Skip to main content
SpringerLink
Log in

cDNA cloning and characterization of UDP-glucose: anthocyanidin 3-O-glucosyltransferase in Freesia hybrida

  • Original Paper
  • Published:
Plant Cell Reports Aims and scope Submit manuscript

Abstract

The enzyme that catalyzes the formation of the first stable anthocyanin in the biosynthesis of natural compounds is UDP-glucose: anthocyanidin 3-O-glucosyltransferase (UF3GT). A cDNA clone (Fh3GT1) encoding UF3GT was isolated from Freesia hybrida. Phylogenetic tree analysis indicated that Fh3GT1 was a novel member of glycosyltransferase, which was classified into monocot subgroups. Semi-quantitative RT-PCR analysis detected transcripts of Fh3GT1 in different organs of F. hybrida and in petals of Freesia cultivars of different colors, and the expression level reached the maximum at the fully opened stage of petals. Characterization of the enzymatic assays indicated that Fh3GT1 had a role in anthocyanin glycoside biosyntheses in vitro. To elucidate the function of Fh3GT1, RNA interference vector (pART-Fh3GT1i) was constructed, and introduced into Petunia grandiflora by Agrobacterium-mediated transformation. Integration of the Fh3GT1 in petunia genome was confirmed by PCR and Southern blotting. SqRT-PCR revealed that the endogenous Ph3GT1 mRNA expression levels decreased in transgenic lines compared with the wild-type. The content of total anthocyanin pigments also decreased with the reduction of mRNA transcript levels, and the transgenic petunia plants had significant changes on their flower colors. In summary, this work identified a UF3GT gene from Freesia hybrida and demonstrated a method to modify plant flower color by redirecting the anthocyanin biosynthesis.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

Abbreviations

Bp:

Base pair

HPLC:

High-performance liquid chromatography

PSPG:

Plant secondary product glycosyltransferase

PTGS:

Post-transcriptional gene silencing

SqRT-PCR:

Semi-quantitative reverse transcription polymerase chain reaction

UF3GT:

UDP-glucose: anthocyanidin 3-O-glucosyltransferase

References

  • Almeida JR, D’Amico E, Preuss A, Carbone F, de Vos CH, Deiml B, Mourgues F, Perrotta G, Fischer TC, Bovy AG, Martens S, Rosati C (2007) Characterization of major enzymes and genes involved in flavonoid and proanthocyanidin biosynthesis during fruit development in strawberry (Fragaria × ananassa). Arch Biochem Biophys 465:61–71

    Article  PubMed  CAS  Google Scholar 

  • Arabidopsis GI (2000) Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408:796–815

    Article  Google Scholar 

  • Eamens A, Wang MB, Smith NA, Waterhouse PM (2008) RNA silencing in plants: yesterday, today, and tomorrow. Plant Physiol 147:456–468

    Article  PubMed  CAS  Google Scholar 

  • Ford CM, Boss PK, Hoj PB (1998) Cloning and characterization of Vitis vinifera UDP-glucose: flavonoid 3-O-glucosyltransferase, a homologue of the enzyme encoded by the maize Bronze-1 locus that may primarily serve to glucosylate anthocyanidins in vivo. J Biol Chem 273:9224–9233

    Article  PubMed  CAS  Google Scholar 

  • Forkmann G, Martens S (2001) Metabolic engineering and applications of flavonoids. Curr Opin Biotechnol 12:155–160

    Article  PubMed  CAS  Google Scholar 

  • Fukuchi-Mizutani M, Okuhara H, Fukui Y, Nakao M, Katsumoto Y, Yonekura-Sakakibara K, Kusumi T, Hase T, Tanaka Y (2003) Biochemical and molecular characterization of a novel UDP-glucose: anthocyanin 3′-O-glucosyltransferase, a key enzyme for blue anthocyanin biosynthesis, from gentian. Plant Physiol 132:1652–1663

    Article  PubMed  CAS  Google Scholar 

  • Gachon CM, Langlois-Meurinne M, Saindrenan P (2005) Plant secondary metabolism glycosyltransferases: the emerging functional analysis. Trends Plant Sci 10:542–549

