Plant Mol Biol (2013) 82:181–192 DOI 10.1007/s11103-013-0053-0
NtPDR1, a plasma membrane ABC transporter from Nicotiana tabacum, is involved in diterpene transport Je´roˆme Crouzet • Julien Roland • Emmanuel Peeters Tomasz Trombik • Eric Ducos • Joseph Nader • Marc Boutry
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Received: 23 July 2012 / Accepted: 26 March 2013 / Published online: 7 April 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract ATP-binding cassette transporters are involved in the active transport of a wide variety of metabolites in prokaryotes and eukaryotes. One subfamily, the Pleiotropic Drug Resistance (PDR) transporters, or full-size ABCG transporters, are found only in fungi and plants. NtPDR1 was originally identified in Nicotiana tabacum suspension cells (BY2), in which its expression was induced by microbial elicitors. To obtain information on its expression in plants, we generated NtPDR1-specific antibodies and, using Western blotting, found that this transporter is localized in roots, leaves, and flowers and this was confirmed in transgenic plants expressing the ß-glucuronidase reporter gene fused to the NtPDR1 promoter region. Expression was seen in the lateral roots and in the long glandular trichomes of the leaves, stem, and flowers. Western blot analysis and in situ immunolocalization showed NtPDR1 to be localized in the plasma membrane. Induction of NtPDR1 expression by various compounds was tested in N. tabacum BY2 cells. Induction of expression was observed with the hormones methyl jasmonate Je´roˆme Crouzet and Julien Roland contributed equally to this work.
Electronic supplementary material The online version of this article (doi:10.1007/s11103-013-0053-0) contains supplementary material, which is available to authorized users. J. Crouzet J. Roland E. Peeters T. Trombik E. Ducos J. Nader M. Boutry (&) Institut des Sciences de la Vie, Universite´ catholique de Louvain, Croix du Sud, 4-5, Box L7-04-14, 1348 Louvain-la-Neuve, Belgium e-mail:
[email protected] J. Crouzet Laboratoire de Stress, De´fense et Reproduction des Plantes, Unite´ de Recherche Vignes et Vins de Champagne, Universite´ de Reims Champagne-Ardenne, Reims, France
and naphthalene acetic acid and diterpenes. Constitutive ectopic expression of NtPDR1 in N. tabacum BY2 cells resulted in increased resistance to several diterpenes. Transport tests directly demonstrated the ability of NtPDR1 to transport diterpenes. These data suggest that NtPDR1 is involved in plant defense through diterpene transport. Keywords ABC transporter Diterpenes Sclareol Trichome Pleiotropic drug resistance
Introduction ATP binding cassette (ABC) proteins are widespread in all living organisms and most are transporters that couple ATP hydrolysis to the transport of a wide variety of substrates across biological membranes (Higgins 2001). In plants, the ABC family is very large: for instance, the Arabidopsis and rice genomes each contain more than 120 members classified into different phylogenetic subfamilies (Rea 2007; Yazaki et al. 2009; Kretzschmar et al. 2011). One of these, the pleiotropic drug resistance (PDR) transporters, or full size ABCG subfamily, is found only in fungi and plants. The encoded proteins consist of two copies of two basic elements, a transmembrane domain and a cytosolic nucleotide-binding domain (Verrier et al. 2008). All Saccharomyces cerevisiae PDR transporters analyzed have been found to be localized in the plasma membrane and one of their most plausible physiological functions is detoxification, since some extrude hundreds of functionally and structurally unrelated cytotoxic compounds or potentially toxic metabolites (Lamping et al. 2010). In plants, the PDR subfamily has been characterized in various species, leading to the identification of 15 PDR genes in Arabidopsis
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(Sanchez-Fernandez et al. 2001; van den Bruˆle and Smart 2002), 23 in rice (Jasinski et al. 2003; Crouzet et al. 2006), and, more recently, 19 in Medicago trucatula (Jasinski et al. 2009). Several PDR genes have been shown to be involved in the biotic stress response, as pathogens or elicitors have been shown to induce their expression. For some of them, it has been shown that gene silencing or allele variation confer increased susceptibility to microbial pathogens (Campbell et al. 2003; Kobae et al. 2006; Stein et al. 2006; Jasinski et al. 2009; Bultreys et al. 2009; Krattinger et al. 2009). PDRs also intervene in symbiosis as the petunia PDR1 transporter controls strigolactone-dependent symbiotic signaling and branching (Kretzschmar et al. 2012). Biotic interactions with insects also concern PDR transporters. Indeed NtPDR5 in N. tabacum is involved in resistance to the herbivory Manduca sexta (Bienert et al. 2012). As expected for genes involved in the plant pathogen response, expression of PDR genes can be induced by defense hormones, such as salicylic acid and methyl jasmonate. Expression of OsPDR9, NpPDR1 and NtPDR5 is induced by methyl jasmonate (Moons 2008; Grec et al. 2003; Bienert et al. 2012), AtPDR8 and OsPDR20 expression is induced by salicylic acid (Kobae et al. 2006; Stein et al. 2006; Moons 2008), and GmPDR12 and AtPDR12 expression is induced by both agents (Eichhorn et al. 2006; Campbell et al. 2003). In addition, hormone response boxes are found in PDR promoters. A cis-regulatory sequence required for methyl jasmonate-mediated expression is present in the NpPDR1 transcriptional promoter (Grec et al. 2003), while W-boxes, elements commonly found in salicylic acid-responsive genes, are found in the GmPDR12 promoter (Eichhorn et al. 2006). In their defense role, PDR transporters are believed to export antimicrobial secondary metabolites. However, direct identification of PDR substrates is often lacking. NpPDR1 expression is induced by pathogens, such as Botrytis cinerea or Pseudomonas syringae pv. tabaci, and plays a role in plant pathogen defense (Stukkens et al. 2005). It is also induced by sclareol, an antifungal diterpene synthesized by, and secreted from, trichomes, which express NpPDR1 (Jasinski et al. 2001). Indirect evidence suggests that NpPDR1 transports sclareol (Jasinski et al. 2001; Stukkens et al. 2005), and AtPDR12 and SpTUR2 also seem to be involved in sclareol transport (van den Bruˆle and Smart 2002; Campbell et al. 2003). Expression of NtPDR1, identified in N. tabacum, is induced in culture cells by microbial elicitors or methyl jasmonate (Sasabe et al. 2002), and the presence in its transcription promoter of putative cis-elements, such as the GCC box, W boxes, and JA-responsive elements, support a role of NtPDR1 in the biotic stress response (Schenke et al. 2003). However, its expression profile and role in the plant
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are not known and no putative substrates have been identified for the encoded transporter. Here, we show that NtPDR1 is expressed in the root epidermis and in the head cells of the long trichomes all over the plant and that suspension cells that constitutively express NtPDR1 can transport several diterpenes. These data support a role of NtPDR1 in biotic stress defense.
