Study On The Quality Formation Of Lonicerae Japonicae Flos Under Salt Stress Based On Multi-omics

Lonicerae Japonicae Flos(LJF)is a classic and commonly used Chinese medicinal material.It is the dried flower buds or first-opening flowers of Lonicera japonica Thunb.with the effect of clearing heat and detoxification,evacuation of wind and heat,and various pharmacological effects such as anti-oxidation,anti-inflammatory,anti-bacterial,antiviral,liver and gallbladder,enhance immunity,hypoglycemic and so on.It also be used for carbuncle swollen furuncle,throat numbness,erysipelas,heat toxin blood dysentery,wind-heat cold,febrile fever and other symptoms.At the same time,clinical practice has also shown that LJF has a preventive effect on SARS,H1N1 influenza virus and COVID-19.The market demand is increasing,but its quality is uneven;moreover,there are many near-source varieties,which are mixed and adulterated.With the increase in the area of soil salinization in the world,the study on plant salt tolerance has attracted the attention of scholars at home and abroad.The main producing areas of LJF,such as Shandong,Henan province and other areas,have large area and wide distribution of saline soil,therefore,the salt stress environment is closely related to the quality of LJF,moderate salt stress can promote the synthesis and accumulation of bioactive constituents in LJF,improving the quality.However,in the quality formation of LJF,how salt stress affects the synthesis and accumulation of the bioactive constituents and the molecular mechanism of LJF quality formation under salt stress is still unclear.Therefore,the quality evaluation and the molecular mechanism of quality formation under salt stress are important parts to control the intrinsic quality and improve the quality.The main research content are as follows:1.Analysis of multiple index constituents In this study,UFLC-QTRAP MS/MS technology combined with multivariate statistical analysis was optimized and established to simultaneously determine the content of multivariate index constituents in LJF,LJC and LF;and comprehensively evaluate the quality of LJF from different habitats and batches.The results showed that there were significant differences in the contents of organic acids,flavonoids,iridoids,saponins,amino acids and nucleosides in different samples.The average contents of organic acids,flavonoids and iridoids in flower buds of LJF were higher than those in open flowers.Principal component analysis(PCR)showed that LJF could be separated from LF and LJC.Discriminant analysis by the least squares method(PLS-DA)were further selected 11 different markers in LJF and LF:proline,serine,glutamic acid,chlorogenic acid,1,3-O-dicaffeonyl quinic acid,protocatechuic acid,secoxyloganin,secologanic acid,morroniside,macranthoidin A and dipsacoside B;and nine markers of difference between LJF and LJC:isochlorogenic acid A,chlorogenic acid,neochlorogenic acid,quinic acid,secologanic acid,loganin,secoxyloganin,morroniside and isoleucine.Gray relational analysis(GRA)was used to rank the quality of flower bud,open flower and LF,respectively.The results showed that the quality of LJF was better than LF,and the flower bud was better than open flower.It provides a new reference method for intrinsic quality control and evaluation of LJF.2.Analysis on the differences of chemical constituents in the ground part of L.Japonica UFLC-triple TOF-MS/MS and multivariate statistical analysis were used to compare the metabolic profiles of the ground parts of L.japonica(leaves,buds,flowers and stems).A total of 71 compounds were identified,including 4 alkaloids,24 organic acids,15 iridoids,23 flavonoids and 5 saponins.PCA results showed that leaves were mixed with buds,and there was overlap between buds,with only vines completely separated from other parts.Compared with PCA,PLS-DA could divide the aerial parts,and lonicerin,kaempferol-3-O-rutinoside,loganin,isochlorogenic acid B,isochlorogenic acid C,secologanic acid,luteoloside,astragalin could be considered as the chemical markers based on variable importance in projection(VIP).It provides a reference method for metabolomic of LJF under salt stress in the next.3.Effects of salt stress on physiology and multiple index constituents of L.japonica Through establishing a growth model of L.japonica,four levels of salt stress were treated.From morphological,physiological and biochemical indicators(including chlorophyll a and b,total chlorophyll,carotenoid,soluble protein,total amino acid content,proline,malondialdehyde,superoxide dismutase(SOD),peroxidase(POD),catalase(CAT)and ascorbic acid peroxidase(APX))and the change of the content of bioactive constituents to evaluate the quality of LJF under salt stress.The results showed that the growth,physiological indexes of LJF under low salt stress were better than other groups.The total contents of 47 index constituents in LJF under low salt stress were higher,which was 1.1 times of that in control group.In addition,Cluster analysis results showed that the between control group and salt stress group was obvious separated,indicating that salt stress had a greater effect on the content of index constituents in LJF.PLS-DA analysis showed that the four groups of samples could be separated obviously,and compounds including glutamic acid,lysine,neochlorogenic acid,sweroside,loganic acid,secoxyloganin,secologanic acid contributed more to the separation of the samples.GRA was used to rank the quality of LJF under different salt stress.The results showed that low salt stress promoted the quality improvement of LJF,while high salt stress had the opposite effect.Pearson correlation coefficient analysis showed that the increase of phenolic acid accumulation could reduce oxidative damage of L.japonica.4.Multi-omics