Primary Study On The Bioactive Compound Accumulation And Mechanism In Astragalus Mongolicus Induced By Hexanal

Objective:Astragali radix with a strong beany flavor is well accepted as good in quality.A growing body of modern research has shown that the beany flavor volatiles function as signal molecule and play roles in the process of plant growth and secondary metabolism.Our preliminary study demonstrated that the application of hexanal,which is a dominant contributor to the beany flavor,inhibited Astragalus mongolicus seedling growth but promoted the accumulation of a few bioactive components in the roots.This paper was to extend its role and to understand underlying mechanism.Firstly,the quantification of astragaloside IV,calycosin-7-O-β-D-glucoside and ononin was established with alkali treatment in small amount of raw materials,providing a simple and feasible method for the quantitative analysis of the components in A.mongolicus tissue cultures and potted seedlings.Secondly,the effect of hexanal treatment on the accumulation of the main bioactive components was examined in A.mongolicus transplanted in the field.Underlying mechanism induced by hexanal treatment was investigated in potted A.mongolicus seedlings treated with hexanal as well as the discovery of terpene synthase genes.This work would be useful for further study on the role and mechanism of the beany flavor signals in the biosynthesis and accumulation of bioactive compounds in medicinal plant A.mongolicus.Methods:1.The sample was ultrasonically extracted with 70%ethanol containing 0,2%,4%and 6%of ammonium hydroxides.The concentration of astragaloside IV in the extracts was determined to optimize the alkali level supplied in the extraction solution.The quantification was performed by UPLC-QTRAP-MS in multi-reaction monitoring mode and BEH C₁₈8 column（2.1 mm×50 mm,1.8μm）was used.And gradient elution was carried out with 0.1%formic acid（A）-acetonitrile（B）as mobile phase at a flow of 0.2 m L·min^-1,the column temperature was maintained at 30℃.The injection volume was 3μL.The quantification of calycosin-7-O-β-D-glucoside and ononin was performed by HPLC with the same conditions described in Pharmacopoeia of the People’s Republic（Version 2015）.And the characterization of acetylated astragaloside IV,calycosin-7-O-β-D-glucoside and ononin was performed by UPLC-Q-TOF-MS.2.Two-year old A.mongolicus plants transplanted in the field were used as materials and soil-borne exposure to hexanal was applied at the dosages of 5μL/seedling（Group 1）and 10μL/seedling（Group 2）.Each group contained 20 seedlings and 2 biological repeats were carried out and the plants with the same interference but with no hexanal application were used as control.The application data were as follows:June 29,July 23,September 13in 2016 and all applications were done at 9:00 a.m.In the early November of the same year,fresh roots were collected,dried in the shade and used for quantification of the bioactive compounds by the method established in the section one.3.With the help of next-generation sequencing technique,the transcriptome library was constructed using A.mongolicus seedlings upon below-ground exposure to 50μM hexanal as well as the one from the normal control.GO and pathway enrichment of differentially expressed genes（DEGs）was performed and comparison of the DEGs associated with the biosynthesis of the primary and secondary terpene compounds,especially the triterpene saponin.4.The genes annotated as terpene synthase was identified and checked by BLASTX.The phylogenetic tree was constructed by the software MEGA4 and other analysis was carried out by online tools.Using A.mongolicus adventitious root as raw material,effects of hexanal on CASs expression was primarily examined by quantitative Real-Time PCR.Results:1.The extraction with 70%ethanol containing 4%ammonia resulted in the highest yields of the three index compounds in trace amount of samples and the linearity ranges were 10.70⁸5.60μg·m L^-1,1.039⁷2.73μg·m L^-11 and 1.051⁷3.57μg·mL^-1,respectively.Intheoptimizedconditions,theconversionofastragalosideI,acylated calycosin-7-O-β-D-glucoside and ononin was relatively complete while astragaloside II was partially transformed.2.Compared with the control,the application of hexanal enhanced the root astragaloside IV accumulation in A.mongolicus transplanted in the field while had no significant effect on the accumulation of isoflavones and polysaccharide.3.82,619 Unigenes were obtained from the transcriptome library,the total nucleotide length was 90,944,019 nt,and the total annotation rate was 75.72%.Of the 5,791 DEGs,2,518 were classified into 125 pathways and those annotated as"metabolic pathways"and"secondary metabolites synthesized"were the most.Based on the annotation of DEGs as"plant hormone signal transduction pathway",the signal transduction pathways were deduced,indicating that multiple growth and defense signal pathways were induced upon hexanal treatment.In addition to the key enzyme genes responsible MVA pathway,the expression of these responsible for chlorophyll,gibberellin and abscisic acid biosynthesis were also induced by hexanal treatment.4.A total of 76 TPSs were identified,of which 13 had full open reading frames（ORFs）.The sequences annotated as TPS10 were the most,followed by the ones annotated as germacrene D synthase and monoterpene synthase.Seventh representative sequences of TPSs were selected for constructing phylogenetic tree,which demonstrated that the TPSs were clustered into three groups,each consisted of classⅠand classⅡterpenoid cyclase.In addition,three CASs responsible for cycloartenol biosynthesis were identified and predicted in endoplasmic reticulum.All of them were expressed in A.mongolicus adventitious roots,and the expression levels of CAS2 and CAS3 were upregulated by suppling of hexanal.Conclusion:1.An alkali treatment method was established for quantitative analysis of the three chemical index compounds in trace amount of Radix Astragali,and the protocol can simultaneously enhance the conversion rates of the three chemicals.2.Hexanal application exerted a positive role on astragaloside IV accumulation in A.mongolicus transplanted in the field.3.Multiple growth and defense signaling pathways were induced by the hexanal.In addition to the enzyme genes associated with the triterpenoid saponin biosynthesis,the gene expression of primary terpene compound biosynthesis from MEP pathway were also regulated.4.17 representative TPSs were identified,in which CASs was located in the endoplasmic reticulum,which was consistent with the cytoplasmic biosynthesis of the triterpenoid saponin by MVA pathway,coinciding with enhanced CAS expression induced by hexanal treatment.