Mol Biol Rep DOI 10.1007/s11033-014-3338-8
Molecular cloning and characterization of genistein 40 -O-glucoside specific glycosyltransferase from Bacopa monniera Ruby • R. J. Santosh Kumar • Rishi K. Vishwakarma Somesh Singh • Bashir M. Khan
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Received: 3 March 2013 / Accepted: 18 March 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Health related benefits of isoflavones such as genistein are well known. Glycosylation of genistein yields different glycosides like genistein 7-O-glycoside (genistin) and genistein 40 -O-glycoside (sophoricoside). This is the first report on isolation, cloning and functional characterization of a glycosyltransferase specific for genistein 40 -Oglucoside from Bacopa monniera, an important Indian medicinal herb. The glycosyltransferase from B. monniera (UGT74W1) showed 49 % identity at amino acid level with the glycosyltransferases from Lycium barbarum. The UGT74W1 sequence contained all the conserved motifs present in plant glycosyltransferases. UGT74W1 was cloned in pET-30b (?) expression vector and transformed into E. coli. The molecular mass of over expressed protein was found to be around 52 kDa. Functional characterization of the enzyme was performed using different substrates. Product analysis was done using LC–MS and HPLC, which confirmed its specificity for genistein 40 -Oglucoside. Immuno-localization studies of the UGT74W1 showed its localization in the vascular bundle. Spatiotemporal expression studies under normal and stressed conditions were also performed. The control B. monniera plant showed maximum expression of UGT74W1 in leaves followed by roots and stem. Salicylic acid treatment causes R. J. Santosh Kumar and Rishi K. Vishwakarma have contributed equally.
Electronic supplementary material The online version of this article (doi:10.1007/s11033-014-3338-8) contains supplementary material, which is available to authorized users. Ruby R. J. Santosh Kumar R. K. Vishwakarma S. Singh B. M. Khan (&) Plant Tissue Culture Division, National Chemical Laboratory, Dr. Homi Bhabha Road, Pune 411008, Maharashtra, India e-mail: [email protected]
almost tenfold increase in UGT74W1 expression in roots, while leaves and stem showed decrease in expression. Since salicylic acid is generated at the time of injury or wound caused by pathogens, this increase in UGT74W1 expression under salicylic acid stress might point towards its role in defense mechanism. Keywords Bacopa monniera Glycosyltransferase Plant secondary product glycosyltransferase motif Immuno-localization Expression analysis Abbreviations HPLC High performance liquid chromatography LC–MS Liquid chromatography–mass spectroscopy cDNA Complementary DNA UGTs UDP-dependent glycosyltransferases qRT-PCR Quantitative real-time polymerase chain reaction RACE Rapid amplification of cDNA ends BCIP/ 5-Bromo-4-chloro-30 -indolyphosphate/nitroNBT blue tetrazolium chloride PBS Phosphate buffer saline IPTG Isopropyl b-D-1-thiogalactopyranoside h Hour (s) X-gal 5-Bromo-4-chloro-3-indolyl-beta-Dgalactopyranoside
Introduction Plants have the ability to produce a wide range of secondary metabolites including alkaloids, terpenoids and flavonoids [1]. These plant secondary metabolites undergo several modifications such as hydroxylation, methylation and glycosylation. Glycosylation is one of the key
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modification reactions and is mostly the final step in the biosynthesis of plant secondary metabolites. Glycosylation is catalyzed by UDP-glycosyltransferases that transfers the sugar moiety from a uridine 50 -diphosphosugars (UDPsugars) to low molecular weight acceptor substrates [2–4]. Glycosylation of secondary metabolites increase water solubility, improves chemical stability, facilitates their storage and accumulation in the plant cells. It has also been shown to alter biological activity as well. One of the most extensively studied classes of plant glycosides is the large group of flavonoids. Flavonoids are polyphenolic compounds found everywhere in plants. Flavonoids are classified, according to chemical structure, into flavonols, flavones, flavanones, isoflavones, catechins, anthocyanidins and chalcones. Among those isoflavones comprise a large and distinctive subclass of the flavonoids. They are more restricted in the plant kingdom than other flavonoids. Genistein, diadzein and glycitein are the predominant isoflavones. These naturally occurring products usually exist in plants as glycosides and their glycosylation has a great influence on their pharmacological properties. Isoflavones and their glycosides have received much attention because of their health benefits such as antioxidative [5–8], anticancerous properties [9–15], lowering the incidence of cardiovascular diseases [16, 17], prevention of osteoporosis and attenuation of post-menopausal problems. They are also known for their anti-insect, antifungal and antimicrobial activities in plants. Genistein, one of the most abundant isoflavone, is the promising agent for cancer chemoprevention and/or treatment and is also known for its estrogenic effects [18, 19]. There are quite a few reports of soy isoflavones which showed some minor side effects like gastrointestinal effects, sleepiness and myalgia but is not associated with increased risk of breast and endometrial cancers [20, 21]. Many plant UGTs can recognize genistein as an acceptor when assayed in vitro. Although certain enzymes show specificity toward one hydroxyl group of the aglycone while others show specificity towards multiple hydroxyl groups. There are several studies about UGTs from different plants which glycosylate genistein resulting in the formation of genistein 7-O-glycoside [22–24] but there is not even a single report regarding the UGT which specifically glycosylates genistein to genistein 40 -O-glucoside to the best of our knowledge. Bacopa monniera is a creeping herb found profoundly in wet and marshy areas. The herb has been used as a memory enhancer since centuries because of the glycosides present in the plant. Despite the importance and medicinal properties of glycosides from B. monniera, to date no cDNA encoding any glycosyltransferases has been isolated from this plant. Therefore studying such glycosyltransferases from B. monniera could be of great pharmaceutical
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important. In this study we report the isolation and cloning of a UDP-glucose:glycosyltransferase (UGT74W1) from B. monniera using degenerate primers. Phylogenetic analysis showed that UGT74W1 is closely related to a UGT from Lycium barbarum glycosylating flavonoids. Functional expression of the gene product in Escherichia coli demonstrated that the recombinant UGT74W1 catalyzed the transfer of sugar unit from UDP-glucose to genistein resulting in the formation of genistein 40 -O-glucoside. Furthermore gene expression profile and immuno-localization studies of UGT74W1 were carried out to gain insight into the gene transcript level in different organs of the plant and its cellular localization.
Materials and methods Chemicals, plant materials and treatments All the chemicals and reagents were procured from Sigma-Aldrich, USA unless stated otherwise. Genistein 40 -O-glucoside and genistein 7-O-glucoside were obtained from Chromadex, USA. B. monniera plants grown under field conditions were used throughout the experiments. For Real time PCR experiments B. monniera plants grown under field conditions were collected and cut into 1 cm-long pieces, surface sterilized and inoculated in the initiation medium (MS basal medium supplemented with 0.5 mg/L 6-benzyl amino purine and 3 % sucrose, solidified with 1 % agar) under aseptic conditions. Cultures were incubated at the temperature 25 ± 1 °C under 16 h photoperiod at 11.7 lmol/m2/s light intensity/8 h dark cycles for about 20 days. The proliferating multiple adventitious shoots were separated and re-inoculated on the proliferation medium (MS medium with 3 % sucrose) for further growth. To determine stress-induced (salt, methyl jasmonate, salicylic acid, mannitol, cold and heat) gene expression in different parts i.e., stem, leaves and roots of the B. monniera plant, the above in vitro proliferated shoots were used by transferring them to MS basal medium supplemented with 50 mM NaCl, 20 lM methyl jasmonate, 30 lM salicylic acid and 2 % mannitol separately. Approximately 0.1–0.2 g of tissue was inoculated on each medium containing salt, methyl jasmonate, salicylic acid and mannitol respectively. The cultures were further incubated under the same conditions as described earlier. Sample tissue was collected from these cultures after 2 months. A separate control was grown for salicylic acid stress since salicylic acid was dissolved in ethanol. In case of cold (-20 °C) and heat (37 °C) treatment, cultures were re-inoculated without any supplement in MS basal medium and grown till 2 months followed by
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heat treatment for 1 day and cold shock treatment for 2 h. Tissue was harvested from heat and cold treated plants and were used for qRT-PCR experiments.
Cloning of B. monniera glycosyltransferase cDNA Total RNA was isolated from the B. monniera plant shoots by using Sigma Aldrich RNA extraction kit as per the manufacturer’s instructions. RNA quality and quantity was checked at 260 and 280 nm with UV–Vis spectrophotometer (NanoVue plus, GE Healthcare, Uppsala, Sweden). First-strand cDNA synthesis was performed by reverse transcription from 1 lg of total RNA using an oligo (dT)15 primer and a first strand cDNA synthesis kit (Promega, USA) following the manufacturer’s protocol. Degenerate primers were made from the conserved amino acid sequences among the glycosyltransferase gene family. The primers used were: NTMF1 (50 TCC (C/A)GC (C/A)CA AGG CCA TAT TAA (C/T)C 30 ) and NTMR2 (50 ACT TTC (G/C)AG T/A)GT CGA ATT CCA TCC AC 30 ). A PCR was performed using these primers and cDNA as template. Amplified product was purified by AxygenTM GEL elution kit (Biosciences, USA) and subcloned into pGEM-T Easy vector (Promega, USA). Sequencing was done using automated DNA sequencer with T7 and SP6 primers. Sequence similarity searches were performed using BLAST program at National Centre for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov). To obtain full length cDNA of the candidate gene, named as UGT74W1, 30 and 50 rapid amplification of cDNA ends (RACE) was performed with the GeneRacer kit (Invitrogen, USA) with the following cycling program: 1 cycle at 95 °C for 5 min; 35 cycles at 95 °C for 1 min, 55–58 °C for 0.45 min and 72 °C for 1.30 min; and a final extension at 72 °C for 5 min. The primer set used for 30 RACE were: 30 GSP (50 TCC CGC CCA AGG CCA TAT TAA TC 30 ) and Nested 30 GSP (50 CCG TCT ACT GCC ATT TGC ATC AAG GT 30 ) whereas 50 GSP (50 TTC CGC AAC CTG AGT CTC CAA GTC AT 30 ) and Nested 50 GSP (50 GCG CTG ACA AAA CAA GAC TGA GTG 30 ) were used for 50 RACE. The amplified fragments were cloned into pGEM-T Easy vector and subjected to sequencing as described above. Once 50 and 30 RACE sequences were determined, the full-length clone of UGT74W1 (Accession no. FJ586244) was amplified from the cDNA with the following extreme forward (50 ATG GAG AGC AAA GGA ACA GGG AAG GA 30 ) and the reverse (50 TCA AGT TAG GAG CAG GGA CAT GTT AAA 30 ) primers. This full length UGT74W1 gene was cloned into pGEM-T Easy vector and was transformed into E. coli XL-1 Blue cells and the recombinant clone was confirmed by sequencing.
