TY - JOUR
AU - Lang,, Minglin
AB - Abstract Organic selenium (Se), specifically Se-methylselenocysteine (MeSeCys), has demonstrated potential effects in human disease prevention including cancer and the emerging ameliorating effect on Alzheimer’s disease. In plants, selenocysteine methyltransferase (SMT) is the key enzyme responsible for MeSeCys formation. In this study, we first isolated a novel SMT gene, designated as BjSMT, from the genome of a known Se accumulator, Brassica juncea L. BjSMT shows high sequence (amino acid) similarity with its orthologues from Brassica napus and Brassica oleracea var. oleracea, which can use homocysteine (HoCys) and selenocysteine (SeCys) as substrates. Similar to its closest homologues, BjSMT also possesses a conserved Thr187 which is involved in transferring a methyl group to HoCys by donating a hydrogen bond, suggesting that BjSMT can methylate both HoCys and SeCys substrates. Using quantitative real-time PCR (qRT-PCR) technology and BjSMT-transformed tobacco (Nicotiana tabacum) plants, we observed how BjSMT responds to selenite [Se(IV)] and selenate [Se(VI)] stress in B. juncea, and how the phenotypes of BjSMT-overexpressing tobacco cultured under selenite stress are affected. BjSMT expression was nearly undetectable in the B. juncea plant without Se exposure, but in the plant leaves it can be rapidly and significantly up-regulated upon a low level of selenite stress, and enormously up-regulated upon selenate treatment. Overexpression of BjSMT in tobacco substantially enhanced tolerance to selenite stress manifested as significantly higher fresh weight, plant height, and chlorophyll content than control plants. In addition, transgenic plants exhibited low glutathione peroxidase activity in response to a lower dose of selenite stress (with a higher dose of selenite stress resulting in a high activity response) compared with the controls. Importantly, the BjSMT-transformed tobacco plants accumulated a high level of Se upon selenite stress, and the plants also had significantly increased MeSeCys production potential in their leaves. This first study of B. juncea SMT demonstrates its potential applications in crop MeSeCys biofortification and phytoremediation of Se pollution. Biofortification, BjSMT, Brassica juncea L, phytoremediation, selenium, Se-methylselenocysteine Introduction Since Swedish scientist Jakob Berzelius discovered selenium (Se) in 1817, it has been known to be an essential micronutrient for humans and animals. Recent studies indicated that supplementation of organic Se in the human diet could produce surprising benefits in a number of intractable diseases, including cancer (Soriano-Garcia, 2004), thyroid disease (Soriano-Garcia, 2004; Ventura et al., 2017), and Alzheimer’s disease (Zhang et al., 2017). Schwarz et al. (2017) found that Se has an antioxidant effect (Schwarz et al., 2017) and, as a component of glutathione peroxidase (GSH-Px), it plays a vital role in free radical scavenging activity (Sors et al., 2009). In the normal concentration range, Se has beneficial effects on health such as improving male fertility and immune function, reducing virus infection, and delaying the aging process (McKenzie et al., 2001; Beck et al., 2003; Soriano-Garcia, 2004). Se also plays an influential role in cancer defense as an anticancer and Se-rich diet can reduce the risk of cancer in humans (Ip et al., 2002). In contrast, Se deficiency is an important cause of Keshan disease (Whanger, 1989) and Kaschin–Beck disease (Peng and Yang, 1991; Li et al., 2016). However, excess amounts can also cause poisoning characterized by skin damage and nervous system abnormalities; in severe cases, growth deformities and even death have occurred in fish, birds, and human (Moxon, 1937; Eisler, 1985; Hu et al., 2016). In higher plants, the essential function of Se has not yet been determined (Zhang and Gladyshev, 2010). However, most plants do not accumulate Se (White et al., 2004) and, on the contrary, the accumulation of Se in the branches of super-Se-enriched plants can reach 1000–15 000 mg Se g−1 DW (0.1–1.5%) when grown on soil with low Se (Virupaksha and Shrift, 1965; Galeas et al., 2007). Therefore, the role of Se in plants should not be overlooked. It is well known that plants can easily absorb and assimilate Se in the form of selenate and selenite through sulfur transport proteins and metabolic pathways (Smith et al., 1995; Terry et al., 2000; Shibagaki et al., 2002; Maruyama-Nakashita et al., 2004; White et al., 2004), and remove it by converting it into volatilized methylated forms. Being an abundant bioavailable form in oxidized soil, selenate that has activity similar to sulfate is transported into the cytoplasm through sulfate transporters that showed differential expression in hyperaccumulators and non-hyperaccumulators (Sors et al., 2009; Schiavon et al., 2015). Both are antagonistic (i.e. compete to bind with permease); however, their selectivity varies depending on the nutritional value of sulfur to the plant (White et al., 2004). Selenite is a dominant form in anaerobic soil, and its transportation mechanism is not well known. There is strong evidence that supports selenite uptake through phosphate (Pi) transporters and reflects the sharing of uptake mechanisms like Pi (Zhang et al., 2014). However, the Se substitution of S in proteins can destroy the molecular function of these proteins, so an increased level of Se is toxic to most organisms. In plants, selenates are reduced and assimilated to organic Se which can be converted to methylselenocysteine (MeSeCys) in addition to selenocysteine (SeCys), selenomethionine (SeMet), and dimethylselenide (DMSe); MeSeCys has the best anticancer effect (Ellis and Salt, 2003; Sors et al., 2005b; Unni et al., 2005). This form of Se can be safely accumulated because it is not incorporated into proteins and it is thought to be the critical mechanism for plant Se detoxification. MeSeCys is also the precursor of dimethyl diselenide (DMDSe) in plants, another form of volatile Se (Neuhierl and Bock, 1996; Terry et al., 2000), and the ability of a plant to produce MeSeCys is closely related to its ability to hyperaccumulate Se (Dunnill and Fowden, 1967; Brown and Shrift, 1982; Sors et al., 2005a). As higher plants may have lost their basic Se metabolic function during evolution, the main accumulated form of Se in the leaves of common concentrated Se plants is SeCys, which may induce toxicity. The key enzyme in the conversion of SeCys to MeSeCys is selenocysteine methyltransferase (SMT) (Neuhierl and Bock, 1996; Neuhierl et al., 1999). The expression level of SMT in high-Se plants is higher than in common plants, leading to high accumulation of MeSeCys thus preventing SeCys being misincorporated into proteins (Brown and Shrift, 1981, 1982; Neuhierl and Bock, 1996). The first attempt at overexpressing the SMT gene from the Se hyperaccumulator Astragalus bisulcatus in the Se non-accumulator Arabidopsis thaliana significantly increased the plant’s selenite tolerance and Se accumulation in MeSeCys form (Ellis et al., 2004). The expression of SMT also increased the volatilization rate and produced more volatile Se in the form of DMDSe. LeDuc et al. (2004) had come to the same conclusion but found that the effect is not significant when plants are supplied with selenates, which may be due to the fact that the conversion of selenate to selenite appears to be a rate-limiting step in the production of SeCys (LeDuc et al., 2004). As the addition of tomato in food processing has good nutritional value, the A. bisulcatus SMT gene was overexpressed in tomato, and selenite or selenate were provided in the medium during the fruit development period (Brummell et al., 2011). The results showed that the content of MeSeCys in fruit increased to 16% of total Se content (Brummell et al., 2011). Plants from the Brassica genus such as mustards and cabbages are good Se accumulators. Indian mustard (Brassica juncea), a metal-rich plant (Salt et al., 1995), can efficiently synthesize MeSeCys, making the plant a good Se accumulator (Wiesner-Reinhold et al., 2017). However, the SMT gene in Indian mustard has not been reported. LeDuc et al. (2004) overexpressed the A. bisulcatus SMT gene in Indian mustard which significantly increased the plant’s Se accumulation and volatilization compared with that of wild-type controls when Na2SeO3 was supplied in the medium (LeDuc et al., 2004). The aforementioned transgenic practices suggest the feasibility of transforming A. bisulcatus SMT into normal plants to creating Se-rich or hyperaccumulating plants which can be used for the purpose of removing excess Se from Se-contaminated soil and waste water (phytoremediation) (Banuelos and Meek, 1990; Hansen et al., 1998; Terry et al., 2000). These practices may also be useful in food fortification and human disease prevention in areas where Se deficiency is widespread (Ellis et al., 2004). All these concerns prompted the further exploration