Download sites of protein synthesis in nucleoli of root meristematic cells of

J. Cell Sci. 2, 473-480 (1967)
Printed in Great Britain
473
SITES OF PROTEIN SYNTHESIS
IN NUCLEOLI OF ROOT MERISTEMATIC
CELLS OF ALLIUM CEP A AS SHOWN BY
RADIOAUTOGRAPHY WITH [3H]ARGININE
L. A. CHOUINARD AND C. P. LEBLOND
Departments of Anatomy, Laval University, Quebec City andMcGill
Montreal, Canada
University,
SUMMARY
The interphase nucleolus in root meristematic cells of Allium cepa may be divided into four
regions, three of which are always present: the fibrillar, granular and lacunar regions, while the
fourth or vacuolar region may be missing. The sites of protein synthesis in nucleoli were investigated by means of light and electron-microscope radioautography after a 5-min immersion of
the roots in a solution of [3H]arginine.
The radioautographs of interphase nucleoli showed many silver grains over both the fibrillar
and the granular regions. Occasional silver grains were also recorded over, or close to, the
lacunar regions, but none were over the vacuolar regions. A 15-min chase period did not change
the radioautographic pattern. It is concluded that the three permanent regions of the interphase
nucleoli, namely the fibrillar, the granular and the DNA-containing lacunar regions, are sites
of protein synthesis.
INTRODUCTION
Light- and electron-microscope observations reveal that the interphase nucleoli in
plant cells are made up of several components segregated into regions distinguishable
by differences in staining properties and ultrastructural characteristics (Lafontaine
& Chouinard, 1963; Hyde, Sankaranarayanan & Birnstiel, 1965; Chouinard, 1966 a, b).
That protein synthesis takes place in plant nucleoli has been shown by the uptake of
labelled amino acids as detected by radioautography in the light microscope (Woodard,
Rash & Swift, 1961; De, 1961; Mattingly, 1963) and by biochemical analyses (Birnstiel, Chipchase & Bonner, 1961; Birnstiel & Hyde, 1963; Birnstiel & Flamm, 1964;
Flamm & Birnstiel, 1964). Yet it is not known which of the structural components
of the nucleolar mass is instrumental in these processes of protein synthesis. The
synthesis of protein has also been observed in the nucleoli of animal cells, as shown
in the review by Stocker (1963). The uptake of labelled amino acids first observed by
Ficq in 1953 was later questioned (Schultze, Oehlert & Maurer, 1958; Carneiro &
Leblond, 1959), but with improvements in technique was clearly demonstrated in the
light microscope (Leblond & Amano, 1962; Kasten & Strasser, 1966) as well as in the
electron microscope (Sandborn, 1963; Droz, 1965).
The present investigation was undertaken to pinpoint the sites of synthesis of
474
•£• A. Chouinard and C. P. Leblond
proteins in interphase nucleoli of root meristematic cells in Allium cepa. Radioautography was used in the light and electron microscopes following pulse-labelling
of the proteins with tritiated arginine.
MATERIALS AND METHODS
Seeds of Allium cepa were germinated on the surface of loosely packed vermiculite
kept moist by daily watering. After i week germination at room temperature, the
primary roots of the seedlings had reached approximately i cm in length. The roots,
still attached to the seedlings, were then immersed for 5 min in distilled water
containing 100 /ic/ml of tritiated arginine. The isotope used, L-[G-3H]arginine monohydrochloride (Radiochemical Centre, Amersham, England), has a specific activity of
1830 mc/mM. At the end of the 5-min pulse treatment, the seedlings were rinsed in
distilled water and the distal 1-5 mm of some root tips were fixed for 2 h at room
temperature in a mixture of formaldehyde and glutaraldehyde (Karnovsky, 1965) in
O-IM Sorensen's phosphate buffer, PH7.2; the remainder of the undetached roots
were re-immersed for a period of 15 min in a solution of non-radioactive L-arginine
monohydrochloride made up to a concentration 100 times that of the radioactive
sample. Following this chase period, the seedlings were rinsed with distilled water
and the root tips fixed as described above. After fixation, all root tips were placed
overnight in O-IM Sorensen's buffer, pH 7-2, at 4 °C; the next day, they were dehydrated over a 2-h period in an ascending series of ethanol concentrations and finally
embedded in Epon 812.
