The accumulation of astragaloside IV is affected by exogenous ethephon treatment in A. membranaceus
To investigate the impact of Ethylene (Eth) treatment on the accumulation of astragaloside IV and determine the optimal treatment concentration, A. membranaceus plants were exposed to different concentrations (0, 50, 200, 500 µmol·L− 1) of Eth. After a 3-hour treatment period, the content of astragaloside IV in A. membranaceus roots was quantitatively analyzed using HPLC. The results demonstrated a significant increase in astragaloside IV accumulation following Eth treatment, with the most pronounced effect observed at a concentration of 200 µmol·L− 1 (Fig. 1A). Therefore, this concentration was selected for subsequent experiments on A. membranaceus.
To further confirm the enhancement of astragaloside IV content in A. membranaceus by moderate Eth treatment, various treatment durations with 200 µmol·L− 1 Eth were investigated. The findings revealed an initial increase followed by a decrease in astragaloside IV accumulation with increasing treatment duration (Fig. 1B). Among the tested durations, a peak in astragaloside IV content was observed after 12 hours of treatment, exhibiting a significant 70% increase compared to the control group (P < 0.05). These findings provide further evidence that moderate Eth treatment can stimulate the accumulation of astragaloside IV in A. membranaceus.
The expression of key genes involved in the biosynthesis of astragaloside IV respond to exogenous ethephon signals
In order to further investigate the impact of Eth on the expression levels of genes associated with Astragaloside IV biosynthesis in A. membranaceus, ten genes involved in the biosynthetic pathway were selected for analysis, including AACT, HMGS, HMGR, IDI, FPS, SS, SE, CAS, CYP88D6, and CYP93E3. The expression levels of these genes were assessed through quantitative polymerase chain reaction (qPCR) at 0 hours, 12 hours, 3 days, and 7 days after Eth treatment (Fig. 2).
The results revealed that the exogenous application of Eth consistently downregulated the expression of HMGS, FPS, CAS, and CYP88D6 genes, maintaining lower levels compared to the control group. The HMGS gene exhibited reductions of 85.7%, 93.6%, and 98.6% at 12 hours, 3 days, and 7 days after Eth treatment, respectively. Similarly, the FPS gene displayed downregulation of 20.9%, 60.2%, and 3.9%, while the CAS gene showed reductions of 68%, 85.2%, and 99.3% at the respective time points. The CYP88D6 gene demonstrated decreases in expression of 40.5%, 90.6%, and 97.0% under the influence of Eth treatment at 12 hours, 3 days, and 7 days, respectively.
In contrast, although the CYP93E3 gene consistently displayed downregulated expression, its levels were higher than the control group at 7 days, with downregulation of 11.6% and 55.7% observed at 12 hours and 3 days, respectively, and upregulation of 5.2% at 7 days under Eth treatment. The AACT and IDI genes exhibited initially increasing, then decreasing, and finally increasing expression patterns, reaching their maximum levels at 12 hours with upregulations of 35.1% and 3.3%, respectively, compared to the control. However, by 3 days, their expression levels had significantly dropped, downregulated by 93.6% and 93.1% compared to the control. The HMGR and SS genes displayed initial increases followed by decreases, reaching their peak expression levels at 12 hours with upregulations of 833.4% and 2.8% compared to the control, and declining to their lowest levels at 7 days, still exhibiting upregulation of 49.9% and 18.6% compared to the control, respectively.Regarding the SE gene, its expression pattern exhibited an initial decrease, followed by an increase, and subsequently another decrease. At 12 hours, the expression level reached its lowest point with a downregulation of 89.4% compared to the control.
The accumulation of astragaloside IV in response to ethylene treatment exhibits a significant correlation with the expression of its key biosynthetic genes
Correlation analysis was conducted to assess the relationship between astragaloside IV content in A. membranaceus roots treated with Eth and the expression levels of key enzyme genes. As depicted in Fig. 3, the findings revealed a significant negative correlation between FPS and astragaloside IV content (P < 0.05). Additionally, highly significant negative correlations were observed between HMGR, IDI, SS, CYP93E3, and astragaloside IV content (P < 0.01). Conversely, SE exhibited a highly significant positive correlation with astragaloside IV content (P < 0.01). These results indicate the involvement of these six genes in the biosynthetic pathway of astragaloside IV and emphasize the need for further investigation into their mechanisms of action. Other genes did not exhibit a significant correlation with astragaloside IV content, suggesting their involvement in astragaloside IV biosynthesis, albeit to a lesser extent.