Plant Molecular Biology Reporter

, Volume 36, Issue 4, pp 632–642 | Cite as

Regulation of Carotenoid Pigmentation in Corollas of Petunia

  • Sanae KishimotoEmail author
  • Chihiro Oda-Yamamizo
  • Akemi Ohmiya
Original Paper


Petunia (Petunia hybrida) is an important ornamental plant, with corolla colors ranging widely from pink to red to purple, owing mainly to anthocyanins. Although there are no bright-yellow-flowered cultivars, some pale-yellow-flowered cultivars accumulate a small amount of carotenoids. To find a key regulatory step that controls carotenoid content in petunia corollas, we compared the expression of carotenoid metabolism genes and carotenoid composition in corollas of white-flowered and pale-yellow-flowered cultivars. Pale yellow corollas tended to have higher expression of biosynthesis genes. The most prominent result was the complete lack of carotenoid cleavage dioxygenase 4a (CCD4a) transcripts in pale yellow corollas. We found two insertions, one in the putative promoter region and the other in the coding region, of the genomic CCD4a sequence of a pale-yellow-flowered cultivar relative to that of a white-flowered cultivar. We consider this the main reason for the lack of CCD4a transcripts. The results suggest that pale yellow corollas have higher carotenoid biosynthesis activity and lower catabolism activity than white corollas. We propose that carotenoid content in petunia corollas is determined by the balance of the degradation and biosynthesis of carotenoids.


Carotenoid Carotenoid cleavage dioxygenase 4 (CCD4) Flower color Gene expression Petunia (Petunia hybrida



Carpet Buttercream


Carotenoid cleavage dioxygenase 1


Carotenoid cleavage dioxygenase 4


California Girl


β-Ring hydroxylase


Cytochrome P450-type β-ring hydroxylase


Cytochrome P450-type ε-ring hydroxylase


Carotenoid isomerase


Carpet White


1-Deoxy-d-xylulose 5-phosphate synthase


Eagle White


Geranylgeranyl pyrophosphate synthase


Isopentenyl diphosphate isomerase


Lycopene β-ring cyclase


Lycopene ε-ring cyclase


Mambo White


9-cis-Epoxycarotenoid dioxygenase


Phytoene desaturase


Prism Sunshine


Phytoene synthase


Reverse transcription quantitative real-time PCR


ζ-Carotene desaturase


Zeaxanthin epoxygenase


15-cis-ζ-Carotene isomerase


Funding Information

This work was supported by JSPS KAKENHI Grant Number JP25450051.

Supplementary material

11105_2018_1107_MOESM1_ESM.pptx (1.4 mb)
Supplementary Fig. S1 Carotenoid analysis in corollas of petunia cultivars. Saponified carotenoid extracts from 0.4 g f.w. of limbs and tubes were analyzed by HPLC. Peaks: 1, (all-E)-neoxanthin; 2, (all-E)-violaxanthin; 3, (9′Z)-neoxanthin; 4, (9Z)-violaxanthin; 5, unknown carotenoid; 6, lutein; 7, antheraxanthin and zeaxanthin; 8, β-carotene. Designations of cultivars are as in Fig. 2. (PPTX 1449 kb)
11105_2018_1107_MOESM2_ESM.pptx (348 kb)
Supplementary Fig. S2 Carotenoid analysis in leaves of CG and EW. Saponified carotenoid extracts from 0.4 g f.w. of leaves were analyzed by HPLC. Peaks: 2, (all-E)-violaxanthin; 3, (9′Z)-neoxanthin; 4, (9Z)-violaxanthin; 5, unknown carotenoid; 6, lutein; 7, antheraxanthin and zeaxanthin; 8, β-carotene. Designations of cultivars are as in Fig. 2. (PPTX 347 kb)
11105_2018_1107_MOESM3_ESM.pptx (44 kb)
Supplementary Fig. S3 Alignment of deduced amino acid sequences of CCD1 and CCD4 of various plant species. AtCCD1, from Arabidopsis thaliana (acc. no. NM_116217); AtCCD4 (AtNCED4), A. thaliana (NM_118036); CcCCD4a, Citrus clementina (DQ309330); CcCCD4b, C. clementina (DQ309331); CmCCD4a, Chrysanthemum morifolium (AB247158); CmCCD4b, C. morifolium (AB247160); CsCCD4a, Crocus sativus (EU523662); CsCCD4b, C. sativus (EU523663); PhCCD1, Petunia hybrida (AY576003); PhCCD4a, P. hybrida (LC335775); PhCCD4b, P. hybrida (LC335772). Numbers at branch points indicate bootstrap values (1000 replicates). (PPTX 43 kb)
11105_2018_1107_MOESM4_ESM.pptx (4.5 mb)
Supplementary Fig. S4 Genomic sequences of CCD4a presumed promoter and coding regions of California Girl (CG) and Eagle White (EW). Red arrows, genomic PCR primers for insertion detection; green arrows, primers for CCD4a RT-qPCR. Blue bar, insertion; pink bar, deletion in CG relative the sequence from EW. (PPTX 4613 kb)
11105_2018_1107_MOESM5_ESM.xlsx (27 kb)
ESM 1 (XLSX 27 kb)


