Download p34 homologue level, cell division, phytohormone responsiveness

p34cdc2 homologue level, cell division, phytohormone responsiveness and
cell differentiation in wheat leaves
P. C. L. JOHN, F. J. SEK, J. P. CARMICHAEL and D. W. McCURDY
Plant Cell Biology Group, Research School of Biological Sciences, Australian National University, Canberra City, GPO Box 475,
ACT 2601, Australia
Summary
Formation of a plant involves generation of new cells
by the division cycle and development in these of
specialised structure and metabolism. Specialisation
is accompanied by a decreasing capacity for division,
which declines with particular rapidity in cells of
monocotyledonous plants such as the cereals. Here
a homologue of the cell
we report that in wheat leaves
cycle control protein p34cdca participates in the
control of these cdc2
developmental programmes. Accumulation of p34
to a maximum level in dividing
cells and the cessation of its accumulation during
subsequent cell growth and expansion indicate that
it contributes cdc2
specifically to division. There is a
decline in p34
level as cell differentiation pro-
ceeds, in close parallel with the previously established decline of cell division in response to auxin
hormones. A basal level of p34cdc2 in fully differentiated cells that is one-sixteenth of that in dividing
cells correlates with their loss of capacity to divide.
We conclude that p34cdc2 level is controlled in diverse
multicellular eukaryotes and suggest that it is an
important element in the switch from cell division to
differentiation.
Introduction
Materials and methods
The cdc2 gene function is necessary for progress through
the two major control points (Nurse and Bisset, 1981;
Nurse, 1990) at which the cell cycle can be delayed until
requirements of cell size and nutrition are met (Nurse and
Fantes, 1981). Control at these points in the fission yeast
cell cycle is exerted by the interaction of the cdc2 gene
product p34cdc2 with stimulatory and inhibitory elements
(Russell and Nurse, 1987; Moreno et al. 1990). The p34cdc2
protein has been identified as a component of maturation
promoting factor (Dunphy et al. 1988; Gautier et al. 1988),
which is part of a universal control mechanism regulating
onset of M phase in proliferating cells (Nurse, 1990).
However, the possible contribution of changing p34cdc2
level to control of cell division during development has
been little studied in any organism. Plants are especially
favourable for such analysis because dividing cells are
spatially restricted to regions termed meristems (reviewed
by Walbot, 1986). For example, leaves of wheat seedlings
provide a linear developmental gradient, from a meristematic region at the base through a series of progressively differentiating leaf blade cells to a mature zone
extending upwards to the tip (Boffey et al. 1979). Distal to
the meristem, the non-dividing cells exhibit a gradient in
their responsiveness to auxin phytohormones. Cells near
the meristem readily re-enter the division cycle but, as
they differentiate and are displaced further up the leaf,
they become unable to resume division (Wernicke and
Milkovits, 1987a).
Plants
Plants were grown exactly as described in an earlier study in this
laboratory (Wernicke and Milkovits, 1987a).
Journal of Cell Science 97, 627-630 (1990)
Printed in Great Britain © The Company of Biologists Limited 1990
Key words: cdc2, p34cdc2, cell cycle, plant cell division,
phytohormone, hormone sensitivity, cell development, cell
differentiation, wheat leaf.
Protein extraction, electrophoreses and blotting
Protein was extracted by grinding in liquid nitrogen and vigorous
mixing at 0°C with RTPA buffer, pH7.4, containing 20 mM
Tris-HCl, 5mM EDTA, 100 mM NaCl, 0.1% Tween 20, lmM
dithiothrietol, 10 /iM leupeptin, 10 /iM pepstatin, 10 JXM. NaF, 1 mM
EGTA, 1 mM sodium pyrophosphate, 1 mM /3-glycerophosphate, to
which was added immediately before use 1 mM sodium orthovanadate and 200 /an PMSF (John et al. 1989). Samples containing
50 /(g of protein were separated by SDS- PAGE on a 10 % to 15 %
gradient gel. The proteins were transferred to nitrocellulose and
probed with anti-EGVPSTAIREISLLKE antibody (EGV antibody) that has been described previously (Lee and Nurse, 1987;
John et al. 1989) after affinity purification of the antibody against
mouse p34cdc2 fusion protein (Snyder et al. 1987). Control
purifications without p34cdc2 gave no reacting antibody (not
shown). Bound antibody was detected with alkaline phosphatase
I-labelled
as described previously (John et al. 1989) or with
anti-rabbit IgG F(ab')2 at O.SmCil"1 (Amersham, IM1340)
employing 24 h autoradiographic exposure.
