Physiological and Biochemical Measurements Reveal How Styrax japonica Seedlings Response to Flooding Stress

Abstract

This study investigated the physiological and biochemical responses of Styrax japonica Sieb. et Zucc. seedlings to normal water and nutrient management (control group, CK), waterlogging (root submerged, T1), and partial submergence (partial stem submergence, T2) over a period of 25 days. Measurements of root activity, malondialdehyde (MDA) content, hydrogen peroxide (H₂O₂) content, antioxidant enzyme (SOD, POD, and CAT), and anaerobic respiratory enzyme (LDH, ADH, and PDC) activities were conducted every 5 days. The results showed the following: the seedlings of the control group maintained high root activity and low oxidative damage levels throughout the experiment; under T1 treatment, root activity initially increased but declined, while MDA and H₂O₂ content gradually increased; T2 seedlings showed little change initially, but root activity briefly increased at Day 20 before rapidly declining. Regarding the antioxidant system, the control group had the highest SOD activity, while seedlings under T1 and T2 treatments exhibited compensatory upregulation of CAT and POD activities (from Day 15 to 20). Additionally, under waterlogging stress, LDH and ADH activities significantly increased, reflecting the activation of anaerobic metabolic pathways, while PDC content continuously decreased, indicating that low-oxygen stress induced the accumulation of LDH and ADH but reduced ethanol fermentation. PCA revealed that the first two principal components explained 61.53% of the total variation, with PC1 (45.76%) reflecting the contrasting relationship between the activation of anaerobic metabolism (increased ADH and LDH activity) and decreased root activity under waterlogging stress, while PC2 (15.77%) primarily captured the responses of oxidative damage (increased MDA) and corresponding antioxidant defense (upregulated CAT and POD activities). Overall, S. japonica seedlings adapt to short-term waterlogging stress by regulating anaerobic respiration and antioxidant systems, but prolonged stress leads to a continued increase in H₂O₂ and a decline in antioxidant enzyme activities. This study provides experimental evidence and theoretical support for understanding the waterlogging tolerance mechanisms of S. japonica. This experiment provides important information on the adaptive mechanisms of plants under waterlogging stress.

1. Introduction

Styrax japonica, a deciduous tree native to China, Japan, and Korea, is widely favored in landscaping due to its ornamental value. Its white, bell-shaped flowers bloom between May and June every year, making it a popular choice for gardens [1,2]. Furthermore, S. japonica contains a variety of compounds that have potential therapeutic uses for treating conditions such as inflammation [3]. However, research on its physiological and biochemical responses to flooding stress remains limited. Therefore, this study aims to fill this knowledge gap by exploring the tolerance mechanisms of S. japonica to flooding stress.

Waterlogging stress is one of the major environmental stresses that plants encounter during their growth and development, severely affecting agricultural and forestry production [4]. The most direct effect of waterlogging stress on plants is hypoxia [5], which triggers a series of physiological and metabolic changes, including the accumulation of reactive oxygen species (ROS) and the toxic compound malondialdehyde (MDA) [6,7].

ROS plays a critical role as signaling molecules in the normal growth and development of plants, regulating various physiological processes [8]. However, when ROS accumulate excessively, they can cause cellular damage either by directly oxidizing cellular components or indirectly activating stress pathways [9]. Furthermore, an increase in ROS levels leads to lipid peroxidation in cell membranes, resulting in altered membrane permeability, impaired ion transport, enzyme activity disturbances, and protein cross-linking [10,11]. Therefore, maintaining a dynamic balance between ROS production and scavenging is crucial for plant survival. Excessive H₂O₂, as a form of ROS, can activate the plant’s antioxidant defense system; however, when its concentration is too high, it can also cause damage to plants, such as inhibiting photosynthesis, disrupting mitochondrial function, and compromising membrane integrity [12,13]. MDA, as a product of lipid peroxidation, is commonly used as an indicator of oxidative stress levels in plants. An increase in MDA content typically indicates significant oxidative damage to the plant [14,15].

To reduce the accumulation of toxic substances and maintain a dynamic balance of ROS content, plants activate a series of response mechanisms, including enhancing the antioxidant enzyme system, adjusting metabolic pathways, and synthesizing non-enzyme antioxidants to mitigate oxidative damage. Superoxide dismutase (SOD), as the first line of defense against ROS, catalyzes the dismutation of superoxide anion (O₂⁻) into H₂O₂ and oxygen, thereby reducing the toxicity of superoxide anions. Peroxidase (POD) alleviates oxidative damage by scavenging H₂O₂ and strengthens the cell wall to improve plant stress resistance, while catalase (CAT) decomposes H₂O₂ into water and oxygen, significantly reducing the toxic effects of H₂O₂ on cells [16,17].

