Synergistic effects of H₂O₂, melatonin, and L-cysteine on biochemical traits and alkaloid accumulation in Catharanthus roseus L. “Ocellatus” in vitro plants

Abstract

Background

Elicitation is a widely used strategy to enhance secondary metabolite production in medicinal plants. However, the comparative effectiveness of different redox-modulating elicitors and their interactions in regulating antioxidant systems and terpenoid indole alkaloid (TIA) biosynthesis remains insufficiently understood.

Objectives

This study aimed to evaluate and compare the effects of hydrogen peroxide (H) in combination with melatonin (MT) or L-cysteine (Cys) on redox stability, antioxidant defense, phenolic metabolism, and TIA accumulation in Catharanthus roseus in vitro plants.

Methods

In vitro–grown plants were treated with H₂O₂ (H: 20 µM) alone or combined with different concentrations of MT (100, 200 and 400 µM) or Cys (200, 400 and 800 µM). Photosynthetic pigments, antioxidant enzyme activities (CAT, POD, SOD), oxidative stress indicators (H₂O₂, MDA), total phenolic content (TPC) and total flavonoid content (TFC), antioxidant capacity (TAC, FRAP), phenylpropanoid enzymes (PAL, PPO), and major alkaloids (vincristine, vinblastine, ajmalicine) were quantified using spectrophotometric and HPLC analyses.

Results

All treatments significantly enhanced antioxidant capacity and secondary metabolite accumulation compared with the control. Cys400 + H treatment was most effective for promoting biomass accumulation, activating antioxidant enzymes and TAC, whereas MT200 + H effectively enhanced TPC and minimized oxidative damage. MT400 + H preferentially improved chlorophyll content, while Cys800 + H strongly promoted carbohydrates accumulation and vincristine production. The highest plant growth, TFC and vinblastine content was recorded under H treatment alone. Ajmalicine content decreased under all elicitation regimes, indicating a redirection of metabolic flux within the TIA pathway.

Conclusion

The results demonstrate that H in combination with MT or Cys differentially modulates redox balance and metabolic flux, enabling selective enhancement of antioxidant enzymes and valuable alkaloids. Optimized elicitor combinations therefore provide an effective strategy for targeted metabolic engineering of C. roseus under in vitro conditions.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12870-026-08230-5.

Introduction

Catharanthus roseus L. (Apocynaceae) is a perennial species native to Madagascar that is renowned for both its medicinal and ornamental importance. The plant is a rich source of diverse phytochemicals, including carbohydrates, flavonoids, saponins, and, most notably, alkaloids. These compounds possess broad potential for application across the pharmaceutical and agrochemical industries. To date, over 200 terpenoid indole alkaloids (TIAs) have been identified in C. roseus, representing some of the most pharmacologically valuable secondary metabolites [1]. Among the therapeutically important alkaloids are vincristine, vinblastine, vindesine, vindeline, tabersonine, ajmalicine, raubasine, and catharanthine, which are present in all organs of the plant, although at very low concentrations [2, 3]. Despite their high pharmaceutical value, the inherently low abundance and complex biosynthetic regulation of these alkaloids continue to limit their large-scale production.

Several pharmacological activities have been reported for this species, including anticancer, antidiabetic, antimicrobial, antioxidant, antiulcer, hypotensive, antidiarrheal, wound-healing, and memory-enhancing properties [3, 4]. Owing to their extensive applications across diverse industries and the increasing demand in recent decades, medicinal plants face significant challenges in large-scale propagation and production. This demand has exposed fundamental limitations of conventional cultivation approaches, particularly for metabolites that are tightly regulated by developmental and environmental cues. Consequently, researchers have increasingly emphasized the development and optimization of biotechnological strategies, particularly plant tissue culture, to ensure a sustainable supply of bioactive compounds. Compared with conventional propagation, plant tissue culture represents a more time-efficient and reliable system for large-scale multiplication. Beyond propagation, in vitro systems offer a powerful experimental platform to manipulate metabolic regulation under controlled conditions. In addition to propagation, in vitro culture techniques can enhance morphological, physiological, and phytochemical attributes, thereby improving the overall quality of medicinal plants [5].

Several limitations inherent to conventional propagation, such as seed dormancy, low seed viability and germination rates, dependence on artificial pollination, susceptibility to pathogens, and reproductive barriers such as herkogamy, can adversely influence the phytochemical profile of C. roseus [6]. To overcome these challenges, plant producers and researchers are prioritizing advanced biotechnological approaches—particularly tissue culture—for the enhanced biosynthesis of secondary metabolites, genetic improvement, and large-scale mass propagation of this economically valuable medicinal plant [6, 7]. Various in vitro techniques, such as precursor feeding, elicitation, and culture medium optimization, have emerged as cost-effective, straightforward, and efficient approaches for improving the biosynthesis of biocompounds [8]. Among these strategies, elicitation is uniquely suited to investigate and manipulate stress-responsive pathways that control secondary metabolite production.

The production and large-scale micropropagation of C. roseus have substantially advanced through in vitro culture techniques, optimized by the application of diverse plant growth regulators [9]. Among the strategies used to increase metabolite yield, elicitation is considered one of the most effective approaches for stimulating the biosynthesis of bioactive compounds [10, 11]. Elicitors are broadly categorized into biotic and abiotic groups, and their ability to promote secondary metabolite accumulation depends on several factors, including concentration, exposure duration, and the developmental stage of the culture [5, 12]. In this respect, elicitation is a method that includes the addition of exogenous elicitor compounds to the growth medium to induce a stress response, resulting in improved production of valuable secondary metabolites, including phenolic compounds and medicinal alkaloids [13]. Hydrogen peroxide (H₂O₂), a key abiotic elicitor, is naturally generated during oxidative stress metabolism and belongs to the group of reactive oxygen species (ROS). Unlike other ROS, H₂O₂ (H) is relatively stable in solution because of its neutral charge [14]. It is synthesized in plants via both enzymatic and non-enzymatic pathways and is considered a non-radical ROS [15, 16]. Its role in plant physiology is concentration-dependent: at moderate levels (1–5 mM g^{− 1} FW), it functions as a signaling molecule regulating adaptation and stress tolerance mechanisms, whereas higher concentrations (> 7 mM g^{− 1} FW) trigger programmed cell death [17]. In addition to stress signaling, H participates in diverse developmental and physiological processes, including the enhancement of photosynthetic efficiency, carbohydrate metabolism, seed germination, flowering, senescence, root and shoot development, and the activation of antioxidative defense mechanisms [18]. As a regulator, growth stimulator, and metabolic modulator, H influences cellular signaling cascades and gene expression [19]. Importantly, it is classified as a GRAS (generally recognized as safe) and is approved for use in organic agricultural systems [15].

Melatonin (MT: N-acetyl-5-methoxytryptamine) has similarly emerged as a multifunctional signaling molecule with profound roles in regulating plant growth and development. The amino acid tryptophan serves as the initial precursor in the biosynthesis pathway of MT [20]. MT contributes to the regulation of photosynthetic pigments and phytohormone biosynthesis (IAA, ABA, GA and SA) and alleviates oxidative stress through its ability to scavenge ROS. Genomic studies further highlight its regulatory influence on transcription factors and plant signaling networks [20, 21]. For example, the application of MT improves the redox state and essential oil biosynthesis in two species of Salvia [22] and confers drought tolerance in Areca catechu [23] by stimulating antioxidant enzyme activities and reducing ROS accumulation.

L-cysteine (Cys), a sulfur-containing amino acid characterized by its thiol (-SH) group, serves as both a signaling molecule and a potent antioxidant. It functions as a precursor for the biosynthesis of various biomolecules, including vitamins and cofactors, and participates in enzymatic reactions that provide antioxidative protection [24, 25]. Exogenous supplementation with Cys and other amino acids has been shown to increase nitrogen assimilation, vitamin C accumulation, growth vigor, and yield, thereby mitigating the adverse effects of environmental stress [26, 27].

