Screening And Preliminary Validation Of Different Metabolites Of Codonopsis Pilosula Under Drought Stress

Codonopsis pilosula(Franch.)Nannf.is a perennial herb of the genus Codonopsis in the family Platycodon,mainly produced in the northern arid and semi-arid mountainous areas of Weiyuan and Longxi in Gansu Province.With global warming,drought occurs frequently,affecting the growth and quality formation of Codonopsis pilosula.Previous research by the research team found that Codonopsis pilosula mainly reduces the damage caused by drought by increasing the activity of osmoregulation substances and antioxidant enzymes in the body.A large number of studies have shown that plants can synthesize many small molecular metabolites when encountering stress,which is not only the basis for the formation of medicinal material quality,but also may play an important role in the resistance of plants to stress.Based on this,this study conducted an outdoor pot water control experiment.Four treatments were set according to the maximum field water capacity: normal water content(CK: 90%-85%),mild drought(LD: 70%-65%),moderate drought(MD: 55%-50%),and severe drought(SD: 40%-35%).Samples were taken at harvest time for metabolomic analysis and transcriptome sequencing to screen differential metabolites and analyze their synthesis pathways and differential genes,Then,the effect of differential metabolites on the drought resistance of Codonopsis pilosula was verified through exogenous application.The preliminary results are as follows:LC-MS analysis of Codonopsis pilosula under different treatments revealed a total of20571 substance peaks,and a total of 8253 metabolites were obtained through compound identification,including 274 significantly different metabolites(142 up-regulated and 132down-regulated)in the LDvs CK group,284(157 up-regulated and 127 down-regulated)in the MDvs CK group,and 317(133 up-regulated and 184 down-regulated)in the SDvs CK group.Then,each group of differential metabolites was annotated with pathway annotation.Among them,the LDvs CK group annotated 26 differential metabolites(12 up-regulated and 14down-regulated),with 21 main enriched metabolic pathways;In the MDvs CK group,there were 25 differential metabolites(20 up-regulated and 5 down-regulated),with 17 main metabolic pathways enriched;In the SDvs CK group,there were 29 differential metabolites(13 up-regulated and 16 down-regulated),with 19 main metabolic pathways enriched.Finally,the differential metabolites annotated in the three comparison groups were classified and analyzed,and it was found that they were mainly concentrated in fatty acyls,carboxylic acids and derivatives,and organooxygen compounds.Based on the identification of differential metabolites,high-throughput sequencing showed that:(1)there were 4410 differential genes(1574 up-regulated,2836 down-regulated) in the LDvs CK group,5428 genes(2464 up-regulated,2964 down-regulated)in the MDvs CK group,and 6660 genes(3085 up-regulated,3575 down-regulated)in the SDvs CK group.(2)The differential genes on the annotated differential metabolite pathway were screened according to the conditions of P<0.05 and FC>2.The common pathways in the three control groups were galactose metabolism,pyrimidine metabolism,glycerol phospholipid metabolism,glyceride metabolism,nicotinic acid and nicotinamide metabolism,and the common pathways in the MDvs CK and SDvs CK groups were flavonoid biosynthesis,arachidonic acid metabolism,amino sugar and nucleotide sugar metabolism,and phenylalanine biosynthesis.(3)Based on literature analysis,galactose metabolism(ko00052)and flavonoid biosynthesis(ko00941)pathways may be related to the drought resistance of Codonopsis pilosula.Through the analysis of genes related to the biosynthesis of flavonoids and sugar differential metabolites of Codonopsis pilosula under drought stress,chalcone synthase gene,chalcone isomerase gene,bifunctional dihydroflavonol 4-reductase/flavaone 4-reductase gene,shikimate hydroxycinnamoyltransferase gene,caffeoyl coenzyme A gene positive regulation metabolite epigallocatechin gallate(EGCG)Synthesis of epigallocatechin(EGC).UTP-glucose-1-phosphate uridyltransferase gene,UDP glycopyro phosphorylase gene α-Galactosidase gene β-fructofuranosidase gene is regulating the synthesis of galactinol(Gal)and raffinose(Raf).Therefore,four key differential metabolites,EGCG,EGC,Gal,and Raf,were screened and their synthesis pathways were analyzed,laying the foundation for the preliminary validation of drought resistance in Codonopsis pilosula in the later stage.In order to determine whether the four differential metabolites play a role in the resistance of Codonopsis pilosula to drought stress,preliminary research through exogenous application methods found that: on the basis of drought inhibiting the photosynthesis of Codonopsis pilosula,after applying Gal,Raf,EGC,and EGCG,the net photosynthetic rate,stomatal conductance,and transpiration rate of Codonopsis pilosula increased to varying degrees,while the intercellular CO2 concentration showed a downward trend.This indicates that the accumulation of four metabolites can improve the effects of drought stress on the photosynthetic characteristics of Codonopsis pilosula.In summary,this study screened out the differential metabolites in Codonopsis pilosula under drought stress,and combined with transcriptome mining of differential metabolite related regulatory genes.Through association analysis and differential metabolite synthesis pathway analysis,it was finally determined that four key differential metabolites may play an important role in the resistance of Codonopsis pilosula to drought stress,and the impact of drought stress on the photosynthetic characteristics of Codonopsis pilosula can be improved by exogenous application of specific key differential metabolites.This result lays a foundation for further studying the mechanism of metabolite accumulation on the drought resistance of Codonopsis pilosula.