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

Transgenic Research volume 27, pages25–38(2018)Cite this article

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.

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References

  1. Ahrazem O, Trapero A, Gómez MD et al (2010) Genomic analysis and gene structure of the plant carotenoid dioxygenase 4 family: a deeper study in Crocus sativus and its allies. Genomics 96:239–250. https://doi.org/10.1016/j.ygeno.2010.07.003

    CAS  Article  PubMed  Google Scholar 

  2. Ahrazem O, Gómez-Gómez L, Rodrigo MJ et al (2016) Carotenoid cleavage oxygenases from microbes and photosynthetic organisms: features and functions. Int J Mol Sci 17:1781. https://doi.org/10.3390/ijms17111781

    Article  PubMed Central  Google Scholar 

  3. Altschul SF, Madden TL, Schäffer AA et al (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402. https://doi.org/10.1093/nar/25.17.3389

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  4. 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

    CAS  Article  PubMed  Google Scholar 

  5. Auldridge ME, McCarty DR, Klee HJ (2006) Plant carotenoid cleavage oxygenases and their apocarotenoid products. Curr Opin Plant Biol 9:315–321. https://doi.org/10.1016/j.pbi.2006.03.005

    CAS  Article  PubMed  Google Scholar 

  6. 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

    CAS  Article  Google Scholar 

  7. 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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  8. Britton G (1995) UV/visible spectroscopy. In: Britton G, Liaaen-Jensen S, Pfander H (eds) Carotenoids, vol 1B. Spectroscopy. Birkhäuser Verlag, Basel, pp 13–62

    Google Scholar 

  9. Buah S, Mlalazi B, Khanna H et al (2016) The quest for Golden Bananas: investigating carotenoid regulation in a Fe’i group Musa cultivar. J Agric Food Chem 64:3176–3185. https://doi.org/10.1021/acs.jafc.5b05740

    CAS  Article  PubMed  Google Scholar 

  10. Bustin SA, Benes V, Garson JA et al (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55:611–622. https://doi.org/10.1373/clinchem.2008.112797

    CAS  Article  PubMed  Google Scholar 

  11. Edwards K, Johnstone C, Thompson C (1991) A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucleic Acids Res 19:1349. https://doi.org/10.1093/nar/19.6.1349

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  12. Fauser F, Schiml S, Puchta H (2014) Both CRISPR/Cas-based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana. Plant J 79:348–359. https://doi.org/10.1111/tpj.12554

    CAS  Article  PubMed  Google Scholar 

  13. Galpaz N, Ronen G, Khalfa Z et al (2006) A chromoplast-specific carotenoid biosynthesis pathway is revealed by cloning of the tomato white-flower locus. Plant Cell 18:1947–1960. https://doi.org/10.1105/tpc.105.039966

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  14. Giuliano G, Bartley GE, Scolnik PA (1993) Regulation of carotenoid biosynthesis during tomato development. Plant Cell 5:379–387. https://doi.org/10.1105/tpc.5.4.379

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  15. Gomez-Roldan V, Fermas S, Brewer PB et al (2008) Strigolactone inhibition of shoot branching. Nature 455:189–194. https://doi.org/10.1038/nature07271

    CAS  Article  PubMed  Google Scholar 

  16. Gonzalez-Jorge S, Ha S-H, Magallanes-Lundback M et al (2013) Carotenoid cleavage dioxygenase4 is a negative regulator of β-carotene content in Arabidopsis seeds. Plant Cell 25:4812–4826. https://doi.org/10.1105/tpc.113.119677

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  17. Higuchi Y, Sage-Ono K, Kamada H, Ono M (2007) Isolation and characterization of novel genes controlled by short-day treatment in Pharbitis nil. Plant Biotechnol 24:201–207. https://doi.org/10.5511/plantbiotechnology.24.201

    CAS  Article  Google Scholar 

  18. Hoshino A, Jayakumar V, Nitasaka E et al (2016) Genome sequence and analysis of the Japanese morning glory Ipomoea nil. Nat Commun 7:13295. https://doi.org/10.1038/ncomms13295

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. Huang F-C, Molnár P, Schwab W (2009) Cloning and functional characterization of carotenoid cleavage dioxygenase 4 genes. J Exp Bot 60:3011–3022. https://doi.org/10.1093/jxb/erp137

