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Role of melatonin in promoting growth attributes, thymol, rosmarinic acid and biochemical properties in Thymus vulgaris L. under water deficiency

BMC Plant Biology volume 25, Article number: 603 (2025) Cite this article

Abstract

Thymus vulgaris L. is a rich source of thymol and rosmarinic acid. Nowadays, water deficiency is one of the critical environmental factors affecting plants performance and metabolites. Melatonin, as a biostimulant, increases the tolerance of plant to environment stressors, especially drought. In order to exploit dry and low-yielding lands, this study was conducted on enhancing T. vulgaris' growth, phytochemical and biochemical traits by means of melatonin foliar spray (0, 50, 100, and 150 µM) under various water deficiencies (100%, 70%, and 40% FC) which were carried out as a factorial completely randomized design. Foliar application of melatonin caused to improve water deficit tolerance, successfully revived the suppressed morphological traits and significantly increased biochemical capacity. The findings of present experiment demonstrated that as water deficiency increased, growing parameters reduced although, the negative effect of water deficit on vegetative parameters such as plant height/diameter, leaf length/width, and shoot fresh/dry weight were ameliorated by melatonin specially at concentration of 100 µM. The maximum essential oil content and yield was recorded respectively at 40% FC and 100% FC in combination with 100 µM melatonin. Based on GC and HPLC analyses, the amount of thymol, carvacrol and rosmarinic acid in the treatment of 40% FC and 150 μM melatonin increased by 42.25%, 81.36%, and 85.34%, respectively, compared to the control. Increase in drought severity led to increase in total phenol content, total flavonoid content, and also antioxidant capacity which were multiplied by 100 µM melatonin application. The least malondialdehyde (MDA) content, and hydrogen peroxide (H2O2) generation as well as proline accumulation was related to the plants treated with 150 µM melatonin under normal conditions while plants grown at 40% FC without melatonin had the most concentrations of the mentioned osmolytes. Regardless to melatonin application, in T. vulgaris under 40% FC conditions the activity of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were considerably more than those in control plants (100% FC) by 4.1, 1.8, and 0.64 times respectively. However, increased levels of melatonin had a promoting effect on POD and CAT activity, although SOD was negatively affected by melatonin concentrations. In conclusion melatonin probably with protection against oxidative damage and scavenging of free radicals through the stimulation of the enzymatic system, enrichment of plasma with phytochemicals, and regulation of osmotic status of the cells through accumulation of various solutes leads to an increase in the T. vulgaris tolerance against drought stress and ultimately increases the growth and phytochemical compounds.

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Introduction

The commercial value and application of medicinal plants because of the biologically active compounds in traditional medicine, pharmaceutical, cosmetic and food industries is expanding worldwide [38]. The quantitative and qualitative attributes of biologically active agents in a medicinal plant is affected by various factors including genetics, geographical factors, soil type, production conditions and environmental stresses, especially drought [67]. Water scarcity is one of the environmental problems of the world due to weather fluctuations, as one of the most common environmental stress, has influenced 25% of the world's agricultural land [3].

Water deficit significantly inhibits plant growth and development, disrupts essential crop functions, and reduces cell turgor potential. This causes to potentially drastic reductions in the quality and performance of crops [35]. During periods of water deficit, leaf morphological processes such as gaseous exchanges, photoassimilation rate, and hydraulic relations are considerably reduced, leading to a 50% reduction in biomass production [25]. Furthermore, drought negatively affects osmotic stability within leaves through the accumulation of various organic and inorganic metabolites, which in turn reduce the average osmotic potential of the plant. Conversely, increased concentrations of these metabolites under drought stress can prevent of protein denaturation and membrane collapse through oxidative stress associated with their elevated amounts [14, 33]. Fluctuations in the activity of antioxidative enzymes can affect plants resilience to water deficit and their capacity to cope on free reactive radicals [15]. In addition, plants combat water deficit-induced oxidative stress because of increased levels of antioxidants either enzymatic or non-enzymatic which scavenge ROS and reduce their harmful effects [35]. Furthermore, water deficiency can alter the amounts of biologically active compounds such as quality and quantity of essential oils, phenols, flavonoids, etc.) in medicinal plants [34].

Given the negative effect of water deficit on morphology and productivity of plants, finding a suitable solution to modulate the mentioned stress seems essential [31]. It is widely known that plant hormones are very important in modulating stress signal transduction pathways which are accompanied with various processes in physiology and biochemistry [24]. External application of bio-stimulants or phytohormones represents an effective strategy to enhance crop resilience and adaptability in response to challenging environmental conditions [30]. Melatonin is a multifunctional compound which has a major effect on optimum mitochondrial electron transport chain, neutralizing reactive oxygen molecules, protection of enzymatic antioxidats against oxidative stress, and enhancing their activities [65, 68]. Various literatures refer to the beneficial effects of melatonin in reducing the negative effects of abiotic stressors in most of plants such as Phaseolus vulgaris [65], Helianthus annuus [40], Gerbera jamosonii [68], Capsicum spp. (Khosravi et al., 2024), and Dracocephalum kotschyi (Mehralian et al., 2023).

