Published July 1, 1965
A LIGHT AND ELECTRON
OF S M A L L , S P H E R I C A L
IN MERISTEMATIC
MICROSCOPE
NUCLEAR
STUDY
BODIES
C E L L S OF A L L I U M
CEPA, VICIA FABA, AND RAPHANUS
J . G. L A F O N T A I N E ,
SATIVUS
Ph.D.
From the Department of Biology, Laval University, Quebec, Canada
ABSTRACT
INTRODUCTION
In the course of an earlier electron microscope
study (1) of nuclear structures in meristematic
cells of Allium cepa and Vicia faba, spherical bodies,
approximately 2 /z in diameter, were observed at
interphase. These structures were lighter than
either the chromosomes or the nucleolus under
electron microscopy, but stained metachromatically with azure B, as the nucleolus, following fixation with formalin. Since then, nuclear bodies of a
similar appearance have been noted in Tetrahymena pyriformis (2, 3), in kidney (4, 5), and
thymus (6).
The purpose of the present paper is to describe
in more detail the distribution of these bodies
during interphase, preprophase, and prophase
and to report certain characteristics which they
share with the nucleolus.
MATERIAL
AND
METHODS
Roots of Allium cepa grown in distilled water and of
Vicia faba and Raphanus sativus L. grown in damp
vermiculite were used for the present study. Portions
0.5 to 1 mm in length were excised from the root
tips and fixed in the following solutions: (a) 1 per
cent osmium tetroxide in distilled water (7), (b)
1 per cent osmium tetroxide buffered with Veronal
(8) or phosphate (9) to a pH of 7.3, (c) 4 per cent
formaldehyde or glutaraldehyde. Other roots were
fixed in one of these aldehydes and postfixed in 1
per cent osmium tetroxide (10). Following dehydration in alcohol the specimens were embedded in
Epon 812.
Sections 0.25 to 0.75 D thick were stained with
Feulgen and methylene blue (11); ultrathin sections
were stained with solutions of heavy metals (12, 13).
Electron micrographs were obtained with a Siemens
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Interphase, preprophase, and prophase nuclei of meristematic cells of AUium ¢epa, Vide faba,
and Raphanus sativus are characterized by the presence of spherical bodies approximately 1
/~ in diameter. These structures are Feulgen-negative but stain metachromatlcaUy with
azure B, as the nucleolus, following fixation with glutaraldehyde. At the ultrastructural
level, they consist predominantly of fibrils estimated to be between 70 and 100 A in diameter
which greatly resemble those found within certain zones of the nucleolus in these plant
species. Moreover, in Allium ¢epa, these spherules often exhibit dense particles which are also
found within the fibrillar zones of the nucleolus in this species. The observations that the
bodies in question are frequently located at the surface of the nucleolus and moreover show
cytochemical and ultrastructural similarities with this organelle suggest that they may originate from the nucleolus. However, the common association of the spherules with chromosome strands may indicate instead that these bodies represent extranucleolar ribonucleoprotein materials synthesized by specific chromosomal loci.
Published July 1, 1965
Elmiskop I or Philips EM-200 microscope operating
at 80 kv.
RESULTS
Morphology
T h e small bodies described in this report are
difficult to recognize u n d e r light microscopy except in r a t h e r thin preparations. T h e e x a m i n a t i o n
u n d e r phase-contrast microscopy of serial sections
0.25 /z thick shows t h a t these nuclear structures
correspond to small spheres a b o u t 1 g in diameter.
