Skip to main content
Download PDF

Intraspecific divergence in essential oil content, composition and genes expression patterns of monoterpene synthesis in Origanum vulgare subsp. vulgare and subsp. gracile under salinity stress

BMC Plant Biology volume 23, Article number: 380 (2023) Cite this article

Abstract

Background

Oregano (Origanum vulgare L.), one of the important medicinal plants in the world, has valuable pharmacological compounds with antimicrobial, antiviral, antioxidant, anti-inflammatory, antispasmodic, antiurolithic, antiproliferative and neuroprotective activities. Phenolic monoterpenes such as thymol and carvacrol with many medical importance are found in Oregano essential oil. The biosynthesis of these compounds is carried out through the methyl erythritol-4 phosphate (MEP) pathway. Environmental stresses such as salinity might improve the secondary metabolites in medicinal plants. The influence of salinity stress (0 (control), 25, 50 and 100 mM NaCl) on the essential oil content, composition and expression of 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR), γ-terpinene synthase (Ovtps2) and cytochrome P450 monooxygenases (CYP71D180) genes involved in thymol and carvacrol biosynthesis, was investigated in two oregano subspecies (vulgare and gracile).

Results

Essential oil content was increased at low NaCl concentration (25 mM) compared with non-stress conditions, whereas it was decreased as salinity stress intensified (50 and 100 mM). Essential oil content was significantly higher in subsp. gracile than subsp. vulgare. The highest (0.20 mL pot−1) and lowest (0.06 mL pot−1) amount of essential oil yield was obtained in subsp. gracile at 25 and 100 mM NaCl, respectively. The content of carvacrol, as the main component of essential oil, decreased with increasing salinity level in subsp. gracile, but increased in subsp. vulgare. The highest expression of DXR, Ovtps2 and CYP71D180 genes was observed at 50 mM NaCl in subsp. vulgare. While, in subsp. gracile, the expression of the mentioned genes decreased with increasing salinity levels. A positive correlation was obtained between the expression of DXR, Ovtps2 and CYP71D180 genes with carvacrol content in both subspecies. On the other hand, a negative correlation was found between the expression of CYP71D180 and carvacrol content in subsp. gracile.

Conclusions

The findings of this study demonstrated that both oregano subspecies can tolerate NaCl salinity up to 50 mM without significant reduction in essential oil yield. Also, moderate salinity stress (50 mM NaCl) in subsp. vulgare might increase the carvacrol content partly via increment the expression levels of DXR, Ovtps2 and CYP71D180 genes.

Peer Review reports

Background

Oregano (Origanum vulgare L.) is an herbaceous perennial plant in the mint family (Lamiaceae), native to Europe and central Asia [1, 2]. Many investigations have demonstrated its antimicrobial, antiviral, antioxidant, anti-inflammatory, antispasmodic, antiurolithic, antiproliferative, and neuroprotective pharmacological activities [3,4,5]. Furthermore, it is an important natural source for preserving foods or cosmetics due to its high antioxidant activity [5,6,7]. A great variety has been reported in the essential oil composition of oregano, which is attributed to the high morphological and chemical diversity within the genus Origanum [8,9,10]. Depending on the growth conditions, growth stage and different organs, the dominant constituents of essential oil in oregano have been recognized such as carvacrol, thymol, p-cymene, γ-terpinene, sabinene, linalool, borneol, β-bisabolene, terpinen-4-ol, β-caryophyllene, caryophyllene oxide, germacrene D and β-ocimene [5, 9, 11,12,13,14,15]. Phenolic monoterpenes (thymol and carvacrol) identified in O. vulgare essential oil, are medically important due to their antioxidant, antimicrobial, antitussive, expectorant, antispasmodic and antibacterial properties [16,17,18,19]. Terpene synthases (TPS) are key enzymes involved in the biosynthesis of monoterpenes and sesquiterpenes, which catalyze the oxidation steps from precursors for each group of terpenes [20, 21]. Terpene synthase genes have been identified in different species of mint family plants such as, Salvia officinalis [22, 23], Mentha spicata [24], O. vulgare [25], and Thymus capitatus [26]. The methyl erythritol-4 phosphate (MEP) pathway provides substrate for producing both mono and di-terpenes in plastids [27]. The induction and activity of 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) is recognized as an important key point in MEP pathway, which has been reported as the first major and separating step of this pathway [21, 28]. Generally, the synthesis of geranyl diphosphate (GDP) from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) (as a precursor of monoterpenes) catalyzes by geranyl diphosphate synthase. The next step of the pathway is the conversion of GDP to γ-terpinene, which is catalyzed by γ-terpinene synthase. In plants such as thyme and oregano, the precursor of thymol and carvacrol is γ-terpinene [25, 29] and cytochrome P450 (CYP) monooxygenases are involved in the conversion of γ-terpinene to thymol and carvacrol (Fig. 1). According to the previous studies, carvacrol is derived from γ-terpinene through the acting of CYP71D180 and CYP71D181, whereas CYP71D178, CYP71D179 and CYP71D182 are likely involved in thymol biosynthesis [30,31,32].

Fig. 1
figure 1

Proposed pathway for carvacrol and thymol biosynthesis in oregano and thyme in plastids (Crocoll, 2011). DXR: 1-deoxy-D-xylulose-5-phosphate reductoisomerase, Ovtps2: γ-Terpinene synthase, CYP71D180

Full size image

About 20% of the world’s land, as well as about half of arable irrigated land in the worlds, is affected by salinity [33]. As a worldwide issue, soil salinization restricts agricultural production due to its adverse effect on the plant growth and production [34, 35]. Soil salinity reduces soil water potential, leaf water potential and turgor pressure of the plant cells, consequently induce osmotic stress [36]. High accumulation of ions (Na+ and Cl−) in saline conditions prevents K+ and Ca+2 uptakes and leads to ion imbalance [37]. Salinity increases reactive oxygen species (ROS) in the plant cells [38] which causes lipid peroxidation, membrane degradation, and DNA and protein damage [39]. To deal with saline conditions, plants use various strategies such as ionic homeostasis and partitioning, ion transport, osmotic adjustment, antioxidant defense system, and polyamine biosynthesis [40]. Furthermore, plant secondary metabolites notably improve plant growth and survival under biotic and abiotic stresses [41, 42] and their biosynthesis and accumulation are influenced by environmental stresses such as salinity [43]. It has been demonstrated that environmental stresses might change both the quality and quantity of the plant secondary metabolites through influencing the expression of the genes involved in their biosynthesis [44]. Studies have shown that soil salinity changes essential oil biosynthesis and composition in several plant species such as Salvia officinalis [45], Satureja hortensis [46], and Melissa officinalis [47].

