| Antarctica is located in the most south of the earth and is itself surrounded by the Southern Ocean,making it isolated with other lands.It is 1.24 billion square kilometers,about one tenth area of the earth.However,only about 850 kinds of plant were found in this area and most of them were cryptogams.Bryophytes,the most ancient terrestrial plants,represent a class of plant taxa that are transferred from aquatic to terrestrial.And they are the dominate producers in the terrestrial flora.UV-B radiation is one of the most important factors that limits the plant growth.Bryophytes adapted the severe surroundings depending on special mechanisms of their metabolic pathways and genes.Flavonoids are extensively distributed in the plants from higher plants to lower plants.They are the most diverse compounds of polyphenol in plant secondary metabolites and they fulfill a variety of functions including anti-UV radiation and anti-oxidant process and so on.The flavonoids biosynthetic pathway in higher plant has been well studied.Flavonoids are synthesized through the phenylpropanoid pathway,transforming phenylalanine into 4-coumaroyl-CoA,which finally enters the flavonoid biosynthesis pathway.Then chalcone synthase(CHS)condenses 4-coumaroyl-CoA and malonyl-CoA to form the open-chain flavonoid naringenin chalcone,which is converted to naringenin by chalcone isomerase(CHI).Naringenin as the precursor of flavanone 3?-hydroxylase(F3H),flavonol synthase(FLS),flavone synthase(FNS)accounts for the synthesis of several classes of flavonoids.However,the flavonoid metabolism pathway in bryophytes remains unclear.Up to now,only the CHS,CHI,and FNS have been cloned and reported.The harsh environment in Antarctic territorial would confer a unique approach for the plants to survive.In this study,we analyzed the phenotypic characteristics and the flavonoids HPLC profiling of Antarctic moss Pohlia nutans under UV-radiation.The CHS family genes of P.nutans were cloned by RACE techniques and Phylogenetic analysis of CHS was also performed.A flavone synthase gene(PnFNS)from P.nutans was also cloned into E.coli and Arabidopsis to investigate the function.This study contributes to clarify the function of the flavonoid metabolism pathway in moss in the processes of acclimatizing and adapting the rigorous climate in Antarctic continent.More details are below.1 Effect of UV-radiation on Antarctic moss Pohlia nutansCompared to a moss(Pohlia proligera,collected from Shandong Province),Antarctic moss Pohlia nutans is more resistant to UV-radiation.After UV treatments for 100 h,the stems of Antarctic moss gametophyte were altered to brown from green,and gradually changed to light brown after recovery for 12 h in the dark without UV-B radiation.While the top of gametophyte stems of Pohlia proligera were withered to death to some extent under the same condition.And then the flavonoids of the Antarctic moss wre extracted and detected by HPLC.We discovered that UV radiation not only promoted the content of the flavonoids but also stimulated to produce new flavonoids.2 Characterize the chalcone synthase family genes in Antarctic moss Pohlia nutansChalcone synthase commits the first key step reaction in the flavonoid biosynthesis pathway,and take an essential role in response to environmental stress.In this paper,we isolated six chalcone synthase genes(Pn021,Pn444,Pn768,Pn088,Pn847 and Pn270)by RACE techniques.The length of these genes is about 1200bp.Amino acid sequence alignment analysis revealed that these genes shared about 24.0%-84.0%similarity to other species homologous genes.Pn444,Pn088,and Pn768 have conserved Cys164,His303 and Asn336(amino acid numbered by alfalfa protein)catalytic triad and GFGPG loop motifs,and two conserved substrate binding pockets.The phylogenetic tree of PnCHS was constructed by the nearest neighbor comparison method.The results showed that the CHS of bryophytes clustered into one branch and located in the middle of fungi and higher plants.Later,the transient expression vector 35S::Pn444::GFP/pB1221 was transferred into Arabidopsis protoplast by PEG-CaCl2 method.The location of the fluorescent signal was mainly located in the membrane and cytoplasm.Real time PCR analysis showed that the gene family expression patterns varied depended on the stress conditions.These genes were up regulated after UV treatment,while they were down regulated in the cold environment.3 Functional analysis of flavone synthase enzyme from Antarctic moss Pohlia nutans in vitro and in vivoFlavone synthase is one of the important 2-Oxoglutarate-dependent dioxygenase(20DD)in the flavonoids biosynthetic pathway,which converts flavanones into flavones directly by introducing a double bond between the C-2 and C-3 residues.A flavone synthase(PnFNS)gene was cloned from Antarctic moss Pohlia nutans and the deduced amino acid sequence shared 20.0-80.0%similarity compared with other species homologous genes.Sequence alignment revealed that it contained the featured conserved domains HXDX?50HX?10RXS motif,Fe2+binding site,2-ketoglutaric acid binding site,and naringenin catalytic sites.Phylogenetic analysis showed that PnFNS formed a clade with Physcomitrella patens.Homology model of PnFNS was constructed based on Arabidopsis ANS structre(2brt).The transient expression 35S::PnFNS::GFP/pBI221 vector was constructed and transferred into Arabidopsis protoplast by PEG-CaCl2 method.The location of the fluorescent signal was mainly located in the membrane and cytoplasm.To get the constitutive expressed protein,PnFNS-pET28a was constructed and transferred into E.coli.The recombinant protein can only catalyze naringenin to apigenin in vitro.The catalytic activity of PnFNS in the reaction solution is very low below pH4.0.The catalytic activity of enzyme elevates with the increase of pH between pH5.0-7.0,and drops rapidly when pH>7.0.The gradient temperature reaction showed that PnFNS was insensitive to low temperature and had higher activity at 0-20 ?.The effects of different irons to the recombinant protein were investigated at pH7.0 and 15?.The results indicated that Mg2+,Ca2+,Mn2+,and Cu2+could promote the enzyme activity to some extent while Co2+ decreased the enzyme activity.The activity was almost inhibited when the Na2EDTA chelator added,which indicated that the enzyme was an ion dependent enzyme.In the substate feeding assay,Arabidopsis seeds(3 mg)were cultured in 1/2MS liquid medium with or without 200?M naringein in the flasks for 10 days.There was no difference among the growth of the wild type(WT)and over expression(OE)strains in the medium without narginein.The growth of OE strains was markedly better than the WT in the medium containing 200 ?M naringein though they were all growth inhibited.The flavonoids were extracted and identified by HPLC.There was no naringein accumulation in WT and OE strains.Interestingly,apegin accumulation was detected in the OE strains,while an unknown flavonoid was detected in the wild type Arabidopsis when cultured in the 200 ?M naringein.This unknown flavonoid compound was further identified by mass spectrometry and discovered that it was a naringein derivative.These results indicated that PnFNS probably catalytic the same reaction both in vivo and vitro.Arabidopsis seedlings(3 weeks old)were cultured with a supplement of UV-Blamp(20 ?W/cm2)for 6 h.After recovery for 2days,the rosette leaf tissue was examined for UV damage and DAB staining was used for detecting the ROS(H2O2).The results indicated over expression PnFNS in Arabidopsis enhanced plant tolerance to UV radiation. |
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