T h e u h r a s t r u c t u r e of the spherules varies with
the fixation used and, to some extent, also with
the species. After fixation with buffered osmium
tetroxide they show a homogeneous texture in
Vicia faba and Raphanus sativus (Figs. 18 to 20) and
consist of tightly packed fibrils the d i a m e t e r of
w h i c h is estimated to be between 70 a n d 100 A
(Fig. 19). I t is not clear how these fibrils are organ-
ized within the bodies or w h e t h e r they represent
their only structural constituent. At high e n o u g h
magnification (Fig. 19) the boundaries of the
spherules are not too clearly delineated due to the
intermingling of their constituent fibrils with those
of the s u r r o u n d i n g nucleoplasm. In Allium cepa
the bodies in question are often further characterized b y the presence of dense granules (Figs. 13
and 14) of the type already described in nucleoli
(1, 14-17) and prenucleolar bodies (1) of this
species as well as of other plants. M a n y cells in
Alliurn cepa contain spherical bodies w h i c h are
slightly larger a n d m a y r e a c h 1.3 /~ in diameter.
T h e r a t h e r loose organization of their fibrils
(Figs. 15 and 16) confers u p o n these bodies a
configuration somewhat reminiscent of a ball of
threads (Fig. 15) and a density sometimes only
slightly higher t h a n t h a t of the s u r r o u n d i n g
nucleoplasm.
A quite similar ultrastructural organization of
ht, heterochromatin
lz, light zone
he, nuclear envelope
nl, nucleolus
nlo, nucleolar organizer
sp. spherule
FIGURE 1 Allium cepa interphase nucleus showing two peripheral heterochromatin masses
(ht), one closely associated with the nucleolar surface. One light circular zone (lz) is located next to the nuclear envelope. Root tip fixed in buffered osmium tetroxide. Section
0.7 # thick, stained by means of the Feulgen reaction and counterstained with methylene
blue. X 4000.
FIGURE ~ Allium cepa interphase nucleus characterized by the presence of a spherule (sp)
adjacent to a heterochromatin mass (ht). A light circular zone (lz) is seen at the nuclear
boundaries. Root tip fixed in buffered osmium tetroxide. Phase-contrast micrograph of a
section 0.5 # thick, stained by means of the Feulgen reaction and counterstained with
methylene blue. X 4000.
FIGURE 8 Nucleus similar to that illustrated in Fig. ~. In this micrograph the spherule
is completely surrounded by heterochromatin. Buffered osnlium tetroxide fixation. The
section 0.5 # thick, stained by means of both the Feulgen reaction and methylene blue,
was photographed under phase-contrast microscopy. X 4000.
FIGURE 4 Allium cepa preprophase nucleus with three nucleoli. Two spherules (sp)
may be recognized on the surface of one of these organelles. The nucleolar organizing
(nlo) regions of two of the nucleoli are seen within the nucleolar mass. Buffered osmium
tetroxide fixation. Preparation stained and photographed as preceding figure. X 4000.
FIGURE 5 Allium cepa preprophase nucleus depicting a light circular zone (lz) located on
the surface of one of the nucleoli. Buffered osmium tetroxide fxation. Section 0.7 # thick,
stained as preceding preparation but photographed under ordinary optics. X 4000.
FIGURE 6 This Allium cepa preprophase nucleus shows one spherule (sp) and a light zone
(lz). Same type of preparation as Fig. 5. X 4000.
2
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ch, chroniosomc
dp, dense particle
fz, fibrillar zones
gz, granular zones
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J. G. LAFONTAINE Nuclear Bodies in Meristematic Cells
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Published July 1, 1965
these nuclear spherules is observed following fixation with formaldehyde, glutaraldehyde, or
aldehyde-osmium
tetroxide.
However, their
boundaries appear more continuous with the surrounding nucleoplasm when a double fixation is
used, presumably as a result of a better preservation of the nucleoplasm itself.
Unbuffered solutions of osmium tetroxide in
distilled water (7) give rise to marked changes in
the density of the chromosomes and nucleoplasm
as compared with that of the nucleolus and spherical bodies (18). The spherules are somewhat more
compactly organized and their fibrillar texture
less evident (Fig. 17) than when standard fixatives
(8, 9) are used.
Staining Characteristics
FIGURE 7 Micrograph of preparation similar to Fig. 6. Here a spherule (sp) and light
zone (lz) are both located next to the nuclear boundaries. X 4000.