Half-micron sections of the root meristematic regions were mounted on glass slides
with a wire loop and coated by dipping in liquid Ilford L-4 emulsion. After 48 h
exposure and development, a drop of filtered 1 % toluidine blue was placed on the
slides, allowed to stand at room temperature for 5 min, and rinsed off with distilled
water. After dehydration, a coverslip was placed over the sections. The emulsioncoated and exposed sections as well as the uncoated half-micron sections from the
same blocks were studied with a Leitz binocular microscope, using a ribbon-filament
lamp, Kohler illumination, and a 100 x 1-32 N.A. apochromatic objective. An orange
Wratten filter was used in the illumination system.
Ultrathin pale gold sections, cut from the same tissue blocks as the o-5-/t sections,
were mounted on celloidin-coated glass slides, coated with a layer of Gevaert 307
emulsion and, after 2 weeks exposure and proper development, stripped and mounted
on 300-mesh copper grids (Salpeter & Bachmann, 1964). Successful double staining
of these grids was achieved by floating, emulsion side down, upon drops of a saturated
solution of uranyl acetate in 50% ethanol (30 min) followed by lead citrate (30 min).
Uncoated sections were also stained in the same manner for electron-microscope
examination. The sections were examined in a Siemens Elmiskop I electron microscope using the double condenser, 80 kV and 50-/6 objective aperture.
Protein synthesis in nucleolus of Allium
475
OBSERVATIONS
Structural organization of the interphase nucleolus
Light microscopy. A. cepa is a diploid species with a single pair of nucleolar chromosomes (Heitz, 1931). Two nucleoli, therefore, are formed at telophase and these,
during interphase, may remain distinct (Fig. 1) or adhere to form a dumbbell-shaped
structure, or fuse into a large spherical organelle which may reach up to 6/t in
diameter (Figs. 3, 4).
In 0-5-/4 sections of meristematic cells stained with toluidine blue, the nucleolar
material exhibits a purplish blue metachromatic colour (Figs. 1-4). The nucleolus
often contains a large, unstained space located centrally or paracentrally, the nucleolar
vacuole (Figs. 3, 4). The vacuole may be missing, or there may be more than one
(Fig. 3). With an orange filter, the metachromatically stained material of the nucleolus
proves to be not homogeneous but made up of two components slightly differing in
staining intensity (Figs. 1-4). The less intensely stained component is located along
the edge and in the centre of the nucleolus as well as in radial areas extending
in between (Figs. 1, 2); this component also surrounds the vacuole when present
(Figs. 3, 4). The more intensely stained component, on the other hand, is arranged in
irregular patches usually located between the edge and the centre of the nucleolus
(Figs. 2, 4). Within these patches, there are a few tiny unstained spaces which will be
referred to as lacunae (Figs. 2, 3, arrows). Such lacunae are less readily identified here
than in sections of osmium-fixed meristematic cells stained by the Feulgen/methylene
blue procedure; they are then seen to be a constant feature of the more intensely
stained component of the nucleolus (Chouinard, 1966a, b).
Electron microscopy. The nucleoli appear as electron-dense bodies containing light
spaces corresponding to the vacuoles and lacunae distinguished in the light microscope. The dense portion shows denser and less dense regions, corresponding respectively to the more and the less intensely stained regions seen in the light microscope
(Fig. 9). The denser regions have irregular boundaries with processes projecting
into the surrounding less dense regions; small isolated patches of denser material
may also be embedded within the less dense regions.
The less dense regions of the nucleolus consist mainly of packed granules about
150 A in diameter (Fig. 9). In addition, fibrils are encountered which also are 150 A
in diameter and, therefore, would appear as granules when cut across. Occasionally,
series of granules are strung like beads; or barely distinguishable granules appear as
periodic thickenings along the 150-A fibrils. Finally, a few fine, parallel fibrils approximately 60 A or more in diameter may also be seen among the granules. These less
dense regions will be referred to as 'granular'.
