J. CellSci. 47, 77-89(1981)
Printed in Great Britain © Company of Biologitts Limited 1981
INORGANIC PHOSPHATE ACCUMULATION
AND PHOSPHATASE ACTIVITY IN THE
NUCLEUS OF MAIZE EMBRYO ROOT CELLS
R. DELTOUR, S. FRANSOLET AND R. LOPPES*
Ddpartement de Botanique, Service de Morphologie vefge'tale et
*Laboratoire de Gdnetique mole'culaire, University de Liege,
Sart Tilman, B4.000 Liige, Belgique/Belgium
SUMMARY
The nucleus of growing root cells of Zea mays contains a high concentration of inorganic
phosphate. In order to verify whether this high nuclear P ( concentration is correlated with the
metabolic activity of the nucleus, the P ( has been visualized in root cells of maize embryos at
the electron-microscope level during 2 different periods which are both characterized by a
spectacular reactivation of the nuclear metabolism, i.e. the early germination and the period
of recovery following a thermal treatment given to the seeds after 48 h of germination. In both
situations the Pj concentration increased in the nucleus during its reactivation.
To verify whether the high nuclear P< concentration could be of endogenous origin, the
phosphatase activities were measured in crude extracts of root tissues during nuclear reactivation. The specific activity was optimal at pH 4 5 and was shown to increase with cellular
reactivation.
The ultrastructural localization of acid phosphatase activity showed that P< may be produced
at 3 distinct sites: plasmalemma, vacuoles and most probably nucleus itself. High acid phosphatase activities were found in nuclei displaying a high metabolism.
Taking these results and previous data into account, we suggest that a correlation may
exist between the rate of nuclear transcription, the level of nuclear acid phosphatase activity
and the nuclear Pt accumulation.
INTRODUCTION
Inorganic P ; is present at high concentration in the nucleus of cells having a high
metabolic activity, namely maize and onion root tips (Tandler & Solari, 1969). The
concentration of the phosphate ion in the sole nucleolus was calculated to be approximately 0-5-0-8 M and to represent 30-50% of the total inorganic phosphate of the
cell (Libanati & Tandler, 1969). The reason why P i accumulates in the nucleus is
unknown. One of the first questions to be solved however would be to determine
whether high P, concentrations in the nucleus are related to high metabolic activities
of the nucleus itself. This problem can be approached by using experimental or
natural systems which allow nuclei of low and high metabolic activity to be compared.
A spectacular nuclear reactivation is known to occur during early germination of
maize which is characterized by the resumption of transcription (Deltour, 1970;
Van de Walle, Bernier, Deltour & Bronchart, 1976), of mitotic activity and of DNA
synthesis (Deltour & Jacqmard, 1974). A similar reactivation can also be observed in
78
R. Deltour, S. Fransolet and R. Loppes
maize embryos during the recovery following a thermal shock at the second day of
germination (Fransolet, Deltour, Bronchart & Van de Walle, 1979).
In order to verify whether the concentration of P i in the nucleus increases during
germination and during the period of recovery after a thermal shock, we have localized
this ion by using ultrastructural cytochemistry. In addition, as inorganic phosphate
may originate from phosphatase activities, we have biochemically determined and
cytochemically localized these enzyme activities. It will be shown that Pi accumulates
and that phosphatase activities increase during nuclear reactivation. These data taken
together with previous results strongly suggest that in the nucleus a relationship
may exist between the acid phosphatase activity, the accumulation of Pf and the rate
of transcription.
MATERIAL AND METHODS
Germination and thermal shock conditions. Kernels of Zea mays var C1V2 were germinated in
Petri dishes on moist cotton and filter paper in the dark in a temperature box at 16 °C.
The thermal shock was given to kernels after 48 h of germination by transfering the Petri
dishes during 5 h to another temperature box at 46 °C. Afterwards the dishes were returned
to 16 °C.