Materials and methods Plant material Nicotiana tabacum cv petit Havana SR1 was grown in compost in a growth chamber with 16 h light (200 lmol s-1 m-2) at 25 °C and 8 h darkness at 19 °C. Nicotiana tabacum L. cv Bright Yellow 2 (BY2) suspension cells (Nagata et al. 1992) were grown in MS medium supplemented with 0.02 % KH2PO4, 5.10-3 % myoinositol, 5.10-4 % thiamine, and 2.10-5 % 2,4-D, pH 5.6 (KOH) at 25 °C in the dark with agitation at 100 rpm. The cultures were diluted 20-fold every week. RNA isolation and cloning of NtPDR1 Total RNA was isolated using a standard guanidine thiocyanate method from N. tabacum BY2 cells treated for 24 h with 500 lM methyl jasmonate. cDNA were generated from total RNA (5 lg) using a 18-oligo (dT) primer and M-MLV reverse transcriptase (Promega) following the manufacturer’s protocol, and the PCR fragment corresponding to NtPDR1 was amplified using the primers 50 TGGTTACAAAATGGAGCCAGC-30 and 50 -TTAAACTCACTTATCTTCTCTGG-30 , cloned into the pGEM-T Easy vector (Promega), and sequenced. Plant transformation A 1.5 kb fragment corresponding to the putative NtPDR1 promoter region (Schenke et al. 2003) was amplified by PCR (50 -AAATTGACTTAAAAAATGAAAC-30 and 50 TCTAGACTCCATTTTGTAACCAAAACAC-30 ), cloned into the pGEM-T Easy vector (Promega), and transferred into the pAUX3131 vector upstream of the gusA reporter gene (Goderis et al. 2002) using NotI and XbaI. The NtPDR1 promoter-gusA cassette was then excised using I-SceI and inserted into the pMODUL binary vector (Goderis et al. 2002). Agrobacterium tumefaciens strain LBA4404 virG N54D (van der Fits et al. 2000) was used to transform N. tabacum L. cv. SR1 plants by the leaf disk method of Horsch et al. (1985). Transformed plants were selected on MS solid medium supplemented with 100 mg/l of kanamycin.
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For NtPDR1 overexpression in N. tabacum BY2 cells, the gene contained in pGEM-T Easy was cloned in two steps. In the first, the 50 part of NtPDR1 was cut with SphI, blunt ended, and cut with BamHI, then was ligated into pBI121 that had been cut with SacI, blunt ended, and cut with BamHI under the control of the NpPMA4 transcription promoter reinforced with two CaMV 35S enhancers (En2pPMA4, De Muynck et al. 2009). In the second, the 30 part of NtPDR1 was excised from pGEM-T Easy with SacII, blunt ended, and cut with BamHI and ligated into the BamHI and SmaI sites of the NtPDR1 50 fragment-containing plasmid, yielding the pBi121-NtPDR1 vector. N. tabacum BY2 cell transformation was performed as described in Navarre et al. (2006). Membrane preparation For N. tabacum BY2 cells, cells in 5 ml of a 7-day culture (about 200 mg fresh cell weight/ml) were harvested by filtration on a layer of Miracloth (Calbiochem) and washed with cold homogenization buffer (250 mM sorbitol, 50 mM Tris–HCl, pH 8.0, 2 mM EDTA, 0.7 % polyvinylpyrrolidone, 5 mM DTT, 1 mM PMSF, and 2 lg/ml each of leupeptin, pepstatin, aprotinin, antipain and chymostatin). The cells were ground for 3 min in tubes containing 600 mg of glass beads (0.8–1 mm diameter, VWR) and 800 ll of cold homogenization buffer using a cell homogenizer (Biospec). The beads and cell debris were then removed by centrifugation for 5 min at 3,500 g at 4 °C and the supernatant centrifuged at 10,000g for 10 min at 4 °C and the resultant supernatant centrifuged at 100,000g for 30 min at 4 °C. The final pellet (microsomal fraction) was suspended in suspension buffer (5 mM KH2PO4, 330 mM sucrose, 3 mM KCl, pH 7.8). For plants, fresh tissue (0.25–10 g) was ground in two volumes of cold homogenization buffer using a 1 ml sintered glass grinder (VWR) for small amounts or a commercial blender (Waring) for large amounts and the microsomal fraction prepared as described above. Plasma membranes were isolated from the microsomal fraction by aqueous polymer phase partition as described by Larsson et al. (1987). Protein concentrations were measured as described by Bradford (1976), using bovine serum albumin as the standard. Antibody generation Two milligrams of a synthetic peptide corresponding to an NtPDR1-specific sequence (VLPEDGENAENGEVSS, residues 792–807) with an additional N-terminal Cys residue was coupled to 2 mg of keyhole limpet hemocyanin (ImjectÒ Maleimide Activated mcKLH kit, Pierce) and the