FIGURE 8 Allium cepa preprophase nucleus characterized by two spherules (sp) much
denser than usually found in this species. Phase-contrast micrograph of section 0.5 # thick.
Same fixation and staining prooedures as previous figures. X 4000.
FIGURE9 Midprophase nucleus from Viciafaba root tip fixed with a 1 per cent unbuffered
solution of osmium tetroxide containing 0.005 per cent CaC12. Note that the chromosomes
are not too conspicuous but that both the nucleolus (nl) and the spherule (sp) are quite
dense. Phase-contrast micrograph of a section 0.7 # thick, stained by means of both the
Feulgen reaction and methylene blue. )< 4200.
FIGURE 10 Allium cepa midprophase nucleus depicting two spherules (sp). Buffered
osmium tetroxide fixation and staining as above figures. Phase-contrast micrograph of a
section 0.7 # thick. X 4000.
FmURE 11 Midprophase nucleus (AUium cepa) with a light circular zone (lz). Fixation
and staining as in Fig. 10. The section 0.7 bt thick was photographed under ordinary optics.
X 4000.
FIGURE 12 Late prophase nucleus (Viciafaba) showing a dense spherule (sp) at a time
when the nucleolus (nl) is dispersing. Phase-contrast micrograph of a section 0.5 # thick.
Same fixation and staining as Figure 9. X 3500.
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The nuclear spherules are Feulgen-negative but
may be brought out with methylene blue staining
(Figs. 2 to 4, 6 to 10). Their density, following
such staining, usually matches quite closely that
of the nucleolus (Figs. 6, 7, 9, and 10). However
the density of these two types of structure relative
to that of the chromosomes varies somewhat with
the species examined and the method of fixation
used.
Following fixation with buffered solutions of
osmium tetroxide, the spherules and nucleoli are
lighter than the chromosomes in Vicia faba preparations stained with the Feulgen procedure and
methylene blue. The same situation also usually
prevails in Allium cepa meristematic cells (Figs.
2 to 4, 6, and 7) but in this species both the
spherules and nucleoli may sometimes stain more
densely than the chromosomes (Fig. 8) ; the reasons
underlying their occasional increased stainability
in certain preparations are not understood.
Instances have also been recorded in Allium cepa
where the spherules are lighter than both the
nucleolus and the chromosomes. This last observation is in accord with the finding that some of
these bodies show a rather loose ultrastructural
organization (Figs. 15 and 16).
In Vicia faba material fixed with unbuffered
solutions of osmium tetroxide in distilled water,
the chromosomes stain very little with the Feulgen
reaction and likewise appear to react to only a
small extent with methylene blue. As a result the
chromosomes are barely distinguishable even
under phase-contrast microscopy. The nucleolus
and the spherical bodies, nevertheless, stain quite
intensely with methylene blue and are therefore
easily seen in thin sections (0.5/z). In fact, due to
their relative density as compared with that of the
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J. G. LAFONTAINE Nuclear Bodies in Meristematlc Cells
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Published July 1, 1965
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FIGURE 13 Electron micrograph of portion of a preprophase nucleus (Allium cepa). T h e nucleolus (nl)
is clearly seen to consist of a peripheral granular zone (gz) and a fibrillar zone (fz) located more centrally.
This latter zone contains most of the dense particles seen in the nucleolus. T h e lighter central area shows
chromatin material corresponding to the nucleolar organizer (n/o). Next to the nucleolus is recognized a
spherule consisting predominantly of fibrils b u t also exhibiting dense particles similar to those found
within the nucleolus. The chromosomes are made up of fibrils mostly and contain no such particle. Fixation with buffered osmium tetroxide. T h e preparation was double-stained with solutions of uranyl acetate
a n d lead hydroxide. X 41,000.
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FIGUI~E 14 Electron micrograph similar to Fig. 13. The majority of the dense particles are restricted to
the central fibrillar zone (fz). The spherule (sp), located within the nucleoplasm, shows a number of identical particles. Note that these particles are not found free in the nucleoplasm. Other less dense particles
are, however, detected in groups (arrows) between the chromosomes. Fixation and heavy metal staining
as in Fig. 13. X 38,000.
