Note This paper is dedicated to the inspirational memory of Dr Jayanti Sen
A. K. Sinha, National Centre for Plant Genome Research, JNU Campus, Aruna Asaf Ali Marg, New Delhi 110 067, India Fax: +91 11 26716658 Tel: +91 11 26735188 E-mail: firstname.lastname@example.org Website: http://www.ncpgr.nic.in
Catharanthus roseus (L.) G. Don produces a number of biologically active terpenoid indole alkaloids via a complex terpenoid indole alkaloid biosynthetic pathway. The final dimerization step of this pathway, leading to the synthesis of a dimeric alkaloid, vinblastine, was demonstrated to be catalyzed by a basic peroxidase. However, reports of the gene encoding this enzyme are scarce for C. roseus. We report here for the first time the cloning, characterization and localization of a novel basic peroxidase, CrPrx, from C. roseus. A 394 bp partial peroxidase cDNA (CrInt1) was initially amplified from the internodal stem tissue, using degenerate oligonucleotide primers, and cloned. The full-length coding region of CrPrx cDNA was isolated by screening a leaf-specific cDNA library with CrInt1 as probe. The CrPrx nucleotide sequence encodes a deduced translation product of 330 amino acids with a 21 amino acid signal peptide, suggesting that CrPrx is secretory in nature. The molecular mass of this unprocessed and unmodified deduced protein is estimated to be 37.43 kDa, and the pI value is 8.68. CrPrx was found to belong to a ‘three intron’ category of gene that encodes a class III basic secretory peroxidase. CrPrx protein and mRNA were found to be present in specific organs and were regulated by different stress treatments. Using a β-glucuronidase–green fluorescent protein fusion of CrPrx protein, we demonstrated that the fused protein is localized in leaf epidermal and guard cell walls of transiently transformed tobacco. We propose that CrPrx is involved in cell wall synthesis, and also that the gene is induced under methyl jasmonate treatment. Its potential involvement in the terpenoid indole alkaloid biosynthetic pathway is discussed.
Catharanthus roseus (L.) G. Don produces a class of secondary metabolites, namely, terpenoid indole alkaloids (TIAs), with antitumor properties. Two of these leaf-specific dimeric alkaloids, vinblastine and vincristine, are used as valuable drugs in cancer chemotherapy. Owing to the medicinal importance of these alkaloids and their low levels in C. roseus in vivo, TIA biosynthesis has been intensively studied in this plant. The TIA biosynthetic pathway (supplementary Fig. S1) is highly complex, involves more than 20 enzymatic steps, and is reported to be stress-induced, mainly due to the increased transcription of biosynthetic genes [1,2]. However, the genes involved in the final dimerizing step of the coupling of monomeric precursors, catharanthine and vindoline, to yield leaf-specific α-3′-4′-anhydrovinblastine (AVLB), and the final step of conversion of root-specific ajmalicine to serpentine, have not yet been identified. Previous studies have led to the finding of a class III basic peroxidase in C. roseus that shows AVLB synthase activity and is localized in vacuoles [3–5].
Plant peroxidases are reported to be involved in various physiological processes [6–9]. Class III plant peroxidases, considered to be plant-specific oxidoreductases, have been found to participate in lignification , wound healing , defense against pathogen attack, including crosslinking of cell wall protein , and aspects of plant growth regulator action . Furthermore, the presence of a separate hydroxylic cycle, which leads to the formation of various radical species, opens a new range of possibilities for this class of enzymes . Plant peroxidases are reported to have many different isoforms; 73 members have so far been identified in Arabidopsis thaliana. The expressed proteins of these genes are reported to be localized either in the cell wall or in the vacuole. In this article, we report the cDNA cloning, characterization and subcellular localization of a novel stress-induced peroxidase (CrPrx) from C. roseus belonging to the class III basic peroxidase family. The observed expression patterns suggest its potential role during stress conditions and elicitor treatment in C. roseus. CrPrx tagged with β-glucuronidase (GUS)–green fluorescent protein (GFP) was expressed in Nicotiana tabacum and C. roseus leaf epidermal cells as well as in xylem cell wall thickening. The possibility of its involvement in the TIA biosynthetic pathway has also been discussed.
