The Preliminarily Function Analysis Of Dopamine ? Hydroxylase(DBH) In Arabidopsis

Dopamine ? hydroxylase(DBH),a biosynthetic enzyme,involved in the metabolism of Catecholamines(CAs).And it catalyzes dopamine(DA)to produce norepinephrine(NE).In mammals,the researches for DBH mainly focus on the genetic polymorphism of DBH.Howveer,there is currently no related research for the function of DBH in plant.CAs mainly include three kinds of substances:DA,NE and epinephrine(E)in plant.Previous studies have shown that CAs participate in important biological processes such as plant growth,development,antioxidant protection and disease resistance.At the same time,they can interact with plant hormones,induce flowering and regulate sugar metabolism.DBH is a key enzyme for CAs metabolism in plant.Therefore,the analysis of DBH protein function will benifit further study for the biological function of CAs in plants.In this paper,Arabidopsis dbh mutants and transgenic materials were used as the research object to explore the regulation of DBH in AB A-mediated stomatal signal pathway and NaCl stress.The interacting proteins of DBH were screened by yeast two-hybrid technology using Arabidopsis cDNA library,which provides important evidence and clues for further study of the molecular function of DBH.There was only one gene encoding DBH in Arabidopsis(Gene number:AT5G54830).By bioinformatics,we preliminarily determined that DBH was a membrane protein,so we selected two fregments from DBH:extracellular fragment(50-650 amino acid)and intracellular fragment(850-907 amino acid),which were constructed into bait vectors,and was used for screening the cDNA library of Arabidopsis by yeast two-hybrid method.Through yeast four-deficiency medium screening,gene sequencing and rotation validation ect.,nine gene-coding proteins interacting with DBH(the bait protein:50-650 DBH)were respectively AT5G48657(DEFENSE PROTEIN-LIKE PROTEIN),AT2G19520(FVE),AT4G14960(TUA6),AT2G20310(RIN13),AT2G27050(EIL1)?AT2G05990(ENR1),AT1G73500(MKK9),AT2G38270(GRX2)and AT1G29990(PFD6).Among them,PFD6 is a chaperone protein,which participates in the regulation of microtubule organiztion and stress response in plants.We confirmed the specific interaction between DBH and PFD6 by protein pull-down and bimolecular fluorescence complementary BiFC experiments.In addition,no interaction between DBH and PFD1,PFD2,PFD3,PFD4,or PFD5 was detected in yeast system.We cloned the promoter of DBH and constructed it into a binary expression vector of GUS(?-glucuronidase),and transferred it into wild-type(WT)Arabidopsis.Results of GUS staining showed that DBH promoter was highly expressed in the axial root,lateral root,cotyledon,guard cells,flowers and siliques of Arabidopsis,and DBH expression was higher in guard cell than epidermal pavement cell.The GUS expression decreased significantly in maturation zone of root after treatment with 10 ?M ABA and 150 mM NaCl.We further constructed the fusion expression vector of DBH-GFP driven by DBH promoter and successfully transferred it into dbh-1 T-DNA insertion mutantsmutant.Confocal observation showed that DBH-GFP protein fluorescence was strongly expressed in root,hypocotyl,seed,stigma,pollen,calyx,petal and guard cells.Using tobacco transient expression technology,we found that DBH protein was primarily located in the plasma membrane and Golgi apparatus.In order to further study the regulatory function of DBH in plant stress response,we purchased two T-DNA insertion mutant lines of DBH from ABRC(Arabidopsis Resource Center):SALK₀89031C(dbh-1)and CS882172(dbh-2).Homozygous mutants were identified by the identification method with three primers and qRT-PCR etc at the genomic level and transcriptional level.Sequencing results showed that the T-DNA insertionl sites of dbh-1 and dbh-2 mutants were localized in the 642 and 625 nucleotide sites from ATG initiation.Next we treated WT and dbh mutants with ABA.In Arabidopsis medium containing 1.5 ?M ABA,the green conversion rate of WT cotyledons was 17%,while dbh-1 and dbh-2 mutants was 75%and 45%,which was significantly higher than that of WT.The epidermis of leaves were treated with 50 ?M ABA and stomatal apertures were measured in WT and dbh mutants.The results showed that compared with WT,ABA-induced reactive oxygen species(ROS)and stomatal closure mutants were significantly inhibited in the dbh-1 and dbh-2,which suggested that DBH may positively regulate stomatal movement in the ABA signal pathway.Meanwhile,TUA6-GFP was used to observe the microtubule organiztion of guard cells in cotyledons of WT and dbh-1 mutant.After 10 ?M ABA treatment,the microtubules of WT were significantly depolymerized and stomata closed in guard cells,while the dbh-1 mutant was insensitive to ABA treatment in terms of microtubule organzition and stomatal movement.It suggested that DBH may respond to ABA signal stimulation by regulating microtubule organiztion,and the interaction of DBH-PFD may be involved in the signal pathway.In addition,we found that the water loss rate was no significant difference beween WT and dbh mutants under drought treatment.In summary,Using molecular biology,biochemistry,cell biology,plant physiology and other experimental techniques,we preliminarily confirm that DBH is located in the plasma membrane and Golgi apparatus,and interacts with molecular chaperone protein PFD6,which might together regulates the Arabidopsis response to NaCl and ABA stimulation.