Phylogenetic tree Deduced amino acid sequences of plant glycosyltransferases available in the NCBI GenBank database were used to construct the phylogenetic tree. These translated amino acid sequences were aligned using Clustal X alignment program at Expasy (www.expasy.ch). The evolutionary history of UGT74W1 protein was inferred using the Neighbor-Joining method [25]. The evolutionary distances were computed using the Poisson correction method [26] and are in the units of the number of amino acid substitutions per site. Phylogenetic analysis was done using MEGA4 software [27]. Heterologous expression To express recombinant protein in E. coli, the full length cDNA of UGT74W1 was amplified by Accu Taq LA DNA polymerase (Sigma, USA) with appropriate restriction sites. The forward and reverse primers used were as follows: the forward primer 50 CAT ATG GAG AGC AAA GGA ACA GGG AAG GA 30 and the reverse primer 50 CTC GAG AGT TAG GAG CAG GGA CAT GTT AAA 30 . The PCR product has NdeI at 50 end and XhoI at the 30 end. This product was then digested with NdeI and XhoI and directionally subcloned into pET-30b (?) vector (Novagen, Germany) to create inframe C-terminal fusion with a 69 His-tag. After transformation into BL21 (DE3) E. coli cells, colonies were selected on LB containing kanamycin (KAN; 50 lg/ml) plates. Individual colonies were grown overnight in 5 ml of LB-KAN medium at 37 °C, and 1 % of the culture was used to inoculate 100 ml of LB-KAN fresh medium. Cells were grown at 37 °C until an OD600 reached 0.6, after which the culture was induced with 0.1 mM IPTG and allowed to grow for 18 h at 15 °C. The cells were harvested by centrifugation at 6,000 rpm for 10 min and resuspended in 6.25 ml of lysis buffer (50 mM Tris, 100 mM NaCl, 5 mM EDTA, 0.5 % TritonX-100, 0.7 mM DTT and 0.1 mM PMSF). Cells were disrupted by sonication at 70 % amplitude on an ultrasonic liquid processor, XL 2000 model (MISONIX) for 5 min. MgSO4, final concentration of 10 mM and lysozyme, final concentration 100 lg/ml was added to the disrupted cells and kept at 4 °C for half an hour. Soluble proteins were then collected after centrifugation at 12,000 rpm for 15 min. The soluble fraction was mixed with Ni2?–NTA (nickel– nitrilotriacetic acid) agarose beads (Qiagen, CA, USA) equilibrated with binding buffer (50 mM Tris–HCl buffer, pH 8, 300 mM NaCl) and incubated for 2 h on 4–7 °C on a rotating shaker. Column was then washed with wash buffer (binding buffer containing 30 mM imidazole) until the absorbance at 280 nm stabilized near zero. Bound UGT74W1 proteins were eluted with the binding buffer
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containing 250 mM imidazole. The partially purified proteins were analysed on 10 % SDS-PAGE. Expressed proteins were visualized by Coomassie Blue staining.
along with 20 mM Tris–HCl pH 8, 600 lM UDP-sugar and 5 mM acceptor substrate. LC–MS analysis
Matrix-assisted laser desorption/ionization (MALDI) MS/MS Partially purified recombinant glycosyltransferase protein fraction was run on a 10 % SDS-PAGE gel. The Coomassie Blue staining was used to stain the gel. Band corresponding to the molecular mass of the recombinant protein was manually excised from Coomassie blue-stained SDS-PAGE gel. The gel pieces were destained by destaining solution (50 % acetonitrile/50 % 50 mM NH4HCO3) till colour was gone. The gel was then dehydrated with 100 % acetonitrile and vacuum dried. The gel pieces were reduced with 10 mM dithiothreitol in 100 mM NH4HCO3 for 45–60 min at 56 °C and then alkylated with 55 mM iodoacetamide in 100 mM NH4HCO3 at room temperature for 45 min in the dark. Gel pieces were washed with 100 mM NH4HCO3 for 5 min, followed by washing with acetonitrile, and dried under vacuum. Then enough trypsin solution was added to cover the gel pieces and the gel pieces were rehydrated at 4 °C for 30 min in buffer containing 50 mM NH4HCO3 and trypsin. This was followed by overnight digestion at 37 °C. After digestion, the supernatants were recovered. Peptides were extracted from the gel pieces with a 2 % formic acid–50 % acetonitrile solution with 10 min each of sonication and vortexing. All extracts were pooled, and the volume was reduced with a Speed Vac. The sample was then ready for loading onto MALDI/MS/MS. Simultaneously MALDI plate was also washed in order to remove the particulate matter deposited if any. Then sample and the MALDI matrix (a-cyano-4-hydroxycinnamic acid) in the proportion of 2:1 ratio were loaded on to the MALDI plate. This was followed by reading of MALDI plate with laser energy of about 280 V using MALDSNYPT equipment (Waters). Enzyme assays and analysis of reaction products The standard reaction mixture (500 ll) containing 20 mM Tris–HCl pH 8, 600 lM UDP-sugar (UDP-glucose, UDPgalactose and UDP-glucuronic acid), 5 mM acceptor substrates and the recombinant enzyme (30 lg of partially purified recombinant protein). The reaction carried out at 30 °C for 2 h was then terminated and extracted with ethyl acetate. The extracted reaction was then completely vacuum dried and dissolved in methanol. The substrates and glycosylated products were analysed by LC–MS and HPLC. The negative control mixture contained the extracts of bacteria transformed with empty pET 30b (?) vector
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The LC–MS analysis of glycosides was performed using a Q-TOF Premier mass spectrometer (Waters, USA) outfitted with an electrospray ion source operated in the V-Optics negative mode. A Develosil C18 column was used for LC on a Shimadzu LC-20AD HPLC. For LC–MS analysis solvent system used was 20 % water and 80 % (v/v) acetonitrile (CH3CN) for 5 min. The mass spectrometer scanned from m/z 0–2000. HPLC and product analysis Analysis of the substrates and their glycosylated products was performed using High performance liquid chromatography (HPLC, Perkin Elmer series 200, USA) connected online to a photodiode array detector on a supelco C18 column (5 lm, 25 cm 9 4.6 mm). The mobile phase consisted of sterile mili Q water with 0.1 % Trifluoroacetic acid. 20 ll of sample dissolved in methanol was injected in HPLC column and was programmed as follows: 10 % acetonitrile for 5 min, 35 % acetonitrile for 5 min 80 % acetonitrile for 5 min and 95 % acetonitrile for 5 min at a flow rate of 1 ml/min. Wavelength recorder set at 270 nm was used to detect the compounds eluting from the column. SDS-PAGE and Western blotting Tissue harvested from the whole B. monniera plant was crushed to a fine powder in liquid nitrogen. Ground tissue was homogenized in buffer (100 mM Tris–HCl pH 7.5, 2 % PVPP, 2 % PEG 4000, DTT 5 mM and PMSF 1 mM). Homogenized tissue was centrifuged at 12,000 rpm at 4 °C and supernatant was collected in fresh microfuge tube. The total protein was quantified using Bradford assay (Bradford reagent, Sigma) and used for Western blotting. The crude extracts (200 lg) were resolved on SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membrane using iBlot Gel Transfer System (Invitrogen, USA) according to the manufacturer’s instructions. The PVDF membrane containing transferred protein samples were processed as per standard procedure, blocking with 1 % BSA at 37 °C for 2 h, treatment with 1:5,000 dilution of primary antibody (polyclonal antibodies raised against purified recombinant UGT74W1 protein in New Zealand white rabbit). Following washing with PBS-T (PBS containing 0.02 % Tween 20), the polypeptide-primary antibody complex were incubated with the secondary antibody i.e., goat anti-rabbit IgG conjugated with alkaline phosphatase (Bangalore Genei, India) at a 1:20,000 dilution. Finally
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washing and visualization of immunoreactive proteins was done with colour developing reagent (BCIP/NBT solution). Histology and immuno-localization For immunolocalization free hand transverse sections of roots, leaves and stem of B. monniera were fixed overnight under vacuum in freshly prepared cold 4 % buffered formaldehyde (4 % paraformaldehyde in 19 PBS). The sections were dehydrated by passages through increasing ethanol: water series (30, 50, 70, 85, 95 and 100 % ethanol) for 30 min each. This was followed by passages through tertiary butanol:ethanol series (25:75, 50:50, 75:25, and 100:0). The sections were rehydrated by treating with 70 %, 50 % ethanol and 0.59 SSC for 2 min. The rehydrated sections were soaked in two changes of 19 PBS for 10 min each. Next, the sections were washed in 19 PBS containing 1 % BSA for 5 min and subjected to 30 min of blocking with 10 % BSA at room temperature in a humidified chamber. Post blocking washes included three washes of 15 min each with 19 PBS containing 1 % BSA. Primary antibody (antibodies raised against purified UGT74W1 protein) incubation was carried out overnight in a humidified chamber at 4 °C using 1:5,000 dilution of primary antibody in 19 PBS containing 1 % BSA. Following the primary antibody incubation, the sections were washed thrice for 15 min each in 19 PBS containing 1 % BSA. A secondary antibody, 1:10,000 dilution of Antirabbit-IgG-goat alkaline phosphate conjugate antibody (diluted in 19 PBS with 1 % BSA), was added to the tissue sections at this stage and incubated at 37 °C in a humidified chamber for 2 h in dark. Post secondary antibody washes were carried out at room temperature using 19 PBS with 1 % BSA. Color was developed using BCIP/NBT solution (Bio-world, USA) and the slides were placed in humidified chamber at room temperature in dark for 45 min. Upon color development, 10 mM EDTA was used to stop the reaction, rinsed with water, air dried and cover slipmounted using glycerol. Samples were examined and images captured with Axioplan 2 Carl Zeiss microscope equipped with a digital camera (AxioCamMRc5). Control sections were treated in the same way as described above, except that the primary antibody was replaced by rabbit pre-immune serum. Analysis of mRNA levels in different tissues and stress conditions Total RNA was extracted from leaves, stem and roots of B. monniera using the RNA extraction kit (Sigma). cDNAs were reverse-transcribed from 1 lg of each total RNA with ImProm cDNA synthesis kit (Promega, Madison, USA). qRT-PCR was performed using a Brilliant SYBRGreen
Q-PCR kit (Stratagene, USA) in a real-time PCR machine (Stratagene Mx3000P, USA). Gene-specific primer set used were BMGT1F: 50 AAA CAG CTC CAT TAG CAT AGA AAG C 30 and BMGT1R: 50 GCC TCT CTC CTA AAG CGG TTG 30 .The 18S rRNA primer set used were 18SF: 50 GCA CGC GCG CTA CAA TGA AAG TAT 30 and 18SR: 50 TGT GTA CAA AGG GCA GGG ACG TAA 30 . The results were normalized to the reference gene 18S rRNA and fold expression was calculated by comparative Ct method (2-DDCt value).
