untitled ACTA BOT. CROAT. 75 (1), 2016 11 Acta Bot. Croat. 75 (1), 11–16, 2016 CODEN: ABCRA 25 DOI: 10.1515/botcro-2016-0015 ISSN 0365-0588 eISSN 1847-8476 Selenium induced selenocysteine methyltransferase gene expression and antioxidant enzyme activities in Astragalus chrysochlorus Özgür Çakir1*, Neslihan Turgut-Kara1, Şule Ari1,2 1 Department of Molecular Biology and Genetics, Faculty of Science, Istanbul University, 34134 Vezneciler Istanbul, Turkey 2 Research and Application Center for Biotechnology and Genetic Engineering, Istanbul University, 34134 Vezneciler, Istanbul, Turkey Abstract – Astragalus sp. are used in folk medicine because of their biological activities and are known for the ability to accumulate high levels of selenium (Se). The purpose of this study was to explore gene expres- sion of selenocysteine methyltransferase (SMT), responsible for forming MeSeCys, and activities of ascor- bate peroxidase (APX), peroxidase (POX), catalase (CAT) and glutathione reductase (GR) enzymes in callus tissues of Astragalus chrysochlorus growing in different Se-containing media. Quantitative real-time poly- merase chain reaction assay was done for quantifi cation of SMT gene transcript and it was normalized to ac- tin gene. It was found that transcript level of callus tissues grown at 5.2 μM and 26.4 μM Se-enriched media was lower than that of the control callus. In contrast, a high level of Se (132.3 μM) in the medium caused an approximately 4.26 times higher level of SMT transcript in callus than the control. APX, POX, CAT and GR enzymes were all effected by different Se concentrations. While POX and APX activities were higher then control, CAT and GR activities decreased. These results show that an increase of SMT gene expression led to a rise in APX and POX, but a suppression of CAT and GR enzymes activities in Astragalus chrysochlorus. This suggests that Se could be involved in the antioxidant metabolism in Astragalus chrysochlorus. Keywords: antioxidant enzymes, callus, real time pcr, selenium, selenocysteine methyltransferase * Corresponding author, e-mail: ozgurckr@istanbul.edu.tr Introduction Antioxidant selenoproteins contain selenium (Se) which is a very important element for animals, microorganisms and some other eukaryotes (Birringer et al. 2002, Berken et al. 2002). These organisms contain the selenocysteine (Se- Cys) that may be incorporated in selenoproteins. In addition to nutritional needs for Se, the element also has health effi - cacy. It is also effective in reducing the incidence of some debilitating disorders (McKenzie et al. 2001, Foresta et al. 2002, Beck et al. 2003, Soriano-Garcia 2004) and there is some evidence that Se plays a role as a cancer preventive agent when given in certain amounts (Ip 1998, Combs and Gray 1998, Fleming et al. 2001, Whanger 2004). Selenium is metabolized through the sulfur (S) assimi- lation pathway. Selenocysteine methyltransferase (SMT) is the key enzyme in Se metabolism and it was isolated from the Se hyperaccumulator Astragalus bisulcatus for the fi rst time (Neuhierl et al. 1999). The SMT enzyme couples the methyl group with selenocysteine (SeCys) to produce Se- methylselenocysteine. A. bisulcatus SMT gene was overex- pressed in Arabidopsis thaliana and Brassica juncea and it was shown that selenium accumulation and tolerance were enhanced in SMT transgenics (Ellis et al. 2004, LeDuc et al. 2004). In our previous work, SMT cDNA sequences were isolated from Astragalus chrysochlorus (Çakir and Ari 2013). SMT was expressed continuously in the tissues of the Se hyperaccumulator Astragalus bisulcatus (Picker- ing et al. 2003). While most plants cannot survive with high concentrations of Se, some of them can tolerate high con- centrations and accumulate Se, like A. bisulcatus. Thus, the amount of Se is of vital importance for plants because it could be toxic at high concentrations. Se can change a plant’s antioxidant capacity. This can be achieved on two ways; fi rst, by changing the antioxidant activity of seleno- compounds, and second, by Se inducing a plant’s antioxi- dants (Hartikainen 2005). Enzymes of antioxidant metabo- lism such as ascorbate peroxidase (APX), peroxidase (POX), glutathione reductase (GR) and catalase (CAT) play an important role against reactive oxygen species (Apel and ÇAKIR Ö., TURGUT-KARA N., ARI Ş. 12 ACTA BOT. CROAT. 