Developmental Dynamics of Bioactive Compound Accumulation During Vegetative Growth in Thymus daenensis Čelak

ABSTRACT

Thymus daenensis is a pharmacologically important species, as its monoterpene‐rich essential oil exhibits strong antibacterial activity. This study examined the developmental regulation of bioactive compounds in T. daenensis during two key vegetative growth stages: an early stage (4 weeks post‐germination, WPG) and a late stage (8 WPG), under controlled laboratory conditions. Using an integrated analytical approach, we quantified hormonal and metabolic changes and observed marked reductions in indole‐3‐acetic acid (59%) and abscisic acid (55%) at 8 WPG relative to 4 WPG. Protein and soluble carbohydrate reserve also declined progressively over this period. Microscopic observations revealed structural maturation of peltate glandular trichomes (PGTs) in 8‐week‐old plants, a change that may be associated with increased secretory capacity. Chromatographic analysis revealed a greater diversity of compounds at 8 WPG; carvacrol was the dominant constituent at both stages (71.65% at 4 WPG and 72.82% at 8 WPG), followed by thymol as the second most abundant compound. Overall, the findings indicate that developmental progression is accompanied by coordinated changes in hormone levels, trichome morphology, and bioactive compound profiles under controlled conditions.

Vegetative development in Thymus daenensis reshapes phytohormone balance, peltate glandular trichome maturation, and secondary metabolite diversity. Between 4 and 8 weeks post‐germination, IAA and ABA decline, trichomes mature structurally, and carvacrol remains the dominant volatile while thymol decreases. These developmental shifts refine the optimal harvest window for bioactive compound production.

1. Introduction

Plants have been indispensable sources of medicinal compounds for millennia, and traditional herbal remedies continue to play a pivotal role in global healthcare [1]. The pharmacological value of medicinal plants is routinely validated through phytochemical screening, antimicrobial assays, and heavy metal profiling—approaches that collectively establish both efficacy and safety for therapeutic use [2]. Among the most pharmacologically significant plant families, the Lamiaceae stand out for their remarkable diversity of species renowned for therapeutic and aromatic properties [3]. These plants are characterized by specialized glandular trichomes that synthesize and sequester bioactive essential oils rich in terpenoids, which underpin a broad spectrum of biological activities, from antimicrobial to antioxidant effects [4, 5]. Within the Lamiaceae, the genus Thymus—comprising over 350 perennial species—is particularly notable for its ecological adaptability and medicinal value, with a center of diversity in the Mediterranean region [6].Thymus species synthesize terpenoid‐rich essential oils primarily in peltate glandular trichomes (PGTs), which serve as biochemical defenses against herbivores and pathogens [7]. The bioactivity of plant‐derived terpenoids extends beyond antimicrobial action; numerous plant extracts have shown significant anti‐inflammatory effects, as evidenced by in vivo suppression of C‐reactive protein and interleukin‐6 in experimental models [8].

T. daenensis Čelak., an endemic Iranian species thriving in the semi‐arid Zagros Mountains, exemplifies the pharmacological potential of this genus. Its essential oil, characterized by high concentrations of the phenolic monoterpenes thymol and carvacrol together with their biosynthetic precursors γ‐terpinene and p‐cymene, displays pronounced antibacterial properties [9]. The antimicrobial action of thymol and carvacrol involves interactions with amine functional groups of bacterial membrane proteins, leading to reduced cellular ATP and ion levels and consequent disruption of membrane potential and pH gradients [10, 11].

Terpenoid production in plants is dynamic and fluctuates with developmental stage, environmental conditions, and tissue‐specific regulation [12]. Monoterpenes and sesquiterpenes found in plant essential oils have recently attracted considerable attention because of their antimicrobial properties and diverse biological roles [13]. Notably, terpenoids also contribute substantially to the antioxidant capacity of medicinal plant extracts, as shown by oxygen radical scavenging assays in terpenoid‐rich species such as Eucalyptus globulus [14]. However, the variation in the terpenoid profile of T. daenensis throughout vegetative development remains poorly understood. Critically, no previous study has integrated three key dimensions of this variation—phytohormonal dynamics, trichome morphological changes, and the volatile metabolome—at defined developmental time points. This knowledge gap limits our ability to optimize harvest timing for maximum essential oil yield and quality, despite the plant's commercial and therapeutic importance. Advances in enzyme‐assisted extraction and gas chromatography–mass spectrometry (GC–MS) based phytochemical profiling have recently enabled more efficient recovery and identification of bioactive compounds from plant tissues, providing methodological frameworks that can be adapted to investigate developmental changes in secondary metabolite accumulation [15].

The novelty of the present study lies in the simultaneous application of hormonal profiling, trichome microscopy (light and scanning electron microscopy), and GC–MS to investigate T. daenensis at precisely defined vegetative stages. Our objective was to determine how the concentration and composition of essential oils—particularly thymol and carvacrol—change between two critical vegetative phases (4 and 8 weeks post‐germination, WPG) under controlled conditions, and to elucidate the accompanying shifts in phytohormonal levels and PGT morphology. We hypothesized that (i) the monoterpene profile and trichome development undergo significant alterations during this period, (ii) these chemical changes correlate with specific phytohormonal signatures, and (iii) plants at 8 WPG exhibit a distinct phytochemical profile compared with those at 4 WPG. By testing these hypotheses, this work provides a mechanistic framework linking developmental timing to phytochemical profile in T. daenensis and offers actionable insights for the precision agronomic management of this medicinally valuable species.

