Download The ABC transporter SpTUR2 confers resistance to the antifungal

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
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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.
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