of the mechanism of the Se metabolic pathway and its functional genes in crops and model plants. Brassicales plants are of particular interest because of their ability to accumulate and synthesize more health-beneficial forms of Se such as MeSeCys (Wiesner-Reinhold et al., 2017) than Se non-accumulators which have shown no SMT activity (Sors et al., 2009). In this study, we cloned and functionally characterized a novel SMT gene named BjSMT from Indian mustard. BjSMT was overexpressed in tobacco to investigate the effect of selenite stress at different concentrations on transgenic tobacco plants. The results showed an increase of MeSeCys production in tobacco leaves and inferred enhancement of tolerance and total Se accumulation upon selenite stress, suggesting that BjSMT has potential use for crop MeSeCys biofortification and phytoremediation of Se pollution. Materials and methods The sequential methodology followed throughout this study is summarized in an experimental methodology flowchart (Fig. 1) which includes the substeps of the main experiments along with the given conditions. Fig. 1. Open in new tabDownload slide A flowchart of the experimental strategy used in this study. The sequential diagram provides an overview of the complete methodology followed. Every part is further articulated with detailed subsequent steps together with the growth medium (e.g. Hoagland solution, MS medium, and addition of selenite) and conditions, namely the days to reshuffle the plants and to do the final experiment, together with the names of methods. The figure explaining the experimenalt results has also been mentioned. (This figure is available in color at JXB online.) Fig. 1. Open in new tabDownload slide A flowchart of the experimental strategy used in this study. The sequential diagram provides an overview of the complete methodology followed. Every part is further articulated with detailed subsequent steps together with the growth medium (e.g. Hoagland solution, MS medium, and addition of selenite) and conditions, namely the days to reshuffle the plants and to do the final experiment, together with the names of methods. The figure explaining the experimenalt results has also been mentioned. (This figure is available in color at JXB online.) BjSMT gene cloning and transformation According to the available SMT nucleotide and amino acid sequences in GenBank (accession nos LOC106396739, AY817737, LOC106342238, LOC103862723, AAM65096.1, ACV03424.1, and LOC104894536), homologous BjSMT forward and reverse primers containing XbaI and BamHI restriction sites were designed as BjSMT-F1: 5'-tgcTCTAGAGAGGTTGTTGGGATGGTGAC-3' and BjSMT-R1: 5'-cgcGGATCCGGAAATCTAATGGTGCGAG-3', respectively. Genomic DNA from B. juncea L. was extracted by the cetyltrimethylammonium bromide (CTAB) method (Springer, 2010). A 50 μl aliquot of PCR buffer was prepared containing 25 μl of 2× PrimeSTAR GXL Premix (Takara), 1 μl of genomic DNA (100 ng μl–1), 1 μl of forward primer (20 μM), and 1 μl of reverse primer (20 μM). The PCR conditions were set at 5 min at 94 °C, 30 s at 94 °C, 30 s at 65 °C, 2 min at 72 °C (30 cycles), and 10 min extension at 72 °C. The BjSMT PCR products were cloned into the pEasy-T1 Cloning vector (TransGen Biotech) for sequencing (Shanghai UGI-SERVICE, China), then the obtained BjSMT genomic sequences were analyzed by BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and deposited in GenBank (GenBank accession no. MH197041). For constructing the plant BjSMT–green fluorescent protein (GFP) expression vector, we designed another reverse primer containing BamHI restriction sites but without the stop codon: BjSMT-R2: 5'-cgcGGATCCATGGTGCGAGGAAG-3', and the BjSMT genomic DNA without the stop codon was PCR amplified by using the BjSMT-F1 and BjSMT-R2 primer pair and re-cloned into the pEasy-T1 vector using the same procedure. The pBI121-GFP vector used for plant transformation was the same as previously described, which contains the 35S promoter of Cauliflower mosaic virus (CaMV), the GFP marker gene, and an NOS (nopaline synthase) terminator, an nptII cassette used for selection in plants under the control of the nos promoter and terminator (Lang et al., 2011). The plasmids pEasy-T1-BjSMT and pBI121-GFP were double digested by XbaI and BamHI enzymes, respectively, and the purified BjSMT genomic DNA with sticky ends produced by XbaI and BamHI was ligated into the digested pBI121-GFP vector. The obtained pBI121-BjSMT-GFP construct was verified by PCR analysis and sequencing, and then it was transformed into tobacco (Nicotiana tabacum L. W38) following the method used previously(Lang et al., 2011). The control pBI121-GFP empty vector-transformed tobacco plants were generated synchronously. The stably inherited transgenic lines were screened firstly on Murashige and Skoog (MS) medium supplemented with 1 mg l−1 6-benzyladenine (6-BA), 0.1 mg l−1 naphthaleneacetic acid (NAA), 100 mg l−1 kanamycin, 500 mg l−1 cefatoxamine, and 8 g l−1 agar, till the regenerated shoots grew to 1.0–1.5 cm in height, then they were screened on 1/2 MS medium supplemented with the same compositions only without 6-BA, till the shoots grew to 4–5 cm in height. The obtained transgenic seedlings were verified with PCR using the BjSMT primers F1 and R2 to detect an 1860 bp BjSMT genomic DNA fragment by using the transgenic plants’ genomic DNA as a template, and a 1068 bp BjSMT cDNA fragment by using the transgenic plants’ cDNA as a template. Seeds of B. juncea were a gift of the North Central Regional Plant Introduction Station (NCRPIS) of the US National Plant Germplasm System (NPGS), which were grown as described previously (Lang et al., 2005). All plants were grown routinely under a 16 h light and 8 h dark cycle at 25 °C. BjSMT sequence analysis The nucleotide and protein sequences of BjSMT were queried in BLASTN and BLASTP of the NCBI database by considering all the default options to determine the gene and protein homologues. The encoded protein was further processed by online tools to examine different signals and properties, such as the TMHMM server (version 2.0) used for cross-membrane structural analysis (Krogh et al., 2001), ProtParam was applied to predict the physical and chemical properties (Gasteiger et al., 2005), and the signal peptide of BjSMT was analyzed by SignalP (version 4.1). The subcellular localization of BjSMT was predicted by TargetP (version 1.1; Emanuelsson et al., 2000) followed by phylogenetic tree construction from SMTs of different plants using the phylogeny.fr server that employs the built-in MUSCLE program for aligning the sequences, Gblocks to refine the alignment, and PhyML for constructing the evolutionary tree. The protein sequences of all the SMTs were input by using the one-click phylogenetic analysis option on the interface, and the tree was generated by the WAG (Whelan and Goldman) amino acid eplacement matrix (Dereeper et al., 2008). 3D structure analysis of the BjSMT protein A 3D model of BjSMT was constructed using the online I-TASSER server that simulates the model by iterative template-based fragment assembly (Yang et al., 2015). It requires the protein sequence and produces five models as output with their confidence score. All these models were further evaluated by Verify 3D (determines the models’ compatibility by amino acid location and environment together with comparison with known structures) and ERRAT (plots error function per position of residue by analyzing the statistics of non-bonded interactions between atoms) which assist in checking the topological quality of the model (Colovos and Yeates, 1993). Together with topology, phi and psi bonds angles were also evaluated by Ramachandran plot that filters the residues with the incorrect angles into the outside region of the graph. Moreover, the ConSurf server was used to observe the conservation of amino acids at specific positions by utilizing the Neighbor–Joining (NJ) algorithm-based phylogenetic tree (Eisenberg et al., 1997; Glaser et al., 2003). BjSMT transcriptional level analysis The seeds of B. juncea L. were sown in a flower pot mixed with vermiculite and nutrient soil (3:1) after soaking and budding. The culture conditions are the same as for tobacco. After 7 d growth, Indian mustard seedlings were rinsed with clean deionized water five times to remove the nutrient soil. After 1 d of culture in Hoagland nutrient solution (see Supplementary Table S1 at JXB online), 40 μM Na2SeO3, 40 μM Na2SeO4, 40 μM Na2SeO3+1 mM MgSO4, or 40 μM Na2SeO4+1 mM MgSO4 was added to the medium. Semi-quantitative reverse transcription–PCR (sqRT–PCR) was used to check the BjSMT mRNA level, following an approach we used before (Lang et al., 2011). Briefly, total RNA was extracted by using the all-Gold TransZolUp total RNA extraction Kit (TransGen Biotech), and the cDNA was reverse transcribed using the full-style Easyscript First Strand cDNA Synthesis Supermix kit (Takara) with 1 μg of total RNA as the template with reference to the