Collectively, the results of previous studies highlight that the individual application of MT, Cys, and H strongly influence the biochemical and physiological traits of medicinal plants, ultimately promoting secondary metabolite biosynthesis. A critical unresolved question remains: how does H-induced signaling interact with distinct redox modulators, such as MT and Cys, to regulate antioxidant capacity and secondary metabolite biosynthesis? We hypothesized that H acts as a primary signaling trigger, while MT and Cys differently adjust cellular redox conditions, leading to distinct physiological responses and phytochemical profiles.

To the best of our knowledge, this investigation is the first to report the effects of the combination of the signaling molecule H with MT and Cys on the phytochemical attributes of C. roseus “Ocellatus” under in vitro conditions. By integrating physiological, biochemical, and metabolic profiles, this work aims to establish a framework for elicitor-driven redox modulation and selective metabolite enhancement in medicinal plants.

Materials and methods

Plant material preparation and growth conditions

Nodal shoot explants (each containing two leaf pairs) were obtained from one-month-old greenhouse-grown seedlings and used as the initial source material for tissue culture. Explants were surface sterilized by sequential washing under running tap water for 6 min with two drops of Tween-20, immersion in 70% ethanol for 1 min, exposure to 2% sodium hypochlorite for 9 min, and finally washing three times with sterile distilled water. The sterilized explants were cultured in 50 mL of Murashige and Skoog [28] medium (MS) in glass jars and maintained in a controlled growth chamber at 27/24°C (day/night), with a photoperiod of 16 h light/8 h dark under white fluorescent lamps (65 µmol m^{− 2} s^{− 1}). Subculturing was carried out at four-week intervals to ensure sufficient plant growth and uniformity. For treatments, three plantlets each were transferred to basal MS medium jars supplemented with 20 µM hydrogen peroxide (30% H₂O₂, analytical grade, Sigma-Aldrich) in combination with MT: 100, 200, or 400 µM (≥ 98%, Sigma-Aldrich) or Cys: 200, 400, or 800 µM (≥ 98.5%, Sigma-Aldrich). Stock solutions were sterilized through 0.22 μm filters and were added to cooling medium before solidification. The cultures were maintained for three months under the same growth conditions. The leaves were harvested and stored at − 80 °C for enzymatic measurements, while dried plant material (40 °C for 48 h) was used for phytochemical characterization, including antioxidant assays and total phenolic and flavonoid content determination.

Measurements

Growth attributes

At the end of the growth period, the whole plants were harvested, and the roots were carefully washed under tap water to remove residual MS medium, after which the roots were gently blotted dry with facial tissues. Plant height, number of leaves and roots, fresh and dry weights were measured in this study. The drying process for determination of dry weight included oven-drying the plant material at 40 °C for 48 h.

Photosynthetic pigments

Photosynthetic pigments were quantified according to previous reports [29, 30]. Briefly, 1000 mg of fresh leaves were homogenized in 10 mL of 80% acetone and centrifuged at 6000 rpm for 10 min. The absorbance was recorded at 663, 645, and 470 nm using a UV–VIS spectrophotometer (Specord). The concentrations of chlorophyll a, chlorophyll b, and carotenoids were calculated using standard Eqs. (1, 2, and 3) and expressed as mg g^{− 1} fresh weight (FW).

1

2

3

Soluble and reductive carbohydrate contents

The carbohydrate contents were determined according to a previously reported protocol [31]. Segments of fresh leaves (200 mg) were homogenized in 10 mL of 96% ethanol and incubated at 80 °C in a water bath for 1 h. For soluble carbohydrate measurement, 1 mL of plant ethanolic extract was mixed with 1 mL of 5% phenol and 5 mL of concentrated sulfuric acid (98%), and the absorbance was recorded at 483 nm after 30 min of incubation [32]. For reducing carbohydrate measurement, 2 mL of ethanolic extract was mixed with 1 mL of a colored reagent (1% dinitrosalicylic acid, 1.6% NaOH, and 25% sodium potassium tartrate), boiled at 100 °C for 10 min, cooled, and diluted with 10 mL distilled water. The absorbance was read at 546 nm [33]. Standard calibration curves were generated using glucose (0, 5, 10, 15, 20, 25, and 50 µg), and values were expressed as µg g^{− 1} FW.

Plant extract preparation

For phytochemical analysis, 20 mg of dried powdered tissue was macerated in 2 mL of 80% methanol at room temperature (approximately 25 °C) for 48 h. The methanolic extracts were centrifuged at 10,000 rpm for 15 min, and the supernatant was stored at 4 °C for subsequent assays. For enzymatic analyses, 600 mg of frozen tissue was homogenized in 2 mL of ice-cold 50 mM sodium phosphate buffer (pH 7) containing 1 mM EDTA and 1% (w/v) polyvinylpyrrolidone. The homogenate was centrifuged at 15,000 rpm for 20 min at 4 °C, and the extract was used for enzyme activity assays.

Total flavonoid content (TFC) and total phenolic content (TPC) assays

Total flavonoids were quantified via the aluminum chloride colorimetric method [34]. The mixture contained 2.75 mL of distilled water, 0.3 mL of plant methanolic extract, 0.1 mL of aluminum chloride (10%) and 0.1 mL of potassium acetate (1 M). After incubation in the dark for 30 min, the absorbance was read at 415 nm, and quercetin was used for standard calibration.

The total phenolic content was estimated via the Folin–Ciocalteu method [35]. The reaction mixture contained 1.95 mL of distilled water, 0.3 mL of methanolic extract, 0.5 mL of Folin–Ciocalteu solution (10%), and 0.5 mL of 7% sodium carbonate. After 30 min of incubation in the dark, the absorbance was measured at 765 nm. The results are expressed as mg quercetin equivalents (mg QE g^{− 1} DW) for total flavonoids and mg gallic acid equivalents (mg GAE g^{− 1} DW) for phenolic compounds.

Enzyme activity assays: phenylalanine ammonia lyase (PAL) and polyphenol oxidase (PPO) measurements

PAL activity was assayed by homogenizing 600 mg of plant tissue in 3 mL of 0.1 M sodium borate buffer and centrifuging it at 15,000 rpm for 20 min. The mixture contained 0.5 mL of the resulting supernatant, 2 mL of 0.1 M sodium borate buffer, and 0.5 mL of 20 mM phenylalanine. The samples were incubated at 37 °C for 60 min, and the absorbance was measured at 290 nm [36].

The PPO activity was determined using 0.9 mL of 100 mM sodium phosphate buffer (pH 7.8), 0.25 mL of catechol (0.5 M), and 0.35 mL of plant extract. The change in absorbance was recorded at 420 nm for 2 min [36].

Total antioxidant capacity (TAC) and ferric reducing antioxidant power (FRAP) assays

The total antioxidant capacity (TAC) was measured via the addition of 1 mL of sulfuric acid (0.6 M), ammonium molybdate (4 mM), and sodium triphosphate to 0.3 mL of plant extract, which was subsequently incubated at 95 °C for 90 min [37]. The absorbance was recorded at 695 nm, and the values are expressed as mg ascorbic acid equivalents (mg AAE g^{− 1} DW).

Ferric reducing antioxidant power (FRAP) was evaluated following a previous study [38]. The extracts were mixed with potassium ferricyanide (1%) and phosphate buffer (0.2 M, pH 6.6), vortexed for 5 min, incubated for 20 min at 50 °C, and then treated with trichloroacetic acid (10%). The absorbance was recorded at 700 nm, and the results are expressed as mg ascorbic acid equivalent (mg AAE g^{− 1} DW).

Antioxidant enzyme activity: catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) measurements

CAT activity was determined by monitoring H₂O₂ decomposition at 240 nm in a mixture containing 2 mL of citrate–phosphate–borate buffer (0.1 M, pH 7), 0.05 mL of plant extract, and 0.026 mL of H₂O₂ (0.1 M). The decrease in absorbance was monitored spectrophotometrically for 2 min and expressed as U g^{− 1} FW [39]. POD activity was estimated by monitoring guaiacol oxidation at 470 nm. The reaction mixture contained 2 mL of citrate–phosphate–borate buffer (0.1 M, pH 7.0), 0.1 mL of guaiacol (480 mM), 0.1 mL of plant extract, and 0.1 mL of H₂O₂ (0.1 M). The absorbance changes were recorded continuously for 2 min and expressed as U g^{− 1} FW [39].

