Download Expression of GFP-fusions in Arabidopsis companion cells reveals

The Plant Journal (2005) 41, 319–331
doi: 10.1111/j.1365-313X.2004.02298.x
Expression of GFP-fusions in Arabidopsis companion cells
reveals non-specific protein trafficking into sieve elements
and identifies a novel post-phloem domain in roots
Ruth Stadler1,†, Kathryn M. Wright2,†, Christian Lauterbach1, Gabi Amon1, Manfred Gahrtz1,‡, Andrea Feuerstein1,
Karl J. Oparka2 and Norbert Sauer1,*
1
Molekulare Pflanzenphysiologie, Universität Erlangen-Nürnberg, Staudtstraße 5, D-91058 Erlangen, Germany, and
2
Cell-cell communication programme, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK
Received 28 May 2004; revised 23 September 2004; accepted 1 November 2004.
*
For correspondence (fax þ49 9131 8528751; e-mail [email protected]).
†
These authors contributed equally to this work.
‡
Present address: Institute of General Botany, Centre for Applied Plant Molecular Biology (AMPII), University of Hamburg, Ohnhorststr. 18, D-22609 Hamburg,
Germany.
Summary
Transgenic Arabidopsis plants were constructed to express a range of GFP-fusion proteins (36–67 kDa) under
the companion cell (CC)-specific AtSUC2 promoter. These plants were used to monitor the trafficking of these
GFP-fusion proteins from the CCs into the sieve elements (SEs) and their subsequent translocation within and
out of the phloem. The results revealed a large size exclusion limit (SEL) (>67 kDa) for the plasmodesmata
connecting SEs and CCs in the loading phloem. Membrane-anchored GFP-fusions and a GFP variant targeted
to the endoplasmic reticulum (ER) remained inside the CCs and were used as ‘zero trafficking’ controls. In
contrast, free GFP and all soluble GFP-fusions, moved from the CCs into the SEs and were subsequently
translocated through the phloem. Phloem unloading and post-phloem transport of these mobile GFP-fusions
were studied in root tips, where post-phloem transport occurred only for the free form of GFP. All of the other
soluble GFP-fusion variants were unloaded and restricted to a narrow zone of cells immediately adjacent to the
mature protophloem. It appears that this domain of cells, which has a peripheral SEL of about 27–36 kDa,
allows protein exchange between protophloem SEs and surrounding cells, but restricts general access of large
proteins into the root tip. The presented data provide additional information on phloem development in
Arabidopsis in relation to the formation of symplasmic domains.
Keywords: AtSUC2, companion cells, GFP, phloem, plasmodesmata, size exclusion limit.
Introduction
The phloem of higher plants facilitates the long-distance
allocation and partitioning not only of organic carbon and
nitrogen compounds, such as sugars, sugar alcohols and
amino acids, but also of macromolecules, including proteins and RNAs (Balachandran et al., 1997; Sjolund, 1997).
In the phloem of angiosperms, the sieve element–companion cell (SE–CC) complex represents a functional unit,
within which the individual cells are descendents of a
common mother cell (Esau, 1969a; Oparka and Turgeon,
1999). After the division of this cell the newly formed
daughter cells undergo different developmental programs,
eventually producing individuals with highly specialized
anatomies and functions. During maturation, SEs lose their
ª 2004 Blackwell Publishing Ltd
nuclei, ribosomes and vacuoles and possess only reduced
numbers or specialized forms of most other cellular
organelles (Behnke, 1989). Consequently, these cells
depend during their entire lifespan on the continuous
supply not only of energy, but also of macromolecules,
such as enzymes, structural proteins and membrane
transporters. All these components seem to be provided
by the closely linked CCs, which have small vacuoles and
are densely packed with mitochondria and ribosomes. SEs
and CCs are intimately linked via specialized plasmodesmata that function as intercellular conduits between these
cell types (Behnke, 1989; van Bel and Kempers, 1996; van
Bel et al., 2002; Oparka and Turgeon, 1999).
319
320 Ruth Stadler et al.
In many plants, phloem loading occurs from the apoplast
via plasma membrane-localized transporters located around
the SE–CC complex (Barth et al., 2003; Williams et al., 2000).
In Arabidopsis the gene for the plasma membrane-localized
AtSUC2 Hþ-sucrose symporter is expressed specifically, and
exclusively in the CCs of the phloem (Stadler and Sauer,
1996; Truernit and Sauer, 1995). Similar expression data
were obtained for genes encoding transporters involved in
the phloem loading in other plants [e.g. Plantago major
(common plantain) sucrose transporter PmSUC2 (Stadler
et al., 1995)] or in the phloem loading of other substrates
[e.g. common plantain sorbitol transporters PmPLT1 and
PmPLT2 (Ramsperger-Gleixner et al., 2004)]. Therefore, the
promoters of these genes represent excellent tools for
analyses of cell-to-cell trafficking between SEs and CCs
(Ayre et al., 2003; Imlau et al., 1999; Oparka et al., 1999).
Studies in Arabidopsis plants expressing GFP under the
control of the CC-specific AtSUC2 promoter revealed, for the
first time, CC–SE trafficking of GFP and its subsequent
unloading in sink tissues (Imlau et al., 1999). This study
showed that plasmodesmata between these two cell types
have a potentially large size exclusion limit (SEL), allowing
the passage of proteins up to 27 kDa, and confirmed earlier
results obtained using microinjection of fluorescent dextrans (Kempers and van Bel, 1997). In addition, Imlau et al.
(1999) showed that GFP was unloaded from terminal SEs,
and that unloaded GFP underwent extensive post-phloem
transport within sink tissues. In contrast, no unloading was
observed in Arabidopsis source tissues. Subsequently,
Oparka et al. (1999) demonstrated that plasmodesmata of
sink-leaf mesophyll cells underwent a downregulation in
their SEL during the sink-to-source transition. These authors
detected a transition from simple (unbranched) plasmodesmata in sink leaf mesophyll cells of tobacco to complex
(branched) plasmodesmata in source leaves, which was
paralleled by a major decrease in the SEL of these plasmodesmata. Since this study, several papers have reported
the movement of free GFP in a range of plant tissues and
organs (Crawford and Zambryski, 2000, 2001; Itaya et al.,
2000).
