The RNA content of the nucleolus and nucleolus-like inclusions in the
anther of Lilium estimated by an improved RNase-gold labelling method
S. SATO
Department of Biology, Faculty of Science, Ehime University, Bunkyo-cho 2-5, Matsuyama, Ehime 790, Japan
C. WILLSON and H. G. DICKINSON
Department of Botany, Plant Science laboratories, University of Reading, Wliitenights, Reading RG6 2AS, UK
Summary
Using RNase-gold labelling a comparative estimation has been made of the RNA content of the
nucleoli and nucleolus-like inclusions in anther
cells of Lilium henryi Thunb. Pretreatment with
glow electric discharge prior to application of the
RNase-gold labelling remarkably lowered the level
of nonspecific adsorption and allowed specific
labelling of RNA-containing structures. In the tapetal cells, the nucleoli, the perichromatin material
and the dense bodies labelled heavily, but both the
interchromatin region and the condensed chromatin did not bind the label. The nucleolus-like inclusions, which were found in the nucleoplasm of
meiotic cells at the tetrad stage, and were termed
'nuclear nucleolus-like bodies (NLBs)' in the present study, also showed a high response compared
with both the loosened and the condensed chromatin; this labelling was some 60% of that observed
over the nucleoli of somatic cells. Another type of
nuclear NLB was differentiated from the predominant type of NLB by a conspicuous electron opacity,
reduced size, and strong labelling with the RNasegold complex, suggesting that some nuclear NLBs
may be highly condensed. The labelling over the
cytoplasmic nucleoloids (nucleolus-like inclusions
found in the cytoplasm) was only 50 % of that over
the nuclear NLBs, although both are similar in
ultrastructural texture.
Introduction
plasmic NLBs appear only from late anaphase to the
beginning of telophase (Sato and Sato, 1984; Sato,
19886). Cytoplasmic NLBs are also found in the dyadand tetrad-stage meiocytes of Lilium and have been
termed cytoplasmic nucleoloids (Dickinson and HeslopHarrison, 1970; Porter et al. 1984; Dickinson and Willson, 1985).
There is some circumstantial evidence that the cytoplasmic nucleoloids in meiotic cells may represent aggregates of ribosomal RNA (Dickinson and Heslop-Harrison, 1970, 1977; Dickinson and Willson, 1985; Williams
et al. 1973; Porter et al. 1984) but this may not be the
case, for apparently identical cytoplasmic nucleoloids
found in egg cells of ferns do not contain RNA molecules
as detected by both [ 3 H]uridine autoradiography and
RNase-gold labelling (Bell and Pennell, 1987). However, biological significance has yet to be confirmed for
most categories of nucleolus-like inclusions. Clearly, it is
of paramount importance to establish whether these
structures contain RNA and, if so, in what quantity. The
RNase—gold labelling technique is probably the most
Plant cells feature small compact inclusions, showing the
same cytohistochemical characteristics as nucleoli. These
nucleolus-like inclusions have been classified into categories according to their location and the juncture of the
cell cycle at which they appear. At the termination of
mitosis, nucleolus-like bodies (NLBs) called 'prenucleolar bodies' form within the nucleus and assemble at the
nucleolus-organizing regions (NORs) presumably thus
forming early nucleoli (De La Torre and GimenezMartfn, 1982; Risueno and Medina, 1986). Smaller
nucleolus-like inclusions, termed 'dense bodies', have
been also found in interphase nuclei of many higher
plants (Barlow, 1981a,6; Williams et al. 1983). It has
been suggested that these are derived from the prenucleolar bodies that have failed to assemble at the NOR
(Barlow, 1981a,b, 1983), or formed directly from the
nucleolus by budding or partial disintegration (Sato et al.
1988). Very similar inclusions have also been detected in
the cytoplasm. In root tip meristematic cells, these cytoJournal of Cell Science 94, 675-683 (1989)
Printed in Great Britain © The Company of Biologists Limited 1989
Key words: cytoplasmic nucleoloids, nuclear NLBs,
nucleolus, RNase-gold labelling.