2. Materials and Methods

2.1. Plant Cultivation

Seeds of Thymus daenensis Čelak. were obtained from Pakan Bazr Esfahan Co. (Isfahan, Iran) on 15 March 2024. Prior to sowing, seeds were surface sterilized with 2% (v/v) sodium hypochlorite for 5 min, rinsed three times with distilled water, and sown in pots containing autoclaved perlite (Agro Perlite, the Netherlands). The plant material was taxonomically identified by Prof. Fatemeh Zarinkamar (Department of Plant Biology, Tarbiat Modares University). A voucher specimen (MPH‐1942) was deposited in the Herbarium of the Research Institute of Forests and Rangelands (RIFR), Tehran, Iran.

Plants were cultivated in a growth chamber (Snijders Scientific, model MLR‐352H) under controlled environmental conditions: a 16 h light/8 h dark photoperiod, a photosynthetic photon flux density of 150 µmol m^{− 2} s^{− 1}, a temperature of 24 ± 1°C, and a relative humidity of 40%. Plants were irrigated twice weekly with 50% Hoagland nutrient solution until harvest [16].

2.2. Sampling and Preservation

The life cycle of T. daenensis and the duration of its vegetative and reproductive growth phases were first determined under the laboratory conditions described above. On this basis, aerial tissues were collected at 4 and 8 WPG, corresponding to the beginning and end of the vegetative growth phase, respectively. For biochemical analyses, samples were immediately frozen in liquid nitrogen and stored at −80°C until use. For microscopic examination, tissues were fixed in formalin–acetic acid–alcohol (FAA; 5:5:90, v/v/v) for 7 d at 4°C.

2.3. Carbohydrates and Protein Quantification

Starch: Dried tissue samples (0.1–0.5 g) were homogenized in 80% (v/v) ethanol, incubated at 80°C for 10 min, and centrifuged at 4000 × g for 10 min. The resulting pellet was extracted with 6% perchloric acid (Sigma‐Aldrich), and starch concentration was determined using the anthrone method. Absorbance was measured at 630 nm with a UV‐1800 spectrophotometer (Shimadzu) and quantified against a glucose standard curve (20–100 µg mL^{− 1}) [17].

Total soluble carbohydrates (TSC): Ethanol‐soluble supernatants were reacted with anthrone reagent, heated at 100°C for 20 min, and quantified spectrophotometrically at 630 nm according to the method of McCready et al. [18].

Cellulose: Dried tissue (0.05 g) was refluxed in acetonitrile (HPLC grade, Merck) at 100°C for 30 min and centrifuged at 5000 × g for 20 min. Cellulose content was then determined using the anthrone method [19].

Total protein: Fresh tissue (0.1 g) was homogenized in ice‐cold 50 mM potassium phosphate buffer (pH 7.8) containing 0.14 mM EDTA and centrifuged at 12,000 × g for 10 min at 4°C. The supernatant was analyzed using Bradford reagent (Bio‐Rad) at 595 nm with bovine serum albumin (BSA; 0–1 mg mL^{− 1}) as the standard [20].

2.4. Chlorophyll Quantification

Fresh leaves (0.1 g) were extracted in 80% (v/v) acetone, centrifuged (5000 × g, 5 min), and absorbance was measured at 663, 645, and 470 nm (UV‐1800 spectrophotometer). Chlorophyll a, chlorophyll b, and total chlorophyll concentrations were calculated according to Sebastian et al. [21].

2.5. Antioxidant Activity Assays

2,2‐diphenyl‐1‐picrylhydrazyl (DPPH): Methanolic extracts (5 mL per 0.5 g dry tissue) were reacted with 63.4 µM DPPH (Sigma‐Aldrich) for 30 min in the dark. Absorbance at 515 nm was converted to Trolox equivalents (0.125–1 mM) [22].

Ferric reducing antioxidant power (FRAP): Extracts (100 µL) were mixed with FRAP reagent (10 mM TPTZ, 20 mM FeCl₃, 300 mM acetate buffer, pH 3.6) and incubated at 37°C for 10 min. Absorbance at 593 nm was compared to FeSO₄ standards (0.164–2.633 mM) [23].

2.6. Phytohormone Profiling

Fresh tissue (0.5 g) was extracted in 3 mL methanol (HPLC grade), sonicated (30 min, 4°C; Branson 3800), and centrifuged at 12000 × g for 15 min. The filtered solution was transferred to conical tubes and protected from light with aluminum foil. Analysis was performed on a Waters 2695 HPLC system equipped with a 2489 UV/Vis detector and a C18 column. The mobile phase consisted of 0.001% acetic acid in water (A) and methanol (B) at a flow rate of 0.8 mL min^{− 1}; the gradient was 20% B (0–10 min) followed by a linear increase to 90% B (10–35 min). Quantification was performed using external standards of indole‐3‐acetic acid (IAA), abscisic acid (ABA), brassinosteroids (BRs), and methyl jasmonate (MJ) (Sigma‐Aldrich) [24].

2.7. Microscopic Analyses

Light microscopy: Plant samples were fixed in FAA solution for at least one week. After overnight maceration in 1 M KOH, the upper leaf epidermis was carefully removed. Sections were examined and photographed using an Olympus BH2 light microscope (Olympus, Tokyo, Japan) at 10 × magnification [25].

Scanning electron microscopy (SEM): Samples were fixed in FAA and dehydrated through a graded ethanol series (60%, 70%, 80%, 90%, and 100%). The dehydrated samples were sputter‐coated with a 20 nm layer of gold and examined using a Philips XL30 scanning electron microscope (PW 6848/00) operated at an accelerating voltage of 20 kV and a magnification of 200 ×  [26]. Trichome density on the adaxial leaf surface was quantified from SEM images and expressed as the number of trichomes mm^{− 2}.

