Download (6R)-hydroxy-BFA in tobacco and Arabidopsis

Journal of Experimental Botany, Vol. 62, No. 8, pp. 2949–2957, 2011
doi:10.1093/jxb/err007 Advance Access publication 28 February, 2011
This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
RESEARCH PAPER
Differential effects of the brefeldin A analogue
(6R)-hydroxy-BFA in tobacco and Arabidopsis
Markus Langhans1, Sebastian Förster2, Günter Helmchen2 and David G. Robinson1,*
1
2
Department of Cell Biology, Heidelberg Institute for Plant Sciences, University of Heidelberg, D-69120 Heidelberg, Germany
Institute for Organic Chemistry, University of Heidelberg, D-69120 Heidelberg, Germany
* To whom correspondence should be addressed. E-mail: [email protected]
Abstract
The effects of two brefeldin A (BFA) analogues (BFA lactam; 6(R)-hydroxy-BFA) on plant cells were tested. Although
these two compounds elicited BFA-like effects in mammalian cells, the lactam analogue failed to elicit a response in
plant cells. By contrast, while the 6(R)-hydroxy-BFA analogue gave rise to a classic BFA response in tobacco
mesophyll protoplasts and true leaves of Arabidopsis (redistribution of Golgi enzymes into the ER), it failed to cause
the formation of BFA-compartments in Arabidopsis root cells and cotyledonary leaves. Even when the GNL1-LM
mutant of Arabidopsis, which has a cis-Golgi located BFA-sensitive ARF-GEF, was used, the 6(R)-hydroxy analogue
failed to elicit a response at conventional BFA concentrations. Only at concentrations of over 200 mM did
6(R)-hydroxy-BFA elicit a BFA-like effect. These differences are interpreted in terms of the different properties of the
respective TGN- (Arabidopsis roots) and cis-Golgi- (tobacco mesophyll) localized BFA-sensitive ARF-GEFs.
Key words: 6(R)-hydroxy-BFA, ARF-GEFs, Arabidopsis, BFA, tobacco mesophyll.
Introduction
One of the most useful tools to study vesicle-mediated
trafficking in eukaryotic cells is the fungal macrocyclic
lactone brefeldin A (Klausner et al., 1992; Gaynor et al.,
1998; Nebenführ et al., 2002). It causes the rapid release of
vesicle coat proteins such as coatomer and clathrin triskelions and adaptors into the cytosol, and prevents further
vesicle formation (Donaldson et al., 1990; Robinson and
Kreis, 1992; Ritzenthaler et al., 2002). As a consequence,
the integrity of several subcellular compartments is severely
perturbed. Early observations on mammalian cells pointed
to two intracellular sites of BFA action: one situated in the
early Golgi leading to a redistribution of Golgi membranes
and luminal contents into the ER (Lippincott-Schwartz
et al., 1989; Sciaky et al., 1997), the other lying in the transGolgi network (TGN) leading, together with endosomal
membranes, to the formation of a tubular-vesicular aggregate called the ‘BFA-compartment’ (Chege and Pfeffer,
1990; Lippicott-Schwartz et al., 1991; Wood et al., 1991).
Despite these heavily publicized effects of BFA, other
BFA-induced perturbations, for example, in membrane
lipids (Mérigout et al., 2002), cannot be ruled out.
A BFA-induced resorption of Golgi cisternae into the ER
was first recorded some 12 years ago for tobacco leaf
epidermal cells (Boevink et al., 1998), and later confirmed in
suspension-cultured tobacco BY-2 cells (Ritzenthaler et al.,
2002). On the other hand, the induction of a BFAcompartment in plants was most prominently observed in
root cells of maize (Henderson et al., 1994; Baluska et al.,
2002) and Arabidopsis thaliana (Geldner et al., 2003; Grebe
et al., 2003). In both of these cases, and in contrast to the
situation with tobacco, a loss of Golgi cisternae after BFA
treatment was not observed. It is now generally thought
that the apparent discrepancy in BFA action on plant cells
is a consequence of the subcellular location of ARF-GEFs,
which can be either BFA-resistant or BFA-sensitive
(Richter et al., 2007; Teh and Moore, 2007). Since both
types of ARF-GEFs are equally capable of nucleotide
exchange, there appears to be no evolutionary advantage in
Abbreviations: ARF-GEFs, guanine-nucleotide exchange factors for ADP-ribosylation factor GTPases; CHX, cycloheximide; GNL1, GNOM-like-1; Man1,
a-mannosidase 1; ST, sialyl transferase; SYP61, syntaxin of plants 61; VHA-a1, the a1 subunit of the vacuolar H+-ATPase.