To our knowledge, the effect of salinity stress on the content of terpenes and expression of their biosynthetic genes has not been evaluated in O. vulgare yet. Due to the presence of valuable compounds in the essential oil of O. vulgare, study the expression of the genes involved in their biosynthesis and their association with the accumulation of the compounds under salinity conditions may be of great interest for pharmaceutical and industrial market. Hence, for the first time, the expression of the genes involved in the biosynthesis of the valuable secondary metabolites (carvacrol and thymol) was compared in two oregano subspecies (gracile and vulgare) under various salinity levels. Moreover, the association between genes expression levels and their corresponded compounds, changes in essential oil content, oil yield and their compounds were also studied under salinity conditions.

Results

Essential oil content and yield

Essential oil content was significantly affected by salinity treatments and subspecies. According to the results, essential oil content was increased at low NaCl concentration (25 mM) compared with non-stress conditions, whereas it was decreased as salinity stress intensified (50 and 100 mM). Briefly, essential oil content was significantly higher in subsp. gracile than subsp. vulgare (Fig. 2). Essential oil yield was significantly influenced by salinity treatments, subspecies and their interaction. In vulgare subspecies, essential oil yield decreased by increasing salinity, but the difference between 0, 25 and 50 mM NaCl was not significant. In gracile subspecies, the essential oil yield increased by enhancing the intensity of salinity, up to 25 mM and then decreased by increasing salinity level. Also, the difference between 0, 25 and 50 mM NaCl was not significant (Fig. 3). A positive relationship was found between essential oil content and yield in both subspecies (Fig. 6a,b).

Fig. 2
figure 2

Effect of salinity stress and Origanum vulgare subspecies (subsp. vulgare and subsp. gracile) on essential oil content. Columns with different letters have significant differences (p < 0.05)

Full size image
Fig. 3
figure 3

Interaction effect of salinity × Origanum vulgare subspecies (subsp. vulgare and subsp. gracile) on essential oil yield. Columns with different letters have significant differences (p < 0.05)

Full size image

Chemical composition of essential oil

The alterations of essential oil compounds in O. vulgare subsp. vulgare and O. vulgare subsp. gracile under salinity stress were presented in Table 1. According to the results of GC–MS analysis, total volatile compounds detected in gracile and vulgare subspecies were 23 and 27, respectively. The dominant constituents of essential oils were carvacrol, carvacrol methyl ether, γ-terpinene, thymol, cis-α-bisabolene and p-cymene in both subspecies. The results revealed the different impact of salinity on the chemical composition of essential oil in two subspecies. The highest percentage of carvacrol (60 and 47.36%) was recorded at non-stress conditions and 50 mM NaCl in gracile and vulgare subspecies, respectively. Although in gracile subspecies, the percentage of carvacrol decreased with the application of salinity stress, no significant difference was observed between salinity treatments in terms of this composition. Conversely, in vulgare subspecies, the percentage of carvacrol raised by increasing salinity levels. Although, the trend of thymol changes in two subspecies does not follow a discrete pattern, but in both subspecies, the amount of thymol in non-stress treatment was higher than salinity treatments. The findings of this research demonstrated that, p-cymene was significantly increased in both subspecies by enhancing salinity stress. However, no significant differences were found between 25, 50 and 100 mM salinity treatments in vulgare subspecies. In both subspecies, the amount of γ-terpinene increased up to 25 mM NaCl and then decreased by increasing salinity. Furthermore, the trend of changes in carvacrol methyl ether and cis-α-bisabolene did not follow a specific pattern, however, vulgare subspecies had higher content of carvacrol methyl ether and cis-α-bisabolene under all salinity treatments (Fig. 4).

Table 1 Essential oil components in O. vulgare subsp. vulgare and O. vulgare subsp.gracile under salinity stress
Full size table
Fig. 4
figure 4

Interaction effect of salinity × Origanum vulgare subspecies (subsp. vulgare and subsp. gracile) on γ-terpinene, ρ-cymene, carvacrol, thymol, carvacrol methyl ether and cis-α-bisabolene content in the essential oil. Columns with different letters have significant differences (p < 0.05)

Full size image

According to the results, monoterpenes were the main groups of the identified components in both subspecies. The essential oil of subsp. gracile contained monoterpenes (93.65%, 93.18%, 93.02% and 94.6%) at different levels of salinity, respectively. Oxygenated monoterpenes had the highest percentage in the subclass of monoterpenes. Of these, carvacrol, carvacrol methyl ether and thymol were the major components. 1,8-cineole, as an oxygenated monoterpene, was detected only at 100 mM NaCl in subsp. gracile. Monoterpene hydrocarbons are the second subclass of the monoterpenes, among which γ-terpinene and ρ-cymene were identified as the dominant components. Sesquiterpene hydrocarbons were the next subclass of compounds found in subsp. gracile oil that reached the highest percentage (4.18%) at 50 mM NaCl, and cis-α- bisabolene was identified as the major component. In addition, oxygenated sesquiterpenes were not detected in subsp. gracile. In contrast, the oil of subsp. vulgare contained monoterpenes (89.19%, 91.53%, 92.23% and 91.57%) and sesquiterpenes (6.05%, 4.91%, 4.78% and 5.76%) at different levels of salinity, respectively. Oxygenated monoterpenes were the most dominant subclass of compounds in subsp. vulgare. Of these, carvacrol, carvacrol methyl ether and thymol were the major components. Furthermore, the highest percentage (61.39%) of oxygenated monoterpenes was found at 50 mM NaCl. The highest monoterpene hydrocarbons (35.07%) as the second subclass of compounds were observed at 25 mM NaCl, of which γ-terpinene and ρ-cymene were identified as predominant components. Sesquiterpene hydrocarbons were another dominant subclass of compounds with cis-α- bisabolene as the major component. Spathulenol and caryophyllene oxide are the only oxygenated sesquiterpenes identified at non-stress treatments in subsp. vulgare. Moreover, α-phellandrene was detected at 25 mM NaCl in subsp. gracile. Whereas, in subsp. vulgare it was not detected only at 50 mM NaCl. Sabinene, as a monoterpene hydrocarbon, was not identified in gracile subspecies but was found at non-stress treatment and 25 mM NaCl in vulgare subspecies (Table 1).

Correlation analysis showed a negative relationship between γ-terpinene and p-cymene in subsp. vulgare. Also in this subspecies, a negative relationship was obtained between γ-terpinene and thymol with carvacrol, whereas the correlation between p-cymene and carvacrol was positive. In addition, the correlation between γ-terpinene and p-cymene with thymol was negative (Fig. 6a). In contrast in subsp. gracile, a negative correlation was observed between γ-terpinene and p-cymene. Also, a negative relationship was obtained between p-cymene and carvacrol, as well as γ-terpinene with thymol. Furthermore, a positive relationship was observed between carvacrol with thymol and γ-terpinene with carvacrol (Fig. 6b).