Recently AtSUC2 promoter::GFP constructs were used for
a functional analyses of different vein classes in developing
leaves of transgenic tobacco plants (Wright et al., 2003). In
these studies the unloading of phloem-mobile GFP into the
sink areas of transition leaves was compared with the
loading of 14C-sucrose into the minor veins of the same
areas. These analyses showed a clear correlation between
the decreasing capacity of veins to unload GFP and their
increasing capacity to load sucrose. In the same study,
transgenic tobacco plants were analyzed that expressed a
GFP variant that was targeted to the endoplasmic reticulum
(ER). This ER-GFP could not traffic into tobacco SEs, was not
phloem-mobile, and the GFP-dependent fluorescence
was detected exclusively in the veins of source leaves. In
sink–source transition leaves, the ER-GFP fluorescence
correlated precisely with the regions exhibiting apoplastic
sucrose loading and lacking GFP-unloading from the
phloem in AtSUC2-promoter::GFP plants (Wright et al.,
2003). These data confirmed that the sites of AtSUC2
promoter-driven GFP expression represent the sites of active
phloem loading into the minor veins. Furthermore, they
showed that the movement of free GFP within the phloem,
and its unloading into sink leaves, is a clear indicator for the
movement and unloading of assimilates.
In the present study, the non-invasive approaches
described above were extended to include seven different
GFP-variants of increasing molecular mass (36–67 kDa).
Transgenic Arabidopsis plants expressing the genes for
five cytosolic [GFP-ubiquitin, apparent molecular weight
(MWapp): 36 kDa; GFP-sporamin, MWapp: 47 kDa; GFP-aequorin, MWapp: 48 kDa; GFP-patatin, MWapp: 67 kDa], two
membrane-anchored (tmGFP9, tmGFP2) and one ER-localized GFP-fusion (ER-GFP) were generated under control of
the AtSUC2-promoter. Plants expressing these constructs
were used to monitor the CC–SE trafficking of the different
fusions, and to study their potential unloading in sink organs
(developing leaves and roots). The data show that under
non-invasive conditions SEs and CCs are connected by
plasmodesmata that exhibit an SEL of up to 67 kDa, allowing
the translocation of all the cytosolic GFP-fusion proteins
produced in CCs. We demonstrate the presence of a
symplasmic domain that encircles the root protophloem,
allowing the escape of free GFP but restricting the widespread post-phloem distribution of macromolecules
>30 kDa. Our results underline the CC-specificity of the
AtSUC2 promoter (Truernit and Sauer, 1995), and provide
new information on phloem function that correlates closely
with the pattern of phloem development in Arabidopsis
roots. Finally, our data underline the potential of the phloem
for non-specific protein trafficking.
Results
Expression of GFP and GFP-fusions in Arabidopsis source
and sink leaves
Plants of Arabidopsis (ecotype C24) were transformed with
the seven GFP fusion constructs (Figure 1), and transgenic
lines were selected for growth in the presence of BASTA.
The soluble proteins used for these GFP-fusions (patatin,
sporamin, aequorin, ubiquitin) were chosen, because they
are not expected to interfere with the cellular metabolism.
Sporamin and patatin, which are normally localized in storage vacuoles, were amplified without their N-terminal signal
sequences responsible for targeting of the proteins to the ER
(Hattori et al., 1989; Mignery et al., 1984).
The soluble fusion proteins have increased molecular masses, and these molecular mass values (kDa) are
ª Blackwell Publishing Ltd, The Plant Journal, (2004), 41, 319–331
Protein trafficking in the phloem 321
Figure 1. Eight different constructs were used for the analyses of size
exclusion limits.
In all constructs expression is driven by the CC-specific AtSUC2-promoter
from Arabidopsis thaliana. In addition to a previously described construct
with free GFP (bottom; 27 kDa; Imlau et al., 1999) four GFP-fusion constructs
(bottom to top) were generated encoding fusions with ubiquitin (Ubi fi
fusion is 36 kDa), sporamin (fi fusion is 47 kDa), aequorin (fi fusion is
48 kDa) or patatin (fi fusion is 67 kDa) to the C-terminus of GFP, one
construct encoding an ER-resident GFP (ER) and two constructs encoding
GFP-variants fused to membrane anchors (tmGFPs for transmembraneGFPs). The membrane-anchored GFP-variants were generated by cloning the
GFP cDNA to the 3¢-end of a truncated AtSTP9 gene (tmGFP9) or to the 3¢-end
of the AtSUC2 gene (tmGFP2). Dashed regions in the constructs for
membrane-bound GFPs represent intron sequences.
generally used to describe the SEL of plasmodesmata.
Obviously, the molecular mass of GFP or of GFP-fusions is
not the ideal unit to measure the SEL. The fusion product of
globular GFP (Stokes radius ¼ 2.82 nm; Terry et al., 1995)
with another globular protein, such as ubiquitin (Stokes
radius about 1.2 nm) or sporamin (Stokes radius about
2.2 nm), will not result in one larger globule, but rather in a
dimer of two globules. Therefore, it is not only the increased
molecular mass, but also the altered shape that influences
the movement of GFP-fusions through plasmodesmata.
However, in the absence of structural information on these
fusion proteins their molecular masses (in kDa) are a crude
but generally accepted unit for describing the SELs of
plasmodesmata.
From each transformation plants from at least 30
independent BASTA-resistant lines were screened in the
T2 generation for GFP fluorescence in their rosette leaves.
Plants transformed with the same construct showed slight
differences with respect to the fluorescence intensities,
but no differences in the expression patterns were detected. Figure 2 (top row) shows the fluorescence in source
leaves from the different transgenic lines in comparison
with a leaf from the previously generated AtSUC2-promoter GFP plants (Imlau et al., 1999). GFP-fluorescence
was detected in all lines analyzed, although at different
intensities. Plants carrying the GFP-ubiquitin, GFP-sporamin and tmGFP9 fusions [AtSTP9 encodes an Arabidopsis monosaccharide transporter (Schneidereit et al., 2003)]
showed similar GFP fluorescence, although the fluorescence in these plants was slightly lower than in plants
with free GFP. Plants of the other lines showed decreasing fluorescence intensities (tmGFP2 > GFP-ER > GFPaequorin > GFP-patatin).