Under waterlogging stress, due to the lack of oxygen, plants gradually shift from aerobic respiration to anaerobic respiration to maintain cellular energy supply. Pyruvate decarboxylase (PDC), alcohol dehydrogenase (ADH), and lactate dehydrogenase (LDH) are key enzymes in anaerobic metabolism, involved in the decarboxylation of pyruvate to acetaldehyde, the reduction of acetaldehyde to ethanol, and the conversion of pyruvate to lactate [18]. Studies have shown that under waterlogging conditions, the activity of these three enzymes increases significantly [19,20], thereby enhancing the ethanol fermentation and lactate fermentation pathways to help plants adapt to hypoxic environments [21]. Thus, the coordinated action of these enzymes is crucial for the development of waterlogging tolerance in plants.

Plants are subjected to different types of waterlogging stress based on the depth of submergence and its effects. Waterlogging refers to root submersion, leading to root hypoxia. Submergence refers to the complete or near-complete submersion of the plant [22]. Partial submergence refers to the submersion of part of the plant’s stem, which increases the oxygen transport distance from the stem to the roots and reduces oxygen concentration at the stem-root junction [23]. This study focused on waterlogging and partial submergence stress conditions, investigating the adaptive mechanisms of S. japonica seedlings’ roots under different waterlogging stress conditions through systematic analysis of various physiological indicators, providing a reference for the cultivation and management of waterlogging-tolerant plants.

In this study, principal component analysis (PCA), a method less commonly used to investigate the effects of flooding stress on plants, was applied to reveal the complex interactions among various physiological responses under flooding stress. PCA helped identify key physiological indicators with significant impacts during the flooding process of Jasminum sambac and revealed the relationships between root activity, oxidative stress markers, antioxidant enzyme activity, and anaerobic respiration enzyme activity. By analyzing the temporal changes of these indicators, this study provides a new perspective for a more comprehensive understanding of plant adaptive responses to flooding stress.

2. Materials and Methods

2.1. Experimental Location and Materials

The experiment was conducted at a forest farm in Jurong County, Zhenjiang City, Jiangsu Province, China. The seeds of S. japonica were collected from the planting base of Nanjing Yangzi Jasmine Valley Cultural and Technology Co., Ltd. in Liuhe District, Nanjing, China. In November 2023, the collected seeds underwent cold stratification, and in March 2024, the seeds were sown in 32-cell trays after dormancy was broken. Once the seedlings reached an average height of 15 cm, they were transplanted into nursery pots measuring 14 cm × 12 cm (diameter × height) for the subsequent waterlogging experiment. The nursery substrate used was a uniform mixture of general-purpose organic nutrient soil (Jiangsu Peilei Substrate Technology Development Co., Ltd., Zhenjiang, China), loess, and perlite (V:V:V = 5:3:2). After transplantation, seedlings were managed with normal water and nutrient management.

2.2. Experimental Design

The waterlogging experiment began when the S. japonica seedlings grew to approximately 25 cm in height (early July 2024). A double-pot waterlogging method was used, with the waterlogging containers being turnover boxes measuring 70 cm × 45 cm × 18 cm (length × width × height). Three moisture gradient treatments (Figure 1) were set in this experiment, namely, the control group (CK, normal water, and nutrient management), the waterlogging group (water level 2 cm below the soil surface, submerging the roots, T1), and the submergence group (water level 4 cm above the soil surface, submerging part of the plant’s above-ground tissues, T2). Each treatment included 45 seedlings, and observations and sampling were conducted at five time points: 5 days, 10 days, 15 days, 20 days, and 25 days after waterlogging. The experiment was conducted outdoors, with the treatments placed in an open field. To ensure the integrity of the experiment during adverse weather conditions, such as rain, the plants were moved into a greenhouse when necessary. Once the weather improved, the plants were returned to the open field. All three treatment groups (CK, T1, T2) were subjected to the same experimental conditions, with each treatment group being exposed to identical environmental conditions (e.g., temperature, light, and humidity). This approach ensured that the environmental conditions were consistent across treatments, minimizing the impact of external factors on the results.