Electron microscopy
Cytochemical visualization of inorganic phosphate. Embryos were excised from kernels after
which the coleorhiza and the root cap were discarded; root tips 1 mm long were fixed at
4 °C in a 2 % glutaraldehyde solution containing 4 % lead acetate ino-i M cacodylate buffer,
pH 7-0, then treated as described by Tandler & Solari (1969). The root tips were finally
embedded in Epon. Ultrathin sections (70-100 nm thick) were cut in cortical cells at about
1 mm below the root apex, mounted on 100-mesh copper grid with a Formvar coat and observed without staining.
Control embryos were treated as described above except that they were fixed with 2 %
glutaraldehyde devoid of lead acetate. Lead precipitates were never observed in root cells of
embryos fixed in this way.
Cytochemical localization of fi-glycerophosphatase activity. Root tips were fixed for 45 min
with 4 % glutaraldehyde in o-i M sodium cacodylate buffer, pH 7-0. Then 2 cross-sections
about 0-2 mm thick were made in each root at about 1 mm from the apex. These cross-sections
were used for the ultrastructural localization of acid phosphatase activity (pH 5'o). The study
was carried out using the lead precipitation procedure with /?-glycerophosphate as a substrate.
This method is described in detail in Poux (1967, 1970). After incubation overnight at 4 then
at 37 °C for 30 or 60 min in the /?-glycerophosphatase staining medium, the samples were
dehydrated and embedded in Epon. Ultrathin sections were cut in the cortical cells. The sections were examined without post-staining with uranyl acetate. They were compared with
sections of samples incubated in a medium containing /?-glycerophosphatase +0'Oi M NaF
or in a medium devoid of /?-glycerophosphate.
All the observations were made in a Siemens Elmiskop 101 at 80 kV.
Biochemistry
Phosphatase activities. T o o-i ml dialysed crude extract in water were successively added
O43 ml of 0-2 M buffer (sodium acetate buffer, pH 4-8, Tris-maleate buffer, pH 7-0 or glycineNaOH buffer, pH 9'o) and 0-3 ml phosphatase substrate (2 X I O " ' M Na-/?-glycerophosphate,
I O ~ 2 M pyrophosphate or io~ a M ATP). T h e mixture was incubated at 37 °C for 30 min
then cooled, 0-7 ml cold 10 % trichloroacetic acid was added and the mixture centrifuged at
PL in maize embryo nuclei
79
1000 g for 5 min. Inorganic phosphate was estimated in the supernatant as described previously
(Matagne, Loppes & Deltour, 1976).
Protein was measured by the method of Lowry, Rosebrough, Farr & Randall (1951). Specific
activities of phosphatases are given as fimol of phosphorus per h, per mg of protein, at 37 °C.
Isolation of nuclei
The isolation of nuclei was performed as described in Greimers & Deltour (in preparation).
Electron-microscope observations of the isolated nuclei showed them to be undamaged and
devoid of cytoplasmic contaminations.
RESULTS
Cytochemical localization of inorganic phosphate during germination and after thermal
shock
Germination. Roots were excised from quiescent embryos or after 8, 24, 48, 72 and
96 h of germination at 16 °C then fixed for inorganic phosphate visualization. Lead
phosphate precipitates were seldom observed in cells of quiescent embryos. A slight
and diffuse deposit was detected in a few ultrathin sections of these cells (Fig. 1).
At 8 h after germination lead deposits were observed in a few nuclei. The deposits
were located exclusively in the extranucleolar space (Fig. 2).
The same P{ localization was also observed at 24 h but in a greater number of
nuclei. From 48 to 96 h after germination numerous microcrystals of lead phosphate
were visible in all the nuclei and at the plasmalemma level. The deposits were especially dense inside the nucleoli. Small areas remained electron-transparent in the
nucleoli; they might correspond to nucleolar vacuoles (Fig. 3).
These observations show that during the first 48 h of germination, the inorganic
phosphate concentration strongly increases inside the nucleus and especially inside
the nucleolus.