Gene expression levels

To partly unravel the molecular mechanism by which salinity stress alters the content of essential oil in two studied oregano subspecies, the expression levels of DXR, Ovtps2 and CYP71D180 genes were investigated under various salinity levels in these subspecies for the first time. The expression levels of studied genes were significantly affected by salinity treatments, subspecies and their interaction. The highest DXR expression was observed at 50 mM NaCl in vulgare subspecies, while the lowest expression of that was obtained at gracile subspecies under salinity stress. Furthermore, the highest expression of Ovtps2 was observed at 50 mM NaCl in vulgare subspecies. However, in gracile subspecies, the relative expression of this gene decreased with increasing salinity. Similar to the DXR gene, the highest relative expression of CYP71D180 was obtained at 50 mM salinity in vulgare subspecies. Whereas, in gracile subspecies, the expression of this gene decreased with increasing salinity up to 50 mM, then increased at 100 mM salinity (Fig. 5).

Fig. 5
figure 5

Relative expression (RE) of DXR, Ovtps2, CYP71D180 genes in Origanum vulgare (subsp. vulgare and subsp. gracile) under salinity stress. Error bars indicate standard error of the mean (n = 3). Columns with different letters have significant differences (p < 0.05)

Full size image

A positive relationship was observed between the expression of DXR, Ovtps2 and CYP71D180 genes with carvacrol in vulgare subspecies, while, the correlation of these genes with thymol content was negative. Also, a negative correlation was found between the relative expression of Ovtps2 gene and γ-terpinene, while the correlation of this gene with p-cymene was positive in this subspecies (Fig. 6a). In contrast, in gracile subspecies, a positive correlation was obtained between the relative expression of Ovtps2 and γ-terpinene, whereas the correlation of this gene with p-cymene was negative. There was a negative relationship between the expression of CYP71D180 and carvacrol, while a positive correlation was obtained between DXR and Ovtps2 genes expression with carvacrol content. Also, in gracile subspecies, the correlation between the three studied genes and thymol content was positive (Fig. 6b).

Fig. 6
figure 6

Pearson’s correlation coefficients between the studied genes, essential oil content and yield and essential oil components in O. vulgare subsp. vulgare (a) and O. vulgare subsp. gracile (b). Positive and negative correlations are displayed in blue and red, respectively

Full size image

Discussion

To deal with salinity, plants adjust their growth and development behaviors along with an organizing between primary and secondary metabolites [48]. The results of the several investigations demonstrate that the biosynthesis of secondary metabolites in medicinal plants is seriously affected by environmental factors [23, 49,50,51]. Furthermore, the difference between the content and composition of essential oil in medicinal plants depends on the various factors such as cultivar, genetics and environmental conditions [52]. However, studies have shown that these changes may be caused through the different expression of the enzymes involved in the production of these compounds under salinity conditions [53, 54]. In this investigation, essential oil content influenced by salinity stress and subspecies. The highest percentage of essential oil was achieved for subsp. gracile at 25 mM salinity. However, the essential oil content decreased at 50 and 100 mM NaCl stress. Under moderate salinity stress, the stimulation of essential oil production can be due to the higher density of essential oil glands [55]. Moreover, the increment of essential oil contents in plants may be due to the reduction of primary metabolites by salinity and the improvement of intermediary products availability for secondary metabolites synthesis [54, 55]. According to the previous studies, the essential oil content increased with the intensity of salinity in Salvia officinalis [56] and Ocimum basilicum [55]. However, the essential oil content decreased by increasing salinity in O. majorana [57] and Mentha piperita [58]. Moreover, the highest essential oil yield was observed at low salinity level in subsp. gracile. The essential oil yield in subsp. vulgare decreased with increasing salinity. Similarly, high salinity levels led to a decline in essential oil yield in some plant species such as, Trachyspermum ammi [59] and Matricaria sp. [60].

The chemical composition of O. vulgare essential oil has been studied in several researches [9, 15, 25, 61]. There is a high variety in essential oil composition of this plant. The main composition of essential oil in O. vulgare is thymol, carvacrol, γ-terpinene, p-cymene, β-myrcene and β-bisabolene [2, 9, 13]. In this study, the main components under salinity treatments and in both subspecies were carvacrol, γ-terpinene, p-cymene, thymol, carvacrol methyl ether and cis-α-bisabolene. It can be considered that the accumulation of some main compounds as a defense mechanism in medicinal plants by inducing changes in cellular metabolism adapts them to stress conditions [62]. Salinity stress can affect the essential oil composition of plants depending on its severity. In previous reports, percentage of main compounds enhanced with severity of NaCl stress in comparison with non-stress conditions, in Salvia officinalis [48, 53, 63], S. mirzayanii [64] and Ocimum basilicum [55].

Monoterpenes in plants have a high commercial value industrially and can be used in the perfume, anti-cancer and pesticide industries [65]. Two dominant components of oregano essential oil are phenolic monoterpenes, thymol and carvacrol, which are well known their anti-vegetarian, antimicrobial, medicinal and antioxidant activities [25]. In the present research, an early gene (DXR), a middle gene (Ovtps2) and a last gene (CYP71D180) in MEP pathway, involved in thymol and carvacrol biosynthesis [30, 31] were evaluated, which showed that salinity stress significantly affected their expression. This might be due to the role of terpenes in defense pathways and signal transduction in oregano. Based on the results, a positive correlation was obtained between the expression of DXR with Ovtps2 and CYP71D180 in both subspecies. According to the previous studies, γ-terpinene and p-cymene are the main precursors of thymol and carvacrol in oregano and thyme, which are synthesized by the γ-terpinene synthase enzyme from geranyl diphosphate [25, 29]. The results of Ovtps2 gene expression in subsp. vulgare indicated that salt stress increased the expression of this gene compared with control. Furthermore, Ovtps2 as an intermediate gene in the pathway of thymol and carvacrol biosynthesis was more affected than DXR and CYP71D180 genes at all salinity levels. In oregano, the contents of thymol and carvacrol in leaves are related to the expression of Ovtps2 [25]. In this study, the relative expression of Ovtps2 was increased in subsp. vulgare at 50 mM salinity, while the percentage of γ-terpinene (as a precursor of thymol and carvacrol) decreased. In other words, a negative correlation was found between the expression of Ovtps2 and γ-terpinene at this salinity level. The lack of congruence between the transcriptional levels of the genes and their corresponded compounds (less gene expression but more compound production) may be due to the effect of stress on the enzymatic activity or some changes in transcription and post-translational processes [50, 66]. Post-translational modifications of proteins are very important factor in regulating the plant response to the stress conditions [67] and can regulate protein function, location, half-life and protein interactions to reduce the potential damage caused by environmental stresses [68]. However, the activity of the enzymes under salinity stress have not been studied in this investigation.

