Preliminary Study On The Relationship Between Tree Peony(Paeonia Suffruticosa)Phenolic Substances And Rooting Of Plantlet In Vitro Based On Metabolomics

In this paper,the stem tissue of the tree peony varieties ‘Fengdanbai’,‘Wulongpengsheng’,‘Taipinghong’plantlet in vitro was used as the experimental materials,and the detection method of non-targeted metabolomics was used in the positive and negative ion modes.The differential metabolites of these three varieties during the critical period of adventitious roots were analyzed.The phenolic differential metabolites were used as the main research object.Based on KEGG and Lipid Maps database,the qualitative and quantitative analysis of the differential phenolic substances between different varieties was carried out whichto explore the metabolic pathways and key enzymes of differential phenolic substances that may play an important role in the formation of root primordia between different varieties,so as to clarify the relationship between phenolic substance metabolism and rooting in the root initiation process of Paeonia suffruticosa plantlets in vitro.The research results would provide theoretical basis and technical support for solving the root of tree peony plantlets in vitro and other problems.The main results were as follows:1)The cytological observation of the stem tissue of the tree peony varieties‘Fengdanbai’,‘Wulongpengsheng’,‘Taipinghong’plantlet in vitro showed that the adventitious roots of the tree peony plantlet in vitro belonged to the type of induced root primordial,The critical period of formation of plantlet in vitro roots was 2-3 d.On the 3rd day of rooting culture,morphologically dense cell clusters were observed in the anatomical structures of the stem tissues of the three varieties,and the vascular bundle formation layer began to appear and had the initial morphology of the root primordium.The adventitious roots formed in the rooting culture for about 10 days,the medullary cavity became larger,and the stem cells became relatively loose.After that,the adventitious roots continued to grow;20 days later,the adventitious roots broke through the epidermis.2)Based on the three values of VIP value,folding change and q-value,85 different phenolic substances were screened,including hydroxycinnamic acid type,hydroxybenzoic acid type and flavonoids.Based on the three screening conditions of VIP≥1,folding change ≥1.2(or ≤0.8333)and q-value<0.05,increasing the VIP value further screened for significant differential phenolic metabolites.Combining the analysis of ‘Fengdanbai’,‘Wulongpengsheng’ and ‘Taipinghong’(ab,ac,bc),screening out 15 significant phenolic compounds that may play an important role in root primordium formation:(-)-epiafzelechin,(-)-epigallocatechin,(+)-gallocatechin,3-o-methylquercetin,5-deoxyleucopelargonidin,afzelechin,apiforol,leucocyanidin,[6]-gingerol,2-hydroxyphenylacetic acid,3,4-dihydroxyphenylacetaldehyde,3-hydroxyphenylacetic acid,vanillin,4-hydroxyphenylacetic acid,3-(2g-xylosylrutinoside).3)The study found that these 15 significant differential phenols are mainly involved in the flavonoid biosynthesis pathway,flavone and flavonol biosynthesispathway,phenylalanine metabolism,tyrosine metabolism,stilbene metabolism and other related metabolic pathways.Among them,7 differential phenols such as(-)-epiafzelechin,(-)-epigallocatechin,(+)-gallocatechin,5-deoxyleucopelargonidin,afzelechin,apiforol,leucocyanidin are all located in the flavonoid biosynthesis pathway.3-o-methylquercetin is located in the biosynthesis pathway of flavone and flavonol.2-hydroxyphenylacetic acid,vanillin biosynthesis are localized in the phenylalanine metabolic pathway,and [6]-gingerol is located in the stilbene metabolic pathway,3,4-dihydroxyphenylacetaldehyde,3-hydroxyphenylacetic acid,4-hydroxyphenylacetic acid are located in the tyrosine metabolic pathway.The key enzymes involved mainly include CHS(chalcone synthase),F3H(flavonol hydroxylase),DFR(dihydroflavonol 4-reductase),ANR(anthocyanin reductase),LAR(colorless flower pigment)Reductase),CHI(chalcone isomerase),TAT(tyrosine transaminase),4CL(4-coumaroyl-Co A-ligase),PAL(phenylalanine ammonia-lyase),4-acyl-Co A 10 kinds of enzyme and so on.4)Among the above 15 significant differential phenols,(-)-epiafzelechin,(-)-epigallocatechin,(+)-gallocatechin,3-o-methylquercetin,5-deoxyleucopelargonidin,afzelechin,apiforol,leucocyanidin were down-regulated between the ‘Fengdanbai’ and ‘Wulongpengsheng’(ab)comparison groups,between the ‘Wulongpengsheng’ and‘Taipinghong’(bc)comparison groups were up-regulation of expression;[6]-gingerol,2-hydroxyphenylacetic acid,3,4-dihydroxyphenylacetaldehyde,3-hydroxyphenylacetic acid,vanillin,4-hydroxyphenylacetic acid,3-(2g-xylosylrutinoside)were down-regulated in both‘Wulongpengsheng’,‘Taipinghong’(bc)and ‘Fengdanbai’,‘Fengdanbai’(ac)comparison groups.Combined with the analysis of‘Fengdanbai’,‘Wulongpengsheng’,‘Taipinghong’ rooting rate,the first eight significant differential phenols may inhibit the root primordium formation,the latter seven promote the formation of root primordium.In this study,in addition to afzelechin(monohydric phenol),(-)-epiafzelechin,(-)-epigallocatechin,(+)-gallocatechin,3-o-methylquercetin,5-deoxyleucopelargonidin,apiforol,leucocyanidin that promote rooting ofseven different phenols are dihydric phenols or polyphenols;and takes root in inhibition of phenols in the seven significantly differences,in addition to3,4-dihydroxyphenylacetaldehyde(dihydric phenol)and quercetin3-(2g-xylosylrutinoside)(polyphenol),[6]-gingerol,2-hydroxyphenylacetic acid,3-hydroxyphenylacetic acid,vanillin,4-hydroxyphenylacetic acidwere all monohydric phenols.Studies have shown that most monophenols inhibit the occurrence of adventitious roots,while diphenols and polyphenols promote the occurrence of adventitious roots.