The Effects of Low-Temperature Stress on the Physiological Characteristics and Active Components of Ginseng Under Different Soil Moisture Conditions

Abstract

Ginseng growth is susceptible to environmental stresses, particularly the frequent occurrence of low temperatures and water fluctuations in spring in Northeast China, which often lead to a decline in medicinal yield and quality. This study systematically analyzed the physiological response characteristics and variation patterns of active components under dual stresses of low temperature and water. The aim was to elucidate the adaptation mechanism of ginseng to abiotic stresses, providing a theoretical basis for optimizing ginseng cultivation management practices and enhancing the quality of medicinal materials. In this study, 2-year-old and 4-year-old ginseng roots were selected as research materials. They were subjected to treatments of low soil moisture (20–30%), medium soil moisture (40–50%), and high soil moisture (60–70%). Low-temperature treatments were conducted at 0 °C for different durations (4 h, 24 h, 33 h, 48 h). Physiological indicators of the ginseng roots were determined at each time point, and the active components of ginseng roots in the control and treatment groups were investigated. The results indicated significant differences in osmotic adjustment substance changes between 2-year-old and 4-year-old ginseng roots. The content of superoxide dismutase (SOD) increased during low-temperature stress in both age groups. An increase or decrease in soil moisture significantly enhanced the accumulation of total ginsenosides. However, low-temperature stress notably reduced the accumulation of total ginsenosides. Nevertheless, after low-temperature treatment, the PPT-type ginsenosides in the high soil moisture group showed a significant increase. The findings of this study provide a scientific basis for improving the medicinal component content of ginseng and offer theoretical support for future water management practices.

1. Introduction

Ginseng (Panax ginseng C.A. Meyer), a perennial herbaceous medicinal plant belonging to the genus Panax in the family Araliaceae, is a geo-authentic medicinal herb in Northeast China. Modern medical research and clinical practice have demonstrated that ginseng exhibits significant therapeutic effects on diseases related to the central nervous system [1], cardiovascular system [2], and respiratory system [3]. Ginsenosides are biosynthesized from acetyl-CoA via the mevalonic acid (MVA) or methylerythritol phosphate (MEP) pathways, which further produce isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). Subsequently, enzymes such as FPS, SS, and SE catalyze the conversion of 2,3-oxidosqualene into various ginsenoside monomers through a series of cyclization, hydroxylation, and glycosylation processes of the cyclic functional groups [4]. Ginsenosides, the primary active components of ginseng, serve as crucial indicators for evaluating the quality of ginseng. These include Rg1, Re, Rg2, Rb1, Rc, Rg3, and Rh2, among others [5]. Ginsenosides exhibit potential medicinal value in various aspects, such as the nervous system, cardiovascular system, immune system, anti-aging, and functional foods [6,7]. For instance, ginsenosides Rb1 and Rg3 demonstrate optimal biological activity in protecting the brain from ischemic injury and treating respiratory diseases [8,9]. Ginsenoside Rh2 and its aglycone aPPD, as primary metabolites of Panax ginseng exhibit significant antitumor potential in prostate cancer treatment [10]. Rg2 ameliorates Alzheimer’s disease by multi-target regulation of the cerebral metabolic network, alleviating cognitive dysfunction and memory decline [11].

Optimal soil moisture not only promotes plant growth, but also mitigates low-temperature damage. When soil moisture is controlled at approximately 80% of field capacity, Panax ginseng exhibits enhanced growth and ginsenoside biosynthesis [12]. Under drought stress, soybean increases the root/shoot ratio by elevating sucrose and starch content alongside related enzyme activity, thereby facilitating sucrose translocation from leaves to roots to sustain root growth and metabolism [13]. Cameron et al. demonstrated that short-term water deficit suppresses nutritional growth, tuber yield, and quality in potato plants, whereas short-term excessive irrigation mildly improves yield and quality [14]. Root system robustness is closely linked to environmental conditions. Research conducted by Zhang revealed that superoxide dismutase (SOD), soluble proteins, and soluble sugars play critical roles in wheat’s defense against low-temperature stress [15]. In a study on Dendrocalamus latiflorus roots subjected to low-temperature treatment for 72 h, research conducted by Zhang [16] found that roots alleviate membrane lipid peroxidation by maintaining high SOD and peroxidase (POD) activity while reducing plasma membrane permeability through increased unsaturated fatty acid ratios. Chu et al. reported elevated malondialdehyde (MDA) levels in soybean roots under low-temperature stress, indicating root injury [17]. To address combined cold-drought stress in harsh environments, the cold-season grass Poa annua was investigated. Under dual stress, it exhibited significant accumulation of reactive oxygen species (ROS: O₂⁻, H₂O₂) and MDA, yet activated stress resistance mechanisms by enhancing antioxidant enzyme activities (SOD, POD, CAT, APX, GR) and osmotic adjustment substances to mitigate adverse effects [18]. Collectively, these studies suggest that moderate environmental stress enhances plant stress tolerance.