675
accurate method available for the detection of RNA
molecules at the electron-microscopic level. However, an
appreciable amount of nonspecific adsorption of the label
occurs when hydrophobic epoxy resin is used for embedding tissues (Sato et al. 1988). Nonspecific adsorption of
antibody proteins also occurs when a hydrophobic
embedding resin is employed (Causton, 1984), and a
similar mechanism may also result in RNase binding. We
have pretreated thin sections with glow electric discharge
prior to RNase-gold labelling, and this pretreatment
strikingly reduces nonspecific adsorption. The present
work reports a comparative estimation of the RNA
content of the nucleoli and nucleolus-like inclusions of
Lilium anther cells using this modified RNase-gold
labelling technique.
Materials and methods
Plants of Lilium henryi Thunb. were grown in the greenhouse at
20°C±5 deg. C. Anthers, excised from a 20mm flower bud,
were cut transversely into small sections and fixed for 3 h in
1.2% glutaraldehyde in 0.03M-phosphate buffer (pH7.0). The
specimens were rinsed in distilled water, dehydrated in an
acetone series and embedded in Epon-812 (Shell Chemical)
using propylene oxide as a transfer reagent.
A colloidal gold suspension was prepared according to Frens
(1973) and the RNase-gold complex was prepared as described
by Bendayan (1981). The final concentration of RNase A (type
III-A, Sigma) in the gold suspension was 25 fig ml~ .
Gold grids bearing thin sections were subjected to glow
electric discharge with an 1B-3 type Ion Coater (Eiko Engineering Co., Japan) at 3 mA for 15, 30 and 60s. The procedure for
the RNase-gold labelling was essentially that of Bendayan
(1981) and is described elsewhere in detail (Sato et al. 1988).
Quantitative estimation of the RNase-gold labelling was
carried out using data obtained from single grids, to avoid any
between-grid variation. Photographs were taken at X20000 to
cover the nucleoli, NLBs or CNLs, and the surrounding
regions of nucleoplasm or cytoplasm. Then, prints were made
at X40 000 and the gold particles over each cell structure were
counted. The area of cell structures involved was estimated by
cutting the structure from the print and weighing. Thus, values
for gold particle number per unit area were obtained for each
cell structure studied.
Some semi-thin sections of anthers embedded in Epon resin
were stained with AgNC>3 according to the method of Sato and
Shigematsu (1985). After photography, the silver-stained specimen was treated in a 1:1 (v/v) mixture of 0.75 % sodium
thiosulfate and 0.5 % potassium ferricyanide for 5 min to
remove the silver from the specimen, which was then stained
with 0.05% Toluidine Blue and photographed.
Results
Effect of pretreatment with glow electric discharge
In non-pretreated control specimens appreciably high
levels of nonspecific adsorption occurred, although nucleoli were found to be covered more densely with gold
particles than other parts of cells (data not shown). Many
gold particles were also located on areas of pure epoxy
resin. Nonspecific adsorption took place irrespective of
incubation time in RNase-gold suspension (15-60min).
Pretreatment with glow electric discharge for 15 s did
676
5. Sato et al.
not markedly decrease nonspecific adsorption. However,
some specimens pretreated for 30 s showed very little
nonspecific adsorption and, consequently, the nucleoli
appeared specifically labelled, while others still displayed
a high level of nonspecific adsorption after 30 s pretreatment. Pretreatment for 60s resulted in a more stable
specific labelling of the nucleoli and, in the work described below, sections were pretreated with glow electric
discharge for 60 s before application of the RNase-gold
label.
Nucleoli, dense bodies and perichromatin material in
the anther somatic cells
Typical nucleoli are known to consist of at least three
major components, a fibrillar centre, and a granular and a
fibrillar component. Since none of these could be discerned in the tetrad stage meiocytes, estimations were
made of the labelling over nucleoli of the somatic cells of
the anther. The tapetal cells were easily distinguished
from other cells, in that the nucleoli were very compact
and the chromatin and perichromatin material was separated into distinct areas. The RNase-gold labelling was
largely restricted to the nucleolus and the perichromatin
material (Fig. 1). Very little labelling occurred over both
the condensed chromatin and the interchromatin area.
Sometimes, small spherical bodies with diameters of less
than 1 fim, previously termed 'dense bodies', were seen in
the interchromatin area and were labelled strongly
A representative picture of the nucleoli in the anther
wall cells outside the layer of tapetal cells is shown in
Fig. 3. Nucleolus-associated chromatin (NAC), which
consists of loosely packed chromatin-like material, bound
very few gold particles, as did the condensed chromatin,
while the nucleoli were heavily labelled.
