The Influence of Methyl Jasmonate on Expression Patterns of Rosmarinic Acid Biosynthesis Genes, and Phenolic Compounds in Different Species of Salvia subg. Perovskia Kar L.

Salvia yangii B.T. Drew and Salvia abrotanoides Kar are two important fragrant and medicinal plants that belong to the subgenus Perovskia. These plants have therapeutic benefits due to their high rosmarinic acid (RA) content. However, the molecular mechanisms behind RA generation in two species of Salvia plants are still poorly understood. As a first report, the objectives of the present research were to determine the effects of methyl jasmonate (MeJA) on the rosmarinic acid (RA), total flavonoid and phenolic contents (TFC and TPC), and changes in the expression of key genes involved in their biosynthesis (phenylalanine ammonia lyase (PAL), 4-coumarate-CoA ligase (4CL), and rosmarinic acid synthase (RAS)). The results of High-performance liquid chromatography (HPLC) analysis indicated that MeJA significantly increased RA content in S. yungii and S. abrotanoides species (to 82 and 67 mg/g DW, respectively) by 1.66- and 1.54-fold compared with untreated plants. After 24 h, leaves of Salvia yangii and Salvia abrotanoides species treated with 150 M MeJA had the greatest TPC and TFC (80 and 42 mg TAE/g DW, and 28.11 and 15.14 mg QUE/g DW, respectively), which was in line with the patterns of gene expression investigated. Our findings showed that MeJA dosages considerably enhanced the RA, TPC, and TFC contents in both species compared with the control treatment. Since increased numbers of transcripts for PAL, 4CL, and RAS were also detected, the effects of MeJA are probably caused by the activation of genes involved in the phenylpropanoid pathway.