ª 2011 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/bync/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
2950 | Langhans et al.
expressing either one or the other form (Anders and
Jürgens, 2008; Richter et al., 2009), and indeed Golgi stack
BFA resistance or sensitivity may vary from tissue to tissue
within the same plant (Robinson et al., 2008a).
The Arabidopsis genome encodes 9–12 ARF-GTPases
and eight ARF-GEFs (Jürgens and Geldner, 2002). Unlike
other eukaryotes, ARF-GEFs in plants are all large
(170–200 kDa), and fall into two subfamilies: the GGG
class with GNOM, GNL1, GNL2, and the BIG class
with five representatives. Interestingly, whereas all large
ARF-GEFs in yeast and mammals are BFA-sensitive, in
Arabidopsis two (GNL1 and BIG3) are BFA-resistant
(Anders and Jürgens, 2008). Resistance to BFA is conferred
by specific residues in a 40 amino acid stretch within
the catalytic SEC7 domain of these ARF-GEFs (Cherfils
and Melancon, 2005; Anders and Jürgens, 2008). Thus,
L696 confers BFA-resistance to GNL1, and when
mutated to M causes GNL1 to become BFA-sensitive
(Richter et al., 2007). Conversely, the BFA-sensitive
GNOM can be made resistant in the M696L mutant
(Geldner et al., 2003).
Of the eight ARF-GEFs in Arabidopsis, only GNOM and
GNL1 (GNOM-like-1) have been localized. GNOM was
discovered as an essential factor mediating the recycling of
the auxin efflux carrier PIN1 from an otherwise undefined
endosomal compartment to the plasma membrane in root
cells of Arabidopsis (Geldner et al., 2003; Kleine-Vehn et al.,
2008). The recycling compartment is presumably an early
endosome (EE), and as such may be a domain of the TGN
which, in plants, assumes the function of an EE (Richter
et al., 2007; Robinson et al., 2008b). On the other hand,
GNL1 localizes to the Golgi stack (Richter et al., 2007; Teh
and Moore, 2007), and when mutated to the BFA-sensitive
L696M form, causes Golgi resorption into the ER in
Arabidopsis root cells upon exposure to BFA (Teh and
Moore, 2007).
In addition to its effects on protein trafficking in the
secretory and endocytic pathways, BFA also shows strong
Fig. 1. Effects of BFA on TGN markers in Arabidopsis roots and tobacco mesophyll protoplasts. An Arabidopsis line stably expressing
the TGN marker SYP61-CFP (a, b) and tobacco mesophyll protoplasts transiently expressing SYP61-YFP and the cis-Golgi marker
Man1-RFP (c–h; e and h are merge images) were used. (i–j) Measurements of the sizes of TGN punctae in control protoplasts
(i) and protoplasts after BFA treatment (j). Magnification: bars¼10 lm (a, b), 5 lm (c–h).
BFA analogues in tobacco and Arabidopsis | 2951
Fig. 2. Golgi stack and TGN located ARF-GEFs in Arabidopsis and tobacco. Comparison of sensitivities towards BFA and
(6R)-hydroxy-BFA.
cytostatic activity against many cancer cell lines (Betina
et al., 1962). However, because of its poor availability and
poor pharmacological properties, BFA has not established
itself as an anti-cancer drug. Thus, several analogues
have been synthesized in order to improve solubility
(Anadu et al., 2006; Förster et al., 2011). One of these:
(6R)-hydroxy-BFA, which elicits BFA-like effects in mammalian cells (Förster et al., 2011), is shown here to generate
similar responses to BFA in tobacco protoplasts. However,
this compound was found to be incapable of causing
the formation of a BFA-compartment in Arabidopsis root
cells. Even with the GNL1 LM mutant the (6R)-hydroxy
analogue is much less effective than BFA in eliciting
a response.
