Exogenous H₂O₂ enhances the quality of Radix Paeoniae Rubra and Radix Paeoniae Alba by activating ROS-mediated secondary metabolism

Abstract

Radix Paeoniae Rubra (RPR) and Radix Paeoniae Alba (RPA) are important medicinal herbs widely used in Asian countries. Although both are derived from the dried roots of Paeonia lactiflora Pall., they exhibit distinct pharmacological properties due to differences in germplasm and post-harvest processing methods. However, growing market demand has led to cultivated materials becoming the primary commercial source, resulting in a significant decline in their medicinal quality. Environmental stress induces the substantial production of reactive oxygen species (ROS), which can modulate enzyme activities and enhance secondary metabolism. This study aimed to address this quality decline by treating fresh roots of two P. lactiflora germplasms—one for RPR production (RPR-germplasm) and one for RPA production (RPA-germplasm)—with H₂O₂ solutions at concentrations of 0.0, 0.5, 1.0, or 2.0 mmol/L to induce ROS bursts, thereby mimicking environmental stress and activating cellular oxidative stress defense and secondary metabolic pathways. In the fresh roots of RPR-germplasm treated with 1.0 mmol/L H₂O₂ on day 2, the contents of the secondary metabolites paeoniflorin, albiflorin, paeonol, catechin, gallic acid, benzoylpaeoniflorin, oxypaeoniflorin, and benzoic acid increased by 23.7%, 22.8%, 77.0%, 58.5%, 31.3%, 29.4%, 37.4%, and 43.8%, respectively. In the fresh roots of RPA-germplasm, the contents of the major secondary metabolites albiflorin, paeoniflorin, oxypaeoniflorin, benzoylpaeoniflorin, gallic acid, and catechin increased by 23.0%, 30.1%, 39.4%, 14.1%, 20.3%, and 19.1%, respectively. Furthermore, co-treatment experiments with H₂O₂ and ROS scavengers confirmed that ROS act as the critical signaling mediator connecting exogenous H₂O₂ to the secondary metabolism of P. lactiflora: scavenging ROS significantly attenuated the H₂O₂-induced accumulation of target secondary metabolites. Collectively, these findings demonstrate that exogenous H₂O₂ activates ROS-mediated secondary metabolism to enhance the quality of cultivated RPR and RPA. This study elucidates a “H₂O₂–ROS–secondary metabolism” regulatory axis and provides a practical strategy for improving secondary metabolite accumulation in medicinal plants via controlled oxidative stress.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12870-026-09182-6.

Introduction

RPR is obtained by direct drying of fresh roots of wild P. lactiflora, whereas RPA is derived from fresh roots of the cultivated variety P. lactiflora ‘Baishao’, which has undergone long-term directional domestication and breeding [1]. After removing root tips and fibrous roots, the fresh roots are processed by water boiling followed by peeling, or peeling prior to boiling [2, 3]. Both are essential traditional medicinal herbs widely used in Asian countries. RPR is rich in paeoniflorin, catechin and paeonol, with potent anti-inflammatory, analgesic, antiviral and antibacterial properties. It is commonly used for adjuvant treatment of hemorheological abnormalities, mild-to-moderate inflammation and infectious diseases. RPA contains high levels of albiflorin and exhibits immunomodulatory, hematopoietic, hepatoprotective and anti-tumor auxiliary effects; it is clinically applicable to patients with immunodeficiency, chronic anemia, liver injury and rehabilitation after chemoradiotherapy [4, 5]. Currently, market demand for RPR and RPA continues to grow, and wild resources can hardly meet market supply, making cultivated materials the dominant commercial source. Nevertheless, cultivated medicinal herbs generally suffer from inferior quality. Therefore, improving the quality of cultivated P. lactiflora is critical to enhancing its clinical therapeutic efficacy.

ROS are pivotal signaling molecules regulating plant metabolism, mainly including O₂·⁻, H₂O₂, ·OH, ¹O₂ and HOO· [6]. At physiological concentrations, ROS participate in protein disulfide bond formation [7], act as essential modulators of enzyme and functional protein conformation, and regulate normal plant growth and development [8]. When plants are exposed to various abiotic stresses, intracellular ROS production increases sharply. Superoxide dismutase (SOD) catalyzes the rapid dismutation of O₂·⁻ into H₂O₂ and O₂, serving as the primary pathway for endogenous H₂O₂ generation in plant cells [9]. Additional H₂O₂ is also produced during photorespiration, where glycolate oxidase catalyzes the oxidation of glycolate to glyoxylate while generating H₂O₂ [10]. Accumulated evidence confirms that environmental stress is the primary cause of massive ROS accumulation. Under adverse conditions, endogenous H₂O₂ content increases by 2- to 10-fold and O₂·⁻ levels rise by 1- to 3-fold [11, 12]. Excessive ROS trigger cascading oxidative damage, disrupting biomembrane stability and molecular structures, impairing DNA integrity and genetic information stability, and further inducing peptide chain cleavage and protein cross-linking. Ultimately, this leads to metabolic disturbance, organelle dysfunction and programmed cell death [13, 14]. As sessile organisms, plants are constantly exposed to adverse environments and tend to accumulate excessive ROS under stress [15]. To cope with oxidative damage, plants have evolved a sophisticated antioxidant defense system. Among these components, secondary metabolites — the major bioactive ingredients of medicinal plants — play an indispensable role in scavenging excess ROS [16]. Accordingly, ROS burst acts as a core signaling hub linking environmental stress to quality formation of medicinal plant materials.

H₂O₂ can diffuse freely within cells and has a relatively long half-life, making it the predominant ROS modulating plant metabolic processes [17]. By oxidatively modifying key proteins in stress signaling pathways, H₂O₂ activates multiple downstream signal transduction cascades and enhances plant stress tolerance [18]. In recent years, exogenous H₂O₂ has been widely recognized as an efficient elicitor for plant secondary metabolism. It can mimic natural stress signals and significantly promote the biosynthesis and accumulation of flavonoids, phenolic acids, phenylpropanoids, terpenoids and saponins in medicinal plants [19, 20]. Accumulating evidence shows that H₂O₂ modulates the expression of secondary metabolism-related genes and the activities of key rate-limiting enzymes, and maintains intracellular redox homeostasis through crosstalk with the antioxidant system [21, 22]. Nevertheless, the regulatory mechanism of exogenous H₂O₂ on secondary metabolism in P. lactiflora remains poorly understood. Given the widespread quality degradation of cultivated P. lactiflora, this study treated fresh roots with exogenous H₂O₂ under the classic “stress–ROS–secondary metabolism” regulatory framework. The aim was to clarify how H₂O₂-mediated stress signals modulate secondary metabolism in P. lactiflora, including the accumulation of key secondary metabolites, changes in metabolic enzyme activities, and the central signaling role of ROS. This study provides a theoretical basis and feasible technical strategy for quality improvement and secondary metabolite enhancement in medicinal plant cultivation.

Materials and methods

Plant materials

The experimental materials were obtained from four-year-old cultivated RPR-germplasm and RPA-germplasm of P. lactiflora, grown at a private medicinal herbs production base in the Greater Khingan Mountains region of Heilongjiang Province, China, with the permission of the landowner, Jinrong Wu. The fresh roots were harvested on September 12, 2025, with more than 15 intact plants of each variety selected as research subjects, totaling 15 kg of fresh roots per germplasm type, authenticated by Professor Xiangcai Meng, Heilongjiang University of Chinese Medicine as fresh roots of P. lactiflora. Voucher specimens (Herbarium of Heilongjiang University of Chinese Medicine, deposition numbers: HLJCM-2025-186 for RPR-germplasm and HLJCM-2025-187 for RPA-germplasm) were deposited and identified by the same authority.

Rationale for using excised fresh roots

Freshly excised roots were used instead of intact plants for the following reasons. First, freshly excised roots retain full metabolic activity, which is a basic tenet of plant physiology, making them a valid and simplified system for studying root-specific responses. Second, the fresh root is precisely the medicinal organ of RPR and RPA; using excised roots directly investigates the material ultimately used for medicinal purposes, without confounding influences from above-ground organs. Third, excised roots eliminate systemic effects such as photosynthesis, source-sink transport, and other whole-plant processes, allowing us to focus solely on root-autonomous responses to H₂O₂. Fourth, this system enables accurate regulation of H₂O₂ concentration, exposure time, and sampling uniformity, which is difficult to achieve with intact plants.

