Plant Physiol.
(1972) 50, 308-310
Short Communication
Mitotic Index and Cell Cycle of Lemna perpusilla
under Different Photoperiods1
RuTH HALABAN2
Department of Biology, Brookhaven National Laboratory, Upton, New York 11973
One of the major problems in the photoperiodic induction of
flowering is the mechanism of time measurement. Bunning has
suggested the involvement of circadian rhythms (4), a model
supported by evidence from several plant systems. (6-8, 13).
Lemna perpusilla is a short day plant in its flowering re-
sponse (9), and its photoperiodic time measurement appears to
be controlled by a circadian rhythm (8), which can be corre-
lated to an overt circadian rhythm of CO2 output (10, 1 1). The
origin of the rhythmicity in CO2 output is not yet clear, nor is it
known what aspects of the endogenous circadian rhythm are
involved in the photoperiodic response. Since synchrony in the
increase of cell number has been documented in several orga-
nisms (3, 12, 15, 19, 20), it seemed worth undertaking a study
to find whether cell division is synchronized in L. perpusilla by
light-dark cycles and what other effects light has on the cell
cycle of this plant.
MATERIALS AND METHODS
Vegetative stocks and experimental plants of Lemna per-
pusilla strain 6746 were cultured on half-strength Hutner's
medium supplemented with 1% (w/v) sucrose. The vegetative
stocks were grown under continuous illumination from cool
white fluorescent lamps (350 ft-c) with the air temperature 24
to 26 C. Experimental plants were grown in perforated vials
immersed in 38- X 200-mm test tubes containing 60 ml of
growth medium. They were transferred to growth chambers
and kept under either 12- or 8-hr inductive photoperiods or
16-hr noninductive photoperiods (450 ft-c) at 25 C.
The mitotic index-defined as the percentage of mitotic fig-
ures-was determined by counting the number of mitotic figures
in samples taken at 3-hr intervals during the 24-hr cycle on the
2nd and 4th day. For this purpose, plants removed for sampling
were fixed overnight in one part glacial acetic acid and three
parts ethanol, hydrolyzed for 20 min at 60 C with 1 N HCl,
and stained with Schiff's reagent (Feulgen stain) overnight at 5
C. The meristematic regions (stained deep purple) were re-
moved from the rest of the frond on a microscopic slide. The
cells were separated with a blunt glass rod in 45% (v/v) acetic
acid, further squeezed under a cover glass, rapidly frozen on
Dry Ice, immersed in 100% ethanol, and permanently fixed
(5, 17). For each sample, three slides were made and 1000 cells
of each slide were scored for mitotic figures.
The mitotic cycle duration was measured by the tritiated
1 This research was carried out under the auspices of the United
States Atomic Energy Commission.
2 Present address: Department of Biological Sciences, State Uni-
versity of New York, Albany, N. Y. 12222.
308
Received for publication January 18, 1972
thymidine technique (14) as described by Van't Hof (17). The
plants were transferred to medium containing 12 ,uc/ml triti-
ated thymidine (T-H3)3 on the 2nd day under the light-dark
cycles. T-H', obtained from Schwarz Bioresearch Inc., specific
radioactivity 6.0 c/mmole, was added to 200 ml of growth
medium. This transfer to the radioactive solution was done at
the onset of light under the 16-hr photoperiods and at the onset
of darkness under the 12-hr photoperiods. At the end of a
30-min pulse label the plants were washed and returned to the
nonradioactive growth medium. Samples were fixed at hourly
intervals, hydrolyzed, stained, and squashed on microscopic
slides as described above. The slides were then hydrated to
water by means of a graduated ethanol series, dipped in Kodak
NTB liquid emulsion, and exposed for 10 days (17).
RESULTS
Cell Division. The mitotic index of samples grown under in-
ductive or noninductive photoperiods (Table I) did not vary in
a periodic manner through the 24 hr day. This indicates that
cell division in L. perpusilla is not synchronized by light-dark
cycles.
The Mitotic Cycle Duration. The intention was to compare
mitotic cycle duration under long night and long day condi-
tions. The curves for T-H3 labeled mitotic figures as a function
of time after labeling are given in Figure 1. The curve for the
12-hr night shows two distinctive peaks of maximally labeled
mitotic figures while in the 16-hr day curve the second peak is
suppressed.
