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Ornithine enantiomers modulate essential oil yield and constituents and gene expression of monoterpenes synthase in Salvia officinalis under well-watered and drought stress conditions

BMC Plant Biology volume 25, Article number: 148 (2025) Cite this article

Abstract

The impact of drought stress on plant growth, development, and productivity presents a significant challenge in various environments worldwide. The exogenous application of polyamines as osmotically active materials plays a crucial role in enhancing plant tolerance to environmental stress. In this study, we examined the effects of L- and D-enantiomers of ornithine (0 and 1 mM) under both well-watered and drought stress conditions on the growth traits, essential oil (EO) yield, and composition, gene expression, and total phenolic and flavonoid content of Salvia officinalis. The experiment was designed as a factorial experiment using a completely randomized design with three replications. The results demonstrated that drought stress led to a decrease in plant biomass and an increase in EO content, chemical profiles of the EO, and total phenolic and flavonoid content compared to the respective control values. However, the exogenous supplementation of ornithine particularly D-ornithine resulted in enhanced stem, leaf, and total plant biomass, a 20% increase in EO content, and a 75% increase in yield. Additionally, these were increases of 11.76% in total phenol and 70%, 105.66%, and 114.28% in flavonoid content when compared to well-watered plants without ornithine supplementation. These improvements were strongly linked to growth enhancement, as evidenced by principal component analysis (PCA). The EO extracted from S. officinalis consisted of 22 compounds, primarily monoterpenes, including α-thujone (18.47–41.65%), camphor (15.05–25.17%), 1,8-cineole (10.12–21.6%), and β-thujone (6.23–21.2%). The percentage of these volatile compounds was found to be highest in D-ornithine-treated stressed plants compared to control conditions. The interaction between water availability and the application of D-ornithine and L-ornithine significantly influenced the expression of borneol diphosphate synthase (BS), sabinene synthase (SS), and cineole synthase (CS) under drought stress, with notable upregulation observed compared to normal growth conditions. Specifically, D-ornithine enhanced the expression of BS and SS by 45.29% and 113.63%, respectively, under drought stress, while both D-ornithine and DL-ornithine significantly increased CS expression. The present results suggest that D-ornithine may serve as a stress-protecting compound, increasing total phenol and flavonoids content, thereby enhancing the capacity of the antioxidant system and increasing EO compounds under drought stress.

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Introduction

Salvia officinalis, commonly known as sage, is a well-known medicinal plant belonging to the Lamiaceae family. Its aromatic properties and therapeutic benefits have contributed to its long-standing use in perfumery and pharmaceuticals [1, 2]. The leaves of S. officinalis are rich in essential oils (EOs), making them a valuable source of bioactive substances that offer various health benefits [3]. The EO of S. officinalis contains several important compounds, including sesquiterpenes like α–humulene, phenolics like phenolic acids and flavonoids, and monoterpenes such as 1,8–cineole, linalool and α-and β-thujone and camphor. These compounds are responsible for its anti-inflammatory, antimicrobial, antispasmodic, carminative, and anticancer properties [3, 4]. Key monoterpene synthases in S. officinalis, including bornyl diphosphate synthase (BS), cineole synthase (CS), and sabinene synthase (SS), are responsible for producing specific compounds like 1,8-cineole and bornyl diphosphate (which is converted to camphor), as well as sabinene (which leads to the production of α- and β-thujone). This highlights the diversity and significance of monoterpene compounds synthesized in S. officinalis [5]. Further molecular and biochemical research on monoterpene synthases and their products in S. officinalis could enhance the production of specific bioactive compounds with desired properties.

The composition and concentrations of bioactive compounds in EOs can be influenced by various ecological factors, including both abiotic and biotic stresses. Environmental stressors, such as drought, can trigger biochemical responses in plants, leading to changes in the production and accumulation of specific secondary metabolites, including monoterpenes and sesquiterpenes. These metabolites serve as protective mechanisms against environmental challenges. Ghasemi et al. [6] reported that the effects of water deficiency stress on specific compounds differ, impacting the active components and ultimately altering the composition of EOs. Similarly, Fallah Imani et al. [7], Safari et al. [8], and Mohammadi et al. [9] found that the content of EOs was significantly enhanced under drought stress. In the cases of S. officinalis and Thymus vulgaris, researchers observed an increased concentration of certain compounds, such as monoterpenes in S. officinalis and 1,8-cineole in Thymus vulgaris, under drought stress conditions as a response mechanism to combat stress-related challenges [10, 11]. Additionally, polyphenolic compounds play a significant role as secondary metabolites in plant stress responses. By modulating oxidative stress and regulating various physiological processes, these compounds contribute to the resilience and acclimation of plants to challenging conditions [12,13,14,15]. Research studies have shown that phenolic compounds assist plants in coping with water deficit stress by regulating water uptake and retention, maintaining cellular balance, and enhancing antioxidant defense systems [16,17,18]. Safari et al. [8], Mohammadi et al. [9], and Sarmadi et al. [19] reported an increase in flavonoid and phenol content under water deficit conditions. The interaction between polyphenols and other secondary metabolites in plants facing stress is a fascinating area of study that reveals the complex mechanisms behind plant responses to environmental challenges [20].