Instruments and reagents

Key instruments used in this study included a digital water bath, refrigerated centrifuge, UV-Vis spectrophotometer, microplate reader, analytical balance, drying oven, ultrasonic cleaner, vacuum pump, HPLC system, constant temperature shaker, and ice maker. All chemicals and assay kits for O₂·⁻, total protein (TP), H₂O₂, malondialdehyde (MDA), catalase (CAT), SOD, peroxidase (POD), phenylalanine ammonia-lyase (PAL), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), 1,3-diphosphoglycerate (1,3-DPG), UDP-glucosyltransferase (UGT), and the eight reference compounds were purchased from standard commercial suppliers (e.g., Beijing Solarbio, Suzhou Grace Biotech, Jiangsu Meimian, Chengdu Alfa). Solvents were of HPLC or analytical grade.

Sample handling

Selection of H₂O₂ concentrations

The concentrations of H₂O₂ used in the main experiment were 0.0 (distilled water, CK), 0.5, 1.0, and 2.0 mmol/L, which were selected based on preliminary experiments. In these pre-tests, fresh roots were treated with a broad concentration series: 0.0, 0.1, 0.25, 0.5, 1.0, 2.0, 4.0, and 8.0 mmol/L H₂O₂. Concentrations below 0.5 mmol/L induced only marginal changes in root viability and secondary metabolite levels, whereas concentrations above 2.0 mmol/L caused visible browning and reduced root viability. Therefore, the range of 0.5–2.0 mmol/L, together with a 0.0 control, was selected to represent sub-lethal to moderately stressful conditions.

Treatment of fresh roots of P. lactiflora with different H₂O₂ concentrations

Fresh, intact roots of RPR-germplasm and RPA-germplasm were divided into four groups based on diameter, length, and weight. After removing surface soil and dirt, the samples were uniformly sprayed with H₂O₂ solutions at concentrations of 0.0, 0.5, 1.0, and 2.0 mmol/L, respectively. Spraying was performed every 8 h until the surfaces of fresh roots reached water saturation, and the entire process was conducted at room temperature in the dark for 4 days. Samples were collected on days 0, 1, 2, 3, and 4 (24-hour intervals) using the following methods: (1) For each sample at each time point, fresh root tissues were collected from at least 8 plants, cut into uniform small pieces, and divided into 50 aliquots. Each aliquot was precisely weighed to 0.30 g and stored at -80 °C. These aliquots were used for the determination of O₂·⁻, H₂O₂, MDA, and 1,3-DPG contents, as well as the activities of SOD, CAT, POD, PAL, HMGR, and UGT. (2) Additional fresh roots > 300 g per group were collected: for RPR-germplasm, after removing root tips, root bases, and fine roots, the roots were sun-dried; for RPA-germplasm, after the same pretreatment, the roots were boiled in water, peeled, and then sun-dried. The dried roots were pulverized and passed through a No. 4 sieve, and these powdered samples were used for the determination of gallic acid, oxypaeoniflorin, catechin, albiflorin, paeoniflorin, benzoic acid, benzoylpaeoniflorin, and paeonol contents. Day 0 samples were collected from untreated fresh roots of RPR-germplasm and RPA-germplasm and served as the blank control.

Combined treatment of fresh roots of RPR-germplasm with H₂O₂ and ROS scavengers

Additional fresh roots of RPR-germplasm meeting the same selection criteria as those used in the preceding section were divided into four groups: water control group (CK), H₂O₂ treatment group, H₂O₂ + α-tocopherol (α-Toc) group, and H₂O₂ + N-acetylcysteine (NAC) group. The CK group was sprayed with distilled water; the H₂O₂ treatment group was sprayed with 1.0 mmol/L H₂O₂ solution; and the combined groups were first sprayed with 1.0 mmol/L H₂O₂ solution, followed by 0.5 mmol/L α-Toc or 2.0 mmol/L NAC solution (2-hour interval between sprays). For all groups, spraying was performed every 8 h until surface run-off, with the entire process conducted in the dark for 4 days. Samples were collected on days 0, 1, 2, 3, and 4 (24-hour intervals), following the sampling protocol in the preceding section with modifications as follows: (1) Each sample was derived from at least 8 plants. 30 aliquots of 0.30 g each were precisely weighed and stored at -80 °C. These aliquots were used only for determining O₂·⁻, H₂O₂, and MDA contents. (2) Fresh roots > 300 g per group were processed by removing root tips, root bases, and fine roots, then sun-drying, pulverizing, and passing through a No. 4 sieve. These powdered samples were used for determining paeoniflorin and seven other components.

Determination of biochemical parameters

Protein content in fresh root homogenates was determined using a TP assay kit. Levels of O₂·⁻ and H₂O₂ were measured using specific kits and expressed as µmol/g and µmol/mL, respectively. MDA content was quantified by the TBA method and expressed as nmol/g. Activities of SOD, CAT, and POD were assessed with kits and expressed as U/g, µmol/min/g, and ΔOD₄₇₀/min/g, respectively. 1,3-DPG content was measured using a plant ELISA kit and expressed as nmol/L. Activities of PAL, HMGR, and UGT were evaluated with kits and expressed as ΔOD₂₉₀/h/g, IU/L, and U/L, respectively.

Determination of secondary metabolite content

Preparation of reference standard solutions

Accurately weighed amounts of gallic acid (2.80 mg), oxypaeoniflorin (1.72 mg), catechin (1.38 mg), albiflorin (5.23 mg), paeoniflorin (10.58 mg), benzoic acid (1.88 mg), benzoylpaeoniflorin (1.31 mg), and paeonol (1.22 mg) were placed in a 10 mL volumetric flask. Approximately 7–8 mL of 50% methanol was added, and the mixture was ultrasonicated (200 W, 40 kHz) for 8 min to facilitate dissolution. After cooling to room temperature, the volume was made up to the mark with the same solvent. The final concentrations of the eight components in the mixed reference standard solution were 0.280, 0.172, 0.138, 0.523, 1.058, 0.188, 0.131, and 0.122 g/L, respectively, corresponding to the order of the components listed above.

Preparation of the test solution

An accurately weighed 0.20 g portion of the medicinal powder, which had been passed through a No. 4 sieve, was transferred into a 50 mL stoppered conical flask. Subsequently, 20 mL of 50% methanol was precisely added, the flask was stoppered tightly, and its initial weight was recorded. The mixture was allowed to stand at room temperature for 4 h, followed by ultrasonication (200 W, 40 kHz) for 30 min. After cooling to room temperature, the flask was shaken well, reweighed, and the lost weight was replenished with 50% methanol. The resulting solution was centrifuged at 9000 r/min for 5 min. The supernatant was collected and filtered through a 0.45 μm microporous membrane, and the subsequent filtrate was used as the test solution.

Chromatographic conditions

The separation was performed on a Diamonsil C₁₈ column (250 mm × 4.6 mm, 5 μm) using a mobile phase consisting of acetonitrile (A) and a phosphoric acid aqueous solution adjusted to pH 2.7 (B) under gradient elution as follows: 0–20 min, 5% A to 15% A; 20–40 min, 15% A to 20% A; 40–50 min, 20% A; 50–80 min, 20% A to 40% A; 80–90 min, 40% A to 5% A; 90–100 min, 5% A. The column temperature was maintained at 25 °C, the flow rate was 1 mL/min, and the injection volume was 10 µL. The detection wavelength was set at 230 nm (chromatograms are shown in Fig. 1).

Fig. 1.

HPLC chromatograms of mixed reference substances (A), RPR (B) and RPA (C)

Methodological investigation

Linearity

The mixed reference stock solution, prepared as described in the section “Preparation of reference standard solutions”, was serially diluted with 50% methanol to obtain a series of concentrations. Each solution was injected in triplicate under the chromatographic conditions specified in the section “Chromatographic conditions”, and the corresponding peak areas were recorded. Linear regression was performed by plotting the peak areas (Y) against the corresponding mass concentrations (X). The regression equations, coefficients of determination (R²), and linear ranges for the eight components were calculated, which demonstrated good linearity within the tested ranges.

Precision

A sample of RPR powder treated with 1.0 mmol/L H₂O₂ for 2 days was prepared as a test solution according to the section “Preparation of the test solution”. To determine intra-day precision, the same solution was injected six times consecutively under the conditions described in the section “Chromatographic conditions”. Inter-day precision was assessed by injecting the same solution in triplicate on three consecutive days. The RSD values for intra-day and inter-day precision of the eight components were 0.44%, 0.54%, 0.71%, 0.46%, 0.58%, 0.85%, 0.31%, and 1.26%; and 1.35%, 1.59%, 1.63%, 0.97%, 1.04%, 0.63%, 1.26%, and 1.70%, respectively. All RSD values were below 2%, indicating excellent precision of the method.

Stability

A test solution was prepared from RPR powder treated with 1.0 mmol/L H₂O₂ for 2 days as described in the section “Preparation of the test solution”. The same solution was injected under the chromatographic conditions of the section “Chromatographic conditions” at 0, 2, 4, 8, 12, and 24 h. The RSD values for the eight components were 1.17%, 1.22%, 1.34%, 0.73%, 0.87%, 0.49%, 1.08%, and 0.61%, respectively. All RSD values were below 2%, demonstrating that the sample solution remained stable for 24 h.

