| The unique geographical environment and extreme climate of Antarctica limit the growth of most terrestrial plants.Bryophytes are widely distributed and the dominate Antarctic vegetation.Researches have shown that Antarctic vegetation may originate from ancient terrestrial flora and has been separated from other continents for thousands of years.Intense ultraviolet radiation has significantly increased the flavonoids content in bryophytes and is also one of the reasons for the high genetic diversity of the Antarctic mosses.Flavonoids were used for filtering UV-B in plants.They play significant roles to adapting terrestrial environment.Flavonoid biosynthesis pathways are highly conserved which is an important branch of the phenylpropanoid metabolic pathway.Phenylalanine is catalyzed by three enzymes to produce the naringenin,which was subsequently mainly catalyzed by 2-oxoglutarate-dependent dioxygenase(2-ODD)family to form a variety of flavonoids.Flavonoids play an important role in various physiological functions such as organ coloration,UV-B resistance,insect resistance,disease resistance and cold resistance.They also have various pharmacological effects such as anti-cancer,anti-oxidation,anti-inflammatory,and softening blood vessels.At present,the 2-oxoglutarate dependent dioxygenase(2-ODD)in flavonoid metabolic pathways has been researched in eukaryotes such as Arabidopsis,maize,petunia,and other higher plants.FNS I from Plagiochasma appendiculatum was the only reported in cryptogam.The naringenin downstream branching pathway of flavonoid biosynthetic pathway is still unclear in bryophytes.In this dissertation,Antarctic moss(Pohlia nutans)differentially expressed genes were analyzed by the Illumina high-throughput sequencing technology after UV-B treatment,and the antioxidant pathway and flavonoid content of Pohlia nutans were also measured.The gene expression level of related pathway was analyzed.In addition,we cloned flavanone 3-hydroxylase(PnF3H)from Antarctic moss(Pohlia nutans),a key enzyme in flavonoids metabolic pathway,and the PnF3H catalytic activity and response to abiotic stress were studied in Arabidopsis.More details are below.Analysis of Antarctic moss Polia nutans to UV-B treatmentHigh-throughtput illumina sequencing was performed to analyze the transcriptome of Pohlia nutans without or with UV-B treatment(260 ?W/cm2,3 h).Using qvalue<0.005&|log2(Fold change)|>1 as the criteria for screening genes,analysis of the differential expression genes(DEGs)revealed that 581 unigenes were significantly up-regulated after UV-B treatment,while 249 unigenes were significantly down regulated.DEGs were involved in different signaling pathways,that they play an important regulatory role in response to UV-B radiation in Pohlia nutans.12 DEGs in the transcriptome were verified by qRT-PCR,and 10 genes expression changes were basically consistent with the results of transcriptome differential expression except for two genes,indicating that the data of the transcriptome was basically reliable.We also analyzed the flavonoid content and antioxidant enzymes activity after UV-B radiation.The content of flavonoids was increased significantly after UV-B treatment for 96 h,which was 1.65-fold than the control.After UV-B for 3 h,the content of malondialdehyde in Pohlia nutans increased,which was 1.67-fold of the control.The catalase activity of catalase(CAT),glutathione reductase(GR),and peroxidase(POD)increased significantly.Compared with the control group,it increased 5.4,2.8 and 1.6 fold,respectively.The response of PnF3H to abiotic StressA high-response UV-B radiation gene was selected from the transcriptome for further study.The gene was annotated as Flavanone 3-hydroxylase(F3H)and named PnF3H.The full-length of PnF3H cDNA contained a 1071 bp open reading frame and encoded flavanone 3-hydroxylase of 356 amino acids with a calculated molecular weight of 39.2 kDa and a pI of 6.21.The PnF3H was activated by 200 mM NaCl,low temperature(4 ?),drought(20%PEG 6000),UV-B radiation,and abscisic acid(ABA)by qRT-PCR.Multiple sequence alignments showed that PnF3H has conserved domains DIOX-N and the 2-OGII-xy,which are ubiquitous in F3H.The multiple alignments result of PnF3H showed that PnF3H was identical to the reported flavanone 3-hydroxylase sequence of other plant.The 3-D model of PnF3H is also similar to F3H.Phylogenetic analysis also showed that PnF3H belongs to F3H.PnF3H/GFP fusion protein by constructing