41 expression of gfp-fusions in arabidopsis companion cells … · 2017-11-29 · expression of...

Expression of GFP-fusions in Arabidopsis companion cellsreveals non-specific protein trafficking into sieve elementsand 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,*

1Molekulare Pflanzenphysiologie, Universitat Erlangen-Nurnberg, Staudtstraße 5, D-91058 Erlangen, Germany, and2Cell-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 pro-

teins and RNAs (Balachandran et al., 1997; Sjolund, 1997).

In the phloem of angiosperms, the sieve element–com-

panion 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

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 plasmodes-

mata 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).

ª 2004 Blackwell Publishing Ltd 319

The Plant Journal (2005) 41, 319–331 doi: 10.1111/j.1365-313X.2004.02298.x

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 dex-

trans (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) plasmodes-

mata 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 plas-

modesmata. 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-aequ-

orin, MWapp: 48 kDa; GFP-patatin, MWapp: 67 kDa], two

membrane-anchored (tmGFP9, tmGFP2) and one ER-locali-

zed 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 wide-

spread 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 stor-

age 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 molecu-

lar masses, and these molecular mass values (kDa) are

320 Ruth Stadler et al.

ª Blackwell Publishing Ltd, The Plant Journal, (2004), 41, 319–331

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 detec-

ted. 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-pro-

moter GFP plants (Imlau et al., 1999). GFP-fluorescence

was detected in all lines analyzed, although at different

intensities. Plants carrying the GFP-ubiquitin, GFP-spor-

amin and tmGFP9 fusions [AtSTP9 encodes an Arabidop-

sis monosaccharide transporter (Schneidereit et al., 2003)]

showed similar GFP fluorescence, although the fluores-

cence in these plants was slightly lower than in plants

with free GFP. Plants of the other lines showed decreas-

ing fluorescence intensities (tmGFP2 > GFP-ER > GFP-

aequorin > GFP-patatin).

Clear differences were observed in the cell-to-cell move-

ment 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 membrane-

anchored GFPs (tmGFP9 or tmGFP2 lines), which is ex-

plained by the source-leaf specific activity of the AtSUC2-

promoter and which confirms data from AtSUC2-promo-

ter::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 Ara-

bidopsis roots than in leaves, which are less accessible to

non-invasive imaging (Oparka et al., 1994). Immunohisto-

chemical studies (Stadler and Sauer, 1996) and GUS-histo-

chemical 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 vas-

cular bundles at the root tips (Imlau et al., 1999), predom-

inantly 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 trans-

port. 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),

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 fifusion 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 transmembrane-

GFPs). 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.

Protein trafficking in the phloem 321


free GFP is unloaded at the tip of Arabidopsis roots from the

two protophloem files (Figure 3a). Reproducibly, we ob-

served 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 fluor-

escence was seen in GFP-ubiquitin plants (Figure 3f) and in

the other plants expressing constructs for soluble GFP-

fusions (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), fluores-

cence 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 GFP-

containing 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 proto-

phloem 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 GFP-

variant.

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.



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

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 pro-

moter are presented. Note that only the free form of GFP

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 GFP-

sporamin 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).



showed extensive post-phloem transport throughout the

root tip. In contrast, the fluorescence of all other phloem

mobile GFP-variants (GFP-ubiquitin, GFP-sporamin, GFP-

aequorin 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 membrane-

anchored 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, GFP-

fusions 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 AtSUC2-

promoter (Imlau et al., 1999; Truernit and Sauer, 1995).

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). GFP-

sporamin 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).



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 cell-

specificity of the AtSUC2 promoter (Stadler and Sauer,

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 GFP-

variants.

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.



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-pata-

tin 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

of the plasmodesmata connecting CCs and SEs in this spe-

cies, where phloem loading occurs into the SEs (Kuhn 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

In none of the transgenic lines expressing soluble GFP-

fusions 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 fluores-

cent, 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 radi-

ally out of the protophloem SEs. Clearly, GFP and GFP-

fusions can move into these adjoining cells, but all GFP-

fusions 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;

Figure 6. Quantitative analysis of the distance between the most distal GFP-

fluorescence 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.



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 (Fig-

ure 3b,c), although this occurred to a much lower extent than

the unloading of free GFP from the SEs in the root proto-

phloem (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 Arabid-

opsis 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.

