AUTHOR=Miranda Simón , Lagrèze Jorge , Knoll Anne-Sophie , Angeli Andrea , Espley Richard V. , Dare Andrew P. , Malnoy Mickael , Martens Stefan 

TITLE=De novo transcriptome assembly and functional analysis reveal a dihydrochalcone 3-hydroxylase(DHC3H) of wild Malus species that produces sieboldin in vivo

JOURNAL=Frontiers in Plant Science

VOLUME=13

YEAR=2022

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2022.1072765

DOI=10.3389/fpls.2022.1072765

ISSN=1664-462X

ABSTRACT=<p>Sieboldin is a specialised secondary metabolite of the group of dihydrochalcones (DHC), found in high concentrations only in some wild <italic>Malus</italic> species, closely related to the domesticated apple (<italic>Malus</italic> × <italic>domestica</italic> L.). To date, the first committed step towards the biosynthesis of sieboldin remains unknown. In this study, we combined transcriptomic analysis and a <italic>de novo</italic> transcriptome assembly to identify two putative 3-hydroxylases in two wild <italic>Malus</italic> species (<italic>Malus toringo</italic> (K. Koch) Carriere syn. <italic>sieboldii</italic> Rehder<italic>, Malus micromalus</italic> Makino) whose DHC profile is dominated by sieboldin. We assessed the <italic>in vivo</italic> activity of putative candidates to produce 3-hydroxyphloretin and sieboldin by <italic>de novo</italic> production in <italic>Saccharomyces cerevisiae</italic>. We found that CYP98A proteins of wild <italic>Malus</italic> accessions (CYP98A195, <italic>M. toringo</italic> and CYP98A196, <italic>M. micromalus</italic>) were able to produce 3-hydroxyphloretin, ultimately leading to sieboldin accumulation by co-expression with PGT2. CYP98A197-198 genes of <italic>M.</italic> × <italic>domestica</italic>, however, were unable to hydroxylate phloretin <italic>in vivo</italic>. CYP98A195-196 proteins exerting 3-hydroxylase activity co-localised with an endoplasmic reticulum marker. CYP98A protein model from wild accessions showed mutations in key residues close to the ligand pocket predicted using phloretin for protein docking modelling. These mutations are located within known substrate recognition sites of cytochrome P450s, which could explain the acceptance of phloretin in CYP98A protein of wild accessions. Screening a <italic>Malus</italic> germplasm collection by HRM marker analysis for CYP98A genes identified three clusters that correspond to the alleles of domesticated and wild species. Moreover, CYP98A isoforms identified in <italic>M. toringo</italic> and <italic>M. micromalus</italic> correlate with the accumulation of sieboldin in other wild and hybrid <italic>Malus</italic> genotypes. Taken together, we provide the first evidence of an enzyme producing sieboldin <italic>in vivo</italic> that could be involved in the key hydroxylation step towards the synthesis of sieboldin in <italic>Malus</italic> species.</p>