SOD activity was evaluated by measuring the inhibition of nitro-blue tetrazolium reduction at 560 nm. The assay mixture comprised 2.3 mL of sodium phosphate buffer (100 mM, pH 7.8), 0.1 mL of EDTA-KCN, 0.1 mL of nitro-blue tetrazolium (75 mM), and 0.1 mL of plant extract. After an 8 min incubation in the darkness, 0.1 mL of riboflavin (0.12 mM) was added, and the mixture was then exposed to light (40 W) for 10 min. Absorbance was measured at 560 nm and expressed as U g^{− 1} FW [40].

Lipid peroxidation (malondialdehyde content)

The malondialdehyde (MDA) content, a marker of lipid peroxidation, was measured using the thiobarbituric acid reaction [41]. A 100 mg frozen leaf sample was homogenized in 5 mL of 0.1% (w/v) trichloroacetic acid (TCA) and centrifuged at 10,000 rpm for 5 min at 4 °C. An aliquot of 0.3 mL of the supernatant was mixed with 1.2 mL of 0.5% thiobarbituric acid (TBA) prepared in 20% (w/v) TCA and incubated at 95 °C for 30 min. The reaction was terminated by placing the samples in an ice bath for 5 min, followed by centrifugation at 10,000 rpm for 10 min at 25 °C. The absorbance was recorded at 532 and 600 nm, and the MDA content was expressed as nmol g⁻¹ fresh weight (FW) using Eq. (4).

4

Hydrogen peroxide (H₂O₂) content

The quantification of hydrogen peroxide (H₂O₂) was performed by homogenizing 100 mg of fresh plant tissue in 5 mL of 0.1% (w/v) TCA and centrifuging at 12,000 rpm for 10 min at 4 °C [42]. The supernatant was subsequently neutralized with 1 mL of 1 M KI and 0.5 mL of 10 mM phosphate buffer (pH 7). The absorbance of the reaction mixture was measured at 390 nm, and the H₂O₂ content was expressed as nmol g⁻¹ FW.

Extract preparation and HPLC properties of alkaloids

The extraction was performed according to a previously described protocol, with slight modifications [43]. Fifty milligrams of dry tissue (oven dried for 48 h at 40 °C) was macerated in 3 mL of 80% methanol and left to stand overnight. The resulting methanolic extract was sonicated for 30 min at 50 °C in an ultrasonic system. The extract was subsequently shaken for 24 h and centrifuged at 3500 rpm for 15 min. The mixture was then acidified with 5 mL of 3% hydrochloric acid, and 5 mL of chloroform was added. The chloroform phase was collected, and the pH was adjusted to 11 with ammonia.

Subsequently, 5 mL of chloroform was added, the mixture was shaken for 5 min, and 5 mL of ammonia was added. The ammonia phase was separated, while the chloroform phase was subjected to rotary evaporation. After evaporation, the residue was dissolved in methanol and analyzed via HPLC (KNAUER system, Germany) using a Eurospher column (250 × 4.6 mm, 5 μm). Separation was achieved using phosphate buffer (pH 6.0) as mobile phase A and acetonitrile as mobile phase B under a gradient program. The gradient elution program was optimized as follows (A/B, v/v): 0–25 min, linear gradient from 75/25 to 25/75; 25–30 min, 20/80; and 30–40 min, 75/25. The flow rate was 1 mL min⁻¹.

Statistical analysis

A completely randomized design (CRD) was used. The data were analyzed by one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test at p ≤ 0.05, and the results are expressed as means ± standard error (SE) from three independent biological replicates (n = 3) using SAS software (version 9.4). The PCA and Pearson correlation coefficient were generated using the Python programming language.

Results

Growth attributes

Morphological growth parameters of C. roseus, including plant height, number of leaves, number of roots, and biomass accumulation (fresh and dry weight), were significantly influenced by elicitor type and concentration (p < 0.01; Table 1). Overall, elicitor treatments markedly improved plant growth compared with the control. Plant height responded most strongly to H and Cys200 + H treatments, exhibiting approximately a three-fold greater height than the control. Intermediate and high Cys concentrations (Cys400 + H and Cys800 + H, respectively) also significantly enhanced plant height, although to a lesser extent, indicating a concentration-dependent growth response to Cys. H alone produced the highest number of leaves, corresponding to an approximately 1.5-fold increase relative to the control, indicating a stimulatory role of H in shoot development. Root number was most strongly enhanced under MT100 + H and H alone, showing approximately 3.3- and 3.1-fold increases compared with the control. In contrast, higher concentrations of MT + H strongly suppressed root formation, indicating an inhibitory effect of excessive MT concentrations on root development. MT200 + H and MT400 + H predominantly induced callus formation rather than a functional root system.

Table 1.

Effect of H₂O₂ in combination with melatonin and L- cysteine on in vitro-cultured Catharanthus roseus L. “Ocellatus” plants

Treatment			Growth attributes
			Height (cm)	Number of leaves	Number of roots	Fresh weight (mg plant⁻¹)	Dry weight(mg plant⁻¹)
Control (MS medium)			4.23 ± 0.7e	50 ± 0d	13.66 ± 0.57f	6011.2 ± 55.6e	539.93 ± 44.1c
H20 µM			12.6 ± 0.5a	76 ± 1.2a	42 ± 1.20b	9394.5 ± 12.8a	641.13 ± 31.1ab
MT100 µM + H20 µM			7 ± 1.0cd	61.66 ± 0.5b	45.66 ± 1.52a	6826.4 ± 18.3d	643.5 ± 26.2ab
MT200 µM + H20 µM			6.50 ± 0.8d	44.33 ± 1.5e	0g	1601 ± 33.3h	185.33 ± 1e
MT400 µM + H20 µM			6.8 ± 0.6cd	62.66 ± 1.1b	0g	3564.3 ± 97.1g	435.77 ± 2d
Cys200 µM + H20 µM			12.6 ± 0.5a	56.66 ± 1.1c	22.33 ± 0.57e	7475.03 ± 13.5c	601.87 ± 7.8b
Cys400 µM + H20 µM			7.5 ± 1.1c	51.33 ± 1.15d	37.33 ± 1.15c	8466.5 ± 62.1b	658.17 ± 21.2a
Cys800 µM + H20 µM			9 ± 0.5b	26.66 ± 1.15f	32 ± 2.21d	5269.7 ± 74.2f	603.37 ± 18.7b
Significant	DF
Treatments	7		**	**	**	**	**
Error	16		0.33	1	0.66	2959.9	626.89
CV			6.95	1.8	3.28	0.89	4.64

Note: Values indicate mean ± SE (n = 3). Different lowercase letters in the columns represent significant differences among the treatments according to one-way ANOVA (Duncan’s test; *, p < 0.05; **p < 0.01; ns, not significant)

Abbreviations: H H₂O_2, MT Melatonin, Cys L-cysteine

Biomass accumulation closely mirrored these growth trends. Fresh weight was maximized under H alone, followed by Cys400 + H and Cys200 + H, representing a 1.6-fold increase compared with the control, followed by Cys400 + H and Cys200 + H, which increased fresh weight by approximately 1.4- and 1.2-fold, respectively. The highest dry weight was recorded under Cys400 + H and H, corresponding to approximately 1.2-fold increases relative to the control, indicating improved structural biomass formation rather than transient water accumulation. MT200 + H and MT400 + H exhibited the lowest fresh and dry weights, further confirming their growth-inhibitory effects at higher doses.

Collectively, these results demonstrate that H alone and Cys + H elicitation—particularly at low to intermediate concentrations—were the most effective strategies for promoting overall plant growth and biomass accumulation, whereas MT exerted concentration-dependent effects, stimulating growth at low doses but inhibiting development at intermediate and high levels.