2.8. GC‐MS Analysis

Because of the limited biomass, ethanol extraction was used instead of hydrodistillation to characterize the volatile and semi‐volatile constituents under controlled laboratory‐scale conditions. Dried tissue (1.25 g) was ground and extracted with 6.25 mL of 96% ethanol on a rotary shaker for 72 h. The extract was centrifuged (5000 × g, 15 min) and analyzed on an Agilent 7000 triple quadrupole GC–MS system (GC 7890A). The mass spectrometer was operated in electron ionization (EI) mode at 70 eV with an ion source temperature of 320°C, quadrupole temperature of 150°C, and a scan range of m/z 25–800. The GC was equipped with a DB‐5 capillary column (30 m × 0.25 mm ID, 0.25 µm film thickness; Agilent). The oven program was 50°C for 2 min, then 10°C min^{− 1} to 300°C held for 5 min. The run time was 32 min, with no equilibration delay [27]. Compounds were identified by matching mass spectra against the NIST library and by comparing experimentally calculated linear retention indices (RI) with literature values obtained on columns of equivalent stationary phase polarity (DB‐5 or HP‐5). RI were calculated using a homologous series of n‐alkanes (C₇–C₂₀) injected under identical conditions, according to the formula RI = 100n + 100[RT(x)−RT(n)]/[RT(n+1)−RT(n)]. Only compounds with a mass spectral match factor ≥90% and an RI deviation <20 units from the literature value (when available) were considered positively identified. Compounds that likely originated from solvent–solute reactions or laboratory contamination were distinguished from genuine plant metabolites based on literature data and the comparison of retention behavior.

2.9. Statistical Analysis

Statistical analyses were performed using SPSS version 23.0 (IBM Corp., Armonk, NY, USA). Data are presented as mean ± standard deviation (SD) based on three biological replicates (n = 3). Differences between developmental stages were evaluated using the Mann–Whitney U test at a significance level of p < 0.05. Given the limited sample size, assumptions of normality could not be reliably assessed; therefore, nonparametric statistical methods were employed throughout the study. Relationships among variables were examined using Spearman's rank correlation analysis, and the resulting correlations were interpreted as descriptive rather than inferential. Hierarchical clustering and heatmap visualization were performed using CIMminer and SRplot. Owing to the limited number of biological replicates, the statistical outcomes should be interpreted with caution and confirmed in future studies with larger sample sizes.

3. Results

3.1. Growth Parameters

At 8 WPG, whole‐plant fresh biomass was approximately threefold greater than that at 4 WPG, indicating substantial vegetative growth during this developmental interval. Fresh weight of the aerial parts was also significantly greater at the later stage (Figure 1A). Similarly, whole‐plant dry weight (0.07 g), as well as the dry weight of the aerial parts and roots, increased significantly from 4 to 8 WPG (Figure 1B). Whole‐plant length (31.11 cm) and stem length (22.61 cm) both increased by approximately threefold between 4 and 8 WPG. Root length also increased, although this change was not statistically significant (Figure 1C).

FIGURE 1.

Growth parameters of Thymus daenensis during the vegetative growth phase. (A) Fresh weight, (B) dry weight, (C) shoot length, (D) shoot‐to‐root ratio. Data are means ± standard deviation (n = 3 replicates). Different letters indicate significant differences (p < 0.05).

The aerial part‐to‐root ratios, calculated on both fresh‐ and dry‐weight bases, did not differ significantly between 4 and 8 WPG, indicating coordinated biomass allocation between shoot and root tissues during vegetative growth (Figure 1D). In contrast, the stem length‐to‐root length ratio decreased by 26.53%, reflecting a relatively greater increase in root elongation than in stem elongation at 8 WPG.

3.2. Carbohydrates and Protein Content

TSC content decreased by 9% from 4 to 8 WPG. Starch concentration declined by 32%, reaching 25 mg g^{− 1} at 8 WPG. Cellulose exhibited the most pronounced reduction, falling by 55% to 1.99 mg g^{− 1}. Total protein also dropped significantly, from 16.85 mg g^{− 1} at 4 WPG to 8.54 mg g^{− 1} at 8 WPG, a decrease of approximately 50% (Figure 2).

FIGURE 2.

Total soluble carbohydrates (TSC), starch, cellulose, and total protein content of Thymus daenensis during the vegetative growth phase. Data are means ± standard deviation (n = 3 replicates). Different letters indicate significant differences (p < 0.05).

3.3. Chlorophyll Content

Chlorophyll a, chlorophyll b, and total chlorophyll concentrations did not differ significantly between the two stages (Figure 3). Although values were slightly higher at 8 WPG, the increase was not statistically significant. The ratio of chlorophyll a to chlorophyll b remained stable at approximately 3:1 throughout vegetative growth.

FIGURE 3.

Chlorophyll content of Thymus daenensis during the vegetative growth phase. Data are means ± standard deviation (n = 3 replicates). Different letters indicate significant differences (p < 0.05).

3.4. Antioxidant Activity

Antioxidant activity measured by the DPPH assay did not change significantly between 4 and 8 WPG (Figure 4A). In the FRAP assay, values were 147.55 mg Fe^{2 +} g^{− 1} at 4 WPG and 108.59 mg Fe^{2 +} g^{− 1} at 8 WPG; although the decline appeared notable, the difference was not statistically significant (Figure 4B).

FIGURE 4.

Antioxidant activity of Thymus daenensis during the vegetative growth phase. (A) 2,2‐Diphenyl‐1‐picrylhydrazyl (DPPH) radical‐scavenging assay, (B) ferric reducing antioxidant power (FRAP) assay. Data are means ± standard deviation (n = 3 replicates). Different letters indicate significant differences (p < 0.05).