Quantification of p34cdc2 homologue
p34cdc2 homologue was quantified on Western biota by probing
with with EGV antibody and iodinated second antibody. Complete
sets of samples were processed on single panels of nitrocellulose
and are therefore directly comparable. Autoradiography was used
to locate the p34 bands, which were then cut out for radioactive
627
counting and corrected for the low background (seen in Fig. 3B)
that was measured in each track.
12345
Results
6
7
8
9
10
11
12
Segment sample number
cdc2
Detection of pS4
homologue
Fig. 1 shows that a homologue of p34cdc2 was detected in
extracts from the cell division region of wheat leaves using
antibody raised against the EGVPSTAIREISLLKE sequence (EGV peptide), which has been perfectly conserved
in p34cdc2 between yeasts and humans (Lee and Nurse,
1987) but not in other protein kinases (Hanks et al. 1988).
The antibody used here has detected p34cdc2 in yeasts and
animal cells (Lee and Nurse, 1987; Gautier et al. 1988) and
a homologous protein that is involved in the cell cycle of
the unicellular plant Chlamydomonas (John et al. 1989).
After affinity purification by binding to mammalian
p34cdc2, the antibody recognised a wheat protein also of Mr
34x10 . Recognition was specific for the conserved EGV
peptide region since it was eliminated by competition with
20 nM EGV peptide (Fig. 1). An analogous peptide encoded
by the PHO85 gene, which has similarities with cdc2 but
no effect on cell division (Toh-E et al. 1988), failed to
compete, indicating that the EGV antibody recognised a
configuration within the largest perfectly conserved
region that is specific for cell cycle function.
Distribution in leaf
Segments were taken from base to tip of the seedling leaf
(Fig. 2A) and cell division was detected only in the lower
12 mm (Fig. 2B), which coincides with the region of
thymidine incorporation into nuclear DNA (Boffey et al.
1979; Wernicke and Milkovits, 1987a). The changes in
abundance of proteins during differentiation (Fig. 3 A) are
illustrated by the appearance of the Mr 55xlO 3 subunit of
RUBISCO beyond 28 mm from the base, which correlates
with the increase in chloroplast number and initiation of
photosynthesis (Boffey et al. 1979). The electrophoretic
mobility of p34cdc2 homologue did not coincide with that of
any of the abundant proteins and its level relative to that
of other proteins declined sharply outside the region of
active cell division (Fig. 3B). This decline in relative level,
quantified in Fig. 4A, is due to accumulation of other
proteins as differentiating cells increase in size, in protein
concentration (Fig. 4B) and therefore in total protein
content (Fig. 4C). The amount of p34cdc2 homologue per
M,
x1(T3
- 94
-67
"~
_ 30
-20
O V T
628
Fig. 1. Detection of p34 cdc2 homologue in
a Western blot of proteins from the cell
division region of wheat leaf by probing
with EGV antibody. Antibody solution was
divided and preincubated, (0) without
addition, (V) with 20 run EGV peptide
EGVPSTAIREISLLKE, or (T) with 200 nM
of altered peptide EGTPSTAIREISLMKE
with substitutions at the third and
fourteenth positions. The locations and
sizes of markers are indicated on the
right.