Each treatment group used five turnover boxes, each measuring 70 cm × 45 cm × 18 cm, with nine potted plants placed in each box. At sampling times on days 5, 10, 15, 20, and 25, nine plant samples were randomly selected from different boxes for analysis at each time point. During the experiment, water lost from the turnover boxes was replenished daily to maintain consistent water levels. For each sampling, 9 randomly selected seedlings were used, and the third to fifth leaves from the top down were collected along with the entire root system. After sampling, root activity was immediately measured, and the remaining samples were stored in a −80 °C refrigerator until use. All measurements were performed on fresh samples at the time of analysis. During root collection, the entire root system was carefully extracted using a small shovel, with efforts made to minimize physical damage to the roots. The roots were then washed gently with slow-running water to remove attached soil and impurities. Immediately after each sampling, root vitality was measured. Fine roots were used as samples for root vitality measurements in all treatment groups. Root volume and mass were not specifically considered in the experimental design, but efforts were made to minimize interference from treatment procedures on the roots to ensure the accuracy of the measurement results.

2.3. Measurement of Indicators

Root activity was measured using the TTC method, as follows: Root tips (0.2 g) were incubated with 5 mL of 1% TTC solution and 5 mL of 0.1 mol·L⁻¹ phosphate buffer at 37 °C for 1 h. The reaction was stopped by adding 2 mL of 1 mol·L⁻¹ H₂SO₄. The roots were ground in ethyl acetate to extract formazan. Absorbance at 485 nm was measured to determine TTC reduction; MDA content was determined using the thiobarbituric acid (TBA) method, as follows: 0.2 g of root tissue was weighed and placed into a mortar in an ice bath. Then, 10 mL of 0.05 mol·L⁻¹ phosphate buffer was added and the mixture was ground into a homogenate. The homogenate was transferred to a test tube; 5 mL of 0.5% thiobarbituric acid was added to the extract and mixed thoroughly. The test tube was placed into a boiling water bath and heated for 10 min. Afterward, the test tube was removed and allowed to cool completely in cold water. Then, the test tube was centrifuged at 3000 rpm for 15 min. The supernatant was collected and its volume was measured. We used 0.5% thiobarbituric acid solution as the blank and measured absorbance at 532 nm, 600 nm, and 450 nm. H₂O₂ content was measured by colorimetry, using an H₂O₂ kit (Shanghai Jining Biotechnology Co., Ltd., Shanghai, China), with absorbance at 450 nm. SOD activity was determined by the NBT method, using a SOD kit (Suzhou Keming Biotechnology Co., Ltd., Suzhou, China), according to the instructions, and absorbance was measured at 560 nm. POD activity was determined by the guaiacol method using a POD kit (Shanghai Jining Biotechnology Co., Ltd.), and absorbance was measured at 470 nm. CAT activity was determined by ultraviolet spectrophotometry using a CAT kit (Shanghai Jining Biotechnology Co., Ltd.) and absorbance was measured at 240 nm. LDH activity was measured by visible spectrophotometry using an LDH kit (Shanghai Jining Biotechnology Co., Ltd.) according to the tetrazolium salt color reaction method, with absorbance measured at 450 nm. ADH activity was determined by ultraviolet spectrophotometry using an ADH kit (Shanghai Jining Biotechnology Co., Ltd.), based on the NADH/NAD⁺ change method, with absorbance measured at 340 nm. PDC activity was determined by ultraviolet spectrophotometry using a PDC kit (Shanghai Jining Biotechnology Co., Ltd.), based on the NADH oxidation method, with absorbance measured at 340 nm.

2.4. Data Analysis

Statistical analysis of experimental data was performed using one-way analysis of variance (ANOVA) to test for significant differences between the different treatments. The significance level was set at p < 0.05, and differences between groups were compared using Duncan’s multiple range test. Data analysis was completed using SPSS 26.0 software. For the differences in indicators between different treatment groups, bar charts were created using Origin 2021 to visually display significant differences between the groups. Significant differences were indicated by different letters in the figures. All data presented are mean values and standard errors (SEs). To comprehensively evaluate and compare the effects of different treatments on S. japonica seedlings’ responses, principal component analysis (PCA) was performed using R language version 4.4.2. All measured indicators (root activity, MDA and H₂O₂ content, SOD, POD, CAT, LDH, ADH, and PDC activity) were included in the analysis.

3. Results and Analysis

3.1. Root Activity

Throughout the experiment (5 d, 10 d, 15 d, 20 d, 25 d), the root activity in the control group (CK) was consistently significantly higher than in the other treatment groups. On Day 5, T1 exhibited significantly higher root activity than T2. However, at subsequent time points (10 d, 15 d, 20 d, and 25 d), there were no significant differences between T1 and T2.

