Exogenous ethephon treatment on the biosynthesis and accumulation of astragaloside IV in Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao

Exogenous ethephon treatment on the biosynthesis and accumulation of astragaloside IV in Astragalus membranaceus Bge. var. mongholicus (Bge.) 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Hsiao Haonan Wu, Yu Tian, Jiawen Wu, Zhenqing Bai, Xiujuan Zhang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3791227/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Background Astragaloside IV, a prominent secondary metabolite found in Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao ( A. membranaceus ), serves as a crucial indicator of A. membranaceus quality. Ethylene, acting as an exogenous signal, plays a role in regulating secondary metabolism in plants. In this study, the application of ethephon (Eth) to hydroponically cultivated A. membranaceus was employed to investigate the biosynthesis of astragaloside IV in the roots, involving both content measurement and analysis of key gene expression. Results The results demonstrated that the significantly accumulation of astragaloside IV was observed on the 3rd day after 200 µmol·L − 1 Eth treatment, reaching 0.269%. Among the 10 key genes involved in astragaloside IV synthesis, HMGS , FPS , CAS , CYP88D6 , and CYP93E3 were found to be insensitive to Eth. On the other hand, the expression levels of AACT , HMGR , IDI , and SS exhibited a significant increase at 12 hours under Eth treatment, followed by a notable decrease at 3rd day. Additionally, SE displayed a significant decrease at 12 hours and a subsequent increase in the 3rd day under Eth treatment. The expression level of FPS , HMGR , IDI , SS , and CYP93E3 exhibited significant negative correlations with astragaloside IV content, while expression level of SE displayed a significant positive correlation. Conclusions These findings suggest that exogenous Eth treatment can potentially influence the synthesis of astragaloside IV by modulating the expression of FPS , HMGR , IDI , SS , CYP93E3 and SE . This study provides a theoretical basis for utilizing molecular strategies to enhance the quality of A. membranaceus . Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao ethylene astragaloside IV gene expression Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao ( A. membranaceus ) is a traditional Chinese medicinal (TCM) renowned for its tonifying properties, diuretic effects, and its use in treating various conditions such as cerebral ischemic diseases and immune system Disorders (National Pharmacopoeia Committee. 2020; Hu et al. 2021 ; Kong et al. 2018 ; Tsai et al. 2019 ). However, excessive wild harvesting led to big challenge in meeting market demands. Therefore, field cultivation serves as the primary approach to address this issue (Liu et al. 2020 ). Nonetheless, the quality of A. membranaceus field cultivation has a decrease in the levels of its active ingredients (Yang et al. 2022 ; Chen et al. 2022 ). To address this issue, hydroponics, a soilless cultivation method, has gained popularity due to its advantages such as shorter growth cycles, high yields, improved quality, and reduced susceptibility to pests and diseases (Barrett et al. 2016 ). Hydroponics has been extensively employed in the organic production of various Chinese medicinal herbs, including dandelion, ligularia, peppermint, and ginseng (Minling et al. 2022 ; Wei et al. 2018 ; Ćavar Zeljković et al. 2022 ; Lee et al. 2022 ). Moreover, it has emerged as a key approach for the cultivation of Chinese medicinal herbs in recent years. Previous studies have indicated that hydroponically grown A. membranaceus exhibits significantly higher levels of Astragaloside IV compared to field cultivation (Chen et al. 2021 ). Astragaloside IV is a major active compound of A. membranaceus and bears a tetracyclic triterpenoid saponin structure. The biosynthetic pathway of tetracyclic triterpenoid saponins primarily involves the mevalonic acid pathway located in the cytoplasm. Extensive research has been conducted on key genes in this pathway. These genes can be categorized into three groups: upstream genes involved in the synthesis of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), including acetoacetyl-cozymeA (CoA) thiolase (AACT) and 3-hydroxy-3-methyl glutaryl coenzyme A reductase (HMGR), which directly catalyze the generation of mevalonate (Tian et al. 2015), the genes involve in the formation of terpene carbon skeletons and intermediates, including squalenesynthase (SS) and squaleneepoxidase (SE), which catalyze the production of 2,3-oxidosqualene leading to triterpene skeleton cyclization, and cycloartenolsynthase (CAS), which catalyzes the 2,3-oxidosqualene cyclization reaction (Yoshioka et al. 2020 ). The downstream genes involved in complex structural modifications of intermediates and terpene compounds, such as cytochrome P450 (CYP450) and UDP-glycosyltransferases (UGT). These genes play a role in modifying the triterpene skeleton, leading to the synthesis of various triterpene compounds (Dai et al. 2015 ; Wang et al. 2020 ). While upstream key genes in tetracyclic triterpenoid saponin metabolism have been extensively studied, the influence of downstream key enzymes on metabolism remains ambiguous. Ethylene is a regulatory molecule in plants, impacting plant secondary metabolite synthesis (Zhao et al. 2005 ; Shahrajabian