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conjugate used to immunize rabbits (Centre d’Economie Rurale, Marloie, Belgium), then antibodies were purified from the serum using the immobilized peptide (SulfoLinkÒ kit, Pierce). To test the specificity of the antibody, constructs coding for the glutathione S-transferase (GST) fused to peptides of NtPDR1, NpPDR1 (Stukkens et al. 2005), NpPDR2 (Trombik et al. 2008), and NtPDR3 (Ducos et al. 2005) that correspond to peptide VLPEDGENAENGEVSS of NtPDR1 were obtained in pGEX-KT (Hakes and Dixon 1991). Western blotting Proteins (20 lg) were incubated for 15 min at 37 °C in Laemmli buffer (80 mM Tris–HCl, pH 6.8, 2 % SDS, 10 % glycerol, 1 % dithiothreitol, and 0.005 % bromophenol blue) containing 1 mM PMSF and protease inhibitors (2 lg/ml each of leupeptin, pepstatin, aprotinin, antipain, and chymostatin), separated by SDS-PAGE (7 % polyacrylamide) using a Mini-protean 3 cell electrophoresis apparatus (Bio-Rad), and transferred onto a PVDF membrane (Millipore) using the semi-dry Bio-Rad system in 50 mM Tris, 40 mM glycine, 0.0375 % SDS, and 10 % methanol. The membranes were then blocked for 30 min at room temperature in Tris-buffered saline (50 mM Tris– HCl, pH 7.6, 150 mM NaCl) containing 3 % low fat dried milk and 0.5 % Tween 80, washed for 3 9 5 min in washing medium (Tris-buffered saline containing 0.1 % low fat dried milk and 0.5 % Tween 80). The membranes were then incubated for 1 h at room temperature in washing medium containing peptide affinity-purified rabbit anti-NtPDR1 antibodies or mouse anti-penta-His antibodies. After 2 9 15 min washes in washing medium, the membranes were incubated for 1 h at room temperature with horseradish peroxidase-conjugated anti-rabbit or antimouse IgG antibodies, then, after 4 9 5 min washes, the antigen–antibody complexes were detected by the enhanced chemiluminescence method (Roche). In situ immunolocalization Three-day-old BY2 cell cultures (1.8 ml) were centrifuged for 10 min at 1000 rpm (Eppendorf 5417C). Cells were resuspended and incubated for 12 min in 5 ml MS medium supplemented with 0.3 M sorbitol. Then, 1 % formaldehyde was added. After incubation for 2 h at 25 °C, cells were centrifuged for 15 min at 700 rpm (Rotofix 32 A, Hettich zentrifugen), washed 3 times with MS medium supplemented with 0.55 M sorbitol and treated for 20 min at 25 °C in 10 ml digestion medium (0.55 M sorbitol, 0.6 % cellulase Onuzuka R-10 (Yakult Pharmaceutical), 0.2 % macerozyme Onuzuka R-10 (Yakult Pharmaceutical), pH 5.6–5.8) under agitation. Cells were centrifuged
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and washed 3 times with MS medium supplemented with 0.55 M sorbitol. Cells were permeabilized for 6 min with 800 ll methanol at -20 °C, centrifuged and saturated with 900 ll PBS T-Milk (PBS supplemented with 0.5 % (v/v) TweenÒ 20 and 2 % (w/v) nonfat milk powder) for 1 h at room temperature. Cells were divided into aliquots of 300 ll, centrifuged and then incubated for 16 h at 37 °C in 40 ll PBS T-milk containing primary antibodies against NtPDR1 (1:20 dilution), centrifuged for 25 min at 1,000 rpm (Eppendorf 5417C) and washed 3 times with PBS T-milk and once with PBS. Cells were incubated for 1 h at 37 °C in 40 ll PBS T-milk containing fluorescein isothiocyanate-coupled goat anti-rabbit IgG antibodies (Invitrogen) (1:100 dilution), centrifuged, washed 3 times with PBS T-milk and once with PBS and then incubated for 1 h at 37 °C in 40 ll PBS T-milk containing antibodies against H?-ATPase coupled to Alexafluor 555 (1:90 dilution). Cells were centrifuged, washed 3 times with PBS T-milk and once with PBS and then examined by confocal laser-scanning microscopy (Carl Zeiss MicroImaging, LSM 710, Software Zen 2010, objective 409). DAPI was excited at 405 nm (emission between 410 and 484 nm), FITC at 488 nm (emission between 498 and 547 nm) and AlexaFluor 555 at 543 nm (emission between 552 and 697 nm). GUS histochemical analysis Hand-cut sections of different plant organs were prepared, prefixed for 30 min under vacuum in 50 mM phosphate buffer pH 7.2 containing 0.05 % Triton X-100 and 4 % formaldehyde, washed for 3 9 5 min in 50 mM phosphate buffer, and incubated in reaction solution (50 mM NaH2PO4 pH 7.2, 0.1 % Triton X-100, 1 mM 5-bromo-4chloro-3-indolyl-b-D-glucuronide, 0.02 % NaN3, and 5 mM b-mercaptoethanol). The sections were then vacuum infiltrated for 5 min, incubated for 2 h at 37 °C in the dark, and the reaction solution discarded. Chlorophyll was solubilized in 100 % ethanol until the tissues became clear, then the tissues were successively soaked in 70 % ethanol for 2 min and 50 % ethanol for 2 min and finally preserved in 50 % glycerol, 0.02 % NaN3. For tissues strongly prone to oxidation, such as the petals, the staining reaction solution contained 15 mM b-mercaptoethanol.