3.5. Phytohormone Content

All quantified phytohormones either declined or remained relatively stable between 4 and 8 WPG (Figure 5). IAA concentration decreased significantly from 1981.33 µg g^{− 1} at 4 WPG to 811.86 µg g^{− 1} at 8 WPG (Figure 5A). ABA also declined significantly, from 161.05 to 72.37 µg g^{− 1} (Figure 5B). BR and MJ concentrations did not change significantly (Figure 5C,D). Among the hormones quantified, MJ was present at the highest concentration and ABA at the lowest.

FIGURE 5.

Phytohormone levels in Thymus daenensis during the vegetative growth phase. (A) Indole acetic acid (IAA), (B) abscisic acid (ABA), (C) brassinosteroids (BRs), (D) methyl jasmonate (MJ). Data are means ± standard deviation (n = 3 replicates). Different letters indicate significant differences (p < 0.05).

3.6. Anatomical Development

Light microscopy revealed that at 4 WPG, diacytic stomata were predominantly observed in the closed state (Figure 6A). Non‐glandular trichomes (NGTs), characterized by opaque walls and the absence of visible secretory structures, were present in both surface and side views, indicating an immature stage of development (Figure 6A–C). At 8 WPG, stomatal density and the proportion of open versus closed stomata remained unchanged. In contrast, PGTs were clearly visible in both surface and side views (Figure 6B–D). Compared with the earlier stage, PGTs exhibited a transparent subcuticular wall, and the secretory cells were readily distinguishable as discrete spots.

FIGURE 6.

Light micrographs of Thymus daenensis leaves during the vegetative growth phase. (A, B) Adaxial surface views; (C, D) cross‐sections showing dorsoventral organisation. (A, C) 4 weeks post‐germination (WPG); (B, D) 8 WPG. Magnification, 10×; scale bar, 0.3 mm. PGT, peltate glandular trichome; st, stoma; st ca, storage cavity; s c, secretory cell; s, stalk; U ep, upper epidermis; L ep, lower epidermis.

Scanning Electron Microscopy (SEM) observations confirmed these developmental changes. At 4 WPG, NGT density on the adaxial leaf surface was 31 ± 4 trichomes mm^{− 2} (Figure 7A). PGT density was 5 ± 1 trichomes mm^{− 2}, and the apical secretory head cells were clearly visible. At 8 WPG, NGT density decreased significantly to 4 ± 1 trichomes mm^{− 2}, whereas PGT density remained unchanged at 4 ± 1 trichomes mm^{− 2} (Figure 7B). In addition, PGTs at 8 WPG displayed a transparent secretory storage compartment enclosing the secretory cells, a morphology distinct from that observed at 4 WPG. Overall, PGT density remained stable throughout development, whereas substantial morphological changes occurred during trichome maturation. These observations suggest that developmental progression primarily affected trichome differentiation and secretory structure formation rather than glandular trichome abundance.

FIGURE 7.

Scanning electron microscopy (SEM) images of the adaxial leaf surface of Thymus daenensis during the vegetative growth phase. (A) 4 weeks post‐germination (WPG), (B) 8 WPG. Magnification, 200×; scale bar, 100 µm. NGT, non‐glandular trichome; PGT, peltate glandular trichome; hc, head cell; sc, secretory cell; s, stalk.

3.7. Bioactive Compounds Profiling

GC–MS analysis of the ethanolic extracts detected a diverse array of compounds, including volatile monoterpenes, sesquiterpenes, and several non‐volatile or semi‐volatile constituents (Table 1). However, some peaks corresponded to compounds considered artefacts or contaminants, such as toluene, while others were potentially derived from interactions between extract constituents and the extraction solvent. Consequently, not all detected compounds were regarded as endogenous metabolites of T. daenensis. The following analysis is therefore restricted to compounds previously reported as plant‐derived metabolites and for which biological activities have been documented.

TABLE 1.

Compounds identified by gas chromatography–Mass spectrometry (GC–MS) in ethanol extracts of Thymus daenensis at 4 and 8 weeks post‐germination (WPG). Data are means ± standard deviation (n = 3 independent experiments). Different lowercase letters within a row indicate significant differences between time points (p < 0.05).

	Compound	Formula	RT (min)	Exp. RI	Lit. RI	Amount (%)
						4 WPG	8 WPG
1	1,1‐Diethoxyethane	C6H14O2	5.25	716	724 b	3.40 ± 0.14 a	nd
2	3‐Nitropropanoic acid	C3H5NO4	5.26	717	—	Nd	2.65 ± 0.09 a
3	Toluene	C7H8	5.98	765	767 b	3.66 ± 0.17 a	2.88 ± 0.08 b
4	3,7,7‐Trimethylcyclohepta‐1,3,5‐triene	C10H14	10.55	1003	1015 b	0.85 ± 0.08 b	1.25 ± 0.06 a
5	γ‐Terpinene	C10H16	11.07	1054	1062 a	1.57 ± 0.09 a	Nd
6	Coniferyl alcohol	C10H12O3	11.09	1081	—	nd	1.31 ± 0.04 a
7	Carvacrol	C10H14O	14.58	1285	1298a b	71.65 ±1.34 a	72.82 ± 1.36 a
8	Thymol	C10H14O	14.74	1287	1289a b	8.52 ± 0.57 a	4.87 ± 0.36 b
9	β‐Caryophyllene	C15H24	16.54	1401	1418 a	2.21 ± 0.26 a	nd
10	(1Z,4Z,6Z,9Z)‐Nonadeca‐1,4,6,9‐tetraene	C19H32	16.57	1432	—	Nd	3.01 ± 0.23 a
11	α‐caryophyllene	C15H24	17.43	1442	1452a b	Nd	1.92 ± 0.14 a
12	2,3‐Dihydroxypropyl (9Z,12Z,15Z)‐octadeca‐9,12,15‐trienoate	C21H36O4	17.81	1456	—	0.99 ± 0.18 a	nd
13	8,11,14‐Eicosatrienoic acid, (Z,Z,Z)	C20H34O2	17.83	1471	—	2.35 ± 0.26 a	nd
14	(Z)‐2‐(Octadec‐9‐en‐1‐yloxy)ethan‐1‐ol	C20H40O2	20.94	1581	—	3.17 ± 0.34 a	nd
15	2‐Tetradecyloxirane	C16H32O	20.98	1634	—	Nd	2.59 ± 0.20 a
16	Oleic acid	C18H34O2	21.39	2088	2097 b	1.58 ± 0.17 a	Nd
17	3',8,8'‐Trimethoxy‐3‐piperidyl‐2,2'‐binaphthalene‐1,1',4,4'‐tetrone	C28H25NO7	23.36	2105	—	Nd	6.65 ± 0.73 a

