Plant Molecular Biology Reporter

, Volume 31, Issue 1, pp 64–74 | Cite as

Identification and Functional Characterization of the Promoter of a Phytoene Synthase from Sweet Orange (Citrus sinensis Osbeck)

  • Wenfang Zeng
  • Ming Huang
  • Xiaopei Wang
  • Charles Ampomah-Dwamena
  • Qiang Xu
  • Xiuxin Deng
Original Paper


The phytoene synthase (PSY) gene product is involved in the carotenoid biosynthesis pathway. In this study, the promoter of the sweet orange gene CsPSY of sweet orange ‘Cara Cara’ (Citrus sinensis Osbeck [Cs]) was isolated and characterized. Its sequence included a number of regulatory elements predicted to be responsive to light and other environmental cues. The 926-bp region upstream of CsPSY was fused to the β-glucuronidase (GUS) reporter gene, and introduced as a transgene into Arabidopsis thaliana. The pattern of GUS expression in the transgenic plants showed that the CsPSY promoter drove expression in the young seedlings, the leaves, the roots and in parts of the flower, in a developmentally regulated fashion. A promoter deletion analysis revealed that the region −479 to −306 nt positively regulated expression, the region −306 to −93 nt was associated with negative regulation of expression and the region −46 to +21 nt maintained basal promoter activity. In the presence of sucrose, gene expression was regulated via a cis-acting promoter element(s). Two highly repetitive sequences (Sp1 and A-box), located in the region from −479 to −306 nt, may act as light-responsive elements regulating CsPSY transcription during photomorphogenesis.


Carotenoid Citrus sinensis GUS activity Phytoene synthase Promoter 



Abscisic acid








High temperature


3-Indoleacetic acid


6-Furfuryl aminopurine


Low temperature


Phytoene synthase


Cardiovascular disease


Age-related macular degeneration


Phytochrome-interaction factors


Rapid amplification of cDNA ends


Transcription start site






Salicylic acid



The research was financially supported by the National Basic research program of China (973 program; No. 2011CB100601), and National Natural Science Foundation of China (Nos. 30921002 and 30830078).