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 plasmodes-

mata opened only after treatment of the root with inhibitors

of transmembrane ion fluxes [cyanide-m-chlorophenylhy-

drazone (CCCP) or probenicid (Cole et al., 1991)], suggesting

that they may function for transient periods in the symplas-

mic 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, photoassimi-

lates 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 GFP-

fusions 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 obser-

vations 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 freeGFP

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 exclu-

sively 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 GFP-

fusions are retained in this unloading domain indicating a smaller SEL

between these cells and the adjacent cells of the root tip.



(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 sev-

eral Arabidopsis sink tissues, one of them being the epi-

dermal 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-to-

cell transport in roots. Clearly, this transport is not unidi-

rectional (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 fu-

sion 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 macromo-

lecular trafficking reflects the behavior of CC-derived pro-

teins 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) radiolabe-

led 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 sig-

nals 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 tran-

scription 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 plas-

modesmata 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 higher-

than-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 Arabid-

opsis, suggesting that they may enter the SE from the CC by

diffusion. A clear challenge for the future will be to demon-

strate 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 symplas-

mic 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.



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 analysesplants were grown under sterile conditions on vertical Petri plateson 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 anear-vertical position under long-day conditions in 16 h light/8 hdark regime at 22�C and 70% relative humidity for 14 days. Formicroscopy of leaves, 3-week-old seedlings were transferred to soiland grown for two more weeks in a growth chamber (long-dayconditions, 22�C, 70% humidity). Agrobacterium tumefaciens strainGV3101 (Holsters et al., 1980) was used for plant transformation. Allcloning 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 AtSUC2promoter (pEPS1) have been described previously (Imlau et al.,1999). For construction of GFP-ubiquitin, GFP-sporamin and GFP-patatin fusions we generated the pUC19-based vector pGA04 thatcontained 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). Atthe very 3¢-end of the GFP open-reading frame (ORF) BglII and XbaIcloning sites were introduced by a polymerase chain reaction (PCR).

For construction of the GFP-ubiquitin fusion the complete ORF ofa 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) cloningsites were introduced. Using these sites the ubiquitin ORF wascloned into the BglII/XbaI sites at the 3¢-end of the GFP-ORF inpGA04, yielding the plasmid pGA04-Ubi. From this plasmid aHindIII/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 signalsequence 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/XbaIsites at the 3¢-end of the GFP-ORF in pGA04, yielding the plasmidpGA04-Spor. From this plasmid a HindIII/SacI fragment wasexcised and cloned into pGPTV-Bar (Becker et al., 1992), yieldingpGPTV-Spor.

For construction of the GFP-patatin fusion the ORF of patatin(accession no.: A24142) lacking the N-terminal 23 amino acidssignal 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 wereintroduced. Using these sites the patatin ORF was cloned into theBglII/XbaI sites at the 3¢-end of the GFP-ORF in pGA04, yielding theplasmid pGA04-pat. From this plasmid the C-terminal XbaI site wasremoved and a new XbaI site was introduced at the 5¢-end of theAtSUC2-promoter yielding plasmid pGA05-Pat. From this plasmidan 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 alreadyexisting fusion was PCR-amplified from the plasmid p7rolB-GFP-

AEQ (C. Plieth, Zentrum fur Biochemie und Molekularbiologie,University of Kiel, Kiel, Germany) and BspHI (5¢-end) and SacI (3¢-end) cloning sites were introduced. Using these sites the GFP-aequorin fusion was cloned into pGA05-pat after removal of theGFP-patatin insert by a NcoI/SacI-digest. From the resulting plasmidpGA05-Aeq an XbaI/SacI fragment was excised and cloned intopGPTV-Bar (Becker et al., 1992), yielding pGPTV-Aeq.

For construction of the ER-resident GFP-variant an alreadyexisting sequence was PCR-amplified from the vector pBIN-m-gfp5-ER [contains the N-terminal signal sequence (21 amino acids)of an Arabidopsis basic chitinase (accession no.: AAM10081)] anda C-terminal HDEL-retention signal; J. Haseloff, Department ofPlant Sciences, University of Cambridge, Cambridge, UK) and aBspHI cloning site was introduced into the start ATG of theresulting PCR-fragment, which was cloned into pGEM-T Easy(Promega, Madison, WI, USA), yielding plasmid pMG002. Fromthis plasmid the modified GFP-ORF was excised with BspHI/SacIand cloned into NcoI/SacI-digested pEP/pUC (Imlau et al., 1999). ABamHI/SacI fragment harboring 2200 bp of AtSUC2-promotersequence and the modified GFP-ORF was excised from theresulting plasmid and cloned into pGPTV-Bar (Becker et al.,1992), yielding the plasmid pMG004.

