Download Transport of virally expressed green fluorescent protein through the

Planta (1999) 208: 392±400
Transport of virally expressed green ¯uorescent protein through
the secretory pathway in tobacco leaves is inhibited
by cold shock and brefeldin A
Petra Boevink1, Barry Martin2, Karl Oparka1, Simon Santa Cruz1, Chris Hawes2
1
Scottish Crop Research Institute, Invergowrie, Dundee, DD2 5DA, UK
School of Biological and Molecular Sciences, Oxford Brooke University, Gipsy Lane, Oxford, OX3 0BP, UK
2
Received: 8 October 1998 / Accepted: 16 November 1998
Abstract. Potato virus X (PVX) has been used as an
expression vector to target the green ¯uorescent protein
(GFP) from the jelly®sh Aequorea victoria to the
endoplasmic reticulum (ER) of tobacco (Nicotiana
clevelandii L.) leaves. Expression of free GFP resulted
in strong cytoplasmic ¯uorescence with organelles being
imaged in negative contrast. Translocation of GFP into
the lumen of the ER was mediated by the use of the
sporamin signal peptide. Retention of GFP in the ER
was facilitated by the splicing of the ER retrieval/
retention tetrapeptide, KDEL to the carboxy terminus
of GFP. Fluorescence of GFP was restricted to a labile
cortical network of ER tubules with occasional small
lamellae and to streaming trans-vacuolar strands. Secretion of ER-targeted GFP was inhibited both by cold
shock and low concentrations of the secretory inhibitor
brefeldin A. However, both prolonged cold and prolonged incubation in brefeldin A resulted in the recovery
of secretory capability. In leaves infected with the GFPKDEL construct, high concentrations of brefeldin A
induced the tubular network of cortical ER to transform
into large lamellae or sheets which reverted to the
tubular network on removal of the drug.
Key words: Endoplasmic reticulum ± Golgi apparatus ±
± Brefeldin A ± Green ¯uorescent protein ± Nicotiana ±
protein secretion
Introduction
A number of di€erent approaches have been taken to
study the transport of secretory products through the
Abbreviations: BFA = brefeldin A; GFP = green ¯uorescent protein; pTXS.P3C2 = potato virus X expression vector; PVX = potato virus X
Correspondence to: C. Hawes;
E-mail: [email protected]; Fax: 44 (1865) 483955
endomembrane system of plant cells. These started with
the pioneering work of Pickett-Heaps (1968) who used
radiolabelled probes in combination with electronmicroscope autoradiography to establish the role of
the Golgi apparatus in cell wall matrix biosynthesis.
More recently, anity labelling of secretory products in
the endoplasmic reticulum (ER), Golgi, storage vacuoles
and at the cell surface (Greenwood and Chrispeels 1985;
Sherrier and VandenBosch 1994), and biochemical
fractionation of the various components of the pathway
(Brummell et al. 1990) have been the techniques of
choice. However, it has been the use of heterologous
expression systems, which permit the observation of
foreign and chimeric proteins in model plant species,
that has revolutionised our understanding of tracking
and targeting of proteins within the secretory system (for
reviews, see Chrispeels 1991; Neuhaus 1996). Thus, it
has been demonstrated that plants use the H/KDEL Cterminal signal peptide for retention of proteins in the
lumen of the ER (Denecke et al. 1992; Napier et al.
1992), and a range of peptide sequences for targeting
proteins to the vacuole (Neuhaus 1996).
Transport of secretory material post-Golgi is by
means of vesicle vectors (Staehelin and Moore 1995;
Hawes et al. 1996) and it is assumed that transport from
the ER to the cis-Golgi is also by vesicles. However,
evidence for the existence of ER-derived transport
vesicles in higher plants is scant (Hawes and SatiatJeunemaitre 1996) with little in-vivo structural or
biochemical data being available. In mammalian cells,
ER-to-Golgi transport has been demonstrated by the
use of the secretory inhibitor brefeldin A (BFA) which
blocks vesicle transport between the ER and Golgi
(Klausner et al. 1992). In plants, BFA has been shown
to block transport from the ER and appears to inhibit
secretion from the trans-Golgi (Matsuoka et al. 1995;
Satiat-Jeunemaitre et al. 1996). To investigate the ERto-Golgi step of the secretory pathway in further detail a
cell system synthesising and secreting a high level of
foreign protein should prove invaluable.
