Environmental and Physiological Effects on Pistillate Flower Production in Silene

noctiflora L. (Caryophyllaceae)
Suzanne H. Folke; Lynda F. Delph
International Journal of Plant Sciences, Vol. 158, No. 5. (Sep., 1997), pp. 501-509.
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FOLKE & DELPH-ENVIRONMENTAL

AND PHYSIOLOGICAL MALE STERILITY

Bud and Branch Removal

Seed-derived sib progeny (replicates)

from each maternal parent
Maternal parents in
each population
Population

503

0control
Treated

Flower Removal

Fig. 2 Diagram showing the design o f the three removal experiments (bud, branch, and flower). See additional details under "Physical
disturbance" in the "Material and methods" section.

partial morph retains a functional lower whorl of anthers but

lacks the upper whorl.
Most flowers produced by individual plants are perfect,
but we have observed transitions to predominantly pistillate
flower production in the greenhouse. Perfect flowers are
completely self-compatible, and the first upper whorl of five
anthers dehisces ca. 1-2 h before the flower opens at dusk.
Silene noctiJEora is therefore at least a partial prior-selfer
because some selfing occurs before any opportunity for outcrossing. Pistillate flowers can be either outcrossed or geitonogamously selfed (if there are perfect flowers open simultaneously on a plant). The principle pollinators for S.
noctiJEora are various species of nocturnal moths seeking
nectar, but bees occasionally visit plants during the day (S.
Folke and L. Delph, personal observation). Fruit maturation
requires ca. 1 mo, at which time the hardened, inflated ovary
with surrounding calyx cracks open to passively release
seeds.

GREENHOUSE
EXPERIMENTS
All greenhouse experiments were conducted from September 1992 to May 1993 at Indiana University, Bloomington. The plants used in each experiment were grown from
seeds collected during the summer of 1992 along the roadside leading to Mountain Lake Biological Station, Giles
County, Virginia (lat. 8032'N, long. 37"211W). Four naturally occurring populations were represented, hereafter
called populations 1, 2, 3, and 4. Seeds were germinated in
5 X 5 cm cells containing Metro Mix and eventually grown
under high-intensity lights (1000 W metal-halide, for 16 hld)
in 6-in clay pots with a mixture of soil, Metro Mix, and sand
(3: 1 : 1). Three main possible causes for the induction of
pistillate flower production were studied: temperature, physical disturbance (by bud, flower, or branch removal), and
exposure to the plant hormones gibberellin and ethylene.

The effect of temperature on pistillate flower production

was observed using two greenhouse rooms in which the average daily temperature differed by ca. 6C (19"C, 25C).
One sib replicate of 18 different maternal parents from populations 1, 2, and 3 were grown in each room, and the number and type of flowers produced per plant were recorded
daily for 2 mo. The number of maternal parents used from
each population were five, seven, and six, respectively, for
a total of 36 plants.