P. C. L. John et al.
£. 6 - OS
2 2
0
J
-o-
I
I
30
60
Distance from base of leaf (mm)
90
Fig. 2. Distribution of (A) leaf segments taken for analysis
and (B) incidence of cells in the mitotic phase of division, in
90 mm long seedling leaves of wheat taken after 8 days of
germination under conditions described previously (Wernicke
and Milkovits, 1987a,6). Segments 4 mm long were taken
contiguously between 0 and 20 mm from the leaf base and
above this at 10 mm intervals. Placing of sample numbers in
the diagram indicates the centre of each segment. Mitotic
index was determined in samples fixed in 4:1, ethanohacetic
acid and stained in acetocarmine. A high incidence of division
in the meristem region is indicated by detection of cells in this
brief phase of the division cycle.
cell (Fig. 4D) was derived by using protein content per cell
to indicate how many cells yielded the protein that was
probed in Figs 3B and 4A. No significant net breakdown of
p34cdc2 occurred during differentiation and the 94%
relative decline is accounted for by cessation of its
accumulation while extensive synthesis of other proteins
occurs during differentiation.
Discussion
An involvement of a homologue of p34cdc2 in higher plant
cell division, as in Chlamydomonas (John et al. 1989), is
now indicated since the level of this protein relative to that
of others is maximal in dividing cells and declines as
divisions cease. Its level also correlates with the capacity
for cell division when leaf segments are stimulated with
auxin, as discussed below in relation to hormone responsiveness. The present circumstantial evidence is supported
by our consistent observation of high p34cdc2 levels in
dividing cells of carrot cotyledons and pea root tips during
both normal development and hormone treatment (J. R.
Gorst, F. Wightman, F. J. Sek and P. C. L. John,
unpublished observations). Although division is restricted
to cells with high levels of p34cdc2, the occurrence of
division in such cells can be controlled. The present data
show that cells adjacent to the meristem retain an
appreciable level of p34cdc2, which can support division if
they are excised and stimulated with auxin (Wernicke and
Milkovits, 1987a), but they are able to abstain from
division in normal leaf development (Fig. 2B). We are
investigating whether the wheat homologue of p34cdc2 is
controlled by interaction with regulatory proteins as in
other eukaryotes (Nurse, 1990).
The full extent of similarity between wheat p34cdc2 and
its homologues in other taxa cannot be deduced from the
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Leaf segment number
Fig. 3. Changes during cell differentiation, in (A) stainable
proteins and (B) level of p34cdc2 homologue, detected in leaf
segments cut as shown in Fig. 2A. Samples containing 50 /jg of
protein were separated by PAGE, transferred to nitrocellulose
and stained with Ponceau S, then p34cdc2 was probed with
affinity-purified EGV antibody and detected using 125I-labelled
anti-rabbit IgG F(ab')2.
present immunological data. However, our cloning of part
of the wheat cdc2 gene by polymerase chain reaction,
using degenerate primers complementary with the sequences detected by antibody in the present study,
confirms the presence in wheat of a cdc2 gene containing
those sequences (S. Baindur, M. G. Lee and P. C. L. John,
unpublished observations). Cloning and sequencing of the
whole gene, which is in progress, will define the full extent
of sequence equivalence.
The differential responsiveness of plant cells to hormones (Trewavas, 1981) has been accepted as a potentially
important element in plant development (Wernicke and
Milkovits, 1987a; Finkelstein et al. 1988; Moore, 1989) and
the wheat leaf has provided the clearest example of a
changing responsiveness to division-inducing hormones,
since a recent study in this laboratory, using the same
tissue and conditions as employed in the present work,
showed that cells increasingly distant from the meristem
declined in capacity for resumption of division in spite of
an equal rate of uptake and metabolism of auxin
(Wernicke and Milkovits, 19876). The loss of potential for
division is typical of monocotyledonous plants, such as the
cereals, which are therefore difficult subjects for trans-
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ell
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40 60
80 100
Distance from leaf base (mnV
'"l
0
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1
20
Fig. 4. Changes in amount of p34cdc2 homologue and total
protein during leaf cell development. (A) Level of p34cdc2
protein in 50 ;(g samples of protein, (B) concentration of total
protein in cells (total protein per gram tissue fresh mass), (C)
average total protein content per cell, (D) amount of p34cdc2
homologue per cell. Total protein in extracts made as in Fig. 1
was estimated by dye-binding, after eightyfold dilution, using
an ovalbumin standard. Protein per cell was calculated by
multiplying the protein concentration shown in B by the
average cell fresh mass, which was derived from earlier
measurements of cell number and fresh mass made in this
laboratory under identical conditions (Wernicke and Milkovits,
19876) and was confirmed by measurement of cell dimensions
in these samples. Quantification of p34cdc2 homologue on
Western blots was done by probing with EGV antibody and
iodinated second antibody. Complete sets of samples were
processed on single panels of nitrocellulose and are therefore
directly comparable.
