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Plant Physiol. (1998) 116: 1585-1591
The Intracellular Localization of Ribulose-1,5-Bisphosphate
Carboxylase/Oxygenase in Chlamydomonas
reinhardtii1
Olga N. Borkhsenious,
Catherine B. Mason, and
James V. Moroney*
Department of Biological Sciences, Louisiana State University,
Baton Rouge, Louisiana 70803
 |
ABSTRACT |
The pyrenoid is a proteinaceous
structure found in the chloroplast of most unicellular algae. Various
studies indicate that ribulose-1,5-bisphosphate carboxylase/oxygenase
(Rubisco) is present in the pyrenoid, although the fraction of Rubisco
localized there remains controversial. Estimates of the amount of
Rubisco in the pyrenoid of Chlamydomonas reinhardtii
range from 5% to nearly 100%. Using immunolocalization, the amount of
Rubisco localized to the pyrenoid or to the chloroplast stroma was
estimated for C. reinhardtii cells grown under different
conditions. It was observed that the amount of Rubisco in the pyrenoid
varied with growth condition; about 40% was in the pyrenoid when the
cells were grown under elevated CO2 and about 90% with
ambient CO2. In addition, it is likely that pyrenoidal
Rubisco is active in CO2 fixation because in vitro activity
measurements showed that most of the Rubisco must be active to account
for CO2-fixation rates observed in whole cells. These
results are consistent with the idea that the pyrenoid is the site of
CO2 fixation in C. reinhardtii and other
unicellular algae containing CO2-concentrating mechanisms.
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INTRODUCTION |
Pyrenoids are electron-dense structures that are found in the
plastids of most algae. When pyrenoids are isolated, the most abundant
protein present is Rubisco (Kuchitsu et al., 1991 ; McKay and Gibbs,
1991; Okada, 1992). However, the amount of Rubisco present in the
pyrenoid is controversial. Estimates of the amount of Rubisco in the
pyrenoid versus the amount in the entire chloroplast range from 60% or
higher (Vladimirova et al., 1982; Lacoste-Royal and Gibbs, 1987; Morita
et al., 1997) to as low as 5% (Süss et al., 1995).
The function of Rubisco in the pyrenoid is also unclear. Lacoste-Royal
and Gibbs (1987) found that a higher percentage of Rubisco was
localized to the pyrenoid in stationary cells rather than in actively
growing cells of Chlamydomonas reinhardtii, an observation
that is consistent with the hypothesis that the pyrenoid is a storage
body. Süss et al. (1995) interpreted their localization data to
indicate that there were two forms of Rubisco in C. reinhardtii: form I, which bound to the thylakoid membranes in the
stroma, and form II, which was found in the pyrenoid. They further
speculated that the pyrenoid-localized form II might function as
part of the CO2-concentrating mechanism.
In photosynthetic organisms that possess a
CO2-concentrating mechanism, Rubisco has a very
specific localization. In higher plants with C4-type photosynthesis,
Rubisco is specifically localized to chloroplasts of bundle-sheath
cells (Hatch, 1992). In unicellular cyanobacteria with
CO2-concentrating mechanisms, Rubisco is
specifically localized to structures known as carboxysomes (Allen,
1984; Codd and Marsden, 1984). Recent studies of the
cyanobacteria's CO2-concentrating mechanism indicate that HCO3
accumulated by the cell is dehydrated specifically in the carboxysome, where the resulting CO2 can be fixed before
leaking out of the cell (Badger and Price, 1994). Models of this
process have also been proposed (Reinhold et al., 1991).