    Article  PubMed  CAS  Google Scholar 

  • Gong Z, Yamazaki M, Sugiyama M, Tanaka Y, Saito K (1997) Cloning and molecular analysis of structural genes involved in anthocyanin biosynthesis and expressed in a forma-specific manner in Perilla frutescens. Plant Mol Biol 35:915–927

    Article  PubMed  CAS  Google Scholar 

  • Goto T, Kondo T, Tamura H, Imagawa H, Lino A, Takeda T (1982) Structure of gentiodelphin, an acylated anthocyanin isolated from Gentiana Makinoi, that is stable in dilute aqueous solution. Tetrahedron Lett 23:3695–3698

    Article  CAS  Google Scholar 

  • Griesser M, Hoffmann T, Bellido ML, Rosati C, Fink B, Kurtzer R, Aharoni A, Munoz-Blañco J, Schwab W (2008) Redirection of flavonoid biosynthesis through the downregulation of an anthocyanidin glucosyltransferase in ripening strawberry fruit. Plant Physiol 146:1528–1539

    Article  PubMed  CAS  Google Scholar 

  • Grotewold E, Davis KM (2008) Trafficking and sequestration of anthocyanins. Nat Prod Commun 3:1251–1258

    CAS  Google Scholar 

  • Harborne JB, Williams CA (2000) Advances in flavonoid research since 1992. Phytochemistry 55:481–504

    Article  PubMed  CAS  Google Scholar 

  • Hodges DM, Delong JM, Forney CF, Prange RK (1999) Improving the thiobarbituric acid-reactive-substances assay for estimating lipid peroxidation in plant tissues containing anthocyanin and other interfering compounds. Planta 207:604–611

    Article  CAS  Google Scholar 

  • Holsters M, de Waele D, Depicker A, Messens E, van Montagu M, Schell J (1978) Transfection and transformation of Agrobacterium tumefaciens. Mol Gen Genet 163:181–187

    Article  PubMed  CAS  Google Scholar 

  • Holton TA, Cornish EC (1995) Genetics and biochemistry of anthocyanin biosynthesis. Plant Cell 7:1071–1083

    Article  PubMed  CAS  Google Scholar 

  • Hondo T, Yoshida K, Nakagawa A, Kawai T, Tamura H, Goto T (1992) Structural basis of blue-color development in flower petals from Commelina communis. Nature 358:515–518

    Article  Google Scholar 

  • Huang JQ, Wei ZM, An HL, Zhu YX (2001) Agrobacterium tumefaciens-mediated transformation of rice with the spider insecticidal gene conferring resistance to leaf folder and striped stem borer. Cell Res 11:149–155

    PubMed  CAS  Google Scholar 

  • Kaminaga Y, Sahin FP, Mizukami H (2004) Molecular cloning and characterization of a glucosyltransferase catalyzing glucosylation of curcumin in cultured Catharanthus roseus cells. FEBS Lett 567:197–202

    Article  PubMed  CAS  Google Scholar 

  • Kapitonov D, Yu RK (1999) Conserved domains of glycosyltransferases. Glycobiology 9:961–978

    Article  PubMed  CAS  Google Scholar 

  • Katsumoto Y, Fukuchi-Mizutani M, Fukui Y et al (2007) Engineering of the rose flavonoid biosynthetic pathway successfully generated blue-hued flowers accumulating delphinidin. Plant Cell Physiol 48:1589–1600

    Article  PubMed  CAS  Google Scholar 

  • Kerschen A, Napoli CA, Jorgensen RA, Müller AE (2004) Effectiveness of RNA interference in transgenic plants. FEBS Lett 566:223–228

    Article  PubMed  CAS  Google Scholar 

  • Kim JH, Kim BG, Ko JH, Lee Y, Hur HG, Lim Y, Ahn JH (2006) Molecular cloning, expression, and characterization of a flavonoid glycosyltransferase from Arabidopsis thaliana. Plant Sci 170:897–903

    Article  CAS  Google Scholar 

  • Klein AO, Hagen CW (1961) Anthocyanin production in detached petals of impatiens balsamina L. Plant Physiol 36:1–9

