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Article

Effects of Growth Phases and Intensification of Light on Secondary Metabolites and Agro-Morphological Traits of the St. John’s Wort (Hypericum perforatum L.)

by
Mina Sadat Tabatabaei
1,2,3,*,
Ahmad Sobhani
4,
Morteza Khanahmadi
4,
Sara Zare
5 and
Stefan Wanke
6,7,8
1
Botany Institute, Dresden University of Technology, 01062 Dresden, Germany
2
Department of Plant Breeding, Agriculture College, Shiraz University, Shiraz 71441-65186, Iran
3
Institute for Breeding Research on Horticultural Crops, Julius Kühn-Institut, 06484 Quedlinburg, Germany
4
Agricultural Biotechnology Research Institute, Isfahan 84156-83111, Iran
5
Department of Agronomy and Plant Breeding Sciences, College of Aburaihan, Tehran University, Pakdasht 14174-66191, Iran
6
Institute of Ecology, Evolution and Diversity, Goethe-University, 60438 Frankfurt am Main, Germany
7
Senckenberg Research Institute and Nature Museum, 60325 Frankfurt am Main, Germany
8
Instituto de Biología, Universidad Nacional Autónoma de México, Mexico City 04510, Mexico
*
Author to whom correspondence should be addressed.
Plants 2026, 15(4), 663; https://doi.org/10.3390/plants15040663
Submission received: 30 December 2025 / Revised: 10 February 2026 / Accepted: 18 February 2026 / Published: 22 February 2026
(This article belongs to the Section Plant Physiology and Metabolism)
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Abstract

Light regime and growth phase are significant determinants of agro-morphological traits and secondary metabolite accumulation in plants. This study evaluated the effects of two light conditions on agro-morphological and phytochemical traits of two Hypericum perforatum genotypes (Topas and Mariana). Natural daylight and an extended 19 h photoperiod with supplemental white fluorescent light were tested at two growth phases (vegetative versus reproductive (flowering)), based on leaf sampling at the respective phases. Analysis of variance showed significant effects of growth phase, light treatment, and genotype on most traits, with growth phase exerting the most decisive influence (p < 0.01). Significant genotype × growth phase interactions were observed for most traits, whereas genotype × light interactions mainly affected phytochemical parameters. Leaf area, gland number, and gland area increased during the reproductive phase, especially in Topas, and were further enhanced under the 19 h photoperiod. Leaf area increased markedly during the reproductive phase, reaching 118.81 mm2 in Topas under the 19 h photoperiod compared with 68.40 mm2 under natural light. Prolonged light exposure increased hypericin, pseudo-hypericin, hyperforin, flavonoids, and total phenolics. The highest sum of hypericins (4.67 mg g−1 DW), flavonoids (143.09 mg QE g−1 DW), and phenolics (242.74 mg GA g−1 DW) was observed in the Topas in the reproductive phase under the 19 h photoperiod, whereas hyperforin content peaked in vegetative Mariana (55.65 mg g−1 DW). In contrast, the lowest sum of hypericins (1.80 mg g−1 DW) occurred in vegetative Mariana under natural light, while the minimum flavonoids (74.45 mg QE g−1 DW) and phenolics (133.22 mg GA g−1 DW) contents were recorded in the vegetative Topas under natural light regim, and the lowest hyperforin concentration (19.65 mg g−1 DW) was found in the Mariana genotype under natural light regime and in the reproductive phase. Principal component analysis associated PC1 with agro-morphological traits and hypericin-related metabolites, and PC2 with hyperforin and white gland traits. Heatmap and correlation analyses supported these patterns. Overall, extended photoperiod and growth phase are critical drivers of medicinal compound accumulation in H. perforatum.

1. Introduction

Plants occupy a central role in modern society as sources of both food and medicine. Hypericum perforatum L. (Hypericaceae), commonly known as St. John’s wort, is a widely used medicinal plant traditionally consumed as herbal tea and a food supplement [1,2]. H. perforatum contains pharmacologically active phytochemical constituents, especially phenolic compounds, including the naphthodianthrones hypericin and pseudo-hypericin, phloroglucinols such as hyperforin, as well as a diverse range of flavonoids [3,4,5,6]. Beyond its well-documented antidepressant effects, H. perforatum has attracted interest for its antioxidant, antianxiety, anticancer, and anti-inflammatory activities, and its extracts can reduce oxidative stress, thereby preventing inflammation, neurotoxicity, and gastrointestinal disorders [7,8,9,10].
Among environmental factors, light is one of the most influential regulators of photosynthesis, plant growth and development, and the biosynthesis and accumulation of secondary metabolites, which are key determinants of product quality [11,12,13]. In contrast to primary metabolites, secondary metabolites such as anthocyanins, carotenoids, and flavones occur at relatively low concentrations. Nevertheless, they play crucial roles in human health and are found in most plant tissues and vegetables, with their production strongly influenced by light [14,15]. Both the quality and quantity of incident light can substantially affect plant morphology, photosynthetic performance, photosystem adaptations, and tolerance to biotic and abiotic stresses by modifying radiation intensity and exposure duration [16,17]. Light strongly shapes plant growth by influencing morphology, pigmentation, and overall developmental patterns. Each species has distinct light requirements, and different growth phases, such as vegetative versus the reproductive phase, may require different light regimes [18,19,20,21].
In H. perforatum, increasing light intensity and exposure duration improve yield and product quality, while strongly affecting secondary metabolites such as phenolics and flavonoids [22]. Notably, unstable protoforms of hypericin are efficiently converted to stable hypericin upon light exposure [23]. Similarly, in coriander, increases in photoperiod and light intensity enhanced most morphological traits and boosted secondary metabolites, including linalool [24]. Plant responses to light vary across developmental phases, and metabolite production may change substantially between them. Previous studies have demonstrated that extending the duration of light exposure enhances photosynthetic rates, promotes leaf expansion, accelerates plant growth, and shortens vegetative and reproductive phases [25].
Given the strong influence of light and developmental phase on plant performance, the present study was designed to achieve the following goals: (a) investigate the effect of extended light exposure on agro-morphological and phytochemical traits; (b) examine the impact of vegetative and reproductive phases on these traits under two light conditions; and (c) analyze the relationships between agro-morphological and phytochemical traits in H. perforatum.