Garden thyme (Thymus vulgaris L.) is a herbaceous, perennial, and also more cultivated medicinal plant of the Labiaceae, distributed in Asia, Mediterranean countries, and Central Europe [54]. T. vulgaris has attracted considerable attention in various food, pharmaceutical, perfumery, and cosmetic industries due to its biological activities, including antioxidant, antimicrobial, anti-inflammatory, and anticancer activities [42]. The chemical composition of T. vulgaris extracts and essential oils is primarily characterized by key secondary metabolites, including phenolic compounds, terpenoids, and saponins [26]. Different T. vulgaris essential oils and extracts exhibit lots of medicinal agents consist of expectorant, antispasmodic, antioxidative, antifungal, antimicrobial, and pesticidal activities. These effects are largely attributed to the accumulation of bioactive compounds including thymol and carvacrol [37, 54].

So far, no study has been conducted to investigate the role of the exogenous application of melatonin on ton morphological, physiological, and biochemical as well as essential oil quality and quantity of T. vulgaris in countering with water deficiency. Therefore, the experiment aimed to study the effect of external spray of melatonin on growth traits, enzymatic and phytochemical activity (essential oil, thymol and phenolic compounds content) of T. vulgaris exposed to water deficiency. The findings of the present work can be used for cultivating T. vulgaris in low yielding and dry regions to provide raw material for the food, medicine, cosmetic and perfume industries.

Materials and Methods

Plant Material and Experimental Setup

The seeds of T. vulgaris were sourced from the gene bank at the Research Institute of Forests and Rangelands in Tehran, Iran. For seed germination, trays were prepared using a growing medium composed of coco peat and perlite mixed in equal proportions by volume (1:1). Once the seedlings reached the four-leaf stage, transferred to the pots measuring 20˟25 cm (diameter/depth), containing sandy loam soil with a composition of 56.80% sand, 34.85% silt, and 8.35% clay. The factorial completely randomized design experiment was done with three replicates, incorporating 12 treatments. The setup included four melatonin levels (0% as control, 50 µM, 100 µM, and 150 µM) alongside three irrigation regimes, corresponding to 100% (control), 70%, and 40% of field capacity (FC). Each replication consisted of four pots per treatment, resulting in a total of 144 pots across the study. The required water for each irrigation regime was calculated by assessing the soil's FC by means of the pot method according to the Pourmeidani et al. [45] protocol. Irrigation cycles for each treatment were established based on the pots'weight and water evaporation, with irrigation conducted every two days. To apply melatonin treatment, melatonin was dissolved in ethanol and phosphate-buffered saline was used to dilute it and the desired concentrations were prepared. Tween-20 was used to increase the consistency of application. Throughout the experimental period, measures were taken to manage pests, diseases, and weeds effectively. The pots were maintained in a greenhouse environment with night/day temperatures of 19 °C and 30 °C, respectively, and a relative humidity range of 60% to 75%.

Evaluation of growth and yield traits

During the full flowering stage, measurements were taken using a ruler to determine plant height (cm), plant diameter (cm), leaf length (mm), and leaf width (mm). Following harvest, the shoot's fresh weight was recorded with a digital scale. Once the shoot had been dried at the ambient temperature, its dry weight was also assayed using the same method.

Phytochemical traits

Essential oil extraction

According to the method described in the British Pharmacopoeia, the essential oil contents (EOC) were extracted from the plants through hydro-distillation. The process involved using 50 g of dried aerial parts and a Clevenger apparatus over a duration of three hours. The essential oil yield (EOY) was calculated in 100 g of aerial parts following the 180-min distillation period.

GC and GC–MS analysis and compound identification

A gas chromatograph equipped with a flame ionization detector (FID) and a DB-5 column was used to analyze the EOs. The temperature of the detector was maintained at 300 °C, while the injector was set at 250 °C. The carrier gas was nitrogen, which moved at 1.1 ml per minute. Starting at 60 °C and, the oven's temperature rose by 5 °C per min until it reached 250 °C, where it stayed for ten min.

We used a Thermoquest-Finnigan gas chromatograph with a fused silica capillary HP-5MS column that was 60 m long and had a 0.25 µm thick film. A Manchester, UK-based TRACE mass spectrometer was connected to this system. We fixed the ionization voltage to 70 electron volts and employed helium as the carrier gas. The temperature of the ion source and interface was kept at 200 °C and 250 °C, respectively, and the mass spectrometer was used to measure atomic masses between 40 and 460. The program utilized for the oven temperature and the GC-FID analysis were identical. We used temperature-programmed analysis with n-alkanes (C6–C24) to determine retention indices to identify the components of the essential oil. We then compared the indices to those of the EOs that were studied on a DB-5 column under identical chromatographic conditions. Mass spectra were compared to recognized standards or an internal reference library (Adams and Wiley 7.0) to identify the compound. By contrasting retention indices with substances that have been verified or with references from the literature, we were able to verify identities. According to Adams [1], quantification was carried out by examining the relative area percentages derived from FID signals without using any correction factors.

Preparation of phenolic extract

About 100 mg of grounded aerial parts of the sample was subjected to extraction with 80% methanol, assisted by ultrasound for a duration of 44 min. Following the extraction process, the mixtures were centrifuged at 4400 × g for 15 min. The resulting supernatants were then collected and utilized for phytochemical analysis.