The diverse roles of polyamines (PAs) in helping plants tolerate water deficit have been the subject of extensive research. Studies have demonstrated a significant link between PAs and water deficit tolerance in various plants including cherry tomatoes [21] and wheat [20, 22,23,24]. Research conducted by Hussain et al. [25] indicated that plants that are more tolerant to stress are better at increasing the production of PA compounds, particularly putrescine, in response to abiotic stresses, compared to those that are intolerant. Furthermore, the supplementation of spermine or spermidine has been shown to enhance antioxidant activity, PA metabolism, and water status in plants, thereby improving drought tolerance in Damask rose [26]. Putrescine is synthesized from ornithine and is further amino-propylated by spermidine and spermine synthases, as well as S-adenosylmethionine decarboxylase, to produce spermidine and spermine, respectively. Additionally, researchers have explored the effects of amino acid enantiomers on how plants respond to drought stress are not yet fully understood. It is believed that D-amino acids might help modulate plant metabolism, influence stress signaling pathways, or enhance antioxidant defense mechanisms during drought conditions [27]. L-ornithine, which is a precursor of PAs essential for plant growth and development, is important in arginine biosynthesis. This process leads to the production of proline, an osmo-protective compound that helps plants cope with stress [28]. PAs are vital for the synthesis of DNA, RNA, and proteins, as well as for scavenging free radicals and reducing oxidative stress [29]. However, the high cost of PAs such as spermidine and spermine has led researchers to look more affordable alternatives. Studies by Ghahremani et al. [30] and Gholami et al. [31] have shown that D-ornithine effectively alleviate the negative effects of salinity stress in tobacco plants.

This study examines the effects of amino acid enantiomers, specifically D- and L-ornithine, on the physiological and biochemical responses of S. officinalis under conditions of water scarcity. Our objective is to understand the molecular mechanisms behind these effects and to explore the potential of D-amino acids as bio-stimulants that can enhance plant tolerance to drought stress. We focus on how the external application of L- and D- ornithine influences the synthesis of key metabolites, including EOs, total phenolic content, flavonoids, and the expression of monoterpene synthase genes. This research aims to provide insights into how different amino acids can help alleviate drought stress, contributing to sustainable agricultural practices and crop improvement strategies.

Material and method

Plant material, growth conditions, and sampling

In this study, cuttings were obtained from one-year-old parent plants of Salvia officinalis L. at the medicinal field of the Institute of Medicinal Plants & Natural Products Research, affiliated to the Iranian Academic Center for Education, Culture & Research (ACECR) (Karaj, Iran). One-year-old rooted cuttings of S. officinalis was planted in plastic pots. Pots contained a mixture of peat moss, soil, and sand to a proportional of 1: 1: 1. The physical and chemical properties of the soil were assessed as follows in Table 1. The experiment was performed in greenhouse under conditions (20 –15 °C, 16/8 h light/dark, and relative humidity of 65–75% and 300 µmol− 2. S− 1 light intensity). The growing conditions of plants in the greenhouse are as follows: 12 h light period with a temperature of 25/15°C (day/night), relative humidity of 65–75%, and a photosynthetic photon flux of 300 µmol m− 2 s− 1 (400–700 nm) at the plant level. The study was designed as a factorial experiment based on a completely randomized design with three replications. Treatments included irrigation regimes (well-watered and drought-stressed), L-ornithine and D-ornithine (two levels each), L-ornithine (0 and 1 mM) and D-ornithine (0 and 1 mM). Each treatment had two pots in each replication and each pot contained two plants. A total of 96 sage plants were sown in 48 plastic pots. After 30 days of growth, plants were sprayed with L-ornithine and D-ornithine. All upper plant parts were evenly sprayed using a calibrated hand sprayer fitted with an 8001E nozzle. The sprayer was held at a distance of 10 cm above the canopy until uniform leaf wetness was achieved, with approximately 30 ml of solution applied per plant.

Table 1 The physical and chemical properties of the potting soil before starting the experiment
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Soil moisture was monitored daily using a TDR (Time Domain Reflectometer) instrument (TRIM-FM 10776, Germany). Volumetric moisture was measured using a 20 cm three-rod probe. The data were confirmed using gravimetric moisture. The following equation was used. The available soil water deficit (ASWD) was quantified using the basic equation:

ASWD = (θFC - θPWP) × D × 100.

Where θ FC is the field capacity moisture content; θ PWP is the percentage of permanent wilting point moisture; and D is the depth of the soil layer.

The calculations involved determining the Maximum Allowable Depletion (MAD) by considering the ASWD percentage. Soil moisture characteristics were carefully assessed using TDR for real-time moisture monitoring. Soil moisture at the field capacity point and wilting point were determined (Table 1). In addition, the soil pF was determined using a pressure plate apparatus, and the soil moisture curve was then plotted. Regular irrigation cycles are guided by accurate technological measurements. The experimental design included well-watered and drought-stressed treatment conditions, with irrigation rates calculated and adjusted based on comprehensive soil moisture assessments. During this phase, the well-watered plants were irrigated until the soil water content reached the critical threshold of 20% ASWD, while the drought-stressed plants received irrigation up to 80% of MAD from ASWD. This meticulous approach was sustained for two weeks, showcasing the meticulous attention to detail in the study’s methodology [11, 15, 30, 32]. After the end of the treatment period, two plants from each treatment were sampled in repetition and leaves were then preserved at -80 °C to facilitate RNA extraction and the assessment of physiological characteristics.