Photosynthetic pigments

Photosynthetic pigment accumulation was significantly influenced by elicitor type and concentration (p < 0.01; Table 2). Overall, MT + H were most effective for enhancing chlorophyll a and b, whereas Cys + H treatments preferentially promoted chlorophyll b and carotenoid accumulation. Chlorophyll a content increased markedly under all treatments compared with the control. The strongest stimulation was observed under MT400 + H, which resulted in the highest chlorophyll a content (2.87 mg g⁻¹ FW), representing a 205% increase relative to the control. Other MT- and Cys-based treatments also significantly increased chlorophyll a, but their effects were consistently lower than those of MT400 + H, indicating that intermediate to high MT concentrations were optimal for chlorophyll biosynthesis. A similar trend was observed for chlorophyll b. All treatments significantly increased chlorophyll b content, with the greatest increases (129% and 125%) recorded under H alone and MT400 + H, respectively. In contrast to chlorophyll a, carotenoid accumulation responded most strongly to Cys-based treatments. The highest carotenoid contents were recorded under Cys400 + H and Cys800 + H, corresponding to approximately 100% increases relative to the control.

Table 2.

Effect of H₂O₂ in combination with melatonin and L- cysteine on in vitro-cultured Catharanthus roseus L. “Ocellatus” plants

Treatment			Photosynthesis pigments			Carbohydrate content
			Chlorophyll a(mg g− 1 FW)	Chlorophyll b (mg g− 1 FW)	Carotenoid(mg g− 1 FW)	Soluble carbohydrate (µg g− 1 FW)	Reductive carbohydrate (µg g− 1 FW)
Control (MS medium)			0.94 ± 0.16f	0.55 ± 0.02e	0.065 ± 0.001bc	1820.9 ± 61.01e	102.067 ± 3.7f
H20 µM			2.19 ± 0.06cd	1.26 ± 0.07a	0.06 ± 0.0009c	3203.8 ± 25.6a	108.733 ± 3.5ef
MT100 µM + H20 µM			1.78 ± 0.14e	0.86 ± 0.05cd	0.051 ± 0.003d	2310.7 ± 238.4d	86.067 ± 2.4g
MT200 µM + H20 µM			2.72 ± 0.06ab	0.99 ± 0.01bc	0.025 ± 0.001e	2449.1 ± 200.1cd	141.4 ± 4.6d
MT400 µM + H20 µM			2.87 ± 0.13a	1.24 ± 0.05a	0.017 ± 0.0005e	2744.7 ± 63.1bc	187.4 ± 11b
Cys200 µM + H20 µM			2.04 ± 0.13de	0.78 ± 0.02d	0.07 ± 0.002b	2717 ± 2.8bc	122.733 ± 4.8e
Cys400 µM + H20 µM			2.05 ± 0.06de	1.18 ± 0.004a	0.13 ± 0.005a	2847.8 ± 28.1ab	164.733 ± 1.3c
Cys800 µM + H20 µM			2.49 ± 0.15bc	1.02 ± 0.09b	0.12 ± 0.001a	3172.3 ± 124.7a	362.067 ± 3.5a
Significant	DF
Treatments	7		**	**	**	**	**
Error	16		0.045	0.008	0.00002	45619.236	79.5
CV			9.91	9.14	6.94	8.03	5.59

Note: Values indicate mean ± SE (n = 3). Different lowercase letters in the columns represent significant differences among the treatments according to one-way ANOVA (Duncan’s test; *, p < 0.05; **p < 0.01; ns, not significant)

Abbreviations: H H₂O_2, MT Melatonin, Cys L-cysteine

Soluble and reductive carbohydrate contents

Changes in photosynthetic pigments were accompanied by pronounced increases in carbohydrate accumulation, indicating coordinated regulation of carbon assimilation and storage. Both soluble and reductive carbohydrate contents were significantly enhanced by all treatments relative to the control (p < 0.01; Table 2). Soluble carbohydrate levels increased consistently across treatments, with the H, Cys800 + H, and Cys400 + H treatments producing the strongest responses, resulting in 1.76, 1.74 and 1.56-fold increases compared with the control. Treatments that induced higher photosynthetic pigments accumulation generally exhibited greater soluble carbohydrate content, indicating a positive association between enhanced photosynthetic capacity and carbon fixation efficiency. Reductive carbohydrate accumulation showed a pronounced concentration-dependent response to both MT and Cys; however, the magnitude of enhancement was substantially greater under Cys-H treatments. The highest reductive carbohydrate content was observed under Cys800 + H, reaching 362.07 µg g⁻¹ FW, which corresponds to a 3.55-fold increase over the control. This marked stimulation suggests that high Cys availability, in combination with H signaling, strongly promotes reductive carbon metabolism and sugar accumulation. Cys + H were the most effective treatments for stimulating both soluble and reductive carbohydrates biosynthesis, particularly at 800 µM concentration. Collectively, these results demonstrate that while all elicitor treatments enhanced carbohydrate content relative to the control, Cys + H—particularly at 800 µM—was the most effective in maximizing both soluble and reductive carbohydrates, highlighting its superior role in strengthening carbon assimilation.

TFC and TPC

Application of H in combination with MT or Cys significantly enhanced TPC and TFC compared with the control (p < 0.01). TPC responded most strongly to MT + H treatments, particularly at intermediate and high MT concentrations (Fig. 1). MT200 + H and MT400 + H produced the greatest increases in TPC (23.64% and 20.49%, respectively), indicating that MT was more effective than Cys in promoting phenolic compound accumulation. Cys200 + H also significantly enhanced TPC, but to a lesser extent (11.0%), suggesting a comparatively weaker stimulation of phenolic biosynthesis. In contrast, H alone preferentially promoted flavonoid biosynthesis. H alone induced the highest flavonoid accumulation, resulting in more than a two-fold increase relative to the control. Cys200 + H and MT200 + H to a lesser extent enhanced TFC (60.6% and 47.0%, respectively). These findings suggest that Cys + H, particularly at low concentration, was effective in promoting flavonoid accumulation, whereas MT + H treatments were more effective for enhancing TPC.

Fig. 1.

Mean comparison of hydrogen peroxide (H: 20 µM) in combination with melatonin (MT: 100, 200, 400 µM) and L-cysteine (Cys: 200, 400, 800 µM) on phenol metabolism (TPC (A), TFC (B), PAL (C), and PPO (D)) on in vitro-cultured Catharanthus roseus L. “Ocellatus” plants. Values presented are the means ± SE (n = 3). Different lowercase letters indicate significant differences among treatments (Duncan’s test, p < 0.05)

Enzyme activity assays: PAL and PPO

The accumulation of phenolics and flavonoids was accompanied by significant changes in the activities of key phenylpropanoid enzymes, phenylalanine ammonia-lyase (PAL) and polyphenol oxidase (PPO). PAL activity increased significantly under all elicitor treatments compared with the control (p < 0.01; Fig. 1), confirming activation of the phenylpropanoid pathway. Overall, both MT and Cys effectively activated phenylpropanoid metabolism, with maximal responses observed at intermediate concentrations. The strongest induction occurred under Cys400 + H and MT200 + H, which enhanced PAL activity by 163.1% and 128.4%, respectively.

PPO activity exhibited an even more pronounced response. Cys400 + H and MT200 + H resulted in approximately 8- and 5-fold increases, respectively, demonstrating strong activation of downstream phenolic oxidation processes. Notably, H alone also induced a substantial increase in PPO activity (7.33-fold) compared with the control.

Total antioxidant capacity (TAC) and ferric reducing antioxidant power (FRAP) assays

Antioxidant capacity, as assessed by TAC and FRAP, was significantly influenced by elicitor type and concentration (Table 3). Overall, Cys + H treatments were more effective than MT + H in enhancing antioxidant capacity. The highest TAC value was recorded under Cys400 + H, representing a 30.6% increase relative to the control. FRAP values increased markedly under all elicitor treatments, with the most pronounced enhancement observed under Cys200 + H, which resulted in a 3.73-fold increase. These results indicate that Cys + H, particularly at low to intermediate concentrations, substantially enhances antioxidant potential.

Table 3.