C. reinhardtii also possesses a
CO2-concentrating mechanism that is inducible
(Badger et al., 1980; Aizawa and Miyachi, 1986; Moroney and Mason,
1991). When C. reinhardtii is grown under elevated CO2, the CO2-concentrating
mechanism is not present, but when the alga is grown under low
CO2, the CO2-concentrating
mechanism is operational. With these observations in mind, we
investigated the distribution of Rubisco in C. reinhardtii
under a variety of growth conditions, including high- and
low-CO2 concentrations. Under all growth
conditions we found that a significant fraction of Rubisco was
localized to the pyrenoid. In particular, under limiting
CO2 more than 90% of the Rubisco was found
within the pyrenoid. In addition, we found that the in vivo rates of
CO2 fixation are similar to the total amount of
Rubisco activity, as estimated by in vitro Rubisco assays. This means
that nearly all of the Rubisco in the cell must be functional to
account for the rates of CO2 fixation observed in
cells. This observation implies that the Rubisco localized to the
pyrenoid is active in CO2
fixation.
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MATERIALS AND METHODS |
Algal Cultures
The wild-type Chlamydomonas reinhardtii strain used in
this study, 137 mt+, was obtained from Dr. R.K.
Togasaki (Indiana University, Bloomington). Strain 18-7G (Spreitzer et
al., 1985), containing a truncation of the large subunit of Rubisco,
was obtained as CC-2653 from the Chlamydomonas Genetics Center (Duke
University, Durham, NC). When grown photoautotrophically, strain 137 was cultured in minimal medium (Sueoka, 1960) in 2.8-L carboys,
illuminated with 200 µE m2
s1 at room temperature, and shaken
continuously. Cultures were bubbled with 5% CO2
in air (final Ci concentration = 2 mm) or in ordinary air (final Ci = 4 µm). When grown with acetate as a C source, Tris-acetate-phosphate medium was used (Sueoka, 1960). Strain 18-7G
was grown with this medium in the dark.
Electron Microscopy
For transmission electron microscopy, various cell strains were
prepared according to Henk et al. (1995). Equal parts of the cell
suspension were mixed with equal parts of 4%
OsO4, 8% formaldehyde, and 2%
glutaraldehyde and fixed for 15 min. The sample was then filtered
and fixed for an additional 15 min in equal parts of 4%
OsO4, 8% formaldehyde, 2% glutaraldehyde, and
0.2 m sodium cacodylate buffer (pH 7.2). The final
concentration of the fixative components were: 1%
OsO4, 2% formaldehyde, 0.5% glutaraldehyde, and
50 mm sodium cacodylate buffer. The subsequent steps for
obtaining sections and treatment of the grids were the same as
described by Henk et al. (1995).
Immunolocalization
The immunocytochemical procedure was similar to the method of
Schroeder et al. (1993), with some modifications. Sections were pretreated with 2% sodium-meta-periodate (Sigma) for 15 min to remove
any glutaraldehyde, then blocked two times for 30 min each in 2% BSA
and 0.1% Tween 20 in PBS. The sections were then incubated for 1 h with diluted primary antibody (1-50 dilutions of anti-Rubisco) or
with preimmune serum diluted similarly as a control, washed with 0.5%
Tween 20 in PBS four times for a total of 20 min, and blocked again in
2% BSA two times for 15 min each. To detect bound antibodies, we used
Protein A conjugated with 15-nm colloidal gold particles (BB
International, Cardiff, UK) and diluted 1:50 with 1% BSA for 40 min.
Evaluation of immunogold labeling was made using the computer program
Image-1/AT release version 4 (Universal Imaging Co., West
Chester, PA). The images from the negatives were frozen and stored in a
frame buffer as an 8-bit, 256-gray-scale image. Once this was
freshholded, the gold particles associated with the pyrenoid were
counted, as were the particles in the same size area in the cytoplasm
and thylakoid regions of the individual cells. Because the size of the
pyrenoid was different from cell to cell, we calculated the number of
particles in a 1-µm2 area as a means for
comparing the labeling intensity for the various cell compartments of
the different strains in the various experimental conditions.
Enzymes Assays
C. reinhardtii Rubisco was purified according to
Spreitzer and Mets (1980). Rubisco activity in vitro was estimated by
the method described by Pierce et al. (1982).