Results Cloning and characterization of glycosyltransferase cDNAs from B. monniera To isolate putative glycosyltransferase genes from B. monniera, degenerate primers were designed from the conserved regions of the UGT genes from the other plant species. These primers were used to amplify the partial (*1 kb) fragment of UGT from B. monniera. Sequencing of the partial PCR product revealed its similarity to UGT gene from Lamiaceae family. The sequence information of the partial clone was used to design the gene specific primers to obtain full-length clone using rapid amplification of cDNA ends (RACE). 50 RACE and 30 RACE were performed using B. monniera cDNA as template. The fulllength sequence contained an open reading frame of 1,392 bp in length. The predicted amino acid sequence of UGT74W1 specifies a polypeptide of 463 amino acids in length with a molecular mass of 52.3 kDa. The deduced amino acid sequence of UGT74W1 showed less degree of overall similarity to other known plant UGTs, with the exception of the conserved donor binding site at the C-terminus. It contained the consensus sequence plant secondary product glycosyltransferase motif (PSPG) box (Fig. 1) as described by [28] which has been proposed to be the sugar donor binding site [29]. According to the CAZy database (http://www.cazy.org/), UGT74W1 belongs to the family 1 of the glycosyltransferases. UGT74W1 (GenBank accession no. FJ586244) showed 49 % identity at amino acid level with the UDP-glucose:glycosyltransferase gene from L. barbarum. A Phylogenetic tree was constructed using MEGA4 software program (www.megasoftware.net/.) after aligning predicted protein coding sequences retrieved from the NCBI GenBank Database using Clustal X2.0. The phylogenetic tree suggests that UGT74W1 belongs to Cluster IV as shown in Fig. 2. Since the glycosyltransferases in this cluster mostly catalyze glycosylation of flavonoid glycosides [30], we predicted that UGT74W1 may show glycosyltransferase activity towards flavonoid glycosides. The
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Mol Biol Rep b Fig. 1 Sequence alignment of deduced amino acid of UGT74W1
with other plant glycosyltransferases: sequence alignment was done using Clustal X2.0 analysis. Conserved region of the donor binding site i.e., PSPG Box is shown within the box. Most conserved motif within the PSPG box i.e., HCGWNS is highlighted in red color. Other conserved residues within the PSPG box which are important for donor binding are W, D & Q as shown in pink color. The conserved residue i.e., Histidine which is reported to be important for catalytic activity within the acceptor binding site is shown in blue color. Accession nos. and name of the sources are as: gi/7385017 (AF190634, Nicotiana tabacum), gi/60649935 (AJ889012, Solanum lycopersicum), gi/209954705 (AB360619, Lycium barbarum) and gi/ 302310821 (FJ586244, Bacopa monniera). (Color figure online)
Phylogenetic tree also provided information that UGT74W1 belongs to a separate Cluster whereas Cluster I, Cluster II, Cluster III and Cluster V included glycosyltransferases glycosylating flavonoids at 7-OH, 2-OH, 5-OH and 3-OH positions respectively. Expression and identification of the partially purified recombinant protein To determine whether the isolated putative glycosyltransferase gene sequence encodes functional enzyme, the Hisfusion protein was expressed in E. coli (Fig. 3b). As a control reaction empty pET 30b (?) vector was also transformed into E. coli (Fig. 3a). The expressed protein (*52 kDa) was partially purified by His-tag affinity chromatography, run on 10 % SDS-PAGE and the expected protein of interest was excised from the gel and analyzed with MALDI/MS/MS. The MALDI/MS/MS analysis was done for confirmation of partially purified recombinant UGT74W1 protein with the sequences already submitted to the Uniprot Database. The MALDI/MS/MS ionization spectra and coverage map of UGT74W1 protein are shown in Fig. 4. The ionization spectra showed mass of different peptides of the query protein and the coverage map showed the number of peptides of query protein which shows exact match with the template (UGT74W1) present in the Uniprot database. From the MALDI/MS/MS analysis it was concluded that the partially purified recombinant protein extract contained UGT74W1 protein. Enzymatic activity of the recombinant protein To examine whether the expressed protein encodes an active flavonoid glycosyltransferase, enzyme assay was performed. The glycosyltransferase activity was checked with the partially purified recombinant protein and different acceptor substrates (Supplementary Table 1) in order to check the specificity or regiospecificity but UGT74W1 protein showed activity only with genistein as an acceptor substrate in the presence of UDP-glucose. The assay reaction was subjected to liquid chromatography-mass
spectrometry (LC–MS) analysis. The peak identified as genistein with m/z 271 (M ? H)? whereas the peak corresponding to genistein glycoside, m/z 455.3 (M ? Na)? was detected in the assay reaction (Fig. 5). The product analysis was further validated by HPLC. The product showed retention time identical to that of standard genistein 40 -O-glucoside (Fig. 6). In control reaction the extracts of bacteria transformed with empty vector did not recognize genistein as acceptor substrate using UDP-glucose as sugar donor molecule. This suggests that bacteria did not give any background activity of its own. These results proved that the recombinant enzyme UGT74W1 incorporates glucose molecule to genistein molecule giving rise to genistein 40 -O-glucoside only. Spatio-temporal expression of UGT74W1 gene under normal and stress induced conditions The expression pattern of UGT74W1 mRNA in different tissues of B. monniera plant was performed using qRTPCR. The qRT-PCR analysis showed differential transcript levels with maximum expression in leaf tissues (Fig. 7a). In order to determine whether the expression levels of UGT74W1 gene were influenced by different types of stresses like methyl jasmonate, salicylic acid, mannitol, salt (NaCl), cold and heat, qRT-PCR was carried out. Relative expression in terms of mean fold expression under mannitol stress conditions has increased almost fivefold in roots and huge dip in fold expression has been seen in leaves. In methyl jasmonate and salt stress, expression level has increased in stem and decreased in leaves whereas in case of heat and cold shock treatments expression has totally decreased as shown in Fig. 7a. On the other hand salicylic acid stress increased fold expression by almost 10 times in roots and has decreased in stem and leaves (Fig. 7b). Identification of endogenous UGT74W1 protein expression by Western blotting To check the endogenous expression of UGT74W1 protein in B. monniera plant, Western blotting was performed. The crude proteins extracted from the whole B. monniera plant were subjected to SDS-PAGE and blotted with the antibodies raised against the purified UGT74W1 protein. A clear single band, corresponding to the molecular mass of the UGT74W1 protein was observed in the plant extract (Fig. 8). Since glycosyltransferases comprise of a large family, the specificity of the antibody was checked using one more UGT protein isolated from B. monniera (data not shown) but only single band of expected molecular mass of UGT74W1 protein was observed on Western blot. This also confirms that the antibodies raised against UGT74W1
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Fig. 2 Phylogenetic tree of UGT74W1 with other plant glycosyltransferases: the evolutionary relationship of UGT74W1 protein with other 26 different plant glycosyltransferases was constructed using the Neighbor-Joining method after alignment of amino acids sequences by Clustal X2.0. The optimal tree with the sum of branch length = 7.26316574 is shown. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The names and accession numbers are: Scutellaria baicalensis (BAA83484), Perilla frutescens (UGT73A7, UGT73A13, BAA19659), Antirrhinum majus (UGT73N1,
UGT73E2, UGT73A9), Malus 9 domestica (UGT71A15), Pyrus communis (UGT71A16), Catharanthus roseus (UGT71E2), Cyclamen persicum (UGT71E3), Dianthus caryophyllus (UGT71F3), Glandularia x hybrid (BAA36423), Perilla frutescens var. crispa (BAA36421), Torenia hybrid cultivar (BAC54093), Petunia 9 hybrid (BAA89009, BAA89008), Lycium barbarum (UGT74P1, UGT74P2, UGT74NI, UGT74N2), Arabidopsis thaliana (NP 564357, 197207), Vitis vinifera (AAB81682), Gentiana triflora (UGT78B1), Forsythia 9 intermedia (AAD21086), Bacopa monniera (UGT74W1)
protein does not provide any non-specific binding to other UGT members present in the B. monniera plant.
protein in the vascular bundles and its absence in the pith and cortex tissues as shown in Fig. 9.