Thermal shock. Roots of embryos submitted to a 5-h thermal shock (between 48
and 53 h of germination) and roots of embryos replaced at 16 °C for 6, 13 or 19 h
were fixed for inorganic phosphate visualization.
In cells of embryos fixed after 53 h of germination, the localization of inorganic
phosphate was similar to that described at 48 h (Fig. 3). At the end of the thermal
shock, lead phosphate microcrystals had almost totally disappeared from the nucleus.
A very slight and diffuse precipitate was visible in the whole cell (Fig. 4). Similar
pictures were obtained after 6 h of recovery at 16 °C. After 13 h of recovery in half of
the radicles under examination the Pf localization was similar to that of control.
After 19 h recovery at 16 °C the nuclei of all the radicles showed a high inorganic
phosphate content (Fig. 5).
Thus, the heat treatment leads to a strong decrease of inorganic phosphate in the
nucleus. The Pf ions are probably scattered in the whole cell and their concentration
becomes insufficient to be clearly detected by the Tandler and Solari test. Loss of
Pj by the cell is also to be considered. Several hours of recovery are required to allow
the nuclei to reconstitute their content of phosphate ions (Fig. 5).
R. Deltour, S. Fransolet and R. Loppes
.«• •
2 *
v
Pi in maize embryo nuclei
81
Fig. 4. Inorganic phosphate visualization, in a cell immediately after thermal shock.
The section is contrasted with uranyl acetate. A diffuse lead phosphate precipitate is
present in the whole cell. The high concentration of inorganic phosphate in the
nucleus has disappeared (compare with Fig. 3). x 5600.
Fig. 5. Inorganic phosphate visualization in the nucleus 19 h after return to 16 °C.
The localization of inorganic phosphate is similar to that found in a cell before the
thermal shock (see Fig. 3). x 11 200.
Phosphatase activities during germination and after a thermal shock
The high increase of intranuclear phosphate at the beginning of germination as well
as during the recovery period following a thermal shock led us to focus our attention
on the phosphatase activities in root cells in those 2 situations using biochemical and
cytochemical methods. A great part of P { in the cell can indeed originate from the
activity of phosphatases.
Fig. 1. Inorganic phosphate visualization in the nucleus of a root cell of quiescent
embryo. In this unstained section a diffuse lead phosphate precipitate is visible on
the whole nucleus, x 15600.
Fig. 2. Phosphate visualization in a nucleus after 8 h of germination. Unstained
section. Lead phosphate microcrystals are exclusively located in the nucleoplasm.
x 10500.
Fig. 3. Root cell after 48 h of germination. Numerous lead orthophosphate microcrystals are visible in the nucleolus. The clear areas (arrow) probably correspond to
nucleolar vacuoles. A less-abundant precipitate is seen in the nucleoplasm. x 11500.
82
R. Deltour, S. Fransolet and R. Loppes
Biochemistry. The phosphatase activity was measured in crude extracts of roots
using 3 different substrates: ATP, /?-glycerophosphate and pyrophosphatc. The
term phosphatase refers here to the common hydrolytic function of these enzymes
and does not imply that the phosphate liberated in the presence of the 3 substrates
results from the activity of a unique enzyme. The phosphatase activities were measured
at pH 4-5, 7-0 and 9-0 after 14 and 72 h of germination (Table 1).
In all samples and regardless of substrate, the highest phosphatase activities were
found at pH 4-5 and 7-0. At pH 9-0, the activities were much lower and even nil
(/?-glycerophosphatase after 14 h of germination). These activities at pH 9-0 were
not increased by the addition of io~ 2 M Mg2 + : these ions are required for optimal
activity of alkaline phosphatase in other systems (Schurr & Yagil, 1971). On the other
hand no alkaline phosphatase activities were found to be associated with cell debris
(results not shown).
