Article pubs.acs.org/JAFC
A Novel Soybean (Glycine max) Gene Encoding a Family 3 β‑Glucosidase Has High Isoflavone 7‑O‑Glucoside-Hydrolyzing Activity in Transgenic Rice Chia-Chen Hsu,† Tsung-Meng Wu,§ Yi-Ting Hsu,# Chih-Wen Wu,⊥ Chwan-Yang Hong,*,† and Nan-Wei Su*,† †
Department of Agricultural Chemistry, National Taiwan University, Taipei 10617, Taiwan Department of Aquaculture, National Pingtung University of Science and Technology, Pingtung 91201, Taiwan # Department of Agronomy, National Chung-Hsing University, Taichung 40227, Taiwan ⊥ Laboratory of Agronomic Crops, Kaohsiung District Agricultural Research and Extension Station, Pingtung 90846, Taiwan §
ABSTRACT: A previous study demonstrated that purified Glycine max β-glucosidase (GmBGL) could hydrolyze glucosyl isoflavone to the aglyconic form. This study reports the cloning and functional characterization of a soybean cDNA encoding the β-glucosidase. GmBGL was isolated by use of a purified soybean N-terminal amino acid sequence and conserved sequences of βglucosidase genes from other plants. Sequence analysis of GmBGL revealed an open reading frame of 1884 bp encoding a polypeptide of 627 amino acids with a calculated molecular mass of 69 kDa. Phylogenetic analysis classified the GmBGL into the glycosyl hydrolase 3 family. In soybean, the GmBGL transcript was predominantly accumulated in roots and leaves. To examine the enzymatic activity and substrate specificity, GmBGL was ectopically expressed in transgenic rice. Purified GmBGL protein from transgenic rice could catalyze the hydrolysis of genistin and daidzin to produce genistein and daidzein, respectively, which confirmed GmBGL as a functional β-glucosidase with isoflavone glucoside-hydrolyzing activity. This paper reveals that GmBGL is a key enzyme in transforming glucosyl isoflavones to aglycones in soybean, which may help in genetic manipulation of aglycone-rich soybean seeds. KEYWORDS: genistein, β-glucosidase, isoflavones, soybean, transgenic rice
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INTRODUCTION Isoflavones are a class of chemical compounds found naturally in various plants, especially the legume family. Isoflavones are called phytoestrogens, which share structural similarities to the human female hormone 17β-estradiol and bind to estrogen receptors.1 Dietary surveys indicated that intake of 1 g of soy protein from traditional soy foods can provide approximately 3.5 mg of isoflavones.2 Isoflavones are involved in hormone regulation, such as preventing postmenopausal bone loss,3 reducing menopausal symptoms.4,5 and beneficial effects for breast, prostate. and colon cancer.6 Isoflavones can also improve cardiovascular risk factors including reducing the level of low-density lipoprotein cholesterol.7 In addition, the biotranformation of isoflavones by intestinal bacteria may also deliver positive effects on their biological activities. For example, the bacterial metabolite equol shows a higher binding affinity to human estrogen receptors than its precursor, daidzein.8,9 Soybean isoflavones are of three major typesdaidzein, genistein, and glyciteinwith four chemical forms, aglycone, glucoside (daidzin, genistin, and glycitin), acetylglucoside (6″O-acetyldaidzin, 6″-O-acetylgenistin, and 6″-O-acetylglycitin), and malonylglucoside (6″-O-malonyldaidzin, 6″-O-malonylgenistin, and 6″-O-malonylglycitin). Recently, the aglyconic form, demonstrated to be absorbed more rapidly in greater amounts than the other forms, has attracted much attention in terms of human health benefits.10,11 The content of the aglyconic form was significantly higher in fermented than nonfermented © XXXX American Chemical Society
soybean products such as miso, natto, and tempeh inoculated with Aspergillus oryzae, Rhizopus oligosporus,12 and Bacillus subtilis.13 The increased aglycone content in microbial fermentation processes may result from isoflavone glucoside hydrolyzed by β-glucosidase. The β-glucosidases (β-D-glucopyranoside glucohydrolases, EC 3.2.1.21) hydrolyze glycosidic bonds to release nonreducing terminal glucosyl residues from glycosides and oligosaccharides.14 These enzymes are widely distributed in living organisms from Archaea and Eubacteria to eukaryotes. β-Glucosidases play important roles in many biological processes such as a defense mechanism against herbivores and pathogens,15 cell wall metabolism,14,16 phytohormone activation,17 and secondary metabolism.18 In chickpea, isoflavone conjugates such as isoflavone glucosides and malonylglucosides serve as pools for the release of aglycone by malonyl esterase and β-glucosidase.19,20 Released aglyconic isoflavones function as pathogen inhibitors, chemoattractants, inducers of microbial growth, and enhancers of nodulation genes in mutualistic rhizobia and bradyrhizobia.21 Several papers have described glucosidase activity in soybean; however, only a few enzymes and corresponding genes have Received: October 7, 2014 Revised: December 6, 2014 Accepted: January 8, 2015
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Figure 1. Glycine max β-glucosidase (GmBGL)-catalyzed hydrolysis of glycosyl isoflavones. Isoflavone 7-O-glucosides including daidzin and genistin are hydrolyzed by GmBGL into the corresponding aglycones. Glc, glucose.