The green ¯uorescent protein (GFP) from the jelly®sh
Aequorea victoria is a powerful tool for the study of gene
P. Boevink et al.: Transport of virally expressed green ¯uorescent protein
expression and for protein location using fusions of GFP
to proteins of interest. The intrinsic ¯uorescence of the
GFP allows non-destructive, in-vivo imaging of events
within cells. Fused to the C-terminus of a neuroendocrine secretory protein, GFP has been used to image the
secretory pathway (Kaether and Gerdes 1995) and the
mobility of Golgi transferases in live, transfected HeLa
cells (Cole et al. 1996). In plant cells, expression of a
signal peptide-GFP-HDEL construct resulted in ¯uorescence of the ER in Arabidopsis thaliana roots (Haselo€ and Amos 1995).
It has recently been shown that very high levels of
foreign-protein expression can be obtained in plants
using virus-based episomal vectors. A potato virus X
(PVX) vector has been used to express both native GFP
and chimeric GFP-protein fusions in tobacco leaves
(Baulcombe et al. 1995; Oparka et al. 1996a). In combination with confocal microscopy this technique is
proving invaluable for studying the targeting of proteins
in vivo (Boevink et al. 1996, 1998; Oparka et al. 1996b).
In a preliminary publication we demonstrated the
targeting and retention of a GFP-KDEL construct to
the ER of tobacco leaf cells using the patatin signal
peptide (Boevink et al. 1996). Here we report on the
increased eciency of the sporamin signal peptide for
insertion of GFP-KDEL into the ER and on the
secretion of GFP, without KDEL, to the apoplast. In
addition, we show that both the inhibitor BFA and cold
shock can inhibit transport of GFP downstream from
the ER with a resulting build up of ¯uorescence in the
ER lumen.
Materials and methods
Constructs. Standard DNA manipulation techniques were used
(Sambrook et al. 1989). Overlap extension polymerase chain
reaction (PCR; Higuchi et al. 1988) using mutagenic oligonucleotides was used to fuse the signal-peptide-encoding sequence (sp)
from the sweet potato (Ipomoea batatas Lam.) storage protein,
sporamin (Hattori et al. 1985), to the 5¢ end of the gfp gene (wild
type) to give sp-GFP. ClaI and NsiI restriction-enzyme recognition
sequences engineered into the mutagenic oligonucleotides allowed
the overlap extension PCR product to be cloned into ClaI/NsiIdigested potato virus ´ expression vector (pTXS.P3C2; data not
shown), giving rise to the plasmid pTXS.sp-GFP. A second
construct, pTXS.sp-GFP-K, was made that also contained the
coding sequence for the carboxy-terminal 11 amino acids from a
heat-shock protein (HSP90) homologue, which include KDEL
(Schroder et al. 1993), fused to the 3¢ end of the gfp gene (Fig. 1).
In-vitro transcription and plant inoculation. Templates for transcription were prepared by digesting pTXS-GFP (Baulcombe et al.
1995), pTXS.sp-GFP and pTXS.sp-GFP-K with the restriction
enzyme SpeI. In-vitro, run-o€ transcripts were synthesised from the
templates using a T7 transcription kit (Ambion, Austin, Tex.,
USA). Leaves of approximately 4-week-old Nicotiana clevelandii L.
plants were inoculated with transcripts by rubbing the transcription
product derived from 0.2 mg of template (per leaf) onto aluminium-oxide-dusted leaves. Note that progeny viruses on plants
inoculated with in-vitro transcripts are referred to by substituting
the pre®x PVX for the plasmid name pTXS.