Photosynthesis Assays
The photosynthetic rate of algal cells was measured with an
O2 electrode (Rank Brothers, Cambridge, UK). Algae were
centrifuged at 5000 rpm for 5 min, and the pellet resuspended at 25 µg chlorophyll mL1 in 4 mL of 25 mm Hepes-KOH (pH 7.3) and transferred to the electrode chamber. Algal cells were assayed at 300 µmol
m2 s1 of PAR. They were
allowed to consume the Ci of the buffer and the
intracellular pool of Ci until no net
O2 exchange was observed, which took between 3 and 10 min. NaHCO3 at the indicated concentrations was
added and the rate of O2 evolution was measured
over the next 30 s to 2 min. Chlorophyll concentrations were
determined spectrophotometrically. The
K0.5(CO2) value is
the CO2 concentration required to give half-maximal rates of O2 evolution.
SDS-PAGE and Immunoblotting
SDS-PAGE was performed on 12.5% polyacrylamide gels as described
by Laemmli (1970). The protocol from Bio-Rad was followed when
immunoblotting. The blots were probed with antisera raised against
C. reinhardtii Rubisco (HTI Bioproducts, Ramona, CA). Immunoblotting was visualized using horseradish peroxidase-conjugated secondary antibodies, as described in the protocol from Bio-Rad.
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RESULTS |
In this study an antibody raised against Rubisco isolated from
C. reinhardtii was used. This antibody reacted strongly
and specifically with C. reinhardtii Rubisco, as
judged by immunoblots (Fig. 1). The
antibody recognized the C. reinhardtii Rubisco large subunit
much better than the small subunit (Fig. 1). For this reason, the
presence of the small subunit precursor should not complicate the
immunolocalization studies.
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| Figure 1.
Specificity of the C. reinhardtii
anti-Rubisco antibody. Different amounts of cell homogenate from high-
and low-CO2-grown cells were separated by SDS-PAGE, blotted
to nitrocellulose, and probed with an anti-Rubisco antibody, as
described in ``Materials and Methods''. Lanes a, 5 µg of total
protein; lanes b, 10 µg of protein; lanes c, 20 µg of protein;
lanes d, 25 µg of protein; and lanes e, 30 µg of protein. The large
and small subunits of Rubisco are indicated with arrows. The weak band
at 44 kD (indicated with *) is a breakdown product of the large subunit
(Rawat, 1994).
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When the anti-Rubisco antibody was used in immunolocalization studies,
immunogold particles were localized preferentially to the chloroplast
pyrenoid (Fig. 2). Two different growth
conditions were used for the initial study: growth on elevated
CO2 or growth on air levels of
CO2. In both cases the pyrenoid had a high
concentration of immunogold particles. In contrast, the immunogold
labeling density in the chloroplast stroma depended on the growth
condition. Growth on low CO2 resulted in a low
density of immunogold particles in the stroma (Fig. 2A), with the
labeling density being similar to that seen in the cytoplasm. In
contrast, cells grown on elevated CO2 had higher
densities of immunogold particles in the stroma (Fig. 2B). In the case
of cells grown on high CO2 the labeling of the
stroma was significantly higher (P < 0.01) than the labeling of
the cytoplasm. Cells grown under either growth condition showed a very
low density of immunogold particles over the entire cell when probed
with the preimmune serum (Fig. 2, C and D).
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| Figure 2.
Immunogold labeling of C. reinhardtii cells grown on high- or low-CO2
conditions. A, Low-CO2-grown wild-type cells grown on minimal medium probed with an antibody raised against C. reinhardtii Rubisco. B, High-CO2-grown cells grown
on minimal medium probed with an antibody raised against C. reinhardtii Rubisco. C, High-CO2-grown wild-type
cells grown on minimal medium probed with a preimmune serum. D,
Low-CO2-grown cells grown on minimal medium probed with a
preimmune serum. Bars indicate 0.5 µm. P, Pyrenoid; Ssh, starch sheath; and St, chloroplast stroma.
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When the relative volumes of the pyrenoid and the stroma are taken into
account (Lacoste-Royal and Gibbs, 1987), it is clear that almost all of
the Rubisco appears to be localized to the pyrenoid in C. reinhardtii cells grown under low levels of
CO2 (Table I).