been characterized. β-Glucosidase has been purified from soy cotyledons that hydrolyze isoflavone glucosides to release aglycones.22,23 Suzuki et al.24 reported cloning a gene encoding isoflavone conjugate-hydrolyzing β-glucosidase (GmICHG). GmICHG is a member of the glycoside hydrolase 1 (GH1) family, which can hydrolyze both malonylated and nonmalonylated forms of isoflavone-7-O-β-D-glucosides. In soybean, although β-glucosidase is believed to play an important role in the transformation and regulation of isoflavones, most of the biochemical and molecular properties remain unclear. In our previous study, we purified a novel βglucosidase with high specificity toward glucosidic isoflavones (Figure 1), but not malonylglucosidic isoflavone from soybean okara, the byproduct of soy milk and tofu-making, and we determined the N-terminal amino acid sequence of the enzyme.25 Here, we obtained and characterized the full-length GmBGL cDNA. Moreover, transgenic rice harboring GmBGL was created, and β-glucosidase activities toward isoflavone conjugate were determined. GmBGL may be a functional βglucosidase for use in producing isoflavone aglycone-rich soybean by genetic engineering.
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PrimerSelect program of Lasergene 6 (DNASTAR, Inc., Madison, WI, USA) for 5′ and 3′ RACE. PCR involved a Mastercycler gradient thermal cycler (Eppendorf, Hamburg, Germany). The high-fidelity Platinum Taq DNA polymerase (Invitrogen, Carlsbad, CA, USA) was used for the amplification of the GmBGL cDNA. PCR products were cloned with use of the pGEM-T Easy Vector System II (Promega, Madison, WI, USA). An ABI Prism 3730XL DNA Sequencer (Applied Biosystems, Foster City, CA, USA) was used to determine the nucleotide sequences of positive clones. After sequencing, we obtained a full-length cDNA of the soybean β-glucosidase gene (GmBGL, GenBank accession no. KF740479). Sequence and Phylogenetic Analysis. DNA sequences were analyzed by use of BLAST (http://www.ncbi.nlm.nih.gov/blast/) and InterProScan (http://www.ebi.ac.uk/Tools/pfa/iprscan/). The amino acid sequences of β-glucosidase from soybean and other plants including Medicago truncatula, Zea mays, Arabidopsis thaliana, Hordeum vulgare, Nicotiana tabacum, Triticum aestivum, Tropaeolum majus, and Oryza sativa deposited in GenBank were used for phylogenetic analysis. The alignment of the deduced protein sequences and phylogenetic tree were computed by use of Megalign in Lasergene 6 (DNASTAR, Inc., Madison, WI, USA) and MEGA5,27 respectively, with standard parameters. Rice Transformation and Growth Conditions. The maize ubiquitin 1 promoter together with its first intron was used to drive the overexpression of GmBGL in rice. The hygromycin phosphotransferase gene was chosen as a selectable marker. The cassette was cloned into the binary vector pCambia1302 and then transferred into Agrobacterium tumefaciens strain EHA101 by electroporation. Embryogenic calli from immature seeds of TNG67 were transfected as described.28 Three independent T2 homozygous lines (oxBGL1, oxBGL2, and oxBGL3) were used for molecular and biochemical analysis. Seeds of wild-type and transgenic rice were hydroponically cultivated at 30/25 °C (day/night) in a phytotron (Agricultural Experimental Station, National Taiwan University, Taipei) with natural sunlight and 90% relative humidity. Semiquantitative RT-PCR. Total RNA was isolated from 2-weekold rice leaves and soybean tissues by use of Trizol reagent (Invitrogen). RNA samples were treated with TURBO DNase (Ambion, Grand Island, NY, USA) at 37 °C for 30 min to avoid DNA contamination. First-strand cDNA was synthesized by use of MMLV high-performance reverse transcriptase (EPICENTRE