et al. 2022). Such as, ethylene boosts secondary metabolite production, exemplified by its capacity to increase total flavonoid levels in sandalwood leaves (Li et al. 2021 ). Ethylene promotes saponin accumulation by upregulating key enzyme genes in squalene biosynthesis, such as squalene synthase and squalene epoxidase. For example, applying the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) to cultured ginseng cells enhances transcription of these genes, leading to increased saponin content (Rahimi et al. 2015 ). Similarly, ethylene treatment elevates the total saponin content in young Gynostemma leaves (Xu et al. 2022). While hormonal regulation of synthetic metabolism is a nascent area of research, further inquiry is necessary to elucidate hormone effects on triterpenoid saponin biosynthesis. To comprehend how exogenous ethylene modulates astragaloside IV biosynthesis and accumulation in A. membranaceus roots, this study utilized hydroponic methodologies, HPLC analysis, and qPCR analysis. The principal aim was to assess the influence of exogenous ethylene on astragaloside IV synthesis, thereby establishing a theoretical basis for advancing hydroponic techniques in A. membranaceus. Materials and Methods Plant materials and growth conditions. A. membranaceus seeds were collected in Chifeng City, Inner Mongolia, China, and then, seeds were sown in nutrient soils (vermiculite and nutrient soil ratio was 1: 3). The seeds were planted with sufficient water. Then, the seedlings were cultured under 16h light/8h darkness and 25°C for 30 days. Watered every two days. Then, the one-month plants were cultivated in a hydroponic device. Ethephon concentration screen and sample collection. On the 30th day of hydroponic cultivation, ethephon was added to the nutrient solution for A. membranaceus seedlings, resulting in a working concentration of 200 µmol·L − 1 . Nutrient solutions without inducers were used as controls, with each treatment group having 3 replicates. Subsequently, samples were taken at 0 h, 12 h, 72 h, and 7 days after treatment. Samples were washed with tap water, surface liquid was filtered, and the samples were then air-dried at 45°C to measure their fresh weight. Afterward, the dried samples were ground into powder for further analysis. Determination of astragaloside IV The High-Performance Liquid Chromatography (HPLC) was used to determine the content of astragaloside IV. The determination method described as previously published by our laboratory (Chen et al. 2021 ). RNA Extraction and Real‑Time PCR Extract Total RNA from the roots of A. membranaceus using the Total RNA extraction kit (TAKARA, Beijing). Total RNA was reverse transcribed into cDNA using the PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) kit (TAKARA, Beijing). Using cDNA from A. membranaceus roots as a template and 18S as an internal reference gene, the AACT , HMGS , HMGR , IDI , FPS , SS , SE , CAS , CYP88D6 and CYP93E3 were amplified by RT-PCR. The experiment was repeated three times. The gene expression levels were analyzed using the 2 − ΔΔCt method. The primer sequences are provided in Table S. The RTPCR reaction program was as follows: 95°C for 30 s, 95°C for 5 s, 60°C for 30 s, 40 cycles. Data Analysis The statistical analysis for all data was conducted using the statistical analysis software Excel 2010 and SPSS software (version 20.0). All data were presented as the mean values and standard deviations of three replicate experiments. The significance differences between means were determined using the Duncan multiple range test. Graphs were generated using Origin 2021. Results 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. 1 A). 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. 1 B). 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. Discussion Astragaloside IV has anti-inflammatory, antioxidant, anti-apoptotic, and tumor-inhibiting biological activities, which make it a highly promising compound with broad clinical potential (Liang et al. 2023 ). Moreover, the content of astragaloside IV serves as an important indicator for assessing the quality of A. membranaceus (Zhang et al. 2020 ). The hydroponically cultivated A. membranaceus exhibits significantly higher levels of astragaloside IV compared to field (Chen et al. 2021 ). Exogenous inducers could modulate the levels of secondary metabolites in plants. Ethylene, as a signaling molecule, plays a regulatory role in plant secondary metabolism (Dubois et al. 2018 ). For instance, under exogenous 50 µM ethylene treatment, root growth and ginsenoside accumulation significantly enhanced in adventitious root of Panax ginseng C.A. Meyer (Bae et al. 2006 ). High concentrations of ethylene can stimulate the biosynthesis of saponins in Calendula officinalis hairy roots (Markowski et al. 2022 ). Similarly, exogenous ethylene treatment has been found to promote the production of ganoderic acid in Ganoderma lucidum (Zhang et al. 2017 ). Under exogenous ethylene treatment, the accumulation of various indole alkaloids enhanced in Chrysanthemum leaves (Pan et al. 2018 ). In the present study, under exogenous 200 mM ethylene treatment, the content of astragaloside IV significantly increased in A. membranaceus roots. Ethylene plays a crucial role in regulating the changes in active ingredient content in medicinal plants by modulating the expression of key genes involved in secondary metabolism pathways (Tahmasebi et al. 2019 ). Understanding the alterations in the expression levels of these key genes is essential for studying the synthesis and accumulation of secondary metabolites (Pan et al. 2015 ). For instance, ethylene has been shown to regulate the expression of genes such as FPS, SS, and SE involved in ginsenoside synthesis, resulting in ginsenoside content the increased in Panax ginseng C.A. Mey (Rahimi et al. 2015 ). Similarly, ethylene treatment upregulates key genes of the jasmonate alkaloid synthesis pathway, leading to the accumulation of jasmonate alkaloids in Catharanthus roseus . In Ganoderma lucidum , the upregulation of genes such as HMGR , SS , and OSC involved in the biosynthesis of ganoderic acid by ethylene treatment, leading to its increased accumulation (Pan et al. 2018 ). Additionally, treatment with an ethylene releaser increases the expression of key genes involved in the biosynthetic pathways of rhynchophylline (RIN) and isorhynchophylline (IRN) in Uncaria rhynchophylla , resulting in higher RIN and IRN content in U. rhynchophylla leaves (Li et al. 2022 ). In this study, treatment with ethephon led to the downregulation of gene expression of AACT , HMGR , IDI , and SS , while SE gene expression was promoted. These genes likely have shared roles and mutually influence each other in the synthesis pathway of astragaloside IV, collectively regulating its synthesis. Furthermore, under ethylene treatment, significant negative correlations were observed between FPS , HMGR , IDI , SS, CYP93E3 and astragaloside IV, whereas SE showed a highly significant positive correlation. This suggests that the SE gene can respond to exogenous ethylene signals, facilitating the synthesis and accumulation of astragaloside IV. In conclusion, exogenous treatment with ethephon can enhance the content of astragaloside IV in hydroponically cultivated A. membranaceus . This effect is achieved through the regulation of key genes involved in the synthesis pathway, with particular emphasis on the critical role of FPS , HMGR , IDI , SS , CYP93E3 and SE gene, which is regulated by ethylene and promotes the synthesis of astragaloside IV (Fig. 4 ). This study provides a foundation for further investigations into the mechanisms by which exogenous inducers affect the secondary metabolism pathway of astragaloside IV, and it offers insights for genetic improvement of A. membranaceus. Declarations Ethics approval and consent to participate Not applicable Consent for publication Not applicable Availability of data and materials The data and material used during the current study are available from the author on reasonable request. Competing interests Not applicable Funding We thank Science and technology projects of Inner Mongolia Autonomous Region (2021GG0342 and 2022YFDZ0015) for financial supports. Authors' information BZQ conceived the research and revised the manuscript. WHN and TY performed the experiments, data analysis and wrote the manuscript. WJW and ZXJ gave the project support and the design guidance of experimental. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3791227","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":266213338,"identity":"01874d82-59de-4fd4-852c-7b004568b765","order_by":0,"name":"Haonan Wu","email":"","orcid":"","institution":"Yan'an University","correspondingAuthor":false,"prefix":"","firstName":"Haonan","middleName":"","lastName":"Wu","suffix":""},{"id":266213339,"identity":"795afb27-9e7c-458f-bf8e-5ab029a32ace","order_by":1,"name":"Yu Tian","email":"","orcid":"","institution":"Yan'an University","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Tian","suffix":""},{"id":266213340,"identity":"a5f4073f-a029-4995-92bb-8ab6360b6a8b","order_by":2,"name":"Jiawen Wu","email":"","orcid":"","institution":"Yan'an University","correspondingAuthor":false,"prefix":"","firstName":"Jiawen","middleName":"","lastName":"Wu","suffix":""},{"id":266213341,"identity":"6b33cd06-a545-4090-b0a8-c7c8d1256d81","order_by":3,"name":"Zhenqing Bai","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAx0lEQVRIiWNgGAWjYDACCRBhcICBgb2x8eEH0rTwHG42liBeCwNQi0R6mwAPMTrkZzc/e8xTcMee7+bDNqB+OzndBgJaDO4cMzecYfCMWfJ2YtuDAoZkY7MDhLRIJJhJfDA4zGZwO7HdQILhQOI2QlrkZ6R/k0gwOMxjcPNgmwQPMVoYbuSAbZEwuMFIpBaDGzllkkC/GEieSQQGsgERfgE6bJs0zx9giB0//vDhhwo7OYJaEACs0oBo5XAto2AUjIJRMAqwAACUAkTYPBUe5QAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-0942-1843","institution":"Yan'an University","correspondingAuthor":true,"prefix":"","firstName":"Zhenqing","middleName":"","lastName":"Bai","suffix":""},{"id":266213342,"identity":"c3b62245-0871-4a78-b3bf-793ff44bb273","order_by":4,"name":"Xiujuan Zhang","email":"","orcid":"","institution":"Inner Mongolia Academy of science and technology","correspondingAuthor":false,"prefix":"","firstName":"Xiujuan","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2023-12-22 09:46:00","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3791227/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3791227/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":49466940,"identity":"d13d7583-eb8f-4f43-8d5b-4d9e01fc0063","added_by":"auto","created_at":"2024-01-11 10:01:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":360628,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of different concentrations of ethylene releaser (A) and different treatment durations (B) on the content of astragaloside IV. \u0026nbsp;** represents \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-3791227/v1/d1852dd5c68a644bb134a1d0.png"},{"id":49466939,"identity":"a789e3ae-8b3c-42aa-97dd-10b878fcafdb","added_by":"auto","created_at":"2024-01-11 10:01:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":617265,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of Ethylene on the expression of key genes in the roots of \u003cem\u003eA. membranaceus\u003c/em\u003e. * and** represent signifcant correlation\u003cem\u003e \u003c/em\u003ein\u003cem\u003e P\u003c/em\u003e\u0026lt;0.05 and \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 respectively.