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Toxicity assay in liquid culture Three-day-old N. tabacum BY2 cell suspensions were adjusted at 0.028 g/ml in MS medium supplemented with putative substrates. Cells were incubated for 48 h at 25 °C under agitation in darkness. Cells were centrifuged at 4,000 rpm on four disks of Miracloth (Calbiochem) (Rotofix 32 A, Hettich zentrifugen) and weighted. Transport of substrates by NtPDR1 BY2 cells (3-day-old cultures) were diluted to 0.1 g fresh cell weight/ml in MS medium and supplemented with the indicated concentrations of putative substrates. After 30 min incubation, 5 ml of cell suspension was filtered on a Whatman 3MM paper disk, transferred into 5 ml of fresh MS medium, and incubated for the indicated periods of time. Then 1 ml of the suspension was filtered and washed once with 1 ml of MS. For 3H-decahydro-2-hydroxy-2,5,5,8a-tetramethyl-1-naphtalene ethanol (*2 mCi/mmol, Jasinski et al. 2001), cell radioactivity was determined by liquid scintillation counting. For the other substrates, cells were ground in microtubes containing 600 mg glass beads (0.8–1 mm diameter, VWR) and 500 ll of CH2Cl2 (1 9 20 s and 2 9 40 s at 5,000 rpm Precellys 24, Bertin). Tubes were centrifuged at 4 °C for 5 min at 14,000 rpm (Eppendorf 5417C). The CH2Cl2 phases were collected and 2 ll were injected in a GC–MS system (Hewlett Packard 5890 Series II Plus gas chromatograph and Finnigan MAT TSQ 7000 mass spectrometer). Separation was performed using an ECTM-5 column (30 m, I.D.: 0.32 mm, film thickness: 0.25 lM) (Grace). The program of oven temperature was adapted to the tested compounds. Helium flow was 1 ml per min, injector was heated at 250 °C, interface GC–MS temperature was at 305 °C and ionization was obtained by electron impact at 70 eV. The peak area of the substrate was determined. Protein analysis by mass spectrometry The protein band of interest was excised from the gel, treated with trypsin, and analyzed by MS/MS, as described in Duby et al. (2010). The acquired spectra were analyzed using Applied Biosystems GPS Explorer (version 3.6) and the Matrix Science MASCOTÒ algorithm in the NCBInr viridiplantae protein database, as described in Duby et al. (2010).
Drug treatment of BY2 cells Results Chemicals were added to 5 ml samples of 3.5-day-old cultures in MS medium, then, after 24 h incubation, the cells were collected by filtration and washed with two volumes of 20 mM KCl, 5 mM Na-EDTA, 10 mM Tris– HCl, pH 8.0.
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NtPDR1 expression pattern NtPDR1 has been previously identified in suspension N. tabacum BY2 cells as a PDR gene induced by elicitors
In situ NtPDR1 expression To examine NtPDR1 expression at the cell level, a 1.5 kb fragment upstream of the NtPDR1 coding region (-1487
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(Sasabe et al. 2002). NtPDR1 is not the N. tabacum ortholog of NpPDR1, which was originally identified in N. plumbaginifolia (Jasinski et al. 2001). Indeed, NpPDR1 and NtPDR1, which share 84 % identity at the amino acid level, are found in both species, and a gene has been identified in N. plumbaginifolia that shares 99 % identity with NtPDR1 and is the NtPDR1 ortholog (Genbank accession KC758855). To examine NtPDR1 expression in the plant, we generated NtPDR1-specific antibodies using a 16 amino acid peptide (VLPEDGENAENGEVSS, residues 792–807) from a region that is highly divergent in PDRs. Only five non-contiguous residues are conserved in the corresponding NpPDR1 sequence (TISDESENNESESSP, residues 796–810). AntiNpPDR1 antibodies were already available (Stukkens et al. 2005) and both antibodies were affinity-purified on the corresponding immobilized peptide. GST fusion proteins containing the peptide sequence from NtPDR1, NpPDR1, NpPDR2 (Trombik et al. 2008), or NtPDR3 (Ducos et al. 2005) corresponding to that from NtPDR1 were tested for reactivity with these two antibodies using Western blotting and the anti-NpPDR1 antibodies were found to react only with NpPDR1 and the anti-NtPDR1 antibodies to react only with NtPDR1 (Supplemental Figure 1). NtPDR1 expression was then analyzed in different organs, using antibodies against plasma membrane H?ATPase as a loading control. Root tissue gave a strong signal, with an apparent molecular mass of 150 kDa (Fig. 1a) close to the predicted molecular mass for NtPDR1 (162 kDa), taking into account the abnormal migration of hydrophobic proteins. Separation of the roots into main root and lateral roots showed stronger NtPDR1 expression in the lateral roots than the main root (Fig. 1c). Coomassie blue staining of the microsomal membrane fraction from the main and lateral roots showed a major band corresponding to the expected size of the PDR in the lateral roots (Fig. 1d). When this band was cut out and analyzed by mass spectrometry, two sets of peptides were identified, corresponding to NtPDR1 and NpPDR1 (Supplemental Table 1). NtPDR1 was also detected in the stem and leaf tissues, in particular the leaf epidermis (Fig. 1a). As shown in Fig. 1b, in flower organs, NtPDR1 was detected at a moderate level in the lower part of the petals (petal bottom) and the sepals and at a higher level in the upper pigmented part of the petals (petal top). This contrasts with NpPDR1 expression, which is restricted to the upper part of the petals (Stukkens et al. 2005). No NtPDR1 signal was found in the reproductive organs and fruit (Fig. 1b).