Table 1. Phosphatase specific activities (/.imol P\ fanned per h at 37 °C per mg protein)
in crude extracts of root tissues after 14 and 72 h of germination
Specific activity
pH A T P
4 •5
7•0
9•0
1 80
1-49
o-oo
14 h of germination
72 h of germination
A
A
Pyrophosphate /?-glycerophosphate A T P Pyrophosphate /?-g!ycerophosphate
3 •09
1 •69
0 •58
1-07
4-75
4-25
648
446
299
0 0 0
I 22
044
010
136
792
Three different substrates were tested at 3 different pH levels.
The comparison of the enzyme activities after 14 and 72 h of germination clearly
shows a significant increase of the acid and neutral phosphatase at the 72nd hour.
After a thermal shock, the acid phosphatase activity was severely reduced with the
3 substrates tested (Table 2). The neutral phosphatase activity was significantly
lower when ATP was used as a substrate.
Ultrastructural cytochemistry. As root tissues of maize embryo display an optimal
phosphatase activity at low pH we have localized the y?-glycerophosphatase activity
in similar conditions. This localization has been performed in meristematic root tissues
of quiescent embryos and after 6, 24, 48, 72, 96 and 220 h of germination at 16 °C.
Three active sites were detected in the cells during germination: the cytoplasmic
vacuoles, the plasmalemma and the nucleus. These sites were not active simultaneously. From o to 24-28 h the /9-glycerophosphatase was exclusively located in
the cytoplasmic vacuoles (Figs. 6, 7). At 48 h the hydrolytic activity of the vacuoles
was reduced (Fig. 8). One day later, a slight activity was still associated with the
tonoplast of a few vacuoles. After 96 h of germination all the vacuoles appeared
inactive (Fig. 9). Comparison of Figs. 6 and 7 with Fig. 9 clearly shows that the
vacuolar system grows intensively during germination and that concomitantly, the
associated phosphatase activity diminishes.
P i in maize embryo nuclei
83
The plasmalemma showed a /?-glycerophosphatase activity only from 72 h onwards. Later (96 and 220 h), the hydrolytic activity of the plasmalemma was very
high (Fig. 9).
Up to 24 h after germination /9-glycerophosphatase activity was very low in the
nucleus. Later, all the nuclei and the nucleoli always showed an abundant lead
phosphate precipitate (Figs. 8, 9).
Table 2. Specific activities of ATPase, pyrophosphatase and ft-glycerophosphatase in
root extracts at 53 h after germination {control) and after a heat shock given from 48 to
53 k after germination
Specific activity
A
Substrate
pH
Control
(53 h of germination)
Heat shock
(48 h of germination
at 16 °C, s h at 46 °C)
ATP
4-5
289
7-0
90
2-28
1 90
i-i5
098
0-84
4'5
4-56
1-64
269
072
077
1 26
068
o-oo
069
064
Pyrophosphate
7-0
90
/?-glycerophosphate
4-5
7-0
90
1 44
000
Table 3. Specific activities at pH 4-8 of ATPase, pyrophosphatase and ft-glycerophosphatase in extracts of isolated root cell nuclei
Substrate
Specific activity
ATP
Pyrophosphate
/?-glycerophosphate
189
2-82
3-08
At the end of a 5-h thermal shock applied 48 h after germination the /?-glycerophosphatase activity in the cytoplasmic vacuoles and in the nuclei was very low
(Figs. 10, 11). Conversely a slight /?-glycerophosphatase activity (not present in
control cells) was detected in the plasmalemma (Fig. 10).
The cellular /?-glycerophosphatase activity was totally inhibited when NaF (o-oi M)
was added to the incubation medium.
Acid phosphatase activity in isolated nuclei
In order to know whether the lead phosphate which deposits in the nuclei during
cytochemical visualization results from acid-phosphatase activities in the nucleus
itself, the enzymes were assayed in crude extracts of nuclei isolated from root cells.
Table 3 shows that at pH 4-8 and with 3 different substrates the isolated nuclei had a
significant acid phosphatase activity.