In recent years, abnormal global climate conditions have frequently resulted in low spring temperatures. If soil moisture is insufficient and not timely adjusted through irrigation, ginseng may experience delayed growth and yellowing of leaves due to water deficit following cold spells. Conversely, excessive soil moisture combined with low temperatures can lead to freezing damage, loss of oxygen in the soil, and frozen or rotten roots, thereby affecting the quality and yield of ginseng. Therefore, the aim of the current study was to investigate the effect of soil moisture adjustments under low-temperature stress on changes in physiological characteristics and active components of ginseng of different ages, thereby enhancing ginseng yield and quality and providing a scientific basis for water management under low-temperature conditions caused by climate change.

2. Materials and Methods

2.1. Overview of the Experimental Site

The experimental site was located in the greenhouse of Jilin Agricultural University in Changchun City, Jilin Province (longitude 125.4238 E, latitude 43.8136 N). During the trial period, the environmental temperature in the greenhouse was controlled between 15 °C and 30 °C (with an average temperature of 24 °C ± 1 °C), and the relative humidity was maintained at 75% ± 5%.

2.2. Experimental Materials and Treatments

The experiment selected 4-year-old ginseng and 2-year-old ginseng plants (Fusong County, Baishan, China) as the test materials. The plants were cultivated in pots with a diameter of 11.5 cm and a height of 14.2 cm, using a soil mixture consisting of peat, vermiculite, soil, and perlite in a ratio of 1:1:1:0.5 as the growing medium. The environmental temperature was maintained between 21 and 25 °C, and the relative air humidity was kept at 60–70%, conditions suitable for ginseng growth. After the ginseng seedlings emerged, plants with uniform growth were selected for different soil moisture treatments until the leaves were fully expanded. During this period, three soil moisture levels were maintained: low soil moisture (20–30%, referred to as the ES group), medium soil moisture (40–50%, referred to as the SW group), and high soil moisture (60–70%, referred to as the LQ group). Soil moisture was monitored daily using a soil moisture detector (QS-SFY-I soil moisture rapid measuring instrument, Shanghai, China), and watering was performed as needed. Thirty pots of ginseng plants were used for each soil moisture treatment.

After the leaves of the ginseng plants under three different soil moisture treatments were fully expanded, they were transferred to a cold light source artificial climate chamber (Shanghai Wanbai Biotechnology Co., Ltd., Shanghai, China) for low-temperature treatment. The temperature was set to 0 °C, the relative air humidity was maintained at 40–60%, and the light/dark cycle was set to 14 h/10 h for 48 h. Root samples were collected at 4 h, 24 h, 33 h, and 48 h under low temperature, respectively. The plants from each soil moisture group (ES, SW, LQ) without low-temperature treatment were used as controls (0 h) and stored in a cold light source artificial climate chamber at a temperature of 21–25 °C and a relative air humidity of 40–60%. For root sampling, the main and lateral roots were completely excavated, and the fibrous roots were retained, immediately rinsed with distilled water to remove soil from the surface, and wiped clean; the cleaned samples were cut into small pieces, placed in liquid nitrogen, and stored at −80 °C for physiological index analysis. The determination of ginsenosides was carried out by sampling before low-temperature treatment and after recovery to normal temperature growth following low-temperature stress until the harvest period. Three independent biological replicates were performed at each time point, and each biological replicate underwent three technical replicate determinations.

2.3. Determination of Physiological Indices

Superoxide dismutase (SOD) activity was determined using the nitroblue tetrazolium method [19]. The anthrone method was employed to measure the soluble sugar content and the sucrose content [13]. The Coomassie brilliant blue G-250 method was used to determine the soluble protein content [20]. The ninhydrin method was adopted for proline content determination [21]. The MDA content was assayed using the thiobarbituric acid method [22].

2.4. Determination of the Ginsenoside Content

2.4.1. Preparation of the Sample Solution

The ginseng root samples were cleaned and then placed in an oven (Shanghai Longyue Instrument & Equipment Co., Ltd., Shanghai, China) to be dried at 40 °C until reaching a constant weight. After that, they were pulverized and passed through an 80-mesh sieve. Then, 20 mg of the powder was weighed and added to 1 mL of methanol (70% v/v) in a 1.5 mL centrifuge tube. Ultrasonic extraction was performed at 50 °C for 30 min, and each sample was centrifuged at 13,500 rpm for 5 min. The solution was filtered through a syringe filter (0.22 μm) and directly injected into the UPLC system.

