Tree Genetics & Genomes

, Volume 6, Issue 1, pp 101–112 | Cite as

Cloning and molecular characterization of R2R3-MYB and bHLH-MYC transcription factors from Citrus sinensis

  • Antonietta CultroneEmail author
  • Paola S. Cotroneo
  • Giuseppe Reforgiato Recupero
Original Paper


Members of the MYB and MYC family regulate the biosynthesis of phenylpropanoids in several plant species. Two sequences, called CsMYB8 and CsMYC2, were identified from Citrus sinensis, and both the cDNA and the genomic clones were isolated and characterized from the flesh of common and blood oranges. Analysis by real-time polymerase chain reaction showed that the expression pattern of CsMYC2 is generally higher in rind than in flesh and in blood oranges than in common ones. In contrast, no significant difference in expression was observed for CsMYB8. The expression pattern of the structural genes chalcone synthase, anthocyanidin synthase, and UDP-glucose–flavonoid 3-O-glucosyltransferase, which code for three enzymes involved in the anthocyanin biosynthetic pathway, was also analyzed and correlated with CsMYC2, in flesh, rind, and leaf of the common and blood oranges, and in leaf of Citrus limon cultivars (characterized by anthocyanin absence or variable content). Surprisingly, CsMYC2 is highly expressed in the leaf and expression is correlated with UFGT expression in this organ. These results suggest that CsMYC2 is involved in the regulation of the flavonoid biosynthetic pathway in Citrus.


Citrus Flavonoid Regulation 



Chalcone synthase


Anthocyanidin synthase


UDP-glucose–flavonoid 3-O-glucosyltransferase


Basic helix-loop-helix


Transcription factor



We thank Paul Bailey (John Innes Institute, Norwich) and Concetta Licciardello (C.R.A.-ACM Centro di Ricerca per l’Agrumicoltura e le Colture Mediterranee, Acireale) who constructed the phylogenetic tree. We also thank Christine Drevet (Universite Paris-XI) and Willem Van De Velde (CNRS Gif sur Yvette) for their helpful advice. This work was supported by FLORA project (Sixth Framework Programmes EEC; Contract no. 007130).