analysis of Lonicerae Japonica Flos under salt stress(1)Untargeted metabolomics analysis UFLC-Triple TOF-MS/MS coupled with multivariate statistical analysis was applied to comparatively investigate the metabolites changes in LJF under different salt stress for 35 days.Ninety compounds were identified and 47 different metabolites were screened out.OPLS-DA analysis showed that the control group and salt stress group could be completely separated under positive and negative ion modes.The relative content(peak area)of bioactive components in low salt stress group was higher than that in other groups.Phenylpropanoid biosynthesis pathway,monoterpenoid biosynthesis,glycolysis,TCA cycle and alkaloid biosynthesis pathway were all disturbed in salt-stress LJF.The results showed that salt stress significantly induced the metabolism of LJF,and the content of difference constituents in LJF was higher in the low salt stress group,which laid a foundation for the proteomic and transcriptomic analysis of LJF in the next study.(2)Proteomics analysis TMT technology combined with bioinformatics analysis was used to study the proteomics of LJF between the low salt stress group and the control group.Based on the proteomic results,the molecular map of protein changes in LJF under low salt stress was drawn,and the key differentially expressed proteins(DEPs)in the synthesis pathway of phenolic acids,flavonoids and iridoids were screened out.The expression of DEPs,including phenyl ammonium lyase(PAL),trans-cinnamate 4-monooxygenase(C4H),4-coumarate-CoA ligase(4CL),hydroxycinnamoly CoA shikimate/quinate hydroxycinnamoly transferase(HCT),p-coumarate 3'-hydroxylase(C3'H),caffeic acid 3-O-methyltransferase(COMT),chalcone isomerase(CHI),secologanin synthase(SLS),DXS(Deoxyxylulose-5-phosphate synthase)and G10H(Geraniol 8-hydroxylase)were significantly enriched in the biosynthetic pathways of phenylpropanoids and monoterpenoids,and the expression was significantly up-regulated in the salt stress group.Protein-protein interaction(PPI)network indicated that energy metabolism and carbohydrate metabolism were the main biological functions of DEPs in LJF under salt stress.Parallel reaction monitoring(PRM)was performed to verify the abundance of candidate proteins.(3)Transcriptomics analysis RNA-seq technology combined with bioinformatics was performed to study the transcriptome of LJF in the low salt stress group and the control group.According to transcriptomics,a total of 78,261 gene sequences were obtained,among which 1,055 differentially expressed genes(DEGs)were involved in the biosynthesis of secondary metabolites.The DEGs involved in biosynthesis of bioactive constituents were screened out,including phenylalanine ammonia-lyase(PAL),shikimate O-hydroxycinnamoyltransferase(HCT),4-coumarate-CoA ligase(4CL),p-coumarate 3'-hydroxylase(C3'H),chalcone synthase(CHS),flavonol synthase(FLS),ferulate-5-hydroxylase(F5H),cinnamyl-alcohol dehydrogenase(CAD),peroxidase,caffeic acid 3-O-methyltransferase(COMT),and cinnamoyl-CoA reductase(CCR).Except for FLS and F5H,the expression of DEGs were up-regulated under salt stress.The expression of 10 DEGs were verified by qRT-PCR.PALs were selected to construct phylogenetic analysis,and multiple sequence alignment.It was found that gene PAL(TRINITY_DN16988_c0_g14)clustered with Arabidopsis thaliana,the homology with ATPAL1,ATPAL2 was 73.96%.Therefore,it can be speculated that PAL(TRINITY_DN16988_c0_g14)gene might play an important role in phenolic biosynthesis of LJF under salt stress.In addition,261 and 122 CYP450 and UDP-glycosyltransferase genes were annotated under salt stress,respectively;transcription factor MYB(210)family is the most abundant family in salt-treated LJF.5.Correlation analysis of multi-omics of Lonicerae Japonicae Flos under salt stressThe metabolic profile,the content of bioactive metabolites and the DEPs,DEG of LJF during salt stress were analyzed,and correlated,to draw the omics map of phenolic acids,flavonoids and iridoids in LJF under salt stress.(1)Correlation analysis of comparative proteomics and bioactive constituents content Correlation analysis of comparative proteomics and the contents of bioactive constituents of LJF during salt stress,drawing the omics map of phenolic acids,flavonoids and iridoids in LJF under salt stress.The results showed that the expression trend of related structural proteins was basically consistent with the content of bioactive constituents of LJF under salt stress.(2)Correlation analysis of transcriptome and untargeted metabolome Through combing analysis of transcriptome and metabolic profile of LJF under salt stress and control group,the results showed that the DEGs was positively correlated with the relative contents of phenolic acid and flavonoids,as well as the contents of chlorogenic acid and luteoloside.At the same time,the expression of 72%glycotransferase gene significantly up-regulated in salt stress group,which was speculated to be closely related to the synthesis and accumulation of downstream flavonoid glycosides.(3)Correlation analysis of transcriptome and proteome Combined analysis of proteome and transcriptome of LJF during salt stress was carried out to screen and identify common differential proteins/genes and glycotransferases.In summary,based on the comprehensive evaluation of the quality of LJF,LF and LJC,a comprehensive method was conducted from the aspects of morphology,physiology,index constituents content,metabolomics,proteomics and transcriptomics to evaluate the quality and elucidate the mechanism of the synthesis and accumulation of bioactive constituents,analyzing the logical relationship between genes,proteins and bioactive constituents of LJF,in order to reveal the molecular mechanisms of quality formation in LJF under salt stress.