Sucrose Induced HMGR to Promote Ginsenoside Biosynthesis in the Growth of Wild Cultivated Ginseng (Panax ginseng)

Wild cultivated ginseng is considered to be of higher quality than field-cultivated ginseng. Many active constituents of wild cultivated ginseng, including ginsenoside, have significant effects on human health. In the development phase of wild cultivated ginseng, we investigated the regulatory factors and pathways of ginsenoside biosynthesis. To determine glycolysis activity, the soluble sugar content, EL activity, and acetyl-CoA content of ginseng at various growth ages were measured. To examine the potential of ginsenoside biosynthesis, the ginsenoside contents and activities of SS, HMGR, and DXR of ginseng were measured at various growth ages. Ginseng cells were treated with various concentrations of sucrose to further test the effect of sugar on ginsenoside biosynthesis. MVA and MEP are the two primary pathways for ginsenoside biosynthesis. The key enzymes HMGR and DXR were detected when the sugar content was changed. The targets and primary pathways of sucrose regulation of ginsenoside biosynthesis in ginseng cells were investigated using MVA and MEP pathway inhibitors. We observed that the glycolysis of older wild cultivated ginseng was increased over that of younger ginseng, suggesting that older ginseng might provide adequate precursors for downstream ginsenoside biosynthesis. Furthermore, the total ginsenoside content and the activities of critical enzymes were increased by the ages of wild cultivated ginseng. Ginsenoside biosynthesis and glycolysis showed a significant linear relationship (R2 = 0.9562). We also verified that sucrose may stimulate glycolysis and ginsenoside biosynthesis at the cellular level. The MVA and MEP pathways were found to contribute to 58.15% and 39.72% of ginsenoside biosynthesis, respectively. The activity of HMGR, the rate-limiting enzyme of the MVA pathway, was increased with the increase of sucrose concentration in a dose-dependent manner (R2 = 0.9579). In contrast, the activity of DXR, the rate-limiting enzyme of the MEP pathway, was unaffected by sucrose concentration (R2 = 0.5414). Our findings suggest that the MVA pathway might be the main source of ginsenoside biosynthesis in wild cultivated ginseng. Sucrose promoted the MVA pathway over the MEP pathway by activating HMGR, resulting in increased ginsenoside biosynthesis year after year. This research contributes to a better understanding of the active constituents found in wild cultivated ginseng as it matures.