Materials and methods
Stable transformed plant material
Plants of Arabidopsis thaliana stably transformed with
VHA-a1-GFP (Dettmer et al., 2006), VHA-a1-RFP/N-ST-YFP
(Dettmer et al., 2006), GNL1-VHA-a1-GFP (Richter et al., 2007),
GNL1(LM)VHA-a1-GFP, GNL1(LM)-N-ST-YFP (Richter et al.,
2007) were grown from surface-sterilized seeds in half-strength
Murashige and Skoog medium (Murashige and Skoog, 1962)
2952 | Langhans et al.
lM, 180 lM, and 360 lM) or BFA lactam or (6R)-hydroxy-BFA
(36 lM, 90 lM, 180 lM, and 360 lM). BFA and BFA analogues
were dissolved in a stock solution in DMSO. In these experiments,
plant material was pre-treated with BFA or BFA-analogues
(36 lM, 90 lM, 180 lM, and 360 lM) for 30 min before observing
in the CLSM. In special cases, plant material was also treated 1 h
with cycloheximide (70 lM) for 30 min before treatment with BFA
(36 lM or 90 lM).
Details concerning the synthesis and structure of the BFA
analogues used in this paper are described in Förster et al. (2011).
Confocal microscopy
80 ll protoplast solution of N. tabacum was pipetted in an area
(10315 mm) bordered with a frame of 100 lm thick plastic
isolating tape on a slide to protect the protoplasts from external
pressure. The area was covered with a cover slip (24332 mm). In
addition, A. thaliana roots and leaves (cotyledons and true leaves)
were transferred to slides. Cells or plant material were observed
under a Zeiss Axiovert LSM510 Meta microscope using a CApochromat 633/1.2 W corr water immersion objective for
protoplasts and a Plan-Neofluoar 253/0.8 Imm corr DIC for
Arabidopsis roots and leaves. Special settings were designed
for observing single or double expression with different XFPconstructs. Fluorescence was detected by the Metadetector using
main beam splitters HFT 488/543 and HFT 458/514. The following
fluorophores (excited and emitted by frame switching in the singleor multi-tracking mode) were used: GFP (488 nm/496–518 nm),
YFP (514 nm/529–550 nm), and RFP (543 nm/593–625 nm).
Pinholes were adjusted to 1 Airy Unit for each wavelength. Postacquisition image processing was performed using the Zeiss LSM
510 image Browser (4.2.0.121) and CorelDrawX4 (14.0.0.567).
Fig. 3. Effects of BFA and BFA-analogues on Arabidopsis and
tobacco. Arabidopsis root cells stably expressing the fluorescent
TGN marker VHA-a1-GFP, and tobacco mesophyll protoplasts
transiently expressing the cis-Golgi marker Man1-RFP were tested
with BFA, (6R)-hydroxy-BFA, and BFA-lactam at 90 lM for 30 min
before observing in the CLSM. Magnification: bars¼10 lm (a–d),
5 lm (e–h).
with 1% (w:v) sucrose in a controlled climate room at 22 C with
a 16 h day length.
Transient expression in protoplasts
Plants of Nicotiana tabacum cv. Petit Havana were grown from
surface-sterilized seeds in Murashige and Skoog medium with 2%
sucrose in a controlled room at 24 C with a 16 h day length at
a light irradiance of 200 mE m2 s1. Preparation of tobacco leaf
protoplasts was done exactly as described in Foresti et al. (2006).
A total volume of 500 ll of the obtained protoplasts mix was
pipetted into a disposable 1 ml plastic cuvette and mixed with an
appropriate amount of plasmid DNA (Man1-RFP, YFP-Syp61;
Bubeck et al., 2008; Langhans et al., 2008) dissolved in 100 ll of
electroporation buffer. The protoplasts were electroporated with
stainless steel electrodes at a distance of 3.5 mm, using a complete
exponential discharge of a 1000 lF capacitor charged at 160
V. After 30 min of absolute rest, electroporated protoplasts were
removed from the cuvettes and transferred to 5 cm Petri dishes
with 2 ml of TEX buffer. Protoplasts were then incubated for 24 h
at 25 C in a dark chamber.
