Plant Physiol. (1998) 116: 101-106
ADP-Glucose Pyrophosphorylase Is Localized to Both the Cytoplasm
and Plastids in Developing Pericarp of Tomato Fruit1
Bing-Yuan Chen,
Yi Wang, and
Harry W. Janes*
Department of Plant Science, Rutgers University, New Brunswick, New
Jersey 08903 (B.-Y.C., H.W.J.); and Institute of Vegetables and
Flowers, Chinese Academy of Agricultural Sciences, Beijing, China
(Y.W.)
 |
ABSTRACT |
The intracellular location of
ADP-glucose pyrophosphorylase (AGP) in developing pericarp of tomato
(Lycopersicon esculentum Mill) has been
investigated by immunolocalization. With the use of a highly specific
anti-tomato fruit AGP antibody, the enzyme was localized in cytoplasm
as well as plastids at both the light and electron microscope levels.
The immunogold particles in plastids were localized in the stroma and
at the surface of the starch granule, whereas those in the cytoplasm
occurred in cluster-like patterns. Contrary to the fruit, the labeling
in tomato leaf cells occurred exclusively in the chloroplasts. These
data demonstrate that AGP is localized to both the cytoplasm and
plastids in developing pericarp cells of tomato.
| |
INTRODUCTION |
AGP converts Glc-1-P and ATP to ADP-Glc and PPi. The product of
this reaction, ADP-Glc, is the major substrate for starch synthases
(Preiss, 1991
). Substantial evidence from the analysis of the
starch-deficient mutants (Tsai and Nelson, 1966; Lin et al., 1988;
Hylton and Smith, 1992), transgenic plants (Müller-Röber et
al., 1992; Stark et al., 1992), control analysis of photosynthate partitioning (Neuhaus and Stitt, 1990), and kinetic models (Pettersson and Ryde-Pettersson, 1989) firmly establish that AGP catalyzes an
essential step for starch biosynthesis in both photosynthetic and
nonphotosynthetic tissues.
AGP in higher plants is a heterotetramer composed of two small and two
large subunits. Recently, multiple forms of both the small and the
large subunits have been found in several plants. Several isoforms of
the large subunit were observed when the purified potato (Solanum
tuberosum L.) tuber AGP was subjected to high resolution 2-D PAGE
(Okita et al., 1990). The identification of three AGP large subunit
cDNAs from potato tuber suggests that multiple polypeptides are not the
result of proteolytic degradation or posttranslational modification
(Cognata et al., 1995). Similarly, multiple AGP polypeptides have been
detected in pea (Pisum sativum L.) and rice endosperm
(Hylton and Smith, 1992; Nakamura and Kawaguchi, 1992). Multiple cDNA
clones for AGP have also been isolated from barley (Hordeum
vulgare L.; Villand et al., 1992), Arabidopsis (Villand et al.,
1993), maize (Zea mays L.; Giroux and Hannah, 1994), broad
bean (Weber et al., 1995), pea (Burgess et al., 1997), and sweet potato
(Bae and Liu, 1997).
Whereas the presence of isoforms seems to be a common feature of plant
AGPs, the significance of their occurrence is not presently known. One
possible explanation is that individual isoforms could have different
intracellular locations. It was proposed that AGP of cereal endosperm
exists in the cytoplasm as well as in the amyloplasts (Hannah et al.,
1993; Villand and Kleczkowski, 1994). Recently, substantial
immunological evidence demonstrated that a plastidial form and a major
cytoplasmic form of the enzyme exist in barley and maize endosperm
(Denyer et al., 1996; Thorbjørnsen, et al., 1996). The occurrence of
this phenomenon in plants other than cereals is unknown.
Purification and characterization of AGP from tomato
(Lycopersicon esculentum L.) fruit revealed the existence of
two small subunit isoforms and three large subunit isoforms (Chen and
Janes, 1997). To determine the subcellular location of AGP isoforms in developing tomato fruit pericarp, we used immunocytochemical techniques at the light and electron microscope levels using a highly specific anti-tomato fruit AGP antibody. This study demonstrates that AGP is
also localized to both the cytoplasm and plastids in developing pericarp cells of tomato.
| |
MATERIALS AND METHODS |
Tomato (Lycopersicon esculentum Mill. var Laura) plants
were grown in the greenhouse under a 16-h light/8-h dark cycle. Fruit were collected 2 weeks postanthesis (fresh weight about 30 g). The
inner pericarp tissue of the fruit and mature fourth leaves were
utilized in this study and processed immediately as described below.
