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Cloning, characterization and localization of a novel basic peroxidase gene from Catharanthus roseus Santosh Kumar, Ajaswrata Dutta, Alok K. Sinha and Jayanti Sen National Centre for Plant Genome Research, JNU Campus, Aruna Asaf Ali Marg, New Delhi, India Keywords Catharanthus roseus; organ specific; peroxidase; terpenoid indole alkaloid; 2 subcellular localization Correspondence 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: alokksinha@yahoo.com Website: http://www.ncpgr.nic.in Note This paper is dedicated to the inspirational memory of Dr Jayanti Sen (Received 1 December 2006, revised 2 January 2007, accepted 3 Januay 2007) doi:10.1111/j.1742-4658.2007.05677.x 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 1 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 b-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 leafspecific 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]. How- ever, the genes involved in the final dimerizing step of the coupling of monomeric precursors, catharanthine and vindoline, to yield leaf-specific a-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 Abbreviations AVLB, a-3¢-4¢-anhydrovinblastine; GFP, green fluorescent protein; GST, glutatione S-transferase; GUS, b-glucuronidase; HRP, horseradish peroxidase; MJ, methyl jasmonate; TIA, terpenoid indole alkaloid. 1290 FEBS Journal 274 (2007) 1290–1303 ª 2007 The Authors Journal compilation ª 2007 FEBS S. Kumar et al. [10], wound healing [11], defense against pathogen attack, including crosslinking of cell wall protein [12], and aspects of plant growth regulator action [13]. 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 [14]. Plant peroxidases are reported to have many different isoforms; 73 members have so far been identified in Arabidopsis thaliana [15]. 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 b-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. Results A novel peroxidase CrPrx from C. roseus 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 [16] 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 [17] revealed significant sequence identity between CrPrx and a number of other class III plant peroxidases (EC 1.11.1.7), 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 [18], 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 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 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 auto- FEBS Journal 274 (2007) 1290–1303 ª 2007 The Authors Journal compilation ª 2007 FEBS 1291 A novel peroxidase CrPrx from C. roseus S. Kumar et al. Fig. 1. The complete CrPrx cDNA sequence and its translation product. The 5¢-UTR and 3¢-UTR are represented in lower case; the 39 stop codon is indicated by w. The putative signal peptide is boxed in gray. A predicted NX-propeptide is boxed. A predicted N-glycosylation site (NESL) is underlined. Nucleotide sequences in red represent predicted polyA signal sequences. radiograph, showing bands of different sizes, revealed that CrPrx occurs as single copy in the Catharanthus diploid genome of C. roseus plants. Phylogenetic analysis 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 considera1292 tion 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 stressinducible. 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. FEBS Journal 274 (2007) 1290–1303 ª 2007 The Authors Journal compilation ª 2007 FEBS S. Kumar et al. A novel peroxidase CrPrx from C. roseus Fig. 2. CLUSTALW 1.82 multiple alignment of translated amino acid sequence of CrPrx with peroxidases retrieved from the NCBI database, i.e. Avicennia (BAB16317), Nicotiana secretory peroxidases (AAD33072), cotton (COTPROXDS) (AAA99868), barley grain (BP1) (AAA32973), Ar. thaliana (ATP2A) A2 (Q42578) and HRP-C (AAA33377). Residue numbers start at the putative mature proteins by analogy with HRP-C. Preprotein sequences are shown in italics, conserved residues are indicated by w, and amino acids forming buried salt bridge are indicated by r. The amino acid side chains involved in Ca2+-binding sites are marked by m; S–S bridge formed by cysteines in is yellow, and heme40 binding sites are highlighted in reverse print. The location of a-helices, A–J, as observed in HRP-C, is indicated