Download Native and Artificial Reticuloplasmins Co

Native and Artificial Reticuloplasmins Co-Accumulate in
Distinct Domains of the Endoplasmic Reticulum and in
Post-Endoplasmic Reticulum Compartments1
Esperanza Torres, Pablo Gonzalez-Melendi, Eva Stöger, Peter Shaw, Richard M. Twyman, Liz Nicholson,
Carmen Vaquero, Rainer Fischer, Paul Christou, and Yolande Perrin2*
Molecular Biotechnology Unit (E.T., P.G.-M., E.S., R.M.T., L.N., P.C., Y.P.) and Cell Biology Department
(P.G.-M., P.S.), John Innes Centre, Colney Lane, Norwich NR4 7UH, United Kingdom; Institut für Biologie I
(Botanic/Molekulargenetik), RWTH Aachen, Worringerweg 1, D–52074 Aachen, Germany (C.V., R.F.); and
Fraunhofer Abteilung für Molekulare Biotechnologie, IUTC, Grafschaft, Auf dem Aberg 1, D–57392
Schmallenberg, Germany (R.F.)
We compared the subcellular distribution of native and artificial reticuloplasmins in endosperm, callus, and leaf tissues of
transgenic rice (Oryza sativa) to determine the distribution of these proteins among endoplasmic reticulum (ER) and post-ER
compartments. The native reticuloplasmin was calreticulin. The artificial reticuloplasmin was a recombinant single-chain
antibody (scFv), expressed with an N-terminal signal peptide and the C-terminal KDEL sequence for retrieval to the ER
(scFvT84.66-KDEL). We found that both molecules were distributed in the same manner. In endosperm, each accumulated
in ER-derived prolamine protein bodies, but also in glutelin protein storage vacuoles, even though glutelins are known to
pass through the Golgi apparatus en route to these organelles. This finding may suggest that similar mechanisms are
involved in the sorting of reticuloplasmins and rice seed storage proteins. However, the presence of reticuloplasmins in
protein storage vacuoles could also be due to simple dispersal into these compartments during protein storage vacuole
biogenesis, before glutelin deposition. In callus and leaf mesophyll cells, both reticuloplasmins accumulated in ribosomecoated vesicles probably derived directly from the rough ER.
Plant proteins targeted to the secretory pathway
can be directed to many different intracellular sites,
including the endoplasmic reticulum (ER), Golgi apparatus (GApp), vacuoles, tonoplast, and plasma
membrane. They may also be directed to the extracellular space (apoplasm) or cell wall matrix, as well
as to the external environment. Therefore, the secretory pathway is of fundamental importance, not only
for those proteins destined ultimately to be secreted,
but also for those with functions in the compartments
en route. The ER is the point of entry into the secretory pathway, and proteins targeted to the ER are
distinguished from cytosolic proteins by the presence
of an N-terminal signal peptide that associates with a
ribonucleoprotein structure called the signal recognition particle (for review, see Vitale and Denecke,
1999). The signal recognition particle enables cytosolic ribosomes to bind to the ER membrane, and pro1
This work was supported in part by the Instituto Colombiano
para el Desarrollo de la Ciencia y la Technologı́a “Francisco José de
Caldas” (COLENCIAS; PhD fellowship to E.T.).
2
Present address: Université de Technologie de Compiègne,
Unité Mixte de Recherche 6022 Centre National de la Recherche
Scientifique, BP 20529 – 60205 Compiègne cedex, France.
* Corresponding author; e-mail [email protected]; fax
33– 0 –344 –234300.
Article, publication date, and citation information can be found
at www.plantphysiol.org/cgi/doi/10.1104/pp.010260.
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teins destined for the secretory pathway are cotranslationally translocated into lumen of the ribosomecoated (rough) ER, a process conserved among all
eukaryotes (for review, see Galili et al., 1998). Once
inside the ER lumen, the signal peptide is removed
from the nascent polypeptide and the cleaved
polypeptide associates with ER-resident enzymes
and molecular chaperones that help to fold it into its
native conformation. Other posttranslational modifications, such as multisubunit assembly and glycosylation, also begin in the ER lumen, with the assistance
of molecular chaperones.
Many proteins entering the ER have a particular
function at specific points in the secretory pathway.
Such proteins are targeted to their intended destinations by signals within the polypeptide chain, in the
form of specific amino acid sequences or particular
structural determinants (for review, see Neuhaus and
Rogers, 1998). For example, most soluble, ERresident proteins (known as reticuloplasmins) contain a C-terminal retrieval signal comprising the consensus tetrapeptide H/KDEL, allowing the proteins
that escape to the GApp to be taken back into the ER
(for review, see Vitale and Denecke, 1999).
In plants, the ER system appears more versatile
than in animals and yeast. The plant ER is, for example, important for the biogenesis of protein storage
organelles and oil bodies. Furthermore, the ER plays
an important role in cell-cell communication because
Plant Physiology, November 2001, Vol. 127, pp. 1212–1223, www.plantphysiol.org © 2001 American Society of Plant Biologists
Reticuloplasmins in the Endoplasmic Reticulum Network
it can form a continuous network among cell populations through plasmodesmata (for review, see Staehelin, 1997). Investigation of the ER system has revealed a complex collection of distinct subdomains
that correspond to particular cellular functions.