Tissue Preparation
Tomato inner pericarp tissue was cut into small blocks (about 2 mm3) and then immediately fixed in 100 mm phosphate buffer (pH 7.2) with 3% (w/v)
paraformaldehyde and 1.25% (v/v) glutaraldehyde for 3 to 4 h at
room temperature. After the tissue blocks were washed with 100 mm phosphate buffer (pH 7.2), they were dehydrated through
a graded ethanol series (10-100%) and infiltrated with London Resin
White (Electron Microscopy Sciences, Fort Washington, PA) according to
the manufacturer's protocol. Polymerization was conducted at 40°C
for 24 h, at 50°C for 24 h, and then at 60°C for 36 h. For carbohydrate-specific staining the inner pericarp tissue was
fixed and embedded in wax as described previously (Wang and Lou, 1994).
Immunolabeling and Observation
For light microscope observation, thin sections (180-200 nm) cut
by a LKB ultramicrotome were mounted onto gelatin-coated glass slides
(Superfrost/plus, Fisher Scientific). The sections were first incubated
with TBST buffer (20 mm Tris, pH 7.4, 500 mm
NaCl, and 0.1% Tween 20) containing 2% (w/v) BSA at room temperature for 1 h and then incubated in either preimmune serum or antiserum (both diluted 1:2000 in TBST buffer containing 0.1% BSA) raised against tomato fruit AGP (Chen and Janes, 1997) for 3 h. Following washes in antibody diluent, sections were incubated for 1 h in goat anti-rabbit IgG antibody conjugated with 20 nm gold (Electron Microscopy Sciences) diluted 1:50 as above, and then rinsed
consecutively in antibody diluent, TBST buffer containing 2% (w/v) BSA
and distilled water. Immunogold particles were enlarged by incubation
with silver-enhancement solution (ICN) following the manufacturer's
recommendations. The sections were counterstained with 0.05% safranin
solution and viewed using a light microscope.
For electron microscope observation, ultrathin sections (70-80 nm)
were collected on nickel grids and processed for immunogold labeling as
described above (without the silver enhancement step). The ultrathin
sections were double stained with uranyl acetate-lead citrate (Wang,
1994) and examined with a JEO100CX electron microscope.
| |
RESULTS AND DISCUSSION |
To establish the intracellular location of the AGP isoforms
revealed by 2-D PAGE analysis of the purified tomato fruit enzyme, a
polyclonal antibody raised against the purified enzyme preparation was
used. The antiserum reacts with each of the AGP isoforms. Moreover,
this reaction was also highly specific, i.e. no other proteins in the
tomato fruit crude extract cross-reacted with the antiserum (Chen and
Janes, 1997). The material used for AGP purification, the inner
pericarp at 2 weeks postanthesis, was also used for immunolocalization
in the present study to ensure that each of the AGP isoforms observed
by 2-D PAGE is present.
Figure 1A shows carbohydrate-specific
staining of the inner pericarp. The cells at this sampling stage
contain a large central vacuole and numerous amyloplasts that contain
starch grains, which stain red by the periodic acid-Schiff reagent.
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| Figure 1.