The root activity in CK peaked on Day 20, increasing by approximately 36% compared to Day 5, and then slightly decreased on Days 20 and 25. T1 showed high root activity in the early stages of the experiment (Day 5), which gradually declined at Days 10 and 15 (decreasing by approximately 23% and 42%, respectively). It slightly recovered on Day 20 (an increase of about 68% compared to Day 15), but declined again on Day 25 (a decrease of about 22% compared to Day 20). Overall, T1 exhibited considerable fluctuations in root activity, showing a significant decline followed by a slight recovery.

In T2, root activity remained relatively low and stable from the early to middle stages of the experiment (Day 5 to Day 15). On Day 20, there was a significant increase (approximately 137% compared to Day 15), but it decreased again on Day 25 (a decrease of about 55% compared to Day 20). T2 showed little variation in the early stages, with a noticeable increase at Day 20, but this trend did not persist in the later stages (Figure 2).

3.2. Lipid Peroxidation Products

3.2.1. MDA Content

In the control group (CK), the MDA content gradually decreased from Day 5 to Day 20 (a decrease of approximately 61.5%), with a slight recovery by Day 25. Overall, the change was minimal, and on Day 20, the MDA content in CK was significantly lower than at other time points, indicating a low level of oxidative damage.

In T1, the MDA content gradually increased from Day 5 to Day 20 (an increase of approximately 73.9%), peaking on Day 20 (0.0233 µmol/g), then slightly decreased by Day 25. The significant increase on Day 20 indicates the cumulative effect of oxidative damage under T1 stress.

In T2, the overall variation in MDA content was smaller, but there was a slight increase on Day 20 (0.0139 µmol/g), which then stabilized. Overall, CK maintained lower and more stable MDA content, indicating the lowest level of oxidative damage. T1 exhibited the highest peak and fluctuation in MDA content, reflecting greater sensitivity to prolonged waterlogging stress. Meanwhile, T2 showed less fluctuation in MDA content (Figure 3).

3.2.2. H₂O₂ Content

Throughout the experiment (5 d, 10 d, 15 d, 20 d, 25 d), the H₂O₂ content in T2 and T1 was consistently significantly higher than in the CK group. Compared to the other two groups, the H₂O₂ content in CK showed a slight increase over the entire experiment, rising from 2.61 μmol/g on Day 5 to 5.55 μmol/g on Day 25.

As shown in Figure 4, in T1, the H₂O₂ content gradually increased from 4.30 μmol/g on Day 5 to 15.70 μmol/g on Day 25, with a total increase of 265.12%. Between Days 10 and 20, the H₂O₂ content continued to rise, with Day 20 showing an increase of 68.46% compared to Day 15. On Day 25, there was a slight increase, but it was small (an increase of 8.61% compared to Day 20).

In T2, the H₂O₂ content steadily increased throughout the experiment, from 4.84 μmol/g on Day 5 to 16.68 μmol/g on Day 25, with a total increase of 244.21%. The increase was more significant on Days 10 and 15 (78.93% and 32.85%, respectively), with a slower increase on Day 20 (5.45% compared to Day 15), and it peaked on Day 25 (a 37.43% increase compared to Day 20).

3.3. Antioxidant Enzyme Activity

3.3.1. SOD Activity

The SOD activity in CK was the highest (84.48 U/g), significantly higher than in T1 (30.22 U/g) and T2 (32.35 U/g), with no significant difference between T1 and T2. As waterlogging continued, the SOD activity in T1 and T2 significantly increased compared to Day 5, with increases of 56.5% and 43.3%, respectively, on Day 10. Despite this, CK still had significantly higher SOD activity than both T1 and T2. On Day 15, the SOD activity in T1 (42.73 U/g) was significantly lower than in CK but higher than in T2 (31.24 U/g).

From Day 20 to Day 25, the SOD activity in all treatment groups decreased, and the differences between the groups were not significant. On Day 20, CK had a value of 43.30 U/g, while T1 and T2 had values of 37.19 U/g and 37.04 U/g, respectively. By Day 25, the SOD activity in all groups further decreased. CK had a value of 30.97 U/g, a decrease of 28.5% compared to Day 20. The SOD activity in T1 (19.37 U/g) was significantly lower than in the waterlogging group (27.59 U/g), and both were significantly lower than CK (Figure 5).

3.3.2. POD Activity

Throughout the waterlogging period, the POD activity in T1 was consistently higher than in CK and T2, while the POD activity in T2 significantly decreased in the later stages of the experiment.

