Introduction

Stevia rebaudiana Bertoni is a plant that is native to Paraguay, which has become widely distributed in the world for its ability to produce non-caloric steviol glycosides (SG) with high sweetening power. There are different SG identified in stevia leaves; however, the most important are stevioside (St) and rebaudioside A (Reb A). SG are mainly used to sweeten soft drinks, soy sauce, yogurt, and other foods in Japan, Korea, and Brazil (Tadhani et al. 2007; Modi et al. 2014), and Europe (Novel Food Catalog, European Commission 2017). Additionally, it has been reported that SG have glucoregulatory, hypotensive, anti-inflammatory, and anticancer biological activities (Momtazi-Borojeni et al. 2017).

Due to the importance of the production of SG from S. rebaudiana, several studies have been reported using in vitro cultures as synthetic seeds (Nower 2014), callus, cells in suspension, and seedlings (Gupta 2013), transformed roots (Fu et al. 2015; Pandey et al. 2016; Michalec-Warzecha et al. 2016), and adventitious roots (Reis et al. 2011; Lopes et al. 2016; Reis et al. 2017; Ahmad et al. 2018; Ghazal et al. 2018).

The culture of adventitious roots as a differentiated system enables the accumulation of secondary metabolites in a higher quantity than the undifferentiated systems (Verpoorte et al. 2002). SG production has been reported from adventitious roots of S. rebaudiana grown in the absence of light at different pH levels, in which pH 5.1 was optimal for SG production (Ahmad et al. 2018).

Light has two important roles in plant development; it provides energy through photosynthesis, and it is a factor that regulates cell differentiation, growth, and metabolism (Escobar-Bravo et al. 2017). Zhang et al. (2002) tested continuous white fluorescent light irradiation (8000–8300 lx) and found that prolonged exposure of cell cultures to white light stimulates photoreceptors, which in turn can regulate the expression of specific genes responsible for growth, development, and secondary metabolism. Root cultures of Acmella oppositifolia (Asteracea) that were grown under continuous light conditions showed plastidial interconversion from amiloplastid to chloroplasts, which were morphologically similar to those present in the leaves of the plants. The authors concluded that photo-autotrophic behavior of the roots could cause important consequences for the secondary metabolic pathways (Flores et al. 1993). Wu et al. (2007) studied adventitious root cultures of Echinacea purpurea and found a direct correlation between the increment in secondary metabolism production and photoperiods. Similar results were found in S. rebaudiana plants that accumulated more SG when grown under photoperiod conditions (Mohamed et al. 2011).

Additionally, elicitation is a strategy widely used in in vitro cultures to increase the production of secondary metabolites. Methyl jasmonate (MeJA) and hydrogen peroxide (H2O2) are signaling molecules in plants, and both are involved in growth, development, and defense (Mittler 2017; Wasternack and Strnad 2019). Moreover, MeJA and H2O2 have been tested as elicitors of in vitro seedlings and callus of S. rebaudiana to favor SG production (Bayraktar et al. 2018; Javed et al. 2018a; Mejía-Espejel et al. 2018), but were still not tested in root cultures. Recently, research studies conducted to investigate stress conditions and the effects on total phenol and flavonoid levels in in vitro culture of Stevia rebaudiana have been on the rise (Ahmad et al. 2016; Javed et al. 2018a; Idrees et al. 2018; Javed et al. 2018b; Lucho et al. 2018).

Under stress conditions, there are two mechanisms of detoxification that are activated to reduce a stress condition: enzymatic and non-enzymatic. The enzymatic mechanism is formed by the enzymes superoxide dismutase (SOD), catalase (CAT), and peroxidase (Px), and the non-enzymatic mechanism is formed by antioxidant molecules within the cell (Apel and Hirt 2004), such as phenolic compounds, flavonoids, and different secondary metabolites with antioxidant activities (Ramos-Valdivia et al. 2012).

The present study explores the response to elicitation by H2O2 and MeJA in green roots of S. rebaudiana, using photoperiod conditions to favor SG accumulation. The increase in the production of phenolic and flavonoid compounds was verified, as well as the activation of enzymatic antioxidant mechanisms, to study the orchestrated response of antioxidant systems in green root culture of S. rebaudiana.

