4.1. Growth parameters
The effect of different light sources and genotypes on growth parameters was significant (Table 1). Table 2 showed that the highest shoot fresh and dry weight and Leaf number were observed under red+blue LED light source in plantlets of both genotypes. Increment in fresh and dry weight, leaf area, and chlorophyll content in lettuce plants in studies conducted by Kim et al. (2004), Johkan et al. (2010) and Yorio et al. (2001) showed that the highest amount of these indicators can be seen in the combination of red and blue light. The combination of red+blue light increased the fresh and dry weight by 3 times compared to the combination of blue and far-red light. It seemed that the combination of red and blue light increased plant growth by increasing the amount of pure photosynthesis because the red+blue light energy distribution corresponded to the absorption spectrum of chlorophyll (Wang et al. 2007). On the other hand, the intensity of photons from LED lamps is higher than other light sources, which is another mechanism of the positive effect of LED lights to improve plant growth indices (Yorio et al. 2001).
4.2. Essential oil content and constituents
The essential oil content of two genotypes of M. officinalis was not significantly affected by different LED light sources when compared to the greenhouse condition (in range of 0.27% to 0.32%, data not shown). However, the effect of different light sources and genotype on major compounds of lemon balm essential oil was significant (Table 1). A total of thirty-eight compounds were identified using GC-MS analysis (Table 3). A considerable difference was observed in the concentration of essential oil constituents in both genotypes of M. officinalis grown under various light sources (Table 3). In plantlets of Ilam genotype, ß-cubebene and citronellal had the lowest and highest amounts among all of the identified constituents under red and red+blue LEDs, respectively with 0.6 and 15.3%, but in the othergenotype, the lowest amount of essential oil content was belonging to bicyclo[2.2.1]heptan-2-one, ß-ionone, and eugenol in plantlets that were subjected to red, blue, and red+blue LEDs (0.8%). Among essential oil compounds, citronellal had the highest percent content (17.2%) under red+blue LED lights in the Isfahan genotype.
It is very important to study the change in the amount of essential oil of medicinal plants because it is related to the change in their nutritional, medicinal and antioxidant properties. Scientists believed that chemical composition of essential oil and production of secondary metabolites were related to the physiology of plant, developmental stage, plant species, and various treatments such as various light sources (Yu et al. 2005; Fernandes et al. 2013; Mashkani et al. 2018). In the study by Batista et al. (2016) on three chemotypes of Lippia alba, they demonstrated that the differences in the composition of essential oil were more influenced by chemotype than light treatments (LED and fluorescent) applied. They and Viccini et al. (2014) stated that this difference between genotype and chemotypes could be depended on the content of plant DNA.
The content and composition of essential oils in two genotypes studied in the present work were variable under different light treatments and the total amounts of compounds in the Ilam genotype were higher than the other genotype. However the observed variation between two genotypes in the essentials oil composition was related to the relative percentage of the constituents and not to the appearance or lack of a specific component. Light quality and its intensity influenced on production of secondary metabolites and essential oils with changing in physiological and morphological properties of plants (Briskin et al. 2001). Light affects the number and morphology of leaves and essential oil storage structures such as trichrome, causing changes in the amount and chemical composition of essential oil in plants (Fernandes et al. 2013). In the present study, in plantlets of Ilam genotype, all LEDs lights had a greater effect on essential oil content than plants grown under greenhouse conditions but in Isfahan genotype, the effect of red+blue and red LEDs treatments were higher than plants grown under greenhouse conditions. Treatments of red and red+blue LEDs on Ilam and Isfahan genotypes have resulted in the greatest essential oil contents. Various studies have suggested the positive effect of LED light on increasing the essential oil content in Mentha species, green vegetables (such as parsley, onion, lettuce and…), and Brassica oleracea (Sabzalian et al. 2014; Žukauskas et al. 2011; Lefsrud et al. 2008).