Similar calculations using data from the
high-CO2-grown cells show that a significant
fraction (approximately 60%) of the Rubisco is localized to the
chloroplast stroma in those cells (Table I). These data imply that the
localization of Rubisco in C. reinhardtii depends in part on
the growth conditions of the organism.
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Table I.
Calculation of Rubisco fraction in both the pyrenoid
and stroma of the chloroplast
To obtain the fraction of Rubisco in the pyrenoid, the average density
of immunogold particles in the cytoplasm was subtracted from the
average density of particles in the stroma or in the pyrenoid. The net
particle density of the pyrenoid or stroma was then multiplied by the
average volume of the compartment (which is 2.4 mm3 and
35.6 mm3, respectively), giving the total particles for
each compartment. To calculate the fraction of Rubisco in the pyrenoid,
the total number of particles in the pyrenoid was divided by the
combined number of particles in the pyrenoid and in the stroma. The
stroma includes the thylakoid area. The data shown are the averages ± sd of 10 samples and 40 different evaluations. Preimmune
sera gave immunogold densities of less than 2 particles
mm2.
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We also grew cells under different conditions to determine whether the
distribution of Rubisco changes with a different C source. The growth
condition tested was mixotrophic growth (Fig. 3). All of the wild-type cultures were
grown at 150 µmol m2
s1 of PAR. In these studies the distribution of
immunogold particles in acetate-grown cells indicated that a
significant fraction of the Rubisco was in the stroma (Figs. 2B and 3).
C. reinhardtii strain 18-7G was also tested as a
negative control. This strain has a stop codon in the rbcL
gene and does not have detectable Rubisco (Spreitzer et al., 1985).
However, strain 18-7G can grow with acetate as a C source under low
light. As shown previously, this strain also does not have a detectable
pyrenoid (Rawat et al., 1996). When probed with a Rubisco antibody,
strain 18-7G had a very low immunogold density over the chloroplast
stroma (Fig. 3A). The low immunogold density seen in this strain is
similar to the immunogold density seen in wild-type cells growing on
low CO2, implying that the amount of Rubisco in
the stroma of wild-type cells is very low. This result from strain
18-7G also supports the contention that the immunogold particle
density in the stroma of wild-type cells grown on high
CO2 or on acetate is higher than background
(Table II).
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| Figure 3.
Immunogold labeling of C. reinhardtii strain 18-7G or wild-type cells grown on acetate.
A, Strain 18-7G. B, Wild-type cells. Sections were probed with an
antibody raised against C. reinhardtii Rubisco, as
described in ``Materials and Methods''. Bars indicate 0.5 µm. P,
Pyrenoid; and St, chloroplast stroma. No pyrenoids were seen in strain
18-7G, in agreement with previous observations (Rawat et al., 1996).
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Table II.
Calculation of the Rubisco fraction in both the
pyrenoid and the stroma of wild-type cells and strain 18-7G grown on
acetate
Both cell lines were grown in moderate light (150 µmol
mm2 s1) on acetate medium. To quantitate
the number of immunogold particles in the stroma and in the pyrenoid
the method described in the legend to Table I was employed. Because the
pyrenoid is absent from 18-7G, the number of particles in the pyrenoid
is not applicable (N.A.). Ten samples of each strain were sampled. The
data shown are the averages ± sd.
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The change in distribution of Rubisco under high- and
low-CO2 growth conditions follows the presence or
absence of the CO2-concentrating mechanism. To
further test this correlation, we determined the localization of
Rubisco while cells were adapting to low-CO2
growth conditions. In this experiment cells were first grown on
elevated CO2 and then switched to low
CO2. Using immunogold, the distribution of
Rubisco between the pyrenoid and stroma was estimated for various times
after the switch in CO2 level (Figs.
4 and 5). As seen from the time course
(Fig. 4), the distribution of Rubisco changes about 2 h after the
change in CO2 supply. This is also the time when
the affinity of the cells for CO2, as estimated
by the K0.5(CO2), is
rapidly changing. Representative electron micrographs showing the
immunogold distributions are shown in Figure
5. The data in this experiment are
consistent with that of Morita et al. (1997) and our earlier experiment
(Fig. 2A), indicating that 90% or more of the Rubisco is in the
pyrenoid when cells are grown on low CO2.