manufacturer’s specifications. The B. juncea actin gene was used as an internal control for the relative quantification of transcript levels. The primer sequences were designed for sqRT–PCR and the subsequent quantitative real-time PCR (qRT–PCR) as follows: 5'-GATTCTGAAGAAGCGGCCTAT-3' and 5'-TGTTGAACAGGCCACGATTCAG-3' for BjSMT to amplify a 165 bp product; and 5'- CAGGAATCGCTGACCGTATG-3' and 5'-GTTGGAAGGTGCTGAGGGAT-3' for BjACTIN to amplify a 141 bp product. Three repeated tests were set up for the experiment, and each group contains roots or leaves of 4–6 plants. PCR band intensities were measured using ImageJ software. For testing the temporal and spatial expression level of BjSMT upon stress with a low dose of selenite, the 20-day-old Indian mustard seedlings were removed from the pots and washed with water to remove residues, then washed five times with deionized water. Thereafter, the seedlings were cultured for 5 d each in 1/4, 1/2, and 1/1 Hoagland nutrient solutions in succession and treated with Se by adding 300 μg l–1 Na2SeO3 in Hoagland nutrient solution. The control group was grown without adding Na2SeO3. The transcriptional levels of BjSMT in the leaves of Indian mustard were detected by qRT-PCR after Na2SeO3 treatment at 0, 4, 12, and 24 h. Three biological repeats were set up for each group containing leaves of 4–6 plants. Total RNA extraction and cDNA synthesis were the same as described for sqRT–PCR. RT-PCRs were monitored on an iCycler (Bio-Rad) by means of the SYBR® Premix Dimer Eraser™ kit (Takara). mRNA expression levels were determined relative to BjACTIN expression by relative quantification. Statistical analysis was performed using the Student’s t-test. Selenium tolerance analysis Transgenic tobacco seeds were first sterilized with 75% alcohol for 30 s and then with 0.5% NaClO for 10 min. After washing thoroughly with sterile water five times for 2 min each, the water on the surface of the seed was removed with sterilized filter paper. By using sterilized toothpicks, seeds were laid on 1/2 MS medium (100 mg l−1 kanamycin, 10 g l−1 agar, and 30 g l−1 sucrose). After growth for 2 weeks, seedlings at the same growth stage were selected and transferred to 1/2 MS medium with or without Na2SeO3 (0, 60, 120, and 240 μM) for 20 d. The cultivation conditions were 28 °C for a 16 h light/8 h dark cycle along with 1000 Lux of light intensity. The plant height, root length, root weight, and fresh weight of the two genotypes were measured at different concentrations. GSH-Px activity and quantification of chlorophyll content Under the above stress conditions (1/2 MS medium with 0, 60, 120, and 240 μM Na2SeO3 addition, treated for 20 d), the GSH-Px activity in tobacco leaves was determined by the 2,4-dinitrothiocyanate benzene (DNTB) method. A 0.1 g aliquot of leaves was ground rapidly in liquid nitrogen, with 1.5 ml of 0.2 mol l–1 phosphate buffer [containing 1 mol l–1 EDTA-Na, 2.5% water-soluble polyvinylpyrrolidone (PVP), pH 6.2] and homogenate, and centrifuged for 4000 rpm for 10 min. The absorptivity of GSH-Px was calculated by measuring the absorptivity of λ 340 nm at 10 s and 190 s. The change in chlorophyll content was determined by the Chlorophyll Assay Kit. In the condition of avoiding light, the leaves were ground and the extract was extracted for 3 h, then the content of chlorophyll was determined by spectrophotometry. Selenium and MeSeCys quantification Tobacco seedlings were grown on the screening medium for 2 weeks, then seedlings with consistent growth were selected for treatment with different concentrations of selenite in pot soil and MS medium, respectively. After 30 d of treatment with 0 μM and 60 μM Na2SeO3 in MS medium, the BjSMT transgenic and control pBI121 vector-transformed tobacco plants were harvested to determine their contents of MeSeCys and total Se. Each group contained 12 seedlings. The seedlings treated with selenite were divided into two parts: roots and shoots, and the samples were dried at 80 °C for 24–48 h. Each sample (0.5 g) was weighed into a centrifuge tube filled with 6 ml of concentrated HNO3 and 2 ml of 30% H2O2. Following 16 h of digestion, the tube was kept at 90 °C for 10 min and at 120 °C for the next 40 min (Ellis et al., 2004). Soon after adding 50 ml of deionized water for dilution, the samples were assayed by inductively coupled plasma mass spectrometry (ICP-MS) (Ellis et al., 2004). Seedlings were treated with 0, 60, and 120 μM Na2SeO3 in the pot soil for 60 d, and their total Se in the roots and leaves was determined in the same way. Each group contained 3–4 plants, and ~2 g of each sample was used for mineralization. For the determination of MeSeCys content of transgenic seedlings grown in MS medium, freeze-dried samples (0.5 g) each dissolved in 5 ml of deionized water were mixed with 100 mg of protease XIV. This mixture was kept at 37 °C for 24 h and then centrifuged at 6000 rpm for 20 min. The supernatant was separated from the residue and filtered through a 0.45 μm polyvinylidene fluoride (PVDF) membrane. The supernatant was kept at 20 °C until morphological analysis of Se was carried out using HPLC-ICP-MS (Vonderheide et al., 2002). Chromatographic separations were accomplished on an Agilent 1100 liquid chromatograph (Agilent Technologies, Palo Alto, CA, USA), and anion-exchange HPLC was performed on a 250 mm×4 mm, 10 μm particle size PRP-X100 column (Hamilton Co., Reno, NE, USA)]. The standard solution was purchased from the China Academy of Metrology with MeSeCys in water, 0.433 μmol g–1. Results Molecular characterization of BjSMT and its transgenic tobacco lines A novel SMT gene from the genome of Indian mustard was cloned by using a homologous cloning method, and was designated as BjSMT. The gene’s ORF is 1830 bp in length (Fig. 2A) including five introns and six exons, and it encodes 346 amino acids. Using the NCBI CD-search tool, it was confirmed that BjSMT belongs to the S-methyltransferase superfamily (Supplementary Fig. S1). Fig. 2. Open in new tabDownload slide Cloning and molecular identification of BjSMT transgenic tobacco. (A) PCR amplification of BjSMT from genomic DNA of Indian mustard. Lane A1 was the amplified PCR products, and the arrow shows the BjSMT genomic DNA band amplified with the BjSMT-F1 and BjSMT-R1 primer pair which was 1866 bp in length. (B) Genome DNA identification of BjSMT transgenic tobacco lines. Lanes from B1 to B5 represent PCR products from five different BjSMT tobacco transgenic lines, lane B6 represents the PCR product from wild-type tobacco plants, lane B7 represents the PCR product from the pBI121-BjSMT-GFP plasmid (positive control), and lane B8 represents the PCR product from water (negative control). The arrow indicates that the BjSMT genomic DNA band amplified with the BjSMT-F1 and BjSMT-R2 primer pair was 1860 bp in length. (C) A representative identification of BjSMT transgenic tobacco lines at the mRNA level. Lane C1 represents the PCR product from genomic DNA of the B3 BjSMT transgenic line, lane C2 represents the product from the pBI121-GFP plasmid, and lane C3 represents the PCR product from the cDNA of the B3 BjSMT transgenic line. Arrows indicate that the BjSMT genomic DNA and cDNA bands amplified with the BjSMT-F1 and BjSMT-R2 primer pair were 1860 bp and 1068 bp in length, respectively. M, DNA ladder marker DL2000. (D) The full length of the BjSMT gene, including six exons and five introns. (This figure is available in color at JXB online.) Fig. 2. Open in new tabDownload slide Cloning and molecular identification of BjSMT transgenic tobacco. (A) PCR amplification of BjSMT from genomic DNA of Indian mustard. Lane A1 was the amplified PCR products, and the arrow shows the BjSMT genomic DNA band amplified with the BjSMT-F1 and BjSMT-R1 primer pair which was 1866 bp in length. (B) Genome DNA identification of BjSMT transgenic tobacco lines. Lanes from B1 to B5 represent PCR products from five different BjSMT tobacco transgenic lines, lane B6 represents the PCR product from wild-type tobacco plants, lane B7 represents the PCR product from the pBI121-BjSMT-GFP plasmid (positive control), and lane B8 represents the PCR product from water (negative control). The arrow indicates that the BjSMT genomic DNA band amplified with the BjSMT-F1 and BjSMT-R2 primer pair was 1860 bp in length. (C) A representative identification of BjSMT transgenic tobacco lines at the mRNA level. Lane C1 represents the PCR product from genomic DNA of the B3 BjSMT transgenic line, lane C2 represents the product from the pBI121-GFP plasmid, and lane C3 represents the PCR product from the cDNA of the B3 BjSMT transgenic line. Arrows indicate that the BjSMT genomic DNA and cDNA bands amplified with the BjSMT-F1 and BjSMT-R2 primer pair were 1860 bp and 1068 bp in length, respectively. M, DNA ladder marker DL2000. (D) The full length of the BjSMT gene, including six exons and five introns. (This figure is available in color at JXB online.) BjSMT transgenic tobacco lines were identified by