Biotechnologies, Madison, WI, USA) according to the manufacturer’s instructions. For amplifying the full-length cDNA of GmBGL, we designed a gene-specific primer pair (GmBGL-F2, 5′-ACTAGTAGTCCACTCAATTTTAGATTTTTAAGTGTCTGGT-3′; GmBGL-R2, 5′-ACTAGTAACAATAGTAATCAAAATGAAAAGTCTGCCCTAG-3′; SpeI underlined for subcloning). To quantify the relative amount of GmBGL transcripts in transgenic rice and soybean, we designed two pairs of gene-specific primers (OsActin-F, 5′-ATGCTCTCCCCCATGCTATC-3′; OsActin-R, 5′-TCTCCTTGCTCATCCTGTC-3′; GmEF-1-F, 5′-ACATCCCAAGCTGACTGTGC-3′; GmEF-1-R, 5′-TACCTGGCCTTGGAATACTTGG-3′) for rice
MATERIALS AND METHODS
Plant Materials and Chemicals. Soybean (Glycine max L.) Kaohsiung No. 12 was obtained from the agricultural research and extension station in Kaohsiung district, Taiwan. Soybean seeds were washed with running water, soaked in tap water overnight, and then ground in liquid nitrogen for RNA extraction. Japonica-type rice (Oryza sativa L.) Tainung 67 (TNG 67) was used for heterologous expression. p-Nitrophenol (pNP), dithiothreitol, and electrophoresis reagents were from Sigma-Aldrich Co. (St. Louis, MO, USA). CMSepharose CL-6B cation exchanger, Hiload 16/60 Superdex 75 columns, and protein standards were from GE Healthcare BioSciences Co. (Piscataway, NJ, USA). Isoflavone standards were purchased from Sigma-Aldrich Co. Liquid chromatography grade acetonitrile, methanol, and n-hexane were from Merck (Darmstadt, Germany). DIAION HP-20 was from Mitsubishi Chemical Co. (Tokyo, Japan). All chemicals used were of analytical grade. Rapid Amplification of cDNA ends (RACE). Total RNA was extracted from soybean seeds as described.26 Purified RNA was used to construct the RACE library by use of the SMART RACE cDNA Amplification kit (Clontech, Palo Alto, CA, USA). Our previous paper25 gave the partial N-terminal amino acid sequence for soybean β-glucosidase. To obtain the full-length cDNA encoding soybean βglucosidase, pairs of degenerate primers were designed on the basis of this partial protein sequence and the conserved sequences of βglucosidase from other plants in Genbank. After obtaining the gene’s partial sequence, gene-specific primers (GmBGL-F1, 5′-GCTGCGACCTGGCAGCAAATGGTGAATC-3′; GmBGL-R1, 5′-CCGCAATGCATGGAGCAAAGACATACGG-3′) were designed by use of the B
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Figure 2. Multiple sequence alignment of deduced amino acid sequences of β-glucosidase genes from Glycine max (GmBGL, KF740479), Arabidopsis thaliana (AtBGL, NM_122104), Gossypium hirsutum (GhBGL, AAQ17461), Hordeum vulgare (HvBGL, U46003), Nicotiana tabacum (NtBGL, AB017502), Oryza sativa (OsBGL, NM_001057810), Triticum aestivum (TaBGL, AY091513), Tropaeolum majus (TmBGL, AJ006501), and Zea mays (ZmBGL, NM_001136824). Conserved stretches and residues are indicated in bold and shading, respectively. N-terminal and Cterminal domains of glycosyl hydrolase 3 (GH3) family members are underlined with continuous and dashed lines, respectively. The positions of potential N-glycosylation sites are denoted by asterisks. Arrows indicate the catalytic nucleophile (Asp) and the catalytic acid/base (Glu) responsible for GH3 activities. C