Immuno-localization studies of UGT74W1 in B. monniera plant
Discussion
Immuno-localization was performed for cellular localization of UGT74W1 protein in different parts of the B. monniera plant. Cross sections of immuno-localized root, leaf and stem showed the presence of UGT74W1
Bacopa monniera belongs to the family Scrophulariaceae, is commonly referred to as brahmi. Since brain is the source of creative activity, any compound that ameliorates brain activity is called brahmi [31]. It has been used for
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Fig. 3 SDS-PAGE analysis of recombinant UGT74W1 protein: coomassie blue staining (a) lane M protein molecular weight marker, lane 1 lysate of empty pET 30 (b) vector transformed in E. coli BL21 DE3 cell line, lane 2 inclusion bodies of empty vector transformed in
E. coli BL21 DE3 cell line. b Lane M protein molecular weight marker, lane 1 partially purified UGT74W1 protein, lane 2 lysate of UGT74W1 protein
Fig. 4 MALDI/MS/MS spectra: MALDI MS/MS analysis of recombinant UGT74W1 protein a ionization spectra, b coverage map of recombinant UGT74W1 protein
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Fig. 5 LC–MS analysis of UGT74W1 activity with genistein: a LC–MS spectra of the control reaction (i.e., bacteria transformed with empty vector) with genistein, b LC–MS spectra of assay reaction with genistein
centuries in Ayurveda (the Hindu system of traditional medicine native to India) for the treatment of anxiety, improving intellect, memory and revitalisation of sensory organs [32, 33]. B. monniera is also reported to possess analgesic, antipyretic, anti-inflammatory, sedative [34], free radical scavenging and anti-lipid peroxidative activities [35]. Apart from that the plant is also found to be useful in the treatment of various respiratory [36], cardiac and neuropharmacological [31] disorders. In view of the importance of this plant in the indigenous system of medicine, systematic chemical examinations of the plant had been carried out by several groups of researchers. The major chemical entities found to be responsible for the medicinal properties of B. monniera were various glycosides present in the plant. These glycosides which were produced by an enzyme glycosyltransferase were present in the plant in very low quantities so an alternative approach to increase the yield of such medicinally important
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glycosides was to isolate the enzyme, glycosyltransferase and over express it in suitable expression system. Therefore the isolation and characterization of such glycosyltransferases is of prime importance for pharmacological applications. In this study we reported the isolation and cloning of cDNA encoding UGT gene, the identity of which was established by sequence comparison with previously isolated and characterized UGTs. Conserved domains of UGT74W1 were detected by conserved domains database (CDD) at NCBI GenBank Database which showed that UGT74W1 belongs to a glycosyltransferase GTB type superfamily. The isolated UGT74W1 also contained the conserved domain (45 amino acid long PSPG box) in the sugar donor binding pocket as well as the important residues like His-22 and Asp-121 [29] known for their role in catalytic activity in the acceptor binding pocket. UGT74W1 protein also does not contain any signal peptide as is the
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Fig. 6 High-performance liquid chromatography (HPLC) elution profiles of UGT74W1 activity with genistein: a authentic standard of genistein, b authentic genistein 40 -O-glucoside, c assay reaction
with genistein, d HPLC profile of control reaction (i.e., bacteria transformed with empty vector) with genistein
property of all the plant UGTs characterized till date [37] which suggests that the enzyme is present in the cytosol. Phylogenetic analysis revealed that UGT74W1 is evolutionary similar to the UGTs from L. barbarum which accepts UDP-glucose as preferred sugar donor and flavonoids as acceptor molecule [30]. The identity of the recombinant UGT74W1 protein expressed in E. coli was confirmed by MALDI MS/MS because of the presence of other minor contaminating bands seen after affinity purification of the protein. MALDI MS/MS ionic spectra and coverage map confirmed its presence in the partially purified fraction. These results were consistent with the Western blotting analysis of the
partially purified fraction of the recombinant UGT74W1 protein (data not shown) using antibodies against it. Plant glycosyltransferases have been reported to exhibit specificity towards various flavonoids giving rise to single or multiple products. Many plant UGTs recognize genistein as an acceptor molecule when assayed in vitro and can glycosylate at 7-OH positions but only rarely glycosylates at 40 -OH position. In the present study we had isolated UGT which specifically glycosylates only at 40 -OH position of genistein resulting in the formation of genistein 40 O-glucoside. These results were in conformity with our previous report wherein homology model of UGT74W1 was docked with UDP-Glucose and different acceptor
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Fig. 8 Western blot analysis of UGT74W1: lane 1 crude plant extract (200 lg), lane 2 purified recombinant UGT74W1 protein expressed in E. coli BL2 (DE3) cell line
Fig. 7 Tissue specific expression of UGT74W1 using qRT-PCR: a relative expression in terms of mean fold expression of UGT74W1 gene under normal (control plant) and stress treated conditions i.e., mannitol, methyl jasmonate (MJ), cold, salt and heat, b relative expression of UGT74W1 gene under salicylic acid (SA) stress with respect to salicylic acid control plant. All values are plotted with standard deviation taken into account
molecules but the preferred acceptor for UGT74W1 was found to be genistein with binding energy of -6.68 kcal/mol [38]. To the best of our knowledge this is the first ever isolated UGT which glycosylates genistein exclusively at the 40 -OH position. qRT-PCR was performed to determine the transcript level of UGT74W1 gene in different parts of the plant under normal and stress treated conditions. The expression level of UGT74W1 was found to be highest in the leaf tissues as compared to the roots and stem under normal conditions. Some glycosyltransferases reported from Arabidopsis thaliana [39] and from Catharanthus roseus [40] also showed maximum expression in leaves whereas the UGT isolated from Solanum aculeatissimum [41] showed maximum expression in roots. It has been reported
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that glycosyltransferases are induced by a variety of abiotic stresses, including cold, salicylic acid [42] and methyl jasmonate [40, 43, 44]. In comparison to the normal plant, a fivefold increase in expression of UGT74W1 was observed in roots of plants under mannitol stress. In case of methyl jasmonate stress expression level of UGT74W1 was found to be induced in stem whereas around 14–15 folds dip in expression was seen in leaves. Many glycosyltransferases reported had been induced on treatment with methyl jasmonate [40, 43, 44]. Additionally gene expression of UGT74W1 was found to be increased by tenfold after induction with salicylic acid, an important molecule in plant defense [45]. Several glycosyltransferases reported till date had been induced by salicylic acid treatment [46– 50]. Few glycosyltransferases that are putatively involved in defense mechanism are also induced by salicylic acid [51]. The fact that UGT74W1 enzyme is also induced by salicylic acid and methyl jasmonate might point towards its role in plant defense. Immuno-localization studies of UGT74W1 protein showed that the protein was localized in the vascular bundle. Since the isolated UGT from B. monniera is a flavonoid glycosyltransferase and flavonoids being polyphenols present in the vascular bundles [52] might be the possible reason why B. monniera GT is localized in the vascular bundle region. This study is also the first time report of immuno-localization of UGTs on cross-sections of different parts of the plant.
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Fig. 9 Immuno-localization of UGT74W1: immuno-localization of UGT74W1 in Bacopa monniera: a–c cross sections of control stem, root and leaf at 920 magnification respectively, d–f cross sections of
stem, root and leaf treated with UGT74W1 antibody at 920 magnification respectively
Acknowledgments We are highly grateful for the support of this work by Networking Project of Council of Scientific and Industrial Research (CSIR), India, via grant no: NWP0008. We are also thankful to CSIR for awarding CSIR-Senior Research fellowship. We are also grateful to Dr. Thomas Vogt, Leibniz Institute of Plant Biochemistry, Germany for the critical reading of the manuscript. We are also thankful to Peter McKenzie for naming Bacopa monniera UGT as UGT74W1 at http://www.flinders.edu.au/medicine/sites/clinical-phar macology/ugt-homepage.cfm.