Also, the highest expression of the studied genes and carvacrol content was observed in vulgare subspecies at 50 mM salt stress. The gene expression levels may be variable depending on the stress and plant species [69]. The higher expression of these genes in retort to moderate salinity stress may reverberate the elevation of phenolic monoterpenes such as carvacrol. Similarly, the higher expression of biosynthesis genes in response to abiotic elicitors has been associated with the increment of the corresponding metabolites in plants such as Tanacetum parthenium (L.) Sch. Bip. [70] and Nigella sativa L. [71]. On the contrary, despite the higher expression of DXR, Ovtps2 and CYP71D180 genes at 50 mM salinity, the content of thymol decreased. In previous studies, high transcription levels and high carvacrol production in thyme and oregano were correlated with genes encoding CYP71D180 and CYP71D181 [72]. Therefore, the reduction of thymol can be attributed to CYP71D. In the present study, severe salinity stress reduced the expression of CYP71D180 in subsp. vulgare, which was consistent with the trend of carvacrol changes. It can be concluded that salinity stress probably reduces the amount of carvacrol in vulgare subspecies through reducing the expression of CYP71D180. However, in plants treated with sever salinity concentrations, despite a decline in the expression of CYP71D180, the biosynthesis of thymol (as a carvacrol isomer) increased, indicating that, other CYP450 homologues are likely involved in increasing thymol production. Noteworthy, 11 sequences of CYP450 gene have been isolated from oregano and thyme by Crocoll et al. [25]. Previous studies have shown that, there is a significant relation between the activity of CYP450 family enzymes and the production of monoterpenes such as carvacrol and thymol in oregano [30, 31]. In the formation of thymol and carvacrol from γ-terpinene, the aromatic hydrocarbon p-cymene has been proposed as an intermediary [73], however, its participation and the nature of the enzymes involved in the formation of the aromatic ring are still unknown [72]. In this study, the trend of Ovtps2 changes was consistent with p-cymene at different salinity levels.

Also, a positive relationship was observed between the relative expressions of the studied genes with carvacrol in subsp. vulgare and inversely, a negative relationship was obtained with thymol production. In addition, a negative relationship was observed between γ-terpinene and carvacrol in vulgare subspecies. Similarly, Morshedloo et al. [31] stated a negative correlation between γ-terpinene and carvacrol in O. vulgare subsp. gracile under drought stress. Also, a negative relationship between carvacrol and thymol in this subspecies was found. Hence, it can be concluded that γ-terpinene is a precursor for carvacrol. On the other hand, carvacrol is an isomer of thymol and they can be converted to each other. In gracile subspecies, a positive correlation was observed between the relative expression of DXR and Ovtps2 with carvacrol, but the correlation between CYP71D180 and carvacrol was negative. Presumably, the negative association between carvacrol and CYP71D180 may be due to the role of other enzymes of cytochrome family in this pathway [31]. It has been demonstrated that Ovtps2, as the main terpene synthase, produces the half of the total terpenes content [30]. In this study, a direct relationship was obtained between the expression of Ovtps2 and thymol synthesis in subsp. gracile. The findings of this investigation are in line with Crocoll et al. [30] who reported that there is a positive correlation between Ovtps2 gene expression and γ-terpinene and thymol production in O. vulgare.

Conclusions

In overall, the result revealed that the essential oil content increased up to 25 mM NaCl and then decreased. Also, gracile subspecies had a higher essential oil content than vulgare subspecies. No significant difference was found between NaCl treatments (0, 25 and 50 mM) in terms of essential oil yield in both subspecies. Carvacrol, as the main component of essential oil, decreased with increasing salinity levels in subsp. gracile but increased in subsp. vulgare. The highest expression of DXR, Ovtps2 and CYP71D180 genes was observed at 50 mM NaCl in subsp. vulgare. A positive relationship was observed between the expression of DXR, Ovtps2 and CYP71D180 with carvacrol content in subsp. vulgare and between the expression of DXR and Ovtps2 with carvacrol content in subsp. gracile. While, a negative association was observed between the expression of DXR, Ovtps2 and CYP71D180 with thymol content in subsp. vulgare. In contrast, the correlation of DXR, Ovtps2 and CYP71D180 with thymol content in subsp. gracile was positive. Therefore, due to the pharmacological properties of carvacrol and its economic value in the food and cosmetics industries, it can be suggested to enhance its production by increasing the expression of DXR, Ovtps2 and CYP71D180 genes under controlled conditions in the future studies. Also, study the expression of salt-inducible genes/transporters in both subspecies and their relationship with genes involved in MEP pathway under salinity conditions, as well a transcriptome analysis using RNA-seq in both subspecies might lead to a comprehensive view regarding the MEP pathway in the studied subspecies and genetically closed genera.

Materials and methods

Plant material and growing conditions

Seeds of two subspecies of Oregano (O. vulgare subsp. vulgare and O. vulgare subsp. gracile) were obtained from the collection of medicinal plants in Department of Horticultural Science, Urmia University (West Azerbaijan province, Iran). The plant samples were identified by Hossien Maroofi (Research Center of Agriculture and Natural Resources of Kurdistan, Sanandaj, Iran). Voucher specimens were deposited at the herbarium in Department of Horticultural Science, Faculty of Agriculture, Urmia University, Iran. The experiment was performed as a factorial in a completely randomized design (CRD) with three replications during 2019–2020. Seeds of two subspecies were planted in plastic pots in research greenhouse of Urmia University. Each pot (diameter: 25 cm and height: 30 cm) was filled with a 3:2 ratio of soil and sand. The physical and chemical characteristics of the soil used in the pots were: pH (8.02), EC (1.27 ds m−1), organic material (0.62%), total nitrogen (0.12%), available P (9.45 ppm), exchangeable K (0.46 meq/100g soil), and texture (sandy loam). The greenhouse temperature was in the range of 20 ± 2 to 28 ± 2°C with 50–60% relative humidity under natural sunlight. After seed germination, the seedlings were thinned and finally 7 plants kept in each pot. The plants were irrigated evenly with ordinary water until reaching the stage of 6–8 leaves. After this stage, they were subjected to salinity stress for 45 days (until the flowering stage). The salinity treatments applied, included four levels of saline irrigation (0, 25, 50 and 100 mM NaCl). To avoid sudden shock from salinity stress, salinity treatments gradually reached the final concentration during the three irrigation stages. At the full flowering stage, 10 fully developed leaves were harvested from each treatment and transferred to a -70 °C freezer to evaluate the relative expression of DXR, Ovtps2 and CYP71D180 genes. Then, the aerial parts of plants were cut from 10 cm above the soil in order to essential oil extraction and analysis.

