PnODD1,a 2-oxoglutarate-dependent Dioxygenase Gene Of Pohlia Nutans,Plays An Important Role In Plant Resistance To Stress

Antarctica is located at the south of the earth.The continent with the highest average altitude in the world,its climate is cold,dry and small annual precipitation,it is known as the "white desert".Extreme environments pose great challenges to the survival of plants,and bryophyte is the main vegetation group in the Antarctic land.The harsh environment produces extremely rich secondary metabolites in their bodies.Flavonoids are one of the main representatives,and they play an important physiological role in plant growth and abiotic stress response.Although the related genes in flavonoid metabolism pathways have been well studied in advanced plants,there are few studies in lower groups of advanced plants.Such as chalcone synthase of Physcomitrella patens,chalcone isomerase of Marchantia polymorpha L.and flavone synthase of Plagiochasma appendiculatum.2-oxoglutarate-dependent dioxygenase(2-ODD)is the second largest family of enzymes in the plant genome,members of which participate in various oxygenation/hydroxylation reactions in plants and animals,and plays an important role in plant and animal development,transcriptional regulation,nucleic acid modification/repair,fatty acid metabolism,and secondary metabolite biosynthesis.2-ODD is one of the key family genes that form the diversity of flavonoids.In this study,a highly responsive gene was screened from the transcriptome of Pohlia nutans under UV-B stress,and bioinformatics analysis was the key 2-ODD family gene of flavonoid metabolic pathway,named PnODD1{Pohlia nutans 2-ODD1).The full-length sequence of the gene was cloned,the structural characteristics of the protein were analyzed,and its biological function was studied.It provided an important reference for revealing the special mechanism of mosses adaptation to the polar environment and enriched the genetic resources of Antarctic plants.1.The effect of strong ultraviolet radiation(UV-B)on Pohlia nutans and Pohlia crudaPrevious studies have found that Antarctic Pohlia nutans has stronger UV radiation resistance than Pohlia proligera,and this advantage is related to flavonoids.This paper further compares the difference in response to ultraviolet radiation between Pohlia nutans and Pohlia cruda at room temperature.After UV-B(70)pW/C1cm2)radiation treatment for 12 h,the whole plant of Pohlia cruda is bent and unkempt,the leaves are shrunk,the color is darkened,and some top leaves are dead and severely damaged.The growth state of Pohlia nutans was slightly yellower than before treatment,but the growth state was better.After UV-B treatment,the content of active oxygen free radicals and MDA in Pohlia nutans was significantly lower than that in Pohlia cruda.The total amount of flavonoids were significantly higher than that of Pohlia cruda.At the same time,the activities of SOD and POD increased in both mosses,but the enzyme activity in Pohlia cruda increased more.The above results indicated that the resistance of Pohlia nutans to ultraviolet radiation is stronger than that of Pohlia cruda.One reason is dependent on the more flavonoids,and the other is mainly dependent on the stable antioxidant enzyme system.2.Cloning,expression analysis and functional study of PnODDl gene from Pohlia nutansIn this study,a highly responsive UV-B gene was selected from the transcriptome of Pohlia nutans,which was cloned,expressed and functionally studied.Bioinformatics analysis found that the gene is a 2-oxoglutarate-dependent dioxygenase gene(PnODDl),it also resembles a LDOX/ANS gene.The length of PnODD1 gene is 1510 bp and contains a 1050 bp ORF,encoding 346 amino acids,its molecular weight is 38.4 kD.Multiple sequence alignment revealed that the sequence similarity between PnODD1 and other 2-ODD superfamily genes was 39.5-48.9%.This gene has the typical N-terminally conserved DIOX N domain and 2-OD-Fel1 Oxy domain.In addition,PnODD1 is consistent with other 2-ODD genes,it has Fe2+ binding site residues(His219,Asp221 and His 275)and AKG(2-oxoglutarate)binding site residues(Tyr2O4,His219,Asp221,His 275,Arg285 and Ser287).In order to present the spatiotemporal structure,the homology