6. Rice-Evans C, Miller N, Paganga G (1997) Antioxidant properties of phenolic compounds. Trends Plant Sci 2:152–159 7. Ru¨fer CE, Kulling SE (2006) Antioxidant activity of isoflavones and their major metabolites using different in vitro assays. J Agric Food Chem 54:2926–2931 8. Naderi GA, Asgary S, Sarraf-Zadegan N, Shirvany H (2003) Anti-oxidant effect of flavonoids on the susceptibility of LDL oxidation. Mol Cell Biochem 246:193–196 9. Peterson G, Barnes S (1991) Genistein inhibition of the growth of human breast cancer cells: independence from estrogen receptors and the multi-drug resistance gene. Biochem Biophys Res Commun 179:661–667 10. Peterson G, Barnes S (1993) Genistein and biochanin A inhibit the growth of human prostate cancer cells but not epidermal growth factor receptor tyrosine autophosphorylation. Prostate 22:335–345 11. Barnes S, Peterson G, Grubbs C, Setchell K (1994) Potential role of dietary isoflavones in the prevention of cancer. Adv Exp Med Biol 354:135–147 12. Barnes S (1995) Effect of genistein on in vitro and in vivo models of cancer. J Nutr 125:777S–783S 13. Sarkar FH, Li Y (2003) Soy isoflavones and cancer prevention. Cancer Invest 21:744–757 14. Adlercreutz H, Honjo H, Higashi A, Fotsis T, Hamalainen E, Hasegawa T, Okada H (1991) Urinary excretion of lignans and isoflavonoid phytoestrogens in Japanese men and women consuming a traditional Japanese diet. Am J Clin Nutr 54:1093–1100 15. Troll W, Wiesner R, Shellabarger CJ, Holtzman S, Stone JP (1980) Soybean diet lowers breast tumor incidence in irradiated rats. Carcinogenesis 1:469–472
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Molecular cloning and characterization of genistein 40 -O-glucoside specific glycosyltransferase from Bacopa monniera Ruby • R. J. Santosh Kumar • Rishi K. Vishwakarma Somesh Singh • Bashir M. Khan
•
Received: 3 March 2013 / Accepted: 18 March 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Health related benefits of isoflavones such as genistein are well known. Glycosylation of genistein yields different glycosides like genistein 7-O-glycoside (genistin) and genistein 40 -O-glycoside (sophoricoside). This is the first report on isolation, cloning and functional characterization of a glycosyltransferase specific for genistein 40 -Oglucoside from Bacopa monniera, an important Indian medicinal herb. The glycosyltransferase from B. monniera (UGT74W1) showed 49 % identity at amino acid level with the glycosyltransferases from Lycium barbarum. The UGT74W1 sequence contained all the conserved motifs present in plant glycosyltransferases. UGT74W1 was cloned in pET-30b (?) expression vector and transformed into E. coli. The molecular mass of over expressed protein was found to be around 52 kDa. Functional characterization of the enzyme was performed using different substrates. Product analysis was done using LC–MS and HPLC, which confirmed its specificity for genistein 40 -Oglucoside. Immuno-localization studies of the UGT74W1 showed its localization in the vascular bundle. Spatiotemporal expression studies under normal and stressed conditions were also performed. The control B. monniera plant showed maximum expression of UGT74W1 in leaves followed by roots and stem. Salicylic acid treatment causes R. J. Santosh Kumar and Rishi K. Vishwakarma have contributed equally.
Electronic supplementary material The online version of this article (doi:10.1007/s11033-014-3338-8) contains supplementary material, which is available to authorized users. Ruby R. J. Santosh Kumar R. K. Vishwakarma S. Singh B. M. Khan (&) Plant Tissue Culture Division, National Chemical Laboratory, Dr. Homi Bhabha Road, Pune 411008, Maharashtra, India e-mail: [email protected]
almost tenfold increase in UGT74W1 expression in roots, while leaves and stem showed decrease in expression. Since salicylic acid is generated at the time of injury or wound caused by pathogens, this increase in UGT74W1 expression under salicylic acid stress might point towards its role in defense mechanism. Keywords Bacopa monniera Glycosyltransferase Plant secondary product glycosyltransferase motif Immuno-localization Expression analysis Abbreviations HPLC High performance liquid chromatography LC–MS Liquid chromatography–mass spectroscopy cDNA Complementary DNA UGTs UDP-dependent glycosyltransferases qRT-PCR Quantitative real-time polymerase chain reaction RACE Rapid amplification of cDNA ends BCIP/ 5-Bromo-4-chloro-30 -indolyphosphate/nitroNBT blue tetrazolium chloride PBS Phosphate buffer saline IPTG Isopropyl b-D-1-thiogalactopyranoside h Hour (s) X-gal 5-Bromo-4-chloro-3-indolyl-beta-Dgalactopyranoside
Introduction Plants have the ability to produce a wide range of secondary metabolites including alkaloids, terpenoids and flavonoids [1]. These plant secondary metabolites undergo several modifications such as hydroxylation, methylation and glycosylation. Glycosylation is one of the key
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modification reactions and is mostly the final step in the biosynthesis of plant secondary metabolites. Glycosylation is catalyzed by UDP-glycosyltransferases that transfers the sugar moiety from a uridine 50 -diphosphosugars (UDPsugars) to low molecular weight acceptor substrates [2–4]. Glycosylation of secondary metabolites increase water solubility, improves chemical stability, facilitates their storage and accumulation in the plant cells. It has also been shown to alter biological activity as well. One of the most extensively studied classes of plant glycosides is the large group of flavonoids. Flavonoids are polyphenolic compounds found everywhere in plants. Flavonoids are classified, according to chemical structure, into flavonols, flavones, flavanones, isoflavones, catechins, anthocyanidins and chalcones. Among those isoflavones comprise a large and distinctive subclass of the flavonoids. They are more restricted in the plant kingdom than other flavonoids. Genistein, diadzein and glycitein are the predominant isoflavones. These naturally occurring products usually exist in plants as glycosides and their glycosylation has a great influence on their pharmacological properties. Isoflavones and their glycosides have received much attention because of their health benefits such as antioxidative [5–8], anticancerous properties [9–15], lowering the incidence of cardiovascular diseases [16, 17], prevention of osteoporosis and attenuation of post-menopausal problems. They are also known for their anti-insect, antifungal and antimicrobial activities in plants. Genistein, one of the most abundant isoflavone, is the promising agent for cancer chemoprevention and/or treatment and is also known for its estrogenic effects [18, 19]. There are quite a few reports of soy isoflavones which showed some minor side effects like gastrointestinal effects, sleepiness and myalgia but is not associated with increased risk of breast and endometrial cancers [20, 21]. Many plant UGTs can recognize genistein as an acceptor when assayed in vitro. Although certain enzymes show specificity toward one hydroxyl group of the aglycone while others show specificity towards multiple hydroxyl groups. There are several studies about UGTs from different plants which glycosylate genistein resulting in the formation of genistein 7-O-glycoside [22–24] but there is not even a single report regarding the UGT which specifically glycosylates genistein to genistein 40 -O-glucoside to the best of our knowledge. Bacopa monniera is a creeping herb found profoundly in wet and marshy areas. The herb has been used as a memory enhancer since centuries because of the glycosides present in the plant. Despite the importance and medicinal properties of glycosides from B. monniera, to date no cDNA encoding any glycosyltransferases has been isolated from this plant. Therefore studying such glycosyltransferases from B. monniera could be of great pharmaceutical
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important. In this study we report the isolation and cloning of a UDP-glucose:glycosyltransferase (UGT74W1) from B. monniera using degenerate primers. Phylogenetic analysis showed that UGT74W1 is closely related to a UGT from Lycium barbarum glycosylating flavonoids. Functional expression of the gene product in Escherichia coli demonstrated that the recombinant UGT74W1 catalyzed the transfer of sugar unit from UDP-glucose to genistein resulting in the formation of genistein 40 -O-glucoside. Furthermore gene expression profile and immuno-localization studies of UGT74W1 were carried out to gain insight into the gene transcript level in different organs of the plant and its cellular localization.
Materials and methods Chemicals, plant materials and treatments All the chemicals and reagents were procured from Sigma-Aldrich, USA unless stated otherwise. Genistein 40 -O-glucoside and genistein 7-O-glucoside were obtained from Chromadex, USA. B. monniera plants grown under field conditions were used throughout the experiments. For Real time PCR experiments B. monniera plants grown under field conditions were collected and cut into 1 cm-long pieces, surface sterilized and inoculated in the initiation medium (MS basal medium supplemented with 0.5 mg/L 6-benzyl amino purine and 3 % sucrose, solidified with 1 % agar) under aseptic conditions. Cultures were incubated at the temperature 25 ± 1 °C under 16 h photoperiod at 11.7 lmol/m2/s light intensity/8 h dark cycles for about 20 days. The proliferating multiple adventitious shoots were separated and re-inoculated on the proliferation medium (MS medium with 3 % sucrose) for further growth. To determine stress-induced (salt, methyl jasmonate, salicylic acid, mannitol, cold and heat) gene expression in different parts i.e., stem, leaves and roots of the B. monniera plant, the above in vitro proliferated shoots were used by transferring them to MS basal medium supplemented with 50 mM NaCl, 20 lM methyl jasmonate, 30 lM salicylic acid and 2 % mannitol separately. Approximately 0.1–0.2 g of tissue was inoculated on each medium containing salt, methyl jasmonate, salicylic acid and mannitol respectively. The cultures were further incubated under the same conditions as described earlier. Sample tissue was collected from these cultures after 2 months. A separate control was grown for salicylic acid stress since salicylic acid was dissolved in ethanol. In case of cold (-20 °C) and heat (37 °C) treatment, cultures were re-inoculated without any supplement in MS basal medium and grown till 2 months followed by
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heat treatment for 1 day and cold shock treatment for 2 h. Tissue was harvested from heat and cold treated plants and were used for qRT-PCR experiments.
