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Flavonoid glucosides, typically generated from aglycones via the action of uridine diphosphate-dependent glycosyltransferases (UGTs), both contribute to plant viability and are pharmacologically active. The properties of UGTs produced by liverworts, one of the basal groups of non-vascular land plants, have not been systematically explored. Here, two UGTs potentially involved in flavonoids synthesis were identified from the transcriptome of Plagiochasma appendiculatum. Enzymatic analysis showed that PaUGT1 and PaUGT2 accepted various flavones, flavonols, flavanones and dihydrochalcones as substrates. A mutated form PaUGT1-Q19A exhibited a higher catalytic efficiency than did the wild type enzyme. When expressed in Escherichia coli, the yield of flavonol 7-O-glucosides reached to over 70 %. Co-expression of PaUGT1-Q19A with the upstream flavone synthase I PaFNS I-1 proved able to convert the flavanone aglycones naringenin and eriodictyol into the higher-yield apigenin 7-O-glucoside (A7G) and luteolin 7-O-glucoside (L7G). The maximum concentration of 81.0 μM A7G and 88.6 μM L7G was achieved upon supplementation with 100 μM naringenin and 100 μM eriodictyol under optimized conditions. This is the first time that flavonoids UGTs have been characterized from liverworts and co-expression of UGTs and FNS Is from the same species serves as an effective strategy to synthesize flavone 7-O-glucosides in E. coli., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The distribution of type I and II chalcone isomerases (CHIs) in plants is highly family specific. We have previously reported that ancient land plants, such as the liverworts and Selaginella moellendorffii, harbor type II CHIs. To better understand the function and evolution of CHI-fold proteins, transcriptomic data obtained from 52 pteridophyte species were subjected to sequence alignment and phylogenetic analysis. The residues determining type I/II CHI identity in the pteridophyte CHIs were identical to those of type I CHIs. The enzymatic characterization of a sample of 24 CHIs, representing all the key pteridophyte lineages, demonstrated that 19 of them were type I enzymes and that five exhibited some type II activity due to an amino acid mutation. Two pteridophyte chalcone synthases (CHSs) were also characterized, and a type IV CHI (CHIL) was demonstrated to interact physically with CHSs and CHI, and to increase CHS activity by decreasing derailment products, thus enhancing flavonoid production. These findings suggest that the emergence of type I CHIs may have coincided with the divergence of the pteridophytes. This study deepens our understanding of the molecular mechanism of CHIL as an enhancer in the flavonoid biosynthesis pathway., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Caffeoyl Coenzyme A 3-O-methyltransferases (CCoAOMTs) catalyze the transfer of a methyl group from S-adenosylmethionine (SAM) to a hydroxyl moiety. CCoAOMTs are important for the synthesis of lignin, which provides much of the rigidity required by tracheophytes to enable the long distance transport of water. So far, no CCoAOMTs has been characterized from the ancient tracheophytes ferns. Here, two genes, each encoding a CCoAOMT (and hence denoted PaCCoAOMT1 and PaCCoAOMT2), were isolated from the fern species Polypodiodes amoena. Sequence comparisons confirmed that the product of each gene resembled enzymes known to be associated with lignin synthesis in higher plants. When either of the genes was heterologously expressed in E. coli, the resulting recombinant protein was able to methylate caffeoyl CoA, along with a number of phenylpropanoids, flavones and flavonols containing two vicinal hydroxyl groups. Their in vitro conversion rate when presented with either caffeoyl CoA or certain flavonoids as substrate was comparable with that of the Medicago sativa MsCCoAOMT. Their constitutive expression in Arabidopsis thaliana boosted the plants' lignin content, but did not affect that of methylated flavonols, indicating that both PaCCoAOMTs contributed to lignin synthesis and that neither was able to methylate flavonols in planta. The transient expression of a PaCCoAOMT-GFP fusion gene in tobacco demonstrated that in planta, PaCCoAOMTs are likely directed to the cytoplasm., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)