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Open Access

Peer-reviewed

Research Article

Effects of water stress on secondary metabolism of Panax ginseng fresh roots

  • Wei Zhang,

    Roles Data curation, Formal analysis, Methodology, Resources, Writing – original draft

    Affiliation Department of Pharmacognosy, Heilongjiang University of Chinese Medicine, Harbin, Heilongjiang, China

    ⨯
  • Wenfei Liu,

    Roles Data curation, Methodology, Resources

    Affiliation Department of Pharmacognosy, Heilongjiang University of Chinese Medicine, Harbin, Heilongjiang, China

    ⨯
  • Liyang Wang,

    Roles Data curation, Methodology

    Affiliation Department of Pharmacognosy, Heilongjiang University of Chinese Medicine, Harbin, Heilongjiang, China

    ⨯
  • Pengcheng Yu,

    Roles Data curation, Investigation, Methodology

    Affiliation Department of Pharmacognosy, Heilongjiang University of Chinese Medicine, Harbin, Heilongjiang, China

    ⨯
  • Xiaowen Song,

    Roles Data curation, Resources

    Affiliation Department of Pharmacognosy, Heilongjiang University of Chinese Medicine, Harbin, Heilongjiang, China

    ⨯
  • Yao Yao,

    Roles Data curation, Investigation

    Affiliation Department of Pharmacognosy, Heilongjiang University of Chinese Medicine, Harbin, Heilongjiang, China

    ⨯
  • Xiubo Liu,

    Roles Funding acquisition, Supervision, Writing – review & editing

    Affiliation Jiamusi College, Heilongjiang University of Chinese Medicine, Jiamusi, Heilongjiang, China

    ⨯
  • Xiangcai Meng

    Roles Funding acquisition, Supervision, Writing – review & editing

    2904755958@qq.com, Mengxiangcai000@163.com

    Affiliation Department of Pharmacognosy, Heilongjiang University of Chinese Medicine, Harbin, Heilongjiang, China

    ⨯

Abstract

The roots and rhizomes of Panax ginseng C.A. Mey are commonly used herbal medicine in Asian countries. These components contain a large number of secondary metabolites known as ginsenosides, which serve as primary active ingredient. Environmental factors significantly influence the production of secondary metabolites, which are crucial for enhancing plant adaptability to ecological stress. P. ginseng is a shady plant that thrives in a constantly humid and temperate environment. However, it cannot withstand excessive moisture, making soil moisture a significant ecological stress affecting P. ginseng survival. In this study, we applied a water spray to maintain a water-saturated surface on 5-year-old fresh P. ginseng roots for a duration of 5 days, to establish a short-term water stress condition. The results revealed a notable increase in superoxide anion (O2·-), hydrogen peroxide (H2O2), and NADPH oxidase (NOX) activity (p < 0.01), as well as malondialdehyde (MDA) contents (p < 0.01) in both the main root and fibrous root of P. ginseng. Additionally, superoxide dismutase (SOD), catalase (CAT), peroxides (POD), ascorbate peroxidase (APX) and glutathione reductase (GR) activities also elevated significantly under water stress (p < 0.01). Ascorbic acid (AsA), glutathione (GSH) and oxidized glutathione (GSSG) contents also showed a marked increase (p < 0.01). The main root treated with water showed the most positive impact on the 5th day. Water stress boosted the activities of key enzymes including 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), farnesyl pyrophosphate synthase (FPS), squalene synthase (SS), squalene epoxidase (SE), and dammarenediol-II synthase (DS) involved in the ginsenoside biosynthesis pathway (p <0.01). This resulted in a significant an increase in the level of ginsenosides Rg1, Rb1, Rf, Rg2+Rh1, Rc, and Rb3, by 42.4%, 21.0%, 15.7%, 157.9%, 18.3%, and 10.6% respectively, and an increase of 40.1% in total saponins content. Similarly, the fibrous root changes in the treated sample showed the most positive impact on the 4th day. Specifically, Rg1, Re, Rb1, Rf, Rg2+Rh1, Rc, Ro, and Rb2 increased by 41.8%, 20.5%, 17.3%, 84.3%, 30.7%, 35.6%, 8.6%, and 7.6%, respectively, and an increase of 4.2% in total saponins content. Furthermore, 1,3-disphosphoglycerate (1,3-DPG) contents and phosphoenolpyruvate carboxylase (PEPC) activities, which are key intermediate of primary metabolism, were significantly elevated under water stress (p < 0.01). This indicates that the primary source of the raw materials used in the biosynthesis of secondary metabolites is sugars. Pharmacodynamic analysis demonstrated that water stress could increase the contents of ginsenosides, improve the quality of ginseng, and enhance the efficacy of ginseng root to a certain extent.

Citation: Zhang W, Liu W, Wang L, Yu P, Song X, Yao Y, et al. (2024) Effects of water stress on secondary metabolism of Panax ginseng fresh roots. PLoS ONE 19(11): e0312023. https://doi.org/10.1371/journal.pone.0312023

Editor: Ying Ma, Universidade de Coimbra, PORTUGAL

Received: March 24, 2024; Accepted: September 25, 2024; Published: November 27, 2024

Copyright: © 2024 Zhang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and its Supporting Information files.

Funding: This research was funded by Joint Funds of the National Natural Science Foundation of China (U20A20407) and Application demonstration of key technologies for the production of authentic medicinal materials such as ginseng in rural industries (2021YFD1600901). The funders have attributed in conceptualization, supervision, decision to publish, and writing-review and editing of this manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Plants cannot move to escape harsh ecological stress such as high temperatures, drought, floods, and other adversities. Under ecological stress, increased levels of abscisic acid levels cause the closure of stomata, leading to a rise in intracellular O2 produced by photosynthesis. Meanwhile, the photosynthetic electron transport system experiences excessive reduction, resulting in an improved conversion of oxygen into O2·-. Subsequently, the O2·- is further converted into H2O2 and other products (Mehler reaction) [1,2]. The over-production of Reactive Oxygen Species (ROS) can cause damage to molecular structures, bio-membrane, DNA and peptide chain, and proteins, ultimately leading to metabolic dysregulation or cell death [3,4]. The antioxidant enzymes, such as SOD, CAT, POD, APX, etc., are the primary substances that against ROS. However, these antioxidant enzymes are also proteins that depend on hydrogen bonds and disulfide bonds to preserve their secondary and tertiary structures, ensuring their optimal functionality. Elevated levels of ROS can disrupt these bonds, causing the enzymes to lose their effectiveness. Plants can survive under severe ecological stress due to the development of a sophisticated secondary metabolism that helps eliminate or neutralize excessive ROS [5]. Secondary metabolites, including polyphenols (flavonoids), saponins, and alkaloids, etc, play critical roles in multiple ecological processes [6–8]. Once environmental stress occurs, plants respond by inducing the production of secondary metabolites to counteract ROS. Therefore, the levels of ROS can be regulated by antioxidant enzymes, secondary metabolites, and other antioxidant systems, ultimately maintained at a stable level [9].