To determine the degree to which physical disturbance

affects pistillate flower production, three experiments were
performed in which newly emerging buds, open flowers (and
any developing fruits at the time of removal), or entire flowering branches were removed, and subsequent flower production was recorded. Two of the experiments (bud and
branch removal) were done sequentially on the same group
of plants: sib replicates, representing control and treatment
plants, of 10 maternal parents (five from population 2, four
from population 3, and one from population 4) for a total of
20 plants (fig. 2). The third experiment (flower removal) was
performed using sib replicates, paired within control and
treatment groups, of 18 maternal parents (six from population 2, seven from population 3, and five from population
4) for a total of 36 plants (fig. 2).
In the bud removal experiment, one replicate plant of each
maternal parent had all of its emerging buds removed for 2
wk while the other replicate was grown undisturbed in the
r roduction was resame greenhouse as control. ~ G w e p
corded for both treatment and control groups during the
2-wk removal period and for an additional 2 wk after bud
removal was stopped. At this point, 4 wk after flowering
started, the branch removal experiment was begun. In order
to control for the effects that the initial bud removal might
have had on the treated plants, half of the plants in the original treatment and control groups were randomly chosen to
represent the new treatment group. The remaining plants
served as controls. In the new treatment group, all developing flowers were removed by cutting the branches down
to the next developing node that would flower. This generally included the uppermost apical meristem and one to three
sets of lateral branches. All plants were then allowed to flower and the type and number of flowers produced were recorded for 2 wk.
The third experiment, flower removal, was performed on
sib replicates of 18 maternal parents (described above). All
36 plants were allowed to flower without disturbance for 2
wk. At this time, 28 plants had produced approximately
equal numbers of flowers, and 14 of these were chosen to
have all of their developing fruits and flowers removed. Because of differences in overall size and flower number between sibs, we were not able to use sibs from each parent
as treatment and control plants. The 14 treatment plants were
chosen by grouping sib replicates together and randomly
choosing seven maternal parents to serve as the treatment

504

INTERNATIONAL JOUR

group. The other 22 plants (from 11 maternal parents) were

used as controls (fig. 2). Any new flowers that were produced by the treatment group were subsequently removed
daily for 1 mo. The type and number of flowers produced
by all plants during flower removal were recorded daily for
4 wk.

.L O F PLANT SCIENCES

Pistillate

801

Perfect
H Total

GIBBERELLIN.
TO determine the effects of gibberellin on
pistillate flower production, small amounts (1 kL) of a 10
mg/L solution of gibberellic acid (GA,) were applied daily
to randomly selected newly emerging buds using a disposable syringe. The GA, solution was made by dissolving 100
mg of crystalline GA, in 5 pL of 100% ethanol and then
diluting to 50% with distilled H,O. Control buds on each
plant were given 1-kL applications of 50% ethanol. Sib replicates of nine maternal parents, one from population 2, five
from population 3, and three from population 4, were tested.
The sex of treated and control buds was recorded daily for
3 wk.
ETHYLENE.TO determine the effects of ethylene on flower production, two types of experiments were performed: (1)
direct hormonal application to developing flowers or (2) indirect exposure to other plants already producing pistillate
flowers. Since ethylene is a gaseous compound, and the
greenhouses are enclosed areas, the second type of experiment tested whether exposure to other plants that might have
been producing large quantities of ethylene gas could induce
pistillate flower production in healthy plants.
Direct hormonal application to developing buds was performed in both the greenhouse (1992) and the field (1993).
In the greenhouse, 10 plants representing 10 maternal parents (three from population 2, two from population 3, and
five from population 4) were given small doses (0.1 rnL) of
a 1 mM aqueous solution of ACC (1-aminocyclopropane-1carboxylic acid), the biochemical precursor to ethylene. This
compound is absorbed through the calyx of the flower and
then converted to ethylene. The solution was applied daily
to all buds from the first day of emergence on randomly
chosen branches of each plant using disposable syringes. The
remaining untreated branches on each plant served as controls. Both the number and type of flower produced per
branch were recorded for 1 mo.
A similar experiment was also conducted in the field during August 1993, near Mountain Lake Biological Station.
Flower production was observed in a common-garden environment using seed replicates of naturally pollinated sibprogeny from five maternal parents (10 total plants) collected from five roadside populations in 1991. The seeds were
germinated in 5 X 5 cm cells containing Metro Mix at
Mountain Lake Biological Station in mid-June. Healthy rosette-stage seedlings were transplanted ca. 3 wk after germination into 10-in plastic pots using a 3 : 1 mixture of soil
and sand. Each pot was then placed at ca. 0.5-m intervals,
in an open field that served as the common-garden site ca.
3.2 km from other naturally occurring populations. All pots
were watered every 2 d unless sufficient rainfall thoroughly
soaked the soil, and a weak solution of 20 : 20 : 20 general
purpose fertilizer was applied once a week.
For this field experiment, entire plants (one sib from each
parent) were given the daily treatments of ACC, and the
solution was applied not only to emerging buds but also at
an earlier stage of development, i.e., to meristematic tissue
of the rosette-stage seedling just before bolting. This early
application tended to inhibit bolting in some plants, and