Immunochemical detection of GFP. Total protein preparations were
made from infected leaves at 10±12 days post inoculate and from
393
Fig. 1. Insertion of modi®ed GFPs into the PVX vector to create
pTXS.sp-GFP and PTX.sp-GFP-K. The PVX vector, pTXS.P3C2,
and modi®ed GFPs are not drawn to scale. The viral cDNA is ¯anked
by T7 RNA promoter and speI recognition sequences. Open boxes
represent coding sequences and lines represent untranslated sequence.
The genes in the PVX vector are, from left to right: the replicase
(REP), the triple-gene-block proteins, represented by their approximate molecular weights in kDa (25, 12, 8), and the coat protein (CP).
pTXS.P3C2 contains a duplication of the coat protein subgenomic
promoter (black triangles) and a multiple cloning site (magni®ed). The
two modi®ed GFP genes with the sporamin signal peptide at the 5¢
terminus (SP) and with or without sequence encoding the KDEL
peptide (K) at the 3¢ terminus were inserted independently into the
vector between the ClaI and NsiI sites to give pTXS.spGFP-K and
pTXS.sp-GFP, respectively
healthy (non-inoculated) leaves by grinding them in one and a half
times (w/v) grinding bu€er [100 mM Tris-HCl (pH 7.5), 10 mM
KCl, 5 mM MgCl2, 0.4 M sucrose, 10% glycerol, 15 lM bmercaptoethanol]. The resulting mixture was centrifuged in a
microfuge and the supernatant collected. Aliquots of the extract
were taken and mixed with equal volumes of loading bu€er (1%
glycerol, 1% SDS, 12.5 bg/ml bromophenol blue, 6.25 mM TrisHCl, 5 mM b-mercaptoethanol, pH 6.8). Intercellular ¯uid extracts
were obtained from infected and healthy leaves by vacuumin®ltrating the leaves with water and centrifuging them at 4000 g
for 2±3 min. Proteins in the extract were concentrated by mixing
with Strataclean resin (Stratagene) and centrifuging. Loading
bu€er was added directly to the precipitated resin. Samples with
loading bu€er added were boiled for 5±10 min and loaded onto an
SDS-PAGE gel. Gels were electrophoresed at 100 V for 4±5 h and
the separated proteins were transferred to nitrocellulose membranes. Membranes were probed with anti-GFP (Santa-Cruz et al.
1996) and anti-PVX-coat-protein antibodies (Oparka et al. 1996a).
Brefeldin A and cold treatments. Brefeldin A (Sigma) was kept as a
10 mg/ml stock solution in methanol. Explants about 3 mm square
were taken from infected areas of leaves from plants inoculated
with pTXS.sp-GFP and pTXS.sp-GFP-K transcripts and ¯oated
on 10 or 100 lg/ml BFA, or 1% methanol in distilled water, for 4±
12 h. E€ects of BFA on the distribution of GFP were monitored by
confocal microscopy. The ecacy of the used BFA was tested on
fresh leaf segments. For cold treatment, plants were held under
continuous light at 4 °C for varying lengths of time and segments
of leaves excised from infected areas for confocal microscopy.
394
P. Boevink et al.: Transport of virally expressed green ¯uorescent protein
Fig. 2. Comparison of the ¯uorescence of
lesions produced by PVX.GFP, PVX.spGFP-K and PVX.sp-GFP. Nicotiana clevlandii leaves infected with PVX.GFP (left),
PVX.sp-GFP-K (centre) or PVX.sp-GFP
(right) at 6 d post infection. The PVX.spGFP lesions are noticeably less bright than
the control PVX-GFP. The PVX.sp-GFP
lesions are visible as darker regions on the
leaf. Some auto¯uorescence due to damage
is also visible on this leaf. Bar = 1 cm
Confocal microscopy. Leaf pieces were viewed, usually at approx.
6±8 days post inoculation, with a Bio-Rad MRC 1000 laser
scanning microscope and all images are of epidermal cells of the
adaxial leaf surface. The methods were as described by Baulcombe
et al. (1995).