RT: Retention time on a non‐polar DB‐5 capillary column.

Exp. RI: Experimental linear retention index calculated against a homologous series of n‐alkanes (C7–C20) using the formula: RI = 100n + 100[RT(x)—RT(n)] / [RT(n+1)—RT(n)].

Lit. RI: Literature retention index obtained on columns with equivalent stationary phase polarity (DB 5 or HP 5).

^aAdams, R. P. (2007). Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry (fourth ed.). Allured Publishing Corporation.

^bNational Institute of Standards and Technology. (n.d.). NIST Chemistry WebBook. Retrieved June 26, 2026, from https://webbook.nist.gov/.

^ab: The literature RI value can be supported by both of the aforementioned references.–: Literature RI not available for this compound in the consulted databases, or identification requires confirmation with an authentic standard.

nd: Not detected. Different lowercase letters within a row indicate statistically significant differences between treatments (p < 0.05).

At both developmental stages, carvacrol was the dominant constituent of the ethanolic extracts, accounting for 71.65% and 72.82% of the total composition at 4 and 8 WPG, respectively (Table 1). Thymol was the second most abundant metabolite but decreased from 8.52% at 4 WPG to 4.87% at 8 WPG. In contrast, γ‐terpinene, a known biosynthetic precursor of thymol and carvacrol, and p‐cymene were detected at relatively low levels (<2%) at both stages.

Developmental changes in the metabolite profile were primarily associated with the appearance and disappearance of minor constituents (Figure 8). Several compounds, including 3',8,8'‐trimethoxy‐3‐piperidyl‐2,2'‐binaphthalene‐1,1',4,4'‐tetrone (6.65%) and (1Z,4Z,6Z,9Z)‐nonadeca‐1,4,6,9‐tetraene (3.01%), were detected exclusively at 8 WPG, whereas 1,1‐diethoxyethane, 2,3‐dihydroxypropyl (9Z,12Z,15Z)‐octadeca‐9,12,15‐trienoate, and oleic acid were restricted to the 4‐WPG stage. Despite these compositional differences, the combined abundance of carvacrol, thymol, p‐cymene, and β‐caryophyllene remained relatively stable, decreasing only slightly from 81.02% at 4 WPG to 78.94% at 8 WPG.

FIGURE 8.

Gas chromatography–mass spectrometry (GC–MS) total ion chromatograms of ethanol extracts from Thymus daenensis during the vegetative growth phase. (A) 4 weeks post‐germination (WPG), (B) 8 WPG.

Overall, the chemical profile was dominated by carvacrol at both developmental stages (Figure 9). Although metabolite diversity was broadly similar between stages, the relative abundance of detected compounds was significantly greater in 8‐week‐old plants.

FIGURE 9.

Heatmap of relative abundance of bioactive compounds in Thymus daenensis during the vegetative growth phase. The color gradient reflects normalized metabolite levels (red, high; blue, low).

3.8. Correlation Analysis

Correlation analysis revealed distinct association patterns among the major metabolites (Figure 10). Thymol was strongly negatively correlated with p‐cymene (ρ = −0.81), 3‐nitropropanoic acid (ρ = −0.96), and coniferyl alcohol. In contrast, carvacrol showed a moderate positive correlation with p‐cymene (ρ = 0.71) and weaker positive correlations with 3‐nitropropanoic acid (ρ = 0.44) and coniferyl alcohol. Despite its dominance in the extract (∼72% of total composition), carvacrol exhibited limited variation across developmental stages.

FIGURE 10.

Heatmap of Spearman's rank correlation coefficients among identified bioactive compounds in Thymus daenensis during the vegetative growth phase. Correlation coefficients range from −1 (red) to +1 (blue).

γ‐Terpinene showed a strong positive correlation with thymol (ρ = 0.98) but only a weak correlation with carvacrol (ρ = 0.35). In addition, γ‐terpinene and p‐cymene were strongly negatively correlated (ρ = −0.90). Oleic acid and β‐caryophyllene were positively correlated with thymol (ρ = 0.99). Overall, thymol displayed the strongest connectivity within the metabolite correlation network (Figure 10).

Correlation analysis integrating metabolites, phytohormones, and physiological traits revealed that thymol was positively associated with all measured phytohormones (IAA, ABA, BRs, and MJ) and primary metabolites (starch, cellulose, total soluble sugars, and protein), whereas it was negatively associated with growth‐related traits (fresh weight, dry weight, and plant height) and chlorophyll content (Figure 11). By contrast, carvacrol showed no consistent relationships with these variables.

FIGURE 11.

Heatmap of Spearman's rank correlation coefficients between major bioactive compounds and quantitative parameters in Thymus daenensis during the vegetative growth phase. Red indicates positive correlations, whereas blue indicates negative correlations.