  1. Alquezar B, Rodrigo MJ, Zacarías L (2008) Regulation of carotenoid biosynthesis during fruit maturation in the red-fleshed orange mutant Cara Cara. Phytochemistry 69:1997–2007PubMedCrossRefGoogle Scholar
  2. Bartley GE, Scolnik PA (1995) Plant carotenoids: pigments for photoprotection, visual attraction, and human health. Plant Cell 7:1027PubMedGoogle Scholar
  3. Bouvier F, Hugueney P, D’Harlingue A, Kuntz M, Camara B (1994) Xanthophyll biosynthesis in chromoplasts: isolation and molecular cloning of an enzyme catalyzing the conversion of 5,6-epoxycarotenoid into ketocarotenoid. Plant J 6:45–54PubMedCrossRefGoogle Scholar
  4. Bramley PM (2002) Regulation of carotenoid formation during tomato fruit ripening and development. J Exp Bot 53:2107–2113PubMedCrossRefGoogle Scholar
  5. Cazzonelli CI, Pogson BJ (2010) Source to sink: regulation of carotenoid biosynthesis in plants. Trends Plant Sci 15:266–274PubMedCrossRefGoogle Scholar
  6. Chen C, Costa MGC, Yu Q, Moore GA, Gmitter FG (2010) Identification of novel members in sweet orange carotenoid biosynthesis gene families. Tree Genet Genomes 6:905–914CrossRefGoogle Scholar
  7. Cheng YJ, Guo WW, Yi HL, Pang XM, Deng X (2003) An efficient protocol for genomic DNA extraction from Citrus species. Plant Mol Biol Rep 21:177–178CrossRefGoogle Scholar
  8. Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743PubMedCrossRefGoogle Scholar
  9. Corona V, Aracri B, Kosturkova G, Bartley GE, Pitto L, Giorgetti L, Scolnik PA, Giuliano G (1996) Regulation of a carotenoid biosynthesis gene promoter during plant development. Plant J 9:505–512PubMedCrossRefGoogle Scholar
  10. DellaPenna D, Pogson BJ (2006) Vitamin synthesis in plants: tocopherols and carotenoids. Annu Rev Plant Biol 57:711–738PubMedCrossRefGoogle Scholar
  11. Fanciullino AL, Dhuique-Mayer C, Luro F, Morillon R, Ollitrault P (2007) Carotenoid biosynthetic pathway in the citrus genus: number of copies and phylogenetic diversity of seven genes. J Agric Food Chem 55:7405–7417PubMedCrossRefGoogle Scholar
  12. Fraser PD, Bramley PM (2004) The biosynthesis and nutritional uses of carotenoids. Prog Lipid Res 43:228–265PubMedCrossRefGoogle Scholar
  13. Fraser PD, Enfissi EMA, Halket JM, Truesdale MR, Yu D, Gerrish C, Bramley PM (2007) Manipulation of phytoene levels in tomato fruit: effects on isoprenoids, plastids, and intermediary metabolism. Plant Cell 19:3194–3211PubMedCrossRefGoogle Scholar
  14. Giuliano G, Bartley GE, Scolnik PA (1993) Regulation of carotenoid biosynthesis during tomato development. Plant Cell 5:379–387PubMedGoogle Scholar
  15. Giuliano G, Tavazza R, Diretto G, Beyer P, Taylor MA (2008) Metabolic engineering of carotenoid biosynthesis in plants. Trends Biotechnol 26:139–145PubMedCrossRefGoogle Scholar
  16. Gomez-Roldan V, Fermas S, Brewer PB, Puech-Pagès V, Dun EA, Pillot JP, Letisse F, Matusova R, Danoun S, Portais JC (2008) Strigolactone inhibition of shoot branching. Nature 455:189–194PubMedCrossRefGoogle Scholar
  17. He X, Tian J, Yang L, Huang Y, Zhao B, Zhou C, Ge R, Shen Y, Huang Z (2011) Overexpressing a glycogen synthase Kinase gene from wheat, TaGSK1, enhances salt tolerance in transgenic Arabidopsis. Plant Mol Biol Rep. doi: 10.1007/s11105-011-0390-6
  18. Higo K, Ugawa Y, Iwamoto M, Korenaga T (1999) Plant cis-acting regulatory DNA elements (PLACE) database: 1999. Nucleic Acids Res 27:297–300PubMedCrossRefGoogle Scholar
  19. Howitt CA, Cavanagh CR, Bowerman AF, Cazzonelli C, Rampling L, Mimica JL, Pogson BJ (2009) Alternative splicing, activation of cryptic exons and amino acid substitutions in carotenoid biosynthetic genes are associated with lutein accumulation in wheat endosperm. Funct Integr Genomic 9:363–376CrossRefGoogle Scholar
  20. Ikoma Y, Komatsu A, Kita M, Ogawa K, Omura M, Yano M, Moriguchi T (2001) Expression of a phytoene synthase gene and characteristic carotenoid accumulation during citrus fruit development. Physiol Plant 111:232–238CrossRefGoogle Scholar
  21. Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901PubMedGoogle Scholar
  22. Kato M, Ikoma Y, Matsumoto H, Sugiura M, Hyodo H, Yano M (2004) Accumulation of carotenoids and expression of carotenoid biosynthetic genes during maturation in citrus fruit. Plant Physiol 134:824–837PubMedCrossRefGoogle Scholar
  23. Kim OR, Cho MC, Kim BD, Huh JH (2010) A splicing mutation in the gene encoding phytoene synthase causes orange coloration in Habanero pepper fruits. Mol Cells 30:569–574PubMedCrossRefGoogle Scholar
  24. Lescot M, Déhais P, Thijs G, Marchal K, Moreau Y, Van de Peer Y, Rouzé P, Rombauts S (2002) PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res 30:325–327PubMedCrossRefGoogle Scholar
  25. Li F, Vallabhaneni R, Wurtzel ET (2008a) PSY3, a new member of the phytoene synthase gene family conserved in the poaceae and regulator of abiotic stress-induced root carotenogenesis. Plant Physiol 146:1333–1345PubMedCrossRefGoogle Scholar
  26. Li F, Vallabhaneni R, Yu J, Rocheford T, Wurtzel ET (2008b) The maize phytoene synthase gene family: overlapping roles for carotenogenesis in endosperm, photomorphogenesis, and thermal stress tolerance. Plant Physiol 147:1334–1346PubMedCrossRefGoogle Scholar