Essential oil extraction

The aerial parts of oregano were shade dried, and then plant material (20 g) was subjected to hydro-distillation (Clevenger apparatus, 2.5 h) for essential oil extraction. The essential oil content was expressed as volume per dry weight percentage (%v/w). The collected essential oils were dehydrated over anhydrous sodium sulfate and stored in dark sealed vials at low temperature (4°C) till analysis.

GC–MS analysis of plant volatiles

Gas chromatography/mass spectrometry (GC–MS) was used for analysis of essential oil components. An Agilent 7890 gas chromatograph paired with a 5975A mass spectrometer equipped with a HP-5 MS capillary column (5% Phenyl Methylpolysiloxane, 30 m length, 0.25 mm i.d., 0.25 μm film thickness) (Agilent Technologies, Wilmington-DE, USA), was used for GC–MS analysis. The oven temperature program was adjusted for 3 min (at 80°C), then raised at 10°C min−1 to 200°C, kept for 15 min at 200°C. The temperatures applied to the injector, transfer line and ion resource were 240°C, 280°C and 230°C, respectively. The carrier gas used (with a flow rate of 1 mLmin−1 and an electron impact (EI) of 70 eV) was helium. The injector was set in a split mode (split ratio of 1:50) and injection volume was 1.0 μL. Mass spectra were scanned in the range of 40–500 amu. The constituents of essential oil were determined by using the calculated linear retention indices (Wiley 2007; NIST 2005) and mass spectra with those reported in the NIST 05 and Wily 07.

RNA isolation and cDNA synthesis

Total RNA of O. vulgare leaves was extracted using RNX plus™ kit according to the manufacturer's instructions (Sinaclon, Iran). After evaluating the quality and quantity of RNA using 1% agarose gel electrophoresis and nanodrop ND-1000, cDNA was synthesized using Revert Aid™ First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, USA) according to the instructions of the manufacturer (Thermo Scientific, USA). Negative control reactions using reverse transcriptase minus (-RT) and non-template control (NTC), was performed to ensure no genomic DNA contamination and for reagent contamination, respectively.

Real time PCR reactions

The relative expression of the genes was investigated using Real time PCR (Rotor gene Q-Pure Detection-Qiagen) in the treated plants compared with the control. Gene specific primer pairs were selected from previous studies [25, 31]. Real time PCR reactions were carried out by considering three biological replications in the final volume of 12.5 μL using Maxima ® SYBR-Green/ROX qPCR Master mix kit (Thermo Fisher Scientific, USA), according to the manufacturer's instructions. Initial activation of the enzyme was done at 95°C for 10 min in one cycle, followed by 40 cycles including denaturation at 95°C for 10 s, annealing at 58–60°C for 15 s and fluorescence data collection at 72°C for 20 s. The actin gene was used as the reference gene to normalize the data. The relative expression of the studied genes was calculated after obtaining Ct by ΔΔCt method [74].

Statistical analysis

The experiment was performed as a factorial experiment in CRD with three replications. Data obtained were subjected to analysis of variance (ANOVA) followed a comparison of the means using Duncan’s multiple range test at p < 0.05 level using SAS 9.2 software. The relevance between the main constituents of essential oil and gene expression level were estimated using the Pearson’s correlation coefficient by R software.

Availability of data and materials

The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.

References

  1. Azizi A, Yan F, Honermeier B. Herbage yield, essential oil content and composition of three oregano (Origanum vulgare L.) populations as affected by soil moisture regimes and nitrogen supply. Ind Crops Prod. 2009;129(2):554–61.

    Article  Google Scholar 

  2. Peter KV, Babu KN. Introduction to herbs and spices: medicinal uses and sustainable production. In: Peter KV, editor. Handbook of Herbs and Spices (Second Edition). Woodhead Publishing; 2012. p. 1–16.

    Google Scholar 

  3. Zhang XL, Guo YS, Wang CH, Li GQ, Xu JJ, Chung H, Ye WC, Li YL, Wang GC. Phenolic compounds from Origanum vulgare and their antioxidant and antiviral activities. Food Chem. 2014;152:300–6.

    Article  PubMed  CAS  Google Scholar 

  4. Elshafie HS, Armentano MF, Carmosino M, Bufo SA, De Feo V, Camele I. Cytotoxic activity of Origanum vulgare L. on hepatocellular carcinoma cell line HepG2 and evaluation of its biological activity. Molecules. 2017;22(9):1435.

    Article  PubMed  PubMed Central  Google Scholar 

  5. Pezzani R, Vitalini S, Iriti M. Bioactivities of Origanum vulgare L: an update. Phytochem Rev. 2017;16(6):1253–68.

    Article  CAS  Google Scholar 

  6. Yan F, Azizi A, Janke S, Schwarz M, Zeller S, Honermeier B. Antioxidant capacity variation in the oregano (Origanum vulgare L.) collection of the German National Genebank. Ind Crops Prod. 2016;92:19–25.

    Article  Google Scholar 

  7. JafariKhorsand G, Morshedloo MR, Mumivand H, EmamiBistgani Z, Maggi F, Khademi A. Natural diversity in phenolic components and antioxidant properties of oregano (Origanum vulgare L.) accessions, grown under the same conditions. Sci Rep. 2022;12(1):1–9.

    Google Scholar 

  8. Giuliani C, Maggi F, Papa F, Maleci BL. Congruence of phytochemical and morphological profiles along an altitudinal gradient in Origanum vulgare subsp. vulgare from Venetian region (NE Italy). Chem Biodivers. 2013;10(4):569–83.

    Article  PubMed  CAS  Google Scholar 

  9. Lukas B, Schmiderer C, Novak J. Essential oil diversity of European Origanum vulgare L. (Lamiaceae). Phytochem. 2015;119:32–40.

    Article  CAS  Google Scholar 

  10. Emrahi R, Morshedloo MR, Ahmadi H, Javanmard A, Maggi F. Intraspecific divergence in phytochemical characteristics and drought tolerance of two carvacrol-rich Origanum vulgare subspecies: Subsp. hirtum and subsp. gracile. Ind Crops Prod. 2021;168:113557.

    Article  CAS  Google Scholar 

  11. Verma RS, Padalia RC, Chauhan A, Verma RK, Yadav AK, Singh HP. Chemical diversity in Indian oregano (Origanum vulgare L.). Chem Biodivers. 2010;7(8):2054–64.