PCR which amplified the whole ORF of BjSMT (Fig. 2B1–B8) and no PCR product was amplified from the genome of wild-type tobacco (Fig. 2B6). These results confirmed the successful transformation of all the tested (B1–B5) BjSMT transgenic lines. For testing the expression of BjSMT in transgenic lines, total RNA of each transgenic line was extracted and the mRNA level of BjSMT was verified after reverse transcription. All the tested (B1–B5) transgenic lines demonstrated that BjSMT was expressed at the mRNA level. Figure 1C shows the testing of of the B3 transgenic line in which the amplified band size (~1000 bp) corresponds to the cDNA size of the BjSMT ORF (1038 bp) (Fig. 2C3). There was no amplified band in the corresponding position in the empty vector-transformed control tobacco plants (Fig. 2C2). The sequencing results for the C3 band also exactly match with BjSMT cDNA compared with the final spliced BjSMT mRNA, in which the five introns of the BjSMT genomic sequences were removed (Fig. 2D). The relative molecular weight of BjSMT is 37.9 kDa (calculated by ProtParam), approximately the same as its closest homologue BoSMT. The theoretical formula of the protein is C1682H2661N451O521S10 with an isoelectric point (pI) of 5.61 and an instability index (II) of 39.71 (<40) which illustrates that BjSMT is a stable protein. Furthermore, no predictions of transmembrane and signal peptide were found during transmembrane (TMHMM) and signal peptide (SignalP4.1) analysis, and its anticipated subcellular localization scored 3.6 for the cytoplasm, indicating that it might be located in the cytoplasm. The evolutionary tree is mainly considered to provide an estimation of conservation and divergence of genes. A maximum likelihood tree was generated with SMTs from several species including Se accumulators and non-accumulators. It gathered all the SMTs of different Brassica species into the same evolutionary clade, making them monophyletic with 100% bootstrap support (Fig. 3), and this close kinship was supported by 99% identity of the amino acid sequence of BjSMT with B. napus (XP_013692667.1), B. oleracea var. italica (AAX20123.1), and B. oleracea var. oleracea (XP_013636561). The SMTs from the Citrus genus formed the basal lineage. SMTs of the Brassica genus in the tree are more close to homocysteine methyltransferases (HMTs) of Eutrema salsugineum, Capsella rubella, A. thaliana, and Oryza sativa, leading to the speculation that it uses both substrates, namely homocysteine (HoCys) and selenocysteine (SeCys). Like previously reported SMTs, BjSMT also possesses highly conserved motifs: G(I/V)NC, YPNSGE, and GGCCR starting at amino acid positions 245, 272, and 313, respectively. Cys248, Cys315, and Cys316 have been implicated in creating such a geometrical structure believed to form a zinc-binding motif near the C-terminus (Millian and Garrow, 1998; Peariso et al., 1998; Koutmos et al., 2008) responsible for zinc binding in enzymes. Fig. 3. Open in new tabDownload slide Evolutionary tree inferred by BLAST results. This tree indicates the evolutionary distance between BjSMT (underlined) and other SMTs. As BjSMT shows 99% identity in BLASTP with Brassica species such as B. oleracea var. italica, B. oleracea var. oleracea, and B. napus, all these belong to one clade in the tree (arrowheads). However, it also shares close homology with the coding region of HMTs (check marks) that put them all (SMTs and HMTs) in one branch. In comparison, the SMTs (arrows) from the Astragalus genus precede BjSMT, so are distant and in another branch of the tree, indicating the time distance between Brassica and the Astragalus genus, and the change of a few conserved residues in them. Accession numbers and species names of each gene are shown in Supplementary Table S2. Fig. 3. Open in new tabDownload slide Evolutionary tree inferred by BLAST results. This tree indicates the evolutionary distance between BjSMT (underlined) and other SMTs. As BjSMT shows 99% identity in BLASTP with Brassica species such as B. oleracea var. italica, B. oleracea var. oleracea, and B. napus, all these belong to one clade in the tree (arrowheads). However, it also shares close homology with the coding region of HMTs (check marks) that put them all (SMTs and HMTs) in one branch. In comparison, the SMTs (arrows) from the Astragalus genus precede BjSMT, so are distant and in another branch of the tree, indicating the time distance between Brassica and the Astragalus genus, and the change of a few conserved residues in them. Accession numbers and species names of each gene are shown in Supplementary Table S2. To identify and analyze the potential amino acids involved in the active site or binding pocket, the structure of BjSMT was predicted by the I-TASSER server which uses iterative modeling (Yang et al., 2015). Due to the unavailability of the crystallized structure of its closest homologues in the PDB, all five predicted models of BjSMT were validated by Verify 3D and ERRAT servers (Colovos and Yeates, 1993; Eisenberg et al., 1997). The model with the highest validation score—93.93% (Verify 3D) and 92.60% (ERRAT)—along with a 0.88 C-score (confidence score) in I-TASSER was selected (Fig. 4A) and further refined by ModFOLD4 to improve disordered psi and phi angles (McGuffin et al., 2013). The quality of angles was also verified by Ramachandran plot which showed 93.99% of amino acids in the favored region, 4.43% in the allowed region and 1.58% (5 amino acids) outliers, demonstrating the high quality of the structure (Fig. 4B). Fig. 4. Open in new tabDownload slide Prediction and validation of the 3D structure of B. juncea SMT (BjSMT) predicted by I-TASSER by using Tm-MetH (1Q7M) as a template. (A) A side view of the predicted BjSMT protein model. (B) Verification of psi and phi angles of the 3D model by Ramachandran plot. Green colored amino acids are in the core region of the plot representing accurate angles in the model. (C) The conserved residues Cys248, Cys315, and Cys316, which are involved in binding of Zn, coordinate homocysteine binding, and methylation, are highlighted. The specific Thr187 (blue) is also conserved in BjSMT as in other HMTs, and has a role in methylation of homocysteine. Fig. 4. Open in new tabDownload slide Prediction and validation of the 3D structure of B. juncea SMT (BjSMT) predicted by I-TASSER by using Tm-MetH (1Q7M) as a template. (A) A side view of the predicted BjSMT protein model. (B) Verification of psi and phi angles of the 3D model by Ramachandran plot. Green colored amino acids are in the core region of the plot representing accurate angles in the model. (C) The conserved residues Cys248, Cys315, and Cys316, which are involved in binding of Zn, coordinate homocysteine binding, and methylation, are highlighted. The specific Thr187 (blue) is also conserved in BjSMT as in other HMTs, and has a role in methylation of homocysteine. Interestingly, the best template used to estimate the structure of BjSMT was the cobalamin-dependent methionine synthase (MetH) from Thermotoga maritima (Evans et al., 2004). MetH, also known as HMT, catalyzes the transfer of the methyl group from the methyl donor tetrahydrofolate (THF) to HoCys to form methionine by using cobalamin as an intermediate methyl carrier. BjSMT shared high homology to the coding region of HMTs as most of the hits in BLASTP were HMTs from different plants and showed identity up to 87%. This high homology to HMTs supports its use of HoCys in addition to SeCys for methylation. Koutmos et al. (2008) found a (βα)8 barrel in the crystallographic structure of T. maritima MetH (Tm-MetH) having a catalytic zinc-binding site at the top of the barrel involved in binding of HoCys (Evans et al., 2004; Koutmos et al., 2008). This post-secondary structure assembled by conserved Cys207, Cys272, and Cys273 residues is believed to form such a geometrical catalytic pocket which will position the sulfur group of HoCys in close proximity to Thr147, responsible for methyl group transfer by donating a hydrogen bond. Protein alignment of BjSMT and Tm-MetH has confirmed the presence of these conserved amino acids in BjSMT at positions Cys248, Cys315, and Cys316, suggesting that BjSMT may also coordinate the binding of Zn (Fig. 4C). Moreover, Thr147, a highly conserved amino acid in HMTs, was found in BjSMT at Thr187 and previously was noted in broccoli, a plant of the same genus (Lyi et al., 2005), explaining how BoSMT can use both substrates. However, substitution of the conserved Thr147 in HMTs to Ala184 in some SMTs isolated from different Astragalus species led to the proteins losing the ability to methylate HoCys, thus making them specific for SeCys methylation (Sors et al., 2009). In order to find polymorphisms between BjSMT and other non-accumulators, multiple sequence alignment (MSA) analysis was performed among four SMT sequences derived from accumulator plants (B. napus, B. juncea, B. oleracea var. italica, and B. oleracea var. oleracea) and six non-accumulators (A. thaliana, T. urartu, O. sativa subsp. Japonica, A. ceramicus, A. chrysochlorus, and A. drummondii). The MSA analysis revealed eight positions which were conserved in accumulators and substituted in non-accumulators, and the substituted positions were well conserved in all non-accumulators (Fig. 5). In addition, evolutionary conservation of these positions in BjSMT was inspected using ConSurf software (Ashkenazy et al., 2010) and, out of eight positions, Ile27, Ser76, and Gly210 were found to be evolutionarily most conserved in BjSMT compared with variable amino acids which include Phe144, Ile182, Phe197, and Glu255. Structural location of the three conserved amino acids was in the core of the protein in the most buried part involved in form the binding pocket, thus directing their conservation in the binding pocket. Fig. 5. Open in new tabDownload slide MSA analysis between accumulators and non-accumulators of Se. The first four sequences are SMTs derived from accumulator plants (B. juncea, B. oleracea var. italica, B. oleracea var. oleracea, and B. napus which are involved in accumulation of Se). The remainder all are taken from non-accumulators of different species (A. drummondii, A. crassicarpus, A. leptocarpus, O. sativa, A. thaliana, and T. urartu). MSA analysis shows eight sites (boxes) where signature amino acids conserved in accumulators have been substituted in non-accumulator sequences. Fig. 5. Open in new tabDownload slide MSA analysis between accumulators and non-accumulators of Se. The first four sequences are SMTs derived from accumulator plants (B. juncea, B. oleracea var. italica, B. oleracea var. oleracea, and B. napus which are involved in accumulation of Se). The remainder all are taken from non-accumulators of different species (A. drummondii, A. crassicarpus, A. leptocarpus, O. sativa, A. thaliana, and T. urartu). MSA analysis shows eight sites (boxes) where signature amino acids conserved in accumulators have been substituted in non-accumulator sequences. BjSMT expression was dramatically up-regulated by selenate treatment in the leaves and also has a rapid up-regulation response upon low dose selenite stress To test whether the BjSMT expression level changes in response to environmental Se in Indian mustard, sqRT–PCR and qRT-PCR were performed to test the BjSMT mRNA transcript level (Fig. 6). First, we analyzed how BjSMT responds to external selenate, selenite, and sulfate supplements in the roots and leaves of Indian mustard (Fig. 6A, B). Comparatively, treating the plants with 40 μM Na2SeO4 dramatically increased the BjSMT mRNA level in the leaves but not in the roots. Only a low increase of BjSMT transcripts was observed in the roots; however, on treatment with 40 μM Na2SeO3, we detected very faint BjSMT transcripts in the roots, and a small increase in BjSMT transcripts in the leaves, similar to the increase in the roots treated with Na2SeO4 (Fig. 6A). Supplementation with 1 mM MgSO4 markedly reduced BjSMT expression in the leaves and roots regardless of whether the plants were treated with selenate or selenite; an ~76.4% and 54% reduction for Na2SeO3 and Na2SeO4 treatments in the leaves, respectively (Fig. 6A, B). The BjSMT transcripts were nearly undetectable in the roots and leaves for plants without Se treatment. Fig. 6. Open in new tabDownload slide sqRT–PCR and qRT-PCR analysis of the mRNA level of BjSMT transcripts upon selenate and selenite treatment. (A) A representative sqRT–PCR analysis of BjSMT transcript accumulation in leaves and roots of Indian mustard plants supplied with 40 μM Na2SeO4, 40 μM Na2SeO3, and 1 μM MgSO4 in the medium as indicated. ‘–’ indicates that no extra compounds were supplied in the medium. (B) The amounts of BjSMT and actin transcripts in each well in (A) were quantified by using ImageJ software, and the data show the amount of BjSMT relative to actin transcripts in roots and leaves of Indian mustard plants. (C) RT-PCR determined the BjSMT mRNA level in the leaves of Indian mustard seedlings after 0, 4, 12, and 24 h treatment with (Na2SeO3) or without (CK) 300 μg l–1 Na2SeO3 addition, respectively. Data are expressed as means ±SE and analyzed by Student’s t-test. n=3 for each genotype, ***P<0.001. (D) Phenotypic changes of Indian mustard seedlings after 7 d treatment with Na2SeO3. B1 and B2 represent the same seedlings before Na2SeO3 treatment, B3 is the seedling treated with 300 μg l–1 Na2SeO3 for 7 d, and B4 is the seedling growing with the same medium but without Na2SeO3. (This figure is available in color at JXB online.) Fig. 6. Open in new tabDownload slide sqRT–PCR and qRT-PCR analysis of the mRNA level of BjSMT transcripts upon selenate and selenite treatment. (A) A representative sqRT–PCR analysis of BjSMT transcript accumulation in leaves and roots of Indian mustard plants supplied with 40 μM Na2SeO4, 40 μM Na2SeO3, and 1 μM MgSO4 in the medium as indicated. ‘–’ indicates that no extra compounds were supplied in the medium. (B) The amounts of BjSMT and actin transcripts in each well in (A) were quantified by using ImageJ software, and the data show the amount of BjSMT relative to actin transcripts in roots and leaves of Indian mustard plants. (C) RT-PCR determined the BjSMT mRNA level in the leaves of Indian mustard seedlings after 0, 4, 12, and 24 h treatment with (Na2SeO3) or without (CK) 300 μg l–1 Na2SeO3 addition, respectively. Data are expressed as means ±SE and analyzed by Student’s t-test. n=3 for each genotype, ***P<0.001. (D) Phenotypic changes of Indian mustard seedlings after 7 d treatment with Na2SeO3. B1 and B2 represent the same seedlings before Na2SeO3 treatment, B3 is the seedling treated with 300 μg l–1 Na2SeO3 for 7 d, and B4 is the seedling growing with the same medium but without Na2SeO3. (This figure is available in color at JXB online.) Due to selenate supplementation resulting in the Se form accumulated in the tissues of Indian mustard being predominantly selenate, while selenite supplementation resulted in the accumulated Se mainly being in reduced organic Se forms (de Souza et al., 1998; LeDuc et al., 2004), which have more potential health benefits to human, we tested whether BjSMT responds to a low dose of external selenite treatment (Fig. 6C), and also because selenite [Se (IV)] is more toxic than selenate [Se (VI)], and its detoxification is more relevant in the phytoremediation approach, we focused on selenite in the following experiments. After 4 h of 300 μg l–1 Na2SeO3 (~1.7 μM) treatment, the BjSMT mRNA level was increased rapidly ~1.5-fold compared with the controls (Fig. 5C) and reached 2.62-fold that in the controls, with the highest level at 12 h of treatment. Subsequently, the BjSMT mRNA level decreased but still maintained a higher expression level at 24 h of treatment. These results explained that BjSMT responds to even a low level of external environmental selenite stress, implying that the gene plays a critical role in detoxification of selenite by the plant. After 7 d of continuing Na2SeO3 treatment, the treated Indian mustard plants (Fig. 6D-B3) grew even better than the control plants without selenite treatment (Fig. 6D-B4), suggesting that a low level of selenite stress could even promote plant growth and the up-regulated BjSMT may actively take part in this process. BjSMT expression markedly enhanced plant tolerance to selenite stress To evaluate the Se tolerance of BjSMT tobacco, we measured the fresh weight and plant height between BjSMT-transformed tobacco and control empty vector-transformed tobacco after 60 d of selenite treatment at concentrations of 0, 60, 120, and 240 μM Na2SeO3. There were significant differences in fresh weight and plant height between BjSMT transgenic tobacco plants and controls at 120 μM selenite treatment (Fig. 7A, B). The fresh weight and the height of BjSMT-transformed tobacco plants were consistent with controls in the absence of Na2SeO3 treatment. However, the fresh weight and height of all tested pBI121 empty vector-transformed tobacco plants decreased with the increase of Se concentration and a sharp decrement was observed when the selenite concentration reached 120 μM, while at this concentration the plant height of BjSMT tobacco was still similar to that with no selenite treatment (Fig. 7A). In addition, although the growth of both transgenic tobacco plants and controls was greatly inhibited upon 240 μM selenite treatment, the BjSMT-transformed tobacco still grew significantly better than controls in terms of fresh weight and plant height (P<0.001). The results showed that overexpression of BjSMT could substantially improve the Se tolerance of tobacco. This is also shown in Fig. 8, which represents the growth status of BjSMT-transformed tobacco and control plants before and after 90 d of treatment with different concentrations of selenite in MS medium, in which 60 μM selenite-treated BjSMT-transformedplants grew even better than controls without selenite treatment. Figure 7C also shows that the pot-grown