    Article  PubMed  CAS  Google Scholar 

  • Ko JH, Kima BG, Kim JH, Kim H, Lim CE, Lim J, Lee C, Lim Y, Ahn JH (2008) Four glucosyltransferases from rice: cDNA cloning, expression, and characterization. J Plant Physiol 165:435–444

    Article  PubMed  CAS  Google Scholar 

  • Kubo A, Arai Y, Nagashima S, Yoshikawa T (2004) Alteration of sugar donor specificities of plant glycosyltransferases by a single point mutation. Arch Biochem Biophys 429:198–203

    Article  PubMed  CAS  Google Scholar 

  • Li Y, Baldauf S, Lim EK, Bowles DJ (2001) Phylogenetic analysis of the UDP-glycosyltransferase multigene family of Arabidopsis thaliana. J Biol chem 276:4338–4343

    Article  PubMed  CAS  Google Scholar 

  • Liang HR, Vuorela P, Vuorela H, Hiltunen R (1997) Isolation and immunomodulating effect of flavonol glycosides from Epimedium hunanense. Planta Med 63:316–319

    Article  PubMed  CAS  Google Scholar 

  • Mackenzie PI, Owens IS, Burchell B et al (1997) The UDP glycosyltransferase gene superfamily: recommended nomenclature update based on evolutionary divergence. Pharmacogenetics 7:255–269

    Article  PubMed  CAS  Google Scholar 

  • Marrs KA, Alfenito MR, Lloyd AM, Walbot V (1995) A glutathione S-transferase involved in vacuolar transfer encoded by the maize gene Bronze-2. Nature 375:397–400

    Article  PubMed  CAS  Google Scholar 

  • Martin C, Gerats T (1992) The control of flower coloration. In: Jordan B (ed) Molecular Biology of Flowering. CIB International, Wallingford, UK, pp 219–255

  • Martin C, Prescott A, Mackay S, Bartlett J, Vrijlandt E (1991) Control of anthocyanin biosynthesis in flowers of Antirrhinum majus. Plant J 1:37–49

    Article  PubMed  CAS  Google Scholar 

  • Modolo LV, Blount JW, Achnine L et al (2007) A functional genomics approach to (iso)flavonoid glycosylation in the model legume Medicago truncatula. Plant Mol Biol 64:499–518

    Article  PubMed  CAS  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  • Offen W, Martinez-Fleites C, Yang M et al (2006) Structure of a flavonoid glucosyltransferase reveals the basis of plant natural product modification. EMBO J 25:1396–1405

    Article  PubMed  CAS  Google Scholar 

  • Ossowski S, Schwab R, Weigel D (2008) Gene silencing in plants using artificial microRNAs and other small RNAs. Plant J 53:674–690

    Article  PubMed  CAS  Google Scholar 

  • Proudfoot NJ, Brownlee GG (1976) 3′ non-coding region sequences in eukaryotic messenger RNA. Nature 263:211–214

    Article  PubMed  CAS  Google Scholar 

  • Ross J, Li Y, Lim EK, Bowles DJ (2001) Higher plant glycosyltransferases. Genome Biol 2(2): reviews 3004 http://genomebiology.com/2001/2/2/reviews/3004

  • Saito K, Yamazaki M (2002) Biochemistry and molecular biology of the late-stage of biosynthesis of anthocyanin: lessons from Perilla frutescens as a model plant. New Phytol 155:9–23

    Article  CAS  Google Scholar 

  • Sharp PA (2001) RNA interference-2001. Genes Dev 15:485–490

    Article  PubMed  CAS  Google Scholar 

  • Smith NA, Singh SP, Wang MB, Stoutjesdijk PA, Green AG, Waterhouse PM (2000) Total silencing by intron-spliced hairpin RNAs. Nature 407:319–320

    Article  PubMed  CAS  Google Scholar 

  • Tanaka Y, Yonekura K, Fukuchi-Mizutani M, Fukui Y, Fujiwara H, Ashikari T, Kusumi T (1996) Molecular and biochemical characterization of three anthocyanin synthetic enzymes from Gentiana triflora. Plant Cell Physiol 37:711–716

    PubMed  CAS  Google Scholar 

  • Tanaka Y, Katsumoto Y, Brugliera F, Mason J (2005) Genetic engineering in floriculture. Plant Cell Tiss Org 80:1–24