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  20. Ilg A, Yu Q, Schaub P et al (2010) Overexpression of the rice carotenoid cleavage dioxygenase 1 gene in Golden Rice endosperm suggests apocarotenoids as substrates in planta. Planta 232:691–699. https://doi.org/10.1007/s00425-010-1205-y

    CAS  Article  PubMed  Google Scholar 

  21. Kikuchi R, Sage-Ono K, Kamada H, Ono M (2005) Efficient transformation mediated by Agrobacterium tumefaciens with a ternary plasmid in Pharbitis nil. Plant Biotechnol 22:295–302. https://doi.org/10.5511/plantbiotechnology.22.295

    CAS  Article  Google Scholar 

  22. Kishimoto S, Ohmiya A (2006) Regulation of carotenoid biosynthesis in petals and leaves of chrysanthemum (Chrysanthemum morifolium). Physiol Plant 128:436–447. https://doi.org/10.1111/j.1399-3054.2006.00761.x

    CAS  Article  Google Scholar 

  23. Kishimoto S, Sumitomo K, Yagi M et al (2007) Three routes to orange petal color via carotenoid components in 9 compositae species. J Jpn Soc Hortic Sci 76:250–257. https://doi.org/10.2503/jjshs.76.250

    CAS  Article  Google Scholar 

  24. Kloer DP, Schulz GE (2006) Structural and biological aspects of carotenoid cleavage. Cell Mol Life Sci 63:2291–2303. https://doi.org/10.1007/s00018-006-6176-6

    CAS  Article  PubMed  Google Scholar 

  25. Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evolut 33:1870–1874. https://doi.org/10.1093/molbev/msw054

    CAS  Article  Google Scholar 

  26. Lätari K, Wüst F, Hübner M et al (2015) Tissue-specific apocarotenoid glycosylation contributes to carotenoid homeostasis in Arabidopsis leaves. Plant Physiol 168:1550–1562. https://doi.org/10.1104/pp.15.00243

    Article  PubMed  PubMed Central  Google Scholar 

  27. Leitner-Dagan Y, Ovadis M, Shklarman E et al (2006) Expression and functional analyses of the plastid lipid-associated protein CHRC suggest its role in chromoplastogenesis and stress. Plant Physiol 142:233–244. https://doi.org/10.1104/pp.106.082404

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  28. Li L, Yuan H (2013) Chromoplast biogenesis and carotenoid accumulation. Arch Biochem Biophys 539:102–109. https://doi.org/10.1016/j.abb.2013.07.002

    CAS  Article  PubMed  Google Scholar 

  29. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2 −ΔΔCT method. Methods 25:402–408. https://doi.org/10.1006/meth.2001.1262

    CAS  Article  PubMed  Google Scholar 

  30. Moehs CP, Tian L, Osteryoung KW, DellaPenna D (2001) Analysis of carotenoid biosynthetic gene expression during marigold petal development. Plant Mol Biol 45:281–293. https://doi.org/10.1023/A:1006417009203

    CAS  Article  PubMed  Google Scholar 

  31. Ohmiya A (2009) Carotenoid cleavage dioxygenases and their apocarotenoid products in plants. Plant Biotechnol 26:351–358. https://doi.org/10.5511/plantbiotechnology.26.351

    CAS  Article  Google Scholar 

  32. Ohmiya A, Kishimoto S, Aida R et al (2006) Carotenoid cleavage dioxygenase (CmCCD4a) contributes to white color formation in chrysanthemum petals. Plant Physiol 142:1193–1201. https://doi.org/10.1104/pp.106.087130

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  33. Ohmiya A, Sumitomo K, Aida R (2009) “Yellow Jimba”: suppression of carotenoid cleavage dioxygenase (CmCCD4a) expression turns white chrysanthemum petals yellow. J Jpn Soc Hortic Sci 78:450–455. https://doi.org/10.2503/jjshs1.78.450

    CAS  Article  Google Scholar 

  34. Ohmiya A, Toyoda T, Watanabe H et al (2012) Mechanism behind petal color mutation induced by heavy-ion-beam irradiation of recalcitrant chrysanthemum cultivar. J Jpn Soc Hortic Sci 81:269–274. https://doi.org/10.2503/jjshs1.81.269