  1. Ariizumi T, Kishimoto S, Kakami R, Maoka T, Hirakawa H, Suzuki Y, Ozeki Y, Shirasawa K, Bernillon S, Okabe Y, Asamizu E, Rothan C, Ohmiya A, Ezura H (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 79:453–465CrossRefPubMedGoogle Scholar
  2. Brazda V, Kolomaznik J, Lysek J, Haronikova L, Coufal J, Stastny J (2016) Palindrome analyser - a new web-based server for predicting and evaluating inverted repeats in nucleotide sequences. Biochem Biophys Res Commun 478:1739–1745CrossRefPubMedGoogle Scholar
  3. Britton G (1995) UV/visible spectrometry. In: Britton G, Liaaen-Jensen S, Pfander H (eds) Carotenoids, vol 1B. Birkhauser Verlag, Basel, pp 13–62Google Scholar
  4. Brown MS, Goldstein JL (1993) Protein prenylation: mad bet for Rab. Nature 366:14–15CrossRefPubMedGoogle Scholar
  5. Camara B (1993) Plant phytoene synthase complex: component enzymes, immunology, and biogenesis. Methods Enzymol 214:352–365CrossRefGoogle Scholar
  6. Campbell R, Ducreux LJ, Morris WL, Morris JA, Suttle JC, Ramsay G, Bryan GJ, Hedley PE, Taylor MA (2010) The metabolic and developmental roles of carotenoid cleavage dioxygenase4 from potato. Plant Physiol 154:656–664CrossRefPubMedPubMedCentralGoogle Scholar
  7. Cazzonelli CI, Pogson BJ (2010) Source to sink: regulation of carotenoid biosynthesis in plants. Trends Plant Sci 15:266–274CrossRefPubMedGoogle Scholar
  8. Falchi R, Vendramin E, Zanon L, Scalabrin S, Cipriani G, Verde I, Vizzotto G, Morgante M (2013) Three distinct mutational mechanisms acting on a single gene underpin the origin of yellow flesh in peach. Plant J 76:175–187PubMedPubMedCentralGoogle Scholar
  9. Felsenstein J (1997) An alternating least squares approach to inferring phylogenies from pairwise distances. Syst Biol 46:101–111CrossRefPubMedGoogle Scholar
  10. Fraser PD, Schuch W, Bramley PM (2000) Phytoene synthase from tomato (Lycopersicon esculentum) chloroplasts–partial purification and biochemical properties. Planta 211:361–369CrossRefPubMedGoogle Scholar
  11. Galpaz N, Ronen G, Khalfa Z, Zamir D, Hirschberg J (2006) A chromoplast-specific carotenoid biosynthesis pathway is revealed by cloning of the tomato white-flower locus. Plant Cell 18:1947–1960CrossRefPubMedPubMedCentralGoogle Scholar
  12. Gonzalez-Jorge S, Ha SH, Magallanes-Lundback M, Gilliland LU, Zhou A, Lipka AE, Nguyen YN, Angelovici R, Lin H, Cepela J, Little H, Buell CR, Gore MA, Dellapenna D (2013) CAROTENOID CLEAVAGE DIOXYGENASE4 is a negative regulator of β-carotene content in Arabidopsis seeds. Plant Cell 25:4812–4826CrossRefPubMedPubMedCentralGoogle Scholar
  13. Goodwin TW (1988) Carotenoids in higher plants. In: The biochemistry of the carotenoids vol 1 Plants. Chapman and Hall, London and New York, pp 143–188CrossRefGoogle Scholar
  14. Hai MTL, Masuda J, Miyajima I, Thien NQ, Mojtahedi N, Hiramatsu M, Kim J-H, Okubo H (2012) Involvement of carotenoid cleavage dioxygenase 4 gene in tepal color change in Lilium brownii var. colchesteri. J Japan Soc Hortic Sci 81:366–373CrossRefGoogle Scholar
  15. Hernandez-Garcia CM, Finer JJ (2014) Identification and validation of promoters and cis-acting regulatory elements. Plant Sci 217–218:109–119CrossRefPubMedGoogle Scholar
