol Rep 34:257–264. https://doi.org/10.1007/s11105-015-0920-8 CrossRefGoogle Scholar
- Brandi F, Bar E, Mourgues F et al (2011) Study of “Redhaven” peach and its white-fleshed mutant suggests a key role of CCD4 carotenoid dioxygenase in carotenoid and norisoprenoid volatile metabolism. BMC Plant Biol 11:24–37. https://doi.org/10.1186/1471-2229-11-24 CrossRefPubMedPubMedCentralGoogle Scholar
- Britton G (1995) UV/visible spectroscopy. In: Britton G, Liaaen-Jensen S, Pfander H (eds) Carotenoids, vol 1B. Spectroscopy. Birkhäuser Verlag, Basel, pp 13–62Google Scholar
- Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425. https://doi.org/10.1093/oxfordjournals.molbev.a040454 PubMedGoogle Scholar
- Vishnevetsky M, Ovadis M, Itzhaki H et al (1996) Molecular cloning of a carotenoid-associated protein from Cucumis sativus corollas: homologous genes involved in carotenoid sequestration in chromoplasts. Plant J 10:1111–1118. https://doi.org/10.1046/j.1365-313X.1996.10061111.x CrossRefPubMedGoogle Scholar
- Watanabe K, Oda-Yamamizo C, Sage-Ono K et al (in press) Overexpression of carotenogenic genes in the Japanese morning glory Ipomoea (Pharbitis) nil. Plant Biotechnol. https://doi.org/10.5511/plantbiotechnology.17.1016a