Exploration Of Phenylpropanoids Biosynthesis Key Enzymes Function And Bisbibenzyls Biosynthesis Regulatory Mechanism In Liverworts

The bryophytes(including liverworts,mosses,hornworts)pioneered the colonization of terrestrial habitats with adaptation from an aqueous to a non-aquatic environment.The bryophytes are phylogenetically placed between the algae and the vascular plants and they synthesize a range of metabolites.Almost all the liverworts contained oil bodies which produce a number of lipophilic terpenoids and aromatic compounds.These metabolites are structurally diverse and display a broad spectrum of biological activity,including toxicity to the bacteria,the fungi,and oxidation resistance,acting as insecticide.Based on the special evolution status and abundance of secondary metabolites with unique structure and diverse biological functions,liverworts are worthy of further study.The research on bryophyte chemistry has experienced a sharp increase,while the investigations on biology lag far behind.Therefore,the article from the viewpoint of molecular biology investigated the key enzymes on secondary metabolic pathway and regulatory mechanisms of the active ingredients in liverworts,by means of gene cloning and metabolic engineering.Then it will not only elucidate the origin and evolution of liverworts,but also lay the theoretical foundation for improving the bio-active ingredients content.Liverworts synthesize a range of metabolites bearing aromatic side chains,and the macrocyclic bisbibenzyls which display a broad spectrum of biological activity are rather specific to the liverworts.Similar to flavonoids,cyclic bisbibenzyls also come from the phenylpropanoid and polyketide pathway,as known through isotope tracking experiments.The biosynthesis of cyclic bisbibenzyls and flavonoids is driven by a general set of enzyme system in liverworts.Higher plant bHLH transcription factors(TFs)are responsible for a number of important biological functions,and in particular,those assigned to subgroup ?f regulate flavonoid metabolism.Hence it was thought likely that bisbibenzyl biosynthesis would be regulated in the liverworts by a member of the bHLH TF family.In previous study,a bHLH TF,PabHLH,was isolated from P.appendiculatum thallus and the preliminary evidence showed that PabHLH was involved in the regulation of bisbibenzyl biosynthesis in P.appendiculatum.The variation in bisbibenzyl content between P.appendiculatum thallus growing in greenhouse,axenically cultured thallus and callus was correlated with the abundance of PabHLH transcript.An important property of plant bHLHs is their transcriptional responsiveness to stress,and PabHLH was strongly induced when the plant material was challenged by exposure to either SA or MeJA.The PabHLH sequence was shown to harbor a conserved bHLH domain,even though the rest of its sequence was only distantly related to those of higher plant bHLHs.A phylogenetic analysis based on the bHLH sequence showed that PabHLH resembled most closely that of various regulators of the subgroup ?f protein of flavonoid synthesis.P.appendiculatum axenically cultured thallus were transformed by A.tumefaciens harboring the OE and RNAi transgenes,and the bisbibenzyl content of transgenic thallus was detected.The ricarrdin D(RD)content of the OE thallus was higher than that in vector control thallus.The knock-down of PabHLH,achieved by introducing an RNAi construct,led to a pronounced fall in the accumulation of bisbibenzyls in thallus samples.As a consequence of the induced variation in the content of RD,OE thallus significantly inhibited the growth of C.albicans hyphae,while the RNAi thallus were more permissive of fungal growth.As was the case with the bisbibenzyls,the flavonoid content of the thallus was higher in the OE than in the WT material,and lower in the RNAi material.In both callus and thallus,the over-expression of PabHLH up-regulatd PAL,4CL1,STCS1 and two genes encoding P450 cytochromes,and its RNAi-induced suppression down-regulated the same set of genes.The abundance of CHS and FNSI transcript was related to flavonoid accumulation in transgenic thallus.In response to hostile environments,the bryophytes have developed their own antioxidant and detoxification mechanisms in addition to producing bioactive molecules.The ?,?-unsaturated double bond reductase was found to catalyze the reduction of cytotoxic products of lipid peroxidation,at higher rates.?,?