Quantification of Phenolic compound by HPLC–DAD

Phenols were extracted and analyzed using a Knauer HPLC system from Germany, featuring dual Wellchrom-K1001 pumps and a K2800 PDA detector. The separation was carried out on an RP-C18 column with dimensions of 4.6 mm internal diameter and 250 mm length, manufactured by Eurosphr. The mobile phase consisted of methanol and HPLC-grade water. Peak detection was performed within a wavelength range of 200–600 nm. The sample injection volume was set at 20 µl, with the system maintained at a constant temperature of 25 °C. The pure phenolic compounds standard was sourced from Sigma Aldrich. Retention times and the analysis of the spiked oil extract, alongside a standard solution, were applied to identify the presence of phenolic acids. Calibration curve was generated by injecting varying concentrations of standard compounds (5, 10, 20, 40, 60, 80, 100, 150, and 200 ppm) to quantify the phenolic acids. The recorded data was expressed as mg/g DW.

Estimation of total phenol content (TPC), total flavonoid content (TFC), and antioxidant activity

Total phenolic compounds turned into quantified the usage of the approach described through Singleton et al., (1965), which includes the Folin-Ciocalteu reagent. Total flavonoids compounds were assayed based at the technique mentioned with the aid of Dewanto et al., [16], utilising aluminium chloride. The absorption wavelengths for phenols and flavonoids were 765 nm and 510 nm, respectively. The approach outlined via Benzie and strain [10] turned into hired. Absorbance readings were taken at 593 nm the use of a spectrophotometer. Various iron sulfate (FeSO4) concentrations became applied to expand the calibration curve. The standard curve changed into created with FeSO4 solutions ranging from 0.05 to 10 mg/ml.

Physiological and Biochemical traits

Relative moisture content (RWC)

First, the sparkling weight of the leaf pattern become measured after which the pattern turned into immersed in dH2O for 12 h at ambient temperature and after this time the turgor weight of the samples became read. Then the samples had been located in an oven at 75 °C for forty eight hours and their dry weight become additionally measured. The relative moisture content material become evaluated through the following equation [22]:

$$\mathrm{RWC}\;\left(\%\right)=\left(\mathrm{Ww}-\mathrm{Wd}/\mathrm{Wt}-\mathrm{Wd}\right)\times100$$

where Ww is the fresh weight, Wd is the dry weight and Wt is the turgor weight.

Total chlorophyll content measurement

The Arnon [6] technique was applied to determine the full chlorophyll amount. Chlorophyll changed into extracted from the leaves the usage of 80% acetone. 2 g of clean leaf tissue from every treatment became weighed. The leaves have been gradually rubbed with acetone until a inexperienced answer became received. Then the quantity of the solution turned into made up to twenty ml with acetone. The solution become centrifuged for 10 min at 4000 rpm at 4 °C. After that, the absorption of the supernatant was recorded by means of a spectrophotometer at wavelength of 645 and 663 nm. Eventually, total chlorophyll amount was assyed in milligrams according to gram of clean weight based on the subsequent formula:

$$\mathrm{Chlorophyll}\;\mathrm{content}\;=\:((20.2\;\times\:\mathrm A645)\;+\:(8.02\;\times\:\mathrm A663)/1000\;\mathrm W)\;\times\:\mathrm V.$$

In which V = quantity of the filtered answer, A = mild absorption at wavelengths of 663 and 645 nm, and W = fresh weight of the sample in grams.

Evaluation of biochemical traits and antioxidant enzyme activity

Enzyme activity become measured the use of a spectrophotometer (name and model). Sparkling leaf sample (0.1 g) has been combined with 1 ml of cold 50 mM phosphate buffer (pH = 7.8) to degree enzymatic antioxidant activity. The ensuing aggregate changed into centrifuged at 12,000 rpm at 4 °C for 20 min. Catalase hobby become calculated the use of the Aebi method (1984), peroxidase activity was decided using the standard Zhang approach (1992), and superoxidase desmutase (SOD) activity become determined the use of the Beauchamp and Fridovich approach (1971). Proline extraction was also accomplished in line with Bates et al. [8] protocol. Hydrogen peroxide (H2O2) content evaluated at a wavelength of 390 nm in step with the technique of Velikova and Loreto [57]. Malondialdehyde (MAD) awareness become also measured regarding to the Wang et al. [58] protocol.

Statistical analyses

Manner evaluation of variance (ANOVA) (factorial experiment using a completely randomized design with 3 replications) changed into performed the usage of SAS 9.1.3 software (SAS Institute Inc). The least considerable difference (LSD) look at a chance degree of 0.05 (p < 0.05) changed into used to evaluate the suggest values of every variable. Origin 2021 software become used to plotting graphs.