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Fig. 1 Expression profile of NtPDR1 in different organs. a, b Western blots of the microsomal fraction isolated from N. tabacum vegetative (a) and reproductive organs (b) using anti-NtPDR1 or antiH?-ATPase antibodies. c, d Western blot (c) or the Coomassie Bluestained gel (d) of the microsomal fraction of the main root and lateral roots
upstream of the start codon, Schenke et al. 2003) was retrieved by PCR from N. tabacum genomic DNA and this promoter region (pNtPDR1) was fused upstream of the b–glucuronidase reporter gene (gusA) in a binary vector for plant transformation using A. tumefaciens. Ten independent transgenic N. tabacum lines showed the same profile of GUS activity. In agreement with the Western blotting analysis (Fig. 1a), strong GUS expression was found in the root (Fig. 2). pNtPDR1-gusA expression was
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localized at the tip and for a few centimeters behind tip of both the lateral and main root (Fig. 2a) and appeared at an early stage of lateral root emergence (Fig. 2b). A longitudinal section of a root tip showed GUS expression around the epidermal cells and in the root cap cells (Fig. 2c). In the aerial parts, GUS staining was observed in the long glandular trichomes of different organs, such as the leaf (Fig. 2d), stem (Fig. 2e), and petal (Fig. 2f). This expression pattern differs from that of NpPDR1 (Stukkens et al. 2005) in two ways: 1/NpPDR1 is expressed in the short, rather than the long, trichome heads in the leaf and 2/NpPDR1 is not expressed in petal trichomes, but is found
in the epidermis cells of the inner face of the upper part of the petal.
Fig. 2 In situ expression profile of NtPDR1. a The root from a plant expressing pNtPDR1-gusA was stained for GUS activity (bar = 1 cm). b Detail of a lateral root at two different developmental stages (bar = 0.5 mm). c Longitudinal section of a lateral root tip
(bar = 0.5 mm). d–f GUS expression in long glandular trichomes of the abaxial leaf face (d), stem (e), and lower part of the petal (f) (bars = 0.1 mm)
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Subcellular localization of NtPDR1 NtPDR1 was found to be expressed in BY2 cells only during the late exponential growth phase (data not shown). Western blotting (Fig. 3) of similar concentrations of a microsomal fraction (M) and a plasma membrane-enriched fraction (PM) prepared from wild-type BY2 cells during late exponential growth phase showed that NtPDR1 was enriched in the plasma membrane-enriched fraction to a similar extent as the H?-ATPase marker.
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Induction of NtPDR1 expression As NtPDR1 expression has been shown to be induced by methyl jasmonate and elicitors, such as yeast extract (Sasabe et al. 2002), we examined if NtPDR1 expression at the protein level was modulated by methyl jasmonate, yeast extract, and other compounds. As shown in Fig. 4, BY2 cells at 4.5 days of culture (middle exponential phase) displayed low or no detectable NtPDR1 expression on Western blotting when treated with water or 0.5 % methanol (vehicles for the added test compounds), but high expression was seen after treatment with the plant defense signaling molecule methyl jasmonate (MeJa), but not salicylic acid (SA), and by treatment with yeast extract. This suggests that NtPDR1 might be associated with the plantpathogen response through the methyl jasmonate pathway. The auxin hormone naphthalene acetic acid (NAA) also induced strong expression, whereas the hormones abscisic acid (ABA), gibberellic acid (GA), the ethylene precursor 1-aminocyclopropane-1-carboxlate (ACC), and epibrassinolide (EpiB), had either no, or only a slight, effect. We also tested various secondary metabolites. Diterpenes are known to be synthesized in trichome heads (Wagner 1991). NtPDR1 expression was slightly induced by the polycyclic labdane diterpenes sclareol and abietic acid and more strongly induced by the macrocyclic duvane diterpene cembrene. No induction of NtPDR1 expression was seen following treatment with the alkaloid berberine or the flavonoid quercetine. Overexpression of NtPDR1 in BY2 cells In order to analyze its transport activity, NtPDR1 was expressed in the yeast Saccharomyces cerevisiae using a constitutive promoter. However, Western blotting showed that expression was very weak and unstable over time and that NtPDR1 was not correctly targeted to the plasma membrane (data not shown). We therefore turned to the homologous system of N. tabacum BY2 cells. Since the N-terminal region is poorly conserved between PDR kDa