2.4.2. Chromatographic Conditions

The UPLC analysis was conducted using a Waters ACQUITY H-Class UPLC system (Agilent Technologies Inc., Santa Clara, CA, USA). An ACQUITY BEH C18 column (2.1 mm × 100 mm; 1.7 μm) was employed. The mobile phase consisted of solvent A (pure water (MilliporeSigma, Burlington, VT, USA) and 0.1% formic acid (Honeywell, Charlotte, VT, USA) (v/v)) and solvent B (acetonitrile (Honeywell, Charlotte, VT, USA) and 0.1% formic acid (v/v)). The elution gradient was as follows: 0–0.5 min, B 15%; 0.5–1 min, B 15–20%; 1–6 min, B 20%; 6–13 min, B 20–30%; 13–23 min, B 30–35%; 23–24 min, B 35–38%; 24–27 min, B 38–60%; 27–31 min, B 60–90%; 31–32 min, B 90–15%; 32–35 min, B 15%. The flow rate was set at 450 μL/min, and the injection volume was 2 μL per run.

2.4.3. Mass Spectrometry Conditions

Mass spectrometry analysis was performed using a Waters Xevo G2-S QTOF MS (Agilent Technologies Inc, Santa Clara, CA, USA). The operating parameters were set as follows: cone voltage, 40 V; capillary voltage, 3.0 kV; source temperature, 120 °C; desolvation temperature, 550 °C; cone gas flow, 30 L/h; desolvation gas flow, 800 L/h. The negative ion mode was used, and the scanning range was set from 100 to 2000 m/z.

2.5. Comprehensive Evaluation of Cold Resistance

2.5.1. Evaluation Method of Cold Resistance

The cold resistance evaluation of the test materials was conducted by the membership function method [23], and the membership function value of each cold resistance index of the test ginseng root was as follows:

$$\mathsf{\mu }(X_j)=(X_j-X_{\min })/(X_{\max }-X_{\min })$$

(1)

where ${{\displaystyle X}}_j$ represents the value of the j^th indicator, ${{\displaystyle X}}_{\min }$ represents the minimum value of the j^th indicator, and ${{\displaystyle X}}_{\max }$ represents the maximum value of the j^th indicator.

The coefficient of standard deviation ${{\displaystyle V}}_j$ is as follows:

$$V_{\mathrm{j}}=\frac{\sqrt{\frac{{\displaystyle \sum _{i=1}^n{(X_{ij}-{\overline{X}}_i)}^2}}{n-1}}}{{\overline{X}}_j}$$

(2)

where ${\stackrel{-}{X}}_j$ represents the average value of the j^th indicator for each material, ${{\displaystyle X}}_{ij}$ represents the membership function value of the material’s trait i in the formula, and ${{\displaystyle V}}_j$ represents the coefficient of variation of the j^th indicator.

The weight W value of each indicator ${{\displaystyle W}}_j$ is as follows:

$${{\displaystyle W}}_j={\displaystyle \frac{{{\displaystyle V}}_j}{{\displaystyle \sum _{j=1}^n{{\displaystyle V}}_j}}}$$

(3)

$$D={\displaystyle \sum _{j=1}^n\left[u\left({{\displaystyle X}}_j\right)\times {{\displaystyle W}}_j\right]}$$

(4)

where ${{\displaystyle W}}_j$ shows the weight of the j^th index and integrated assessment value D indicates the comprehensive evaluation value of cold resistance of each material; the larger the D value, the stronger the cold resistance.

2.5.2. Classification Standards for Cold Resistance Evaluation

The comprehensive cold resistance D value of plants can be classified into three levels: level I: D value above 0.7, indicating strong cold resistance; level II: D value between 0.5 and 0.7, indicating moderate cold resistance; and level III: D value below 0.5, indicating weak cold resistance [23].

2.6. Data Processing

Data analysis was conducted using SPSS version 27.0 (IBM Corporation, Chicago, IL, USA). The results were expressed as the means of three independent measurements. Statistical evaluation of the experimental data was performed using one-way ANOVA followed by Duncan’s multiple range test. A p-value less than 0.05 was considered statistically significant. Pearson correlation analysis and principal component analysis were used to assess correlations between the various physiological indices of the plants. Graphical representations were created using Origin 2024b software (OriginLab Corporation, Northampton, MA, USA).

