Functional Characterization Of O-Methyltransferases From Ferns

As the earliest vascular plant,ferns are a large group of higher plants.Ferns are very special in the evolution of plants:between lower plants and higher plants.Furthermore,due to the diversity of morphological structure,ferns are often used in the study of plant evolution and phylogeny.Lignin constitute the main component of plant cell wall,which plays an important role in plant growth and development.Lignin is synthesized by the polymerization of monolignol.O-methylation is an important step in the biosynthesis of lignin monomers.Two types of S-adenosyl-L-methionine(SAM)-dependent O-methyltransferases(OMTs)involved in lignin biosynthesis in plants,which are the caffeic acid 3-OMTs(COMTs)and the caffeoyl co-enzyme A OMTs(CCoAOMTs)and belong to SAM as methyl donors O-methyltransferases,respectively.Studies have shown that CCoAOMTs were mainly found in higher plants.At present,a COMT was identified from Selaginella moellendorffii.The CCoAOMTs involved in lignin biosynthesis have not been identified in ferns.In order to study the evolution and function of type I OMTs in ferns,51 species of ferns were selected from Eusporangiopsida,Protolyptosporangiopsida and Leptosporangiopsida.The gene annotated CCoAOMT was searched in the transcriptome database of these pteridophytes by using MsCCoAOMT gene sequence as a query sequence.The results showed that there was no CCoAOMTs gene in Eusporangiopsida.Four genes belonged to the Protolyptosporangiopsida and 3 7 genes belonged to Leptosporangiopsida.The phylogenetic tree analysis showed that two CCoAOMT genes in each of Polypodiodes amoena and Adinatum Capillus-veneris located in special evolutionary status in pteridophytes.One was located at the root of the evolutionary branch of the Protolyptosporangiopsida and another was located at the middle part of the evolutionary branch of the Leptosporangiopsida.Therefore,these four genes were chosen for further investigation.Firstly,the four genes were designated as PaCCoAOMTl,PaCCoAOMT2,AcCCoAOMT1 and AcCCoAOMT2,respectively.The sequences were submitted to NCBI website for BLAST,the results showed that they all belong to type I O-methyltransferase superfamily.The amino acid sequences were aligned with MsCCoAOMT and phylogenetic analysis was performed with the OMTs from other plants.These four OMT genes are evolutionarily located at the roots of the true CCoAOMT clade of higher plants.The sequence similarity with MsCCoAOMT was above 60%.The ORFs of four CCoAOMT genes were cloned from the cDNA of corresponding plants and were constructed into prokaryotic expression vectors by protein recombination technology.After induced by IPTG,soluble recombinant proteins were obtained and enzyme activities were carried out in vitro.The activity of MsCCoAOMT was used as a control.Four CCoAOMT genes showed high conversion efficiency for flavonoids.They can specifically methylate 3’-hydroxyl groups in the B ring and 6-hydroxyl groups in the A ring of flavonoids.They have no catalytic properties for apigenin,naringenin and kaempferol which contain single hydroxyl groups in the B ring.The proteins were able to methylate flavonoids containing two vicinal hydroxyl groups.For phenylpropanoids,four CCoAOMTs had the highest catalytic activity towards caffeoyl-CoA.Their in vitro conversion rate when presented with either caffeoyl CoA or certain flavonoids as substrate was comparable with that of the MsCCoAOMT.The transient expression of a PaCCoAOMT-GFP fusion gene in tobacco demonstrated that PaCCoAOMTs are likely localized in the cytoplasm in planta.In order to further research the function of CCoAOMTs gene in vivo,PaCCoAOMT1 and PaCCoAOMT2 were constructed into plant expression vector and transformed into model plant Arabidopsis thaliana,respectively.The contents of lignin and flavonol in overexpressed transgenic plants were determined and compared with wild type plants.The results indicated that the content of lignin in transgenic plants increased significantly,but there was no significant difference in the content of flavonol,indicating that both PaCCoAOMTs contributed to lignin synthesis and that neither was able to methylate flavonols in planta.Four CCoAOMT genes involved in the methoxylation of lignin monomers and flavonoids were identified in this study.It will contribute to understand the evolution of CCoAOMT involved in the biosynthesis of lignin.