| Astragali Radix in Hunyuan,Shanix is well recognized as a genuine and high-quality Chinese herbal medicine in Chinese medicine.The formation of the high-quality herb is closely related to the unique ecological geography,cultural background,planting and processing patterns,and the theory of traditional Chinese theory.The genuine herb of Astragali Radix possesses a strong “beany” odor,which is one of the important sensory characters for indicating its good quality.At present,the chemical substances closely related to the “beany” odor have been basically clarified,which results from a variety of aldehydes,alcohols,and esters,collectively known as green leaf volatiles(GLVs).Limited works have shown that GLVs play important signaling roles in plant chemical defense.In the present study,we used adventitious root cultures of Astragalus membranaceus(Fisch.)Bge.var.mongholicus(Bge.)as plant material and explored the effects of certain GLVs dominantly contained in Astragali Radix in Hunyuan on the biosynthesis and accumulation of calycosin-7-O-β-D-glucoside by adding into the culture medium and underlying mechanism as well.The volatiles exmianed included hexanal,hexanol,and E-2-hexanal and the concentration of 50 μM was applied.The methods involved are tissue culture,UPLC-MS,transcriptome sequencing,and real-time PCR.The main results are as follows:1.Effects of hexanal,hexanol,and E-2-hexanal on the biosynthesis and accumulation of calycosin-7-O-β-D-glucoside(CG)in adventient roots:(1)the CG contents in hexanal-and hexanol-treated roots at the 7-day point were significantly higher than they were in the normal control of the same point,and the corresponding increase folds were 56.07% and 65.18%,respectively;(2)the CG contents in hexanol-treated roots at the 14-day point was significantly incresed and kept at the same level as it was at the 7-day point;(3)the onone content in hexanol-treated group at the 14-day point was obviously higher than they were in the control and treated groups with hexanal and E-2-hexanal;(4)the root yield in hexanol treatment at the 14-day point was the highest and its growth rate was 1.25 ± 0.34;(5)among the treatments with different concentrations of hexanol in second batch experiment,50 μM hexanol simultaneously increased the contents of CG and ononin in the 2-week root cultures,while 100 and 200 μM hexanol resulted in the highest production of CG and ononin in the 6-week root cultures.2.Promoting mechanism of green leaf volatiles on the CG biosynthesis and accumulation in adventient roots of A.membranaceus var.mongholicus:(1)with respect to the transcriptional profiles,the application of hexanol induced a different gene expression pattern from E-2-hexenal,50% ethanol,and the normal control;(2)along with the extended period of the root culture,the amount of the genes with FPKM values between 1 to 10 was incresed in hexanol treatment,so did the amount of differentially expressed genes enriched in the biosyntheis of CG;(3)hexanol treatment down-regulated the relative expression levels of the enzyme genes involved in the biosynthesis of the scafffolds of CG such as 4CL,IFS1.1,and IFS1.2,and upregulated the transcription of the enzyme genes responsible for the glycosylation and acylation of calycosin;(4)exposure to hexanol significantly increased the expression level of the gene IFS1.1 in the 5-week root cultures;(5)hexanol treatment reduced the contents of formononetin and calycosin in the 1-week root cultures as well as the content of formononetin in the 2-week roots.Taken together,GLVs expecially hexanol exerted promoting effects on the production of CG and ononin as well as the biomass yield in adventitious root cultures.And the promotion of hexanol was mediated via the inhibition of the biosynthesis of the precursors formononetin and calycosin at the transcriptional and metabolic levels,and the upregulated expression of the enzyme gene responsible for the glycosylation and acylation. |
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