Ginseng (Panax. ginseng C. A. Meyer) is a perennial herb derived from the genus Panax in the family Araliaceae. Its medicinal parts are roots and rhizomes, which are called "the king of herbs". They possess various effects such as anti-aging, regulation of the nervous system, improvement of blood circulation, and regulation of the endocrine system, etc. [10–12].

Flavonoids have a strong ability to eliminate ROS as a result of their higher concentration of -OH and -OCH3 groups [13–15]. However, ginseng’s primary active components are secondary metabolite saponins, including Rg1, Re, Rg2, Rb1, Rc, and Rb2, etc. [16]. Consequently, because flavonoids are sparsely present in ginseng, they demonstrate a relatively limited ability to adapt to diverse environments [17,18]. The biosynthesis of these ginsenosides proceeds as follows: acetyl-CoA generating isopentenyl pyrophosphate (IPP) and dimethylallyl diphosphate (DMAPP) through the mevalonate (MVA) or methylerythritol phosphate (MEP) pathways. This is followed by the catalysis of 2,3-oxidosqualene by enzymes such as FPS, SS, and SE, resulting in the formation of various ginsenoside monomers through a sequence of cyclization, hydroxylation, and ring functional group glycosylation processes [19]. It has been reported that soil moisture is the main stress for ginseng. Both excessive drought and excessive humidity cause root damage [20]. P. ginseng can only survive in an environment with stable temperature and humidity. The growth of ginseng and the synthesis of ginsenosides are enhanced when the relative soil water content ranges from 60% to 80% [21]. Additionally, a field water holding capacity of around 30% ± 5% boosts the total ginsenosides content as well as ginsenosides Rg1 and Re. On the other hand, a temporary field capacity of 90% ± 5% increases the ginsenoside Rb1 content [22], suggesting that water stress leads to an increase in ginsenoside content.

The secondary metabolism of plants is closely related to the environment. Appropriate stress can enhance plant secondary metabolism, which usually are medicinal components. Consequently, short-term ecological stress can increase the quality of the medicinal materials. The post-harvest ginseng fresh root is a living organ with a complete metabolic system. This study focuses on exploring the relationship among ROS, antioxidant system, and secondary metabolites of ginseng under excessive water conditions. The fresh root of ginseng is used as the subject of study in vitro, to uncover the biological mechanism of water stress on ginsenosides. This research provides both a theoretical foundation and practical techniques to produce high-quality ginseng.

Materials and methods

Plant materials

Fresh 5-year-old roots of cultivated ginseng, identified by Prof. Xiang-cai Meng of Heilongjiang University of Chinese medicine, were collected in Ji’an County, Jilin Province, China, collected in May 2023 and wrapped in moss to maintain freshness.

Instrument

CP225D Electronic balance (Shanghai Precision Instrument Co., Ltd.), SD40 Ice making Machine (Guangzhou Guangkun Electric Appliance Manufacturing Co., Ltd.), 752 UV-vis Spectrophotometer (Shanghai Jinghua Science and Technology Instrument Co., Ltd.), 98-1-B Electronic temperature regulating Electric heating sleeve (Tianjin Tester instrument Co., Ltd.), TGL- 16LM desktop high-speed freezing centrifuge (Hunan Star Science Instrument Co., Ltd.), JulaboTW20 digital display constant temperature water bath pot (Julabo Co., Ltd., Germany). QL-902 Vortex oscillator (Haimen Qilin Bell instrument Manufacturing Co., Ltd.); Thermo enzyme labeling instrument (Thermo Co., Ltd.); DHG- 9015A blast drying oven (Shanghai-Heng Scientific instrument Co., Ltd.); Shimadzu High performance liquid Chromatography (LC2010A).

Reagent

O2·- free radical determination kit (NO.2309002), protein quantification (TP) kit (20230420), H2O2 kit (20230726), MDA kit (20230422). SOD kit (20230624), CAT kit (20230503), POD kit (20230616). 1,3-DPG (202307), PEPC kit (202306), NOX kit (202303), APX kit (20230521), AsA kit (NO.1012304011), GR kit (NO.1012304011), GSH determination kit (NO.1012303305), GSSH determination kit (NO.1012304041), HMGCR kit (202303), FPS kit (202303), SS kit (202303), SE kit (202303), DS kit (202303), HPLC grade acetonitrile (R142190), phosphoric acid chromatograph pure (TEDIA Co., Ltd.); The ginsenoside standards, Rg1, Re and Rb1 were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China), ginsenoside standards, Rf, Rg2, Rh1, Rc, Ro, Rb2, Rb3, were purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China), the purity is more than 98%. D101 macroporous adsorption resin (Tianjin Huida Chemical Co., Ltd.), vanillin (Tianjin Guangfu Fine Chemical Research Institute), perchloric acid (Tianjin Fuyu Fine Chemical Co., Ltd.), glacial acetic acid (Tianjin Tianli Chemical Reagent Co., Ltd.).

Experimental method

Selected fresh ginseng roots with a similar diameter to main root was divided into two groups. The treatment group sprayed water uniformly to the surface until saturated, replenished water once a day for 5 days. In contrast, the control group was covered with moss to maintain its freshness. The samples of two groups of ginsengs were placed in an artificial intelligence incubator at 15°C/10°C. Root samples were collected at 0, 1, 2, 3, 4 and 5 d of treatment, with several roots (n ≥ 6) being collected simultaneously. First, the main root phloem and fibrous roots were cut into separate pieces of 0.30 g × 30 samples per tissue type. The samples were used to evaluate the activities of various enzymes, including SOD, CAT, POD, NOX, PEPC, HMGCR, FPS, SS, SE, DS, and the content of 1,3-DPG. Additionally, 0.1 g × 30 samples of each piece were taken for the determination of APX and GR activities, as well as the content of O2·-, H2O2, MDA, AsA, GSH, and GSSG. All samples were sealed with aluminum foil and stored at -80°C. Next, more than 30 g of the main roots and fibrous roots were dried in an oven at 55°C, then crushed by an ultra-fine grinder and sifted through a 65-mesh sieve for the determination of Rg1, Re, Rf, Rg2+Rh1, Rb1, Rc, Ro, Rb2 and Rb3. Each index measurement was repeated three times.