Introduction
Ginseng (Panax ginseng C. A. Meyer) has been widely used as Chinese herbal medicine for thousands of years. Ancient Chinese medical books have documented that ginseng can be used to fight fatigue, delay aging, regulate the central nervous system, improve immunity, and improve cardiovascular and cerebrovascular functions (Yang and Wu 2021). Wild ginseng has been harvested and used as an edible herb for a long time. The increasing demand for wild ginseng resulted in the overexploitation and scarcity of wild ginseng, and the ginseng has been artificially planted since the fifteenth century (Park et al. 2012). Since then, wild cultivated ginseng has been sown in the mountains or forest areas using the wild imitation planting technique. It takes 20 years to mature without human intervention and has a long life span, with some species living up to 100 years (Park et al. 2012). Wild cultivated ginseng has become an important source of revenue for forest communities as supply and demand have increased. Wild cultivated ginseng was considered to have a higher therapeutic value than field cultivated ginseng (Choi et al. 2007). Furthermore, elder ginseng had a higher quality than younger ginseng Dai et al. 2017;Jeong et al. 2016). The dynamic alteration of bioactive constituents with increasing years caused the variance in ginseng quality. Saponins (also known as ginsenosides) and non-saponins (phenolic compounds, acidic polysaccharides, peptides, and amino acid derivatives) are the primary active constituents of ginseng (Shin et al. 2021). Ginsenoside, one of the most significant bioactive constituents in ginseng, has significant anti-oxidant, anticancer, anti-obesity, and anti-inflammation activities (Yu et al. 2015;Jang et al. 2015;Hwang and Jung 2018;Lee and Son 2011;Lee et al. 2017). Consequently, ginsenoside accumulation was thought to be associated with ginseng quality improvement Han et al. 2007). Previous research has shown that wild ginseng contains much more ginsenoside content and has a greater variety of ginsenosides than cultivated ginseng (Choi et al. 2007;Liang et al. 2019). The quantities of seven metabolites, ginsenosides Ra3, Rd, 20(S)-Rg3, Rh1, Rh2, 20(S)-F1, and notoginsenoside R1, were higher in wild cultivated ginseng than that in field cultivated ginseng (Jing et al. 2018). Total ginsenoside, Rb1, Re, Rg1, Rb2, Rc, Rd, Rf, and Rg2 contents in wild cultivated ginseng rose as the plants became older (Wang et al. 2021). Ginsenosides Rb1, Rg1, and Re had a propensity to rise with time (Jing et al. 2018;Liang et al. 2019;Zhu et al. 2021). Climate, environment, and development stage have all been demonstrated to alter ginseng's intrinsic metabolic dynamics, particularly the accumulation of ginsenoside, which may be related to the expression of key genes involved in ginsenoside biosynthesis. (Dongmin et al. 2018;Lee et al. 2019;Kim et al. 2018;Zhang et al. 2018;Lee et al. 2019). In addition to metabolomics, it was found that the expression patterns of important enzymes in the genome, transcriptome, and proteome had a direct impact on ginsenoside biosynthesis (Wang et al. 2015;Ma et al. 2016;Jiang et al. 2017).
Two biosynthetic pathways for ginsenoside biosynthesis have been discovered (Zhao et al. 2014;Jin and Zhao 2013). One of them involves the decomposition of glucose into acetyl-CoA via the glycolysis pathway, followed by catalysis to isopentenyl pyrophosphate (IPP) by 3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMGR) via mevalonic acid (MVA) pathway (Omura et al. 2007;Liang et al. 2014). The other pathway involves the conversion of glyceraldehyde triphosphate, an intermediate product of the glycolysis pathway, to IPP by 1-Deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) via 2-C-Methyl-D-Erythritol-4-phosphate (MEP) pathway (Carretero-Paulet 2002). These two pathways begin by producing the triterpenoid carbon skeleton, and then proceed to synthesize ginsenosides, that catalyzed by farnesyl diphosphate synthase (FDPS), squalene synthase (SS), squalene cyclooxygenase (SQE), and several other enzymes (Jin and Zhao 2013;Dai et al. 2014;Liang and Zhao 2010). The regulatory targets of ginsenoside production were HMGR and DXR, the important enzymes in the MVA and MEP pathways. In our previous proteomic research, we reported that the ginsenosides biosynthesis in wild cultivated ginseng increased with age. At the same time, the ability of wild cultivated ginseng to perform glycolysis was improved. Continuously increasing activity of key enzymes, such as amylase, glucose-6-phosphate dehydrogenase, and enolase (EL), which are all involved in glucose metabolism, and FDPS, SQE, and SS, which are all involved in ginsenoside biosynthesis, has been observed . According to the UPLC-Q-TOF-MS metabonomic analysis, it was found that the concentrations of fructose, maltose, sucrose, and different ginsenosides in wild cultivated ginseng increased significantly with the growing years, which was consistent with the results of a proteomics analysis (Jing et al. 2018). Furthermore, during the process of ginseng cell culture, it was observed that the quantity of sugar was directly related to the content of ginsenoside present (Wang et al. 2012(Wang et al. , 2011. These findings suggest that sugar may play a beneficial role in the production of ginsenoside. The effect of sugar two ginsenoside synthesis pathways, particularly the regulatory impacts of HMGR and DXR activities, remained unknown.
In this work, the variations in glycolysis and ginsenoside biosynthesis in the growth phase of the wild cultivated ginseng, and sugar regulation on MVA and MEP pathways at the cell level were investigated. This study will enable the researchers to get more insight into the regulation of ginsenoside biosynthesis in plants.