75 (1), 2016 Hirt 2004, Habibi and Hajiboland 2012). Because of the bi- ological activities like hepatoprotective, antioxidative, im- munostimulative and antiviral properties, Astragalus spe- cies have been used in traditional medicine (Ionkova et al. 1997). The chemical contents of them have been broadly investigated because of their economic and medicinal im- portance (Calis et al. 1997, Bedir et al. 1998, Yahara et al. 2000). One of the Turkish endemics, A. chrysochlorus, has been determined as a secondary Se accumulator and a cDNA sequence of the SMT gene (accession number: GQ844862) named AchSMT was isolated (Ari et al. 2010). The AchSMT cDNA has an open reading frame (ORF) of 1020 bp and this ORF encodes 339 amino acid residues with a 36.94 kD molecular mass (Çakir and Ari 2013). The aim of this work was to investigate the expression pattern of the SMT gene and determine the alteration of ac- tivities of enzymes related to the antioxidant metabolism of A. chrysochlorus treated with Se. Here we report the activi- ties of APX, POX, CAT and GR enzymes in callus tissues of A. chrysochlorus grown in different concentrations of Se and the expression of a crucial gene related to Se metabo- lism by real-time quantitative PCR. Materials and methods Plant material Plants and seeds were collected from Sertavul, Kara- man, Turkey and were identifi ed by Prof. Dr. Tuna Ekim (Istanbul University, Faculty of Science, Department of Botany, ISTF no: 40006, Istanbul University, Faculty of Science Herbarium). Germination of seeds and callus induction Seeds were surface-sterilized in 70% alcohol for 1 min, then in 5% commercial bleach for 15 min, followed by three rinses for 15 min with sterile distilled water. Seeds were germinated aseptically in Petri dishes containing 25 ml of growth-regulator-free MS (Murashige and Skoog 1962) medium supplemented with 3% (w/v) sucrose and solidifi ed with 0.8% agar (w/v). The pH of the MS medium was adjusted to 5.8 before sterilization by autoclaving at 121 °C at 105 kPa for 20 min. The pH of the MS medium was confi rmed after sterilization. For callus induction, 30-day-old hypocotyl parts of seedlings were used. Callus proliferation was achieved with 0.5 mg L–1 2,4-dichloro- phenoxyacetic acid (2,4-D) and was subcultured every three weeks. The calli used in this study were grown in MS medium supplemented with 5.2 μM, 26.4 μM and 132.3 μM sodium selenate (Sigma, S8295). Seed germination, callus induction and subculture were carried out in a growth chamber illuminated with fl uorescent light (ca. 1400 μmol m–2 s–1) over a 16/8 day and night at 25 ± 2 ºC. Calli were treated with Se for three weeks, and after treat- ment, the calli were harvested, and their fresh weights were determined and samples were taken for various analyses. Enzyme extractions and assays Catalase (CAT, EC 1.11.1.6) activity was done accord- ing to Aebi (1984). Callus samples (0.5 g) were homoge- nized in 50 mM sodium phosphate buffer (pH 7.0) with pre- chilled mortar and pestle. The extract was centrifuged at 15000 g for 15 min at 4 °C, and the supernatant was used as enzyme extract. The reaction mixture contained 3% H2O2 and 0.1 mM EDTA in 50 mM sodium phosphate buffer. To measure the activity, absorbance was read at 240 nm, and it was calculated as μmol per minute. Peroxidase (POX, EC 1.11.1.7) activity was determined according to Putter (1974), using the guaiacol oxidation method in a 3 mL reaction mixture containing 50 mM sodi- um phosphate buffer (pH 7.0), 20 mM guaiacol and 20 mM H2O2. The increase in absorbance was recorded at 470 nm within 20 s through 2 min after H2O2 was added. A unit of peroxidase activity was expressed as μmol H2O2 decom- posed per minute. Ascorbate peroxidase (APX, EC 1.11.1.11) activity was determined according to the method of Nakano and Asada (1981). 