Growth parameters

To determine the dry weight of plant parts (leaves and stem), the foliage of the S. officinalis was cut at the soil surface and then oven dried at 70 °C (48 h).

Essential oil (EO) extraction and identification its composition

EOs were extracted from dried aerial parts of the plants by hydro distillation. For each treatment, 20 g of dried samples were extracted in triplicate using a Clevenger apparatus according to British Pharmacopoeia protocols. The extraction was carried out for 2 h. The extracted EOs were dehydrated over anhydrous Na2SO4 and stored in sealed at 4 °C for analysis of GC and GC/MS. The EOs were analyzed using an Agilent model 7890 A GC equipped with a DB-5 fused silica column (30 m × 0.25 mm i.d., and a film thickness of 0.25 μm). The detector (FID) and injector temperature were 280 °C. Also, carrier gas with a linear velocity (0.7 ml/min) was Nitrogen. Quantification data (without application correction agents) was obtained from GC/FID area percentages. The oven was programmed with the following temperature settings: starting at 60 °C, it was rapidly raised to 220 °C at a rate of 3 °C per minute. Subsequently, the temperature was further increased to 260 °C at a rate of 20 °C per minute, where it remained constant for 5 min. The transfer line and injector temperatures were set at 280 °C and 260 °C, respectively. Helium was used as the carrier gas with a linear velocity of 30.6 cm/s. The ionization energy was set at 70 eV, scan time at 1 s, split ratio at 1:100, and the mass range for analysis was set between 40 and 300 atomic mass units (a.m.u). The constituents of the EO were analyzed by evaluating the retention index of a series of n-alkanes and the oil on a Ph-4 column under identical chromatographic conditions. Through comparisons of their mass spectra fragmentation patterns with standard references in the literature from Wiley libraries, individual constituents were successfully identified [33].

RNA extraction, cDNA synthesis, and real-time quantitative polymerase chain reaction (qRT-PCR)

The RNA extraction from S. officinalis leaves was performed using the RNeasy Plant Mini Kit from QIAGEN GmbH. The RNA concentration and quality were evaluated using a NanoDrop spectrophotometer and agarose gel electrophoresis, respectively. Subsequently, cDNA was synthesized using the SuperScript III Reverse Transcriptase kit provided by Thermo Fisher Scientific. Designs for qRT-PCR primers targeting the desired genes were then developed based on Mohammadi-Cheraghabadi et al. [11] method and the qRT-PCR was conducted using Azure biosystems (Table 2). The qRT-PCR reaction mixture for amplification of the target gene transcripts consisted of MgCl2, PCR buffer, dNTPs, Taq polymerase, SYBR green I, diluted cDNA, and reverse and forward primers, with a final volume of 25 µL. The PCR conditions included denaturation, primer annealing, extension, and final extension steps. Melting curve analysis was also performed. For normalization and control purposes, the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase C2 (GapC2) was utilized based on previous studies by Mafra et al. [34] and Zhang et al. [35].

Table 2 Description of reference genes and the monoterpene synthase primer sequences for qRT-PCR
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Total phenolic and flavonoid content

Folin-Ciocalteu reagent was applied to measure total phenolic content. 200 mg of fresh harvested leaf samples underwent extraction using 80% methanol, followed by centrifugation at 12,000 rpm for 15 min. Subsequently, the resulting crude extract was combined with 7.5% Na2CO3 and 10% Folin-Ciocalteu reagent. After incubating the samples for 1.5 h in the dark, their absorbance was measured using a Spectrophotometer at a wavelength of 760 nanometers [36]. According to the method of Krizek et al. [37], flavonoid concentration was measured using the spectrophotometric procedure. In this procedure, 200 mg of fresh leaf samples were prepared and ground in mortars filled with ethanol and acetic acid in 99:1 v/v. The extract was treated for 10 min in a warm bath of 80 °C upon centrifugation (12000 rpm at 15 min). The absorption was recorded using a UV-visible spectrophotometer (the wavelengths of 270, 300, and 330 nm). Quercetin was used as a standard to plot the calibration curve. The content of flavonoids was expressed as quercetin equivalents (QE).

Statistical analysis

The Pfaffl [38] mathematical model offers a quantitative approach to determine the relative expression of a target gene compared to a reference gene using real-time PCR data. The formula for calculating the relative expression ratio (R) considers the PCR efficiency (E) and the cycle threshold (Cp) deviation of the target gene and reference gene between control and sample conditions. In this equation, R represents the relative expression ratio of the target gene in comparison to the reference gene. E Reference and E Target denote the real-time PCR efficiencies of the reference gene and target gene transcripts, respectively. ΔCP Reference and ΔCP Target represent the cycle threshold deviations between control and sample conditions for the reference gene and target gene transcripts. The real-time PCR data was analyzed using SAS 9.4 software to perform analysis of variance (ANOVA) and assess the statistical significance of differences between samples. The PROC UNIVARIATE within SAS was used to test the assumptions of ANOVA, and the residuals were normally distributed, so the data did not need transformation. Mean comparisons were conducted using the LSD Test at a 5% probability level to identify significant differences in gene expression levels. Principal component analysis (PCA) was used to simplify the complexity of the obtained data and visualize their similarity under different treatments using Origin software. Additionally, Pearson’s correlation coefficients were calculated using the CORR method to determine the strength and direction of relationships between variables, providing insights into the associations between gene expression levels and experimental conditions.