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Figure 3. Phylogenetic tree of plant β-glucosidases. The tree was generated with the full-length BGL protein sequences by use of Clustal W and the neighbor-joining method (MEGA 5.0). The bar beneath the dendrogram represents a distance of 0.05 change per amino acid. Branches are labeled with species names as follows: G. max (GmBGL, KF740479; GmICHG, NP_001237501), A. thaliana (AtBGL, NP 197595), Brachypodium distachyon (BdBGL, XP 00355; BdBGL1, Bradi1g42690), Cicer arietinum (CaBGL_X1, XP_004507133), G. hirsutum (GhBGL, AAQ17461), H. vulgare (HvBGL, AAC49170), Medicago truncatula (MtBGL, XP 003601350; MtBGL_G2, ABW76287), O. sativa (OsBGL, Os03g0749300; OsBGL24, LOC_Os06g21570), Populus trichocarpa (PtBGL, XP 00231363), Solanum lycopersicum (SlBGL, Solyc08g044510), Theobroma cacao (TcBGL, EOY33198), T. aestivum (TaBGL, AAM13694), and Z. mays (ZmBGL, NP 001130296). Actin1 and soybean elongation factor-1α, respectively, as normalization controls. Enzyme Extraction and Activity Assay. Shoots from 2-week-old rice plants were ground to powder in liquid nitrogen, and 2 mM EDTA aqueous solution was added to a volume of approximately 10 mL g−1 fresh weight. After centrifuging at 13000g for 15 min, the supernatant was discarded to remove water-soluble parts. The residue was then extracted twice with 10 mL of 3% NaCl aqueous solution for 30 min at room temperature. The homogenates were centrifuged at 13000g for 15 min at 4 °C. The supernatant as crude β-glucosidase was assayed for GmBGL activity and protein content. β-Glucosidase activity was assessed by measuring pNP release from p-nitrophenyl-βD-glucopyranoside (pNPG) at pH 9.5 by absorption spectrophotometry at 405 nm absorbance.25 The enzyme reaction was initiated by mixing 20 μL of crude β-glucosidase with 25 μL of 10 mM pNPG and 55 μL of 50 mM acetate buffer, pH 4.5. After incubation at 45 °C for 15 min, 100 μL of 1 M sodium carbonate solution was added to stop the reaction, and the released pNP was measured. A quantitative curve was prepared for authentic pNP at 0−0.5 mM. β-Glucosidase activity was calculated from the angular coefficient of the linear slope and normalized to protein content by using the Bradford method. Antiserum Preparation. The full-length coding region of GmBGL was cloned into the pET32a vector at the NcoI and XhoI sites for protein expression in the Escherichia coli BL21 (DE3) strain by induction with isopropyl-D-thiogalactopyranoside. For purification of recombinant protein, bacterial extracts were initially passed through an Ni2+ column (GE Healthcare Life Sciences) according to the manufacturer’s protocol, and then purified recombinant GmBGL was used as the rabbit antigen for antibody production (LTK BioLaboratories, Taoyuan, Taiwan). Western Blot Analysis. In total, 2 μg of crude extract from shoots was separated by 10% SDS-PAGE, and proteins were transferred to an Immobilon-P PVDF transfer membrane (Merck Millipore, Darmstadt, Germany), which was blocked with 5% skim milk for 1 h and incubated with primary antibody at a 1:500 dilution at 4 °C for 12 h and then with horseradish peroxidase-conjugated secondary goat antirabbit IgG antibody (GeneTex, Irvine, CA, USA) at 1:50000 dilution for 1 h. Immunoreactivity was detected by enhanced chemiluminescence (Amersham International PLC, Gloucester, UK). The positive control from soybean was purified as described.25 Briefly, 200 g of okara was homogenized in 1 L of 3% (w/v) NaCl aqueous solution; after filtration, the filtrate was concentrated by ultrafiltration (Hollow Fiber Cartridge, MWCO 5 kDa, GE Healthcare Life Science), treated
with dithiothreitol, and then further purified by CM-Sepharose CL-6B cation-exchange chromatography to obtain soybean β-glucosidase. Hydrolyzing Test of Malonylglucosidic and Glucosidic Forms of Isoflavone. Malonylglucosidic and glucosidic forms of isoflavone were prepared as described.29 Briefly, isoflavones were extracted from soybean with 60% methanol, and then solvents were removed under reduced pressure to obtain the crude extracts of isoflavone, which were further fractionated by passing through a column prepacked with Diaion HP20 resins. The HP20 adsorbed fraction was eluted