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

Abstract

Introduction

Flavonoid biosynthesis, a branch of the phenylpropanoid pathway, is a central specialised metabolite pathway of land plants. Flavonoids are key to how plants interact with the terrestrial environment, helping in tolerance of diverse abiotic stresses and attacks from pests and pathogens and providing pigmentation to flowers, fruit, seeds and vegetative tissues (Agati & Tattini, 2010; Cheynier et al., 2013; Landi et al., 2015; Davies et al., 2018). The flavonoid pathway commences with the production of chalcones by the polyketide synthase (PKS) enzyme CHALCONE SYNTHASE (CHS). The subsequent pathway steps lead to diverse, distinct flavonoid classes, including flavones, flavonols, isoflavonoids, aurones, auronidins, anthocyanins and proanthocyanidins (condensed tannin). The core biosynthesis genes for the major flavonoid classes have been extensively characterized across many plant species (Yonekura-Sakakibara et al., 2019; Ferreyra et al., 2021; Davies et al., 2022). However, recent studies have brought to light possible important roles in flavonoid production for additional nonenzymatic proteins, the modes of action of which are generally not understood. The best studied is CHALCONE ISOMERASE-LIKE (CHIL), which is necessary for efficient flavonoid biosynthesis in diverse land plant lineages (Morita et al., 2014; Berland et al., 2019). CHIL can bind with and alter the specificity of CHS and at least some other PKS enzymes, notably STILBENE SYNTHASE (Waki et al., 2020). As the PKSs direct substrate flow into the alternative phenylpropanoid pathway sections, such as flavonoids, stilbenes, dihydrochalcones and bibenzyls, CHIL acts at a key biosynthetic step where enzyme specificity and efficacy are particularly important.

Relatively unexamined candidates for other nonenzymatic proteins involved in flavonoid biosynthesis are some members of the pathogenesis-related 10 (PR10) sub-class of pathogenesis-related proteins (PR proteins). Pathogenesis-related proteins represent diverse protein families, often with unknown functions, whose corresponding genes are induced in response to pathogen attacks, environmental stresses or certain physiological processes (van Loon, 1985). Following identification of PR10 in parsley (PcPR10 of Petroselinum crispum; Somssich et al., 1986) and as the major allergen present in birch pollen (Bet v1 of Betula spp.; Breiteneder et al., 1989), PR10 genes have been identified in various gymnosperm and angiosperm species (Liu & Ekramoddoullah, 2006). Yet, no conserved role for PR10 family members has been identified, although they have commonly been linked to defence against pathogens (Park et al., 2004; Andrade et al., 2010; Fan et al., 2015; Longsaward et al., 2023). An association with specialized metabolite pathways has also often been noted, including for flavonoids (Hjernø et al., 2006; Muñoz et al., 2010), sporopollenin (Huang et al., 1997), benzylisoquinoline alkaloids (Hagel & Facchini, 2013), Amaryllidaceae alkaloids (Singh et al., 2018), iridoids (Lichman et al., 2020) and thebaine (Chen et al., 2018).

The mode of action of PR10 proteins is unresolved. Some PR10 proteins have been found to directly catalyse specialized metabolite biosynthetic reactions (Hagel & Facchini, 2013; Chen et al., 2018; Singh et al., 2018), while other PR10 proteins may guide pathway stereochemistry and product selectivity (Lichman et al., 2020). However, the ability to bind small metabolites may be key to all PR10 functions. Pathogenesis-related 10 proteins have a compact, stable conformation, characterised by a seven-stranded antiparallel β-sheet wrapped around an α-helix, along with two additional short helices (Hoffmann-Sommergruber et al., 1997; Wen et al., 1997; Handschuh et al., 2007; Lebel et al., 2010; Fernandes et al., 2013). These elements collectively form a hydrophobic cavity that is the site of ligand binding (Aglas et al., 2020; Morris et al., 2021). A glycine-rich loop L4 is preserved even in distant homologues among seed plants, and thus recognised as a signature motif (Fernandes et al., 2013). A diverse array of molecules has been shown to be able to be bound by different PR10 proteins, ranging from invading viral RNAs to varied specialized metabolites (Park et al., 2004; Chadha & Das, 2006; Sliwiak et al., 2016; Aglas et al., 2020). Notably, the PR10 of strawberry (Fra a of Fragaria × ananassa) can bind flavonoids, including quercetin-3-O-glucuronide, myricetin and (+)-catechin (Casañal et al., 2013). Additionally, Fra a genes are downregulated in an anthocyanin-lacking strawberry line (Hjernø et al., 2006), and transient RNAi-mediated silencing of the Fra a genes in strawberry fruits reduced amounts of anthocyanins and upstream metabolites and the transcript abundance for PHENYLALANINE AMMONIA LYASE (PAL) and CHS. Based on the available data, there are various proposals for how PR10 might contribute to flavonoid biosynthesis: chemical chaperones assist intercellular transportation of compounds (Casañal et al., 2013), participation in multi-protein flavonoid biosynthetic complexes (metabolons) to restrict diffusion and protect unstable substrates (Morris et al., 2021), finely adjusting metabolic flux by differential binding of intermediates and channelling them between enzymes (Casañal et al., 2013) and transcriptional regulation of flavonoid biosynthesis genes (Muñoz et al., 2010). Thus, the mode of action of PR10 in the flavonoid pathway remains equivocal.