CrPrx cDNA is 1197 bp long
Degenerate oligonucleotide primers, PF1 and PR1, were designed on the basis of the conserved amino acid sequences of proteins (RLHFHDC and VALLGAHSVG) encoded by the class III peroxidase gene family and used to amplify cDNA fragments from different tissues of C. roseus var. Pink. A 394 bp partial peroxidase cDNA (CrInt1; accession number AY769111) was amplified from the internodal stem tissue by RT-PCR; upon sequencing, this showed similarity with a truncated class III peroxidase ORF. Full-length C. roseus peroxidase cDNA (CrPrx) was isolated by screening a leaf-specific cDNA library with the 394 bp partial CrInt1 as a probe. A single positive plaque that was identified after tertiary screening revealed a 1357 bp full-length cDNA with a 5′-UTR and a 3′-UTR upon sequencing (accession number AY924306) (Fig. 1). The complete coding region for CrPrx was then amplified using a primer pair complementary to the 5′-UTR and 3′-UTR regions of CrPrx that was 1197 bp in length, excluding part of the 3′-UTR and the polyA tail (accession number DQ415956).
CrPrx encodes a class III peroxidase
Computational analysis of the CrPrx nucleotide sequence showed that it encodes a 330 amino acid polypeptide (Fig. 1). The molecular mass of this deduced protein is calculated to be 37.43 kDa, and it has a theoretical pI of 8.68. The analysis of CrPrx protein using signal p v3.0 software  identified a putative 21 amino acid signal peptide that was cleaved between Ala21 and Glu22. CrPrx protein showed an N-terminal extension of eight amino acids (Glu-Asn-Glu-Ala-Glu-Ala-Asp-Pro) before the start of the mature protein as an NX-propeptide (Fig. 1). blast searches  revealed significant sequence identity between CrPrx and a number of other class III plant peroxidases (EC 22.214.171.124), notably secretory peroxidases from Avicennia marina (accession number AB049589) and Nicotiana tabacum (accession number AF149252) (Fig. 2). The amino acid sequences of seven mature peroxidases, including CrPrx, were all close to 300 residues (Fig. 2). They showed 33–86% amino acid identity and share 67 conserved residues. When compared with horseradish peroxidase (HRP)-C , the translated polypeptide showed that it contains all the eight conserved cysteines for disulfide bonds, and all the indispensable amino acids required for heme binding, peroxidase function, and coordination of two Ca2+ ions (Fig. 2).
CrPrx contains three introns and four exons
To obtain an insight into the complete sequence of CrPrx, PCR was performed using primer pair PFLF1 and PFLR1, designed to anneal to conserved 5′-UTR and 3′-UTR regions (accession number DQ415956), with genomic DNA of C. roseus as template. The amplified product upon cloning and sequencing was found to be 1793 bp long (accession number DQ484051). CrPrx consists of four exons (268 bp, 189 bp, 172 bp, 405 bp, stop at UAG) and three introns (95 bp, 435 bp, 79 bp) (Fig. 3A,B). The first and third introns were more or less similar in size. The second intron in CrPrx was found to be the largest, and was even larger in size than the exons. This CrPrx structure supports the concept of origin of peroxidases from a common ancestral gene of peroxidases with three introns and four exons.
CrPrx is present in single copy in the C. roseus genome
Southern blot analysis was performed on genomic DNA of C. roseus plants (obtained by self-pollination), digested with BglII, EcoRV and HindIII (with 0, 1 and 0 cut site, respectively) and probed with full-length CrPrx cDNA at high stringency (Fig. 4). The autoradiograph, showing bands of different sizes, revealed that CrPrx occurs as single copy in the Catharanthus diploid genome of C. roseus plants.