sequentially with a bed volume of 40% methanol, 70% methanol, and pure methanol to obtain the corresponding fractions that contained the dominant constituent with malonylglucosidic, glucosidic, and aglycone forms of isoflavone, respectively. The isolated isoflavones in each of the eluents was identified by comparison with authentic isoflavone standards through high-performance liquid chromatography (HPLC). After evaporation to dryness under vacuum, the isolated fraction with dominant glucosyl isoflavone was used as the substrate for analyzing the enzyme activity of recombinant βglucosidase. For evaluating the substrate affinity of enzyme, both malonylglucosidic and glucosidic forms of isoflavone were used as substrates. The hydrolysis was initiated by adding 100 μL of the substrate isoflavone at 3000 μg/mL into the reaction mixture, which consisted of 10 μg of crude extract protein and 100 μL of 50 mM acetate buffer, pH 4.5, with a final volume of 350 μL. After incubation at 45 °C for 60 min, the reaction was stopped by adding 350 μL of methanol containing 1000 μg/mL benzoic acid. The insoluble substances were removed by centrifugation, and the supernatant was used for analyzing isoflavone content by HPLC. HPLC involved the model 584 Solvent Delivery Module (ESA Biosciences, Chelmsford, MA, USA) equipped with a YMC-Pack ODS-AM C18 column (4.6 × 250 mm, 5 μm) and a Thermo SpectroMonitor 3200 digital UV−vis detector. The linear mobile phase gradient was obtained with 0.1% acetic acid in H2O (solvent A) and 0.1% acetic acid in acetonitrile (solvent B). After injection of 20 μL of sample, solvent B was increased from 15 to 20% over 20 min, to 24% within the next 10 min, isocratic at 24% over 6 min, increased to 35% within the next 8 min, then isocratic at 35% over 6 min, and finally to decreased to 15% within 5 min and held at that percentage for the next 15 min. The flow rate was set at 1 mL/min. The eluted components were detected at 254 nm. Statistical Analysis. Data are expressed as mean ± SE (n = 4). Comparisons involved ANOVA, and means were compared by Duncan’s multiple-range test (P < 0.05). D
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Analysis of Transgenic Rice Plants Harboring the GmBGL Gene. Figure 5a illustrates the plasmid construct used for heterologous expression of GmBGL in rice. After Agrobacterium-mediated transformation, 15 independent transgenic lines were obtained. Three randomly selected transgenic lines were analyzed. RT-PCR analysis revealed increased GmBGL transcript in oxBGL1- and oxBGL3-overexpressing lines and markedly increased GmBGL transcript in the oxBGL2-overexpressing line, with no GmBGL transcript detected in nontransformants (WT) and plants transformed with empty vector (Figure 5b). To examine the translation of GmBGL in transgenic rice, we produced a polyclonal antibody to detect GmBGL protein. GmBGL protein highly accumulated in oxBGL1 and oxBGL2 lines and slightly in oxBGL3. Moreover, GmBGL antibody strongly bound to the purified soybean BGL protein, with no signal detected in the WT or rice plants transformed with empty vector (Figure 5c). β-Glucosidase Activity in Transgenic Rice and Substrate Specificity toward Isoflavones. To examine the βglucosidase activity of recombinant GmBGL in transgenic rice, we performed a chromogenic assay with pNPG as a substrate. As compared with the WT, oxBGL1 and oxBGL2 showed 6and 31-fold increased activity, respectively (Figure 6). The βglucosidase activity of oxBGL3 and the WT was similar. The substrate specificities of recombinant GmBGLs toward glucosidic and malonylglucosidic isoflavones are in Figure 7. Figure 7a shows the chromatogram for isoflavone substrate before hydrolysis. After