Effect of H₂O₂ in combination with melatonin and L- cysteine on in vitro-cultured Catharanthus roseus L. “Ocellatus” plants

Treatment			Antioxidant activity assay
			TAC (mg AAE g− 1 DW)		PFRAP (mg AAE g− 1 DW)
Control (MS medium)			158.75 ± 3.3 b		21.48 ± 0.2 e
H20 µM			159.89 ± 19.4 b		48.97 ± 5.08 bc
MT100 µM + H20 µM			161.82 ± 0.4 b		45.99 ± 0.2 cd
MT200 µM + H20 µM			161.29 ± 15.3 b		55.37 ± 0.7 b
MT400 µM + H20 µM			158.31 ± 8.5 b		51.86 ± 0.15 bc
Cys200 µM + H20 µM			174.27 ± 7.2 b		80.02 ± 4.8 a
Cys400 µM + H20 µM			207.34 ± 5.3 a		55.46 ± 1.5 b
Cys800 µM + H20 µM			179.18 ± 4.8 ab		39.5 ± 1.49 d
Significant	DF
Treatments	7		*		**
Error	16		301.61		20.57
CV			10.2		9.1

Note: Values indicate mean ± SE (n = 3). Different lowercase letters in the columns represent significant differences among the treatments according to one-way ANOVA (Duncan’s test; *, p < 0.05; **p < 0.01; ns, not significant)

Abbreviations H H₂O_2, MT Melatonin, Cys L-cysteine

Antioxidant enzyme activity: catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) measurements

Antioxidant enzyme activities were significantly influenced by the treatments (p < 0.01; Table 4). Overall, Cys + H treatments were the most effective in activating antioxidant enzymes, whereas MT + H treatments were more efficient in limiting oxidative damage. Among all treatments, Cys400 + H induced the strongest enzymatic response. Under this treatment, CAT activity increased by approximately 400%, indicating a marked enhancement of H₂O₂-scavenging capacity. POD activity increased 9.36-fold, highlighting strong stimulation of ROS-detoxifying pathways at intermediate Cys concentration. SOD activity was also significantly elevated, with Cys400 + H and Cys800 + H resulting in an 18.5% increase relative to the control.

Table 4.

Effect of H₂O₂ in combination with melatonin and L- cysteine on in vitro-cultured Catharanthus roseus L. “Ocellatus” plants

Treatment			Antioxidant enzymes activity
			CAT(U g− 1 FW)		POD(U g− 1 FW)		SOD(U g− 1 FW)		H2O2(nmol g− 1 FW)		MDA(nmol g− 1 FW)
Control (MS medium)			1.73 ± 0.00 e		0.36 ± 0.01 h		72.1 ± 0.5 b		1.29 ± 0.02 c		0.24 ± 0.01 a
H20 µM			2.59 ± 0.003 d		2.33 ± 0.06 b		69.2 ± 4.4 b		1.7 ± 0.02a		0.19 ± 0.003 c
MT100 µM + H20 µM			5.19 ± 0.013 c		1.31 ± 0.07 e		76.5 ± 8.3ab		1.73 ± 0.17 a		0.22 ± 0.01 b
MT200 µM + H20 µM			2.88 ± 0.21 d		1.94 ± 0.09 c		77.83 ± 9.8 ab		0.5 ± 0.36 e		0.048 ± 0.008 g
MT400 µM + H20 µM			1.77 ± 0.07 e		1.44 ± 0.03 d		71.6 ± 1.3 b		1.5 ± 0.05 b		0.126 ± 0.002 e
Cys200 µM + H20 µM			6.39 ± 0.46 b		1.01 ± 0.06 f		76.5 ± 6.8ab		1.5 ± 0.13 b		0.15 ± 0.001 d
Cys400µM + H20 µM			6.92 ± 0.26 a		3.37 ± 0.08 a		85.5 ± 0.45 a		1.57 ± 0.1b		0.08 ± 0.002 f
Cys800 µM + H20 µM			2.59 ± 0.13 d		0.73 ± 0.06 g		85.5 ± 0.85 a		1.57 ± 0.14 b		0.08 ± 0.009 f
Significant	DF
Treatments	7		**		**		*		**		**
Error	16		0.046		0.0047		29.34		0.027		0.00006
CV			5.75		4.39		7.04		11.05		5.40

Note: Values indicate mean ± SE (n = 3). Different lowercase letters in the columns represent significant differences among the treatments according to one-way ANOVA (Duncan’s test; *, p < 0.05; **p < 0.01; ns, not significant)

Abbreviations: H H₂O_2, MT Melatonin, Cys L-cysteine

Lipid peroxidation (MDA) and H₂O₂ content

Lipid peroxidation and endogenous H₂O₂ levels were significantly reduced by elicitor treatments compared with the control (p < 0.01; Table 4), indicating effective alleviation of oxidative stress. Overall, MT + H—particularly at intermediate concentration—was the most effective strategy for minimizing oxidative damage, whereas Cys + H treatments provided substantial but comparatively lower protection. Malondialdehyde (MDA) content, an indicator of membrane lipid peroxidation, was markedly suppressed under all elicitor treatments. The most pronounced reduction was observed under MT200 + H, where MDA levels decreased by approximately 80% relative to the control. This was followed by Cys400 + H and Cys800 + H, which reduced MDA accumulation by 67% and 66%, respectively. These results indicate that both elicitor strategies effectively limited membrane oxidative damage, with MT exhibiting superior efficacy. Endogenous H levels showed a similar response pattern. All treatments significantly decreased H accumulation relative to the control, with the greatest reduction (76%) recorded under MT200 + H. In contrast, H₂O₂ levels remained relatively higher under H alone and less effective MT treatments.

Medicinal alkaloids

HPLC analysis revealed that elicitor application significantly altered the accumulation of vincristine, vinblastine, and ajmalicine (Fig. 2). Overall, all treatments markedly increased vincristine and vinblastine contents compared with the control, confirming effective stimulation of terpenoid indole alkaloid (TIA) biosynthesis. Cys + H and H alone were the most effective treatments for enhancing vincristine accumulation. According to Fig. 2A, vincristine content increased progressively with increasing Cys concentration, reaching a maximum under Cys800 + H, which produced the highest vincristine level (1.10 µg g⁻¹ DW) and the greatest increase relative to the control (14.32%). The highest vinblastine content was recorded under H treatment alone (3.47 µg g⁻¹ DW; Fig. 2B). Significant vinblastine enhancement was observed at higher concentrations of Cys (Cys800 + H) and to a lesser extent at all MT concentrations. Overall, the treatments consistently stimulated a pronounced increase in either vincristine or vinblastine levels relative to those in the control. Ajmalicine content exhibited an inverse response pattern relative to vincristine and vinblastine, with the highest levels detected in the control plants, while all treatments reduced ajmalicine accumulation (Fig. 2C).

Fig. 2.

Mean comparison of hydrogen peroxide (H: 20 µM) in combination with melatonin (MT: 100, 200, 400 µM) and L-cysteine (Cys: 200, 400, 800 µM) on vincristine content (A), vinblastine content (B) and Ajmalicine content (C) on in vitro-cultured Catharanthus roseus L. “Ocellatus” plants. Values presented are the means ± SE (n = 3). Different lowercase letters indicate significant differences among treatments (Duncan’s test, p < 0.05)

Correlation analysis

To elucidate the relationships among photosynthetic pigments; soluble and reducing carbohydrates; MDA; hydrogen peroxide contents; TAC; FRAP; antioxidant enzyme activities (CAT, POD, and SOD); phenolic metabolism parameters (TPC, TFC, PAL, and PPO); and alkaloid accumulation (vincristine, vinblastine, and ajmalicine) in C. roseus extracts, a correlation analysis was performed. The correlation heatmap is presented in Fig. 3.

Fig. 3.

Pearson correlation coefficient between different traits on in vitro-cultured Catharanthus roseus L. “Ocellatus” plants

Significant positive correlations were detected between the chlorophyll a and chlorophyll b contents, soluble and reducing carbohydrates, TPC, vinblastine content, PAL activity, and PPO activity, with correlation coefficients of 0.71^**, 0.57^*, 0.44^*, 0.65^**, 0.59^**, 0.53^**, and 0.40^*, respectively. The vincristine and vinblastine contents also showed strong positive correlations with the photosynthetic pigments, carbohydrate fractions, TFC, and PPO activity. Similarly, TAC was positively correlated with carotenoid content (r = 0.61, p < 0.01). In contrast, the ajmalicine content was negatively correlated with the carotenoid, soluble carbohydrate, TFC, vincristine, vinblastine, and CAT contents (r = -0.59^**, -0.58^**, -0.50^**, -0.65^**, -0.51^*, and − 0.57^**, respectively). Principal component analysis (PCA) further demonstrated that the first two principal components (PCs) explained 55.27% of the total variance (Fig. 4).

