Protection of naringenin chalcone by a pathogenesis‐related 10 protein promotes flavonoid biosynthesis in Marchantia polymorpha

Pathogenesis-related (PR) proteins are diverse stress- or pathogen-induced proteins. Some are associated with specialised metabolism, including proposed functions for anthocyanin biosynthesis. However, data are limited to a few angiosperm species, and the mode(s) of action are uncertain. Using the liverwort Marchantia polymorpha (Marchantia), we examined whether pathogenesis-related 10 (PR10) contributes to flavonoid biosynthesis in other land plant lineages and investigated its mode of action. Marchantia produces two major flavonoid types: flavones and the pigment auronidin. MpPR10.5 is a target of the auronidin regulator MpMYB14; therefore, Mppr10.5 mutants were generated using CRISPR/Cas9 and analysed for transcript abundance (via RNA sequencing) and for metabolite content. Recombinant MpPR10.5 protein was used for metabolite binding and stabilisation assays. Mppr10.5 mutants had reduced auronidin and flavone content, demonstrating that MpPR10.5 promotes flavonoid biosynthesis. Flavone and auronidin biosynthesis share a single flavonoid intermediate, naringenin chalcone (NC), suggesting MpPR10.5 acts on this compound. MpPR10.5 protein binds NC strongly (micromolar affinity), preventing spontaneous self-cyclisation in vitro. Several phenylpropanoid and flavonoid genes were downregulated in Mppr10.5 and Mpchalcone isomerase-like plants. This suggests PR10 proteins promote flavonoid biosynthesis by selectively binding unstable intermediates (NC), protecting them from degradation or undesirable nonenzymatic conversions and facilitating their transport to subsequent pathway steps.