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The Plant Journal (2002) 30(6), 649±662 The ABC transporter SpTUR2 confers resistance to the antifungal diterpene sclareol Sybille van den BruÃle1, Axel MuÈller2, Andrew J. Fleming1 and Cheryl C. Smart1,* 1 Department of Plant Biochemistry and Physiology, Institute of Plant Sciences, Swiss Federal Institute of Technology (ETH) ZuÈrich, UniversitaÈtsstrasse 2, CH-8092 ZuÈrich, Switzerland, and 2 Lehrstuhl fuÈr P¯anzenphysiologie, Ruhr-UniversitaÈt, D-44780 Bochum, Germany Received 28 January 2002; revised 5 March 2002; accepted 8 March 2002. * For correspondence (fax +41 1 632 1044; e-mail [email protected]). Summary PDR5-like proteins represent one group of the ABC superfamily of transporters. Members of this group are present in plants and, due to the function of PDR5-related proteins in fungi in the excretion of xenobiotics (including antifungal agents), it has been proposed that they might play a similar role in plants in the response to and detoxi®cation of herbicides and fungicides. However, until now no functional data has been presented showing an altered plant response to any herbicide or fungicide as a result of manipulating the expression of a PDR5-like gene in plants. In this paper, we show that the plant SpTUR2 PDR5-like ABC transporter is localised to the plasma membrane and that expression of this protein in Arabidopsis leads to the acquisition of resistance to the diterpenoid antifungal agent sclareol. These data both de®ne a possible endogenous substrate for this transporter and highlight the potential of manipulating plant chemical resistance via modulating the expression of speci®c PDR5-like transporters. Keywords: PDR5, SpTUR2, Arabidopsis thaliana, Spirodela polyrrhiza, abscisic acid, terpenoid. Introduction The ATP-binding cassette (ABC) superfamily consists of a large group of related proteins whose members mediate a wide range of transport processes in organisms ranging from bacteria to man (Higgins, 1992). Common to these proteins is the presence of one or two copies each of two core structural elements: a hydrophobic transmembrane spanning (TMS) domain consisting of multiple (usually six) membrane-spanning segments and a cytosolically orientated highly conserved ATP-binding cassette (ABC) containing Walker A, Walker B, and ABC signature motifs. This molecular architecture allows the proteins to transport substrates across biological membranes, usually against a concentration gradient, via the binding and hydrolysis of ATP. The transmembrane domains are believed to form a chamber and to determine the substrate speci®city of the transporter (Chang and Roth, 2001; Egner et al., 2000). A feature of ABC transporter proteins is the diversity of substrates that can be transported, often even by one member of a family. Thus, they have been implicated in the movement of alkaloids, amino acids, carbohydrates, ã 2002 Blackwell Science Ltd inorganic ions, lipids, peptides, pigment molecules, steroids and a variety of xenobiotics. This diversity in substrate has led to a similar diversity in proposed function of the different members of the superfamily, with ABC transporters being implicated in roles ranging from the maintenance of physiological ion balance (Higgins, 1995) through hormone transport (Edelmann et al., 1999) to the view of some transporters as lipid ¯ippases or membrane hoovers moving xenobiotics out of the cell (Eytan and Kuchel, 1999; Higgins and Gottesman, 1992; van Helvoort et al., 1996). The superfamily of eukaryotic full-length ABC transporters can be subdivided into four major subfamilies based on the conservation of molecular structure. Full-length ABC transporters contain two TMS domains and two ABC domains and these can be subclassi®ed into those that have a forward (TMS-ABC)2 con®guration such as the human multidrug resistance protein HsMDR1 (Chen et al., 1986) and those that have the reverse (ABC-TMS)2 con®guration such as the yeast pleiotropic drug resistance protein ScPDR5 (Balzi et al., 1994). Multidrug resistance 649 650 Sybille van den BruÃle et al. related proteins (MRPs), have the forward orientation but are distinguished by having additionally an N-terminal extension and an internal regulatory domain (Borst et al., 1999; Rea et al., 1998; Rea, 1999). ABCA subfamily members, such as the human ABCA1 protein (responsible for Tangier disease when mutated) contain a hydrophobic segment within a very large regulatory domain (Broccardo et al., 1999; Oram, 2000). It has now been shown that plants contain genes encoding members of all four of these subfamilies. Thus, sequencing of the Arabidopsis genome has led to the identi®cation of 22 genes encoding MDR-like proteins, 15 encoding MRP-like proteins, 12 encoding PDR5-like proteins and 1 encoding an ABCA1-like protein (Davies and Coleman, 2000; Martinoia et al., 2002; SaÂnchezFernaÂndez et al., 2001; Theodoulou, 2000). However, the potential substrates for most of these transporters remain totally unknown and, indeed, the subcellular localisation has only been substantiated for one member each of the MDR-like (Sidler et al., 1998) and PDR5-like (Jasinski et al., 2001) family. The greatest progress has been made in the characterisation of the MRP-like proteins in plants for which a function in the transport of glutathione conjugates, glucuronides and glucuronide conjugates and chlorophyll catabolites into the vacuole has been shown in a heterologous yeast expression system (Liu et al., 2001; Lu et al., 1997, 1998; Tommasini et al., 1998), as well as a potential role in endogenous hormone transport (Gaedeke et al., 2001). With respect to plant MDR-like transporters, evidence indicates that the Arabidopsis HsMDR1 homolog AtPGP1 leads to altered hypocotyl elongation under low light levels when over- and underexpressed (Sidler et al., 1998), suggesting that it might transport an endogenous compound involved in a hypocotyl cell elongation pathway active in dim light. However, the nature of this compound remains unknown. Although AtPGP1 is MDR-like in structure it appears to be a functional homolog of the yeast PDR5 transporter in that it can complement an ScPDR5 deletion mutant resulting in restoration of cycloheximide resistance (Thomas et al., 2000). These authors additionally showed that overexpression of AtPGP1 confers resistance to cycloheximide and the cytokinin N6-(2isopentenyl)adenine in Arabidopsis and also suggested that the protein might transport ATP. Very recently it has been shown by using reverse genetics that the related gene AtMDR1 encodes an NPA-binding protein required for normal auxin distribution and auxin-mediated development and it is proposed that AtMDR1 is indeed an IAAH ef¯ux transporter (Noh et