Fig. 4.

Classification diagram of different properties on in vitro-cultured Catharanthus roseus L. “Ocellatus” plants

Variables including soluble and reducing carbohydrate contents, TFC, vinblastine content, TAC, CAT, and PPO were closely associated and contributed strongly to PC1 and PC2, highlighting their functional interdependence.

Discussion

The effects of foliar application of Cys and MT on C. roseus under greenhouse conditions have been studied previously [44]. However, the combined elicitation effects of H with Cys and MT still need to be elucidated. In the current investigation, under well-controlled tissue culture conditions, the relationships among these important signaling molecules can be addressed. Although the exogenous application of H in combination with MT and Cys has not been well studied in the literature, their individual applications have been reported to study biological responses of plants.

Hydrogen peroxide, N-compounds, such as melatonin, and S-compounds, such as cysteine, exert multidimensional influences on plant phytochemical profiles, modulating the quantity and quality of secondary metabolites through complex interactions involving enzymatic activity and metabolic pathways [45, 46]. The main focus of the present work was to elucidate the effects of MS medium supplemented with combined elicitation of hydrogen peroxide together with different concentrations of MT or Cys on the diverse characteristics of C. roseus.

The treatments generally caused a significant increase in the measured parameters. H alone markedly promoted plant height, number of leaves, number of roots, and biomass accumulation, supporting its role as a signaling molecule that stimulates growth-related pathways at controlled concentrations [47, 48]. Low to moderate H levels are known to enhance cell division, cell elongation, and vascular development through activation of mitogen-activated protein kinase (MAPK) cascades and hormonal crosstalk [49]. Additionally, the individual application of H at a 20 µM concentration resulted in an increase in the chlorophyll a and b contents without a significant influence on the carotenoid content compared with the control plants. In another study, the application of 1–10 mM H to lettuce genotypes promoted growth and increased the contents of photosynthetic pigments such as chlorophyll a, chlorophyll b and total chlorophyll [47]. In contrast, the foliar application of H (at 200, 480, and 610 ppm) to amaranth plants did not significantly increase the chlorophyll content compared with that of untreated controls [50]. These results indicate that the effects of H on photosynthetic pigments and photosynthetic performance vary depending on its concentration and application method. In this respect, soil application of 5- and 20-mM H to potted melon plants enhanced photosynthetic activity, whereas higher concentrations (50 mM) failed to exert significant effects compared with those of the control plants [51].

According to the results, MT100 + H promoted growth parameters, whereas higher MT concentrations suppressed these characteristics (Table 1). Under MT200 + H and MT400 + H, callus formation predominated over root initiation. Thus, MT + H elicitation exhibited a clear concentration-dependent morphological effect, stimulating growth at low concentration while suppressing root formation and biomass accumulation at intermediate and high levels. MT is known to function as both a growth regulator and an antioxidant, with its effects strongly dependent on dosage and developmental context [52, 53]. At low concentrations, MT enhances root initiation and shoot development by modulating auxin signaling and ROS distribution [54]. However, excessive MT may decrease ROS signaling below optimal thresholds, thereby limiting growth processes that rely on controlled oxidative cues [55]. In the present study, intermediate and high levels of applied MT + H increased chlorophyll a and b, indicating a synergistic interaction between H and MT. However, MT treatments resulted in a reduction in carotenoid content compared with that of the control (Table 2). MT is known to regulate chlorophyll biosynthesis by modulating the tetrapyrrole pathway genes and preserving chloroplast ultrastructure under oxidative conditions. MT upregulates Rubisco small subunits, Rubisco-interacting protein, and the photosystem I reaction center subunit, which correlates with improved photosynthetic activity [52, 56]. In another study, the exogenous application of MT at a concentration of 100 µM increased the levels of photosynthetic pigments, including chlorophyll a and carotenoids, and improved the performance of photosystems I and II in Prunella vulgaris [57]. According to other reports [20, 58], MT primarily protects photosynthetic pigments from degradation under stressful environmental conditions. In the current study, the reduced carotenoid levels observed—particularly at higher MT—may reflect a metabolic shift, whereby MT partially replaces carotenoids as a photoprotective antioxidant, allowing greater resource allocation toward chlorophyll synthesis. MT therefore likely plays a critical role in mitigating H toxicity. Furthermore, MT functions as both a direct antioxidant and a regulator of ROS signaling, enabling controlled H accumulation that favors photosynthetic efficiency rather than oxidative stress [47, 55]. Consequently, MT application below 100 µM may favor higher carotenoid accumulation, whereas higher concentrations prioritize chlorophyll protection in C. roseus.

According to An et al. [58], exogenous MT at 100 µM under normal conditions did not significantly influence photosynthetic pigments in tomato. However, under heat stress, MT effectively mitigated stress-induced damage and reduced the degradation of photosynthetic pigments, including chlorophyll a and b. Under stress conditions, the indole amine structure of MT safeguards chlorophyll, enhances photosystem II efficiency, and subsequently increases the total chlorophyll content in developing Hyoscyamus pusillus calli [59]. It has been reported that the application of MT, by supporting sulfur assimilation, can stimulate the production of Cys, thereby contributing to enhanced growth and improved photosynthetic efficiency under adverse conditions [60]. In addition, MT has been reported to modulate the expression of genes responsible for pigment protection in plant cells [61]. Cys + H elicitation, particularly at low to intermediate concentrations, significantly enhanced growth indices such as plant height and biomass. Cys maintains cellular redox balance and protects growth-related enzymes from oxidative damage [62]. The decline in growth at higher Cys concentrations indicates that excessive thiol availability may disrupt metabolic balance or impose feedback inhibition on sulfur assimilation pathways [63].

According to Table 2, the application of Cys + H increased the chlorophyll b and carotenoid contents, particularly at 400 and 800 µM. Cys contributes to photosynthetic performance and stress tolerance. In addition to its central metabolic functions, Cys is also involved in the formation of chloroplastic S-sulfocysteine, thereby protecting photosystems from oxidative stress [64]. Cys application on flax plants at 0.8–1.6 mM concentrations significantly improved photosynthetic pigment contents and phenolic compound accumulation [46].

The present study revealed synergistic effects of MT, Cys, and H, which contributed to increased photosynthetic pigment accumulation. On the one hand, a fairly low concentration of applied H (20 µM) can mimic oxidative stress to initiate antioxidant enzymes; on the other hand, MT protected photosynthetic pigments from degradation, and Cys also improved photosynthetic performance in response to H elicitation. Elevated concentrations of MT (200–400 µM) combined with H significantly increased the chlorophyll a content, whereas intermediate and high levels of Cys (400–800 µM) in combination with H increased chlorophyll b content as well as carotenoid accumulation (Table 2). These findings suggest that the interplay between signaling compounds plays an essential role in sustaining photosynthetic capacity and pigment stability. On the basis of the correlation data (Fig. 3), chlorophyll a and b were positively correlated with each other as well as with carbohydrates, TFC, TPC, FRAP, vinblastine, vincristine, PAL, PPO, POD and SOD, whereas they were negatively correlated with carotenoids, ajmalicine, MDA and H₂O₂.