Repeatability

Six individual test solutions were prepared from RPR powder treated with 1.0 mmol/L H₂O₂ for 2 days by the same operator under identical conditions, following the procedure in the section “Preparation of the test solution”. Each solution was then injected in triplicate as described in the section “Chromatographic conditions”. The RSD values for the eight components were 0.92%, 0.94%, 0.67%, 1.41%, 0.80%, 0.73%, 1.63%, and 0.52%, respectively. All RSD values were below 2%, indicating excellent repeatability of the analytical method.

Spike recovery assay

The initial contents of the eight components in RPR powder treated with 1.0 mmol/L H₂O₂ for 2 days were first determined. Subsequently, six portions of this powder were accurately weighed, and appropriate amounts of mixed reference standards were spiked into each portion. The spiked samples were then processed according to the section “Preparation of the test solution” to prepare test solutions and analyzed using the method described in the section “Chromatographic conditions”. The total concentrations were calculated using the external standard method. The average recovery rates for the eight components were 98.6%, 101.1%, 99.7%, 98.5%, 100.5%, 100.9%, 99.1%, and 98.4%, all within the range of 95%–105%. The corresponding RSD values were 1.58%, 1.63%, 1.44%, 1.28%, 1.47%, 1.70%, 1.32%, and 1.27%, all below 2%. These results demonstrate that the method exhibits satisfactory accuracy and is suitable for quantitative analysis.

Data processing methods

Microsoft Excel 2021 (Microsoft, Redmond, WA, USA) was used to organize the raw data. Prism 9 (GraphPad Software, La Jolla, CA, USA) was used for generating graphs. All data were expressed as the mean ± standard deviation (SD). IBM SPSS Statistics 28.0 (IBM, Armonk, NY, USA) was used for statistical analysis. The normality of data distribution was verified using the Shapiro-Wilk test, and the homogeneity of variances was assessed using Levene’s test. An independent-samples t-test was applied for two-group comparisons, while one-way analysis of variance (ANOVA) followed by Dunnett’s post-hoc test was used for multiple-group comparisons. Differences were considered statistically significant at *p < 0.05 and **p < 0.01.

Results

ROS level in fresh roots of P. lactiflora

Compared with day 0, the O₂·⁻ level in the control group of RPR-germplasm remained relatively unchanged, while all H₂O₂-treated groups showed an initial increase followed by a subsequent decrease. The 2.0 mmol/L group showed the most pronounced elevation. Specifically, the O₂·⁻ levels in the 0.5, 1.0, and 2.0 mmol/L H₂O₂ groups peaked on days 2, 1, and 1, respectively, representing increases of 87.4%, 142.8%, and 178.6% compared with day 0 (Fig. 2-A).

Fig. 2.

ROS level changes in RPR (A–B) and RPA (a–b) under different H₂O₂ concentrations. Data are presented as±s (n = 3, error bars: SD). Significance: #/## vs. 0.0 mmol/L H₂O₂ (#:P < 0.05, ##: P < 0.01); */** vs. day 0 (*: P < 0.05, **: P < 0.01). Groups: 0.0 mmol/L H₂O₂ (CK, blue), 0.5 mmol/L H₂O₂ (orange), 1.0 mmol/L H₂O₂ (green), 2.0 mmol/L H₂O₂ (brown).A/a: O₂·⁻ contents (µmol/g); B/b: H₂O₂ contents (µmol/mL).The complete versions of Figs. 2, 3, 4, 5, 6, 7, 8 and 9 with all significance markers are provided in Supplementary Information

For H₂O₂ levels in RPR-germplasm, the control and 0.5 mmol/L groups showed a slight upward trend compared with day 0, while all other treatment groups exhibited an initial increase followed by a subsequent decrease. Again, the 2.0 mmol/L group showed the most pronounced elevation. The H₂O₂ levels in the 1.0 and 2.0 mmol/L groups peaked on days 2 and 3, respectively, representing increases of 134.4% and 149.1% compared with day 0 (Fig. 2-B). The ROS levels in all H₂O₂-treated groups were significantly higher than those in the control group, indicating that exogenous H₂O₂ can induce ROS production in RPR-germplasm.

In RPA-germplasm, all groups showed an initial increase followed by a subsequent decrease in O₂·⁻ levels compared with day 0, with the 2.0 mmol/L group exhibiting the most pronounced elevation. The O₂·⁻ levels in the control, 0.5, 1.0, and 2.0 mmol/L groups peaked on days 2, 3, 1, and 2, respectively, representing increases of 89.7%, 104.7%, 138.3%, and 160.7% compared with day 0 (Fig. 2-a).

Similarly, all groups in RPA-germplasm showed an initial increase followed by a subsequent decrease in H₂O₂ levels compared with day 0, with the 2.0 mmol/L group again showing the most pronounced elevation. The H₂O₂ levels in the control, 0.5, 1.0, and 2.0 mmol/L groups peaked on days 3, 2, 2, and 1, respectively, representing increases of 151.8%, 122.5%, 202.0%, and 264.5% compared with day 0 (Fig. 2-b).

Cross-germplasm comparison revealed that at high H₂O₂ concentrations, RPR-germplasm exhibited an earlier O₂·⁻ peak: both 1.0 and 2.0 mmol/L groups peaked on day 1, whereas in RPA-germplasm, the 1.0 mmol/L group peaked on day 1 and the 2.0 mmol/L group peaked on day 2. However, RPA-germplasm exhibited a greater magnitude of H₂O₂ accumulation at all treatment concentrations, with a 264.5% increase in the 2.0 mmol/L group compared with 149.1% in RPR-germplasm. These results indicate that, with prolonged treatment time, the ROS levels in both germplasms generally exhibited an initial rise followed by a decline, and the magnitude of the increase was positively correlated with H₂O₂ concentration. Overall, RPA-germplasm exhibited a stronger H₂O₂ accumulation response, whereas RPR-germplasm showed a faster O₂·⁻ response to exogenous H₂O₂ treatment.

MDA level in fresh roots of P. lactiflora

Compared with day 0, the MDA content in the control group of RPR-germplasm showed a slight upward trend, whereas all H₂O₂-treated groups showed significantly greater increases than the control group. The 2.0 mmol/L group showed the most pronounced elevation. The MDA contents in the 0.5, 1.0, and 2.0 mmol/L H₂O₂ groups peaked on days 4, 3, and 4 of treatment, respectively, representing increases of 62.5%, 74.9%, and 102.9% compared with day 0 (Fig. 3-A). These results indicate that exogenous H₂O₂ application can cause considerable cellular damage in RPR-germplasm, thereby mimicking the physiological state of plants under stress conditions.

Fig. 3.

MDA content changes in RPR(A) and RPA(a) under different H₂O₂ concentrations. Data are presented as±s (n = 3, error bars: SD). Significance: #/## vs. 0.0 mmol/L H₂O₂ (#:P < 0.05, ##: P < 0.01); */** vs. day 0 (*: P < 0.05, **: P < 0.01). Groups: 0.0 mmol/L H₂O₂ (CK, blue), 0.5 mmol/L H₂O₂ (orange), 1.0 mmol/L H₂O₂ (green), 2.0 mmol/L H₂O₂ (brown). A/a: MDA contents (nmol/g)

In RPA-germplasm, compared with day 0, the MDA contents in the control group and 0.5 mmol/L H₂O₂ group showed a slight upward trend, while all other groups showed an initial increase followed by a subsequent decrease. The 2.0 mmol/L group showed the most significant increase. The MDA contents in both the 1.0 and 2.0 mmol/L H₂O₂ groups peaked on day 3 of treatment, representing increases of 157.2% and 187.5%, respectively, compared with day 0 (Fig. 3-a).

Cross-germplasm comparison revealed that RPA-germplasm exhibited a much stronger MDA response to exogenous H₂O₂ than RPR-germplasm. The peak MDA increase reached 187.5% in the 2.0 mmol/L group of RPA-germplasm, nearly double the 102.9% increase observed in RPR-germplasm. Additionally, RPA-germplasm reached its MDA peak earlier, with both the 1.0 and 2.0 mmol/L groups peaking on day 3, compared with day 4 for the 0.5 and 2.0 mmol/L groups in RPR-germplasm. These results indicate that exogenous H₂O₂ application causes more severe cellular damage in RPA-germplasm than in RPR-germplasm at the same concentration.