BjSMT-transformed tobacco (Fig. 7C-a) grew significantly better than vector controls (Fig. 7C-b) after 15 d of low dose selenite treatment (300 μg l–1 Na2SeO3), and they grew even better than wild-type tobacco W38 receiving no selenite treatment (Fig. 7C-c). Fig. 7. Open in new tabDownload slide Effects of selenite treatment on BjSMT-overexpressing tobacco. After 20 d of treatment with 0, 60, 120, and 240 μM Na2SeO3, the plant height (A) and fresh weight (B) were recorded. Error bars in (A) and (B) denote the SE between replicate plants of the same genotype (n=4–6); values for BjSMT transgenic plants that are statistically significantly different from control plants are denoted (t-test, ***P<0.001). (C) A representative of the phenotypes of BjSMT tobacco growing under the conditions indicated, in which plant c was not exposed to Na2SeO3. a, BjSMT tobacco; b, pBI121 empty vector-transformed tobacco; c, wild-type W38. BjSMT, BjSMT transgenic tobacco; pBI121, empty vector transformants. (This figure is available in color at JXB online.) Fig. 7. Open in new tabDownload slide Effects of selenite treatment on BjSMT-overexpressing tobacco. After 20 d of treatment with 0, 60, 120, and 240 μM Na2SeO3, the plant height (A) and fresh weight (B) were recorded. Error bars in (A) and (B) denote the SE between replicate plants of the same genotype (n=4–6); values for BjSMT transgenic plants that are statistically significantly different from control plants are denoted (t-test, ***P<0.001). (C) A representative of the phenotypes of BjSMT tobacco growing under the conditions indicated, in which plant c was not exposed to Na2SeO3. a, BjSMT tobacco; b, pBI121 empty vector-transformed tobacco; c, wild-type W38. BjSMT, BjSMT transgenic tobacco; pBI121, empty vector transformants. (This figure is available in color at JXB online.) Fig. 8. Open in new tabDownload slide BjSMT-transformed tobacco plants exhibited marked tolerance to selenite stress. The photographs are representative of the performance of BjSMT transgenic tobacco and controls transformed with pBI121 empty vector on days 0 and 90 after planting on MS medium with the addition of 0, 60, 120, and 240 μM Na2SeO3, respectively. BjSMT, BjSMT transgenic tobacco plants; Control, pBI121 empty vector-transformed tobacco plants. (This figure is available in color at JXB online.) Fig. 8. Open in new tabDownload slide BjSMT-transformed tobacco plants exhibited marked tolerance to selenite stress. The photographs are representative of the performance of BjSMT transgenic tobacco and controls transformed with pBI121 empty vector on days 0 and 90 after planting on MS medium with the addition of 0, 60, 120, and 240 μM Na2SeO3, respectively. BjSMT, BjSMT transgenic tobacco plants; Control, pBI121 empty vector-transformed tobacco plants. (This figure is available in color at JXB online.) BjSMT plants maintain a higher level of GSH-Px activity and chlorophyll content under severe selenite treatment To analyze the mechanism of enhanced plant tolerance to selenite stress by BjSMT overexpression, the GSH-Px activity and chlorophyll content of transgenic tobacco plants were analyzed at the same time (Fig. 9). There was no difference in the GSH-Px activity of BjSMT-transformed tobacco plants and controls under normal growth conditions without selenite addition, while when the medium was supplied with 60 μM selenite, the GSH-Px activity of the controls increased by nearly 0.5-fold compared with normal conditions, significantly higher than BjSMT-transformed tobacco plants (P<0.001) in which no significant change in GSH-Px activity was observed (Fig. 9A). When the selenite concentration increased to 120 μM, the GSH-Px activity of controls again showed a >0.5-fold increase, significantly higher than BjSMT-transformed tobacco plants (P<0.001). Although it was a large increase, when the supplied selenite increased to 240 μM, the GSH-Px activity of BjSMT-transformed tobacco continued to increase markedly and was significantly higher than controls, and the controls showed almost no change. The GSH-Px activity is not only a useful marker of free radical-mediated damage and oxidative stress, but it also plays a role to protect the organism from oxidative damage. The sharp increase of the GSH-Px activity in control plants at a lower dose of selenite accompanied by great phenotype damage indicates that the control tobacco is more sensitive to Se stress in the absence of BjSMT overexpression (Fig. 8). Under the severe toxic dose of 240 μM selenite treatment, the BjSMT-transformed tobacco can have a large increase in GSH-Px activity to rescue the oxidative stress-induced damage, while the GSH-Px activity up-regulation mechanism of control plants without BjSMT expression was likely to be paralyzed. Fig. 9. Open in new tabDownload slide Effects of BjSMT overexpression on the GSH-Px activity of plants and chlorophyll content in leaves of tobacco. After 20 d of treatment with 0, 60, 120, and 240 μM Na2SeO3, the GSH-Px activity (A) and chlorophyll content (B) of BjSMT tobacco and control plants were determined. Error bars in (A) and (B) denote the SE between three replicate groups of plants of the same genotype (n=3); values for BjSMT transgenic plants that are statistically significantly different from controls are denoted (t-test, **P<0.01, ***P<0.001). BjSMT, BjSMT transgenic tobaccos; pBI121, empty vector transformants. Fig. 9. Open in new tabDownload slide Effects of BjSMT overexpression on the GSH-Px activity of plants and chlorophyll content in leaves of tobacco. After 20 d of treatment with 0, 60, 120, and 240 μM Na2SeO3, the GSH-Px activity (A) and chlorophyll content (B) of BjSMT tobacco and control plants were determined. Error bars in (A) and (B) denote the SE between three replicate groups of plants of the same genotype (n=3); values for BjSMT transgenic plants that are statistically significantly different from controls are denoted (t-test, **P<0.01, ***P<0.001). BjSMT, BjSMT transgenic tobaccos; pBI121, empty vector transformants. There was a significant negative correlation between chlorophyll content and senescence, so we measured the chlorophyll content of transgenic tobacco plants. The chlorophyll content of BjSMT-transformed tobacco was not very different from that of the controls when treated with 0–60 μM Se (Fig. 9B), and was even slightly lower than the control tobacco. The chlorophyll content was significantly different between the two groups at 120 μM selenite treatment (P<0.01) and it was very different at 240 μM selenite treatment (P<0.001). Despite the consistency of the chlorophyll content with tobacco phenotypes, the chlorophyll content was decreased with the increase of Se concentration, resulting in white or yellowish green leaves; however, the BjSMT tobacco plants could still maintain a higher level of chlorophyll content in leaves compared with the controls (Fig. 9B). BjSMT plants accumulate significantly more Se at a lower dose of selenite supply The total Se content of transgenic tobacco plants was determined by ICP-MS (Fig. 10). The results showed that the concentration of total Se in the roots was much higher than that in leaves after 60 d of treatment with different concentrations of selenite. In leaves, the total Se content in BjSMT-transformed and control tobacco plants without Na2SeO3 stress was 0.84 mg kg–1 and 0.71 mg kg–1, respectively. Under 60 μM Na2SeO3 stress, the content of total Se in BjSMT transgenic tobacco plants was significantly different from that of the controls (P<0.001) and it was 1.68 times higher than that measured in controls (Fig. 10A). Consistent results were found under 120 μM Na2SeO3 stress, and the total Se content in BjSMT transgenic tobacco plants was 1.11 times higher (Fig. 10A) than in control tobacco (P<0.05). BjSMT-transformed tobacco plants grew well at a concentration of 60 μM selenite, and grew even better than they did without selenite stress, while the controls showed some defects in growth, suggesting that the gene has excellent potential for use in Se biofortification. Fig. 10. Open in new tabDownload slide Total Se accumulation by BjSMT plants after selenite treatment. After 60 d treatment with 0, 60, and 120 μM Na2SeO3, respectively, the contents of total Se in the leaves (A) and roots (B) of BjSMT plants and controls were determined as mg kg–1 DW. Error bars in (A) and (B) denote the SE between three replicate group of plants of the same genotype (n=3); values for BjSMT transgenic plants that are statistically significantly different from controls are denoted (t-test, *P<0.05, ***P<0.001). BjSMT, BjSMT transgenic tobaccos; pBI121, empty vector transformants. Fig. 10. Open in new tabDownload slide Total Se accumulation by BjSMT plants after selenite treatment. After 60 d treatment with 0, 60, and 120 μM Na2SeO3, respectively, the contents of total Se in the leaves (A) and roots (B) of BjSMT plants and controls were determined as mg kg–1 DW. Error bars in (A) and (B) denote the SE between three