Antioxidant Potential in Stevia Rebaudiana Callus

Antioxidant potential in Stevia rebaudiana callus in response to polyethylene glycol, paclobutrazol and gibberellin treatments

Abstract

Stevia callus culture has the potential to advance drought resistance science by allowing study of antioxidant mechanisms in absence of potent antioxidants of steviolglycosides. The effect of polyethylene glycol (PEG; 0 and 4% w/v) in combination with paclobutrazol (PBZ; 0 and 2 mg l^-1) and gibberellin (GA; 0 and 2 mg l^-1) were studied on Stevia rebaudiana callus. PEG treatment led to an oxidative stress, as indicated by increased H₂O₂ level. The combination of PBZ and/or GA with PEG prevented high accumulation of H₂O₂. All treatments of PEG, PBZ and GA increased the total antioxidant capacity. The super oxide dismutase activity increased in PEG treatment alone or in combination with PBZ and/or GA. Interaction of PBZ and/or GA with PEG increased the catalase and ascorbate peroxidase activity to a level more than PEG treatment, which resulted in H₂O₂ scavenging. All treatments of PEG, PBZ and GA led to an increase in proline content, with the highest amount in PEG+PBZ. Although PEG increased proteins and amino acids, the interaction of PBZ and/or GA with PEG increased them more than PEG alone. The pyrroline-5-carboxylate synthetase and proline dehydrogenase activity remained similar to control in none of treatments. Collectively, our results demonstrated that PBZ and GA increased reactive oxygen species scavenging and osmolytes of Stevia callus to alleviate negative effects of PEG. These findings will enable us to design effective genetic engineering strategies in callus culture to generate some somaclonal variation that may be useful in enhancing drought resistance in Stevia.

Keywords: Callus; Drought stress; Gibberellin; Paclobutrazol; Polyethylene glycol; Stevia rebaudiana

Abbreviations: APX, ascorbate peroxidase; CAT, catalase; FRAP, ferric reducing antioxidant power; H₂O₂, hydrogen peroxide; KO, kaurene oxidase; KS, kaurene synthase; MS, Murashige and Skoog; P5CS, pyrroline-5-carboxylate synthetase; PDH, proline dehydrogenase; PEG, polyethylene glycol; ROS, reactive oxygen species; SVglys, steviol glycosides; SOD, superoxide dismutase; SV, Steviol.

1. Introduction

Many environmental stresses are responsible for limiting plant growth and production worldwide, including drought stress, high temperature and salinity. These adverse conditions induce osmotic stress in plant cells by decreasing water availability and increasing turgor pressure, thus leading to the accumulation of reactive oxygen species (ROS) that are detrimental to plant growth and development . Enhancement of antioxidant defense system is an important strategy to proficiently scavenge ROS with enzymatic and non-enzymatic antioxidants (Gupta & Prakash 2009; Anjum et al. 2012). In enzymatic systems, superoxide dismutase (SOD) scavenges O^2- with reaction product H₂O₂, whereas peroxidase, catalase (CAT) and ascorbate peroxidase (APX) decompose H₂O₂ to H₂O at different cellular locations (Noctor & Foyer 1998). ROS reacts with lipid membranes, proteins and DNA results in cell damage, following by cell death (Torres-Franklin et al. 2008). The accumulation of compatible solutes such as proline, proteins and amino acids protects the cell by balancing the osmotic pressure of cytosol, which in turn, protects cell from injury caused by maintaining turgor pressure during water deficiency (Anjum et al., 2012).

Stevia rebaudiana L. is a valuable medicinal plant originated from Paraguay with a subtropical climate and average rainfall of 1500 mm per year (Hajihashemi & Ehsanpour 2013). Stevia has a huge demand in industries of food and beverage as a source of high potency natural sweetener with low calorie. Today, its cultivation has spread to several regions of the world crop, including Iran, since consumer demand for this plant is increasing. Stevia cultivation in Iran with a hot and dry weather has some problems associated with drought stress. S. rebaudiana showed a little resistance to water stress under in vitro and greenhouse condition (Hajihashemi & Ehsanpour 2013; Hajihashemi & Geuns 2016; Hajihashemi & Ehsanpour 2014). Hence, the most practical approach for increasing Stevia yield in drought condition is to investigate features involved in cell response to drought stress.