above the aligned sequences. FEBS Journal 274 (2007) 1290–1303 ª 2007 The Authors Journal compilation ª 2007 FEBS 1293 A novel peroxidase CrPrx from C. roseus S. Kumar et al. Table 1. References used for sequence and expression data pre42 sented in Fig. 5. for phylogenetic analysis. NA, not available. A B Fig. 3. Intron mapping of CrPrx gene. (A) Lanes M show size markers in base pairs. Lanes 2, 4, 6 and 8 show PCR reactions run on plasmid DNA harboring CrPrx cDNA, and lanes 1, 3, 5 and 7 show the same using genomic DNA of C. roseus. Primer pairs were: #GSP-4 and #PFLF1 (lanes 1 and 2); #GSP-2 and #GSP-4 (lanes 3 and 4); #GSP-2 and #PFLR-1 (lanes 5 and 6); and #PFLF-1 and #PFLR-1 (lanes 7 and 8). (B) Schematic organization of the CrPrx gene. The asterisk indicates the position of the codon encoding the first amino acid of the mature protein, and the regions of the distal and proximal histidines are indicated by dHis and pHis. 1 2 3 8.9kb 6kb 4kb 3kb Fig. 4. DNA gel blot of C. roseus probed with full-length CrPrx cDNA. Lanes 1, 2 and 3 show the genomic DNA digested with BglII, EcoRV and HindIII restriction enzymes, respectively. 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 1294 Label Accession no. MIPS Reference Glycine Prx2b Cicer peroxidase Avicennia peroxidase Nicotiana peroxidase CrPrx Arabidopsis ATP1a Arabidopsis prx5 Arabidopsis prx Marchantia MpPOD1 Oryza prx71 prx97 TPA inf Triticum POX7 Hordeum BP1 WSP1 Arabidopsis RCI3A Arabidopsis BT024864 Senecio SSP5 Spinacia PC42 Spinacia PB11 Euphorbia prx Vigna prx Catharanthus prx1 Medicago prx Zinnia ZPO-C Glycine GMIPER1 Spinacia PC23 Quercus POX2 Ipomoea swpb3 AtPrx Asparagus prx3 Picea SPI2 Picea px17 Picea px16 Nicotiana PER4 Dimocarpus POD1 Ipomoea swpb1 Ipomoea swpb2 Spinacia prx14 AF145348 AJ271660 AJ271660 AF149251 AY924306 X98189 X98317 AY087458 AB086023 BN000600 BN000626 BN000568 AY857761 M73234 AF525425 U97684 BT024864 AJ810536 Y10464 Y10462 AY586601 D11337 AM236087 X90693 AB023959 AF007211 Y10467 AY443340 AY206414 AY065270 AJ544516 AJ250121 AM293547 AM293546 AY032675 DQ650638 AY206412 AY206413 AF244923 NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA At5g40150 NA NA NA NA NA NA NA NA NA NA NA NA At5g05340 NA NA NA NA NA NA NA NA NA Unpublished Unpublished [25] [7] Present study [43] [43] [44] Unpublished [14] [14] [14] [45] [46] Unpublished [47] Unpublished [48] [49] [49] [50] [51] Unpublished [52] [53] [54] [49] [55] [56] Unpublished [57] [58] Unpublished Unpublished Unpublished Unpublished [56] [56] Unpublished 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 FEBS Journal 274 (2007) 1290–1303 ª 2007 The Authors Journal compilation ª 2007 FEBS S. Kumar et al. A novel peroxidase CrPrx from C. roseus Fig. 5. Phylogenetic relationships between peroxidase cDNA, CrPrx and other related class III peroxidases. Alignment consists of the nucleotide sequences of coding regions. Bootstrap values mark the percentage frequency at which sequences group in 100 resampling replicates. The expression pattern is represented by semi-color circles indicating: floral, vegetative and stress-inducible (abiotic and biotic) expression. Information on expression is referenced in Table 1, gathered from published and unpublished sources and from NCBI databases. 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 lm 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 lm MJ (Fig. 7C). FEBS Journal 274 (2007) 1290–1303 ª 2007 The Authors Journal compilation ª 2007 FEBS 1295 A novel peroxidase CrPrx from C. roseus Youn g lea ve s Matu re lea ve s Old l eaves Flow er bu ds Flow ers Fruit s Root s Is t In terno de IInd Inter node A S. Kumar et al. CrPrx 28S rRNA B kDa M 1 2 97.4 66 3 63 kD 43 B OL M L YL FL FL kDa FR C PP GX IN T R 29 79 47 33 Fig. 6. (A) Northern blot analysis. Upper panel shows CrPrx expression, with each lane containing 20 lg of total RNA. (B) Large-scale purification of GST fusion CrPrx protein; the mobility of the fusion protein matches its predicted molecular weight. Lanes M, 1, 2 and 3 show molecular weight markers, total protein from uninduced bacterial culture, induced bacterial lysate, and purified eluted CrPrx fusion protein, respectively. (C) Immunoblot analyses of CrPrx expression in various tissue types; denaturing SDS ⁄ PAGE of total proteins extracted from various organs, followed by immunoblotting using the antibodies to CrPrx. The blot was imaged on X-ray film using chemiluminescent substrate. PPGX is CrPrx cloned in PGEX 4T-2 fusion vector as a purified GST fusion protein. 