    Article  PubMed  CAS  Google Scholar 

  12. Pirigharnaei M, Zare S, Heidary R, Khara J, EmamaliSabzi R, Kheiry F. The essential oils compositions of Iranian Oregano (Origanum vulgare L.) populations in field and provenance from Piranshahr district, West Azarbaijan province. Iran Avicenna J Phytomed. 2011;1(2):106–14.

    Google Scholar 

  13. Moradi M, Hassani A, Sefidkon F, Maroofi H. Chemical composition of leaves and flowers essential oil of Origanum vulgare subsp. gracile growing wild in Iran. J Essent Oil-Bear Plants. 2015;18(1):242–7.

    Article  CAS  Google Scholar 

  14. Morshedloo MR, Mumivand H, Craker LE, Maggi F. Chemical composition and antioxidant activity of essential oils in Origanum vulgare subsp. gracile at different phenological stages and plant parts. J Food Process Preserv. 2018;42(2):13516.

    Article  Google Scholar 

  15. Moradi M, Hassani A, Sefidkon F, Maroofi H. Qualitative and quantitative changes in the essential oil of Origanum vulgare L. subsp. gracile as affected by different harvesting times. J Agric Sci Technol. 2021;23(1):179–86.

    Google Scholar 

  16. Höferl M, Buchbauer G, Jirovetz L, Schmidt E, Stoyanova A, Denkova Z, Slavchev A, Geissler M. Correlation of antimicrobial activities of various essential oils and their main aromatic volatile constituents. J Essent Oil Res. 2009;21(5):459–63.

    Article  Google Scholar 

  17. Youdim KA, Damien Dorman H, Deans SG. The antioxidant effectiveness of thyme oil, α-tocopherol and ascorbyl palmitate on evening primrose oil oxidation. J Essent Oil Res. 1999;11(5):643–8.

    Article  CAS  Google Scholar 

  18. Dorman H, Deans SG. Antimicrobial agents from plants: antibacterial activity of plant volatile oils. J Appl Microbiol. 2000;88(2):308–16.

    Article  PubMed  CAS  Google Scholar 

  19. Rodríguez J, Ortuno C, Benedito J, Bon J. Optimization of the antioxidant capacity of thyme (Thymus vulgaris L.) extracts: Management of the drying process. Ind Crops Prod. 2013;46:258–63.

    Article  Google Scholar 

  20. Tholl D. Terpene synthases and the regulation, diversity and biological roles of terpene metabolism. Curr Opin Plant Biol. 2006;9(3):297–304.

    Article  PubMed  CAS  Google Scholar 

  21. Tholl D. Biosynthesis and biological functions of terpenoids in plants. In: Schrader, J, Bohlmann J. editors. Biotechnology of Isoprenoids. Advances in Biochemical Engineering/Biotechnology. Cham: Springer; 2015. p. 63–106.

  22. Wise ML, Savage TJ, Katahira E, Croteau R. Monoterpene synthases from common sage (Salvia officinalis) cDNA isolation, characterization, and functional expression of (+)-sabinene synthase, 1, 8-cineole synthase, and (+)-bornyl diphosphate synthase. J Biol Chem. 1998;273(24):14891–9.

    Article  PubMed  CAS  Google Scholar 

  23. Aslani Z, Hassani A, Abdollahi Mandoulakani B, Barin M, Maleki R. Effect of drought stress and inoculation treatments on nutrient uptake, essential oil and expression of genes related to monoterpenes in sage (Salvia officinalis). Sci Hortic. 2023;309: 111610.

    Article  CAS  Google Scholar 

  24. Wang G, Tian L, Aziz N, Broun P, Dai X, He J, King A, Zhao PX, Dixon RA. Terpene biosynthesis in glandular trichomes of hop. Plant Physiol. 2008;148(3):1254–66.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  25. Crocoll C, Asbach J, Novak J, Gershenzon J, Degenhardt J. Terpene synthases of oregano (Origanum vulgare L.) and their roles in the pathway and regulation of terpene biosynthesis. Plant Mol Biol. 2010;73(6):587–603.

    Article  PubMed  CAS  Google Scholar 

  26. Lima AS, Schimmel J, Lukas B, Novak J, Barroso JG, Figueiredo AC, Pedro LG, Degenhardt J, Trindade H. Genomic characterization, molecular cloning and expression analysis of two terpene synthases from Thymus caespititius (Lamiaceae). Planta. 2013;238(1):191–204.

    Article  PubMed  CAS  Google Scholar 

  27. Verma N, Shukla S. Impact of various factors responsible for fluctuation in plant secondary metabolites. J Appl Res Med Aromat Plants. 2015;2(4):105–13.

    Google Scholar 

  28. Ramak P, Osaloo SK, Ebrahimzadeh H, Sharifi M, Behmanesh M. Inhibition of the mevalonate pathway enhances carvacrol biosynthesis and DXR gene expression in shoot cultures of Satureja khuzistanica Jamzad. Plant Physiol. 2013;170(13):1187–93.

    Article  CAS  Google Scholar 

  29. Grosso C, Figueiredo AC, Burillo J, Mainar AM, Urieta JS, Barroso JG, Coelho JA, Palavra AM. Composition and antioxidant activity of Thymus vulgaris volatiles: comparison between supercritical fluid extraction and hydrodistillation. J Sep Sci. 2010;33(14):2211–8.

    Article  PubMed  CAS  Google Scholar 

  30. Crocoll C. Biosynthesis of the phenolic monoterpenes, thymol and carvacrol, by terpene synthases and cytochrome P450s in oregano and thyme. Germany: Academic Dissertation: der Biologisch-Pharmazeutischen Fakultät der Friedrich-Schiller- Universität Jena; 2011.

  31. Morshedloo MR, Craker LE, Salami A, Nazeri V, Sang H, Maggi F. Effect of prolonged water stress on essential oil content, compositions and gene expression patterns of mono-and sesquiterpene synthesis in two oregano (Origanum vulgare L.) subspecies. Plant Physiol Biochem. 2017;111:119–28.

    Article  PubMed  CAS  Google Scholar 

  32. Jan S, Mir JI, Shafi W, Faktoo SZ, Singh DB, Wijaya L, Alyemeni M, Ahmad P. Divergence in tissue-specific expression patterns of genes associated with the terpeniod biosynthesis in two oregano species Origanum vulgare L., and Origanum majorana. Ind Crops Prod. 2018;123:546–55.

    Article  CAS  Google Scholar 

  33. Arif Y, Singh P, Siddiqui H, Bajguz A, Hayat S. Salinity induced physiological and biochemical changes in plants: An omic approach towards salt stress tolerance. Plant Physiol Biochem. 2020;156:64–77.

