| Flavonoids and coumarin are important active ingredients of Aesculus chinensis.They play a significant role in defense of plants and human health.Flavonoids compounds take part in plant regulation of plant growth,protection of plants from UV damage,control the color of flower,and the form of plant-microbial interactions.Coumarin compounds have double physiological activities on plants that they promote plant growth at low concentrations and inhibit plant growth when the concentration reaches a certain value.And they have a high bioavailability in plants and humans.The two active ingredients will be modified by glycosyltransferase after they are synthesized.The specificity of the glycosylation site,the difference of connection sequence,and the variety of number of quantities and types of the linked glycosyl all contribute to the diversity of the structure of the compound,which determines the diversity of functions in vivo in turn.What's more,glycosylation can enhance the stability of metabolites in plants,and facilitate the accumulation and storage of metabolites in plants.There are a large number of flavonoid and coumarin glycoside compounds in A.chinensis.The process of identifying and analyzing their corresponding glycosyltransferases is a key step in explain the biosynthesis pathway of flavonoid and coumarin compounds clearly.Moreover,the process provides a theoretical basis for the synthetic biology of flavonoid and coumarin,improve the medicinal value of A.chinensis ultimately.In this paper,UPLC-MS was used to analyze the metabolic profile of flavonoids and coumarin components in different parts of A.chinensis.It showed that the flavonoid aglycones,flavonoid glycosides,coumarin aglycones and coumarin were mainly concentrated in flower organ of A.chinensis,some were concentrated in the leaf,and the contents in the seed were bare.The content of astragalin in flower organ reached 0.352 mg/g,which was 14 times and 16 times of the content of astragalin in leaf and seed organ,respectively.The content of isoquercitrin was 0.103 mg/g,which was 2.1 times and 2.7 times of the content of isoquercitrin respectively.The content of kaempferol in Isoquercitrin was 0.011 mg/g,which was 25 and 72 times of the content of kaempferol in leaves and seeds,respectively.And the content of quercetin was 0.229 mg/g,which was 3.8 times and 3.1 times higher than that of leaves and seeds of A.chinensis.However,the content of quercitrin in the leaves of A.chinensis reached 0.59 mg/g,and the content of quercitrin in flowers and seeds was 0.019 mg/g and 0.008 mg/g,respectively.Another compound of coumarins—fraxetin?fraxin?fraxetin?esculetin are low in various tissues of A.chinensis.The contents of fraxin in the flowers,leaves and seeds of A.chinensis were 10.74 ?g/g,8.98?g/g and 1.37 ?g/g respectively.The content of fraxetin in flowers,leaves and seeds were 0.56 ?g/g and 0.04 ?g/g,0.32 ?g/g respectively.The accumulation of esculetin in flowers,leaves and seeds were 0.46?g/g,0.07 ?g/g,0.18 ?g/g respectively.While the content of esculin in leaves was 0.60 ?g/g,0.20 ?g/g in flowers organ,0.07 ?g/g in the seeds organ.(All plant materials used are dry weight)Five flavonoid and coumarin glycosyltransferases were identified finally,combining the transcriptome data of A.chinensis,PSPG analysis,protein enzyme activity tests and q RT-PCR analysis.It was found that UGT84A56?UGT73C29 can catalyze the glycosylation of flavonol and flavonoid compounds,and UGT92G7?UGT71A47 can catalyze the glycosylation of esculetin and fraxetin among twenty-five full-length glycosyltransferases by enzyme activity experiment in vitro.The catalytic properties of these five active UGTs are of the difference.Among them,UGT84A56 can glycosylate both flavonol and flavonoids;UGT73C29 can glycosylate some flavonol compounds.UGT92G7 holds the catalytic ability on both esculetin and fraxetin.UGT71A47 only holds the catalytic ability on fraxetin.UGT84A57 has an activity on esculetin and has weak activity on fraxetin.q RT-PCR analysis showed that UGT84A56 was specifically expressed in flowers,UGT73C29 was specifically expressed in flowers and seeds;UGT92G7,UGT71A47 and UGT84A57 were highly expressed in flowers,among which UGT92G7 and UGT71A47 were highly expressed in flowers and leaves,and UGT84A57 was specifically expressed in flowers.The relative expression of UGT84A56 in flowers was 9.14 times in leaves and 14.17 times in seeds.The relative expression of UGT73C29 in flowers was 1.30 times in leaves and 3.37 times in seeds.In flowers,the transcriptional levels of UGT84A56 and UGT73C29 are consistent with flavonol glycoside enrichment,suggesting that they are involved in the synthesis of flavonol glycosides in flowers.The expression of three glycosyltransferase genes of UGT92G7,UGT71A47 and UGT84A57 in the three organs was consistent with the accumulation of fraxin and esculin.It indicated that UGT92G7 and UGT71A47 played a key role in the enrichment of coumarin glycosides flowers and leaves,and UGT84A57 was associated with the accumulation of coumarin glycosides in flowers of A.chinensis.This study has identified and analyzed the flavonoids and coumarin UGTs in A.chinensis,which will assist the study on biological pathways of these two compounds,provide theoretical basis for the synthetic biology of flavonoids and coumarin,and enrich the synthetic and metabolic pathways of flavonoids and coumarins in plants. |
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