Interaction of PKR with STCS1: an indispensable step in the biosynthesis of lunularic acid in Marchantia polymorpha.

Unlike bibenzyls derived from the vascular plants, lunularic acid (LA), a key precursor for macrocyclic bisbibenzyl synthesis in nonvascular liverworts, exhibits the absence of one hydroxy group within the A ring. It was hypothesized that both polyketide reductase (PKR) and stilbenecarboxylate synthase 1 (STCS1) were involved in the LA biosynthesis, but the underlined mechanisms have not been clarified. This study used bioinformatics analysis with molecular, biochemical and physiological approaches to characterize STCS1s and PKRs involved in the biosynthesis of LA. The results indicated that MpSTCS1s from Marchantia polymorpha catalyzed both C2→C7 aldol-type and C6→C1 Claisen-type cyclization using dihydro-p-coumaroyl-coenzyme A (CoA) and malonyl-CoA as substrates to yield a C6-C2-C6 skeleton of dihydro-resveratrol following decarboxylation and the C6-C3-C6 type of phloretin in vitro. The protein-protein interaction of PKRs with STCS1 (PPI-PS) was revealed and proved essential for LA accumulation when transiently co-expressed in Nicotiana benthamiana. Moreover, replacement of the active domain of STCS1 with an 18-amino-acid fragment from the chalcone synthase led to the PPI-PS greatly decreasing and diminishing the formation of LA. The replacement also increased the chalcone formation in STCS1s. Our results highlight a previously unrecognized PPI in planta that is indispensable for the formation of LA.

Molecular Basis for Chemical Evolution of Flavones to Flavonols and Anthocyanins in Land Plants.

During the course of evolution of land plants, different classes of flavonoids, including flavonols and anthocyanins, sequentially emerged, facilitating adaptation to the harsh terrestrial environment. Flavanone 3ß-hydroxylase (F3H), an enzyme functioning in flavonol and anthocyanin biosynthesis and a member of the 2-oxoglutarate-dependent dioxygenase (2-ODD) family, catalyzes the hydroxylation of (2S)-flavanones to dihydroflavonols, but its origin and evolution remain elusive. Here, we demonstrate that functional flavone synthase Is (FNS Is) are widely distributed in the primitive land plants liverworts and evolutionarily connected to seed plant F3Hs. We identified and characterized a set of 2-ODD enzymes from several liverwort species and plants in various evolutionary clades of the plant kingdom. The bifunctional enzyme FNS I/F2H emerged in liverworts, and FNS I/F3H evolved in Physcomitrium (Physcomitrella) patens and Selaginella moellendorffii, suggesting that they represent the functional transition forms between canonical FNS Is and F3Hs. The functional transition from FNS Is to F3Hs provides a molecular basis for the chemical evolution of flavones to flavonols and anthocyanins, which contributes to the acquisition of a broader spectrum of flavonoids in seed plants and facilitates their adaptation to the terrestrial ecosystem.

Identification and evolutionary analysis of chalcone isomerase-fold proteins in ferns.

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.

Asperunguisins A-F, Cytotoxic Asperane Sesterterpenoids from the Endolichenic Fungus Aspergillus unguis.

Six new asperane-type sesterterpenoids, asperunguisins A-F (1-6), were isolated from the endolichenic fungus Aspergillus unguis, together with a known analogue, aspergilloxide (7); these are rare asperane-type sesterterpenoids, characterized by a unique hydroxylated 7/6/6/5 tetracyclic system. The structures of asperunguisins A-F (1-6) were elucidated on the basis of spectroscopic methods (NMR and HRESIMS), X-ray single-crystal diffraction analysis, ECD calculations, and biogenetic considerations. Asperunguisin C (3) showed cytotoxicity against the human cancer cell line A549 with an IC50 value of 6.2 µM. Further investigation revealed that the observed cell death was a result of G0/G1 cell cycle arrest via DNA damage followed by cellular apoptosis.

Isolation and functional characterization of four microbial type terpene synthases from ferns.

Typical plant terpene synthases (TPSs) are responsible for the production of terpenes, a major class of plant secondary metabolites. However, various nonseed plants also harbor genes encoding microbial terpene synthase-like (MTPSL) enzymes. Here, a scan of 31 ferns transcriptomes revealed 40 sequences putatively encoding MTPSLs. Two groups of sequences were recognized based on the key conserved motifs. Four representative genes were isolated from each of the four species Adiantum capillus-veneris, Cyclosorus parasiticus, Drynaria bonii and Microlepia platyphylla. Following their heterologous expression in E. coli, the recombinant proteins were tested for monoterpene synthase and sesquiterpene synthase activity. These enzymatic products were typical monoterpenes and sesquiterpenes that have been previous shown to be generated by classical plant TPSs when provided with GPP and FPP as substrates. Subcellular localization experiments in the leaf epidermis of Nicotiana benthamiana and onion (Allium cepa) inner epidermal cells indicated that AcMTPSL1 and DbMTPSL were deposited in both the cytoplasm and nucleus, whereas CpMTPSL1 and MpMTPSL were localized in the cytoplasm, chloroplasts and nucleus. AcMTPSL1 was up-regulated in plants exposed to methyl jasmonate treatment, suggesting a role for this gene in host defense. This study provides more information about the catalytic function of MTPSLs in nonseed plants and for the first time, the subcellular localization of MTPSLs was experimentally characterized.

Functional characterization of UDP-glycosyltransferases from the liverwort Plagiochasma appendiculatum and their potential for biosynthesizing flavonoid 7-O-glucosides.

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.

Isolation and functional characterization of two Caffeoyl Coenzyme A 3-O-methyltransferases from the fern species Polypodiodes amoena.

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.