Results

Growth and yield traits

The interaction effect of various levels of melatonin and water deficiency on the morphological traits of T. vulgaris have been illustrated in Fig. 1 and Table S1. Foliar spray of Melatonin considerably alleviated the adverse effect of water deficiency on plant height and diameter in the increasing trend at concentration of 0 to 100 µM but not at 150 µM (Fig. 1a & b). The highest plant height (29.20 cm) and plant diameter (39.06 cm) were observed in the control plant at 100% FC in treated with 100 µM melatonin, while the lowest plant height (10.11 cm) and plant diameter (17.15 cm) were recorded in drought-treated plants under 40% FC without any melatonin treatment. Similarly, leaf length/width, shoot fresh weight/dry weigh were also decreased in parallel with decrease in percentage FC as T. vulgaris under 40% FC without any melatonin application showed the least amounts of the mentioned traits (Fig. 1). In addition, foliar spray of melatonin caused to a significant increment in leaf length/width and shoot fresh/dry weight. However, melatonin mitigated the negative effects of water deficiency. Even also the ameliorative effect of melatonin was so drastic under 100% FC as there was a significant difference between T. vulgaris treated with 100 µM and untreated plants up to 49.66% in plant height (Fig. 1a), 28.05% in plant diameter (Fig. 1b), 35.94% in leaf length (Fig. 1c), 52.04% in leaf width (Fig. 1d),52.03% in shoot fresh weight (Fig. 1e), and 50.59% in shoot fresh weight (Fig. 1f).

Fig. 1
figure 1

Effect of different levels of melatonin on growth and performance traits of Thymus vulgaris under different irrigation regimes. The mean comparisons were performed using the LSD test at P ≤ 0.05 significant level. Data are presented as means ± SE (n = 3). Means followed by the same letter(s) are not significantly different

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Phytochemical traits

EOC and EOY

According to the resulted data the drought stress of 40% FC resulted in a 34.51% increment in the amount of T. vulgaris essential oils. Of course, the percentage of essential oil of this plant under water deficiency and lack of water deficiency are significantly influenced by treatment with different concentrations of melatonin (Fig. 2a). So that, under normal conditions (100% FC) and treatment with 100 µM melatonin caused to 17.69% increase in the T. vulgaris essential oil compared to the plants without any melatonin treatment while foliar application with 100 and 150 µM melatonin under water deficit as much as 40% FC, increase the percentage of essential oil by 61.06% and 22.55%, respectively.

Fig. 2
figure 2

Effect of different levels of melatonin on essential oil content (a) and yield (b) of Thymus vulgaris under different irrigation regimes. The mean comparisons were performed using the LSD test at P ≤ 0.05 significant level. Data are presented as means ± SE (n = 3). Means followed by the same letter(s) are not significantly different

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Regarding to the to the of EOE, results was different from EOC. As illustrated in Fig. 2b, the EOY was more affected by melatonin treatment as there was no significant different between all un-treated plants with melatonin under eighter druoght stress or lack of drought stress. Although the amount of EOY reduced in water deficit conditions, treatment with melatonin at 100 µM concentration caused to a maximum EOY (0.94 g/plant) at 100% FC followed by the second increment up to 0.79 g/plant under 40% FC compared to the control conditions. Furthermore, stress-induced plants (40% and 70% FC) as well as unstressed plants (100% FC) without melatonin treatment showed the least essential oil yield (Fig. 2b).

GC and GC–MS analysis and compounds identification

The results of GC–MS and GC-FID analysis of thyme EOs are presented in Table 1. Among all identified 22 compounds, thymol, p-cymene, and carvacrol were the major components of the EO. The percentage of thymol (64.61%) and carvacrol (9.83%) reached the highest values under the influence of 150 µM melatonin and 40% FC. The amount of thymol and carvacrol derived from essential oils increased by 42.25% and 81.36%, respectively, in comparison with the untreated plants. In all drought treatments, the percentage of p-cymene in the EO decreased. The highest (15.33%) and lowest (5.78%) percentage of p-cymene was observed in the controls and 150 µM melatonin and 40% FC treatment.

Table 1 Effect of drought stress and melatonin application on the composition of Thymus vulgaris essential oil
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Phenolic compounds

The content of phenolics extracted from T. vulgaris was significantly influenced by melatonin application and water deficiency (P < 0.01). Rosmarinic acid (RA) was the major factor of the methanol extract of T. vulgaris (Fig. 3). The highest level of RA (8.17 mg/g DW) was obtained in the treatment of melatonin 150 µM and 40% FC. Under stress conditions of 40% FC, foliar spraying with 150 μM melatonin advanced the level of RA by 85.34% compared to the control. With increasing drought and melatonin levels, the results showed higher levels of RA.

Fig. 3
figure 3

Effect of different levels of Melatonin on phenolic compounds of Thymus vulgaris under different irrigation regimes. DS: Drought stress; DS100: 100% FC; DS70: 70% FC; DS40: 40% FC. M: Melatonin; M0: without melatonin; M50: 50 µM; M100: 100 µM; M150: 150 µM

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Total phenolic content, total flavonoid content and antioxidant activity

Results of the Fig. 4 showed the same trend in TPC and TPC as different intensities of water deficiency enhanced the mentioned compounds in T. vulgaris. Furthermore, this increment was also affected by exogenous application of melatonin and enhanced more compared to the no application of melatonin. The maximum total phenols (15.31 mg GAE/g DW) and total flavonoids (10.22 mg QE/g DW) were evaluated in plants under water stress as much as 40% FC accompanied with 100 µM melatonin treatment (Fig. 4a). Application of exogenous melatonin at 150 µM concentrations had also simulative effect on TPC and TFC production but not as much as 100 µM concentration. However, the concentration of 150 µM was more effective than 50 µM and less than 100 µM (Fig. 4b). The antioxidant activity of T. vulgaris was also assayed via the ferric-reducing antioxidant power (FRAP) method. Regarding to the data an increasing trend in antioxidant activity in parallel with increase in water deficiency as the antioxidant potential of un-stressed plants without treatment with melatonin increased up to 33.75 µmol Fe/ml in plants under 40% FC and without melatonin treatment (Fig. 4c).

