Effect of sucrose on the fatty acid metabolism of adventitious root cultures in vitro of Stevia rebaudiana

In this study, the effect of sucrose on the neutral lipid profile of adventitious root cultures of Stevia rebaudiana was evaluated. The cultures were obtained employing a roller bottle system. In this system, Schott-type flasks were used, which contained Murashige and Skoog liquid medium at 33.3% strength (MS/3) supplemented with 30, 60, and 80 g L of sucrose, respectively, and 10.7 mM 1-naphthaleneacetic acid (NAA). The spectroscopic analyzes showed that the portion of polyunsaturated fatty acids (PUFAs) was highest in roots treated with 30 g L of sucrose. The spectrometric analyzes showed that the palmitic acid was found to be present in relatively higher amounts in the roots submitted to the MS/3-30 g L (31.9%) and MS/3-60 g L (29.5%) sucrose treatments, and lower in the treatment with MS/3-80 g L (28.8%) of sucrose. Also, the treatment using 30 g L of sucrose was the best for obtaining unsaturated fatty acids (UFAs) in the culture, with a relative percentage of 62.9%. Our results indicate that the MS medium that received 30 g L of sucrose induced a lesser abiotic stress condition, which favored PUFAs production in the adventitious root cultures of S. rebaudiana.

S. rebaudiana is a species of nutritional importance, it has been applied industrially since the 1970s, during which Japanese researchers optimized techniques to extract glucoside steviosides (Dacome et al., 2005). The main producers of Stevia are China and Southeast Asian countries. Currently, this plant has been gaining importance since 2011 when steviol glucosides obtained from the leaves were recognized as a sweetener by some important regulatory agencies around the world (Tavarini, & Angelini, 2013). In addition, recently, studies have shown that its roots are a rich source of inulin, a compound used as a prebiotic source (Lopes et al., 2016).
The use of plant tissue cultures and micropropagations in plant biotechnology are techniques that maintain the genetic stability of plants, which aim to produce primary and secondary metabolites for pharmacological and nutritional purposes (Wu et al., 2009;Thiyagarajan, & Venkatachalam, 2012;Lopes et al., 2016).Thus, with the fast root growth that happens through in vitro procedures, it is possible to acquire sufficient raw material for the extraction and commercial production of bioactive metabolites without the destruction of natural biodiversity. The adventitious roots of S. rebaudiana represent an important and promising biotechnological alternative for obtaining bioactive substances, given their good propagation, conservation of the species can be achieved, as shown in our previous work using adventitious roots cultured in vitro (Reis et al., 2011;Lopes et al., 2016).
Some environmental stresses, such as temperature, sunlight, and variations in the composition of nutrients, can induce changes in the lipid composition of these root cells (Oksman-Caldentey et al., 1994;Singer, Zou, & Weselake, 2016;Zhai et al., 2017). One reason is that neutral lipids(NLs), made up mainly of triacylclycerols (TAGs) and free fatty acids (FAs), provide not only energy and the building blocks for secondary metabolite biosynthesis by plants, but also play important roles in plant growth/survival when submitted to biotic and abiotic stresses. Thus, understanding all the physiological attributions of TAGs and FAs will be a crucial point for the future design of biotechnological strategies to overcome bottlenecks in increasing Research, Society andDevelopment, v. 10, n. 10, e182101018651, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i10.18651 3 the production of metabolites of interest. Therefore, the present study aimed to evaluate the effect of sucrose concentration on the neutral lipid profile in the adventitious root cultures of S. rebaudiana by gas-liquid chromatography coupled to mass spectrometry (GC-MS) and nuclear resonance spectroscopy (NMR).

Methodology
The work corresponded to laboratory activities of semi-quantitative and qualitative character of the effect of sucrose on adventitious roots of Stevia rebaudiana cultured in vitro. The first one related to the establishment of the cultivation process of adventitious roots of S. rebaudiana. The second, in turn, is linked to the qualitative and quantitative assessment of the effect of different concentrations of sucrose on the pattern of neutral lipids and fatty acids. The research was carried out experimentally based on the procedures proposed by Jacomini et al. (2015) and using a comparative research method as described by Pereira et al. (2018), took into account the observation, recording and analysis of the objects of study and their relationship with other phenomena.