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 1296 Fig. 7. Northern blot and immunoblot analysis of CrPrx transcript and protein, respectively. (A, B) Transcript regulation of CrPrx under different abiotic stress conditions and 100 lM MJ; the lower panel shows methylene blue-stained 28S RNA as loading control. (C) Immunoblot analysis of CrPrx after wounding and 100 lM MJ treatment with antibodies to CrPrx. Blots were imaged on X-ray film using chemiluminescent substrate. C, untreated control; W, wounding. 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). Discussion 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 FEBS Journal 274 (2007) 1290–1303 ª 2007 The Authors Journal compilation ª 2007 FEBS S. Kumar et al. A novel peroxidase CrPrx from C. roseus D A E B C F Fig. 8. GUS and GFP fluorescence patterns of CrPrx expression in N. tabacum leaf. (A) GUS staining and (B) GFP fluorescence patterns of the same. (C–E) GFP fluorescence patterns of stomatal guard cells, leaf epidermal cells and (F) xylem cells of transiently transformed N. tabacum with CrPrx–GUS–GFP. In epidermal and stomatal guard cells, CrPrx–GFP is restricted to the cell wall and associated structures, 41 the membranes of the central vacuole, and the wall thickening of xylem cells (fi). 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 [15]. 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) [23]. The full-length CrPrx gene, like most of the plant peroxidase genes, contains three introns, which differ in their sizes [24]. 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 [25] and N. tabacum [7] 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 organspecific, 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 [28]. The induction of secondary metabolite accumulation is an FEBS Journal 274 (2007) 1290–1303 ª 2007 The Authors Journal compilation ª 2007 FEBS 1297 A novel peroxidase CrPrx from C. roseus B A C S. Kumar et al. D E important stress response that depends on jasmonate as a regulatory signal [2]. 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. 1298 Fig. 9. GUS and GFP fluorescence patterns of CrPrx expression in C. roseus leaf. (A) GUS staining and (B) GFP fluorescence patterns of stomatal guard cells of C. roseus. (C) GUS staining and (D) GFP fluorescence patterns of leaf sections of C. roseus. (B, D, E) CrPrx–GFP is restricted to the leaf epidermal cells (B), guard cell walls (D) and the wall thickening of xylem tissues (E) of transiently transformed C. roseus with CrPrx–GFP. Experimental procedures Plant materials 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 nurserygrown 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. Stress treatments 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 FEBS Journal 274 (2007) 1290–1303 ª 2007 The Authors Journal compilation ª 2007 FEBS S. Kumar et al. A novel peroxidase CrPrx from C. roseus detached from plants and kept on paper soaked in gency with 0.1 · NaCl ⁄ Cit and 0.1% SDS at 65 C. The 3 1 ⁄ 10 Murashige Skoog (MS) basal medium by painting on 1359 bp full-length clone was identified after in vivo the adaxial surface of the leaves, and the tray containing excision in the phagemid vector pBSK+ (Clontech, Palo the leaves was sealed with saran wrap. In control experi- 8 Alto, CA, USA). The complete cDNA coding region was PCR amplified ments, similar leaves were painted with double-distilled using forward primer PFLF1 (5¢-CACGAGCTGACCTTwater containing the same amount of ethanol required for CACTGTC) and reverse primer PFLR1 (5¢-GCTCACCACdissolving MJ. For UV treatment, young leaves were CATTACATTGC), designed to anneal with the 5¢-UTR detached from the plants and kept on 1 ⁄ 10 MS media. A and 3¢-UTR regions. PCR amplification consisted of 2 lL short-term exposure (2 min) of leaves under a UV lamp of cDNA template in a reaction volume of 50 lL, (kmax 312 nm; 28 JÆm2Æs)1) was given, and this was followed by incubation on 1 ⁄ 10 MS medium for various time peri1 · ThermoPol buffer, 1.5 mm MgCl2, 0.4 mm dNTPs, 0.2 lm each primer, and 1 U of Deep VentR DNA Polymods before harvesting. For each treatment, young leaves, the first to the third from the shoot apex, were used. The 9 erase (NEB, Beverly, MA, USA). Thermal cycling was carried out on an MJ Research Master Cycler (Global leaves were harvested at different time points by snap freezing in liquid nitrogen, and stored at ) 80 C for further 10 Medical Instrumentation, Ramsey, MN, USA) with the following conditions: initial denaturation at 94 C for 2 min, analyses. 