replicate group of plants of the same genotype (n=3); values for BjSMT transgenic plants that are statistically significantly different from controls are denoted (t-test, *P<0.05, ***P<0.001). BjSMT, BjSMT transgenic tobaccos; pBI121, empty vector transformants. In roots, the backgrounds of total Se content in BjSMT tobacco (~3.8-fold higher) and control plants growing without Na2SeO3 stress were 1.87 mg kg–1 and 0.49 mg kg–1, respectively, suggesting that BjSMT overexpression can lead to more Se accumulation in the roots even with no additional Se supply. The total Se content in BjSMT-transformed tobacco roots was significantly higher (~1.11-fold) than that of controls under 60 μM Na2SeO3 stress (P<0.01) and it was even much higher (~1.37-fold) than that of controls under 120 μM Na2SeO3 stress (P<0.01) (Fig. 10B). The results showed that after treatment with a higher dose of selenite, BjSMT-transformed plants can accumulate significantly more Se in the roots than controls without BjSMT expression. BjSMT plants convert significantly more MeSeCys in the leaves To analyze the effect of BjSMT on MeSeCys conversion in transgenic tobaccos, we used HPLC-ICP-MS to determine the contents of total Se and MeSeCys in the leaves and roots after 30 d of 60 μM Na2SeO3 treatment (Table 1; Supplementary Fig. S2). The total Se content in the roots was more than in the leaves, and no significant difference in roots was detected between the two tobacco genotypes. However, the Se content was much higher in leaves of BjSMT-transformed tobacco (~252–845 mg kg–1 DW) than in those of controls (P<0.01). MeSeCys was only detected in leaves of BjSMT-transformed tobacco (~0.35–1.13 mg kg–1 DW) and its content in roots and leaves of controls was below the limit of detection, indicating that BjSMT overexpression could substantially enhance MeSeCys conversion and accumulation in the leaves of tobacco, and it was found to significantly reduce the damage to plants produced by treatment with a high selenite concentration. Table 1. Accumulation of selenium and MeSeCys in the leaves and roots of BjSMT transgenic tobacco seedlings grown in medium Leaf Root pBI121 BjSMT pBI121 BjSMT Total Se (mg kg−1 FW) 5.96 9.46** 44.3 43.7 MeSeCys (mg kg−1 FW) <0.7×10−3a 12.6×10−3*** <0.7×10−3a <0.7×10−3a Leaf Root pBI121 BjSMT pBI121 BjSMT Total Se (mg kg−1 FW) 5.96 9.46** 44.3 43.7 MeSeCys (mg kg−1 FW) <0.7×10−3a 12.6×10−3*** <0.7×10−3a <0.7×10−3a a Below the detection limit. Open in new tab Table 1. Accumulation of selenium and MeSeCys in the leaves and roots of BjSMT transgenic tobacco seedlings grown in medium Leaf Root pBI121 BjSMT pBI121 BjSMT Total Se (mg kg−1 FW) 5.96 9.46** 44.3 43.7 MeSeCys (mg kg−1 FW) <0.7×10−3a 12.6×10−3*** <0.7×10−3a <0.7×10−3a Leaf Root pBI121 BjSMT pBI121 BjSMT Total Se (mg kg−1 FW) 5.96 9.46** 44.3 43.7 MeSeCys (mg kg−1 FW) <0.7×10−3a 12.6×10−3*** <0.7×10−3a <0.7×10−3a a Below the detection limit. Open in new tab Discussion Se is essential for human health, and many studies along with clinical trials have demonstrated that the increased consumption of Se compounds is beneficial not only for maintaining human health but also for prevention of disease, including some intractable diseases such as cancers of the liver, breast, stomach, prostate, and esophagus (Medina et al., 2001; Soriano-Garcia, 2004; Combs, 2005). MeSeCys is one of the most effective anticarcinogenic Se compounds (Medina et al., 2001; Unni et al., 2005; Tsai et al., 2013), and a very recent study showed that this compound has potential ameliorating effects on Alzheimer’s disease (Xie et al., 2018), underlining its possible applications for human health and disease prevention. Indian mustard, being a model plant of multimetal accumulators for phytoremediation, is also a secondary Se accumulator which could accumulate more MeSeCys in its leaves, while the mechanism of Se accumulation and MeSeCys conversion is still not well known in this plant. In this study, we identified a novel SMT gene from B. juncea by using a homology cloning strategy. BjSMT is a key enzyme involved in the conversion of SeCys to MeSeCys, allowing us to examine its structure, evolution, and expression in relation to the production of MeSeCys and tolerance to Se stress in plants. The study showed that BjSMT expression could be tremendously up-regulated in leaves by external selenate but not selenite, demonstrating that transgenic tobacco expressing the BjSMT protein had markedly enhanced tolerance to selenite stress, total Se accumulation, and MeSeCys conversion. The full-length amino acid sequence of BjSMT shares high identity (99%) with other SMTs reported in different species of the Brassica genus, being distributed in the same clade in the evolutionary tree and with HMTs from several other species anticipating similarity in function. BjSMT does not possess transmembrane and signal peptides, appearing to be a cytosolic protein. Although its cytosolic location is not confirmed, its predicted pI (5.61) comes within the pI range (5–6) of cytosolic proteins (Schwartz et al., 2001). In addition, previously reported data related to methylation of SeCys and SeMet including a mechanism for production of S-Met most probably occurs in the cytoplasm (Bourgis et al., 1999; Ranocha et al., 2000; Plateau et al., 2017). Moreover, BjSMT has some remarkable motifs conserved in reported methyltransferases such as G(I/V)NC and GGCCR, the latter of which possibly forms a zinc-binding motif and can be stimulated by a zinc cofactor, as its closest homologue previously showed enzymatic activity (Lyi et al., 2005), and these motifs may also have a role in the methylation process because a full-length cDNA without GGCCR which shares 87% sequence identity with BoSMT showed no SMT activity (Lyi et al., 2005). The close homology of BjSMT to HMTs which specifically methylate HoCys also suggests some sharing of function and conserved amino acids, especially the conserved Thr147 residue (in Tm-MetH which corresponds to Thr187 in BjSMT) in HMTs, as this single residue controls the ability of enzymes to methylate HoCys. The threonine side chain possesses a β-carbon hydroxyl group which is donated to the Zn-bound thiol group of HoCys to facilitate the methylation reaction (Koutmos et al., 2008). In AbSMT, functionally specific for SeCys methylation, Ala184 replacement by Thr184 through a mutagenesis approach enabled this enzyme to gain an activity to methylate HoCys even more efficiently than the HMTs (Neuhierl et al., 1999; Sors et al., 2009). Moreover, YagD, an enzyme from Escherichia coli possessing Thr at the same position, can perform methylation of both SeCys and HoCys (Neuhierl et al., 1999). As the same residue was found to be conserved in BjSMT at Thr187, this suggests that BjSMT can execute a dual function as an SMT/HMT. Previous studies concluded that the pathway for conversion of SeCys to MeSeCys is a basic mechanism for Se hyperaccumulation and detoxification in Se hyperaccumulators (Brown and Shrift, 1981; Pickering et al., 2003). Several transgenic studies on overexpressing AbSMT in Arabidopsis (LeDuc et al., 2004), Indian mustard (LeDuc et al., 2004), and tomato (Brummell et al., 2011) have shown the development of Se tolerance in these plants, thus confirming its role in Se detoxification. Later studies on a secondary Se-accumulator broccoli (Brassica oleracea var. italica) showed that BoSMT expression could also be substantially induced when treated with selenate but not with selenite, resulting in significant accumulation of MeSeCys. The transgenic studies of overexpression of AbSMT in Arabidopsis (LeDuc et al., 2004), Indian mustard (LeDuc et al., 2004), tobacco (McKenzie et al., 2009), and tomato (Brummell et al., 2011) showed that all the SMT plants accumulated significantly more MeSeCys and total Se than controls after treatment with selenate and/or selenite. Further studies on the SMT-like protein AdSMT from the Se non-accumulator A. drummondii revealed that the protein lacks SMT activity in vitro, and the sequence analysis along with that of two SMT-like proteins, AcSMT (A. crassicarpus) and AlSMT (A. leptocarpus) derived from non-accumulators, and with those from hyperaccumulators showed that all the SMT-like proteins contain three conserved sites (A24, Y148, -334) which are different in the three SMTs (G24, F148, T334) from those in hyperaccumulators. In BjSMT, the three corresponding conserved sites are A32, Y151, and S341 in which the third site is different from that of Se non-accumulators (Fig. 4). The experimental introduction of the corresponding AbSMT amino acids into the three conserved sites of AdSMT greatly increased the protein’s SMT activity but is still not be comparable with that of AbSMT (Sors et al., 2009), suggesting that there are still other sites contributing to the SeCys methyltransferase enzymatic activity of SMT. The result explained why Se non-accumulators cannot accumulate more Se in their bodies, and it also emphasized the importance