hydrolyzation by crude GmBGLs from oxBGL1 and oxBGL2 at 45 °C and pH 4.5 for 30 min, the contents of daidzin and genistin were decreased to 42 and 46% of the original content with β-glucosidase from oxBGL1 (Figure 7b) and to 97 and 80% with β-glucosidase from oxBGL2 (Figure 7c), but the corresponding contents of daidzein and genistein with both treatments were increased. Daidzin and genistin contents were not reduced in oxBGL3, the WT, or the empty vector control (Figure 7d−f). Thus, both GmBGLs from transgenic rice oxBGL1 and oxBGL2 could effectively hydrolyze glucosyl-conjugated isoflavones into their corresponding aglycones. The content of malonylglucosidic isoflavones in all treatments remained relatively stable. Thus, the β-glucosidase activity of GmBGL from transgenic rice, which possesses high substrate specificity toward glycosyl isoflavones rather than malonylglucosidic isoflavones, could be markedly increased by overexpressing GmBGL.
RESULTS Characterization of GmBGL Gene. After RACE analysis, we obtained a 2190 bp DNA fragment containing a poly-A signal region in the 3′-UTR. Sequence analysis revealed a single open reading frame of 1884 bp encoding a polypeptide of 627 amino acids. The open reading frame was designated the G. max β-glucosidase gene (GmBGL). The amino acid sequences for GmBGL shared 90% similarity with β-D-glucosidase of Gossypium hirsutum (GhBGL, GenBank accession no. AAQ17461). The cDNA and amino acid sequence of GmBGL have been deposited at GenBank (accession no. KF740479) (Figure 2). GmBGL Is a Member of the Glycosyl Hydrolase 3 Family. A BLAST search of the soybean genome database (http://www.phytozome.net/soybean) revealed four highly homologous genes for GmBGL. Soybean clones Glyma10g15980.1, Glyma02g33550.1, Glyma02g43990.1, and Glyma14g04940.2 share 99, 96.5, 86.1, and 86.0% identity, respectively, to GmBGL, and all genes were annotated as members of the β-glucosidase family. However, N-terminal amino acid sequence analysis demonstrated that only the Glyma10g15980.1 was identical to the N-terminal sequence we identified previously.25 As compared with Glyma10g15980.1, two single-nucleotide polymorphisms were found in the GmBGL cDNA sequence, which may result from different soybean genetic backgrounds. Multiple sequence alignment of β-glucosidase among G. max, A. thaliana, G. hirsutum, H. vulgare, N tabacum, O. sativa, T. aestivum, T. majus, and Z. mays demonstrated that the GH3 family shared high similarity of the N-terminal domain (105Leu−Val340) and C-terminal domain (412Leu−Thr621) (Figure 2). Aspartic acid, located in the conserved motif 303 GFVISDW309 of all GH3 family members, was thought to be one of the active sites of amino acid residues. Another putatively catalytic residue of glutamic acid was located in E514. The phylogenetic tree shown in Figure 3 summarizes the βglucosidases identified in the soybean genome and other species. It revealed a clear divergence between GH1 and GH3 isoforms (Figure 3). Both GH1 and GH3 isoforms could be divided into dicot and Poaceae subgroups. The novel GmBGL belongs to the dicot GH3 subgroup. The sequences in this group are restricted to dicot plants and have been identified in G. hirsutum, Theobroma cacao, Populus trichocarpa, M. truncatula, and A. thaliana. No isoform from this group was found in the Poaceae genome. In contrast, another soybean βglucosidase, GmICHG, is included in GH1 (Figure 3). Expression of GmBGL Gene in Soybean Plants. We analyzed the spatial GmBGL gene expression in soybean by semiquantitative RT-PCR. The GmBGL transcript was present in both vegetative and reproductive tissues. Roots and leaves accumulated more GmBGL transcript than did other tissues (Figure 4).