The denser regions of the nucleolus are composed of an amorphous ground substance
containing tightly packed, electron-dense fibrils (Fig. 9). Most of them are thin with
a diameter of 60 A. Larger ones may also be observed up to 150 A. The denser regions
will be referred to as 'fibrillar'.
Of the lighter spaces, some are vacuoles. These contain loosely and uniformly
dispersed granules and fibrils. The vacuoles are enclosed within the granular region.
476
L. A. Chouinard and C. P. Leblond
On the other hand, within the fibrillar region, the lacunae appear as light staining
spaces usually smaller than the vacuoles but variable in size and shape and usually
exhibiting a loose fibrillar texture (Fig. 9, arrows); in the lacunae it is often possible
to distinguish a core of dense material staining with the same intensity as the chromatin
(Figs. 9-13, double arrows). In this connexion, evidence has recently been obtained
indicating that, as in the case of the large Spirogyra nucleoli (O'Donnel, 1961; Godward
& Jordan, 1965), the lacunae in question contain DNA. It is likely that these lacunae
are cross-sections of a channel containing the nucleolar organizing region of the
nucleolar chromosome (Chouinard, 1966 c).
In summary, the interphase nucleolus in root meristematic cells of A. cepa is made
up of 4 structural components segregated into regions distinguishable by differences
in staining properties and ultrastructural characteristics: 2 dense ones referred to as
granular and fibrillar regions, and 2 light ones, as vacuolar and lacunar regions.
In previous electron-microscope observations based on osmium-fixed material, evidence had been presented suggesting that the 2 main structural components of the
dense portion of the nucleolus (i.e. the granular and the fibrillar) correspond respectively to what has been referred to in animal nucleoli as nucleolonema (or nucleolonematic network) and pars amorpha (Chouinard, 1966 a, b). Indeed, in several places
within the granular regions and more particularly on the fluffy outer surface as well
as at the surface of the vacuoles, light irregular narrow spaces may be detected suggesting that the granules and fibrils are assembled into a coarse network, the meshes of
which are approximately o-1 ju, in diameter (Fig. 9); this would correspond to the
nucleolonema.
Sites of incorporation of tritiated arginine into interphase nucleoli
Light microscopy. As early as 5 min after immersion of the primary roots of A. cepa
into a solution containing [3H] arginine, a radioautographic reaction was observed over
all cells of the meristematic region. Representative light-microscope radioautographs
of interphase cells (Figs. 5-8) showed many silver grains over the nucleolus, a few
over the rest of the nucleus, that is, mainly over chromatin and, finally, a few over
cytoplasm. Careful examination of the nucleoli showed grains over both the denser
and less densely stained regions (Figs. 5, 6), but not over the vacuoles (Figs. 7, 8).
Electron microscopy. In the cytoplasm, a radioautographic reaction was found over
the ribosome-rich regions, and, occasionally, over mitochondria. In the nucleus, the
silver grains overlay the nucleolus, to a lesser extent the chromatin material, and to a
still lesser extent the nuclear sap. Examination of electron-microscope radioautographs
of the nucleolus (Figs. 10-13) showed silver grains over both fibrillar (Fig. 11) and
granular regions (Fig. 10). Grains were located not only over the main fibrillar regions
but also over the small and apparently isolated patches of similar fibrillar material
embedded in the granular regions (Fig. 12). Significant numbers of silver grains overlay
the irregular and often diffuse boundaries where fibrillar and granular regions merged
(Fig. 13). Finally, a few silver grains were located over, or in the immediate vicinity
of, the DNA-containing lacunar regions (Fig. 12), whereas there was no significant
labelling of the vacuolar regions.
Protein synthesis in nucleolus of Allium
477
Pulse-chase experiment. In this case, the roots were immersed first in a solution of
[3H]arginine for 5 min and then in a solution of non-radioactive arginine for 15 min.
Again light- and electron-microscope radioautographs showed labelling in all meristematic cells. Except for a 10-20 % reduction in the number of silver grains over the
nucleoli, the distribution pattern of the labelling appeared essentially the same as that
described in the experiments without chase.