R. Deltour, S. Fransolet and R. Loppes
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11
Figs. 10, n . Visualization of acid phosphatase activity in root cells submitted to
thermal shock.
Fig. 10. Portions of the plasmalemma react positively whereas the vacuoles are
unstained, x 23 500.
Fig. 11. Staining of the nucleolus and nucleoplasm is strongly reduced by thermal
treatment (compare with Fig. 8). x 5800.
DISCUSSION
Using the Tandler and Solari test we have shown that during early germination,
the nucleus and especially the nucleolus progressively accumulate P t . The question
therefore arises of the origin of this P i . As phosphatase activities lead to the liberation
of P { in the cell, it may be supposed that a direct relation exists between these activities
Figs 6-9. Portions of root cells stained for acid phosphatase at pH 50.
Fig. 6. Quiescent embryo cell. The stain is intense inside the vacuoles. The
nucleus appears very slightly stained, x 10000.
Fig. 7. Cell 24 h after germination. As in quiescent cells the positive reaction for
acid phosphatase is restricted to the vacuoles. In some of these the /?-glycerophosphatase activity is clearly associated with the tonoplast. x 8500.
Fig. 8. Cells 48 h after germination. Note intense staining of the nucleolus and
nucleoplasm. x 5700.
Fig. 9. Cells 96 h after germination. The reaction is very strong in the plasmalemma; note the presence of microcrystals outside the cells, x 3700.
86
R. Deltour, S. Fransolet and R. Loppes
and the accumulation of P i inside the nucleus. Using different substrates we have
shown biochemically that the phosphatase activities actually increase in root tissues
during germination. By cytochemical methods we have localized the sites of /?glycerophosphatase activity in optimal conditions (pH 5-o). The /?-glycerophosphatase activity was present at several cellular sites. The nucleus itself shows intense
activity. However, staining of the nucleus by the method of lead salt precipitation is
frequently considered as an artifact due to non-specific lead deposit or to diffusion
of the reaction products (Barka & Anderson, 1962; Washitani & Sato, 1976). The
following considerations establish that with our material and in our working conditions, the lead staining of the nucleus most probably corresponds to direct visualization of an acid phosphatase activity. (1) The diffusion artifact is generally characterized by contamination of the cytoplasm with lead deposits (Vorbrodt, 1974). In
our observations the cell sections displayed strong nuclear staining but very low
cytoplasmic contamination. (2) Isolated nuclei of root tissues showed acid phosphatase
activity with different substrates namely with /?-glycerophosphate and pyrophosphate. (3) Several methods known to eliminate the artifactual lead deposits in the
nucleus (fixation with formol calcium, changes of lead nitrate concentration in the
reaction medium or changes in buffers) (Barka & Anderson, 1962; Pfeifer, Poehlmann
& Witschel, 1974) gave less precise localization than the method of Poux (1967, 1970)
and never completely abolished the nuclear staining (results not shown). (4) It has
been shown (Tandler & Solari, 1969) that fixation of tissues with glutaraldehyde
eliminates the inorganic phosphate of the cells. Accordingly the presence of phosphate
in the nucleus can be ascribed to phosphatase activity. (5) When NaF was added to
the incubation medium, deposits of lead phosphate were not formed in the nucleus
although Pb2 + ions were present. This rules out the possibility of a non-specific lead
deposit in the nucleus. (6) Striking differences were observed in lead phosphate
deposit between nuclei from quiescent and germinated embryos, and between cells
before and after a thermal shock. These can hardly be explained by a non-specific
lead staining.
Accordingly it seems that there is an increase of /9-glycerophosphatase activity in
the nucleus during early germination. It may thus be suggested that part of the Fi
accumulated in this organelle directly results from a phosphatase activity located both
in the nucleoplasm and in the nucleolus.