On Day 5, the POD activity in T1 was 430 U/g, higher than CK (290 U/g) and the waterlogging group (320 U/g), although the differences between the three groups were not significant. On Day 10, the POD activity in T2 significantly increased to 410 U/g, while T1 had a POD activity of 386.67 U/g, slightly lower than on Day 5, but the difference between the two was not significant. The POD activity in CK remained at 286.67 U/g, which was nearly the same as Day 5.

On Day 15, the POD activity in T1 significantly increased to 510 U/g, while T2’s POD activity was 486.67 U/g, which was an increase of 18.68% compared to Day 10. The POD activity in CK was 293.33 U/g, which was significantly lower than in T1.

On Day 20, the POD activity in T1 further increased to 606.67 U/g, the peak value during the experiment. The POD activity in CK also significantly increased to 460 U/g (a 56.83% increase compared to Day 15). T2’s POD activity was 580 U/g, slightly lower than T1’s, but the difference was not significant.

On Day 25, the POD activity in T1 decreased to 520 U/g, while the POD activity in CK decreased to 423.33 U/g (a 7.97% decrease compared to Day 20). T2’s POD activity significantly decreased to 193.33 U/g (a 66.67% decrease compared to Day 20), which was significantly lower than both T1 and CK (Figure 6).

3.3.3. CAT Activity

Throughout the experiment (5 d, 10 d, 15 d, 20 d, 25 d), the CAT activity in T1 and T2 was consistently higher than in CK (p < 0.05). Although the CAT activity in CK significantly increased over time, its value was consistently significantly lower than in T1 and T2, particularly in the later stages of the experiment (Day 20 and Day 25), where the differences were more pronounced.

On Day 5, the CAT activity in T2 was the highest (102.63 µmol/min/mL), significantly higher than in T1 (94.08 µmol/min/mL) and CK (66.99 µmol/min/mL). There was no significant difference between T1 and CK.

On Day 10, the CAT activity in T2 and T1 was 163.93 µmol/min/mL and 162.50 µmol/min/mL, respectively, with no significant difference between the two, but both were significantly higher than in CK (81.25 µmol/min/mL). On Day 15, the CAT activity in T2 significantly increased to 209.54 µmol/min/mL, an increase of about 27.83% compared to Day 10, marking the highest value among all treatments. T1’s CAT activity remained relatively unchanged (162.50 µmol/min/mL), significantly higher than in CK (82.68 µmol/min/mL).

On Day 20, the CAT activity in T1 significantly increased to 256.58 µmol/min/mL (a 57.88% increase compared to Day 15), while the CAT activity in T2 increased to 219.52 µmol/min/mL (a 4.77% increase compared to Day 15). Both values were significantly higher than in CK (86.95 µmol/min/mL).

On Day 25, the CAT activity in all treatment groups reached their respective peak values. The CAT activity in T2 was the highest, at 292.21 µmol/min/mL (a 32.97% increase compared to Day 20), while T1 had a value of 276.54 µmol/min/mL (a 7.78% increase compared to Day 20). CK significantly increased to 175.33 µmol/min/mL (a 101.58% increase compared to Day 20), but it remained significantly lower than the other two groups (Figure 7).

3.4. Anaerobic Respiratory Enzyme Activity

3.4.1. LDH Activity

On Day 5, the LDH activity in T2 was the highest (302.22 nmol/min/g), significantly higher than in CK (169.11 nmol/min/g) and T1 (104.62 nmol/min/g).

By Day 15, T1 showed a sharp increase to 510.75 nmol/min/g, significantly higher than CK (224.72 nmol/min/g) and T2 (400.83 nmol/min/g). In the early stages (Day 5 to Day 10), the LDH activity in CK remained stable, increasing by approximately 32.8% from Day 10 to Day 15, and further rising by about 31.7% from Day 15 to Day 20. However, from Day 20 to Day 25, it decreased by nearly 38.6%. This decline suggests that, under normal water and nutrient management, cellular metabolism may weaken in the later stages due to natural decline or adaptive regulation.

In the T1 group, LDH activity increased by more than 100% from Day 5 to Day 10, and by approximately 140% on Day 15 compared to Day 10. It then decreased by approximately 25.5% on Day 20 and by approximately 33.2% on Day 25.

In T2, the LDH activity started at a relatively high level on Day 5 and gradually increased from Day 10 to Day 20 (an increase of about 23.3% from Day 5 to Day 10, 7.5% from Day 10 to Day 15, and 12.7% from Day 15 to Day 20). By Day 25, the activity decreased by about 17.9%. Under T2 conditions, root metabolism was more active and relatively stable, but after prolonged stress, there was some functional decline (Figure 8).