4. Discussion

4.1. Growth Parameters

The substantial increase in biomass at 8 WPG is consistent with the rapid vegetative expansion reported in aromatic perennials [28]. The relatively greater root growth at the later stage may indicate a developmental shift toward resource sequestration that often precedes or accompanies the onset of reproductive development [29]. In Thymus species, such allocation patterns have been linked to a trade‐off between vegetative growth and the metabolic investment in glandular‐trichome‐borne terpenoids, suggesting that the late vegetative stage may represent an inflection point for specialized metabolism. Recent phytochemical screening approaches have demonstrated the value of tracking these developmental transitions to optimize harvest timing in medicinal plants [30].

4.2. Carbohydrates and Protein

TSC declined only modestly, whereas starch, cellulose, and total protein decreased significantly at 8 WPG. These trends align with the principle that during active vegetative growth, photoassimilates are directed toward structural and catalytic components, while later stages involve the remobilization of reserves [31]. The reduction in starch is consistent with increased metabolic demand during late vegetative development and may contribute to the carbon supply available for secondary metabolism [32]. The decline in cellulose is consistent with the cessation of rapid cell expansion [33], and the loss of total protein mirrors the age‐dependent proteolysis documented in tomato and other species [34]. The reduction in these primary metabolites is compatible with a reallocation of resources toward secondary metabolism, a pattern observed across diverse medicinal plant systems when profiled using integrated biochemical and bioactivity assays [35].

4.3. Chlorophyll

Chlorophyll levels remained statistically unchanged, and the chlorophyll a/b ratio was stable at approximately 3. This stability indicates that the photosynthetic machinery is maintained through late vegetative development and that the observed metabolic shifts are not attributable to a decline in photosynthetic capacity. The persistence of chlorophyll is consistent with the non‐senescent phenotype expected under controlled, stress‐free conditions [36]. The unchanged ratio also suggests that chlorophyll degradation, which typically begins with the preferential loss of chlorophyll b has not been initiated [37]. Thus, the plants retain a fully functional photosynthetic apparatus at 8 WPG, implying that the metabolic differences between stages are developmentally programmed rather than stress‐induced.

4.4. Antioxidant Activity

Neither the DPPH nor the FRAP assay revealed significant developmental changes in antioxidant capacity. This outcome is consistent with the absence of imposed abiotic or biotic stress, which is often a major driver of antioxidant upregulation [38]. In Achillea millefolium, antioxidant activity declined with age while flavonoid levels remained stable [39], indicating that developmental trends in redox‐related traits are species‐specific. Recent work on Thymus species has shown that both the extraction method and the phenological stage can influence antioxidant activity and related phytochemical traits [40, 41], underscoring the need for methodologically harmonized studies when comparing developmental stages. Likewise, Kiran et al. [42] demonstrated that ethanolic extracts of A. millefolium contained higher concentrations of phenolics, flavonoids, and tannins and exhibited stronger antioxidant and antiproliferative activities than n‐hexane extracts, highlighting the importance of phytochemical composition and extract polarity in determining antioxidant potential. Similarly, Arshad et al. [43] reported that Phyllanthus niruri extracts mitigated CCl₄‐induced oxidative stress and liver injury through mechanisms associated with their rich phytochemical profile and antioxidant properties. Collectively, these findings indicate that antioxidant capacity is influenced not only by developmental stage but also by the qualitative and quantitative composition of bioactive metabolites. Therefore, the stable antioxidant capacity observed here does not preclude developmental changes in individual antioxidant compounds, which may follow independent accumulation trajectories and contribute differently to the overall antioxidant profile.

4.5. Phytohormones

All measured phytohormones either declined or remained relatively stable between 4 and 8 WPG, with the largest decreases observed for IAA and ABA. The reduction in IAA is consistent with the transition from active cell division and expansion to tissue maturation [44]. A marked decline in ABA was also observed. Given that exogenous ABA has been reported to promote the accumulation of phenolic compounds and monoterpenes in several Lamiaceae species [45, 46], the lower ABA levels detected at 8 WPG may be associated with the absence of further increases in terpenoid accumulation despite the presence of morphologically mature PGTs. However, the present data do not establish a direct regulatory role for ABA in this response.

BR and MJ levels remained largely unchanged between developmental stages. Jasmonates have been shown to induce the expression of genes involved in thymol and carvacrol biosynthesis in thyme [47, 48], suggesting that the absence of significant changes in MJ concentrations coincided with the relatively stable terpenoid profile observed during development. Nevertheless, additional molecular analyses would be required to verify such a relationship in T. daenensis.

Overall, the hormonal profile appears to reflect a developmental shift from active growth toward tissue maturation. This interpretation is supported by the decline in IAA and ABA and is consistent with the relatively stable terpenoid profile observed between 4 and 8 WPG. The integration of phytohormonal and metabolomic datasets provides a useful framework for exploring developmental regulation of specialized metabolism and is increasingly employed in medicinal plant research [49, 50].

4.6. Anatomical Development

The anatomical observations indicate substantial maturation of PGTs during vegetative development. The formation of a transparent subcuticular storage cavity and the increased visibility of secretory cells at 8 WPG are consistent with developmental patterns reported for other glandular trichome‐bearing species, including Perilla frutescens and Lonicera japonica [51, 52]. These structural changes likely reflect progressive differentiation of the secretory apparatus and potentially increased capacity for metabolite storage.

Notably, PGT density remained relatively stable throughout development, whereas NGT density declined markedly. This pattern suggests that developmental changes in secretory function may be driven more by trichome maturation than by changes in glandular trichome abundance. Similar observations have been reported in Thymus and other Lamiaceae species, where the metabolic activity and developmental status of glandular trichomes appear to be more closely associated with specialized metabolite accumulation than trichome density alone [53, 54].