  27. Li F, Tsfadia O, Wurtzel ET (2009) The phytoene synthase gene family in the grasses: subfunctionalization provides tissue-specific control of carotenogenesis. Plant Signal Behav 4:208PubMedCrossRefGoogle Scholar
  28. Lintig J, Welsch R, Bonk M, Giuliano G, Batschauer A, Kleinig H (1997) Light-dependent regulation of carotenoid biosynthesis occurs at the level of phytoene synthase expression and is mediated by phytochrome in Sinapis alba and Arabidopsis thaliana seedlings. Plant J 12:625–634CrossRefGoogle Scholar
  29. Liu Q, Xu J, Liu Y, Zhao X, Deng X, Guo L, Gu J (2007) A novel bud mutation that confers abnormal patterns of lycopene accumulation in sweet orange fruit (Citrus sinensis L. Osbeck). J Exp Bot 58:4161–4171PubMedCrossRefGoogle Scholar
  30. Lu S, Li L (2008) Carotenoid metabolism: biosynthesis, regulation, and beyond. J Integr Plant Biol 50:778–785PubMedCrossRefGoogle Scholar
  31. Martínez-García JF, Huq E, Quail PH (2000) Direct targeting of light signals to a promoter element-bound transcription factor. Science 288:859PubMedCrossRefGoogle Scholar
  32. Nambara E, Marion-Poll A (2005) Abscisic acid biosynthesis and catabolism. Annu Rev Plant Biol 56:165–185PubMedCrossRefGoogle Scholar
  33. Rodríguez-Villalón A, Gas E, Rodríguez-Concepción M (2009) Phytoene synthase activity controls the biosynthesis of carotenoids and the supply of their metabolic precursors in dark-grown Arabidopsis seedlings. Plant J 60:424–435PubMedCrossRefGoogle Scholar
  34. Tao N, Hu Z, Liu Q, Xu J, Cheng Y, Guo L, Guo W, Deng X (2007) Expression of phytoene synthase gene (Psy) is enhanced during fruit ripening of Cara Cara navel orange (Citrus sinensis Osbeck). Plant Cell Rep 26:837–843PubMedCrossRefGoogle Scholar
  35. Télef N, Stammitti-Bert L, Mortain-Bertrand A, Maucourt M, Carde JP, Rolin D, Gallusci P (2006) Sucrose deficiency delays lycopene accumulation in tomato fruit pericarp discs. Plant Mol Biol 62:453–469PubMedCrossRefGoogle Scholar
  36. Toledo-Ortiz G, Huq E, Rodríguez-Concepción M (2010) Direct regulation of phytoene synthase gene expression and carotenoid biosynthesis by phytochrome-interacting factors. Proc Natl Acad Sci USA 107:11626Google Scholar
  37. Umehara M, Hanada A, Yoshida S, Akiyama K, Arite T, Takeda-Kamiya N, Magome H, Kamiya Y, Shirasu K, Yoneyama K (2008) Inhibition of shoot branching by new terpenoid plant hormones. Nature 455:195–200PubMedCrossRefGoogle Scholar
  38. Von Gromoff ED, Schroda M, Oster U, Beck CF (2006) Identification of a plastid response element that acts as an enhancer within the Chlamydomonas HSP70A promoter. Nucleic Acids Res 34:4767–4779CrossRefGoogle Scholar
  39. Welsch R, Beyer P, Hugueney P, Kleinig H, von Lintig J (2000) Regulation and activation of phytoene synthase, a key enzyme in carotenoid biosynthesis, during photomorphogenesis. Planta 211:846–854PubMedCrossRefGoogle Scholar
  40. Welsch R, Medina J, Giuliano G, Beyer P, Von Lintig J (2003) Structural and functional characterization of the phytoene synthase promoter from Arabidopsis thaliana. Planta 216:523–534PubMedGoogle Scholar
  41. Welsch R, Maass D, Voegel T, DellaPenna D, Beyer P (2007) Transcription factor RAP2. 2 and its interacting partner SINAT2: stable elements in the carotenogenesis of Arabidopsis leaves. Plant Physiol 145:1073–1085PubMedCrossRefGoogle Scholar
  42. Welsch R, Wüst F, Bär C, Al-Babili S, Beyer P (2008) A third phytoene synthase is devoted to abiotic stress-induced abscisic acid formation in rice and defines functional diversification of phytoene synthase genes. Plant Physiol 147:367–380PubMedCrossRefGoogle Scholar
  43. Welsch R, Arango J, Bär C, Salazar B, Al-Babili S, Beltrán J, Chavarriaga P, Ceballos H, Tohme J, Beyer P (2010) Provitamin A accumulation in cassava (Manihot esculenta) roots driven by a single nucleotide polymorphism in a phytoene synthase gene. Plant Cell 22:3348–3356PubMedCrossRefGoogle Scholar
  44. Yin H, Zhao X, Bai X, Du Y (2010) Molecular cloning and characterization of a Brassica napus L. MAP kinase involved in oligochitosan-induced defense signaling Plant Mol Biol Rep 28:292–301Google Scholar
  45. Zhang J, Tao N, Xu Q, Zhou W, Cao H, Xu J, Deng X (2009) Functional characterization of Citrus PSY gene in Hong Kong kumquat (Fortunella hindsii Swingle). Plant Cell Rep 28:1737–1746PubMedCrossRefGoogle Scholar
  46. Zhang F, Liu X, Zuo K, Zhang J, Sun X, Tang K (2011) Molecular cloning and characterization of a novel Gossypium barbadense L. RAD-like gene. Plant Mol Biol Rep 29:324–333CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Wenfang Zeng
    • 1
  • Ming Huang
    • 1
  • Xiaopei Wang
    • 1
  • Charles Ampomah-Dwamena
    • 2
  • Qiang Xu
    • 1
  • Xiuxin Deng
    • 1
  1. 1.Key Laboratory of Horticultural Plant Biology National Key Laboratory of Crop Genetic ImprovementHuazhong Agricultural UniversityWuhanChina
  2. 2.New Zealand Institute for Plant and Food Research LimitedAucklandNew Zealand

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