Fig. 4
figure 4

Effect of different levels of melatonin on TPC (a), TFC (b) and antioxidant activity (c) of Thymus vulgaris under different irrigation regimes. The mean comparisons were performed using the LSD test at P ≤ 0.05 significant level. Data are presented as means ± SE (n = 3). Means followed by the same letter(s) are not significantly different

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Physiological and biochemical traits

Leaf RWC were increased as increase in %FC as plants grown under 100% FC had the highest Leaf RWC in comparison with the plants exposed to 40% FC without melatonin treatment (Fig. 5a). Melatonin treatment caused to increase in water absorption of leaves in a dosage dependent manner as plants under 100% FC and sprayed with 150 µM melatonin indicated the maximum RWC compared to the same plants without any melatonin treatment. Furthermore, the lowest relative water content was belonged to the drought-stressed plants (40% FC) without any melatonin treatment. Regarding to the Fig. 5b it was found the similar results to relative water content. Decrease in water supply up to 40% FC, reduced chlorophyll content in T. vulgaris leaves by 18.27% compared to the same plants under 100% FC without melatonin treatment. Melatonin application prevented of chlorophyll reduction in a dosage manner under 40% and 70% FC, although it was a slightly decrease in 150 µM concentration of melatonin compared to the 100 µM melatonin in T. vulgaris samples grown in 100% FC conditions.

Fig. 5
figure 5

Effect of different levels of melatonin on relative water content (a) and chlorophyll (b) of Thymus vulgaris under different irrigation regimes. The mean comparisons were performed using the LSD test at P ≤ 0.05 significant level. Data are presented as means ± SE (n = 3). Means followed by the same letter(s) are not significantly different

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The interaction effect of different levels of water deficit and melatonin application on H2O2 content showed an increasing trend along with increased in drought stress severity, although exogenous application of melatonin specially at 150 µM concentration prevented of H2O2 accumulation in plants (Fig. 6a). Regarding to the SOD activity data, the effects of water deficit and melatonin were significant as the maximum (145.73 U/g FW) and minimum (23.05 U/g FW) activity of SOD were observed respectively in stressed- plants (40% FC) without melatonin and plants grown at 100% FC treated with 150 µM melatonin. Indeed, different concentrations of melatonin not only did not have an intensifying effect on SOD activity, but also decreased its activity as illustrated in Fig. 6b. However, the negative effect of melatonin was more considerable in 70% FC and 40% FC compared to the normal conditions (100% FC). In contrast, melatonin influenced the increased activity of POD and CAT positively (Fig. 6c & d) so that the maximum and minimum enzyme activities were belonged to the T. vulgaris respectively exposed to 40% FC conditions and treatment with 150 µM melatonin and normal conditions (100% FC) without melatonin treatment.

Fig. 6
figure 6

Effect of different levels of melatonin on H2O2 content (a), superoxide dismutase (b), peroxidase (c), catalase (d), proline (e) and Malondialdehyde (f) of Thymus vulgaris under different irrigation regimes. The mean comparisons were performed using the LSD test at P ≤ 0.05 significant level. Data are presented as means ± SE (n = 3). Means followed by the same letter(s) are not significantly different

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The interaction effects of different irrigation regimes and melatonin foliar spray on proline and MDA value demonstrated an increasing trend in their production along with increased in drought stress severity, although treatment with melatonin decreased the amount of proline and MDA either in stress conditions or normal conditions (Fig. 6e & f). T. vulgaris grown under %40 FC without melatonin treatment had the highest proline and MDA production while the least amount of proline and MDA was observed in plants grown at 100% FC in combination with 150 µM melatonin as illustrated in Fig. 6e and 6f, although different among various dosages of melatonin was more drastic in 70% FC and 40% FC compared to the normal conditions.

Pearson correlation analysis

The analysis of the relationships among growth, biochemical, and phytochemical characteristics (Fig. 7) indicated that EOC exhibited strong positive correlations with POD (r = 0.91), TPC (r = 0.93), FRAP (r = 0.94), TFC (r = 0.95), CAT (r = 0.91), SOD (r = 0.80), proline (r = 0.75), H2O2 (r = 0.70), and MAD (r = 0.79). Additionally, TPC (r = 0.98), TFC (r = 0.93), and EOC (r = 0.88) all demonstrated significant positive correlations with antioxidant activity. Furthermore, a robust positive correlation was identified between RWC and plant height (r = 0.89), plant diameter (r = 0.90), leaf length (r = 0.87), leaf width (r = 0.96), shoot fresh weight (r = 0.88), shoot dry weight (r = 0.85), and total chlorophyll content (r = 0.87). The shoot dry weight, an economically significant trait, was positively correlated with plant height (r = 0.93), plant dimeter (r = 0.94), leaf length (r = 0.90), leaf width (r = 0.89), shoot fresh weight (r = 0.95), RWC (r = 0.79), EOY (r = 0.81), and total chlorophyll (r = 0.87), while exhibiting a negative correlation with antioxidant enzymes. The efficacy of each medicinal plant regarding its desired metabolites is derived from the functionality of its medicinal parts. Given that secondary metabolites such as thymol, carvacrol, and rosmarinic acid in T. vulgaris primarily stem from dry weight, any factors that enhance this weight could potentially lead to increased metabolite production. Consequently, these traits warrant consideration by plant breeders in their selection processes.