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-3791227/v1/ddd9a22c3375cb1128146be2.png"},{"id":49467183,"identity":"5cd8ccf3-841a-4c5c-8b5d-3dcef9e2acd2","added_by":"auto","created_at":"2024-01-11 10:09:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":908171,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation analysis between astragaloside IV and key genes under Ethylene treatment. * and** represent signifcant correlation\u003cem\u003e \u003c/em\u003ein\u003cem\u003e P\u003c/em\u003e\u0026lt;0.05 and \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 respectively.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-3791227/v1/2d1a5c3b923f33f31ec46740.png"},{"id":49466938,"identity":"0e062e23-a25a-4e55-ac1f-f1906ca5d73e","added_by":"auto","created_at":"2024-01-11 10:01:52","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":352251,"visible":true,"origin":"","legend":"\u003cp\u003eGene-metabolite correlation network under ETH treatment.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-3791227/v1/11568c976631786657d257fe.png"},{"id":49467680,"identity":"53437ba4-ad56-4848-8e66-2201cffb117e","added_by":"auto","created_at":"2024-01-11 10:17:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":915910,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3791227/v1/66f53da5-fed6-4e2a-9390-5982b958edb6.pdf"},{"id":49466937,"identity":"bfadaa26-9f67-4ad6-b4bc-63ba5d4cec52","added_by":"auto","created_at":"2024-01-11 10:01:52","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":16174,"visible":true,"origin":"","legend":"","description":"","filename":"TableS.docx","url":"https://assets-eu.researchsquare.com/files/rs-3791227/v1/03d4ffc19f43fb31386780a2.docx"}],"financialInterests":"","formattedTitle":"Exogenous ethephon treatment on the biosynthesis and accumulation of astragaloside IV in Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao","fulltext":[{"header":"Background","content":"\u003cp\u003e \u003cem\u003eAstragalus membranaceus\u003c/em\u003e Bge. var. mongholicus (Bge.) Hsiao (\u003cem\u003eA. membranaceus\u003c/em\u003e) is a traditional Chinese medicinal (TCM) renowned for its tonifying properties, diuretic effects, and its use in treating various conditions such as cerebral ischemic diseases and immune system Disorders (National Pharmacopoeia Committee. 2020; Hu et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Kong et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Tsai et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). However, excessive wild harvesting led to big challenge in meeting market demands. Therefore, field cultivation serves as the primary approach to address this issue (Liu et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Nonetheless, the quality of \u003cem\u003eA. membranaceus\u003c/em\u003e field cultivation has a decrease in the levels of its active ingredients (Yang et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Chen et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). To address this issue, hydroponics, a soilless cultivation method, has gained popularity due to its advantages such as shorter growth cycles, high yields, improved quality, and reduced susceptibility to pests and diseases (Barrett et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Hydroponics has been extensively employed in the organic production of various Chinese medicinal herbs, including dandelion, ligularia, peppermint, and ginseng (Minling et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Wei et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Ćavar Zeljković et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Lee et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Moreover, it has emerged as a key approach for the cultivation of Chinese medicinal herbs in recent years. Previous studies have indicated that hydroponically grown \u003cem\u003eA. membranaceus\u003c/em\u003e exhibits significantly higher levels of Astragaloside IV compared to field cultivation (Chen et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAstragaloside IV is a major active compound of \u003cem\u003eA. membranaceus\u003c/em\u003e and bears a tetracyclic triterpenoid saponin structure. The biosynthetic pathway of tetracyclic triterpenoid saponins primarily involves the mevalonic acid pathway located in the cytoplasm. Extensive research has been conducted on key genes in this pathway. These genes can be categorized into three groups: upstream genes involved in the synthesis of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), including acetoacetyl-cozymeA (CoA) thiolase (AACT) and 3-hydroxy-3-methyl glutaryl coenzyme A reductase (HMGR), which directly catalyze the generation of mevalonate (Tian et al. 2015), the genes involve in the formation of terpene carbon skeletons and