  16. Hirschberg J (2001) Carotenoid biosynthesis in flowering plants. Curr Opin Plant Biol 4:210–218CrossRefPubMedGoogle Scholar
  17. Ilg A, Yu Q, Schaub P, Beyer P, Al-Babili S (2010) Overexpression of the rice carotenoid cleavage dioxygenase 1 gene in golden rice endosperm suggests apocarotenoids as substrates in planta. Planta 232:691–699CrossRefPubMedGoogle Scholar
  18. Kishimoto S, Ohmiya A (2006) Regulation of carotenoid biosynthesis in petals and leaves of chrysanthemum (Chrysanthemum morifolium Ramat.). Physiol Plant 128:437–447CrossRefGoogle Scholar
  19. Kishimoto S, Maoka T, Nakayama M, Ohmiya A (2004) Carotenoid composition in petals of chrysanthemum (Dendranthema grandiflorum (Ramat.) Kitamura). Phytochemistry 65:2781–2787CrossRefPubMedGoogle Scholar
  20. Kishimoto S, Sumitomo K, Yagi M, Nakayama M, Ohmiya A (2007) Three routes to orange petal colour via carotenoid component in 9 Compositae species. J Japan Soc Hortic Sci 76:250–257CrossRefGoogle Scholar
  21. Liu Y-G, Whittier RF (1995) Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking. Genomics 25:674–681CrossRefPubMedGoogle Scholar
  22. Liu H, Kishimoto S, Yamamizo C, Fukuta N, Ohmiya A (2013) Carotenoid accumulations and carotenogenic gene expressions in the petals of Eustoma grandiflorum. Plant Breed 132:417–422CrossRefGoogle Scholar
  23. Meier S, Tzfadia O, Vallabhaneni R, Gehring C, Wurtzel ET (2011) A transcriptional analysis of carotenoid, chlorophyll and plastidial isoprenoid biosynthesis genes during development and osmotic stress responses in Arabidopsis thaliana. BMC Syst Biol 5:77CrossRefPubMedPubMedCentralGoogle Scholar
  24. Moehs CP, Tian L, Osteryoung KW, DellaPenna D (2001) Analysis of carotenoid biosynthetic gene expression during marigold petal development. Plant Mol Biol 45:281–293CrossRefPubMedGoogle Scholar
  25. Murakami Y, Fukui Y, Watanabe H, Kokubun H, Toya Y, Ando T (2003) Distribution of carotenoids in the flower of non-yellow commercial petunia. J Hortic Sci Biotechnol 78:127–130CrossRefGoogle Scholar
  26. Muszyński S (1964) A survey of anthocyanidins in Petunia. Physiol Plant 17:975–979CrossRefGoogle Scholar
  27. Nielsen KM, Bloor SJ (1997) Analysis and developmental profile of carotenoid pigments in petals of three yellow petunia cultivars. Sci Hortic 71:257–266CrossRefGoogle Scholar
  28. Ohmiya A (2009) Carotenoid cleavage dioxygenases and their apocarotenoid products in plants. Plant Biotechnol 26:351–358CrossRefGoogle Scholar
  29. Ohmiya A (2011) Diversity of carotenoid composition in flower petals. JARQ 45:163–171CrossRefGoogle Scholar
  30. Ohmiya A (2013) Qualitative and quantitative control of carotenoid accumulation in flower petals. Sci Hortic 163(2013):10–19CrossRefGoogle Scholar
  31. Ohmiya A, Kishimoto S, Aida R, Yoshioka S, Sumitomo K (2006) Carotenoid cleavage dioxygenase (CmCCD4a) contributes to white color formation in chrysanthemum petals. Plant Physiol 142:1193–1201CrossRefPubMedPubMedCentralGoogle Scholar
  32. Okada K, Saito T, Nakagawa T, Kawamukai M, Kamiya Y (2000) Five geranylgeranyl diphosphate synthases expressed in different organs are localized into three subcellular compartments in Arabidopsis. Plant Physiol 122:1045–1056CrossRefPubMedPubMedCentralGoogle Scholar