ª Blackwell Publishing Ltd, The Plant Journal, (2004), 41, 319–331
Clear differences were observed in the cell-to-cell movement of the different GFP-fusions in the sink leaves of these
transgenic lines. The previously described influx of source
leaf-synthesized free GFP into Arabidopsis sink leaves
(Figure 2; first leaf of second row) was not detected in sink
leaves from lines expressing constructs for membraneanchored GFPs (tmGFP9 or tmGFP2 lines), which is explained by the source-leaf specific activity of the AtSUC2promoter and which confirms data from AtSUC2-promoter::GUS plants (Truernit and Sauer, 1995). No synthesis of
membrane-bound GFP variants was expected to occur in the
sink leaves of these plants, and the resulting GFP-fusion
protein was not expected to be phloem-mobile. An identical
result was obtained in sink leaves of ER-GFP plants. This
soluble GFP variant made in the source leaves of these
plants was targeted to the ER and could not move from the
CCs into the SEs.
In contrast, GFP fluorescence was detected in the sink
leaves of GFP-ubiquitin and of GFP-sporamin plants
(Figure 2; bottom row) showing that these GFP-fusions were
able to traffic from their site of synthesis (the CCs) into the
SEs. Inside the SEs these GFP-fusions moved with the mass
flow of assimilates and were eventually imported into the
sink leaves. It is not clear if these GFP-fusions, like the free
form of GFP, were unloaded into the surrounding mesophyll
cells, or if post-phloem transport had occurred. Certainly,
the extent of unloading was much lower than for free GFP
(Figure 2, bottom row). Due to the even lower expression of
GFP-aequorin and the GFP-patatin in transgenic plants, no
data are presented relating to these transformants.
Trafficking of GFP and GFP-fusions in Arabidopsis roots
Analyses of sink-specific GFP-fluorescence is easier in Arabidopsis roots than in leaves, which are less accessible to
non-invasive imaging (Oparka et al., 1994). Immunohistochemical studies (Stadler and Sauer, 1996) and GUS-histochemical analyses of AtSUC2-promoter::GUS plants had
previously shown that the AtSUC2-promoter is active in the
root phloem (Truernit and Sauer, 1995). Moreover, analyses
of roots from AtSUC2-promoter::GFP plants had revealed
that GFP synthesized under the control of this promoter is
symplastically released via plasmodesmata from the vascular bundles at the root tips (Imlau et al., 1999), predominantly from the protophloem sieve tubes. It appears that
these protophloem files undertake the bulk of phloem
unloading in the root tip (Oparka et al., 1994; Schulz, 1994;
Zhu et al., 1998).
We investigated, which of the GFP variants could be
unloaded from the protophloem SEs into the sink tissues at
the root tips and subsequently undergo post-phloem transport. All root analyses shown in Figure 3 were performed by
confocal laser scanning microscopy (CLSM). As shown
before by epifluorescence microscopy (Imlau et al., 1999),
322 Ruth Stadler et al.
Figure 2. GFP fluorescence in source and sink leaves from transgenic plants expressing constructs for GFP or GFP-fusion proteins.
Source leaves (top row) and sink leaves (bottom row) from transgenic Arabidopsis plants expressing constructs shown in Figure 1 were photographed under a
stereomicroscope with an excitation wavelength of 460–500 nm. Emitted fluorescence of GFP (green) and chlorophyll (red) was monitored at detection wavelengths
longer than 510 nm. Bars ¼ 2 mm in the top row and 0.1 mm in the bottom row.
free GFP is unloaded at the tip of Arabidopsis roots from the
two protophloem files (Figure 3a). Reproducibly, we observed symplastic unloading of free GFP also along the
transport phloem (Figure 3b), although the extent of this
unloading was significantly lower than in the root tips. This
was unexpected, because in previous papers (Oparka et al.,
1994; Wright and Oparka, 1997) no unloading of the small
fluorescent probe 5(6) carboxyfluorescein (CF) had been
observed from the root transport phloem. The fluorescence
resulting from symplastically unloaded GFP was observed in
the nuclei of all cell layers of the root cortex and in the nuclei
of the root epidermal cell layer. This unloading from the root
transport phloem is also seen in an optical cross section
(Z-axis), where GFP-fluorescence is seen in the two strands
of root vascular bundles, but with decreasing intensities also
in cells of the root cortex and of the root epidermis
(Figure 3c).
No symplastic unloading was seen from the transport
phloem (Figure 3d) or from the more distal unloading
phloem in the roots of GFP-sporamin plants (Figure 3e). In
these plants the fluorescence is confined to the vascular
strands, and nuclei in the cortex or the root epidermis do not
show any GFP-labeling. The identical distribution of fluorescence was seen in GFP-ubiquitin plants (Figure 3f) and in
the other plants expressing constructs for soluble GFPfusions (data not shown). The sloping contact sites between
the labeled cells shown at higher magnification identify
these cells as SEs (Figure 3h; Stadler and Sauer, 1996)
confirming that the GFP-fusions moved from CCs into SEs.
In the GFP-sporamin plants shown in Figure 3(e), fluorescence terminated at the ends of the two mature protophloem
poles, and there was no evidence of post-phloem transport
toward the root tip. However, closer examination of the root
tips from these plants revealed the GFP-fusion protein also
in a domain of cells surrounding the mature protophloem
files (Figure 4a–c). To more clearly delineate this GFPcontaining domain from the conducting protophloem SEs,
we first imaged the root tip for GFP (Figure 4a), and
subsequently for aniline blue staining to reveal the sieve
plates of the protophloem SEs (Figure 4b). Clearly, the
fluorescence in these root tips was confined to the protophloem and to this ‘protophloem domain’ (Figure 4c and
insert). In contrast to plants synthesizing free GFP, no
unloading beyond this domain and no GFP-labeling in the
nuclei of the cortex or the root epidermis was observed
(Figures 3e and 4). An identical distribution of fluorescence
was seen in all other GFP-fusion plants examined (data not
shown).
As expected from the sink-leaf analyses (Figure 2), no
trafficking of GFP out of the CCs was detected in the roots of
ER-GFP plants, where the label was restricted to intracellular
structures of the CCs, most likely the ER, which is suggested
by the ring-like structures labeled inside these cells (possibly
the labeled nuclear envelopes; Figure 3g). Similarly, no
trafficking of GFP fluorescence was seen in tmGFP2 plants
(Figure 3i), where individual CCs but no SEs were labeled as
a consequence of the membrane anchor fused to this GFPvariant.
ª Blackwell Publishing Ltd, The Plant Journal, (2004), 41, 319–331
Protein trafficking in the phloem 323
Figure 3. Analysis of GFP-fluorescence in roots of transgenic Arabidopsis plants.