3. Results

3.1. Response of the Malondialdehyde (MDA) and Superoxide Dismutase (SOD) Contents in Ginseng Roots of Different Ages to Low Temperature

In the 2-year-old ginseng group (Figure 1), changes in the MDA content showed that the ES group remained below the 0 h level during low-temperature stress, reaching a peak at 48 h, but with no significant difference. The SW group reached its maximum value at 48 h, increasing by 10.22% compared to 0 h. The LQ group exhibited an initial increase followed by a decrease, peaking at 24 h with a 32.65% increase compared to 0 h. Changes in the SOD content indicated that both the ES and SW groups followed a similar pattern of initial increase and subsequent decrease. The ES group reached its maximum at 24 h (33 h), increasing by 106.87% (104.42%) compared to 0 h, while the SW group had the highest content at 33 h, increasing by 111.83% compared to 0 h.

In the 4-year-old ginseng group (Figure 2), the changes in the MDA content demonstrated that the increase in MDA levels across the three soil moisture treatment groups was significantly lower compared to that observed in 2-year-old plants. Regarding the SOD activity, both the ES and SW groups exhibited a reduced SOD content during low-temperature stress relative to the initial 0 h, while the LQ group delayed membrane damage by enhancing the SOD activity.

3.2. Response of Osmotic Adjustment Substance Content in Ginseng Roots of Different Years to Low Temperature

In 2-year-old ginseng plants, the LQ group generally exhibited smaller increases in osmoregulatory substances compared to the ES and SW groups with prolonged low-temperature exposure. Soluble protein content analysis revealed a significant decreasing trend across all three groups during the 24 h to 48 h low-temperature period (Figure 3a). Following low-temperature stress, soluble sugar content showed marked increases in all treatment groups, reaching peak levels at 33 h (Figure 3b). Sucrose content measurements demonstrated that both ES and SW groups maintained significantly lower sucrose levels than the initial 0 h values during cold stress, whereas the LQ group attained the maximum sucrose content at 24 h, representing a 22.39% increase compared to 0 h (Figure 3c). The proline content in all the three groups displayed differential increases under cold stress: the ES group peaked at 24 h (81.99% increase) and 33 h (62.35% increase), the SW group reached the maximum levels at 48 h (70.45% increase), and the LQ group showed peaks at 4 h (32.71% increase) and 33 h (40.69% increase) relative to the baseline 0 h measurements (Figure 3d).

In 4-year-old ginseng plants, soluble protein content analysis revealed distinct patterns: the ES group exhibited a continuous increasing trend during low-temperature treatment, peaking at 48 h with a 148.60% increase compared to 0 h. In contrast, both SW and LQ groups showed that the ES group increased continuously during the low-temperature treatment and reached the maximum at 48 h, with an increase of 148.60% compared to 0 h. The SW and LQ groups both showed an initial increase, followed by a decrease, with the maximum values at 33 h and 24 h, respectively. Compared to 0 h, the content increased by 71.58% and 69.21%, respectively (Figure 4a). Determination of the soluble sugar content indicated that the ES group increased continuously from 4 h to 48 h under low temperature, while the SW group showed a gradually decreasing trend. The LQ group reached the maximum at 48 h, with an increase of 54.36% compared to 0 h (Figure 4b). Determination of the sucrose content showed that changes in the sucrose content in the ES and SW groups were consistent with those of soluble sugar. The LQ group had a sucrose content higher than that at 0 h from 4 h to 48 h, but the changes were not significant during this period (Figure 4c). Determination of the proline content revealed that the ES group reached the maximum at 33 h, with an increase of 60.84% compared to 0 h. The SW group showed a gradually increasing trend during the low-temperature stress period. The LQ group reached the maximum at 4 h and 48 h, with an increase of 61.26% and 74.39%, respectively, compared to 0 h (Figure 4d).

3.3. Correlation Analysis of Cold Resistance Physiological Indices in Ginseng of Different Ages Under Low-Temperature Stress

In 2-year-old ginseng roots, Pearson analysis of six physiological indicators was conducted (Figure 5a–c). In the ES group (Figure 5a), SOD showed highly significant positive correlations with soluble sugar and proline (p < 0.001). In the LQ group (Figure 5c), MDA exhibited a highly significant positive correlation with sucrose (p < 0.001), while significant positive correlations were observed between MDA and soluble protein, soluble protein and sucrose, soluble sugar and proline, and soluble sugar and sucrose. Principal component analysis further revealed (Figure 5d) that the SOD activity had the highest loading coefficient in the first principal component P1, while proline showed the highest loading in the second component P2. The PCA results were highly consistent with those from Pearson analysis.