2. Results

2.1. Primary Data for Agro-Morphological and Phytochemical Traits

After testing the normality of the data, the mean of all traits across both genotypes (Topas and Mariana) was calculated to investigate the effect of extended day length on phytochemical and agromorphological traits, and the results were presented in Appendix A, Table A1. These data demonstrate that the extended 19 h photoperiod substantially enhanced most parameters compared to natural light. Detailed interaction effects of genotype × growth phase × light treatment were analyzed using the LSD post hoc test and are shown in Table 1. Key phytochemical traits (phenolics, hypericin, hyperforin, flavonoids) are visualized in Figure 1.
Under natural light, leaf area (LA) increased from the vegetative to the reproductive phase. Both genotypes exhibited the highest LA during the reproductive phase (Topas: 84.54 mm2; Mariana: 83.89 mm2), while Mariana showed the lowest value during the vegetative phase (57.55 mm2). Other morphological traits including white gland number (WN), dark gland number (DN), white gland area (WA), dark gland area (DA), dark gland number/leaf area (DN/LA), dark gland area/leaf area (DA/LA), white gland area/dark gland area (WA/DA), and white gland number/dark gland number (WN/DN), also increased from the vegetative phase to the reproductive phase under natural light. Topas in the reproductive phase exhibited maximum dark gland parameters (DA: 0.22 mm2; DN: 15.21; DA/LA: 0.0026; WN/DN: 9.04), while Mariana in the vegetative phase displayed minimum values (DA: 0.13 mm2; DN: 12.76; DA/LA: 0.0017; WN/DN: 4.10). The greatest WA, WN, WN/LA, WA/LA, and WA/DA were found in the reproductive phase of Mariana (0.79 mm2, 137.85, 1.64, 0.009, and 5.38), whereas the lowest values were detected in vegetative Topas (0.35 mm2, 53.70, 0.93, 0.005, and 2.18) (Table 1).
Under the 19 h photoperiod, all morphological traits increased relative to the natural light (control) (Table 1). The highest LA, DN, DA, DA/LA, WA/DA, and WN/DN were recorded in Topas in the reproductive phase (118.81 mm2, 17.10, 0.30 mm2, 0.0026, 2.78, and 10.14), while the lowest values occurred in Mariana in the vegetative phase (76.85 mm2, 13.98, 0.14 mm2, 0.0018, and 5.98). The highest WA, WN, WN/LA, and WA/LA were observed in Mariana in the reproductive phase (0.84 mm2, 173.53, 1.63, and 0.007), whereas Topas exhibited the lowest values in the vegetative phase (0.43 mm2, 82.99, 1.07, and 0.006).
For phytochemical traits under natural light, the maximum pseudohypericin (PsH), hypericin (Hy), sum hypericin (SH), flavonoids (Fla), and total phenolics (Ph) were observed in Topas in the reproductive phase (2.29, 1.41, 3.79, 114.45, and 233.70 mg g−1, respectively). In contrast, hyperforin (HF) and DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging assay activity peaked in Mariana in the vegetative phase (35.65 mg g−1 and 60.57%). Minimum PsH, Hy, and SH were recorded in Mariana in the vegetative phase (1.12, 0.46, and 1.80 mg g−1), minimum Fla (74.45 mg g−1) and Ph (133.22 mg g−1) in Topas in the vegetative phase, and minimum HF (19.65 mg g−1) and DPPH (40.26%) in Mariana in the reproductive phase.
The extended photoperiod increased secondary metabolite accumulation in both genotypes, particularly during the reproductive phase, except for hyperforin (Figure 1). Most phytochemical traits peaked in Topas in: PsH (2.83 mg g−1), Hy (1.79 mg g−1), SH (4.67 mg g−1), Fla (143.09 mg g−1), and Ph (242.74 mg g−1). HF and DPPH reached their highest values in Mariana in the vegetative phase (55.65 mg g−1 and 69.68%). Minimum PsH (1.81 mg g−1), Hy (0.74 mg g−1), and SH (2.52 mg g−1) occurred in Mariana in the vegetative phase, while HF (34.18 mg g−1) and DPPH (50.64%) reached minimum values in Mariana in the reproductive phase. The lowest Fla and Ph (116.34 and 201.63 mg g−1) were recorded in Topas in the vegetative phase (Table 1).
Figure 1 demonstrates that extending the photoperiod increased phenolics, hypericin, hyperforin, and flavonoids accumulation in both genotypes, particularly during the reproductive phase, except for hyperforin. Phenolic content in both genotypes during both vegetative and reproductive phases increased under a 19h photoperiod, reaching its highest levels in Topas under the reproductive phase. Increasing phenolic content using extended photoperiod is observed in the vegetative phase more than the reproductive phase (Table 1, Figure 1A). Hypericin, a key metabolite in Hypericum, responded vigorously to extended photoperiod in Topas, nearly doubling in the reproductive phase, whereas the lowest values were observed in Mariana in the vegetative phase (Table 1, Figure 1B). Under natural light, Topas in the reproductive phase had the highest hypericin (1.41 mg g−1) while vegetative Mariana had the lowest (0.46 mg g−1). Extended the light photoperiod increased them to 1.79 mg g−1 and 0.74 mg g−1, respectively, representing increases of 27% and 61%. In contrast, hyperforin peaked in the vegetative phase; although increased by prolonged light exposure, its maximum concentration occurred in vegetative Mariana, and its minimum appeared in Mariana in the reproductive phase (Table 1, Figure 1C). Extended light increased from 35.65 mg g−1 to 55.65 mg g−1 (56% increase). Flavonoid levels responded moderately to an extended photoperiod, showing the highest accumulation in Topas under a 19 h photoperiod and reproductive phase, and the lowest in vegetative Topas under natural light (Table 1, Figure 1D).

2.2. Analysis of Variance for Main Effects and Interactions

The variance analysis of the examined traits across genotypes (Topas, Mariana), growth phases (vegetative, reproductive), and light environments (natural, 19 h photoperiod) is presented in Table 2.
ANOVA results revealed that the growth-phase effect was significant (p < 0.01) for all traits studied. Light treatment was significant (p < 0.01) for all traits except WA/DA and DA/LA, and significant (p < 0.05) for sum hypericin (SH), pseudo-hypericin (PsH), WA/LA, DA, WN/LA, and WA. Genotype effects were significant (p < 0.01) for most traits and non-significant only for DN/LA and WA/DA.
According to ANOVA (Table 2), the interaction between genotype and growth phase was significant (p < 0.01) for most agro-morphological traits, including white gland, number WN, WN/LA, WA/LA, and (WN/DN), and significant at p < 0.05 for WA and DA, but non-significant for dark gland number (DN), DN/LA, DA/LA, and WA/DA. Similarly, the genotype × light interaction was significant for most traits except DN/LA, DA/LA, and WN/DN. The light × growth phase interaction was substantial for WA, WN/LA, DA, WA/LA, and LA, but non-significant for the remaining traits. Notably, the genotype × light × growth phase interaction was highly significant (p < 0.01) for all morphological traits.
ANOVA results (Table 2) indicated a significant genotype × growth phase interaction (p < 0.01) for Hy, SH, HF, and DPPH, and for PsH, Fla, and Ph at p < 0.05. The genotype × light interaction was significant (p < 0.01) for most traits except PsH, Ph, and DPPH, which were significant at p < 0.05. The light × growth-phase interaction was significant for all traits except flavonoids. The three-way interaction (genotype × light × growth phase) was highly significant for all traits except Ph, which was substantial at p < 0.05.

2.3. Correlation Patterns Among Traits

Correlation analysis under natural light (Table 3) indicated that LA had positive and significant correlations with WN, WA, DN, DA, and WA/LA, and a negative correlation with DN/LA. WN was positively and significantly correlated with WA and WN/LA. WA exhibited positive correlations with WN/LA, WA/LA, and WA/DA. DN was positively correlated with DA and DN/LA, while DA correlated positively with DA/LA.
Under a 19 h photoperiod, LA again showed significant positive relationships with WN, WA, DN, DA, and WN/DN, and a negative correlation with DN/LA. WN and WA were positively correlated with WN/LA and WA/LA; DN and DA demonstrated positive correlations with each other and with DN/LA and DA/LA (Table 3).
Correlation analysis under natural light (Table 3) showed strong positive correlations between PsH and DN, DA, DA/LA, Hy, SH, and Ph. Hy correlated positively with LA, DN, DA, PsH, SH, Ph, and Fla. SH, representing combined hypericins, exhibited positive correlations with LA, DN, DA, DA/LA, PsH, Hy, and Fla. In contrast, HF showed significant negative correlations with DN, DA/LA, PsH, SH, Hy, and Fla, but positive correlations with WN and WA. Fla showed positive correlations with DN, SH, DPPH, and Ph. Ph correlated positively with LA, WA, Hy, PsH, HF, Fla, and DPPH. DPPH showed positive correlations with WN (0.75), HF (0.88), Fla (0.84), and Ph (0.78), and negative correlations with PsH (−0.85), SH (−0.75), and LA (−0.98).
Under 19 h photoperiod (Table 3), PsH was positively correlated with LA, DA, DN, SH, Hy, Ph, and Fla. Hy showed positive correlations with LA, DN, DA, WN/DN, WA/DA, SH, Ph, and Fla. SH was positively correlated with DN, DA, PsH, Hy, and Ph. HF exhibited strong negative correlations with PsH (−0.98), Hy (−0.89), SH (−0.94), Fla (−0.80), and with DN (−0.83) and DA (−0.86), but showed positive correlations with WA (0.93), WN (0.94), Ph (0.89), and DPPH (0.70). DPPH showed negative correlations with all agromorphological traits except WN (0.82) and WA (0.74), and with phytochemical traits except HF (0.70), Fla (0.73), and Ph (0.64). Ph correlated positively with LA (0.80), DN (0.82), DA (0.83), PsH (0.90), Fla (0.94), and HF (0.89).