3.4.2. ADH Activity

In CK, the ADH activity increased from 3005.35 nmol/min/g on Day 5 to 3641.58 nmol/min/g by Day 15 (a 21.1% increase), and then slightly decreased to 3541.35 nmol/min/g on Day 20, further decreasing to 3471.67 nmol/min/g on Day 25 (a 2.7% decrease compared to Day 20). Overall, CK exhibited a relatively stable change, with the anaerobic metabolism level remaining stable under normal water and nutrient management conditions.

In T1, the ADH activity increased from 3709.12 nmol/min/g on Day 5 to 4178.12 nmol/min/g on Day 10 (a 12.6% increase). On Day 15, it further rose (a 9.8% increase compared to Day 10), reaching its peak on Day 20 at 5667.13 nmol/min/g (a 23.7% increase compared to Day 15). However, on Day 25, it sharply declined to 2819.90 nmol/min/g (a 50.3% decrease compared to Day 20). The waterlogging treatment showed a significant increase in ADH activity in the middle stage, which may reflect a marked enhancement in anaerobic metabolism. However, prolonged hypoxia likely led to metabolic disturbances, causing a significant decrease in the later stages.

In T2, the ADH activity increased from 3547.25 nmol/min/g on Day 5 to 4152.39 nmol/min/g on Day 10 (a 17.0% increase), and then dramatically increased to 6664.62 nmol/min/g on Day 15 (a 60.6% increase compared to Day 10). By Day 20, the activity remained relatively stable at 6623.89 nmol/min/g (with small changes), but it decreased to 3499.54 nmol/min/g on Day 25 (a 47.3% decrease compared to Day 20). T2 showed the highest ADH activity in the middle stages, suggesting that under T2 conditions, the cells may better maintain energy supply through enhanced ethanol fermentation. However, prolonged stress still led to a decline in anaerobic respiration function (Figure 9).

3.4.3. PDC Activity

Throughout the experiment, the PDC activity in CK was significantly higher than in T1 and T2, with the differences between groups reaching statistical significance at all time points except Day 5. This suggests that, under non-stress conditions, the root system is able to maintain high metabolic activity without being limited by external environmental factors.

The PDC activity in T1 and T2 was generally significantly lower than in CK, with some differences observed between the two treatment groups. On Day 5, the PDC activity in T2 was slightly higher than in the waterlogging group, but the difference was not significant.

At subsequent time points (Day 10 to Day 25), the PDC activity in both T1 and T2 gradually decreased. Between Days 10 and 20, the PDC enzyme activity in T1 was slightly higher than in the waterlogging group. By Day 25, the PDC activity in T1 had decreased to 47% of its initial value, while T2’s PDC activity decreased to 49%.

In CK, the PDC content gradually increased over time, rising from its initial value on Day 5 to the final value on Day 25, with a cumulative increase of 62%. This gradual increase indicates that, under suitable conditions, the metabolic activity of the plant roots continues to enhance (Figure 10).

3.5. PCA Analysis

3.5.1. Analysis Process

Principal component analysis (PCA) was used to analyze the relationships between physiological and biochemical indicators under waterlogging stress and their influence on the adaptation of S. japonica seedlings. Using the statistical software R 4.4.2, the measured physiological and biochemical indicators (root activity, MDA content, H₂O₂ content, CAT, POD, SOD activity, LDH, ADH, PDC activity) were standardized to eliminate dimensional differences. PCA was then performed to obtain the variance contribution, variable loadings, and PCA score scatter plots.

3.5.2. Analysis Results

As shown in

Table 1. Variance explanation and cumulative variance of principal component analysis.

Principal Component	Explained Variance	Cumulative Variance
PC1	0.457616	0.457616
PC2	0.157731	0.615348
PC3	0.10985	0.725198
PC4	0.092654	0.817852
PC5	0.077278	0.89513
PC6	0.043128	0.938258
PC7	0.03111	0.969368
PC8	0.029685	0.999053
PC9	0.000947	1

, the first two principal components, PC1 and PC2, explained 45.76% and 15.77% of the data variation, respectively, with a cumulative explanation of 61.53% of the total variation. This indicates that PC1 and PC2 effectively capture the main variation characteristics in the experimental data, representing the physiological responses of S. japonica under different waterlogging conditions.

The main contributing variables for PC1 include root activity (loading −0.35), SOD (loading −0.30), and metabolic enzymes (e.g., ADH 0.30, LDH 0.32). A high PC1 score reflects higher metabolic activity and lower antioxidant capacity in S. japonica, which may correspond to the T1 and T2 treatment groups.