The quantitative estimation of the labelling over the
four cell structures is summarized in Table 1. For this
statistical estimation the anther wall cells outside the
layer of tapetal cells were used. The labelling over the
nucleoli was remarkably high, being five to six times that
recorded over other cell structures. From the histogram it
is clear that the labelling over each nucleolus fell in a very
wide range from about 50 to over 300 gold particles /im~
(Fig. 4). The ratio between mininum and maximum
labelling exceptionally reached 6 and this variation was
found to be strongly correlated with labelling over both
the loosened and the condensed chromatin (Fig. 5).
Nuclear NLBs and cytoplasmic nucleoloids in the
meiocytes
In tetrad stage meiocytes, spherical bodies with diameters of up to about 2 /.im were seen in both the cytoplasm
and the nucleoplasm. These two types of nucleolus-like
inclusions possessed very similar cytohistochemical
characteristics. They responded positively both to basic
dyes and to silver staining (Figs 6, 7), but the nucleoluslike inclusions in the cytoplasm (termed the cytoplasmic
nucleoloids) could be distinguished from the nuclear
nucleolus-like inclusions, in that the former frequently
possessed small lacunae while those in the nucleus did
not. Hence, we have termed the nucleolus-like inclusions
Fig. 1. Portion of a tapetal cell nucleus labelled with RNase-gold complex. The nucleolus (no) and perichromatin material
(arrows) are most strikingly labelled, c, chromatin. X38 000. Bar, 0.5 fun.
Fig. 2. Dense body in a tapetal cell, heavily labelled with the RNase-gold complex. X38000. Bar, 0.5 jum.
Fig. 3. Portion of an anther wall cell nucleus. Gold particles are predominant over the two nucleoli. The nucleolus-associated
chromatin (nac) the condensed (cc) and the loosened chromatin (lc) labelled weakly. X40000. Bar, 0.5^/m.
Nucleolar RNA in Lilium anthers
677
Table 1. Quantitative estimation of RNase-gold labelling over anther wall cells
Student's /-test
NO
S.D.
NO
L-chr.
NAC
C-chr.
64.90
14.35
14.35
9.59
136.20
28.20
28.03
23.50
25
25
13
25
L-chr.
NAC
C-chr.
7.43
5.38
0.03
7.82
1.34
1.19
**#
###
###
NS
NS
NS
.v, mean number of gold particles/(m - 2 ; s.D., standard deviation; n, number of samples; NO, nucleolus; L-chr., loosened chromatin; NAC,
nucleolus-associated chromatin; C-chr., condensed chromatin; ***, / 3 <0.001; NS, not significant.
A
40300-
3020-
it
0-
40
a.
~fcq
arti
10-
120 200 280
old
o.
30-
B
O
o
<u
200
o
3
C
<U
s
100-
——
20-
0
10-
f—1 ] \
00>
T1
0
, i
3
a-
10
20
30
40
50
60
Gold particles /<m~2
over condensed (•) and loosened chromatin (O)
40
c
30 H
20
i
10-
0
5040-
D
3020100
20 40
60 80 100 120 140 160
Gold particles ,um~2
Fig. 4. Frequency distributions of RNase-gold labelling on
the nucleolus (A), nuclear NLBs (B), cytoplasmic nucleoloids
(C) and cytoplasm (D). Numbers of specimens used for each
frequency distribution are indicated in Tables 1 and 2. For
details see text.
found in the nucleoplasm as 'nuclear nucleolus-like
bodies (NLBs)' to distinguish them from the cytoplasmic
nucleoloids. The nuclear NLBs were easily distinguished
from the nucleoli because they were not associated with
the NORs, and also did not show differentiation into
fibrillar and granular components, which are characteristics of typical nucleoli.
678
S. Sato et al.
Fig. 5. Correlations between RNase-gold labelling over the
nucleoli and that over the condensed ( • ) or over the loosened
chromatin (O). The equations are v=25.54+4.08.v (/=0.89,
P<0.001) and v=-1.00+6.06x (/•=().78, P<0.001),
respectively.