Green fluorescence resulting from symplastic phloem unloading of GFP in AtSUC2-promoter::GFP plants is seen in all cells of the root tip (a). AtSUC2-promoter::GFP
plants show symplastic unloading of free GFP also into the cortical and rhizodermal cell layers in the more proximal regions of the root; arrows mark some of the
labeled nuclei (b). The cross-section (optical Z-section) through the root section shown in (b) is presented in (c). Unloading of GFP is seen into several cells adjacent
to the vascular strands (arrow with asterisk) and low amounts of GFP can even be detected in epidermal and subepidermal cells (arrows). A similar optical Z-section
section as in (c) but from the root of an AtSUC2-promoter::GFP-sporamin plant shows no unloading of GFP-sporamin and fluorescence is restricted in the two
phloem files (d). Longitudinal section through the root of an AtSUC2-promoter::GFP-sporamin plant (e). As in (d) and in contrast to (a) and (b) no unloading of GFPsporamin is observed in the transport phloem and into the root tip of (e). Longitudinal section through the root of an AtSUC2-promoter::GFP-ubiquitin plant with no
detectable unloading of GFP-ubiquitin from the phloem (f). Labeling of intracellular structures in the root of an AtSUC2-promoter::ER-GFP plant. No fluorescence is
detected outside the phloem (g). Phloem tissue (longitudinal) close to the root tip of an AtSUC2-promoter::GFP-ubiquitin plant is shown in (h). The contact sites
between two of the GFP-labeled cells (arrow) characterize these cells as SEs. The other fluorescent cells are SEs or CCs. In (i) individual CCs are labeled in the
longitudinal section through the root of an AtSUC2-promoter::AtSUC2-GFP plant. All photos were taken under the CLSM and represent Z-stacks. Red fluorescence of
cell walls results from staining with propidium iodide [not in (b), (e) and (h)]. For (b) and (e) several Z-stacks were assembled. Bars ¼ 50 lm for (a), 40 lm for (b) and
(d), 25 lm for (c), (f) and (g), 100 lm for (e), 10 lm for (h) and 20 lm for (i).
GFP translocation correlates with root phloem development
The trafficking behavior of soluble GFP-fusion proteins, and
the lack of trafficking of the two membrane-bound GFPs,
provides an excellent tool for the analysis of functional
ª Blackwell Publishing Ltd, The Plant Journal, (2004), 41, 319–331
phloem development, and of the SELs between the cells
involved. In Figure 5, typical confocal images of the tips of
main roots from all eight transgenic lines expressing free
GFP or GFP-variants under the control of the AtSUC2 promoter are presented. Note that only the free form of GFP
324 Ruth Stadler et al.
Figure 4. Unloading of GFP-sporamin from the protophloem into a specific domain of cells.
A confocal GFP image of the root tip from a GFP-sporamin plant is shown (a). This root was also stained with aniline blue (for callose detection) and an
epifluorescence image of the aniline blue-derived fluorescence is presented in (b). Aniline-stained sieve plates in the single protophloem pole are stained pink
[arrow; false color representation for better differentiation of aniline-blue staining (bluish green) and GFP fluorescence (green) in the merged picture]. For the image
shown in (c) and for the magnification shown in the insert of (c) images (a) and (b) were merged (Adobe Photoshop; Adobe Systems Inc., San Jose, CA, USA). GFPsporamin is unloaded from the very thin protophloem sieve tube (identified by the callose stained sieve plates; arrow) into a discrete domain of surrounding cells.
One of the two differentiating protophloem SEs (marked with an asterisk) is seen in the center of the longitudinal section from an Arabidopsis root tip shown in (d).
Vacuoles (V) and other organelles are still seen in the early SEs of the protophloem at the lower end of the SE-file, but are absent in the mature SEs in the upper end.
In contrast, sieve plates (SP), which are clearly visible in the fully differentiated SEs (upper end of the file), are just being formed in the SEs of the protophloem (lower
end). Bars ¼ 25 lm for (a) to (c), 10 lm for the insert in (c) and 4 lm for (d).
showed extensive post-phloem transport throughout the
root tip. In contrast, the fluorescence of all other phloem
mobile GFP-variants (GFP-ubiquitin, GFP-sporamin, GFPaequorin and GFP-patatin) ended at a similar distance from
the root tip (approximately 250 lm). This distance was
significantly greater in the root tips of the lines expressing
the genes for the two membrane-bound GFPs (STP9-GFP
or AtSUC2-GFP) and the ER-GFP (approximately 500 lm).
Because of the CC-specific localization of the membraneanchored GFP probes (Figure 3g,i), these GFP-variants
should be trapped inside the CCs, suggesting that the
extended GFP/tip-distances in these three lines is due to the
onset of expression of the AtSUC2 promoter in the most
distal CCs of the metaphloem.
Figure 6 shows a quantitative analysis of this observation
in the main roots of up to 12 plants from each of the different
lines. The average GFP/tip-distances are presented, and
confirm the qualitative observation made in Figure 5 that the
transgenic plants fall into three classes: the membrane-
anchored class (STP9-GFP, AtSUC2-GFP, ER-GFP) which are
expressed in the first CCs of the metaphloem, the soluble
GFP-fusions (GFP-ubiquitin, GFP-sporamin, GFP-aequorin,
GFP-patatin) which are translocated to the ends of the
protophloem files, and the free (unfused) GFP which is
transported out of the protophloem domain into the extreme
root tip.
Discussion
The approach presented in this paper uses free GFP, GFPfusions similar to those previously used by Oparka et al.
(1999; GFP-sporamin and GFP-patatin) and new GFP-fusions
in a non-destructive approach for analyses of symplastic
domains along the phloem path of Arabidopsis and in the
terminal sink of the root tip. To this end cDNAs encoding free
GFP (27 kDa) or one of seven different GFP-fusions was
expressed under the control of the CC-specific AtSUC2promoter (Imlau et al., 1999; Truernit and Sauer, 1995).
ª Blackwell Publishing Ltd, The Plant Journal, (2004), 41, 319–331
Protein trafficking in the phloem 325
Figure 5. Comparison of cell-to-cell trafficking of free GFP and of different GFP-fusions in Arabidopsis root tips.
The three images on the top show the fluorescence emitted from the root tip of a tmGFP9 plant, of a GFP-sporamin plant and of a plant expressing free GFP.
