Molecular cloning and functional analysis of 4-coumarate: CoA ligases from Marchantia paleacea and their roles in lignin and flavanone biosynthesis.

Phenylpropanoids play important roles in plant physiology and the enzyme 4-coumarate: coenzyme A ligase (4CL) catalyzes the formation of thioesters. Despite extensive characterization in various plants, the functions of 4CLs in the liverwort Marchantia paleacea remain unknown. Here, four 4CLs from M. paleacea were isolated and functionally analyzed. Heterologous expression in Escherichia coli indicated the presence of different enzymatic activities in the four enzymes. Mp4CL1 and Mp4CL2 were able to convert caffeic, p-coumaric, cinnamic, ferulic, dihydro-p-coumaric, and 5-hydroxyferulic acids to their corresponding CoA esters, while Mp4CL3 and Mp4CL4 catalyzed none. Mp4CL1 transcription was induced when M. paleacea thalli were treated with methyl jasmonate (MeJA). The overexpression of Mp4CL1 increased the levels of lignin in transgenic Arabidopsis. In addition, we reconstructed the flavanone biosynthetic pathway in E. coli. The pathway comprised Mp4CL1, co-expressed with chalcone synthase (CHS) from different plant species, and the efficiency of biosynthesis was optimal when both the 4CL and CHS were obtained from the same species M. paleacea.

Efficient Production of Flavonoid Glucuronides in Escherichia coli Using Flavonoid O-Glucuronosyltransferases Characterized from Marchantia polymorpha.

As a model liverwort, Marchantia polymorpha contains various flavone glucuronides with cardiovascular-promoting effects and anti-inflammatory properties. However, the related glucuronosyltransferases have not yet been reported. In this study, two bifunctional UDP-glucuronic acid/UDP-glucose:flavonoid glucuronosyltransferases/glucosyltransferases, MpUGT742A1 and MpUGT736B1, were identified from M. polymorpha. Extensive enzymatic assays found that MpUGT742A1 and MpUGT736B1 exhibited efficient glucuronidation activity for flavones, flavonols, and flavanones and showed promiscuous regioselectivity at positions 3, 6, 7, 3', and 4'. These enzymes catalyzed the production of a variety of flavonoid glucuronides with medicinal value, including apigenin-7-O-glucuronide and scutellarein-7-O-glucuronide. With the use of MpUGT736B1, apigenin-4'-O-glucuronide and apigenin-7,4'-di-O-glucuronide were prepared by scaled-up enzymatic catalysis and structurally identified by NMR spectroscopy. MpUGT742A1 also displayed glucosyltransferase activity on the 7-OH position of the flavanones using UDP-glucose as the sugar donor. Furthermore, we constructed four recombinant strains by combining the pathway for increasing the UDP-glucuronic acid supply with the two novel UGTs MpUGT742A1 and MpUGT736B1. When apigenin was used as a substrate, the extracellular apigenin-4'-O-glucuronide and apigenin-7,4'-di-O-glucuronide production obtained from the Escherichia coli strain BB2 reached 598 and 81 mg/L, respectively. Our study provides new candidate genes and strategies for the biosynthesis of flavonoid glucuronides.

Functional specialization of two UDP-glycosyltransferases MpUGT735A2 and MpUGT743A1 in the liverworts Marchantia polymorpha.

Family 1 UDP-glycosyltransferases (UGTs) are known to glycosylate multiple secondary plant metabolites and have been extensively studied. The increased availability of plant genome resources allows the identification of wide gene families, both functional and organizational. In this investigation, two MpUGT isoforms were cloned and functionally characterized from liverworts marchantia polymorpha and had high glycosylation activity against several flavonoids. MpUGT735A2 protein, in particular, tolerates a wide spectrum of substrates (flavonols, flavanones, flavones, stilbenes, bibenzyls, dihydrochalcone, phenylpropanoids, xanthones, and isoflavones). Overexpression of MpUGT735A2 and MpUGT743A1 in Arabidopsis thaliana enhances the accumulation of 3-O-glycosylated flavonol (kaempferol 3-O-glucoside-7-O-rhamnose), consistent with its in vitro enzymatic activity. Docking and mutagenesis techniques were applied to identify the structural and functional properties of MpUGT735A2 with promiscuous substrates. Mutation of Pro87 to Ser, or Gln88 to Val, substantially altered the regioselectivity for luteolin glycosylation, predominantly from the 3'-O- to the 7-O-position. The results were elucidated by focusing on the novel biocatalysts designed for producing therapeutic flavonoids. This investigation provides an approach to modulate MpUGT735A2 as a candidate gene for diverse glycosylation catalysis and a tool to design GTs with new substrate specificities for biomedical applications.