J. G. LAFONTAINE Nuclear Bodies in Merlstematic Cells
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Published July 1, 1965
chromosomes, these bodies are more easily detected in such preparations than in material fixed
with buffered osmium tetroxide. When calcium
ions are added in increasing concentrations to the
fixing solution, the chromosomes progressively
gain density (18) and become more stainable with
both the Feulgen procedure and methylene blue
(Fig. 9).
U n d e r electron microscopy it is observed that
the staining behaviour of the small spherical
bodies likewise closely corresponds to that of the
nucleolus. Following fixation with buffered
osmium tetroxide and after staining with a 1 per
cent aqueous solution of uranyl acetate for 2
hours, the density of the spherules equals that of
the nucleolus but both types of structures are
lighter than the chromosomes. Subsequent to
counterstaining with lead hydroxide, an increase
in the density of the two first structures is observed
and it then matches that of the chromosomes
(Figs. 13, 14, and 18).
In a given section 0.5/z thick or under electron
microscopy, the majority of nuclei characterized
by the presence of spherules show only one such
body (Figs. 2, 3, 6, 7, 9, 12 to 14, 18, and 20) but,
occasionally, some nuclei exhibit two such bodies
(Figs. 4, 8, and 10). A survey of consecutive sections reveals, moreover, that a proportion of
nuclei, much higher than suggested by the examination of single preparations, possess at least one
spherule. Taking into account the portion of a
FIGURE 15 Micrograph depicting a light spherule (sp) next to two thick segments of
chromosomes (ch) at interphase (Allium cepa). Note that the chromatin material is intimately associated with the surface of the spherule and, at places (arrows), closely follows
its contours. Fixation with buffered osmium tetroxide and staining with uranyl acetate
and lead hydroxide. X 36,000.
FIGURE 16 Portion of an interphase nucleus (Allium cepa) illustrating a light spherule.
At this magnification, the body appears to consist predominantly of fibrillar material.
Some of these fibrils are coarser, or perhaps more densely stained, and stand out quite
clearly. They are loosely organized and confer the appearance of a ball of threads to the
body in question. Other fibrils are less distinct, and form a sort of greyish, more amorphous
matrix. At several places (arrows), small chromatin projections are continuous with the
surface of the spherule and merge imperceptibly with its fibrillar constituents. Groups of
large granules (double arrows) may be seen in the nucleoplasm. Fixation and staining as in
Fig. 15. )< 44,500.
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Occurrence and Distribution in Interphase,
Preprophase and Prophase Nuclei
nucleus represented in a section 0.5/~ thick as well
as the frequency of occurrence of these structures
per section, it is estimated that approximately half
of the nuclei contain one or two spherical bodies.
A similar conclusion is arrived at when series of
consecutive sections are examined. Indications
have also been obtained that certain nuclei are
characterized by three and sometimes four similar
bodies.
No attempt was made in the course of the present work to investigate the possibility of variations
in the frequency of occurrence of these bodies,
from interphase to late prophase, in a given tissue
of the root tip or of the influence of cell differentiation on this frequency.
During interphase, the nuclear spherical bodies
are quite often closely associated with heterochromatin masses (Figs. 2 and 3) which sometimes
surround them completely (Fig. 3). Such associations are observed both when the spherules are
found on the surface of the nucleolus and when
they are located elsewhere within the nucleoplasm. By preprophase, presumably due to the
disappearance of these chromatin masses themselves (11), the spherules are no longer associated
with heterochromatin. Spherical nuclear bodies
are also found throughout prophase (Figs. 9, 10,
and 18), and some are still detected at the end of
that stage when the nucleolus has already begun
to disperse (Fig. 12). Their size, staining characteristics, and ultrastructure (Figs. 18 and 19) do
not appear to differ from the situation observed at
interphase or preprophase. No such spherules are
detected at metaphase, anaphase, or telophase in
Vicia faba. Prenucleolar bodies similar to those
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J. G. LAFONTAINE Nuclear Bodies in Meristematic Cells
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DISCUSSION
Possible Relation of the Spherical Bodies
with the Nucleolus
O u r observations show that the nuclear
spherules share a number of morphological and
staining characteristics with the nucleolus. Both
types of structure react identically with methylene
blue, azure B, or to solutions of heavy metals.