Electron microscopy. Segments of leaf 2 mm square were excised
under ®xative containing 2% glutaraldehyde and 1% paraformaldehyde in 0.1 M sodium cacodylate bu€er (pH 6.9), transferred to
fresh ®xative for 1 h under vacuum and post-®xed for 2 h in 1%
aqueous osmium tetroxide. After dehydration in a graded water/
ethanol series, material was embedded in Spurr resin (Spurr 1969),
sectioned with an MT7000 ultramicrotome (RMC, Tucson, Arizona), stained with uranyl acetate and lead citrate and observed with
a 1200EXII (JEOL, Tokyo, Japan) transmission electron microscope.
Results
Expression of GFP constructs in tobacco leaves. To target
GFP into the secretory pathway the coding sequence for
the 21-amino-acid signal peptide of the sweet-potato
storage protein sporamin (Hattori et al. 1985) was fused
to the amino-terminus of the gfp gene. A second
construct was made, to retain the GFP in the ER, with
the sporamin signal peptide fused to the amino-terminus
and a KDEL retrieval/retention signal fused to the
carboxy-terminus of the gfp gene (Boevink et al. 1996).
The recombinant gfp genes were cloned into the PVX
expression vector pTXS.P3C2 to give pTXS.sp-GFP and
pTXS.sp-GFP-K respectively (Fig. 1). Circular, green
¯uorescent lesions containing cells expressing GFP were
visible under a 395-nm UV lamp on Nicotiana clevelandii
leaves inoculated with pTXS.sp-GFP-K after 4 d. The
pTXS.sp-GFP-K lesions showed a lower level of ¯uorescence than PVX.GFP control leaves (Fig. 2), whilst
lesions of PVX.sp-GFP leaves were only visible as
slightly darker regions on the leaf (Fig. 2). The viral
lesions increased in size with time and systemic symptoms, and also systemic ¯uorescence in the case of
PVX.sp-GFP-K, were observed at about 7 d post-
inoculation, indicating that the modi®ed viruses were
not impaired in cell-to-cell or long-distance movement.
Leaf epidermal cells infected with PVX.sp-GFP and
viewed by confocal microscopy, showed very faint GFP
¯uorescence that appeared to be ER (Fig. 3C). This can
be compared with the very high levels of cytoplasmic
GFP ¯uorescence in PVX-GFP cells with no ER signal
peptide, where the same microscope contrast and
brightness settings were used to record the image
(Fig. 3A). In these PVX-GFP cells cytoplasmic organelles could be seen in negative contrast (Fig. 3B).
Occasionally, in some older, heavily infected areas of
leaves, apoplastic GFP ¯uorescence was also detected,
indicating secretion of the sp-GFP (Fig. 3D). Cells
infected with PVX.sp-GFP-K, had the same subcellular
distribution of GFP as that previously described for the
construct, PVX.p-GFP-K containing the patatin signal
peptide (Boevink et al. 1996). Fluorescence was restricted to the ER, which had two predominant distributions:
(i) in a labile cortical network of tubules with occasional
small lamellae (Fig. 3E,F) and (ii) in bright transvacuolar strands (not shown). Within both these systems
small motile ¯uorescent inclusions were observed. The
cortical network of ER is restricted to a thin layer of
cortical cytoplasm approximately 0.1±0.3 lm thick
(Fig. 4). Visually, the level of ¯uorescence in the ER of
PVX.sp-GFP-K-infected cells was substantially higher
than that previously reported with PVX.p-GFP-K
(Boevink et al. 1996) although with the viral expression
system this could not be quanti®ed. This result was
con®rmed by the observation that PVX.sp-GFP-K
lesions were visually ¯uorescent under a hand-held U/
V lamp, whereas PVX.p-GFP-K lesions were not (data
not shown). The total amount of GFP ¯uorescence
observed by confocal microscopy associated with
PVX.sp-GFP was much lower than in PVX.sp-GFPK- or PVX.GFP-infected cells. This was in agreement
with the di€erences in ¯uorescence seen at the macroscopic level with UV illumination (Fig. 2).