intermediates, including squalenesynthase (SS) and squaleneepoxidase (SE), which catalyze the production of 2,3-oxidosqualene leading to triterpene skeleton cyclization, and cycloartenolsynthase (CAS), which catalyzes the 2,3-oxidosqualene cyclization reaction (Yoshioka et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The downstream genes involved in complex structural modifications of intermediates and terpene compounds, such as cytochrome P450 (CYP450) and UDP-glycosyltransferases (UGT). These genes play a role in modifying the triterpene skeleton, leading to the synthesis of various triterpene compounds (Dai et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). While upstream key genes in tetracyclic triterpenoid saponin metabolism have been extensively studied, the influence of downstream key enzymes on metabolism remains ambiguous.\u003c/p\u003e \u003cp\u003eEthylene is a regulatory molecule in plants, impacting plant secondary metabolite synthesis (Zhao et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Shahrajabian et al. 2022). Such as, ethylene boosts secondary metabolite production, exemplified by its capacity to increase total flavonoid levels in sandalwood leaves (Li et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Ethylene promotes saponin accumulation by upregulating key enzyme genes in squalene biosynthesis, such as squalene synthase and squalene epoxidase. For example, applying the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) to cultured ginseng cells enhances transcription of these genes, leading to increased saponin content (Rahimi et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Similarly, ethylene treatment elevates the total saponin content in young Gynostemma leaves (Xu et al. 2022). While hormonal regulation of synthetic metabolism is a nascent area of research, further inquiry is necessary to elucidate hormone effects on triterpenoid saponin biosynthesis.\u003c/p\u003e \u003cp\u003eTo comprehend how exogenous ethylene modulates astragaloside IV biosynthesis and accumulation in \u003cem\u003eA. membranaceus\u003c/em\u003e roots, this study utilized hydroponic methodologies, HPLC analysis, and qPCR analysis. The principal aim was to assess the influence of exogenous ethylene on astragaloside IV synthesis, thereby establishing a theoretical basis for advancing hydroponic techniques in \u003cem\u003eA. membranaceus.\u003c/em\u003e\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e \u003cb\u003ePlant materials and growth conditions.\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eA. membranaceus\u003c/em\u003e seeds were collected in Chifeng City, Inner Mongolia, China, and then, seeds were sown in nutrient soils (vermiculite and nutrient soil ratio was 1: 3). The seeds were planted with sufficient water. Then, the seedlings were cultured under 16h light/8h darkness and 25\u0026deg;C for 30 days. Watered every two days. Then, the one-month plants were cultivated in a hydroponic device.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEthephon concentration screen and sample collection.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eOn the 30th day of hydroponic cultivation, ethephon was added to the nutrient solution for \u003cem\u003eA. membranaceus\u003c/em\u003e seedlings, resulting in a working concentration of 200 \u0026micro;mol\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Nutrient solutions without inducers were used as controls, with each treatment group having 3 replicates. Subsequently, samples were taken at 0 h, 12 h, 72 h, and 7 days after treatment. Samples were washed with tap water, surface liquid was filtered, and the samples were then air-dried at 45\u0026deg;C to measure their fresh weight. Afterward, the dried samples were ground into powder for further analysis.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of astragaloside IV\u003c/h2\u003e \u003cp\u003eThe High-Performance Liquid Chromatography (HPLC) was used to determine the content of astragaloside IV. The determination method described as previously published by our laboratory (Chen et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eRNA Extraction and Real‑Time PCR\u003c/h2\u003e \u003cp\u003eExtract Total RNA from the roots of \u003cem\u003eA. membranaceus\u003c/em\u003e using the Total RNA extraction kit (TAKARA, Beijing). Total RNA was reverse transcribed into cDNA using the PrimeScript\u0026trade; RT reagent Kit with gDNA Eraser (Perfect Real Time) kit (TAKARA, Beijing). Using cDNA from \u003cem\u003eA. membranaceus\u003c/em\u003e roots as a template and \u003cem\u003e18S\u003c/em\u003e as an internal reference gene, the \u003cem\u003eAACT\u003c/em\u003e, \u003cem\u003eHMGS\u003c/em\u003e, \u003cem\u003eHMGR\u003c/em\u003e, \u003cem\u003eIDI\u003c/em\u003e, \u003cem\u003eFPS\u003c/em\u003e, \u003cem\u003eSS\u003c/em\u003e, \u003cem\u003eSE\u003c/em\u003e, \u003cem\u003eCAS\u003c/em\u003e, \u003cem\u003eCYP88D6\u003c/em\u003e and \u003cem\u003eCYP93E3\u003c/em\u003e were amplified by RT-PCR. The experiment was repeated three times. The gene expression levels were analyzed using the 2\u0026thinsp;\u0026minus;\u0026thinsp;ΔΔCt method. The primer sequences are provided in Table S. The RTPCR reaction program was as follows: 95\u0026deg;C for 30 s, 95\u0026deg;C for 5 s, 60\u0026deg;C for 30 s, 40 cycles.