-unsaturated reductase plays an important role in the antioxidation and detoxification because of its hydrogenation of the ?,?-double bond.A search of the thallus transcriptome sequence datasets for M.paleacea,identified two candidate ?,?-unsaturated double bond reductase(DBR)homologs,namely MpDBR1 and MpDBR2.The full sequence clones of two DBRs were obtained using 3'-RACE,5'-RACE and other molecular cloning methods.Both sequences of MpDBR1 and MpDBR2 were shown to harbor conserved AXXGXXG and GXXS domain,even though their overall similarity was relatively low.Phylogenetic analysis showed that MpDBR1 was categorized into a subclade along with other characterized plant DBRs,while MpDBR2 failed into the other subclade along with some putative fungus DBRs,and the ?,?-unsaturated double bond reductase classified like MpDBR2 has never been found in other plants.The prokaryotic expression vectors of MpDBR1 and MpDBR1 were constructed and expressed in BL21(DE3),and the purified proteins were used to clarify their enzymatic characteristics.The recombinant MpDBR1 and MpDBR2 proteins both displayed typical activities of hydrogenation of p-coumaryl,caffeyl,coniferyl and 5-hydroxyconiferyl aldehyde to form corresponding reduced phenylpropenes,while the optimal temperature of MpDBR1 for the maximum activity was 37?,and the optimum pH value was 6.5;the optimal temperature of MpDBR2 for the maximum activity was 20?,and the optimum pH value was 7,and MpDBR2 was particularly sensitive to the reaction temperature.It revealed that there existed at least two subtypes of ?,?-unsaturated double bond reductase in liverwort M.paleacea,and the gene diversity of liverwort was also elucidated.In order to adapt to the transition from aquatic to terrestrial environment,corresponding physiological changes have taken place in bryophytes.The appearance of lignin during evolution is considered a crucial adaptation for plants to live on land.Athough the existence of lignin in bryophytes remains a matter of debate until further evidence,it is believed that bryophytes form 'uncondensed' monolignol-like molecules.At the same time,lignans have also been isolated from bryophytes.In higher plants,hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase(HCT)activity has been shown to be critical for the synthesis of lignin,however,HCTs have never been studied in liverworts.A search of the thallus transcriptome sequence datasets for M.paleacea and P.appendiculatum,identified two candidate HCT homologs,namely MpHCT and PaHCT.Both MpHCT and PaHCT harbored an HXXXD and a DFGWG motif,which were characteristic in the BAHD superfamily members.Although both sequences exhibited a considerable extent of similarity with the higher plant HCTs,each also featured a sequence of>100 residues in their central region which was not represented in their homologs.A phylogenetic analysis revealed that both of the liverwort enzymes were closely related to the P.patens homolog PpHCT and Selaginella moellendorfii homolog SmHCT.The sequences lay at the periphery of the cluster,which suggested they likely represent an ancestral out-group for the bona fide HCTs.The truncated forms of MpHCT and PaHCT along with SmHCT were constructed to clarify the role,if any,of these additional residues.No matter the short HCTs or long HCTs were able to accept p-coumaroyl-,caffeoyl-and feruloyl-CoA as their hydroxycinnamoyl donor and shikimate,quinate as hydroxycinnamoyl receptor.However they all prefer p-coumaroyl-CoA and shikimate as their substrates and the five forms of HCT behaved almost identically with one another on the catalytic efficiency of substrates,when transiently expressed in tobacco leaves,both MpHCT and PaHCT enhanced the total production of CGA and 4-CQA,as did that of their truncated forms MpHCT-Tr and PaHCT-Tr,and when four HCTs were stably expressed in liverworts,the lignin-like contents were also accumulated.In conclusion,the redundant stretch of HCTs in liverworts does not participate in the catalysis function.The sub-cellular localization of the HCTs was explored by fusing each of MpHCT,PaHCT,MpHCT-Tr,PaHCT-Tr and SmHCT in frame to the GFPN terminus,and introducing these constructs into N.tabacum leaf epidermal cells.While the truncated forms of HCT were deposited in the nucleocytoplasm,the full length versions were found in the cytoplasm only.The conclusion was that the liverworts produce bona fide HCTs,and their redundant stretchs are realated to the sub-cellular localization.