Fluorescence was monitored with a CLSM (cell walls stained with propidium iodide; maximal projection of 20 scans). Fluorescence was observed in the CCs of the
transport phloem (tmGFP9), in the SE–CCs of the transport phloem plus in the protophloem (GFP-sporamin) or in the SE–CCs of the transport phloem, in the
protophloem plus in all cells of the root tip (free GFP).
The bottom row of pictures shows that similar differences in the distribution of fluorescence are detected with all eight transgenic lines. These confocal images were
taken without the propidium iodide staining that was used for the top row images. The horizontal insert shows the GFP-fluorescence in the two vascular strands of
the main root of a GFP-ubiquitin plant (merged presentation of GFP-fluorescence and transmitted light picture). Scale bars are 100 lm for the root tips in the top row,
50 lm for the root tips in the bottom row and 40 lm for the horizontal insert.
The obtained results demonstrate that ER-GFP, STP9-GFP
and AtSUC2-GFP do not traffic into the SEs and are restricted
to the CCs (Figures 2, 3g,i and 5) confirming the cellspecificity of the AtSUC2 promoter (Stadler and Sauer,
ª Blackwell Publishing Ltd, The Plant Journal, (2004), 41, 319–331
1996). These plants represent ‘zero trafficking controls’ for
the analyses of the established cell-to-cell trafficking of free
GFP, and the potential cell-to-cell trafficking of soluble GFPvariants.
326 Ruth Stadler et al.
of the plasmodesmata connecting CCs and SEs in this species, where phloem loading occurs into the SEs (Kühn et al.,
1997) and not into the CCs as shown for Arabidopsis (Stadler
and Sauer, 1996; Truernit and Sauer, 1995). Alternatively,
this observation in tomato may result from the extremely
low activity of the rolC promoter used in these analyses.
Data supporting a different SEL in solanaceous plants were
also obtained by Itaya et al. (2002). These authors found no
movement of a GFP–GFP-fusion from the CCs into the SEs of
transgenic tobacco plants.
GFP-fusions show restricted movement out of the
protophloem domain
Figure 6. Quantitative analysis of the distance between the most distal GFPfluorescence and the surface of the tip in the main root of the different
transgenic Arabidopsis lines.
Distances were determined under the confocal microscope in up to 12 plants
from each transgenic line. Plants were analyzed as shown in the bottom row
in Figure 5. The individual results (a) or the average distances (b) between the
most distal GFP-fluorescence and the root tips (mean SD) are presented.
All soluble GFP-fusions can move from CCs into SEs
Analyses of leaves (Figure 2) and/or roots (Figure 3) from
the different transgenic Arabidopsis lines showed that all
soluble GFP-fusions synthesized in the cytoplasm of the CCs
were able to traffic into the phloem SEs. Green fluorescence
resulting from GFP-ubiquitin or GFP-sporamin fusions was
detected in sink leaves, where the AtSUC2-promoter is not
active (Stadler and Sauer, 1996; Truernit and Sauer, 1995;
see also Figure 2). Green fluorescence resulting from the
GFP-ubiquitin, GFP-sporamin, GFP-aequorin and GFP-patatin fusions was also seen in the root phloem. The root tips
analyzed in this study represent terminal sinks, where
fluorescent proteins released from the CCs into the SEs
accumulate with time, and can thus be detected even in
plants with lower expression levels. Our data demonstrate
that in Arabidopsis plasmodesmata connecting CCs and SEs
have a SEL of at least 67 kDa. A recent report describing the
complete lack of GFP movement out of the tomato CCs
(Lalonde et al., 2003) may be explained with a different SEL
In none of the transgenic lines expressing soluble GFPfusions were we able to detect significant transport of the
fusions beyond the protophloem terminus. In Arabidopsis,
the protophloem is formed by two separate files of SEs,
each composed of a single, narrow sieve tube (Bonke et al.,
2003; Dolan et al., 1993). Figure 4(d) shows a longitudinal
section of one of these two protophloem SE files. Its
diameter of approximately 2–3 lM fits well to the diameter
of the aniline blue-stained SEs shown in Figure 4(b,c).
Comparison of these protophloem files and the fluorescent, GFP-labeled cells in this part of the root (Figure 4b,c)
revealed that the regions showing GFP fluorescence were
too wide to represent just this single protophloem SE file.
All of the soluble GFP-fusions had exchanged laterally
between the protophloem SEs and one or two surrounding
cell layers (Figure 4c). This ‘protophloem domain’ is
represented diagrammatically in Figure 7.
It appears that the protophloem of many plant species is
devoid of true CCs (Eleftheriou and Tsekos, 1982), a feature
that may be related to its short lifespan (see discussion in
Sjolund, 1997). In Lemna roots, however, protophloem SEs
and CCs form from the division of a common phloem
mother cell (Melaragno and Walsh, 1976), although other
species appear to lack such an obvious ontogeny (Esau
and Gill, 1973; Schulz, 1994). Our observation that AtSUC2
promoter activity is absent from the root protophloem files
in Arabidopsis supports observations that protophloem
SEs are not accompanied by CCs. Nevertheless, cells
adjoining these protophloem SEs might perform CC-like
functions, such as the exchange of macromolecules radially out of the protophloem SEs. Clearly, GFP and GFPfusions can move into these adjoining cells, but all GFPfusions are restricted from passing beyond this nucleate
cell layer. We, therefore, suggest that this protophloem
domain may play a role in phloem unloading, limiting the
passage of macromolecules into the main body of the
root. It may also play a role as a ‘checkpoint’ for
macromolecular trafficking (see also Foster et al., 2002).
As mature protophloem SEs are enucleate (van Bel and
Knoblauch, 2000; Esau, 1969b; Oparka and Turgeon, 1999;
ª Blackwell Publishing Ltd, The Plant Journal, (2004), 41, 319–331
Protein trafficking in the phloem 327
Figure 7. Model illustrating the unloading zone of an Arabidopsis root.
The model summarizes the data obtained on the cell-to-cell trafficking of GFP
or GFP-fusions and relates the expression pattern (tmGFP2, tmGFP9, ER-GFP)
and the trafficking distances (all other constructs) to the anatomy of
protophloem and metaphloem in the developing Arabidopsis root tip.
