The identification and functional characterization of three liverwort class I O-methyltransferases.

Previously it has been shown that the caffeoyl coenzyme A O-methyltransferase (CCoAOMT) type enzyme PaF6OMT, synthesized by the liverwort Plagiochasma appendiculatum Lehm. & Lindenb., (Aytoniaceae), interacts preferentially with 6-OH flavones. To clarify the biochemistry and evolution of liverwort OMTs, a comparison was made between the nucleotide sequence and biological activity of PaF6OMT and those of three of its homologs MpOMT1 (from Marchantia paleacea Bertol., (Marchantiaceae)), MeOMT1 (Marchantia emarginata Reinw et al., (Marchantiaceae)) and HmOMT1 (Haplomitrium mnioides (Lindb.) Schust., (Haplomitriaceae)). The four genes shared >60% level of sequence identity with one another but a <20% level of similarity with typical CCoAOMT or CCoAOMT-like sequences; they clustered with genes encoding animal catechol methyltransferases. The recombinant OMTs recognized phenylpropanoids, flavonoids and coumarins as substrates, but not catechol. MpOMT1 and PaF6OMT exhibited some differences with respect to their substrate preference, and the key residues underlying this preference were identified using site-directed mutagenesis. The co-expression of MpOMT1 and the Arabidopsis thaliana gene encoding S-adenosyl-L-methionine synthase in Escherichia coli was shown to be an effective means of enhancing the production of the pharmacologically active compounds scopoletin and oroxylin A. Liverwort OMTs are thought likely to represent an ancestral out-group of bona fide higher plant CCoAOMTs in evolution and have the potential to be exploited for the production of methylated flavones and coumarins.

Isolation and functional characterization of hydroxycinnamoyltransferases from the liverworts Plagiochasma appendiculatum and Marchantia paleacea.

Hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase (HCT, EC: 2.3.1.133) is a key metabolic entry point for the synthesis of monolignols in vascular plants; however, little is known about HCT in liverworts. Here, the isolation and characterization of HCTs encoded by the two liverwort species, Plagiochasma appendiculatum and Marchantia paleacea, are described. The sequences of the two enzymes harbor features typical of BAHD family members, except for the presence of a stretch of >100 residues that are not represented in higher plant HCTs. When truncated versions of both genes, which were constructed to clarify the significance of these extra residues, were investigated, it became apparent that the full-length and the truncated gene products shared similar catalytic activity and recognized the same substrates in vitro. They also functioned equivalently in vivo either when transiently expressed in tobacco to cause a higher total production of CGA (5-CQA) and 4-CQA or stably expressed in liverworts to accumulate the lignin-like contents. A structural model of MpHCT suggests that its active site bind to its substrate similar to that of Arabidopsis thaliana HCT. While truncated forms of HCT were deposited in the nucleocytoplasm, the full-length versions occurred exclusively in the cytoplasm. The conclusion is that liverworts produce bona fide HCTs that represent a point of departure in studying the evolution of lignin synthesis in plants.

The Functional Characterization of a Site-Specific Apigenin 4'-O-methyltransferase Synthesized by the Liverwort Species Plagiochasma appendiculatum.

Apigenin, a widely distributed flavone, exhibits excellent antioxidant, anti-inflammatory, and antitumor properties. In addition, the methylation of apigenin is generally considered to result in better absorption and greatly increased bioavailability. Here, four putative Class II methyltransferase genes were identified from the transcriptome sequences generated from the liverwort species Plagiochasma appendiculatum. Each was heterologously expressed as a His-fusion protein in Escherichia coli and their methylation activity against apigenin was tested. One of the four Class II OMT enzymes named 4'-O-methyltransferase (Pa4'OMT) was shown to react effectively with apigenin, catalyzing its conversion to acacetin. Besides the favorite substrate apigenin, the recombinant PaF4'OMT was shown to catalyze luteolin, naringenin, kaempferol, quercetin, genistein, scutellarein, and genkwanin to the corresponding 4'-methylation products. In vivo feeding experiments indicated that PaF4'OMT could convert apigenin to acacetin efficiently in E. coli and approximately 88.8 µM (25.2 mg/L) of product was synthesized when 100 µM of apigenin was supplemented. This is the first time that a Class II plant O-methyltransferase has been characterized in liverworts.

Cloning and functional characterization of a phenolic acid decarboxylase from the liverwort Conocephalum japonicum.

Some commercially important vinyl derivatives are produced by the decarboxylation of phenolic acids. Enzymatically, this process can be achieved by phenolic acid decarboxylases (PADs), which are able to act on phenolic acid substrates such as ferulic and p-coumaric acid. Although many microbial PADs have been characterized, little is known regarding their plant homologs. Transcriptome sequencing in the liverworts has identified seven putative PADs, which share a measure of sequence identity with microbial PADs, but are typically much longer proteins. Here, a PAD-encoding gene was isolated from the liverwort species Conocephalum japonicum. The 1197 nt CjPAD cDNA sequence was predicted to be translated into a 398 residue protein. When the gene was heterologously expressed in Escherichia coli, its product exhibited a high level of PAD activity when provided with either p-coumaric or ferulic acid as substrate, along with the conversion of caffeic acid and sinapic acid to their corresponding decarboxylated products. Both N- and C-terminal truncation derivatives were non-functional. The transient expression in tobacco of a GFP/CjPAD fusion gene demonstrated that the CjPAD protein is expressed in the cytoplasm. It is first time a PAD was characterized from plants and the present investigation provided a candidate gene for catalyzing the formation of volatile phenols.