DISCUSSION
The present observations revealed that, as early as 5 min after immersion of primary
roots of A. cepa in a solution of [3H]arginine, a radioautographic reaction was obtained
over the cytoplasm and nucleus of meristematic cells. This indicated that the radioactive arginine must have reached even the cells in the centre of the meristem and been
taken up into their cytoplasm and nucleus. Because of the high solubility of arginine
in water, it was assumed that any free arginine present in the cells would be washed out
during fixation and the subsequent 12-h immersion in buffer. However, when the
labelled specimens were subjected to large doses of non-labelled arginine in the
pulse-chase experiment, the grain count was decreased by 10-20 %. Hence, of the
label taken up at 5 min, 10-20% may consist of free arginine which is adsorbed
strongly enough not to be washed out during processing. [It is possible that the 10-20 %
decrease in radioactivity after the 15-min pulse chase is not due to removal of free,
labelled arginine. An alternative explanation would be that some of the newly synthesized proteins of the nucleolus have completely turned over during the 15 min period
as a result of emigration or breakdown.] But the radioautographic pattern was not
visibly modified by the pulse chase. Consequently, the pattern would be caused by
the rest of the label, that is, the 80-90 % which was not adsorbed and must therefore
have been incorporated into newly synthesized tissue components. Indeed, it was
known that, after injection of a labelled amino acid, only that fraction taken up into
newly synthesized proteins was retained in the sections (Droz & Warshawsky, 1963;
Leblond, 1965). Hence, the distribution pattern of radioactivity after glutaraldehydeformaldehyde fixation indicated the location of protein synthesized during the 5 min
of exposure to the labelled amino acid. If it is assumed that this time is too short for
significant migration of the new protein, then the radioactive regions are the sites of
protein synthesis. It may therefore be concluded that organelles of cytoplasm (ribosomes, mitochondria) and of nucleus (chromatin material, Figs. 5,1 o, 13, and nucleolus,
Figs. 5-8 and 10-13) were concomitantly the seats of protein synthesis.
Incorporation of [3H]arginine was observed in all interphase nucleoli. Hence, it
would appear that protein synthesis in that organelle is a continuous process throughout interphase. These observations confirm previous cytological findings that interphase nucleoli in root meristematic cells are sites of continuous protein synthesis
(Woodard et al. 1961; De, 1961; Mattingly, 1963). Judging from the relative concentration of silver grains in Figs. 5-8, it also seems as if the rate of protein synthesis
in the nucleolus of A. cepa is more rapid than that occurring elsewhere within the cell
nucleus, a conclusion confirming previous biochemical observations (Birnstiel et al.
478
L. A. Chouinard and C. P. Leblond
1961; Birnstiel & Hyde, 1963; Birnstiel & Flamm, i964;Flamm&Birnstiel, 1964). The
nature of nucleolar proteins which were being synthesized during the 5-min exposure
to [3H]arginine is not known. They could be any of the four types of proteins which
biochemists claim are present in both plant and animal nucleoli, since all four have
been shown to contain arginine and become labelled after administration of [3H]amino
acids (Birnstiel, Chipchase & Flamm, 1964; Grogan, Desjardins & Busch, 1966).
In the present study, a radioautographic reaction was observed over both the
granular and the fibrillar regions of the nucleolus. Hence, it may be concluded that
these two regions are sites of protein synthesis. In addition, a few silver grains were
located over, or in the immediate vicinity of, the DNA-containing lacunar regions,
thus raising the possibility that the intranucleolar DNA also may somehow be involved
in protein synthesis. These observations may be interpreted in relation to what is
known of RNA metabolism in the nucleolus. A number of authors have observed that
RNA synthesis takes place in the fibrillar regions of animal nucleoli and subsequently
the newly synthesized RNA migrates to the surrounding granular regions (Granboulan
& Granboulan, 1965; Karasaki, 1965; Geuskens & Bernhard, 1966). In root meristematic cells RNA synthesis was also associated with the fibrillar regions of the nucleolus
(La Cour & Crawley, 1965). According to Karasaki (1965) and Granboulan & Granboulan (1965) chromatin material present in the fibrillar regions of the nucleolus
participates in nucleolar RNA synthesis. It is conceivable that the newly synthesized
RNA is in the form of nucleoprotein fibrils (Marinozzi, 1964), which in the course of
migration to the granular region become beaded and transform into granules. It is
tempting to speculate further that the RNA molecules which are being synthesized
by the intranucleolar chromatin and which subsequently invade the rest of the
nucleolar mass, are, during their migration, somehow instrumental in the elaboration
of protein moieties in both the fibrillar and the granular regions of the nucleolus.