Determination of related indexes of ROS metabolism and antioxidant system.

Determination of ROS in fresh roots of ginseng. The content of homogenate protein in fresh root of P. ginseng was determined with the commercially available TP assay kit (Nanjing Jiancheng Bioengineering, Nanjing, China) and expressed in g/L. The O2·-and H2O2 contents were measured using a commercially available assay kit (Beijing Solarbio Science & Technology, Beijing, China and Nanjing Jiancheng Bioengineering, Nanjing, China), expressed in μmol/g and mmol/g, respectively.

Determination of MDA content. The MDA contents were measured using a commercially available assay kit (Nanjing Jiancheng Bioengineering, Nanjing, China) and expressed in nmol/g.

Determination of ROS-related enzyme activity. The activity of NOX was determined with a commercially available assay kit (Jiangsu jingmei Science & Technology, Yancheng, China) and expressed in ng/L. Evaluation of the SOD, CAT, and POD activities was expressed as U/g FW according to the protocol of a commercial assay kit (Jiancheng Bioengineering, Nanjing, China).

Determination of parameters related to AsA-GSH cycle. The AsA, GSH, and GSSG contents were measured using a commercially available assay kit (Beijing Boxbio, Beijing, China), expressed as μmol/g, μg/g, and μg/g, respectively. Evaluation of APX and GR activities were expressed as U/g FW according to the protocol of a commercial assay kit (Jiancheng Bioengineering, Nanjing, China).

Determination of secondary metabolism-related enzyme activity.

The activities of HMGCR, FPS, SS, SE, and DS were determined with a commercially available assay kit (Jiangsu jingmei Science & Technology, Yancheng, China) and expressed in ng/L.

Determination of the ginsenosides contents in the fresh roots of P. ginseng.

Monomer ginsenosides. The contents of ginsenosides Rg1, Re, Rf, Rg2, Rh1, Rb1, Rc, Ro, Rb2, and Rb3 were determined by HPLC, following the method described by Lin et al. [23]. 1.0 g of both the main root and fibrous root powder of ginseng were precisely measured and placed in a 50 ml cork triangle container. The extraction process involved using methanol and ultrasonic at 100 Hz for 40 min and filtered by 0.22 μm filter for HPLC analysis. Each sample was repeated three times. The ginsenoside standards of Rg1, Re, Rf, Rg2, Rh1, Rb1, Rc, Ro, Rb2, and Rb3 were weighed precisely, then placed in a 5 ml centrifuge tube, dissolved with methanol, and adjusted volume to prepare a ginsenoside mixed standard solution for use.

The prepared samples were analyzed using an Agilent 1260 HPLC system (Agilent, United States) and separated in a C18 column (250 mm × 4.6 mm, ID 5 μm). The column temperature was 30°C, the flow rate was 1.0 mL/min, the injection volume was 10 μL, and UV measurements were obtained at 203 nm. The mobile phase was composed of A (0.1% phosphoric acid water) and B (acetonitrile), the gradient elution was performed as follows: 0~20 min, 18%~20% B; 20~60 min, 20%~24% B; 60~80 min, 24%~30% B; 80~110 min, 30%~34% B.

Total ginsenosides. The total ginsenosides were determined by using the vanillin-glacial acetic acid-perchloric acid colorimetric method reported by Kubo et al [24]. And ginsenoside Re was employed as standard. The absorbance (Y) was ordinate and the solution mass (X) was Abscissa. The standard curve was drawn to calculate the total ginsenosides content.

Method validation for quantitation. The linear relationship of 10 active components, the precision of the instrument, the stability of the solution within 24 h, the repeatability of the method, and the recovery rate were investigated respectively.

Determination of the intermediate metabolite contents of glycolysis PEPC activity.

1,3-DPG contents and PEPC activities were determined with a commercially available assay kit (Jiangsu jingmei Science & Technology, Yancheng, China) and expressed in nmol/L and ng/L respectively.

Pharmacodynamic verification.

Drug preparation. The method described by Song et al was referenced [25]. The 0th day ginseng main root and the ginseng main root on the 5th day with water stress were dried to constant weight and crushed into coarse powder. An appropriate amount of ginseng was taken, and 10 times the amount of water was added. The mixture was soaked for 12 h, then boiled for 1 h, and filtered, the extraction process was repeated three times. The filtrates were then combined and concentrated to achieve 0.039 g/ ml crude drug for further use.

Animal experiments. The 8-week-old male Kunming mice (weighing 18~24 g), provided by Liaoning Changsheng Biotechnology Co., Ltd with Certificate of Quality No. SCXK (Liao)-2020-0001 (Benxi, China), were raised in a ventilated and dry environment, with a temperature of 20~23°C, a relative humidity of 50~60%, and a 12 h light/dark cycle. They had free access to food and water. All animals’ treatments and experiments were carried out after being approved by the Animals Laboratory Ethical Committee of Heilongjiang University of Chinese Medicine (Approval Number: SYXK2023-122913).

After suitable feeding for 7 days, the mice were randomly divided into 6 groups (n = 10): the blank control group (administrated sterile saline and injected with saline); the model group (D-galactose (120 mg/kg/d) was subcutaneously injected with a volume of 0.01 ml/g, qd×42); the positive control group (from the 16th day of the establishment of D-galactose model, the mice were administrated with donepezil hydrochloride (1.3 mg/kg/d) qd×27); the ginseng main root group on the 0th day (from the 16th day of the establishment of D-galactose model, the mice were administrated with 0-day ginseng decoction (0.39 g/kg/d) qd×27); the ginseng main root group with water stress on the 5th day (from the 16th day of the establishment of D-galactose model, the mice were administrated with the 5th ginseng decoction (0.39 g/kg/d) qd×27).

