Plant Molecular Biology 44: 733–745, 2000. © 2000 Kluwer Academic Publishers. Printed in the Netherlands.
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Molecular cloning and functional expression of a stress-induced multifunctional O-methyltransferase with pinosylvin methyltransferase activity from Scots pine (Pinus sylvestris L.) H´el`ene Chiron1,2 , Alain Drouet2 , Anne-Catherine Claudot2 , Christoph Eckerskorn3,4 , Monika Trost1, Werner Heller1 , Dieter Ernst1,∗ and Heinrich Sandermann Jr1 1 GSF-National
Research Center for Environment and Health, Institute of Biochemical Plant Pathology, 85764 Neuherberg, Germany (∗ author for correspondence; e-mail:
[email protected]); 2 Biology Department, University of Orl´eans, BP 6749, 45067 Orl´eans Cedex 2, France; 3 MPI für Biochemie, 82512 Martinsried, Germany; 4 Present address: Top-Lab, 82512 Martinsried, Germany Received 18 February 2000; accepted in revised form 7 August 2000
Key words: cDNA cloning, elicitor, ozone, pinosylvin O-methyltransferase, Pinus sylvestris, stilbene
Abstract Formation of pinosylvin (PS) and pinosylvin 3-O-monomethyl ether (PSM), as well as the activities of stilbene synthase (STS) and S-adenosyl-l-methionine (SAM):pinosylvin O-methyltransferase (PMT), were induced strongly in needles of Scots pine seedlings upon ozone treatment, as well as in cell suspension cultures of Scots pine upon fungal elicitation. A SAM-dependent PMT protein was purified and partially characterised. A cDNA encoding PMT was isolated from an ozone-induced Scots pine cDNA library. Southern blot analysis of the genomic DNA suggested the presence of a gene family. The deduced protein sequence showed the typical highly conserved regions of O-methyltransferases (OMTs), and average identities of 20–56% to known OMTs. PMT expressed in Escherichia coli corresponded to that of purified PMT (40 kDa) from pine cell cultures. The recombinant enzyme catalysed the methylation of PS, caffeic acid, caffeoyl-CoA and quercetin. Several other substances, such as astringenin, resveratrol, 5-OH-ferulic acid, catechol and luteolin, were also methylated. Recombinant PMT thus had a relatively broad substrate specificity. Treatment of 7-year old Scots pine trees with ozone markedly increased the PMT mRNA level. Our results show that PMT represents a new SAM-dependent OMT for the methylation of stress-induced pinosylvin in Scots pine needles. Abbreviations: CA, caffeic acid; COMT, caffeate O-methyltransferase; CCoA, caffeoyl-coenzyme A; CCoAOMT, caffeoyl-CoA O-methyltransferase; PS, pinosylvin; OMT, O-methyltransferase; PMT, pinosylvin 3-Omethyltransferase; PSM, pinosylvin 3-O-monomethyl ether; Que, quercetin; RACE, rapid amplification of cDNA ends; SAM, S-adenosyl-L-methionine; STS, stilbene synthase Introduction Stilbenes have so far been detected in relatively few plant families where they contribute to the resistance of woody tissues to degradation, and act as phytoalexins (Gorham, 1995). In Scots pine trees, the stilbenes pinosylvin (PS) and pinosylvin 3-O-methyl The PMT cDNA sequence has been applied for a patent: Deutsche Patentanmeldung 19836774.0.
