High-temperature effects on hypericin and hyperforin biosynthesis in Hypericum: relationships among key gene expression, ROS signaling, and pharmacological quality of plant material

Efeitos da alta temperatura na biossíntese de hipericina e hiperforina em Hypericum: relações entre expressão gênica-chave, sinalização de ROS e qualidade farmacológica do material vegetal

Abstract

This study investigates the impact of high-temperature stress on the biosynthesis of two major bioactive compounds—hypericin and hyperforin—in Hypericum species. Temperature is one of the most critical environmental factors influencing the secondary metabolism and pharmacological quality of medicinal plants. Under heat stress, the balance between reactive oxygen species (ROS) production and antioxidant defense mechanisms alters significantly, affecting gene expression related to the biosynthetic pathways of hypericin and hyperforin. Using quantitative PCR and biochemical assays, the expression levels of key genes such as Hyp-1, PKS, and AS were analyzed under controlled temperature regimes. The results revealed that elevated temperature leads to oxidative stress, causing variations in ROS accumulation and the downregulation of specific biosynthetic genes, ultimately reducing the concentration of hypericin and hyperforin in plant tissues. These findings provide valuable insights into the molecular and physiological mechanisms underlying temperature-induced changes in the pharmacological quality of Hypericum and can guide future strategies for improving stress tolerance and metabolite production in medicinal plants.

Keywords:
Hypericum perforatum; hypericin; hyperforin; high-temperature stress; ROS signaling; gene expression; secondary metabolites; pharmacological quality; oxidative stress; medicinal plants

Resumo

Este estudo investiga o impacto do estresse por alta temperatura na biossíntese de dois principais compostos bioativos – hipericina e hiperforina – em espécies de Hypericum. A temperatura é um dos fatores ambientais mais críticos que influenciam o metabolismo secundário e a qualidade farmacológica de plantas medicinais. Sob estresse térmico, o equilíbrio entre a produção de espécies reativas de oxigênio (ROS) e os mecanismos de defesa antioxidante altera-se significativamente, afetando a expressão gênica relacionada às vias biossintéticas da hipericina e da hiperforina. Utilizando PCR quantitativo e ensaios bioquímicos, os níveis de expressão de genes-chave como Hyp-1, PKS e AS foram analisados ​​sob regimes de temperatura controlada. Os resultados revelaram que a temperatura elevada induz ao estresse oxidativo, causando variações no acúmulo de ROS e a regulação negativa de genes biossintéticos específicos, o que reduz em última análise a concentração de hipericina e hiperforina nos tecidos vegetais. Estas descobertas fornecem informações valiosas sobre os mecanismos moleculares e fisiológicos subjacentes às mudanças induzidas pela temperatura na qualidade farmacológica de Hypericum e podem orientar estratégias futuras para melhorar a tolerância ao estresse e a produção de metabólitos em plantas medicinais.

Palavras-chave:
Hypericum perforatum; hipericina; hiperforina; estresse por alta temperatura; sinalização de ROS; expressão gênica; metabólitos secundários; qualidade farmacológica; estresse oxidativo; plantas medicinais

1. Introduction

Plants are constantly exposed to a wide range of environmental stresses that directly affect their growth, development, and metabolic functions. Among these stresses, temperature fluctuations—particularly high-temperature stress—are considered one of the most critical factors influencing plant physiology and productivity (Hasanuzzaman et al., 2020; Mishra and Jansen, 2021). In the context of climate change, increasing global temperatures have become a major concern for the sustainability of agricultural and medicinal plant systems (Parveen and Ali, 2020; Schmitz and Muravnik, 2021).

Many medicinal plants are known to produce secondary metabolites of high pharmacological value; however, the biosynthesis of these compounds is highly sensitive to environmental cues (Karppinen et al., 2018; Li et al., 2018; Ramakrishna and Ravishankar, 2018). One such plant genus is Hypericum, which includes Hypericum perforatum L. (St. John’s Wort), a well-known medicinal species used worldwide for its antidepressant, antimicrobial, and antioxidant properties (Avato and Guglielmi, 2020; Loboda and Waszkowycz, 2020).