Steviol glycosides (SVglys) are the compounds responsible for sweet taste of S. rebaudiana. Besides its sweet taste, antioxidant activities have been reported for this compound (Hajihashemi and Geuns, 2013). In SVglys biosynthesis pathway, four consecutive enzymes convert geranylgeranyl-diphosphate into steviol. Then, the glycosyltransferase enzymes produce different SVglys by glycosylation of steviol (Brandle & Telmer 2007). Swanson et al. (1992) reported that Callus of Stevia is not capable of biosynthesis of steviol (SV). They suggested that SVglys biosynthesis is a function of tissue differentiation (Swanson et al. 1992). Gibberellic acid (GA) and SV have similar tetra-cyclic diterpene skeletons with a common precursor for them. The enzymes of kaurene oxidase (KO) and kaurene synthase (KS) are involved in both GA and SV biosynthesis (Brandle & Telmer 2007). GA treatment significantly increased the transcription of ent-KS1, ent-KO in S. rebaudiana while the GA biosynthesis inhibitor ofpaclobutrazol (PBZ) reduced their transcription (Hajihashemi & Geuns 2016; Hajihashemi et al. 2013).

High crop production under unfavorable growth condition is one major target for researches in 21^th century. Knowledge of cell response to drought stress could be used to create new varieties of crops with higher yield under water deficient. Our previous study on Stevia plants showed that PEG-induced drought stress had negative effect on SVglys biosynthesis, plant growth and yield (Hajihashemi & Ehsanpour 2013; Hajihashemi et al. 2013; Hajihashemi & Geuns 2016).  Subsequently, we decided to investigate callus of Stevia to better understand unorganized cell responses to water deficit. In order of identifying traits that can decrease the cell sensitivity to dehydration, analysis of antioxidant capacity and osmolytes of callus exposed to PEG-induced drought stress seems to be a promising approach. In order to evaluate antioxidant system and osmolytes, the enzymes of SOD, CAT and APX, total antioxidant capacity (FRAP), H₂O₂, proteins, proline, total amino acids and activities of proline metabolizing enzymes (pyrroline-5-carboxylate synthetase, proline dehydrogenase) were studied. Study on Stevia showed that PBZ and GA treatments could partly alleviate negative effect of PEG treatment on plant (Hajihashemi & Ehsanpour 2013; Hajihashemi & Ehsanpour 2014; Hajihashemi & Geuns 2016). In a follow-up study, the interaction of PBZ or GA with PEG treatments was examined in which we figured out if these treatments can reduce negative effects of PEG-induced drought stress in callus. A comprehensive examination of the antioxidants mechanisms underlying in Stevia callus in response to interaction of PEG with GA or PBZ allowed the characterization of some antioxidant factors modulating the negative effects of drought stress and provided evidence that can be used in crop improvement.

2. Materials and Methods

2.1. Callus culture

S. rebaudiana (Bert.) seeds were provided from Prairie Oak Publishing, 221 South Saunders Street, Marville MO 64468. The seeds were cultured in MS (Murashige & Skoog 1962) medium. Stevia propagation was carried out on in vitro plants. Leaves of Stevia accumulate up to 30% of SVglys (Geuns 2003) so the plant leaves were used to generate callus. The plants leaves were cut in 1 cm² pieces. These explants were placed in MS medium containing 2, 4 D and kinetin (each at 2 mg l^-1), which is referred to as callus medium. The emerged calluses with 1 g weight were transferred to callus medium containing PEG (molecular weight 6000; 0 and 4% w/v), PBZ (0 and 2 mg l^-1) and GA (0 and 2 mg l^-1). In this experiment, there are 8 treatments including: control, PBZ, GA, GA+PBZ, PEG, PEG+PBZ, PEG+GA and PEG+PBZ+GA. All cultures were incubated at 26 ± 1 °C under 2500 lux light under a photoperiod of 16 h day/ 8 h night for one month. The treated calluses were used for further analysis.

2.2. Total antioxidant capacity (FRAP assay)

The antioxidant power of samples were measured according to Szôllôsi and Varga (2002).

2.3. H₂O₂ content measurement

The content of H₂O₂ was quantified using Velikova et al. (2000) method.

2.4. Protein and antioxidant enzymes measurement

Fresh leaves were extracted using sodium phosphate buffer (pH 7.8) for protein and enzyme analysis. Proteins were measured by Bradford (1976) method. The SOD activity assay was based on  Beauchamp and Fridovich (1971) method. The CAT activity measurement was done according to Aebi (1984). The APX activity was determined according to Asada (1992).

2.5. Total amino acids, Free proline and proline metabolism enzymes

The total amino acids contents were determination based on the Yemm et al. (1955) method. Free proline was determined according to Bates et al. (1973) method. The Pyrroline-5-carboxylate synthetase (P5CS; EC 1.2.1.41) activity was determined by Stines et al. (1999) method. The Proline dehydrogenase (PDH; EC 1.5.1.2) activity was assayed according to (Rena & Splittstoesser 1975) method.