50 mM sodium phosphate buffer (pH 7.0), 0.5 mM ascorbate, 0.1 mM H2O2, and 0.1 mL enzyme extract in a fi nal assay volume of 1 mL were the components of the re- action. Ascorbate oxidation was measured by reading ab- sorbance at 290 nm. To calculate the oxidized ascorbate concentration, the extinction coeffi cient (ε = 2.8 mM−1 cm−1) was used. 1 μmol ascorbate oxidized per minute was described as one unit APX. Glutathione reductase (GR, E.C. 1.6.4.2) activity was determined according to the method of Foyer and Halliwell (1976). GR activity was determined spectrophotometrically at 340 nm in a reaction mixture containing 50 mM Tris-HCl buffer (pH 7.6), 1 mM GSSG, and 10 mM NADPH. 1 μmol NADPH oxidized per minute was used as one unit GR. Protein concentration was determined according to Bradford (1976), using bovine serum albumin as a stan- dard. The specifi c enzyme activity for all enzymes was ex- pressed as units (U) per mg of proteins. RNA isolation and cDNA synthesis For RNA isolation 5.2 μM, 26.4 μM and 132.3 μM so- dium selenate-treated calli and control without sodium sele- nate were used. At the end of third week of treatment, total RNA was extracted (TRIzol Reagent, Invitrogen, Carlsbad, CA) and reverse-transcribed (SuperScript First-Strand Syn- thesis System, Invitrogen, 11904-018) from A. chrysochlo- rus. RNA isolation and fi rst strand cDNA synthesis were done according to the manufacturers instructions. Real-time PCR conditions and analysis Quantitative real time PCR (qPCR) was performed with the DyNAmo™ HS SYBR® Green qPCR Kit (Thermo Scientifi c) following the manufacturer’s instructions. Prim- ers are given in Table 1. The qPCR conditions were set as follows: 2 min at 50 °C (uracil-DNA glycosylase incubati- on), 5 min at 94 °C (pre-incubation), followed by 40 cycles of 30 s at 94 °C, 30 s at 64 °C and 30 s at 72 °C. To obtain melting curve data collection, a gradual temperature increa- se from 55 °C to 95 °C at the rate of 1 °C/10 s was added as a fi nal step. A non-template control was run alongside this, and serial dilutions (1, 1:5, 1:25 and 1:125) of the reference (actin) and SMT gene were included with every assay. SELENIUM INDUCED ANTIOXIDATIVE METABOLISM CHANGES IN ASTRAGALUS ACTA BOT. CROAT. 75 (1), 2016 13 Amplifi cation specifi city of each reaction was verifi ed by melting curve analysis. Actin was used to normalize expres- sion levels. MX3000 Stratagene software (Agilent Techno- logies, Inc., Santa Clara, USA) was used to determine rela- tive gene expression. All qPCR experiments were done in triplicate experiments for each sample. Statistical analysis All tests were done in triplicates. Analysis of variance was performed using Graphpad prism ver. 5.0 trial version. The data were presented as the means for each treatment. Data were compared using the one-way ANOVA test with post Tukey test at the 5% probability level. Results Establishment of callus cultures The hypocotyl parts of 30 day old seedlings produced friable and green callus on MS medium supplemented with 0.5 mg L–1 2,4-D after a 15-day culture period (Fig. 1A). After induction of callus, 2,4-D was used for proliferation purpose and A. chrysochlorus callus was maintained for over ten years by subculture at three week intervals on MS medium supplemented with 0.5 mg L–1 2,4-D. The calli were then taken and grown in media containing 5.2 μM, 26.4 μM and 132.3 μM sodium selenate for 21 days (Fig. 1B, C, D). The color red was observed when the concentra- tion of selenium was 132.3 μM (Fig. 1D). Effect of selenium treatments on antioxidant enzyme activities The activity of CAT decreased by 13%, 22% and 27% in the 5.2 μM, 26.4 μM and 132.3 μM Se-treated calli of A. chrysochlorus, respectively (Fig. 2A). In all calli, the activ- ity of this enzyme was not modifi ed and the decrease was not signifi cant statistically according to treatment with Se. The POX activity increased 4, 3 and 2 fold when compared to the control calli, respectively, after treatment with Se (Fig. 2B). Lower doses of Se (5.2 μM and 26.4 μM) have increased the POX activity more than 132.3 μM Se. The increase in the POX activity was statistically signifi cant in 5.2 μM and 26.4 μM Se-treated calli. The APX activity was signifi cantly increased in 5.2 μM and 26.4 μM Se-treated calli (Fig. 2C). The activity of this enzyme was higher in 132.3 μM Se treated callus tissue than in control tissues