The relationship between CrPrx cDNA and other cDNAs encoding class III peroxidases was investigated using a parsimonious phylogenetic analysis. blast searches were used to identify other full-length peroxidase cDNA sequences showing close similarity to CrPrx. The varying degrees of expression patterns of peroxidase cDNAs in different tissues in different plant systems under stress was taken into consideration during this study (Table 1). Phylogenetic analysis was performed on the aligned nucleotide sequences corresponding to the cDNA ORFs (Fig. 5). The tree was rooted with the Spinacea prx14 sequence, which may be distantly related to the CrPrx sequence. Most of these cDNAs, with a few exceptions, are expressed in both vegetative and reproductive tissues, and are stress-induced. CrPrx expression was also noted in all the tissues tested and found to be stress-inducible. After its origin from Spinacea prx14, the tree showed a divergence from a liverwort peroxidase, indicating a distant relationship of ancestral Marchantia peroxidase with this angiosperm CrPrx sequence.
Table 1. References used for sequence and expression data presented in Fig. 5. for phylogenetic analysis. NA, not available.
Internodal stem tissue shows maximum CrPrx expression
Northern blot analysis revealed expression of CrPrx in different organs of C. roseus, i.e. leaves (young, mature and old), flower buds, open flowers, fruits, roots, and internodal stem tissue (Fig. 6A). Among vegetative tissues, the transcript was maximal in internodal stem tissues, followed by roots, young leaves, and mature leaves. Among reproductive tissues, the transcript was most abundant in fruits, followed by young buds. CrPrx expression was not detected in old leaves and flowers.
In order to purify CrPrx for preparation of antibody, a glutathione S-transferase (GST)–CrPrx fusion protein was constructed in pGEX 4T-2 vector with CrPrx ORF (PPGX) and expressed in a bacterial system. As the protein was repeatedly found in inclusion bodies, different concentrations of glutathione, sarcosyl and Triton X-100 were tested to achieve purification of the fusion protein (Fig. 6B). The purified protein was used for preparation of polyclonal antibodies against CrPrx in rabbit. Immunoblot analysis performed using different organs of C. roseus revealed differential accumulation of CrPrx in different organs, with a maximum level of accumulation in the internodes (Fig. 6C). CrPrx was detected at 37 kDa, whereas heterologously expressed GST–CrPrx was detected at 63 kDa (Fig. 6C, first lane).
CrPrx transcript is induced by various abiotic stresses and methyl jasmonate
Many plant peroxidase genes are reported to be induced in vegetative tissues by stress, particularly wounding [19,20]. To investigate whether CrPrx expression is stress-induced, leaves of C. roseus were subjected to different stress conditions as well as methyl jasmonate (MJ) treatment, and analyzed for CrPrx transcript regulation over a time course of 24 h (Fig. 7A,B). An increase in the level of CrPrx expression was noted with increasing time when leaves were either wounded or exposed to UV and cold treatments. The expression level reached its peak after 6 h of wound treatment, following an initial decline during the first hour. In the case of UV and cold exposure, the maximum transcript level was observed at 12 and 24 h, respectively. On the other hand, a gradual steady-state increase in the expression level of CrPrx was noted with increasing time in response to application of 100 µm MJ on leaves. This was later confirmed by immunoblot analysis, which revealed accumulation of CrPrx in C. roseus leaves after 6 h of wound stress and 6–12 h of treatment with 100 µm MJ (Fig. 7C).
Subcellular localization of GUS–GFP fused CrPrx
To examine the subcellular localization of CrPrx in N. tabacum and C. roseus, the CrPrx coding region was fused in-frame to the coding region for the N-terminal side of GUS and GFP under the control of the 35S promoter of cauliflower mosaic virus (CaMV) in pCAMBIA 1303. When the construct CrPrx–GUS–GFP was expressed in transformed tobacco and in C. roseus, GUS staining and green fluorescence were observed in the epidermal parenchymatous cells, stomatal guard cells, and vascular tissues (xylem tissue) (Figs 8A–F and 9A–E). However, in epidermal parenchymatous and stomatal guard cells, CrPrx–GUS–GFP was found to be accumulated mostly in the cell walls, outer cell membranes and associated structures (Figs 8A,B and 9A,B). On detailed examination, CrPrx–GFP fluorescent dots were visible in the part of the epidermal cell wall abutting a mature guard cell in tobacco leaf tissue (Fig. 8B). In xylem tissue, CrPrx–GFP fluorescence was observed specifically in the secondary wall thickenings both in tobacco and in C. roseus (Figs 8F and 9D,E).