Results

Total plant, stem, and Leaf dry weight

Based on the ANOVA results there was a statistically significant three-way interaction between water availability, D-ornithine, and L-ornithine affecting on total plant (p < 0.01), stem dry weight (p < 0.05), and leaf dry weight (p < 0.01) (Table 3). The interaction indicates that the effect of D-ornithine and L-ornithine on plant biomass varies significantly depending on the water availability conditions. This complex interaction suggests that the combined effects of these three factors are not simply additive, but show an intricate interdependence in plant biomass accumulation. The current result showed the drought stress significantly reduced total plant, stem, and leaf dry weight compared to well-watered plants (Fig. 1A and B, and 1C). Further results indicate that D-ornithine (200, 168.08, 178.57, %), L-ornithine (152.17, 100, 117.14%), and DL- ornithine (139.13, 36.17, 70%) increased dry weight of the total plant, stem, and leaf dry weight under drought stress compared to the control group. D-ornithine was the most effective in increasing plant biomass under drought stress (Fig. 1A and B, and 1C). D-ornithine was also able to increase total plant, stem, and leaf dry weight (Fig. 1A and B, and 1C).

Table 3 Analysis of variance (mean square) of measured characteristics of Salvia officinalis influenced by water availability (W), D-ornithine (D), L-ornithine (L), water availability and D-ornithine (W×D), water availability and L-ornithine (W×L), D-ornithine and L-ornithine (D×L), water availability, D-ornithine and L-ornithine (W×D×L)
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Fig. 1
figure 1

The three-way interaction between water availability, D-ornithine, and L-ornithine on leaf dry weight (A), stem dry weight (B) and total plant dry weight (C). The different letters show significant differences at the level of 0.01. The error bars represent standard error

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Essential oil (EO) content and yield

The results of the ANOVA showed that the interaction (three-way) between water availability, D-ornithine, and L-ornithine significantly influenced EO content and yield (p < 0.01). (Table 3). The interaction indicates that the effect of D-ornithine and L-ornithine on EO content and yield varied significantly with water availability conditions. The result showed that EO content and yield were increased under drought stress compared to well- watered plants (Fig. 2A and B). Similarly, D-ornithine was able to increase EO content (up to 20%) under drought stress compared to its respective control plants (Fig. 2A) which were increased from 0.1 to 0.12% respectively. The EO content was also found to be 1.50 and 1.71-folds, higher in D-ornithine-treated plants than in L-ornithine- and DL-ornithine-treated plants, respectively, under drought conditions. In addition, D-ornithine application improved EO yield by 66.66 and 75% both under well-watered and drought-stressed conditions compared to, that of control plants, respectively (Fig. 2B). Overall, the highest EO content and EO yield were obtained with the application of D-ornithine under drought stress (Fig. 2A and B). The EO yield in D-ornithine treated plants were increased by 2 and 2.8 folds higher compared to L-ornithine and DL-ornithine treated plants, respectively (Fig. 2B).

Fig. 2
figure 2

The three-way interaction between water availability, D-ornithine, and L-ornithine on essential oil (EO) content (A) and essential oil (EO) yield (B). The different letters show significant differences at the level of 0.01. The error bars represent standard error

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Chemical profiles of the essential oil (EO)

GC–MS analysis resulted in the recognition of 22 compounds based on their retention indices and mass spectra, representing 99.07% of the total EO components of S. officinalis aerial part. Five classes of chemical groups that characterize S. officinalis EO were also recognized, consisting that were composed of oxygenated monoterpenes > sesquiterpene hydrocarbons > oxygenated sesquiterpenes > oxygenated diterpene > monoterpene hydrocarbons. Monoterpene hydrocarbons were composed of α-pinene (0.2–2.57%), camphene (0.1–2.11%), β-pinene (0.1–2.63%), limonene (0.1–1.2%), ɣ-terpinene (0.11–0.75%) and myrcene (0.1–0.97%). Oxygenated monoterpenes were composed 1,8-cineole (10.12–21.6%), α-thujone (18.47–41.65%), β-thujone (6.23–21.2%), camphor (15.05–25.17%), cis-pinocamphone (0.23–0.73%), borneol (1.28-4%), terpinen-4-ol (0.4–0.69%) and bornyl acetate (1.27–3.8%). Sesquiterpene hydrocarbons were composed of e-caryophyllene (3.54–7.71%) aromadendrene (0.52–1.22%), α-humulene (2.92–10.64%), and viridiflorene (0.27–0.78%). Oxygenated sesquiterpenes were composed of caryophyllene oxide (0.1–0.62%), viridifloral (2.74–7.9%), and humulene epoxide II (0.51–2.3%). oxygenated diterpene was composed of manool (3.14–12.9%).