The mitotic cycle time under the long dark period, measured
from the time interval between two successive maxima, is about
6 hr. The post-DNA synthetic period (G2) and mitosis (M) are
equivalent to about 3.25 hr measured by the interval from
hour zero to the time when the ascending portion of the curve
is one-half maximum. The DNA synthetic period (S) is about
3 hr as measured by the interval between the ascending and
descending portions of the curve at the one-half maximum
value (14).
These are rough estimates and they leave a very short time
for pre-DNA synthetic period (G1) duration. Similar values can
be calculated for the light conditions, except that one has diffi-
culties in measuring the cell cycle duration, based on these
data.
Average nuclear diameter was measured at interphase in the
primordia of 4 fronds utilizing 10 cells per tissue. The perma-
nent slides used for these measurements were prepared by
microtome sectioning and hematoxyline-erythrosin staining.
'Abbreviation: thymidine-3H: T-H3.
Plant Physiol. Vol 50, 1972
- CELL DIVISION AND PHOTOPERIODS IN LEMNA 309
The nuclear volume, based on diameter measurements, in
interphase, is about 48 /2. By extrapolation, based on this w
value and the curve given by Baetcke et al. (1) demonstrating
the relation of DNA content to nuclear volume, one gets a 80
value of 3 picograms DNA per cell for L. perpusilla. The cellu-
lar DNA content of 3 picograms so estimated and the mitotic
cycle time of 6 hr fit nicely into Van't Hof's (16) data showing O
260 -
a relationship between the two.

DISCUSSION w
-J 40-
One cannot explain the overt circadian rhythm of CO, out- -LJ
put in L. perpusilla on the basis of synchrony in cell division,
since the mitotic index of this plant does not change in a z -20-
rhythmical manner over the 24 hr day; rather, it is constant w//
at the level of 3 to 4%. Therefore, one must look to other ci:
processes as the source for this rhythmicity as well as for the a.C00 2 4 6 8 10 12 14
understanding of endogenous rhythms involvement in photo-
periodism. HOURS AFTER H3T LABELING
The results indicate that under long night conditions, the FIG. 1: Change in the percentage of radioactive mitotic figures
population of cells synthesizing DNA at the onset of darkness with time after 30 min pulse label with T-H'. 0: L. perpusilla
passes through at least two cycles having identical mitotic was grown under 16-hr photoperiods. Pulse label was given at the
cycles duration. Under long day conditions however, the curve onset of light on the 2nd day of the non-inductive photoperiod. 0:
becomes progressively irregular, a kind of response predicted The plants were grown under 12-hr photoperiods, and the pulse
for heterogeneous populations of cells by Van't Hof and Mc- label was given at the onset of darkness on the 2nd day under the
Millan (18). Thus light may induce heterogeneity in the average inductive photoperiod.
cell cycle duration in at least two ways. First, the cycle of cells
proliferating in the dark may be altered by exposure to light alternative exists. Bernier et al. (2) showed that increasing the
so as to increase the average duration. Second, exposure to day length from 8 to 20 hr for 1 day was accompanied by the
light may cause previously nondividing cells to enter the mi- division of cells previously "held" in G2 for an undetermined
totic cycle, thus perturbing the cell population kinetics estab- length of time. The division of these G2 cells represents a re-
lished in the dark. At the present time, evidence of the second entry into the mitotic cycle and would, of course, change the
cell population kinetics established during the previous 8 hr
Table I. Mitotic Division under Differentt Photoperiods day growth period.
Each value is a mean of three counts. For each count 1000 cells Further experiments are also needed to determine whether
were scored. LD: light dark cycles of 8, 12, or 16 hr light, respec- the continuous presence of light is required to induce this kind
tively. Time: hr after lights were turned on. of response, or whether light pulses such as those used to in-
hibit the photoperiodic flowering response are sufficient.
No. of Cells in 'Mitotic Division Further experiments should also determine the location of cells
Time affected by the light conditions and the relationship to the
LD 8:16 LD 12:12 morphogenetic response of flower development.
Acknowledgments-I am indebted to Dr. Jack Van't Hof for his continuous
0 28.0 interest in this work, his helpful suggestions and discussions, and his generosity
1 58.3 in allowing me to use his laboratory equipment. I am also thankful to Dr. William
2 30.0 52.8 S. Hillman for grammatical corrections of this manuscript and his encouragement
3 34.6 to proceed in this project.
4 37.6 38.3
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