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DISCUSSION We previously purified and characterized a β-glucosidase with isoflavone glucoside-hydrolyzing activity from soybean okara.25 In this study, we isolated GmBGL, a novel gene encoding βglucosidase, and validated the enzyme activity and substrate specificity in transgenic rice. GmBGL encodes for a polypeptide of 627 amino acids with molecular mass of 69 kDa. The deduced amino acid sequence had a high isoelectric point at pH 8.98, which agrees with our previous finding of native GmBGL from soybean okara being capable of separation by adsorption to CM-Sepharose CL-6B cation-exchange resin.25 GmBGL was transformed into the rice genome and could be transmitted to progenies. Among three randomly selected transgenic lines overexpressing GmBGL, oxBGL2 expressed the highest mRNA levels of β-glucosidase, and oxBGL1 and oxBGL3 expressed lower levels (Figure 5b). As well, oxBGL2 expressed the most abundant GmBGL protein, followed by oxBGL1, with the least by oxBGL3. Both oxBGL1 and oxBGL3
Figure 4. Semiquantitative RT-PCR of GmBGL transcript levels in root, stem, leaf, pod, and mature seeds. G. max elongation factor-1α (GmEF-1α) was used as an internal control. E
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Figure 5. Heterologous expression of GmBGL in rice. (a) Schematic representation of the expression cassette used for rice transformation. LB, left border; RB, right border; Pubi, maize ubiquitin 1 promoter together with its first intron; nos, terminator of the nopaline synthase gene; P35S, CaMV 35S promoter; hpt, coding sequence of hygromycin phosphotransferase; tml, terminator of a tumor morphology large gene; oxBGL, overexpressing BGL line. (b) Semiquantitative RT-PCR of expression of GmBGL in the wild type (WT) and transformants. Semiquantitative RT-PCR was performed with gene-specific primers of GmBGL and the rice Actin1 as a control. (c) Western blot analysis of GmBGL protein level in the WT and transformants. Equal loading of extracted proteins (2 μg each lane) is shown by Coomassie brilliant blue (CBB) R250 staining. Partially purified GmBGL protein from soybean was used as a positive control to demonstrate antibody specificity.
glycosylation sites in the GmBGL amino acid sequence (Figure 2), so the higher molecular mass of GmBGL in transgenic rice than in soybean okara may be due to glycosylation. The β-glucosidases have been characterized into five familiesGH1, GH3, GH5, GH9, and GH30by amino acid sequence similarity.32 Most of the identified genes encoding β-glucosidases belong to the GH1 family. The first isoflavone-7-O-glucoside-specific β-glucosidase was isolated from chick pea (Cicer arietinum). Three isozymes, including R3, H1, and L1, were isolated from roots, hypocotyls, and leaves, respectively.19 Among the isoflavone-related leguminous β-glucosidases, two members of the GH1 family, dalcochinin8′-O-β-glucoside β-glucosidase (dalcochinase) from Dalbergia cochinchinensis and Dalbergia nigrescens β-glucosidase, have been cloned and characterized. Dalcochinase, showing high aglycone specificity for isoflavonoids, can hydrolyze both β-glucosides and β-fucosides, and D. nigrescens β-glucosidase can efficiently remove disaccharides from dalpatein 7-O-β-D-apiofuranosyl(1→6)-β-D-glucopyranoside and 7-hydroxy-2′,4′,5′,6-tetramethoxy-7-O-β- D -apiofuranosyl-(1→6)-β- D -glucopyranoside. They can also remove a single glucose residue from isoflavonoid 7-O-glucosides such as daidzin and genistin.33,34 Our phylogenetic analysis revealed that GmBGL is a novel GH3 protein in soybean (Figure 3). GmBGL is preferentially expressed in roots and leaves (Figure 4), which is a similar expression pattern as for the soybean GH1 gene GmICHG.29 However, soybean GH1 and GH3 β-glucosidases show divergence in substrate specificity. The GmICHG (GH1 family) can hydrolyze both malonylated and nonmalonylated
Figure 6. β-Glucosidase activity in transgenic rice plants. Proteins were extracted from shoots of WT and transgenic rice plants. Data are expressed as specific activity. β-Glucosidase activity was assayed with pnitrophenyl-β-D-glucopyranoside (pNPG) as the substrate, and activity was calculated from measuring pNP release by absorbance at 405 nm. Data are the mean ± SE (n = 4). Bars with the same letter are not significantly different at P < 0.05.