    CAS  Article  Google Scholar 

  35. Ono M, Sage-ono K, Kawakami M et al (2000) Agrobacterium-mediated regeneration and transformation of Pharbitis nil. Plant Biotechnol 17:211–216. https://doi.org/10.5511/plantbiotechnology.17.211

    CAS  Article  Google Scholar 

  36. Park S, Kim HS, Jung YJ et al (2016) Orange protein has a role in phytoene synthase stabilization in sweetpotato. Sci Rep 6:33563. https://doi.org/10.1038/srep33563

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  37. Puchta H (2017) Applying CRISPR/Cas for genome engineering in plants: the best is yet to come. Curr Opin Plant Biol 36:1–8. https://doi.org/10.1016/j.pbi.2016.11.011

    CAS  Article  PubMed  Google Scholar 

  38. Rodrigo MJ, Alquézar B, Alós E et al (2013) A novel carotenoid cleavage activity involved in the biosynthesis of Citrus fruit-specific apocarotenoid pigments. J Exp Bot 64:4461–4478. https://doi.org/10.1093/jxb/ert260

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  39. Saito N, Cheng J, Ichimura M et al (1994) Flavonoids in the acyanic flowers of Pharbitis nil. Phytochemistry 35:687–691. https://doi.org/10.1016/S0031-9422(00)90588-0

    CAS  Article  Google Scholar 

  40. 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

    CAS  PubMed  Google Scholar 

  41. Schwartz SH, Tan BC, Gage DA et al (1997) Specific oxidative cleavage of carotenoids by VP14 of maize. Science 276:1872–1874. https://doi.org/10.1126/science.276.5320.1872

    CAS  Article  PubMed  Google Scholar 

  42. Schwartz SH, Qin X, Zeevaart JAD (2001) Characterization of a novel carotenoid cleavage dioxygenase from plants. J Biol Chem 276:25208–25211. https://doi.org/10.1074/jbc.M102146200

    CAS  Article  PubMed  Google Scholar 

  43. Schwartz SH, Qin X, Zeevaart JAD (2003) Elucidation of the indirect pathway of abscisic acid biosynthesis by mutants, genes, and enzymes. Plant Physiol 131:1591–1601. https://doi.org/10.1104/pp.102.017921

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  44. Tan BC, Schwartz SH, Zeevaart JAD, McCarty DR (1997) Genetic control of abscisic acid biosynthesis in maize. Proc Natl Acad Sci USA 94:12235–12240. https://doi.org/10.1073/pnas.94.22.12235

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  45. Tan BC, Joseph LM, Deng WT et al (2003) Molecular characterization of the Arabidopsis 9-cis epoxycarotenoid dioxygenase gene family. Plant J 35:44–56. https://doi.org/10.1046/j.1365-313X.2003.01786.x

    CAS  Article  PubMed  Google Scholar 

  46. Tanaka Y, Ohmiya A (2008) Seeing is believing: engineering anthocyanin and carotenoid biosynthetic pathways. Curr Opin Biotechnol 19:190–197. https://doi.org/10.1016/j.copbio.2008.02.015

    CAS  Article  PubMed  Google Scholar 

  47. Umehara M, Hanada A, Yoshida S et al (2008) Inhibition of shoot branching by new terpenoid plant hormones. Nature 455:195–200. https://doi.org/10.1038/nature07272

    CAS  Article  PubMed  Google Scholar 

  48. Vallabhaneni R, Bradbury LMT, Wurtzel ET (2010) The carotenoid dioxygenase gene family in maize, sorghum, and rice. Arch Biochem Biophys 504:104–111. https://doi.org/10.1016/j.abb.2010.07.019

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  49. van der Fits L, Deakin EA, Hoge JHC, Memelink J (2000) The ternary transformation system: constitutive virG on a compatible plasmid dramatically increases Agrobacterium-mediated plant transformation. Plant Mol Biol 43:495–502. https://doi.org/10.1023/A:1006440221718

    Article  PubMed  Google Scholar 

  50. 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

    CAS  Article  PubMed  Google Scholar 

  51. Walter MH, Floss DS, Strack D (2010) Apocarotenoids: hormones, mycorrhizal metabolites and aroma volatiles. Planta 232:1–17. https://doi.org/10.1007/s00425-010-1156-3