4.2.1. Monoterpenes content under various light treatments in two genotypes of M. officinalis
The number of identified monoterpenes in the two genotypes of lemon balm was 21 compounds. The amount of these compounds was different under various light treatments. In Ilam genotype, the highest amount of monoterpenes was observed in incubators with white, red+blue, and red LEDs compared to Isfahan genotype,but in Isfahan genotype, these compounds were higher under blue LED and greenhouse condition than the other genotype. The red+blue LED light treatment in both genotypes produced the highest amount of monoterpene compounds compared to the greenhouse condition (Table 3).
In Ilam genotype, monoterpene compounds ranged from 0.9% for myrcene under red+blue LED to 15.3% for citronellal under the same light treatment (Table 3). Under LED light treatments, monoterpenes such as myrcene, alpha-phellandrene, para cymene, eucalyptol, γ-terpinene, linalool, cis-sabinene, trans-carveol, citronellal, citral, thymol, carveol, and carveol acetate were more affected than greenhouse condition. In this genotype, myrcene (0.9%) and para cymene (1%) had the lowest identified monoterpenes, regardless of light treatment. In this genotype, the red+blue LED treatment resulted in the highest amount of major essential oils namely citronellal (15.3%), trans-carveol (10.2%), linalool (8.9%), and citral (8.2%) compared to the other light.
The range of monoterpene composition in Isfahan genotype changed from 1% for thymol under the white LED light to citronellal with 17.2% under red+blue LED light (Table 3). Myrcene, limonene, eucalyptol (1.2%), and thymol (1%) had the lowest values among known monoterpenes, regardless of light treatments. Alpha-pinene, myrcene, γ-3-carene, limonen, eucalyptol, linalool, cis-sabinene, β-thujone, trans-carveole, citronellal, citral, thymol, and carveol had the most contents in plantlets grown in incubators with LED lamps compared to greenhouse condition. Among light treatments, red+blue LED light had the highest contribution to increasing the amounts of major essential oils including citronellal (17.2%), trans-carveole (11.1%), citral (9.1%), and linalool (8.9%).
The chemical composition of lemon balm essential oil (that is 0.02 - 0.3% of DW) had been investigated. The main combination was citronellal (2-40%) and citral (10-30%) with ß-caryophyllene, germacrene-D, ocimene and citronellol (Schultze et al. 1989; Adzet et al. 1992; Kreis and Mosandl 1994; Moradkhani et al. 2010). Citronellal, as one of the major components in the essential oil of lemon balm, is a monoterpene aldehyde (Chung et al. 2010). In the present study, citronellal percentage varied from 10.2-17.2%, that its highest amount (17.2%) was detected in plantlets of both genotypes grown in incubators containing red+blue LED. In many studies, citral after citronellal was the most commonly reported composition of lemon balm essential oil, but as was seen in the present study, the highest essential oil content after citronellal was citronellol with a varied range of 6.5–11.8%. In the present study, citral after citronellal, citronellol, and trans-carveol compounds has the highest amount of essential oil in two genotypes under different light treatments.
Light quality can change the composition of essential oils in medicinal plants (Amaki et al. 2011). In a study by Batista et al. (2016), different qualities of light caused a change in the pattern of essential oil in Lippia alba. These researchers showed changes in the number of monoterpenes such as eucalyptol and linalool in two L. alba chemotypes under LED and fluorescent lamps. Noguchi and Amaki (2016) and Nguyen and Saleh (2019) reported an increase in monoterpenes such as alpha-pinene, beta-pinene, limonene and carvone in mint plants under red LED light.
According to the findings of the present study, it was found that in the two genotypes of lemon balm, light treatments play an effective role in changing the number of monoterpenes. Since most of the essential oil components of lemon balm in this study were monoterpenes, it is concluded that the quality and property of essential oil will probably change significantly. Ghaffari et al. (2019) and Tohidi et al. (2019) reached the same results with the current study about increase in the number of monoterpenes under LED lamps. In the present study, red+blue LED light was the major contributor to the increase in monoterpenes, but in studies by Ghaffari et al. (2019) and Tohidi et al. (2019), blue light played this role. Therefore, this difference reflects the different effects of LED light on the essential oil of different plants.