Plants with non-mobile GFP-fusions (tmGFP2, tmGFP9, ER-GFP) label exclusively the CCs of the mature metaphloem that ends about 500 lm behind the
root tip. All soluble GFP-fusions (GFP-ubiquitin, etc.) as well as free GFP can
move from these metaphloem CCs into the metaphloem SEs and enter
eventually the two single protophloem SE files that end about 250 lm behind
the root tip. From there they can be unloaded into one or two cell layers
forming the protophloem unloading domain, which allows further movement
into all cells of the root tip only for free GFP. In contrast, all soluble GFPfusions are retained in this unloading domain indicating a smaller SEL
between these cells and the adjacent cells of the root tip.
Sjolund, 1997), such a role would be best served by living
cells surrounding the phloem.
GFP is unloaded from the transport phloem
An unexpected observation was the limited but reproducible
unloading of free GFP from the transport phloem (Figure 3b,c), although this occurred to a much lower extent than
the unloading of free GFP from the SEs in the root protophloem (Figure 3a). In contrast, no unloading from the
transport phloem was observed for GFP-ubiquitin or for any
of the larger GFP-fusions (Figure 3d,e). In previous analyses,
Oparka et al. (1994) studied the unloading of CF in Arabidopsis roots after application of the dye to the cotyledons. In
these studies, unloading of CF was detected mainly from the
root tip but not from the transport phloem, suggesting that
the number of plasmodesmata in the root transport phloem
is low, or that these plasmodesmata are non-functional.
ª Blackwell Publishing Ltd, The Plant Journal, (2004), 41, 319–331
Wright and Oparka (1997) also showed that in Arabidopsis
the root transport phloem functions as an isolated domain
that is connected to surrounding cells by plasmodesmata
that are ‘held’ in a closed configuration. These plasmodesmata opened only after treatment of the root with inhibitors
of transmembrane ion fluxes [cyanide-m-chlorophenylhydrazone (CCCP) or probenicid (Cole et al., 1991)], suggesting
that they may function for transient periods in the symplasmic supply of assimilates to the root stele and cortex (Wright
and Oparka, 1997). In a similar approach Itaya et al. (2002)
showed that the fluorescent tracer fluorescein, which was
loaded into the phloem of a mature leaf, was confined to the
SE–CC complex in stems and source leaves of tobacco. From
these data they suggested that the SE–CC in the transport
phloem represents a symplastically isolated domain.
The plants used in the present study were transgenic,
rather than pulse labeled [as in Wright and Oparka (1997) or
in Itaya et al. (2002)], and the observed limited movement of
free GFP out of the transport phloem of mature roots is a
clear indication for the existence of a symplastic path for the
lateral unloading of assimilates. In addition, photoassimilates may be unloaded into the apoplast of the transport
phloem by plasma membrane-localized transport proteins.
This process has previously been postulated to be catalyzed
by the AtSUC2 sucrose transporter (Truernit and Sauer,
1995).
Phloem differentiation in the root tip
Quantitative analyses of the trafficking of GFP and GFPfusions into the root tip (Figure 6) demonstrated that GFP
and all GFP-fusions passing from the CCs into the SEs
trafficked toward the very end of the protophloem. The
protophloem, by definition, is the ‘first formed’ phloem
(Esau, 1977), and in the roots of most species occurs as
vertical files of SEs. In Arabidopsis the most mature SEs are
found at about 250 lm from the root tip (Zhu et al., 1998).
This mature protophloem sieve tube appears to conduct
the bulk of phloem unloading in root tips (Giaquinta et al.,
1983; Oparka et al., 1994; Schulz, 1994; Zhu et al., 1998)
and, although short lived, appears to be specialized for this
function. Proximal to the protophloem files, and internal to
them, the metaphloem (‘late-formed’ phloem) develops
and contains the first true CCs (Esau, 1969a). Our observations of GFP movement into the root protophloem, and
the appearance of AtSUC2 expression in metaphloem CCs,
correlates perfectly with the progressive development of
protophloem and metaphloem (see Figure 7).
The root tip represents a large symplastic domain, where
non-targeted cell-to-cell movement of proteins can occur
Our results on the cell-to-cell transport of free GFP in roots
confirm a recent report of Meyer et al. (2004), where free GFP
328 Ruth Stadler et al.
(together with a membrane-anchored GFP-variant; tm-GFP
for transmembrane-GFP) was expressed under the control of
the AtSUC3-promoter. This promoter is very active in several Arabidopsis sink tissues, one of them being the epidermal cell layer of the root tip. This cell layer was labeled
with high specificity in AtSUC3-promoter::tm-GFP plants. In
contrast, a massive movement of free GFP out of this cell
layer into the other cells of the root tip was observed in
AtSUC3-promoter::GFP plants. These data and the result
from the present paper show that GFP can undergo cell-tocell transport in roots. Clearly, this transport is not unidirectional (e.g. from the phloem toward the root surface). It
rather follows the concentration gradient of GFP, can occur
in either direction and is, therefore, driven by diffusion.
Implications for macromolecular signaling and transport in
the phloem
Another important feature of the presented work is the
demonstration that CC-synthesized proteins of up to 67 kDa
can enter the translocation stream non-specifically. The fusion proteins used here are ‘xenobiotic’ in the sense that
they are not normal constituents of the CC cytoplasm.
Therefore, it is reasonable to ask whether such macromolecular trafficking reflects the behavior of CC-derived proteins that enter the phloem for long-distance signaling
purposes. Several publications have drawn attention to the
entry of macromolecular signals into SEs. Such signals
include mRNAs, gene silencing signals and transcription
factors that have been suggested to pass selectively through
the plasmodesmata connecting CC and SE by a specific,
chaperone-mediated transport process (Citovsky and
Zambryski, 2000; Crawford and Zambryski, 1999; Lucas
et al., 2001; Ruiz-Medrano et al., 2001).
An alternative view is that the transport of many proteins
and solutes in SEs is regulated not at the level of CC–SE
plasmodesmata, but rather within the conducting phloem
itself (Ayre et al., 2003; Fisher et al., 1992; Oparka and Santa
Cruz, 2000). In an early study, Fisher et al. (1992) radiolabeled amino acids in wheat leaves and detected a large range
of radiolabeled proteins (10–79 kDa) in sieve tube exudate.
They proposed a highly selective regulation of protein
removal from SEs of the pathway phloem and non-selective
protein removal from the SEs in sink tissues. As a third
model, Imlau et al. (1999) suggested that movement of
soluble CC proteins into the SE may represent the ‘default’
pathway, unless a given protein has a retention signal that
locates it to a given domain within the CC or, alternatively,
targets it to a specific site within the SE. The implication of
this hypothesis is that molecules synthesized within CCs
may be continually lost to the translocation stream.