al., 2001). Finally, recent work on a PDR5-like protein from Nicotiana plumbaginifolia (Jasinski et al., 2001) showed that induction of the gene in cell cultures by the diterpenes sclareol and sclareolide was correlated with a decreased accumulation of a synthetic sclareolide analogue, suggesting that the protein might function to transport this substrate. Sclareolide is a close analogue of sclareol, which is a plant-growth-inhibitory and fungitoxic terpenoid metabolite (Cutler et al., 1977) excreted onto the leaf surface by the trichomes of many species of Nicotiana (Guo and Wagner, 1995) where it is thought that it functions to restrict fungal growth (Bailey et al., 1975; Kennedy et al., 1992). Interest in ABC transporters has particularly been awakened by their involvement in the extrusion of cytotoxic compounds. Thus, ABC transporters are associated with the acquisition of multiple drug resistance by pathogenic organisms (Prasad et al., 1995; Sanglard et al., 1997), as well as the occurrence of multidrug resistant tumours (Endicott and Ling, 1989; Gottesman and Pastan, 1993; Kane, 1996). This ability of at least some ABC transporters to excrete xenobiotics (Bauer et al., 1999; Kolaczkowski et al., 1996, 1998) has led to the proposal that in plants they are likely to be involved in the response to and detoxi®cation of herbicides and fungicides, as well as the transport of plant defence compounds (Davies and Coleman, 2000). In this respect, the PDR5-like family of ABC transporters is of particular interest (Bauer et al., 1999; Decottignies and Goffeau, 1997). In plant pathogenic fungi, members of this transporter group play a role in resistance to antifungals (Nakaune et al., 1998; Schoonbeek et al., 2001; Vermeulen et al., 2001) or have been shown to be necessary for pathogenicity (Urban et al., 1999), and in yeast expression of PDR5-like genes is associated with antifungal drug resistance (Bauer et al., 1999; Kolaczkowski et al., 1998). Taking into account the work described above on NpABC1 intimating a function in the excretion of the antifungal agent, sclareol, a speci®c role for PDR5-like transporters in plants in the response to fungicides seems possible. Speci®cally, if PDR5-like proteins transport antifungals one might predict that overexpression of these transporters would confer resistance to antifungal agents. However, to date no functional data have been presented showing an altered plant response to any herbicide or fungicide as a result of manipulating expression of an ABC transporter. Our previous work led to the identi®cation of the ®rst PDR5-like ABC transporter in plants, SpTUR2 (Smart and Fleming, 1996). The cDNA encoding this protein was isolated from the water plant Spirodela polyrrhiza and we could show that the expression of the gene at the transcript level was subject to a complex hormonal and environmental regulation. In particular, although transcripts were present at low levels in control plants, addition of the hormone ABA led to the rapid and high accumulation of SpTUR2 mRNA. In addition to ABA, a number of factors associated with plant stress led to induction of SpTUR2 gene expression (such as high salt ã Blackwell Science Ltd, The Plant Journal, (2002), 30, 649±662 SpTUR2 confers resistance to sclareol a 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 wt - + 651 and cold), leading us to propose that SpTUR2 might play a role in a general plant response to conditions inhibiting plant growth. In this paper, we show that the SpTUR2 protein is localised to the plasma membrane and that expression of SpTUR2 in Arabidopsis plants confers resistance against the antifungal plant metabolite sclareol. 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 wt - + 160 kDa c TU R2 -35 wt -ve cto r TU R2 -11 160 Spirodela kDa wt -35 R2 TU R2 -14 wt -17 TU wt -14 b -17 Results control ABA Arabidopsis wt TUR2-14 203 118 Figure 1. Analysis of expression of SpTUR2 in transgenic Arabidopsis plants and in Spirodela polyrrhiza. (a) Autoradiograph of a northern blot analysis of the ®rst 36 independent transformants (T1) obtained by expressing the SpTUR2 cDNA in Arabidopsis plants under the control of the CaMV 35S promoter. RNA (3 mg) from Arabidopsis SpTUR2 lines 1±36 was compared with RNA from a wild-type Arabidopsis plant (wt) and with RNA from untreated (±) and ABA-treated (250 nM) Spirodela plants (+). A 32P-labelled 1.4 kb EcoRI fragment of the SpTUR2 cDNA was used as probe. The size of the RNA produced in the transgenic plants is approximately 4.8 kb. (b) Western blot analysis of total protein (6 mg) extracted from homozygous T5 SpTUR2 expressing Arabidopsis 11-day-old seedlings of four independent lines (TUR2-14, TUR2-17, TUR2-35 and TUR2-11) and in the case of the ®rst three lines, their corresponding wild-type segregant lines (wt-14, wt-17 and wt-35), and a line transformed with the empty vector (wt-vector). The blots were probed with an af®nity-puri®ed SpTUR2-speci®c rabbit antiserum directed against a peptide corresponding to the amino acids 30±44 of SpTUR2, detected with a goat antirabbit IgG alkaline phosphatase conjugate, and processed with the Bio-Rad Immun-Star chemiluminescent detection system. The approximate size of the revealed SpTUR2 protein is given on the left in kilodaltons. (c) Western blot analysis of total protein extracted from untreated (control) and ABA-treated (250 nM, 24 h) S. polyrrhiza plants (ABA), and wild-type (wt) and kanamycin resistant T1 SpTUR2 expressing Arabidopsis 12-day-old seedlings of line 14 (TUR2-14). Twenty mg of total protein were loaded for Spirodela and 10 mg for the Arabidopsis samples. The blot was immunodetected with the SpTUR2 peptide antibody as described in (b) but processed with the Bio-Rad Immun-Blot colorimetric detection system. The sizes of marker proteins are given on the left in kilodaltons. ã Blackwell Science Ltd, The Plant Journal, (2002), 30, 649±662 Generation of transgenic Arabidopsis plants expressing the SpTUR2 protein The SpTUR2 cDNA (Smart and Fleming, 1996) was cloned behind the CaMV 35S constitutive promoter in the vector pRT104 and transformed into Arabidopsis using the Agrobacterium-mediated vacuum in®ltration method (Bechtold et al., 1993). An initial northern blot analysis of the ®rst 36 T1 plants obtained from the transformation experiment revealed an accumulation of SpTUR2 transcripts (approximately 4.8 kb) in all samples (Figure 1a). There was a relatively high accumulation of SpTUR2 transcripts in many of the transgenic plants compared with the endogenous level of SpTUR2 mRNA attained by Spirodela plants treated with ABA (Figure 1a). To ascertain whether the high level of SpTUR2 transcript accumulation led to an accumulation of the SpTUR2 protein, we generated