| Objective: Focusing on the excavation and utilization of the microbial resources in Astragali Radix distributed in Shanxi,Astragali Radix in Hunyuan was used as raw materials for its strong bean odor that was imparted by green leaf volatiles(GLVs).Using GLVs as chemical clues,the present study was performed to characterize the composition and functions of the bacterial endophytes residing the root tissue of Astragalus mongholicus,the dried roots of which are authorized as Astragali Radix in Chinese medicine.Some interesting results have been obtained,which should be of great significance for the development and utilization of the microbial resources in plants with important economical and/or medicinal values in practice,the development of green and sustainable cultivation of Chinese herbal plants medicinal material planting industry and the sustainable utilization of Chinese herbal resources as well.Research contents and methods:(1)Composition and functions of the endophytic bacteria residing the root tissue of Astragalus mongholicus in Hunyuan: The fresh root tissue was collected from 7-year old plants of Astragalus mongholicus at the traditional harvest season for isolation of the bacterial endophytes;subsequent identification of the species was performed by 16S rRNA sequencing while the screening of the candidate plant growth-promoting strains was carried out by adding exogenous substrates such as tryptophan,K-feldspar and GLVs that existed in the herb,and the quantification of GLVs in the herb was performed by headspace GC-MS;(2)Mechanism of the growth promotion of E-2-hexenal on the strain H65: the growth promotion of E-2-hexenal was firstly characterized by cold pretreatment of the inoculant then being cultured in the medium supplied with 50 ?M E-2-hexenal;afterwards,the cells at the log phase in the control and treated groups with E-2-hexenal were collected for high-throughput sequencing to identified differentially expressed genes(DEGs),the cell response to the chemical and metabolism were deduced based on the GO and pathway enrichments of the DEGs and verified by q RT-PCR and GC-MS.(3)Effects of E-2-hexenal and 2,3-butanediol on the biosynthesis and accumulation of isoflavone and triterpenoid saponin compounds: the liquid adventitious root cultures of A.mongholicus were used as raw material and randomly divided into four groups: normal control,E-2-hexenal,2,3-butanediol and a combination of E-2-hexenal,2,3-butanediol;After supplied into the medium,a dynamic sampling strategy was applied and UHPLC-MS in a selected ion manner was used to determine the contents of formononeyin-7-O-?-D-glucoside,formononeyin,calycosin,calycosin-7-O-?-D-glucoside and astragaloside ?.Results:(1)Composition and function of endophytic bacteria residing the root tissue of Astragalus mongholicus in Hunyuan: A total of 85 bacterial strains were obtained and classified into the follow phyla: Proteobacteria,Firmicutes,actinobacteria and Bacteroides.At the genus level,they were categorized into 13 genera and the stains of Pseudomonas,Pantoea and Staphylococcus were more abundant(80.00%)than the ones of the other genera.Among candidate isolates with plant growth-promoting activities,these that can synthesize indoleacetic acid(IAA)shared the biggest proportion(69.41%),followed by these with ACC deaminase(40.00%)and nitrogen fixation activity(31.76%),and these that can solubilize phosphorus and potassium shared relatively lower proportions(i.e.,14.12% and 7.06%).Among the isolates with double plant growth-promoting activities,the IAA-producing strains containing ACC deaminase shared the biggest proportion(37.65%),followed by the IAA-producing strains with nitrogen fixation activity and the strains with ACC deaminase and nitrogen fixation activity,accounting for 28.24% and24.71%,respectively,while these that not only contain ACC deaminase but can solubilize potassium bearing minerals shared the smallest part(1.18%).Besides,low concentrations of GLVs(i.e.,2-50 ?mol/L of hexanal and Z-3-hexenal,and 5-125 ?mol/L hexanol)exerted positive effects on the growth of a few strains with ACC deaminase.(2)Mechanism of the growth promotion of E-2-hexenal on the strain H65: the growth-promotion of E-2-hexenal was tightly associated with the cold pretreatment of the inoculants and the pretreatment for 12 h resulted in significantly increased cell densities relative to the control,coinciding with dysregulated genes associated with environmental information processing,carbohydrate metabolism and xenobiotics biodegradation and metabolism.Based on the annotation of the DEGs,the nem A and DAR genes encoding the enzymes encoding N-ethylmaleimide reductase and diacetyl reductase should be involved in the reduction of E-2-hexenal and biosynthesis of 2,3-butanediol.Changes in their expression levels and the contents of 2,3-butanediol and E-2-hexenal in the culture medium indicated that E-2-hexenal should be transported into the cells at the early growth stage and reduced by NEM reductase then utilized for the biosynthesis of 2,3-butanediol at the stationary phase when carbon sources such as glucose are insufficient.(3)Effects of E-2-hexenal and 2,3-butanediol on the biosynthesis and accumulation of isoflavone and triterpenoid saponin compounds: The results showed that green leaf volatile E-2-hexenal and microbial-derived 2,3-butanediol exerted positive effects on the biosynthesis and accumulation of multiple bioactive compounds described above.In comparison,the induction of E-2-hexenal on the isoflavone compounds was stronger and more persistent while the induced biosynthesis and accumulation of astragaloside ? by2,3-butanediol was more rapid and shorter.Conclusion:(1)There are GLV-promoting bacterial endophytes in the root tissue of A.mongholicus in Hunyuan;(2)These bacterial cells can use green leaf volatiles as an alternative nutrient source by the reduction and conversation to 2,3-butanediol at the stationary phase;(3)GLVs and corresponding converted products exhibited positive effects on the biosynthesis and accumulations of bioactive isoflavone and triterpenoid compounds in different patterns,and the induction of microbial converted products was more rapid and the effect of GLVs was more persistent as far as astragaloside ? was concerned. |
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