but the increase was not signifi cant. Treatment with Se resulted in an increase of GR activity in 5.2 μM-treated callus tis- sues and a decrease in 26.4 μM and 132.3 μM-treated calli tissues, respectively (Fig. 2D). However, in the 5.2 μM Se- treated callus tissues, this enzyme activity did not signifi - cantly change. Isolation and quality detection of total RNA Electrophoresis of isolated RNA on 1% agarose gel stained with EtBr showed distinct 28S and 18S rRNA bands, indicating a good quality of total RNA (On-line Sup- pl. Fig. 1). The A260/A230 absorbance ratio is usually calcu- lated to check polysaccharide or polyphenolic contamina- tion, and the A260/A280 ratio for protein contamination. We determined that A260/A230 ratios were greater than 2, and the A260/A280 ratios between 1.9 and 2.1. This result showed that the isolated RNAs were intact and not contaminated. Expression analysis of SMT gene To evaluate the expression of the transcript, the reverse- transcribed cDNAs of samples (control, and treated tissues with 5.2 μM, 26.4 μM and 132.3 μM of Se) were used as templates. SMT was amplifi ed by the method for an SYBR Green qPCR assay. We can conclude that PCR amplifi ca- tion curves show that amplifi cation has good reproducibili- ty in each sample. The Se metabolism pathway was studied extensively, and the conservation level of this pathway is high in plants. The expression of SMT were observed by qPCR in all sam- ples of selenium-treated Astragalus plant (Fig. 3). From the results obtained, we conclude that SMT transcript levels for 5.2 μM and 26.4 μM selenium-treated callus were lower than for the control callus tissues. In contrast, the SMT ex- pression level of 132.3 μM in selenium-treated callus was 4.26 times higher than in the control callus. Discussion Selenium hyperaccumulation is involved with SMT en- zymatic activity and accumulation of S-methylcysteine (MeCys) and MeSeCys (Sors et al. 2005). In this study, our Tab. 1. Primers used in quantitative real time PCR reactions. SMT – selenocysteine methyltransferase. Target Sequences (5′-3′) SMT Forward Revers ACGGAGCTTGAACGTCATGGTG ATCAAGATGCACCTGGCGAATGAG Actin Forward Revers TCAAGACGAAGGATG TTGGATTCTGGTGAT Fig. 1. Astragalus chrysochlorus callus: untreated control (A), and selenium-treated tissue: 5.2 μM (B), 26.4 μM (C), and 132.3 μM (D). ÇAKIR Ö., TURGUT-KARA N., ARI Ş. 14 ACTA BOT. CROAT. 75 (1), 2016 main object was to investigate the expression profi le of the SMT gene (AchSMT, Accession number:GQ844862) in- volved in selenium metabolism in A. chrysochlorus callus under increasing concentrations of selenium. We used qPCR to measure SMT gene expression. As a result, SMT expres- sions in 5.2 μM and 26.4 μM selenium-treated callus were slightly lower than in the control callus. In contrast, the SMT expression level of 132.3 μM selenium-treated callus was found to be higher and the fold change was 4.26. In our previous work (Çakir and Ari 2013), A. chrysochlorus plantlets were treated with 0, 5.2 μM, 26.4 μM and 132.3 μM sodium selenate and analysed with semi-quantitative reverse transcription PCR. According to the results, diffe- rentially, SMT expression sodium selenate treated plantlets were constitutively expressed like AbSMT from hyperaccu- mulator A. bisulcatus in all selenate treatments. SMT gene has been isolated from a Se hyperaccumula- tor A. bisulcatus (Neuhierl et al. 1999, Neuhierl and Bock 1996). It was thought that SMT is the key player in the ac- cumulation of selenium and protection of the plants from the toxic effects (Neuhierl et al. 1999). Pickering et al. (2003) concluded that SMT gene expression was continu- ous in tissues of A. bisulcatus. In contrast, Lyi et al. (2005) reported that broccoli SMT (BoSMT) expression is up-regu- lated by selenate treatment. Its expression in leaf has been determined to be involved in development. Tao et al. (2012) reported that in young leaves of selenium-treated tea plant, the expression level of SMT gene was similar to mature le- aves, and it was the highest in roots. These fi ndings sugges- ted that selenite is immediately converted to organic Se and remains in the root tissues. In the present study, we