Gold particles were particularly predominant over the
nuclear NLBs (Fig. 9). Although the distribution histogram of gold particles over the nuclear NLBs showed that
labelling varied from 40 to 130 particles jitrn"2, most of
that fell in the range of 80-90 particles jxm~2 (Fig. 4).
The quantitative estimation of labelling, which represented the average number of gold particles per jitm ,
indicated the labelling over the nuclear NLBs to be twice
as strong as that over the loosened chromatin, and four
times as strong as that over the condensed chromatin
(Table 2). As shown in Fig. 8, the intensity of labelling
over the nuclear NLBs was correlated with the loosened
chromatin, but not with the condensed chromatin
(r=0.22, P>0.05).
A further type of nuclear NLB was also distinguished;
these were mostly smaller in size, less than 1 ,um in
diameter, and showed extremely high electron opacity.
We have termed this type of nuclear NLB as electronopaque NLB. The most prominent feature of the electron-opaque NLBs is that they show a significantly
higher level of RNase-gold labelling (Figs 9, 10).
Although their RNase-gold labelling could not be estimated statistically, owing to the small sample available, a
total of 11 electron-opaque NLBs were examined. Results from this study showed that gold particles bound at
•w •-•
Fig. 6. Light micrograph of a tetrad cell stained with silver nitrate. Nucleolus-like inclusions are seen in both the cytoplasm
and nucleoplasm. Arrows indicate the nuclear NLBs and arrowheads the cytoplasmic nucleoloids. X1500.
Fig. 7. Light micrograph of tetrad cell shown in Fig. 6, stained with Toluidine Blue after the removal of silver nitrate. X1500.
Bar, O
Table 2. Quantitative estimation of RNase-gold labelling over tetrad stage meiocytes
Student s Mest
NLB
L-chr.
C-chr.
CNL
Cyt.
Vac.
•x
S.D.
H
NLB
L-chr.
C-chr.
CNL
Cyt.
Vac.
82.50
38.46
19.04
44.31
20.26
5.34
22.91
10.92
5.68
13.11
7.72
3.11
32
26
26
29
29
29
##
#*#
###
*#*
###
3.28
21.96
8.20
***
NS
##*
10.88
1.77
8.83
###
#*#
21.69
7.46
0.68
8.39
##*
29.20
15.77
10.70
14.65
9.78
_
—
###
NS
###
###
NLB, nuclear nucleolus-like body; CNL, cytoplasmic nucleoloid; Cyt, cytoplasm; Vac, vacuole; •*, P<0.01.
140
120
u
'= ffl 100
'£ i
2
>-
o
4>
80-
O § 60
CO
40-
0
60
20
30
40
50
Gold particles/im""
over condensed ( • ) and loosened chromatin (O)
a minimum value of 200 particles ,ttm , and maximum of
over 1000 particles /im~ .
Gold particles were more highly concentrated over the
cytoplasmic nucleoloids than over the cytoplasm, and
almost no gold particles were distributed over cell structures lacking RNA, such as the cell wall and vacuoles
(Fig. 11). Labelling over the cytoplasm was generally
very low. The distribution histogram showed that labelling over the cytoplasmic nucleoloids varied from about
20 to 70 particles /xm~ (Fig. 4). The average labelling
over the cytoplasmic nucleoloids was twice that of the
cytoplasm and nine times that of the vacuoles (Table 2).
There was no linear correlation between the levels of the
labelling over the cytoplasmic nucleoloids and the cytoplasm (Fig. 12).
10
Fig. 8. Correlations between RNase-gold labelling over the
NLBs and that over the condensed ( • ) and loosened
chromatin (O). The equation is_v=25.23 + 1.41x (/•=().64,
P<0.00\). No significant correlation was found between the
labelling over the NLBs and the condensed chromatin.
Discussion
When colloidal gold-conjugated antibodies are applied to
thin sections, hydrophobic resins cause nonspecific adsorption (Causton, 1984). By subjecting the sections to
an oxidising agent, the level of nonspecific adsorption
Nucleolar RNA in Lilium anthers
679
/
•
Fig. 9. Portion of a tetrad nucleus labelled with the RNase-gold complex. Gold particles are predominant over the nuclear
NLBs, especially over the electron-opaque NLB (arrow). XS3 000. Bar, 0.5 fan.