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Fig. 3 Subcellular localization of NtPDR1 in N. tabacum BY2 cells. Western blots of a microsomal (M) or plasma membrane-enriched (PM) fraction prepared from a 7-day-old culture of N. tabacum BY2 cells using anti-NtPDR1 or anti-H?-ATPase antibodies
transporters, cDNA encoding NtPDR1 was provided with a 6-His tag between codons 2 and 3 and fused to the constitutive promoter NpPMA4 reinforced with two copies of the CaMV 35S enhancer (De Muynck et al. 2009) ligated into a binary vector and introduced into A. tumefaciens, which was used to transform BY2 cells. BY2 cells in the late exponential phase show NtPDR1 expression. This might be related to a possible response to the toxicity of metabolites that accumulate in the growth medium. Ectopic NtPDR1 expression was thus evaluated using anti-His antibodies. Of the twenty transformants tested (see Fig. 5a for 6 representative clones), fourteen gave a positive signal of the expected apparent molecule mass, but of variable intensity, presumably because of the position effect of the inserted transgene. Using a plasma membrane-enriched fraction prepared from the microsomal fraction by phase partition, we examined whether NtPDR1 was correctly targeted to the plasma membrane and found that it was enriched in the plasma membrane fraction to the same extent as H?-ATPase, a plasma membrane marker (Fig. 5b). In addition, we performed in situ immunolocalization of wild-type and NtPDR1-His-expressing cells (Fig. 5c). A strong signal was observed only around the NtPDR1-His-expressing cells and it co-localized with the plasma membrane marker, H?-ATPase. NtPDR1 confers tolerance to diterpenes Nicotiana species secrete antimicrobial diterpenes from their trichomes (Wagner 1991) and, since NtPDR1 was expressed in the long secretory trichomes, we hypothesized that it might transport diterpenes. If this were the case, we might expect that BY2 cells overexpressing NtPDR1 would be able to extrude diterpenes more efficiently from the cell. BY2 cells do not synthesize diterpenes, but these can be rapidly loaded into the cells by diffusion from the external medium because of their high hydrophobicity. Actually the equilibrium was reached within a few minutes (data not shown). Since high concentrations of diterpenes such as sclareol are toxic for the cell, toxicity would be expected to decrease if diterpenes entering the cell by diffusion were transported back into the external medium by NtPDR1. We therefore performed toxicity growth tests. Cells were incubated with putative substrates for 48 h and growth was scored by measuring the cell mass after filtration. The ratio between cell growth in the presence and in the absence of putative substrates was compared between lines. Preliminary assays were performed to determine inhibiting concentrations. No difference in growth was observed between the wild-type and two transgenic lines in the standard MS medium. However, both transgenic lines were able to growth better than the wild-type line in the presence
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kDa
Ab ie ti M ca eJ cid a SA 50 50 µ 0 2 µ M AB 50 M µ A M N 50 AA µ M G 25 A 0 1 µ Sc 00 M la µM C reo em l 2 AC bre 50 n C e µM Ep 25 25 iB 0 µ 0 µ Be 12 M M rb 5 µ Q erin M ue e r M cet 250 et in ha 2 µM Ye no 50 as l 0 µM W t ex .5% at tr er ac t5 m g/ m l
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100 Fig. 4 Induction of NtPDR1 expression in N. tabacum BY2 cells by various chemicals. Cultures (3.5-day-old) of N. tabacum BY2 were incubated with the indicated compounds for 24 h, then a microsomal fraction was prepared and analyzed by Western blotting using anti-
NtPDR1 or anti-H?-ATPase antibodies. Except for ACC, which was added as an aqueous solution, the chemicals tested were added as a methanol solution (0.5 % final concentration)
of four diterpenes tested: sclareol, manool, abietic acid and dehydroabietic acid (Fig. 6). A macrocyclic diterpene, cembrene, was also tested but because of its limited solubility, we could not reach a concentration high enough to be toxic for the wild-type cells. No growth difference was found between wild-type and NtPDR1-expressing cells for the linear diterpene, geranylgeraniol, and artemisinin, a sesquiterpene (Fig. 6).
eucalyptol, which could be monitored by GC–MS, and in this case, no significant difference was found between NtPDR1-expressing and wild-type cells.