Introduction
Medicinal and aromatic plants are used in pharmaceuticals, cosmetics, personal care items, incense, and food to treat diseases, maintain health, and prevent them [1]. These plants are becoming more popular as public interest in natural resources increases daily [2,3].
One of the most significant genera in the Lamiaceae family, Salvia (garden sage), has around 1000 species worldwide [4]. Numerous Salvia species are used to make herbal tea, as well as in cosmetics, fragrances, and medicines [5]. The biological properties of plants in antidiabetic, and anxiolytic characteristics, further set them apart [6][7][8][9].
Several perennial, shrubby, and fragrant species with possible medical found in the subgenus Perovskia Kar of the Salvia expanded genus [10]. Salv Drew (formerly Perovskia atriplicifolia Benth.) and Salvia abrotanoides Kar known as Perovskia abrotanoides Kar) are the two most common among them, in extensive regions including the western regions of Iran, Pakistan, Tibet, in China [11][12][13][14].
Both S. abrotanoides and S. yangii are used as traditional medicines in where they grow naturally. Diabetes, diarrhea, scabies, fever, wound h antibiotic therapy are among the conditions that S. yangii is used to treat [15-has been described as a potent analgesic and parasiticide in traditional Chinese medicine [20,21]. Locally, S. abrotanoides, also known as "Brazambol parlance, is used as a sedative, analgesic, and antiseptic as well as a therapy f typhoid, fever, headache, cardiovascular disorders, gonorrhoea, vomiting, l painful urination, and cough [22][23][24][25]. Moreover, S. abrotanoides was show cytotoxic, anti-plasmodial, and anti-inflammatory pharmacological activities have been employed in Iranian traditional medicine to cure leishmaniasis [26 Rosmarinic acid (RA) has been found to be the main phenylpropanoid p medicinal plant Slavia [28]. This molecule has many roles, such as antio inflammatory, and antibacterial activities [29][30][31][32]. The main active phenolic RA, chemically, is an ester of 3,4-dihydroxyphenyllactic acid and caffeic acid is a substance that occurs naturally and is present in many medicinal plant found to be an active component in a number of Lamiaceae-related medicinal may also be found in plants from other floras [34,35]. The RA biosynthesis pathway genes in two species of Salvia subgenus P yangii and S. abrotanoides) treated with MeJA have not yet been assessed, de that their expression has been investigated in a range of plant species. Pro accumulation of these molecules are influenced by various time scales, de phases, and responses to biotic and abiotic stimuli [36][37][38][39]. Elicitors are important factors that trigger plant defense mechanisms and cause the accu certain secondary metabolites [40][41][42]. Methyl jasmonate (MeJA) as a signal is essential for the signal transduction pathway. Exogenous application o abiotic elicitor in plants is employed to encourage the production of the desire compounds [43,44]. Several medicinal plants, such as Salvia miltiorrhiza Bunge The RA biosynthesis pathway genes in two species of Salvia subgenus Perovskia (S. yangii and S. abrotanoides) treated with MeJA have not yet been assessed, despite the fact that their expression has been investigated in a range of plant species. Production and accumulation of these molecules are influenced by various time scales, developmental phases, and responses to biotic and abiotic stimuli [36][37][38][39]. Elicitors are one of the important factors that trigger plant defense mechanisms and cause the accumulation of certain secondary metabolites [40][41][42]. Methyl jasmonate (MeJA) as a signaling molecule is essential for the signal transduction pathway. Exogenous application of this potent abiotic elicitor in plants is employed to encourage the production of the desired secondary compounds [43,44]. Several medicinal plants, such as Salvia miltiorrhiza Bunge [45], Satureja khuzistanica Jamzad [46], Capparis spinosa L. [47,48], Thymus migricus Klokov and Des.-Shost [49], Melissa officinalis L. [50], Foeniculum vulgare Mill. [51], and Coriandrum sativum L. [52], have been the subject of extensive MeJA research into the regulation of secondary metabolism. Numerous studies [53][54][55] have shown that MeJA has a positive influence on RA levels and the expression levels of related essential genes in plants.
Despite the fact that many studies have looked into the effects of elicitors, such as MeJA, on the formation of phenolic acids, we are unaware of any research that has looked into RA and gene expression patterns in RA-related genes in S. yangii and S. abrotanoides species. Hence, the main objective of the present study was to investigate changes in RArelated gene expression (PAL, 4CL, and RAS), phenolic compound accumulation, and RA concentration in two species of Salvia (S. yangii and S. abrotanoides) in response to the MeJA treatment. Furthermore, we looked for an association between the expression patterns of genes involved in the RA biosynthesis pathway and the RA content in the leaves in two species of Salvia during the vegetative growth stage after the MeJA treatment.

RA Changes at Different MeJA Concentrations
As shown in Figure 2, the different MeJA treatments significantly affected RA, a key component of two Salvia species. Additionally, Figure 2 illustrates how, after 24 h, the RA content in both treated Salvia species dramatically increased under the MeJA elicitor. In general, RA accumulation was greater in the S. yangii than the S. abrotanoides at all MeJA concentrations. In the S. yangii species, the concentration of RA increased by 1.24, 1.38, 1.66, and 1.54 times greater than in untreated plants when they were exposed to 10, 100, 150, and 200 µM MeJA, respectively. The RA quantity in the S. abrotanoides species also increased in accordance with the application of various MeJA treatments and was 1.19, 1.23, 1.54, and 1.35 times more than their control ( Figure 2). According to the results, the RA values of two Salvia species were different, which indicates that RA production is affected by genotype-specific responses to stimuli in abiotic stresses.
metabolism. Numerous studies [53][54][55] have shown that MeJA has a positive RA levels and the expression levels of related essential genes in plants.
Despite the fact that many studies have looked into the effects of elici MeJA, on the formation of phenolic acids, we are unaware of any research th into RA and gene expression patterns in RA-related genes in S. yangii and S species. Hence, the main objective of the present study was to investigate ch related gene expression (PAL, 4CL, and RAS), phenolic compound accumula concentration in two species of Salvia (S. yangii and S. abrotanoides) in response treatment. Furthermore, we looked for an association between the expressio genes involved in the RA biosynthesis pathway and the RA content in the l species of Salvia during the vegetative growth stage after the MeJA treatmen