P. Boevink et al.: Transport of virally expressed green ¯uorescent protein
Fig. 3A±F. Expression of GFP in tobacco leaf epidermal cells.
A Epidermal cell in a leaf infected with PVX-GFP demonstrating
high a level of expression of cytoplasmic GFP (compare with C). B
Detail of a PVX-GFP epidermal cell showing that organelles (arrows)
can be observed in negative contrast. C Epidermal cell in a leaf
infected with PVX.sp-GFP. This construct results in extremely low
levels of ¯uorescence in the ER which can only be observed with high
laser power and high gain settings on the confocal microscope. Note
that for comparative purposes the contrast and brightness settings for
A and C were identical. D A heavily infected PVX.sp-GFP leaf
epidermis showing apoplastic accumulation of GFP. E Low
magni®cation of a leaf infected with PVX.sp-GFP-K showing GFP
¯uorescence of the cortical network of ER in several epidermal cells.
Uninfected guard cells with auto¯uorescent central cell walls and
chloroplasts can be seen in the centre of the image. F Epidermal cells
of a leaf infected with PVX.sp-GFP-K showing detail of GFP
¯uorescence in the cortical network of ER. Bars = 15 lm (A±C, F),
30 lm (E), 50 lm (D)
395
Western Blotting. Further con®rmation that the GFP
expressed from PVX.sp-GFP was secreted into the
extracellular space was obtained from western blotting
of intercellular ¯uid extracts from infected leaves
(Fig. 5). Intercellular ¯uid extracts were prepared by
vacuum-in®ltrating leaves with water followed by lowspeed centrifugation to extract the in®ltrated water.
Proteins in the extracts were concentrated using a
protein-binding resin. Relative to the total amount of
GFP in PVX.sp-GFP-infected leaves (a very faint GFP
band was visible in the total protein extract on the
original membrane), the majority of GFP appeared to be
extracellular (Fig. 5b, lanes 3 and 7). Direct quantitative
comparisons between the di€erent constructs were not
possible because of variation in the levels of viral
infection from plant-to-plant and leaf-to-leaf. Blots of
viral coat protein from the di€erent infections indicated
that PVX-GFP plants contained the highest levels of
396
P. Boevink et al.: Transport of virally expressed green ¯uorescent protein
Fig. 4. Transmission electron micrograph
of a tobacco leaf epidermal cell demonstrating the thin layer of cortical cytoplasm.
120 kV, bar = 4 lm Insert: high magni®cation showing cortical endoplasmic reticulum (ER) in epidermal cell cytoplasm.
´45 000
coat protein, and therefore virus, followed by PVX.spGFP-K plants with PVX.sp-GFP plants showing the
lowest levels (Fig. 5a, lanes 5±3 and 9±7). There also
appeared to be substantially less GFP in PVX.sp-GFPand PVX.sp-GFP-K-infected tissue relative to the
amount of virus present and compared to the PVX.GFP
control. It was thought that a proportion of the GFP in
the endomembrane system or the extracellular matrix
may have been precipitated with the cell debris in total
protein extractions, thus preventing true quantitative
comparisons between the di€erent viral constructs.
Cold-induced inhibition of secretion. To determine whether cold shock could inhibit secretion in tobacco cells,
PVX.sp-GFP-infected plants showing systemic symptoms were placed at 4 °C. The GFP ¯uorescence was
visible in the ER after 8±12 h (Fig. 6A compare with
Fig. 3C). The ER-localised ¯uorescence gradually diminished when plants were returned to room temperature until, after 4±6 h, the cells had similar ¯uorescence
to the untreated PVX.sp-GFP-infected cells (Fig. 6B
compare with Fig. 3C). The ER-localised ¯uorescence
also diminished in plants left in the cold for more than
12±18 h (data not shown). Incubation at 10 °C had no
e€ect on PVX.sp-GFP leaves (data not shown).