Plant Materials
The samples of wild cultivated ginseng were collected from Lushuihe Town, Fusong County, Jilin Province. The area is located at 127 degrees, 46 min east longitude, and 42 degrees, 48 min north latitude with an altitude of 520 m. It is a mountainous area and has a dark brown soil form as well as a humid climate. Ten wild cultivated ginsengs were selected each, which had grown for 10 years, 15 years, 20 years, 25 years, and 30 years in the same slope, the same slope position, and the same slope direction (Fig. 1). Wild cultivated ginseng was washed with ultrapure water, cut into pieces, put into a pre-cooled mortar, ground into a fine powder by adding liquid nitrogen, and frozen at -80°C.

Culture of Ginseng Cells
Fresh ginseng roots of various ages were used to produce callus. In a 1-L conical flask, 200 mL liquid medium (4.302 g L -1 Murashige & Skoog medium, 30 g L -1 sucrose, 2 mg L -1 2, 4-dichlorophenoxyacetic acid) was crushed and inoculated with light yellow callus with loose texture. The shaking incubator was set at 120 rpm at 22 o C and kept in dark conditions. The large tissue and cell masses were removed after two weeks, and the medium containing single cells and small cell masses was transferred to a sterile culture flask for continuous oscillation. The optimal initial inoculum of ginseng cells was 35-40 g L -1 . Every 20 days, ginseng cells were subcultured four times before reaching a stable number of cells. The cells of 3, 10, and 20 years old ginseng were studied for their growth rate, sugar, and ginsenoside contents. The physiological activity of ginseng cells did not alter significantly across years ( Supplementary Fig. 1). The 5th generation of three-year-old ginseng cells was used as experimental materials to facilitate the sampling process. The cells were collected by filtration for subsequent experiments (Kochan and Chmiel 2011).

Treatment of Ginseng Cells
In suspension culture, the cells were weighed, and an 8 g fresh culture was injected into a 200-mL medium. MS media with 10 g L -1 , 20 g L -1 , and 30 g L -1 sucrose was added and cultured for 6 days, respectively. Sugar influences the ginsenoside production of ginseng cells. HMGR is inhibited by lovastatin (Rupasinghe et al. 2000). The produced lovastatin solution was added to the MS medium to make final concentrations of 5 μmol L -1 , 10 μmol L -1 , and 15 μmol L -1 and incubated for 3 days. The influence of HMGR activity on ginsenoside production by ginseng cells was studied. Fosmidomycin is a DXR inhibitor (Ortmann et al. 2010). The fosmidomycin solution was added to the MS medium to make final concentrations of 5 μmol L -1 , 10 μmol L -1 , and 20 μmol L -1 , and the cells were cultured for 3 days. The effect of DXR activity on ginseng cell ginsenoside production was studied. Two enzyme inhibitors were added to MS medium containing 10 g L -1 , 20 g L -1 , and 30 g L -1 sucrose and co-cultured for 6 days. Sugar affects the activity of the enzymes HMGR and DEX.

Determination of Ginsenosides Content
One gram of dried sample was combined with 30 mL methanol and extracted with 150 W ultrasound for 1 h. The extract was dried with methanol using a rotary evaporator at 50 o C, after three rounds of extraction. Ginsenosides were dissolved in methanol and concentrated to 10 mL. The extract was filtered using a 0.22-μm membrane before subsequent testing. 0.5 mL of extract was combined with 1% vanillin-perchloric acid reagent in equal volumes and maintained at 60 o C for 15 min, and then 5 mL of 77% sulfuric acid solution was added after cooling for 2 min with ice water, and the absorbance of the combination was measured at 540 nm. The following formula was used to calculate the total ginsenoside content: Total ginsenosides (%) = (standard (mg)* Sample absorbance/ Standard absorbance/ Sample weight) (Seifzadeh et al. 2019).
The ginsenosides' content was evaluated by LC-2030C Plus system equipped at 203 nm with a C18 column (5 μm, 4.6 × 250 mm; Kromasil, Sweden). Rb1, Re, and Rg1 were used as standards. The mobile phase composed of water (A) and acetonitrile (B), and the following elution program was used:

Extraction and Estimation of Soluble Sugars
0.2 g fresh weight (FW) of samples was homogenized with 5-10 ml distilled water and boiled twice for 30 min. The extract was filtered and the absorbance of the combination was measured at 540 nm. The soluble sugars were determined using the phenol-sulfuric acid method (Siqueira et al. 2011). A 0.5 ml extract was treated with 1 ml 9% phenol and 5 ml 98% sulfuric acid, left to stand for 30 min and then the absorbance at 490 nm was determined using a spectrophotometer (Biochrom 210; Biochrom, China). By fitting into the standard curve, the sugar content was determined and represented as mg/g FW.

Determination of Acetyl-CoA Content
According to the test kit's instructions, a sample of around 0.1 g was obtained to determine the content of acetyl-CoA using UV spectrophotometry. The growing rate of absorbance value at 340 nm represents the concentration of acetyl CoA which is formed following the coupling reaction of malate dehydrogenase and citrate synthase. Under standard conditions, the standard regression equation (y = 0.0331x + 0.1434, R 2 = 0.9994) was generated, using the absorbance values as the abscissa and the standard concentrations as the ordinate. The concentration of acetyl-CoA was calculated by fitting the absorbance values into the standard curve and expressed as nmol g -1 FW.

Determination of Enzyme Activity
0.5 g of fresh samples was crushed to powder using liquid nitrogen, then 2.5 mL tissue lysate was added and shaken for 1 h on ice. Following 5 times repeated freeze-thaw process, the sample was centrifuged at 4°C, 12000 rpm for 15 min, and the supernatant was taken for later use. The ELISA kits were used to detect the activities of DXR, HMGR, FDPS, SS, SQE, and EL. According to the specific operation steps in the manual, a standard curve was drawn. The extracted samples of the crude enzyme solution of wild cultivated ginseng and ginseng cells were accurately added. The value was read on the microplate reader at 450 nm to calculate the enzyme activity of each sample.

Statistical Analysis
All test data in this study are represented as the mean of three replicas. Statistical comparisons between the test and control groups were performed using the Student t-test. Statistical evaluation was performed using GraphPad Prism, version 5.0 (GraphPad Software, San Diego, CA, USA). We considered p < 0.05 to be statistically significant.

The Glycolysis of Wild Cultivated Ginseng Increased Year by Year
During the growth and development stage, the morphology of wild cultivated ginseng altered progressively (Fig. 1). Ginseng root requires active energy metabolism to produce the required substance and energy due to its increased thickness and weight. The sugar content of wild cultivated ginseng continues to rise with time ( Fig. 2A). The glycolysis route, as the primary mechanism for sugar decomposition, would necessarily shift as a result. EL is the rate-limiting enzyme in the glycolysis pathway and its activity increases during the growth phase (Fig. 2B). As the final product of glycolysis, acetyl-CoA content increased with the number of growth years of the wild cultivated ginseng (Fig. 2C).
In 30 years, the content of acetyl-CoA in wild cultivated ginseng is 3.68 times that in 10 years. This revealed that the ability of wild cultivated ginseng to perform glycolysis grew over time.

