Functional Characterization Of O-methyltransferases For Flavonoids And Phenylpropanoids From Liverworts

Bryophytes are the most primitive terrestrial taxa in higher plants. With the adaption from an aqueous to a non-aquatic environment, abundant secondary metabolites are accumulated in bryophytes, including bibenzyls, flavonoids, phenylpropanoids and coumarins, whose alkylation need the catalysis of O-methyltransferase (OMTs). It is generally believed that the appearance of lignin represents the evolutionary event which allowed plants to adapt to a non-aquatic environment. And the existence of G or G/S moieties of lignin in cell wall is a primitive character of land plants. The prediction is that at least one CCoAOMT is involved in the synthesis of lignin-like compounds or lignans in liverworts.Methylation performed by S-adenosyl-L-methionine (SAM)-dependent O-methyltransferases is a common alkylation reaction in the biosynthesis of secondary metabolites. Plant OMTs have been categorized into two major classes. Class I OMTs require a Mg2+ for activity and are typically 26-29 kDa. The Class I OMTs were considered to methylate caffeoyl-CoA specifically and named caffeoyl CoA O-methyltransferases (CCoAOMTs). While CCoAOMT-like enzymes are able to methylate various flavonoids, anthocyanins, coumarins and aromatic esters. Class II OMTs are not Mg2+ dependant and commonly referred to as caffeic acid O-methyltransferases (COMTs). Their substrates include caffeic acid, coumarins, as well as various flavonoids and alkaloids. CCoAOMT involved in the methylation of phenylpropanoids and the biosynthesis of lignin which wide spread in higher plants, such as Mesembryanthemum crystallinum, Arabidopsis thaliana, ice plant, poplar and Vanilla planifolia. Most of the investigated CCoAOMT derived from higher plants with vascular tissue, while CCoAOMTs from bryophytes and ferns are rarely characterized.The cDNA library and the transcriptome sequencing of the Plagiochasma appendiculatum were constructed. Two genes encoding putative CCoAOMT were identified from the databases and isolated from plants and designated as PaOMTl and PaOMT2. The N-terminals of PaOMTl and PaOMT2 are about 150 bp longer than other plant CCoAOMTs. A phylogenetic analysis shows that PaOMTs were intermediate between CCoAOMT and CCoAOMT-like sub-clades, indicating that PaOMTs belongs to the family of Class I OMTs. The PaOMTl and PaOMT2 from P. appendiculatum may represent ancient O-methyltransferases. The proteins encoded by PaOMT1 and PaOMT2 were expressed as polyhistidine-tagged fusions in E. coli BL21(DE3) and purified for the investigation of their in vitro activity. PaOMT1 and PaOMT2 displayed activity against a broad range of substrates with aromatic vicinal dihydroxyl groups, while the compounds with single aromatic hydroxyl were not accepted as substrates. CCoA and phenylpropanoids are methylated in meta-position; Coumarin (esculetin) is methylated in meta-position or para-position; Flavonoids can be methylated in the position of 6-OH,3’-OH,4’-OH or 5’-OH. The preferred substrate of PaOMTl and PaOMT2 are quercetin and esculetin, respectively. PaOMTs display unusual 4-O-methylation specificities. PaOMTl and PaOMT2 share high sequence identity, but the preferred substrates are different. To investigate the influence on substrate preference of domains and key amino acids, domain exchange and site-directed mutagenesis are carried out on PaOMT2. It is suggested that while the N-terminus and the insertion loop can influence selectivity for esculetin, they are not the decisive factor. The truncated form of PaOMT1 and PaOMT2(PaOMT1-Tr and PaOMT2-Tr) obtained by PCR were 150 bp and 160 bp shorter, respectively. PaOMTs-Tr displayed a relatively lower activity with most of the compounds. Kinetic analysis shows that the longer N-terminal enhanced the turnover rate and did not affect enzyme affinity or subcellular localization. This is the first time the O-methyltransferases were characterized in liverworts and finding an O-methyltransferases with a preference for esculetin.Liverworts are in a special evolution statue in the plant group. The characteristic of CCoAOMTs in P. appendiculatum is different from that in other higher plants. To investigate the common sequence and catalysis characterization of CCoAOMTs in liverworts, transcriptome sequencing database of the Marchantia paleacea were analyzed and three unigenes showed high sequence homology with genes encoding CCoAOMT were identified and designated as MPaOMTl, MPaOMT2 and MPaOMT3. The phylogenetic tree revealed that MPaOMTs were in the root of the Class I OMTs sub-clades. MPaOMTl and PaOMT1 are homologous genes; The deduced MPaOMT2 sequence shared less 20％identity with OMTs from other plants, while a higher identity with mammalian catechol OMTs, with a preference for esculetin; The predicted MPaOMT3 sequence encoded a 28.7 kDa protein (lie in the Class I OMTs size range), which has high catalytic efficiency with caffeoyl alcohol and myricetin. Compared with other OMTs in liverworts, MPaOMT3 has higher turn over rate with phenylpropanoids. The N-terminal of MPaOMT3 may be a chloroplast transit peptide which is necessary for plastid location but inhibiting catalytic efficiency. To study the in vivo function of MPaOMTs, three genes were subcloned into the overexpression vector and then transformed into the Arabidopsis thaliana. The content of lignin monomer, phenylpropanoids and flavonoids of the transgenic lines and wild type will be analyzed. By studying the in vivo and in vitro function of liverworts OMTs systematically, the origin and evolutionary relationships of plant OMTs can be analyzed.