ether (PSM) are known to be major constitutive phenolic constituents of heartwood and root tissues (Billek, 1964). Both compounds accumulate in Scots pine upon biotic and abiotic stress (Gehlert et al., 1990; Rosemann et al., 1991; Bonello et al., 1993; Lieutier et al., 1996; Langebartels et al., 1998). The pinosylvin backbone is synthesized from cinnamoylCoA and malonyl-CoA by a specific stilbene synthase (STS) (Preisig-Müller et al., 1997). Enzymatic methy-
734 lation by O-methyltransferases (OMTs) (EC 2.1.1.6) generally involves the transfer of the methyl group of S-adenosyl-l-methionine (SAM) to phenolic hydroxyl groups. This is thought to play an important role in processes such as inactivation of reactive hydroxyl groups, plant structural support, chemical defence, signalling, lipophilicity increase and antimicrobial activity (Luckner, 1990; Ibrahim et al., 1998). Plant OMTs generally possess strict substrate and position specificities for various methyl acceptor molecules (Ibrahim et al., 1987). To date, there are more than 40 plant OMT DNA sequences that can be retrieved from sequence databases. Most sequences encode proteins of 27–43 kDa involved in the methylation of substrates such as phenylpropanoids (Bugos et al., 1991; Busam et al., 1997), flavonoids (Maxwell et al., 1993; Gauthier et al., 1996), benzylisoquinoline alkaloids (Takeshita et al., 1995; Frick and Kutchan, 1999) and myoinositol (Vernon and Bonhert, 1992). Five highly conserved regions comprising 36 amino acid residues rich in glycine are located in the second half of the protein sequence (Ibrahim et al., 1998). Two clones of multifunctional caffeic acid (CA) and caffeoyl-CoA (CCoA) OMTs have been reported from loblolly pine (Li et al., 1997, 1999); however, no DNA clone of OMT for stilbenes has yet been described. Here, we have purified and partially sequenced the PMT protein and measured the contents of PS and PSM and the activities of STS and PMT in ozone-treated Scots pine seedling needles and cell suspension cultures treated with fungal elicitor. Moreover, we describe the molecular cloning of a cDNA from ozone-treated Scots pine seedlings that encodes a PMT. The recombinant protein catalysed the methylation of diverse phenolic substrates. RT-PCR analysis revealed an ozone-induced PMT mRNA accumulation in 7-year old pine trees.
Materials and methods Plant material and treatments Cell cultures Cell suspension cultures were obtained from Scots pine callus cultures and grown at 25 ◦ C at 120 rpm in a modified MS medium containing 2 mg/l 2,4dichlorophenoxyacetic acid (2,4-D) and 1 mg/l N 6 benzylaminopurine (BAP). Cell cultures were elicited with cell wall preparations (0.6 g/l glucose equivalents) of Lophodermium seditiosum as described
(Lange et al., 1994). Cells were collected at different times after elicitation, immediately frozen in liquid nitrogen and stored at −80 ◦ C. Seedlings Seedlings were cultivated and exposed to ozone as described by Rosemann et al. (1991). Four-week old Scots pine seedlings were treated with 0.15 and 0.3 µl/l ozone for 8 h per day during 25 days. Needles were collected at 0, 24 and 48 h after the start of ozone treatment, immersed in liquid nitrogen and stored at −80 ◦ C. Trees Seven-year old Scots pines grown at the Bauchery nursery (Crouy sur Cosson, France) acclimated (April 1998) for 6 days in the GSF phytotron walk-in chambers (Thiel et al., 1996) in a 14 h/10 h day/night cycle including UV-B radiation at 22/16 ◦ C and 70/85% relative humidity. Ozone treatment (0.15 or 0.3 µl/l) was for 10 h per day during 2 days. Needles were harvested after the beginning of ozone treatment, immersed in liquid nitrogen, freeze-dried and stored under vacuum in the dark. Stilbene contents Pinosylvin and pinosylvin monomethyl ether contents were determined by HPLC and fluorescence detection as described (Rosemann et al., 1991). Enzyme extraction and purification The purification of PMT enzyme was performed by standard procedures. Frozen cell suspension-cultured Scots pine cells (250 g), collected 24 h after elicitation with Polytran N (0.25 g/l), were homogenized with 500 ml buffer 1 (0.1 M potassium phosphate, containing 1 mM DTE, 1 mM PMSF pH 7.5), containing 10% w/w PVP and 10% w/w sea sand. After filtration and centrifugation the resulting supernatant was used as crude extract (step 1). After ammonium sulfate fractionation between 35 and 60% (saturation) (step 2), the resuspended pellet was cleared by centrifugation (27 000 × g) and subjected to hydrophobic interaction chromatography on a Butyl Sepharose CL-4B column (Amersham Pharmacia). Elution was performed with 20 mM Tris-HCl pH 7.5, 1 mM DTE using a decreasing ammonium sulfate gradient from 0.7 to 0 M (step 3). Active fractions were collected and precipitated with ammonium sulfate (80% saturation). After centrifugation the pellet was dissolved