Ginsenosides Accumulate During the Older Wild Cultivated Ginseng
With the extension of the growth years of wild cultivated ginseng, the total ginsenoside content increased (Fig. 3A). At the same time, there was a good linear relationship between ginsenoside content and age (R 2 = 0.9562). Moreover, Rb1, Re, and Rg1 were the important components of ginsenosides, which were used as physical and chemical indexes to evaluate the quality of wild ginseng in The National Standard of China (GB/T 18765-2008). We found that the contents of Rb1, Re, and Rg1 were increased with ginseng age (Fig. 3B), indicating that the accumulation of ginsenoside was positively correlated with their growth years and promoted the quality development of wild cultivated ginseng. The MVA pathway, which uses HMGR as a major enzyme, is one of the two main ginsenoside biosynthesis processes. The MEP pathway, whose main enzyme is DXR, is the other. The enzyme activities of HMGR and DXR were detected respectively in the different growth years of wild cultivated ginseng ( Fig. 3C and D). It was found that the activities of both enzymes manifest an increase with the increase of ages. Compared with 10-year old ginseng, the were compared to the control group of 10-year-old wild cultivated ginseng. Graphical representation of data calculated using the Student t-test. Error bars show the standard error between three replicates performed, ( # P < 0.05, ## P < 0.001, ### P < 0.0001). HMGR, 3-Hydroxy-3-methylglutaryl coenzyme-A reductase; DXR, 1-Deoxy-D-xylulose 5-phosphate reductoisomerase; FDPS, Farnesyl diphosphate synthase; SQE, Squalene cyclooxygenase; SS, Squalene synthase activities of HMGR and DXR in 30-year old ginseng were increased by 2.24 times and 4.48 times, respectively. Subsequently, the carbon skeleton of ginsenoside was synthesized by a series of enzymes. FDPS is a branch of diterpenoid and triterpene biosynthesis. This enzyme activity of 30-year-old wild cultivated ginseng in terms of FDPS was 1.82 times higher than that of a 10-year-old one (Fig. 3E). SS and SQE are the key enzymes for squalene synthesis, and their content and activity determine the yield of ginsenoside. SS activity was 3.16 times higher in 30-yearold wild cultivated ginseng than in 10-year-old plants (Fig. 3F). Similarly, SQE activity was 1.84 times higher in 30-year-old wild cultivated ginseng than in 10-year-old plants (Fig. 3G). The apparent discrepancy in the activity of these important enzymes suggested that ginsenoside biosynthesis was active in older wild cultivated ginseng, promoting ginsenoside accumulation year after year.

Sucrose Promotes the Glycolysis and Ginsenoside Biosynthesis in Ginseng Cells
The ginseng cells were given varying doses of sucrose to test the link between glycolysis and ginsenoside production. The intracellular sugar content increased as sucrose concentration increased (Fig. 4A). EL activity and the amount of acetyl-CoA in the body both rose ( Fig. 4B and C). These findings revealed that sucrose could boost the glycolysis process, resulting in an increased generation of ginsenoside precursors. We also found that as the sucrose concentration increased, the total ginsenoside content increased (Fig. 4D). In addition, the contents of Rb1, Re, and Rg1 in ginseng cells were increased under a high concentration of sucrose (Fig. 4E), suggesting that sucrose could promote ginsenosides biosynthesis. Increasing the sucrose concentration, on the other hand, increased the activity of HMGR in a dose-dependent manner (R 2 = 0.9495) (Fig. 4F). HMGR activity was 1.69 times higher than the blank group at a concentration of 30 g L -1 . Although DXR activity was higher in the experimental group than in the control group, there was no dose-dependent relationship between sucrose concentration and DXR activity (R 2 = 0.6231) (Fig. 4G). Sucrose may thereby promote ginsenoside biosynthesis through the MVA pathway.

Sucrose Regulates the Ginsenoside Biosynthesis by HMGR Induction
The HMGR activity is inhibited by lovastatin. HMGR activity reduced when the quantity of inhibitor was increased after adding lovastatin to MS medium. The ginsenoside content was reduced by 58.15% when the inhibition rate of its activity reached 79.21% (Fig. 5A-B). Despite this, the addition of sucrose lowered the inhibition of lovastatin. With increasing exogenous sucrose concentration, HMGR activity was re-enhanced, revealing a good linear connection Graphical representation of data was calculated using the Student t-test. Error bars show the standard error between three replicates performed, (*P < 0.05, **P < 0.001, ***P < 0.0001). EL, Enolase; HMGR, 3-Hydroxy-3-methylglutaryl coenzyme-A reductase; DXR, 1-Deoxy-D-xylulose 5-phosphate reductoisomerase (R 2 =0.9579) (Fig. 5C). The content of ginsenoside was also increased (Fig. 5D). These findings suggested that sucrose could increase HMGR activity and cause a boost in ginsenoside production.