We report here the cloning, characterization and localization of a novel C. roseus peroxidase, CrPrx, for the first time. This particular full-length CrPrx cDNA (1359 bp) and its functional product were noted to be localized and expressed in different tissues of the plant tested. Computational analysis revealed that the translated polypeptide sequence of CrPrx contains eight conserved cysteine residues forming disulfide bridges, two Ca2+-binding ligands, and distal and proximal heme-binding domains, in common with other plant peroxidases [18,21,22]. The inclusion of Ser96 and Asp99 in a salt bridge motif at the beginning of helix D and its connection to the following long loop by a tight hydrogen bonding network with Gly121-Arg122 was also an important feature in CrPrx . The presence of a signal peptide and the lack of a carboxyl extension identifies CrPrx as a secretory (class III) plant peroxidase, rather than a vacuolar plant peroxidase. Unlike other class III peroxidases, the mature CrPrx polypeptide starts with a glycine (G) residue and not with glutamine (Q) residue. This feature will possibly make the CrPrx polypeptide unable to generate a pyrrolidone carboxylyl residue (Z) .
The full-length CrPrx gene, like most of the plant peroxidase genes, contains three introns, which differ in their sizes . Phylogenetic analysis grouped CrPrx cDNA with the ancestral Marchantia peroxidase cDNA. The two peroxidase cDNAs that were found to be structurally most closely related to CrPrx are Av. marina and N. tabacum peroxidase cDNAs.
The CrPrx transcript and its translated product were found to be differentially expressed in different vegetative as well as reproductive tissues of C. roseus under normal conditions and upon exposure to stress as well as MJ treatment, confirming that it is organ-specific, developmentally regulated, and stress-inducible as well as elicitor-inducible. The subcellular localization study using CrPrx–GUS–GFP is indicative of a correlation between the accumulation of CrPrx fusion protein and the parenchymatous as well as xylem cell wall thickening, both in tobacco and in C. roseus. The classical plant peroxidases (class III) are ascribed a variety of functional roles in plant systems, which include lignification, suberization, auxin catabolism, defense, stress, and developmentally related processes [6,15,26,27]. The stress-inducible nature of CrPrx cDNA and the localization of its functional product in cell walls in the present study suggest its apoplastic nature and its involvement in the stress-related as well as developmental processes in C. roseus.
Jasmonic acid and its volatile derivative, MJ, collectively called jasmonates, are plant stress hormones that act as regulators of defense responses . The induction of secondary metabolite accumulation is an important stress response that depends on jasmonate as a regulatory signal . In the present study, CrPrx was found to be expressed upon elicitation by MJ. A number of TIA biosynthetic pathway genes have also been shown to be regulated by jasmonate-responsive AP2 domain transcription factor (ORCAs) [29–31]. These findings demonstrate that, like that of other TIA biosynthetic pathway genes, expression of CrPrx falls under an MJ-responsive control mechanism that operates in C. roseus under stress conditions. However, it is difficult to ascertain from the present investigation whether CrPrx has a similar function to that of AVLB synthase in C. roseus, because CrPrx was found to lack a vacuolar targeting signal and to be apoplastic in nature.
In conclusion, we report the cloning of a novel CrPrx gene from C. roseus that encodes a functional product and is localized in epidermal cells as well as vascular cell walls in leaves of tobacco and C. roseus. All the accumulated evidence suggests that it encodes a ‘three intron’ class III secretory peroxidase that shows organ-specific and stress-inducible as well as MJ-inducible expression. Accordingly, we assume its involvement during stress regulation and developmental processes in C. roseus. The possibility of using CrPrx for manipulation of the TIA pathway needs further experimental investigation.
Seeds of C. roseus var. Pink were obtained from Rajdhani nursery, New Delhi and grown in the experimental nursery of the National Centre for Plant Genome Research, New Delhi, India. Different parts of the plant, i.e. young (first to third from the shoot apex), mature (fourth to sixth from shoot apex) and old (eighth and ninth from shoot apex) leaves, internodal segments, flower buds, open flowers, pods and roots (branched side roots) from 6-month-old nursery-grown plants were used as plant materials. Leaves of 1-month-old aseptically grown plantlets of N. tabacum and C. roseus were used as explants for transformation experiments.