The predominance of open stomata at 8 WPG, together with reduced ABA levels, is consistent with developmental adjustments in leaf physiology reported in other plant species [55]. However, because stomatal behavior is regulated by multiple endogenous and environmental factors, the present data do not allow direct attribution of this response to hormonal changes.

Taken together, the anatomical and hormonal data support the view that PGT development is closely linked to leaf maturation. This interpretation is consistent with recent studies highlighting the coordinated regulation of glandular trichome differentiation by developmental programs and phytohormonal signaling networks [56, 57]. Although the underlying regulatory mechanisms remain to be elucidated, the observed structural maturation of PGTs appears to represent a normal developmental transition rather than a response to external stress or environmental variation.

4.7. Bioactive Compounds

The essential oil composition of T. daenensis was dominated by carvacrol at both developmental stages (∼72%), with thymol representing the second most abundant constituent. This carvacrol‐dominant profile is consistent with previous reports for T. daenensis [9] and highlights the remarkable stability of the species’ major terpenoid profile during vegetative development. Carvacrol is widely recognized for its antimicrobial, antioxidant, anti‐inflammatory, and anticancer activities [58, 59]. Therefore, its consistently high abundance suggests that the pharmacological potential of T. daenensis is largely maintained across the investigated harvest stages. Although thymol declined at 8 WPG, it remained an important constituent and may contribute additional anti‐inflammatory and immunomodulatory effects [60].

The exclusive detection of γ‐terpinene at 4 WPG is consistent with its established role as a biosynthetic precursor in the thymol–carvacrol pathway. The disappearance of this intermediate at the later stage is consistent with developmental changes in monoterpene metabolism, although metabolic flux was not directly assessed in the present study. In contrast, several minor constituents, including 3‐nitropropanoic acid, coniferyl alcohol, and long‐chain hydrocarbons such as (1Z,4Z,6Z,9Z)‐nonadeca‐1,4,6,9‐tetraene were detected only at 8 WPG, indicating greater chemical complexity during later vegetative development. Some of these compounds have been associated with stress responses and defensive functions in plants [61]. Nevertheless, because the biological activities of many trace constituents have been inferred primarily from studies of complex extracts rather than purified compounds, their individual contributions to the bioactivity of T. daenensis remain uncertain [62, 63].

Several stage‐specific constituents merit particular attention. At 4 WPG, 1,1‐diethoxyethane represented 3.40% of the detected compounds. This acetal is generally regarded as an extraction‐derived artifact formed through acid‐catalyzed reactions between acetaldehyde and ethanol and is unlikely to represent a genuine metabolite of T. daenensis. Consequently, its contribution to the intrinsic biological properties of the plant is expected to be negligible. Another relatively abundant compound at this stage was 8,11,14‐eicosatrienoic acid (Z,Z,Z) (dihomo‐γ‐linolenic acid; DGLA; 2.35%). In mammalian systems, DGLA serves as a precursor of anti‐inflammatory eicosanoids and has attracted considerable attention for its therapeutic potential [63, 64]. Although the physiological significance of DGLA accumulation in thyme remains unclear, it may be associated with membrane remodeling processes or lipid‐derived signaling pathways during active vegetative growth.

Likewise, 2‐tetradecyloxirane was detected predominantly at 4 WPG. While specific biological studies on this compound are scarce, epoxide‐containing metabolites are frequently reported as components of bioactive plant extracts exhibiting antimicrobial, antioxidant, and cytotoxic properties. However, direct evidence linking these activities to 2‐tetradecyloxirane itself is currently lacking, and any functional interpretation should therefore be considered tentative [62].

At 8 WPG, the most abundant non‐terpenoid constituent was identified as 3′,8,8′‐trimethoxy‐3‐piperidyl‐2,2′‐binaphthalene‐1,1′,4,4′‐tetrone (6.65%). This compound belongs to a broader class of naphthoquinone‐related metabolites, whose members frequently exhibit antimicrobial, cytotoxic, and anti‐inflammatory activities [65]. Recent in silico analyses have suggested potential antifungal activity through interactions with key proteins of Rhizoctonia solani, including β‐1,3‐glucan synthase [66]. Nevertheless, because compound identification was based on GC–MS profiling and biological activity has not yet been experimentally validated in T. daenensis, these findings should be interpreted cautiously. Additional structural confirmation and bioactivity assays are required before definitive functional roles can be assigned.

The reduction in thymol abundance and the appearance of several stage‐specific metabolites at 8 WPG indicate that substantial qualitative changes occur within the secondary metabolic network during vegetative maturation. Similar developmental shifts in thymol and carvacrol accumulation have been reported in T. vulgaris and T. quinquecostatus, where essential oil composition varies according to developmental stage, environmental conditions, and chemotype [67, 68]. Our results suggest that, in T. daenensis, the thymol‐producing branch of the pathway may be more developmentally responsive than the carvacrol‐producing branch, potentially reflecting differential regulation of CYP71D‐family monooxygenases involved in monoterpene biosynthesis [69, 70]. From an applied perspective, harvesting at 4 WPG may be preferable when a relatively thymol‐enriched and chemically simpler extract is desired, whereas harvesting at 8 WPG provides access to a broader spectrum of secondary metabolites while maintaining high carvacrol content. Integrating developmental metabolite profiling with bioactivity‐guided fractionation, as demonstrated in recent studies of Lamiaceae species [35, 50], could further refine harvest timing and optimize the pharmaceutical and industrial utilization of T. daenensis.