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| Figure 4.
Change in the apparent affinity of cells for
Ci and the change in distribution of Rubisco after cells
are switched to low CO2. At 0 h cells were switched
from high- to low-CO2 conditions. The apparent affinity of
the cells was determined by estimating the
K0.5(CO2) of the cells ( ) as
described in ``Materials and Methods''. The percentage of Rubisco in
the pyrenoid ( ) is calculated from the distribution of immunogold
particles.
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| Figure 5.
Immunogold labeling of C. reinhardtii cells after being switched from high- to
low-CO2 conditions. A, Cells just before transfer to low
CO2 (0 h). B, Cells 2 h after the switch to low
CO2. C, Cells 6 h after the switch to low
CO2. D, Cells 12 h after the switch to low
CO2. Bars indicate 0.5 µm. P, Pyrenoid; Ssh, starch sheath; and St, chloroplast stroma.
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Since 90% of the Rubisco is localized to the pyrenoid, we compared the
in vitro carboxylation activity of Rubisco isolated from
low-CO2-grown wild-type cells with the in vivo
14CO2-fixation rate of
these same cells. The in vitro Rubisco carboxylation rate measured
under saturating CO2 (186 ± 50 µmol h
1 mg chlorophyll 1)
closely matched the maximal in vivo rate of
14CO2 fixation (199 ± 44 µmol h 1 mg chlorophyll
1). This result supports the suggestion that
the majority of Rubisco within the low-CO2-grown
cell must be active to support the rates of CO2
fixation observed in whole cells. Since most of the Rubisco is
localized to the pyrenoid, it follows that the Rubisco within the
pyrenoid is active and that the pyrenoid is the actual location of
CO2 fixation in C. reinhardtii.
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DISCUSSION |
One aim of this report was to resolve the present controversy
concerning the localization of Rubisco in C. reinhardtii.
Lacoste-Royal and Gibbs (1987) reported that between 60 and 70% of
Rubisco appeared to be localized to the pyrenoid. Recently, however,
Süss et al. (1995) challenged that earlier finding by reporting
that only 5% of the Rubisco was localized in the pyrenoid. In
addition, Morita et al. (1997) have recently reported than 99% of the
Rubisco is localized to the pyrenoid, illustrating the wide range of
estimates in the literature. Süss et al. (1995) used
cryo-techniques to avoid spatial dislocation of soluble enzymes such as
Rubisco and other C3 enzymes. They cited this as a possible difference
between their results and those of Lacoste-Royal and Gibbs (1987).
However, Morita et al. (1997) used similar techniques in their study.
Süss et al. (1995) also argued that Lacoste-Royal and Gibbs
(1987) did not take into account the relative volumes of the pyrenoid and stroma in their calculations. The data presented in this report are
consistent with the data of Lacoste-Royal and Gibbs (1987) and Morita
et al. (1997). The small differences between the reports of
Lacoste-Royal and Gibbs (1987), Morita et al. (1997), and this present
work are likely due to growth-condition differences. For example, many
of the cultures used by Lacoste-Royal and Gibbs (1987) were
mixotrophically grown, whereas Morita et al. (1997) grew cells
phototrophically on low CO2.
Our results are in agreement with all earlier studies that showed a
high level of immunogold particles in the pyrenoid when anti-Rubisco
antibodies were used. However, we disagree with the conclusions of
Süss et al. (1995). The primary reason for our different
conclusions is the method of counting the immunogold particles. In this
report and in the paper by Lacoste-Royal and Gibbs (1987), the number
of immunogold particles is reported as the number of particles per unit
area. Süss et al. (1995) counted the total number of particles
per section in each subcellular location and did not take into account
the area of each organelle in the section sampled.