of SMT expression and activity for Se hyperaccumulation or accumulation. In broccoli, BoSMT expression was remarkably up-regulated upon treatment with a low concentration of selenate (10–75 μM Na2SeO4) and dramatically decreased with increased concentrations up to 100 μM (Lyi et al., 2005). Similarly, BjSMT expression is also inducible, which was nearly undetectable in plants not exposed to Se, but up-regulated tremendously in leaves by selenate treatment but not selenite treatment (Fig. 6A). The result suggests that selenate reduction is not a rate-limiting step for Se assimilation in Indian mustard plants. The low level of selenite induction of SMT expression in leaves could be attributed to the inefficiency of selenite translocation from the root to the shoot (Terry et al., 2000; Lyi et al., 2005). However, our qRT-PCR analysis showed that BjSMT transcripts in leaves substantially increased (~2.5-fold increase) upon a very low dose selenite stress (~1.73 μM Na2SeO3) (Fig. 6C). These results indicated that the two SMT genes are inducible, unlike the constitutively expressed AbSMT in the hyperaccumulator (Pickering et al., 2003). As selenite is more toxic than selenate, the remarkable up-regulation of BjSMT in Indian mustard with a low concentration of selenite might provide an explanation for why the Indian mustard plant can accumulate a higher concentration of MeSeCys and displays more tolerance to selenite stress than broccoli. Moreover, better growth of Indian mustard plants under low dose selenite stress (Fig. 6D) indicates the BjSMT up-regulation is beneficial for plant growth and Se assimilation. In addition to Se, recent studies provided clues that SMT expression could be regulated by exogenous cytokinin (Jiang et al., 2018) and salicylic acid (Smolen et al., 2016), suggesting an alternative strategy for increasing accumulation and tolerance of Se in non-transgenic plants. Because uptake of selenate in plants is through sulfate transporters and channels, and sulfate and selenate compete directly for transport (Terry et al., 2000; Gupta and Gupta, 2016), it is reasonable that a high level of sulfate exposure would decrease the selenate-up-regulated SMT expression level, due to the reduced Se level in plant tissues. Lyi et al. (2005) found that 10 mM sulfate exposure dramatically reduced selenate-up-regulated BoSMT expression in broccoli leaves, but 1 mM sulfate exposure had little effect (Lyi et al., 2005). However, our testing in Indian mustard found that 1 mM sulfate exposure still dramatically reduced BjSMT expression in both leaves and roots in the case of either 40 μM selenate or selenite treatment (Fig. 6A). The results suggested that sulfate may inhibit Se assimilation more efficiently in Indian mustard than in broccoli. To further characterize BjSMT functions in Se metabolism, we overexpressed BjSMT in tobacco plants under the control of the CaMV 35S promoter. The BjSMT-transformed plants showed remarkable tolerance to exposure to a higher concentration of selenite (120–240 μM) compared with empty vector-transformed control tobacco plants (Figs 7, 8). A distinct difference in plant height and fresh weight between transgenic and control tobacco plants was observed at 60 μM and 120 μM. Both the plant height and fresh weight decrease in control plants might be due to the reduction of protein synthesis in the Se-inhibited plants (Banuelos et al., 1997). On the other hand, a possible reason for greater plant height and fresh weight in the transgenic plant could be that the increased expression of BjSMT interacts with a substrate with greater turnover to form MeSeCys, thus preventing the Se-induced inhibition of protein synthesis and Se-amino acid misincorporation into proteins. Moreover, the BjSMT-transfomed plants grew even better when exposed to ~1.7 μM or 60 μM selenite than they did without selenite treatment (Figs 5D, 6C). Presently, there is no experimental report about the effects of BoSMT overexpression in tobacco or other plants. However, overexpression of AbSMT in tobacco showed almost no significant differences in shoot weight compared with controls when plants were watered with different concentrations of selenate (McKenzie et al., 2009). Meanwhile, overexpression of AbSMT in A. thaliana and Indian mustard significantly increased plant fresh weight and root length (LeDuc et al., 2004) when exposed to selenite, which is consistent with our findings. Furthermore, we tested the total Se and MeSeCys contents of BjSMT plants and noticed a significantly greater accumulation of Se in BjSMT plants than in controls especially when they were exposed to 60 μM selenite treatment. The total Se in leaves of BjSMT plants increased >60–80% compared with control plants, and the growth of BjSMT plants was not inhibited but was even better than without selenite treatment. Likewise, we also detected significantly more MeSeCys accumulation in leaves of BjSMT plants when exposed to selenite treatment. The same findings were reported in previously published data obtained from AbSMT overexpression in Arabidopsis, Indian Mustard, tomato, and tobacco (LeDuc et al., 2004; McKenzie et al., 2009; Brummell et al., 2011), where SMT plants treated with selenate accumulate even more total Se than plants treated with selenite, due to the fact that selenite is poorly transported from the root to the shoot (Zayed et al., 1998; Terry et al., 2000). However, in the leaves of AbSMT-transformed tomato plants treated with a higher concentration of selenite (5 mM), there was no detection of MeSeCys, and lower accumulation of total Se (134 mg kg–1 DW) (Brummell et al., 2011), suggesting more potential applications for BjSMT. In addition, BjSMT was possibly able to produce γ-GluMeSeCys, MeSeMet, and volatile forms of dimethyl selenide (DMSe) and dimethyl diselenide (DMDSe) in the leaves of transgenic tobacco (Ellis et al., 2004; LeDuc et al., 2004), which might also contribute to the plants’ tolerance to selenite stress. The tolerance to oxidative stress and plant growth status in transgenic plants tested by GSH-Px activity (Jiang et al., 2016) and chlorophyll content (Ptushenko et al., 2014) revealed an increase in both GSH-Px activity and chlorophyll content at a higher dose of selenite than controls, and vice versa, in which the GSH detoxification pathway of BjSMT plants could still be up-regulated upon a high dose of selenite stress. This may explain partial mechanisms for how BjSMT plants could tolerate a high dose of selenite stress. In summary, we cloned a novel BjSMT gene from Indian mustard which has high amino acid sequences similarity to BoSMT in broccoli. We demonstrated that BjSMT transcripts could be substantially up-regulated by exposure to a low dose of selenite, and overexpression of BjSMT in tobacco could significantly increase the plant’s tolerance to selenite stress. The enhanced total Se accumulation and MeSeCys conversion suggests the potential applications of BjSMT in MeSeCys biofortification of crops and phytoremediation of Se pollution. Supplementary data Supplementary data are available at JXB online. Fig. S1. Conserved domain analysis of BjSMT amino acid sequences. Fig. S2. A typical HPLC-ICP-MS elution profile of MeSeCys. Table S1. The formula of the Hoagland solution used in this study. Table S2. The gene names, including SMTs and different types of HMTs, their accession number, and the plant species used to construct the phylogenetic tree. Acknowledgments This work was supported by the National Natural Science Foundation of China (31571042), Key Basic Research Project of Hebei Applied Basic Research Program (18966315D, 14966318D), and One Hundred of Outstanding Creative Talents Support Program of Hebei (BR2-218). We thank the North Central Regional Plant Introduction Station (NCRPIS) of the US National Plant Germplasm System (NPGS) who provided seeds of B. juncea, Dr Kai Xiao for donating seeds of Nicotiana tabacum L. W38, Dr Jingao dong for providing Gel-imaging analysis instruments, and Miss Wenqian Shao for giving some techniques assistant. References Ashkenazy H , Erez E , Martz E , Pupko T , Ben-Tal N . 2010 . ConSurf 2010: calculating evolutionary conservation in sequence and structure of proteins and nucleic acids . Nucleic Acids Research 38 , W529 – W533 . Google Scholar Crossref Search ADS PubMed WorldCat Bañuelos GS , Ajwa HA , Wu L , Guo X , Akohoue S , Zambrzuski S . 1997 . 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Google Scholar Crossref Search ADS PubMed WorldCat Author notes These authors contributed equally to this work. © The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Identification and functional characterization of a novel selenocysteine methyltransferase from Brassica juncea L.
JF - Journal of Experimental Botany
DO - 10.1093/jxb/erz390
DA - 2019-11-18
UR - https://www.deepdyve.com/lp/oxford-university-press/identification-and-functional-characterization-of-a-novel-dePGb0fqpe
SP - 6401
VL - 70
IS - 21
DP - DeepDyve
ER -