RA Changes at Different MeJA Concentrations
As shown in Figure 2, the different MeJA treatments significantly affect component of two Salvia species. Additionally, Figure 2 illustrates how, after content in both treated Salvia species dramatically increased under the MeJ general, RA accumulation was greater in the S. yangii than the S. abrotanoide concentrations. In the S. yangii species, the concentration of RA increased 1.66, and 1.54 times greater than in untreated plants when they were expose 150, and 200 μM MeJA, respectively. The RA quantity in the S. abrotanoides increased in accordance with the application of various MeJA treatments a 1.23, 1.54, and 1.35 times more than their control ( Figure 2). According to th RA values of two Salvia species were different, which indicates that RA p affected by genotype-specific responses to stimuli in abiotic stresses. Our results imply that MeJA treatments may increase the amount of R Salvia species, which in turn has an effect on the gene expression patte implicated in RA synthesis. On the other hand, our findings support tho research [47][48][49]56] and suggest that Salvia plants' RA levels vary depend genotype and dose. It has been reported that the treatments with 100-150 μM in the accumulation of high levels of RA in Lamiaceae families [50,57]. Moreo caper plants with 150 μM MeJA might stimulate the production of flavon Our results imply that MeJA treatments may increase the amount of RA in various Salvia species, which in turn has an effect on the gene expression patterns of genes implicated in RA synthesis. On the other hand, our findings support those of earlier research [47][48][49]56] and suggest that Salvia plants' RA levels vary depending on their genotype and dose. It has been reported that the treatments with 100-150 µM MeJA result in the accumulation of high levels of RA in Lamiaceae families [50,57]. Moreover, treating caper plants with 150 µM MeJA might stimulate the production of flavonoids [20,58]. According to some studies, MeJA stimulates the PAL enzyme's activity, which may contribute to the growth of RA in diverse plants [46,54]. In fact, these results imply that MeJA could affect the increase in RA amount under 100-to 150-M therapy [50]. According to our findings, both ecotypes' metabolic characteristics were noticeably impacted by the MeJA that was delivered. Moreover, the increase in RA accumulation through the MeJA may be due to the stimulation of biosynthetic pathways and the activation of related genes to induce radical scavenging through phenolic components. Both species saw a rise in RA after MeJA, although the S. yangii species experienced this increase more quickly.
Previous studies corroborate our findings about the effect of MeJA at varying doses on RA buildup and gene expression in Salvia spp. Satureja khuzistanica nodal cultures, Agastache rugosa Kuntze cell cultures, and Coleus forskohlii hairy root cultures all showed that MeJA is the most efficient elicitor for the development of RA, supporting this concept [57][58][59]. Park et al. [60] reported that both abiotic and biotic elicitors stimulated RA production in A. rugosa callus suspension cultures. A previous study conducted by Kintzios et al. [61] showed that cell suspension cultures increased RA at a rate of up to 10 mg/g dry weight in Ocimum basilicum. After MeJA treatment, Lithospermum erythrorhizon cells showed a 10-fold increase in RA content, which was in accordance with Mizukami et al. [62].