Six-month-old potted mature plants of C. roseus var. Pink were subjected to different stress conditions in the following manner.
Wounding stress was performed by puncturing the young leaves attached to plants several times across the apical lamina with a surgical blade, which effectively wounded ∼ 40% of the leaf area. For cold stress, whole plants were kept at 4 °C, and control plants were maintained in the greenhouse at 25 °C. MJ treatment was applied on leaves detached from plants and kept on paper soaked in 1/10 Murashige Skoog (MS) basal medium by painting on the adaxial surface of the leaves, and the tray containing the leaves was sealed with saran wrap. In control experiments, similar leaves were painted with double-distilled water containing the same amount of ethanol required for dissolving MJ. For UV treatment, young leaves were detached from the plants and kept on 1/10 MS media. A short-term exposure (2 min) of leaves under a UV lamp (λmax 312 nm; 28 J·m2·s−1) was given, and this was followed by incubation on 1/10 MS medium for various time periods before harvesting. For each treatment, young leaves, the first to the third from the shoot apex, were used. The leaves were harvested at different time points by snap freezing in liquid nitrogen, and stored at − 80 °C for further analyses.
Cloning of CrPrx cDNA and gene
Total RNA was isolated from vegetative tissue (roots, stem, leaves) as well as reproductive tissues (flower buds, open flowers and pods) of C. roseus using the LiCl precipitation method . First-strand cDNA synthesis was carried out with 5 µg of total RNA using oligo-dT15 primer (Promega, Madison, WI, USA) and Powerscript reverse transcriptase (BD Biosciences, Palo Alto, CA, USA) following the manufacturer's instruction, and used as the template for PCRs.
PCR amplifications were performed with degenerate oligonucleotide primers PF-1 (5′-AGRCTTCAYTTYCATGAYTGC), PF-2 (5′-AGRCTTCAYTTYCATGAYTGT′), PR-1 (5′-GTGNSCMCCDRRSARRGCDAC), and PR-2 (5′-CATYTCDGHYCAHGABAC), which were designed on the basis of highly conserved amino acid sequences of proteins encoded by the peroxidase gene family, namely, RLHFHDC, VALLGAHSVG, and VSCSDI. PCR conditions used were initial denaturation at 94 °C for 2 min, followed by 29 cycles of denaturation at 94 °C for 45 s, annealing at 45 °C for 30 s, and extension at 72 °C for 1 min, with a final extension at 72 °C for 10 min. Amplified products of the expected size were gel purified using the MinElute Gel Extraction Kit (Qiagen, Hilden, Germany), and cloned directly into the pGEM-T Easy cloning vector (Promega), following the manufacturer's instructions. Clones were sequenced using Big Dye terminator v3.1 cycle sequencing (Applied Biosystems, Foster City, CA, USA) chemistry on an ABI prism DNA sequencer (DNA sequencing facility, National Centre for Plant Genome Research, New Delhi, India).
In order to clone complete CrPrx cDNA, a λ-ZapII-oriented leaf-specific cDNA library was screened under high-stringency conditions with modified church buffer at 60 °C . The 394 bp (CrInt1) PCR product obtained using degenerate PCR primers was used as a probe (accession number AY769111). One positive plaque was obtained after a final wash of the membrane at high stringency with 0.1 × NaCl/Cit and 0.1% SDS at 65 °C. The 1359 bp full-length clone was identified after in vivo excision in the phagemid vector pBSK+ (Clontech, Palo Alto, CA, USA).
