Journal of Experimental Botany, Vol. 48, No. 310, pp. 1147-1150, May 1997
Journal of
Experimental
Botany
SHORT COMMUNICATION
Gibberellin does not accelerate rates of cell division in
the dwarf pea shoot apical meristem
Alison Daykin1'2, Ian M. Scott2'3, David R. Causton 2 and Dennis Francis1
1
School of Pure and Applied Biology, University of Wales Cardiff, PO Box 915, Cardiff CF1371, UK
2
Institute of Biological Sciences, University of Wales Aberystwyth, Dyfed SY233DA, UK
Received 4 December 1996; Accepted 16 January 1997
Abstract
Although GA3 doubled the numbers of cells in dwarf
pea internodes, it caused no significant acceleration
of cell division rates in the apical meristem, estimated
using cell doubling times, mitotic indices, or percentage labelled mitoses data. Increased cell numbers in
GAg-treated pea stems must be generated within the
extending internodes.
Key words: Cell division cycle, gibberellin, pea, Pisum,
shoot apical meristem.
Introduction
An important component of the growth promotion caused
by gibberellin in stems of pea (Pisum sativum L.) is an
increase in cell number (Araey and Mancinelli, 1966;
Yang et al., 1996). A question requiring investigation is
whether GA 3 application has an effect on the rate of cell
division in the dwarf pea apical meristem, as discussed
by Arney and Mancinelli (1966). The reactions of the
apical meristem cells in GA 3 -induced stem elongation
are not well established (Besnard-Wibaut et al., 1983).
Therefore, cell cycle techniques were applied in order to
examine whether GA 3 affected cell division in the apical
meristem of dwarf peas during the stimulation of stem
growth.
Materials and methods
Plant material
Seeds of Pisum sativum L. cultivar 'Meteor' (Sharpes
International Seeds, U K ) were selected for uniformity and
3
imbibed with aeration at 20 °C for 24 h in either distilled water
(the - G A 3 treatment) or in 0.29 mM GA 3 (Sigma, U K ) . Fully
imbibed seeds were sown (day 0) in Levington's compost
(Fisons, U K ) , 4 cm apart at a depth of 1 cm, and fed weekly
with Hoagland's solution. The growth conditions were: 20 °C,
16 h light (cool white fluorescent tubes; fluence rate of
2
11
photosynthetically active radiation =
), 8 h
darkness. On day 7, the + G A 3 seedlings were sprayed with
0.29 mM GA 3 containing 0.01% (v/v) Silwet L77 wetting agent
(supplied by Dr JC Ormrod, Zeneca Agrochemicals, U K ) .
Estimation of rates of ceil division
When the seedlings were 10-d-old, [methyl- 3 H]thymidine (185
GBq mmol" 1 , 37 kBq ml" 1 , Amersham, U K ) was applied as a
20 JU.1 droplet to the shoot apex. As is usual in this technique
(Gonthier et al., 1987), some of the leaves shielding each apex
were removed to permit application of the droplet, which was
held in place by the young leaves surrounding the meristem.
After 4 h, excess radioactivity was removed using serrated filter
paper and each apex was washed with 20 /*1 of a 0.75% (v/v)
solution of Silwet L77. Unlabelled thymidine (10~ 5 M ) was
then applied in an identical manner for 4 h.
Five plants were sampled every 4 h for 80-84 h from the end
of the unlabelled thymidine chase. Shoot tips, comprising the
apical dome (AD) and 5-6 unexpanded leaf primordia, were
fixed in 3:1 absolute ethanol:glacial acetic acid, and dehydrated
in an alcohol series prior to wax-embedding, sectioning (5 ^m
thickness), and autoradiography (Ormrod and Francis, 1985).
From the three most median sections, the percentage of labelled
mitoses (PLM) was determined at each sampling time in defined
apical regions (Fig. 1). All mitotic figures were scored for the
presence or absence of silver grains overlying the chromosomes.
Random checks on background labelling were made by assessing
the number of silver grains in background areas equal in size
to the mitotic figures (background < 5 grains).
For the cell doubling time estimations, the shoot apices of
unlabelled 10-d-old plants (n = 5) from both treatments were
fixed and processed as longitudinal sections as outlined above.
Median sections, stained by the Feulgen reaction and counter-
To whom correspondence should be addressed. Fax: + 441970 622350. E-mail: iaseaber.ac.uk
Abbreviations: AD, apical dome; CDT, cell doubling time; GA,, gibberellic acid; PLM, percentage labelled mitoses; SAR, sub-apical region; YP, youngest
primordium.
0 Oxford University Press 1997
1148
Daykin et al.
to the mean CDT of 36.4 h reported for various zones of
the shoot apex of the pea cultivar Lincoln (calculated
from Table 3 of Lyndon, 1970). Similarly, mitotic indices
in the AD were not significantly different between the
- G A 3 (6.1 ±0.7%) and +GA 3 plants (5.8±0.6%)
(n = 9).
An additional, independent method was used to examine any effect of GA3 on the cell cycle in the shoot apex.
The 'percentage labelled mitoses' (PLM) curves (Quastler
and Sherman, 1959) obtained for both the — GA3 and
+ GA3 apices (Fig. 2) yielded patterns that indicated cell
cycle durations of 36-40 h, which were similar to the
estimated CDTs. Thus, the PLM experiment also indicated that GA3 treatment did not accelerate the rates of
cell division in the apical meristem.