TPC and TFC as a Function of MeJA Concentration
The TPC and TFC levels of two Salvia sub-genus Perovskia Kar leaves are obviously impacted by all concentrations of MeJA ( Figure 3). It is probable that the pattern of RA content changes during the MeJA treatment was reflected in the pattern of TPC and TFC changes in both S. yangii and S. abrotanoides species (Figures 2 and 3A,B). Additionally, as shown by our findings, MeJA had a distinct degree of influence on phenol and flavonoid accumulation in the S. yangii species as compared to the S. abrotanoides species. In accordance with previous studies, our results revealed that MeJA as a signaling molecule plays a key role in flavonoid formation [63,64]. Hence, these genes' elevated expression in response to MeJA is almost certainly a result of the subsequent rise in phenolic compounds.
Concentrations of 10, 100, 150, and 200 µM MeJA raised the TPC in S. yangii by 1.14, 1.27, 1.53, and 1.41 times, respectively, compared to the untreated plants ( Figure 3A). Accordingly, TPC values for the S. abrotanoides treated with the various doses of MeJA were tested and were, correspondingly, 1.21, 1.5, 2.02, and 1.7 times higher than the controls ( Figure 3A). Moreover, S. yangii species treated with different doses of MeJA produced TFCs of up to 12.68, 17, 27.52, and 20.59 mg QUE/g DW, which were 1.30, 1.74, 2.82, and 2.11 times higher than the control plants, respectively ( Figure 3B). TFC levels in the S. abrotanoides species were increased by MeJA treatments; these increases were about 1.27, 1.53, 2.07, and 1.64 times greater than the levels in untreated plants ( Figure 3B). These results corroborate those of another study [65], which discovered species-specific differences in Salvia leaf TPC and TFC. In both ecotypes, TPC values were higher than TFC in both untreated and all treated leaves, supporting previous findings [66]. Recent investigations [67][68][69] agree with ours in showing that MeJA stimuli greatly influenced TPC in several plants. Researchers found that MeJA increased secondary metabolite accumulation in various plant species [70][71][72].
Higher quantities of polyphenolic compounds may come from the more rapid breakdown of larger phenolic compounds into smaller molecules, as shown by the research of Jaafar et al. [73]. The major regulating enzyme in phenylpropanoid metabolism, PAL, has been linked to the induction of TPC and TFC in two species of the subgenus Perovskia after treatment with MeJA [71]. Many plants' expression of the phenylpropanoid biosynthesis genes (PAL, C4H, and 4CL) is inversely proportional to their flavonoid concentration [74,75]. Hence, the increased synthesis of RA and phenolic compounds after MeJA treatments was consistent with the expression of genes involved in the RA biosynthetic pathway. Genes 2023, 14, x FOR PEER REVIEW 5 of 14 Concentrations of 10, 100, 150, and 200 μM MeJA raised the TPC in S. yangii by 1.14, 1.27, 1.53, and 1.41 times, respectively, compared to the untreated plants ( Figure 3A). Accordingly, TPC values for the S. abrotanoides treated with the various doses of MeJA were tested and were, correspondingly, 1.21, 1.5, 2.02, and 1.7 times higher than the controls ( Figure 3A). Moreover, S. yangii species treated with different doses of MeJA produced TFCs of up to 12.68, 17, 27.52, and 20.59 mg QUE/g DW, which were 1.30, 1.74, 2.82, and 2.11 times higher than the control plants, respectively ( Figure 3B). TFC levels in the S. abrotanoides species were increased by MeJA treatments; these increases were about 1.27, 1.53, 2.07, and 1.64 times greater than the levels in untreated plants ( Figure 3B). These results corroborate those of another study [65], which discovered species-specific differences in Salvia leaf TPC and TFC. In both ecotypes, TPC values were higher than TFC in both untreated and all treated leaves, supporting previous findings [66]. Recent investigations [67][68][69] agree with ours in showing that MeJA stimuli greatly influenced TPC in several plants. Researchers found that MeJA increased secondary metabolite accumulation in various plant species [70][71][72].
Higher quantities of polyphenolic compounds may come from the more rapid breakdown of larger phenolic compounds into smaller molecules, as shown by the research of Jaafar et al. [73]. The major regulating enzyme in phenylpropanoid

