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Front. Plant Sci. | doi: 10.3389/fpls.2018.00149

Great cause – small effect: Undeclared genetically engineered orange petunias harbour an inefficient dihydroflavonol 4-reductase

Christian Haselmair-Gosch1, Silvija Miosic1,  Daria Nitarska1, Barbara L. Roth1, Benjamin Walliser1,  Renate Paltram1, Rares C. Lucaciu2, Lukas Eidenberger1,  Thomas Rattei2,  Klaus Olbricht3,  Karl Stich1 and  Heidi Halbwirth1*
  • 1Institute of Chemical, Environmental and Biological Engineering, Vienna University of Technology, Austria
  • 2Department of Microbiology and Ecosystem Science, University of Vienna, Austria
  • 3Thaer-Institute of Agricultural and Horticultural Sciences, Humboldt-Universität zu Berlin, Germany

A recall campaign for commercial, orange flowering petunia varieties in spring 2017 caused economic losses worldwide. The orange varieties were identified as undeclared genetically engineered (GE)-plants, harbouring a maize dihydroflavonol 4-reductase (DFR, A1), which was used in former scientific transgenic breeding attempts to enable formation of orange pelargonidin derivatives from the precursor dihydrokaempferol (DHK) in petunia. How and when the A1 cDNA entered the commercial breeding process is unclear. We provide an in-depth analysis of three orange petunia varieties, released by breeders from three countries, with respect to their transgenic construct, transcriptomes, anthocyanin composition, and flavonoid metabolism at the level of selected enzymes and genes. The two possible sources of the A1 cDNA in the undeclared GE-petunia can be discriminated by PCR. A special version of the A1 gene, the A1 type 2 allele, is present, which includes, at the 3’-end, an additional 144 bp segment from the non-viral transposable Cin4-1 sequence, which does not add any functional advantage with respect to DFR activity. This unequivocally points at the first scientific GE-petunia from the 1980s as the A1 source, which is further underpinned e.g. by the presence of specific restriction sites, parts of the untranslated sequences, and the same arrangement of the building blocks of the transformation plasmid used. Surprisingly, however, the GE-petunia cannot be distinguished from native red and blue varieties by their ability to convert DHK in common in vitro enzyme assays, as DHK is an inadequate substrate for both the petunia and maize DFR. Recombinant maize DFR underpins the low DHK acceptance, and, thus, the strikingly limited suitability of the A1 protein for a transgenic approach for breeding pelargonidin-based flower colour. The effect of single amino acid mutations on the substrate specificity of DFRs is demonstrated. Expression of the A1 gene is generally lower than the petunia DFR expression despite being under the control of the strong, constitutive p35S promoter. We show that a rare constellation in flavonoid metabolism - absence or strongly reduced activity of both flavonol synthase and B-ring hydroxylating enzymes - allows pelargonidin formation in the presence of DFRs with poor DHK acceptance.

Keywords: DFR substrate specificity, genetically engineered petunia, pelargonidin based flower colour, A1-type 2 from Zea mays, Flavonoid metabolism

Received: 11 Sep 2017; Accepted: 29 Jan 2018.

Edited by:

Mariana Mondragón-Palomino, University of Regensburg, Germany

Reviewed by:

Michal Oren-Shamir, Agricultural Research Organization (Israel), Israel
Xiang Gao, Key Laboratory of Molecular Epigenetics of MOE, Northeast Normal University, Changchun, China, China
Teemu H. Teeri, University of Helsinki, Finland  

Copyright: © 2018 Haselmair-Gosch, Miosic, Nitarska, Roth, Walliser, Paltram, Lucaciu, Eidenberger, Rattei, Olbricht, Stich and Halbwirth. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence: Prof. Heidi Halbwirth, Vienna University of Technology, Institute of Chemical, Environmental and Biological Engineering, Getreidemarkt 9/1665, Vienna, Austria, hhalb@mail.zserv.tuwien.ac.at