2.4. Multivariate Analysis: Principal Component and Heatmap Analysis

Multivariate approaches, including Principal component analysis (PCA) and hierarchical heat-mapping, reveal trait relationships and genotype responses beyond univariate comparisons. Based on univariate analyses, both H. perforatum genotypes (Topas and Mariana) responded significantly to the photoperiod and growth phase. Trait distributions under both natural and extended 19 h photoperiod were therefore visualized using biplot analysis (Figure 2).
The first two PCA components explained more than 83% of the total variance: PC1 accounted for 57.23%, and PC2 for 26.18%. PC1 was positively correlated with LA, DN, DA, gland ratios, PsH, SH, Hy, Fla, and Ph, while PC2 showed positive associations with WA, WN, HF, and DPPH. Consequently, PC1 was designated as the agro-morphological and hypericin component, while PC2 represented the hyperforin component. Genotypes located in the first quadrant (positive for PC1 and PC2) were superior for the studied traits (Figure 2).
Trait line angles in the biplot reflect the strength of the correlations. A narrow angle between phenolics, flavonoids, and hypericin-related traits indicated strong positive associations, supporting the observation that increases in hypericin were accompanied by increases in phenolics and flavonoids. Biplot results showed that in both photoperiods, Topas during the reproductive phase under the 19 h photoperiod exhibited the highest PC1 values and the best overall trait performance. Extended photoperiod increased agronomic and phytochemical traits in both genotypes, with stronger effects during the reproductive phase, except for hyperforin. Extended photoperiod induced trait shifts were most significant in Topas in the reproductive phase and Mariana in the vegetative phase. In contrast, plant leaves in the vegetative phase exhibited higher PC2 values (WA, WN, HF, DPPH), with vegetative Mariana being superior in this component. The reproductive phase (flowering) enhanced hypericin-like metabolites, particularly under a 19 h photoperiod.
Heatmap clustering was used to classify genotypes by trait patterns (Figure 3a,b). Un-der natural light (Figure 3a), three clusters were identified: (1) Topas duringreproductive phase, showing highest values for most traits except HF, DPPH, WN, and WA, which were lowest; (2) Marianaand Topas during vegetative phase, showing minimum values for most traits except HF in Mariana; and (3) Mariana during the reproductive phase, characterized by high DPPH, WN, WA, and Fla, and intermediate values for other traits.
Under the 19 h photoperiod (Figure 3b), three clusters again emerged. Topas inreproductive phase formed the group with the highest hypericin-associated traits (DA, DN, Hy, SH, PsH, Ph, LA, DN/LA, Fla). Mariana inreproductive phase formed a second cluster distinguished by high WN and flavonoid content. Both genotypes in the vegetative phase formed the third cluster, showing the lowest values for most traits and similar to natural light highest levels of hyperforin, DPPH, and WA.

3. Discussion

The primary aim of this study was to elucidate the influence of photoperiod extension and growth phase on morphological and phytochemical traits in H. perforatum. In this study, only leaves were selected as the target tissue because they contain both dark and white glandular structures, which are the principal sites of hypericin and hyperforin accumulation. By restricting analysis to a single organ, the study isolates the effects of the photoperiod and developmental phase while eliminating confounding factors arising from differences in organ-specific metabolite composition. This approach reveals how the physiological transition to flowering affects secondary metabolite accumulation in vegetative tissues, independent of contributions from reproductive organs. In general, extended photoperiods stimulate photosynthetic carbon assimilation, increasing both biomass and the accumulation of secondary metabolites such as phenolics and flavonoids [26,27,28]. Consistent with this, the ANOVA results demonstrated significant effects of growth phase, light regime, and genotype on nearly all investigated traits. The growth phase exerted the most significant influence, with most traits showing higher values during the reproductive phase than during the vegetative phase. Light conditions also played a decisive role, with most traits increasing under the 19 h photoperiod. Significant genotype effects were observed in nearly all parameters. These findings align with previous observations in medicinal and aromatic plants, where genotypic background and photoperiodic dynamics substantially affect morphological development and secondary metabolite biosynthesis [29,30,31].

3.1. Morphological Plasticity Under Extended Photoperiods

In terms of morphological traits, prolonged photoperiods markedly increased leaf area, gland number, and gland area. This response was especially pronounced in the Topas genotype under 19 h light conditions during the reproductive phase. Leaf expansion under prolonged illumination is generally attributed to enhanced photosynthetic performance and carbon acquisition, leading to increased vegetative vigor. Photoperiods enhance leaf expansion and glandular development in Cannabis sativa L., ultimately increasing metabolic productivity [32]. Furthermore, glandular structures in Hypericum function as specialized secretory organs that synthesize, store, and accumulate phloroglucinols, hypericins, and pigments [22]. Their expansion under extended light suggests that the photoperiod regulates structural organogenic processes, possibly through photoreceptor-mediated hormonal regulation such as phytochrome or cryptochrome pathways. These findings corroborate those of Kuo and coauthors, who showed that increased light duration promoted LA expansion and biomass accumulation in H. perforatum [33]. The photoperiod-driven increases in glandular traits provide a mechanistic link to metabolite production, as the quantity and area of glands directly define the metabolic storage and biosynthetic capacity of Hypericum tissues.

3.2. Light-Induced Modulation of Phytochemical Profiles

Extending the photoperiod notably increased secondary metabolite content, especially during the reproductive phase. Hypericin, pseudohypericin, phenolics, and flavonoids reached their highest concentrations under long-day conditions in the Topas genotype. This pattern aligns with broader evidence that elevated light exposure upregulates carbon flux into the phenylpropanoid and naphthodianthrone biosynthetic pathways, which drive the formation of DPPH compounds [22,24,31,34]. Notably, the marked increase in hypericin, pseudohypericin, flavonoids, and phenolics under the extended 19 h photoperiod using fluorescent white light in this study suggests that photoperiod-dependent regulation of secondary metabolite biosynthetic pathways plays a central role in H. perforatum. In line with this interpretation, studies in other plant species have shown that extended daylength can upregulate key phenylpropanoid and flavonoid pathway genes (such as PAL, CHS, CHI, 4CL, F3H, and F3′H) via sustained activation of light-responsive transcription factors, including HY5 and MYB, which accumulate and remain active throughout prolonged light periods [13,35,36,37,38]. Although gene expression was not investigated, these findings provide a mechanistic framework that may explain the coordinated increase in hypericin-related metabolites, phenolics, and flavonoids observed under the 19 h photoperiod in H. perforatum. Furthermore, light-mediated photoconversion of hypericin precursors and the light-dependent activation of antioxidants [23] mechanistically explain why extended photoperiod enhances the production of these secondary metabolites. Unlike hypericin derivatives, hyperforin accumulation was preferentially enhanced during the vegetative phase, particularly in the Mariana genotype. This divergence suggests distinct biosynthetic regulation of the two prominent metabolite families in Hypericum. Hypericins derive from polyketide metabolism within dark glands, while hyperforin originates from phloroglucinol pathways predominantly associated with white glands [39,40,41]. Therefore, physiological developmental phases appear to favor different biosynthetic machinery. This observation is supported by Lazzara and coauthors, who reported strong positive associations between hypericin and pseudohypericin, confirming their shared precursors and organ specificity [42]. The heightened hypericin levels in the reproductive phase indicate functional specialization of dark glands during reproductive development. Conversely, Mariana’s vegetative-phase hyperforin dominance indicates that white-gland metabolism is less dependent on floral induction and may be more responsive to photoperiodic stress.
The correlation analysis further supported these observations. Correlation analyses revealed strong positive associations between glandular traits and metabolite content. Increased LA, dark gland number, and dark gland area were closely associated with higher levels of hypericin and pseudohypericin. These associations highlight a central physiological rule in medicinal plants: morphological expansion and structurally specialized tissues drive metabolite output. Li and coauthors similarly reported that morphological traits predict bioactive compound accumulation in medicinal plant species [43]. Similarly, white glands showed a positive correlation with hyperforin content under both light regimes. This is consistent with anatomical and chemical evidence demonstrating that white glands act as reservoirs for phloroglucinols and lipophilic compounds, including hyperforin [39,41]. Thus, the architecture of glandular tissue can be used as a phenotypic proxy for secondary metabolic capacity, providing a selection target for breeding.
Phenolic compounds and flavonoids also demonstrated strong positive correlations with hypericin across both photoperiod environments. This correlation aligns with findings by Gadzovska and coauthors [44], who reported that these metabolites are coordinately regulated and may respond to common upstream signaling pathways controlling secondary metabolism in H. perforatum. Taken together, these findings indicate that the 19h photoperiod and reproductive phase strongly modulate metabolite biosynthesis and can be leveraged to improve phytochemical yield. Furthermore, the positive correlations between LA, gland traits, and metabolite content suggest that larger leaf surface areas and greater gland densities facilitate higher secondary metabolite accumulation, findings consistent with [43,45], who showed that leaf morphology correlates with active compound concentrations in medicinal plants.

