Identification of a Hydroxycinnamoyl-CoA Double Bond Reductase (HDR) Affirms Multiple Pathways for Dihydrochalcone Formation in Apple

Authors

• Susan Schröpfer, Julius Kühn-Institute (JKI) - Federal Research Centre for Cultivated Plants, Institute for Breeding Research on Fruit Crops, Dresden, Germany.
• Christian Haselmair-Gosch, Institute of Chemical, Environmental and Bioscience Engineering, Technische Universität Wien, Vienna, Austria.
• Christoph Böttcher, Julius Kühn-Institute (JKI) - Federal Research Centre for Cultivated Plants, Institute for Ecological Chemistry, Plant Analysis and Stored Product Protection, Berlin, Germany.
• Christian Molitor, Institute of Chemical, Environmental and Bioscience Engineering, Technische Universität Wien, Vienna, Austria.
• Jens Keilwagen, ulius Kühn-Institute (JKI) - Federal Research Centre for Cultivated Plants, Institute for Biosafety in Plant Biotechnology, Quedlinburg, Germany.
• Lukas Eidenberger, Institute of Chemical, Environmental and Bioscience Engineering, Technische Universität Wien, Vienna, Austria.
• Silvija Marinovic, Institute of Chemical, Environmental and Bioscience Engineering, Technische Universität Wien, Vienna, Austria.
• Matthias Hackl, Institute of Chemical, Environmental and Bioscience Engineering, Technische Universität Wien, Vienna, Austria.
• Andreas Spornberger, Institute of Viticulture and Pomology, BOKU University, Vienna, Austria.
• Christopher Schlosser, Institute of Chemical, Environmental and Bioscience Engineering, Technische Universität Wien, Vienna, Austria.
• Karl Stich, Institute of Chemical, Environmental and Bioscience Engineering, Technische Universität Wien, Vienna, Austria.
• Annette Rompel, Fakultät für Chemie, Institut für Biophysikalische Chemie, Universität Wien, Vienna, Austria.
• Henryk Flachowsky, Julius Kühn-Institute (JKI) - Federal Research Centre for Cultivated Plants, Institute for Breeding Research on Fruit Crops, Dresden, Germany.
• Heidi Halbwirth, Institute of Chemical, Environmental and Bioscience Engineering, Technische Universität Wien, Vienna, Austria.

Document Type

Article

Publication Date

10-1-2025

Abstract

Dihydrochalcones are secondary metabolites with manifold dietary and pharmaceutical properties, but with a yet unclear function in plants. The abundance of dihydrochalcones, particularly phloridzin, makes Malus ssp. unique in the plant kingdom. The gene(s)/enzyme(s) for the key reaction in phloridzin biosynthesis, the reduction in the C3-bridge connecting the aromatic rings A and B of chalcones, have long been searched for. To date, two possible pathways to phloridzin have been described, one via the reduction in p-coumaroyl-CoA and the other via the reduction in naringenin chalcone. In this work, an enzyme from apple leaves, which catalyses the reduction in p-coumaroyl-CoA, was purified and partially sequenced. The newly identified hydroxycinnamoyl-CoA double bond reductase (HDR) has not been mentioned in the context of phloridzin biosynthesis so far. Long-read sequencing and sequence analyses in search of transcriptional and translational variants predicted a MdHDR proteoform potentially formed by alternative translational initiation. Heterologous expression of the two HDR proteoforms in E. coli showed that recombinant HDR is able to reduce p-coumaroyl-CoA beside other hydroxycinnamic acid CoA esters, whereas other substrates including the corresponding free hydroxycinnamic acids and naringenin chalcone were not accepted. Heterologous expression of the HDR proteoforms in A. thaliana resulted in the formation of O-hexosylated dihydrocinnamic acids, confirming the functional activity of the HDR as a reductase in planta, but no phloretin derivatives were detected. Our findings support the presence of an alternative pathway to the recently described reduction in naringenin chalcone in the biosynthesis of dihydrochalcones.

Recommended Citation

Schröpfer S, Haselmair-Gosch C, Böttcher C, Molitor C, Keilwagen J, Eidenberger L, Marinovic S, Hackl M, Spornberger A, Walliser B, Schlosser C, Stich K, Rompel A, Flachowsky H, Halbwirth H. Identification of a hydroxycinnamoyl-CoA double bond reductase (HDR) affirms multiple pathways for dihydrochalcone formation in apple. Plant Biotechnol J. 2025 Oct;23(10):4522-4535. doi: 10.1111/pbi.70225. Epub 2025 Jul 9. PMID: 40635315; PMCID: PMC12483985.