expressed similar mRNA levels of GmBGL, but a very low protein level in oxBGL3 (Figure 5c). This suggests that mRNA levels of GmBGL in transgenes may, but not necessarily, contribute to corresponding protein levels. The molecular mass of GmBGL in transgenic rice was slightly higher than that for β-glucosidase purified from soybean okara (Figure 5c). An explanation for the difference in molecular mass could be that recombinant GmBGL underwent post-translational modification such as glycosylation. Many foreign proteins, including soybean proteins, overexpressed in rice are glycosylated.30,31 We found five potential NF
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conversion of isoflavone conjugates into bioactive aglycones in soybean. In conclusion, the cloning and identification of functional βglucosidase helps us investigate its physiological role and provides the opportunity for genetic engineering of soybean seeds with high levels of aglycone.
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AUTHOR INFORMATION
Corresponding Authors
*(N.-W. Su) Phone: +886 2 33664819. E-mail:
[email protected]. tw. *(C.-Y. Hong) Phone: +886 2 33663839. E-mail: cyhong@ntu. edu.tw. Author Contributions ∥
C.-C. Hsu and T.-M. Wu have equally contributed to this work. Notes
The authors declare no competing financial interest.
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REFERENCES
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Figure 7. High-performance liquid chromatograms of substrate isoflavones after hydrolysis by crude β-glucosidases from transgenic rice oxBG lines at 45 °C and pH 4.5: (a) original substrate before hydrolysis as a blank control; (b−f) isoflavones were hydrolyzed for 30 min by the crude enzyme from oxBGL1, 2, 3, the WT (TNG 67), and the empty vector control, respectively. Peaks: 1, daidzin; 2, genistin; 3, internal standard (benzoic acid); 4, malonyl-genistin; 5, daidzein; 6, genistein.
forms of isoflavone-7-O-β-D-glucosides, the malonylated form being the preferred substrate. In contrast, the GmBGL (GH3 family) has strict substrate specificity toward glucosyl but not malonylglucosidic isoflavones. GmBGL shares 72% sequence identity with a barley β-Dglucan exohydrolase isoenzyme ExoI, which also belongs to the GH3 family. The 3D structure of ExoI demonstrated that its first 357 residues represent an (α/β)8 barrel domain and the 374−559 residues, an (α/β)6 sheet.35 Protein engineering predicted that GmBGL likely shares similar protein folding. According to Varghese et al.,35 Asp285 and Glu491, representing two conserved residues of the barley β-D-glucan exohydrolase, were suggested to be involved in the nucleophilic catalytic sites within general acid/base catalysis. We found these two conserved amino acids in GmBGL (Asp308 and Glu514) and also in other GH3 family members (Figure 2). Many studies have described the efficient transformation of isoflavone glycosides to aglycones by exogenous enzymatic hydrolysis and microbial fermentation, but only a few have characterized β-glucosidase from soybean. Because aglyconic isoflavones are considered practically to be the bioactive form of soy isoflavones, which could have a role as chemoattractants to attract nodule bacteria occurring in soy plant rhizosphere,21,36 the amount of aglyconic forms of isoflavone in mature soybean seeds is actually negligible; the predominant forms of isoflavone are malonylglucosides and glucosides.37 Therefore, most isoflavones in soybean have to undergo a hydrolysis process for their function while soybean seeds are sprouting and growing. We found greater GmBGL transcript level in roots and leaves than in other tissues of soybean seedlings (Figure 4), so GmBGL may play an important role in G
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