3.3. Multivariate Interpretation and Genotype Selection

PCA results confirmed two principal functional trait axes. PC1, the “agro-morphological and hypericin axis,” captured LA, dark-gland traits, hypericin derivatives, phenolics, and flavonoids. PC2 formed a distinct “hyperforin axis,” driven by white gland traits, hyperforin, and DPPH activity. Kimáková and coauthors similarly demonstrated PCA’s power to discriminate phytochemical chemotypes, facilitating targeted genotype selection for hypericin and hyperforin optimization [46].
The biplot indicated that Topas, during the reproductive phase, under a 19 h photoperiod, is the optimal genotype × growth phase combination for targeting hypericin-related traits. Conversely, Mariana is superior during the vegetative phase for hyperforin production.
Heat map clustering supported the PCA results. Under both photoperiod regimes, Topas in the reproductive phase formed a cluster characterized by high levels of hypericin, phenolics, and flavonoids, and by dark-gland traits, but low levels of hyperforin. Vegetative Mariana clustered oppositely, with elevated hyperforin and DPPH, and lower hypericin traits. These opposing chemotype trajectories suggest pathway specialization.
This result aligns with Kusari and coauthors, who documented differential metabolite signatures across Hypericum species, and with Sirvent and coauthors and Noormohammadi and coauthors, who demonstrated differential elicitor sensitivity in hyperforin vs. hypericin biosynthesis [47,48,49]. Such divergence reflects H. perforatum’s metabolic compartmentalization and validates targeted cultivation strategies.

3.4. Critical Gaps and Outlook

Despite the strong evidence, several research gaps must be acknowledged. First, the present findings were obtained in controlled growth conditions. Real agricultural systems involve fluctuating light intensity, spectral composition, temperature, humidity, and nutrient availability. Photoperiod alone rarely drives metabolic shifts in natural environments; instead, it interacts with environmental variables in synergistic or antagonistic ways [50,51]. Thus, future tests might want to evaluate these findings under natural field conditions prior to agricultural field implementation. Second, although the results indicated correlations between phytochemical and morphological traits, the molecular underpinnings of metabolite biosynthesis and gland development were not investigated. Therefore, comprehensive proteomic and transcriptomic analyses are warranted to further elucidate these relationships. Third, the study analyzed only two genotypes, which limits genetic generalizability. A broader sampling of ecotypes and wild accessions might reveal whether metabolite responses are additive, epigenetic, or genotype-specific [52]. Fourth, prolonged photoperiods may induce metabolic stress rather than only enhancing growth. Photoperiod stress alters carbon allocation, ROS generation, and antioxidant protective responses [53,54]. Measuring stress biomarkers (e.g., MDA, antioxidant enzyme activity) would help distinguish photoperiodic optimization from stress-driven metabolic compensation. Finally, this study did not investigate spectral quality. Light wavelength strongly alters gland development and metabolite pathways. UV-A, blue, or red-shifted light spectra differentially impact hypericin and phenolic accumulation [22,30]. Integrating photoperiod with spectrum tuning may be necessary for maximal phytochemical output.

4. Materials and Methods

4.1. Plant Material, Experimental Site, and Design

The plant material used in this experiment consisted of two H. perforatum genotypes, Topaz and Mariana, obtained from Pakanbazr Company (Isfahan, Iran). Seeds of H. perforatum were surface sterilized by immersion in 70% ethanol for 2 min, rinsed with sterile water, and germinated on Murashige and Skoog (MS) medium [55] in a growth chamber. Eight-week-old seedlings were transplanted into pots (18 cm height, 13 cm cross-sectional diameter, 20 cm opening diameter). The growth medium consisted of 50% agricultural soil and 50% peat moss. Pots were then transferred to a greenhouse with controlled environmental conditions of 24–26 °C and 70% relative humidity.
The experiment was conducted under greenhouse conditions using a split-plot design over time, with trait measurements taken at two growth phases (vegetative and reproductive). The split-plot arrangement was implemented within a completely randomized design (CRD) with three replications. Two light environments were applied as the main plots: natural summer photoperiod (daylight) and an extended 19 h photoperiod with supplemental fluorescent white light. Genotype and growth phase were considered as sub-plot factors. For each genotype × light × growth phase combination, 30 leaves were sampled per replication for agro-morphological and phytochemical analyses.
Notably, in this study, only leaves were used as the experimental material. The terms “vegetative” and “flowering” refer to the growth phase of the plant at the time of sampling and not to the plant organ analyzed.

4.2. Light Condition

H. perforatum is a long-day plant; therefore, the effect of photoperiod extension was investigated by increasing day length to 19 h using supplemental fluorescent white light. The light intensity at canopy level was approximately 2300 lux, corresponding to 35–45 µmol m−2 s−1 photosynthetic photon flux density (PPFD), and the treatment was applied for two months. All other greenhouse conditions were kept constant across treatments (25 °C and 70% relative humidity). Both genotypes (Topas and Mariana) were grown under two light regimes: (i) a natural summer photoperiod in Iran (control, approximately 14 h) and (ii) an extended 19 h photoperiod with supplemental fluorescent white light.

4.3. Agro-Morphological Trait Evaluation

To evaluate agromorphological and phytochemical traits, mature leaf samples were collected at two developmental phases: (1) the vegetative phase (45 days after planting, prior to visible flower bud initiation) and (2) the reproductive (flowering) phase. Importantly, only leaf tissue was sampled for all analyses; flowers were not included in this study. The terms ‘vegetative’ and ‘flowering’ refer to the plant’s overall developmental phase at the time of sampling, not to the tissue type examined.

4.3.1. Leaf Area

Leaf area was quantified using a digital leaf area meter (Model GA-5, OSK Company, Tokyo, Japan). Thirty leaves were randomly selected from each plant, and their green leaf area was measured. The average LA was calculated from these thirty measurements.

4.3.2. White and Dark Gland Number and Area

Since hyperforin and hypericin concentrations correlate with the density of white and dark glands, gland traits were evaluated as key parameters. Approximately 30 mature leaves were collected per growth phase (vegetative and reproductive), light treatment (natural summer photoperiod and extended 19 h photoperiod), and replication. Each leaf is placed between two microscope slides. Leaves were photographed with a DSLR camera equipped with a macro lens, using transmitted illumination from an under-leaf light source to clearly visualize glands (Figure 4).

4.4. Phytochemical Trait Evaluation

To examine potential genotype responses to natural summer and extended 19 h photoperiods, the following phytochemical traits were analyzed in both growth phases.

4.4.1. Methanol Extraction

Sample extraction was performed according to the method described by Sobhani and coauthors with slight modifications [54]. Dried, powdered tissue (100 mg) from each sample (CLL, LM, and FB) was mixed with 1 mL of methanol. The mixture was subjected to ultrasonication (Sonorex, Bandelin Electronic, Berlin, Germany) for 30 min, followed by incubation in a thermomixer (Eppendorf) at room temperature (RT) for 30 min with shaking at 200 rpm. The suspension was centrifuged at 10,000 rpm for 5 min at RT, and the supernatant containing target metabolites was collected. The residual pellet underwent a second extraction with 1 mL of methanol under identical shaking and centrifugation conditions, excluding the ultrasonication step. Supernatants from both extraction steps were pooled to form the final sample for analysis.