This kind of passive, diffusional cell-to-cell trafficking
through plasmodesmata has recently been shown for
the non-cell-autonomous Arabidopsis transcription factor
LEAFY (LFY) (Sessions et al., 2000; Wu et al., 2003) and for
transcription factors from maize (Lucas et al., 1995) or
Antirrhinum majus (Perbal et al., 1996). Wu et al. (2003)
provided strong evidence that the movement of LFY and
LFY-GFP fusions from L1 into underlying cell layers of the
Arabidopsis apex is non-targeted. Specific movement signals could not be identified and the observed cell-to-cell
movement of LFY is thus likely to be driven by diffusion (Wu
et al., 2003). Based on parallel analyses of the trafficking of
APETALA1 (AP), of AP-GFP fusions and of GFP–GFP dimers
these authors suggested that diffusion-driven, non-targeted
cell-to-cell movement of proteins may represent a general
mechanism and that the extent of this trafficking may only
be reduced by subcellular trapping, e.g. in the nuclear
compartment or by the formation of large complexes.
In contrast, plasmodesmata-trafficking of the maize transcription factor KNOTTED1 (KN1) seems to be targeted, and
it has been shown that KN1 can increase the SEL of
plasmodesmata and enables the transport of its own mRNA
(Lucas et al., 1995). Similarly, the Arabidopsis SHORT-ROOT
protein (SHR) moves from the stele into a single layer of
adjacent cells, where it enters the nucleus (Nakajima et al.,
2001). Analyzes with in frame-fusions of GFP to the coding
region of SHR showed that SHR-GFP fusion does traffic from
the stele into this single, adjacent cell layer representing the
endodermis. The authors concluded that SHR (60 kDa)
might need a special transit signal that widens the plasmodesmata or initiates a transient unfolding/refolding of the
protein.
Also some of the macromolecules shown to traffic from
CC to SE appear to depend on targeted trafficking and to be
transported no further than into the SE parietal layer.
Examples are specific enzymes of the alkaloid biosynthetic
pathway (Bird et al., 2003). Moreover, the phloem exudate of
Cucurbits appears to be replete with proteins that can ‘gate’
the plasmodesmata present in mesophyll cells to a higherthan-normal SEL (Balachandran et al., 1997), suggesting
that many phloem proteins have plasmodesmata-modifying
functions. However, most of the proteins translocated in the
phloem (Fisher et al., 1992) appear to be smaller than the
passive SEL ‘cutoff’ (67 kDa) demonstrated here for Arabidopsis, suggesting that they may enter the SE from the CC by
diffusion. A clear challenge for the future will be to demonstrate whether or not or to what extent the specialized
plasmodesmata that connect SE and CC (Mezitt and Lucas,
1996; Oparka and Turgeon, 1999; Schulz, 1998; Sjolund,
1997) require to be gated in order to permit macromolecular
trafficking in the phloem.
Our analyses demonstrate that GFP and GFP-fusions are
powerful tools for studying phloem transport and symplasmic domains under non-invasive conditions in intact plants.
In the future, it will be interesting to induce biotic and abiotic
stresses in such transgenic plants and to examine the effects
on macromolecular trafficking via the phloem.
ª Blackwell Publishing Ltd, The Plant Journal, (2004), 41, 319–331
Protein trafficking in the phloem 329
Experimental procedures
Strains and growth conditions
If not otherwise indicated, Arabidopsis thaliana plants (ecotype C24)
were grown in potting soil in the greenhouse. For root tip analyses
plants were grown under sterile conditions on vertical Petri plates
on Murashige–Skoog medium (pH 5.8) containing 1% phyto agar
(Duchefa, Haarlem, the Netherlands; Murashige and Skoog, 1962).
Roots were grown on the agar surface by incubating the plates in a
near-vertical position under long-day conditions in 16 h light/8 h
dark regime at 22C and 70% relative humidity for 14 days. For
microscopy of leaves, 3-week-old seedlings were transferred to soil
and grown for two more weeks in a growth chamber (long-day
conditions, 22C, 70% humidity). Agrobacterium tumefaciens strain
GV3101 (Holsters et al., 1980) was used for plant transformation. All
cloning steps were performed in Escherichia coli strain DH5a
(Hanahan, 1983).
Construction of vectors for stable plant transformation
Plants expressing free GFP under the control of the 900-bp AtSUC2
promoter (pEPS1) have been described previously (Imlau et al.,
1999). For construction of GFP-ubiquitin, GFP-sporamin and GFPpatatin fusions we generated the pUC19-based vector pGA04 that
contained the 900-bp AtSUC2 promoter fragment from pEPS1
(Imlau et al., 1999) as an SphI/NcoI fragment, followed by GFP
(NcoI/SacI) and the nopaline synthase terminator (SacI/EcoRI). At
the very 3¢-end of the GFP open-reading frame (ORF) BglII and XbaI
cloning sites were introduced by a polymerase chain reaction (PCR).
For construction of the GFP-ubiquitin fusion the complete ORF of
a Plantago major ubiquitin 1 (PmUBI1; accession no.: AJ841743)
was PCR-amplified from the vector pPP35 (N. Sauer and M. Gahrtz,
unpublished data) and BamHI (5¢-end) and XbaI (3¢-end) cloning
sites were introduced. Using these sites the ubiquitin ORF was
cloned into the BglII/XbaI sites at the 3¢-end of the GFP-ORF in
pGA04, yielding the plasmid pGA04-Ubi. From this plasmid a
HindIII/SacI fragment was excised and cloned into pGPTV-Bar
(Becker et al., 1992), yielding pGPTV-Ubi.
For construction of the GFP-sporamin fusion the ORF of sporamin
(accession no.: P14715) lacking the N-terminal 37 amino acids signal
sequence responsible for targeting to the ER (Hattori et al., 1989)
was PCR-amplified from the vector pIM023 (Hattori et al., 1985)
and BglII (5¢-end) and XbaI (3¢-end) cloning sites were introduced.
Using these sites the sporamin ORF was cloned into the BglII/XbaI
sites at the 3¢-end of the GFP-ORF in pGA04, yielding the plasmid
pGA04-Spor. From this plasmid a HindIII/SacI fragment was
excised and cloned into pGPTV-Bar (Becker et al., 1992), yielding
pGPTV-Spor.