E€ects of BFA on the ER and on secretion. To determine
whether the secretory inhibitor BFA would also induce a
build up of GFP ¯uorescence in the ER, sp-GFPexpressing leaf segments were treated with 10 lg/ml of
the drug for 8±12 h. These leaf segments showed an
accumulation of GFP ¯uorescence in the ER (Fig. 7A),
and on removal of the drug the ER-localised ¯uorescence gradually faded over 8 h (Fig. 7B). However,
prolonged incubation of leaf segments in BFA (>18 h)
also resulted in a gradual loss of the accumulated
¯uorescence in the ER, indicating a recovery of secretory
ability in the presence of the drug (Fig. 7C). A similar
treatment of PVX.sp-GFP-K-expressing leaves had little
e€ect on the morphology of the ER. However, in leaf
segments treated for 1±4 h with 100 lg/ml BFA, dramatic but reversible morphological changes to the ER
were observed. The cortical ER gradually changed from
the network of tubules typically observed, to large sheets
or lamellae punctured by irregularly sized holes
(Fig. 7D,E). On removal of the BFA, the tubular ER
network, as seen in untreated cells, re-formed in 1±2 h
(Fig. 7F).
Discussion
The signal peptide plus a KDEL sequence was required
for the insertion and retention of GFP in the ER,
although it is likely that some protein escaped the
retention mechanism and was secreted. Green ¯uorescent protein that was targeted to the ER by the fused
signal peptide, but which did not contain KDEL was
barely visible in the ER and appeared to be largely
secreted. We would not expect any retention mechanism
for GFP other than the added KDEL peptide, as it is a
protein foreign to the plant and when expressed without
a signal sequence is normally found in the cytoplasm and
nucleus (Baulcombe et al. 1995). The PVX signal
peptide-GFP-KDEL construct made with the sporamin
signal peptide was markedly brighter than the construct
previously made that used the patatin signal peptide
(Boevink et al. 1996). This suggests that the two signal
peptides may di€er in the eciency with which they
direct protein translocation into the ER.
In PVX.sp-GFP-infected cells the lack of overall
¯uorescence may indicate that, in the majority of the
GFP molecules, the ¯uorophore had not had time to form
in order for the protein to become ¯uorescent before it was
P. Boevink et al.: Transport of virally expressed green ¯uorescent protein
Fig. 5a,b. Western blots of total protein and intercellular ¯uid
extracts from tobacco leaves. a Western blot probed with anti-PVX
coat protein antibody. Lanes 1±9 contain: the PVX coat protein
standard (1), total protein extract from a healthy plant (2), plants
infected with PVX.sp-GFP (3), PVX.sp-GFP-K (4), and PVX.GFP
(5), and intercellular ¯uid extracts from a healthy plant (6), and plants
infected with PVX.sp-GFP (7), PVX.sp-GFP-K (8), and PVX.GFP
(9). Equal amounts of extract were loaded per lane, except for lane 5
where half as much was loaded. b Western blot of the same extracts
shown in a but probed with anti-GFP antibody. Lanes 1±9 contain:
GFP standard (1), total protein extracts from a healthy plant (2), and
plants infected with PVX.sp-GFP (3), PVX.sp-GFP-K (4), and PVXGFP (5), plus intercellular ¯uid extracts from a healthy plant (6), and
plants infected with PVX.sp-GFP (7), PVX.sp-GFP-K (8), and
PVX.GFP (9). Twice as much extract, compared with blot a, was
loaded per lane. Note that in plants infected with PVX.sp-GFP, GFP
can only be detected in intercellular ¯uid extracts
secreted. It is known that for native GFP to fold and
mature into the ¯uorescent form the chromophore has to
cyclise, be oxidised and be encapsulated within the folded
protein, a process which can take from 2±4 h for wild-type
GFP (Cubitt et al. 1995). However, it is possible that the
concentration of GFP in the ER lumen was extremely low
because it was continually being secreted. The very low
levels of ¯uorescence in PVX.sp-GFP-infected leaves may
indicate that most GFP molecules which are secreted into
the apoplast before the ¯uorophore has cyclised and the
protein folded, are unable to mature in the apoplastic
environment. In some older leaves with substantial viral
infection, when high levels of GFP production would be
expected, extracellular ¯uorescence of GFP could be
detected (Fig. 3D). This, combined with the fact that GFP
has an inherent stability (Cubitt et al. 1995), makes the
alternative explanation that the protein is proteolytically
cleaved in the cell wall and/or apoplastic space less likely.