Cloning of B. monniera glycosyltransferase cDNA Total RNA was isolated from the B. monniera plant shoots by using Sigma Aldrich RNA extraction kit as per the manufacturer’s instructions. RNA quality and quantity was checked at 260 and 280 nm with UV–Vis spectrophotometer (NanoVue plus, GE Healthcare, Uppsala, Sweden). First-strand cDNA synthesis was performed by reverse transcription from 1 lg of total RNA using an oligo (dT)15 primer and a first strand cDNA synthesis kit (Promega, USA) following the manufacturer’s protocol. Degenerate primers were made from the conserved amino acid sequences among the glycosyltransferase gene family. The primers used were: NTMF1 (50 TCC (C/A)GC (C/A)CA AGG CCA TAT TAA (C/T)C 30 ) and NTMR2 (50 ACT TTC (G/C)AG T/A)GT CGA ATT CCA TCC AC 30 ). A PCR was performed using these primers and cDNA as template. Amplified product was purified by AxygenTM GEL elution kit (Biosciences, USA) and subcloned into pGEM-T Easy vector (Promega, USA). Sequencing was done using automated DNA sequencer with T7 and SP6 primers. Sequence similarity searches were performed using BLAST program at National Centre for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov). To obtain full length cDNA of the candidate gene, named as UGT74W1, 30 and 50 rapid amplification of cDNA ends (RACE) was performed with the GeneRacer kit (Invitrogen, USA) with the following cycling program: 1 cycle at 95 °C for 5 min; 35 cycles at 95 °C for 1 min, 55–58 °C for 0.45 min and 72 °C for 1.30 min; and a final extension at 72 °C for 5 min. The primer set used for 30 RACE were: 30 GSP (50 TCC CGC CCA AGG CCA TAT TAA TC 30 ) and Nested 30 GSP (50 CCG TCT ACT GCC ATT TGC ATC AAG GT 30 ) whereas 50 GSP (50 TTC CGC AAC CTG AGT CTC CAA GTC AT 30 ) and Nested 50 GSP (50 GCG CTG ACA AAA CAA GAC TGA GTG 30 ) were used for 50 RACE. The amplified fragments were cloned into pGEM-T Easy vector and subjected to sequencing as described above. Once 50 and 30 RACE sequences were determined, the full-length clone of UGT74W1 (Accession no. FJ586244) was amplified from the cDNA with the following extreme forward (50 ATG GAG AGC AAA GGA ACA GGG AAG GA 30 ) and the reverse (50 TCA AGT TAG GAG CAG GGA CAT GTT AAA 30 ) primers. This full length UGT74W1 gene was cloned into pGEM-T Easy vector and was transformed into E. coli XL-1 Blue cells and the recombinant clone was confirmed by sequencing.
Phylogenetic tree Deduced amino acid sequences of plant glycosyltransferases available in the NCBI GenBank database were used to construct the phylogenetic tree. These translated amino acid sequences were aligned using Clustal X alignment program at Expasy (www.expasy.ch). The evolutionary history of UGT74W1 protein was inferred using the Neighbor-Joining method [25]. The evolutionary distances were computed using the Poisson correction method [26] and are in the units of the number of amino acid substitutions per site. Phylogenetic analysis was done using MEGA4 software [27]. Heterologous expression To express recombinant protein in E. coli, the full length cDNA of UGT74W1 was amplified by Accu Taq LA DNA polymerase (Sigma, USA) with appropriate restriction sites. The forward and reverse primers used were as follows: the forward primer 50 CAT ATG GAG AGC AAA GGA ACA GGG AAG GA 30 and the reverse primer 50 CTC GAG AGT TAG GAG CAG GGA CAT GTT AAA 30 . The PCR product has NdeI at 50 end and XhoI at the 30 end. This product was then digested with NdeI and XhoI and directionally subcloned into pET-30b (?) vector (Novagen, Germany) to create inframe C-terminal fusion with a 69 His-tag. After transformation into BL21 (DE3) E. coli cells, colonies were selected on LB containing kanamycin (KAN; 50 lg/ml) plates. Individual colonies were grown overnight in 5 ml of LB-KAN medium at 37 °C, and 1 % of the culture was used to inoculate 100 ml of LB-KAN fresh medium. Cells were grown at 37 °C until an OD600 reached 0.6, after which the culture was induced with 0.1 mM IPTG and allowed to grow for 18 h at 15 °C. The cells were harvested by centrifugation at 6,000 rpm for 10 min and resuspended in 6.25 ml of lysis buffer (50 mM Tris, 100 mM NaCl, 5 mM EDTA, 0.5 % TritonX-100, 0.7 mM DTT and 0.1 mM PMSF). Cells were disrupted by sonication at 70 % amplitude on an ultrasonic liquid processor, XL 2000 model (MISONIX) for 5 min. MgSO4, final concentration of 10 mM and lysozyme, final concentration 100 lg/ml was added to the disrupted cells and kept at 4 °C for half an hour. Soluble proteins were then collected after centrifugation at 12,000 rpm for 15 min. The soluble fraction was mixed with Ni2?–NTA (nickel– nitrilotriacetic acid) agarose beads (Qiagen, CA, USA) equilibrated with binding buffer (50 mM Tris–HCl buffer, pH 8, 300 mM NaCl) and incubated for 2 h on 4–7 °C on a rotating shaker. Column was then washed with wash buffer (binding buffer containing 30 mM imidazole) until the absorbance at 280 nm stabilized near zero. Bound UGT74W1 proteins were eluted with the binding buffer
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containing 250 mM imidazole. The partially purified proteins were analysed on 10 % SDS-PAGE. Expressed proteins were visualized by Coomassie Blue staining.
along with 20 mM Tris–HCl pH 8, 600 lM UDP-sugar and 5 mM acceptor substrate. LC–MS analysis
Matrix-assisted laser desorption/ionization (MALDI) MS/MS Partially purified recombinant glycosyltransferase protein fraction was run on a 10 % SDS-PAGE gel. The Coomassie Blue staining was used to stain the gel. Band corresponding to the molecular mass of the recombinant protein was manually excised from Coomassie blue-stained SDS-PAGE gel. The gel pieces were destained by destaining solution (50 % acetonitrile/50 % 50 mM NH4HCO3) till colour was gone. The gel was then dehydrated with 100 % acetonitrile and vacuum dried. The gel pieces were reduced with 10 mM dithiothreitol in 100 mM NH4HCO3 for 45–60 min at 56 °C and then alkylated with 55 mM iodoacetamide in 100 mM NH4HCO3 at room temperature for 45 min in the dark. Gel pieces were washed with 100 mM NH4HCO3 for 5 min, followed by washing with acetonitrile, and dried under vacuum. Then enough trypsin solution was added to cover the gel pieces and the gel pieces were rehydrated at 4 °C for 30 min in buffer containing 50 mM NH4HCO3 and trypsin. This was followed by overnight digestion at 37 °C. After digestion, the supernatants were recovered. Peptides were extracted from the gel pieces with a 2 % formic acid–50 % acetonitrile solution with 10 min each of sonication and vortexing. All extracts were pooled, and the volume was reduced with a Speed Vac. The sample was then ready for loading onto MALDI/MS/MS. Simultaneously MALDI plate was also washed in order to remove the particulate matter deposited if any. Then sample and the MALDI matrix (a-cyano-4-hydroxycinnamic acid) in the proportion of 2:1 ratio were loaded on to the MALDI plate. This was followed by reading of MALDI plate with laser energy of about 280 V using MALDSNYPT equipment (Waters). Enzyme assays and analysis of reaction products The standard reaction mixture (500 ll) containing 20 mM Tris–HCl pH 8, 600 lM UDP-sugar (UDP-glucose, UDPgalactose and UDP-glucuronic acid), 5 mM acceptor substrates and the recombinant enzyme (30 lg of partially purified recombinant protein). The reaction carried out at 30 °C for 2 h was then terminated and extracted with ethyl acetate. The extracted reaction was then completely vacuum dried and dissolved in methanol. The substrates and glycosylated products were analysed by LC–MS and HPLC. The negative control mixture contained the extracts of bacteria transformed with empty pET 30b (?) vector
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The LC–MS analysis of glycosides was performed using a Q-TOF Premier mass spectrometer (Waters, USA) outfitted with an electrospray ion source operated in the V-Optics negative mode. A Develosil C18 column was used for LC on a Shimadzu LC-20AD HPLC. For LC–MS analysis solvent system used was 20 % water and 80 % (v/v) acetonitrile (CH3CN) for 5 min. The mass spectrometer scanned from m/z 0–2000. HPLC and product analysis Analysis of the substrates and their glycosylated products was performed using High performance liquid chromatography (HPLC, Perkin Elmer series 200, USA) connected online to a photodiode array detector on a supelco C18 column (5 lm, 25 cm 9 4.6 mm). The mobile phase consisted of sterile mili Q water with 0.1 % Trifluoroacetic acid. 20 ll of sample dissolved in methanol was injected in HPLC column and was programmed as follows: 10 % acetonitrile for 5 min, 35 % acetonitrile for 5 min 80 % acetonitrile for 5 min and 95 % acetonitrile for 5 min at a flow rate of 1 ml/min. Wavelength recorder set at 270 nm was used to detect the compounds eluting from the column. SDS-PAGE and Western blotting Tissue harvested from the whole B. monniera plant was crushed to a fine powder in liquid nitrogen. Ground tissue was homogenized in buffer (100 mM Tris–HCl pH 7.5, 2 % PVPP, 2 % PEG 4000, DTT 5 mM and PMSF 1 mM). Homogenized tissue was centrifuged at 12,000 rpm at 4 °C and supernatant was collected in fresh microfuge tube. The total protein was quantified using Bradford assay (Bradford reagent, Sigma) and used for Western blotting. The crude extracts (200 lg) were resolved on SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membrane using iBlot Gel Transfer System (Invitrogen, USA) according to the manufacturer’s instructions. The PVDF membrane containing transferred protein samples were processed as per standard procedure, blocking with 1 % BSA at 37 °C for 2 h, treatment with 1:5,000 dilution of primary antibody (polyclonal antibodies raised against purified recombinant UGT74W1 protein in New Zealand white rabbit). Following washing with PBS-T (PBS containing 0.02 % Tween 20), the polypeptide-primary antibody complex were incubated with the secondary antibody i.e., goat anti-rabbit IgG conjugated with alkaline phosphatase (Bangalore Genei, India) at a 1:20,000 dilution. Finally