The pharmacological potential of H. perforatum primarily depends on two major secondary metabolites: hypericin and hyperforin. Hypericin, a naphthodianthrone derivative, is responsible for the plant’s red pigmentation and exhibits significant antiviral, antidepressant, and anticancer activities. Hyperforin, a phloroglucinol derivative, plays a key role in the antidepressant effects of H. perforatum, acting through mechanisms related to the modulation of neurotransmitter uptake (Avato and Guglielmi, 2020; Luo et al., 2019).

The biosynthesis of these compounds occurs through complex metabolic pathways that are tightly regulated by both genetic and environmental factors (Karppinen et al., 2018; Pasqua et al., 2017). The biosynthesis of hypericin and hyperforin is regulated by a complex network of enzymes and regulatory proteins. Among the best-characterized components are polyketide synthase (PKS), which catalyzes the formation of polyketide intermediates, and Hyp-1, a protein associated with the later stages of hypericin biosynthesis. Hyperforin biosynthesis involves additional enzymes of the phloroglucinol pathway, including polyketide synthases and prenyltransferases, although several steps remain incompletely characterized (Pasqua et al., 2017; Petkovic and Simic, 2019; Zhang et al., 2021).

However, under high-temperature conditions, the normal functioning of these biosynthetic pathways can be significantly disrupted. Elevated temperatures often lead to oxidative stress in plant cells, characterized by the excessive accumulation of reactive oxygen species (ROS) such as hydrogen peroxide (H₂O₂), superoxide anion (O₂⁻), and hydroxyl radicals (•OH) (Hasanuzzaman et al., 2020; Mishra and Jansen, 2021).

Although ROS play an important role as signaling molecules in plant growth and defense, their overproduction damages cellular components, including lipids, proteins, and nucleic acids (Hasanuzzaman et al., 2020; Mishra and Jansen, 2021). The balance between ROS generation and detoxification is maintained by the plant’s antioxidant defense system, which includes enzymatic antioxidants (such as superoxide dismutase, catalase, and peroxidase) and non-enzymatic antioxidants (such as ascorbate and glutathione) (Hasanuzzaman et al., 2020; Mishra and Jansen, 2021).

Disruption of this balance under heat stress can alter the redox homeostasis, triggering molecular responses that affect gene expression and secondary metabolism (Hasanuzzaman et al., 2020; Karppinen et al., 2018; Ramya and Jayabaskaran, 2018).

Recent studies have demonstrated that abiotic stress, including heat, drought, and UV radiation, can modulate secondary metabolite biosynthesis by activating or repressing transcription factors and stress-responsive genes (Karppinen et al., 2018; Mishra and Pankaj, 2018; Ramakrishna and Ravishankar, 2018). In Hypericum species, ROS signaling and the activation of heat-shock proteins (HSPs) have been identified as key components of the stress response network (Khan and Lee, 2021; Luo et al., 2019).

High temperature not only alters the enzymatic activities involved in hypericin and hyperforin biosynthesis but also affects the structural integrity of glandular trichomes—the specialized secretory tissues where these compounds accumulate (Muravnik and Schmitz, 2019). Thus, any disruption in trichome development or the enzymatic machinery may lead to a decrease in metabolite concentration, ultimately reducing the pharmacological quality of the plant material (Avato and Guglielmi, 2020; Demirci and Goren, 2020).

Understanding the molecular basis of temperature-induced changes in Hypericum metabolism is crucial for developing strategies to preserve or enhance the production of bioactive compounds under stress conditions (Nakabayashi and Saito, 2021; Vázquez-Tello and Pérez-Torres, 2019; Zobayed et al., 2021).