    Article  PubMed  CAS  Google Scholar 

  34. Munns R, Tester M. Mechanisms of salinity tolerance. Annu Rev Plant Biol. 2008;59:651–81.

    Article  PubMed  CAS  Google Scholar 

  35. Acosta-Motos JR, Ortuño MF, Bernal-Vicente A, Diaz-Vivancos P, Sanchez-Blanco MJ, Hernandez JA. Plant responses to salt stress: adaptive mechanisms. Agronomy. 2017;7:18.

    Article  Google Scholar 

  36. Parida AK, Das AB. Salt tolerance and salinity effects on plants: a review. Ecotoxicol Environ Saf. 2005;60(3):324–49.

    Article  PubMed  CAS  Google Scholar 

  37. Isayenkov SV, Maathuis FJ. Plant salinity stress: many unanswered questions remain. Front Plant Sci. 2019;10:80.

    Article  PubMed  PubMed Central  Google Scholar 

  38. Bistgani ZE, Hashemi M, DaCosta M, Craker L, Maggi F, Morshedloo MR. Effect of salinity stress on the physiological characteristics, phenolic compounds and antioxidant activity of Thymus vulgaris L. and Thymus daenensis Celak. Ind Crops Prod. 2019;135:311–20.

    Article  CAS  Google Scholar 

  39. El Ghazali GEB. Structural characteristics and adaptations to salinity and drought: A review. Int J Sci. 2020;9(2):28–33.

    Google Scholar 

  40. de Freitas PAF, de Carvalho HH, Costa JH, de Souza MR, de Cruz Saraiva KD, de Oliveira FDB, Gomes-Filho E. Salt acclimation in sorghum plants by exogenous proline: physiological and biochemical changes and regulation of proline metabolism. Plant Cell Rep. 2019;38(3):403–16.

    Article  PubMed  Google Scholar 

  41. Kliebenstein DJ. Making new molecules evolution of structures for novel metabolites in plants. Curr Opin Plant Biol. 2013;16(1):112–7.

    Article  PubMed  CAS  Google Scholar 

  42. Zandalinas SI, Mittler R, Balfagón D, Arbona V, Gómez-Cadenas A. Plant adaptations to the combination of drought and high temperatures. Physiol Plant. 2018;162(1):2–12.

    Article  PubMed  CAS  Google Scholar 

  43. Ksouri R, Megdiche W, Debez A, Falleh H, Grignon C, Abdelly C. Salinity effects on polyphenol content and antioxidant activities in leaves of the halophyte Cakile maritima. Plant Physiol Biochem. 2007;45(3):244–9.

    Article  PubMed  CAS  Google Scholar 

  44. Debnath M, Pandey M, Bisen P. An omics approach to understand the plant abiotic stress. Omics J Integr Biol. 2011;15(11):739–62.

    Article  CAS  Google Scholar 

  45. Tounekti T, Khemira H. NaCl stress-induced changes in the essential oil quality and abietane diterpene yield and composition in common sage. J Intercult Ethnopharmacol. 2015;4(3):208.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  46. Najafi F, Khavari-Nejad R. The effects of salt stress on certain physiological parameters in summer savory (Satureja hortensis L.) plants. J Stress Physiol Biochem. 2010;6(1):13–21.

    Google Scholar 

  47. Bonacina C, Trevizan CB, Stracieri J, dos Santos TB, Gonçalves JE, Gazim ZC, de Souza SGH. Changes in growth, oxidative metabolism and essential oil composition of lemon balm (Melissa officinalis L.) subjected to salt stress. Aust J Crop Sci. 2017;11(12):1665–74.

    Article  CAS  Google Scholar 

  48. Kulak M, Gul F, Sekeroglu N. Changes in growth parameter and essential oil composition of sage (Salvia officinalis L.) leaves in response to various salt stresses. Ind Crops Prod. 2020;145:112078.

    Article  CAS  Google Scholar 

  49. AbdollahiMandoulakani B, Eyvazpour E, Ghadimzadeh M. The effect of drought stress on the expression of key genes involved in the biosynthesis of phenylpropanoids and essential oil components in basil (Ocimum basilicum L.). Phytochem. 2017;139:1–7.

    Article  CAS  Google Scholar 

  50. Majroomi Senji B, Abdollahi MB. The impact of cold stress on genes expression pattern of mono-and sesquiterpene biosynthesis and their contents in Ocimum basilicum L. Phytochem. 2018;156:250–6.

    Article  CAS  Google Scholar 

  51. Hancioglu NE, Kurunc A, Tontul I, Topuz A. Irrigation water salinity effects on oregano (Origanum onites L.) water use, yield and quality parameters. Sci Hortic. 2019;247:327–34.

    Article  CAS  Google Scholar 

  52. Muráriková A, Ťažký A, Neugebauerová J, Planková A, Jampílek J, Mučaji P, Mikuš P. Characterization of essential oil composition in different basil species and pot cultures by a GC-MS method. Molecules. 2017;22(7):1221–32.

    Article  PubMed  PubMed Central  Google Scholar 

  53. Taarit MB, Msaada K, Hosni K, Marzouk B. Changes in fatty acid and essential oil composition of sage (Salvia officinalis L.) leaves under NaCl stress. Food Chem. 2010;119(3):951–6.

    Article  Google Scholar 

  54. Sarmoum R, Haid S, Biche M, Djazouli Z, Zebib B, Merah O. Effect of salinity and water stress on the essential oil components of rosemary (Rosmarinus officinalis L.). Agronomy. 2019;9(5):214.

    Article  CAS  Google Scholar 

  55. Farsaraei S, Moghaddam M, Pirbalouti AG. Changes in growth and essential oil composition of sweet basil in response of salinity stress and superabsorbents application. Sci Hortic. 2020;271: 109465.

    Article  CAS  Google Scholar 

  56. Taarit MB, Msaada K, Hosni K, Hammami M, Kchouk ME, Marzouk B. Plant growth, essential oil yield and composition of sage (Salvia officinalis L.) fruits cultivated under salt stress conditions. Ind Crops and Prod. 2009;30(3):333–7.

    Article  Google Scholar 

  57. Baâtour O, Kaddour R, Mahmoudi H, Tarchoun I, Bettaieb I, Nasri N, Mrah S, Hamdaoui G, Lachaâl M, Marzouk B. Salt effects on Origanum majorana fatty acid and essential oil composition. J Sci Food Agric. 2011;91(14):2613–20.

    Article  PubMed  Google Scholar 

  58. Çoban Ö, Baydar NG. Brassinosteroid effects on some physical and biochemical properties and secondary metabolite accumulation in peppermint (Mentha piperita L.) under salt stress. Ind Crops Prod. 2016;86:251–8.