Analysis of Morris water maze. The evaluation of the spatial learning and memory abilities of mice referred to the method described by Li et al [26] with minor modifications. In detail, training mice to find a circular platform (10 cm in diameter, 1 cm underwater) located in the middle of any quadrant in 2 min. After the beginning of the experiment, the instrument automatically recorded the time and trajectory of the mice till finding the platform, the time of the platform being found was the incubation period. All the indicators are averaged.

Histopathological staining analysis. After the water maze experiment, mice were anesthetized by intraperitoneal injection of urethane (0.008 ml/g) as reported by Yu et al [27], the brain tissue was perfused with 4% paraformaldehyde and fixed for 24 h, rinsed with the slow flow for 12 h, dehydration with gradient ethanol and xylene, then soaked in melted wax and embedded, sliced to 5~8 μm. The sections were stained with HE reagent and the pathological changes in the hippocampus of mice were observed by light microscope (Nikon, Eclipse Ci-L, Japan).

Analysis of oxidative stress indexes in brain tissues. The method described by Zhang et al. was referenced [28]. The mice brain tissues of appropriate size in each group were rinsed quickly in precooled normal saline, and the residual fluid on the surface of the brain tissue was discarded with filter paper. After preparing the 10% brain tissue homogenate, the supernatant was collected at 3500 rpm centrifugation for 10 min. SOD, glutathione peroxidase (GSH-PX) activities and MDA contents of the brain tissue were determined using commercial kits (Jiancheng Bioengineering, Nanjing, China), expressed in U/mg, U/mg, and nmol/mg, respectively.

Statistical analysis

Microsoft Office Excel 2007 (Microsoft Corp., Redmond, WA, United States) was used to sort the original data. Origin 2021 (OriginLab, Northampton, MA, United States) was used for graphic illustration. The whole data was expressed in mean ± standard deviation (Mean ± S.D.) and IBM SPSS 27.0 (IBM Corp., Armonk, NY, United States) was used for the independent samples t-test. The statistically significant differences were thought to be *p < 0.05 or **p < 0.01.

Results

Effect of water stress on ROS

O2·- and H2O2 contents.

Compared with the 0 d, the contents of O2·- and H2O2 in the control group remained essentially unchanged at all times. However, in the water stress group, the levels of O2·- and H2O2 increased to various degrees during the stress period, indicating that excessive water has produced stress to ginseng.

The contents of O2·- in the main root exhibited a pattern of decrease-increase-decrease. It peaked on the 3rd day of stress, showing a 14.4% increase over the 0th day. The content of H2O2 firstly decreased and then increased, and peaked on the 5th day, with a 28.3% enhancement compared to that on the 0th day.

The contents of O2·- in the fibrous root of stress increased, and peaked on the 4th day, with a 21.8% higher level than that on the 0th day. H2O2 exhibited an increase-decrease course, and peaked on the 3rd day, with a 56.7% higher level than that on the 0th day (Fig 1).

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Fig 1. O2·- and H2O2 contents in P. ginseng.

(A) main root, (B) fibrous root.

https://doi.org/10.1371/journal.pone.0312023.g001

NOX activity.

Compared with the 0 d, the NOX activities in the main root of the control group had a little change, but they increased slightly in the fibrous root, reaching their highest on the 3rd day with a growth of 33.0% compared to the control group. The concentration of NOX in the water stress group increased notably during the duration of stress.

The activities of NOX in the ginseng main root showed a linear increase from 2 to 5 days, peaking on the 5th day, with a 104.1% elevation from that on the 0th day. The fibrous roots showed a gradual growth, reaching their highest on the 4th day with a 50.2% rise compared to the 0th day (Fig 2).

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Fig 2. NOX activities in P. ginseng.

(A) main root, (B) fibrous root.

https://doi.org/10.1371/journal.pone.0312023.g002

MDA content.

Compared with the 0 d, the contents of MDA in the root of the control group remained relatively stable over time. However, in the late stages, there was a little increase in the levels of MDA in the fibrous root, reaching a level that was 46.1% higher than that in the control group on the 4th day.

The MDA contents in the main roots showed a modest increase in the initial phase of water stress. However, it increased significantly after the 2nd day (p < 0.01) and peaked on the 5th day, with a 72.2% increase compared with the 0th day. The MDA contents in fibrous root first increased, and showed a maximum significant difference on the 4th day (p < 0.01), with an 87.1% higher value than that on the 0th day (Fig 3). These indicated that water stress did damage to ginseng.

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Fig 3. MDA contents in P. ginseng.

(A) main root, (B) fibrous root.

https://doi.org/10.1371/journal.pone.0312023.g003

Antioxidant enzyme activity.

Compared with the 0 days, the activities of SOD, CAT, and POD in the control group remained constant over time. However, in the water stress group, the activities of all three antioxidant enzymes in both the ginseng main root and fibrous root increased.

The SOD activities in the ginseng main root exhibited a trend of decrease-increase-decrease during stress, peaking on the 4th day. The CAT activities increased, peaking on the 4th day. The POD activities were enhanced continuously and peaked on the 5th day. Compared with the 0 days, the activities of SOD, CAT, and POD in ginseng main root grew by 14.5%, 39.8% and 19.3%, respectively.

The activities of SOD in stressed fibrous root increased significantly on the first day (p < 0.01), peaking on the 3rd day, and subsequently remained rather stable. Overall, the activities of CAT and POD showed an increase-decrease trend, and both peaked on the 4th day. The activities of three antioxidant enzymes in fibrous root were increased by 66.9%, 49.3% and 60.1% respectively compared to 0 days (Fig 4).

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Fig 4. SOD, CAT and POD activities in P. ginseng.

(A) main root, (B) fibrous root.

https://doi.org/10.1371/journal.pone.0312023.g004

AsA-GSH cycle.

Compared with the 0 days, the control group showed minimal change in the AsA-GSH cycle, but water stress caused distinct modifications in the parameters of the AsA-GSH cycle in the ginseng main root and fibrous root.

During the stress, the AsA contents and the APX activities exhibited a decrease-increase-decrease trend, peaking on the 3rd day, with an increase of 55.5% and 51.7% compared with the 0 days, respectively. The contents of GSH increased gradually and peaked on the 5th day, increasing by 32.4% compared with the 0 days. The contents of GSSG and the activities of GR decreased at first and then increased, peaking on the 5th day, with a rise of 37.7% and 58.1%, respectively, compared with the 0 days. The ratio of GSH/GSSG increased at first and then decreased, peaking on the 2nd day, with a 37.1% higher than that on the 0 days.