In the negative direction of PC1 (e.g., CK)—S. japonica maintains high root activity and antioxidant capacity, with relatively stable metabolic activity. In the positive direction of PC1 (e.g., waterlogging and submergence groups)—the activity of metabolic enzymes (ADH, LDH) increases, likely as a compensatory mechanism for hypoxic stress. However, root activity decreases, reflecting reduced stress tolerance.

The main contributing variables for PC2 include MDA (loading −0.57), CAT (loading 0.43), and POD (loading 0.43). A high PC2 score indicates a higher level of oxidative stress, which is more apparent in the T2 treatment group. In the negative direction of PC2—low levels of membrane lipid peroxidation (MDA) and oxidative stress indicate that the plants are in a relatively healthy state (e.g., CK). In the positive direction of PC2—MDA levels significantly increase, and the antioxidant system (e.g., CAT and POD) may respond to stress with a compensatory response.

3.5.3. Dynamic Changes in the Temporal Dimension

As shown in Figure 11, in CK, the sample distribution remained stable, indicating that under normal water and nutrient management, there were no significant changes in the plant’s root physiological indicators. In T1: early stage (Day 5 to Day 15)—the movement in the positive direction of PC1 suggested an enhancement in plant metabolic activity, possibly as an initial compensatory mechanism in response to waterlogging stress. In the late stage (Day 20 to Day 25)—the spread in the positive direction of PC2 indicated a significant accumulation of oxidative stress. The accumulation of MDA and overload of the antioxidant system may lead to further damage to root function. In T2: early stage—the samples quickly deviated from CK, indicating that the waterlogging treatment had a more severe impact on the plant’s metabolism and antioxidant system. In the late stage: the sample distribution was further scattered, especially on Day 25, showing that sustained stress may lead to irreversible physiological damage.

Waterlogging and submergence treatments significantly reduced root vitality, while temporarily alleviating oxidative stress by enhancing the activity of metabolic enzymes (such as ADH and LDH) and antioxidant enzymes (such as CAT and POD). However, the accumulation of MDA and the overload of the antioxidant system caused by prolonged stress eventually led to severe damage to root function.

4. Discussion

Plants generally exhibit a decline in root activity under waterlogging stress [24,25,26,27]. In this study, compared to CK, the root activity in T1 and T2 was significantly lower at the early stages of waterlogging, indicating that S. japonica seedlings are quite sensitive to waterlogged environments. Plants adopt various strategies to resist waterlogging stress, and tolerant varieties can recover their roots more quickly through modifications in the structure of aerial tissues, enhanced energy metabolism, and antioxidant defenses [28,29,30]. While S. japonica lacks aerial structures, its root vitality did not continuously decline but instead showed fluctuations during the mid-waterlogging period. Previous studies have shown that under long-term waterlogging stress, plants activate anaerobic metabolic pathways and secrete antioxidant enzymes and secondary metabolites to mitigate oxidative damage caused by stress [31]. The significant increase in antioxidant enzyme (POD, CAT) activity, along with the elevated anaerobic respiration enzyme activity, indicates that S. japonica actively resists stress during waterlogging. The significant decline in root vitality at the end of waterlogging indicates that prolonged stress still leads to root function impairment [32].

MDA and H₂O₂, as indicators of membrane lipid peroxidation and reactive oxygen species (ROS), reflect the oxidative damage levels within plants [33]. In CK, MDA content remained low and stable overall, while in T1, MDA significantly increased on Day 20, suggesting that prolonged hypoxia triggered ROS accumulation and cellular damage [34]. At the same time, the H₂O₂ content in both T1 and T2 was significantly higher than in CK and continuously increased with the stress. This aligns with previous research showing that waterlogging stress leads to a continuous rise in ROS content [35,36,37].

Previous studies have shown that the changes in the activity of SOD, POD, and CAT during stress follow a certain sequence, where SOD responds quickly in the early stages, while CAT and POD continue to act during medium- and long-term stress [38]. In the early stages of waterlogging stress (Day 5), SOD activity was suppressed, while POD and CAT activity began to rise, possibly because SOD had already passed the rapid response phase to waterlogging stress. Due to the compensatory mechanism between antioxidant enzymes, the activities of POD and CAT increase [39]. With time, the SOD activity recovered, possibly because the roots and cells gradually adapted to the waterlogging environment. During the mid-phase (Days 15–20), the activity of all three enzymes peaked, indicating that the plant maximized ROS scavenging by enhancing antioxidant enzyme activity. This highlights the synergistic effect of these three antioxidant enzymes during waterlogging [40]. In the later stages (Day 25), the POD activity in T2 significantly decreased and was notably lower than in T1 and CK. CAT activity remained high, suggesting that the ROS level had exceeded the capacity of POD to clear it, leading to severe root damage and potentially rendering the antioxidant enzymes ineffective against the waterlogging stress. Over time, the activity of SOD recovers, possibly because the roots and cells gradually adapt to the flooded environment. The plant’s antioxidant system gradually becomes ineffective due to overworking and accumulated oxidative damage. Although CAT still maintains high activity, its antioxidant capacity may decrease due to overreaction, while the reduced activity of POD may indicate that the plant has entered a state of decline and is unable to maintain long-term antioxidant defense.