Light micrographs showing immunolocalization of
AGP in developing pericarp of tomato fruit. A, Carbohydrate-specific
staining. Starch grains stained red. B and C, Anti-tomato fruit AGP
serum detected a signal (gray dots) in both plastids and cytoplasm
(arrows). D, The preimmune control serum detected no signal. S, Starch
granule; and V, vacuole. A, ×351; B, ×1560; and C and D, ×1950.
|
|
The intracellular localization of AGP was first studied at the light
microscope level. Because the immunogold particles (20 nm in diameter)
are beyond the resolution limit of the light microscope, they were
enlarged by the silver-enhancement technique, which facilitates
visualization of the dark-gray dots. Gray deposits were observed in
both the plastids and cytoplasm of tomato pericarp cells (Fig. 1, B and
C). The signal was not evenly distributed in the cytoplasm, with denser
particles clustered near plastids (Fig. 1C). The preimmune serum
control showed no gray particles (Fig. 1D), indicating that the
antibody is specific. The intracellular distribution of AGP was further
examined by an immunoelectron microscope. Consistent with the light
microscope findings, immunogold particles were observed in both the
plastids (Fig. 2, A and C) and cytoplasm
(Fig. 2, A and B). The labeling in the plastids was not uniformly
distributed. No immunogold particles were observed inside the starch
granule, contrary to what was found in the maize (Zea mays
L.) endosperm (Miller and Chourey, 1995). Some of the particles
occurred at or near the surface of the starch grains (Fig. 2C), whereas
others were localized mostly in the stroma (data not shown). Similar to
the plastids, labeling in the cytoplasm was not uniform, and most was
localized in cluster-like patterns. Overall, the signal was not as
strong as that observed in amyloplasts of potato (Solanum
tuberosum L.) tuber (Kim et al., 1989) and maize endosperm (Miller
and Chourey, 1995). This is consistent with the very low specific
activity of AGP in tomato fruit crude extract (Chen and Janes, 1997).
No immunogold signal was detected with preimmune control serum (Fig.
2D).
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| Figure 2.
Electron micrographs showing immunolocalization of
AGP in developing pericarp of tomato fruit. Anti-tomato fruit AGP serum was used for immunolocalization. A, Immunogold particles reside in both
plastids and cytoplasm. B, Immunogold particles reside in the
cytoplasm. C, Immunogold particles reside at or near the starch granule
boundary. D, Preimmune serum control. P, Plastid; S, starch granule;
St, stroma; and Cyt, cytoplasm. A, ×24,180;B, ×31,200;C, ×30,420;
and D, ×28,860.
|
|
Reliability of immunolocalization results depends on the following
factors: (a) specificity of the antiserum, (b) absence of artifacts
caused by antigen mobility during sample preparation and labeling, and
(c) absence of nonspecific binding of antiserum. The specificity of the
antiserum is high, as suggested by western-blot analysis (Chen and
Janes, 1997). To minimize artifacts caused by antigen movement,
immunolocalization was done at both the light and electron microscope
levels and the results were consistent with each other. Furthermore, as
mentioned above, the particles were not randomly distributed. No
labeling was observed in the central vacuole or inside the starch
grains. To test the specificity of the cytoplasmic labeling, tomato
leaf tissue served as a control. It is well documented that the AGP
protein is located exclusively in the chloroplasts of leaf tissue
(Echeverria and Boyer, 1986; Robinson and Preiss, 1987). We used the
same immunolocalization procedure as for tomato pericarp, and labeling
occurred exclusively in the chloroplasts of leaf cells (Fig.
3A). This provides evidence that the
cytoplasmic labeling of developing pericarp cells is fruit specific and
that they are not likely to be artifacts of sample preparation.
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| Figure 3.
Electron micrographs showing immunolocalization of
AGP in tomato leaves. Anti-tomato fruit AGP serum was used for
immunolocalization. A, Immunogold particles reside in the stroma of
chloroplasts. B, Preimmune serum control. Labels are as in Figure 2. A,
×54,600; B, ×42,900.
|
|
This immunolocalization study clearly establishes that AGP is both
plastidial and cytoplasmic in developing pericarp cells of tomato
fruit, thereby extending the existence of cytoplasmic AGP to plant
tissues other than cereal endosperm. However, its occurrence appears to
be both species and tissue dependent. Potato tuber immunocytological
studies (Kim et al., 1989) and transgenic plant experiments (Stark et
al., 1992) indicate that AGP is located exclusively within the
amyloplast. In contrast to barley (Hordeum vulgare L.) and
maize endosperm, the majority of tomato pericarp AGP appears to reside
within the plastid. We observed a greater degree of labeling in the
plastids than in the cytoplasm.