Fig. 7
figure 7

Network correlation representation of growth, biochemical and phytochemical traits of Thymus vulgaris under melatonin and drought treatments. PL, Plant length; LL, Leaf length; LW, Leaf width; PD, Plant diameter; SFW, Shoot fresh weight; SDW, Shoot dry weight; TFC, Total phenol content; TFC, Total flavonoid content; FRAP, Antioxidant power assay; EOC: Essential oil content; EOY: Essential oil yield; RWC: Relative water content; MDA: Malondialdehyde; H2O2: Hydrogen peroxide; TChl: Total chlorophyll; POD: Peroxidase; SOD: Superoxide dismutase; Pro: Proline; CAT: Catalase

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Discussion

Water deficiency is the major limiting factor that adversely effect on plant morphology and reproductively [23]. Moreover, it is suggested that melatonin is responsible for controlling plant mechanisms via regulation of plant physiology and biochemistry, finally resulted in increased tolerance to water deficiency conditions [13]. In this research, morphological analyses of T. vulgaris revealed that through water shortage, growth-related traits including plant height/diameter, leaf length/width, and shoot fresh/dry weight were considerably stunted by drought stress as much as % FC. However, treatment with melatonin significantly increased the mentioned traits compared to the plants subjected to sever drought stress (40% FC) without any melatonin treatment. In this case, the best melatonin treatment for improvement in morphological traits was observed at 100 µM compared to the other concentrations. The same results were suggested by Ebtedaei et al. [17] in pear rootstocks. However, the positive effect of melatonin was suppressed at higher concentration (150 µM). Since melatonin is structurally similar to auxin, which both of them are indoleamine-derivatives and can performed its effect through auxin receptors. Low concentrations of melatonin stimulate the biosynthesis of indole acetic acid and thus stimulate other plant hormones (Falcon et al., 2009). Therefore, that is why in most of the evaluated factors in our study, the ameliorative function of 100 µM melatonin was more considerable in comparison with 10 µM concentration. The same results in in Lupinus albus [4] and T. vulgaris [12] demonstrated that melatonin increased growing factors is dependent to the applied melatonin concentration, as well. Based on our findings, it was determined that the most increment in the percentage of T. vulgaris essential oil was obtained when plants exposed to 40% FC conditions and sprayed with 100 µM melatonin followed by 150 µM melatonin. So, the lowest T. vulgaris essential oil (%) was obtained in under no water deficit and no spraying of melatonin. Treatment with melatonin upheld the yield of essential oils in T. vulgaris regardless of drought severities. The same findings were obtained by Hosseini et al. [27] in Lippia citriodora and Mazrou et al. [43] in Pelargonium graveolens. The ability of melatonin in promoting secondary metabolites is likely because of its simulative effect on meristemic tissues improvement which are responsible for production of various chemical compounds of volatiles [11]. Also, reduced amounts of essential oils yield, which is observed in the T. vulgaris under water deficiency, can be because of reduction in herb yield [11, 32] as depicted in Fig. 1.

Drought stress as well as higher concentrations of melatonin increased amount of TPC, TFC, and antioxidant capacity of T. vulgaris (Fig. 4). The role of melatonin specially at 100 µM concentration was so obvious in un-stressed plants. The extract derived from T. vulgaris is characterized by a high concentration of phenolic compounds, with RA being particularly prevalent. The levels of RA are affected by a lot of parameters including the type of plant, cultivar, growth phase, and growing circumstances (Esmaeili et al., 2014). In our investigation, we found that water deficiency considerably promoted the RA levels. Additionally, foliar spray of melatonin resulted in a considerable enhancement in RA content and improved the antioxidant capacity in T. vulgaris. Furthermore, the greatest concentration of RA was recorded in Ocimum basilicum L. under drought conditions [7] when treated with the highest dosage of MT. As an abiotic elicitor, melatonin stimulates the production of reactive oxygen species (ROS), modulates tolerance mechanisms, enhances the antioxidative enzyme activities, and promotes the concentration of phenolics via the activation of signal transduction pathway at molecular level and finally regulate the expression of genes, and triggering plant defense responses [44]. The mentioned compound also up-regulates the expression of key genes responsible for synthesizing phenylalanine ammonialyase, one of the essential enzymes in the phenylpropanoid biosynthetic pathway that facilitates the concentration of phenolics such as RA, in plant [56]. The Plant encountering with oxidative damage, equipped themselves with antioxidative enzymes and antioxidants compounds to cope undesired conditions. Total phenol and flavonoid are the part of non-enzymatic defense, which is accumulated under environmental stress. It seems that the simulative function of melatonin can be appropriated to the increment in the activity of H+-ATPase pump and stabilization of the cell membranes [59, 61]. The antioxidative ability of phenols and flavonoids is greatly appropriated to the quantity of -OH groups (Samec et al., 2021) which arrest free radicals and preserve membrane integrity. Plants phenolics are potent antioxidative reagents which are able to scavenge reactive free radicals in plants which are affected by various environmental stresses. Higher activity of antioxidative systems in plants seems to be because of the up-regulation of phenylpropanoid pathway which is responsible for biosynthesis of phenolics including caffeic, cinnamylmalic, gallic, ferulic, and vanillic acids [12]. These results were obtained also in our experiment when plants subjected to drought stress as well as melatonin application. Total antioxidant capacity was assayed by FRAP assay (Fig. 11). Results showed that FRAP ability greatly intensified in water deficit conditions and affected positively by melatonin concentrations, in comparison with the controls. It is well known that melatonin has the ability for regulating the plant antioxidative mechanisms by phenolic compounds, which protects plants subjected to different environmental stressors such as water deficiency [50]. Additionally, melatonin treatment has been demonstrated to promote the plant antioxidative mechanisms, by means of melatonin direct antioxidative function [64] because of increment in phenolic and flavonoid compounds as depictde in Fig. 4.