Photosynthesis is a vital process that determines the amount of carbohydrates required for plant growth and development [65]. The strong growth response observed under H treatment alone is consistent with the simultaneous enhancement of photosynthetic pigments and carbohydrate availability reported in this study, indicating that improved carbon assimilation underlies enhanced biomass formation. Moreover, the significant enhancement of pigments and sugars across all elicitor-treated plants compared with the control suggests that exogenous H acted as a metabolic signal, which together with MT or Cys, promoted photosynthetic performance and carbohydrate metabolism [66, 67]. In agreement with our results, salt-stressed plants supplemented with 20 and 50 µM MT presented increased levels of total soluble carbohydrates by reversing the adverse effects of heat stress on photosynthesis [60, 68]. Similarly, foliar application of MT on peach trees and soil application on apple trees induced carbohydrate accumulation, thereby increasing the contents of total soluble sugars [69, 70]. In other studies, sugar contents were significantly increased by the application of H in lettuce, amaranth and melon [71 50, 51]. In soil-treated melon, the concentrations of glucose, fructose, and starch in both leaf and fruit tissues were elevated at H concentrations below 50 mM [51]. H, in combination with brassinosteroids, significantly inhibited the expression of the BAM and UGDH genes, which encode β-amylolytic enzymes and UDP-glucose 6-dehydrogenases, thereby regulating carbohydrate metabolism, leading to increased metabolic flux toward sugar accumulation [72].

Our data revealed that higher concentrations of Cys (400 and 800 µM) in combination with H produced the highest carbohydrate contents via increased in photosynthetic pigments. These results indicate that a synergistic interaction between Cys and H exists that maximizes carbohydrate accumulation (Table 2). Cys, in combination with H, further supports to this response by enhancing glutathione-dependent redox capacity, thereby sustaining photosynthetic carbon assimilation [62]. The pronounced increase in carotenoid contents, which function as accessory light-harvesting pigments, under high Cys concentrations suggests enhanced photoprotection of the photosystems, which likely preserved electron transport efficiency and enabled sustained sugar production [73]. Enhanced sugar accumulation under Cys + H treatments may therefore result from prolonged photosynthetic activity and redox-regulated carbon allocation toward soluble and reductive carbohydrate pools. Chlorophyll enrichment enhances photosynthetic capacity, while adequate antioxidant protection prevents ROS-induced inhibition of carbon metabolism [74]. However, the differential allocation of pigments and sugars between MT- and Cys-treated plants indicates elicitor-specific metabolic prioritization. MT primarily enhances chloroplast performance and pigment stability, whereas Cys strengthens photoprotection through carotenoid biosynthesis, sustained carbon fixation, and sugar accumulation [52].

In the present study, there was a considerable positive correlation between carbohydrate content and chlorophyll a, b, carotenoid, vinblastine, vincristine, PPO and SOD contents. Conversely, a negative relationship between carbohydrates and ajmalicine, MDA and H₂O₂ was observed (Fig. 3).

Secondary metabolite production can be assessed by monitoring the TFC, TPC, and the activities of PAL and PPO enzymes. These secondary metabolites frequently display potent antioxidant activities, conferring protection against oxidative stress and contributing to the reinforcement of plant defense mechanisms [72, 75]. In this study, H alone strongly enhanced TFC and PPO activity, suggesting that ROS signaling is sufficient to activate flavonoid biosynthesis and phenolic compound production. As presented in Fig. 1, combinations of H with either Cys or MT also increased TPC. Combined MT + H treatments were particularly effective in enhancing TPC, especially at intermediate MT concentration, indicating a synergistic interaction. Consistent with our findings, it has been reported that low concentrations of H activate the expression of genes encoding enzymes in the phenylpropanoid pathway, activating redox-sensitive transcription factors and MAPK cascades, thereby increasing the biosynthesis of flavonoids, stilbenes, and other phenolic compounds [49, 76].

In line with the current study, Salvia miltiorrhiza, PAL activity increased progressively with increasing H concentrations ranging from 1 to 10 mM [77]. Similarly, in lettuce, exogenous H treatment stimulated the accumulation of diverse phenolic compounds and upregulated key enzymes involved in phenolic biosynthesis pathways, including PAL and flavanone 3-hydroxylase [71]. MT regulates secondary metabolism by modulating ROS homeostasis while acting as a signaling molecule that upregulates phenylpropanoid pathway genes [52, 78]. The pronounced increase in PAL activity under MT200 + H supports the conclusion that MT enhances phenolic accumulation by stimulating carbon flux into the phenylpropanoid pathway [78]. MT has also been identified as an effective elicitor of phenolic metabolism in other plant species. In tomato, MT application resulted in the accumulation of three flavonoids and six additional phenolic compounds [79]. Moreover, MT was shown to transcriptionally upregulate the PAL and PPO genes, further promoting phenolic metabolism and antioxidant capacity [80]. In contrast, Cys + H treatments showed comparatively stronger effects on PPO activity, particularly at intermediate Cys concentration. Cys plays a central role as a precursor in the biosynthesis of sulfur-containing amino acids, vitamins, glucosinolates, and glutathione, acting as an abundant antioxidant that maintains cellular redox balance in plant cells [81, 82]. Enhanced glutathione pools can stabilize enzyme activities and protect phenylpropanoid intermediates from oxidative degradation. The strong induction of PPO under Cys400 + H suggests that Cys-mediated redox regulation favors active phenolic oxidation, contributing to enhanced flavonoid accumulation. Collectively, these results support an integrated model in which H serves as the primary signaling trigger, while MT and Cys differentially modulate phenylpropanoid metabolism. MT preferentially promotes phenolic biosynthesis via PAL activation, whereas Cys strengthens redox regulation, favoring elevated PPO activity. The concentration-dependent nature of these responses highlights the importance of balanced ROS signaling rather than maximal antioxidant capacity in optimizing secondary metabolite production [74]. In the present study (Fig. 3), the activity of the PAL enzyme was positively correlated with the activities of chlorophyll a and b, carbohydrates, TPC, TAC, FRAP, vincristine, CAT, PPO, POD and SOD. On the other hand, PAL was negatively correlated with ajmalicine, MDA and H₂O₂.

In the present study, H alone significantly enhanced the FRAP value relative to the control, but to a lesser extent than Cys200 + H, indicating activation of reducing power–related antioxidant systems. This response aligns with the role of H in inducing redox-sensitive enzymes and metabolites [49, 83]. MT + H treatments produced moderate FRAP increases but did not markedly enhance TAC, consistent with MT’s role in maintaining ROS at signaling-competent levels rather than promoting excessive antioxidant accumulation [55]. Such balanced regulation may be advantageous for maintaining cellular homeostasis rather than maximizing total antioxidant capacity. Conversely, Cys + H—particularly at 200 and 400 µM—was the most effective strategy for enhancing antioxidant capacity, as reflected by TAC and FRAP. Cys serves as a key cellular reducing power and therefore contributes to higher ferric reducing capacity [62]. The strong TAC response observed at intermediate Cys concentration further suggests that Cys + H interactions promote accumulation of non-enzymatic antioxidants, including phenolics to maintain redox homeostasis [74].

It is reported that, exogenous MT and Cys enhanced the antioxidant capacity and TPC in raspberry and litchi fruits, respectively [84, 85]. As shown in Fig. 3, FRAP and TFC were positively correlated with chlorophyll a, b, vincristine, PPO and POD. Conversely, these genes were negatively correlated with ajmalicine, MDA and H₂O₂. Notably, MDA exhibited a positive correlation only with H₂O₂, and vice versa.

In the present study, although H alone increased antioxidant enzyme activities, it also elevated endogenous H levels, indicating activation of defense responses without optimal redox control. This supports previous findings that exogenous H acts as a signaling trigger rather than solely as a stress-inducing agent, initiating downstream antioxidant and metabolic responses [48, 49]. In agreement with our results, a previous study demonstrated that exogenous treatment with H in soybean increased the activity of antioxidant enzymes such as CAT and SOD, along with an increase in TPC [86].