Therefore, the secretory pathway of plants has been
the subject of intensive investigation, particularly
with respect to seed storage proteins because of their
nutritional importance for both plants and animals
(for review, see Müntz, 1998). Furthermore, because
the stable expression of heterologous proteins at high
levels can depend on their accumulation in appropriate organelles, such studies may also assist the optimization of recombinant protein synthesis in transgenic plants. In this respect, much remains to be
learned about the functional organization of the ER,
e.g. pertaining to the distribution of reticuloplasmins
and the mechanisms of protein trafficking from the
ER along the rest of the secretory pathway. Ultrastructural studies have shown that reticuloplasmins,
such as molecular chaperones, can also be found in
post-ER compartments of the seed, such as protein
bodies (PBs) or protein storage vacuoles (Levanony
et al., 1992; Robinson et al., 1995). The presence of
chaperones such as binding protein (BiP) in PBs or
protein storage vacuoles (PSVs; where storage proteins are assembled) raises questions about the functional significance of chaperone distribution in
post-ER compartments.
To determine how reticuloplasmins are distributed
among the post-ER compartments and whether this
distribution is governed entirely by their function, or
whether passive diffusion also plays a role, we investigated the distribution of two reticuloplasmins:
a recombinant mammalian protein targeted to the
ER (an artificial reticuloplasmin, with no function in
the plant cell) and a native plant reticuloplasmin
with an important biological role. The artificial reticuloplasmin was a single-chain antibody fragment
(scFvT84.66) carrying the KDEL signal (scFvT84.66KDEL) for ER retrieval (Torres et al., 1999; Stöger et
al., 2000). ScFvT84.66-KDEL is derived from
mAbT84.66, a monoclonal antibody specific for the
NA3 epitope of the carcionoembryonic antigen
(Tsang et al., 1995). We compared its distribution
with that of the ER-resident molecular chaperone
calreticulin. Rice (Oryza sativa) was chosen as a model
system due to the existence of differential sorting
pathways for the two major seed storage proteins,
prolamines and glutelins, reflecting the existence of
functionally distinct subdomains of the ER (Okita et
al., 1998). Prolamines accumulate in PBs inside the
rough ER, whereas glutelins accumulate in protein
storage vacuoles, and are conveyed to these organelles by transport vesicles budding from the
GApp. We also followed the distribution of reticuloplasmins in two other tissues, leaf and callus, allowing us to investigate any possible links between the
distribution of reticuloplasmins and the metabolic
Plant Physiol. Vol. 127, 2001
function of the plant tissue. We showed that the
artificial and native reticuloplasmins have identical
distribution profiles within the ER network of each
tissue. In endosperm, both reticuloplasmins are
found alongside prolamines accumulating in ER prolamine PBs. However, they are also found alongside
glutelins in PSVs. Glutelins pass through the GApp
before accumulating in PSVs; therefore, the presence
of reticuloplasmins in such vacuoles was unexpected.
We discuss the implications of our results in terms of
functional subdivision of the ER network in plants,
and possible mechanisms by which the accumulation
of reticuloplasmins in post-ER organelles could occur.
RESULTS
Subcellular Localization of scFvT84.66-KDEL and
Calreticulin in the Endosperm of Rice Seeds
Ultrastructural analysis of rice endosperm from
14 d after anthesis (DAA) immature embryos revealed an extensive cisternal ER network distributed
homogeneously through the cytoplasm. Endosperm
cells also contained a large number of membranedelimited structures filled with electron dense deposits resembling glutelin and prolamine PBs (Fig. 1).
The fixation method using 4% (w/v) formaldehyde
was chosen to compromise between a good structural
preservation and antigen preservation. Fixation using 2% (w/v) glutaraldehyde with 4% (w/v) formaldehyde permitted a slightly better preservation of the
tissues (Fig. 2) but was not compatible with immunogold labeling. To provide the best possible structural preservation, the processing of the samples was
carried out at low temperature. With such a fixation
protocol, the classical structures of the different tissues analyzed were clearly recognizable.
Immunogold labeling showed that the antibody
fragment did not accumulate in the cisternal ER, but
did accumulate in the two types of PBs observed. The
labeling was visible inside spherical bodies delimited
by ribosome-associated membranes (Fig. 3) previously described by others as prolamine PBs (Bechtel
and Juliano, 1980; Krishnan et al., 1986; Li et al.,
1993a). The recombinant antibody fragment was also
localized in dense, irregularly shaped structures (Fig.
3), resembling glutelin PBs (Krishnan et al., 1986).
The distribution of scFvT84.66-KDEL in the endosperm secretory system was further characterized
by double immunogold labeling with antisera specific for the ER-resident chaperone calreticulin. The
secondary antibody used to visualize calreticulin was
coupled to 15-nm gold particles to distinguish these
from the 10-nm particles used to label scFvT84.66KDEL. This experiment showed that calreticulin was
also found predominantly in prolamine and glutelin
PBs (Fig. 4A). Labeling of these PBs with the anticalreticulin antiserum was observed not only in the
endosperm of transgenic seeds but also in the endosperm of wild-type seeds (Fig. 4B). A light labeling
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Figure 1. Immunolocalization of scFvT84.66-KDEL in rice endosperm cells 14 DAA. The 10-nm gold particles reveal that
scFvT84.66-KDEL is localized in two types of PBs: spherical prolamine PBs (Pro-PB) and amorphous glutelin PBs (Glu-PB).