Adventitious root cultures of Stevia rebaudiana
Aseptic plants of S. rebaudiana were obtained according to the protocol of Reis et al. (2011). Root cultures of S. rebaudiana were then started from root tip explants about 1.5 cm long derived from in vitro rooting nodal explants on solid MS media supplemented with 10.7 mM of 1-naphthaleneacetic acid (NAA) at 25±1 ºC, with 2 months of cultivation. The MS media (pH 5.8) was incorporated with 0.8% (w/v) agar. These cultures were successfully grown and maintained with regular subcultures every 4 weeks.
After 2 months of cultivation, roots were aseptically isolated from the explants, and the nutrient medium present was completely eliminated. Approximately 0.2 g of fresh roots were withdrawn and transferred to 500 mL Schott-type flasks containing 25 mL of MS liquid medium (pH 5.8), with 33.3% strength (MS/3) that was supplemented, respectively, with 30, 60 and 80 g L -1 of D-sucrose and 10.7 mM 1-NAA. The adventitious roots of S. rebaudiana were cultivated in a roller bottle system at 2 rotations per minute (RPM) at 25±1ºC under dark conditions. These roots were maintained under this culture condition for 15 days, after which they were transferred to 1 L Schott-type flasks containing 50 mL of MS/3 liquid medium that was supplemented, respectively, with 30, 60 and 80 g L -1 D-sucrose and 10.7 mM 1-NAA. The roots were then cultivated for another 30 days in the same conditions described above, as shown in Figure 1.

Neutral lipids extraction
The lyophilized adventitious roots obtained from the different treatments (SRR-30, SRR-60, and SRR-80) were extracted under reflux with hexane for 4 h. After the extracts were filtered, the solvent was evaporated in a rotary evaporator (35 °C), yielding the crude extracts of neutral lipids (NLs). These samples were stored at -20 °C until the respective analyzes.
Two milliliters of 5% methanolic HCl was then added, followed by heating for 15 min at 55 °C and the addition of 1 mL H2O ultrapure. FAMEs were extracted with hexane (3×1 mL) and evaporated to dryness in an evaporator. Samples were redissolved in 200 µL hexane and stored at -4 °C in glass vials until analysis by GC-MS (Kumari, Reddy, & Jha, 2011).

Gas-liquid chromatography coupled to mass spectrometry (GC-MS) analysis
Gas-liquid chromatography coupled to mass spectrometry (GC-MS) analysis was carried out using a Gas Chromatography Agilent 7890B coupled with a Mass Spectrometer Agilent 5977A MSD and an HP5-MS UI-Agilent fused silica capillary (30 × 0.25 mm × 0.25 mm; Agilent Technologies). The He was carrier gas used at a flow rate of 1 mL min -1 .
The temperature of the split injector (split ratio 1:30) was 250 °C and temperature of the transfer lines was maintained at 250 °C. The operating specifications of the column were as follows: the initial temperature of the oven was 60 °C maintained for 4 min, then 60 to 250 °C at 10 °C min -1 , and the final temperature was sustained for 28 min. The mass spectrometer was operated in the electron impact mode, with electron energy of 70 eV for the positive mode. Ionization source and quadrupole temperature were maintained at 230 °C and 150 °C respectively (Jacomini et al., 2015). The verification of the identity of the compounds was performed by checking their mass spectra with data from NIST 11.0 libraries (National Institute of Standards and Technology, US).

Nuclear magnetic resonance spectroscopy (NMR)
Nuclear magnetic resonance spectroscopy (NMR) spectra were recorded on a Bruker Avance HD III spectrometer operating at 500.13 MHz for 1 H and 125.0 MHz for 13 C nucleus. Samples of the NL crude extract (10 mg

Statistical analysis
The results were presented as mean ± standard deviation (SD), followed by the statistical analysis employing the statistical software SISVAR version 5.3. ANOVA and Tukey's test (p<0.05).