Fig. 10. Part of a tetrad cell nucleus showing the two types of nuclear NLBs. The electron-opaque NLB is labelled more
heavily than the less electron-opaque NLB. X53 000. Bar, 0.S fan.
Fig. 11. Portion of tetrad cytoplasm with a cytoplasmic nucleoloid labelled with RNase-gold complex. The cytoplasmic
nucleoloid is labelled weakly, cw, cell wall; v, vacuole. X53 000. Bar, 0.5 fan.
680
5. Sato et.al.
50
i. «
40-
o
o
8
ti .a
C 6 30
S. ii
ooQ}
O
10
20
30
40
Gold particles fim'2
over cytoplasm
50
Fig. 12. Correlation between RNase-gold labelling over the
cytoplasmic nucleoloids and the cytoplasm. There is no linear
correlation between them (r=0.23, P>0.05).
may be considerably reduced. Oxidizing agents, such as
hydrogen peroxide, oxidize the hydrophobic alkane sidechains to hydrophilic alcohols, aldehydes and acids. The
glow electric discharge applied in the present study is
widely used to make grids or supporting membranes
hydrophilic. Althouch there is no evidence available as to
how glow discharge affects the embedded material and
the resin itself, the present studies indicate that it
strikingly lowers the level of nonspecific adsorption. It
therefore seems reasonable to assume that glow electric
discharge induces changes in the resin that render it
hydrophilic.
• Tapetal cells are well suited to the study of RNase-gold
labelling of nuclear structures, because all the compartments of the nucleus, i.e. nucleolus, perichromatin
material, dense bodies, chromatin and interchromatin
area, are very clearly identifiable. The results indicate
that the nucleolus, perichromatin material and dense
bodies all bind the label strongly, while the complex has
little affinity for both the condensed chromatin and the
interchromatin area. Autoradiographic studies at the
electron microscopic level indicate that, in mammalian
cells, transcription takes place first in the perichromatin
area (Fakan and Bernhard, 1971). These authors related
this transcriptional activity to the rapid synthesis of
heterogeneous nuclear (Hn)RNA, and the incorporation
of radiolabel is often associated with small clumps of
fibrillar material, resembling perichromatin fibrils
(Fakan and Deltour, 1981). In plant cells, preferential
staining techniques for ribonucleoprotein have revealed
staining of structures corresponding in their morphology
with perichromatin fibrils of animal cells (Deltour et al.
1979; Risueno et al. 1979; Sato and Shigematsu, 1985).
These findings are compatible with the present results
from tapetal cells, which indicate that perichromatin
material is specifically labelled with RNase-gold complex.
The major aim of this work was to determine the RNA
content of the nucleolus-like inclusions. The labelling of
the nuclear NLBs in meiotic cells is about twice as
strong as that of the loosened chromatin, and four times
that of the condensed chromatin. The labelling of the
nuclear NLBs exceeds by 15-fold that of the vacuoles,
which would not be expected to contain RNA. The
nuclear NLBs would thus appear to contain higher levels
of RNA. However, the concentration of RNA molecules
in the nuclear NLBs seems to be lower than that in
nucleoli. The nuclear NLBs are very similar in appearance, dimension and cytohistochemical characteristics to
the prenucleolar bodies that are formed at anaphase in
some plants. It has been demonstrated that prenucleolar
bodies do not migrate to the NORs and remain free in the
nucleoplasm when the activity of the NOR is inhibited by
chemicals (Philips and Philips, 1973; Gime'nez-Martfn et
al. 1974; Risueno et al. 1976; Morcillo and de la Torre,
1980) or laser irradiation (Berns et al. 1972). Some
authors have shown that the prenucleolar bodies do not
assemble in nuclei from which NORs are absent (Phillips
and Phillips, 1979; Hernandez-Verdun et al. 1979; Sato,
1988a). Interestingly, RNA polymerase I antibodies also
inhibit the telophasic coalescence of the prenucleolar
bodies with the NOR (Benavente et al. 1987). Thus, in
the case of the tetrad cells, the appearance of nucleoluslike bodies, called the nuclear NLBs here, may result
from the inactivity of NORs. Certainly, the three major
components of the nucleoli could not be distinguished in
these cells. If this is the case, the nuclear NLBs should
perhaps be regarded as identical with the prenucleolar
bodies.
In the present study another category of nuclear
NLBs, the electron-opaque NLBs, has been described.