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eData Analysis\u003c/h2\u003e \u003cp\u003eThe statistical analysis for all data was conducted using the statistical analysis software Excel 2010 and SPSS software (version 20.0). All data were presented as the mean values and standard deviations of three replicate experiments. The significance differences between means were determined using the Duncan multiple range test. Graphs were generated using Origin 2021.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eThe accumulation of astragaloside IV is affected by exogenous ethephon treatment in\u003c/b\u003e \u003cb\u003eA. membranaceus\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate the impact of Ethylene (Eth) treatment on the accumulation of astragaloside IV and determine the optimal treatment concentration, \u003cem\u003eA. membranaceus\u003c/em\u003e plants were exposed to different concentrations (0, 50, 200, 500 \u0026micro;mol\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) of Eth. After a 3-hour treatment period, the content of astragaloside IV in \u003cem\u003eA. membranaceus\u003c/em\u003e 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 \u0026micro;mol\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Therefore, this concentration was selected for subsequent experiments on \u003cem\u003eA. membranaceus\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further confirm the enhancement of astragaloside IV content in \u003cem\u003eA. membranaceus\u003c/em\u003e by moderate Eth treatment, various treatment durations with 200 \u0026micro;mol\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Eth were investigated. The findings revealed an initial increase followed by a decrease in astragaloside IV accumulation with increasing treatment duration (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). 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 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These findings provide further evidence that moderate Eth treatment can stimulate the accumulation of astragaloside IV in \u003cem\u003eA. membranaceus\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe expression of key genes involved in the biosynthesis of astragaloside IV respond to exogenous ethephon signals\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn order to further investigate the impact of Eth on the expression levels of genes associated with Astragaloside IV biosynthesis in \u003cem\u003eA. membranaceus\u003c/em\u003e, ten genes involved in the biosynthetic pathway were selected for analysis, including \u003cem\u003eAACT\u003c/em\u003e, \u003cem\u003eHMGS\u003c/em\u003e, \u003cem\u003eHMGR\u003c/em\u003e, \u003cem\u003eIDI\u003c/em\u003e, \u003cem\u003eFPS\u003c/em\u003e, \u003cem\u003eSS\u003c/em\u003e, \u003cem\u003eSE\u003c/em\u003e, \u003cem\u003eCAS\u003c/em\u003e, \u003cem\u003eCYP88D6\u003c/em\u003e, and \u003cem\u003eCYP93E3\u003c/em\u003e. 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.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe results revealed that the exogenous application of Eth consistently downregulated the expression of \u003cem\u003eHMGS\u003c/em\u003e, \u003cem\u003eFPS\u003c/em\u003e, \u003cem\u003eCAS\u003c/em\u003e, and \u003cem\u003eCYP88D6\u003c/em\u003e genes, maintaining lower levels compared to the control group. The \u003cem\u003eHMGS\u003c/em\u003e 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 \u003cem\u003eFPS\u003c/em\u003e gene displayed downregulation of 20.9%, 60.2%, and 3.9%, while the \u003cem\u003eCAS\u003c/em\u003e gene showed reductions of 68%, 85.2%, and 99.3% at the respective time points. The \u003cem\u003eCYP88D6\u003c/em\u003e 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.\u003c/p\u003e \u003cp\u003eIn contrast, although the \u003cem\u003eCYP93E3\u003c/em\u003e 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 \u003cem\u003eAACT\u003c/em\u003e and \u003cem\u003eIDI\u003c/em\u003e 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 \u003cem\u003eHMGR\u003c/em\u003e and \u003cem\u003eSS\u003c/em\u003e 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 \u003cem\u003eSE\u003c/em\u003e 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.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe accumulation of astragaloside IV in response to ethylene treatment exhibits a significant correlation with the expression of its key biosynthetic genes\u003c/b\u003e \u003c/p\u003e \u003cp\u003eCorrelation analysis was conducted to assess the relationship between astragaloside IV content in \u003cem\u003eA. membranaceus\u003c/em\u003e roots treated with Eth and the expression levels of key enzyme genes. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, the findings revealed a significant negative correlation between \u003cem\u003eFPS\u003c/em\u003e and astragaloside IV content (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Additionally, highly significant negative correlations were observed between \u003cem\u003eHMGR\u003c/em\u003e, \u003cem\u003eIDI\u003c/em\u003e, \u003cem\u003eSS\u003c/em\u003e, \u003cem\u003eCYP93E3\u003c/em\u003e, and astragaloside IV content (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Conversely, \u003cem\u003eSE\u003c/em\u003e exhibited a highly significant positive correlation with astragaloside IV content (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;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.