In 4-year-old ginseng roots (Figure 6), in the ES group (Figure 6a), MDA showed significant positive correlations with soluble protein, soluble sugar, and sucrose. Soluble protein demonstrated significant positive correlations with soluble sugar and sucrose, and soluble sugar was significantly positively correlated with sucrose. In the SW group (Figure 6b), SOD exhibited a significant positive correlation with soluble sugar (p < 0.01) and a highly significant positive correlation with sucrose (p < 0.001). Soluble protein showed a significant positive correlation with proline (p < 0.05). In the LQ group (Figure 6c), SOD displayed highly significant positive correlations with soluble sugar (p < 0.001) and proline (p < 0.01), while soluble sugar and proline showed a highly significant positive correlation (p < 0.001). Principal component analysis (Figure 6d) yielded consistent results with those from 2-year-old samples. Notably, the highly significant positive correlation (p < 0.001) between soluble sugar and proline in the LQ group corresponded with proline’s high loading coefficient in P2, suggesting potential synergistic mechanisms in osmotic regulation pathways. Comprehensive analysis indicates that SOD and proline play crucial roles in stress resistance evaluation for both 2-year-old and 4-year-old ginseng, serving as the key physiological indicators for assessing ginseng’s adversity stress responses.

3.4. Comprehensive Evaluation of Cold Resistance in Ginseng of Two Different Ages

A single physiological index cannot fully reflect the adaptability and physiological response of plants to low-temperature conditions. Therefore, to more comprehensively evaluate the cold resistance of plants, it is generally necessary to consider multiple indices. Overall, the 4-year-old ginseng demonstrated stronger cold resistance compared to the 2-year-old ginseng. Among them, the SW group of the 4-year-old ginseng showed the highest cold resistance, followed by the ES group, and finally the LQ group (

Table 1. Membership function values of each index and comprehensive evaluation values of cold resistance after low-temperature stress of ginseng roots.

Test Material	Subordinative Function Value						Cold Resistance Comprehensive Evaluation Value	Order
Number	Soluble Protein	Soluble Sugar	Sucrose	Proline	MDA	SOD
Y2ES	0.22	0	0.25	0.41	0.74	0	0.17	6
Y2SW	0.14	0.76	1	0.16	0.56	0	0.27	4
Y2LQ	0	1	0	0.68	0.57	0	0.22	5
Y4ES	0.82	0.7	0.59	1	1	0	0.80	2
Y4SW	1	0.72	0.19	0	0.43	0	0.94	1
Y4LQ	0.54	0.46	0.55	0.99	0	0	0.52	3

Note: Y2 refers to 2-year-old ginseng seedlings and Y4 refers to 4-year-old ginseng seedlings.

).

3.5. Analysis of the Ginsenoside Content in Ginseng Roots of Different Ages

This study utilized established UPLC–QTOF/MS to analyze ginsenosides in 2-year-old and 4-year-old ginseng roots, identifying significant variations in 28 PPT-type (protopanaxatriol), 16 PPD-type (protopanaxadiol), 1 OT-type (ocotillol), and 3 OA-type (oleanolic acid) ginsenosides.

In the 2-year-old ginseng roots, both increases and decreases in soil moisture significantly enhanced the total ginsenoside accumulation. The control group showed that the low soil moisture group (ES, 20–30%) exhibited a significantly higher total ginsenoside content than the medium (SW, 40–50%) and high (LQ, 60–70%) soil moisture groups, with the LQ group content also surpassing the SW group. In the treatment groups, the LQ group displayed a significantly higher total ginsenoside content than the ES and SW groups. In the ES group (20–30%), treatment groups demonstrated markedly elevated levels of the PPT-G-G-R-Ace_1, 13.35@PPT-G-G-G, PPT-G-X_1, PPD-20R-Rg2, and OT-G-R ginsenosides compared to the controls, with increases of 2.0-fold, 2.01-fold, 3.85-fold, 3.08-fold, and 1.59-fold, respectively. In the SW group (40–50%), treatment groups showed significant increases in OT-G-R (1.62-fold), PPT-G-G-R-Ace_1 (1.58-fold), 13.35@PPT-G-G-G (1.55-fold), PPT-G-X_1 (1.24-fold), and PPD-20R-Rg2 (1.61-fold). In the LQ group (60–70%), treatment groups exhibited elevated levels of OT-G-R (2.33-fold), PPT-G-G-R-Ace_1 (2.77-fold), 13.35@PPT-G-G-G (1.72-fold), PPT-G-X_1 (2.02-fold), and PPD-20R-Rg2 (1.37-fold). Notably, the five monomeric ginsenosides—PPT-G-G-R-Ace_1, 13.35@PPT-G-G-G, PPT-G-X_1, PPD-20R-Rg2, and OT-G-R—showed significant increases across all soil moisture groups under low-temperature treatment, indicating that their accumulation was more influenced by cold stress than by soil moisture stress (

Table 2. The fold changes in ginsenoside percentages across different soil moisture groups under low-temperature stress.