The results of GC/MS and ANOVA showed that the interaction (three-way) between water availability, D-ornithine, and L-ornithine significantly affected all the EO compounds (Table 3). The current result showed that all the EO compounds were increased under drought stress compared to well-watered plants. L-ornithine, D-ornithine, and DL-ornithine were able to increase the production of several terpenoids compared to the control group without drought stress. From the data presented, it appears that L-ornithine (160.71, 100, 263.33, 77.14, 33.67, 27.42, 21.84, 8.37, 33.14, 36.20, 31.70, 41.28, 43.75, 92.54%), D- ornithine (453.57, 278.78, 390, 74.28, 54.31, 70.28, 21.16, 24.02, 24.71, 79.31, 31.50, 7.95, 67.5, 30.28%) and DL-ornithine (314.28, 221.21, 513.33, 120, 36.53, 25.25, 23.53, 22.34, 12.35, 25.86, 25.24, 24.77, 63.75, 162.98%) were able to increase α-pinene, β-pinene, camphene, α-thujone, limonene, β-thujone, camphor, borneol, bornyl acetate, aromadendrene, α-humulene, viridifloral, humulene epoxide II, and manool compared to the control without drought stress, respectively (Fig. 3A, B, C, D, F, G, H, I, G, L, M, N and O, and 3P). Furthermore, D-ornithine (8.28%) and DL-ornithine (17.36%) were able to increase 1,8-cineole compared to normal growth condition (no stress) (Fig. 3E). In addition, D-ornithine (168.88, 60.97, 103.41, 9.58, 32.84, 76.64, 63.38, 14.14, 6.20, 73.33, 20.3%) and DL-ornithine (86.66, 130.48, 59.82, 31.50, 18.09, 27.91, 46.47, 10.64, 62.30, 33.33, 12.8%) were able to increase α-pinene, camphene, β-pinene, limonene, β-thujone, borneol, aromadendrene, α-humulene, viridifloral, humulene epoxide II, and manool compared to the control group under drought stress, respectively (Fig. 3A, B, C, D, G, I, L, M, N and O, and 3P). Furthermore, the L-ornithine (25.47, 7.51, 12.35%), D- ornithine (39.34, 53.99, 25.69%), and DL-ornithine (31.16, 19.71, 30.87%) were able to increase 1,8-cineole, bornyl acetate, and e-caryophyllene than the control group under drought stress, respectively (Fig. 3E and G, and 3K). Overall, the highest α-pinene, β-pinene, 1,8-cineole, α-thujone, camphor, borneol, bornyl acetate, aromadendrene, humulene epoxide II, and manool were obtained with the application of D-ornithine under drought stress (Fig. 3A, C, E, F, H, I, J, L and O, and 3P). The highest camphene, limonene, e-caryophyllene, and viridifloral were obtained with the application of DL-ornithine under drought stress (Fig. 3B, D and K, and 3N). In addition, β-thujone and α-humulene were obtained with the application of D-ornithine and DL-ornithine under drought stress (Fig. 3M and G).

Fig. 3
figure 3

The three-way interaction between water availability, D-ornithine, and L-ornithine on α-pinene (A), camphene (B), β-pinene (C), limonene (D), 1,8-cineole (E), α-thujone (F), β-thujone (G), camphor (H), borneol (I), bornyl acetate (J), e-caryophyllene (K), aromadendrene (L), α-humulene (M), viridifloral (N), humulene epoxide II (O) and manool (P). The different letters show significant differences at the level of 0.01. The error bars represent standard error

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Gene expression assay

It is evident that the interaction between water availability, D-ornithine, and L-ornithine significantly influenced the gene expression levels of BS, SS, and CS under drought stress conditions (Table 3). The results indicate that these genes were up-regulated in response to water deficit condition compared to normal growth. Specifically, the gene expression levels of BS and SS were significantly increased when treated with L-ornithine (323.52%, 1010%), D-ornithine (276.47%, 1440%), and DL-ornithine (317.64%, 970%) under well-watered conditions compared to the control group without stress (Fig. 4A and B). Moreover, under drought stress, D-ornithine was particularly effective in increasing the gene expression levels of BS and SS by 45.29% and 113.63%, respectively, compared to the control group under stress conditions (Fig. 4A and B). In addition, D-ornithine (66.66%) and DL-ornithine (103.33%) showed significantly increased the gene expression levels of CS compared to the control group (Fig. 4C). Also, D-ornithine (165.62%, 113.63%, and 152%), and DL-ornithine (63.38%, 57.00%, and 68.85%) showed significant increases in the gene expression levels of BS, SS, and CS respectively, under drought stress compared to those under well-watered conditions. Overall, the application of D-ornithine demonstrated the highest enhancement in the gene expression levels of BS, SS, and CS under drought stress conditions (Fig. 4A and B, and 4C). These results suggest that D-ornithine may play a crucial role in modulating gene expression related to terpenoid biosynthesis in response to environmental stresses such as drought.