4.8. Correlation Analysis

The correlation analysis provided descriptive evidence of coordinated metabolic changes during vegetative development, although these relationships should not be interpreted as evidence of causation. The strong positive correlation between γ‐terpinene and thymol (ρ = 0.98), together with the negative correlation between thymol and p‐cymene (ρ = –0.81), is consistent with the established monoterpene biosynthetic pathway in Thymus species [71]. In this pathway, γ‐terpinene serves as a key precursor of phenolic monoterpenes, whereas p‐cymene represents a side‐product or intermediate associated with alternative reaction routes. The concurrent decline in γ‐terpinene and thymol is consistent with developmental changes in the thymol‐associated branch of monoterpene metabolism; however, pathway flux cannot be inferred directly from correlation analysis alone.

Although the observed metabolite patterns are compatible with altered activity of enzymes involved in monoterpene biosynthesis, including terpene synthases (TPSs), the present data do not permit direct conclusions regarding enzyme activity or gene expression. Nevertheless, previous studies have demonstrated that expression of TPS and CYP71D‐family genes is closely associated with thymol and carvacrol accumulation in Thymus species [69, 72]. In particular, Alipour et al. [69] reported that the TPS2 gene exhibits greater responsiveness to environmental conditions than several CYP71D isoforms, suggesting that developmental or environmental regulation of upstream pathway components may contribute to the metabolite shifts observed here.

Interestingly, the relationship between p‐cymene and the major phenolic monoterpenes indicates that pathway regulation is unlikely to be governed solely by precursor availability. Despite the reduction in γ‐terpinene abundance at 8 WPG, changes in p‐cymene did not fully mirror those of its presumed precursor, suggesting that flux partitioning among competing enzymatic reactions may vary during development. Because p‐cymene can arise through alternative reaction routes involving unstable intermediates generated during monoterpene oxidation [69, 71], developmental changes in the relative activities of downstream enzymes, including members of the CYP71D family, may influence the balance between thymol‐, carvacrol‐, and p‐cymene‐related metabolites. However, this interpretation remains speculative and requires validation through targeted transcriptomic, proteomic, or metabolic flux analyses.

The absence of a strong correlation between carvacrol and γ‐terpinene (ρ = 0.35) further suggests that carvacrol accumulation may be regulated differently from thymol accumulation during vegetative development. This observation is consistent with the relatively stable carvacrol concentrations detected across growth stages and supports the hypothesis that the carvacrol branch of the pathway is less sensitive to developmental modulation than the thymol branch. Similarly, the strong negative correlation between thymol and coniferyl alcohol (ρ = −0.96) may reflect broader metabolic reallocation between terpenoid and phenylpropanoid metabolism. Such interactions have been reported in plants where these pathways compete for carbon resources, reducing power, and metabolic precursors [73, 74]. Nevertheless, the existence and biological significance of such trade‐offs in T. daenensis remain to be experimentally verified.

Thymol also exhibited positive correlations with several phytohormones, including IAA, ABA, BRs, and MJ, as well as with numerous primary metabolites, while showing negative correlations with growth‐related traits and chlorophyll content. Collectively, these relationships suggest that thymol decline forms part of a broader developmental reprogramming of metabolism rather than an isolated biochemical event. Similar integrative analyses combining metabolite and hormone profiling have been proposed as valuable exploratory approaches for understanding secondary metabolism in medicinal plants [30]. However, given the observational nature of correlation analyses, these associations should be regarded as hypothesis‐generating rather than mechanistic evidence. Future studies integrating metabolomics with gene expression profiling and enzyme activity measurements will be necessary to establish causal links among developmental signals, hormone dynamics, and essential oil biosynthesis in T. daenensis.

5. Conclusion

This study provides an integrated characterization of developmental changes occurring between the early (4 WPG) and late (8 WPG) vegetative stages of T. daenensis, encompassing anatomical, physiological, hormonal, and phytochemical traits. Significant reductions in cellulose, starch, and total protein content were observed at 8 WPG, whereas total soluble carbohydrates remained relatively stable. Concurrently, peltate glandular trichomes underwent marked structural differentiation, culminating in fully developed secretory tissues during late vegetative growth. Developmental progression was accompanied by substantial changes in phytohormone profiles, particularly declines in ABA and IAA concentrations, indicating a coordinated reprogramming of growth and secondary metabolism. Despite these physiological changes, the essential oil profile remained strongly dominated by carvacrol, which accounted for more than 71% of total volatiles at both developmental stages. Thymol remained the second most abundant constituent, although its concentration declined substantially during vegetative maturation. The relatively stable abundance of major monoterpenes demonstrates that the characteristic carvacrol‐rich chemotype of T. daenensis is maintained throughout vegetative development. While the medicinal potential of compounds such as oleic acid, 1,1‐diethoxyethane (C₆H₁₄O₂), and 2,3‐dihydroxypropyl (9Z,12Z,15Z)‐octadeca‐9,12,15‐trienoate (C₂₁H₃₆O₄), which were detected at 4 WPG, is better documented, the transition to the end of the vegetative phase was accompanied by an increase in the diversity and abundance of specialized metabolites, including 3‐nitropropanoic acid and coniferyl alcohol. Correlation analyses further indicated coordinated shifts among phytohormones, primary metabolites, and terpenoid compounds, highlighting the dynamic regulation of secondary metabolism during plant development. Harvesting at 4 WPG yields a profile enriched in thymol and other well‐characterized bioactive constituents, whereas harvesting at 8 WPG maintains a high carvacrol content while also providing a broader spectrum of secondary metabolites. Collectively, these findings improve our understanding of developmental regulation in T. daenensis and offer a framework for optimizing harvest strategies and guiding future research on essential oil biosynthesis and medicinal value in this economically important species.

Author Contributions

Ehsan Bakhshy: conceptualization, methodology, formal analysis, validation, writing – original draft, and project administration. Fatemeh Zarinkamar: validation, resources, investigation, and writing – review and editing. Mehrdad Nazari: formal analysis, investigation, writing – original draft, and writing – review and editing.

Funding

The authors have nothing to report.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.