an antibody raised against a synthetic peptide corresponding to a portion of the N-terminal amino acid sequence of SpTUR2. As shown in Figure 1(b), the antibody recognised an approximately 160 kDa protein in total protein extracts from a series of T5 homozygous transgenic plants expressing the SpTUR2 cDNA (lines TUR2-14, TUR2-17, TUR2-35 and TUR2-11), whereas the antigen was not detectable in Arabidopsis plants transformed with the empty vector or sister wild-type plants of TUR2-14, TUR2-17, and TUR2-35 obtained as T5 kanamycin sensitive segregants (wt-14, wt-17 and wt-35). That the antibody speci®cally recognised the SpTUR2 protein was also indicated by western blot analysis of Spirodela plants. In good agreement with the theoretically calculated mass of 162.7 kDa for SpTUR2, an approximately 160 kDa protein was faintly detected in total protein extracts of untreated plants and the protein accumulated to a high level following treatment with ABA (Figure 1c). The SpTUR2 protein is localised to the plasma membrane SpTUR2 encodes an ABC transporter that is predicted to localise to a membrane system within the plant cell. To investigate to which membrane system SpTUR2 localised, we performed a series of continuous sucrose gradient centrifugation experiments, both with Spirodela plants induced to accumulate SpTUR2 by treatment with ABA 652 Sybille van den BruÃle et al. (Figure 2a) and transgenic Arabidopsis plants expressing the SpTUR2 cDNA (Figure 2b). Gradient fractions were analysed by western analysis. Antibodies against the ER lumen binding protein BiP (HoÈfte and Chrispeels, 1992) and the plasma membrane integral protein PIP (Kammerloher et al., 1994) were used as markers for the endoplasmic reticulum and plasma membrane, respectively, and either the A-subunit of the vacuolar H+translocating ATPase V-ATPase (Fischer-Schliebs et al., 1997) or the vacuolar membrane 23 kDa aquaporin VM23 (Maeshima, 1992) as markers for the tonoplast. In Spirodela the pattern of SpTUR2 protein distribution clearly correlated with that shown by the PIP antigen, suggesting a plasma membrane localisation (Figure 2a). Although SpTUR2 protein distribution in transgenic Arabidopsis was similar to the PIP antigen, and not at all a Antibody 1 3 5 7 9 Fractions 11 13 15 17 19 kDa 21 23 212 SpTUR2 122 PIP 28.4 83 BiP 83 % sucrose V-ATPase 50 40 30 20 10 1 3 5 7 9 11 13 15 17 19 21 23 Fractions b Antibody 1 3 5 7 Fractions 9 11 13 15 kDa 17 19 21 212 SpTUR2 122 PIP 28.4 BiP 83 VM23 28.4 c Antibody S M OM PM kDa SpTUR2 160 PIP 30 similar to the BiP antigen, we could not rule out a tonoplast location (Figure 2a). Therefore, we also analysed SpTUR2 protein distribution in transgenic Arabidopsis plants using an aqueous two-phase partitioning method for the enrichment of plasma membrane. The results shown in Figure 2(c) con®rm that the SpTUR2 protein does not accumulate in the soluble protein fraction and is enriched in the plasma membrane fraction to a similar extent as PIP. As a further con®rmation that the SpTUR2 protein is localised to the plasma membrane, we performed an immunolocalisation of the protein using a ¯uorescentlabelled secondary antibody and confocal laser scanning microscopy, as shown in Figure 3. Using protoplasts obtained from Arabidopsis plants expressing the SpTUR2 protein, a signal corresponding to the SpTUR2 antigen was visualised exclusively around the circumference of the protoplast, as expected for a plasma membrane localised protein (Figures 3a,b). No signal was obtained in either transgenic protoplasts incubated with second antibody alone (Figures 3c,d) or protoplasts obtained from wild-type plants (Figures 3e,f). A signal corresponding to the SpTUR2 antigen was also visualised in intact mesophyll cells of hand-cut leaf sections taken from transgenic plants expressing the SpTUR2 protein (Figure 3g), whereas similar sections from wild-type plants revealed no signal (Figure 3h). This signal in the transgenic plants was limited to the circumference of the cells and was exterior to the Figure 2. Localisation of SpTUR2 by protein gel blot analysis of membrane fractions. (a) Membrane vesicles prepared from S. polyrrhiza plants grown for 24 h in the presence of 250 nM ABA were centrifuged on a continuous sucrose density gradient. The gradient was fractionated and 20 ml aliquots were separated on SDS-PAGE gels and blotted. The blots were probed with the af®nity-puri®ed SpTUR2 antiserum or speci®c antisera to the plasma membrane protein PIP, the endoplasmic reticulum protein BiP and the tonoplast protein V-ATPase, detected with the appropriate second antibody alkaline phosphatase conjugate and processed with the Bio-Rad Immun-Blot colorimetric detection system. The sucrose concentration of the gradient fractions was measured with a refractometer and is shown below. Numbers on the right indicate the sizes of marker proteins in kilodaltons. (b) A similar experiment was performed to that in (a) but with membrane vesicles from leaves of Arabidopsis plants expressing SpTUR2. The blots were probed with the af®nity-puri®ed SpTUR2 antiserum or speci®c antisera to the plasma membrane protein PIP, the endoplasmic reticulum protein BiP, and the tonoplast protein VM23, detected with the appropriate second antibody alkaline phosphatase conjugate and processed as in (a). Numbers on the right indicate the sizes of marker proteins in kilodaltons. (c) Membrane vesicles (M) from Arabidopsis seedlings expressing SpTUR2 were separated from the soluble protein fraction (S), and then subjected to aqueous two-phase partitioning to enrich for plasma membrane (PM) over the other membranes (OM). Protein samples (1 mg) of each fraction were separated by SDS-PAGE, blotted and immunodetected with the af®nity-puri®ed SpTUR2 speci®c antiserum or speci®c antisera to the plasma membrane protein PIP as in (b). Numbers on the right indicate the approximate size of the immunodetected protein in kilodaltons. ã Blackwell Science Ltd, The Plant Journal, (2002), 30, 649±662 SpTUR2 confers resistance to sclareol plastids, again consistent with a plasma membrane localisation. Similarly, in sections of Spirodela a signal corresponding to SpTUR2 protein could be detected after ABA treatment and this signal was localised to the cell periphery (Figure 3i,j). SpTUR2 expression leads to resistance against the fungicide sclareol To search for substrates transported by the SpTUR2 ABC transporter, we proceeded to use the transgenic Arabidopsis plants expressing the SpTUR2 protein in a screening procedure to test for the ability of the plants to maintain root growth on concentrations of compounds that retarded the growth of wild-type plants. In this assay, root growth was assayed after transfer of seedlings to either