Antioxidant enzyme activities in fresh roots of P. lactiflora

Compared with day 0, the SOD activity in the control group of RPR-germplasm remained relatively unchanged, while all other treatment groups showed an initial increase followed by a subsequent decrease. The 1.0 mmol/L group showed the most pronounced rise. The SOD activities in the 0.5, 1.0, and 2.0 mmol/L groups peaked on day 1 of treatment, representing increases of 37.4%, 48.2%, and 29.3%, respectively, compared with day 0 (Fig. 4-A).

Fig. 4.

Antioxidant enzyme activities changes in RPR(A–C) and RPA(a–c) under different H₂O₂ concentrations. Data are presented as ±s (n = 3, error bars: SD). Significance: #/## vs. 0.0 mmol/L H₂O₂ (#:P < 0.05, ##: P < 0.01); */** vs. day 0 (*: P < 0.05, **: P < 0.01). Groups: 0.0 mmol/L H₂O₂ (CK, blue), 0.5 mmol/L H₂O₂ (orange), 1.0 mmol/L H₂O₂ (green), 2.0 mmol/L H₂O₂ (brown). A/a: SOD activities (U/g); B/b: CAT activities (µmol/min/g); C/c: POD activities (ΔOD470/min/g)

Similarly, the CAT activity in the control group of RPR-germplasm remained relatively unchanged compared with day 0, whereas all other groups showed an initial increase followed by a subsequent decrease. The 2.0 mmol/L group showed the most pronounced elevation. The CAT activities in the 0.5, 1.0, and 2.0 mmol/L groups reached their peaks on days 2, 2, and 1 of treatment, respectively, representing increases of 103.9%, 143.5%, and 150.1% relative to day 0 (Fig. 4-B).

For POD activity in RPR-germplasm, the control and 0.5 mmol/L groups exhibited minor fluctuations compared with day 0, while the other groups showed an initial increase followed by a subsequent decrease. The 2.0 mmol/L group showed the most pronounced rise. The POD activities in the 1.0 and 2.0 mmol/L groups peaked on days 2 and 3 of treatment, respectively, representing increases of 62.4% and 69.8% compared with day 0 (Fig. 4-C). These results indicate that, in contrast to the nearly stable activities in the control group, antioxidant enzyme activities in all H₂O₂-treated groups of RPR-germplasm were significantly elevated at the early stage (days 1–2), suggesting that antioxidant enzymes respond rapidly and play a crucial role in the initial phase of H₂O₂ treatment.

In RPA-germplasm, the SOD activities in the control and 0.5 mmol/L groups exhibited minor fluctuations compared with day 0, while all other groups showed an initial increase followed by a subsequent decrease. The 1.0 mmol/L group showed the most pronounced rise. The SOD activities in the 1.0 and 2.0 mmol/L groups peaked on days 2 and 1 of treatment, respectively, representing increases of 53.1% and 42.4% compared with day 0 (Fig. 4-a).

For CAT activity in RPA-germplasm, the control group exhibited minor fluctuations compared with day 0, whereas all other groups showed an initial increase followed by a subsequent decrease. The 2.0 mmol/L group showed the most pronounced elevation. The CAT activities in the 0.5, 1.0, and 2.0 mmol/L groups peaked on days 3, 2, and 1 of treatment, respectively, representing increases of 26.4%, 40.9%, and 53.0% relative to day 0 (Fig. 4-b).

For POD activity in RPA-germplasm, the 0.5 mmol/L group showed minor fluctuations compared with day 0, while the control group displayed a slight gradual increase, and the remaining groups showed an initial increase followed by a subsequent decrease. The 1.0 mmol/L group showed the most pronounced rise. The POD activities in the control, 1.0, and 2.0 mmol/L groups peaked on days 4, 1, and 2 of treatment, respectively, representing increases of 43.9%, 74.7%, and 68.5% compared with day 0 (Fig. 4-c).

Cross-germplasm comparison revealed distinct differences in antioxidant enzyme responses. RPR-germplasm exhibited a much stronger CAT response to H₂O₂ treatment, with peak increases ranging from 103.9% to 150.1%, compared with only 26.4%–53.0% in RPA-germplasm. In contrast, RPA-germplasm showed a slightly higher SOD response in the 1.0 mmol/L group (53.1% increase versus 48.2% in RPR-germplasm), but with a delayed peak at day 2 compared with day 1 in RPR-germplasm. POD responses were relatively similar between the two germplasms, with RPA-germplasm showing a 74.7% peak increase in the 1.0 mmol/L group versus 62.4% in RPR-germplasm. These results indicate that RPR-germplasm possesses a more rapid and robust antioxidant defense response, particularly in CAT activity, which may contribute to its greater tolerance to oxidative stress. Overall, RPR-germplasm exhibited a faster and stronger antioxidant enzyme response to exogenous H₂O₂ treatment than RPA-germplasm, especially with respect to CAT activity.

Key enzyme activities in secondary metabolism in fresh roots of P. lactiflora

Compared with day 0, the PAL activity in the control group of RPR-germplasm remained relatively unchanged, while all other groups showed an initial increase followed by a subsequent decrease. The 1.0 mmol/L group showed the most pronounced rise. The PAL activity in the 1.0 mmol/L group peaked on day 2 of treatment, representing an increase of 72.7% compared with day 0. The 0.5 and 2.0 mmol/L groups peaked on day 1 of treatment, representing increases of 28.8% and 56.1%, respectively (Fig. 5-A).

Fig. 5.

Changes in key enzyme activities in secondary metabolism in RPR (A–C) and RPA (a–c) under different H₂O₂ concentrations. Data are presented as±s (n = 3, error bars: SD). Significance: #/## vs. 0.0 mmol/L H₂O₂ (#:P < 0.05, ##: P < 0.01); */** vs. day 0 (*: P < 0.05, **: P < 0.01). Groups: 0.0 mmol/L H₂O₂ (CK, blue), 0.5 mmol/L H₂O₂ (orange), 1.0 mmol/L H₂O₂ (green), 2.0 mmol/L H₂O₂ (brown). A/a: PAL activities (ΔOD₂₉₀/h/g); B/b: HMGR activities (IU/L); C/c: UGT activities (U/L)

Similarly, the HMGR activities in the control and 2.0 mmol/L groups of RPR-germplasm remained relatively unchanged compared with day 0, while the 0.5 and 1.0 mmol/L groups showed an initial increase followed by a subsequent decrease. The 1.0 mmol/L group showed the most pronounced elevation. The HMGR activities in the 0.5 and 1.0 mmol/L groups peaked on day 2 of treatment, representing increases of 25.0% and 35.6%, respectively, compared with day 0 (Fig. 5-B).

For UGT activity in RPR-germplasm, the control and 0.5 mmol/L groups exhibited minor fluctuations compared with day 0, while the 1.0 and 2.0 mmol/L groups showed an initial increase followed by a subsequent decrease. The 1.0 mmol/L group showed the most pronounced rise. The UGT activities in the 1.0 and 2.0 mmol/L groups peaked on days 2 and 1 of treatment, respectively, representing increases of 19.0% and 15.0% compared with day 0 (Fig. 5-C).

In RPA-germplasm, the PAL activity in the 0.5 mmol/L group remained relatively unchanged compared with day 0, while all other groups showed an initial increase followed by a subsequent decrease. The 1.0 mmol/L group showed the most pronounced rise. The PAL activities in the control, 1.0, and 2.0 mmol/L groups peaked on days 2, 2, and 1 of treatment, respectively, representing increases of 116.4%, 158.6%, and 98.3% compared with day 0 (Fig. 5-a).

Similarly, the HMGR activities in the 0.5 and 2.0 mmol/L groups of RPA-germplasm exhibited minor fluctuations compared with day 0, while the control and 1.0 mmol/L groups showed an initial increase followed by a subsequent decrease. The 1.0 mmol/L group showed the most pronounced elevation. The HMGR activities in the control and 1.0 mmol/L groups peaked on day 1 of treatment, representing increases of 40.0% and 57.7%, respectively, compared with day 0 (Fig. 5-b).

For UGT activity in RPA-germplasm, the 0.5 mmol/L group remained relatively unchanged compared with day 0, while all other groups showed an initial increase followed by a subsequent decrease. The 1.0 mmol/L group showed the most pronounced rise. The UGT activities in the control, 1.0, and 2.0 mmol/L groups peaked on days 1, 2, and 1 of treatment, respectively, representing increases of 18.6%, 27.0%, and 15.8% compared with day 0 (Fig. 5-c).

