Abstract
Questions about in vivo substrates 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 fern Azolla filiculoides. In vitro assay 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% in A. filiculoides, and 8% in A. pinnata biomass dry weight. Consistently, a LAR active in vitro was highly expressed in A. 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 of Azolla ferns, a floating plant–microbe mat massively fixing CO2 and N2, shaped their metabolism in which PA biosynthesis predominates and employs novel fern LAR enzymes. Characterisation of in vivo substrates of these LAR, will help to shed light on the recently assigned and surprising dual catalysis of LAR from seed plants.
Chemical and gene‐expression analyses were combined to characterise PA biosynthesis, leveraging genome annotation from the floating fern Azolla filiculoides. In vitro assay 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% in A. filiculoides, and 8% in A. pinnata biomass dry weight. Consistently, a LAR active in vitro was highly expressed in A. 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 of Azolla ferns, a floating plant–microbe mat massively fixing CO2 and N2, shaped their metabolism in which PA biosynthesis predominates and employs novel fern LAR enzymes. Characterisation of in vivo substrates of these LAR, will help to shed light on the recently assigned and surprising dual catalysis of LAR from seed plants.
| Original language | English |
|---|---|
| Pages (from-to) | 1118-1132 |
| Number of pages | 15 |
| Journal | New Phytologist |
| Volume | 229 |
| Issue number | 2 |
| Early online date | 28 Aug 2020 |
| DOIs | |
| Publication status | Published - Jan 2021 |
Bibliographical note
Funding Information:We thank Jean Chaudi?re from Bordeaux University for sharing the Vitis vinifera DFR recombinant expression vector. We thank Adrie van der Werf from Wageningen University & Research for recruiting IvdM and RdV for the task of running the LC-MS analyses of methanolic extracts from our ferns, and Caroline M. Preston (Pacific Forestry Centre, Victoria BC, Canada) for providing the purified black spruce tannins. We further thank three anonymous reviewers for their very helpful comments. We acknowledge funding for EB from the NWO-TTW grant AZOPRO (Project no. 16294), PB by the LPP foundation and LWD by the NWO-ALW project 2016/ALW/00127599.
Funding Information:
We thank Jean Chaudière from Bordeaux University for sharing the DFR recombinant expression vector. We thank Adrie van der Werf from Wageningen University & Research for recruiting IvdM and RdV for the task of running the LC‐MS analyses of methanolic extracts from our ferns, and Caroline M. Preston (Pacific Forestry Centre, Victoria BC, Canada) for providing the purified black spruce tannins. We further thank three anonymous reviewers for their very helpful comments. We acknowledge funding for EB from the NWO‐TTW grant AZOPRO (Project no. 16294), PB by the LPP foundation and LWD by the NWO‐ALW project 2016/ALW/00127599. Vitis vinifera
Publisher Copyright:
© 2020 The Authors New Phytologist © 2020 New Phytologist Trust
Keywords
- Azolla
- Nostoc
- PIP-family reductases
- RNA-sequencing
- flavonoids
- leucoanthocyanidin reductase
- phenolics
- proanthocyanidins