Identification and Functional Characterization of UDP-Glycosyltransferases Involved in Isoflavone Biosynthesis in Astragalus membranaceus.

Isoflavones are rich natural compounds present in legumes and are essential for plant growth and development. Moreover, they are beneficial for animals and humans. Isoflavones are primarily found as glycoconjugates, including calycosin-7-O-ß-d-glucoside (CG) in Astragalus membranaceus, a legume. However, the glycosylation mechanism of isoflavones in A. membranaceus remains unclear. In the present study, three uridine diphosphate (UDP)-glycosyltransferases (UGTs) that may be involved in the biosynthesis of isoflavone were identified in the transcriptome of A. membranaceus. Enzymatic analysis revealed that AmUGT88E29 and AmUGT88E30 had high catalytic activity toward isoflavones in vitro. In addition, AmUGT88E29 and AmUGT88E30 could accept various flavones, flavanones, flavonols, dihydroflavonols, and dihydrochalcones as substrates. AmUGT71G10 was only active against phloretin and dihydroresveratrol. Overexpression of AmUGT88E29 significantly increased the contents of CG, an isoflavone glucoside, in the hairy roots of A. membranaceus. This study provided candidate AmUGT genes for the potential metabolic engineering of flavonoid compounds in plants and a valuable resource for studying the calycosin glycosides biosynthesis pathway.

Molecular identification of a flavone synthase I/flavanone 3ß-hydroxylase bifunctional enzyme from fern species Psilotum nudum.

The enzyme flavone synthase Is (FNS Is) converts flavanones to flavones, whereas flavanone 3ß-hydroxylases (F3Hs) catalyze the formation of dihydroflavonols, a precursor of flavonols and anthocyanins. Canonical F3Hs have been characterized in seed plants, which are evolutionarily related to liverwort FNS Is. However, as important evolutionary lineages between liverworts and seed plants, ferns FNS Is and F3Hs have not been identified. In the present study, we characterized a bifunctional enzyme PnFNS I/F3H from the fern Psilotum nudum. We found that PnFNS I/F3H catalyzed the conversion of naringenin to apigenin and dihydrokaempferol. In addition, it catalyzed five different flavanones to generate the corresponding flavones. Site-directed mutagenesis results indicated that the P228-Y228 mutant protein displayed the FNS I/F2H activity (catalyzing naringenin to generate apigenin and 2-hydroxynaringenin), thus having similar functions as liverwort FNS I/F2H. Moreover, the overexpression of PnFNS I/F3H in Arabidopsis tt6 and dmr6 mutants increased the content of flavones and flavonols in plants, further indicating that PnFNS I/F3H showed FNS I and F3H activities in planta. This is the first study to characterize a bifunctional enzyme FNS I/F3H in ferns. The functional transition from FNS I/F3H to FNS I/F2H will be helpful in further elucidating the relationship between angiosperm F3Hs and liverwort FNS Is.

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.

Spatial Distribution, Antioxidant Capacity, and Spore Germination-Promoting Effect of Bibenzyls from Marchantia polymorpha.