Ginsenoside	Ginsenoside Retention Time, min	Two-Year-Old Ginseng Roots			Four-Year-Old Ginseng Roots
Group		ES/CK	SW/CK	LQ/CK	ES/CK	SW/CK	LQ/CK
08.11@PPT-G-G-G	8.11	0.08	0.09	0.12	0.81	0.37	0.37
PPT-G-G-X_2	8.59	0.41	0.37	0.54	0.41	0.44	0.34
PPT-G-G-X@Noto_R1	8.70	0.03	0.12	0.03	9.89	1.55	4.50
PPT-G-G-R-X	8.88	0.33	0.25	0.57	0.42	0.63	0.37
PPT-G-G-But_1	9.66	0.13	0.12	0.14	0.39	0.24	0.18
PPT-G-G-R-Ace_1	9.67	2	1.58	2.77	0.44	0.47	0.53
PPT-G-G-R-36_2	9.82	0.70	0.63	0.90	0.67	0.59	0.52
PPT-G-G-R@GinsenoNJ_1	9.83	0.67	0.60	0.88	0.63	0.53	0.45
PPT-G-G-R_1	9.84	0.53	0.48	0.91	0.42	0.48	0.41
PPT-G-G-R-Ace_2	9.84	0.61	0.42	0.71	0.53	0.37	0.30
841@PPT-G-G-Ace_2	11.67	0.07	0.07	0.12	0.36	0.24	0.27
887@PPT-G-G-Ace_2	11.67	0.12	0.12	0.13	0.79	0.51	0.43
PPT-G-G-Mal_2	11.70	0.14	0.14	0.14	0.83	0.56	0.48
PPT-G-G-But-Mal_2	11.70	0.13	0.13	0.14	0.78	0.53	0.45
PPT-G-G-diMal_3	11.70	0.10	0.10	0.12	0.82	0.55	0.49
PPT-G-R-X	12.54	0.75	0.76	1.09	0.63	0.83	0.55
13.35@PPT-G-G-G	13.35	2.01	1.55	1.72	0.42	0.50	0.49
PPT-G-G-R-A	13.88	2.00	1.58	2.77	0.44	0.47	0.53
PPT-G-G@GinsenosiNJ_3	14.70	0.23	0.17	0.36	0.46	0.34	0.21
PPT-G-X_1	15.98	3.85	1.24	2.02	0.45	0.30	0.47
PPT-G-R-36_1	16.62	0.59	0.52	1.14	0.46	0.60	0.42
PPT-G-G-G-G-A	19.50	0.18	0.20	0.42	1.70	1.78	1.58
PPT-G-R-B	16.62	0.59	0.52	1.13	0.43	0.55	0.37
PPT-G-R	16.63	0.75	0.76	1.09	0.63	0.83	0.55
PPT-G-G-Ace_6	16.63	0.59	0.52	1.15	0.44	0.58	0.40
PPT-G-G-R-36_5	16.63	0.38	0.37	1.34	0.36	0.29	0.58
PPT-G-G-G-X_4	20.11	0.49	0.35	0.62	0.11	0.15	0.20
PPT-G-G-R-36_6	20.11	0.47	0.35	0.63	0.11	0.15	0.17
PPD-20S-Rg2	16.62	0.58	0.51	1.12	0.53	0.55	0.38
PPD-20R-Rg2	17.00	3.08	1.61	1.37	1.12	1.13	1.39
1033@PPD-G-G-G-Ace_1	20.40	0.29	0.30	0.50	0.17	0.21	0.23
PPD-G-G-G-X-Mal_3	19.19	0.13	0.19	0.32	0.36	1.08	0.42
PPD-G-G-G-X-But-NJ_17	19.19	0.12	0.18	0.32	0.35	1.06	0.42
PPD-G-G-G-X-But-NJ_18	19.19	0.14	0.19	0.33	0.50	1.19	0.43
987@PPD-G-G-G-Ace_1	20.40	0.22	0.21	0.57	0.11	0.15	0.19
PPD-G-G-G@GinsenoNJ_23	20.09	0.46	0.35	0.61	0.10	0.14	0.15
PPD-G-G-G-B-B	20.10	0.47	0.34	0.63	0.10	0.13	0.15
PPD-G-G-G-But_1	20.11	0.48	0.35	0.61	0.10	0.14	0.15
PPD-M	20.41	0.31	0.32	0.52	0.20	0.23	0.27
PPD-G-G-G-B-B-B-M	20.41	0.31	0.31	0.50	0.20	0.21	0.28
PPD-G-G-G-Mal_1	20.40	0.29	0.30	0.49	0.17	0.21	0.23
PPD-G-G-G-Mal_4	21.69	0.36	0.34	0.47	0.39	0.37	0.47
PPD-G-G-X	22.66	7.80	0.92	3.50	0.57	0.81	1.11
PPD-G-G-X-Mal_2	22.77	0.39	0.29	0.51	0.45	0.56	0.40
OT-G-R	9.06	1.59	1.62	2.33	0.82	1.16	0.68
OA-Ga-G-G@GinsenoNJ_6	18.15	0.05	0.07	0.13	0.90	0.46	0.60
OA-GlcA-G-G-But-NJ_25	20.40	0.29	0.31	0.50	0.19	0.22	0.26
OA-GlcA-G-G-But-NJ_26	20.40	0.28	0.28	0.47	0.19	0.20	0.28
Total saponins		0.39	0.37	0.59	0.46	0.40	0.36