T1 showed higher SOD, POD, and CAT activity than CK throughout the treatment, with significant enhancement in the later stages, reflecting the strong stimulus of complete waterlogging stress on the plant’s antioxidant system. T2 had slightly lower SOD and POD activity in the early stages compared to T1, but CAT activity remained higher, likely because T2 needed to use oxygen metabolism more efficiently to resist the more severe waterlogging stress.

Principal component analysis (PCA) results showed that the root vitality and changes in antioxidant enzymes (such as SOD) of S. japonica were primarily concentrated in the early stages of waterlogging, with oxidative stress gradually increasing over time. This response is different from that of some waterlogging-tolerant plants, such as P. deltoides, which show a continuous increase in antioxidant enzyme activity during waterlogging, with H₂O₂ levels rising initially and then decreasing, demonstrating a clear antioxidant capacity [41]. In contrast, while S. japonica showed an initial increase in antioxidant enzyme activity, root vitality gradually declined as waterlogging continued, and oxidative damage progressively worsened, indicating its weaker adaptation to long-term waterlogging.

Regarding anaerobic respiration enzymes, the activities of LDH, ADH, and PDC showed significant changes in the stressed groups. Both T1 and T2 treatments caused a noticeable increase in ADH and LDH activity. This result is consistent with previous studies. For example, the root systems of Lupinus luteus (L.) seedlings showed increased ADH and LDH activity under hypoxic stress [42]. In flood-tolerant Sorghum bicolor varieties, ADH activity was much higher than in a sensitive variety [43]. Similar responses have also been documented in flood-tolerant plants like rice [44]. However, as the stress duration increased, ADH activity in both T1 and T2 decreased sharply. This decrease, along with the decline in root vitality, may reflect the gradual collapse of cellular energy metabolism under long-term hypoxia. It suggests that the root function is exhausted over time. Although T1 and T2 exhibited similar trends, T1 showed a certain lag. This may be because T2, facing more severe waterlogging stress, utilized anaerobic respiration enzymes earlier to resist the stress.

The single indicator analysis presented specific changes in physiological parameters at different time points and treatments, such as the decline in root vitality, the increase in MDA and H₂O₂, and the changes in anaerobic respiration and antioxidant enzyme activities. PCA, through dimensionality reduction, integrated these changes and separated the following two principal components: PC1 (primarily reflects energy compensation mechanisms and root function loss) and PC2 (reveals the overall level of oxidative stress and defense responses). Through PCA, it is clear that root vitality and SOD activity play key roles in maintaining plant stress resistance, while anaerobic respiration enzymes (ADH, LDH) and oxidative damage indicators (MDA) reflect the compensatory and damage pressures plants face under stress. This provides a basis for further exploration of regulatory signaling pathways at the molecular level. In the temporal dimension, CK exhibited relatively stable indicators, corresponding to a more concentrated distribution of samples along PC1 and PC2; whereas T1 and T2 exhibited distinct time-dependent dynamics, with the early phase mainly characterized by enhanced metabolic enzyme activity (positive movement along PC1), and the later phase showing accumulated oxidative damage (positive diffusion along PC2). This global change aligns with the significant changes observed in single indicators on Days 20 and 25, further demonstrating the advantages of PCA in revealing the overall stress response of plants.

5. Conclusions and Outlook

Overall, this study revealed the spatiotemporal changes in the root physiological indicators of S. japonica under T1 and T2 stress, demonstrating that plants activated anaerobic respiration and antioxidant defense systems in response to flooding. However, prolonged stress led to the accumulation of oxidative damage and the decline of root function. The PCA results provided intuitive evidence for understanding the interactions between various indicators, confirming the multidimensional regulatory mechanisms of stress responses. Future research should explore changes in signaling molecules and regulatory gene expression under stress, combining molecular biology and systems biology methods to further reveal the mechanisms of flood tolerance in plants.