4.4.2. Pseudo-Hypericin, Hypericin, and Hyperforin Quantification

Hypericin, pseudohypericin, and hyperforin contents were determined using a high-performance liquid chromatography (HPLC) system (S 7131, Sykam, Eresing, Germany) equipped with a fluorescence detector (Shimadzu RF-10A) and a Reprosphere 100 C18 column (5 μm, 250 × 4.6 mm). Hypericin and pseudohypericin were detected using a fluorescence detector (Shimadzu RF-10A) at excitation and emission wavelengths of 315 nm and 593 nm, respectively, whereas hyperforin was detected using a UV detector (Sykam S 3210) at 274 nm. The mobile phase consisted of ethyl acetate, methanol, and sodium dihydrogen phosphate (39:160:41, v/v/v). A 20 μL aliquot of the methanol extract was injected, and detection was performed at excitation and emission wavelengths of 315 nm and 593 nm, respectively. Quantification was achieved using an external calibration curve prepared with analytical standards of hypericin (Sigma-Aldrich, Burlington, MA, USA, CAS number 548–04–9) and hyperforin (Sigma-Aldrich, Burlington, MA, USA, CAS No. 11079-53-1) [54].

4.4.3. Determination of Total Phenolic and Flavonoid Content

Total phenolic content was evaluated using the Folin–Ciocalteu method [55]. Briefly, 100 μL of methanolic extract was mixed with 1580 μL of distilled water and 100 μL of Folin–Ciocalteu reagent (Merck, Darmstadt, Germany). After a 6 min incubation, 300 μL of 7% (w/v) sodium carbonate was added. The mixture was incubated at room temperature for 90 min, and absorbance was measured at 760 nm using a spectrophotometer (Beckman DU 530, Stanwood, WA, USA). Results were expressed as mg of gallic acid (GA) per gram of dry weight.
The total flavonoid content was quantified using the aluminum chloride colorimetric method [56]. An aliquot of 50 μL of extract was mixed with 2.8 mL of distilled water, 0.1 mL of 10% aluminum chloride, and 0.1 mL of 1 M potassium acetate. Following incubation in the dark at room temperature for 30 min, absorbance was recorded at 415 nm. Quercetin was used as the standard, and results were expressed as mg of quercetin per gram of dry weight.

4.4.4. DPPH Radical Scavenging Activity Assay

Free radical scavenging activity was assessed using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay [22,57,58,59]. Ascorbic acid was first tested over a concentration range of 0–800 mg/L to identify an appropriate sample concentration range. Based on these preliminary tests, 1.96 mL of 0.01 mM DPPH solution was mixed with 0.04 mL of the methanolic extract (50 mg DW leaves/mL methanol, equivalent to 667 μg dry leaf weight/mL final concentration). The reaction mixture was incubated in the dark at room temperature for 30 min, and absorbance was measured at 517 nm (Beckman DU 530, Stanwood, WA, USA). A control solution contained 1.96 mL of DPPH solution and 0.04 mL of methanol. Separate blanks were prepared without DPPH to correct for the background absorbance of the solvent and extracts. DPPH radical scavenging activity was calculated as follows:
DPPH   scavenging   ( % )   =   ( A C − A C b ) − ( A S − A S b ) A c − A c b × 100
where AC is the absorbance of the control, ACb is the absorbance of the control blank, AS is the absorbance of the sample, and ASb is the absorbance of the sample blank. Therefore, the assay quantifies the percentage of DPPH radical scavenging by compounds present in the methanolic leaf extracts, and is not used here as a direct measure of total antioxidant capacity.

4.5. Statistical Analysis

After normality testing, the data were analyzed using analysis of variance with a split-plot composite structure across two environments, based on a randomized complete block design with three replications. Analysis of variance (ANOVA) was performed in SAS (version 9.4; SAS Institute Inc., Cary, NC, USA) to assess the effects of light environment, growth phase, and genotype on each trait [60]. Mean comparisons and genotype performance across treatments were analyzed using GraphPad Prism (version 9.3.1). Significant differences among means were determined using the least significant difference (LSD) test at p < 0.05.
Principal component analysis (PCA), biplots, and heatmaps were generated to explore trait–genotype relationships using Statgraphics Centurion XVIII (http://www.statgraphics.com (accessed on 25 October 2022)). For genotype and trait clustering, heat maps were generated using JMP software (version 16).

5. Conclusions

The findings in this study indicate that the combined effects of the photoperiod regime and genetic variation can significantly impact both morphogenesis and the biosynthesis of secondary metabolites in H. perforatum. Higher leaf area, greater development of glandular structures, and increased production of secondary metabolites, such as hypericin, pseudo-hypericin, total flavonoids, and total phenolic compounds, were observed when the photoperiod was extended to 19 h during the reproductive phase. The Topas genotype produced the highest levels of hypericin and phenolic compounds. In contrast, the Mariana genotype had the greatest accumulation of hyperforin during the growing period. Positive correlations exist between leaf area and glandular density (both dark and light) and levels of secondary metabolite accumulation; therefore, morphological traits play an integral role in determining the overall potential for secondary metabolite production in plants. This research shows that increased photoperiod positively affects photosynthesis and metabolic quality, thereby influencing both plant performance and chemical composition. Multivariable statistical tools (PCA and heat maps) were used to demonstrate the superiority of Topas during the reproductive phase and of Mariana during the vegetative phase under the two different photoperiods. As such, these findings support the concept that manipulating both photoperiod and growth phase, as well as strategically selecting genotypes, can increase hypericin yields from Hypericum species.

Author Contributions

A.S. and M.S.T. conceived the research idea. M.K. and S.W. supervised the project. A.S. oversaw the methodological design. M.S.T. conducted the experiments. M.S.T. and S.Z. performed the statistical analyses and drafted the manuscript. S.W. critically reviewed and edited the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the Agricultural Biotechnology Research Institute, Isfahan, Iran; the Ministry of Science, Research and Technology of Iran; the German Academic Exchange Service (DAAD); the Erasmus Program; and the Graduate Academy of Technische Universität Dresden, Germany.

Data Availability Statement

All data supporting the findings of this study are provided within the article. Additional information can be requested from the corresponding author.

Acknowledgments

This work was carried out at the Agricultural Biotechnology Research Institute, Isfahan, Iran. The authors also sincerely thank Hooman Razi, Abbas Alemzadeh, and Ali Niazi at Shiraz University for their support and constructive guidance throughout this study. During the preparation of this manuscript, the authors used AI-based tools for English grammar and spelling optimization. The authors have reviewed and edited the output and take full responsibility for the content of this publication.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
WAWhite gland area
DADark gland area
LALeaf area
WNWhite gland number
DNDark gland number
PsHPseudo-hypericin
HyHypericin
SHSum hypericin
HFHyperforin
FlaFlavonoids
PhPhenolics
DPPH2,2-diphenyl-1-picrylhydrazyl scavenging assay
PCAPrincipal component analysis