The contents of AsA, GSH, and the activities of GR in ginseng fibrous root increased at first and then decreased, and all peaked on the 4th day. Compared to the initial day, these levels were elevated by 65.5%, 27.3% and 58.1%, respectively. The contents of GSSG increased gradually and peaked on the 4th day, with a significant increase of 47.3% compared to the 0 days. The ratio of GSH/GSSG in fibrous roots decreased. Overall, the activities of APX were increased, peaking on the 4th day, with a 90.4% higher than that on the 0 days (Fig 5).

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Fig 5. AsA, GSH, GSSH contents, ratio of GSH/GSSH and APX, GR activities in P. ginseng.

(A) main root, (B) fibrous root.

https://doi.org/10.1371/journal.pone.0312023.g005

Effects on the activity of key enzymes of secondary metabolites.

Compared with the 0 days, the activities of key enzymes relating to the ginsenoside synthesis pathway had no significant change in the ginseng main root and fibrous root in the control group, but water stress enhanced their activities during the later stage of stress.

The activities of HMGR, FPS, SS, SE and DS decreased at first and then increased under water stress, increased significantly after the 2nd day (p < 0.01) and reached their highest levels on the 5th day, with increases of 35.7%, 33.2%, 24.6%, 30.5% and 35.1%, compared with the 0 days, respectively.

The activities of HMGCR, FPS, SS and SE in fibrous root exhibited similar trends. However, the activities of HMGR peaked on the 2nd day, while FPS, SS and SE peaked on the 4th day, with increases of 64.6%, 50.8%, 33.3% and 27.7%, respectively, compared with the 0 days. On the other hand, the activities of DS decreased at first and then increased, reaching its peak on the 4th day, which was 28.6% higher than that on the 0 days (Fig 6).

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Fig 6. Activities of HMGCR, FPS, SS, SE and DS in P. ginseng.

(A) main root, (B) fibrous root.

https://doi.org/10.1371/journal.pone.0312023.g006

The above results showed that the changes in the contents or activities of the indicators in the fibrous root occurred before the alterations in the main root.

Effect on the content of ginsenosides.

Monomer ginsenosides. The levels of individual ginsenoside monomers in fresh ginseng roots varied dynamically in response to extended stress, however, the pattern of each monomer ginsenoside was not consistent.

In the main root, except ginsenoside Re and Rb2, the contents of Rg1, Rf, Rg2+Rh1, Rb1, Rc, Ro and Rb3 were significantly higher than those in the control group after water stress (p < 0.01) at the later stages of stress. The contents of Rb1 and Ro peaked on the 3rd day, measuring 1.93 mg/g and 1.42 mg/g, respectively. This represents an increase of 22.9% and 8.4% compared with the 0 days. The contents of Rf peaked at 1.08 mg/g on the 4th day, which was 30.1% higher than that on the 0 days. The contents of Rg1, Rg2+Rh1, Rc and Rb3 reached their highest level on the 5th day, measuring 5.07 mg/g, 0.49 mg/g, 0.84 mg/g and 1.25 mg/g, respectively, increased by 42.4%, 157.9%, 18.3% and 10.6% compared with the 0 days (Fig 7A).

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Fig 7. Content of monomer ginsenosides in P. ginseng.

(A) main root, (B) fibrous root.

https://doi.org/10.1371/journal.pone.0312023.g007

In the fibrous root, the contents of Rc and Rb3 peaked on the first day, were 5.87 mg/g and 1.12 mg/g, respectively. This represents a rise of 40.4% and 45.5% compared with the 0 days. The contents of Rg1 peaked at 5.46 mg/g on the 3rd day, which was 52.1% higher than that on the 0 days. The contents of Re, Rf, Rg2+Rh1, Rb1 and Ro peaked on the 4th day, were 8.99 mg/g, 2.82 mg/g, 2.30 mg/g, 7.61 mg/g and 6.98 mg/g, respectively. These concentrations increased by 20.5%, 84.3%, 30.7%, 17.3% and 8.6% compared with the 0 days. The contents of Rb2 peaked at 0.72 mg/g on the 5th day, which was 9.1% higher than that on the 0 days (Fig 7B).

Rg1, Re and Rb1 are the main indicators to evaluate the quality of ginseng. The treated main root showed the highest improvement on the 5th day. Rg1+Re and Rb1 increased by 42.4% and 21.0%, respectively. Additionally, Rb3, Rf, Rc, and Rg2+Rh1 also surged by 10.6%, 15.7%, 18.3%. and 157.9%, respectively. The treated ginseng fibrous root showed the highest improvement on the 4th day. Rg1, Re and Rb1 increased by 41.8%, 20.5% and 17.3%, respectively. Additionally, Ro, Rc, Rf, Rg2+Rh1, and Rb2 also increased by 8.6%, 35.6%, 84.3%, 30.7%, and 7.6%, respectively.

Total ginsenosides. The total ginsenosides of the ginseng fresh roots were calculated by the regression equation, Y = 0.0043X+0.0247 (R2 = 0.999), where Y stood for absorbance and X for the Re concentration (mg/ml). Compared with the 0 days, the control group showed minimal variation in the total ginsenosides, while the stressed roots exhibited varying degrees of growth. In the main root, the total ginsenosides decreased first and then increased, with the highest content of 35.1 mg/g on the 5th day, which was 40.1% higher than that on the 0 days. The fibrous root exhibited a decreasing-increase-decrease course, and reached its highest point at 73.97 mg/g on the 3rd day, which was 7.5% higher than that on the 0 days (Fig 8).

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Fig 8. Contents of total ginsenosides in P. ginseng.

(A) main root, (B) fibrous root.

https://doi.org/10.1371/journal.pone.0312023.g008

Effects of Water stress on 1,3-DPG content and PEPC activity of P. ginseng.

When comparing the 0 days, there was no significant change in the levels of 1,3-DPG contents and PEPC activities in the main root and fibrous root of the control group. However, these levels rose under water stress.

The contents of 1,3-DPG in the main root decreased first and then increased during the stress, peaking on the 5th day, with a 29.9% increase compared to that on the 0 days. PEPC activities increased gradually and also peaked on the 5th day, with a 39.6% increase compared to the 0 days. However, the fibrous root observed an initial increase followed by a subsequent drop, reaching its highest point on the 4th day. The increase was measured at 15.0% and 46.7% compared to the first measurement on day 0 (Fig 9).