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, Cloning of CrPrx cDNA and gene and a final extension at 72 C for 10 min. The corresponding genomic sequence for CrPrx was PCR-amplified using Total RNA was isolated from vegetative tissue (roots, stem, the same primer pair PFLF1 and PFLR1. The PCR prodleaves) as well as reproductive tissues (flower buds, open uct was cloned into the vector pGEM-T Easy (Promega), flowers and pods) of C. roseus using the LiCl precipitation and sequenced as mentioned above. Gene-specific primers method [36]. First-strand cDNA synthesis was carried out GSP2 (5¢-CCCTTGAAAGGGAGTGTCCTGGAGTTGG) with 5 lg of total RNA using oligo-dT15 primer (Promega, and GSP4 (5¢-GAGGCTCTCATTGTGGTCTG-GGA4 Madison, WI, USA) and Powerscript reverse transcriptase GATG) were designed from the 380 bp and 532 bp posi5 (BD Biosciences, Palo Alto, CA, USA) following the manutions of the cDNA sequence, respectively, for subcloning facturer’s instruction, and used as the template for PCRs. the CrPrx gene. PCR amplifications were performed with degenerate oligonucleotide primers PF-1 (5¢-AGRCTTCAYTTYCAT GAYTGC), PF-2 (5¢-AGRCTTCAYTTYCATGAYTGT¢), Southern blot analysis PR-1 (5¢-GTGNSCMCCDRRSARRGCDAC), and PR-2 Catharanthus roseus genomic DNA was purified using the (5¢-CATYTCDGHYCAHGABAC), which were designed on the basis of highly conserved amino acid sequences of 11 hexadecyltrimethyl ammonium bromide method [32]. Thirty micrograms of BglII-, EcoRV- and HindIII-digested genomproteins encoded by the peroxidase gene family, namely, ic DNA was separated on 0.7% agarose 1 · TAE gel at RLHFHDC, VALLGAHSVG, and VSCSDI. PCR condi40 V for 8 h. DNA was then transferred to a Hybond-N tions used were initial denaturation at 94 C for 2 min, folmembrane, following the manufacturer’s instructions. Prelowed by 29 cycles of denaturation at 94 C for 45 s, hybridization and hybridization of membranes were carried annealing at 45 C for 30 s, and extension at 72 C for out at 60 C in modified church buffer (7% SDS, 0.5 m 1 min, with a final extension at 72 C for 10 min. Amplified NaPO4, 10 mm EDTA, pH 7.2) [33]. Blots were probed products of the expected size were gel purified using with [32P]dCTP[aP] CrPrx cDNA. Blots were finally the MinElute Gel Extraction Kit (Qiagen, Hilden, Ger6 many), and cloned directly into the pGEM-T Easy cloning washed in 1 · NaCl ⁄ Cit and 0.1% SDS at 60 C [33]. Membranes were wrapped in Klin Wrap (Flexo film wraps, vector (Promega), following the manufacturer’s instructions. Clones were sequenced using Big Dye terminator 12 Aurangabad, India) and exposed to XBT-5 CAT film v3.1 cycle sequencing (Applied Biosystems, Foster City, 13 (Kodak, Mumbai, India). 7 CA, USA) chemistry on an ABI prism DNA sequencer (DNA sequencing facility, National Centre for Plant GenNorthern blot analysis ome Research, New Delhi, India). In order to clone complete CrPrx cDNA, a k-ZapIITotal RNA (20 lg) was separated on a 1.2% denaturing oriented leaf-specific cDNA library was screened under agarose gel at 60 V for 6 h and blotted onto Hybond-N high-stringency conditions with modified church buffer at 14 membrane (Amersham-Pharmacia, Piscataway, NJ, USA) 60 C [36]. The 394 bp (CrInt1) PCR product obtained using standard procedures [34]. Following transfer, blots using degenerate PCR primers was used as a probe (acces- 15 were rinsed briefly in diethylpyrocarbonate-treated water, sion number AY769111). One positive plaque was and the RNA was immobilized on the membrane by UVobtained after a final wash of the membrane at high strincrosslinking using a Stratalinker (Model 1800; Stratagene, FEBS Journal 274 (2007) 1290–1303 ª 2007 The Authors Journal compilation ª 2007 FEBS 1299