Liverworts, considered to be the first plant type to successfully make the transition from water to land, can resist different oxidative stress. As characteristic constituents of liverworts, the bibenzyls are efficient antioxidants. In this study, spatial distributions of the bibenzyls within Marchantia polymorpha L., the model species of liverworts, were mapped using airflow-assisted desorption electrospray ionization imaging mass spectrometry. Bibenzyls were found to largely exist in the female receptacle of M. polymorpha, where lunularic acid was found to focus in the central region and bisbibenzyls were enriched in the periphery. The region-specific gene expression and antioxidant activities were characterized. In line with the spatial feature of bibenzyls, higher MpSTCS1A and Mp4CL expression levels and antioxidant ability were exhibited in the archegoniophore. The expression level of MpSTCS1A, and the content of total phenolic acid was increased after UV-B irradiation, suggesting bibenzyls play an important role in UV-B tolerance. Moreover, lunularic acid and extract of archegoniophore at a certain concentration can stimulate the spore germination under normal conditions and UV-B stress. These works broaden our understanding of the significance of bibenzyls in spore propagation and environmental adaptation.

Identification of a flavonoid C-glycosyltransferase from fern species Stenoloma chusanum and the application in synthesizing flavonoid C-glycosides in Escherichia coli.

BACKGROUND: Flavonoid C-glycosides have many beneficial effects and are widely used in food and medicine. However, plants contain a limited number of flavonoid C-glycosides, and it is challenging to create these substances chemically. RESULTS: To screen more robust C-glycosyltransferases (CGTs) for the biosynthesis of flavonoid C-glycosides, one CGT enzyme from Stenoloma chusanum (ScCGT1) was characterized. Biochemical analyses revealed that ScCGT1 showed the C-glycosylation activity for phloretin, 2-hydroxynaringenin, and 2-hydroxyeriodictyol. Structure modeling and mutagenesis experiments indicated that the glycosylation of ScCGT1 may be initiated by the synergistic action of conserved residue His26 and Asp14. The P164T mutation increased C-glycosylation activity by forming a hydrogen bond with the sugar donor. Furthermore, when using phloretin as a substrate, the extracellular nothofagin production obtained from the Escherichia coli strain ScCGT1-P164T reached 38 mg/L, which was 2.3-fold higher than that of the wild-type strain. Finally, it is proved that the coupling catalysis of CjFNS I/F2H and ScCGT1-P164T could convert naringenin into vitexin and isovitexin. CONCLUSION: This is the first time that C-glycosyltransferase has been characterized from fern species and provides a candidate gene and strategy for the efficient production of bioactive C-glycosides using enzyme catalysis and metabolic engineering.

Molecular and structural characterization of a promiscuous chalcone synthase from the fern species Stenoloma chusanum.

The key enzymes involved in the flavonoid biosynthesis pathway have been extensively studied in seed plants, but relatively less in ferns. In this study, two 4-Coumarate: coenzyme A ligases (Sc4CL1 and Sc4CL2) and one novel chalcone synthase (ScCHS1) were functionally characterized by mining the Stenoloma chusanum transcriptome database. Recombinant Sc4CLs were able to esterify various hydroxycinnamic acids to corresponding acyl-coenzyme A (CoA). ScCHS1 could catalyze p-coumaroyl-CoA, cinnamoyl-CoA, caffeoyl-CoA, and feruloyl-CoA to form naringenin, pinocembrin, eriodictyol, and homoeriodictyol, respectively. Moreover, enzymatic kinetics studies revealed that the optimal substrates of ScCHS1 were feruloyl-CoA and caffeoyl-CoA, rather than p-coumaroyl-CoA, which was substantially different from the common CHSs. Crystallographic and site-directed mutagenesis experiments indicated that the amino acid residues, Leu87, Leu97, Met165, and Ile200, located in the substrate-binding pocket near the B-ring of products, could exert a significant impact on the unique catalytic activity of ScCHS1. Furthermore, overexpression of ScCHS1 in tt4 mutants could partially rescue the mutant phenotypes. Finally, ScCHS1 and Sc4CL1 were used to synthesize flavanones and flavones with multi-substituted hydroxyl and methoxyl B-ring in Escherichia coli, which can effectively eliminate the need for the cytochrome P450 hydroxylation/O-methyltransferase from simple phenylpropanoid acids. In summary, the identification of these important Stenoloma enzymes provides a springboard for the future production of various flavonoids in E. coli.

Identification and Characterization of Two Bibenzyl Glycosyltransferases from the Liverwort Marchantia polymorpha.