NtPDR1 transports diterpenes To directly demonstrate transport of diterpenes, we loaded the cells for 30 min with a labeled sclareol analog, 3 H-decahydro-2-hydroxy-2,5,5,8a-tetramethyl-1-naphatalene ethanol (Jasinski et al. 2001), then incubated them in diterpene-free medium and measured the residual internal concentration, which reflects the equilibrium between passive and active transport. If sclareol were indeed exported by NtPDR1, we would expect a lower internal concentration for cells in which the transporter was expressed and this was found to be the case (Fig. 7a). After 15 min, a new equilibrium was reached but the level was significantly lower for the NtPDR1-His-expressing lines than for the wild-type or an antibody-expressing line used as another control. Addition of vanadate or glibenclamide, compounds known to inhibit ABC transporters, increased the sclareol internal concentration (Fig. 7b), indicating that the difference observed in the presence and absence of those inhibitors is most probably linked to an ABC transporter activity. We extended the transport analysis to other diterpenes: sclareol, manool and the macrocyclic cembrene. As these compounds are not available as labeled products, we quantified the intracellular concentration of putative substrates by GC–MS. All three had a lower concentration in NtPDR1-His-expressing cells than in wild-type cells (Fig. 8). As a control, we tested the monoterpene
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Discussion NtPDR1 had been identified in N. tabacum BY2 cells, in which its expression is induced by methyl jasmonate and elicitors (Sasabe et al. 2002). However, its expression profile in the plant, its subcellular localization and its substrates were unidentified. In the present report, we have addressed these unknowns. We analyzed NtPDR1 expression in the plant and found it was constitutively expressed in various organs. High expression was found in the root epidermis and in the long glandular trichomes of the aerial part. This observation, together with the plasma membrane localization of NtPDR1, indicates that this transporter is most probably involved in the secretion of metabolites playing a role in the extracellular medium. BY2 cells expressing NtPDR1 were more resistant to, and accumulated less of, several cyclic diterpenes tested. These diterpenes differ according to their degree of saturation, number of cycles (three, two or a macrocycle) and ring substitutions (Supplemental Fig. 2). This suggests that NtPDR1 does not have a high substrate specificity. However, a linear diterpene (geranylgeraniol), a sesquiterpene (artemisinin) and a monoterpene (eucalyptol) did not discriminate wild-type and NtPDR1-expressing cells, suggesting that they are not transported. Diterpenes are likely to be physiological substrates, as they are synthesized in N. tabacum trichomes and secreted at the leaf surface where they constitute a major component of the leaf exudate and are involved in plant protection against biotic threats (Wagner 1991). These hydrophobic compounds are expected to diffuse freely through membranes. Since they are toxic for plant cells, an efficient
Plant Mol Biol (2013) 82:181–192
(a)
NtPDR1-His transformants 4
5
6
100
170 -
His
(b) PM 2 4
*
170 -
** **
His 130 -
er lg ny
er a
in in
io l an
ac id et ic
dr eh y
NtPDR1
WT
D
G
ol an o
A
M
re Sc la
on e N
(c)
ol
0
H+-ATPase
100 -
ac id
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**
bi
2
**
50
oa
kDa
**
bi et ic
M
%Control
H+-ATPase
100 -
WT NtPDR1-2 NtPDR1-4
**
130 -
is
3
em
2
rt
WT 1
A
kDa
189
1
e
a
I DA P + d ge
+ H
int
er
ti-
an
as TP A -
M
DR
P Nt
Fig. 6 Terpenoid sensitivity of wild-type and NtPDR1-His-expressing N. tabacum BY2 cells. Cells from a wild-type line (WT) and two NtPDR1-His-expressing lines (NtPDR1-2 and NtPDR1-4) were incubated for 48 h as indicated in ‘‘Materials and methods’’ in liquid MS cultures containing or not 85 lM sclareol, 850 lM manool, 130 lM abietic acid, 65 lM dehydroabietic acid, 1 mM geranylgeraniol or 500 lM artemisinin. The ratios of cell weights in the presence or absence of terpenoids are displayed (means and standard errors of three independent experiments. Student’s t test: *P \ 0.05; **P \ 0.01)
Fig. 5 Ectopic expression of NtPDR1-His in transgenic N. tabacum BY2 cells. a Western blots of a microsomal fraction from wild-type N. tabacum BY2 cells (WT) or the indicated NtPDR1-His BY2 transformants using anti-His tag or anti-H?-ATPase antibodies. b Western blots of a microsomal (M) and plasma membrane-enriched fraction (PM) of NtPDR1-His BY2 transformants (lines 2 and 4) using anti-His tag or anti-H?-ATPase antibodies. c In situ immunolocalization of NtPDR1. Wild-type (WT) and NtPDR1-His-expressing (NtPDR1) BY2 cells (cultures at day 3) were submitted to in situ immunodetection as indicated in ‘‘Materials and methods’’. Cells were successively incubated with anti-NtPDR1 antibodies, secondary fluorescein-conjugated antibodies, and finally anti-H?-ATPase antibodies coupled with AlexaFluor 555. Representative figures are shown. Left NtPDR1 localization (green); middle: H?-ATPase localization (red); right differential interference contrast image with overlay of NtPDR1 and H?-ATPase localization (yellow), and nucleus (blue). Bars 20 lm
transport system is required to keep the intracellular concentration low. This might explain the involvement of an ABC-type transporter powered by ATP hydrolysis. Whether diterpenes are also NtPDR1 substrates in roots and petals is unknown. Exudates from these organs in
N. tabacum have not been characterized in great detail. However, it is possible that NtPDR1 transports other types of molecules in these organs. Indeed some ABC transporters, including yeast PDR transporters, are able to transport various substrates with unrelated structures. This feature, which is unusual for an enzyme, has been explained in the case of the mammalian P-glycoprotein, which belongs to the multiple drug resistance ABC subfamily, as the high resolution structure revealed the existence of multiple binding sites in a very large internal cavity that can bind several substrates (Aller et al. 2009). The search for NtPDR1 substrates should therefore not be limited to diterpenes. However, testing the direct transport of metabolites that might be secreted at the root or petal level is difficult, as most have not been characterized and, once identified, will probably not be available as labeled compounds. An alternative would consist in using a transportomics approach as performed for the identification of the substrate spectrum of mouse ABC transporters (Krumpochova et al. 2012). The observation that NtPDR1 expression is upregulated by methyl jasmonate indicates that this transporter might