modeling of PnODD1 protein was performed by SWISS-MODEL,the spatial structure showed that the PnODD1 protein consisted of α-helix,β-sheet and random coil in the central part to form a cavity,which is the Fe2+ binding site.Phylogenetic tree indicated that PnODDI clustered with the Plagiochasma appendiculatum FNS I(Flavone synthase I),showed the close relationship between them.The 35S::PnODD1::GFP/pBI221 transient expression vector was constructed and transferred into Arabidopsis protoplasts,the subcellular localization of the protein was studied.It was found that PnODDl was mainly distributed in the cell membrane and cytoplasmic matrix.The response of PnODDl gene to abiotic stress was analyzed by RT-qPCR.The results showed that PnODDl gene was up-regulated under drought,UV-B and salt stress,suggesting that PnODDl helped Pohlia nutans for inhibitting the stress.The PnODD1 gene was constitutively expressed in Arabidopsis to study its biological function.Under normal growth conditions,overexpression of the PnODD1 gene had no effect on the growth and development of Arabidopsis.Overexpression of PnODDl enhanced the resistance of Arabidopsis to high salt stress.Under 125 mM NaCl stress,the seed germination numbers of WT,AtOEl and AtOE2 were 46.7%,91.7%and 86.7%,respectively.The seed germination numbers of WT,AtOEl and AtOE2 under stress of 150 mM NaCl were 35.0%,71.6%and 75.3%;The root length of AtOEs was significantly longer than that of WT under treatment with 125 mM or 150 mM NaCl.At the same time,overexpression of PnODDl enhanced the resistance to osmotic stress for Arabidopsis.AtOEs showed higher seed germination rate,longer main root length,heavier fresh weight and more number of lateral root after treatment with 0.1 M or 0.3 M D-mannitol.After 12 h of UV-B treatment,the WT was significantly inhibited,showing small stems,leaf wrinkles and small leaf surface area,while AtOEs had no difference compared with previous growth conditions.Overexpression of PnODD1 attenuated the inhibition of Arabidopsis growth by UV-B.Stress can lead to a sharp increase of ABA in plants,which can inhibit seed germination and affect plant growth.Overexpression of PnODDl reduced the sensitivity of Arabidopsis to ABA.After 1.0μM ABA treatment,the seed germination of AtOEl and AtOE2 was 93.3%and 91.7%,respectively,which was significantly higher than 85%of WT The root lengths of AtOE1 and AtOE2 were 1.27 and 1.36 times of WT,respectively.The total flavonoids metabolism profile of AtOEs revealed that this gene affects the species and content of flavonoids in Arabidopsis and provides a material basis for enhancing abiotic stress.In addition,under drought stress,PnODDI enhanced the drought resistance of overexpressing Arabidopsis by affecting the expression of drought response genes(P5CS1 and AtKIN1)and ABA signaling pathway-related genes(AtABF3,AtMYB2,AtRD22 and AtDREB2A).By constructing the prokaryotic expression vector,the PnODD1 gene was heterologously expressed and the expression conditions were optimized,and the three induction conditions with the highest expression of recombinant protein were determined(induction temperature 16℃,IPTG concentration 0.1 mM,induction time 24 h).The HLPC was used to analyze the products of recombinant proteins with various substrates,and it was found that PnODDl protein can catalyze the formation of new compounds from Naringenin,consistent with the NAR feeding experiment in vitro of overexpressing PnODD1 Arabidopsis.In conclusion,Pohlia nutans showed greater resistance to UV-B radiation due to its many flavonoids and its stable antioxidant enzyme system.Bioinformatics analysis shows that PnODD1 contain the typical DIOX N domain,2-OG-FeII_Oxy domain.Fe2+ binding site and 2-oxoglutarate binding site,belongs to the 2-ODD protein family in the flavonoid metabolic pathway.It lays a theoretical foundation for revealing its research on catalytic decomposition of NAR.The PnODD1 gene enhances the resistance of plants to abiotic stress by affecting the content of flavonoids and stress-responsive genes in Arabidopsis.This study provides an important reference for the special mechanism of moss adaptation to the polar environment,enriching the genetic resources of Antarctic plants.