CER, Cisternal ER; CT, cytosol; N, nucleus. Bar ⫽ 0.5 ␮m.
for calreticulin was also observed inside the cisternal
ER (Fig. 4A).
Double labeling for scFvT84.66-KDEL and the storage proteins prolamine and glutelin was used to
characterize the distribution of the recombinant an1214
tibody in protein storage organelles. Again, 15-nm
gold particles were used to label the storage proteins.
Double immunogold labeling for scFvT84.66-KDEL
and prolamine showed that the 10- and 15-nm gold
particles colocalized in the spherical structures
Plant Physiol. Vol. 127, 2001
Reticuloplasmins in the Endoplasmic Reticulum Network
Figure 2. Section of an endosperm cell from tissue fixed with a mixture of 4% (w/v) formaldehyde and 2% (w/v)
glutaraldehyde. The addition of glutaraldehyde in the fixation solution allowed a slightly better fixation of the tissue in
comparison with a fixation protocol using only formaldehyde as for the tissue shown in Figure 1. However, the use of
glutaraldehyde was not compatible with immunogold labeling. A, Amyloplast; CER, cisternal ER; Glu-PB, glutelin PB; n,
nucleus; Pro-PB, prolamine PB. Bar ⫽ 0.5 ␮m.
thought to be prolamine PBs (Fig. 5A). This result
confirmed that the recombinant antibody had accumulated in prolamine PBs, which are known to be
formed inside the ER. In addition, double labeling for
scFvT84.66-KDEL and glutelin showed colocalization
of 10- and 15-nm gold particles in the amorphous
structures thought to represent glutelin PBs (Fig. 5B).
Finally, Golgi-associated vesicles (SV-GApps) that
were clearly labeled by the antiglutelin antiserum
(Fig. 5C) were not labeled with the antiprolamine
(Fig. 5A), the anti-scFvT84.66 (Fig. 5, A and C), or the
anticalreticulin antisera. These vesicles appeared
closely associated with lamellar structures (Fig. 5C)
clearly identified as belonging to the GApp using a
rabbit anti-Lewis antiserum (Fitchette et al., 1999;
data not shown). No background was observed for
the scFvT84.66-KDEL antibody and no labeling of
Plant Physiol. Vol. 127, 2001
scFvT84.66-KDEL
samples.
was
observed
in
wild-type
Localization of scFvT84.66-KDEL and Calreticulin in
Callus and Mesophyll Cells
Callus and leaf mesophyll cells were also examined
to determine the distribution of scFvT84.66-KDEL
and the endogenous reticuloplasmin in non-storage
tissues. Callus cells had a dense cytoplasm rich in
ribosomes, but only a sparse cisternal ER and Golgi
network (Fig. 6A). They also contained numerous
vesicles filled with dense deposits (Fig. 6B). These
vesicles appeared to form a network within the cytoplasm and we observed interconnection between
vesicles, suggesting that large vesicles might be
formed by the fusion of smaller ones (Figs. 6B and
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Figure 3. Immunolocalization of scFvT84.66-KDEL in prolamine and glutelin PBs. Glutelin and prolamine PBs are labeled
with anti-scFvT84.66 (10-nm gold particles). Note one prolamine PB being formed inside an ER cistern. Arrows highlight
areas of ribosome-studded membranes surrounding the prolamine PBs. CER, Cisternal ER; Glu-PB, glutelin PB; Pro-PB,
prolamine PB. Bar ⫽ 0.5 ␮m.
7A). Ribosomes appeared associated with the membrane delimiting these vesicles in certain areas (Figs.
6B and 7A). The labeling of scFvT84.66-KDEL on
sections from transgenic material was restricted to
the vesicles (Fig. 6B), whereas no labeling was observed in the cisternal ER or in other cell
compartments.
In the mesophyll cells, most of the cytosolic volume
was occupied by a single large vacuole, whereas
other organelles were restricted to the cell periphery.
The mesophyll cells, unlike those of callus tissue,
contained fully differentiated chloroplasts (Fig. 6C).
Vesicles filled with electron-dense deposits, similar
to those seen in the callus cells, were present in the
mesophyll. However, the mesophyll vesicles did not
appear to form extensive interconnections, so that the
vesicle size was more homogeneous. Furthermore,
the vesicles generally appeared in close proximity to
plasmodesmata (Fig. 6C). Immunolabelling with
scFvT84.66-specific antiserum revealed that the
scFvT84.66-KDEL had accumulated in the same type
of vesicles as seen in callus cells (Fig. 6, B and C).
Gold particles were also observed occasionally between the inner and outer membranes of the nucleus
(Fig. 6D).
Double labeling using anti-scFvT84.66 and anticalreticulin antisera clearly showed colocalization of the
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two proteins in the vesicles present in callus (Fig. 7A)
and mesophyll cells (Fig. 7B).