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Fig 9. 1,3-DPG contents and PEPC activities in P. ginseng.

(A) main root, (B) fibrous root.

https://doi.org/10.1371/journal.pone.0312023.g009

Principal componnent analysis.

The results of Principal Component Analysis (PCA) showed that the ginseng control and stress groups were separated, indicating that there were significant differences between the control and stress groups and a high level of reproducibility. The cumulative contribution of the two principal components (PC1 and PC2) in the main root was 92.8%, with PC1 accounting for 87.4% and PC2 accounting for 5.4%. The percentage of fibrous roots was 96.2%, with PC1 accounting for 89.7% and PC2 accounting for 6.5% (Fig 10). The correlation chart displayed the positive or negative correlations among the analyzed parameters (Fig 11). The degree of correlation between analyzed parameters is differentiated based on Pearson’s value. Correlation coefficients with absolute values greater than 0.8 are classified as strong correlations, those less than 0.3 are classified as weak correlations, and others are moderate correlations.

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Fig 10. PCA of P. ginseng.

(A) main root, (B) fibrous root.

https://doi.org/10.1371/journal.pone.0312023.g010

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Fig 11. Pearson correlation chart.

(A) main root, (B) fibrous root.

https://doi.org/10.1371/journal.pone.0312023.g011

Pharmacological effects verification.

Morris water maze experiment. Compared with the ginseng main root group on the 0th day, the escape latency of the stressed ginseng main root on the 5th day was shortened by 21.4% (p = 0.228), the number of times crossing the original platform and the residence time of the target area was increased by 0.4 (p = 0.255) and 7.3% (p = 0.486), respectively (Fig 12), indicating that water-treated ginseng has the potential to partially cure memory impairment in rats.

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Fig 12. Results of Morris water maze experiment in mice.

(A) blank control group. (B) model group. (C) positive control group. (D) ginseng main root group on the 0th day. (E) stressed ginseng main root group on the 5th day. All values were expressed as Mean ± S.D. (n = 10). **p < 0.01 vs. blank control group (A); ##p < 0.01 vs. model group (B).

https://doi.org/10.1371/journal.pone.0312023.g012

Histopathological staining. Compared with the model group, the degree of lesions in the hippocampus of mice in the positive control group, main root group on the 0th day and stressed main root group on the 5th day were significantly reduced. The hippocampus lesion degree of the stressed group on the 5th day was less severe than that on the 0th day (Fig 13).

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Fig 13. Mice brain tissues stained with H&E dye kits (200×).

(A) blank control group. (B) model group. (C) positive control group. (D) main root group on the 0th day. (E) stressed the main root group on the 5th day.

https://doi.org/10.1371/journal.pone.0312023.g013

Oxidation-associated indexes in mice. Compared with the model group, the GSH-PX activity in the positive control group, main root group on the 0th day, and stressed main root group on the 5th day were increased by 103.4%, 21.0% and 35.5% (p < 0.01). Similarly, SOD activities increased by 71.8%, 40.0% and 55.7% (p < 0.01), respectively. On the other hand, MDA contents decreased by 47.9%, 29.5% and 35.4%, respectively (p < 0.01). Compared with the main root group on the 0th day, the activities of GSH-PX and SOD in the stressed main root group on the 5th day were increased by 12.0% (p = 0.119) and 11.2% (p = 0.009), respectively, while the content of MDA was decreased by 8.3% (p = 0.155) (Fig 14). Although P > 0.05, it also showed that the quality of the medicine was significantly improved.

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Fig 14. GSH-PX, SOD activities and MDA contents in mice brain tissues.

(A) blank control group. (B) model group. (C) positive control group. (D) main root group on the 0th day. (E) stressed the main root group on the 5th day. All values were expressed as Mean ± S.D. (n = 10). **p < 0.01 vs. blank control group (A); ##p < 0.01 vs. model group (B).

https://doi.org/10.1371/journal.pone.0312023.g014

Discussion

Plant stress often leads to a burst of ROS, which in turn triggers an increase in antioxidant substances such as antioxidant enzymes and secondary metabolites [29]. The active components of medicinal plants are usually secondary metabolites, which play a crucial role in the plant’s ability to adapt to its environment. Therefore, the process by which plants adapt to their environment is closely linked to the development of high-quality therapeutic ingredients.

Effects of water stress on the contents of ROS and MDA in ginseng

Water stress leads to a gradual decrease in oxygen concentration and an imbalance in redox potential in P. ginseng. This, in turn, leads to a decrease in photosynthetic electron transport and PSⅡefficiency, induces the surge of H2O2-centered ROS in plants, causes membrane peroxidation, damage the cell membrane and increases membrane permeability [30–32]. Fig 1 shows that O2·-and H2O2 in the main root of P. ginseng initially decreased in the early stage of water stress. The hypoxia caused by water stress can be attributed to the destruction of the mitochondrial site of ROS generation, as the plant undergoes both aerobic and anaerobic respiration. O2·- contents peaked on the 3rd day and then decreased, while H2O2 increased gradually after the 2nd day and peaked on the 5th day. This pattern suggests that to maintain a balance between oxidation and antioxidants, plants initiate their own antioxidant system to eliminate excessive ROS, albeit with a certain delay in activation. In general, O2·- and H2O2 in fibrous roots demonstrate a higher sensitivity to water stress, possibly because the fibrous roots are more susceptible to water uptake.

NOX located in the plasma membrane, is the main source of ROS [33], which catalyzes the transfer of electrons from NADPH to O2, generating O2·- [34]. Previous studies have found that stress can enhance NOX activity in ginseng cell membranes and promote H2O2 production in ginseng suspension cells [35,36]. The identical behavior was observed in fresh ginseng root in this study. In the main root, the activity of NOX increased linearly on the 2nd day of stress and peaked on the 5th day. NOX activity in fibrous roots increased dramatically right after stress, peaked on day 4, and gradually decreased, although still staying considerably elevated compared to day 0 (Fig 2). It could be attributed to the relatively large surface area of fibrous roots, which makes them more vulnerable to water stress. This indicated that excessive water could stress the fresh roots of ginseng. The MDA in ginseng gradually increased, which correlated with the change in ROS content. This observation further confirmed that ROS is responsible for the damage inflicted on fresh ginseng roots (Fig 3).