The complete cDNA coding region was PCR amplified using forward primer PFLF1 (5′-CACGAGCTGACCTTCACTGTC) and reverse primer PFLR1 (5′-GCTCACCACCATTACATTGC), designed to anneal with the 5′-UTR and 3′-UTR regions. PCR amplification consisted of 2 µL of cDNA template in a reaction volume of 50 µL, 1 × ThermoPol buffer, 1.5 mm MgCl2, 0.4 mm dNTPs, 0.2 µm each primer, and 1 U of Deep VentR DNA Polymerase (NEB, Beverly, MA, USA). Thermal cycling was carried out on an MJ Research Master Cycler (Global Medical Instrumentation, Ramsey, MN, USA) with the following conditions: initial denaturation at 94 °C for 2 min, followed by 29 cycles of denaturation at 94 °C for 45 s, annealing at 60 °C for 30 s, extension at 72 °C for 1 min, and a final extension at 72 °C for 10 min. The corresponding genomic sequence for CrPrx was PCR-amplified using the same primer pair PFLF1 and PFLR1. The PCR product was cloned into the vector pGEM-T Easy (Promega), and sequenced as mentioned above. Gene-specific primers GSP2 (5′-CCCTTGAAAGGGAGTGTCCTGGAGTTGG) and GSP4 (5′-GAGGCTCTCATTGTGGTCTG-GGAGATG) were designed from the 380 bp and 532 bp positions of the cDNA sequence, respectively, for subcloning the CrPrx gene.
Southern blot analysis
Catharanthus roseus genomic DNA was purified using the hexadecyltrimethyl ammonium bromide method . Thirty micrograms of BglII-, EcoRV- and HindIII-digested genomic DNA was separated on 0.7% agarose 1 × TAE gel at 40 V for 8 h. DNA was then transferred to a Hybond-N membrane, following the manufacturer's instructions. Prehybridization and hybridization of membranes were carried out at 60 °C in modified church buffer (7% SDS, 0.5 m NaPO4, 10 mm EDTA, pH 7.2) . Blots were probed with [32P]dCTP[αP]CrPrx cDNA. Blots were finally washed in 1 × NaCl/Cit and 0.1% SDS at 60 °C . Membranes were wrapped in Klin Wrap (Flexo film wraps, Aurangabad, India) and exposed to XBT-5 CAT film (Kodak, Mumbai, India).
Northern blot analysis
Total RNA (20 µg) was separated on a 1.2% denaturing agarose gel at 60 V for 6 h and blotted onto Hybond-N membrane (Amersham-Pharmacia, Piscataway, NJ, USA) using standard procedures . Following transfer, blots were rinsed briefly in diethylpyrocarbonate-treated water, and the RNA was immobilized on the membrane by UV-crosslinking using a Stratalinker (Model 1800; Stratagene, La Jolla, CA, USA) at an energy of 12 000 µJ·cm−2 for approximately 2 min, and then air-dried.
Blots were prehybridized and hybridized in modified church buffer at 60 °C [32,34]. Blots were probed as described for Southern blot analysis.
Purification of GST-fused CrPrx protein from Escherichia coli and production of antibodies to CrPrx
The 330 amino acid ORF of the CrPrx clone was amplified by PCR using Deep VentR DNA Polymerase (NEB) and primers GSTPF2 (5′-GGAATTCCCATGGCTTCCAAAAC) and GSTPR1 (5′-GGTCGACCTCACCACCATTACA), according to the manufacturer's instructions. The amplified fragment was restricted with EcoRI and SalI endonucleases, and inserted in the corresponding restriction sites of the pGEX4T-2 expression vector in the reading frame to obtain the N-terminal GST fusion product (Amersham). Clone PPGX (pGEX 4T-2 with CrPrx ORF) was transformed to BL21-CodonPlus-RP competent cells (Stratagene). The fusion protein was induced at 37 °C by adding 0.05 mm isopropyl thio-β-d-galactoside at a growth stage at D600 of 0.5. Purification of insoluble fusion protein was performed using the method as described in Frangioni & Neel , with slight modifications. Two hundred milliliters of induced culture of bacteria was pelleted at 3000 g at 4 °C for 15 min using a Sorvall RC 5C centrifuge (Global Medical Instrumentation) with GSA rotor, and washed twice with 1 × NaCl/Pi (8.4 mm Na2HPO4, 1.9 mm NaH2PO4, pH 7.4, 150 mm NaCl). The pelleted bacteria were dissolved in STE buffer (10 mm Tris/HCl, pH 8.0, 1 mm EDTA, 150 mm NaCl) containing 1 mm phenylmethanesulfonyl fluoride as protease inhibitor; this was followed