genie improvement (Ozias-Akins and Lorz, 1984). We now
show that differences in capacity for division are directly
predicted by a falling cellular concentration of p34cdc2
homologue relative to other proteins. Cells from the
division region, which we have shown to contain maximum levels of p34cdc2, readily continued division in
medium with 10 ,UM of the natural auxin indole-3-acetic.
Fewer cells responded at 25 mm from the base, where
p34cdcJ homologue level in wheat leaf
629
p34cdc2 level had fallen by 56 % and, beyond 40 mm, where
levels had fallen by more than 85 % (Fig. 4A), cells were
unable to respond to auxins at any concentration (Wernicke and Milkovits, 19876). We cannot eliminate the
possibility that more than one close variant of p34cdc2 was
present and was detected by the antibody. However, it is
clear that the switch to cell differentiation involves a
decline of all such p34cdc2 homologous protein. The
correlation of division capacity with the ratio of p34cdc2 to
total protein, rather than the amount of p34cdc2 per cell, is
striking. Since p34cdc2 is a protein kinase (Simanis and
Nurse, 1986; Draetta and Beach, 1988; Gould and Nurse,
1989), a decline in its ratio with phosphatase activity may
be an important factor, as in extracts ofXenopus (Verde et
al. 1990), but a reduced rate of encounter with target
proteins due to dilution with other proteins may also be
significant. Dilution may be slightly offset by the
increasing proportion of cell volume occupied by the
chloroplast, which rises from 6 % to 14 % by 60 mm from
the leaf base (Ellis and Leech, 1985), but dilution in the
extra-chloroplast compartment will still be more than
thirteenfold. Previously the possibility that differences in
responsiveness to hormone might be important in plant
development has been limited in application by lack of
established mechanisms. In this case the declining
division response can be directly correlated with declining
levels of the essential (Nurse and Bisset, 1981; Nurse,
1990) cell cycle control protein.
A switch from division to differentiation in cells outside
the meristem region of plants is essential in the formation
of organised tissue (see Walbot, 1985, for a review) and can
now be recognised in wheat leaf as involving a decline to
basal levels of the cell cycle control protein. Equivalent
data, for amount of p34
and of other proteins per cell
during the switch to differentiation, are not available for
differentiating animal cells. However, in those animal
cells that have been investigated, we deduce that low
levels of p34cdc2 do correlate with exit from the cell cycle,
because restorative increases in p34cdc2 level accompany
resumption of division when stimulated by serum (Lee et
al. 1988) or by adenovirus infection (Draetta et al. 1988),
and there is direct evidence in chick (Krek and Nigg, 1989)
that only embryonic tissues, containing dividing cells,
have high levels of p34cd . Our evidence from the
taxonomically remote plant kingdom now indicates that
modulation of key division protein levels is employed by
widely diverse eukaryotes during commitment to differentiation.
We thank Dr Melanie Lee for mouse cdc2+ in Agtll and Drs J.
C. Cavadore and M. Dor6e for a peptide containing a sequence
present in PHO85.
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P. C. L. John et al.
(Received 19 July 1990 - Accepted 21 August 1990)