Fig. 4
figure 4

The three-way interaction between water availability, D-ornithine, and L-ornithine on borneol diphosphate synthase (A), sabinene synthase (B) and cineole synthase (c). The different letters show significant differences at the level of 0.01. The error bars represent standard error

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Total phenolic and flavonoid contents

Based on the ANOVA results, there was a statistically significant three-way interaction between water availability, D-ornithine, and L-ornithine for total phenolic (p < 0.05) and flavonoid content (p < 0.01) (Table 3). The current result showed that the total phenolic content was increased by 4.37% under drought stress compared to non-stressed (Fig. 5). However, the flavonoid contents (270, 300, and 330) showed no significant increase under drought stress compared to without stress (Fig. 6). Furthermore, the application of DL-ornithine and D-ornithine resulted in 7.90% and 11.76% increase in total phenolic content, respectively compared to the control group under drought stress conditions. Moreover, demonstrated a significant increase in total phenolic content Notably, the highest total phenolic content was achieved with the application of D-ornithine under drought stress conditions, indicating its potential role in promoting phenolic compound accumulation in S. officinalis (Fig. 5). The current results showed that the application of D-ornithine resulted in a substantial enhancement of flavonoid levels, with increases of 70%, 105.66%, and 114.28% for flavonoid content of 270, 300, and 330, respectively, compared to the control group under drought stress conditions (Fig. 6).

Fig. 5
figure 5

The three-way interaction between water availability, D-ornithine, and L-ornithine on total phenolic content. The different letters show significant differences at the level of 0.01. The error bars represent standard error

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Fig. 6
figure 6

The three-way interaction between water availability, D-ornithine, and L-ornithine on flavonoid content (270, 300, 330). The different letters show significant differences at the level of 0.01. The error bars represent standard error

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Principal component analysis (PCA)

Principal component analysis (PCA) of leaf dry weight (LDW), stem dry weight (SDW), total plant dry weight (TDW), EO percentage (EO%), EO yield (EOY), α-pinene (α-PN), camphene (CP), β-pinene (β-PN), limonene (LMN), 1,8-cineole (1,8-CIN), α-thujone (α-THU), β-thujone (β-THU), camphor (CPH), borneol (BRN), bornyl acetate (BA), β-caryophyllene (BCP), aromadendrene (AMD), α-humulene (α-HUM), viridifloral (VRF), humulene epoxide II (HE-II), manool (MO), borneol diphosphate synthase (BS), sabinene synthase (SS), cineole synthase (SC), flavonoid 270 (FLA 270), flavonoid 300 (FLA 300), flavonoid 330 (FLA 330), total phenolic content (TPC) of S. officinalis under different water availability and exogenous ornithine enantiomers were assessed in this study. In the study of secondary metabolites and physiological traits under varying water stress conditions, the analysis revealed the extraction of two PCs with eigenvalues greater than 1. The first component (PC I) accounted for 58.90% of the variance, while the second component (PC II) contributed to 40.91% of the variance (Fig. 7). The loading plot (Fig. 7) showed that PC I gave flavonoid 270 (FLA270), percentage essentioal oil (EO%), bornyl diphosphate synthase (BS), and limonene (LMN) as dominant variables, which appeared to be positive PC I values. Leaf dry weight (LDW), total plant dry weight (TDW), stem dry weight (SDW), sabinene synthase (SS), and flavonoid 330 (FLA 330) seemed to be negative values for PC (I) However, α-thujone (a-THU), α-humulene (α -HUM), camphor (CPH), β-caryophyllene (β-CP), flavonoid 300 (FLA300), viridifloral (VRF), and camphene (CP) are introduced as dominant positive variables for the PC (II) The dominant negative variables of PC II were β-pinene (β-PN), α-pinene (α-PN), and yield EO (EOY).

Fig. 7
figure 7

Principal component analysis (PCA) of leaf dry weight (LDW), stem dry weight (SDW), total plant dry weight (TDW), EO (EO%), EO yield (EOY), α-pinene (α-PN), camphene (CP), β-pinene (β-PN), limonene (LMN), 1,8-cineole (1,8-CIN), α-thujone (α-THU), β-thujone (β-THU), camphor (CPH), borneol (BRN), bornyl acetate (BA), β-caryophyllene (BCP), aromadendrene (AMD), α-humulene (α-HUM), viridifloral (VRF), humulene epoxide II (HE-II), manool (MO), borneol diphosphate synthase (BS), sabinene synthase (SS), cineole synthase (SC), flavonoid 270 (FLA 270), flavonoid 300 (FLA 300), flavonoid 330 (FLA 330), total phenolic content (TPC) of Salvia officinalis under different water availability and exogenous ornithine enantiomers

Full size image

Discussion

Our results indicate that drought stress leads to a decrease in total plant dry weight, as well as the dry weight of stems and leaves, compared to conditions without stress. This finding is consistent with previous studies by Abd Elbar et al. [39] on Thymus vulgaris, Mohammadi et al. [9] in Origanum majorana and Origanum vulgare, and Fallah Imani et al. [7] in S. mirzayanii. The reduction in dry weight can be attributed to a decline in cell turgor, which inhibits growth and cell elongation, as suggested by Alishah et al. [40]. Furthermore, we observed significant positive correlations between leaf dry weight and both total plant and stem dry weight, indicating that an increase in leaf dry weight is associated with higher total plant and stem dry weights. Additionally, our study found that D-ornithine positively effects the reduction of growth inhibition in S. officinalis plants under drought stress, leading to improved plant growth. This aligns with previous research indicating that ornithine could act as a signaling regulator, helping to maintain cellular balance under drought stress. Other studies suggest that PAs enhance plant stress tolerance by stabilizing membranes and cell walls, as well as providing acid neutralizing and antioxidant effects [41]. Proline, an important osmolyte and stress indicator, is highly soluble and assists in osmoregulation by protecting enzymes and cellular structures. Additionally, it functions as a free radical scavenger and energy source, helping to regulate redox potential and maintain nitrogen levels [42]. Proline biosynthesis occurs via two pathways: glutamate (Glu) and ornithine. The enzyme ornithine aminotransferase (OAT), which relies on pyridoxal-5-phosphate, is essential for the ornithine pathway, especially under drought stress conditions [43]. Research has shown that exogenous application of ornithine can increase OAT activity and proline levels in salt-stressed cashew plants [42]. Overall, the evidence indicates that D-ornithine and other PAs may serve as valuable tools for reducing the negative effects of drought stress on plant growth and productivity, providing promising strategies to enhance plant resilience and performance in challenging environments.