  33. Page RDM (1996) TREEVIEW: an application to display phylogenetic trees on personal computers. Comput Appl Biosci 12:357–358PubMedGoogle Scholar
  34. Robert B, Horton P, Pascal AA, Ruban AV (2004) Insights into the molecular dynamics of plant light-harvesting proteins in vivo. Trends Plant Sci 9:385–390CrossRefPubMedGoogle Scholar
  35. Ruban AV, Berera R, Ilioaia C, van Stokkum IHM, Kennis ITM, Pascal AA, van Amerongen H, Robert B, Horton P, van Grondelle R (2007) Identification of a mechanism of photoprotective energy dissipation in higher plants. Nature 450:575–578CrossRefPubMedGoogle Scholar
  36. Rubio-Moraga A, Ahrazem O, Rambla JL, Granell A, Gómez-Gómez L (2013) Crocins with high levels of sugar conjugation contribute to the yellow colours of early-spring flowering crocus tepals. PLoS One 8:e71946CrossRefPubMedPubMedCentralGoogle Scholar
  37. Santangelo TJ, Artsimovitch I (2011) Termination and antitermination: RNA polymerase runs a stop sign. Nat Rev Microbiol 9:319–329CrossRefPubMedPubMedCentralGoogle Scholar
  38. Schwartz SH, Tan BC, Gage DA, Zeevaart JAD, McCarty DR (1997) Specific oxidative cleavage of carotenoids by VP14 of maize. Science 276:1872–1874CrossRefPubMedGoogle Scholar
  39. Simkin AJ, Schwartz SH, Auldridge M, Taylor MG, Klee HJ (2004a) The tomato carotenoid cleavage dioxygenase 1 genes contribute to the formation of the flavor volatiles β-ionone, pseudoionone, and geranylacetone. Plant J 40:882–892CrossRefPubMedGoogle Scholar
  40. Simkin AJ, Underwood BA, Auldridge M, Loucas HM, Shibuya K, Schmelz E, Clark DG, Klee HJ (2004b) Circadian regulation of the PhCCD1 carotenoid cleavage dioxygenase controls emission of β-ionone, a fragrance volatile of petunia flowers. Plant Physiol 136:3504–3514CrossRefPubMedPubMedCentralGoogle Scholar
  41. Tornielli G, Koes R, Quattrocchio F (2008) The genetics of flower color. In: Gerats T, Strommer J (eds) Petunia: evolutionary, developmental and physiological genetics. Springer, New York, pp 325–342Google Scholar
  42. Vitte C, Fustier M-A, Alix K, Tenaillon MI (2014) The bright side of transposons in crop evolution. Brief Funct Genomics 13:276–295CrossRefPubMedGoogle Scholar
  43. Yamagishi M, Kishimoto S, Nakayama M (2010) Carotenoid composition and changes in expression of carotenogenic genes in tepals of Asiatic hybrid lily. Plant Breed 129:100–107CrossRefGoogle Scholar
  44. Yamamizo C, Kishimoto S, Ohmiya A (2010) Carotenoid composition and carotenogenic gene expression during Ipomoea petal development. J Exp Bot 61:709–719CrossRefPubMedGoogle Scholar
  45. Yoshioka S, Aida R, Yamamizo C, Shibata M, Ohmiya A (2012) The carotenoid cleavage dioxygenase 4 (CmCCD4a) gene family encodes a key regulator of petal color mutation in chrysanthemum. Euphytica 184:377–387CrossRefGoogle Scholar
  46. Zhang B, Liu C, Wang Y, Yao X, Wang F, Wu J, King GJ, Liu K (2015) Disruption of a CAROTENOID CLEAVAGE DIOXYGENASE 4 gene converts flower colour from white to yellow in Brassica species. New Phytol 206:1513–1526CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Institute of Vegetable and Floriculture ScienceNational Agriculture and Food Research Organization (NARO)TsukubaJapan

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