DISCUSSION
Distribution of Reticuloplasmins in the Cisternal ER of
Endosperm Cells
Endosperm cells contained an abundant cisternal
ER network, very different from the limited network
seen in callus and leaf cells as discussed below. This
probably reflected the differential metabolic activity
of the tissues: Developing seeds are engaged in intensely active protein biosynthesis, requiring a more
extensive ER network, whereas the other tissues we
examined would be expected to show less active
protein biosynthesis. Despite the abundance of the
cisternal ER, there was a consistent lack of
scFvT84.66-KDEL labeling, although some light labeling for calreticulin was observed. It is unlikely
that this reflects genuinely different sorting of the
endogenous reticuloplasmin and recombinant antibody. Rather, it is likely that some scFvT84.66-KDEL
does remain in the cisternal ER, but at a level that is
below the detection threshold of the immunological
technique used. The detection of only small amounts
of calreticulin probably indicates that the resolution
Plant Physiol. Vol. 127, 2001
Reticuloplasmins in the Endoplasmic Reticulum Network
Figure 4. Double immunolocalization of scFvT84.66-KDEL with calreticulin in endosperm cells and immunolocalization of
calreticulin in wild-type tissue. A, Glutelin and prolamine bodies are labeled with anti-scFvT84.66 (10-nm gold particles)
and anticalreticulin (15-nm gold particles). Arrows highlight areas of ribosome-studded membranes surrounding the
prolamine PBs. B, Labeling of prolamine and glutelin PBs with the anticalreticulin antiserum in endosperm cells of wild-type
seeds. CW, Cell wall; Glu-PB, glutelin PB; Pro-PB, prolamine PB. Bar ⫽ 0.5 ␮m.
of our detection methodology only allows the detection of soluble proteins when they concentrate into
aggregates such as in PBs or PSVs.
Accumulation of Reticuloplasmins in Prolamine PBs
Immunogold labeling of endosperm tissue clearly
showed accumulation of the recombinant antibody
inside prolamine PBs. We propose that the antibody
is incorporated into the PBs due to its presence in the
subdomain of the ER from which the PBs arise. The
PBs are formed inside the ER following aggregation
of prolamine in the lumen, with no specific transport
mechanism involved (Li et al., 1993a). The native
chaperone calreticulin was also detected inside the
prolamine PBs. Considering the strong biological and
Plant Physiol. Vol. 127, 2001
physical associations between calreticulin and another soluble chaperone BiP (Crofts et al., 1998), the
presence of calreticulin inside prolamine PBs is in
accordance with the presence of BiP in these structures as previously shown (Li et al., 1993b; Muench et
al., 1997). The presence of BiP at the periphery of
prolamine PBs has been linked clearly with its involvement in the biogenesis of these structures
through a role in polypeptide translocation across the
ER membrane and polypeptide folding. The presence
of another chaperone, calreticulin, also involved in
protein folding, is also likely to reflect its biological
role. However, the accumulation of a recombinant
mammalian protein such as scFvT84.66-KDEL inside
prolamine PBs almost certainly reflects a passive
trapping phenomenon during PB biogenesis because
this protein has no function in the plant cell.
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Figure 5. Double immunolocalization of scFvT84.66-KDEL with prolamine and glutelin in endosperm cells. A, Prolamine
PBs were labeled with anti-scFvT84.66 (10-nm gold particles) and antiprolamine (15-nm gold particles). Note that no
labeling is detected in small vesicles closely associated with the GApp (SV-GApp). B, Glutelin PBs are labeled with both
anti-scFvT84.66 antiserum (10-nm gold particles) and antiglutelin antiserum (15-nm gold particles). C, The SV-GApp are
only labeled by antiglutelin antiserum after double labeling with anti-scFvT84.66 and antiglutelin antisera. CER, Cisternal
ER; Glu-PB, glutelin PB; Pro-PB, prolamine PB. Bar ⫽ 0.5 ␮m.
Accumulation of Reticuloplasmins in Glutelin Protein
Storage Vacuoles
The detection of scFvT84.66-KDEL in glutelin storage vacuoles was unexpected because glutelins are
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known to pass through the GApp before deposition
into vacuoles (for review, see Muench and Okita,
1997). The colocalization of scFvT84.66-KDEL with
glutelins could reflect imperfect retention of the recombinant antibody, leading to a partial release from
Plant Physiol. Vol. 127, 2001
Reticuloplasmins in the Endoplasmic Reticulum Network
Figure 6. Immunolocalization of scFvT84.66-KDEL in rice callus and leaf mesophyll cells. A, Ultrastructure of callus cells
at low magnification; B, higher magnification picture showing a network of small vesicles surrounded by ribosome-coated
membranes (see arrows) where scFvT84.66-KDEL accumulates. C, In mesophyll cells, scFvT84.66-KDEL labeling is detected
in similar vesicles that are found mainly in close proximity to plasmodesmata. D, Section of mesophyll cell revealing gold
particles between the inner and outer membranes of the nucleus (see arrows). C, Chloroplast; CT, cytosol; CW, cell wall;
LV, lytic vacuole; N, nucleus; NE, nuclear envelope; PLA, plasmodesmata; Vs, vesicles. Bar ⫽ 0.5 ␮m.
the ER despite the presence of a KDEL signal.