by lysozyme (1 mg·mL−1) treatment and incubation on ice for 30 min. The lysate was sonicated using a sonicator (UP 200S Ultrasonic Processor; Hielscher Ultrasound Technology, Ringwood, NJ, USA) three times separately on ice for 30 s each (amplitude 1, 20% duty cycle). After sonication, the lysate was clarified by centrifugation for 20 min at 37 000 g at 4 °C using an Eppendorf 5415R centrifuge (Westbury, NY, USA) with standard 24 × 1.5 mL/2.0 mL aerosol-tight rotor. The supernatant was transferred to another tube, and Triton X-100 (final concentration of 2%) was added from a 10% stock in STE and well mixed. In addition, 400 µL of washed 50% GST beads were also added and agitated on rocker for 1 h at 4 °C. The beads were washed 10–12 times with ice-cold 1 × NaCl/Pi by repeated centrifugation at 500 g for 5 min at 4°C (Eppendorf 5415R with standard 24 × 1.5 mL/2.0 mL rotor), and resuspended in five volumes of elution buffer [10 mm reduced l-glutathione (G4251; Sigma Aldrich, St Louis, MO, USA) dissolved in 50 mm Tris/HCl, pH 8.0] in different fractions. Each fraction was checked on SDS/PAGE (10% resolving gel). The purified protein was dialyzed and supplied to a company (Banglore Genie, Bangalore, India) for raising polyclonal antibodies in rabbit. The preimmune serum and sera after inoculation were collected and tested for binding to C. roseus proteins by immunoblotting analysis. The preimmune serum did not lead to the detection of any protein band specific to C. roseus by immunoblotting (data not shown).
Protein extraction and immunoblot analysis
Frozen tissues (2 g fresh weight) were ground to a fine powder in a chilled mortal and pestle in the presence of liquid nitrogen. Half of the sample was used for protein extraction, and the other half was used for RNA extraction. Crude protein extracts were prepared by adding protein extraction buffer (100 mm sodium phosphate, pH 7.5, 2 mm dithiothreitol, 5% w/v polyvinylpolypyrrolidone) at a 1 : 4 (w/v) ratio, as described previously . The homogeneous mixture was centrifuged at 17 500 g for 30 min at 4 °C using an Eppendorf 5415R centrifuge with standard 24 × 1.5 mL/2.0 mL aerosol-tight rotor to separate the protein fraction from cell debris. The supernatant containing the total soluble protein was analyzed by means of immunoblot analysis. Protein concentration was determined following the method described by Bradford , using BSA as standard. All steps of protein extraction were performed at 4 °C. Extracted protein was electrophoresed in 12% SDS/PAGE . Samples (20 µg of each) were boiled for 10 min in an equal volume of 2 × SDS/PAGE sample buffer with 0.2 m dithiothreitol. Insoluble materials were removed by centrifugation at 10 000 g using an Eppendorf 5415R centrifuge with standard 24 × 1.5 mL/2.0 mL aerosol-tight rotor. Prestained protein molecular weight markers (MBI Fermentas, Hanover, MD, USA) were used in gels to visualize the size of protein and efficiency of transfer onto the nylon membrane (Hybond C-extra; Amersham). The proteins were electroblotted overnight at 90 mA in a Bio-Rad (Hercules, CA, USA) mini trans-blot system. The blotting buffer was 192 mm glycine and 25 mm Tris (pH 8.3), containing 10% (v/v) methanol. For immunodetection, blotted nylon membrane was blocked with blocking buffer, i.e. 5% decreamed milk in TBS (10 mm Tris pH 7.6 and 0.15 m NaCl) for 1 h. The blocked nylon membrane was incubated with CrPrx antibodies at 1 : 1000 dilution in buffer containing 1% decreamed milk in TTBS (10 mm Tris, pH 7.6, 150 mm NaCl, 0.05% w/v Tween-20) for 1 h. Unbound primary antibodies were removed by washing in TTBS buffer, and the membrane was then incubated for 1 h at room temperature in TBS buffer containing HRP-conjugated goat anti-(rabbit IgG) (diluted to 1 : 100 000). Following the removal of unbound secondary antibody, peroxidase activity of HRP was determined using SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL, USA).
