The Roles Of Brassinosteroids In Promoting Petiole Elongation And Regulating Cellulose Synthesis In Carrot

Carrot(Daucus carota L.),an important kind of root vegetable,is a member of Apiaceae family.The flesh straight roots of carrot contain abundant nutrition,are good foods and scientific research object.Nutrients in carrot roots include carotenoid,anthocyanin,dietary fiber,vitamins,mineral and lipid.Study on carotenoid and anthocyanin is more,while report on dietary fiber in carrot is less.Dietary fiber can improve the gastrointestinal function of human.In carrot,insoluble fiber cellulose is the main type of dietary fiber.Cellulose is the main component of cell walls,can provide mechanical support for plants.Celluloses are catalyzed by cellulose synthase complex on the plasma membrane.Cellulose synthase complex is always constituted by cellulose synthase enzyme(CESA)heterotrimer and some other proteins.As the main component of cell walls,cellulose will be synthezed and recombined during the cell progresses of cell division,cell expansion and cell elongation.Brassinosteroids(BRs)is an important plant hormone,and play critical roles in affecting cell division,cell expansion,and cell elongation.To date,some studies on how BRs regulating cellulose synthesis have been reported,while the quantity is less.Related report in carrot is much less.In this study,exogenous 2,4-epibrassinolide(2,4-EBL)was used to treat carrot seedlings,and results showed that exogenous 2,4-EBL can significantly improve the elongation of carrot petioles.Exogenous 2,4-EBL was found enhancing the biosynthesis of gibberellins(GA)and cellulose when promoting the elongation of carrot petioles.DCAR₀09214 is a BRs metablish gene in carrot.The phenotype of DCAR 009214 over-expression lines is BRs-deficient,and the height and cellulose content of the over-expression lines are obviously lower than that of wild Arabidopsis.These results suggested that defect of BRs will reduce the cellulose synthesis in primary cell walls.By genome analyzing the cellulose synthase gene family,two DcBEH4(DCAR₀19605 and DCAR₀05165)were found to regulate cellulose synthesis by regulating the expression of CESA genes.The main results are as follows:1.Measure the contents of BRs at different developmental stages by ELISA.Contents of BRs keep changing at different developmental stages and in different organs,which suggested that the function forms of BRs are depended on developmental stages and organs.By foliar spraying,elongation of carrot petioles was obviously promoted by 2,4-EBL.In addition,the concentration of 0.5 mg/L was found to be the suitable concentration for treating carrot seedlings.2.0.5 mg/L 2,4-EBL was applied to treat carrot,and the GA and cellulose content in the petioles of treated carrots were measured.Results showed that exogenous BRs obviously promoted the synthesis of GA and cellulose in carrot petioles.Cell structure analysis revealed that exogenous BRs obviously promoted the elongation of carrot petiole cells.To found genes have potential relationship with DcBZR1/BES1,weighted gene co-expression network analysis(WGCNA)was adopted.The cis elements in the promoter regions of these genes were also analyzed.3 hours after treating,the expression level of GA-related gene(DCAR₀09411)and cellulose synthase-like genes(DCAR₀19754 and DCAR₀13973)were significantly induced.3.Based on carrot genome sequencing data,a BRs metabolish gene(DCAR₀09214)was identified.By over-expressing DCAR₀09214 in Arabidopsis,the DCAR₀09214 over-expression line was found to be BRs-deficient.These results indicated that DCAR 009214 is the gene codes the BRs metabolish enzyme in carrot.The cell walls of the over-expression lines were thinner than that of wild Arabidopsis.Cellulose synthesis in over-expression lines was also significantly inhibited.Through quantitative real-time PCR(RT-qPCR),CESA genes involved in cellulose synthesis in primary cell walls were found being obviously inhibited.4.A total of 12 CESA genes were identified from carrot genome.Through WGCNA,3 BR-related genes were found,which have co-expression with CESA genes.After analyzing the RT-qPCR data with pearson correlation coefficient,2 DcBEH4(DCAR₀19605 and DCAR₀05165)gene were found to have high correlation with DCAR₀05199,DCAR₀12579,and DCAR 019410.DCAR₀05199,DCAR₀12579,and DCAR₀19410 have high homology with AtCESA8,AtCESA4,and AtCESA7,respectively.AtCESA8,AtCESA4,and AtCESA7 are the genes that control the cellulose synthesis in Arabidopsis secondary cell walls.However,results of yeast two-hybrid revealed that there was no direct interaction between these genes.By over-expressing DcBEH4 in Arabidopsis,DcBEH4 genes were found significantly enhancing cellulose synthesis by promoting the expression of AtCESA4,AtCESA7,and AtCESA8.These results suggested that DcBEH4 genes may regulate cellulose synthesis in secondary cell walls by indirectly regulating the expression level of DCAR₀05199,DCAR₀12579 and DCAR₀19410 in carrot.