H/KDEL retrieval signals are sufficient to confer ER
localization when fused to non-ER proteins but their
effectiveness can vary (Zagouras and Rose, 1989;
Herman et al., 1990; Denecke et al., 1993; Pueyo et al.,
1995; Gomord et al., 1997). It is possible that adjacent
sequences or the overall three-dimensional structure
of the protein may influence retention efficiency by
influencing the accessibility of the H/KDEL signal to
the receptors involved in the retrieval mechanism.
Plant Physiol. Vol. 127, 2001
We consider this “leaky retention” model insufficient to explain the presence of scFvT84.66-KDEL in
PSVs because the endogenous reticuloplasmin, calreticulin, was also localized to glutelin storage vacuoles. The presence of calreticulin in glutelin PSVs is
not associated with the expression of scFvT84.66KDEL because the same localization pattern was observed in wild-type material. There have been no
reports of leaky retention of calreticulin except when
the protein was heavily overexpressed, in which case
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Torres et al.
Figure 7. Double immunolocalization of scFvT84.66-KDEL with calreticulin in rice callus and leaf mesophyll cells.
ScFvT84.66-KDEL (labeled with 10-nm gold particles) and calreticulin (labeled with 15-nm gold particles) colocalize in the
vesicles in both callus (A) and mesophyll (B) cells. Arrows highlights areas where a ribosome-coated membrane surrounding
the vesicles can be seen. CW, Cell wall; LV, lytic vacuole; Vs, vesicles. Bar ⫽ 0.5 ␮m.
it is likely that the retrieval machinery in the cell
became saturated (Crofts et al., 1999). In our case,
protein overexpression cannot explain the presence
of both native and artificial reticuloplasmins inside
post-ER compartments. In the transgenic lines,
scFvT84.66-KDEL was expressed at only 30 ␮g g⫺1
fresh weight in endosperm, corresponding to 0.1%
of the total soluble proteins, which is too low to be
described as “overexpression” (Stöger et al., 2000).
In addition, there is no indication that the distribution of calreticulin is linked to its overexpression in
transgenic tissue expressing scFvT84.66-KDEL because the distribution pattern of the chaperone was
found to be identical in corresponding wild-type
tissues.
Our data could suggest the existence of a Golgiindependent route to PSV formation in rice endosperm. Such a pathway has been proposed for
prolamine accumulation in developing wheat (Triticum aestivum) endosperm (Levanony et al., 1992) and
proglobulin accumulation in maturing pea (Pisum
sativum) and pumpkin (Cucurbita pepo) seeds (Robinson et al., 1995; Hara-Nishimura et al., 1998). Wheat
prolamines and pea and pumpkin globulins are
known to pass through the GApp, but the presence of
BiP in the resulting PSVs has led to suggestions that
an alternative pathway may bypass the Golgi. Like
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wheat prolamines and most pea and pumpkin globulins, rice glutelins are not glycosylated (Shewry et
al., 1995; Müntz, 1998) and passage through the Golgi
may not be obligatory for correct posttranslational
modification. Despite these reports, we found no direct evidence for an alternative pathway for glutelin
transport. For example, we did not observe the presence of storage protein aggregates inside the ER lumen, as described for pea and pumpkin, or specialized transport vesicles closely associated with the ER,
as described for pumpkin. Another possible explanation is the incorporation of both native and artificial
reticuloplasmins in PSVs independently from glutelin transport. Considering the interconnection between the different ER subdomains, it is conceivable
that some reticuloplasmins could escape their site of
synthesis and biological activity—the rough ER—by
a simple dispersal phenomenon. This would allow
them to reach adjacent regions of the smooth ER from
where PSVs are thought to originate (Staehelin,
1997). The absence of labeling for scFv and calreticulin inside Golgi-associated vesicles, which appear to
transport glutelin from the Golgi to the PSVs, could
represent indirect evidence for the independent
transport of glutelins and reticuloplasmins to PSVs.
We cannot, however, eliminate the possibility that
calreticulin and scFvT84.66-KDEL could be present
Plant Physiol. Vol. 127, 2001
Reticuloplasmins in the Endoplasmic Reticulum Network
in these vesicles at a level below the detection limit of
the immunological technique used.
Distribution of Reticuloplasmins in Callus and Leaf
Mesophyll Cells
In callus and leaf mesophyll, there was a muchreduced cisternal ER network reflecting the lower
metabolic activity of the callus and leaf mesophyll
compared with endosperm. In both tissues, comparison of the distribution of scFvT84.66-KDEL and calreticulin indicated colocalization of the proteins in
ribosome-coated vesicles, and their absence from the
limited cisternal ER. In a few mesophyll samples, the
antibody was also detected inside the nuclear envelope, confirming the continuity of this nuclear domain with the ER in many cell types (Staehelin, 1997).
This phenomenon of nuclear retention, which is attributable to partial redirection due to the retrieval
effect of KDEL, has been described for other proteins
fused to KDEL, such as a recombinant phytohaemagglutinin (Herman et al., 1990) and overexpressed
calreticulin (Crofts et al., 1999).