The study revealed that the EO content and yield of S. officinalis increased when exposed to drought stress compared to normal growth conditions, aligning with previous research findings. Exogenous PAs help improve leaf water potential and relative water content in plants experiencing drought. For example, applying putrescine to drought-affected Trachyspermum ammi not only alleviates stress but also boosts EO production [44]. Our results confirm earlier studies that indicate drought stress enhances EO content as medicinal plants adjust their synthesis to counteract reactive oxygen species (ROS) [45]. Additionally, drought stress can stimulate EO biosynthesis due to changes in redox potential. Therefore, implementing drought stress during cultivation can significantly enhance both the quality and quantity of yields in medicinal and aromatic plants. The positive correlations observed between total plant dry weight, stem dry weight, and EO yield further support the relationship between plant growth parameters and EO production (Table 4). According to Selmar et al. [46], the passive shift in redox potential in medicinal plants under stress can lead to an upregulation of enzymes involved in the biosynthetic pathway of EO, particularly under drought conditions. This includes terpene synthases (TPS) such as sesquiterpene and monoterpene synthases and cytochrome P450 monooxygenases responsible for the oxidative modification of terpene compounds, along with geranyl diphosphate synthase (GPPS), which is essential for constructing the terpenoid backbone [47, 48]. Other plant species, such as S. officinalis [49] and Thymus vulgaris [50], suggest that this mechanism may be conserved across various plant families. Our findings align with previous research highlighting the complex role of EOs in plant physiological adaptation to stress. Furthermore, the synthesis and accumulation of EOs can serve as a defense mechanism against drought stress, as noted by Ramakrishna & Ravishankar [51]. The study highlighted the positive effects of D-ornithine on EO content by alleviating the negative impacts of drought stress. This led to an increase in both EO content and yield in S. officinalis. Similar results have been reported by Bettaieb et al. [52], Rebey et al. [53], and Hassan & Ali [54] regarding S. officinalis, Cuminum cyminum, and Coriandrum sativum, respectively, where compounds like putrescine and GA3 were used. Research has indicated that putrescine can enhance the content and yield of EOs in plants, suggesting the potential for manipulating EO content to improve the medicinal properties of plants under stress conditions. Oxygenated monoterpenes predominantly constitute of the EOs, influenced by growth conditions and agricultural practices. Among the identified compounds, α-humulene is noted as the predominant sesquiterpene while manool is recognized as a key diterpene, both of which are associated with inhibition cancer cell growth. Additionally, the investigation of the major monoterpenes of S. officinalis EO under drought stress revealed the presence of 1,8-cineole, α-thujone, β-thujone, and camphor. The study suggests that the highest levels of these compounds, along with the maximum expression of the main monoterpene synthase genes, were achieved through are application of exogenous D-ornithine and exposure to drought stress. Our study also found significant positive correlations between the expression levels of the major monoterpene synthase genes (BS, SS and CS) and EO content, EO yield and EO chemical profiles, including α-pinene, camphene, β-pinene, 1,8-cineole, α-thujone, β-thujone, camphor, borneol, e-caryophyllene, α-humulene, viridifloral and manool. BS produces bornyl diphosphate, which is then converted to camphor and borneol. This suggests that D-ornithine increases the expression levels of BS under drought stress. CS directly catalyzes the formation of 1,8-cineole and our study found the D-ornithine also increased the transcript abundance of CS under drought stress. Additionally, D-ornithine elevated the levels of 1,8-cineole during drought conditions. For SS, it is responsible for the producing sabinene, which serve as the precursor for α-thujone and β-thujone. Our results indicated that D-ornithine increased the expression of SS under drought stress, leading to elevated levels of the corresponding metabolites, α-thujone and β-thujone.