397
Fig. 6A,B. E€ect of cold on GFP secretion in tobacco leaves. A After
12 h at 4 °C, GFP ¯uorescence in the ER of PVX.sp-GFP infected
leaves is apparent, indicating inhibition of export of GFP from the
ER. B After returning cold-shocked plants to room temperature for
12 h most ¯uorescence is lost from the ER, indicating a resumption of
secretion. Note the bright GFP-containing viral inclusion in the cell.
Bars = 20 lm (A), 10 lm (B)
Cold shock. A short period of cold shock appeared to
block transport from the ER to the Golgi, allowing GFP
¯uorescence to build up to easily detectable levels in the
ER of PVX.sp-GFP-infected cells in a manner similar to
BFA treatment of the same leaves. Thus, it appears that
one response of tobacco plants to cold is an inhibition of
post-ER secretion. However, when left at low temperatures for longer periods the plants acclimatised to the
new conditions and resumed secretion. Whilst temperature blocks to secretion are widely reported in mammalian cell systems (Roman and Garo€ 1985; Presley
et al. 1997), there is little information of the e€ect of
temperature on the higher-plant secretory pathway.
Some reports have suggested that a block of vesicle
and lipid transfer between the ER and Golgi may take
place at a temperature as high as 12 °C (SturboisBalcerzak et al. 1995), although when PVX.sp-GFP
tobacco plants were incubated at 10 °C we detected no
increase in ER ¯uorescence.
E€ects of BFA on secretion and ER morphology.
Treatment of PVX.sp-GFP-infected cells with relatively
low concentrations of BFA resulted in accumulation of
GFP in the ER whereas in control leaves minimal
¯uorescence in the ER was detected. This is a clear
398
P. Boevink et al.: Transport of virally expressed green ¯uorescent protein
Fig. 7A±F. E€ects of BFA on secretion of GFP and ER morphology
in tobacco leaves. A Fluorescence of GFP in the lumen of the ER of a
PVX.sp-GFP-infected leaf after incubation in 10 lg/ml BFA for 12 h,
indicating inhibition of secretion of GFP from the ER. Note several
bright viral inclusions are also present in the cortical cytoplasm. B Leaf
epidermal cell after removal of BFA and incubation in distilled water
for 8 h. Secretory activity has resumed and ER ¯uorescence is lost.
Compare with Fig. 3B. C Leaf epidermal cell after prolonged
incubation (18 h) in 10 lg/ml BFA. Note that ER ¯uorescence is
again lost, indicating recovery of secretory capability in the presence
of the drug. Note the guard cells to the left of the micrograph. D Early
stages of morphological changes to the cortical ER network of a
PVX.sp-GFP-K-infected leaf after treatment for 2±3 h with 100 lg/ml
BFA. Note the formation of lamellae of ER compared to untreated
leaf cells (Fig. 3D). E An epidermal leaf cell after 4 h treatment with
100 lg/ml BFA. Note the conversion of the cortical ER network in
two cells into fenestrated sheets of membrane. Small ¯uorescent
bodies can be seen on the surface of the membrane. F Recovery of the
cortical ER network 6 h after removal of the BFA. Bars = 15 lm
in-vivo demonstration of the inhibition of secretion in
plant cells by BFA. Thus, the lack of substantial ER
¯uorescence in PVX.sp-GFP-infected cells (Fig. 3C)
must re¯ect the fact that GFP was indeed secreted. We
assume that the block is in a transport step between the
ER and the cis-Golgi, although from these experiments a
trans-Golgi blockage of secretion cannot be ruled out.