In callus and leaf mesophyll cells, vesicles were the
main sites of scFvT84.66-KDEL and calreticulin accumulation. These structures were filled with electrondense deposits, and in callus cells they appeared to
fuse together to generate larger structures. Both these
observations indicated a role in protein deposition. In
mesophyll cells, the presence of a large central vacuole may have prevented the fusion of these smaller
vesicles. Although probably involved in protein deposition, these structures were not typical PSVs because PSVs are only found in vegetative tissues specially adapted for protein accumulation (for review,
see Marty 1999). Moreover, the presence of calreticulin inside the vesicles, and of ribosomes coating their
membrane in some locations, suggests the vesicles
are derived directly from the rough ER and are involved in protein synthesis as well as deposition.
Protein accumulation in the rough ER has been
reported in vegetative tissues following the overexpression of recombinant proteins, leading to the formation of ER-derived PBs (Wandelt et al., 1992;
Bagga et al., 1995, 1997). However, the vesicles we
observed are not generated in response to
scFvT84.66-KDEL expression because identical structures were present in wild-type mesophyll cells. We
are unaware of previous reports describing the emergence of organelles from the ER of vegetative tissues,
with the exception of specific examples concerning
rubber and oil bodies (Galili et al., 1998). The identification of ER-derived organelles combining synthetic and storage activities in callus and mesophyll
cells is an interesting finding that could add to the
already extensive list of functional ER subdomains
(Staehelin, 1997).
In summary, we have investigated the distribution
of an artificial reticuloplasmin, a recombinant singlePlant Physiol. Vol. 127, 2001
chain antibody carrying a KDEL signal for ER retrieval, in the secretory pathway of rice endosperm
and two other tissues, callus and leaf. We showed
that the distribution of the antibody was very similar
to that of the endogenous reticuloplasmin calreticulin, whose function is of great importance to the plant
cell. We found that the antibody and calreticulin
were deposited into both ER-derived prolamine PBs
and glutelin PSVs, but we did not find any clear
evidence for an alternative glutelin transport pathway bypassing the Golgi that could explain the presence of reticuloplasmins in post-ER compartments.
Therefore, it is possible that the simple dispersal of
reticuloplasmins within the ER network, and more
precisely within the smooth ER, could lead to their
trapping inside emerging PSVs. The codistribution of
the artificial reticuloplasmin and calreticulin strongly
indicates that the distribution of reticuloplasmins
within, and even outside, the ER network is not
necessarily driven by their biological function, but
could also result to some extent from a simple dispersal phenomenon.
MATERIALS AND METHODS
Plant Material
Transgenic rice (Oryza sativa) callus cultures and plants
expressing the single chain recombinant antibody fragment
scFvT84.66-KDEL were generated by particle bombardment as described previously (Torres et al., 1999; Stöger et
al., 2000). The transformation construct was designed such
that the recombinant antibody was expressed under the
control of the constitutive maize ubiquitin-1 promoter
(plus first intron) and contained an N-terminal signal peptide derived from the murine immunoglobulin heavy-chain
cDNA, and a C-terminal ER retrieval signal, KDEL. These
two peptides ensured that recombinant antibodies were
targeted to the secretory system and retained in the ER
lumen.
Specimen Processing for Electron Microscopy
Immunolocalization experiments were performed on ultrathin tissue sections from plant material isolated from
our best expressing scFvT84.66-KDEL transgenic lines
(Torres et al., 1999; Stöger et al., 2000). Endosperm was
dissected from developing immature seeds, 14 DAA. Callus tissue was sampled after 3 weeks in subculture, and
leaves were harvested when fully expanded. Controls were
taken from wild-type plant tissue at the equivalent developmental stages.
Tissue samples were cut into small pieces with a razor
blade in phosphate-buffered saline (PBS) buffer (140 mm
NaCl, 3 mm KCl, 4 mm Na2HPO4, and 2 mm KH2PO4 pH
7.4). They were then fixed overnight in 4% (w/v) formaldehyde in PEM buffer (50 mm PIPES [1,4-piperazinediethanesulfonic acid]/KOH, pH 6.9; 5 mm EGTA, and 5 mm MgSO4,
pH 7.4) at 4°C. After washing in PBS, the tissue samples
were transferred to 30% (v/v) ethanol for 1 h at 4°C and
1221
Torres et al.
then to a cold box (Thor Industrial Cryogenics, Oxford) for
further processing at ⫺20°C. The specimens were dehydrated through an ethanol series (50%, 70%, and 100%
[v/v], with a 1-h incubation in each solution) and infiltrated with LR white resin containing 0.5% (w/v) benzoin
methyl ether as the catalyst (London Resin Company Ltd.,
Berkshire, UK). The resin was diluted 1:1, 1:2, and 1:3
(ethanol:resin, v/v) and a 1-h incubation was allowed for
each dilution. The specimens were then incubated overnight in undiluted resin and transferred to precooled Beem
capsules (Agar Scientific, Stansted, UK) filled with freshly
prepared resin. The resin was allowed to polymerize for
24 h under indirect UV light at ⫺20°C followed by 16 h
under indirect UV light at room temperature. From these
specimens, 100-nm sections were prepared using an Ultracut E ultramicrotome (Leica Microsystems, Wetzlar,
Germany). The sections were collected on 4% (w/v) pyroxylin and carbon-coated 200-mesh grids. Ultrastructural observations were carried out using a JEOL 1200 transmission
electron microscope running at 80 kV.
