Biosynthetic Mechanism Exploration Of Phenylpropenes And Bis(Bibenzyls)in Plagiochasma Appendiculatum

In higher plants, bryophytes are the most primitive terrestrial taxa with adaptation from an aqueous to a non-aquatic environment. Liverworts are rich in structurally diverse secondary metabolites, and most of them show good biological activities. Therefore, the investigation of the biosynthetic pathway and regulatory mechanisms of the active ingredients in liverworts will not only lay the foundation for improving the content of bio-active ingredients, but also contribute to the elucidation of the origin and evolution of these valuble plant secondary metabolites.Plants generated α,β-unsaturated carbonyl compounds derived from the phenylpropanoid pathway when subjected to abiotic or biotic stress, and the compounds would produce toxic effects to cells. α,β-unsaturated reductase plays an important role in the detoxification of plants because of its capability of reducing alkenal double bond. In addition, this kind of enzymes have been become one of the important enzymes in chiral molecules synthesis because of its stereoselectivity in reduction of carbon-carbon double bond. In the present investigation, two cDNAs with high homology encoding DBRs were obtained from a Chinese liverwort Plagiochasma appendiculatum library and were designated as PaDBRl and PaDBR2. They were ligated into prokaryotic expression vector, expressed in BL21(DE3) and purified. Purified proteins were used for enzyme characterization. Moreover, the product standards were chemically synthetized. The recombinant PaDBR proteins displayed different level activities when using p-coumaryl, caffeyl, coniferyl and5-hydroxyconiferyl aldehyde as substrates to form corresponding reduced phenylpropenes. The enzyme kinetics results showed that PaDBRl and PaDBR2both used p-coumaryl aldehyde as the favorite substrate. Sequence alignment and protein molecular modeling showed that the imparity in catalytic rates of PaDBRs may be caused by the difference of a key amino acid, cysteine in PaDBR1while, tyrosine in PaDBR2. To elucidate this mechanism, site-directed mutagenesis technique was used to mutate cysteine-56in PaDBR1to aromatic amino acids and aliphatic amino acid. The results demonstrate that a mutation to one of the two coding region polymorphisms was sufficient to swap functionality, and it was further shown that the phenol ring stacking interaction between the aromatic residue acid and the bound p-coumaryl aldehyde was responsible for the difference in catalytic rates between the two homologs.Flavonoids and bis(bibenzyls) widely found in liverworts are with important physiological functions. Bis(bibenzyls) with biological activities can be used as lead compounds for drug research. Transcription factors play an important role in regulating the production of plant secondary metabolites. Ⅲf subfamily of bHLH transcription factors have been reported to regulate the biosynthesis of flavonoids in various plant species. To investigate if Ⅲf subfamily of bHLH transcription factors regulate the biosynthesis of both bis(bibenzyls) and flavonoids which derived from the same phenylpropanoid pathway in liverworts, in this study, a bHLH transeription factor, PabHLH, was isolated using5’-RACE method and its genomic DNA sequence was also obtained. The plant transformation vector fused GFP reporter gene was constructed and transformed into onion epidemal cells by Agrobacterium-mediated method. Laser scanning confocal microscope observation showed that PabHLH was targeted to the nuclear. The transcriptional activation ability was demonstrated by the yeast one hybridization using full length cDNA of PabHLH and its truncations. The response of PabHLH expression under MeJA and SA was examined, and it indicated that the PabHLH expression can be induced by abiotic stress. PabHLH gene was subcloned into the overexpression vector and RNAi vector and then transformed into the callus of Plagiochasma appendiculatum. Gene expression and the chemical contents of transgenic clones were analyzed. qRT-PCR showed that the PabHLH transcription level was higher in the over-expression callus while was decreased in the RNAi callus than that of the wilde type callus. Chemical contents analysis showed that Perrottetin E, iso-Marchantin C and neo-Marchantin A could be regulated by PabHLH.