In the present study, no significant labelling of the vacuolar regions of the nucleolus
was observed, a fact indicating that synthesis of proteins does not take place, or is
negligible, there.
The general conclusion to be drawn from the above observations is that all three of
the permanent structural components of the interphase nucleolus in root meristematic
cells of A. cepa, namely, the fibrillar, granular and DNA-containing lacunar regions,
are sites of protein synthesis.
The authors gratefully acknowledge the assistance of Dr Beatrix Kopriwa in preparation of
the radioautographs. We also thank Mr P. Koen for skilled technical assistance. This investigation was supported by grants from the Medical Research Council of Canada.
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Figs. 1-4. Representative light micrographs of interphase nuclei showing the nucleolus
in the midst of a network of chromatin. The stainable portion of the nucleolus has 2
components differing slightly in staining intensity. The less intensely stained component is located along the edge and in the centre of the nucleolus as well as in radial
areas extending in between (Figs. 1, 2); this component also surrounds the unstained
vacuoles (Figs. 3, 4, v). The more intensely stained component is arranged in irregular
patches usually located between the edge and the centre of the nucleolus (Figs. 2, 4);
these patches contain unstained, barely resolvable lacunae, two of which (arrows) may
be recognized in Figs. 2 and 3. x 4000.
Figs. 5-8. Representative light microscope radioautographs of interphase nuclei
after 5 min immersion of the roots in [3H]arginine solution. Many silver grains are
located over the nucleolus, a few over the rest of the nucleus, and, finally, a few over
the surrounding cytoplasm. In the nucleolus, the unstained vacuoles (seen in Figs. 7, 8)
are not labelled, whereas silver grains overlie both the more intensely and the less
intensely stained regions of the rest of the nucleolus. (Ilford L-4 emulsion, 48 h exposure.) x 4000.
Journal of Cell Science, Vol. 2, No. 4
t
5
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L. A. CHOUINARD AND C. P. LEBLOND
'IV
Fig. 9. Electron micrograph depicting an interphase nucleolus. Two types of structural
components, one granular, the other fibrillar, each segregated into regions distinguishable by differences in electron density, are found within the dense portion of the
nucleolus. The less-dense granular regions are located along the edge and in the
centre of the nucleolus with radial areas extending in between. Light, irregular narrow
spaces within these regions suggest that the granular elements are assembled into a
coarse network. The denser fibrillar regions, circumscribed by a solid black line,
appear as irregular patches located between the edge and the centre of the nucleolus.
These regions contain several small light-staining lacunae (arrows). These lacunae
show a core of dense chromatin-like material, which is clearly visible at the double
arrows, x 27000.
Journal of Cell Science, Vol. 2, No. 4
L. A. CHOUINARD AND C. P. LEBLOND
Figs, io, I I . Electron-microscope radioautographs of interphase nucleoli after 5 min
immersion of the roots in [3H]arginine solution. The radioautographic reaction occurs
over the dense fibrillar regions (Figs, io, 11) and the less dense granular regions, as is
well shown in Fig. 10. Some of the lacunae (double arrows) show a core of denser
chromatin-like material. (Gevaert 307 emulsion; 2 weeks exposure.) x 22000.
Journal of Cell Science, Vol. 2, No. 4
L. A. CHOUINARD AND C. P. LEBLOND
Figs. 12, 13. Electron-microscope radioautographs of interphase nucleoli after 5 min
immersion of the roots in [3H]arginine solution. The radioautographic reaction occurs
over the dense fibrillar and the less-dense granular regions of the nucleolus. Many
silver grains also overlie the irregular and in places diffuse boundaries between fibrillar
and granular regions. A few silver grains are located over or adjacent to some of the
lacunar regions (arrows). Some lacunae (double-arrows) show a core of denser chromatin-likc material. (Gevaert 307 emulsion; 2 weeks exposure.) x 22000.
Journal of Cell Science, Vol. 2, No. 4
L. A. CHOUINARD AND C. P. LEBLOND