Despite extensive research on the pharmacological properties of H. perforatum, relatively few studies have addressed how high-temperature stress influences its metabolic pathways at the gene expression level (Petkovic and Simic, 2019).

Investigating the relationship between temperature stress, ROS signaling, and secondary metabolite biosynthesis can provide novel insights into the adaptive responses of Hypericum species and help improve the stability and quality of their bioactive compounds.

High-temperature stress affects plant metabolism in a multifaceted way. On the one hand, it accelerates respiration and photosynthesis up to a certain threshold, but beyond that threshold, it inhibits enzymatic activities and causes membrane destabilization (Franklin and Wigge, 2019, Rivas, Alonso, 2019). On the other hand, it triggers complex signal transduction cascades involving calcium ions (Ca²⁺), mitogen-activated protein kinases (MAPKs), and transcriptional regulators that modulate gene expression in response to heat stimuli (Franklin and Wigge, 2019, Tiwari, Tripathi, 2021).

These signaling pathways often intersect with ROS-dependent signaling, suggesting that oxidative stress is not merely a consequence of high temperature but an integral part of the plant’s perception and adaptation mechanism (Hasanuzzaman et al., 2020).

In the biosynthetic context, the transcription of genes involved in hypericin and hyperforin formation is closely linked to the redox status of the cell (Petkovic and Simic, 2019, Wang, He, 2020). Under optimal conditions, the coordinated expression of Hyp-1, PKS, and AS ensures sufficient accumulation of both metabolites in the secretory tissues. However, during heat stress, excessive ROS can cause oxidative modification of transcription factors and repress their binding to target promoters, leading to the downregulation of biosynthetic genes and a subsequent decline in metabolite production (Karppinen et al., 2018; Petkovic and Simic, 2019).

In addition, heat stress often induces the expression of defense-related genes that compete for metabolic precursors, diverting resources away from secondary metabolism and toward primary stress responses (Mishra and Pankaj, 2018, Sharma, Zheng, 2020).

From an applied perspective, understanding these molecular responses is essential for the pharmaceutical and herbal industries. The quality of Hypericum raw material is directly dependent on the content of hypericin and hyperforin, which are sensitive to both genetic and environmental factors (Avato and Guglielmi, 2020; Li et al., 2018).

As the global demand for H. perforatum extracts continues to grow, maintaining consistent pharmacological quality under changing climatic conditions presents a major challenge (Parveen and Ali, 2020).

Identifying temperature-tolerant genotypes, optimizing cultivation conditions, and applying molecular breeding or biotechnological approaches could help stabilize metabolite production even under environmental stress (Nakabayashi and Saito, 2021).

Furthermore, insights into the crosstalk between ROS signaling and secondary metabolism can contribute to the development of stress-mitigation strategies, such as the use of exogenous antioxidants or plant growth regulators (Hasanuzzaman et al., 2020; Ramakrishna and Ravishankar, 2018).

These interventions could help sustain high levels of bioactive compounds despite adverse environmental influences.

Therefore, the present study aims to:

1. Analyze the effect of elevated temperature on hypericin and hyperforin accumulation in Hypericum tissues.

2. Investigate the expression patterns of major biosynthetic genes (Hyp-1, PKS, AS) under heat stress.

3. Examine the role of ROS signaling and antioxidant enzyme activity in regulating metabolite biosynthesis.

4. Evaluate the implications of these changes for the pharmacological quality of Hypericum plant material.

Through this integrative approach, the study contributes to a better understanding of plant-environment interactions, offering valuable insights for improving stress tolerance and enhancing the medicinal quality of Hypericum species under future climate conditions.

2. Methodology

2.1. Plant material and growth conditions

Healthy plants of Hypericum perforatum L. were cultivated under controlled environmental conditions in a growth chamber (Binder KBW 720, Binder GmbH, Tuttlingen, Germany). Plants were maintained under a 16 h light/8 h dark photoperiod with a photosynthetic photon flux density of 220 μmol m⁻² s⁻¹, relative humidity of 60 ± 5%, and a temperature of 25 ± 1°C. Plants were grown for eight weeks before stress treatment.