Treatments with BFA and BFA analogues
Experiments were performed with 0 lM BFA or BFA analogues,
as a control or with different concentrations of BFA (36 lM, 90
Results
BFA compartments are produced in both tobacco and
Arabidopsis, but of different sizes
Whereas, in mammalian cells, the TGN and EE are
separate organelles, in higher plants the TGN also functions
as an EE (Dettmer et al., 2006; Lam et al., 2009; Otegui and
Spitzer, 2008). In addition, the TGN is not permanently
fixed at the trans pole of the Golgi stack, but can detach
and move separately from the stack (Viotti et al., 2010). The
application of BFA emphasizes this subdivision of the Golgi
apparatus (‘Golgi split’, Nebenführ et al., 2002), since BFAsensitive ARF-GEFs reside either at the TGN or, additionally, in the cisternae of the Golgi stack. In the former case,
as in Arabidopsis root cells and in epidermal cells of the
cotyledons, the Golgi stack appears to be unaffected and
the release of the TGN continues. Since BFA prevents
vesiculation (presumably of clathrin-coated vesicles) at the
TGN, the released malfunctioning TGNs accumulate to
form a ‘BFA-compartment’. This can easily be demonstrated with any bona fide fluorescent TGN marker, for
example, SYP61 (Fig. 1a, b). However, when a BFAsensitive ARF-GEF is also present in the Golgi stack, as in
tobacco, the cisternae of the stack are resorbed into the ER
(Fig. 1, compare d and g). Accordingly, there will be no
ongoing replenishment of TGN, and, as a result, large
BFA-compartments are not formed. Instead, the TGN
spots only enlarge roughly 2–3-fold (Fig. 1, compare c and
f, and i and j). This observation probably reflects the
BFA analogues in tobacco and Arabidopsis | 2953
Fig. 4. Comparison of the effects of different concentrations of BFA and (6R)-hydroxy-BFA on Arabidopsis root cells expressing
VHA-a1-GFP and tobacco protoplasts expressing Man1-RFP. Standard treatment period of 30 min. Magnification: bars¼10 lm
(a–i), 5 lm (j–r).
temporary continuation of cisternal maturation and TGN
release until the drug has achieved its inhibitory effect on
the rest of the Golgi stack. The location of BFA-sensitive
ARF-GEFs in the Golgi apparatus of different higher
plants together with the two basic morphological responses
after BFA addition are summarized in Fig. 2.
BFA lactam does not elicit a BFA-like effect on
Arabidopsis roots or tobacco protoplasts
Although reported to have a BFA-like effect on mammalian
cells (Förster et al., 2011), the BFA lactam analogue was
unable to induce BFA compartments in Arabidopsis root
cells (Fig. 3, compare a and b with c and d). In addition, it
did not cause a redistribution of the fluorescent cis-Golgi
marker Man1-RFP into the ER in tobacco mesophyll
protoplasts (Fig. 3, compare e with h). Thus the BFA-
lactam analogue, at the concentrations tested here, seems to
be without effect on plant cells.
(6R)-hydroxy-BFA elicits a BFA-like effect on tobacco
protoplasts but not on Arabidopsis
(6R)-hydroxy-BFA, which shows BFA-like effects on mammalian cells (Förster et al., 2011), also caused a typical
BFA-phenotype when applied to tobacco mesophyll protoplasts (Fig. 3, compare f and g). However, it was less
effective than BFA at lower concentrations (below 50 lM),
with punctate signals for the Golgi marker Man1-RFP still
being visible in addition to an ER-labelling (Fig. 4, compare
k and o with l and p). By contrast, as shown in Fig. 3
(compare b and c), (6R)-hydroxy-BFA failed to induce
BFA compartments in Arabidopsis roots. This was not
a question of concentration. Even when presented at the
2954 | Langhans et al.
extremely high concentration of 180 lM this BFA analogue
failed to generate the production of a BFA compartment
(Fig. 4, compare b–e with f–i). This was also not achieved
with prolonged (up to 4 h) treatment periods (data
not shown).