Subsequent to such staining procedures, the
spherules show a density which matches that of
the nucleolus; however, the loosely organized
bodies sometimes found in Allium cepa (Figs. 15
and 16) remain lighter. This similarity in staining
behaviour is especially striking when sections are
treated for increasing lengths of time with solutions of uranyl acetate. The densities of both the
spherules and nucleoli are then found to be equal
(Figs. 13, 14, and 18) and to increase at a similar
rate.
It is observed, moreover, that the spherical
bodies consist predominantly of tightly packed
fibrils the diameter of which is between 70 and
100 A (Fig. 19). Such fibrillar material is indistinguishable from that observed within the nongranular zones of the nucleolus (11) (Figs. 13, 14,
and 20). The ultrastructural similarity between
spherules and nucleoli is further illustrated in
Allium cepa where the former bodies and the
fibrillar zones of nucleoli are characterized by the
presence of dense granules some 150 to 400 A in
diameter (Figs. 13 and 14). These particles have
thus far been noted only within the fibrillar zones
of the nucleolus (14) and the telophase prenucleolar bodies (1).
FmUaE 17 Freprophase nucleus (Viciafaba) from a root tip fixed with a 1 per cent unbuffered solution of osmimn tetroxide containing no divalent ions. The chromosomes are
barely distinguishable due to their low density as compared to that of the nucleoplasm.
The nucleolus (nl) and spherules (sp), however, are quite dense and stand out clearly.
It will be noted that the outer portion of the nueleolus is more compactly organized,
especially within the peripheral zone which is usually (compare with Fig. 14) distinctly
granular. The fibrillar nucleolar zones are not easily recognized due, apparently, to a
coarsening of their texture which now resembles that of the granular zones.
The spherules are likewise somewhat more compact in structure and their fibrils less
evident. Their surface is seen to merge more gradually with the surrounding nucleoplasm
than in preparations (Figs. 13 and 14) fixed with buffered solutions of osmium tetroxide.
This is due primarily to the greater density of the nucleoplasm in the present figure.
Preparation double-stained with solutions of uranyl acetate and lead hydroxide. X 37,000.
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already described (1) have been observed at early
and midtelophase in Allium cepa; they are of various sizes and shapes and, moreover, show a fibrillogranular uhrastructure. Such bodies are thus, to
all appearances, not closely related to the spherules
under discussion, which are essentially fibrillar in
texture (Figs. 19 and 20).
There appears to be a definite pattern in the
distribution of the spherules: they are sometimes
either found at the surface of the nucleolus
(Figs. 4, 13, and 20) or in the immediate proximity
of the nuclear envelope (Figs. 7, 9, and 16), but
are more commonly observed in the nucleoplasm
at intermediate positions between these two sites
(Figs. 2, 3, 6, 8, 10, 12, 14, 15, 17, and 18).
U n d e r light microscopy, interphase, preprophase, and prophase nuclei quite frequently show
circular, unstained areas only slightly larger than
the bodies themselves. These light zones are characterized by a distribution matching that of the
spherical bodies: they are frequently observed at
the surface of the nucleolus (Fig. 5), somewhere in
the middle of the nucleus (Figs. 6 and 11), or at
its boundaries (Figs. 1, 2, and 7). Such circular
zones most likely correspond to the very loosely
organized light bodies observed under electron
microscopy (Fig. 16), and it is conceivable that
they are in the process of disintegrating. However,
it must also be noted that such bodies may remain
compact in structure even though located next to
the nuclear envelope (Figs. 7 and 9). Also,
spherules similarly located within a given nucleus
may show quite different densities (Figs. 6 and 7).