control medium or medium containing a speci®c test compound. The rationale of this screening procedure was that if SpTUR2 extruded a particular cytotoxic compound, then expression of the SpTUR2 protein should confer a growth advantage to the transgenic compared with wild-type plants when grown on this compound. While we were performing this screen, M. Boutry and colleagues reported that the induction of a related ABC transporter in cell cultures of Nicotiana plumbaginifolia (NpABC1) by the diterpenes sclareol and sclareolide correlated with the ability of the cells to exclude a labelled sclareolide derivative (Jasinski et al., 2001). We therefore incorporated sclareol and sclareolide into our screening procedure, the results of which are shown in Figure 4 and Table 1. After transfer to control medium, roots of both SpTUR2 expressing lines and wild-type plants (which were sister plants that had lost the transgene) displayed comparable increases in root length (Figure 4a). However, after transfer to medium containing sclareol (75 mM), the increase in root length of the wild-type plants was signi®cantly less than that assayed in the SpTUR2 expressing lines (Figure 4b). This difference in response to sclareol was observed in all three independent transgenic lines tested with a statistically signi®cant difference in growth compared with wild type of P < 0.001. This maintenance of root growth in SpTUR2 expressing plants led to the transgenic plant roots attaining a visibly greater length than wild-type counterparts in the presence of sclareol (Figure 4c). While the sclareol derivative sclareolide also inhibited root elongation in Arabidopsis, the transgenic plants expressing SpTUR2 responded identically to the wild-type controls (data not shown). Our screening procedure incorporated the analysis of a number of compounds covering a range of hormones, stress factors, xenobiotics and known ScPDR5 substrates listed in Table 1. SpTUR2 expressing plants showed no signi®cant difference with respect to root growth compared with wild-type plants after transfer to medium ã Blackwell Science Ltd, The Plant Journal, (2002), 30, 649±662 653 containing these compounds except with sclareol, supporting the speci®city of the response. In addition to the quantitative change in root elongation, plants expressing SpTUR2 showed a qualitative difference in response to sclareol compared with wild-type plants. Thus, as shown in Figure 5, after growth on sclareol at 75 mM wild-type plants showed a stress response, including accumulation of anthocyanins and limited leaf expansion (Figure 5a). In contrast, under the same conditions, plants expressing SpTUR2 grew robustly and showed no visible accumulation of anthocyanins (Figure 5b). Again, this resistance to sclareol was displayed by all three transgenic lines analysed (compare Figure 5c,d, 5e,f). Interestingly, a similar resistance to the detrimental effects of sclareol was observed in Spirodela plants treated with ABA, a known inducer of SpTUR2 gene expression in this plant (Smart and Fleming, 1996). Thus, within 1 day of the addition of 100 mM sclareol to untreated Spirodela, the plants show clear signs of a stress response, most notably chlorosis (compare Figure 5g,h). If the plants are pretreated with ABA (250 nM) for 24 h before the addition of sclareol, then the stress response is almost totally abrogated (compare Figure 5i,j). Regulation of SpTUR2 expression by sclareol The expression of many PDR5-like ABC transporters has been shown to be induced by the substrates that they transport (Del Sorbo et al., 1997; Hirata et al., 1994; Miyahara et al., 1996; Piper et al., 1998). We therefore investigated whether SpTUR2 expression in Spirodela was induced by sclareol. As shown in Figure 6(a), treatment of Spirodela fronds with 100 mM sclareol did indeed lead to an increased accumulation of SpTUR2 protein. However, this induction is not speci®c to sclareol in that rhodamine 6G (a substrate for ScPDR5) leads to a comparable accumulation of SpTUR2. Our previous data (Smart and Fleming, 1996) had identi®ed a number of factors (such as ABA, cycloheximide, salt and cold) that increased the accumulation of transcripts encoding SpTUR2. We therefore investigated whether these changes in transcript level were re¯ected by changes in protein level. As shown in Figure 6(b), both ABA and NaCl led to an accumulation of SpTUR2 protein. However, as shown in Table 1, of the several factors shown to increase the expression of SpTUR2 in Spirodela (Figure 6a,b), only sclareol led to a signi®cant difference in the root growth assay with SpTUR2 expressing Arabidopsis plants. No signi®cant induction of SpTUR2 was observed upon treatment of Spirodela with a spectrum of potential substrates including the ScPDR5 antifungal substrate ketoconazole and the diterpene plant hormone GA3 (Figure 6a), the auxins IAA and NAA, the auxin transport inhibitors NPA and TIBA, the herbicide atrazine and cadmium (data not shown). 654 Sybille van den BruÃle et al. An analysis of the time course and concentration dependence of SpTUR2 accumulation in response to sclareol is shown in Figure 6(c). These data indicate that sclareol at concentrations as low as 100 mM is suf®cient to induce the accumulation of SpTUR2 and that this induction Figure 3. Figure 4. ã Blackwell Science Ltd, The Plant Journal, (2002), 30, 649±662 SpTUR2 confers resistance to sclareol occurs within 8 h. The induction of SpTUR2 expression by sclareol can be compared with the effect of treating the plants with the related compound sclareolide (Figure 6d). As shown in Figure 6(c), sclareolide treatment has no in¯uence on SpTUR2 accumulation, again indicating the speci®city of the response observed to sclareol. To test whether Spirodela plants synthesise sclareol, we performed a GC±MS analysis of extracts of Spirodela plants. The results indicate that sclareol is indeed present in Spirodela (Figure 7). Discussion SpTUR2 is a plasma membrane localised ABC transporter which confers resistance to the antifungal diterpene sclareol A potential role for ABC transporters in the extrusion of xenobiotics (especially herbicides and fungicides) has long been suggested (Davies and Coleman, 2000), but causal data has been lacking. We have shown that expression of the PDR5-like gene SpTUR2 in Arabidopsis leads to resistance to the antifungal diterpene sclareol. This resistance leads to maintained growth of root tissue and robust shoot growth at concentrations of sclareol that are severely detrimental to the growth of Arabidopsis plants not expressing the SpTUR2 protein. These data further the key observation by Jasinski et al. (2001) that up-regulation of the expression of a related PDR5-like gene from Nicotiana plumbaginifolia (NpABC1) by sclareol and sclareolide is associated with a decreased accumulation of a sclareolide derivative. The simplest interpretation of our results is, thus, that expression of the SpTUR2 protein leads to transport of sclareol out of the plant tissue and, as Figure 3. Immunocytochemical localisation of SpTUR2 protein. (a±j) Confocal laser scanning microscopy was performed either with protoplasts or leaf sections probed with the SpTUR2 speci®c antibody. Detection was achieved either with an FITC-conjugated (a±f) or a DTAFconjugated (g±j) second antibody (shown in green). The auto¯uoresence of chloroplasts is shown in red. The grey colour is the cell structure shown by using transmission light where indicated. (a±f) show Arabidopsis leaf protoplasts, (g) and (h) show Arabidopsis leaf sections and (i) and (j) show sections of Spirodela plants treated for 24 h with 250 nM ABA. (a) Protoplast from Arabidopsis expressing SpTUR2. (b) Transmission light image of the protoplast shown in (a). (c) Protoplast from Arabidopsis expressing SpTUR2 (no primary antibody control). (d) Transmission light image of the protoplast shown in (c). (e) Protoplast from wild-type Arabidopsis. (f) Transmission light image of the protoplast shown in (e). (g) Combined confocal and transmission image of a leaf section from Arabidopsis expressing SpTUR2. (h) Combined confocal and transmission image of a leaf section from wild-type Arabidopsis. (i) Confocal image of tissue section from ABA-treated Spirodela. (j) Same as (i) with the inclusion of transmission light to show cell walls. ã Blackwell Science Ltd, The Plant Journal, (2002), 30, 649±662 655 a consequence, decreased toxicity of the fungicide. This is the ®rst demonstration of fungicide resistance in plants mediated by manipulation of the expression of a plant ABC transporter. Although PDR5-like proteins from yeast are also associated with the ability to confer resistance to antifungal agents (Bauer et al., 1999; Kolaczkowski et al., 1998), it is unclear whether this re¯ects a true functional homology between plant and yeast PDR5-like ABC transporters. In fact the plant MDR-like transporter AtPGP1 was shown to complement a yeast PDR5 mutant (Thomas et al., 2000). Attempts to complement either a yeast PDR5 deletion mutant (Katzmann et al., 1994), a double mutant deleted for PDR5 and SNQ2 (Egner et al., 1998), or a mutant deleted for seven ABC transporters (Decottignies et al., 1998) with SpTUR2 were unsuccessful (C. C. Smart et al., unpublished results) and no complementation of a yeast PDR5-like mutant with a plant PDR5-like protein has been reported. SpTUR2 certainly does not appear to confer resistance to typical ScPDR5 substrates like cycloheximide, ketoconazole and rhodamine 6G, and, in fact, seems to possess a high speci®c substrate speci®city, distinguishing between sclareol and sclareolide. In contrast, fungal PDR5-like transporters tend to transport a relatively broad spectrum of substrates. Additionally, although plant PDR5-like transporters are structurally conserved with SpTUR2 and NpABC1 having 70% amino acid identity, they only have 33% amino acid identity to fungal transporters like ScPDR5. Taken together with phylogenetic analyses, which reveal that the plant PDR5-like transporters represent a class of proteins distinct from the classically de®ned fungal PDR5-like transporters (Martinoia et al., 2002; C. C. Smart, unpublished data), it seems likely that plant PDR5like proteins represent a speci®c family of ABC transporters with distinct substrate speci®cities and function compared with fungal PDR5 group members, that is, that they have evolved plant-speci®c functions. Such plant-speci®c features might have evolved to allow for the excretion of speci®c antifungal plant defence compounds, whereas fungal transporters might have undergone selection for a broader substrate speci®city to allow for the extrusion of Figure 4. Arabidopsis transformed with SpTUR2 shows resistance to sclareol. Seeds of three independent lines of Arabidopsis expressing SpTUR2 (SpTUR2) and their corresponding wild-type lines (wt) were sown on control plates and allowed to grow vertically for 6 days. The seedlings were then transferred either to fresh control plates (a) or to plates containing 75 mM sclareol (b)for a further 2 days and the increase in root length measured. The error bars indicate the standard error of the mean and 35±40 seedlings were used per line and treatment. In (b) the transgenic plants were signi®cantly more resistant to sclareol than the wild-type plants in Students t-tests at the following probability levels; line 14 < 5 3 10±9; line 17 < 0.0006; line 35 < 0.0002. (c) Phenotype of seedlings 5 days after transfer to either control or sclareol plates as described in (b) and (c). 656 Sybille van den BruÃle et al. Table 1 Putative SpTUR2 substrates tested in root elongation assaysa Name of compound Description Concentration(s) tested Root growth assay ABA (abscisic acid) EBR (epibrassinolide) CHX (cycloheximide) 2,4-D (2,4-dichlorophenoxyacetic acid) NaCl CdCl2 Chloramphenicol NAA (1-naphthalene-acetic acid) IAA (indole-3-acetic acid) Oligomycin Atrazine NPA (1-N-naphthylphthalamic acid) TIBA (triiodobenzoic acid) Rhodamine 6G Sclareol Sclareolide GA4 (gibberellic acid 4) Taxol Ketoconazole sesquiterpene phytohormone triterpene phytohormone protein synthesis inhibitor synthetic auxin, herbicide salt heavy metal antibiotic synthetic auxin phytohormone inhibitor of oxidative phosphorylation photosynthesis-inhibiting herbicide auxin transport inhibitor auxin transport inhibitor dye antifungal diterpene sclareol analogue norditerpene phytohormone anticancer diterpene antifungal azole 3, 30 mM 100 nM 50 nM 50 nM 50 mM 50 mM 30 mM 100, 300 nM 30, 100 nM 8 mM 1, 3 mM 3 mM 10, 30 mM 300 nM 75 mM 100 mM 100 mM 6 mM 56 mM ± ± ± ± ± ± ± ± ± ± ± ± ± ± 3 ± ± ± ± a Seeds of three independent homozygous lines of Arabidopsis expressing SpTUR2 and their corresponding wild type segregant lines were sown on plates and allowed to grow vertically until the roots were approximately 10 mm long (4±6 days). The seedlings were then transferred either to fresh control plates or to plates containing the putative substrate for a further 2 days and the increase in root length measured. Concentrations were chosen to give between 30% and 70% inhibition of root growth (3). Signi®cant difference between wt and transgenic lines, Ð no signi®cant difference between wt and transgenic lines. many (including plant-derived) antifungal agents (Urban et al., 1999). Our data show that both the endogenous SpTUR2 protein in Spirodela and the SpTUR2 protein expressed in transgenic Arabidopsis plants become localised to the plasma membrane. This was indicated by three independent