MeJA's Effects on the Expression of the PAL, 4CL, and RAS Genes
Changes in the transcript abundance of genes involved in the process that creates RA were investigated using real-time PCR, and the relationship between RA accumulation and gene expression in two species of Salvia that were subjected to varying dosages of MeJA was also analyzed (Figure 4). Results show that three genes investigated in Salvia plants had significantly different mRNA transcript levels in response to different MeJA treatments. It is important to note that MeJA-treated S. yangii had higher transcriptional levels of RA biosynthetic genes than MeJA-treated S. abrotanoides. Possible explanation for increased RA production in MeJA-treated plants, especially at the beginning and end of the RA biosynthetic pathway and during the expression of PAL and RAS.
Our results showed that the expression level of PAL in the S. yangii species enhanced from 8.42-fold at MeJA 10 M to 11.83-fold at MeJA 150 µM and remained almost the same at 10.67-fold at MeJA 200 µM ( Figure 4A). At MeJA 10 M and 100 µM, the transcript level of 4CL significantly increased in comparison to untreated plants (7-and 7.8-fold, respectively), progressively increasing to 8.31-fold at MeJA 150 µM, and subsequently decreasing to 6.49-fold ( Figure 4B). RAS expression also increased significantly to 12-fold at MeJA 100 µM, peaked quickly at 17.1-fold with MeJA 150 µM, and then sharply decreased to 14.38-fold at MeJA 200 µM ( Figure 4C). and gene expression in two species of Salvia that were subjected to varying dosages of MeJA was also analyzed (Figure 4). Results show that three genes investigated in Salvia plants had significantly different mRNA transcript levels in response to different MeJA treatments. It is important to note that MeJA-treated S. yangii had higher transcriptional levels of RA biosynthetic genes than MeJA-treated S. abrotanoides. Possible explanation for increased RA production in MeJA-treated plants, especially at the beginning and end of the RA biosynthetic pathway and during the expression of PAL and RAS.  As MeJA 10 µM was applied to the S. abrotanoides plants, the expression level of PAL greatly increased (6.37-fold) when compared to the control plant, but there was no discernible difference among MeJA treatments. MeJA 150 µM significantly increased PAL expression, which was 8.72-fold greater than in control plants ( Figure 4A). After 24 h of treatment with MeJA 10 µM, 4CL significantly increased (5.51-fold), and with MeJA 100 µM, it increased even more, to 6.36-fold. At MeJA 150 and 200 µM, 4CL expression started to decline after reaching its greatest level at MeJA 100 µM (6.36-fold), although it was still 4.51and 4.37-fold greater than the level before treatment ( Figure 4B (Figure 4). The MeJA treatment increased the expression of the PAL, 4 CL, and RAS genes so that their expression patterns were in accordance with the pattern of RA accumulation. Furthermore, the current study showed that spraying subgenus Perovskia species with high MeJA concentrations (200 µM) resulted in a decrease in the examined gene expression. These results are consistent with those found by Kianersi et al. [20][21][22]50], who found that large quantities of exogenously given MeJA suppressed expression. Examining the impact of external stimuli on the synthesis of secondary metabolites is useful for determining the highest performance of secondary metabolites and for clarifying their biosynthetic pathway(s) [76][77][78][79][80].
Both species showed similar patterns of PAL upregulation in response to MeJA therapy that paralleled those of RA accumulation. Likewise, a clear tread-off was observed between over-expression of the RA production-involved genes in several organs of Ocimum basilicum cultivars, lemon balm ecotypes, and Agastache rugosa [29,50,81]. Since PAL activity is rising in species belonging to the Lamiaceae family before RA accumulation [36,82], it has been hypothesized that it is a key enzyme for entrance into the phenylpropanoid pathway. Our research showed that PAL is essential for the synthesis of RA. Stress and plant species may affect the speed with which transcript levels rise and genes are induced. The expression profile of PAL in Salvia miltiorrhiza was analyzed, and it was shown to be upregulated in response to a wide variety of treatments [83,84]. Researchers have shown that activating MeJA increases PAL activity, a key enzyme in the phenylpro-panoid pathway [85,86]. Although our results unambiguously demonstrate the importance of PAL and RAS expression in RA generation in both species, the observed discrepancies in PAL and RAS expression as well as RA accumulation quantities are likely species-specific to the genus Salvia.
The strong connection between RA biosynthesis and RAS expression was highlighted by our findings. In both Salvia species treated with MeJA, there was a correlation between the pattern of RAS expression and the development of RA (Figures 2 and 4). After treatment with MeJA 150 µM, RAS expression was 17.1 and 10.87 times higher in S. yangii and S. abrotanoides, respectively, compared to control plants. Similarly, the RA levels at this concentration were 1.66 times higher in S. yangii than in usual plants and 1.54 times higher in S. abrotanoides. The RAS in the studied species was reduced to 200 µM MeJA compared to 150 µM MeJA, and this was followed by a decrease in the synthesis of RA, phenol, and flavonoids. This gene may play a crucial role in regulating RA production because of the correlation between RAS expression and RA levels.
In general, our results were consistent with those of Kim et al. [55], who demonstrated that MeJA treatment enhanced the expression of the phenylpropanoid biosynthesis-related genes ArPAL, Ar4CL, and ArC4H in A. rugosa, ultimately leading to higher levels of RA accumulation. Plants exhibited rapid induction of PAL activity in response to MeJA elicitation, as reported by Mizukami et al. [62]. MeJA increases bioactive substance accumulation and changes the mRNA expression of genes involved in secondary metabolite manufacture in several plant species, as proven by numerous studies [46][47][48]76,77,87]. Similarly, Belhadj et al. [88] showed a rapid upregulation of the PAL gene expression in grape leaves using the MeJA treatment. Moreover, MeJA can alter CAD, PAL, and PPO activities as well as their relative mRNA levels [89].
Based on our results, MeJA at 150 µM was found to have the greatest levels of PAL and RAS expression, whereas control plants had the lowest levels, suggesting there is a strong association between amount of MeJA and RA. However, compared to the other dosages, leaves treated with MeJA at 100 µM generated the highest levels of the 4CL transcript in the S. abrotanoides species. Finally, our results show that MeJA treatments directly regulated the accumulation of RA and total phenolic and flavonoid compounds, as well as the expression of key genes involved in RA biosynthesis (PAL, 4CL, and RAS) in Salvia leaves.