Whether the plastidial and cytoplasmic AGP in tomato pericarp represent
two distinct isoforms is not presently known. In barley and maize
endosperm these two forms are distinct, as revealed by differences
in the size of the small subunit (Denyer et al., 1996;
Thorbjørnsen et al., 1996). Tomato pericarp contains two isoforms of
the small subunit and three isoforms of the large subunit (Chen and
Janes, 1997). The data presented here establish that AGP isoforms exist
in both plastids and cytoplasm. However, the localization of each
specific isoform within the plastid or cytoplasm remains unknown. It is
also possible that the cytoplasmic AGP we observed is simply
untransported precursors of AGP subunits resulting from inefficiency of
the protein-translocation machinery on the plastid membranes.
Isoform-specific antibodies are needed to answer these questions.
Recently, we isolated four cDNAs coding for AGP in tomato fruit
(B.-Y. Chen and H. W. Janes, unpublished data), which will
facilitate the production of these antibodies.
If we assume that the role of the cytoplasmic AGP is for starch
biosynthesis, ADP-Glc, the product of the enzyme, must then be
transported into the amyloplasts. It was found by in vitro experiments
that an adenylate translocator in the amyloplasts can transport
ADP-Glc, which is utilized for starch synthesis (Liu et al., 1991;
Pozueta-Romero et al., 1991a, 1991b). This adenylate translocator is
present in all plastid types (Ardila et al., 1993). In vivo evidence
for the presence of the putative ADP-Glc transporter comes from the
maize brittle1 (bt1) mutant (Mangelsdorf, 1926;
Wentz, 1926). Mutant bt1 kernels have a brittle texture and
accumulate about 80% less starch than normal kernels (Tobias et al.,
1992). The BT1 gene was cloned (Sullivan et al., 1991) and its encoded
proteins were localized in the amyloplast membrane of maize endosperm
cells (Cao et al., 1995; Sullivan and Kaneko, 1995). Compared with
the normal endosperm, the level of ADP-Glc in the cytoplasm of
bt1 endosperm was at least 13-fold higher (Shannon et al.,
1996), whereas amyloplasts isolated from bt1 endosperm
were less active in ADP-Glc uptake and conversion to starch (Liu et
al., 1992). These data suggest that the BT1 protein could function as
an ADP-Glc translocator and transport ADP-Glc from the cytoplasm into
amyloplasts in vivo. Therefore, this putative ADP-Glc translocator may
play a pivotal role in linking the cytoplasmic form of AGP and starch
biosynthesis in amyloplasts.
Recently, it was shown that the isolated amyloplasts from potato tuber
are able to synthesize starch from ADP-Glc (Naeem et al., 1997). If
this is of physiological significance, then results from the transgenic
plant experiment (Stark et al., 1992), in which plant expression of an
Escherichia coli AGP gene in the amyloplasts increased
starch content but expression in the cytoplasm did not, may
alternatively indicate that the putative ADP-Glc or adenylate
translocator may be rate-limiting. As a result any increase of ADP-Glc
in the cytoplasm may have no effect on starch content without
corresponding changes in this ADP-Glc or adenylate translocator on the
amyloplast membrane. Inconsistent with this argument, BT1 homologs were
not detected in potato tubers and other starchy tissues by the BT1
antibody (Cao and Shannon, 1997). Therefore, whether the putative
ADP-Glc translocator exists in the amyloplast membrane of tomato
pericarp cells may be a key toward understanding the function of the
cytoplasmic form of AGP in tomato fruit.
| |
FOOTNOTES |
1
This work was supported by the National
Aeronautics and Space Administration.
*
Corresponding author; e-mail janes{at}aesop.rutgers.edu; fax
1-908-932-9441.
Received July 18, 1997;
accepted October 3, 1997.
| |
ABBREVIATIONS |
Abbreviations:
AGP, ADP-Glc pyrophosphorylase.
2-D, two-dimensional.
| |
ACKNOWLEDGMENT |
We thank Dr. Bruce Wasserman for critical reading of the
manuscript.
| |
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Top
Abstract
Introduction