Effect of Sucrose on DXR Activation
Fosmidomycin is an inhibitor of DXR activity. DXR activity was decreased by 84.62% with an increased in fosmidomycin HMGR activity (A) and ginsenoside content (B) in ginseng cells treated with different concentrations of lovastatin for 3 days were compared to the control group of cells treated with 0 μmol L -1 lovastatin; sucrose can relieve the inhibitory effect of lovastatin on HMGR activity (C) and ginsenoside content (D) in ginseng cells.
The lovastatin was added to MS medium containing 0 g L -1 , 10 g L -1 , 20 g L -1 , and 30 g L -1 sucrose and co-cultured for 6 days. Cells treated with 0 g L-1 sucrose were used as a control group. Graphical representation of data calculated using the Student t-test. Error bars show the standard error between three replicates performed, (*P < 0.05, **P < 0.001, ***P < 0.0001). MVA, mevalonic acid; HMGR, 3-Hydroxy-3-methylglutaryl coenzyme-A reductase Fig. 6 Effect of sucrose on MEP pathway in ginseng cell. DXR activity (A) and ginsenoside content (B) in ginseng cells treated with different concentrations of fosmidomycin for 3 days were compared to the control group of cells treated with 0 μmol L -1 fosmidomycin; sucrose can relieve the inhibitory effect of fosmidomycin on DXR activity (C) and ginsenoside content (D) in ginseng cells. The fosmidomycin was added to MS medium containing 0 g L -1 , 10 g L -1 , 20 g L -1 , and 30 g L -1 sucrose and co-cultured for 6 days. Cells treated with 0 g L -1 sucrose were used as a control group. Graphical representation of data calculated using the Student t-test. Error bars show the standard error between three replicates performed, (***P < 0.0001). MEP, 2-C-Methyl-D-Erythritol-4-phosphate ; DXR, 1-Deoxy-D-xylulose 5-phosphate reductoisomerase concentration, and the ginsenoside content was decreased by 39.72% (Fig. 6A-B). The DXR activity, which was inhibited by fosmidomycin, did not change significantly after adding sucrose in a dose-dependent manner (R 2 = 0.5414) (Fig. 6C). As a result, we concluded that DXR activity is unaffected by sucrose. At the transcriptional level, the MEP and MVA pathways have been shown to have the same influence on ginsenoside biosynthesis in ginseng root (Xue et al. 2019;Sun et al. 2017). When the MEP pathway was blocked, the level of ginsenoside still increased (Fig. 6D). The MVA route may be responsible for the increased ginsenoside content, indicating that sucrose promotes ginsenoside accumulation mostly via the MVA pathway.