Plant Materials and Environmental Factors
The seeds of S. yangii and S. abrotanoides, two species of Salvia subg. Perovskia kar, were cultivated in a glasshouse under controlled illumination (16 h day/8 h night, with a photosynthetic photon flux density of 310 mol m −2 s −1 ) and temperature (25/18 • C day/night) conditions.

MeJA Treatments
In the present study, Salvia plants were treated with MeJA at concentrations of 10, 100, 150, and 200 µM throughout their third month of development in a container. Distilled water served as a control (i.e., only possessing root, stem, and leaf components). The 0.22-m MILLIPORE pore-size filter membrane was used for full sterilisation of the MeJA (SIG-MA-ALDRICH) solutions. Final concentrations of MeJA and distilled water (control) were sprayed over the aerial tissues of two Salvia species until runoff. The experimental data were collected from three separate plants for each treatment. After the first day of treatment, both the control and treated leaves were sampled, frozen in liquid nitrogen, and maintained at −80 • C.

Real-Time PCR Analysis
Following the manufacturer's instructions [20], total RNA was extracted from uniform young leaves of Salvia species, and cDNA was synthesized using the cDNA Synthesis Kit. As described before [56], we used gene-specific primers and the actin gene (as a housekeeping gene) to determine how varying amounts of MeJA affected the mRNA transcript levels of PAL, 4CL, and RAS ( Table 1). The fold-change (2 −∆∆Ct ) approach, which has been described earlier [90], was used for this investigation. Three technical and biological replicates were also employed for the gene expression study.