Construction of GFP fusion protein for expression in tobacco and C. roseus leaf discs
The coding region of CrPrx was amplified by PCR with the oligonucleotide primers GSTPF2 (5′-GGAATTCCCATGGCTTCCAAAAC) and PGFPR1 (5′-GGACTAGTATGTAACTTATTAGCT-ACATAT) using Deep VentR DNA Polymerase (NEB). The amplified product contains NcoI and SpeI restriction enzyme cut sites, respectively. After digestion with NcoI and SpeI, the PCR product was directly integrated into pCAMBIA1303 (35S-GUS-mGFP5) vector to generate a CrPrx–GUS–GFP fusion protein transformation vector. The resulting plasmids were used to transform Agrobacterium tumefaciens strain GV3101. A standard leaf-disk transformation method  was used to generate transformants of tobacco and C. roseus expressing CrPrx–GUS–GFP and GUS–GFP via Agrobacterium-mediated transformation. Transformed tobacco and C. roseus leaf disks were grown on MS basal medium supplemented with 1-naphthaleneacetic acid 1 p.p.m. and 6-benzylaminopurine 0.1 p.p.m. for tobacco, and 2,4-dichlorophenoxyacetic acid 1.0 p.p.m and 6-benzylaminopurine 0.1 p.p.m. for C. roseus. After 1 week of incubation at 25 °C ± 2 °C, leaf tissues were harvested for histochemical studies.
Histochemical GUS staining and fluorescence microscopy
Histochemical localization of GUS activity was analyzed after incubating the samples in X-Gluc buffer (50 mm sodium phosphate buffer, pH 7.0, 10 mm EDTA, 0.1% Triton X-100, 5 mm potassium ferrocyanide, 3.8 mm 5-bromo-4-chloro-3-indolyl glucuronide) at 37 °C for 12 h. For sectioning, leaf disks stained with GUS were mounted in Jung tissue freezing medium (Leica CM 1510S, Leica Microsystems GmbH, Wetzlar, Germany). Frozen sections of 30 µm were layered on glass slides with a cryomicrotome (CM 1510S; Leica Instruments) adjusted to − 16 °C for microscopy. Sections (30 µm) were placed under a coverslip and viewed by Diascopic microscopy (Nikon Eclipse 80i, Tokyo, Japan) for histochemical GUS staining. GFP localization was determined by Epifluorescence microscopy (Nikon Eclipse 80i) using cubes of dichroic mirror with excitation filter and barrier filter combination sets for detection of fluorescein isothiocyanate. Images were captured with a digital camera (model DXM 1200C; Nikon) and saved using image-capturing software act-1c (Nikon), and further processed using image-pro express image-analysis software (Media Cybernetics, Silver Spring, MD, USA).
Bioinformatics analysis of CrPrx
The initial design of degenerate primers was done using wise2  and clustalw 1.82 alignment software, freely available at the bioinformatics server of the European Bioinformatics Institute (http://ebi.ac.uk). Similarity searches were performed using BLAST analysis methods . Predictions based on translated amino acid sequences were generated by software programs available at the EXPASY proteomics server of the Swiss Institute of Bioinformatics (http://www.expasy.org). The nucleotide alignment of peroxidases for making the phylogenetic tree was done using the mafft version 5.667 program . The phylogenetic tree was constructed following the maximum parsimony method using the mega2 program . A parameter of close-neighbor interchanges (CNI) with a search level of 3 and 100 bootstrap replicates were considered for this purpose.
Senior Research Fellowships to SK and AD from the Council of Scientific and Industrial Research (CSIR) India are gratefully acknowledged. We thank the Department of Biotechnology (DBT), Government of India for its financial support. SK, AD and AKS pay their tribute to Jayanti Sen, who passed away while the manuscript was under consideration for publication.