Azollaferns testify: seed plants and ferns share a common ancestor for leucoanthocyanidin reductase enzymes

Questions aboutin vivosubstrates for proanthocyanidin (PA) biosynthesis and condensation have not been resolved and wide gaps in the understanding of transport and biogenesis in 'tannosomes' persist. Here we examined the evolution of PA biosynthesis in ferns not previously reported, asking what PAs are synthesised and how. Chemical and gene-expression analyses were combined to characterise PA biosynthesis, leveraging genome annotation from the floating fernAzolla filiculoides.In vitroassay and phylogenomics of PIP-dehydrogenases served to infer the evolution of leucoanthocyanidin reductase (LAR). Sporophyte-synthesised (epi)catechin polymers, averaging only seven subunits, accumulated to 5.3% inA. filiculoides, and 8% inA. pinnatabiomass dry weight. Consistently, a LAR activein vitrowas highly expressed inA. filiculoides. LAR, and paralogous fern WLAR-enzymes with differing substrate binding sites, represent an evolutionary innovation of the common ancestor of fern and seed plants. The specific ecological niche ofAzollaferns, a floating plant-microbe mat massively fixing CO(2)and N-2, shaped their metabolism in which PA biosynthesis predominates and employs novel fern LAR enzymes. Characterisation ofin vivosubstrates of these LAR, will help to shed light on the recently assigned and surprising dual catalysis of LAR from seed plants.

Automated summary

Questions regarding in vivo substrates for proanthocyanidin biosynthesis and condensation remain unanswered, with gaps in understanding transport and biogenesis in 'tannosomes'. This study examined the evolution of proanthocyanidin biosynthesis in ferns, revealing (epi)catechin polymers synthesized in the sporophyte of Azolla ferns. The research also identified LAR and WLAR enzymes with differing substrate binding sites as an evolutionary innovation of the common ancestor of fern and seed plants.