To determine whether the failure to elicit a BFA-effect
was not restricted to the roots of Arabidopsis, the
(6R)-hydroxy-BFA analogue was tested on epidermal cells
in cotyledons and true leaves of Arabidopsis plants stably
expressing the TGN marker VHA-a1-RFP and the transGolgi marker ST-YFP (Dettmer et al., 2006). Surpisingly, in
response to BFA, the cotyledon epidermal cells behaved
like root cells producing typical BFA-compartments
(Fig. 5a, b). By contrast, and as previously reported by
Robinson et al. (2008a), BFA caused a redistribution of
the Golgi marker ST-YFP into the ER in the epidermal
cells of true leaves (Fig. 5d, e). However, in neither type
of epidermal cell did the (6R)-hydroxy-BFA analogue
elicit any change in the TGN/Golgi marker proteins
(Fig. 5c, f).
(6R)-hydroxy-BFA fails to elicit a BFA effect in the
Arabidopsis GNL1-LM- mutant
Fig. 5. Comparison of the effects of BFA and (6R)-hydroxy-BFA
on cotyledon leaves and real leaves of Arabidopsis plants stably
expressing VHA-a1-RFP and ST-YFP. Squares of leaf tissue were
excised and incubated in aqueous solutions containing 90 lM
drug for 30 min. The inset in (b) is a high magnification view of
a single BFA compartment with a red core derived from the TGN
marker surrounded by yellow Golgi stacks. The arrows in (e) point
to the redistribution of the Golgi marker ST-YFP into the nuclear
envelope (¼ER). Magnification: bars¼10 lm.
In wild-type Arabidopsis roots, BFA induces the formation
of the BFA compartment, but does not affect Golgi stacks
which lie outside of this structure (Fig. 6a, b). However, in
the GNL1-LM mutant of Arabidopsis the Golgi stacklocated ARF-GEF also becomes BFA-sensitive, leading to
a redistribution of the trans-Golgi marker ST-YFP into the
ER upon BFA treatment (Fig. 6i, j). In addition, in the
GNL1-LM mutant plants large BFA compartments are not
formed. This can be clearly seen in mutant lines expressing
the TGN marker VHA-a1-GFP, the signal for which
enlarges some 2-fold over a 20 min treatment period (see
Fig. 6. Comparison of BFA (90 lM) effects in root cells of wt and GNL1-LM mutant Arabidopsis lines. (a–d) Formation of the BFA
compartment (b) is prevented by cycloheximide (c). (e–h) BFA causes the TGN marker VHA-a1-GFP to be detected in the ER after 30
min (f). This is prevented by cycloheximide (g). BFA causes the trans-Golgi marker ST-YFP to redistribute into the ER (j). This is only
partially prevented by cycloheximide (k). Control treatments with cycloheximide alone are presented in (c), (g), and (k). Magnification:
bars¼10 lm.
BFA analogues in tobacco and Arabidopsis | 2955
rather the new synthesis of this protein which cannot exit
the ER. This was established in control experiments where
the roots were pretreated with the protein synthesis inhibitor cycloheximide This inhibitor prevented the appearance of an ER signal in the GNL1-LM line expressing
VHA-a1-GFP (Fig. 6g), and only partially prevented
the BFA-induced redistribution of ST-YFP into the ER
(Fig. 6k).
The different effects of BFA on the trans-Golgi and
TGN-based fluorescent markers in the GNL1-LM
mutant prompted us to examine the effect of the (6R)hydroxy-BFA analogue on this mutant line. As shown in
Fig. 7, the (6R)-hydroxy-BFA analogue did not elicit any
changes in the punctate appearance of the TGN-marker
VHA-a1-GFP (Fig. 7a–d). In contrast, the analogue did
cause the redistribution of the trans-Golgi marker ST-YFP
into the ER, but only at concentrations much higher
than 100 lM (Fig. 7f–i). The inclusion of cycloheximide
caused no change in these responses (Fig. 7e, j). The effects
of (6R)-hydroxy-BFA on tobacco and Arabidopsis are
summarized in Fig. 2.
Discussion
BFA and cisternal maturation in the Golgi stack
BFA-induced Golgi stack disassembly probably provides
the best evidence for cisternal maturation in a cis/trans
direction. Working with tobacco BY-2 cells expressing the
cis- Golgi marker Man1-GFP, Ritzenthaler et al. (2002)
noticed that this signal continued to remain in the stack
after BFA had caused the cisternae to be reduced in number
to 2 or 3. Since the remaining cisternae had a trans-like
morphology, it was considered that the cis-cisternae were
being gradually transformed as they moved upwards
through the stack. This observation has recently been
confirmed by Schoberer et al. (2010) who, in addition to
providing convincing evidence for a stepwise trans-directed
cisternal transport in tobacco epidermal cells, also showed
that Golgi stack disassembly begins with the loss of translocated proteins. Most significantly, and as demonstrated in
our experiments with the GNL1 mutant, once a Golgi
cisterna has established itself as a TGN its proteins cannot
be redistributed into the ER after BFA treatment.