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Plant Mol Biol (2013) 82:181–192 b Fig. 7 Transport of a sclareol analog by N. tabacum BY2 cells
(a)
100
%Control
WT AB NtPDR1-2 NtPDR1-4
50
*
* *
*
**
expressing NtPDR1-His. a Cells from a wild-type line (WT), a line expressing the antibody Lo-BM2 (De Muynck et al. 2009) (AB) and two NtPDR1-His-expressing lines (NtPDR1-2 and NtPDR1-4) were loaded with 3H-decahydro-2-hydroxy-2,5,5,8a-tetramethyl-1-naphatalene ethanol, a sclareol analog (Jasinski et al. 2001), as described in the ‘‘Materials and methods’’, then the cells were transferred to medium without the radioactive analog for the indicated period of time, when the intracellular content of the labeled substrate was quantified. The figure shows the ratios (means and SE; Student’s t test: *P \ 0.05) of the labeled substrate at the time indicated compared to time 0 for three independent experiments. Values (cpm) at time 0 were as follows. WT: 636 ± 25; AB: 664 ± 15; NtPDR1-2: 627 ± 21; NtPDR1-4: 615 ± 34. b Cells from the NtPDR1-2 line were treated as in a except that 3 mM vanadate or 200 lM glibenclamide were added in the indicated samples during both incubation periods. Means and SE for three experiments are shown (Student’s t test: *P \ 0.05)
Sclareol
Manool
150
50
12
W T
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tP
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tP
D R
D R
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12
W T N
%Control
Cembrene
14
0
Eucalyptol
1000
*
*
100
0
100
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200
N
150
(b)
**
D R
Min
*
100
Manool peak area
60
30
0
15
0
Sclareol peak area
300
50
**
500
be involved in the pathogen response. Recently, transgenic N. tabacum plants partly silenced for NtPDR1 expression were shown to be slightly more susceptible to the bacterial pathogen Ralstonia solanacearum. However, this phenotype was only seen in the presence of sclareol, which activates the expression of NtPDR1. The authors therefore suggested that NtPDR1, like AtPDR12, an Arabidopsis gene belonging to the same PDR subfamily, might
123
40
30
20
10
14 tP D R
D R tP N
N
14
12 D R tP N
W T N tP D R 12
0
0
W T
+G
30
30
Time (min)
Eucalyptol peak area
ad lib at e en cl am id e
30
*
+V an
0
0
Cembrene peak area
50
Fig. 8 Transport of diterpenes by N. tabacum BY2 cells expressing NtPDR1-His. Cells from a wild-type line (WT) and two NtPDR1expressing lines (NtPDR1-2 and NtPDR1-4) were loaded with 12 lM sclareol, 7 lM manool, 190 lM cembrene or 35 lM eucalyptol as described in the ‘‘Materials and methods’’, then the cells were transferred to medium without the substrate for 30 min, when the intracellular content of the substrate was quantified. The figure shows the peak area (means and SE; Student’s t test: *P \ 0.05; **P \ 0.01) of the substrate after 30 min compared to time 0 for three independent experiments
Plant Mol Biol (2013) 82:181–192
transport abscisic acid, resulting in activation of a pathway that leads to defense (Seo et al. 2012). This contrasts with a previous observation that N. plumbaginifolia plants silenced for NpPDR1, a gene belonging to the same PDR subfamily, are more susceptible to a variety of fungal pathogens without requiring the addition of sclareol (Bultreys et al. 2009). Although the NpPDR1 gene also shows constitutive expression in leaves, roots and flowers (Stukkens et al. 2005), this occurs in different cell types to those shown in this study to express NtPDR1. NpPDR1 is expressed in the head cell of the leaf short glandular trichomes (Stukkens et al. 2005), whereas NtPDR1 was found to be expressed in the head cells of the long glandular trichomes. The respective roles of these two types of glandular trichomes are unclear. For instance, a proteomic analysis of a mixture of the two types showed the presence of both the cytosolic mevalonate and plastid MEP pathways (Van Cutsem et al. 2011). The question whether both pathways are found in both trichome types or whether each is found in a different type will only be resolved by the establishment of a method for separating these two trichome types. In the petal, NpPDR1 is expressed in the inner epidermal cell layer (Stukkens et al. 2005), while NtPDR1 was found to be expressed in the long glandular trichomes. In the root, NpPDR1 is expressed in most root cells, while NtPDR1 expression was concentrated in the epidermis. So clearly, the expression profiles of NpPDR1 and NtPDR1 do not overlap. Methyl jasmonate-treated N. plumbaginifolia cells show more extrusion of sclareol and this transport was originally attributed to NpPDR1, which shows increased expression in these treated cells (Jasinski et al. 2001). However, at that time, NtPDR1 had not been identified and we have now shown that sclareol also induces NtPDR1 expression in BY2 cells and that NtPDR1 can transport sclareol. We also previously showed that sclareol is more toxic for protoplasts from NpPDR1-silenced plants than for those from wild-type plants (Stukkens et al. 2005). However, a more direct transport assay is required to unambiguously demonstrate that NpPDR1 transports sclareol. Even if it turns out that NpPDR1, like NtPDR1, is able to transport sclareol, as explained above, these ABC transporters might also recognize other substrates, which could differ for the two transporters. To try to compare NtPDR1 and NpPDR1, we attempted to overexpress NpPDR1 in N. tabacum BY2 cells in the same way as we did in this study with NtPDR1, but failed because the cDNA was always reorganized in E. coli during vector construction (data not shown). In conclusion, we have shown that NtPDR1 is constitutively expressed in cell types (root epidermis and long glandular trichomes) that are in contact with the external medium and transports antimicrobial diterpenes. This, together with the upregulation of NtPDR1 expression by methyl jasmonate, supports the hypothesis that this ABC
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transporter is involved in plant defense against biotic threats. Acknowledgments This work was supported by the Interuniversity Poles of Attraction Program (Belgian State, Scientific, Technical, and Cultural Services), the ‘‘Communaute´ franc¸aise de Belgique–Actions de Recherches Concerte´es’’ and the Belgian National Fund for Scientific Research.
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