The models used for elucidating the genetics and biochemistry of the flavonoid pathway have traditionally been a relatively small number of angiosperm species. However, recently Marchantia polymorpha (hereafter, Marchantia), a member of the liverwort lineage of bryophytes, has emerged as a powerful system for addressing questions of flavonoid genetics and for identifying which aspects of flavonoid biosynthesis and regulation are conserved across land plants (Albert et al., 2018; Clayton et al., 2018; Kubo et al., 2018; Berland et al., 2019; Bowman et al., 2022; Zhu et al., 2023; Zhou et al., 2024). The two main flavonoid classes produced in Marchantia are the colorless flavones, which assist with tolerance of UVB light exposure, and the red pigment auronidin (Albert et al., 2018; Clayton et al., 2018; Kubo et al., 2018; Berland et al., 2019). The phenylpropanoid enzymes that convert phenylalanine to the flavonoid precursor p-coumaroyl CoA are conserved across all land plants, including Marchantia, being PAL, CINNAMATE 4-HYDROXYLASE (C4H) and 4-COUMAROYL CoA LIGASE (4CL). From there, the pathway in Marchantia branches into two trajectories: one leading to flavonoids and the other to bibenzyls, a group of antimicrobial compounds for which > 125 different structures have been reported from liverworts (Asakawa et al., 2022). The initial bibenzyl-specific step is catalysed by the PKS STILBENECARBOXYLATE SYNTHASE and a POLYKETIDE REDUCTASE. For flavonoids, the naringenin chalcone (NC) formed by CHS/CHIL is subsequently transformed into flavones by the actions of CHALCONE ISOMERASE (CHI) and FLAVONE SYNTHASE (FNS) or auronidins by AUREUSIDIN SYNTHASE (AUS) and uncharacterized enzymes (Berland et al., 2019; Davies et al., 2020) (Fig. 1). The biosynthesis of auronidin is transcriptionally activated by the R2R3MYB transcription factor MpMYB14 (Albert et al., 2018; Kubo et al., 2018).

Fig. 1

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Schematic of the phenylpropanoid biosynthetic pathway in Marchantia polymorpha. 4CL, 4-COUMAROYL COA LIGASE; AUS, AUREUSIDIN SYNTHASE; C4H, CINNAMATE 4-HYDROXYLASE; CHI, CHALCONE ISOMERASE; CHIL, CHALCONE ISOMERASE-LIKE; CHS, CHALCONE SYNTHASE; DBR, double bond reductase; FNS, FLAVONE SYNTHASE; PAL, PHENYLALANINE AMMONIA LYASE; PKR, POLYKETIDE REDUCTASE; STCS, STILBENECARBOXYLATE SYNTHASE.

Previous studies on auronidin biosynthesis in Marchantia identified MpPATHOGENESIS-RELATED10.5 (MpPR10.5/Mp8g00860) as being tightly associated with MpMYB14 expression. MpPR10.5 transcript levels increased over 30-fold in 35S:MYB14 plants and decreased over sevenfold in Mpmyb14 mutants compared to wild-type(WT) (Albert et al., 2018; Berland et al., 2019). Furthermore, in an independent study conducted by Kubo et al. (2018), MpPR10.5 was significantly upregulated (> 50-fold) in their MpMYB14 overexpression line, compared to the WT control (full dataset available in the Marpolbase Expression database: https://mbex.marchantia.info/diffexp). This suggested that MpPR10.5 may have a role in auronidin biosynthesis and that Marchantia could be an excellent model system to elucidate the mode of action of PR10 proteins in the flavonoid pathway.

In this study, using a multidisciplinary combination of reverse genetics, RNAseq analysis and in vitro protein assays, we establish that MpPR10.5 promotes flavonoid biosynthesis in Marchantia, identify a novel feedback regulation mechanism that downregulates phenylpropanoid pathway gene expression and propose a mode of action through binding of MpPR10.5 protein to specific flavonoid pathway intermediates.