735 in 3.7 ml 20 mM Tris-HCl, 1 mM DTE pH 7.5, and then fractionated on a Sephacryl S 200 Superfine column (Amersham Pharmacia). Elution was performed with 50 mM Bis-Tris-HCl, 1 mM DTE, 10% w/v glycerol pH 7 (step 4). Active fractions were subjected to anion exchange chromatography (Fractogel EMD-TMAE, Merck). Elution was carried out with 50 mM Bis-Tris-HCl, 1 mM DTE, 10% w/v glycerol, 1 M NaCl pH 6.5 using a two-step NaCl gradient (0 to 0.3 M and then 0.3 to 1.0 M) (step 5). Active fractions were collected and equilibrated with 25 mM Bis-Tris-HCl, 1 mM DTE, 10% w/v glycerol, 5% w/v betaine pH 6.4, and then applied to dye ligand chromatography (Matrex Red A column, Amicon) (step 6). Unadsorbed fractions were loaded onto a Mono P HR 5/20 column (Amersham Pharmacia). Elution was performed with diluted polybuffer 74 (Amersham Pharmacia), containing 1 mM DTE, 10% w/v glycerol, 5% w/v betaine pH 4.5. Active fractions were collected and brought to pH 7 with 0.5 M Tris-HCl, 1 mM DTE, 10% w/v glycerol (step 7), and then stored at −20 ◦ C. SDS-PAGE and peptide sequencing Active fractions of purification step 7 were separated by 2D-PAGE. The first dimension was carried out in a 7.5% Protogel (National Diagnostics) pH 8.8 at 40 mA for 2 h. After equilibration the second dimension was performed in 10% SDS-PAGE with a constant current of 80 mA for 2.5 h. The gels were run in a Midget Gelelectrophoresis Unit (Amersham Pharmacia). Two protein spots, at 37 kDa and 40 kDa, were blotted onto a glass fibre membrane. Tryptic digestion, HPLC separation of peptides and amino acid sequence analysis were performed as described (Eckerskorn and Lottspeich, 1989). SDS-PAGE of in vitro expressed PMT was carried out under denaturing conditions in a 12.5% acrylamide gel, using a vertical minigel system at 30 mA. Enzyme assays The standard assay for STS activity was as described by Rosemann et al. (1991). Crude extract (80 µl) or partially purified enzyme was assayed for PMT activity with 14 C-SAM (4.9 kBq, 28 µM) and PS and analogues at 500 µM in 0.16 M Hepes-KOH buffer pH 7.7 for 1 h at 30 ◦ C. The reaction was stopped by the addition of 20 µl 6 M HCl and products extracted with ethyl acetate. For seedlings lowmolecular-weight compounds were removed from the
crude extract on a PD-10 column (Amersham Pharmacia). In vitro expressed PMT was assayed with different concentrations of CA, CCoA, quercetin (Que) and PS in 0.1 M Hepes-KOH buffer pH 7.7 and 24 µM 14 C-labelled SAM (4.6 kBq) for 30 min at 30 ◦ C. All other substrates were tested at a concentration of 100 µM. Reactions were stopped with 20 µl 6 M HCl. Products were extracted into ethyl acetate and the radioactivity of the ethyl acetate phase was determined by liquid scintillation counting, except for CCoA as substrate. With CCoA as substrate, the incubation mixture was first extracted with ethyl acetate. This step removes products of free CAs. A 20 µl aliquot of 5 M NaOH were then added to the aqueous residue and the mixture was kept at 56 ◦ C for 30 min to hydrolyse the CoA ester (Pakusch et al., 1991). A 15 µl portion of 6 M HCl was then added, the ferulic acid obtained extracted with ethyl acetate and the extract counted. TLC was carried out on cellulose plates (Merck). For product identification by TLC reference compounds (5 µg) were added to the extracted radioactivity. The solvent system used for more polar compounds was chloroform/acetic acid/water (50:45:5) and 2% v/v formic acid in two dimensions, respectively. For the less polar stilbenes and catechol, toluene/acetic acid/water (50:47:3) and 50% v/v acetic acid was used, respectively. After chromatography, plates were dried, autoradiographed, and analysed under UV light or after exposure to iodine vapour. Nucleic acid extraction Total RNA was isolated from freeze-dried needles (70–90 mg) as described (Kiefer et al., 2000). Total DNA was isolated from fresh needles according to protocol 1 in Csaikl et al. (1998). PMT cloning and characterization cDNA library screening A pSPORT1 (Gibco) cDNA library constructed from ozone-treated Scots pine seedlings (Wegener et al., 1997) was screened as follows. Plasmid DNA was isolated with the Qiagen Midiprep kit. PCR reactions were carried out with an upstream primer, designed from a highly conserved region in plant OMTs (50 (C/T)TIGTIGA(C/T)GTIGGIGGIGG-30) (Gauthier et al., 1996). The SP6 sequencing primer was employed as downstream primer (Gibco). The PCR fragments obtained were sequenced with a Thermo Sequenase kit (Amersham Pharmacia).