Cross-germplasm comparison revealed that RPA-germplasm exhibited a significantly stronger response in key enzymes of secondary metabolism than RPR-germplasm. The peak PAL increase reached 158.6% in the 1.0 mmol/L group of RPA-germplasm, more than double the 72.7% increase observed in RPR-germplasm. Similarly, the peak HMGR increase reached 57.7% in the 1.0 mmol/L group, which was also higher than the 35.6% increase in RPR-germplasm. UGT responses were relatively similar between the two germplasms, with RPA-germplasm showing a 27.0% peak increase in the 1.0 mmol/L group versus 19.0% in RPR-germplasm. These results indicate that exogenous H₂O₂ exerts a more pronounced inductive effect on key enzymes of secondary metabolism in RPA-germplasm than in RPR-germplasm. Overall, RPA-germplasm exhibited stronger upregulation of key enzymes of secondary metabolism in response to exogenous H₂O₂ treatment than RPR-germplasm.

Secondary metabolite levels in fresh roots of P. lactiflora

Compared with day 0, the paeoniflorin content in the control group of RPR-germplasm remained relatively unchanged, while all other groups showed an initial increase followed by a subsequent decrease. The 1.0 mmol/L group showed the most pronounced rise. The paeoniflorin contents in the 0.5, 1.0, and 2.0 mmol/L groups peaked on days 2, 2, and 1 of treatment, respectively, representing increases of 15.6%, 23.7%, and 17.0% compared with day 0 (Fig. 6-A).

Fig. 6.

Changes in secondary metabolites levels in RPR(A–H) and RPA(a–g) under different H₂O₂ concentrations. Data are presented as ±s (n = 3, error bars: SD). Significance: #/## vs 0.0 mmol/L H₂O₂ (#: P < 0.05, ##: P < 0.01); */** vs day 0 (*: P < 0.05, **: P < 0.01). Groups: 0.0 mmol/L H₂O₂ (CK, blue), 0.5 mmol/L H₂O₂ (orange), 1.0 mmol/L H₂O₂ (green), 2.0 mmol/L H₂O₂ (brown). A/a: Paeoniflorin content; B/b: Albiflorin content; C/c: Catechin content; D/d: Gallic acid content; E/e: Benzoylpaeoniflorin content; F/f: Oxypaeoniflorin content; G/g: Benzoic acid content; H: Paeonol content (all units: mg/g)

For albiflorin in RPR-germplasm, the 1.0 mmol/L group showed an initial increase followed by a subsequent decrease compared with day 0, peaking on day 2 of treatment and representing an increase of 22.8%, whereas all other groups remained relatively unchanged throughout the treatment period (Fig. 6-B).

For catechin in RPR-germplasm, the control group showed a gradual decrease over time compared with day 0, while all other groups showed an initial increase followed by a subsequent decrease. The 1.0 mmol/L group showed the most pronounced rise. The catechin contents in the 0.5, 1.0, and 2.0 mmol/L groups peaked on days 2, 2, and 1 of treatment, respectively, representing increases of 31.2%, 58.5%, and 41.0% compared with day 0 (Fig. 6-C).

For gallic acid in RPR-germplasm, the control group exhibited a slight gradual increasing trend compared with day 0, while other groups showed an initial increase followed by a subsequent decrease. The 1.0 mmol/L group showed the most pronounced rise. The gallic acid contents in the 0.5, 1.0, and 2.0 mmol/L groups peaked on days 1, 2, and 2 of treatment, respectively, representing increases of 24.0%, 31.3%, and 25.9% compared with day 0 (Fig. 6-D).

For benzoylpaeoniflorin in RPR-germplasm, the 2.0 mmol/L group remained relatively unchanged compared with day 0. In contrast, the control group showed an initial increase followed by a subsequent decrease, peaking on day 2 of treatment and representing an increase of 33.2%. Meanwhile, the 0.5 and 1.0 mmol/L groups exhibited a gradual increasing trend, both peaking on day 4 and representing increases of 64.4% and 50.9%, respectively, compared with day 0 (Fig. 6-E).

For oxypaeoniflorin in RPR-germplasm, the control and 0.5 mmol/L groups exhibited minor fluctuations compared with day 0, while other groups showed an initial increase followed by a subsequent decrease. The 2.0 mmol/L group showed the most pronounced rise. The oxypaeoniflorin contents in the 1.0 and 2.0 mmol/L groups peaked on days 2 and 1 of treatment, respectively, representing increases of 37.4% and 54.3% compared with day 0 (Fig. 6-F).

For benzoic acid in RPR-germplasm, the control group remained relatively unchanged compared with day 0. The 0.5 and 1.0 mmol/L groups exhibited a gradual increasing trend, peaking on day 4 and representing increases of 88.9% and 73.3%, respectively, compared with day 0. In contrast, the 2.0 mmol/L group showed an initial increase followed by a subsequent decrease, with benzoic acid content peaking on day 3 of treatment and representing an increase of 102.3% compared with day 0 (Fig. 6-G).

Finally, for paeonol in RPR-germplasm, the control and 2.0 mmol/L groups remained relatively unchanged compared with day 0. The 0.5 mmol/L group showed a gradual decreasing trend, while the 1.0 mmol/L group showed an initial increase followed by a subsequent decrease, with paeonol content peaking on day 2 of treatment and representing an increase of 77.0% compared with day 0 (Fig. 6-H).

In RPA-germplasm, the paeoniflorin contents in the control and 2.0 mmol/L groups remained relatively unchanged compared with day 0, while other groups showed an initial increase followed by a subsequent decrease. The 1.0 mmol/L group showed the most pronounced rise. The paeoniflorin contents in the 0.5 and 1.0 mmol/L groups both peaked on day 2 of treatment, representing increases of 22.1% and 30.1%, respectively, compared with day 0 (Fig. 6-a).

For albiflorin in RPA-germplasm, the 0.5 and 2.0 mmol/L groups exhibited minor fluctuations compared with day 0, while the remaining groups showed an initial increase followed by a subsequent decrease. The 1.0 mmol/L group showed the most pronounced rise. The albiflorin contents in the control and 1.0 mmol/L groups both peaked on day 2 of treatment, representing increases of 14.8% and 23.0%, respectively, compared with day 0 (Fig. 6-b).

For catechin in RPA-germplasm, the 0.5 mmol/L group showed a gradual decreasing trend compared with day 0, while all other groups showed an initial increase followed by a subsequent decrease. The 1.0 mmol/L group showed the most pronounced rise. The catechin contents in the control, 1.0, and 2.0 mmol/L groups peaked on days 1, 2, and 2 of treatment, respectively, representing increases of 13.9%, 19.1%, and 11.5% compared with day 0 (Fig. 6-c).

For gallic acid in RPA-germplasm, the control and 2.0 mmol/L groups remained relatively unchanged compared with day 0, while other groups showed an initial increase followed by a subsequent decrease. The 1.0 mmol/L group showed the most pronounced rise. The gallic acid contents in the 0.5 and 1.0 mmol/L groups peaked on days 3 and 2 of treatment, respectively, representing increases of 13.0% and 20.3% compared with day 0 (Fig. 6-d).

For benzoylpaeoniflorin in RPA-germplasm, the 1.0 mmol/L group showed an initial increase followed by a subsequent decrease compared with day 0, peaking on day 2 of treatment and representing an increase of 14.1%. The 2.0 mmol/L group exhibited a gradual increasing trend, peaking on day 4 and representing an increase of 10.5% compared with day 0. The remaining groups remained relatively unchanged with minor overall fluctuations (Fig. 6-e).

For oxypaeoniflorin in RPA-germplasm, all groups showed an initial increase followed by a subsequent decrease compared with day 0. The 1.0 mmol/L group showed the most pronounced rise. The oxypaeoniflorin contents in the control, 0.5, 1.0, and 2.0 mmol/L groups peaked on days 1, 1, 2, and 2 of treatment, respectively, representing increases of 31.3%, 19.6%, 39.4%, and 21.7% compared with day 0 (Fig. 6-f).

Finally, for benzoic acid in RPA-germplasm, the 0.5 mmol/L group remained relatively unchanged compared with day 0, while other groups generally showed an initial increase followed by a subsequent decrease and then a secondary increase. The 2.0 mmol/L group showed the most pronounced rise. The benzoic acid contents in the control, 1.0, and 2.0 mmol/L groups peaked on days 3, 1, and 1 of treatment, respectively, representing increases of 10.4%, 8.0%, and 10.8% compared with day 0 (Fig. 6-g).