A nuclear acid phosphatase activity has already been detected in plant (De Jong,
Olson & Jansen, 1967) and in animal cell nuclei (Love, Studzinski & Walsh, 1969;
Soriano & Love, 1971; Vorbrodt, 1974; Buchwalow & Unger, 1977).
On the other hand, comparison of previous results (Van de Walle et al. 1976) with
the present data suggests that there could be a relation between nuclear transcription
and the accumulation of P i . Both activities indeed appear during germination first in
the extranucleolar space then in the nucleolus.
That RNA synthesis is correlated to the accumulation of P i is also supported by
the fact that when transcription is inhibited by cordycepin, the inorganic phosphate
cannot be further visualized in the nucleus of root cells of AUium cepa (Risuefio,
Moreno Dfas de la Espina, Fernandez-G6mez & Gimenez-Martin, 1976). The
Pi in maize embryo nuclei
87
observations on Phaseolns vulgaris embryos (Walbot, 1971) are also consistent with
our proposal. In this species, the decrease of RNA synthesis which occurs at the
time of seed maturation is parallel to the decrease of orthophosphate in the embryos.
Conversely, the resumption of RNA synthesis at the beginning of germination is
accompanied by an increase of inorganic phosphate.
How could this relation be explained? When transcription takes place, pyrophosphate is liberated from the nucleotides triphosphate incorporated in growing RNA
molecules. This liberation is proportional to the transcription rate. We have detected
pyrophosphatase activity in crude extracts of isolated nuclei. Thus, the pyrophosphate
produced during transcription may be hydrolysed in the nucleus. We failed, however,
to localize an acid pyrophosphatase in the nucleus because all pyrophosphate was
precipitated in the incubation medium in the presence of lead nitrate or acetate.
The observation made after a thermal shock also lead to the same correlation
between nuclear P4 accumulation, phosphatase activity and transcription. In these
circumstances we have observed a strong decrease of the intranuclear P i correlatively
with a strong inhibition of nuclear /?-glycerophosphatase activity and of transcription.
During the period of recovery, the concentration of nuclear P i increases only when
transcription resumes at a high rate (Fransolet, unpublished).
The correlation proposed here above does not exclude other possibilities to explain
the increase of nuclear Pi content. The phosphatases located at the plasmalemma level
or in the vacuoles produce P i which could be transported into the nucleus. Extracellular origin of the Pi might also be considered since during germination phytin
stocked in the endosperm is hydrolysed (Mayer & Poljakoff-Mayber, 1965). However
this last possibility appears improbable because even when germination occurs at an
optimal temperature, the P t coming from the reserves of the endosperm does not
reach the embryo before the second day after the start of germination (Ergle & Guinn,
1959; Hall & Hodges, 1966).
Another point of interest is that 3 main ultrastructural changes may be observed
in the nucleolus of maize root cell when its inorganic phosphate content is low, i.e.
during early germination (Deltour & Bronchart, 1971; de Barsy, Deltour & Bronchart,
1974; Deltour, Gautier & Fakan, 1979) and after a thermal shock (Fransolet et al.
1979): (1) intense vacuolation; (2) low numerical density of the granular elements;
(3) appearance of ribonucleoprotein granules of high size.
These 3 ultrastructural particularities of the nucleolus disappear with transcription
resumption and correlative increase of P i content. This suggests that a relatively high
concentration of unbound phosphate ions is necessary for optimal nucleolar functioning.
In conclusion we propose the following hypothetical sequence of events summarizing our results: nuclear transcription results in liberation of P-P the hydrolysis of
which by pyrophosphatase gives P4 which is in turn required for optimal nucleolar
functioning.
This work was supported by grant of the Fonds de la Recherche Fondamentale Collective
(grant no. 2.4505.78). The authors are very grateful to Mrs Michelyne Dejace for help with
preparation of the manuscript and to Mrs Simone Roos for photographic assistance. R. L. is
chercheur qualify du Foyds National Beige de la Recherche Scientifique.
R. Deltour, S. Fransolet and R. Loppes
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{Received 26 June 1980)