Materials and methods

Plant material

Stevia rebaudiana Bertoni adventitious root cultures were derived from micropropagated wild-type plantlets (Sanchéz-Cordova et al. 2019). Complete roots were cut from in vitro plantlets and 1.2 g dry weight (DW) were placed in 125-mL Erlenmeyer flasks containing 50 mL of half-strength MS medium (Murashige and Skoog 1962), supplemented with 20 g L−1 of sucrose and 0.5 mg L−1 of indolebutyric acid (IBA). The pH of the culture medium was adjusted to 6.3 prior to sterilization. Roots were maintained in two lighting conditions, which included complete darkness, and a photoperiod of 16 h with a light intensity of 26 μmols−1 m−2 at 25 ± 2°C, and shaken at 110 rpm on an orbital shaker. Roots were subcultured every 15 d.

Elicitation experiment with hydrogen peroxide (H2O2)

Erlenmeyer flasks (125 mL) that contained 50 mL of liquid culture medium were inoculated with 1.2 g fresh weight (FW) of young root meristems obtained from previously established cultures. These root meristems were cut in 1–1.5-cm fragments. After 10 d of growth, two concentrations of H2O2 (250 μM and 500 μM) were added, and samples were taken on days 1, 3, and 5 after the additions. The experiment was carried out in triplicate.

Elicitation experiment with methyl jasmonate (MeJA)

Erlenmeyer flasks (125 mL) that contained 50 mL of liquid culture medium were inoculated with 1.2 g FW of roots, which were fragmented as described in the previous section. After 10 d of growth, two concentrations of MeJA (250 and 500 μM) were added. The methyl jasmonate (392707; Sigma-Aldrich®, St Louis, MO) was dissolved in cold absolute ethanol and filtered-sterilized with 0.45-μm nylon filters prior to use. Samples were taken on days 1, 3, and 5 after elicitation. The experiment was carried out in triplicate.

Extraction of steviol glycoside (SG)

The extraction of SG from S. rebaudianain vitro roots was carried out following the methodology of Bondarev et al. (2001). The biomass was lyophilized and pulverized using a mortar and pestle, and the SG were successively extracted twice with methanol to H2O (80:20 v/v) in a ratio of 10 mL of extraction mixture for each gram of biomass (10:1) and sonicated for 30 min. Subsequently, the mixture was centrifuged for 15 min at 4116 g, the supernatant was recovered and all of the samples were concentrated to their constant weight. The crude extracts of SG (CESG) were stored at 4°C until analysis.

Thin-layer chromatography

Standards of stevioside (S3572; Sigma-Aldrich®) and rebaudioside A (1432; Sigma-Aldrich®) were used as references to identify the presence of SG. Standards and CESG from S. rebaudiana roots were dissolved in methanol to H2O (80:20 v/v) and applied on 60 F254 silica gel plates, with ethyl acetate to ethanol to acetone to H2O (15:3:6:6, v/v/v/v), as the mobile phase (Londhe and Nanaware 2013). The plates were developed with a solution of 0.5% orcinol in methanol to sulfuric acid (95:5, v/v) and heated to 120°C for 2 min on a heating plate until the bands corresponding to the SG were observed (Reis et al. 2011).

Identification of SG

The CESG were cleaned by solid-phase extraction (SPE), following the methodology of Wöelwer-Rieck et al. (2010) with some modifications. Chromabond® C18 ec cartridges (500 mg) were used, and the fraction retained was air-dried for 15 min. The fraction with the SG was eluted with acetonitrile to H2O (80:20 v/v). Mass spectral data was obtained through the direct flow infusion of the samples on an LCQ Fleet ion-trap mass spectrometer (Thermo Fisher Scientific®, San Jose, CA), with an electrospray interface (Thermo Fisher Scientific®) (DFI-ESI-IT-MSn), in negative mode. The system used a fused silica capillary tube at 280°C, a spray voltage of 5.00 kV, a capillary voltage of − 35 V, a tube lens of − 100 V, a 10 μL/min flow, and collision energy of 20–30%. Samples were further analyzed by an MS/MS experiment with collision energy of 20–30% and 30-ms activation time until fragmentation ceased. The resulting ions were identified using their mass spectra compared to standards and data previously reported. The compounds were identified by comparing mass spectra of the components with standards and data from the literature.