Note: the control group (CK) represents plants that were not subjected to low-temperature treatment and the treatment group represents plants that were exposed to low-temperature stress and then allowed to recover and grow at normal temperature until harvest. The ginsenosides in green represent those shared by the three soil moisture groups of 2-year-old ginseng, while those in gray indicate the common ginsenosides among the three soil moisture groups of 4-year-old ginseng. Values less than 1 signify lower content after low-temperature treatment compared to the control group, whereas values greater than 1 denote a higher content after low-temperature exposure relative to the control.

).

In the 4-year-old ginseng roots, both the control and treatment groups showed that the total ginsenoside content in the ES group (20–30%) was significantly higher than in the SW (40–50%) and LQ groups (60–70%), with the LQ group (60–70%) also surpassing the SW group (40–50%). In the ES group (20–30%), the treatment group exhibited significantly higher levels of the PPT-G-G-X@Note_R1, PPD-20R-Rg2, and PPT-G-G-G-G-A ginsenosides compared to the control group, with increases of 9.89-fold, 1.12-fold, and 1.70-fold, respectively. In the SW group (40–50%), the treatment group showed significant increases in the PPT-G-G-X@Note_R1 (1.55-fold), PPD-20R-Rg2 (1.13-fold), and PPT-G-G-G-G-A (1.78-fold) ginsenosides. In the LQ group (60–70%), the treatment group displayed elevated levels of PPT-G-G-X@Note_R1 (4.50-fold), PPD-20R-Rg2 (1.39-fold), and PPT-G-G-G-G-A (1.58-fold) compared to the controls. Both increases and decreases in soil moisture significantly enhanced the total ginsenoside accumulation in the 4-year-old ginseng roots. However, cold stress markedly reduced the total ginsenoside accumulation. Notably, PPT-type ginsenosides were significantly elevated under cold stress in both the low soil moisture (ES, 20–30%) and high soil moisture (LQ, 60–70%) groups (Table 2 and Figure 7).

4. Discussion

The fold changes in ginsenoside percentages under cold stress across different soil moisture groups were analyzed. Water and temperature stresses are critical abiotic factors limiting plant growth, and these stresses often co-occur in nature, generating additive effects that inhibit plant growth and reduce quality. This study investigated the impacts of combined soil moisture and cold stress on the physiology and ginsenoside accumulation in ginseng of different ages, aiming to elucidate its adaptive response mechanisms. Cold stress induces reactive oxygen species (ROS) accumulation, and the enhanced activity of SOD, a key antioxidant enzyme, represents a crucial strategy for plants to counteract oxidative stress [24]. Combined cold and high soil moisture stress significantly increased the SOD activity in 2-year-old and 4-year-old ginseng roots, while combined cold and low soil moisture stress markedly reduced the SOD activity. In the 2-year-old ginseng, the soluble protein content decreased significantly in both low- and high-moisture groups under combined stress, aligning with Li et al.’s findings on the sensitivity of SOD and soluble proteins to cold/high-moisture and cold/low-moisture combined stresses [25]. However, the lack of consistent increases in the SW group suggests that responses may depend not only on cold stress, but also on moisture constraints. MDA, a marker of lipid peroxidation, serves as a critical indicator of oxidative damage, reflecting the extent of biomembrane injury [26]. In the 4-year-old ginseng, the MDA content increased significantly under combined stress, indicating that these dual stressors promote free radical formation and adversely affect cell membranes. Osmotic regulators, such as soluble sugars, soluble proteins, and proline, play vital roles in protecting plants against cold stress by reducing osmotic pressure and preserving cellular integrity [23,27]. Notably, the 2-year-old ginseng exhibited distinct response patterns under combined stress: compared to single moisture stress, the addition of cold stress to varying moisture gradients led to a significant decline in the soluble protein content but a marked increase in proline levels, suggesting that combined stress exerts more severe effects than isolated moisture stress, consistent with Kanchan et al.’s observations [28].