According to Weibel (1979) a section is a two-dimensional
projection of the volume of the organelle or tissue being examined in
the electron microscope. As such, the average area of an organelle or
cell observed in many sections is proportional to the volume of that
object. Therefore, the quantitative method employed by Süss et
al. (1995) accounts for the volume difference twice, first in the
counting of the particles and a second time when they multiply their
number by the relative volumes of the pyrenoid and the stroma. If the
number of immunogold particles reported by Süss et al. (1995) is
expressed on a particles per area basis, their data are much more in
line with our results and those of Lacoste-Royal and Gibbs (1987) and
Morita et al. (1997). From these studies it appears that at a minimum
60 to 70% of the Rubisco in C. reinhardtii is localized to
the pyrenoid.
A second aim of this report was to determine whether Rubisco is
mobilized or relocated when cells are switched from high to low
CO2. It appears that growth conditions do affect
the intracellular localization of Rubisco. We looked at cells grown on
high CO2, low CO2, or
acetate. We consistently found significant levels of Rubisco in the
chloroplast stroma if the cells were grown on high
CO2 or acetate. In the extreme case of strain
18-7G, where no Rubisco is produced, no pyrenoid was observed and the
immunogold density in the stroma was at background levels. Our results
are consistent with the pyrenoid being the primary location of Rubisco in C. reinhardtii under low-CO2 growth
conditions when the components of the
CO2-concentrating mechanism are being expressed.
The shift in Rubisco localization correlates well with the formation of the starch sheath, which also forms shortly after cells are switched to
low CO2 (Ramazanov et al., 1994). Süss et
al. (1995) also reported that Rubisco was associated with thylakoid
membranes in the chloroplast stroma and the pyrenoid. For the pyrenoid
we found no particular association of Rubisco with the tubules that penetrate the pyrenoid matrix. The concentration of Rubisco is such
that it is likely to be found throughout the matrix of the pyrenoid.
Our immunolabeling of the stroma was not dense enough to determine
whether there is a particular association of Rubisco with the stromal
lamellae.
The finding of Rubisco in the pyrenoid has raised the question of
whether pyrenoidal Rubisco is active in CO2
fixation. Clearly, if 100% of the Rubisco is localized to the
pyrenoid, then that Rubisco must be active. However, even if a lower
estimate of the amount of Rubisco in the pyrenoid is used, our activity
measurements are consistent with the idea that pyrenoidal Rubisco is
active in vivo. Rubisco activity measurements indicate that there is not an excess of Rubisco activity in the cell, and that most of the
Rubisco in the cell must be active to account for the levels of
CO2 fixation observed in whole cells. Because
90% of the Rubisco is localized to the pyrenoid when cells are grown
on low CO2 (Figs. 2A and 4D; Table I) (Morita et
al., 1997), these results are consistent with the idea that the primary
location of CO2 fixation in C. reinhardtii is the pyrenoid.
A specific localization of Rubisco to the pyrenoid is compatible with
the view that organisms with CO2-concentrating
mechanisms package Rubisco in very specific fashions. The most
analogous situation is found in cyanobacteria, in which Rubisco is
found in carboxysomes (Codd and Marsden, 1984; Reinhold et al., 1991). In cyanobacteria it appears that the CO2 level is
elevated within the carboxysome, thus favoring the carboxylation
activity of Rubisco over the oxygenase activity. The pyrenoid may serve
the same function in C. reinhardtii and other eukaryotic
algae.
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FOOTNOTES |
1
This work was supported by the
National Science Foundation (grant no. IBN-9632087).
*
Corresponding author; e-mail btmoro{at}unix1.sncc.lsu.edu; fax
1-504-388-8459.
Received October 6, 1997;
accepted January 20, 1998.
| |
ABBREVIATIONS |
Abbreviations:
Ci, inorganic carbon.
high
CO2, air supplemented with CO2 so that the
final CO2 concentration is 5% (v/v).
low CO2, air containing ambient (350 ppm) CO2.
| |
ACKNOWLEDGMENTS |
We thank Ronald Bouchard for his help with the Image-1/AT,
and Dr. William Henk and M.C. Henk for their valuable
suggestions.
| |
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Abstract
Introduction