It is well known that the increase in the production of secondary metabolites is related to plant growth conditions such as temperature, light regime, stress, nutrition source, etc. (Selmar and Kirinwächter 2013). If the light energy that is absorbed by the photosynthetic apparatus exceeds compared to the energy required for CO2 fixation, large amounts of this energy according to the energy dissipation mechanism proposed by Selmar and Kirinwächter (2013) ultimately led to the production of secondary metabolites. In the present study, since the intensity of light in LEDs (300 μmol.m-2.s-1) is continuously higher than the greenhouse conditions, these light sources, especially red+blue LED light, can be considered as severe light stress and increased monoterpenes according to the mechanism described above. Recently, researchers have found that terpenoids biosynthesis in addition to the mevalonic Acid (MVA) pathway is made from primary metabolites through another pathway called methylerythritol phosphate (MEP), which is present in plastids and chloroplasts (Lichtenthaler et al. 1999). Therefore, it can be stated that under the treatment of LED lights, especially under red+blue LED light, due to the matching of these wavelengths to the absorption peak of photosynthetic pigments, it resulted in increase of photosynthesis and production of photosynthetic intermediates, especially IPP (isopentenyl diphosphate). This process may have resulted in the activation of the MEP pathway more and more and produced high levels of these terpenoids.
4.2.2. Sesquiterpenes content under various light treatments in two genotypes of M. officinalis
According to the results shown in Table 3, the number of sesquiterpene compounds in lemon balm genotypes was also significantly affected by light treatments. In Ilam and Isfahan genotypes, the highest amount of these compounds were obtained under blue and white LED and greenhouse conditions, respectively. In Ilam genotype, LED light treatments increased the level of sesquiterpene such as ß-caryophyllene, α-humulene, germacrene-D, ß-ionone, and ß-cubebene but decreased the amount of caryophyllene epoxide, calamenene, and α-muurolene compared to greenhouse condition. In contrast, in Isfahan genotype, only α-humulene, ß-ionoe, and germacrene-D levels were higher in most LED treatments compared to the greenhouse condition. The reduction in the amount of ß-caryophyllene, caryophyllene epoxide, and calamenene treated with LED light was significantly higher than greenhouse condition. In plants of two genotypes, caryophyllene epoxide, germacrene-D and ß-caryophyllene had the highest sesquiterpene compounds, respectively, regardless of light treatments. As in the present study, in Jalal et al.'s study (2015), the main sesquiterpene of lemon balm was ß-caryophyllene epoxide (11%) and in the study of Kittler et al. (2018) ß-caryophyllene, germacrene-D and ß-caryophyllene epoxide constituted the main sesquiterpene compounds of lemon balm genotypes. However, in the study conducted by Chung et al. (2010), caryophyllene (0.8%) and farnesene (0.1%) were the most common sesquiterpene. So, it can be said that the essential oil compositions of species and genotypes of a plant are different under different conditions. ß-cariophyllene is a natural bicyclic sesquiterpene that has also been involved in the creation of a spicy taste of black pepper and many essential oils such as Rosmarinus officinalis, Syzygium aromaticum, and Cannabis sativa. This compound has received much attention because of its cyclobutane ring, that is scarce in nature (Taherpour et al. 2012).