Appendix A

Table A1. The effects of two photoperiods (natural summer photoperiod (daylight, 14h) and extended 19h photoperiod with supplemental fluorescent white light) and growth phases (vegetative and reproductive (flowering)) on studied agro-morphological and biochemical phytochemical traits in the leaf tissue of Hypericum perforatum.
Table A1. The effects of two photoperiods (natural summer photoperiod (daylight, 14h) and extended 19h photoperiod with supplemental fluorescent white light) and growth phases (vegetative and reproductive (flowering)) on studied agro-morphological and biochemical phytochemical traits in the leaf tissue of Hypericum perforatum.
LightNatural 19h
gpVFVF
T
LA (mm2)78.55 ± 0.93104.69 ± 1.6285.55 ± 0.99109.39 ± 1.07
WN (N)82.23 ± 1.23127.30 ± 1.8790.63 ± 1.04140.56 ± 2.18
WA (mm2)0.16 ± 0.0020.16 ± 0.0010.19 ± 0.0040.22 ± 0.03
DN (N)13.62 ± 0.3215.09 ± 0.6716.92 ± 0.7617.09 ± 0.02
DA (mm2)0.15 ± 0.0030.21 ± 0.0340.17 ± 0.030.25 ± 0.001
WN/LA1.15 ± 0.021.18 ± 0.041.50 ± 0.0451.70 ± 0.003
WA/LA0.002 ± 0.00010.005 ± 0.00010.004 ± 0.00030.008 ± 0001
DN/LA0.37 ± 0.0030.59 ± 0.030.39 ± 0.0010.66 ± 0.003
DA/LA0.002 ± 0.00040.001 ± 00020.002 ± 0.00010.002 ± 0.0002
WA/DA2.25 ± 0.042.82 ± 0.062.15 ± 0.012.35 ± 0.03
WN/DN6.41 ± 0.457.90 ± 1.227.30 ± 0.858.43 ± 1.04
PsH (mg g−1 DW)2.45 ± 0.323.33 ± 0.452.94 ± 0.833.89 ± 0.88
Hy (mg g−1 DW)0.51 ± 0.0340.81 ± 0.0680.80 ± 0.021.31 ± 0.03
SH (mg g−1 DW)1.73 ± 0.072.18 ± 0.042.24 ± 0.092.46 ± 0.09
HF (mg g−1 DW)33.96 ± 0.9432.35 ± 1.0350.70 ± 0.8934.59 ± 0.79
Fla (mg QE g −1 DW)89.05 ± 1.90126.23 ± 2.87123.04 ± 2.13136.71 ± 1.56
Ph (mg GA g−1 DW)148.66 ± 1.47224.53 ± 3.17207.18 ± 2.09238.55 ± 3.18
DPPH (%)46.21 ± 1.1148.21 ± 1.0161.85 ± 0.9653.40 ± 1.12
LA: Leaf area (mm2), WN: White gland number, WA: White gland area (mm2), DN: Dark gland number, DA: Dark gland area (mm2), WN/LA: White gland number/Leaf area, WA/LA: White gland area/Leaf area, DN/LA: Dark gland number/Leaf area, DA/LA: Dark gland area/Leaf area, WA/DA: White gland area/Dark gland area, WN/DN: White gland number/Dark gland number, PsH: Pseudo-hypericin (mg g−1 DW), Hy: Hypericin (mg g−1 DW), SH: Sum hypericin (mg g−1 DW), HF: Hyperforin (mg g−1 DW), Fla: Flavonoid (mg QE g−1 DW), Ph: Phenol (mg GA g−1 DW), DPPH: 2,2-diphenyl-1-picrylhydrazyl scavenging assay (%), gp: Growth phase, V: Vegetative, F: Reproductive (Flowering). T: Treat. Mean values are presented across both Topas and Mariana genotypes ± standard error.

