Biological Function Of Flavonoid Biosynthesis 2-Oxoglutarate-Dependent Dioxygenase From Antarctic Moss Pohlia Nutans

Flavonoids are the largest class of polyphenolic secondary metabolites in plants.Flavonoids biosynthesis pathway starts with p-coumaroyl-CoA and malonyl-CoA to form naringenin,a key immediate precursor of flavonoids biosynthesis.Then,naringenin can be catalyzed by flavone synthase(FNSⅠ/Ⅱ)and isoflavone synthase(IFS)to generate flavone and isoflavones,respectively,or it can be hydroxylated to form dihydrokaempferol(DHK)by flavanone-3hydroxylase(F3H).The resulting DHK can serve as immediate precursors for the flavonols biosynthesis by flavonol synthase(FLS)and the biosynthesis of anthocyanins by dihydroflavonol reductase(DFR)and anthocyanin synthase(ANS).Among them,flavone synthase Ⅰ(FNSI),flavanone-3-hydroxylase(F3H),flavonol synthase(FLS)and anthocyanin synthase(ANS)all belong to 2-oxoglutarate/Fe(Ⅱ)-dependent dioxygenases(2-ODDs).Flavonoids play important roles in plants land colonization from aquatic to terrestrial lifestyle,but the downstream branch pathways of naringenin in the earliest terrestrial plant bryophytes remain unclear.Whether the terminal product anthocyanins can be synthesized in bryophytes is still controversial.Extreme climate conditions restrict the growth and development of Antarctic terrestrial plants.Mosses,one of the dominant terrestrial plants,are widely distributed in Antarctica,which have evolved a series of special physiological mechanisms to adapt tough environment.In this study,the biological functions of three 2-ODDs genes(Pn2-ODD1,Pn2ODD2 and PnFNSI)in flavonoid biosynthesis pathway were investigated,revealing the role in the adaptation of Antarctic moss to extreme environments.The main results are as follows:The full-length cDNA sequences of Pn2-ODD1 and Pn2-ODD2 encode 361 and 365 amino acids,respectively.Multiple sequence alignment analysis showed that Pn2-ODD1 shared 28.3%identity with Pn2-ODD2,which both possessed the conserved domains of the 2-ODDs:Fe2+binding site HxDxnH and the 2-oxoglutarate(2-OG)binding domain RxS.Pn2-ODD1/Pn2ODD2 were heterologously expressed in the moss model plant Physcomitrella patens to illustrate their roles in plant abiotic stress resistance.Under normal BCD medium,Pn2ODD1/Pn2-ODD2 had no significant effect in the growth of P.patens.However,when exposed to stress conditions,Pn2-ODD1/Pn2-ODD2 increased the resistance of P.patens to salt and drought stress,and decreased the sensitivity to the plant hormone abscisic acid(ABA),exhibited the larger gametophyte or protonema diameters.Arabidopsis thaliana is a widely used model organism in the plant kingdom,due to the easily quantitative stress-resistance indicators,clear flavonoids biosynthesis pathway and regulatory network.To explore the effects on plant flavonoid synthesis and abiotic stress resistance,we constructed the Arabidopsis heterologous expression vector and obtained the expressed-Pn2ODD1/Pn2-ODD2 Arabidopsis(AtOE).Constant light can induce the accumulation of plant flavonoids.We found that Pn2-ODD1/Pn2-ODD2 contributed to the accumulation of total flavonoids and anthocyanin in 5-day-old A rabidopsis(spectrophotometry analysis),which was 1.30 and 1.35 times,1.20 and 1.25 times,in contrast with WT plants,respectively.UPLCMS/MS was used to analyze the influence of Pn2-ODD1 on the flavonoid metabolomes in Arabidopsis seedlings.A total of 48 differential metabolites were screened.Among them,flavonols were the most abundant compounds.Notably,3 anthocyanins were detected,all upregulated.Since the enzyme which catalyzes the anthocyanin biosynthesis in bryophytes has not been reported,the effect of Pn2-ODD1/Pn2-ODD2 on anthocyanin in Arabidopsis was mainly studied.When induced by exogenous sucrose,Pn2-ODD1/Pn2-ODD2 enhanced the levels of anthocyanin in 17-day-old Arabidopsis.The targeted anthocyanin metabolomics showed that transgenic Pn2-ODD1/Pn2-ODD2 Arabidopsis had a 17.6%and 17.1%increase in the levels of total anthocyanins than that of the WT plants,respectively.In WT vs Pn2-ODD1,7 upregulated differential metabolites were identified,including 4 cyanidin compounds.In WT vs Pn2-ODD2,7 up-regulated(5 cyanidin compounds)and 1 down-regulated differential metabolites(1 petunidin)were screened.Meanwhile,we found that