  1. Azuma A, Kobayashi S, Mitani N, Shiraishi M, Yamada M, Ueno T, Kono A, Yakushiji H, Koshita Y (2008) Genomic and genetic analysis of Myb-related genes that regulate anthocyanin biosynthesis in grape berry skin. Theor Appl Genet 117:1009–1019CrossRefPubMedGoogle Scholar
  2. Bagchi D, Bagchi M, Stohs SJ, Das DK, Ray SD, Kuszynsky CA, Joshi SS, Pruess HG (2000) Free radicals and grape seed proanthocyanidin extract: importance in human health and disease prevention. Toxycology 148:187–197CrossRefGoogle Scholar
  3. Bagchi D, Sen CK, Bagchi M, Atalay M (2004) Anti-angiogenic, antioxidant, and anti-carcinogenic properties of a novel anthocyanin-rich berry extract formula anti-angiogenic, antioxidant, and anti-carcinogenic properties of a novel anthocyanin-rich berry extract formula. Biochemistry (Mosc) 69:75–80 ReviewCrossRefGoogle Scholar
  4. Beld M, Martin C, Huits H, Stultje AR, Gerats AGM (1989) Flavonoid synthesis in Petunia hybrida: partial characterization of dihydroflavonol 4-reductase genes. Plant Mol Biol 13:491–502CrossRefPubMedGoogle Scholar
  5. Broun P (2004) Transcription factors as tools for metabolic engineering in plants. Curr Opin Plant Biol 7:202–209CrossRefPubMedGoogle Scholar
  6. Castellarin SD, Matthews MA, Di Gaspero G, Gambetta GA (2007) Water deficits accelerate ripening and induce changes in gene expression regulating flavonoid biosynthesis in grape berries. Planta 227:101–112CrossRefPubMedGoogle Scholar
  7. Christie P, Alfenito MR, Walbot V (1994) Impact of low-temperature stress on general phenilpropanoid and anthocyanin pathways: enhancement of transcript abundance and anthocyanin pigmentation in maize seedlings. Planta 194:541–549CrossRefGoogle Scholar
  8. Coffman JA, Kirchhamer CV, Harrington MG, Davidson EH (1997) SpMyb functions as an intramodular repressor to regulate spatial expression of Cylla in sea urchin embryos. Development 124:4717–4727PubMedGoogle Scholar
  9. Cotroneo PS, Russo MP, Ciuni M, Reforgiato Recupero G, Lo Piero AR (2006) Quantitative real time reverse transcriptase-PCR profiling of anthocyanin biosynthetic genes during orange fruit ripening. J Amer Soc Hort Sci 13:537–543Google Scholar
  10. Davies KM, Schwinn KE (2003) Transcriptional regulation of secondary metabolism. Funct Plant Biol 30:913–925CrossRefGoogle Scholar
  11. Dooner HK, Robbins TP, Jorgensen RA (1991) Genetic and developmental control of anthocyanin biosynthesis. Annu Rev Genet 25:173–199CrossRefPubMedGoogle Scholar
  12. Dubos C, Le Gourrierec J, Baudry A, Huep G, Lanet E, Debeaujon I, Routaboul J-M, Alboresi A, Weisshaar B, Lepiniec L (2008) MYBL2 is a new regulator of flavonoid biosynthesis in Arabidopsis thaliana. Plant J 55:940–953CrossRefPubMedGoogle Scholar
  13. Espley RV, Hellens RP, Putterill J, Stevenson DE, Kutty-Amma S, Allan AC (2007) Red coloration in apple fruit is due to the activity of the MYB transcription factor, MdMYB10. Plant J 49:414–427CrossRefPubMedGoogle Scholar
  14. Fan C, Purugganan MD, Thomas DT, Wiegmann BM, Qiu-Yun Xiang J (2004) Heterogeneous evolution of the MYC-like anthocyanin regulatory gene and its phylogenetic utility in Cornus L. (Cornaceae). Mol Phylog Evol 33:580–594CrossRefGoogle Scholar
  15. Felsenstein J (1989) PHYLIP-Phylogeny inference package (version 3.2). Cladistics 5:164–166Google Scholar
  16. Ferré-D’Amaré AR, Prendergast GC, Ziff EB, Burley SK (1993) Recognition by Max of its cognate DNA through a dimeric b/HLH/Z domain. Nature 363:38–45CrossRefPubMedGoogle Scholar
  17. Ferré-D’Amaré AR, Pognonec P, Roeder RG, Burley SK (1994) Structure and function of the b/HLH/Z domain of USF. EMBO J 13:180–189PubMedGoogle Scholar
  18. Gould KS (2004) Nature’s Swiss army knife: the diverse protective roles of anthocyanins in leaves. J Biomed Biotechnol 2004:314–320CrossRefPubMedGoogle Scholar