2.6. Statistical analysis

The experimental set-up was designed based on a Randomized Block Designs with 3 biological replicates and each replication comprised up to 12 jar.The data were analyzed by the ANOVA test’s SPSS (version 16) statistical package to assess significant differences (at the 5 % level) between means. The statistical analysis results are shown by superscripted letters to reveal significant differences.

3. Results

The ferric reducing antioxidant power (FRAP) method was used to measure the total antioxidant capacity. The results showed a significant increase in total antioxidant capacity of PBZ, GA and PEG-treated calluses (Fig. 1). FRAP in PBZ and GA treatments was more than PEG (with or without PBZ and GA) treatment.  As the results show, the H₂O₂ content was not affected by PBZ and PBZ treatments, while PEG (with or without PBZ and GA) significantly increased it (Fig. 1).  The highest H₂O₂ content was observed in PEG-treated callus, by about 2-folds more than control. Fig. 2 shows that PBZ, GA and PEG treatments significantly enhanced protein contents in calluses. The highest and lowest increase in protein content was observed in PBZ and PEG treatments, respectively. The activity of three key antioxidant enzymes of SOD, CAT and APX were evaluated in Stevia callus. PBZ and GA treatments showed no significant effect on SOD activity, while PEG (with or without PBZ and GA) in MS medium significantly increased SOD activity (Fig. 2). The SOD activity increased in Stevia callus exposed to PEG by about 2.5-fold more than control. Similar to SOD enzyme, PEG (with or without PBZ and GA) treatment significantly increased CAT and APX activity, while no significant changes in their activity was observed in PBZ and GA (without PEG) treatments (Fig. 2).

The results showed that the total amino acids content significantly increased in PBZ, GA and PEG-treated calluses (Fig. 3). The increase in PBZ treatment alone or with GA and/or PEG was more than GA and/or PEG treatments without PBZ treatments. In presence of PBZ in the medium culture, the total amino acids content was almost 5-fold more than control. Proline accumulation in PBZ, GA and PEG-treated calluses was significantly more than control callus (Fig. 3). The data showed that the effect of PBZ treatment on the proline content was similar to the total amino acids content, with the highest increase more than GA and/or PEG treatments. As shown in Fig. 3, PBZ, GA and PEG treatments had no significant effect on the activity of P5CS and PDH which are involved in proline metabolism.

4. Discussion

The first objective of present study was to evaluate the effects of PEG-induced drought stress on the leaves-derived callus of S. rebaudiana. The second aim of study was to understand if PBZ (a GA biosynthesis inhibitor) and GA treatments could reduce negative effects of PEG on Stevia calluses. Our previous study showed that PBZ treatment reduced the negative effects of PEG on physiology and growth of Stevia plants but it could not reduce negative effect of PEG on the transcription of SVglys biosynthetic genes and SVglys content. In contrast to PBZ, GA treatment reduced the adverse effects of PEG on the transcription of SVglys biosynthetic genes and SVglys content in Stevia plant (Hajihashemi & Ehsanpour 2013; Hajihashemi et al. 2013; Hajihashemi & Ehsanpour 2014).  Therefore, it became imperative to us to study the effect of GA or PBZ treatments and their interaction with PEG on calluses of Stevia without SVglys biosynthesis capacity.

The results demonstrate that unorganized cell masses of S. rebaudiana callus accumulated high amount of H₂O₂ in response to PEG which supports our previous finding in Stevia plant (Hajihashemi & Ehsanpour 2014a; Hajihashemi & Ehsanpour 2013). PBZ and GA treatments reduced H₂O₂ accumulation in PEG-treated calluses which confirms results of PBZ and PEG interaction in Stevia plant (Hajihashemi & Ehsanpour 2014). Water stress is inevitably associated with increased oxidative stress due to enhanced accumulation of ROS, particularly H₂O₂ in chloroplasts, mitochondria, and peroxisomes. As a result, the induction of antioxidant enzyme activities is a general adaptation strategy which plants use to overcome oxidative stresses (Foyer & Noctor 2003). PEG provoked a severe drought stress in Stevia plant that could partly be restored by antioxidant capacity associated with active enzymatic and non-enzymatic defense systems (Hajihashemi & Ehsanpour 2013; Hajihashemi & Ehsanpour 2014). Non-enzymatic antioxidants of ST and Reb A showed very potent radical scavenging activity towards both ROS and reactive nitrogen species (Hajihashemi & Geuns 2013). Hence, antioxidant mechanism in PEG-treated calluses (in absence of SVglys) maybe different from Stevia Plant. The total antioxidant activity of calluses (FRAP) significantly increased in PBZ, GA and PEG treatments and their interactions. On contrary to callus results, PBZ treatment did not affect FRAP in Stevia plant (Hajihashemi & Ehsanpour 2013). It shows a variation between antioxidants capacity of Stevia calluses and plants.