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAstragaloside IV has anti-inflammatory, antioxidant, anti-apoptotic, and tumor-inhibiting biological activities, which make it a highly promising compound with broad clinical potential (Liang et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Moreover, the content of astragaloside IV serves as an important indicator for assessing the quality of \u003cem\u003eA. membranaceus\u003c/em\u003e (Zhang et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The hydroponically cultivated \u003cem\u003eA. membranaceus\u003c/em\u003e exhibits significantly higher levels of astragaloside IV compared to field (Chen et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Exogenous inducers could modulate the levels of secondary metabolites in plants. Ethylene, as a signaling molecule, plays a regulatory role in plant secondary metabolism (Dubois et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). For instance, under exogenous 50 \u0026micro;M ethylene treatment, root growth and ginsenoside accumulation significantly enhanced in adventitious root of \u003cem\u003ePanax ginseng\u003c/em\u003e C.A. Meyer (Bae et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). High concentrations of ethylene can stimulate the biosynthesis of saponins in \u003cem\u003eCalendula officinalis\u003c/em\u003e hairy roots (Markowski et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Similarly, exogenous ethylene treatment has been found to promote the production of ganoderic acid in \u003cem\u003eGanoderma lucidum\u003c/em\u003e (Zhang et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Under exogenous ethylene treatment, the accumulation of various indole alkaloids enhanced in \u003cem\u003eChrysanthemum\u003c/em\u003e leaves (Pan et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In the present study, under exogenous 200 mM ethylene treatment, the content of astragaloside IV significantly increased in \u003cem\u003eA. membranaceus\u003c/em\u003e roots.\u003c/p\u003e \u003cp\u003eEthylene plays a crucial role in regulating the changes in active ingredient content in medicinal plants by modulating the expression of key genes involved in secondary metabolism pathways (Tahmasebi et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Understanding the alterations in the expression levels of these key genes is essential for studying the synthesis and accumulation of secondary metabolites (Pan et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). For instance, ethylene has been shown to regulate the expression of genes such as FPS, SS, and \u003cem\u003eSE\u003c/em\u003e involved in ginsenoside synthesis, resulting in ginsenoside content the increased in \u003cem\u003ePanax ginseng\u003c/em\u003e C.A. Mey (Rahimi et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Similarly, ethylene treatment upregulates key genes of the jasmonate alkaloid synthesis pathway, leading to the accumulation of jasmonate alkaloids in \u003cem\u003eCatharanthus roseus\u003c/em\u003e. In \u003cem\u003eGanoderma lucidum\u003c/em\u003e, the upregulation of genes such as \u003cem\u003eHMGR\u003c/em\u003e, \u003cem\u003eSS\u003c/em\u003e, and \u003cem\u003eOSC\u003c/em\u003e involved in the biosynthesis of ganoderic acid by ethylene treatment, leading to its increased accumulation (Pan et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Additionally, treatment with an ethylene releaser increases the expression of key genes involved in the biosynthetic pathways of rhynchophylline (RIN) and isorhynchophylline (IRN) in \u003cem\u003eUncaria rhynchophylla\u003c/em\u003e, resulting in higher RIN and IRN content in \u003cem\u003eU. rhynchophylla\u003c/em\u003e leaves (Li et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In this study, treatment with ethephon led to the downregulation of gene expression of \u003cem\u003eAACT\u003c/em\u003e, \u003cem\u003eHMGR\u003c/em\u003e, \u003cem\u003eIDI\u003c/em\u003e, and \u003cem\u003eSS\u003c/em\u003e, while \u003cem\u003eSE\u003c/em\u003e gene expression was promoted. These genes likely have shared roles and mutually influence each other in the synthesis pathway of astragaloside IV, collectively regulating its synthesis. Furthermore, under ethylene treatment, significant negative correlations were observed between \u003cem\u003eFPS\u003c/em\u003e, \u003cem\u003eHMGR\u003c/em\u003e, \u003cem\u003eIDI\u003c/em\u003e, SS, \u003cem\u003eCYP93E3\u003c/em\u003e and astragaloside IV, whereas \u003cem\u003eSE\u003c/em\u003e showed a highly significant positive correlation. This suggests that the \u003cem\u003eSE\u003c/em\u003e gene can respond to exogenous ethylene signals, facilitating the synthesis and accumulation of astragaloside IV.\u003c/p\u003e \u003cp\u003eIn conclusion, exogenous treatment with ethephon can enhance the content of astragaloside IV in hydroponically cultivated \u003cem\u003eA. membranaceus\u003c/em\u003e. This effect is achieved through the regulation of key genes involved in the synthesis pathway, with particular emphasis on the critical role of \u003cem\u003eFPS\u003c/em\u003e, \u003cem\u003eHMGR\u003c/em\u003e, \u003cem\u003eIDI\u003c/em\u003e, \u003cem\u003eSS\u003c/em\u003e, \u003cem\u003eCYP93E3\u003c/em\u003e and \u003cem\u003eSE\u003c/em\u003e gene, which is regulated by ethylene and promotes the synthesis of astragaloside IV (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). This study provides a foundation for further investigations into the mechanisms by which exogenous inducers affect the secondary metabolism pathway of astragaloside IV, and it offers insights for genetic improvement of \u003cem\u003eA. membranaceus.