Quantification of SG

For quantification, CESG were cleaned by SPE following the methodology of Wöelwer-Rieck et al. (2010), using 100 mg Strata® C18E cartridges, and subsequently quantified by UPLC-TOF-MS in an Acquity Class I (Waters) chromatographic system coupled with a Synapt G2-Si (Waters) high-resolution mass spectrometer, according to Tada et al. (2013) and Gardana et al. (2010), with minor modifications. The chromatographic separation of SG was carried out using an isocratic method with 22% (v/v) acetonitrile and 0.1% (v/v) formic acid at 0.4 mL per min and 25°C on a Kinetex 5-μm EVO C18 column (150 × 2.1 mm, Phenomenex, Torrance, CA). The ionization was carried out by electrospray in negative mode, with a dwell time of 0.5 s and capillary voltage of 3 kV, and the spectra were captured from 100 to 1200 m/z. For the quantification of stevioside and rebaudioside A, the ions of m/z 839.3468 and 1001.3996, respectively, corresponding to the adducts [M + Cl35]− (10 to 100 ng mL−1, R2 = 0.9998), were monitored. SG were reported as the sum of stevioside and rebaudioside A.

Total phenolic and flavonoid content

The determination of the total phenolic content (TPC) was carried out according to the methodology described by Singleton et al. (1999), using catechin (C1251; Sigma-Aldrich®) as the standard in a calibration curve. The TPC was expressed in equivalent micrograms of catechin per milligram of dry extract. The quantification of the total flavonoid content (TFC) was performed according to Dewanto et al. (2002) methodology, using rutin (78,095; Sigma-Aldrich®) as a standard. The TFC was expressed in micrograms equivalent to rutin per milligram of dry extract.

Protein extraction and quantification

Roots were frozen and pulverized in liquid nitrogen, and an extraction buffer (100 mM phosphate buffer pH 7.0, 0.1 mM EDTA, 1% (w/v) PVPP and 5 mM ascorbate) in a 1:10 ratio (w/v) was added and mixed to obtain a homogeneous slurry. The crude root extract was mixed carefully and centrifuged at 33600g for 10 min. All of the last steps were performed at 4°C. The supernatant was preserved for catalase (CAT) and guaiacol peroxidase (Px) activity analyses. The total protein was determined using Bradford reagent (Bradford 1976) (500-0205; BioRad, Hercules, CA), using bovine serum albumin (BSA; 500-0209 BioRad) as a standard.

Enzymatic activity assays

Catalase activity (CAT) was determined according to the protocol described by Aebi (1984). It was calculated using the extinction coefficient of H2O2 (40 M cm−1) and expressed as μmol mg protein−1 min−1. The enzymatic activity of guaiacol peroxidase (Px) was quantified by the oxidation of guaiacol using the method proposed by Pütter (1971). The activity was expressed as the amount of protein that produces 1 mmol of tetraguaiacol min−1.

Statistical analysis

The effects of elicitor concentrations and exposure time were assessed by a two-way ANOVA using the general linear model and Tukey’s post-hoc test with P < 0.05 to establish significance for both analyses with Minitab 16.

Results and discussion

Effect of photoperiod on SG production in S. rebaudiana root culture

To optimize the light regime, an experiment was designed to grow S. rebaudiana adventitious roots in darkness and photoperiod conditions. Figure 1 shows S. rebaudiana adventitious roots grown in darkness (Fig. 1a), and in 16/8-h photoperiod (light-dark) (Fig. 1b). It was observed that the roots that grew in darkness were pale (Fig. 1a) compared to those that grew in the photoperiod (Fig. 1b), which exhibited a green color. The combination of light-dark conditions allowed the formation of photosynthetic pigments, and therefore, SG accumulated in the roots (Fig. 2). This behavior may be due to the fact that the photoperiod conditions induce the transition of amyloplasts to chloroplasts, as reported by Flores et al. (1993), which allows the SG accumulation in green roots of S. rebaudiana. The last argument is consistent with data reported by Ceunen and Geuns (2013) in S. rebaudiana plants. They found enhanced SG accumulation in leaves and roots in photoperiods of 16/8 h (light-dark).