Results revealed a positive correlation between H₂O₂ and SOD activity in the PEG-treated calluses which was not found out in Stevia plants (Hajihashemi & Ehsanpour 2014). H₂O₂, which resulted from the action of SOD, is toxic to cells. Therefore, it is important to scavenge H₂O₂ rapidly by the antioxidative defense system to water and oxygen (Guo et al. 2006). It should be mentioned that SOD activity in PEG, PEG+PBZ, PEG+GA and PEG+PBZ+GA treatments was almost the same while H₂O₂ contents in PEG+PBZ, PEG+GA and PEG+PBZ+GA treatments is significantly less than PEG treatment. It suggests that all of these treatments produce the same amounts of H₂O₂ whereas PBZ and GA treatments activated some ROS scavengers to reduced oxidative stress. The overexpression of SOD, if accompanied by enhanced H₂O₂ scavenging mechanisms like CAT enzyme, has been considered as an important anti-drought mechanism to cope with oxidative stress during water deficit conditions (McKersie et al. 1999). Interestingly, CAT and APX played important role in H₂O₂ detoxification in calluses which was followed by less accumulation of H₂O₂ in PEG, PEG+PBZ, PEG+GA and PEG+PBZ+GA treatments comparing to PEG treatment.

A common adverse effect of drought stress is ROS production which react with proteins, resulting in protein denaturation (Torres-Franklin et al. 2008). Improvement in protein accumulation in response to drought stress not only helped in maintaining tissue water status but also protected from drought induced ROS (Anjum et al. 2012). According to the results of present study, PEG treatment alone increased proteins of calluses, while PBZ and/or GA treatments significantly increased proteins accumulation in PEG-treated plants. Therefore, higher protein accumulation in PBZ and GA in combination of PEG is a promising approach to increase drought resistance in calli. More study on proteins is a promising approach to better understand the responsible mechanism that can be used in biotechnology to produce drought resistance cells.

The osmotic adjustment in drought stress can be achieved by accumulation of amino acids, proline, proteins, and other solutes in the cytoplasm improving water uptake from drying soil (Anjum et al., 2012). The total amino acids and proline contents significantly increased in PEG, PBZ and GA-treated calluses, with the highest accumulation in PBZ treatment alone or in its interaction with GA or PEG.  It is reported that free amino acid accumulation in chickpea is due to protein hydrolysis (Khalil et al. 2014). However, in the present study it cannot be correlated to protein degradation because protein contents increased under different treatments. Thus, it can be suggested that they serve as an organic nitrogen pool to be used for protein synthesis. Proline accumulation in response to water-deficit preserves the structure of complex proteins, maintains membrane integrity, reduces injury to proteins and cells, oxidation of lipid membranes or photo-inhibition, and increases drought-tolerance (Anjum et al. 2012). P5CS catalyzes proline biosynthesis, while PDH mediates proline oxidation (Raymond & Smirnoff 2002). In our experiment, eventhough,the P5CS and PDH activity were not affected by none of PEG, PBZ and GA treatments, the proline content significantly increased in response to PEG, PBZ and GA treatments. In contrast, there is a repot of  increasing PDH and P5CS activity in response to water deficit in mulberry, which was followed by increased proline content (Chaitanya et al. 2009). These results of proline accumulation. confirm our previous observations in S. rebaudiana plants under PEG and PBZ treatments (Hajihashemi & Ehsanpour 2014; Hajihashemi & Ehsanpour 2013) which shows the ability of Stevia plants and calluses to maintain drought tolerance by proline accumulation. Also, it can be suggested that PBZ treatment is more effective than GA treatment in reducing PEG-induced turgor pressure by higher accumulation of proline and amino acids.

5. Conclusion

In conclusion, PEG-induced drought stress resulted in oxidative stress in callus of Stevia by ROS accumulation, supporting the results of PEG treatment in Stevia Plant. The PBZ and GA treatments increased proteins and the activity of antioxidant enzymes against PEG treatment to remove toxic oxygen radicals. Moreover, the PBZ and GA treatments increased total amino acids and proline in PEG-treated calluses to reduce drought-induced turgor pressure, more effectively PBZ treatment. These results provided information that could be used for producing drought resistance crops from calluses using somaclonal variation techniques. To this aim, genome wide analyses are suggested to achieve a comprehensive outlook of the traits involved in antioxidants mechanisms.

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