190C

25oC

N=18

N=18

Mean daytime greenhouse temperature

Fig. 3 Mean (ITSE) pistillate, perfect, total, and percentage of
pistillate flower production per plant by Silene noctijorn in a lowvs. high-temperature greenhouse.

therefore treatments to these plants were only given for 1

wk and then temporarily stopped until bolting occurred. The
other sib replicate of each parent was left untreated and
served as the control. Flower number and type were recorded on all plants for a period of 3 wk.
Indirect exposure to ethylene gas from surrounding plants
was performed in the greenhouse in the following manner.
Sib replicates of 18 maternal parents (six from population 2,
seven from population 3, and five from population 4) were
placed in two adjacent greenhouses separated by a glass
door. In one greenhouse, the plants were surrounded by other
plants that had been heavily pruned and were making large
numbers of pistillate flowers. The type and number of flowers produced per plant in each greenhouse was recorded daily for 2 wk.

All statistical analyses were performed using the General

Linear Models (GLM) nested ANOVA procedure for unequal sample sizes (Type I11 SS) from the SAS statistical
program (SAS Institute 1985). Each induction factor (temperature, removal treatment, or hormonal application) was
analyzed with the genotypic factors of maternal parent
andor population for their effects on the total percentage of
pistillate flowers produced per plant. The induction factor
was considered a fixed effect, population a random effect,
and maternal parent was a nested effect within population.

FOLKE & DELPH-ENVIRONMENTAL

Treatment

AND PHYSIOLOGICAL MALE STERILITY

5 05

group

Mean per plant

Total

Control group

Mean per plant

Total

Bud

Flower*

Branch

Bud

Flower*

Branch

N=10

N=7 N = l l

N=10

Type of biomass removed

fig. 4 Mean ( i S E ) percentage of pistillate (A) and total (B) flower production per plant by Silene noctijora in three different types of
removal experiments (bud, flower, and branch). * = individual values obtained as the sum of two sibs from one maternal parent.

No significant treatment by population interaction effects

were found, so the treatment effect F-ratio was calculated
using the error MS in the denominator.

Results
Average daytime temperature was 19C in the lowtemperature greenhouse (LTG) and 25C in the-high
temperature greenhouse (HTG). Maximum temperatures reached 29C (LTG) and 38C (HTG). The overall 6C difference in temperature caused a significant
increase in the percentage of pistillate flowers produced by HTG plants (9.3% + 1.5% [X ? SE], n =
18) compared with LTG plants (3.2%
0.9%, P =
0.0011; fig. 3). Plants in high temperatures had increased flower production overall, but this difference
was not significant (P = 0.095). The percentage of
pistillate flower production was not significantly affected by genotype, either through population (P =
0.092) or through maternal parent ( P = 0.28) differences. However, total flower production was significantly affected by populational differences (P < 0.05)
but not by maternal parent differences ( P = 0.92).

Pistillate flower production increased between experiments as greater amounts of biomass were removed (fig. 4). Plants that had only newly emerging

buds removed expressed the lowest percentage of pistillate flower production per plant (0.5% + 0.3% [X +SE], n = lo), whereas those with entire branches removed had the highest expression (6.0% ? 2.9%, n =
10). However, treatment groups tended to have a lower
percentage of pistillate flower production per plant in
all experiments relative to the controls. This difference
was significant only for the removal of emerging buds
(P < 0.001). Moreover, the reduction in pistillate flowers occurred despite a significant increase in total flower production for removal plants (bud and flower removal only, P = 0.0001 for both). The branch-removal
group was the only group that produced a higher percentage of pistillate flowers overall. For the percentage
of pistillate flower production, no significant effects of
genotype through maternal parent differences were
found in any of the treatments, but a populational effect was found for the percentage of pistillate flower
production during bud removal ( P < 0.05; table 1).
GIBBERELLIN.
The gibberellin solution applied to
developing buds had no apparent effect on increasing
pistillate flower production. Of the 50 buds that were
treated, neither the control nor GA,-treated buds produced any pistillate flowers.
ETHYLENE.The possible indirect exposure to gas-