Figure 1.
figure 1

Stevia rebaudiana Bertoni root cultures growing in (a) dark; and (b) light conditions.

Full size image
Figure 2.
figure 2

Thin-layer chromatography of methanolic extracts from (a) Stevia rebaudiana Bertoni root cultures growing in (1) photoperiod; (2) dark conditions; and (3) St and Reb A standards; (b) Stevia rebaudiana root cultures growing in light conditions (1) crude extracts of SG (CESG); (2 and 3) after solid-phase extraction; (4) St standard; and (5) Reb A standard. Solvent system ethyl acetate:ethanol to acetone to H2O (15:3:6:6 v/v/v/v), detection with orcinol.

Full size image

The clean-up strategy of the CESG of S. rebaudiana roots by SPE, and the use of a 30 ppm concentration of the SG fraction obtained by SPE, allowed the identification of different SG by DFI-ESI-IT-MSn through the acquisition of the pseudomolecular ions [M-H]− 965, 803, and 317, and their fragmentation patterns compared to the standard analyte and bibliographic references (Gardana et al. 2010; Shafii et al. 2012). The compounds identified in the green roots of S. rebaudiana in this study were stevioside, rebaudioside A, and a mixture of the isomers steviol/isosteviol (Table 1). It has been reported through in vitro and in vivo studies that the accumulation of SG is related to the development of the chloroplast membrane, and the content of photosynthetic pigments in S. rebaudiana (Ladygin et al. 2008). Libik-Konieczny et al. (2018) found a strong direct correlation between the photosynthetic efficiency and SG accumulation in S. rebaudiana plants. Other authors have reported that undifferentiated cultures grown in the presence of light, such as S. rebaudiana callus and cells in suspension, are also capable of producing SG (Janarthanam et al. 2010; Gupta et al. 2014; Javed et al. 2018b). The results from the Stevia root cultures in this study concur with previous observations and suggest that the biosynthetic machinery involved (genes and enzymes) for SG accumulation is active under light conditions. To further support the effect of light on the biosynthetic properties of Stevia green roots, Chen et al. (2018) reported that Ent-kuarene, which is a precursor of the SG pathway is regulated by light.

Table 1. Precursor ions and their corresponding fragments obtained by ESI-IT-MSn (negative ion mode) analyses of the extracts from in vitro root cultures of Stevia rebaudiana Bertoni
Full size table

In this study, adventitious roots of S. rebaudiana grown under dark conditions (Fig. 2a) were not able to accumulate SG, similar to what was reported by Reis et al. (2011, 2017) and Fu et al. (2015), in which roots failed to produce SG.

Effect of elicitors on growth and SG production in S. rebaudiana root culture

To evaluate the elicitation effect on growth and SG production, two concentrations of H2O2 and MeJA were added to the culture medium. Figure 3 shows the effect of 250 and 500 μM H2O2 and MeJA on the growth of the green adventitious roots of S. rebaudiana during 5 d of exposure. Roots without elicitors weighed 4.6 ± 0.67 gDW L−1 on the first day and reached a biomass accumulation of 7.04 ± 0.71 gDW L−1 after 5 d of cultivation (Fig. 3a), while for roots treated with 250 and 500 μM H2O2, the maximum biomass after 5 d of elicitation was 4.7 ± 0.06 and 4.02 ± 0.34 gDWL−1, respectively (Fig. 3b). Similarly, with 250 and 500 μM MeJA, the maximum biomass was 4.06 ± 0.01 and 3.99 ± 1.13 gDW L−1, respectively (Fig. 3c). The elicitation using MeJA and H2O2 showed inhibition of the root growth up to 40%, compared to the control treatment at day 5, which was an effect reported in other plants (Vera-Reyes et al. 2013; Faizal and Sari 2019). The inhibition of root growth is a common phenomenon observed by MeJA and H2O2 treatments due to the interference auxin flux pathway (Yan et al. 2016) and abscisic acid signaling (Bai et al. 2007), respectively. Growth reduction in MeJA-treated cultures was accompanied with a color change to brown at day 5, though the effect was not observed in the H2O2 treatment (Fig. 4). This response could be associated with the phenolic compound accumulation. It has also been reported (Neill et al. 2002) that H2O2 is rapidly neutralized by the different antioxidant systems within the cell, which prevents extensive damage, unlike MeJA, which could be exerting stress conditions in a constant and prolonged way, and leads to physiological degeneration as observed in the roots in this study. The different responses between treatments could be associated with the fact that the antioxidant systems prevent exogenous H2O2 transport far through the cell and is rapidly metabolized (Neill et al. 2002).