Liverworts are rich in bibenzyls and related O-glycosides, which show antioxidant activity. However, glycosyltransferases that catalyze the glycosylation of bibenzyls have not yet been characterized. Here, we identified two bibenzyl UDP-glucosyltransferases named MpUGT737B1 and MpUGT741A1 from the model liverwort Marchantia polymorpha. The in vitro enzymatic assay revealed that MpUGT741A1 specifically accepted the bibenzyl lunularin as substrate. MpUGT737B1 could accept bibenzyls, dihydrochalcone and phenylpropanoids as substrates, and could convert phloretin to phloretin-4-O-glucoside and phloridzin, which showed inhibitory activity against tyrosinase and antioxidant activity. The results of sugar donor selectivity showed that MpUGT737B1 and MpUGT741A1 could only accept UDP-glucose as a substrate. The expression levels of these MpUGTs were considerably increased after UV irradiation, which generally caused oxidative damage. This result indicates that MpUGT737B1 and MpUGT741A1 may play a role in plant stress adaption. Subcellular localization indicates that MpUGT737B1 and MpUGT741A1 were expressed in the cytoplasm and nucleus. These enzymes should provide candidate genes for the synthesis of bioactive bibenzyl O-glucosides and the improvement of plant antioxidant capacity.

Molecular cloning and characterization of two distinct caffeoyl CoA O-methyltransferases (CCoAOMTs) from the liverwort Marchantia paleacea.

Caffeoyl CoA O-methyltransferases (CCoAOMTs) catalyze the transfer of a methyl group from S-adenosylmethionine to a hydroxyl moiety of caffeoyl-CoA as part of the lignin biosynthetic pathway. CCoAOMT-like proteins also catalyze to a variety of flavonoids, coumarins, and phenylpropanoids. Several CCoAOMTs that prefer flavonoids as substrates have been characterized from liverworts. Here, we cloned two CCoAOMT genes, MpalOMT2 and MpalOMT3, from the liverwort Marchantia paleacea. MpalOMT3 has a second ATG codon downstream and the truncated version that lacks 11 amino acids was named MpalOMT3-Tr. Phylogenetic analysis placed MpalOMT3 at the root of the clade with true CCoAOMTs from vascular plants and placed MpalOMT2 between the CCoAOMT and CCoAOMT-like proteins. Recombinant OMTs methylated caffeoyl CoA, phenylpropanoids, and flavonoids containing two or three vicinal hydroxyl groups. MpalOMT3 showed higher catalytic activity for phenylpropanoids than MpalOMT2, but MpalOMT2 showed more promiscuous towards eriodictyol and myricetin. The lignin content in Arabidopsis thaliana stems increased with constitutive heterologous expression of MpalOMT3-Tr, but not MpalOMT2. Subcellular localization experiments indicated that the N-terminus of MpalOMT3 probably served as a chloroplast transit peptide and inhibited its enzymatic activity. Combining the phylogenetic analysis and functional characterization, we conclude that the liverwort M. paleacea harbors true CCoAOMT and CCoAOMT-like genes.

Functional and Structural Investigation of Chalcone Synthases Based on Integrated Metabolomics and Transcriptome Analysis on Flavonoids and Anthocyanins Biosynthesis of the Fern Cyclosorus parasiticus.

The biosynthesis of flavonoids and anthocyanidins has been exclusively investigated in angiosperms but largely unknown in ferns. This study integrated metabolomics and transcriptome to analyze the fronds from different development stages (S1 without spores and S2 with brown spores) of Cyclosorus parasiticus. About 221 flavonoid and anthocyanin metabolites were identified between S1 and S2. Transcriptome analysis revealed several genes encoding the key enzymes involved in the biosynthesis of flavonoids, and anthocyanins were upregulated in S2, which were validated by qRT-PCR. Functional characterization of two chalcone synthases (CpCHS1 and CpCHS2) indicated that CpCHS1 can catalyze the formation of pinocembrin, naringenin, and eriodictyol, respectively; however, CpCHS2 was inactive. The crystallization investigation of CpCHS1 indicated that it has a highly similar conformation and shares a similar general catalytic mechanism to other plants CHSs. And by site-directed mutagenesis, we found seven residues, especially Leu199 and Thr203 that are critical to the catalytic activity for CpCHS1.