approaches: sucrose density centrifugation, plasma membrane enrichment by aqueous two-phase partitioning and immunolocalisation. This is the ®rst localisation of a PDR5-like protein in the native plant tissue and is consistent with our proposed role of SpTUR2 function in exporting sclareol from the cytoplasm into the extracellular space. The proposed transport of sclareol by SpTUR2 is speci®c in that the growth response of transgenic plants expressing SpTUR2 to the closely related derivative sclareolide was no different from that of control plants. In this respect SpTUR2 is distinct from the related PDR5like protein, NpABC1, which does not seem to distinguish between these two compounds, at least with respect to substrate induction (Jasinski et al., 2001). Such substrate speci®city shown by SpTUR2 is relatively rare for PDR5like ABC transporters, which (especially in yeast) tend to be promiscuous (Bauer et al., 1999; Kolaczkowski et al., 1998). Previous work on PDR5-like ABC transporters has shown that substrates tend to induce the expression of genes encoding their relevant transporter (Del Sorbo et al., 1997; Hirata et al., 1994; Miyahara et al., 1996; Piper et al., 1998). This was true for SpTUR2 in that treatment of Spirodela plants with sclareol led to the accumulation of the SpTUR2 protein, whereas sclareolide (apparently not transported by SpTUR2) did not affect SpTUR2 levels. However, SpTUR2 protein level was also induced by other compounds (such as ABA, salt and rhodamine 6G) to which the SpTUR2-expressing Arabidopsis plants showed no difference in growth response compared with wild-type plants. Thus, although expression analysis of ABC transporters can provide a useful pointer to potential substrates, the correlation is not absolute. Thus, it seems that although the expression of SpTUR2 represents one of the downstream elements induced by several factors inhibiting plant growth, its transport function is rather speci®c. It thus represents one of a battery of responses required by the plant for an appropriate response to stress conditions. SpTUR2-mediated sclareol transport and Spirodela polyrrhiza Expression of SpTUR2 confers resistance to sclareol, not only in transgenic Arabidopsis, but it is also correlated with the acquisition of resistance to sclareol in S. polyrrhiza. The question therefore arises whether sclareol represents an endogenous substrate for SpTUR2 in Spirodela. No complete taxonomic analysis of sclareol accumulation has been made, but it has been found to ã Blackwell Science Ltd, The Plant Journal, (2002), 30, 649±662 SpTUR2 confers resistance to sclareol accumulate to high levels in various species of the Solanaceae, Lamiaceae, Asteraceae, Verbenaceae, Euphorbiaceae and even gymnosperms and liverworts, suggesting a broad taxonomic occurrence (Dev and Misra, 1985; Seigler, 1998). Our data provide the ®rst identi®cation of this diterpene in S. polyrrhiza (a member of the 657 Lemnaceae) and indicate that sclareol could indeed function as an endogenous substrate for the SpTUR2 transporter. In such a scenario, elevated levels of SpTUR2 under conditions of environmental stress would provide the plant with an increased potential to excrete sclareol (either synthesised within the plant or entering the plant from the growth medium) and thus provide the plant with a competitive advantage. Our data thus provide both an indication of the endogenous substrate of an ABC transporter and the potential biological signi®cance of the transporter activity. Experimental procedures Plant growth conditions Spirodela polyrrhiza L. was grown aseptically on 100 ml of halfstrength Hutner's medium in 250-ml Erlenmeyer ¯asks as described previously (Smart and Fleming, 1996). Arabidopsis thaliana Col-2 plants were either grown in soil under long day conditions of 16 h light at 21°C and 8 h darkness at 17°C or aseptically on half-strength Murashige and Skoog (MS) medium (Sigma, Buchs, Switzerland), 1% sucrose, 0.8% agar under the same light conditions. For analysis of seedling root growth plates were incubated vertically. For liquid culture, Arabidopsis seeds were germinated in half-strength MS medium, 1% sucrose in Erlenmeyer ¯asks and incubated under the same temperature and lighting conditions with rotation at 70 r.p.m. All Arabidopsis seeds were treated at 4°C for 2 days before transfer to germination conditions. Chemicals (obtained from Aldrich, Fluka or Sigma, Buchs, Switzerland) were dissolved either in water, methanol (ABA, atrazine, TIBA), ethanol (EBR, GA4, IAA, NAA, NPA, oligomycin, rhodamine 6G) or DMSO (¯uconazole, sclareol, sclareolide, taxol), diluted further in growth medium and ®lter-sterilised. They were introduced to the growth media as small volumes of concentrated ®lter-sterilised stock solutions in the appropriate growth medium. Control medium was treated with an equal volume of fresh medium or solvent. Figure 5. SpTUR2 confers resistance to sclareol. (a±f) Seeds of three independent lines of Arabidopsis expressing SpTUR2 (b), (d) and (f) and their sibling wild-type lines (a), (c) and (e) were sown on plates and allowed to grow vertically for 6 days. The seedlings were then transferred to plates containing 75 mM sclareol for a further 5 days and photographed. (a) Line wt-14 (b) Line TUR2-14 (c) Line wt-17 (d) Line TUR2-17 (e) Line wt-35 (f) Line TUR2-35 (g) Spirodela plants treated with water (control for ABA) for 24 h, followed by DMSO (control for sclareol) for an additional 24 h. (h) Spirodela plants treated with water (control for ABA) for 24 h, followed by 100 mM sclareol for an additional 24 h. (i) Spirodela plants pretreated with 250 nM ABA for 24 h, followed by DMSO (control for sclareol) for an additional 24 h. (j) Spirodela plants treated with 250 nM ABA for 24 h, followed by 100 mM sclareol for an additional 24 h. ã Blackwell Science Ltd, The Plant Journal, (2002), 30, 649±662 658 Sybille van den BruÃle et al. Root elongation test for resistance to putative substrates Co ld Time (h) 0 1 2 4 8 24 sclareol sclareolide d O OH O H OH H The SpTUR2 cDNA lacking its 5¢ UTR was inserted into the expression vector pRT104 (ToÈpfer et al., 1987) that contains the 35S promoter and polyadenylation signal of the cauli¯ower mosaic virus (CaMV). A synthetic double-stranded oligonucleotide with NcoI-KpnI sticky ends (top strand, 5¢±cATGGAGATCGCCGGGTAC; bottom strand, 5¢±CCGGCGATCTC), corresponding to the ®rst 18 nucleotides of the coding region of SpTUR2, was inserted into pRT104. The rest of the SpTUR2 cDNA was inserted as a 4.5-kb KpnI fragment from pCS3.94.13 (Smart and Fleming, 1996). The pRT104-SpTUR2 expression cassette was isolated as a 5.2-kb fragment from a partial digestion with HindIII relative intensity (%) 10 0 30 0 30 