Otherwise, there were some differences between the
PLM curves for the two treatments. The PLM data for
the — GA3 seedlings indicated a shorter cell cycle in both
the AD and SAR (Fig. 2a, c) of 24 h, in addition to the
Fig. 1. Median longitudinal section through the pea shoot apex,
showing the positions of: the apical dome (AD); the sub-apical region
longer cell cycle of 36-40 h. In the PLM curves for the
(SAR) and youngest leaf primordium (YP). Magnification x 200
+ GA3 apices, the second peak was smaller, and evidence
for a shorter cell cycle was not discernible (Fig. 2b, d).
stained by Fast Green (Gurr BDH, UK) were used for counting
These shorter cell cycles were not localized to any particuof cells within each apical zone. Statistical significance was
lar part of the AD; when the PLM data for the peripheral
analysed by the Student's /-test. Linear regressions of leaf and
zone
and pith rib meristem were plotted separately (not
primordia number on time were obtained to estimate the rate
of leaf production.
shown), they had similar patterns to those presented in
Fig. 2a, b for the whole AD, i.e. multiple peaks in the
controls but only two peaks in the + GA3 plants.
Results and discussion
The multiple peaks seen in the — GA3 PLM curves
Treatment with GA3, starting at seed imbibition, resulted
indicated a heterogeneity of cell cycles in these shoot
in a 5-fold increase in height of the main stem axis of
apices. Gonthier et al. (1987) described the apical merisMeteor dwarf pea plants on the tenth day after sowing.
tem of Sinapis as a mosaic of rapid-cycling and slowThe GA3 treatment caused a mean 2.1-fold increase in
cycling cells. Such a complex situation hinders explanation
the numbers of epidermis, cortex and pith cells along
of the differences in the PLM curves for the — GA3 and
the fully grown length of the seventh internode. The
+ GA3 apices. The duration of the G2 phase plus half
regression equation for the rate of leaf production for the
the M phase (taken as the interval from 0 h to the time
- G A 3 plants was y = 0J]x + 4.%6 (Z'<0.001); and for when the PLM curve reached 50% of the peak value) was
the +GA3 plants was j^0.79.x+4.70 (/><0.001), where
about 28-32 h ± GA3, assuming that the rate of penetrax is time (d) and y is number of leaves and leaf primordia:
tion of radioactive solution was the same in both treatthere was no significant effect of GA3 on the rate of
ments. The S-phase, measured as the 50% point of the
appearance of the first 12 leaf primordia. The plastochron
ascending and descending limbs of the first peak (minus
was 24 h up to the eighth day after sowing, but then
the labelling time) was also similar 6—8 h±GA 3 , as was
lengthened to 48 h at the time of the cell cycle measurethe duration of the M-phase (from where the ascending
ments. (The seeds possessed 5-6 leaf primordia prior to
limb rises to where it reaches a plateau). If each of these
germination.)
phases was not affected substantially, then the difference
The pea shoot apex as defined in Fig. 1. is known to
between the short and long cell cycles would have been
be an exponentially growing system (Lyndon, 1968).
in the duration of the Gl phase. One interpretation would
Therefore, the relative growth rate (r) of the shoot
then be that GA3 eliminated the cell cycles with a short
apex could be obtained as In (cell number in
[AD + YP + SAR])-ln (cell number in AD)-duration of
the plastochron. This calculation enabled estimates of the
Fig. 2. Changes in time (h) of percentage labelled mitoses (±SE)
mean cell doubling time (In2 — r). Mean cell doubling following a 4 h pulse label with 3H-TdR, in sections through the shoot
apex of plants which were 10-d-old at the start of the experiment: (a)
times (CDTs) in the shoot apex on day 10 were not
control AD, (b) GA3-treated AD, (c) control SAR, (d) GA3-treated
significantly different between the - G A 3 (37.1 ±4.2 h)
SAR. Where error bars are absent, the variation about the mean was
less than the diameter of the symbol.
and +GA 3 (40.4 ±2.1 h) plants. The CDTs were similar
GA3 and apical meristem
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Gl. Alternatively, GA3 may have lengthened all cell
cycles, so that the 2nd peak (at 56 h) in the - G A 3 PLM
curves was displaced to 12-1Ah in the +GA 3 curves,
and the 3rd peak was displaced off the x axis. Most of
the PLM curves, including the SAR of the + GA3 apices,
showed late rises indicating additional, still longer cell
cycles (Fig. 2).
The heterogeneous cell cycle times, revealed by the
PLM curves, nevertheless averaged out to give mean
CDTs in the whole apical meristem that were not significantly different ±GA 3 . A further variable to consider is
the 'growth fraction', which is the proportion of actively
cycling cells. Treatments which alter the growth rate of
plants have been found to have a concomitant effect on
the growth fraction in the shoot apex, suggesting that in
a meristem the proportion of dividing cells, as opposed
to division rate per se, is particularly sensitive to external
treatments (Moses et ai, 1997). The growth fraction may
be calculated by dividing the cell cycle time by the CDT.
Thus, in the +GA 3 apices the similarity of these two
parameters would indicate that most of the cells were
cycling at a uniform, albeit slower rate. In the — GA3
apices the heterogeneous cell cycles complicate this calculation, but it could be that the most rapidly cycling cells
in these apices comprised a lower growth fraction.
The absence of a stimulatory effect of GA3 on cell
division in the dwarf pea apical meristem is in accordance
with the classical experiments on other species by Sachs
and colleagues, who considered that although 'the apical
meristem proper is the ultimate source of all cells of the
shoot, its direct contribution to stem histogenesis, ie. to
the actual formation of the cells which are going to
constitute the mature stem, is negligible' (Sachs et ai,
1960). The pea apical meristem is separated from the
expanding internodes by several primordia and young
leaves. It would appear that the increased numbers of
cells in GA3-treated dwarf pea internodes are generated
almost entirely within these expanding internodes.
80
Acknowledgements
We are grateful to the BBSRC for support of this project (grant
reference P107), and to Dr RF Lyndon and Dr PW Barlow for
suggestions during the preparation of the paper.
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