a transient expression vector revealed that PnF3H is mainly distributed in the cytoplasm.PnF3H was transformed and constitutively expressed in Arabidopsis(AtOEl-3,called transgenes).Overexpression of PnF3H enhanced the resistance of transgenic plants to high salt stress during seed germination and early seedling development.PnF3H overexpression lines enhanced the resistance to high salinity stress during seed germination and early seedling development.On 1/2 MS agar medium containing 125 mM NaCl,the average germination rate of transgenic plants was 26%,while that of wild-type was 15%.The root length of the transgenes plants was also significantly longer than the control under 1 mM H2O2 treatment.The wild-type root length was 0.62 cm,and the average root length of over-expressed lines was 2.17 cm.The expression analysis of stress-response pathway-associated genes in Arabidopsis under stress treatament showed that AtAPXl,AtP5CS1,AtHKT1 and AtCAT1 were up-regulated in transgenic Arabidopsis after 200 mM NaCl treatment for 2 h.Compared with wild-type Arabidopsis,the expression levels of these genes in transgenic Arabidopsis were increased by 1.3-fold,1.9-fold,1.6-fold and 2.5-fold,respectively.Meanwhile,we also analyzed antioxidative enzyme activities after high salt stress.The results showed that the activities of SOD and CAT in transgenic lines were higher than those of wild type.The above results speculated that overexpression of PnF3H in Arabidopsis enhanced plants tolerance to high salinity and oxidative stress.Abscisic acid(ABA)is one of the important hormones in plants which related to seed germination,plant development,response to biotic stress and abiotic stress.Therefore,we studied the transgenic Arabidopsis response to ABA.The results showed that overexpression of PnF3H reduced the sensitivity to ABA in the plant seed germination and early seedling development stages.The germination rates of AtOEl,AtOE2 and AtOE3 in ABA medium containing 0.75?M ABA were 37.50%,43.75%and 52.50%,respectively.However,the wild type seed germination rate was only 18.75%.In the presence of ABA,the roots of transgenes plants were significantly longer than the wild type.On 1/2 MS agar medium containing 0.5 ?MABA medium,the root length of the wild-type plants was 1.1 cm,and the root length of the PnF3H-overexpressing plants was over 1.5 cm.The ABA content using LC-MS/MS revealed that the ABA content in transgenes lines was significantly lower than that of wild type plants.After 50 ?M ABA for 2 h,the key regulatory genes ABI4 and ABI5 in the ABA pathway were significantly down-regulated in PnF3H overexpressing lines.The results showed that overexpression of PnF3H inhibited ABA biosynthesis and signal transduction pathways in transgenes Arabidopsis.Naringenin,a precursor of flavonoids,can be catalyzed by flavanone 3-hydroxylase leading to produce dihydrokaineol.Exogenously added naringenin inhibits the roots growth and geotropism by inhibiting auxin transport.We studied the growth of Arabidopsis in liquid cultures containing 200 p.M naringenin.There is no difference between the wild-type and the transgenic Arabidopsis without naringenin treatment.But the transgenic Arabidopsis was significantly stronger than the wild-type Arabidopsis with 200 ?M naringenin treatment.The transgenic lines have longer root length and larger leaves than these of the wild-type line.Flavonoid metabolism was also analyzed by HPLC and the results showed that flavonoid extracts of transgenic Arabidopsis produced two new substances compared to the wild type.The results showed that PnF3H can reduce the inhibition of on metabolize plant growth by metabolizing naringenin in transgenes lines.In conclusion,P.nutans adapt to ultraviolet radiation by accumulating flavonoids,enhancing antioxidant enzyme activities and abiotic stress related genes expression.A flavanone 3-hydroxylase from P.nutans,a key emzye of flaviods systhesis pathway,significantly increased the resistance of Arabidopsis to salt,oxidative stress,and ABA.Meanwhile,PnF3H can catalyze and decompose naringenin in Arabidopsis.The result indicated that the branch metabolic pathway of flavanone 3-hydroxylase may present in bryophytes and is involved in plant stress resistance.This paper contributed to reveal the adapting to extreme environments mechanism of Antarctic mosses and promote utilization of polar genetic resources. |
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