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Metabolomic and genetic analyses of flavonol synthesis in Arabidopsis thaliana support the in vivo involvement of leucoanthocyanidin dioxygenase

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Abstract

Flavonol synthase (FLS) (EC-number 1.14.11.23), the enzyme that catalyses the conversion of flavonols into dihydroflavonols, is part of the flavonoid biosynthesis pathway. In Arabidopsis thaliana, this activity is thought to be encoded by several loci. In addition to the FLAVONOL SYNTHASE1 (FLS1) locus that has been confirmed by enzyme activity assays, loci displaying similarity of the deduced amino acid sequences to FLS1 have been identified. We studied the putative A. thaliana FLS gene family using a combination of genetic and metabolite analysis approaches. Although several of the FLS gene family members are expressed, only FLS1 appeared to influence flavonoid biosynthesis. Seedlings of an A. thaliana fls1 null mutant (fls1-2) show enhanced anthocyanin levels, drastic reduction in flavonol glycoside content and concomitant accumulation of glycosylated forms of dihydroflavonols, the substrate of the FLS reaction. By using a leucoanthocyanidin dioxygenase (ldox) fls1-2 double mutant, we present evidence that the remaining flavonol glycosides found in the fls1-2 mutant are synthesized in planta by the FLS-like side activity of the LDOX enzyme.

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Abbreviations

ANR:

Anthocyanidin reductase

CHS:

Chalcone synthase

CHI:

Chalcone isomerase

DFR:

Dihydroflavonol 4-reductase

DHF:

Dihydroflavonol

DPBA:

Diphenylboric acid 2-aminoethylester

EBG:

Early biosynthetic gene

ESI:

Electro spray ionization

F3H:

Flavanone 3β-hydroxylase

F3′H:

Flavonoid 3′-hydroxylase

FLS:

Flavonol synthase

GT:

Glycosyltransferase

GUS:

β-Glucuronidase

GUS′:

Standardized specific β-glucuronidase activity

HPLC:

High performance liquid chromatography

HPTLC:

High performance thin layer chromatography

LBG:

Late biosynthetic gene

LC:

Liquid chromatography

LUC:

Luciferase

LDOX:

Leucoanthocyanidin dioxygenase

MS:

Mass spectrometry

MU:

4-Methylumbelliferone

PDA:

Photo-diode array

QTOF:

Quadrupole-time-of-flight

3GT:

Anthocyanidin 3-O-glycosyltransferase

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Acknowledgements

We thank Melanie Kuhlmann for excellent technical assistance and the Sequencing Core Facility at the Bielefeld University for doing an excellent job in determining DNA sequences. This project was funded in part by the EU project FLAVO (FOOD-CT-2004-513960) and the GABI program of the Bundesministerium für Bildung und Forschung (BMBF)/Projektträger Jülich (PTJ).

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Authors and Affiliations

  1. Chair of Genome Research, Bielefeld University, 33594, Bielefeld, Germany

    Ralf Stracke, Lutz Bartelniewoehner, Hirofumi Ishihara, Martin Sagasser & Bernd Weisshaar

  2. Plant Research International, 6700 AA, Wageningen, The Netherlands

    Ric C. H. De Vos

  3. Centre for Biosystems Genomics, 6700 AB, Wageningen, The Netherlands

    Ric C. H. De Vos

  4. Institute of Pharmaceutical Biology, Philipps University, 35037, Marburg, Germany

    Stefan Martens

Authors
  1. Ralf Stracke
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  2. Ric C. H. De Vos
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  3. Lutz Bartelniewoehner
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  4. Hirofumi Ishihara
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  5. Martin Sagasser
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  6. Stefan Martens
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  7. Bernd Weisshaar
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Corresponding author

Correspondence to Ralf Stracke.

Additional information

Nucleotide sequence database accession numbers: GenBank accession EU287457 and EU287459.

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425_2008_841_MOESM1_ESM.pdf

Fig. S1 Multiple alignment of FLS polypeptides. Result from ClustalW2 multiple sequence alignment done at EMBL-EBI (http://www.ebi.ac.uk/Tools/clustalw2/) using default parameters (Chenna et al. 2003). Protein sequence data were obtained from GenBank (http://www.ncbi.nlm.nih.gov/): Petroselinum crispum PcFLS (AAP57395), Petunia x hybrida PhFLS (CAA80264), Solanum tuberosum StFLS (CAA63092), Eustoma grandiflorum EgFLS (AAF64168), Citrus unshiu CuFLS (BAA36554), Malus domestica MdFLS (AAX89401) and Camellia sinensis CsFLS (ABM88786). Amino acid residues, identical in all polypeptides are black boxed. Amino acids found at least in all polypeptides with proven FLS activity are marked in grey. The three regions of high similarity found in 2 oxoglutarate-dependent- and related enzymes (Britsch et al. 1993; Wellmann et al. 2002) are underlined. Amino acid residues, involved in iron binding (Myllylä et al. 1992; Britsch et al. 1993; Lukacin and Britsch 1997; Clifton et al. 2006) are marked with squares (■). The HxD...H motif, defining amino acids involved in 2 oxoglutarate binding (Britsch et al. 1993; Roach et al. 1995; Lukacin and Britsch 1997), is marked with circles (●). A diamond (♦) marks conserved amino acids of unknown function (Lukacin and Britsch 1997; Wellmann et al. 2002). At the bottom of the sequence alignment, the following symbols denote the degree of conservation in each column: (*) identical in all sequences, (:) conserved substitutions, (.) semi-conserved substitutions (PDF 337 kb)

425_2008_841_MOESM2_ESM.xls

Table S1 Summary of LC-PDA-QTOF-MS derived data showing differential metabolites in seedlings of analysed genotypes. Results of three independent biological samples are given. RT, retention time [min]; observed mass [m/z], averaged accurate mass ([M H] ), as mass-to-charge ratio; calc mass, calculated accurate mass; D mass [ppm], deviation between the averages of observed and calculated accurate masses, in parts per million; UV/Vis, absorbance maxima in the UV/Vis range (nd, not detectable absorbance); MS fragments, MS/MS fragments obtained through increased collision energy on indicated parent mass; mol formular, molecular formula of the metabolite; metabolite name, common name of putatively identified metabolite; mean, data represent means of results from the three given separate experiments; SD, standard deviation (XLS 68 kb)

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Stracke, R., De Vos, R.C.H., Bartelniewoehner, L. et al. Metabolomic and genetic analyses of flavonol synthesis in Arabidopsis thaliana support the in vivo involvement of leucoanthocyanidin dioxygenase. Planta 229, 427–445 (2009). https://doi.org/10.1007/s00425-008-0841-y

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