Single Immunogold Labeling
The grids were initially floated on drops of PBS and then
transferred to 3% (w/v) bovine serum albumin in PBS for
5 min at room temperature to block nonspecific binding
sites. The grids were then incubated for 1 h at room temperature with anti-scFvT84.66, anticalreticulin, antiprolamine, or antiglutelin antisera applied at dilutions of 1:400,
1:2,500, 1:30,000, and 1:40,000, respectively, in PBS. AntiscFvT84.66 antiserum was obtained from immunized
chickens as described (Vaquero et al., 1999). Antiprolamine
and antiglutelin antisera were kindly provided by Dr.
Thomas Okita (University of Washington, Pullman) and
were obtained from rabbits injected with rice prolamines or
rice glutelins, respectively (Krishnan and Okita, 1986). Anticalreticulin antiserum from rabbits was a gift from Dr.
Jürgen Denecke (University of Leeds, UK) and was obtained as described (Crofts et al., 1999). To remove any
cross-reacting antibodies prior to the labeling procedure,
the antisera were preabsorbed using an acetone extract of
proteins from wild-type tissue according to Sambrook et al.
(1989). We then carried out controls by western blots on
protein extracts from all three types of tissue obtained from
wild-type rice plants to confirm the absence of any crossreaction of the antisera with rice proteins other than the
ones against which they were directed. After incubation in
the appropriate primary antiserum, the grids were rinsed
briefly in PBS and transferred to the appropriate rabbit
anti-chicken or goat anti-rabbit secondary monoclonal antibodies conjugated to 10-nm-diameter gold particles (BioCell, Cardiff, UK). The secondary antibodies were diluted
1:25 in PBS, and incubation was carried out at room temperature for 45 min. The sections were rinsed briefly in PBS
and subsequently distilled water, air dried, and then
stained with 2% (w/v) uranyl acetate for 20 min.
1222
Double Immunogold Labeling
Double immunogold labeling experiments were carried
out using a combination of anti-scFvT84.66 antiserum and
either the antiprolamine, antiglutelin, or anticalreticulin
antisera. The same concentrations and incubation conditions were used as described for the single labeling experiments above. After washing off the primary antisera with
PBS, the grids were incubated for 45 min at room temperature with the secondary goat anti-rabbit antibody conjugated to 15-nm gold particles (diluted 1:25 in PBS) to label
the antiprolamine, antiglutelin, or anticalreticulin primary
antibodies. The grids were rinsed in PBS and incubated for
a further 45 min at room temperature with the secondary
rabbit anti-chicken antibody conjugated to 10-nm gold particles (diluted 1:25 in PBS) to label the anti-scFvT84.66
primary antibodies. The grids were then washed and
stained as described above.
ACKNOWLEDGMENTS
The authors would like to thank Dr. Jürgen Denecke for
the anticalreticulin antibody, Dr. Thomas Okita for the
antiglutelin and antiprolamine antibodies, and Dr. Loı̈c
Faye for the anti-Lewis antibody. Dr. Neil Emans and Mr.
Markus Sack are acknowledged for helpful discussion.
Received March 15, 2001; returned for revision May 14,
2001; accepted July 8, 2001.
LITERATURE CITED
Bagga S, Adams HP, Kemp JD, Sengupta-Gopalan C
(1995) Accumulation of 15-kilodalton zein in novel protein bodies in transgenic tobacco. Plant Physiol 107:
13–23
Bagga S, Adams HP, Rodriguez FD, Kemp JD, SenguptaGopalan C (1997) Coexpression of maize ␦-zein and
␤-zein genes results in stable accumulation of ␦-zein in
endoplasmic reticulum-derived protein bodies formed
by ␤-zein. Plant Cell 9: 1683–1696
Bechtel DR, Juliano BO (1980) Formation of protein bodies
in the starchy endosperm of rice (Oryza sativa L.): a
re-investigation. Ann Bot 45: 503–509
Crofts AJ, Leborgne-Castel N, Pesca M, Vitale A, Denecke
J (1998) BiP and calreticulin form an abundant complex
that is independent of endoplasmic reticulum stress.
Plant Cell 10: 813–823
Crofts AJ, Leborgne-Castel N, Hillmer S, Robinson DG,
Phillipson B, Carlsson LE, Ashford DA, Denecke J
(1999) Saturation of the endoplasmic reticulum retention
machinery reveals anterograde bulk flow. Plant Cell 11:
2233–2247
Denecke J, Ek B, Caspers M, Sinjorgo KMC, Palva ET
(1993) Analysis of sorting signals responsible for the
accumulation of soluble reticuloplasmins in the plant
endoplasmic reticulum. J Exp Bot 44: 213–221
Fitchette AC, Cabanes-Macheteau M, Marvin L, Martin B,
Satiat-Jeunemaitre B, Gomord V, Crooks B, Lerouge P,
Faye L, Hawes C (1999) Biosynthesis and immunolocalPlant Physiol. Vol. 127, 2001
Reticuloplasmins in the Endoplasmic Reticulum Network
ization of Lewis a-containing N-glycans in the plant cell.