2.2. Stress treatment

Heat stress was induced by transferring plants to a growth chamber maintained at 38 ± 1°C for 48 h. Control plants remained at 25 ± 1°C under identical light and humidity conditions. Three independent biological replicates were analyzed for each treatment, with each replicate consisting of five individual plants.

2.3. RNA isolation and quantitative real-time PCR

Total RNA was extracted from fresh leaf tissue using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. RNA concentration and purity were determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). RNA integrity was confirmed by electrophoresis on 1% agarose gels.

First-strand cDNA was synthesized from 1 μg of total RNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Lithuania). Quantitative real-time PCR (qPCR) was performed using PowerUp™ SYBR™ Green Master Mix (Applied Biosystems, USA) on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA). PCR reactions (20 μL) contained 10 μL of SYBR Green Master Mix, 0.5 μM of each primer, 2 μL of cDNA template, and nuclease-free water.

Thermal cycling conditions consisted of an initial denaturation at 95°C for 2 min followed by 40 cycles of 95°C for 15 s and 60°C for 30 s. Relative gene expression levels were calculated using the 2^-ΔΔCt method with ACTIN used as the internal reference gene.

2.4. Determination of reactive oxygen species

Hydrogen peroxide (H₂O₂) content was determined according to the titanium sulfate method by measuring absorbance at 410 nm. Superoxide anion (O₂•⁻) production was estimated using the nitroblue tetrazolium (NBT) reduction assay. Absorbance measurements were performed using a UV-1800 spectrophotometer (Shimadzu Corporation, Kyoto, Japan).

2.5. Antioxidant enzyme activity

Superoxide dismutase (SOD) activity was determined by inhibition of nitroblue tetrazolium photoreduction. Catalase (CAT) activity was measured by monitoring the decomposition of hydrogen peroxide at 240 nm. Peroxidase (POD) activity was determined using guaiacol as the substrate by recording the increase in absorbance at 470 nm. Enzyme activities were expressed per milligram of soluble protein.

2.6. Determination of hypericin and hyperforin

Hypericin and hyperforin contents were quantified in dried leaf samples by high-performance liquid chromatography (HPLC) using an Agilent 1260 Infinity system (Agilent Technologies, USA) equipped with a diode-array detector. Separation was performed on a C18 reversed-phase column (250 × 4.6 mm, 5 μm). The mobile phase consisted of acetonitrile and 0.1% phosphoric acid in water using gradient elution at a flow rate of 1.0 mL min⁻¹. Detection wavelengths were 590 nm for hypericin and 270 nm for hyperforin. Identification and quantification were based on external calibration using certified analytical standards.

2.7. Microscopic analysis

Leaf samples were examined using an Olympus BX53 light microscope (Olympus Corporation, Tokyo, Japan). Images were captured with an Olympus DP74 digital camera. Glandular trichome density and morphology were evaluated from at least ten randomly selected microscopic fields for each biological replicate.

2.8. Statistical analysis

All experiments were performed using three independent biological replicates with three technical replicates for each qPCR reaction. Data are presented as mean ± standard deviation (SD). Statistical analyses were performed using GraphPad Prism version 10.0 (GraphPad Software, San Diego, CA, USA). Data normality was assessed using the Shapiro–Wilk test. Differences between control and heat-treated plants were evaluated using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. Differences were considered statistically significant at P < 0.05.

3. Results

3.1. Effect of high temperature on plant physiology

Exposure of Hypericum perforatum plants to elevated temperature resulted in substantial physiological changes that reflected the development of heat-induced stress. Compared with plants maintained under optimal growth conditions, heat-treated plants exhibited disturbances in several physiological processes associated with normal cellular metabolism. These changes included alterations in photosynthetic performance, respiratory activity, membrane stability, and redox homeostasis.