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J. G. LAFON'rAINE Nuclear Bodies in Meristematie Cells
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Nature and Fate of the Spherical Bodies
With respect to the problem of the nature of the
spherical bodies, a n u m b e r of possibilities must be
envisaged. A frst hypothesis is that the bodies in
question represent remnants of the prenucleolar
substance observed at telophase in Viciafaba (1 l)
and of the prenucleolar bodies characterizing
corresponding nuclei in Allium cepa (1). Since,
however, this material shows a fibrillogranular
texture in both species, it would have to undergo
some transformation at early interphase to account
for the predominantly fibrillar ultrastructure of the
spherules at that stage as well as during prophase
(Figs. 13, 14, 18, and 19). The available evidence,
in Viciafaba (11) at least, suggests on the contrary
that the prenucleolar material retains its fibrillogranular texture till late telophase and is completely incorporated into the maturing nucleolus.
No spherical bodies are ever seen while this condensation process is taking place.
One could also conjecture that the spherules
are analogous to the parachromatin material
identified cytochemically within certain types of
animal cells. It is believed that these nucleolar
parachromatin bodies, or nucleolini, are extruded
into the nucleoplasm (25, 26) and from there to
the cytoplasm (26, 28). A similar migration phenomenon may account for the observed distribution of the spherules in nuclei of Vicia faba and
Allium cepa, but a n u m b e r of facts remain which
indicate that the parachromatin bodies do not
correspond to the bodies under discussion. First,
as far as we know, nucleolini have not been demonstrated cytochemically in plant meristematic cells
and, moreover, no corresponding structures are
recognized under the electron microscope (1, 11,
14, 15) within the nucleolus. Secondly, the parachromatin bodies, contrary to the spherules, are
thought to increase in size within the nucleoplasm
during prophase and to persist in the cytoplasm
(27, 28) throughout metaphase, anaphase, and
sometimes telophase. Till these inclusions are
characterized at the ultrastructural level, it will
be difficult to ascertain their relationship to the
various components of the nucleus.
Further studies will also be required to verify
whether the spherules described here are related
to the micronucleoli seen in plant cells by certain
authors (29, 30).
In keeping with their frequent localisation on
the surface or in the immediate proximity of the
nucleolus during interphase and prophase, it is
conceivable that the spherules originate from this
organelle. This hypothesis derives support from
FIGURE 18 Portion of a midprophase nucleus (Vicia faba). Several chromosomes (ch)
have been cut obliquely. The spherule (sp) consists of material which appears similar to
that found within the fibrillar zones (fz) of the nueleolus. Buffered osmium tetroxide
fixation. Double staining with solutions of uranyl acetate and lead hydroxide. X 38,000.
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Finally, it should be recalled that following fixation with unbuffered solutions of osmium tetroxide
the chromosomes are barely visible under light
and electron microscopes, but that both the
spherules and the nucleoli remain quite conspicuous (Figs. 9 and 17). A similar situation prevails
in Rhaphanus sativus material fixed with buffered
solutions of aldehydes, osmium tetroxide (Fig. 20),
or both.
T h e above series of observations taken as a
whole, together with the frequent presence of
spherules on the nucleolar surface, (Figs. 4, 13,
and 20) suggest the existence of some sort of relationship between the bodies under discussion and
the nucleolus. Furthermore, on the basis of the
ultrastructural characteristics of the nuclear
spherules, this relationship may be assumed to
exist most particularly with the fibrillar zones of
the nucleolus.
J u d g i n g from the binding of uranyl ions to
R N A and proteins (19) and from the progressive
increase in the density of the spherules as a result
of staining with aqueous solutions of uranyl
acetate, it appears likely that the spherules consist
mainly of R N A and proteins. This, at least, is concordant with available cytochemical data concerning the nature of the different nucleolar zones
(19-24) as well as that of very similar bodies observed in Tetrahymena pyriformis (2, 3). The presence of R N A within the spherules is further indicated by their metachromatic staining with azure
B in preparations fixed with glutaraldehyde and
embedded in glycol methacrylate.