RWC were and chlorophyll increased along with increase in % FC as plants grown under 100% FC had the highest leaf RWC and chlorophyll compared to the plants under 40% FC. Furthermore, exogenous application of melatonin caused to enhancement of water absorption of leaves as well as chlorophyll in a dosage- dependent manner as treatment with 150 µM melatonin had better efficiency in all conditions even in normal conditions. Proof of this claim refers to the protective effect of melatonin on chlorophyll of the leaves, which sometimes has been about 2-folded after 24 h of treatment in comparison with the untreated plants (Arnao and Hernández‐Ruiz, 2009). On the other hand, melatonin caused to increase in leaf turgor pressure and preserve plant hydraulic relationships by means of various related techniques [28]. Drought stress accelerates leaf senescence and cell damage [21], finally reduces chlorophyll content, and finally, the amount of photoassimilation reduces significantly, and consequently, the growth and yield of the crop also decreases [51]. The main reason for the decline in chlorophyll index under water deficiency could be because of the competition of glutamyl kinase and glutamate ligase under drought stress [41] which causes glutamate precursor to consume more for proline synthesis and as a result chlorophyll biosynthesis reduced. On the other hand, melatonin caused to increase in Mg content, carotenoid, anthocyanin, and flavonoid, that are favorable for chlorophyll biosynthesis, protect chlorophyll from ROS and lead to improvement of plant dry weight [20]. Also, melatonin significantly reduces the expression of pheophorbide oxygenase which is responsible for degradation of chlorophyll, aging associated genes, and down regulation of chlorophyllase [60, 62].

The interaction effects of different irrigation regimes and melatonin application on H2O2, proline, and MDA content showed an increasing trend in their production along with increased in drought stress severity, although exogenous application of melatonin decreased their production either in stress conditions or normal conditions (Fig. 6). Application of melatonin specially in water deficiency situations prevent of increased levels of the mentioned biomarkers compared to the normal condition as melatonin at 150 mM concentration had better function compared to the other concentrations. Despite to H2O2, the use of melatonin in non-stress conditions had influenced the content of MDA slightly in plant, but under water deficit conditions, it considerably decrease the level of MDA, H2O2, and also proline. However, the reduced levels of MDA and H2O2 in plants subjected to water deficiency because of the treatment with melatonin (specially 150 µM) may related to the repairing capacity of melatonin in preserving membranes integrity and reducing lipids peroxidation resulted of high concentrations of ROS under water deficit [39]. The same results were suggested also by Campos et al. [13] rely on the reduction in proline content, which is equal to decrease in stress, if plants treated with melatonin under drought stress. Proline is the most common compatible solute in adaptation to drought stress through osmotic regulation. in addition to the osmo-adjuster role, it acts as cell structure protective, antioxidant action, energy transfer, carbon and nitrogen reserve, which are necessary for cell stability. Proline which is called protein-compatible hydrotrope [53], caused to preserve the least required hydration in the cells [47] and tolerate the plant under water deficit. Melatonin, probably due to its effect in preserving relative water content of cells as illustrated in Fig. 13, protecting the membranes, and protein reduces the intensity of drought stress and caused to produce less proline [55], although each of them was affected differently by melatonin treatment. Along with increase in the level of water deficiency, superoxide dismutase, peroxidase, and catalase activities increased and exogenous application of melatonin also multiplied the effect of water deficiency. Indeed, melatonin caused to increase in the activity of antioxidative enzymes in both peroxidase and catalase except for superoxide dismutase (Fig. 6). Melatonin as an antioxidant inhibits active oxygen species much more than vitamin E, as well as activation of antioxidative enzymes such as catalase, peroxidase, and superoxide dismutase [46]. In the present experiment except for SOD activity plants treated with melatonin, the same findings rely on the promoting effect of melatonin on antioxidative enzymes when applied even post-harvest were also previously demonstrated by Seyed Hajizadeh et al. (2024) in gerbera. However, regarding the activation behaviour of antioxidative enzymes in water deficit situations, there are many different results. In some literatures increased levels of antioxidative enzymes were reported [59, 61] while some of the researchers showed the reduced enzyme activity during water deficiency [28]. Ascorbate peroxidase, catalase, and peroxidase are identified as the main factors in detoxification of H2O2 and protective mechanism of plant against various environmental stress. In the present work, increase in the activity of catalase, peroxidase, and also superoxide dismutase in water deficiency could be influenced by the reflection of additive role of them in scavenging the free radicals under abiotic stresses. Our results rely on the increased levels of catalase and peroxidase in melatonin-treated plants subjected to the water deficit were in line with [60] in apple leave, although SOD was an exception to this rule (Fig. 8).