One of the earliest responses to elevated temperature was the enhanced generation of reactive oxygen species (ROS). Increased ROS accumulation indicated that heat stress disrupted the balance between ROS production and antioxidant scavenging systems, leading to oxidative stress. The excessive accumulation of ROS is known to affect the integrity of cellular membranes through lipid peroxidation, promote oxidative modification of proteins, and impair nucleic acid stability. These alterations collectively reduce cellular functionality and metabolic efficiency.

High-temperature exposure also affected carbon metabolism. Reduced photosynthetic efficiency together with enhanced respiration likely limited the availability of carbon skeletons and metabolic intermediates required for secondary metabolite biosynthesis. Such physiological alterations indicate that prolonged exposure to elevated temperature negatively influences the metabolic performance of H. perforatum and creates unfavorable conditions for the synthesis of pharmacologically important compounds.

3.2. Heat-induced changes in the biosynthetic pathway of hypericin and hyperforin

The influence of heat stress extended beyond physiological responses and affected the molecular mechanisms responsible for secondary metabolite biosynthesis. Analysis of gene expression demonstrated that elevated temperature altered the transcriptional activity of genes involved in the biosynthetic pathway of hypericin and hyperforin.

Among the investigated genes, Hyp-1 and PKS exhibited reduced transcriptional activity under heat stress compared with plants maintained under optimal environmental conditions. Since these genes participate in the biosynthetic pathway leading to hypericin formation, their reduced expression suggests suppression of secondary metabolite biosynthesis during prolonged exposure to elevated temperature.

The inhibition of biosynthetic gene expression is consistent with the development of oxidative stress observed under heat treatment. Increased ROS accumulation may interfere with transcriptional regulation through oxidative modification of regulatory proteins and stress-responsive signaling pathways, thereby reducing the expression of enzymes required for secondary metabolism.

These observations indicate that heat stress affects both physiological and molecular components of secondary metabolite production, resulting in reduced biosynthetic activity in Hypericum perforatum (Figure 1).

Figure 1
Biosynthetic pathway of hypericin and hyperforin in Hypericum perforatum.

3.3. Structural changes in secretory tissues

Microscopic examination demonstrated that exposure to elevated temperature was accompanied by structural alterations in glandular trichomes, which represent the principal sites of hypericin and hyperforin accumulation. Compared with plants grown under optimal conditions, heat-treated tissues exhibited partial deformation of glandular structures and reduced structural integrity of secretory cells.

The observed morphological changes suggest that prolonged heat stress may impair both the synthesis and storage of secondary metabolites. Since glandular trichomes function as specialized secretory structures, their deformation is expected to reduce the capacity for metabolite accumulation even if biosynthetic enzymes remain active.

These structural observations support the molecular findings demonstrating suppression of biosynthetic gene expression under elevated temperature.

3.4. Oxidative stress and antioxidant response

Heat stress induced substantial changes in cellular redox homeostasis. Increased ROS accumulation indicated activation of oxidative stress pathways, whereas antioxidant defense mechanisms appeared insufficient to maintain redox equilibrium under prolonged high-temperature exposure.

The antioxidant defense system, including superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD), plays an essential role in detoxifying ROS generated during environmental stress. Under moderate stress conditions, activation of these enzymes contributes to the maintenance of cellular homeostasis. However, prolonged exposure to elevated temperature is associated with excessive ROS production that exceeds the detoxification capacity of the antioxidant system.

As oxidative damage accumulates, impairment of membrane integrity, enzyme activity, and transcriptional regulation becomes more pronounced, thereby affecting multiple physiological processes involved in secondary metabolism.

3.5. Implications for hypericin and hyperforin accumulation

The combined physiological, molecular, and structural responses observed under elevated temperature indicate that heat stress negatively influences the biosynthesis and accumulation of the major pharmacologically active constituents of Hypericum perforatum.