Plant Physiol 121: 333–343
Galili G, Sengupta-Gopalan C, Ceriotti A (1998) The endoplasmic reticulum of plant cells and its role in protein
maturation and biogenesis of oil bodies. Plant Mol Biol
38: 1–29
Gomord V, Denmat L-A, Fichette-Lainé A-C, SatiatJeunemaitre B, Hawes C, Faye L (1997) The C-terminal
HDEL sequence is sufficient for retention of secretory
proteins in the endoplasmic reticulum (ER) but promotes
vascular targeting of proteins that escape the ER. Plant J
11: 313–325
Hara-Nishimura I, Shimada T, Hatano K, Takeuchi Y,
Nishimura M (1998) Transport of storage proteins to
protein storage vacuoles is mediated by large precursoraccumulating vesicles. Plant Cell 10: 825–836
Herman EM, Tague BW, Hoffman LM, Kjemtrup SE,
Chrispeels MJ (1990) Retention of phytohemagglutinin
with carboxyterminal tetrapeptide KDEL in the nuclear
envelope and the endoplasmic reticulum. Planta 182:
305–312
Krishnan HB, Franceschi VR, Okita TW (1986) Immunochemical studies on the role of the Golgi complex in
protein-body formation in rice seeds. Planta 169: 471–480
Levanony H, Rubin R, Altschuler Y, Galili G (1992) Evidence for a novel route of wheat storage proteins to
vacuoles. J Cell Biol 119: 1117–1128
Li X, Franceschi VR, Okita TW (1993a) Segregation of
storage protein mRNAs on the rough endoplasmic reticulum membranes of rice endosperm cells. Cell 72:
869–879
Li X, Wu Y, Zhang D, Gillikin JW, Boston RS, Franceschi
VR, Okita TW (1993b) Rice prolamine protein body biogenesis: a BiP-mediated process. Science 262: 1054–1056
Marty F (1999) Plant vacuoles. Plant Cell 11: 587–599
Muench DG, Okita TW (1997) The storage proteins of rice
and oat. In BA Larkins, IK Vasil, eds, Cellular and Molecular Biology of Plant Seed Development. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp
289–330
Muench DG, Wu Y, Zhang Y, Li X, Boston RS, Okita TW
(1997) Molecular cloning, expression and subcellular localization of a BiP homolog from rice endosperm tissue.
Plant Cell Physiol 38: 404–412
Müntz K (1998) Deposition of storage proteins. Plant Mol
Biol 38: 77–99
Neuhaus JM, Rogers JC (1998) Sorting of proteins to vacuoles in plant cells. Plant Mol Biol 38: 127–144
Okita T, Choi SS, Ito H, Muench DG, Wu Y, Zhang F
(1998) Entry into the secretory system –the role of mRNA
localization. J Exp Bot 50: 1081–1090
Plant Physiol. Vol. 127, 2001
Pueyo JU, Chrispeels MJ, Herman EM (1995) Degradation
of transport-competent destabilized phaseolin with a signal for retention in the endoplasmic reticulum occurs in
the vacuole. Planta 196: 586–596
Robinson DG, Bäumer M, Hinz G, Jeong BK (1995) One
vacuole or two vacuoles: do protein storage vacuoles
arise de novo during pea cotyledon development? J Plant
Physiol 145: 645–664
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A Laboratory Manual, Ed 2.. Cold Spring Harbor
Press, Cold Spring, NY
Shewry PR, Napier JA, Tatham A (1995) Seed storage
proteins: structures and biosynthesis. Plant Cell 7:
945–956
Staehelin A (1997) The plant ER: a dynamic organelle
composed of a large number of discrete functional domains. Plant J 11: 1151–1165
Stöger E, Vaquero C, Torres E, Sack M, Nicholson L,
Drossard J, Williams S, Keen D, Perrin Y, Christou P et
al. (2000) Cereal crops as viable production and storage
system for pharmaceutical scFv antibodies. Plant Mol
Biol 42: 583–590
Torres E, Vaquero C, Nicholson L, Sack M, Stöger E,
Drossard J, Christou P, Fischer R, Perrin Y (1999) Rice
cell culture as an alternative production system for functional diagnostic and therapeutic antibodies. Transgenic
Res 8: 441–449
Tsang KY, Zaremba S, Nieroda CA, Schlom J (1995) Generation of human cytotoxic T-cells specific for human
carcinoembryonic antigen epitopes from patients immunized with recombinant vaccinia-CEA vaccine. J Natl
Cancer Inst 87: 982–989
Vaquero C, Sack M, Chandler J, Drossard J, Schuster F,
Monecke M, Schillberg S, Fischer R (1999) Transient
expression of a tumor-specific single-chain fragment and
chimeric antibody in tobacco leaves Proc Natl Acad Sci
USA 96: 11128–11133
Vitale A, Denecke J (1999) The endoplasmic reticulum:
gateway of the secretory pathway. Plant Cell 11:
615–628
Wandelt CI, Khan MRI, Craig S, Schroeder HE, Spencer
D, Higgins TJV (1992) Vicilin with carboxy-terminal
KDEL is retained in the endoplasmic reticulum and accumulates to high levels in the leaves of transgenic
plants. Plant J 2: 181–192
Zagouras P, Rose JK (1989) Carboxy-terminal SEKDEL
sequences retard but do not retain two secretory proteins in the endoplasmic reticulum. J Cell Biol 109:
2633–2640
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