Cross-germplasm comparison revealed significant differences in secondary metabolite accumulation patterns. RPR germplasm exhibited substantially higher increases in catechin, gallic acid, benzoylpaeoniflorin, and benzoic acid. In the 1.0 mmol/L group, their increases reached 58.5%, 31.3%, 50.9% and 73.3% in RPR germplasm, compared with 19.1%, 20.3%, 14.1% and 8.0% in RPA germplasm. Additionally, RPR germplasm uniquely accumulated paeonol, which was undetectable in RPA germplasm, with a 77.0% increase observed in the 1.0 mmol/L group. In contrast, RPA germplasm showed slightly higher increases in paeoniflorin and oxypaeoniflorin under 1.0 mmol/L treatment, with peak values of 30.1% and 39.4%, respectively, versus 23.7% and 37.4% in RPR germplasm. Meanwhile, albiflorin increases were comparable between the two germplasms, at 23.0% in RPA germplasm and 22.8% in RPR germplasm. These results indicate that exogenous H₂O₂ exerts differential inductive effects on secondary metabolite accumulation in RPR and RPA germplasms, with RPR germplasm displaying a broader and stronger response across multiple metabolites. Overall, RPR germplasm exhibited a more pronounced secondary metabolite accumulation response to exogenous H₂O₂ treatment, particularly for catechin, benzoylpaeoniflorin, and the unique metabolite paeonol.

1,3-DPG level in fresh roots of P. lactiflora

Compared with day 0, the 1,3-DPG content in the control group of RPR-germplasm remained relatively unchanged, while all other groups showed an initial increase followed by a subsequent decrease. The 1.0 mmol/L group showed the most pronounced rise. The 1.0 mmol/L group peaked on day 2 of treatment, representing an increase of 38.0% compared with day 0. The 0.5 and 2.0 mmol/L groups peaked on days 2 and 1 of treatment, respectively, representing increases of 23.0% and 25.4% compared with day 0 (Fig. 7-A).

Fig. 7.

Changes in 1,3-DPG levels in RPR(A) and RPA(a) under different H₂O₂ concentrations. Data are presented as±s (n = 3, error bars: SD). Significance: #/## vs. 0.0 mmol/L H₂O₂ (#:P < 0.05, ##: P < 0.01); */** vs. day 0 (*: P < 0.05, **: P < 0.01). Groups: 0.0 mmol/L H₂O₂ (CK, blue), 0.5 mmol/L H₂O₂ (orange), 1.0 mmol/L H₂O₂ (green), 2.0 mmol/L H₂O₂ (brown). A/a: 1,3-DPG contents (nmol/L)

In RPA-germplasm, the 0.5 mmol/L group exhibited a gradual increasing trend in 1,3-DPG content compared with day 0, while the other groups showed an initial increase followed by a subsequent decrease. The 1.0 mmol/L group showed the most pronounced rise. The 1,3-DPG contents in the control, 0.5, 1.0, and 2.0 mmol/L groups peaked on days 3, 4, 2, and 1 of treatment, respectively, representing increases of 36.6%, 25.1%, 40.6%, and 27.8% compared with day 0 (Fig. 7-a).

Cross-germplasm comparison showed that 1,3-DPG responses were relatively similar between the two germplasms. Both germplasms exhibited the highest increase in the 1.0 mmol/L group, with RPA-germplasm showing a slightly higher peak increase of 40.6% versus 38.0% in RPR-germplasm, and both reaching their peak on day 2. These results indicate that exogenous H₂O₂ exerts a similar inductive effect on 1,3-DPG accumulation in both RPR- and RPA-germplasms. Overall, no significant difference was observed in 1,3-DPG response patterns between the two germplasms.

Verification of the physiological effects of H₂O₂

Compared with the 1.0 mmol/L H₂O₂ treatment alone, the combined application of ROS scavengers, namely 0.5 mmol/L α-Toc and 2.0 mmol/L NAC, significantly decreased the levels of O₂·⁻, H₂O₂, and MDA in fresh roots of RPR-germplasm, returning them to levels close to those in the control group. These results demonstrated that α-Toc and NAC could effectively scavenge the ROS induced by H₂O₂ in fresh roots of RPR-germplasm (Fig. 8). Furthermore, compared with the 1.0 mmol/L H₂O₂ treatment alone, the combined application of ROS scavengers, namely 0.5 mmol/L α-Toc and 2.0 mmol/L NAC, significantly decreased the contents of paeoniflorin, albiflorin, catechin, gallic acid, oxypaeoniflorin, and paeonol in fresh roots of RPR-germplasm, returning them to levels close to those in the control group. These results indicated that the inductive effect of H₂O₂ on secondary metabolism in RPR-germplasm is dependent on ROS accumulation (Fig. 9).

Fig. 8.

ROS Levels and MDA Changes in RPR (A–C). Data are presented as±s (n = 3, error bars: SD). Significance: #/## vs. 0.0 mmol/L H₂O₂ (#:P < 0.05, ##: P < 0.01); */** vs. day 0 (*: P < 0.05, **: P < 0.01). Groups: 0.0 mmol/L H₂O₂ (CK, blue), 1.0 mmol/L H₂O₂ (green), 1.0 mmol/L H₂O₂ + 0.5 mmol/L α-Toc(purple), 1.0 mmol/L H₂O₂ + 2.0 mmol/L NAC (gray). A: O₂·⁻ contents (µmol/g); B: H₂O₂ contents (µmol/mL); C: MDA contents (nmol/g)

Fig. 9.

Changes in Secondary Metabolites Levels in RPR (A–H). Data are presented as±s (n = 3, error bars: SD). Significance: #/## vs. 0.0 mmol/L H₂O₂ (#:P < 0.05, ##: P < 0.01); */** vs. day 0 (*: P < 0.05, **: P < 0.01). Groups: 0.0 mmol/L H₂O₂ (CK, blue), 1.0 mmol/L H₂O₂ (green), 1.0 mmol/L H₂O₂ + 0.5 mmol/L α-Toc(purple), 1.0 mmol/L H₂O₂ + 2.0 mmol/L NAC (gray). A: Paeoniflorin content; B: Albiflorin content; C: Catechin content; D: Gallic acid content; E: Benzoylpaeoniflorin content; F: Oxypaeoniflorin content; G: Benzoic acid content; H: Paeonol content (all units: mg/g)

Discussion

When plants are subjected to environmental stress, cells maintain ROS homeostasis through multiple pathways, including osmotic adjustment, regulation of enzymatic activities, and enhancement of secondary metabolism [23]. Among these, secondary metabolites serve dual roles as ROS scavengers and cytoprotective agents, acting not only as core defensive compounds for plant adaptation to environmental stress but also as key pharmacologically active components in medicinal herbs [24, 25]. Quality formation of medicinal materials is essentially a physiological adaptation process whereby plants perceive environmental stress cues and trigger the biosynthesis of secondary metabolites. This environment-gene regulatory interaction renders the quality of medicinal materials an ecological signature of plant physiological stress responses, thereby providing a theoretical basis for strategies to directionally improve the accumulation of pharmacologically active components in medicinal plants via controlled stress-induced cultivation.

Effects of H₂O₂ on ROS and MDA levels

Various abiotic stresses, such as salt, low temperature, and heat, have been reported to significantly elevate ROS levels in plants [26–28]. High concentrations of ROS can damage plant cell ultrastructure, leading to membrane denaturation and disruption of cellular compartmentalization [29]. MDA, a key product of membrane lipid peroxidation, serves as a core indicator of oxidative damage [30]. In the present study, fresh roots of RPR and RPA treated with different H₂O₂ concentrations exhibited a rapid increase in O₂·⁻ content, followed by a rise in H₂O₂ content (Fig. 2), and a significant increase in MDA levels (Fig. 3). These results indicate that exogenous H₂O₂ treatment successfully mimics stress conditions and recapitulates the physiological state of plants under stress. The survival of plants under severe stress relies on both enzymatic scavenging by antioxidant enzymes and non-enzymatic scavenging via secondary metabolites [31]. These two antioxidant subsystems form a complementary antioxidant network [32].

Effects of H₂O₂ stress on antioxidant enzyme activities

SOD is a key enzyme that specifically catalyzes the conversion of O₂·⁻ to H₂O₂, while CAT and POD are major H₂O₂-scavenging enzymes [33–35]. The catalytic mechanisms of these antioxidant enzymes have been well documented [36–39]. Their activities are finely regulated under oxidative stress and directly determine ROS-scavenging efficiency [40]. Excessive H₂O₂ can inactivate proteins and impair the antioxidant system [41]. Phenolic secondary metabolites can act as electron donors for POD, thereby enhancing ROS scavenging [42]. The dynamic balance between enzymatic and non-enzymatic antioxidant systems ultimately governs cellular redox homeostasis. The enzymatic system, with SOD, POD, and CAT as core components, operates synergistically to convert ROS into non-toxic metabolites [43].