    CAS  Article  PubMed  Google Scholar 

  52. Watanabe K, Kobayashi A, Endo M et al (2017) CRISPR/Cas9-mediated mutagenesis of the dihydroflavonol-4-reductase-B (DFR-B) locus in the Japanese morning glory Ipomoea (Pharbitis) nil. Sci Rep 7:10028. https://doi.org/10.1038/s41598-017-10715-1

    Article  PubMed  PubMed Central  Google Scholar 

  53. 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

  54. Wei Y, Wan H, Wu Z et al (2016) A comprehensive analysis of carotenoid cleavage dioxygenases genes in Solanum lycopersicum. Plant Mol Biol Rep 34:512–523. https://doi.org/10.1007/s11105-015-0943-1

    CAS  Article  Google Scholar 

  55. Willems E, Leyns L, Vandesompele J (2008) Standardization of real-time PCR gene expression data from independent biological replicates. Anal Biochem 379:127–129. https://doi.org/10.1016/j.ab.2008.04.036

    CAS  Article  PubMed  Google Scholar 

  56. Winter D, Vinegar B, Nahal H et al (2007) An “electronic fluorescent pictograph” Browser for exploring and analyzing large-scale biological data sets. PLoS ONE 2:1–12. https://doi.org/10.1371/journal.pone.0000718

    Article  Google Scholar 

  57. Yamada T, Ichimura K, Kanekatsu M, Van Doorn WG (2007) Gene expression in opening and senescing petals of morning glory (Ipomoea nil) flowers. Plant Cell Rep 26:823–835. https://doi.org/10.1007/s00299-006-0285-4

    CAS  Article  PubMed  Google Scholar 

  58. Yamamizo C, Kishimoto S, Ohmiya A (2010) Carotenoid composition and carotenogenic gene expression during Ipomoea petal development. J Exp Bot 61:709–719. https://doi.org/10.1093/jxb/erp335

    CAS  Article  PubMed  Google Scholar 

  59. Yamazaki Y, Akashi R, Banno Y et al (2009) NBRP databases: databases of biological resources in Japan. Nucleic Acids Res 38:D26–D32. https://doi.org/10.1093/nar/gkp996

    Article  PubMed  PubMed Central  Google Scholar 

  60. Zhang B, Liu C, Wang Y et al (2015) Disruption of a CAROTENOID CLEAVAGE DIOXYGENASE 4 gene converts flower colour from white to yellow in Brassica species. New Phytol 206:1513–1526. https://doi.org/10.1111/nph.13335

    CAS  Article  PubMed  Google Scholar 

  61. Zhou X, Welsch R, Yang Y et al (2015) Arabidopsis OR proteins are the major posttranscriptional regulators of phytoene synthase in controlling carotenoid biosynthesis. Proc Natl Acad Sci USA 112:3558–3563. https://doi.org/10.1073/pnas.1420831112

    CAS  Article  PubMed  PubMed Central  Google Scholar 

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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.

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  1. Chihiro Oda-Yamamizo

    Present address: Biological Resources and Post-Harvest Division, Japan International Research Center for Agricultural Sciences, Tsukuba, Ibaraki, 305-8686, Japan

Affiliations

  1. Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, 305-8572, Japan

    Kenta Watanabe & Michiyuki Ono

  2. Institute of Vegetable and Floriculture Science, National Agriculture and Food Research Organization, 2-1 Fujimoto, Tsukuba, Ibaraki, 305-0852, Japan

    Chihiro Oda-Yamamizo & Akemi Ohmiya

  3. Faculty of Life and Environmental Sciences, Gene Research Center, Tsukuba Plant Innovation Research Center (T-PIRC), University of Tsukuba, Tsukuba, Ibaraki, 305-8572, Japan

    Kimiyo Sage-Ono & Michiyuki Ono

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  1. Kenta Watanabe
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Correspondence to Michiyuki Ono.

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Watanabe, K., Oda-Yamamizo, C., Sage-Ono, K. et al. Alteration of flower colour in Ipomoea nil through CRISPR/Cas9-mediated mutagenesis of carotenoid cleavage dioxygenase 4 . Transgenic Res 27, 25–38 (2018). https://doi.org/10.1007/s11248-017-0051-0

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Keywords

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