Effects of water stress on main antioxidant enzymes

The ROS produced in plants interact with other signal molecules to activate antioxidant mechanisms for regulation [37]. SOD, CAT, POD and APX are inductive enzymes. Their activities are enhanced when ROS levels increase within a certain amount. Conversely, their activity decreases when ROS levels fall. This mechanism plays a crucial role in defending plant cells against ROS injury. SOD directly converts O2·- into H2O2 and H2O, whereas POD and CAT reduce ROS damage by generating H2O from H2O2 through catalytic catabolism [38]. In this study, SOD in the ginseng main root showed a decreasing trend at the beginning of stress. A large number of studies have shown that SOD activity decreases in plants at the initial stage of stress [39–41]. Although the mechanism is unclear, the decrease in SOD activity in the early stage can promote the accumulation of ROS and lead to an increase in antioxidants, ultimately improving adaptability. In the middle and late stages of stress, SOD, CAT and O2·- initially increased and then decreased, while POD and H2O2 continued to rise (Figs 1 and 4). This suggests that ginseng exhibits a protective effect against water stress in plants. Nevertheless, as the duration of stress increased, there was a rapid accumulation of H2O2, accompanied by a general drop in the activity of antioxidant enzymes. This led to an increase in membrane lipid peroxidation and increased MDA content (Fig 3). Thus, the enhancement of antioxidant enzyme activity in response to water stress was not sufficient to scavenge excessive ROS and repair oxidative damage.

Effect of water stress on AsA-GSH cycle

The AsA-GSH cycle clears H2O2 driven by NADPH and exists in many plant subcellular compartments. Its main function is to convert AsA and GSH into their oxidative form in order to eliminate ROS and maintain intracellular redox balance. GSH content and GSH/GSSG ratio are the key factors to determine the operation efficiency of this cycle [42]. High GSH/GSSG ratio can inhibit the formation of intermolecular disulfide bonds, form a reduction environment in cells, and maintain the proper structure and activity of protein molecules [43]; conversely, low levels of GSH/GSSG ratio trigger the activation of resistance gene signals in response to adverse environmental conditions [42]. APX utilizes AsA as an electron donor to catalyze the decomposition of H2O2 into H2O and O2 [44], whereas GR utilizes NADPH as an electron donor to reduce GSSG to GSH [45]. The activity of both enzymes serves as a key indicator of plant stress tolerance [46].

In this study, a similar trend of AsA content and the APX activity in the main root and fibrous root of ginseng was observed. These values were significantly higher than those of the control in the early and middle stages of stress (Fig 5). AsA is a crucial low-molecular-weight, water-soluble, non-enzymatic antioxidant. Under water stress conditions, AsA is the primary molecule utilized to supply electrons to APX, facilitating the detoxification of excess H₂O₂ in plants. GSH is a low-molecular water-soluble mercaptan compound that exerts its antioxidant effect by directly scavenging ROS or indirectly scavenging H2O2 through the AsA-GSH cycle. In this study, GSH, GSSG and GR all showed a steady rising trend in the later stage of stress, indicating a regular change in response to water stress (Fig 5). However, the effect was relatively slight in the early stage of stress. These findings suggest that GR did not participate in ROS clearance initially, but its function was replaced by other antioxidant enzymes such as APX over this period. However, the continuous accumulation of H2O2 induced the high activity of GR. Still, it did not impede the regeneration of GSH. As a result, the cycle of GR-GSH remained reduced, effectively eliminating the excessive ROS generated in the cells. The decrease of GSH/GSSG ratio may be attributed to the reduced rate of GSH regeneration relative to GSSH or lack of NADPH in cells. At this point, the balance of ROS in the cell was disrupted, which further led to the blockage of the AsA cycle, ultimately leading to cell damage.

Previous investigations on Vigna radiata L roots have revealed a similar outcome in terms of APX activity, as well as a further reduction in the efficiency of the ASA-GSH cycle [47].

Effects of water stress on the synthesis of metabolites

Ginsenosides not only directly scavenge ROS, but also indirectly increase the activities of antioxidant enzymes such as SOD and CAT, acting as membrane stabilizers and ROS scavengers [48]. The biosynthesis of ginsenosides consists of three stages, including more than 20 consecutive enzymatic reactions: (1) In the stage of terpene precursor biosynthesis, HMGCR is the key rate-limiting enzyme. When the HMGCR level increases, it promotes the MVA pathway, which in turn promotes ginsenoside synthesis [49]. (2) During the prolonged stage of parent nucleotide C skeleton, FPS, SS and SE are the key enzymes. (3) In the skeleton structuring of parent nucleotide and its post-modification stage, DS, β-AS, CYP450 and GT are key enzymes [50]. The activities of DS and GT were positively correlated with ginsenoside content [51]. The results showed a significant correlation (p < 0.05) between soil water potential and the expressions of key ginsenoside synthesis enzymes, namely HMGCR, SS, β-AS, DS and CYP716A47 genes. These results indicate that increasing soil water potential can significantly promote the expression of key enzyme genes in ginseng roots [52].

The accumulation of monomer ginsenosides and total ginsenosides in the main and fibrous root of P. ginseng showed a time-dependent effect on water stress in this study. The activities of HMGCR and SE in the main root of P. ginseng increased significantly on the 3rd and 4th day after stress (p < 0.01), respectively, and peaked on the 4th day. On the other hand, the activities of FPS, SS and DS all markedly increased on the 3rd day (p < 0.01) and peaked on the 5th day. The activity of HMGR in fibrous root significantly climbed and peaked on the 2nd day, and the activity of FPS, SE, SS and DS all reached a peak on the 4th day (Fig 6). The results showed that the activities of five key enzymes in the ginsenoside biosynthesis pathway exhibited a consistent increase during the whole stress period. This increase in enzyme activity is likely to promote the biosynthesis of ginsenosides.