determi- ned that SMT expression level was differentially affected upon treatements with increasing concentrations of Se. In low concentrations (5.2 μM and 26.4 μM) SMT expression was lower than in control tissues (0.397 and 0.597 times, respectively). Our results revealed that the transcriptional levels of SMT gene fluctuated after Se treatment. The chan- ges or variations in expression levels of SMT gene after tre- atment with different concentrations of Se may be due to the delayed response of Se metabolism. Se assimilation and hyperaccumulation pathway are a very complicated meta- bolic process. One of the important approaches is to investi- gate SMT gene expression. Also further research into SMT at genetic translation level and the content of related amino acids in different parts of the A. chrysochlorus plant is nee- ded to validate SMT status in the process of selenium meta- bolism. Selenium is an important micronutrient for many orga- nisms. A. chrysochlorus has been found to be secondary Se Fig. 2. Antioxidant enzyme activities in callus tissue of Astragalus chrysochlorus treated with different concentrations of selenium: A) catalase (CAT), B) peroxidase C), ascorbate peroxidase (APX), D) glutathione reductase (GR). Asterisk (*) means statistically signifi cant (p < 0.05); bars indicate ± standard deviation. Fig. 3. Relative expression levels of selenocysteine methyltransfe- rase gene in callus tissue of Astragalus chrysochlorus treated with different concentrations of selenium in comparison to untreated control. The ratio of the relative quantity of the target gene and the relative quantities of reference genes (actin) of each treatment was normalized against the untreated control (value = 1) and plotted using arbitrary units. Each data point represents the mean ± SE, n = 3. SELENIUM INDUCED ANTIOXIDATIVE METABOLISM CHANGES IN ASTRAGALUS ACTA BOT. CROAT. 75 (1), 2016 15 accumulator in our previous study (Ari et al. 2010). Se is very important for antioxidant metabolism and response. In this study the activities of APX, POX, CAT and GR enzy- mes were also investigated. Antioxidant responses of A. ch- rysochlorus callus tissues varied in a concentration-depen- dent manner. CAT levels decreased gradually in increasing concentrations of Se, nevertheless this decrease was not signifi cant when compared to control. POX and APX showed similar activity patterns. At 5.2 μM level, there was a signifi cant increase, on the other hand at 26.4 μM and 132.3 μM levels the activities were decreased compared to the 5.2 μM level. GR activity was increased at 5.2 μM and decreased signifi cantly at higher concentrations. Under the concentration of 132.3 μM, Se stimulated antioxidant meta- bolism. As shown in fi gure 2, despite the decrease in CAT and GR activity, POX and APX activities were increased signifi cantly (p < 0.05). It is thought that the activities of POX and APX are more convenient to reduce the oxidative stress caused by Se. The results indicate that the increase in POX and APX activities were suffi cient to protect callus cells. At the higher concentration of 132.3 μM, growth rate of callus cultures was decreased and pigmentation started. It is thought that this pigmentation is related to biosynthesis of secondary metabolites and also it is an environmental stress response of A. chrysochlorus. However, POX activity was still higher than in control callus cultures. In our previ- ous studies, crude ethanol and ethyl acetate extracts of this plant exhibited cytotoxic activity and apoptosis-inducing properties (Karagoz et al. 2007) and also antioxidant effects (Hasancebi, unpublished data). Besides the biotechnologi- cal importance of Astragalus species, in callus tissues of the secondary Se accumulator A. chrysochlorus it was found that antioxidant enzymes and SMT gene expression were affected by Se treatment in callus tissues. Therefore, it is thought that it could be related and important for accumula- tion and tolerance to Se of these species. In conclusion, Se triggered significant transcriptional responses in A. chrysochlorus. The SMT gene was repressed by low concentrations of Se. 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