For construction of the GFP-patatin fusion the ORF of patatin
(accession no.: A24142) lacking the N-terminal 23 amino acids
signal sequence responsible for targeting to the ER (Mignery et al.,
1984) was PCR-amplified from the vector pPATB2 (Stiekema et al.,
1988) and BglII (5¢-end) and XbaI (3¢-end) cloning sites were
introduced. Using these sites the patatin ORF was cloned into the
BglII/XbaI sites at the 3¢-end of the GFP-ORF in pGA04, yielding the
plasmid pGA04-pat. From this plasmid the C-terminal XbaI site was
removed and a new XbaI site was introduced at the 5¢-end of the
AtSUC2-promoter yielding plasmid pGA05-Pat. From this plasmid
an XbaI/SacI fragment was excised and cloned into pGPTV-Bar
(Becker et al., 1992), yielding pGPTV-Pat.
For construction of the GFP-aequorin (accession no. for aequorin:
P07164) fusion under the control of the AtSUC2 promoter an already
existing fusion was PCR-amplified from the plasmid p7rolB-GFPª Blackwell Publishing Ltd, The Plant Journal, (2004), 41, 319–331
AEQ (C. Plieth, Zentrum für Biochemie und Molekularbiologie,
University of Kiel, Kiel, Germany) and BspHI (5¢-end) and SacI (3¢end) cloning sites were introduced. Using these sites the GFPaequorin fusion was cloned into pGA05-pat after removal of the
GFP-patatin insert by a NcoI/SacI-digest. From the resulting plasmid
pGA05-Aeq an XbaI/SacI fragment was excised and cloned into
pGPTV-Bar (Becker et al., 1992), yielding pGPTV-Aeq.
For construction of the ER-resident GFP-variant an already
existing sequence was PCR-amplified from the vector pBIN-mgfp5-ER [contains the N-terminal signal sequence (21 amino acids)
of an Arabidopsis basic chitinase (accession no.: AAM10081)] and
a C-terminal HDEL-retention signal; J. Haseloff, Department of
Plant Sciences, University of Cambridge, Cambridge, UK) and a
BspHI cloning site was introduced into the start ATG of the
resulting PCR-fragment, which was cloned into pGEM-T Easy
(Promega, Madison, WI, USA), yielding plasmid pMG002. From
this plasmid the modified GFP-ORF was excised with BspHI/SacI
and cloned into NcoI/SacI-digested pEP/pUC (Imlau et al., 1999). A
BamHI/SacI fragment harboring 2200 bp of AtSUC2-promoter
sequence and the modified GFP-ORF was excised from the
resulting plasmid and cloned into pGPTV-Bar (Becker et al.,
1992), yielding the plasmid pMG004.
For construction of the AtSTP9-GFP fusion (tmGFP9) a genomic
AtSTP9 fragment [encoding the 232 N-terminal amino acids of
AtSTP9 (Schneidereit et al., 2003) and harboring the first two
introns of At1g50310] was PCR-amplified and NcoI cloning sites
were introduced on both ends. Using these NcoI sites the genomic
AtSTP9 fragment was cloned into the unique NcoI site of plasmid
pAF12 yielding the plasmid pMH4. pAF12 represents a pUC19based plasmid that harbors the 900-bp AtSUC2-promoter of pEPS1
(Imlau et al., 1999) followed by GFP with a unique NcoI site in its
start ATG. The AtSUC2-promoter/AtSTP9-GFP fragment was excised from pMH4 with HindIII/SacI and cloned into the respective
sites of pAF16, which represents a modified version of pGPTV-bar
(Becker et al., 1992), where the GUS-reporter gene has been
removed. The resulting plasmid was named pMH5a.
For construction of the AtSUC2-GFP fusion (tmGFP2) the entire
AtSUC2 gene (At1g22710; including all three introns) was PCRamplified and BspHI cloning sites were introduced on both ends.
Using these BspHI sites the AtSUC2 gene was cloned into the
compatible NcoI site in the start ATG of GFP in pEPS/pUC (Imlau
et al., 1999), yielding plasmid pTF5004. In this plasmid the unique
SphI site at the 5¢-end of the AtSUC2-promoter was replaced by
SacI, yielding plasmid pTF5008. From this plasmid the AtSUC2promoter/AtSUC2-GFP fragment was excised by SacI and cloned
into pAF16, yielding the plasmid pTF5010.
Plant transformation
The seven plasmids (pGPTV-Ubi, pGPTV-Spor, pGPTV-Aeq, pGPTVPat, pMG004, pMH5a and pTF5010) were used to transform Agrobacterium tumefaciens strain GV3101 (Holsters et al., 1980) and
finally for transformation of Arabidopsis thaliana C24 WT by dipping (Clough and Bent, 1998).
Epifluorescence and confocal laser scanning microscopy
Green fluorescent protein in leaves was detected using a stereofluorescence microscope (SV11; Carl Zeiss, Jena, Germany) after
excitation with light of 460–500 nm wavelengths. Emitted fluorescence was monitored using a filter permeable for wavelengths
>510 nm. Photos were taken with a Sony 3CCD color video camera
and a Carl Zeiss Vision KS200, 3.0 imaging software.
330 Ruth Stadler et al.
Roots were imaged in situ using a confocal laser scanning
microscope (CLSM, Leica TCS SP II; Leica Microsystems, Bensheim,
Germany). For cell wall staining, roots grown on the agar surface
were covered with a drop of 0.5% propidium iodide and incubated
for 10 min at room temperature. After two washes with water, roots
were imaged while still in their Petri dishes. GFP was excited by
488 nm light produced by an Argon laser and observed using a
detection window from 497 to 526 nm. Propidium iodide-stained
cell walls were detected with the argon laser 488 nm line and a
detection window of 595–640 nm.
Callose was detected by staining roots with 0.01% aniline blue
made up in 0.07-M phosphate buffer (pH 7.5) and imaging using the
epifluorescence microscope by excitation at 330–380 nm, with
emitted light being monitored above 420 nm.
For GFP/tip-distance measurements, seedlings were grown on
plates for 10 days and main root tips were imaged as described
above. Distances of the GFP signals from the root tip were
calculated using the Leica Confocal Software.
Acknowledgements
We thank Marina Henneberg and Anja Schillinger for excellent
technical assistance. This work was supported by the Deutsche
Forschungsgemeinschaft (Grant Sa 382/8 to N.S.) and the Scottish
Executive Environment and Rural Affairs Department (SEERAD;
grant-in-aid to K.J.O.).
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