In the present study, H₂O₂ treatment elevated ROS levels and initially triggered a marked increase in antioxidant enzyme activities (Figs. 2 and 4). In fresh roots of both RPR and RPA, SOD activities increased significantly within 0–2 days (Fig. 4), which enhanced O₂·⁻ elimination and led to a gradual decrease in O₂·⁻ levels during days 2–4 (Fig. 2). CAT and POD acted in a synergistic and complementary manner: in RPR, CAT and POD peaked on days 1–2 and 2–3, respectively; in RPA, they peaked on days 1–3 and 1–2, respectively. Notably, under 2.0 mmol/L H₂O₂, POD maintained relatively high activity during days 3–4 (Fig. 4), indicating its tolerance to high-intensity stress. Exogenous H₂O₂ enhanced antioxidant enzyme activities and ROS scavenging capacity, resulting in decreased ROS levels after 1–2 days. However, with increasing stress intensity or exposure duration, enzyme activities declined within 3–4 days (Fig. 4), likely attributed to oxidative damage to these proteinaceous enzymes [44]. These findings suggest that under severe stress, antioxidant enzymes may not be the primary ROS scavengers, implying that non-enzymatic systems, such as secondary metabolites, play a crucial role.

Effects of H₂O₂ stress on key enzymes and metabolites in secondary metabolism

Low concentrations of ROS serve as signaling molecules involved in the physiological regulation of plants, whereas excessive accumulation triggers severe cellular oxidative damage [45]. When ROS levels exceed the scavenging capacity of the enzymatic system, plants enhance their non-enzymatic defense system, eliminating excess ROS via secondary metabolites to mitigate oxidative stress injury [46]. The major secondary metabolites of P. lactiflora include paeoniflorin, albiflorin, catechin, gallic acid, benzoylpaeoniflorin, oxypaeoniflorin, and paeonol, encompassing both monoterpenoids and phenolic compounds. Among these, catechin, gallic acid, and paeonol belong to distinct subclasses of phenolic compounds, all bearing phenolic hydroxyl groups. Through multiple pathways—such as scavenging free radicals including O₂·⁻ and ·OH, inhibiting NADPH oxidase activity, and chelating transition metal ions such as Fe²⁺ and Cu²⁺ to block the Fenton reaction—these metabolites suppress the generation of ROS such as ·OH, alleviate oxidative damage to intracellular DNA and proteins, and thereby reduce the ROS-scavenging burden on enzymatic antioxidants [47–49]. Among monoterpenoid compounds, paeoniflorin exhibits electrophilicity due to its conjugated benzene-ring system and β-D-glucopyranosyl moiety, enabling it to directly capture and neutralize free radicals. Its isomer albiflorin contains an ester-glycoside group that confers strong electron-donating capacity; in addition to directly reacting with ROS such as O₂·⁻ and ·OH, it also maintains mitochondrial membrane potential and electron-transport-chain function, thereby reducing ROS production derived from electron leakage [50, 51]. Biosynthesis of secondary metabolites consumes substantial quantities of NADPH and ATP; continuous synthesis under non-stress conditions would markedly repress plant growth and development [52]. When stress occurs, plants prioritize activation of defense response mechanisms, and their energy metabolism patterns are reallocated to support the production of defensive compounds, promoting the substantial synthesis of secondary metabolites [53]. The biosynthesis of such compounds is highly dependent on the activation of specific enzyme systems. Among them, PAL, as a key rate-limiting enzyme in the synthesis of flavonoids, is tightly regulated by ROS levels in plants [54, 55]. When plants are subjected to environmental stresses such as pathogen infection, mechanical wounding, or UV radiation, ROS levels rapidly increase, thereby inducing PAL activity, activating the phenylpropanoid pathway, promoting the synthesis of flavonoids, and enhancing plant stress tolerance [56–59]. For instance, under salinity stress in Brassica oleracea L., the PAL gene is activated, thereby promoting the synthesis of flavonoid compounds such as rutin and quercetin [60]. HMGR, a rate-limiting enzyme of the mevalonate pathway, participates in monoterpenoid skeleton biosynthesis [61, 62]; its activity is modulated by ROS and is positively correlated with the contents of paeoniflorin, albiflorin, benzoylpaeoniflorin, and oxypaeoniflorin in P. lactiflora [63, 64]. Furthermore, UGT, as a key modifying enzyme in the secondary metabolism of P. lactiflora, catalyzes glucosyltransferase reactions that modify monoterpenoid precursors into bioactive glycosides, serving as the core enzyme for the synthesis of glycosidic components in Paeonia [65]. Its expression and activity are also regulated by stress-induced ROS. When plants encounter stress, ROS bursts trigger defense signal cascades, activating transcription factor networks, regulating the expression of genes encoding key enzymes in secondary metabolism, substantially enhancing catalytic efficiency, and ultimately promoting the rapid accumulation of defense-related secondary metabolites. For example, under drought stress, the contents of prim-O-glucosylcimifugin, 5-O-methylvisammioside, and cimifugin in Saposhnikovia divaricata are significantly elevated [66], while organic acids, terpenoids, and alkaloids rapidly accumulate in Glycyrrhiza uralensis [67]. For medicinal plants, secondary metabolites often constitute their pharmacologically active components, such as baicalin in Scutellaria baicalensis [68], ginsenosides in Panax ginseng [69], and berberine in Coptis chinensis [70]. Under stress conditions, elevated ROS levels are the fundamental cause of enhanced secondary metabolism, stimulating massive metabolite biosynthesis [71]. Moderate environmental stress promotes the reallocation of resources within plants, leading to a significant increase in the contents of pharmacologically active components over a short timeframe [72].

Our experimental findings demonstrated that spraying H₂O₂ solution on fresh roots of P. lactiflora enhanced the activities of key enzymes PAL, HMGR, and UGT (Fig. 5), resulting in increased contents of flavonoids and monoterpenoids in all treatment groups (Fig. 6). During H₂O₂ treatment, intracellular ROS levels exhibited a trend consistent with the activities of PAL, HMGR, and UGT, all of which increased first and then decreased. Upon treatment with 1.0 mmol/L H₂O₂, the contents of various secondary metabolites initially increased rapidly, which was attributable to the activation of secondary metabolic defense pathways by early stress signals to compensate for the inadequacy of the enzymatic antioxidant system; the subsequent decline in contents resulted from substantial consumption of metabolites to scavenge ROS during the late stage of prolonged stress, accompanied by decreased activities of their synthetic enzymes due to oxidative damage or energy limitation. The differences in secondary metabolite contents between the two germplasms were determined both by the dynamic changes in enzyme activities induced by different concentrations of H₂O₂ treatment and by the metabolic basis and stress response potential governed by their genetic backgrounds. In fresh roots of RPR-germplasm, the 2.0 mmol/L H₂O₂ treatment showed weak inductive effects, possibly because the higher ROS concentration in this group damaged key synthetic enzymes PAL, HMGR, and UGT, inhibiting their activities and consequently limiting the increase in secondary metabolite contents. In contrast, the distilled-water control and 0.5 mmol/L H₂O₂ treatment groups had relatively low ROS concentrations, insufficient to fully activate the self-protective system; antioxidant enzyme potential was not fully exploited, preventing full activation of this resource-intensive defensive secondary metabolic program, as manifested by limited induction of PAL, HMGR, and UGT activities and reduced secondary metabolite synthesis. By comparison, only the 1.0 mmol/L treatment achieved an optimal balance between stress stimulation and oxidative damage, maximizing the activation of PAL, HMGR, and UGT activities and maximizing secondary metabolite accumulation. As shown in Fig. 6, distilled-water treatment of fresh roots of RPA-germplasm also induced ROS accumulation and enhanced antioxidant enzyme activities, and could elevate the contents of certain secondary metabolites such as oxypaeoniflorin, catechin, and benzoic acid. However, its inductive effect on core pharmacologically active components, including paeoniflorin and albiflorin, was weak, with increases significantly lower than those in the 1.0 mmol/L treatment group (P < 0.01). This indicates that although water treatment can activate general metabolic stress responses, it cannot effectively drive the synthesis and accumulation of core pharmacologically active components; rather, specific redox signaling (H₂O₂) serves as the primary trigger for activating their complete biosynthetic pathways. Therefore, water treatment fails to substantially improve medicinal material quality. According to Figs. 2, 4, 5, and 6, during the treatment of fresh roots of both RPR- and RPA-germplasms, as antioxidant enzyme activities declined, the activities of PAL, HMGR, and UGT—key enzymes for flavonoid and monoterpenoid synthesis—were upregulated, accelerating the synthesis of related antioxidants and promoting a rapid decrease in ROS levels. This suggests that secondary metabolites play a crucial compensatory role in plant adaptation to severe stress.

Phenylalanine and isopentenyl pyrophosphate are both intermediates linking primary and secondary metabolism [73]; the upregulated activities of PAL, HMGR, and UGT (Fig. 5) indicate that more primary metabolites are channeled into secondary metabolic pathways. 1,3-DPG, as a high-energy metabolic intermediate in glycolysis, reflects enhanced glycolytic flux and basal metabolic activity when its content increases [74]. This study employed excised fresh roots of P. lactiflora lacking photosynthetic carbon fixation; thus, the increased 1,3-DPG provided sufficient carbon skeletons and energy for secondary metabolism (Fig. 7). The elevated contents of paeoniflorin, albiflorin, oxypaeoniflorin, catechin, and other components mainly stemmed from the targeted conversion of primary metabolic substrates such as carbohydrates, rather than from the reallocation of pre-existing pharmacologically active components, thereby ensuring the improvement of medicinal material quality.