  19. Grotewold E (2006) The genetics and biochemistry of floral pigments. Annu Rev Plant Biol 57:761–780CrossRefPubMedGoogle Scholar
  20. Heim MA, Jakoby M, Werber M, Martin C, Weisshaar B, Bailey PC (2003) The basic helix-loop-helix transcription factor family in plants: a genome-wide study of protein structure and functional diversity. Mol Biol Evol 20:735–747CrossRefPubMedGoogle Scholar
  21. Holton TA, Cornish EC (1995) Genetics and biochemistry of anthocyanin biosynthesis. Plant Cell 7:1071–1083CrossRefPubMedGoogle Scholar
  22. Jaakola L, Maatta K, Pirttila AM, Torronen R, Karenlampi S, Hohtola A (2002) Expression of genes involved in anthocyanin biosynthesis in relation to anthocyanin, proanthocyanidin, and flavonol levels during bilberry fruit development. Plant Physiol 130:729–739CrossRefPubMedGoogle Scholar
  23. Jones DT, Taylor WR, Thornton JM (1992) The rapid generation of mutation data matrices from protein sequences. Comp Appl Biosci (CABIOS) 8:275–282Google Scholar
  24. Ju Z, Liu C, Yuan Y (1995) Activities of chalcone synthase and UDPGal:flavonoid-3-o-glycosyltransferase in relation to anthocyanin synthesis in apple. Sci Hortic 63:175–185CrossRefGoogle Scholar
  25. Kanner J, Frankel EN, Grant R, German JB, Kinsella JE (1994) Natural antioxidants in grapes and wines. J Agric Food Chem 42:64–69CrossRefGoogle Scholar
  26. Kawaii S, Tomono Y, Katase E, Ogawa K, Yano M, Koizumi M, Ito C, Furukawa H (2000) Quantitative study of flavonoids in leaves of Citrus plants. J Agric Food Chem 48:3865–3871CrossRefPubMedGoogle Scholar
  27. Kim SH, Lee JR, Hong ST, Yoo YK, An G, Kim SR (2003) Molecular cloning and analysis of anthocyanin biosynthesis genes preferentially expressed in apple skin. Plant Sci 165:403–413CrossRefGoogle Scholar
  28. Kobayashy S, Ishimaru M, Hiraoka K, Honda C (2002) Myb-related genes of the Kyoho grape (Vitis labruscana) regulate anthocyanin biosynthesis. Planta 215:924–933CrossRefGoogle Scholar
  29. Koes R, Quattrocchio F, Mol J (1994) The flavonoid biosynthetic pathway in plants: Function and evolution. BioEssays 16:123-132CrossRefGoogle Scholar
  30. Koes R, Verweij W, Quattrocchio F (2005) Flavonoids: a colorful model for the regulation and evolution of biochemical pathways. Trends Plant Sci 10:236–242CrossRefPubMedGoogle Scholar
  31. Lila MA (2004) Anthocyanins and human health: an in vitro investigative approach. J Biomed Biotechnol 2004:306–313CrossRefPubMedGoogle Scholar
  32. Lo Piero AR, Puglisi I, Rapisarda P, Petrone G (2005) Anthocyanins accumulation and related gene expression in red orange fruit induced by low temperature storage. J Agric Food Chem 53:9083–9088CrossRefPubMedGoogle Scholar
  33. Lockington RA, Sealy-Lewis HM, Scazzocchio C, Davies RW (1985) Cloning and characterization of the ethanol utilization in Aspergillus nidulans. Gene 33:137–149CrossRefPubMedGoogle Scholar
  34. Mahe A, Grisvard J, Dron M (1992) Fungal and plant specific gene markers to follow the bean anthracnose infection process and normalize a bean chitinase mRNA induction. Mol Plant–Microbe Inter 5:242–248CrossRefGoogle Scholar
  35. Martin C, Paz-Ares X (1997) MYB transcription factors in plant. TIG 13:67–73CrossRefPubMedGoogle Scholar
  36. Martin C, Prescott A, Mackay S, Bartlett J, Vrijlandt E (1991) Control of anthocyanin biosynthesis in flowers of Antirrhinum majus. Plant J 1:37–49CrossRefPubMedGoogle Scholar
  37. Mol J, Grotewold E, Koes R (1998) How genes paint flowers and seeds. Trends Plant Sci 3:212–217CrossRefGoogle Scholar
  38. Offen W, Martinez-Fleites C, Yang M, Kiat-Lim E, Davis BG, Tarling CA, Ford CM, Bowles DJ, Davies GJ (2006) Structure of a flavonoid glucosyltransferase reveals the basis for plant natural product modification. EMBO J 25:1396–1405CrossRefPubMedGoogle Scholar
  39. Park J-S, Kim J-B, Cho K-J, Cheon C-I, Sung M-K, Choung M-G, Roh K-H (2008) Arabidopsis R2R3-MYB transcription factor AtMYB60 functions as a transcriptional repressor of anthocyanin biosynthesis in lettuce (Lactuca sativa). Plant Cell Rep 27:985–994CrossRefPubMedGoogle Scholar