However, we did not observe a build up of ¯uorescent
structures that could be attributed to GFP accumulation
in the Golgi stacks, the bright spots of ¯uorescence on
the ER being smaller than would be expected for Golgi
bodies (Fig. 7E). This result is also supported by the fact
that recently we have shown that a similar BFA
treatment can result in the destruction of GFP-tagged
tobacco leaf Golgi (Boevink et al. 1998). It has previously been shown that BFA treatment inhibits ER-toGolgi transport (Matsuoka et al. 1995) and secretion in
plant cells (Driouich et al. 1993; Satiat-Jeunemaitre and
P. Boevink et al.: Transport of virally expressed green ¯uorescent protein
Hawes 1994; Satiat-Jeunemaitre et al. 1996) and also
results in a trans-Golgi accumulation of secretory
products (Satiat-Jeunemaitre and Hawes 1993). However, this is the ®rst in-vivo visual indication that
blockage of the secretory pathway can lead to an
accumulation of secretory product in the lumen of the
ER. It is also interesting to note that prolonged
incubation of leaf segments in BFA resulted in eventual
loss of ¯uorescence of the ER. It is assumed that this is
attributable to the secretory machinery being re-established in the presence of the drug. This time-dependent
acquisition of resistance to BFA has also been reported
in maize roots where Golgi stacks vesiculated in the
presence of a high concentration of BFA (2 h in 100 lg/
ml BFA) but with time eventually re-formed (Steele
1997). This work also demonstrated that BFA retained
activity after material had been incubated in the drug for
24 h, supporting our conclusion that in tobacco leaves
we are observing some form of induced resistance to
prolonged exposure to BFA.
The application of high concentrations of BFA
induced rapid and dramatic changes to the morphology
of the ER which were reversible on removal of the drug.
The change from the cortical network of tubules to large
¯at membrane lamellae was di€erent from changes seen in
the ER of BFA-treated root cells (Satiat-Jeunemaitre
et al. 1996). Here, it was shown that the ER in meristematic root tips cells, when visualised after immuno¯uorescence detection with an HDEL antibody, aggregated
into tubular and lamellar clusters in the cytosol. It has
previously been demonstrated that the morphology of
cortical ER is extremely sensitive to pharmacological (e.g.
calcium chelators, cytoskeletal inhibitors) and environmental perturbations (e.g. heat stress), showing considerable pleomorphy between tubular and lamellar forms
(Quader et al. 1996). However, such a dramatic morphological change in response to a secretory inhibitor has not
previously been reported. One explanation is that in the
viral expression system, high levels of GFP-KDEL
accumulate in the lumen of the ER, saturating the
retention/retrieval mechanism of the cell. Treatment with
BFA would then block this secretion of excess protein and
lead to a further build up in the ER, resulting in a dramatic
change in morphology and lumen volume to accommodate the GFP-KDEL and any other secretory proteins.
On removal of the drug, secretion resumes and the
morphology of the ER reverts to the normal state.
Alternatively, a direct action of the drug on the ER
membrane cannot be excluded as it has been reported that
BFA can in¯uence the synthesis and transport of lipids
from the ER (Slomiany et al. 1993).
Associated with the sheets of ER in BFA-treated cells
were small irregular ¯uorescent structures. It is assumed
that these are either aggregations of GFP or virus
protein associated with GFP. It is unlikely that they
represent Golgi stacks, or the remains of Golgi stacks, as
in the tobacco leaf system the Golgi is extremely
sensitive to shorter (less than 30 min) treatments of
BFA (Boevink et al. 1998).
399
We thank Mr. Denton Prior (SCRI) for help with confocal
microscopy, Dr. L. Faye (CNRS, Rouen, France) for supplying
sporamin constructs and Dr. B. Satiat-Jeunemaitre (CNRS, Gifsur-Yvette, France) for critical reading of the manuscript. This
work was supported by a grant from the Leverhulme Trust.
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