Effect of sucrose on the accumulation of biomass in adventitious root cultures in vitro of S. rebaudiana
One of the main arguments to invest time and effort into the search for biotechnological alternatives for the production of bioactive metabolites is because the yields of these are normally low in the wild specimens, and often only found in specific plant tissues or species. However, natural production is rarely a viable option for the pharmaceutical and food industry. Therefore, several methodologies are being used to optimize the production of plant-derived metabolites, such as two-part culture systems, genetic engineering, and the use of bioreactor systems (Tocci et al., 2011;Georgiev, & Weber, 2014;Wilson, Cummings, & Roberts,2014;Simonetti et al., 2016). The elicitation technique, used to enhance the synthesis of metabolites in order to ensure plant survival, persistence or competitiveness, has proven to be a competent approach both in the ex vitro and in in vitro root systems (Tocci et al., 2012;Yin et al., 2012).
This bioreactor allows the flow of the culture medium to be uniform within the flasks, allowing the roots to properly absorb nutrients from the medium. This system was successfully used in the adventitious root cultures of S. rebaudiana by Reis et al. (2011) and Lopes et al. (2016).In this work, the effect of increasing concentrations of sucrose of 30 g L -1 , 60 g L -1 and 80 g L -1 onroot growth and the production of NLs in the adventitious root cultures of S. rebaudiana was evaluated.
The MS nutrient medium containing 30 g L -1 of sucrose resulted in the greatest root biomass, as the values of fresh weight (FW), dry weight (DW), and the growth index (GI) were better when compared to other sucrose treatments, as shown in Table 1.The lower root biomass in the treatments with 60 and 80 g L -1 of sucrose may be associated to the osmotic stress

Effect of sucrose stress on the neutral lipid composition of S. rebaudiana adventitious root cultures in vitro
In this study, the yields of the hexane extracts, basically the neutral lipids (free FAs, TAGs, DAGs and MAGs), increased as the supply of sucrose in the nutrient medium increased, see Table 1. It was already expected that the increased supply of sucrose would increase levels of NLs, as the sugar conversion (ATP) obtained through photosynthesis is closely related to the production of FAs in plant cultures, providing basic carbon skeletons (Xu, & Shanklin, 2016;Zhai et al., 2017). However, our results demonstrate that the sucrose concentration of the culture medium did not significantly influence the production of FAs. Oksman-Caldentey et al. (1994) also demonstrated that FA production was not greatly affected by the increment of sucrose concentration in root cultures in vitro of Hyoscyamus muticus.

1 H and 13 CNMR neutral lipid analysis
Through the analysis of 1 H NMR spectra of NLs from adventitious root cultures of S. rebaudiana, it was possible to detect the presence of FAs, TAGs, and DAGs, as shown in Figure 2 A, B, and Table 2. Table 3 shows the relative percentages of the PUFAs, MUFAs, and SFAs through the integration of the signals from the different proton environments. All signals were attributed by comparison with literature data (Thoss et al., 2012;Jacomini et al. 2015). Research, Society andDevelopment, v. 10, n. 10, e182101018651, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i10.18651 8 In addition to the 1 H NMR spectrum, the 13 C NMR spectra were also obtained, as shown in the Figure 2B and Tablr 2, further confirming the presence of NLs in the extracts. The chemical shifts to the triacylglycerides carbon atoms of NLs from S. rebaudiana adventitious roots were similar to literature data described for NLs from other plants (Thoss et al., 2012;Jacomini et al., 2015).
After the spectroscopic analysis, it was determined that SFAs predominate in all extracts regardless of the sucrose concentration to which the roots were submitted. Furthermore, the fraction of PUFAs was higher in roots treated with 30 g L -1 of sucrose and lower in roots treated with 60 g L -1 of sucrose. The fraction of MUFAs was lower in roots treated with 60 g L -1 of sucrose than in the other treatments, Table 3.