    Article  CAS  Google Scholar 

  • 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 Acids Res 22:4673–4680

    Article  PubMed  CAS  Google Scholar 

  • Tsuda S, Fukui Y, Nakamura N et al (2004) Flower color modification of Petunia hybrida commercial varieties by metabolic engineering. Plant Biotechnol 21:377–386

    Article  CAS  Google Scholar 

  • Vaucheret H, Béclin C, Fagard M (2001) Post-transcriptional gene silencing in plants. J Cell Sci 114:3083–3091

    PubMed  CAS  Google Scholar 

  • Vogt T, Jones P (2000) Glycosyltransferase in plant natural product synthesis: characterization of a supergene family. Trends Plant Sci 5:380–386

    Article  PubMed  CAS  Google Scholar 

  • Wesley SV, Helliwell CA, Smith NA et al (2001) Construct design for efficient, effective and high-throughput gene silencing in plants. Plant J 27:581–590

    Article  PubMed  CAS  Google Scholar 

  • Winkel-Shirley B (2001) Flavonoid biosynthesis. A colorful model for genetics, biochemistry, cell biology, and biotechnology. Plant Physiol 126:485–493

    Article  PubMed  CAS  Google Scholar 

  • Wise RP, Rohde W, Salamini F (1990) Nucleotide sequence of the Bronze-1 homologous gene from Hordeum vulgare. Plant Mol Biol 14:277–279

    Article  PubMed  CAS  Google Scholar 

  • Yamazaki M, Yamagishi E, Gong Z et al (2002) Two flavonoid glucosyltransferases from Petunia hybrida: molecular cloning, biochemical properties and developmentally regulated expression. Plant Mol Biol 48:401–411

    Article  PubMed  CAS  Google Scholar 

  • Yonekura-Sakakibara K, Nakayama T, Yamazaki M, Saito K (2009) Modification and stabilization of anthocyanins. In: Gould K, Davies K, Winefield C (eds) Anthocyanins biosynthesis functions and applications. Springer, New York, pp 169–190

    Google Scholar 

  • Yoshida K, Toyama Y, Kameda K, Kondo T (2000) Contribution of each caffeoyl residue of the pigment molecule of gentiodelphin to blue color development. Phytochemistry 54:85–92

    Article  PubMed  CAS  Google Scholar 

  • Yoshihara N, Imayama T, Fukuchi-Mizutanic M, Okuhara H, Tanaka Y, Ino I, Yabuya T (2005) cDNA cloning and characterization of UDP-glucose: anthocyanidin 3-O-glucosyltransferase in Iris hollandica. Plant Sci 169:496–501

    Article  CAS  Google Scholar 

  • Yu O, Jez JM (2008) Nature’s assembly line: biosynthesis of simple phenylpropanoids and polyketides. Plant J 54:750–762

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This research was supported by the National Natural Science Foundation of the People’s Republic of China (grant# 30970280) and Jilin provincial Government of the People’s Republic of China (grant# 20085030). We also want to thank Professor Lixia Liu for providing the plant expression vector pKANNIBAL and pART27 and Agrobacterium tumefaciens strain EHA105, and Professors Chuanbin Mao and Xingzhi Wang for helpful comments on the manuscript.

Author information

Authors and Affiliations

  1. Key Laboratory of Molecular Epigenetics of Ministry of Education, Institute of Genetics and Cytology, Northeast Normal University, Changchun, 130024, People’s Republic of China

    Xin Sui, Xiang Gao, Man Ao, Qinmei Wang, Dan Yang, Meng Wang, Yang Fu & Li Wang

Authors
  1. Xin Sui
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xiang Gao
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Man Ao
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Qinmei Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Dan Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Meng Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Yang Fu
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Li Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Li Wang.

Additional information

Communicated by B. Li.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sui, X., Gao, X., Ao, M. et al. cDNA cloning and characterization of UDP-glucose: anthocyanidin 3-O-glucosyltransferase in Freesia hybrida . Plant Cell Rep 30, 1209–1218 (2011). https://doi.org/10.1007/s00299-011-1029-7

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00299-011-1029-7

Keywords

Search

Navigation