  40. Pattanaik S, Xie CH, Yuan L (2008) The interaction domains of the plant Myc-like bHLH transcription factors can regulate the transactivation strength. Planta 227:707–715CrossRefPubMedGoogle Scholar
  41. Paz-Ares J, Wienand U, Peterson PA, Saedler H (1986) Molecular cloning of the c locus of Zea Mays: locus regulating the anthocyanin pathway. Embo J 5:829–833PubMedGoogle Scholar
  42. Paz-Ares J, Ghosal D, Wienand U, Peterson PA, Saedler H (1987) The regulatory c1 locus of Zea mays encodes a protein with homology to myb proto-oncogene products and with structural similarities to transcriptional activators. Embo J 6:3553–3558PubMedGoogle Scholar
  43. Quattrocchio F, Wing JF, Leppen HTC, Mol JNM, Koes RE (1993) Regulatory genes controlling anthocyanin pigmentation are functionally conserved among plant species and have distinct sets f target genes. Plant Cell 5:1497–1512CrossRefPubMedGoogle Scholar
  44. Quattrocchio F, Wing J, van der Woude K, Souer E, de Vetten N, Mol J, Koes R (1999) Molecular analysis of the anthocyanin2 gene of petunia and its role in the evolution of flower color. Plant Cell 11:1433–44CrossRefPubMedGoogle Scholar
  45. Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425PubMedGoogle Scholar
  46. Sambrook J, Russell DW (2001) Molecular cloning a laboratory manual, 3rd edn. Cold Spring Harbour, New YorkGoogle Scholar
  47. Shimada S, Inoue YT, Masaaki S (2005) Anthocyanidin synthase in non-anthocyanin-producing Caryophyllales species. Plant J 44:950–959CrossRefPubMedGoogle Scholar
  48. Shvarts M, Borochov A, Weiss D (1997) Low temperature enhances petunia flower pigmentation and induces chalcone synthase gene expression. Physiol Plant 99:67–72CrossRefGoogle Scholar
  49. Solfanelli C, Poggi A, Loreti E, Alpi A, Perata P (2006) Sucrose-specific induction of the anthocyanin biosynthetic pathway in Arabidopsis. Plant Physiol 140:637–646CrossRefPubMedGoogle Scholar
  50. Specht CA, Dirusso CC, Novotny CP, Ullrich RC (1982) A method for extracting high molecular weight deoxyribonucleic acid from fungi. Anal Biochem 119:158–163CrossRefPubMedGoogle Scholar
  51. Sun Y, Hrazdina G (1991) Isolation and characterization of a UDPglucose:flavonol 03-glucosyltransferase from illuminated red cabbage (Brassica oleracea cv Red Danish) seedlings. Plant Physiol 95:570–576CrossRefPubMedGoogle Scholar
  52. Takos AM, Jaffé FW, Jacob SR, Bogs J, Robinson SP, Walker AR (2006) Light-induced expression of a MYB gene regulates anthocyanin biosynthesis in red apples. Plant Physiol 142:1216–1232CrossRefPubMedGoogle Scholar
  53. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acid Res 22:4673–4680CrossRefPubMedGoogle Scholar
  54. Tsukaya H, Ohshima T, Naito S, Chino M, Komeda Y (1991) Sugar-dependent expression of the CHS-A gene for chalcone synthase from petunia in transgenic Arabidopsis. Plant Physiol 97:1414–1421CrossRefPubMedGoogle Scholar
  55. Vinson JA, Jang J, Dabbagh YA, Serry MM, Cai S (1995) Plant polyphenols exhibit lipoprotein-bond antioxidant activity using an in vitro oxidation model for heart disease. J Agric Food Chem 43:2798–2799CrossRefGoogle Scholar
  56. Walker AR, Lee E, Bogs J, MacDavid DA, Thomas MR, Robinson SP (2007) White grapes arose through the mutation of two similar and adjacent regulatory genes. Plant J 49:772–785CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Antonietta Cultrone
    • 1
    • 2
    • 3
    Email author
  • Paola S. Cotroneo
    • 1
  • Giuseppe Reforgiato Recupero
    • 1
  1. 1.C.R.A-ACM Centro di Ricerca per l’Agrumicoltura e le Colture MediterraneeAcirealeItaly
  2. 2.Parco Scientifico e Tecnologico della SiciliaCataniaItaly
  3. 3.INRA UEPSDJouy-en-Josas cedexFrance

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