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Plants 15 00663 g001
Figure 1. Bars show phenolic content (A), hypericin (B), hyperforin (C), and flavonoids (D) in leaves of Hypericum perforatum genotypes (Topas and Mariana) under two photoperiods (natural summer photoperiod, 14 h daylight, and an extended 19 h photoperiod with supplemental fluorescent white light) and two growth phases (vegetative and reproductive). Values are mean ± standard error. Letters V and F denote vegetative and reproductive (Flowering) phases, respectively. Red and blue bars represent the natural summer photoperiod and the extended 19 h photoperiod, respectively. In each population diagram, different lowercase letters above the bars indicate statistically significant differences among treatments at p < 0.05. The same letter indicates that values are not statistically different (p < 0.05).
Figure 1. Bars show phenolic content (A), hypericin (B), hyperforin (C), and flavonoids (D) in leaves of Hypericum perforatum genotypes (Topas and Mariana) under two photoperiods (natural summer photoperiod, 14 h daylight, and an extended 19 h photoperiod with supplemental fluorescent white light) and two growth phases (vegetative and reproductive). Values are mean ± standard error. Letters V and F denote vegetative and reproductive (Flowering) phases, respectively. Red and blue bars represent the natural summer photoperiod and the extended 19 h photoperiod, respectively. In each population diagram, different lowercase letters above the bars indicate statistically significant differences among treatments at p < 0.05. The same letter indicates that values are not statistically different (p < 0.05).
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Figure 2. Principal component analysis of the relationship among the studied traits in leaf tissue of Hypericum perforatum and the distribution of genotypes of Topas and Mariana relative to these traits under two photoperiods (natural summer photoperiod and extended 19h photoperiod with supplemental fluorescent white light) and growth phases (vegetative and reproductive phase (flowering)). LA: Leaf area (mm2), WN: White gland number, WA: White gland area (mm2), DN: Dark gland number, DA: Dark gland area (mm2), WN/LA: White gland number/Leaf area, WA/LA: White gland area/Leaf area, DN/LA: Dark gland number/Leaf area, DA/LA: Dark gland area/Leaf area, WA/DA: White gland area/Dark gland area, WN/DN: White gland number/Dark gland number, PsH: Pseudo-hypericin (mg g−1 DW), Hy: Hypericin (mg g−1 DW), SH: Sum hypericin (mg g−1 DW), HF: Hyperforin (mg g−1 DW), Fla: Flavonoids (mg QE g−1 DW), Ph: Phenolics (mg GA g−1 DW), DPPH: 2,2-diphenyl-1-picrylhydrazyl scavenging assay (%). V: Vegetative, F: Reproductive phase (Flowering), c: Control (natural photoperiod), h: 19 h photoperiod, dashed lines are the distance between the genotypes in the natural light and 19 h photoperiod.
Figure 2. Principal component analysis of the relationship among the studied traits in leaf tissue of Hypericum perforatum and the distribution of genotypes of Topas and Mariana relative to these traits under two photoperiods (natural summer photoperiod and extended 19h photoperiod with supplemental fluorescent white light) and growth phases (vegetative and reproductive phase (flowering)). LA: Leaf area (mm2), WN: White gland number, WA: White gland area (mm2), DN: Dark gland number, DA: Dark gland area (mm2), WN/LA: White gland number/Leaf area, WA/LA: White gland area/Leaf area, DN/LA: Dark gland number/Leaf area, DA/LA: Dark gland area/Leaf area, WA/DA: White gland area/Dark gland area, WN/DN: White gland number/Dark gland number, PsH: Pseudo-hypericin (mg g−1 DW), Hy: Hypericin (mg g−1 DW), SH: Sum hypericin (mg g−1 DW), HF: Hyperforin (mg g−1 DW), Fla: Flavonoids (mg QE g−1 DW), Ph: Phenolics (mg GA g−1 DW), DPPH: 2,2-diphenyl-1-picrylhydrazyl scavenging assay (%). V: Vegetative, F: Reproductive phase (Flowering), c: Control (natural photoperiod), h: 19 h photoperiod, dashed lines are the distance between the genotypes in the natural light and 19 h photoperiod.
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Figure 3. Heat-map diagram related to the studied traits in the leaf tissue of Hypericum perforatum genotypes (Topas and Mariana) under two photoperiods (a): natural summer photoperiod and (b): extended 19h photoperiod with supplemental fluorescent) and growth phases (vegetative versus reproductive (flowering)). The dark blue color indicates the lowest value of the characters. The dark red color indicates the highest value, LA: Leaf area (mm2), WN: White gland number, WA: White gland area (mm2), DN: Dark gland number, DA: Dark gland area (mm2), WN/LA: White gland number/Leaf area, WA/LA: White gland area/Leaf area, DN/LA: Dark gland number/Leaf area, DA/LA: Dark gland area/Leaf area, WA/DA: White gland area/Dark gland area, WN/DN: White gland number/Dark gland number, PsH: Pseudo-hypericin (mg g−1), Hy: Hypericin (mg g−1), SH: Sum hypericin (mg g−1), HF: Hyperforin (mg g−1), Fla: Flavonoids (mg QE g−1), Ph: Phenolics (mg GA g−1), DPPH: 2,2-diphenyl-1-picrylhydrazyl scavenging assay (%). V: Vegetative, F: reproductive (Flowering), c: Control (natural photoperiod), h: 19 h photoperiod, dashed lines are the distance between the genotypes under the natural and 19 h photoperiods.
Figure 3. Heat-map diagram related to the studied traits in the leaf tissue of Hypericum perforatum genotypes (Topas and Mariana) under two photoperiods (a): natural summer photoperiod and (b): extended 19h photoperiod with supplemental fluorescent) and growth phases (vegetative versus reproductive (flowering)). The dark blue color indicates the lowest value of the characters. The dark red color indicates the highest value, LA: Leaf area (mm2), WN: White gland number, WA: White gland area (mm2), DN: Dark gland number, DA: Dark gland area (mm2), WN/LA: White gland number/Leaf area, WA/LA: White gland area/Leaf area, DN/LA: Dark gland number/Leaf area, DA/LA: Dark gland area/Leaf area, WA/DA: White gland area/Dark gland area, WN/DN: White gland number/Dark gland number, PsH: Pseudo-hypericin (mg g−1), Hy: Hypericin (mg g−1), SH: Sum hypericin (mg g−1), HF: Hyperforin (mg g−1), Fla: Flavonoids (mg QE g−1), Ph: Phenolics (mg GA g−1), DPPH: 2,2-diphenyl-1-picrylhydrazyl scavenging assay (%). V: Vegetative, F: reproductive (Flowering), c: Control (natural photoperiod), h: 19 h photoperiod, dashed lines are the distance between the genotypes under the natural and 19 h photoperiods.
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Figure 4. Representative leaf images used for gland quantification. The images shown are representative examples that were subsequently used for quantitative analysis. Dark and white glands on the leaf surface were individually detected and measured using ImageJ software (v1.53d), with validation performed in MATLAB (v9.8). Quantification included gland number and total gland area per leaf. The background grid was used for spatial calibration; each grid square corresponds to 1 × 1 cm2.
Figure 4. Representative leaf images used for gland quantification. The images shown are representative examples that were subsequently used for quantitative analysis. Dark and white glands on the leaf surface were individually detected and measured using ImageJ software (v1.53d), with validation performed in MATLAB (v9.8). Quantification included gland number and total gland area per leaf. The background grid was used for spatial calibration; each grid square corresponds to 1 × 1 cm2.
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Table 1. The interaction effects of two photoperiods (natural summer photoperiod (daylight, 14h) and extended 19h photoperiod with supplemental fluorescent white light) and growth phases (vegetative versus reproductive (flowering)) on studied agro-morphological and phytochemical traits in the leaf tissue of Hypericum perforatum genotypes. Letters V and F denote vegetative and reproductive (flowering) phases, respectively.
Table 1. The interaction effects of two photoperiods (natural summer photoperiod (daylight, 14h) and extended 19h photoperiod with supplemental fluorescent white light) and growth phases (vegetative versus reproductive (flowering)) on studied agro-morphological and phytochemical traits in the leaf tissue of Hypericum perforatum genotypes. Letters V and F denote vegetative and reproductive (flowering) phases, respectively.
Light Natural Photoperiod (Control) LSD
(0.05) 		19h LSD
(0.05)
Genotype Topas Topas Mariana Mariana Topas Topas Mariana Mariana
T V F V F V F V F
gp
LA (mm2)68.40b84.55a57.55c83.89a7.3480.24c118.81a76.85cd95.94b6.33
WN (N)53.70c81.05b82.82b137.85a15.6482.99c107.59b98.26b173.53a17.56
WA (mm2)0.35d0.63b0.43c0.79a0.070.28d0.46b0.41b0.84a0.03
DN (N)13.73c15.21a12.76d14.03b1.0214.86c17.10a13.98d16.07b1.09
DA (mm2)0.16b0.22a0.13c0.14b0.030.18b0.30a0.14c0.19b0.03
WN/LA0.93d0.96c1.21b1.64a0.031.07d1.12c1.33b1.63a0.06
WA/LA0.005c0.008ab0.007b0.009a0.00090.006b0.006b0.006b0.007a0.001
DN/LA0.20b0.17c0.23a0.18c0.0130.18a0.14c0.18a0.16b0.01