The most striking feature of these NLBs is that they show
very strong affinity for the RNase-gold complex. The
finding that they are sfflall in size, of higher electron
opacity and bind higher levels of label indicates that they
may simply differ from the predominant type in their
packing intensity.
The cytoplasmic nucleoloids in Lilium tetrad cells may
be derived from the nucleolar material associated with
chromosomes (Dickinson and Heslop-Harrison, 1977).
Since nuclear NLBs are also thought to originate from a
substance coating the chromosomes, it is likely that the
nuclear NLBs contain some of the same components as
the cytoplasmic nucleoloids. The cytoplasmic nucleoloids, however, differ from the nuclear NLBs in two
respects. First, the former frequently possess lacunae;
second, the cytoplasmic nucleoloids do not differentiate
in their electron-density.
The RNase-gold labelling of the cytoplasmic nucleoloids was twice that of the cytoplasm. In the previous
study we also have examined the RNase-gold labelling
over the cytoplasmic NLBs in the root tip meristematic
cells of Vicia{aba, which are similar in appearance to the
cytoplasmic nucleoloids (Sato et al. 1988). The results
show that the labelling of the cytoplasmic NLBs is weak
compared with that of the cytoplasm. Cytoplasmic nucleoloids thus seem to differ superficially from the
cytoplasmic NLBs found in V. faba mitotic cells in their
RNA content. It should be emphasized, however, that
the labelling values obtained for these two types of
nucleolus-like inclusions are not absolute values, and are
related to cytoplasmic labelling. The ribosome population is strikingly low in the cytoplasm of the tetrad cells
when compared with that of the V.faba somatic cells and
Nucleolar RNA in Lilium anthers
681
in situ hybridization using [ 3 H]poly(U) probe has
demonstrated that the concentration of mRNA is also low
in the cytoplasm of the tetrad cells (Porter et al. 1983).
This low level of both species of RNA in the cytoplasm of
these cells may thus give rise to the differential between
cytoplasmic and nucleoloidal RNA levels. In the V.faba
somatic cells, the large population of cytoplasmic RNA
molecules would tend to obscure RNA molecules in the
cytoplasmic NLBs. The conclusion that the cytoplasmic
nucleoloids differ from the cytoplasmic NLBs of somatic
cells in their RNA content may thus be fallacious.
The RNase-gold labelling of the cytoplasmic nucleoloids was only twice that of the cytoplasm. This value
seems to be too small for the cytoplasmic nucleoloids to
be regarded as aggregates of ribosomes. These structures
contain little or no mRNA (Dickinson and Willson, 1985)
and evidence thus seems to be accumulating that they are
primarily proteinaceous. Their reaction with the silver
stain indicates that they may contain proteins previously
associated with the nucleoli or chromatin. An intriguing
possibility is that they represent the residuum of the
synaptonemal complex, lost from the chromosomes in
late meiotic prophase. Further studies are clearly
required before any firm conclusions can be drawn.
There was a highly significant correlation between the
labelling of the nucleolus and that of the condensed or
loosened chromatin. Two possibilities could explain this
relationship: first, the enzyme-substrate reaction may
not be uniform over the sections; the other, less probable
alternative, is that the RNA content of each structure is
heterogeneous within an individual nucleus, and the
RNA content of the nucleoli changes with that of the
condensed and the loosened chromatin. Although no
pointers have been obtained from the present study, it
seems unlikely that sixfold differences in label binding
could result from heterogeneity of RNA content.
BENDAYAN, M. (1981). Ultrastructural localization of nucleic acids by
the use of enzyme gold complexes. J. Histochem, Cvtochem. 29,
531-541.
The authors thank Drs C. Elleman and K. Jones for their
helpful advice and Miss S. Mitchell for technical assistance. We
also express our thanks to Dr Y. Yanagisawa for help in
preparing statistical data. One of the authors (S.S.) is grateful
to the ministry of Education of Japan for providing financial
support for study in United Kingdom. This study was supported in part by the Grant-in-Aid for Scientific Research from
the Ministry of Education, Science and Culture of Japan, no.
01S40S80.
PHILLIPS, D. M. AND PHILLIPS, S. G. (1973). Repopulation of
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(Received 30 June 1989 -Accepted 4 September 1989)
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