Figure 3.
figure 3

Growth kinetics after H2O2 and MeJa elicitation in Stevia rebaudiana Bertoni root culture during 5 d of elicitation. (a) Roots growing without elicitors; (b) Roots growing with hydrogen peroxide. Open and close square, 250 and 500 μM of H2O2, respectively. (c) Roots growing with methyl jasmonate. Open and close rhombus, 250 and 500 μM of MeJA, respectively. Error bars indicate standard deviation from the mean (n = 3).

Full size image
Figure 4.
figure 4

Stevia rebaudiana Bertoni green roots after 5 d of exposure to (a) 500 μM of MeJA and (b) 500 μM of H2O2.

Full size image

To analyze the elicitation effect on SG levels, the same elicitors were tested. Figure 5 shows the production of SG as a function of the exposure time after elicitation with H2O2. It was observed that the addition of 250 μM H2O2 significantly increased the accumulation of SG (stevioside + rebaudioside A, 32 μgSG per gDW), compared to the green roots without elicitation treatment (22.48 μgSG per gDW), from the first day of exposure. After 3 d of exposure, the SG increased up to a 2.4-fold accumulation of the control and reached the maximum SG content in the experiment (50.8 μgSG per gDW). A similar result of increased SG was also reported by Javed et al. (2018a), in shoots of S. rebaudiana elicited with 10 and 20 mM of H2O2. In these results, the root cultures treated with 500 μM H2O2 decreased their SG production. This result suggests that H2O2 favors the production of SG, but the positive effect is dose dependent. Hydrogen peroxide, as a secondary messenger, could be inducing genes involved in SG synthesis at 250 μM H2O2, as observed in tomato (Orozco-Cárdenas et al. 2001), and Uncaria tomentosa (Vera-Reyes et al. 2013), which induced secondary metabolism. Otherwise, higher concentrations and the stress caused by the elicitor affect the biosynthetic capacity of the SG. Another scenario could use the SG present in the roots as antioxidant compounds to counteract the stress. In another study, in vitro oxidative degradation was demonstrated by adding hydrogen peroxide to stevioside (Martono et al. 2018). Libik-Konieczny et al. (2018) proposed that SG could act as protective molecules for the photosynthetic apparatus against adverse conditions. Contrasting cellular responses with the same elicitor, H2O2 in this case, could be attributed to several factors such as the site of molecule perception, degradation rates, and the initial concentration of the elicitor (Neill et al. 2002; Smirnoff and Arnaud 2019).

Figure 5.
figure 5

Effect of 250 and 500 μM exogenous H2O2 on steviol glycoside accumulation (SG) in Stevia rebaudiana Bertoni root culture over 5 d. Error bars indicate standard deviations from the mean (n = 3).

Full size image

On the other hand, in the root cultures treated with 250 μM MeJA, SG were detected on the first day (average 5.5 μgSG per gDW), but at a concentration of 500 μM MeJA, SG were not detected (1 to 5 d). This effect was also observed by Bayraktar et al. (2016), in which an absence of SG was reported with the addition of 100 and 200 μM MeJA to S. rebaudiana shoots. It is possible that higher stress conditions caused by the dose and exposure time of the MeJA affect the photochemical efficiency and cause morphological changes in the chloroplasts. This effect was also observed by Zhang and Xing (2008), in which they demonstrated that MeJA increased reactive oxygen species (ROS) production and changed chloroplast structures, and photochemical efficiency declined in protoplasts or seedlings of Arabidopsis thaliana. A similar response could occur with the MeJA concentrations used in this study, and negatively affect SG biosynthesis.