FOLKE & DELPH-ENVIRONMENTAL

AND PHYSIOLOGICAL MALE STERILITY

507

Table 2
SUMMARY
OF

% Pistillate

NESTED ANALYSIS OF VARIANCE FOR ETHYLENE

EXPERIMENTS

Ei Total

Application, variable,
and source

Control group
El % Pistillate
Total

Meristem

~ u d
N=10

N=12

N=12

df

MS

Young bud:
Percentage of pistillate:
Treatment. ................
Population. ...............
Maternal parent
(population). ...........
Error ......................
Total flower production:
Treatment. ................
Population. ...............
Maternal parent
(population). . . . . . . . . . . .
Error ......................
Rosette stage:
Percentage of pistillate:
Treatment. . . . . . . . . . . . . . . . .
Population. ...............
Maternal parent
.. .
(population).. . . . . . .
Error ......................
Total flower production:
Treatment. ................
Population. ...............
Maternal parent
(population). ...........
Error . . . . . . . . . . . . . . . . . . . . . .
N=l8

Application of ACC (ethylene precursor)

Fig. 5

Mean (?SE) percentage of pistillate and total flower production by Silene noctiJlora when given daily 0.1-mL applications
of ACC (1-aminocyclopropane-1-carboxcylic acid) to either young
buds (first day of emergence) or to meristematic tips of rosette-stage
seedlings.

increasing temperature tends to increase growth and

the development of aerial plant parts, such as leaves,
the overall ratio of gibberellins relative to other hormones may increase, thereby increasing maleness. We
tested the direct application of gibberellins on S. noctiflora in this study, and no effect on pistillate flower
production was observed. However, there is evidence
that significantly higher concentrations of gibberellins
than the one we used (10 mg/L) are necessary to induce femaleness over maleness in some species. For
example, Pharis et al. (1970) found that as much as
2500 mg/L of GA was necessary in conifers. Although
our experiments indicate that gibberellins are not involved in pistillate flower production in S. noctiJlora,
additional experimentation using higher concentrations
of GA, need to be explored before the role of gibberellins is dismissed.
In addition, since sex determination occurs at a very
early stage of development in most species (Grant, in
press), hormonal applications at the time when buds
are visible may be too late to affect the sex of the
flower. This idea is supported by the results from our
experiments using ethylene. When applications of

ACC were given to the emerging calyx of visible buds,

pistillate flower production was induced but at a much
lower percentage (7.2%) compared with applications
given at both the rosette stage and throughout the bolting process (38.9%, fig. 5). This indicates that, if sex
expression is to be altered, it is critical for hormones
to be present as the initial sexual primordia are formed.
Ethylene is thought to be involved in several phases
of plant development, including sex expression and responses to various types of external stimuli such as
temperature and wounding (Field 1985; Hyodo 1991;
Abeles et al. 1992). A number of studies have demonstrated the induction of femaleness in several species using ethylene-related compounds (Chailakhyan
and Khrianin 1987; Sawhney and Shukla 1994; Yin
and Quinn 1995; Trebitsh et al. 1997), and we found
in this study that applications of ACC to both buds
and developing meristematic tissue of S. noctiflora
caused a significant increase in the percentage of pistillate flowers produced (fig. 5). Since both temperature and heavy pruning (wounding) also appear to be
involved in pistillate flower production for this species,
it seems likely that an ethylene-induced response to
these factors may be part of the induction mechanism.
Given the apparent relationship between ethylene
and femaleness in S. noctiflora, the results from the
organ-removal experiments were somewhat surprising.
Our previous observations of heavily pruned greenhouse plants indicated that flower production following heavy pruning was primarily pistillate. Therefore,