As a result of the changes in the activity of the five ginsenosides synthase mentioned above, the content of monomer ginsenosides in ginseng also changed dramatically. These changes were generally in line with the activity of DS, which is downstream of ginsenosides synthesis. Among the ten monomers, tetracyclic triterpenoid ginsenosides Rg1, Re, Rf, Rg2 and Rh1 belong to protopanaxatriol type (PPT), Rb1, Rc, Rb2 and Rb3 belong to protopanaxadiol type (PPD), and Ro belongs to oleanane type of pentacyclic triterpenoid ginsenosides. In the main root, the contents of all monomer ginsenosides except for ginsenosides Re and Rb2 increased to different degrees. The highest increase was observed in the levels of ginsenoside Rg2+Rh1 in the main root, which reached a maximum of 157.9% on the 5th day. This is followed by a 42.4% increase in the levels of Rg1. All 10 monomer ginsenosides in the fibrous roots were increased and all showed highly significant changes from day 0 (p < 0.01). Among the fibrous roots on day 4, Rf was the most elevated, at 84.3%, followed by Rg2+Rh1 at 30.7% (Fig 7). PPT ginsenosides Rg1, Rf and Rg2+Rh1 are more sensitive to water stress than PPD ginsenosides Rb1, Rc and Rb3, which may be attributed to the extra c-3 hydroxyl group. There was no obvious change in Re in the ginseng main root, possibly because the main root experienced mild stress that was insufficient for Rg1 to combine with rhamnose to form Re. The hemicellulose and pectin of plant cell walls contain a large amount of L-arabinose [53], and the 20th position of ginsenoside Rb2 is connected to arabinopyranose. Therefore, it is speculated that Rb2 in ginseng main root is consumed as a supplement to the cell wall during stress. It was recently reported that UDP-arabinose mutase often catalyzes the formation of UDP-arabinosylfuranose from UDP-arabinosylpyranose in plants [54]. It appears to clarify the notable rise in ginsenoside Rc, which is linked to arabofuranose at the C-20 position, although additional experimental confirmation is required. The content of total saponins in the main root of P. ginseng increased continuously and peaked on the 5th day. In contrast, the content of total saponins in fibrous root increased initially, peaked on the 3rd day, and then decreased. The rate of increase in fibrous root was significantly lower than in the main root (Fig 8). The fibrous root is more delicate than the main root, and it responds quickly to stress, which hinders the production of total ginsenosides. The significant decrease in its content may be attributed to the ingestion of ginsenosides as defensive compounds [55].

Glycolysis and tricarboxylic acid cycle are essential energy and raw material pathways for plants to synthesize most secondary metabolites. 1,3-DPG is an important intermediate product of glycolysis and the Calvin cycle, serving as an indicator of glucose catabolism [56]. PEPC plays an important role in regulating the operation of the tricarboxylic acid cycle. It can catalyze the reaction of phosphoenolpyruvate with CO2 to form oxaloacetic acid (the starting material of the tricarboxylic acid cycle), and the subsequent synthesis of malic acid and its derivatives [57]. In this study, the 1,3-DPG content and PEPC activity in fresh roots of P. ginseng rose to different extents under stress (Fig 9). It shows that water stress can promote the conversion of sugar in fresh ginseng root into subsequent material, energy metabolism and ginsenosides., This indicates that ginsenosides are derived from sugars rather than other active saponins, resulting in a significant increase in the content of various ginsenosides.

Taken together, water stress can induce fresh ginseng roots to up-regulate the activities of antioxidant enzymes, enhance AsA-GSH cycle metabolism and trigger the accumulation of secondary metabolites. This helps reduce the negative effects of water stress on themselves. Fig 15 illustrates the mechanism via which ROS enhances the production of ginsenoside under conditions of water stress.

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Fig 15. ROS promote ginsenoside biosynthesis under water stress.

Green boxes represent enzymes and red dashed arrows represent the relationship between ROS and the activities of key enzymes of the ginsenoside synthesis pathway.

https://doi.org/10.1371/journal.pone.0312023.g015

Validation of pharmacodynamics

The content of active ingredients is an important indicator for evaluating the quality of herbal medicine. However, herbal medicines contain a wide range of active components with varying levels and activities, making it difficult to evaluate the quality based on chemical component content [25]. Ginsenosides, especially Rg1 and Rg2 have anti-aging effect [28,58,59]. The D-galactose-induced subacute aging model has been widely used in experimental anti-aging studies because of its similarity to natural aging [60]. In this study, the ginseng main root on the 5th day showed better efficacy (Fig 11). The escape latency was reduced by 21.4% (p = 0.228), the number of times crossing the original platform and the residence time in the target area increased by 0.4 times (p = 0.255) and 7.3% (p = 0.486), respectively, compared with the 0 days. It suggests that ginseng can intervene in the aging process and significantly antagonized the impairment of learning memory function in mice by D-galactose. SOD and GSH-PX have significant functions in antioxidant, anti-aging and anti-injury. The MDA content is positively correlated with the formation of free radicals. In this study, the activities of GSH-PX and SOD on the 5th day were higher than those on the 0 days. Specifically, SOD increased by 11.2% (p = 0.009). The MDA showed a decreasing trend, although there was no significant difference between them (p = 0.155) (Fig 14). It suggests that ginseng can effectively attenuate D-galactose-induced oxidative damage in mice brain tissues by activating antioxidant enzyme activities, it is consistent with the study of Hao et al [61]. Combined with the results of morphological staining (Fig 13), the present study showed that water stress significantly improved the quality of ginseng.

Conclusions

Short-term water stress can trigger a physiological state of oxidative stress in ginseng fresh roots, stimulate the antioxidant defense system in ginseng, and lead to increased synthesis of secondary metabolites. In the main root, the ginsenosides Rg1+Re, Rb1, Rf, Rg2+Rh1, Rc and Rb3 increased by 42.4%, 21.0%, 15.7%, 157.9%, 18.3% and 10.6%, respectively, and the total ginsenosides by 40.1%. In fibrous roots, Rg1, Re, Rb1, Rf, Rg2+Rh1, Rc, Ro and Rb2 increased by 41.8%, 20.5%, 17.3%, 84.3%, 30.7%, 35.6%, 8.6% and 7.6%, respectively, the total ginsenosides increased by 4.2%. The activities of antioxidant enzymes were also enhanced, and they acted synergistically with secondary metabolites to minimize the damage caused by ROS. The effectiveness of the main root of ginseng was somewhat increased on the 5th day due to improved secondary metabolites. In summary, the quality of ginseng could be improved by water stress. Ginseng has a narrow ecological amplitude due to the lack of flavonoids and prefers humidity to waterlogging. Therefore, this study offers novel insights into ginseng’s ability to withstand water stress and its impact on quality formation.

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