Effect of elicitors on antioxidant systems

The increase in the enzymatic antioxidant capacity, which is defined as the enzymatic activity of CAT and Px, under the stress conditions caused by 250 and 500 μM H2O2 and MeJA, was compared with the activities in the green roots without elicitation (Table 2).

Table 2. Effect of elicitation on the enzymatic antioxidant activities of catalase (CAT) and peroxidase (Px) in the cultivation of green roots of Stevia rebaudiana Bertoni for 5 d; 250 and 500 μM H2O2 and MeJA were evaluated. Values are expressed as the mean ± SE (n = 3). The letters identify equivalent means
Full size table

Figure 6 shows the total phenolic content (TPC) as a function of the elicitation time. According to Fig. 6, the elicited green roots produce phenols on the first day at concentrations up to 55.77 ± 0.97 μgCE per mgExt. Under elicitation with 250 μM H2O2, the phenolic content increased by 0.25-fold compared to green roots without elicitation and gradually decreased until the level was similar to the control roots. Using 500 μM H2O2, there was no difference from the control roots. As described previously, the increase in H2O2 beyond 250 μM affects the accumulation of secondary metabolites (SG + phenols), in S. rebaudiana green roots. It could be possible that both types of metabolites are simultaneously counteracting the stress. Alternatively, with 250 and 500 μM MeJA, the total phenolic accumulation was higher compared to roots without elicitation. In addition, the phenolic content increased in green-elicited roots as a function of time, and increased to the fifth day of elicitation by up to 0.90- and 1.38-fold more than the control (42.19 ± 0.78 μgCE per mgExt), respectively.

Figure 6.
figure 6

Total phenol content (TPC) in the roots of Stevia rebaudiana Bertoni as a function of the elicitation time with H2O2 and MeJA. Error bars indicate standard deviations from the mean (n = 3).

Full size image

The total flavonoid content (TFC; Fig. 7) was also modified by the presence of the elicitors. The maximum accumulation of flavonoids in the treatments with 250 and 500 μM H2O2 was presented on third day after their addition (42.99 ± 0.32 and 29.49 ± 0.03 μgRE per mgExt, respectively). This resulted in 3.87- and 2.34-fold increases with 250 and 500 μM, respectively, compared to roots without elicitation. The highest flavonoid content was reached on the fifth day after the addition of 250 and 500 μM MeJA, and increased 8.2- and 4.4-fold, respectively, compared to the control (3.46 ± 0.35 μgRE per mgExt). As noted with TPC, the TFC also increased by exposure time with MeJA elicitation.

Figure 7.
figure 7

Total flavonoid content (TFC) in the roots of Stevia rebaudiana Bertoni as a function of the elicitation time with H2O2 and MeJA. Error bars indicate standard deviations from the mean (n = 3).

Full size image

These results demonstrate that both types of metabolites increased during stress conditions in S. rebaudiana roots, but the production was related to the type of stress applied, dosage, and exposure time.

In contrast to what was observed with H2O2, MeJA activated CAT and Px (Table 2), and joined with the non-enzymatic antioxidant defense mechanisms to mitigate the effect of oxidative stress and damages. Phenols and flavonoids serve as ROS scavengers by locating and neutralizing radicals before they damage the cell and constitute an important defense mechanism for plants under adverse environmental conditions (Mehla et al. 2017).

The results suggest that the elicitors, primarily MeJA, cause high stress conditions in adventitious roots, and promote higher antioxidant activity within the cell. Consequently, the roots produce phenols and flavonoids to maintain the redox balance in the cell instead of SG production.

Conclusions

In this study, the SG production from green roots of S. rebaudiana was investigated, which was favored by the exposure of the roots to light. The results indicated that the accumulation of SG in the roots is enhanced under stress conditions, as long as the level of stress applied does not compromise the SG biosynthetic capacity of the root culture. Moreover, the application of exogenous MeJA maintained the root function under stress by increasing the phenolic and flavonoid contents and enhancing antioxidant enzyme activities.