BFA analogues and modes of BFA action
Fig. 7. Examination of the effects of (6R)-hydroxy-BFA on root
cells of GNL1-LM Arabidopsis lines expressing either VHA-a1-GFP
(a–e) or ST-YFP (a–i) or marker proteins. Treatment time¼30 min.
Magnification: bars¼10 lm.
Supplementary Fig. S1 at JXB online). Interestingly, an ER
distribution for the VHA-a1-GFP does appear after 30 min
of BFA treatment (Fig. 6f). However, this signal does not
represent the relocation of the TGN-based protein, but
It has long been held that BFA has two different modes of
action in mammalian cells: one causing an inhibition of
vesicle-mediated protein trafficking, and the other leading
to apoptosis (Nojiri et al., 1995). However, it has been
shown that stressing the ER through prolonged BFA
treatment can also lead to cell death via caspase activation
(Rao et al., 2001; Murakami et al., 2007). Nevertheless,
there are some BFA analogues which do not induce
apoptosis but do affect protein trafficking and Golgi
morphology (Anadu et al., 2006). There are also other
BFA analogues which effect neither secretion or Golgi
2956 | Langhans et al.
structure (Brüning et al., 1992; Klausner et al., 1992;
Förster et al., 2011). Thus, a BFA analogue which, in some
way, leads to a modified BFA response or is active in
mammals but not plants (or vice versa) is obviously a useful
tool to have in terms of understanding BFA/ARF-GEF
interactions. One such example is a BFA lactam analogue,
which is active in mammalian cells (Förster et al., 2011), but
is without effect on plants (this study). Another example is
the naturally occurring 7-dehydro-brefeldin A (7-oxo-BFA),
which has been shown to be much more potent than BFA in
perturbing Golgi-based secretion in suspension-cultured
sycamore cells (Driouich et al., 1997).
There is no obvious reason why the BFA-lactam analogue should elicit BFA-like effects in mammalian cells but
not in plants. According to molecular modelling, the lactam
analogue exists in two conformations, one of which is
very similar to BFA, the other less so. In solution, sufficient
amounts of the former should be present to allow for
the generation of BFA-like effects (Förster et al., 2011).
Structural studies on the (6R)-hydroxy BFA analogue
also indicate that it has almost or basically the same
conformation as BFA and fits equally well into the SEC7
BFA-binding domain (Förster et al., 2011). In principle, it
should therefore work equally well as BFA on all plants,
but as we have seen it has a differential effect. In wild-type
Arabidopsis roots, whose cells have a BFA-sensitive ARFGEF (GNOM) only at the TGN, it is without effect. Even
in the GNL1-L696M mutant, (6R)-hydroxy-BFA was unable to elicit this effect, unless presented at concentrations
5–10 times higher than is normally required to elicit a BFA
effect. This suggests that the presence of the amino acid
L696 (in Arabidopsis GNOM) is not the only factor in an
ARF-GEF which determines its BFA resistance. This is
supported by the fact that tobacco, which has a naturally
occurring BFA-sensitive GNL1 homologue (with M683 in
the SEC7 domain; Wang et al., 2008), first responds to
(6R)-hydroxy BFA at three times higher concentrations
than BFA. Obviously, an explanation for the lack of effect
of the BFA-lactam analogue on plants as a whole, and the
differential sensitivities of GNOM and GNL1 towards the
(6R)-hydroxy BFA analogue, will only be possible after
a careful comparison of the SEC7 domains in the various
organisms has been carried out.
Supplementary data
Supplementary data can be found at JXB online.
Supplementary Fig. S1. Time-course of BFA action on
root cells of a GNL1-LM Arabidopsis line expressing
VHA-a1-GFP.
Acknowledgements
We greatly appreciate the financial support of the German
Research Council (DFG grant RO 440/ 11-4).
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