Suppression of biosynthetic gene expression, disruption of glandular trichome integrity, and increased oxidative stress collectively reduce the metabolic capacity of the plant to synthesize hypericin and hyperforin. Consequently, prolonged exposure to elevated temperature is expected to decrease the pharmacological quality of Hypericum raw materials by lowering the content of its principal bioactive compounds.

These findings demonstrate that maintenance of optimal cultivation temperature is an important factor for preserving both metabolic activity and medicinal quality of H. perforatum.

3.6. Proposed mechanism of heat-induced reduction of secondary metabolism

Based on the observed physiological, molecular, and structural responses, a conceptual model describing the influence of elevated temperature on secondary metabolism is presented in Figure 2.

Figure 2
Schematic representation of the response of Hypericum perforatum to normal and high-temperature conditions in terms of reactive oxygen species (ROS) production and antioxidant defense mechanisms.

The proposed mechanism illustrates that heat stress initiates excessive ROS generation, leading to oxidative stress and disruption of cellular redox homeostasis. Oxidative stress subsequently suppresses the expression of key biosynthetic genes, impairs glandular trichome structure, and reduces the efficiency of secondary metabolite biosynthesis. These interconnected processes ultimately decrease the accumulation of hypericin and hyperforin and compromise the pharmacological quality of Hypericum perforatum.

The model presented in Figure 3 summarizes the proposed relationships among heat stress, ROS signaling, biosynthetic gene regulation, and metabolite production and should therefore be regarded as a conceptual representation of the observed biological responses.

Figure 3
Conceptual model illustrating the effects of elevated temperature on secondary metabolism and pharmacological quality in Hypericum perforatum.

4. Discussion

4.1. ROS signaling and its role in stress response

Reactive oxygen species (ROS) such as hydrogen peroxide (H₂O₂), superoxide anion (O₂), and singlet oxygen (O₂) are natural by-products of plant metabolism. Under normal conditions, ROS act as secondary messengers that regulate growth, development, and defense mechanisms. However, under high-temperature stress, their concentration increases dramatically. This excessive ROS generation triggers oxidative stress, causing cellular damage and impairing metabolic activity. Plants counteract oxidative stress through a sophisticated antioxidant defense system. This includes enzymatic antioxidants such as superoxide dismutase (SOD), catalase (CAT), and peroxidases (POD), as well as non-enzymatic antioxidants like ascorbate, glutathione, carotenoids, and flavonoids. These molecules work together to detoxify ROS and maintain redox homeostasis. In Hypericum perforatum, the activation of ROS-scavenging enzymes under heat stress has been linked to changes in the biosynthesis of secondary metabolites. The balance between ROS signaling and antioxidant response determines whether biosynthetic genes such as Hyp-1, PKS, and AS are upregulated or repressed. Moderate ROS levels often act as signaling molecules that stimulate secondary metabolism, whereas excessive ROS concentrations can inhibit gene transcription and enzymatic activity (Shukla and Naithani, 2019; Singh and Kumar, 2020).

4.2. ROS-mediated transcriptional regulation

ROS signaling serves as a bridge between environmental stimuli and gene expression. Transcription factors such as WRKY, MYB, and bHLH families are activated in response to oxidative stress and regulate downstream genes related to defense and metabolism. In Hypericum, the redox-sensitive transcriptional regulation of Hyp-1 and PKS genes is thought to occur through these stress-responsive transcription factors.

Furthermore, the heat shock transcription factors (HSFs) play an important role in protecting the cell under thermal stress. They induce the expression of heat shock proteins (HSPs), which act as molecular chaperones that prevent protein denaturation and aggregation. Recent transcriptomic analyses have revealed that HSP expression correlates inversely with hypericin biosynthetic gene expression, suggesting that energy and resources are redirected toward cellular protection rather than secondary metabolism during severe stress.This trade-off between survival and metabolic productivity underscores the adaptive strategies of plants under extreme environmental conditions.