H-activating antioxidant mechanisms may be further amplified with MT and Cys, resulting in increased levels of FRAP, CAT, SOD and POD (Tables 3 and 4). In litchi fruit, Cys supplementation markedly elevated CAT and SOD activities, thereby strengthening antioxidant defense pathways [85]. In the present study, Cys application not only improved enzymatic antioxidant activity but also contributed to maintaining the structural integrity of cell membranes, thereby reducing lipid peroxidation, as reflected by MDA accumulation (Table 4). Cys + H treatment resulted in the strongest activation of CAT, POD, and SOD activities, particularly at intermediate concentration. This effect is linked to the role of Cys as a precursor for glutathione, a pivotal component of the ascorbate–glutathione cycle. Glutathione functions synergistically with MT to increase the GSH/GSSG ratio, thereby enhancing the cellular redox equilibrium and conferring greater tolerance to oxidative stress [60, 87]. Therefore, the results indicate that Cys, particularly at 400 µM, strengthens the antioxidant network, enhancing the plant’s capacity to defend against H-induced oxidative stress, leading to efficient reduction of endogenous H (Table 4). In contrast to Cys, MT-based elicitation produced a more selective antioxidant response. Although MT + H treatments did not consistently induce the highest antioxidant enzyme activities, they were particularly effective in reducing endogenous H and MDA contents, especially at intermediate MT concentration. This reflects MT’s multifunctional role as both a direct radical scavenger and a regulator of ROS signaling [52, 55]. Our findings (Table 4) demonstrate that MT, particularly at an optimal concentration of 200 µM, was most effective in lowering H₂O₂ accumulation and MDA levels, the two well-known coregulating parameters involved in membrane instability [88]. Moreover, the inverse relationship between antioxidant enzyme activity and MDA accumulation across treatments highlights the effectiveness of elicitor-induced defense mechanisms in mitigating membrane lipid peroxidation. Taken together, the combined action of MT + H and Cys + H may strengthen the enzymatic antioxidant mechanism and improve cellular redox homeostasis, resulting in more efficient mitigation of H-induced oxidative stress. Collectively, under stress-like conditions, such as elicitation, both enzymatic antioxidants (e.g., CAT, SOD and POD) and non-enzymatic metabolites (e.g., phenolics, flavonoids, glutathione, free amino acids and alkaloids) contribute to the mitigation of oxidative stress by scavenging ROS.

Elicitation with abiotic or biotic stresses is known to enhance antioxidant enzymes responses in C. roseus leading to altered TIA biosynthesis [1, 89]. The increased activities of SOD, POD, and CAT facilitate the conversion of superoxide radicals into hydrogen peroxide, a non-radical ROS, and its subsequent decomposition [16]. On the basis of our findings (Fig. 3), there was a positive correlation between antioxidant enzymes (PPO, POD and SOD) and chlorophyll a, b, carotenoid, TAC, FRAP, vinblastine and vincristine. However, these indices were negatively correlated with ajmalicine, MDA and H₂O₂.

The synergistic interaction of hydrogen peroxide, MT, and Cys, as illustrated by the increase in phenylpropanoid pathway enzymes, phenolic compounds and antioxidant enzymes (Tables 3 and 4; Fig. 1), is expected to increase alkaloid production. These signaling and antioxidant molecules are actively involved in various physiological processes that regulate secondary metabolite biosynthesis, including those responsible for the production of medicinally important alkaloids. In the present study, elicitation with H alone or in combination with MT or Cys effectively reprogrammed TIA metabolism, enhancing vincristine and vinblastine accumulation while reducing ajmalicine content. This pattern indicates selective redirection of metabolic flux within the TIA pathway, rather than uniform stimulation of all alkaloids. Our data confirmed that vinblastine and vincristine accumulation was highest in plants treated with H alone and Cys800 + H. Compared with the control treatment, all other treatments significantly increased either the vincristine or vinblastine content. These two alkaloids are recognized as essential chemotherapeutic agents that are widely used to treat various cancers and are classified as downstream metabolites of the TIA biosynthetic pathway, in contrast to ajmalicine, which is considered a midstream metabolite of this pathway [90, 91].

Hydrogen peroxide functions as a signaling molecule that triggers stress-responsive pathways in plants, thereby stimulating the transcription of genes involved in secondary metabolite production [92]. MT, as a signaling molecule, protects plants through MAPK signaling. In C. roseus, MAPK upregulation has been shown to stimulate genes associated with alkaloid biosynthesis [93]. Cys plays a protective role against oxidative stress and supports the biosynthesis of secondary metabolites by maintaining redox balance in plant cells [94]. Although it was expected that combined elicitation would maximize alkaloid accumulation, the results showed that the highest vinblastine and vincristine contents were achieved under H treatment alone. The strong enhancement of vinblastine observed under H treatment alone supports the role of ROS signaling as a primary trigger of TIA pathway activation, particularly for late-stage alkaloid formation. This finding supports the concept that oxidative signaling, rather than maximal antioxidant suppression, is essential for late-stage TIA biosynthesis. Accordingly, neither MT + H nor Cys + H surpassed H alone in promoting vinblastine or vincristine accumulation. Consistent with this interpretation, ajmalicine content was highest in control plants and declined under all elicitor treatments, reflecting its role as a competing branch within the TIA pathway. Reduced ajmalicine accumulation suggests redirection of shared precursors toward the biosynthesis of more complex, high-value alkaloids [1, 95].

Calli treated with Cys (300 mg L⁻¹) presented a significant increase in the production of paclitaxel compared with the untreated control [96]. In Ficus deltoidea, the vitexin content increased under the H treatment (60 mM), whereas the isovitexin content was highest under 30 mM H treatment, confirming that metabolite production is concentration dependent [97]. Compared with the control, MT treatment increased galanthamine and lycorine biosynthesis in Leucojum aestivum L. by 58.6- and 1.5-fold, respectively, and induced the production of additional alkaloids such as tazettine and chlidanthine [98]. According to our findings (Fig. 3), vinblastine and vincristine were positively correlated with each other and with chlorophyll a, b, carotenoids, carbohydrates, TFC, PPO, POD and SOD. Conversely, these indices were negatively correlated with ajmalicine and MDA.

Conclusions

This study demonstrates that elicitation with H in combination with MT or Cys does not simply enhance metabolite accumulation, but instead selectively reprograms physiological performance and secondary metabolism in a concentration- and elicitor-dependent manner. By comparing single and combined elicitation strategies, our results reveal that the treatments differentially regulate photosynthetic efficiency, antioxidant capacity, phenylpropanoid activity, and TIA biosynthesis, leading to variable metabolic outcomes. A central conclusion of our results is that alkaloid biosynthesis in C. roseus is governed by coordinated regulation of carbon assimilation, controlled oxidative signaling, and activation of antioxidant enzymes. Cys + H elicitation primarily strengthened enzymatic antioxidant and carbon availability, thereby favoring vincristine accumulation, whereas H more effectively promoted both vincristine and vinblastine biosynthesis through controlled oxidative signaling. The inverse response of ajmalicine further indicates that elicitation redirects metabolic allocation within the TIA pathway rather than uniformly increasing all alkaloids. Importantly, the strong positive associations between photosynthetic pigments, carbohydrate pools, antioxidant activity, PAL/PPO activities, and alkaloid accumulation indicate that enhanced metabolic capacity and redox regulation are key drivers of high-value alkaloid production.

Overall, this work provides a framework for rational elicitor selection and dose optimization in C. roseus and offers practical insights for improving targeted alkaloid production in in vitro cultures, precursor-feeding systems, and metabolic engineering strategies in medicinal plants.

Abbreviations

H

H2O2

MT

Melatonin

Cys

L-cysteine

PAL

Phenylalanine ammonia lyase

CAT

Catalase

POD

Peroxidase

PPO

Polyphenol oxidase

SOD

Superoxide dismutase

MDA

Malondialdehyde

TIAs

Terpenoid indole alkaloids

GRAS

Generally recognized as safe

MAPK

Mitogen-activated protein kinase

TFC

Total Flavonoid Content

TPC

Total Phenolic Content

TAC

Total antioxidant capacity

FRAP

Ferric reducing antioxidant power

Authors’ contributions

E.J.: Formal Analysis, Investigation, Data curation, Project administration, Software, Writing original draft. M.S.: Conceptualization, Methodology, Validation, Supervision, Writing, Review and preparation of final draft. A.K.: Conceptualization, Methodology, Validation, Supervision, Writing original draft. F.R.: Software, Writing original draft. All authors have read and agreed to the final version of the manuscript.

Funding

Not applicable.

Data availability

All the data generated or analyzed during the current study were included in the manuscript. The raw data is available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

We all declare that manuscript reporting studies do not involve any human participants, human data, or human tissue. So, it is not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.