Role of ROS signaling in secondary metabolism under H₂O₂ stress

To further elucidate the signaling role of ROS in H₂O₂-induced secondary metabolism, four treatment groups were established based on the optimal concentration of 1.0 mmol/L H₂O₂: the water control group, 1.0 mmol/L H₂O₂ alone, H₂O₂ + 0.5 mmol/L α-Toc, and H₂O₂ + 2.0 mmol/L NAC. Experimental results showed that H₂O₂ treatment alone not only triggered the bursts of O₂·⁻ and H₂O₂ and induced MDA accumulation (Fig. 8), but also significantly promoted the biosynthesis of a series of characteristic secondary metabolites, including paeoniflorin, paeonol, catechin, and gallic acid (Fig. 9). However, when ROS scavengers were applied, the H₂O₂-induced ROS accumulation and oxidative damage were effectively alleviated, and the accumulation of the aforementioned secondary metabolites was simultaneously and significantly reduced (Figs. 8 and 9). These results, from both positive and negative perspectives, collectively indicate that the inducing effect of exogenous H₂O₂ on secondary metabolism in fresh roots of P. lactiflora is not a direct action, but rather depends on the intracellular accumulation of ROS signals triggered by H₂O₂. Effective ROS scavenging blocked this metabolic response, suggesting that under H₂O₂ stress, elevated ROS levels serve as a key upstream signal for initiating secondary metabolic defense programs. Accordingly, the combined application of ROS scavengers maintained the contents of secondary metabolites at levels close to those in the control group with no significant fluctuations. This can be attributed to the disruption of the stress signaling pathway caused by ROS scavenging, which prevented the induction of secondary metabolite synthesis; simultaneously, the alleviation of oxidative stress avoided the defensive consumption of metabolites, thereby maintaining their stable levels. In summary, this study elucidates from a signal transduction perspective that H₂O₂ regulates the biosynthesis of secondary metabolites in P. lactiflora roots precisely through the critical node of ROS accumulation.

Although our results clearly demonstrate that ROS act as a core regulator in activating secondary metabolism, the underlying signaling network is more complex than a simple linear cascade. Previous studies have reported that other signaling molecules, such as Ca²⁺, jasmonic acid (JA), and salicylic acid (SA), crosstalk with ROS to modulate plant defense responses [75]. For instance, H₂O₂ can activate Ca²⁺ channels to trigger cytosolic Ca²⁺ bursts, further regulating antioxidant and secondary metabolic enzyme activities; in turn, Ca²⁺ signaling also modulates ROS generation [76, 77]. JA and SA are well-known stress-responsive phytohormones whose biosynthesis can be triggered by ROS [78]. Based on our time-series data—where ROS rapidly peaked at days 1–2 after H₂O₂ treatment, whereas secondary metabolites generally accumulated continuously at later time points—we propose that under acute oxidative stress, ROS function as an initial trigger signal, while Ca²⁺ and phytohormones act as downstream amplifiers, forming a ROS-initiated cascade (ROS → Ca²⁺/JA/SA → ROS → Ca²⁺/JA/SA → ROS … → secondary metabolism activation). This model integrates multiple signaling pathways rather than excluding them.

Differential responses of germplasms to H₂O₂ stress

A plant species with a broad distribution range can form multiple ecotypes due to heterogeneous growth conditions across its distribution areas, leading to germplasm differentiation [79]. In response to environmental stresses such as high temperature, saline-alkaline stress, drought, and pests or diseases, different germplasms exhibit distinct stress responses [80]. Through long-term natural selection and artificial domestication, plants have evolved unique physiological and biochemical adaptation mechanisms, a process directly linked to changes in their metabolic pathways. This subsequently leads to variations in the composition and content of secondary metabolites, ultimately manifested in their chemical profiles [81, 82].

Consistent with our antioxidant enzyme activity data, RPR-germplasm possessed a faster and stronger enzymatic antioxidant defense system under H₂O₂ stress, mainly reflected by markedly higher CAT activity with a peak increase ranging from 103.9% to 150.1% and an earlier SOD peak at day 1, which efficiently eliminated excess ROS at the early stress stage. Notably, RPR-germplasm showed more extensive and prominent accumulation of phenolic secondary metabolites under exogenous H₂O₂ treatment, particularly catechin, benzoylpaeoniflorin, and the germplasm-specific metabolite paeonol. As vital non-enzymatic antioxidants in plants, these phenolic compounds mitigate oxidative cellular damage by directly scavenging ROS radicals and inhibiting lipid peroxidation chain reactions. This dual antioxidant capacity from enhanced enzymatic and non-enzymatic systems partly explains why RPR-germplasm exhibited a much lower MDA increase and slighter oxidative injury than RPA-germplasm under identical H₂O₂ treatments. In contrast, RPA-germplasm displayed stronger induction of upstream rate-limiting enzymes for secondary metabolism, namely PAL and HMGR, yet the elevated enzyme activity failed to drive greater accumulation of downstream phenolic metabolites, which may be attributed to differences in post-translational modifications, metabolite transport, or feedback regulation between the two germplasms. Previous studies have indicated that differential expression of antioxidant- and secondary-metabolism-related genes may underlie germplasm-specific stress responses in medicinal plants, while direct molecular evidence remains to be further investigated [83–85]. In this study, the contents of catechin, paeoniflorin, and paeonol in fresh roots of RPR-germplasm treated with 1.0 mmol/L H₂O₂ were increased by 58.5%, 23.7%, and 77.0%, respectively, while the contents of albiflorin, paeoniflorin, and oxypaeoniflorin in fresh roots of RPA-germplasm under the same treatment were increased by 23.0%, 30.1%, and 39.4%, respectively. Notably, these significantly altered components are precisely the pharmacologically active components most closely linked to their clinical efficacy. Such differential responses of specific chemical components to environmental stress not only serve as a direct manifestation of plant adaptation but also constitute an important foundation for crop improvement and medicinal plant development using germplasm resources. By modulating the secondary metabolic pathways of P. lactiflora through ROS signaling, this study has identified a core strategy for enhancing medicinal material quality and revealed the intrinsic differences in secondary metabolite biosynthesis among different germplasms.

Limitations of the present study

This study artificially simulated plant oxidative stress conditions via exogenous H₂O₂ treatment to explore the physiological responses of peony roots. However, basal endogenous ROS levels varied across individual plant samples, which may affect their responsiveness to H₂O₂-induced stress. In addition, plant materials were collected from a single growing season and geographical location, potentially limiting the generalizability of our findings to natural field-grown populations under complex environmental conditions. Although our physiological and biochemical analyses clearly established the “H₂O₂–ROS–secondary metabolism” regulatory axis, direct transcriptional-level evidence for gene expression of key enzymes including PAL, HMGR, and UGT was lacking due to severe RNA degradation of stored root samples, restricting the mechanistic depth of this study. Moreover, we only focused on core secondary metabolites and key synthetic enzymes, without further investigating post-translational modifications, phytohormone dynamics or untargeted metabolomic reprogramming. Nevertheless, this work verifies the feasibility of artificial intervention in secondary metabolism, providing a solid basis for future research on medicinal quality formation through multi-omics strategies such as metabolomics and transcriptomics, as well as systematic molecular pathway analysis.

Conclusion

Exogenous H₂O₂ treatment triggers a ROS burst in fresh roots of P. lactiflora, activating antioxidant enzymes and key secondary metabolic enzymes. These responses synergistically scavenge excess ROS and alleviate oxidative damage. With the extension of stress duration and the decline in antioxidant enzyme activities, secondary metabolism takes on the primary defensive role, leading to a marked accumulation of pharmacologically active compounds, including paeoniflorin, albiflorin, and paeonol. The use of isolated root tissue offers distinct advantages over field-grown treatments, including experimental controllability and lower risk to whole plants, thus serving as a feasible model for targeted induction of secondary metabolism. This study elucidates a “H₂O₂–ROS–secondary metabolism” regulatory axis and provides a practical strategy for enhancing the quality of cultivated medicinal plants through controlled oxidative stress.

Authors’ contributions

KZ: Conceptualization, Data curation, Formal analysis, Methodology, Software, Writing - original draft, Writing - review & editing. XM: Funding acquisition, Supervision, Validation, Writing - review & editing. All authors read and approved the final manuscript.

Funding

This study was supported by the National Natural Science Foundation of China (Grant No. U20A20407).

Data availability

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.