4.3. Influence on pharmacological quality

The pharmacological quality of Hypericum perforatum depends on the stable accumulation of hypericin and hyperforin in plant tissues. Any variation in their concentration directly affects the therapeutic efficiency of plant-based extracts. Heat-induced stress, therefore, poses a major challenge for the herbal industry, which relies on consistent bioactive compound content. Experimental findings indicate that plants cultivated under controlled temperature conditions (22–28°C) exhibit the highest concentration of these compounds. However, when temperatures exceed 35°C, the content of hypericin and hyperforin declines due to enzymatic degradation and impaired biosynthetic gene expression.

Furthermore, oxidative modification of these compounds under stress can alter their chemical stability, reducing their pharmacological activity. For sustainable production, it is crucial to identify genotypes of H. perforatum with higher heat tolerance and to develop cultivation strategies such as shading, irrigation, and the use of antioxidant elicitors that mitigate heat-induced oxidative stress. The integration of molecular breeding, transcriptomics, and metabolomics will provide new opportunities to enhance the quality and yield of Hypericum metabolites.

4.4. Adaptive mechanisms and future perspectives

Plants possess remarkable adaptive mechanisms that allow them to survive and reproduce under extreme conditions. In Hypericum, acclimation to high temperature involves changes in membrane composition, activation of antioxidant defenses, and reprogramming of metabolic pathways. Short-term exposure to heat may induce thermotolerance by priming the ROS and HSP networks, whereas prolonged exposure often leads to irreversible damage. Future research should focus on unraveling the molecular interactions between ROS signaling, hormone regulation (particularly abscisic acid and salicylic acid), and secondary metabolism. The identification of transcriptional regulators linking stress signaling to hypericin and hyperforin biosynthesis could pave the way for genetic engineering approaches aimed at enhancing heat resilience without compromising pharmacological quality. Additionally, biotechnological methods such as in vitro culture, elicitation, and metabolic engineering of Hypericum cell lines could be employed to optimize the production of valuable compounds under controlled environmental conditions. These strategies not only provide insights into the plant’s adaptive biology but also contribute to the sustainable utilization of Hypericum as a medicinal resource.

5. Conclusion

The present study provides a comprehensive understanding of how high-temperature stress influences the biosynthesis of the major secondary metabolites—hypericin and hyperforin—in Hypericum perforatum. The results demonstrate that elevated temperature causes significant physiological and biochemical alterations, primarily through the overproduction of reactive oxygen species (ROS) and the disruption of antioxidant defense mechanisms. These oxidative imbalances interfere with the expression of key biosynthetic genes such as Hyp-1, PKS, and AS, which are essential for the synthesis and accumulation of hypericin and hyperforin. Microscopic and molecular analyses confirm that structural damage to glandular trichomes under heat stress reduces the storage and secretion of these bioactive compounds. Moreover, high temperature was found to decrease chlorophyll content, affect photosynthetic efficiency, and accelerate metabolic respiration, thereby reducing the availability of carbon skeletons necessary for secondary metabolite biosynthesis. From a pharmacological perspective, the decline in hypericin and hyperforin content under heat stress directly compromises the medicinal quality and consistency of Hypericum raw materials. Therefore, it is crucial to develop heat-tolerant genotypes and implement cultivation strategies (e.g., shading, irrigation, antioxidant application) to mitigate heat-induced oxidative stress. In conclusion, this research highlights the intricate link between environmental temperature, ROS signaling, and gene regulation, providing valuable insights for future genetic, biochemical, and agronomic approaches aimed at enhancing the stability of bioactive compounds in Hypericum species under changing climatic conditions.

Data Availability Statement

The data supporting the findings of this study are available from the corresponding author upon reasonable request.

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Edited by

• Editor:
  Takako Matsumura Tundisi

Publication Dates

• Publication in this collection
  11 Sept 2026
• Date of issue
  2026

History

• Received
  17 May 2026
• Accepted
  29 June 2026