Fig. 8
figure 8

The multiple mechanisms of melatonin-induced alleviation of drought stress responses in plants based on the present study's findings and related literature [20]. The negative impacts of drought stress are mainly due to the over-accumulation of reactive oxygen species [(ROS) including superoxide (O2), hydroxyl radical (OH), hydrogen peroxide (H2O2, and alkoxy radical (RO], and the inhibition of cellular processes like photosynthesis, and catabolic of chlorophyll, resulting in decreased growth and metabolism. The disturbance of cellular redox regulation caused by drought stress induces further ROS production. This process is followed by damage to DNA, proteins, and lipids via oxidative burst. Several mechanisms behind the exogenous melatonin-mediated alleviation of drought stress can be discussed: (i regulates the activation of enzymatic and non-enzymatic (i.e., secondary metabolites such as carvacrol and rosmarinic acid antioxidants, (ii enhances the accumulation of osmoprotectants through the maintenance of organic compounds such as amino acids, which further affect the retention of water in the leaf tissue. Moreover, exogenously applied melatonin may also act directly as an antioxidant agent against ROS and lipid peroxidation, ensuring the survival of plants under drought stress conditions

Full size image

It seems that melatonin is able to balance the homeostasis of tissue redox via the activation of antioxidants agents and ultimately induces drought resistance in plants [29]. Generally, according to the obtained results in this experiment, it was proved that the effectiveness of melatonin in regulation of plants morphological and physiological processes, even in the same plant species, depends on the concentration [63] as resulted that the concentration between 100 and 150 µM melatonin likely had the same simulative effect on all evaluated parameters of T. vulgaris in the present study.

Conclusions

Our study suggested the considerable effects in the morphological and physico-chemical responses of T. vulgaris subjected to melatonin application under drought stress. Although T. vulgaris morphological characteristics were decreased as well as decrease in water supply up to the level of 40%FC even in combination with 100 µM melatonin, increment in the level of essential oils in the mentioned conditions was considerable. Furthermore, foliar spray of 100 µM melatonin compensated the reduced level of essential oil yield under 40% FC. However, the higher concentration of melatonin (150 µM) was more effective in phenolic compounds, physiological and biochemical traits. Generally, the direct function of melatonin as an antioxidant causes growth indices improvement, chemical detoxification, and plasma osmotic adjustment but the response of melatonin as a plant hormone depends on its concentration, which increases growth indicators in low doses and it is necessary to do comprehensive studies to identify the accurate concentration of melatonin. Since melatonin is a safe and low-cost substance, it can be considered as a practical method to improve the functional traits and phytochemical compounds (especially essential oil content) as an effective elicitor in T. vulgaris plant under water-deficit stress. Further research efforts exploring the complexities of this method and focusing on clarifying the effects of externally administered MT on changes in particular molecular and biochemical pathways (including the thymol and rosmarinic acid biosynthesis pathway) will provide new perspectives on the direct and indirect mechanisms of melatonin's effectiveness in plant systems.

Data Availability

All data generated or analyzed during this study are included in this article. Further enquiries can be directed to the corresponding author.

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Acknowledgements

The authors are grateful to the Shahid Beheshti University Research Center for their support in providing research facilities.

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Authors and Affiliations

  1. Department of Agriculture, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, Tehran, 1983969411, Iran

    Ghasem Eghlima & Fateme Aghamir

  2. Department of Horticulture, Faculty of Agriculture, University of Maragheh, Maragheh, 55136 553, Iran

    Hanifeh Seyed Hajizadeh

  3. Department of Horticultural Science, College of Agriculture, Shiraz University, Shiraz, Iran

    Saeedeh Zarbakhsh

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  1. Ghasem Eghlima
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  2. Fateme Aghamir
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  3. Hanifeh Seyed Hajizadeh
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Contributions

GE: methodology, conceptualization, supervision, data curation, data analysis and writing-original draft, FA: lab work, data curation, analysis data; HSH: reviewing, and editing; SZ: methodology and editing.

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Eghlima, G., Aghamir, F., Hajizadeh, H.S. et al. Role of melatonin in promoting growth attributes, thymol, rosmarinic acid and biochemical properties in Thymus vulgaris L. under water deficiency. BMC Plant Biol 25, 603 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12870-025-06667-8

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Keywords

BMC Plant Biology

ISSN: 1471-2229

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