Molecular Mechanism Of Anthocyanin Biosynthesis In Purple Celery

Celery(Apium graveolens L.),an annual or perennial herb of the Apiaceae family,is one of the most important vegetable crops.Celery is widely cultivated in the world.Celery is mainly used as leaf vegetables and has potential medicinal effects.Celery contains abundant nutrients such as vitamins,apigenins,and carotenoids.Purple celery accumulated amounts of anthocyanins in the petioles.Anthocyanins are natural plant pigments and belong to flavonoid compounds.There are many kinds of anthocyanins in plants,including pelargonidin,delphinidin,cyanidin,malvidin,petunidin,and peonidin.Anthocyanins play important roles in plant growth and development,and they are also widely used in food and medical industries.There are many researches on the biosynthesis of anthocyanins in higher plants.However,the anthocyanin components,glycosylation process,and regulatory genes in celery are still unclear.In this study,a celery genome database,CeleryDB,was constructed.LC-MS was used to determine the components of anthocyanins in celery,and the structural genes involved in anthocyanin biosynthesis pathway were identified from the celery genome.The anthocyanin glycosylation process was analyzed according to the specific components of anthocyanins.AgUCGalT1 gene was cloned from celery and and glycosylation activity of AgUCGalT1 was determined by using protein purification and enzyme activity assays.Based on genomic data,R2R3-MYB family transcription factors in celery were analyzed.Anthocyanin-related S6 subgroup in R2R3-MYB transcription factors of celery was identified.The functions of AgMYB1 and AgMYB2 transcription factors were verified by using bioinformatics and transgenic technology.This study identified glycosyltransferases and R2R3-MYB transcription factors related to anthocyanin biosynthesis in celery,which have important significance for the research on the regulation mechanism of celery anthocyanin biosynthesis and the cultivation of crops with high anthocyanin content through genetic engineering.The main results of this study are as follows:1.CeleryDB,a celery genome database,was constructed based on the celery genome sequencing data(http://apiaceae.njau.edu.cn/celerydb).Home,BLAST,Genome Browser,Transcription Factor,and Download interfaces composed of the organizational structure of CeleryDB.The sequences of the whole genome,nucleotide sequences of the predicted genes,and amino acid sequences of the predicted proteins are available online on Celery DB.Users can search the celery genomic data by using two user-friendly query tools:basic local alignment search tool(BLAST)and Genome Browser.2.The total anthocyanin contents of different celery varieties were determined.The components of anthocyanin in purple celery were analyzed by using LC-MS.The total anthocyanins contents of in purple celery were significantly higher than that in non-purple celery.LC-MS indicated that the anthocyanins in purple celery were cyanidin glycosylated or acylated with different moieties,mainly including cyanidin 3-xylosylgalactoside,cyanidin 3-xylosyl(feruloylglucosyl)galactoside,and cyanidin 3-xylosyl(coumaroylglucosyl)galactoside.The structural genes of anthocyanin biosynthesis pathway were identified from celery genome,including PAL,C4H,4CL,CHS,CHI,F3H,F3'H,DFR,LDOX and UCGT.3.The glycosylation of anthocyanins in purple celery was investigated.AgUCGalT1,a gene encoding galactosyltransferase,was isolated from purple celery and expressed in Escherichia coli BL21(DE3).Sequence alignments revealed that the AgUCGalT1 protein contained a highly conserved putative secondary plant glycosyltransferase(PSPG)motif.The glycosylation product catalyzed by AgUCGalT1 was detected using UPLC equipment.These results indicated that the purified AgUCGalT1 possessed glycosylation activity,which could catalyze the glycosylation of cyanidin to cyanidin 3-galactoside.The recombinant AgUCGalT1 had an optimal enzyme activity at 35? and pH=8.0,and AgUCGalT1 showed highest enzyme activity toward cyanidin among the enzyme activities involving other substances,namely,peonidin,quercetin,and kaempferol.The expression level of AgUCGalT1 gene and glycosylation ability of crude enzyme extracted from purple celery was significantly higher than that of non-purple celery.4.Based on the genome data in CeleryDB,154 R2R3-MYB transcription factors were identified from celery.R2R3-MYB transcription factors are widely distributed in different species,and the number in higher plants is higher than that in lower plants.This may be due to the duplication and expansion of transcription factors during plant evolution.According to the evolutionary relationship of R2R3-MYB family transcription factors between celety and celery R2R3-MYB transcription factors were divided into different subgroups.The S6 subgroup of Arabidopsis R2R3-MYB factors plays a regulatory role in the anthocyanin biosynthesis pathway in plants.Agr10145 and Agr41800 in S6 subgroup may be related to the regulation of anthocyanin in celery,namely AgMYB1 and AgMYB2,respectively.5.AgMYB1 and AgMYB2 genes were cloned from celery and the bioinformatics analysis and functional verification assays were performed.Sequence analysis indicated that the R2R3 domain was highly conserved in AgMYB1 and AgMYB2,and they contained many anthocyanin-related motifs.Transient expression of AgMYB1 and AgMYB2 in tobacco leaves promoted the accumulation of anthocyanins and produced the red pigments in leaves.The expression levels of structural genes in anthocyanin pathway were also increased.Overexpression of AgMYBl and AgMYB2 in Arabidopsis plants enhanced the anthocyanin contents,antioxidant capacities,and expression levels of anthocyanin biosynthesis genes in transgenic plants.Y2H assay showed that AgMYBl and AgMYB2 could bind to bHLH protein,and the binding activity of AgMYB2 was stronger than that of AgMYB1.