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washing and visualization of immunoreactive proteins was done with colour developing reagent (BCIP/NBT solution). Histology and immuno-localization For immunolocalization free hand transverse sections of roots, leaves and stem of B. monniera were fixed overnight under vacuum in freshly prepared cold 4 % buffered formaldehyde (4 % paraformaldehyde in 19 PBS). The sections were dehydrated by passages through increasing ethanol: water series (30, 50, 70, 85, 95 and 100 % ethanol) for 30 min each. This was followed by passages through tertiary butanol:ethanol series (25:75, 50:50, 75:25, and 100:0). The sections were rehydrated by treating with 70 %, 50 % ethanol and 0.59 SSC for 2 min. The rehydrated sections were soaked in two changes of 19 PBS for 10 min each. Next, the sections were washed in 19 PBS containing 1 % BSA for 5 min and subjected to 30 min of blocking with 10 % BSA at room temperature in a humidified chamber. Post blocking washes included three washes of 15 min each with 19 PBS containing 1 % BSA. Primary antibody (antibodies raised against purified UGT74W1 protein) incubation was carried out overnight in a humidified chamber at 4 °C using 1:5,000 dilution of primary antibody in 19 PBS containing 1 % BSA. Following the primary antibody incubation, the sections were washed thrice for 15 min each in 19 PBS containing 1 % BSA. A secondary antibody, 1:10,000 dilution of Antirabbit-IgG-goat alkaline phosphate conjugate antibody (diluted in 19 PBS with 1 % BSA), was added to the tissue sections at this stage and incubated at 37 °C in a humidified chamber for 2 h in dark. Post secondary antibody washes were carried out at room temperature using 19 PBS with 1 % BSA. Color was developed using BCIP/NBT solution (Bio-world, USA) and the slides were placed in humidified chamber at room temperature in dark for 45 min. Upon color development, 10 mM EDTA was used to stop the reaction, rinsed with water, air dried and cover slipmounted using glycerol. Samples were examined and images captured with Axioplan 2 Carl Zeiss microscope equipped with a digital camera (AxioCamMRc5). Control sections were treated in the same way as described above, except that the primary antibody was replaced by rabbit pre-immune serum. Analysis of mRNA levels in different tissues and stress conditions Total RNA was extracted from leaves, stem and roots of B. monniera using the RNA extraction kit (Sigma). cDNAs were reverse-transcribed from 1 lg of each total RNA with ImProm cDNA synthesis kit (Promega, Madison, USA). qRT-PCR was performed using a Brilliant SYBRGreen
Q-PCR kit (Stratagene, USA) in a real-time PCR machine (Stratagene Mx3000P, USA). Gene-specific primer set used were BMGT1F: 50 AAA CAG CTC CAT TAG CAT AGA AAG C 30 and BMGT1R: 50 GCC TCT CTC CTA AAG CGG TTG 30 .The 18S rRNA primer set used were 18SF: 50 GCA CGC GCG CTA CAA TGA AAG TAT 30 and 18SR: 50 TGT GTA CAA AGG GCA GGG ACG TAA 30 . The results were normalized to the reference gene 18S rRNA and fold expression was calculated by comparative Ct method (2-DDCt value).
Results Cloning and characterization of glycosyltransferase cDNAs from B. monniera To isolate putative glycosyltransferase genes from B. monniera, degenerate primers were designed from the conserved regions of the UGT genes from the other plant species. These primers were used to amplify the partial (*1 kb) fragment of UGT from B. monniera. Sequencing of the partial PCR product revealed its similarity to UGT gene from Lamiaceae family. The sequence information of the partial clone was used to design the gene specific primers to obtain full-length clone using rapid amplification of cDNA ends (RACE). 50 RACE and 30 RACE were performed using B. monniera cDNA as template. The fulllength sequence contained an open reading frame of 1,392 bp in length. The predicted amino acid sequence of UGT74W1 specifies a polypeptide of 463 amino acids in length with a molecular mass of 52.3 kDa. The deduced amino acid sequence of UGT74W1 showed less degree of overall similarity to other known plant UGTs, with the exception of the conserved donor binding site at the C-terminus. It contained the consensus sequence plant secondary product glycosyltransferase motif (PSPG) box (Fig. 1) as described by [28] which has been proposed to be the sugar donor binding site [29]. According to the CAZy database (http://www.cazy.org/), UGT74W1 belongs to the family 1 of the glycosyltransferases. UGT74W1 (GenBank accession no. FJ586244) showed 49 % identity at amino acid level with the UDP-glucose:glycosyltransferase gene from L. barbarum. A Phylogenetic tree was constructed using MEGA4 software program (www.megasoftware.net/.) after aligning predicted protein coding sequences retrieved from the NCBI GenBank Database using Clustal X2.0. The phylogenetic tree suggests that UGT74W1 belongs to Cluster IV as shown in Fig. 2. Since the glycosyltransferases in this cluster mostly catalyze glycosylation of flavonoid glycosides [30], we predicted that UGT74W1 may show glycosyltransferase activity towards flavonoid glycosides. The
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with other plant glycosyltransferases: sequence alignment was done using Clustal X2.0 analysis. Conserved region of the donor binding site i.e., PSPG Box is shown within the box. Most conserved motif within the PSPG box i.e., HCGWNS is highlighted in red color. Other conserved residues within the PSPG box which are important for donor binding are W, D & Q as shown in pink color. The conserved residue i.e., Histidine which is reported to be important for catalytic activity within the acceptor binding site is shown in blue color. Accession nos. and name of the sources are as: gi/7385017 (AF190634, Nicotiana tabacum), gi/60649935 (AJ889012, Solanum lycopersicum), gi/209954705 (AB360619, Lycium barbarum) and gi/ 302310821 (FJ586244, Bacopa monniera). (Color figure online)
Phylogenetic tree also provided information that UGT74W1 belongs to a separate Cluster whereas Cluster I, Cluster II, Cluster III and Cluster V included glycosyltransferases glycosylating flavonoids at 7-OH, 2-OH, 5-OH and 3-OH positions respectively. Expression and identification of the partially purified recombinant protein To determine whether the isolated putative glycosyltransferase gene sequence encodes functional enzyme, the Hisfusion protein was expressed in E. coli (Fig. 3b). As a control reaction empty pET 30b (?) vector was also transformed into E. coli (Fig. 3a). The expressed protein (*52 kDa) was partially purified by His-tag affinity chromatography, run on 10 % SDS-PAGE and the expected protein of interest was excised from the gel and analyzed with MALDI/MS/MS. The MALDI/MS/MS analysis was done for confirmation of partially purified recombinant UGT74W1 protein with the sequences already submitted to the Uniprot Database. The MALDI/MS/MS ionization spectra and coverage map of UGT74W1 protein are shown in Fig. 4. The ionization spectra showed mass of different peptides of the query protein and the coverage map showed the number of peptides of query protein which shows exact match with the template (UGT74W1) present in the Uniprot database. From the MALDI/MS/MS analysis it was concluded that the partially purified recombinant protein extract contained UGT74W1 protein. Enzymatic activity of the recombinant protein To examine whether the expressed protein encodes an active flavonoid glycosyltransferase, enzyme assay was performed. The glycosyltransferase activity was checked with the partially purified recombinant protein and different acceptor substrates (Supplementary Table 1) in order to check the specificity or regiospecificity but UGT74W1 protein showed activity only with genistein as an acceptor substrate in the presence of UDP-glucose. The assay reaction was subjected to liquid chromatography-mass
spectrometry (LC–MS) analysis. The peak identified as genistein with m/z 271 (M ? H)? whereas the peak corresponding to genistein glycoside, m/z 455.3 (M ? Na)? was detected in the assay reaction (Fig. 5). The product analysis was further validated by HPLC. The product showed retention time identical to that of standard genistein 40 -O-glucoside (Fig. 6). In control reaction the extracts of bacteria transformed with empty vector did not recognize genistein as acceptor substrate using UDP-glucose as sugar donor molecule. This suggests that bacteria did not give any background activity of its own. These results proved that the recombinant enzyme UGT74W1 incorporates glucose molecule to genistein molecule giving rise to genistein 40 -O-glucoside only. Spatio-temporal expression of UGT74W1 gene under normal and stress induced conditions The expression pattern of UGT74W1 mRNA in different tissues of B. monniera plant was performed using qRTPCR. The qRT-PCR analysis showed differential transcript levels with maximum expression in leaf tissues (Fig. 7a). In order to determine whether the expression levels of UGT74W1 gene were influenced by different types of stresses like methyl jasmonate, salicylic acid, mannitol, salt (NaCl), cold and heat, qRT-PCR was carried out. Relative expression in terms of mean fold expression under mannitol stress conditions has increased almost fivefold in roots and huge dip in fold expression has been seen in leaves. In methyl jasmonate and salt stress, expression level has increased in stem and decreased in leaves whereas in case of heat and cold shock treatments expression has totally decreased as shown in Fig. 7a. On the other hand salicylic acid stress increased fold expression by almost 10 times in roots and has decreased in stem and leaves (Fig. 7b). Identification of endogenous UGT74W1 protein expression by Western blotting To check the endogenous expression of UGT74W1 protein in B. monniera plant, Western blotting was performed. The crude proteins extracted from the whole B. monniera plant were subjected to SDS-PAGE and blotted with the antibodies raised against the purified UGT74W1 protein. A clear single band, corresponding to the molecular mass of the UGT74W1 protein was observed in the plant extract (Fig. 8). Since glycosyltransferases comprise of a large family, the specificity of the antibody was checked using one more UGT protein isolated from B. monniera (data not shown) but only single band of expected molecular mass of UGT74W1 protein was observed on Western blot. This also confirms that the antibodies raised against UGT74W1
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Fig. 2 Phylogenetic tree of UGT74W1 with other plant glycosyltransferases: the evolutionary relationship of UGT74W1 protein with other 26 different plant glycosyltransferases was constructed using the Neighbor-Joining method after alignment of amino acids sequences by Clustal X2.0. The optimal tree with the sum of branch length = 7.26316574 is shown. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The names and accession numbers are: Scutellaria baicalensis (BAA83484), Perilla frutescens (UGT73A7, UGT73A13, BAA19659), Antirrhinum majus (UGT73N1,
UGT73E2, UGT73A9), Malus 9 domestica (UGT71A15), Pyrus communis (UGT71A16), Catharanthus roseus (UGT71E2), Cyclamen persicum (UGT71E3), Dianthus caryophyllus (UGT71F3), Glandularia x hybrid (BAA36423), Perilla frutescens var. crispa (BAA36421), Torenia hybrid cultivar (BAC54093), Petunia 9 hybrid (BAA89009, BAA89008), Lycium barbarum (UGT74P1, UGT74P2, UGT74NI, UGT74N2), Arabidopsis thaliana (NP 564357, 197207), Vitis vinifera (AAB81682), Gentiana triflora (UGT78B1), Forsythia 9 intermedia (AAD21086), Bacopa monniera (UGT74W1)
protein does not provide any non-specific binding to other UGT members present in the B. monniera plant.