DA/LA0.0022b0.0026a0.0017c0.0022b0.00050.0023b0.0026a0.0018d0.0021bc0.003
WA/DA2.18c3.51b2.68c5.38a0.762.30b2.78a1.99c1.91c0.22
WN/DN6.20b9.04a4.10d5.28c0.226.60b10.14a5.98c6.69b0.98
PsH (mg g−1)1.71b2.29a1.12c1.73b0.342.06b2.83a1.81c2.09b0.37
Hy (mg g−1)0.69 b1.41a0.46c0.68b0.140.75c1.79a0.74c0.82b0.05
SH (mg g−1)2.46b3.79a1.80c2.44b0.673.36b4.67a2.52c3.09b0.41
HF (mg g−1)32.23b29.70c35.65a19.65d2.1245.74b34.99c55.65a34.18cd3.22
Fla (mg QE g−1)74.45d114.45 a103.64c112.77b1.74129.73d143.09a116.34c130.33b4.14
Ph (mg GA g−1)133.22d233.70 a164.09c215.70b15.71201.63d242.74a212.73c231.68b6.13
DPPH (%)51.98b40.47c60.56a40.26c4.8154.01b55.64b69.68a50.64c2.07
Means in each column for each experimental factor, followed by similar letter(s), are not significantly different at a 0.05 probability level, using LSD’s test, LA: Leaf area (mm2), WN: White gland number, WA: White gland area (mm2), DN: Dark gland number, DA: Dark gland area (mm2), WN/LA: White gland number/Leaf area, WA/LA: White gland area/Leaf area, DN/LA: Dark gland number/Leaf area, DA/LA: Dark gland area/Leaf area, WA/DA: White gland area/Dark gland area, WN/DN: White gland number/Dark gland number, PsH: Pseudo-hypericin (mg g−1 DW), Hy: Hypericin (mg g−1 DW), SH: Sum hypericin (mg g−1 DW), HF: Hyperforin (mg g−1 DW), Fla: Flavonoids (mg QE·g−1 DW), Ph: Phenolics (mg GA·g−1 DW), DPPH: 2,2-diphenyl-1-picrylhydrazyl scavenging assay (%). gp: Growth phase, V: Vegetative phase, F: Reproductive phase (Flowering). T: Treat, (N): Number. Different lowercase letters in each photoperiod indicate statistically significant differences among genotypes and growth phase at p < 0.05. The same letter indicates that values are not statistically different (p < 0.05).
Table 2. Analysis of variance (ANOVA) for agro-morphological and phytochemical characteristics in the leaf tissue of Hypericum perforatum genotypes under two photoperiods (natural summer photoperiod (daylight) and extended 19h photoperiod with supplemental fluorescent white light) and two growth phases (vegetative and reproductive (flowering) phase).
Table 2. Analysis of variance (ANOVA) for agro-morphological and phytochemical characteristics in the leaf tissue of Hypericum perforatum genotypes under two photoperiods (natural summer photoperiod (daylight) and extended 19h photoperiod with supplemental fluorescent white light) and two growth phases (vegetative and reproductive (flowering) phase).
S.O.VMean Square
DFLAWNWADNDAWN/LWA/LDN/LDA/LWA/DAWN/DNPsHHySHHFFlaPhDPPH
Growth phase (gp) 13512.9 **12,453 **0.63 **33.46 **0.01 **0.23 **0.021 *0.003 **0.01 **5.92 **23.34 **5.38 **0.43 **5.38 **249.9 **1792 **15,991 **270.03 **
R (gp)494.06174.610.0012.530.0010.0060.00030.0010.0010.220.090.050.0030.0545.4992.32142.540.021
Genotype (G)1633.24 **10,475 **0.15 **8.48 **0.01 **0.69 **0.021 **0.0002 ns8.43 *0.10 ns33.03 **4.83 **0.33 **4.86 **20.39865 **1729.1 **337.33 **
G×gp159.73 ns2302 **0.01 *0.67 ns0.002 *0.09 **0.003 **0.0001 ns9.37 ns0.08 ns8.26 **0.68 *0.08 **0.68 **1483 **474.4 *196.09 *175.5 **
G×r (gp)410.7238.620.0072.630.0020.040.00090.00032.060.160.680.120.0070.1227.3615.48314.237.89
Light (L)11385.8 **4290 **0.09 **3.16 **0.005 *0.04 *0.001 *0.005 **3.37 ns0.34 ns17.03 **0.89 *0.36 **0.89 *295.6 **4945 **5375 **520.01 **
G×L1370.62 **17.29 **0.06 **4.19 *0.006 **0.16 **0.002 **0.0003 ns2.02 ns1.95 **0.45 ns0.67 *0.07 **2.53 **476.8 **849.1 **695.27 *60.52 *
L×gp1177.83 **148.7 ns0.06 **3.44 ns0.006 **0.09 *0.003 **0.0001 ns1.83 ns0.84 ns1.48 ns0.91 *0.11 **0.91 *433.4 **167.1 ns4905.9 **240.14 **
G×L×gp1805.67 **342.1 **0.04 **0.05 **0.08 **0.06 *0.002 **0.003 **7.01 **1.08 ns1.45 ns1.18 **0.25 **1.18 **402.6 **437.7 **923.93 *660.2 **
Error1523.9330.650.0011.550.0070.0080.0040.0021.040.230.480.0110.0080.1124.4769.58141.9511.7
CV 5.835.416.148.409.387.418.429.134.110.107.268.173.172.178.27.275.845.64
ns: Non-significant. * Significant at the 0.05 probability level. ** Significant at the 0.01 probability level. DF: degree freedom, R: replication, LA: Leaf area (mm2), WN: White gland number, WA: White gland area (mm2), DN: Dark gland number, DA: Dark gland area (mm2), WN/LA: White gland number/Leaf area, WA/LA: White gland area/Leaf area, DN/LA: Dark gland number/Leaf area, DA/LA: Dark gland area/Leaf area, WA/DA: White gland area/Dark gland area, WN/DN: White gland number/Dark gland number, PsH: Pseudo-hypericin (mg g−1 DW), Hy: Hypericin (mg g−1 DW), SH: Sum hypericin (mg g−1 DW), HF: Hyperforin (mg g−1 DW), Fla: Flavonoids (mg QE·g−1 DW), Ph: Phenolics (mg GA g−1 DW), DPPH: 2,2-diphenyl-1-picrylhydrazyl scavenging assay (%).
Table 3. Correlation coefficients among the measured traits in the leaf tissue in Hypericum perforatum genotypes in two photoperiods (natural summer photoperiod (daylight) below the diagonal and extended 19h photoperiod with supplemental fluorescent white light above the diagonal).
Table 3. Correlation coefficients among the measured traits in the leaf tissue in Hypericum perforatum genotypes in two photoperiods (natural summer photoperiod (daylight) below the diagonal and extended 19h photoperiod with supplemental fluorescent white light above the diagonal).
TraitLAWNWADNDAWN/LWA/LDN/LDA/LWA/DAWN/DNPsHHySHHFFlaPhDPPH
LA (mm2) 0.98 **0.90 **0.96 **0.96 **0.830.76−0.93 **0.770.710.94 **0.79 *0.92 **0.84 **−0.690.510.80 **−0.73 *
WN (N)0.99 ** 0.98 **0.390.580.91 **0.87 **−0.400.490.99 **0.97 **0.680.700.73 *0.94 *0.210.47−0.82 **
WA (mm2)0.97 **0.75 * 0.630.710.95 **0.93 **−0.240.600.99 **0.97 **−0.560.440.650.93 *0.190.42−0.74 **
DN (N)0.93 **0.340.48 0.78 *0.480.420.81 **0.120.340.640.90 **0.83 **0.82 **−0.83 **0.80 **0.82 **−0.77 *
DA (mm2)0.97 **0.650.500.91 ** 0.230.420.75 *0.82 **−0.230.610.79 *0.91 **0.86 **−0.86 **0.78 *0.83 **−0.77 *
WN/L0.510.96 **0.82 **0.660.68 0.98 **−0.040.79 **0.480.97 **0.530.630.75 *−0.51−0.100.12−0.69
WA/L0.95 **0.350.87 **0.680.440.49 0.080.84 **0.91 **0.93 **0.53−0.750.76 *−0.53−0.060.12−0.57
DN/L−0.94 **−0.34−0.550.90 **0.65−0.76 *−0.86 ** 0.56−0.30−0.25−0.51−0.20−0.33−0.40−0.40−0.600.70
DA/L0.470.540.710.660.85 **−0.75−0.27−0.16 0.560.640.050.500.35−0.09−0.42−0.34−0.17
WA/DA0.320.340.87 **0.610.410.450.84 **−0.14−0.56 0.97 **−0.77 *0.93 **0.91 **0.74 *0.190.43−0.77 *
WN/DN0.670.530.700.780.86 **0.92 **0.27−0.750.460.28 0.620.84 **0.99 **−0.570.87 **0.24−0.82 **
PsH (mg g−1)0.85 *0.530.380.76 **0.92 **0.700.23−0.200.87 **0.100.46 0.88 **0.95 **−0.98 **0.76 **0.90 **−0.52
Hy (mg g−1)0.70 *0.600.640.92 **0.97 **0.86 *0.46−0.370.94 **0.350.630.92 ** 0.98 **−0.89 **0.450.61−0.55
SH (mg g−1)0.76 *0.630.530.86 **0.98 **0.89 *0.23−0.570.79 *0.140.570.96 **0.98 ** −0.94 **0.670.73 *−0.57
HF (mg QE g−1)−0.630.86 **0.91 **−0.85 **−0.130.090.480.56−0.72 *0.36−0.28−0.89 **−0.73 *−0.80 ** −0.80 **0.89 **0.70 **
Fla (mg g−1)0.440.390.610.71 **0.330.260.53−0.52−0.110.640.400.400.670.78 *0.72 ** 0.94 **0.73 **
Ph (mg GA·g−1)0.79 *0.510.88 **0.70 *0.520.510.86 **−0.440.350.91 **0.480.470.420.450.86 **0.88 ** 0.64 *
DPPH (%)−0.98 **−0.75 *0.300.22−0.17−0.560.69−0.62−0.54−0.62−0.40−0.85 **−0.64−0.75 *0.88 **0.84 **0.78 *
* Significant at the 0.05 probability level. ** significant at the 0.01 probability level, LA: Leaf area (mm2), WN: White gland number, WA: White gland area (mm2), DN: Dark gland number, DA: Dark gland area (mm2), WN/L: White gland number/Leaf area, WA/LA: White gland area/Leaf area, DN/AL: Dark gland number/Leaf area, DA/LA: Dark gland area/Leaf area, WA/DA: White gland area/Dark gland area, WN/DN: White gland number/Dark gland number, PsH: Pseudo-hypericin (mg g−1 DW), Hy: Hypericin (mg g−1 DW), SH: Sum hypericin (mg g−1 DW), HF: Hyperforin (mg g−1 DW), Fla: Flavonoids (mg QE g−1 DW), Ph: Phenolics (mg GA g−1 DW), DPPH: 2,2-diphenyl-1-picrylhydrazyl scavenging assay (%).
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Tabatabaei, M.S.; Sobhani, A.; Khanahmadi, M.; Zare, S.; Wanke, S. Effects of Growth Phases and Intensification of Light on Secondary Metabolites and Agro-Morphological Traits of the St. John’s Wort (Hypericum perforatum L.). Plants 2026, 15, 663. https://doi.org/10.3390/plants15040663

AMA Style

Tabatabaei MS, Sobhani A, Khanahmadi M, Zare S, Wanke S. Effects of Growth Phases and Intensification of Light on Secondary Metabolites and Agro-Morphological Traits of the St. John’s Wort (Hypericum perforatum L.). Plants. 2026; 15(4):663. https://doi.org/10.3390/plants15040663

Chicago/Turabian Style

Tabatabaei, Mina Sadat, Ahmad Sobhani, Morteza Khanahmadi, Sara Zare, and Stefan Wanke. 2026. "Effects of Growth Phases and Intensification of Light on Secondary Metabolites and Agro-Morphological Traits of the St. John’s Wort (Hypericum perforatum L.)" Plants 15, no. 4: 663. https://doi.org/10.3390/plants15040663

APA Style

Tabatabaei, M. S., Sobhani, A., Khanahmadi, M., Zare, S., & Wanke, S. (2026). Effects of Growth Phases and Intensification of Light on Secondary Metabolites and Agro-Morphological Traits of the St. John’s Wort (Hypericum perforatum L.). Plants, 15(4), 663. https://doi.org/10.3390/plants15040663

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