GC-MS analysis
The FAME profile of S. rebaudiana adventitious root cultures was established by GC-MS analysis ( Figure 3, Table   4). The peaks corresponding to the FAME content of the samples were compared and identified using the NIST library match software.
All mass spectra of FAMEs showed the peak at m/z 74 responsive to saturated FAMEs, monounsaturated FAMEs show a base peak at m/z 55 and polyunsaturated fatty acids was confirmed by the characteristic base peak at m/z 67 compared to literature data (Tariq et al., 2011;Gunawan et al., 2013).
GC-MS data analysis showed that SFAs were the major FAMEs in all extracts (Table 4), which palmitic acid was the main FA detected. Interestingly, the relative content of stearic acid, a SFA, increased significantly in the NL extract of roots grown in medium supplemented with 80 g L -1 of sucrose, with a relative percentage of 56.8%, in contrast to those grown with the opposite treatments of 60 g L -1 (5.90%) and 30 g L -1 of sucrose (5.10%). This means there was a gradual reduction in the relative concentration of UFAs with sucrose increase in the culture medium (Table 4).
The medium supplemented with 30 g L -1 of sucrose was the most favorable for UFA production in the roots when compared with the results of the other treatments, with a relative percentage of 62.9% as describe in the Table 4.
The application of osmotic stress by an increment of sucrose concentration did not appear to possess a negative influence on the relative amount of PUFA production; however, the sucrose concentration did not have a cumulative boosting effect on PUFA production either. Research, Society andDevelopment, v. 10, n. 10, e182101018651, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i10.18651 TAGs in cytosolic lipid droplets are not only involved in membrane remodeling, but they also provide a source for the production of bioactive compounds derived from PUFAs involved in the plant defense mechanism. According to Shimada et al. (2014) and Shimada, Takano, & Hara-Nishimura (2015) during fungal infections, leaf TAGs accumulate in lipid droplet types. These TAGs contain an abundance of PUFAs, which are consecutively arranged for the formation of oxylipins as defense compounds . Equivalent to the chloroplast plastoglobules, the profusion of cytosolic lipid droplets also increases during adverse environmental conditions and senescence (Singer, Zou, &Weselake, 2016). The amount of TAGs in the leaves appears to be linked to the stress tolerance of plants. Experiments that induced the suppression of TAG and FA degradation pathways, using mutant plants, resulted in an increment in leaf TAG content, but also caused necrosis and bleaching during dark and age-induced senescence (Slocombe et al., 2009). Together, this highlights the essentiality of TAG homeostasis in plant tissue. The effect of sucrose in the nutrient medium has been to increase the osmotic pressure causing oxidative stress in the plant cell with the formation of reactive oxygen species (ROS), such as hydrogen peroxide (H2O2). ROS, in turn, cause lipid peroxidation of PUFAs with the formation of by-products such as malondialdehyde (MDA) (Baque et al., 2012;Cui et al. 2010). This phenomenon could be occurring in our study and would explain the reduction of PUFAs in the roots treated with a higher concentration of sucrose. Thus, to prove the oxidative stress described it would be interesting in future studies to evaluate the production of these compounds. In addition, changes in sucrose concentration also alter the production of secondary metabolites in plants, such as phenolic (Baque et al., 2012;Cui et al. 2010) and terpenoid compounds (Yin et al., 2013;Manuhara et al., 2015). Future perspectives of this work would be to evaluate the production of other groups of substances like phenolic and terpenoid compounds in the adventitious roots of S.rebaudiana, as well as their respective biological activities.

Conclusion
The results of this work demonstrated that the MS/3 medium supplemented with 30 g L -1 of sucrose, for inducing a minor abiotic stress condition, is the more adequate for PUFA production/accumulation using the adventitious root cultures of

S. rebaudiana.
Using this adventitious root culture methodology, new results can be achieved by studying the effects of different carbon sources on the production of polyunsaturated fatty acids as well as other primary and secondary metabolites. Abiotic stress conditions that can improve the results presented here will also be used.