508

INTERNATIONAL JOURNAL O F PLANT SCIENCES

we expected to find a significant increase in the percentage of pistillate flowers for plants that experienced
physical disturbance. Instead, two of the three removal
experiments, (bud and flower removal) seemed to
slightly inhibit pistillate flower production rather than
enhance it, even though the removal treatment caused
a significantly greater number of total flowers to be
produced per plant (fig. 4A, B). Branch removal apparently neither enhanced nor inhibited pistillate flower production per plant as there were no significant
differences between control and removal groups in either percentage of pistillate or total flower production.
However, this treatment group did show a higher percentage of pistillate flowers produced overall for the
removal plants (8.2%) compared with the controls
(4.4%; fig. 4A).
The results from the removal experiments indicate
one or more of the following: (1) larger amounts of
biomass must be removed before the response for pistillate production is initiated (this idea is supported by
the tendency of increased pistillate flower production
under branch removal compared with bud and flower
removal); (2) an entirely different response mechanism
is initiated during branch removal as opposed to flower
removal; and/or (3) there are age-related effects for
pistillate production in response to pruning. Since
pruning events generally involved several branches being removed after a majority of flowers had begun to
mature their fruit, it is possible that the timing and
degree of removal in the experiments were not sufficient to produce an effect.
The removal of flowers and/or leaves has been
shown to alter the sex ratios of later-developing flowers in other species (Whitham and Mopper 1985; Chailakhyan and Khrianin 1987; Spears and May 1988),
but this type of disturbance does not appear to favor
one particular sex. Rather, it enhances the production
of either bisexual flowers or opposite sexed flowers;
i.e., males become more female and vice versa. The
experiments described here using S. noctijora support
the expectation of bisexual flower production under
flower removal, but additional studies must be performed to demonstrate experimentally the increased

pistillate production that has been observed to occur

after heavy pruning.
Flexibility for sexual expression is not an unexpected phenomenon in sessile organisms that maintain
a modular framework such as plants. If an individual
cannot choose the environment it inhabits, and if the
relative success of an individual through either male
or female function can differ depending on environmental conditions (Willson 1983), then it seems likely
that labile sex expression might be selected for to maximize overall fitness. For example, because producing
ovules is thought to be more costly than producing
pollen, evolutionary theory predicts that femaleness
should increase under favorable (high-quality) conditions as opposed to unfavorable conditions (Charnov
1982; Lloyd and Bawa 1984; Lovett Doust et al.
1987). For S. noctijora, favorable conditions for successful seed production in pistillate flowers are probably related to pollinator activity. If higher temperatures increase pollinator activity, as is likely for
night-flying insect pollinators, then plants that produce
pistillate flowers in response to increasing temperature
would have a higher probability of having those flowers successfully outcrossed.
The results of this study of the inductive mechanisms involved in pistillate flower production indicate
that the production of pistillate flowers in S. noctiJEora
is a plastic trait. External environmental factors can
cause significant changes in the proportion of pistillate
flowers produced by individuals of this species. One
of the factors influencing this plasticity appears to be
temperature, and a probable hormonal mechanism via
ethylene also appears to exist. The reasons why this
species possesses such a response have yet to be determined, but the response may have fitness consequences related to obtaining pollinator visitation.
Acknowledgments

We thank the greenhouse staff at Indiana University

and the staff at Mountain Lake Biological Station for
their help, and S. Davis, E. Garber, J. I? Gibson, and
D. Marr for their comments on an earlier version of
the manuscript. This research was supported in part by
NSF grant BSR-9010556 to L. E Delph.

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