Pn2-ODD1/Pn2-ODD2 did not alter the flavonol compositions in A rabidopsis,mainly quercetin and kaempferol.However,heterologous expression of Pn2-ODD1/Pn2-ODD2 significantly increased the levels of flavonol.The contents of total flavonols,quercetin and kaempferol were dramatically increased in transgenic Pn2-ODD1/Pn2-ODD2 Arabidopsis,which was about 50.0%,65.0%and 53.0%,29.0%,30.0%and 25.0%higher than that of the WT plants,respectively.Naringenin inhibits the growth of plant roots and limits plant development by affecting the transportation of auxin.In contrast with the WT plants,Pn2-ODD1/Pn2-ODD2 significantly reduced the growth inhibitory effect on Arabidopsis,with the better root growth and leaf development,and promoted the accumulation of anthocyanins.On the normal medium,there was no significant difference in germination and root growth between WT and Pn2-ODD1/Pn2-ODD2-expressed Arabidopsis.Drought tolerance assay suggested that Pn2-ODD1/Pn2-ODD2 enhanced the survival rate,and contributed to the seed germination and root growth in Arabidopsis.When exposed to 0.2 M D-Mannitol,the germination rate and root length in transgenic Pn2-ODD1/Pn2-ODD2 Arabidopsis were about 1.50 and 1.20 folds,1.20 and 1.55 folds compared with WT plants.Pn2-ODD1/Pn2-ODD2 also markedly improved the tolerance of Arabidopsis to exogenous(H2O2)and endogenous oxidative stress(3-AT,a CAT inhibitor),displayed the longer root length and the greater lateral root numbers,the reduced reactive oxygen species(ROS)levels,and the enhanced total flavonoids and anthocyanins accumulation.Meanwhile,Pn2-ODD1/Pn2-ODD2 decreased the ABA sensitivity in Arabidopsis.Additionally,Pn2-ODD1 conferred the resistance of Arabidopsis to salt and UV-B stress by improving the antioxidant ability.Previously,the catalytic activity assay in vivo and in vitro showed that PnFNSI was a type Ⅰflavone synthase,which catalyzed naringenin to form apigenin.Here,the functions of PnFNSI in plant abiotic stresses were further investigated in P.patens and Arabidopsis.Under normal BCD medium,there was no detectable difference in the growth of gametophytes between the WT and transgenic PnFNSI plants.However,when grown in stress conditions,heterologous expression of PnFNSI increased the tolerance of P.patens and Arabidopsis to salt,drought stress and ABA.Furthermore,under UV-B radiation,PnFNSI decreased ROS and malondialdehyde(MDA)levels,increased chlorophyll and total flavonoids contents,and improved glutathione reductase(GR)activity.Additionally,the expression patterns of ROS scavenger genes were markedly up-regulated in transgenic PnFNSI Arabidopsis.The flavonoid metabolomes analysis of Arabidopsis under UV-B radiation suggested that 7 flavonoid differential metabolites(all up-regulated)were identified,and most of them were flavones.Additionally,in the presence of H2O2 treatment,when compared with the WT plants,transgenic PnFNSI Arabidopsis exhibited the longer root length and more lateral roots,the decreased ROS and MDA accumulation,the down-regulated ROS-generating genes expression and the increased antioxidant enzyme genes expression.In conclusion,the heterologous expression of both Pn2-ODD1,Pn2-ODD2 and PnFNSI in P.patens could enhance the resistance to NaCl and drought stress,and reduce the sensitivity to ABA.In Arabidopsis,Pn2-ODD1 and Pn2-ODD2 significantly increased the accumulation of anthocyanins and flavonols in plants,and enhanced the tolerance of plants to abiotic stresses.Pn2-ODD1 conferred the tolerance of Arabidopsis to salt,drought,UV-B and oxidative stress and ABA by improving the ion transportation levels and antioxidant ability.Pn2-ODD2 only increased the resistance of Arabidopsis to drought and oxidative stress and ABA,which was correlated with reducing ROS production and improving antioxidant capacity.In addition,PnFNSI promoted the resistance of Arabidopsis to drought,UV-B and oxidative stress by increasing ROS-scavenging ability in Arabidopsis.In this study,we explored the biological functions of three key genes in flavonoid synthesis pathway in P.patens and Arabidopsis,which provided a theoretical basis for revealing their roles in the adaptation of Antarctic moss to the environment and had a potential significance in seeking excellent stress resistance genes of Antarctic moss for crop improvement.