Many studies have been shown that LED lights induced several metabolic changes in plants, including increased levels of β-farnecene, germacrene-D, and elmene in Mexican mint grown under blue light (Noguchi and Amaki 2016). Alvarenga et al. (2015) showed that high light intensities, such as 47 and 69 μmol.m-2.s-1, increased monoterpene compounds content in Acillea millefolium L., but these intensities reduced the number of sesquiterpenes. Sesquiterpenes such as ß-caryophyllene and ß-cubebebene have the highest values at 13 μmol.m-2.s-1 intensity. Blue LED light in Mentha spicata increased ß-caryophyllene compared to control condition. The concentration of beta-bourbonene as a sesquiterpene was also higher in mint cultivated under LED light compared to control (Nguyen and Saleh 2019.). Increment in trans-caryophyllene and α-humulene in two species of Perovskia has also been reported under the white LED light (Ghafari et al. 2019). Tohidi et al. (2019) study showed an increase in sesquiterpene levels of three species of thyme under red+blue and red LED lights.
4.3. Radical Scavenging Activity analysis
The results of the analysis of variance of free radical scavenging activity (RSA or antioxidant activity) showed that there was no significant difference between two genotypes in terms of this activity, while the effect of different lights on RSA was significant (p≤0.01) (Table 1). In both genotypes, the highest RSA% was obtained for plantts grown under red+blue LED light (80.77 and 82.09% for Ilam and Isfahan genotypes, respectively) and the lowest was for plants grown under greenhouse condition (34.29 and 25.67% for Ilam and Isfahan genotypes, respectively) (Fig. 1).
Synthesis of secondary metabolites in plants is an important part of the defense response to stress. Oxidative stress and reactive oxygen species (ROS) cause extensive damage to plants. Antioxidant systems that show antioxidant capacity in plants including flavonoids, ascorbate, carotenoids, phenolic compounds, and essential oils, protect plants against photo-oxidative damage (Shohael et al. 2006). Li and Kubota (2009) demonstrated the beneficial role of LEDs in improving antioxidant activity in baby leaf lettuce. In addition, Samuolienė et al. (2012) reported that LED supplementation with HPS lamps altered the antioxidant and nutritional properties of lettuce due to increased metabolic system activity to protect plantlets against moderate photo-oxidative stress induced by the use of LEDs. In the present study, it was observed that the red+blue LED light treatment produced the highest amount of major monoterpene components such as citronellal, trans-carveol, citronellol, linalool, and citral toghether with the highest RSA% in two genotypes. Therefore, it can be stated that the antioxidant capacity of these two genotypes under light treatments is directly related to the essential oil components, especially monoterpenes. Confirming this result, researches has shown that DPPH's scavenging activity is directly related to the levels of phenolic and antioxidant compounds of plants such as essential oils (Wojciechowska et al. 2015; Tohidi et al. 2017 and 2019).
4.4 Principal component analysis
Principal component analysis (PCA) was used to identify the relationship between growth parameters and essential oil components, antioxidant activity, and different wavelengths applied in two genotypes of lemon balm (Fig. 2). The results of PCA analysis identified three main groups and these results also indicated that the first and second groups accounted for 78.39% of the total variation. The first PC (PC1) and the second PC (PC2) revealed 64.61% and 13.77% of the total variation, respectively. The plants grown under LED light sources were completely separated based on the growth parameters, essential oil constituents and antioxidant activity, from the plant grown in the greenhouse condition. In terms of caryophyllene, only plants of Isfahan genotype that were grown in incubators containing red and blue LEDs were similar to greenhouse light. In the other group, Ilam genotype that was under red+blue and red LEDs, and Isfahan genotype grown under red+blue, red and blue LEDs, there was the highest levels of main monoterpene compounds such as citronellal, trans-carveol, linalool, and citral and highest antioxidant activity plus shoot fresh and dry weights. The third group, Ilam genotype grown under white and blue LEDs and Isfahan genotypes with white LEDs also had the same and the highest amount of monoterpene compounds such as citronellol and γ-3-caren and sesquiterpene compounds such as caryophyllene epoxide and germacrene-D. Results showed that two genotypes of lemon balm were differentially affected by LEDs and greenhouse light, successfully distinguished by the main constituents of essential oil and antioxidant activity.