Furthermore, soluble carbohydrates such as sucrose not only contribute to osmotic adjustment, but also protect membrane integrity and enhance antioxidant capacity [29]. As cold stress duration increased, ginseng accumulated osmotic regulators to maintain cellular stability, though response patterns differed between plant ages. The four-year-old ginseng demonstrated stronger environmental adaptability, with the medium soil moisture group (SW) exhibiting optimal cold resistance, likely due to mature root structural features such as well-developed lignified tissues and aerenchyma [30,31]. In contrast, the 2-year-old plants were more sensitive to combined stress, showing less efficient osmotic regulation and antioxidant responses, highlighting the critical influence of developmental stage on stress resilience.

The production and accumulation of secondary metabolites in ginseng are influenced by temperature conditions, and changes in the ginsenoside content exhibit certain correlations with temperature. Xie et al. reported that temperature was significantly negatively correlated with major ginsenosides (Rg1, Re, and Rb1), while moderate water stress positively regulated ginsenoside accumulation [32]. In this study, both low and high soil moisture treatments increased the total ginsenoside content. Yang et al. [33] demonstrated that temperature was significantly negatively correlated with the levels of ginsenosides Rb1 and Rd (p < 0.05), and although negative correlations were observed with other ginsenosides, they did not reach statistical significance. Zhang et al. used fresh roots of 5-year-old ginseng and found that high moisture treatment enhanced the content of PPT-type ginsenosides (Rg1, Rf, Rg2, Rh1) [34]. In the current study, although cold stress generally suppressed ginsenoside synthesis, the high soil moisture group promoted PPT-type ginsenoside biosynthesis. Short-term water stress may trigger oxidative stress in fresh ginseng roots, stimulating the antioxidant defense system and driving the synthesis of secondary metabolites.

5. Conclusions

This study investigated the response mechanisms of two-year-old and four-year-old ginseng root systems to low-temperature stress under different soil moisture conditions, as well as the variation patterns of their medicinal components. The key findings are as follows. (1) Among the two-year-old ginseng roots, the low/medium soil moisture groups exhibited the most significant membrane lipid peroxidation damage (the highest MDA content) under 48 h of low-temperature exposure. Conversely, the high soil moisture group delayed membrane damage by increasing the SOD activity and simultaneously enhancing osmotic substances to adapt to the low-temperature environment. However, this group was unable to maintain the level of osmotic adjustment substances as the low-temperature stress continued. (2) Among the four-year-old ginseng roots, the medium soil moisture group maintained the highest SOD activity and levels of osmotic adjustment substances (soluble proteins, proline). The high soil moisture group, on the other hand, demonstrated a stronger low-temperature tolerance than the two-year-old plants by continuously accumulating osmotic substances to resist low-temperature damage. (3) Soil moisture regulation exhibited a dual effect on saponin metabolism: moderate increases or decreases in soil moisture could promote the accumulation of total saponins, but low-temperature stress inhibited this process overall. It is worth noting that the high soil moisture group significantly increased the PPT-type saponin content after low-temperature treatment, providing a new insight for targeted regulation of medicinal components. This study holds significant implications for ginseng cultivation management. For four-year-old cultivations, adopting medium soil moisture management yields the best cold resistance effects. Two-year-old plants should avoid low soil moisture conditions to prevent exacerbated membrane system damage. In medicinal production, implementing phased high soil moisture treatment can enhance the quality of medicinal materials by utilizing its ability to induce PPT-type saponin accumulation. These discoveries provide a theoretical basis for establishing a precise water management model based on growth duration and formulating climate-adaptive cultivation strategies.