\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data and material used during the current study are available from the author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Science and technology projects of Inner Mongolia Autonomous Region (2021GG0342 and 2022YFDZ0015) for financial supports.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; information\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBZQ conceived the research and revised the manuscript. WHN and TY performed the experiments, data analysis and wrote the manuscript. WJW and ZXJ gave the project support and the design guidance of experimental.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eNational Pharmacopoeia Committee, Pharmacopoeia of the People\u0026apos;s Republic of China, China Medical Science and Technology Press, Beijing, China, 2020.\u003c/li\u003e\n \u003cli\u003eHu S, Zheng W, Jin L (2021) Astragaloside IV\u0026nbsp;inhibits cell proliferation and metastasis of breast cancer via promoting the\u0026nbsp;long noncoding RNA\u0026nbsp;TRHDE-AS1.\u0026nbsp;Journal of natural medicines 75(1):156-166. https://doi.org/10.1007/s11418-020-01469-8\u003c/li\u003e\n \u003cli\u003eKong X, Wang F, Niu Y, Wu X, Pan Y (2018) A comparative study on the effect of promoting the osteogenic function of osteoblasts using isoflavones from Radix Astragalus.\u0026nbsp;Phytother Res 32(1):115-124. https://doi.org/10.1002/ptr.5955\u003c/li\u003e\n \u003cli\u003eTsai CC, Wu HH, Chang CP, Lin CH, Yang HH (2019) Calycosin-7-O-\u0026beta;-D-glucoside reduces myocardial injury in heat stroke rats. 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Journal of plant research 135(3), 485\u0026ndash;500. https://doi.org/10.1007/s10265-022-01387-8\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003eTable S is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"botanical-studies","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bost","sideBox":"Learn more about [Botanical Studies](http://as-botanicalstudies.springeropen.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bost/default.aspx","title":"Botanical Studies","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao, ethylene, astragaloside IV, gene expression","lastPublishedDoi":"10.21203/rs.3.rs-3791227/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3791227/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eAstragaloside IV, a prominent secondary metabolite found in \u003cem\u003eAstragalus membranaceus\u003c/em\u003e Bge. var. mongholicus (Bge.) Hsiao (\u003cem\u003eA. membranaceus\u003c/em\u003e), serves as a crucial indicator of \u003cem\u003eA. membranaceus\u003c/em\u003e quality. Ethylene, acting as an exogenous signal, plays a role in regulating secondary metabolism in plants. In this study, the application of ethephon (Eth) to hydroponically cultivated \u003cem\u003eA. membranaceus\u003c/em\u003e was employed to investigate the biosynthesis of astragaloside IV in the roots, involving both content measurement and analysis of key gene expression.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe results demonstrated that the significantly accumulation of astragaloside IV was observed on the 3rd day after 200 \u0026micro;mol\u0026middot;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e Eth treatment, reaching 0.269%. Among the 10 key genes involved in astragaloside IV synthesis, \u003cem\u003eHMGS\u003c/em\u003e, \u003cem\u003eFPS\u003c/em\u003e, \u003cem\u003eCAS\u003c/em\u003e, \u003cem\u003eCYP88D6\u003c/em\u003e, and \u003cem\u003eCYP93E3\u003c/em\u003e were found to be insensitive to Eth. On the other hand, the expression levels of \u003cem\u003eAACT\u003c/em\u003e, \u003cem\u003eHMGR\u003c/em\u003e, \u003cem\u003eIDI\u003c/em\u003e, and \u003cem\u003eSS\u003c/em\u003e exhibited a significant increase at 12 hours under Eth treatment, followed by a notable decrease at 3rd day. Additionally, \u003cem\u003eSE\u003c/em\u003e displayed a significant decrease at 12 hours and a subsequent increase in the 3rd day under Eth treatment. The expression level of \u003cem\u003eFPS\u003c/em\u003e, \u003cem\u003eHMGR\u003c/em\u003e, \u003cem\u003eIDI\u003c/em\u003e, \u003cem\u003eSS\u003c/em\u003e, and \u003cem\u003eCYP93E3\u003c/em\u003e exhibited significant negative correlations with astragaloside IV content, while expression level of \u003cem\u003eSE\u003c/em\u003e displayed a significant positive correlation.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThese findings suggest that exogenous Eth treatment can potentially influence the synthesis of astragaloside IV by modulating the expression of \u003cem\u003eFPS\u003c/em\u003e, \u003cem\u003eHMGR\u003c/em\u003e, \u003cem\u003eIDI\u003c/em\u003e, \u003cem\u003eSS\u003c/em\u003e, \u003cem\u003eCYP93E3\u003c/em\u003e and \u003cem\u003eSE\u003c/em\u003e. This study provides a theoretical basis for utilizing molecular strategies to enhance the quality of \u003cem\u003eA. membranaceus\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Exogenous ethephon treatment on the biosynthesis and accumulation of astragaloside IV in Astragalus membranaceus Bge. var. mongholicus (Bge.) 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