Photoredox-Catalyzed Cascade Reactions Involving Aryl Radical: Total Synthesis of (±)-Norascyronone A and (±)-Eudesmol.

Herein, we have developed two types of photoredox-catalyzed cascade reactions using diaryliodonium salts for the concise synthesis of norascyronone A and ß-eudesmol. A rationally designed photoredox-catalyzed arylation/cyclization/Friedel-Crafts cascade reaction of enone was exploited to generate the norascyronone polycyclic skeleton. A visible-light-induced radical cyclization/acyloxy-migration reaction was explored to forge the decalin skeleton of eudesmol, and mechanistic studies indicated the reaction was initiated by one-electron oxidation of the enol ester.

Cloning and functional characterization of three flavonoid O-glucosyltransferase genes from the liverworts Marchantia emarginata and Marchantia paleacea.

Flavonoid glycosides are important plant secondary metabolites with broad pharmacological activities. Flavonoid glycosides are generated from aglycones, in reactions catalyzed by typical uridine diphosphate-dependent glycosyltransferases (UGTs). Liverworts produce various types of flavonoid glycosides; however, only two UGTs have been characterized from liverworts to date. Here, we isolated three genes encoding UGTs (MeUGT1, MeUGT2, and MpalUGT1) from the liverwort species Marchantia emarginata and Marchantia paleacea through transcriptome sequencing. Recombinant MeUGT1, MeUGT2, and MpalUGT1 proteins heterologously produced in Escherichia coli exhibited catalytic activity towards multiple flavonoids. MeUGT1 and MpalUGT1 catalyzed the glycosylation of flavonols into the corresponding 3-O-glucosides with UDP-glucose as the sugar donor, while MeUGT2 exhibited a wider substrate specificity that included flavonols, flavones, and flavanones. When MeUGT2 was expressed in E. coli, the yield of flavonol 3-O-glucosides reached to 40-60% with feeding of the substrates kaempferol or quercetin under optimal conditions. Furthermore, heterologous expression of MeUGT1 in Arabidopsis thaliana increased the flavonol glycoside contents in the plants. Therefore, the UGTs characterized in this study could provide new data that will be useful for examining flavonoid biosynthesis in liverworts.

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.

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 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.

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.

Functional characterization of a liverworts bHLH transcription factor involved in the regulation of bisbibenzyls and flavonoids biosynthesis.

BACKGROUND: The basic helix-loop-helix (bHLH) transcription factors (TFs), as one of the largest families of TFs, play important roles in the regulation of many secondary metabolites including flavonoids. Their involvement in flavonoids synthesis is well established in vascular plants, but not as yet in the bryophytes. In liverworts, both bisbibenzyls and flavonoids are derived through the phenylpropanoids pathway and share several upstream enzymes. RESULTS: In this study, we cloned and characterized the function of PabHLH1, a bHLH family protein encoded by the liverworts species Plagiochasma appendiculatum. PabHLH1 is phylogenetically related to the IIIf subfamily bHLHs involved in flavonoids biosynthesis. A transient expression experiment showed that PabHLH1 is deposited in the nucleus and cytoplasm, while the yeast one hybrid assay showed that it has transactivational activity. When PabHLH1 was overexpressed in P. appendiculatum thallus, a positive correlation was established between the content of bibenzyls and flavonoids and the transcriptional abundance of corresponding genes involved in the biosynthesis pathway of these compounds. The heterologous expression of PabHLH1 in Arabidopsis thaliana resulted in the activation of flavonoids and anthocyanins synthesis, involving the up-regulation of structural genes acting both early and late in the flavonoids synthesis pathway. The transcription level of PabHLH1 in P. appendiculatum thallus responded positively to stress induced by either exposure to UV radiation or treatment with salicylic acid. CONCLUSION: PabHLH1 was involved in the regulation of the biosynthesis of flavonoids as well as bibenzyls in liverworts and stimulated the accumulation of the flavonols and anthocyanins in Arabidopsis.

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.