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J. G. LAFONTAINE Nuclear Bodies in Meristematic Cells
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the observed similarities in uhrastructure between
the spherules and the fibrillar zones of the nucleolus (Figs. 14 and 20) as well as from reports that
vacuoles and amorphous substances (31-34) are
extruded from the nucleolus in animal and plant
cells. O u r own data do not demonstrate unequivocally that the spherules seen in Allium cepa, Vicia
faba, and Rhaphanus sativus actually originate from
the nucleolar mass. These observations are
equally compatible with the formation of such
bodies directly on the surface of the nucleolus as a
result of the activity either of the nucleolus itself
or of the nucleolus-associated chromatin.
A final assumption which must be considered is
14
that these nuclear bodies represent extranucleolar
material synthesized by specific chromosomal loci.
In view of the apparent production of these bodies
throughout interphase (Figs. 1 to 8, 13 to 16) and
prophase (Figs. 9 to 12, 18), it must also be conjectured, if this hypothesis is correct, that we are
dealing with loci which become synthetically
active subsequent to the formation of the nucleolus
at telophase. The striking ultrastructural resemblance of these bodies with the fibrillar zones of
the nucleolus could thus reflect that these two
types of nuclear bodies represent different forms
of chromosomal products which consist predominantly of similar biochemical constituents. The
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FmcaE 19 Higher magnification micrograph of portion of Fig. 18. The spherule (sp) is seen to be made
up mostly of lightly packed fibrils. The latter appear somewhat finer, perhaps, than those found within
the chromosomes (eh). At this magnification the surface of the spherule is less clearly delineated, due to
intermingling of some of its constituent fibrils with those present within the surrounding nucleoplasm.
X 68,000.
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FIGURE ~0 Portion of Raphanus sailvus nucleus illustrating a spherical body (sp) located on the surface
of the nucleolus (nl). At this magnification, the texture of the spherule is seen to resemble t h a t of the
fibrillar zones (fz) of the nucleolus. I n this species the nucleolus also consists of granular zones (gz) intermingled with fibrillar areas.
T h e nucleoplasm appears homogeneous and the chromosomes are indistinguishable. X 48,000.
J. G. LAFONTAINE Nuclear Bodies in Meristematic Cells
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Published July 1, 1965
available cytochemical d a t a p e r t a i n i n g to nucleoli
(19-24), as well as our p r e l i m i n a r y observations
with azure B staining, point to R N A as being one
of the constituents w h i c h they contain in common.
I t m a y well be, therefore, t h a t the spherules
described in the present report are analogous in
certain respects to the ribonucleoprotein materials
elaborated b y l a m p b r u s h (35) a n d salivary gland
(36, 37) chromosomes. F u r t h e r studies have been
initiated with cytochemical a n d a u t o r a d i o g r a p h i c
techniques in c o m b i n a t i o n w i t h electron microscopy to characterize further the chemical n a t u r e of
the spherules a n d to verify t h e i r possible relationship to the nucleolar constituents.
Now, concerning the fate of the spherules, it
appears likely t h a t they are eventually secreted
into the cytoplasm. T h e y are indeed frequently
observed at the periphery of the nucleus, a n d m a n y
of o u r micrographs are best i n t e r p r e t e d by assuming t h a t they disperse in this area (Figs. 1, 2, 7,
a n d 16). T h e fact t h a t in the present study such
bodies have never been seen in the process of migrating across the nuclear envelope most likely
indicates t h a t transport of material takes places at
the m a c r o m o l e c u l a r level c o n t r a r y to the situation
observed in Tetrahymena pyriformis (2, 3) where
similar structures are extruded b y m e a n s of blebs
of this envelope.
This investigation was supported by grant T-1018
of the National Research Council of Canada.
Received for publication, April 14, 196"4.
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