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The B-ring hydroxylation pattern of anthocyanins can be determined through activity of the flavonoid 3′-hydroxylase on leucoanthocyanidins

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Abstract

Main conclusion

In contrast to current knowledge, the B -ring hydroxylation pattern of anthocyanins can be determined by the hydroxylation of leucoanthocyanidins in the 3′ position by flavonoid 3’-hydroxylase.

Abstract

The cytochrome P450-dependent monooxygenases flavonoid 3′-hydroxylase (F3′H) and flavonoid 3′,5′-hydroxylase (F3′5′H) are key flavonoid enzymes that introduce B-ring hydroxyl groups in positions 3′ or 3′ and 5′, respectively. The degree of B-ring hydroxylation is the major determinant of the hue of anthocyanin pigments. Numerous studies have shown that F3′H and F3′5′H may act on more than one type of anthocyanin precursor in addition to other flavonoids, but it has been unclear whether the anthocyanin precursor of the leucoanthocyanidin type can be hydroxylated as well. We have investigated this in vivo using feeding experiments and in vitro by studies with recombinant F3′H. Feeding leucoanthocyanidins to petal tissue with active hydroxylases resulted in anthocyanidins with increased B-ring hydroxylation relative to the fed leucoanthocyanidin, indicating the presence of 3′-hydroxylating activity (in Petunia and Eustoma grandiflorum Grise.) and 3′,5′-hydroxylating activity (in E. grandiflorum Grise.). Tetcyclacis, a specific inhibitor of cytochrome P450-dependent enzymes, abolished this activity, excluding involvement of unspecific hydroxylases. While some hydroxylation could be a consequence of reverse catalysis by dihydroflavonol 4-reductase (DFR) providing an alternative substrate, hydroxylating activity was still present in fed petals of a DFR deficient petunia line. In vitro conversion rates and kinetic data for dLPG (a stable leucoanthocyanidin substrate) were comparable to those for other flavonoids for nine of ten recombinant flavonoid hydroxylases from various taxa. dLPG was a poor substrate for only the recombinant Fragaria F3′Hs. Thus, the B-ring hydroxylation pattern of anthocyanins can be determined at all precursor levels in the pathway.

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Abbreviations

CY:

Cyanidin

DFR:

Dihydroflavonol 4-reductase

dLCy:

5-deoxyleucocyanidin

dLPG:

5-deoxyleucopelargonidin

DP:

Delphinidin

F3´H:

Flavonoid 3´-hydroxylase

F3´5H:

Flavonoid 3´5´-hydroxylase

HBR:

Heidi Blue Rim

LCY:

Leucocyanidin

LPG:

Leucopelargonidin

PG:

Pelargonidin

TET:

Tetcyclacis

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Acknowledgments

This work was supported by the Austrian Science Fund FWF [Project P24331-B16]. We thank C. Seitz from the former Chair of Floriculture Crops and Horticultural Plant Breeding, Technical University of Munich, Freising for providing some of the F3′H cDNA clones, and W. Rademacher, BASF, for the gift of tecyclacis. We thank C. Martin for the suggestion of including a DFR mutant in the feeding experiments.

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Authors and Affiliations

  1. New Zealand Institute for Plant and Food Research Limited, Private Bag 11 600, Palmerston North, New Zealand

    Kathy Schwinn & Kevin Davies

  2. Institut für Verfahrenstechnik, Umwelttechnik und Technische Biowissenschaften, Technische Universität Wien, Getreidemarkt 9/166, 1060, Vienna, Austria

    Silvija Miosic, Jana Thill, Tek Prasad Gotame, Karl Stich & Heidi Halbwirth

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  1. Kathy Schwinn
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  2. Silvija Miosic
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  3. Kevin Davies
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  6. Karl Stich
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  7. Heidi Halbwirth
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Correspondence to Heidi Halbwirth.

Additional information

Special topic: Anthocyanins. Guest editor: Stefan Martens.

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Schwinn, K., Miosic, S., Davies, K. et al. The B-ring hydroxylation pattern of anthocyanins can be determined through activity of the flavonoid 3′-hydroxylase on leucoanthocyanidins. Planta 240, 1003–1010 (2014). https://doi.org/10.1007/s00425-014-2166-3

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  • DOI: https://doi.org/10.1007/s00425-014-2166-3

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