    Article  Google Scholar 

  59. Ashraf M, Noman A. Influence of applied nitrogen on growth and tissue nutrient concentration in the medicinal plant Ajowain (Trachyspermum ammi). Aust J Exp Agric. 2006;46(3):425–8.

    Article  CAS  Google Scholar 

  60. Ghanavati M, Kazemi K, Ghaderian R. The effects of salt stress on yield and some components in Chamomile genotypes during growth stage. Planta Med. 2010;76(12):P021.

    Article  Google Scholar 

  61. Mechergui K, Jaouadi W, Coelho JP, Khouja ML. Effect of harvest year on production, chemical composition and antioxidant activities of essential oil of oregano (Origanum vulgare subsp glandulosum (Desf.) Ietswaart) growing in North Africa. Ind Crops Prod. 2016;90:32–7.

    Article  CAS  Google Scholar 

  62. Ekren S, Sönmez Ç, Özçakal E, Kurttaş YSK, Bayram E, Gürgülü H. The effect of different irrigation water levels on yield and quality characteristics of purple basil (Ocimum basilicum L.). Agric Water Manag. 2012;109:155–61.

    Article  Google Scholar 

  63. Aziz EE, Sabry RM, Ahmed SS. Plant growth and essential oil production of sage (Salvia officinalis L.) and curly-leafed parsley (Petroselinum crispum subsp. crispum L.) cultivated under salt stress conditions. World Appl Sci J. 2013;28(6):785–96.

    Google Scholar 

  64. Valifard M, Mohsenzadeh S, Kholdebarin B, Rowshan V. Effects of salt stress on volatile compounds, total phenolic content and antioxidant activities of Salvia mirzayanii. S Afr J Bot. 2014;93:92–7.

    Article  CAS  Google Scholar 

  65. Pavela R, Morshedloo MR, Mumivand H, Khorsand GJ, Karami A, Maggi F, Desneux N, Benelli G. Phenolic monoterpene-rich essential oils from Apiaceae and Lamiaceae species: insecticidal activity and safety evaluation on non-target earthworms. Entomol Gen. 2020;40(4):421–35.

  66. Ryšlavá H, Doubnerová V, Kavan D, Vaněk O. Effect of posttranslational modifications on enzyme function and assembly. Proteomics. 2013;92:80–109.

    Article  PubMed  Google Scholar 

  67. Blom N, Sicheritz-Pontén T, Gupta R, Gammeltoft S, Brunak S. Prediction of post-translational glycosylation and phosphorylation of proteins from the amino acid sequence. Proteomics. 2004;4(6):1633–49.

    Article  PubMed  CAS  Google Scholar 

  68. Yin X, Komatsu S. Quantitative proteomics of nuclear phosphoproteins in the root tip of soybean during the initial stages of flooding stress. Proteomics. 2015;119:183–95.

    Article  PubMed  CAS  Google Scholar 

  69. Kianersi F, Amin Azarm D, Fatemi F, Pour-Aboughadareh A, Poczai P. Methyl jasmonate induces genes involved in linalool accumulation and increases the content of phenolics in two Iranian coriander (Coriandrum sativum L.s) ecotypes. Genes. 2022;13(10):1717.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  70. Majdi M, Abdollahi MR, Maroufi A. Parthenolide accumulation and expression of genes related to parthenolide biosynthesis affected by exogenous application of methyl jasmonate and salicylic acid in Tanacetum parthenium. Plant Cell Rep. 2015;34:1909–18.

    Article  PubMed  CAS  Google Scholar 

  71. Elyasi R, Majdi M, Bahramnejad B, Mirzaghaderi G. Spatial modulation and abiotic elicitors responses of the biosynthesis related genes of mono/triterpenes in black cumin (Nigella sativa). Ind Crops Prod. 2016;79:240–7.

    Article  CAS  Google Scholar 

  72. Krause ST, Liao P, Crocoll C, Boachon B, Förster C, Leidecker F, Degenhardt J. The biosynthesis of thymol, carvacrol, and thymohydroquinone in Lamiaceae proceeds via cytochrome P450s and a short-chain dehydrogenase. Proc Natl Acad Sci. 2021;118(52): e2110092118.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  73. Poulose AJ, Croteau R. Biosynthesis of aromatic monoterpenes: Conversion of γ-terpinene to p-cymene and thymol in Thymus vulgaris L. Arch Biochem Biophys. 1978;187(2):307–14.

    Article  PubMed  CAS  Google Scholar 

  74. Pfaffl MW, Tichopad A, Prgomet C, Neuvians TP. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper–Excel-based tool using pair-wise correlations. Biotechnol Lett. 2004;26(6):509–15.

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

We thank Dr. Ramin Maleki, Research Department of Chromatography, Iranian Academic Center for Education, Culture, and Research (ACECR), Urmia, Iran, for performing GC-MS analyses.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Author information

Authors and Affiliations

  1. Department of Horticultural Science, Faculty of Agriculture, Urmia University, Urmia, Iran

    Zahra Azimzadeh & Abbas Hassani

  2. Department of Plant Production and Genetics, Faculty of Agriculture, Urmia University, Urmia, Iran

    Babak Abdollahi Mandoulakani

  3. Department of Soil Science, Faculty of Agriculture, Urmia University, Urmia, Iran

    Ebrahim Sepehr

  4. Department of Horticultural Science, Faculty of Agriculture, University of Maragheh, Maragheh, Iran

    Mohammad Reza Morshedloo

Authors
  1. Zahra Azimzadeh
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Abbas Hassani
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Babak Abdollahi Mandoulakani
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ebrahim Sepehr
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Mohammad Reza Morshedloo
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

AH and BAM, designed and supervised the study; ZA, carried out the experiment; ZA, AH, BAM and ES, Collected of data and laboratory analysis; ZA and BAM, analyzed the data; AH, BAM, ES and MRM, interpreted the data; AH, ZA and BAM, wrote the manuscript. All authors have read and approved the manuscript.

Corresponding author

Correspondence to Abbas Hassani.

Ethics declarations

Ethics approval and consent to participate

The seeds of Oregano subspecies were obtained from the collection of medicinal plants in Department of Horticultural Science, Urmia University, Iran, for doing the present study. All methods were performed in accordance with the relevant guidelines and regulations.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Azimzadeh, Z., Hassani, A., Mandoulakani, B.A. et al. Intraspecific divergence in essential oil content, composition and genes expression patterns of monoterpene synthesis in Origanum vulgare subsp. vulgare and subsp. gracile under salinity stress. BMC Plant Biol 23, 380 (2023). https://doi.org/10.1186/s12870-023-04387-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s12870-023-04387-5

Keywords

BMC Plant Biology

ISSN: 1471-2229

Contact us