Functional characterization of a Mg(2+)-dependent O-methyltransferase with coumarin as preferred substrate from the liverwort Plagiochasma appendiculatum.

Coumarins (1,2-benzopyrones), which originate via the phenylpropanoid pathway, are found ubiquitously in plants and make an essential contribution to the health of the plant. Some natural coumarins have been used as human therapeutics. However, the details of their biosynthesis are still largely unknown. Scopoletin is derived from either esculetin or feruloyl CoA according to the plant species involved. Here, a gene encoding a O-methyltransferase (PaOMT2) was isolated from the liverwort species Plagiochasma appendiculatum (Aytoniaceae) through transcriptome sequencing. The purified recombinant enzyme catalyzed the methylation of esculetin, generating scopoletin and isoscopoletin. Kinetic analysis shows that the construct from the second Met in PaOMT2 had a catalytic efficiency for esculetin (Kcat/Km) of about half that of the full length PaOMT2, while the Kms of two enzymes were similar. The catalytic capacities of the studied protein suggest that two routes to scopoletin might co-exist in liverworts in that the enzyme involved in the methylation process participates in both paths, but especially the route from esculetin. The transient expression of a PaOMT2-GFP fusion in tobacco demonstrated that PaOMT2 is directed to the cytoplasm.

Functional characterization of a plastidal cation-dependent O-methyltransferase from the liverwort Plagiochasma appendiculatum.

Caffeoyl CoA O-methyltransferases (CCoAOMTs), known to be involved in phenylpropanoid metabolism and lignin synthesis, have been characterized from several higher plant species, which also harbor CCoAOMT-like enzymes responsible for methylation of a variety of flavonoids, anthocyanins, coumarins and phenylpropanoids. Here, a gene encoding a CCoAOMT (PaOMT1) was isolated from a sequenced cDNA library of the liverwort species Plagiochasma appendiculatum, a species belonging to the Family Aytoniaceae. The full-length cDNA sequence of PaOMT1 contains 909 bp, and is predicted to encode a protein with 302 amino acids. The gene products were 40-50% identical to CCoAOMT sequences of other plants. Experiments based on recombinant PaOMT1 showed that the enzyme was able to methylate phenylpropanoids, flavonoids and coumarins, with a preference for the flavonoid quercetin (19). Although the substrate selectivity and biochemical feature of PaOMT1 is similar to CCoAOMT-like enzymes, the sequence alignment results indicated PaOMT1 is closer to true CCoAOMT enzymes. A phylogenetic analysis indicated that PaOMT1 is intermediate between true CCoAOMTs and CCoAOMT-like enzymes. The transient expression of a PaOMT1-GFP fusion in tobacco demonstrated that PaOMT1 is directed to the plastids. PaOMT1 may represent an ancestral form of higher plant true CCoAOMT and CCoAOMT-like enzymes. This is the first time an O-methyltransferase was characterized in liverworts.

A single amino acid determines the catalytic efficiency of two alkenal double bond reductases produced by the liverwort Plagiochasma appendiculatum.

Alkenal double bond reductases (DBRs) catalyze the NADPH-dependent reduction of the α,ß-unsaturated double bond of many secondary metabolites. Two alkenal double bond reductase genes PaDBR1 and PaDBR2 were isolated from the liverwort species Plagiochasma appendiculatum. Recombinant PaDBR2 protein had a higher catalytic activity than PaDBR1 with respect to the reduction of the double bond present in hydroxycinnamyl aldehydes. The residue at position 56 appeared to be responsible for this difference in enzyme activity. The functionality of a C56 to Y56 mutation in PaDBR1 was similar to that of PaDBR2. Further site-directed mutagenesis and structural modeling suggested that the phenol ring stacking between this residue and the substrate was an important determinant of catalytic efficiency.

Production of arachidonic and eicosapentaenoic acids in plants using bryophyte fatty acid Delta6-desaturase, Delta6-elongase, and Delta5-desaturase genes.

The liverwort Marchantia polymorpha L. synthesizes arachidonic (ARA) and eicosapentaenoic acids (EPA) from linoleic and alpha-linolenic acids respectively by a series of reactions catalyzed by Delta6-desaturase, Delta6-elongase, and Delta5-desaturase. Overexpression of the M. polymorpha genes encoding these enzymes in transgenic M. polymorpha plants resulted in 3- and 2-fold accumulation of ARA and EPA respectively, as compared to those in the wild type. When these three genes were introduced and co-expressed in tobacco plants, in which long-chain polyunsaturated fatty acids (LCPUFAs) are not native cellular components, ARA and EPA represented up to 15.5% and 4.9% respectively of the total fatty acid in the leaves. Similarly in soybean, C20-LCPUFAs represented up to 19.5% of the total fatty acids in the seeds. These results suggest that M. polymorpha can provide genes crucial to the production of C20-LCPUFAs in transgenic plants.