Table 4 Pearson’s correlation coefficients among Y1:Leaf dry weight, Y2: stem dry weight, Y3: total plant dry weight, Y4: essential oil percentage, Y5: essential oil yield, Y6: α-pinene, Y7: Camphene, Y8: β-pinene, Y9: Limonene, Y10: 1,8-cineole, Y11: α-thujone, Y12: β-thujone, Y13: Camphor, Y14: Borneol, Y15: Bornyl acetate, Y16: E-caryophyllene, Y17: Aromadendrene, Y18: α-humulene, Y19: Viridifloral, Y20: Humulene epoxide II, Y21: Manool, Y22: Borneol diphosphate synthase, Y23: Sabinene synthase, Y24: cineole synthase, Y25: flavonoid 270, Y26: flavonoid 300, Y27: flavonoid 330, Y28: total phenolic content of Salvia officinalis influenced different water availability and exogenous ornithine enantiomers ns: non-significant, *: α ≤ 0.05, **: α ≤ 0.01
Full size table

In the current study, PCA identified two principal components (PCs) that accounted for a significant portion (99.81%) of the overall variation in water stress, influenced by various plant traits (Fig. 7). Among these components, PC I exhibited a strong negative correlation between total plant dry weight (TDW) and bornyl diphosphate synthase (BS). In contrast, PC II demonstrated a positive relationship with β-caryophyllene (BCP), bornyl acetate (BA), camphor (CPH), α-humulene (α-HUM), and flavonoid 300 (FLA300) (Fig. 7). These results indicate that drought stress primarily affected shoot biomass, as well as the yield and composition of EO and secondary metabolites, leading to a reduction in these parameters. Conversely, a direct correlation was observed between D-ornithine and growth-related parameters, suggesting that D-ornithine may play a protective role in alleviating the damaging effects of drought on the growth and development of S. officinalis by enhancing the production of secondary metabolites. Furthermore, this study reveals an increase in phenolic compounds in S. officinalis under drought stress. The activity of phenylalanine ammonia-lyase (PAL) and tyrosine ammonia-lyase (TAL), which are regulatory enzymes in the phenylpropanoid pathway, appears to increase under drought stress. This increase is related to the enhancement and accumulation of phenolic compounds [55]. These findings are consistent with previous studies by Weidner et al. [16], Gharibi et al. [18], and Al Hassan et al. [56] which explored the effects on grapevine roots, Achillea species, and cherry tomatoes, respectively. Moreover, Vakili et al. [57] discovered that applying putrescine during drought stress leads to a rise in total phenolic compounds. Their research also established a positive correlation between phenolic compounds and plant defense mechanisms against environmental stresses, as seen in the positive relationships between EO content and flavonoids. Additionally, the study suggests that D-ornithine, PA, and putrescine contribute to the enhancement of phenolic compounds under drought stress, potentially through the shikimic acid pathway. On the other hand, drought stress and the application of ornithine influence the direction of PAs metabolism by regulating the levels of PAs and other regulatory metabolites such as abscisic acid (ABA). These changes collectively lead to increase activity of PAL and TAL, enhance the shikimic acid pathway, and ultimately contribute to the production of various phenolic compounds that help improve the plants response to drought stress [55]. Therefore, it appears that S. officinalis has developed a complex metabolic network and multiple signaling pathways to cope with drought stress. Overall, this study emphasizes the significance of phenolic compounds in plant responses to stress and their potential role in maintaining redox balance in plants facing challenges like drought stress.

Conclusion

The current investigation highlights the negative effects of drought stress on the biomass of S. officinalis. D-ornithine has been identified as a helpful in mitigating stress damage and enhancing drought tolerance in S. officinalis plants. This improvement is reflected in better growth parameters and higher levels of phenolic compounds and EO, particularly α-thujone, camphor, 1,8-cineole, and β-thujone. The increase in total phenolic and EO content likely indicates a response to the production of reactive oxygen species, which plays a crucial role in improving the drought tolerance of S. officinalis. Overall, plant growth regulators like D-ornithine demonstrate the ability to promote the production of compounds that help plants tolerate drought stress.

Data availability

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

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Acknowledgements

The authors are grateful for to support of the Iranian National Science Foundation (INSF) through grant no: 4012913. We would like to express further gratitude to Tarbiat Modares University (TMU) for providing facilities.

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Authors and Affiliations

  1. Department of Plant Biology, Faculty of Biological Science, Tarbiat Modares University, PO Box 14115-154, Tehran, Iran

    Maryam Mohammadi-Cheraghabadi & Faezeh Ghanati

  2. Department of Biology, Faculty of Science, Razi University, Kermanshah, Iran

    Naser Karimi

  3. Department of Medicinal Plants, Faculty of Agriculture and Natural Resources, Arak University, Arak, 38156-8-8349, Iran

    Mansour Ghorbanpour

  4. Department of Agronomy and Plant Breeding, Faculty of Agriculture, Azarbaijan Shahid Madani University, Tabriz, Iran

    Saeid Hazrati

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  1. Maryam Mohammadi-Cheraghabadi
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Contributions

M.M-Ch. performed the material preparation, data collection, and analysis, and wrote the primary draft of the manuscript. F.Gh. a supervisor, devised the project, planned the main conceptual ideas, and reviewed and edited the manuscript. N.K. and M.Gh. performed methodology, conceptualization, and writing review and editing. S.H. performed the writing of reviews and editing.

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Correspondence to Faezeh Ghanati.

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The one-year-old mother plants of Salvia officinalis used in this study were collected with the approval of local regulatory authorities. All methods were carried out in compliance with regional and national regulations.

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Mohammadi-Cheraghabadi, M., Ghanati, F., Karimi, N. et al. Ornithine enantiomers modulate essential oil yield and constituents and gene expression of monoterpenes synthase in Salvia officinalis under well-watered and drought stress conditions. BMC Plant Biol 25, 148 (2025). https://doi.org/10.1186/s12870-025-06156-y

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