Treatment Plasmid construction and Arabidopsis transformation Concentration µM Co ntr ol DM SO 10 c Na Cl Co ntr ol AB A b Cy clo he xim 2,4 ide -D Co ntr ol Sc lar eo l Sc lar eo lid Ke e toc Rh onaz od ole a GA min e 3 6G a Seeds of three independent homozygous lines of Arabidopsis expressing SpTUR2 and their corresponding wild-type segregant lines were sown on plates and allowed to grow vertically until the primary roots were approximately 10 mm long (usually 4±6 days). The seedlings were then transferred either to fresh control plates or to plates containing the putative substrate for a further 2 days and the increase in primary root length was measured. Concentrations of chemicals were chosen to give between 30% and 70% inhibition of root growth. Usually between 30 and 40 seedlings were used per line and genotype and statistical analysis was performed using the Students t-test. Only statistically signi®cant differences below the P < 0.05 level shown by all three transgenic lines were considered biologically signi®cant. a 187 100 201 50 145 173 159 sclareolide Figure 6. Induction of SpTUR2 protein by various stress factors. (a) Western blot analysis of total protein (40 mg) extracted from Spirodela plants either untreated (control) or treated for 24 h with sclareol (100 mM), sclareolide (100 mM), ketoconazole (10 mg ml-1), rhodamine 6G (3 mM), or GA3 (100 mM). The blots were probed with the af®nity-puri®ed SpTUR2speci®c peptide, detected with a second antibody alkaline phosphatase conjugate, and processed with the Bio-Rad Immun-Star chemiluminescent detection system. (b) Western blot analysis of total protein extracted from Spirodela plants either untreated (control) or treated for 24 h with ABA (250 nM), cycloheximide (1 mM), 2,4-D (10 mM), NaCl (100 mM) or for 6 days at 11°C (cold). The gel was loaded with 6 mg tissue FW equivalents. The blots were immunodetected as in (a) but processed with the Bio-Rad ImmunBlot colorimetric detection system. (c) Concentration dependence and time course of SpTUR2 induction by sclareol. Western blot analysis of total protein (25 mg) extracted from Spirodela plants either untreated (control) or treated for 24 h with DMSO (0.2%) as a solvent control, or sclareol and sclareolide at concentrations between 10 and 300 mM. For the time course plants were treated with 100 mM sclareol or sclareolide for the indicated times. The blots were immunodetected as in (a). (d) Chemical structure of the diterpenes sclareol and sclareolide. relative intensity (%) 0 sclareol b 187 100 50 0 125 145 159 150 201 173 175 200 225 mass to charge ratio (m:z) Figure 7. Identi®cation of sclareol in Spirodela polyrrhiza by GC-MS. Sclareol was detected by GC-MS, indicated by the fragment pattern obtained from ms/ms process. Mass spectra represent the relative intensity (%) of fragment ions relative to the base peak of 187 versus the mass to charge ratio (m : z) of the fragment ions. (a) Spirodela extract (150 mg FW tissue equivalents). Relative intensity 100% is equivalent to 388 ion counts. (b) Pure sclareol (10 pmol). Relative intensity 100% is equivalent to 3411 ion counts. ã Blackwell Science Ltd, The Plant Journal, (2002), 30, 649±662 SpTUR2 confers resistance to sclareol and was inserted into the HindIII cloning site of the plant transformation vector pBIN19 (Bevan, 1984) and transformed into Agrobacterium tumefaciens C58 (Holsters et al., 1978). Arabidopsis Col-2 plants were transformed by the vacuum in®ltration method of Bechtold et al. (1993). T1 seeds were sown on 50 mg ml-1 kanamycin to select the transformants, and surviving plantlets were transferred to soil to set seed (T2). The segregation frequency of the T2 generation with regard to kanamycin resistance was determined on selective media, and T3 seeds of lines segregating for kanamycin resistance in a Mendelian ratio of 3 : 1 were sown on soil. The seed (T4) from 16 progeny plants (T3) of a T2 heterozygous parent from each line was tested for segregation on kanamycin to identify plants homozygous for the SpTUR2 transgene, and for three lines sister wild-type plants were isolated as controls for further analysis (Meissner et al., 1999). These plants were selfed to isolate the T5 generation. RNA isolation and Northern blot analysis Cauline leaves of kanamycin-resistant T1 plants were frozen in liquid nitrogen before RNA isolation using the method of Logemann et al. (1987). RNA was fractionated on 1.1% agaroseformaldehyde gels, transferred to Hybond N nylon membranes (Amersham, DuÈbendorf, Switzerland), hybridised to a 32P-radiolabelled 1.4 kb EcoRI fragment of SpTUR2 cDNA and washed to a ®nal stringency of 0.1X SSC, 0.1% SDS for 15 min at 65°C using standard procedures (Sambrook et al., 1989). Generation of SpTUR2-speci®c antiserum The peptide H2N-STSDVFGRSSREEDDC-CONH2 corresponding to amino acids 30±44 of SpTUR2 with a C-terminal cysteine for coupling to keyhole limpet hemocyanin was synthesised. Peptide synthesis, immunisation of rabbits and af®nity puri®cation of antiserum were performed by Eurogentec (Seraing, Belgium). Protein extraction and SDS-PAGE Arabidopsis seedlings and Spirodela plants were homogenised on ice with 2 volumes of 100 mM Tris±Cl, pH 8, 50 mM KCl, 10 mM MgCl2 and 20 mM DTT with a mortar and pestle and centrifuged at 10 000 g for 1 min to remove debris. The protein concentration was determined using the Bio-Rad (Reinach, Switzerland) protein assay and aliquots were denatured by the addition of an equal volume of 2X sample buffer (125 mM Tris±Cl pH 6.8, 20% glycerol, 4% SDS, 200 mM DTT, 0.05% bromophenol blue), and incubation at 60°C for 15 min. In some cases Spirodela plants were directly homogenised with four FW equivalent volumes of sample buffer to a ®nal sample buffer concentration of 1X, and the protein extract was denatured at 95°C for 5 min and clari®ed by centrifugation at 10 000 g for 1 min. Proteins were separated on 7.5% SDS-PAGE gels, and blotted onto nitrocellulose ®lters (BioRad, 0.45 mm pore size). For immunodetection of SpTUR2, the blots were blocked and incubated with a 1 : 500 dilution of the af®nity-puri®ed SpTUR2 speci®c antiserum. Blots were washed, incubated with a 1 : 3000 dilution of goat antirabbit alkaline phosphatase-conjugated secondary antibody, and developed using either the Bio-Rad colorimetric Immun-Blot or the chemiluminescent Immun-Star immunodetection system according to the manufacturer's instructions. ã Blackwell Science Ltd, The Plant Journal, (2002), 30, 649±662 659 Density gradient fractionation of membrane vesicles The method described by Sidler et al. (1998) was used with the following modi®cations. Rosette leaves from 30-day-old-soilgrown Arabidopsis plants and Spirodela plants 7 days after subculture (5 g) were homogenised