Study On The Mechanism Of Flavonoids Metabolism And Accumulation In Response To Low Nitrogen In Coreopsis Tinctoria

The flowers of snow chrysanthemum（Coreopsis tinctoria Nutt.）are rich in flavonoids and have high medicinal value.The content and types of flavonoids in Coreopsis tinctoria are important factors affecting its medicinal value,so it is necessary to improve the cultivation measures to further increase the accumulation of flavonoids in Coreopsis tinctoria.In production,the excessive application of nitrogen fertilizer of C.tinctoria is a common phenomenon,which not only leads to the decline of nitrogen fertilizer utilization rate and environmental pollution,but also inhibits the accumulation of flavonoids,which are medicinal active substances of C.tinctoria,and affects the greenness of industry high-quality development.At present,there is no report on improving the metabolism and accumulation of C.tinctoria flavonoids by reducing nitrogen application.In this study,hydroponic experiments were used to determine and analyze the content of flavonoids in Coreopsis tinctoria under different low nitrogen levels.The physiological characteristics and molecular mechanism of flavonoid accumulation in Coreopsis tinctoria under low nitrogen treatment were systematically studied.The main findings are as follows:（1）The test results in 2020 and 2021 both showed that 0.625 m M nitrogen concentration,as an appropriate concentration for evaluating the ability of C.tinctoria to tolerate low nitrogen and maintaining the accumulation of flavonoids,maintained the related activities of carbon and nitrogen metabolism and further ensured the flowering number and biomass of C.tinctoria.The 0.625 m M nitrogen level promoted the carbon/nitrogen ratio inside the leaves,and the total flavonoid content increased in the range of 7.6%-17.3%and 7.9%-28.1%in the four harvest stages of 2020-2021,respectively.Consistent with the accumulation pattern of total flavonoids in leaves,the increasing ranges of total flavonoids in inflorescence were 5.45-13.28%and 3.57-21.71%,respectively.PAL,C4H,4CL and CHS were the key enzymes regulating the accumulation of total flavonoids in leaves.In the analysis of free amino acids in all leaves,reducing nitrogen application increased the contents of Asn,Ser,Glu,Ala,Val,Tyr and Phe and decreased the contents of Thr,Gly,Leu,Ile,Lys and Pro.The carbon and nitrogen metabolism in C.tinctoria plants under nitrogen control can not only control the synthesis of carbon in amino acids and carbohydrates,but also change the biosynthesis of flavonoids in C.tinctoria.（2）The low nitrogen treatment group（0.625 m M Ca（NO₃）₂）and the control group（5.000 m M Ca（NO₃）₂）were set,and the flavonoid metabolomics method based on UPLC-MS/MS was used to analyze the low nitrogen treatment group and the control group.The content of flavonoids in inflorescences of Coreopsis tinctoria buds,early flowering,full flowering and late flowering stages of the group was determined and analyzed.A total of 299 flavonoids were detected in the two treatment groups,and the contents of flavonoids in the two treatments were significantly different.The period with the greater difference was the flower bud stage,and the flavonoids with greater difference included 6’’-O-Malonyl genistein,quercetin,zelan flavone（6-methoxyluteolin）,3,5,7,3’,5’-pentahydroxy-4’-methoxyflavone,etc.The above information indicated from the physiological level that low nitrogen treatment affected the content and types of flavonoids in Coreopsis tinctoria.（3）Transcriptome sequencing was performed on 24 samples of C.tinctoria in the low nitrogen group and the control group,and genes significantly related to low nitrogen tolerance were identified from the data,including SUS,AAM,Hx K,EG in starch and sucrose synthesis pathway and PP2C,Sn RK2,AUX/IAA,AUX/IAA LBD,JAR1,CRE and A-ARR in plant signal transduction pathways.In addition,the related metabolic pathways that affect the accumulation of flavonoids in Coreopsis tinctoria were identified.The main pathways involved were the phenylpropane pathway and the flavonoid pathway.The related genes identified were PAL,4CL,CCR,HCT,C3’H,CHS,DFR,FLS,CYP75B1 and ANR.（4）Combined analysis of flavonoid metabolomic and transcriptomic data found that the up-regulated expression of PAL,4CL,HCT and CHS under low nitrogen treatment was the main reason for improving the accumulation of Coreopsis tinctoria flavonoids.The correlation analysis between the selected flavonoids and antioxidant activity indicators showed that kaempferol-3-O-（6’’-p-coumaroyl）galactoside,quercetin-3-O-（2’’-acetyl）glucuronide,3,7-dimethoxyquercetin marigold,isotasalin,apigenin-8-C-arabino-side,quercetin-5-O-glucuronide and the accumulation of flavonoids such as cyanidin-3-O-（6’’-O-p-coumaroyl-2’’-O-xylosyl）glucoside is the main reason for enhancing the antioxidant activity of Coreopsis tinctoria.