Functional Characterization Of Two BHLH Transcription Factors And An O-methyltransferase From Liverworts

Bryophytes,which locate the special status in the evolution history of plant,harbor an abundance of secondary metabolites,including flavonoids,bibenzyls,terpenoids,lignin,and coumarin when evolved during the transition of marine to terrestrial plants.Bibenzyls,the specialized metabolites in liverworts,possess a wide spectrum of biological activities and pharmacological properties,which will be a lead compound for drug development from natural medicine.Flavonoids are widely distributed secondary plant metabolites and play key roles in human health and plant growth due to their extensive biological activities.Bibenzyls biosynthesis is derived from the phenylpropanoid pathway which has been demonstrated by isotope labeling method.Many structural genes in the biosynthetic pathway and their transcriptional regulator may be common to the early biosynthesis pathway of bibenzyls and flavonoid in liverworts.In the past several decades,the regulatory mechanism involved in the flavonoid biosynthesis has been well characterized in land plants,including many key structural genes and transcriptional regulator.For example,several bHLH transcriptional factors which belong to the subgroup ?f involved in regulation of the flavonoid biosynthesis have been characterized from Arabidopsis thaliana(TT8),Vitis vinifera(MYC1),Petunia hybrid(AN1)and Zea mays(IN1).In this study,PabHLH1 and MebHLH1 which belong to bHLH-MYC-N superfamily,was derived from the transcriptome sequencing database of Plagiochasma appendiculatum and Marchantia emarginata.The missing 3' sequence of PabHLH1 was recovered by 3'-RACE method,and then the full length cDNA of PabHLH1 and MebHLHl was isolated from the cDNA of the thallus of P.appendiculatum and M.emarginata,respectively.The analysis of the deduced amino acid sequence revealed that PabHLHl and MebHLH1 contain the conserved bHLH domain with strong similarity to the AtTT8 and VvMYC1,and gathered to the cluster of bHLH ?f subgroups in phylogenetic analysis.Both PabHLH1 and MebHLHl are transcriptional activators,locating in their N-terminal respectively,as demonstrated by transactivation assays using yeast cells.When 3 5 S::PabHLH1-GFP and 35S::MebHLH1-GFP were transiently expressed in Nicotiana benthamiana leaves,it was observed that they located in the nuclear.The overexpression vector of PabHLHl and MebHLH1 was introduced into Agrobacterium tumefaciens strain EHA105,and transformed into Arabidopsis using the floral dip method.The transcript levels of PAL,CHS,CHI,F3H,DFR and FLS were increased in both overexpression Arabidopsis lines than the wild type.Transgenic Arabidopsis plants that overexpress PabHLH1 exhibit an activation of flavonoid and anthocyanin biosynthesis than the wild type,without the impact on the biosynthesis of lignin illustrated by Wiesner's stained method and Acetyl bromide lignin assay.Moreover,there is no obvious change in flavonoids content between MebHLH1 transgenic A.thaliana and the wild type,but the lignin content of MebHLHl transgenic plants was higher than that of the wild type by stained with Wiesner's method.In the thallus of Pappendiculatum overexpressing PabHLH1,both the transcript levels of PaHLH and bibenzyls content were up-regulated.These results showed that PabHLH1 may regulate bibenzyls synthesis in P.appendiculatum.The functional identification of liverworts transcription factors is of great significance to elucidate the biosynthetic pathway before higher plant differentiation,and also provides a new way to improve the active components of natural products.The functional diversity of the flavonoids results in part from further modification to the hydroxyl groups via methylation,acetylation,or glycosylation.Methylation,which is particularly prominent,induces the physiological properties diversity and alters the chemical reactivity of flavonoids more hydrophobic.Here,a Mg2+-dependent flavone 6-O-methyltransferase has been identified in the liverwort species P.appendiculatum.The phylogenetic analysis showed that PaF60MT belong to Class ? OMT and gathered to the cluster of Rattus norvegicus catechol OMT.Enzyme activity analysis revealed PaF60MT shows a substrate preference for the flavone 6-OH group,like baicalein and scutellarein.It provides a safe,clean,and rapid means of converting baicalein and scutellarein into,respectively,the valuable products oroxylin A and hispidulin.