HPLC Analysis
To assess the impact of different MeJA concentrations on the formation of RA, Skendi et al.'s [91] approach was slightly modified. In a nutshell, 95 mg of the powdered dry leaf was mixed with 85% methanol (1:10 w/v), sonicated for 45 min, and then centrifuged at 3000 rpm for 15 min. The supernatant was mixed with sterile water to create the 20 mL reaction volume. To separate the RA, the resultant solution was filtered using a 0.45 M syringe filter before being injected onto an Agilent Technologies 1100 series HPLC system (C18 column (250 × 4.6 mm)). One milliliter of methanol/water (50/50 v/v) was produced with various concentrations of the RA standard, ranging from 1 to 350 mg/L. The calibration curve was created using the peak regions discovered from the injections. Mobile phase eluents A (acetonitrile) and B (water-acetic acid, 97.5:2.5, v/v) were used at a flow rate of 1 mL/min. The gradient of the solvent composition was 80A/20B for five minutes, followed by 50A/50B for ten minutes, and then 100% B for another fifteen minutes. Finally, the concentration of RA was determined as mg/g of dry weight after the RA molecule was detected at a wavelength of 330 nm ( Figure S1A-C). Each sample was examined three times. Validation of the chromatographic peak of RA was conducted using the retention time of the reference standard. Agilent ChemStation software was used to measure the peak regions as part of the quantitative analysis with external standardization. The results were presented as mg/g DW.

Total Phenolic and Flavonoid Contents Analysis
After being suspended in 80% methanol (25 mL) and shaken for 24 h at room temperature in a shaker, 1000 mg of dried and crushed Salvia leaf samples were turned into methanolic extracts to assess their total phenolic content (TPC) (150 rpm). The TPC was then calculated using the Folin-Ciocalteu reagent, as previously described [50], after the extracts had been filtered through two layers of Whatman paper. To begin, 2.5 mL of the Folin-Ciocalteu reagent (10-fold diluted) and 2 mL of sodium carbonate (7.5%) were added to 0.5 mL of the methanolic extract from each sample. The next step was to measure the absorbance at 765 nm after 15 min of heating at 45 • C. TPC was then determined as mg tannic acid equivalent/g dry weight (DW) ( Figure 5A). the total flavonoid content (TFC). First, 1.25 mL of water and 0.75 mL of sodium nitrate were mixed with 0.25 mg of each sample extract, and then the mixture was allowed to sit for 6 min in the dark. After 300 s of dark incubation, 0.15 mL of aluminum chloride (10%) was added to the liquid to complete the reaction. Each sample (5%) was then given 0.275 mL of water and 0.5 mL of sodium hydroxide solution. The TFC was shown as mg quercetin equivalent/g DW after reading the adsorption of the reaction solution at 510 nm ( Figure 5B).  Additionally, Zhang et al.'s [92] chloride colorimetric technique was used to assess the total flavonoid content (TFC). First, 1.25 mL of water and 0.75 mL of sodium nitrate were mixed with 0.25 mg of each sample extract, and then the mixture was allowed to sit for 6 min in the dark. After 300 s of dark incubation, 0.15 mL of aluminum chloride (10%) was added to the liquid to complete the reaction. Each sample (5%) was then given 0.275 mL of water and 0.5 mL of sodium hydroxide solution. The TFC was shown as mg quercetin equivalent/g DW after reading the adsorption of the reaction solution at 510 nm ( Figure 5B).

Data Analysis
The experimental data were analyzed using a factorial experiment based on a completely randomized design (CRD) with three replicates. The Duncan's multiple range test was used to compare the means (DMRT). The statistical analysis was computed using SPSS ver. 16 software.

Conclusions
As the first report, our results revealed that exogenous MeJA treatment increases the contents of RA, TP, and TF, as well as the expression of key genes in the RA (phenylpropanoid) pathway such as PAL, 4CL, and RAS in two species of Salvia. Hence, further research is required to understand how the accumulation of phenolic compounds in response to other treatments involving abiotic stresses interacts with the regulation of other genes engaged in this pathway. In the future, this process may be amenable to genetic modification in order to boost Salvia's production of crucial chemicals.

Conflicts of Interest:
The authors declare no conflict of interest.