736 The 50 region was obtained with a T7 sequencing primer (Gibco) as upstream primer, and 50 GCATCGGCCACCACATGCGCCATGTC-30 derived from the above-mentioned PCR sequence. The PCR fragment was again sequenced. A final PCR reaction between the START and the STOP regions was carried out. Amplified product was purified by electrophoresis on agarose, excised and eluted by standard methods (Sambrook et al., 1989). Cloning and sequencing DNA fragments were ligated into the pGEM-T vector (Promega) and transferred into competent E. coli cells. The amplified DNA plasmids were purified as described above and sequenced in both directions. Three independent clones were used for sequencing. Subcloning and expression in E. coli NdeI and NotI sites were introduced by PCR at the 50 and 30 ends of the cDNA clone with primer 50 -GGATTCCATATGGGATCGGCTTCC-30 and 50 CATGTCGCGGCCGCCGTGGAAG-30 , respectively. The PCR reaction was performed on pGEM-T plasmid containing the PMT cDNA. The PCR product was purified, digested with NdeI and NotI, and cloned into NdeI/NotI-digested expression vector pET21(a)+ (Novagen). Competent E. coli JM 109 (DE 3) (Promega) were transformed and grown at 37 ◦ C at 220 rpm in 50 ml LB medium, containing 100 µg/ml ampicillin. When cultures reached an optical density at 590 nm of 0.6, 1 mM IPTG was added to induce expression of the fusion protein, and the cultures were incubated for an additional 3 h. Cells were collected at 5000 × g and resuspended in 2 ml 50 mM TrisHCl pH 7.5, containing 2 mM DTT and 10% w/v glycerol. Cells were lysed by sonication at 0 ◦ C at high power (4 × 30 s) and the resulting extracts were centrifuged at 15 000 × g for 15 min at 4 ◦ C. The supernatants were utilized for SDS-PAGE, FPLC and for PMT activity measurements.
and 0.2 µM of the required 30 and 50 primers. For both primer pairs amplification was performed during 35 cycles of 1 min denaturation at 94 ◦ C, 1 min primer annealing at 58 ◦ C and 2 min elongation at 72 ◦ C. Blank RT-PCR reactions were carried out by omitting either RNA, reverse transcriptase or cDNA. The reaction products were analysed by electrophoresis (1% agarose gel), DNA bands were visualized under UV light after ethidium bromide staining. PCR products were sequenced and identified as PMT or cab (X14506), and an increase in PCR product amounts was proportional to an increased amount of cDNA. Southern blot hybridization Genomic DNA (10 µg) was digested with HindIII, NheI and SpeI, fractionated in a 0.7% agarose gel in 0.5× TBE, and blotted onto a Hybond N+ nylon membrane (Amersham Pharmacia). The filter was pre-hybridized in ExpressHyb hybridization solution (Clontech) at 60 ◦ C for 1 h. The PMT cDNA was labelled with [α-32 P]dCTP using the Nonaprimer kit (Appligene) and added to the hybridization solution. After overnight incubation, the filter was washed twice in 2× SSC and 0.05% SDS at room temperature for 20 min, and twice in 0.1× SSC and 0.1% SDS at 50 ◦ C for 20 min. The filter was exposed for 7 h to an imaging plate and analysed with a PhosphorImager (FUJIX, BAS 1000, Fuji). Statistical analysis Numerical values generally are means of three replicates ± SD. Statistical analysis was determined by using Student’s t-test of Snedecor and Cochran (1972) (P