For construction of the AtSTP9-GFP fusion (tmGFP9) a genomicAtSTP9 fragment [encoding the 232 N-terminal amino acids ofAtSTP9 (Schneidereit et al., 2003) and harboring the first twointrons of At1g50310] was PCR-amplified and NcoI cloning siteswere introduced on both ends. Using these NcoI sites the genomicAtSTP9 fragment was cloned into the unique NcoI site of plasmidpAF12 yielding the plasmid pMH4. pAF12 represents a pUC19-based plasmid that harbors the 900-bp AtSUC2-promoter of pEPS1(Imlau et al., 1999) followed by GFP with a unique NcoI site in itsstart ATG. The AtSUC2-promoter/AtSTP9-GFP fragment was ex-cised from pMH4 with HindIII/SacI and cloned into the respectivesites of pAF16, which represents a modified version of pGPTV-bar(Becker et al., 1992), where the GUS-reporter gene has beenremoved. The resulting plasmid was named pMH5a.

For construction of the AtSUC2-GFP fusion (tmGFP2) the entireAtSUC2 gene (At1g22710; including all three introns) was PCR-amplified and BspHI cloning sites were introduced on both ends.Using these BspHI sites the AtSUC2 gene was cloned into thecompatible NcoI site in the start ATG of GFP in pEPS/pUC (Imlauet al., 1999), yielding plasmid pTF5004. In this plasmid the uniqueSphI site at the 5¢-end of the AtSUC2-promoter was replaced bySacI, yielding plasmid pTF5008. From this plasmid the AtSUC2-promoter/AtSUC2-GFP fragment was excised by SacI and clonedinto pAF16, yielding the plasmid pTF5010.

Plant transformation

The seven plasmids (pGPTV-Ubi, pGPTV-Spor, pGPTV-Aeq, pGPTV-Pat, pMG004, pMH5a and pTF5010) were used to transform Agro-bacterium tumefaciens strain GV3101 (Holsters et al., 1980) andfinally for transformation of Arabidopsis thaliana C24 WT by dip-ping (Clough and Bent, 1998).

Epifluorescence and confocal laser scanning microscopy

Green fluorescent protein in leaves was detected using a stereo-fluorescence microscope (SV11; Carl Zeiss, Jena, Germany) afterexcitation with light of 460–500 nm wavelengths. Emitted fluores-cence was monitored using a filter permeable for wavelengths>510 nm. Photos were taken with a Sony 3CCD color video cameraand a Carl Zeiss Vision KS200, 3.0 imaging software.



Roots were imaged in situ using a confocal laser scanningmicroscope (CLSM, Leica TCS SP II; Leica Microsystems, Bensheim,Germany). For cell wall staining, roots grown on the agar surfacewere covered with a drop of 0.5% propidium iodide and incubatedfor 10 min at room temperature. After two washes with water, rootswere imaged while still in their Petri dishes. GFP was excited by488 nm light produced by an Argon laser and observed using adetection window from 497 to 526 nm. Propidium iodide-stainedcell walls were detected with the argon laser 488 nm line and adetection window of 595–640 nm.

Callose was detected by staining roots with 0.01% aniline bluemade up in 0.07-M phosphate buffer (pH 7.5) and imaging using theepifluorescence microscope by excitation at 330–380 nm, withemitted light being monitored above 420 nm.

For GFP/tip-distance measurements, seedlings were grown onplates for 10 days and main root tips were imaged as describedabove. Distances of the GFP signals from the root tip werecalculated using the Leica Confocal Software.

Acknowledgements

We thank Marina Henneberg and Anja Schillinger for excellenttechnical assistance. This work was supported by the DeutscheForschungsgemeinschaft (Grant Sa 382/8 to N.S.) and the ScottishExecutive Environment and Rural Affairs Department (SEERAD;grant-in-aid to K.J.O.).

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