Discussion
It was reported that the quality of wild cultivated ginseng is due to the accumulation of multiple active components with ages (Kim et al. 2018;Jang et al. 2015;Hwang and Jung 2018;Hang et al. 2016;Zhang et al. 2013). Ginsenoside, the principal active component of ginseng, is one of the most important standards used to assess its quality. Our findings confirmed that the amount of total ginsenosides and main ginsenosides monomer (Rb1, Re, and Rg1) in wild cultivated ginseng grew year after year, as reported in the literature Zhang et al. 2008).
Year after year, the activity of key enzymes in the ginsenoside production pathway grew. To better understand the medicinal effects of wild cultivated ginseng, it is vital to uncover ginsenoside biosynthesis regulatory elements and mechanisms. Previously, we have discovered using proteomics technology that glycolysis and ginsenoside synthesis have been increased year by year in the growth of wild cultivated ginseng Hang et al. 2016). In this study, we observed that the glycolysis of older wild cultivated ginseng was enhanced over that of younger ginseng, suggesting that older ginseng might provide adequate precursors for downstream ginsenoside biosynthesis. Additionally, the ginsenoside content of wild cultivated ginseng increased with age. Ginsenoside biosynthesis and glycolysis showed a strong linear correlation. Previous research has also demonstrated this tendency. At the metabonomic level, the contents of various sugars and ginsenosides in wild ginseng were found to be positively associated (Jing et al. 2018;Lee et al. 2019). Transcriptome analysis revealed that gene expression levels in basic metabolisms, such as glycolysis, may contribute to the accumulation of various ginsenosides in various ginseng varieties . In secondary metabolic engineering, the sugar may help to promote the biosynthesis of ginsenosides (Kochan and Szymańska 2014;Follett et al. 2004). As a result, we hypothesized that the rise in ginsenoside content in aged ginseng could be influenced by enhanced glycolysis. To confirm this finding, researchers looked at the effect of glycolysis on ginsenoside production in ginseng suspension cells. The activity of critical enzymes for glycolysis and ginsenoside biosynthesis was increased with increased sucrose concentration. This means that the addition of sucrose enhanced glycolysis and promoted ginsenoside biosynthesis.
The ginsenoside is synthesized by two independent pathways located in the cytoplasm (MVA pathway) and the plastids (MEP pathway) (Wei et al. 2020;Henry et al. 2018). There is a difference between the two pathways related to the initial raw material of the reaction. The starting material of the former pathway is acetyl-CoA, the end product of the glycolysis pathway, whereas, the starting material of the latter pathway is glyceraldehyde triphosphate, the intermediate product of the glycolysis pathway (Mao et al. 2020;Vranová et al. 2013). The content of acetyl-CoA was found to increase with the increase of sucrose concentration, which would provide more ginsenoside synthesis precursor substances to activate the MVA pathway. The activity of HMGR, the rate-limiting enzyme of the MVA pathway, was increased with the increase of sucrose concentration in a dose-dependent manner.
In contrast, the activity of DXR, the rate-limiting enzyme of the MEP pathway, was insensitive to sucrose concentration. We added lovastatin and fosmidomycin, the respective inhibitors of the ginsenoside biosynthesis in MVA and MEP pathways, to the ginseng cells (Rather et al. 2019). It was found that after the addition of the inhibitor, the activities of key enzymes in the corresponding pathway were decreased and the ginsenoside synthesis pathway was inhibited. When the MVA pathway in ginseng cells was inhibited, the ginsenoside content was decreased more than that in the MEP pathway. This may be because of the reason that the MVA pathway has a major contribution to ginsenoside synthesis. However, when we added different concentrations of sucrose to ginseng cells treated with the inhibitor, we found that with the increase of sucrose concentration, the inhibition of HMGR activity was alleviated, and the change of DXR activity was not obvious.
The results demonstrated that sucrose activates the MVA pathway via modulating HMGR activity and promoting ginsenoside production (Fig. 7). Although prior research has shown that the MVA and MEP pathways are involved in ginsenoside biosynthesis, simultaneously (Xue et al. 2019;Sun et al. 2017). However, the MVA pathway in ginseng roots was primarily responsible for regulating ginsenoside biosynthesis in response to biotic and abiotic stresses (Bian et al. 2021;Wang et al. 2016). In ginseng leaves, the MEP pathway contributed more to ginsenoside biosynthesis than the MVA pathway (Xue et al. 2019). In this research, cell growth and ginsenoside biosynthesis were shown to be partly dependent on the crosstalk between the MVA and MEP pathways, which are becoming more active in the development phase of wild cultivated ginseng root (Yang et al. 2012). According to our findings, the MVA pathway was the primary source of ginsenoside biosynthesis, whereas the MEP pathway was critical for ginseng growth.

Conclusion
The continuous accumulation of ginsenosides improved the quality of ginseng. Sucrose, a key molecule involved in the transport and partitioning of carbon resources, was found to enhance ginsenoside biosynthesis through the mevalonic acid pathway. When the carbon source in the soil depleted while ginseng develops, homeostatic regulation of the carbon source in the soil might be the key to attaining highquality ginseng. We'll keep looking at the factors that influence ginsenoside expression patterns and diversity in wild and farmed ginseng. These findings will aid in comprehending the implication that wild cultivated ginseng becomes more valued as it matures. More significantly, this study may have a significant impact on future research on ginseng cultivation, breeding, and associated functional candidate genes.