in 3 volumes of 50 mM Hepes-KOH pH 7.5, 5 mM EDTA, 2 mM DTT, 250 mM sucrose and 1X Complete Protease inhibitor Cocktail (Roche, Rotkreuz, Switzerland) using a mortar and pestle. The homogenate was centrifuged at 7000 g for 15 min to remove debris and the microsomal membrane fraction was pelleted from the supernatant by ultracentrifugation at 100 000 g for 30 min. The pellet was resuspended in 1 ml of gradient buffer (10 mM Tris-MES pH 7, 1 mM DTT, 250 mM sucrose and 1X Complete Protease inhibitor Cocktail) and centrifuged at 30 000 r.p.m. overnight on a 15±45% continuous sucrose gradient in the same buffer using a Beckman SW41Ti rotor. Gradient fractions (0.5 ml) were collected, the sucrose concentration of samples determined with a refractometer, and 20 ml aliquots from each fraction were denatured by addition of an equal volume of 2X sample buffer and incubation at 60°C for 15 min. SDS-PAGE, blotting and immunodetection of SpTUR2 was performed as described above. The antibodies against marker proteins were gifts of A. R. SchaÈffner, GSF Research Centre, MuÈnich, Germany (chicken anti-PIP 1 : 1000; Kammerloher et al., 1994), M. Chrispeels, University of California, San Diego (rabbit anti-BiP 1 : 1000; HoÈfte and Chrispeels, 1992), M. Maeshima, Nagoya University, Japan (rabbit anti-VM23 1 : 1000; Maeshima, 1992), and R. Ratajczak, University of Technology Darmstadt, Germany (rabbit anti-V-ATPase 1 : 1000; Fischer-Schliebs et al., 1997). SDS-PAGE gels of 15% were used to detect PIP and VM23, 10.5% gels were used for V-ATPase, and 7.5% gels were used for BiP. The antisera were used at the dilutions indicated above and detection was achieved with a 1 : 3000 dilution of the corresponding alkaline phosphataseconjugated secondary antibody (antirabbit, Bio-Rad; antichicken, Promega (Wallisellen, Switzerland). Plasma membrane puri®cation Arabidopsis seedlings expressing SpTUR2 (T5 homozygous line 11) were grown in liquid culture with 0.5% sucrose for 17 days and 10 g were used to prepare microsomes as described by Nagpal and Quatrano (1999). Plasma membranes were puri®ed from the microsomes by two-phase partitioning according to Kjellbom and Larsson (1984). Fractions were measured for protein concentration and prepared for SDS-PAGE as described above. Subcellular localisation of SpTUR2 Leaves of 3-week-old wild-type plants and plants of Arabidopsis expressing SpTUR2 (T5 homozygous line 11) grown in liquid culture were used to prepare protoplasts. Protoplasts were isolated according to Fitzpatrick and Keegstra (2001) except that digestion was carried out with 2% cellulase `Onozuka' R-10 and 0.04% Macerozyme R-10 for 15 h and the protoplasts were resuspended in 500 mM mannitol, 4 mM MES pH 5.7, 2 mM KCl. Immuno¯uorescence labelling of the protoplasts was carried out as described by A.G. von Arnim (http://www.arabidopsis.org/cshlcourse/7-gene_expression.html). The af®nity-puri®ed SpTUR2 speci®c antiserum was used at a dilution of 1 : 20 and an FITCconjugated goat antirabbit secondary antibody (Pierce, Socochim, Lausanne, Switzerland) was used at a dilution of 1 : 100. 660 Sybille van den BruÃle et al. For the localisation in tissue, hand sections were cut from the leaves of wild-type seedlings and seedlings of Arabidopsis expressing SpTUR2 (T5 homozygous line 11) grown in liquid culture for 3 weeks and from Spirodela plants treated with 250 nM ABA for 24 h. Sections were ®xed and processed for immunocytochemistry as described by Sidler et al. (1998), except that the sections were incubated in a 1 : 500 dilution of the af®nitypuri®ed SpTUR2 speci®c antiserum and a 1 : 200 dilution of a DTAF-conjugated goat antirabbit secondary antibody (Jackson Immuno Research Laboratories, West Grove, PA, USA). The sections were examined with a confocal laser scanning microscope (Leica, Heidelberg, Germany) with excitation by an argon 488 nm laser and detection via a 505±530 nm barrier ®lter for FITC/DTAF ¯uorescence and a 660±690 nm ®lter for chlorophyll auto¯uorescence. Extraction of plant tissue for GC±MS analysis Spirodela plants (3 g) were treated with 250 nM ABA for 24 h and homogenised in 80% methanol. The extract was clari®ed by centrifugation, dried under vacuum, resuspended in 2 ml 50% (v/ v) methanol and sonicated. The extract was recentrifuged and applied to a 1-g column of Bondesil C18 (Varian, Darmstadt), washed with 0.5 ml of 50% methanol and eluted with 5 ml of diethyl ether. The eluate was dried under nitrogen, redissolved in 0.5 ml of chloroform: methanol (95 : 5, v/v), applied to a 0.5-g silica column (IST, Hengoed, UK), and eluted in 0.5 ml steps using the same solvent. The second eluate was dried and treated for a few seconds with tri¯uoroacetic acid anhydride. After the reactant was removed under nitrogen the sample was dissolved in 20 ml of chloroform. An aliquot of 1 ml was subjected to GC-MS. Spectra were recorded on a Varian Saturn 2000 ion-trap mass spectrometer operated in EI-MS/MS mode with the following settings: GC, splitless injection of 1 ml (injector temp. 260°C) using a ZB-50 fused silica capillary column (30 m, 0.25 mm i. d., 0.25 mm ®lm thickness; Phenomenex (Aschaffenburg, Germany) with 1 ml min±1 He carrier gas ¯ow; chromatographic conditions: 1 min 50°C, linear ramp at 20°C min±1 to 250°C; transfer line 260°C; mass spectrometric conditions: 2 scans (50±300 amu) s±1, multiplier offset 200 V, resonant waveform type, excitation amplitude 0.8 V. Sclareol was detected by storing the ion m/z 257 (Rt = 11.69 min) and observing the fragment ions m/z 187 (100% relative intensity), 201, 145, 159 and 173. Acknowledgements We thank Drs Maarten J. Chrispeels, Anton R. SchaÈffner, Masayoshi Maeshima and Rafael Ratajczak for providing antibodies to BiP, PIP, VM23, and V-ATPase, respectively. Drs Michel Ghislain, Karl Kuchler, Reinhard ToÈpfer and Philip Rea kindly provided us with yeast deletion mutants and expression vectors and we are especially grateful to Dr Scott Moye-Rowley and Sara Max who performed some of the unpublished yeast complementation studies. We are indebted to Prof Elmar Weiler of the Ruhr University at Bochum for help with the GC-MS analysis, Dr Robert Dudler of the University of ZuÈrich for advice on immunolocalisation methods, and Dr Roger Kuhn for help on the use of the confocal laser scanning microscope. We thank Prof N. Amrhein for his support and encouragement. This work was supported by a grant (31±54303.98) from the Swiss National Science Foundation to C. S. References Bailey, J.A., Garter, G.A., Burden, R.S. and Wain, R.L. (1975) Control of rust diseases by diterpenes from Nicotiana glutinosa. Nature, 255, 328±329. Balzi, E., Wang, M., Leterme, S., Van Dyck, L. and Goffeau, A. 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