protein in the vascular bundles and its absence in the pith and cortex tissues as shown in Fig. 9.
Immuno-localization studies of UGT74W1 in B. monniera plant
Discussion
Immuno-localization was performed for cellular localization of UGT74W1 protein in different parts of the B. monniera plant. Cross sections of immuno-localized root, leaf and stem showed the presence of UGT74W1
Bacopa monniera belongs to the family Scrophulariaceae, is commonly referred to as brahmi. Since brain is the source of creative activity, any compound that ameliorates brain activity is called brahmi [31]. It has been used for
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Fig. 3 SDS-PAGE analysis of recombinant UGT74W1 protein: coomassie blue staining (a) lane M protein molecular weight marker, lane 1 lysate of empty pET 30 (b) vector transformed in E. coli BL21 DE3 cell line, lane 2 inclusion bodies of empty vector transformed in
E. coli BL21 DE3 cell line. b Lane M protein molecular weight marker, lane 1 partially purified UGT74W1 protein, lane 2 lysate of UGT74W1 protein
Fig. 4 MALDI/MS/MS spectra: MALDI MS/MS analysis of recombinant UGT74W1 protein a ionization spectra, b coverage map of recombinant UGT74W1 protein
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Fig. 5 LC–MS analysis of UGT74W1 activity with genistein: a LC–MS spectra of the control reaction (i.e., bacteria transformed with empty vector) with genistein, b LC–MS spectra of assay reaction with genistein
centuries in Ayurveda (the Hindu system of traditional medicine native to India) for the treatment of anxiety, improving intellect, memory and revitalisation of sensory organs [32, 33]. B. monniera is also reported to possess analgesic, antipyretic, anti-inflammatory, sedative [34], free radical scavenging and anti-lipid peroxidative activities [35]. Apart from that the plant is also found to be useful in the treatment of various respiratory [36], cardiac and neuropharmacological [31] disorders. In view of the importance of this plant in the indigenous system of medicine, systematic chemical examinations of the plant had been carried out by several groups of researchers. The major chemical entities found to be responsible for the medicinal properties of B. monniera were various glycosides present in the plant. These glycosides which were produced by an enzyme glycosyltransferase were present in the plant in very low quantities so an alternative approach to increase the yield of such medicinally important
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glycosides was to isolate the enzyme, glycosyltransferase and over express it in suitable expression system. Therefore the isolation and characterization of such glycosyltransferases is of prime importance for pharmacological applications. In this study we reported the isolation and cloning of cDNA encoding UGT gene, the identity of which was established by sequence comparison with previously isolated and characterized UGTs. Conserved domains of UGT74W1 were detected by conserved domains database (CDD) at NCBI GenBank Database which showed that UGT74W1 belongs to a glycosyltransferase GTB type superfamily. The isolated UGT74W1 also contained the conserved domain (45 amino acid long PSPG box) in the sugar donor binding pocket as well as the important residues like His-22 and Asp-121 [29] known for their role in catalytic activity in the acceptor binding pocket. UGT74W1 protein also does not contain any signal peptide as is the
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Fig. 6 High-performance liquid chromatography (HPLC) elution profiles of UGT74W1 activity with genistein: a authentic standard of genistein, b authentic genistein 40 -O-glucoside, c assay reaction
with genistein, d HPLC profile of control reaction (i.e., bacteria transformed with empty vector) with genistein
property of all the plant UGTs characterized till date [37] which suggests that the enzyme is present in the cytosol. Phylogenetic analysis revealed that UGT74W1 is evolutionary similar to the UGTs from L. barbarum which accepts UDP-glucose as preferred sugar donor and flavonoids as acceptor molecule [30]. The identity of the recombinant UGT74W1 protein expressed in E. coli was confirmed by MALDI MS/MS because of the presence of other minor contaminating bands seen after affinity purification of the protein. MALDI MS/MS ionic spectra and coverage map confirmed its presence in the partially purified fraction. These results were consistent with the Western blotting analysis of the
partially purified fraction of the recombinant UGT74W1 protein (data not shown) using antibodies against it. Plant glycosyltransferases have been reported to exhibit specificity towards various flavonoids giving rise to single or multiple products. Many plant UGTs recognize genistein as an acceptor molecule when assayed in vitro and can glycosylate at 7-OH positions but only rarely glycosylates at 40 -OH position. In the present study we had isolated UGT which specifically glycosylates only at 40 -OH position of genistein resulting in the formation of genistein 40 O-glucoside. These results were in conformity with our previous report wherein homology model of UGT74W1 was docked with UDP-Glucose and different acceptor
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Fig. 8 Western blot analysis of UGT74W1: lane 1 crude plant extract (200 lg), lane 2 purified recombinant UGT74W1 protein expressed in E. coli BL2 (DE3) cell line
Fig. 7 Tissue specific expression of UGT74W1 using qRT-PCR: a relative expression in terms of mean fold expression of UGT74W1 gene under normal (control plant) and stress treated conditions i.e., mannitol, methyl jasmonate (MJ), cold, salt and heat, b relative expression of UGT74W1 gene under salicylic acid (SA) stress with respect to salicylic acid control plant. All values are plotted with standard deviation taken into account
molecules but the preferred acceptor for UGT74W1 was found to be genistein with binding energy of -6.68 kcal/mol [38]. To the best of our knowledge this is the first ever isolated UGT which glycosylates genistein exclusively at the 40 -OH position. qRT-PCR was performed to determine the transcript level of UGT74W1 gene in different parts of the plant under normal and stress treated conditions. The expression level of UGT74W1 was found to be highest in the leaf tissues as compared to the roots and stem under normal conditions. Some glycosyltransferases reported from Arabidopsis thaliana [39] and from Catharanthus roseus [40] also showed maximum expression in leaves whereas the UGT isolated from Solanum aculeatissimum [41] showed maximum expression in roots. It has been reported
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that glycosyltransferases are induced by a variety of abiotic stresses, including cold, salicylic acid [42] and methyl jasmonate [40, 43, 44]. In comparison to the normal plant, a fivefold increase in expression of UGT74W1 was observed in roots of plants under mannitol stress. In case of methyl jasmonate stress expression level of UGT74W1 was found to be induced in stem whereas around 14–15 folds dip in expression was seen in leaves. Many glycosyltransferases reported had been induced on treatment with methyl jasmonate [40, 43, 44]. Additionally gene expression of UGT74W1 was found to be increased by tenfold after induction with salicylic acid, an important molecule in plant defense [45]. Several glycosyltransferases reported till date had been induced by salicylic acid treatment [46– 50]. Few glycosyltransferases that are putatively involved in defense mechanism are also induced by salicylic acid [51]. The fact that UGT74W1 enzyme is also induced by salicylic acid and methyl jasmonate might point towards its role in plant defense. Immuno-localization studies of UGT74W1 protein showed that the protein was localized in the vascular bundle. Since the isolated UGT from B. monniera is a flavonoid glycosyltransferase and flavonoids being polyphenols present in the vascular bundles [52] might be the possible reason why B. monniera GT is localized in the vascular bundle region. This study is also the first time report of immuno-localization of UGTs on cross-sections of different parts of the plant.
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Fig. 9 Immuno-localization of UGT74W1: immuno-localization of UGT74W1 in Bacopa monniera: a–c cross sections of control stem, root and leaf at 920 magnification respectively, d–f cross sections of
stem, root and leaf treated with UGT74W1 antibody at 920 magnification respectively
Acknowledgments We are highly grateful for the support of this work by Networking Project of Council of Scientific and Industrial Research (CSIR), India, via grant no: NWP0008. We are also thankful to CSIR for awarding CSIR-Senior Research fellowship. We are also grateful to Dr. Thomas Vogt, Leibniz Institute of Plant Biochemistry, Germany for the critical reading of the manuscript. We are also thankful to Peter McKenzie for naming Bacopa monniera UGT as UGT74W1 at http://www.flinders.edu.au/medicine/sites/clinical-phar macology/ugt-homepage.cfm.
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