Blackwell Science, LtdOxford, UK

PCEPlant, Cell and Environment0016-8025Blackwell Science Ltd 2001

25
807
Reactions of bell pepper to elevated temperature
A. N. Erickson & A. H. Markhart
10.1046/j.0016-8025.2001.00807.x
Original ArticleBEES SGML

Plant, Cell and Environment (2002) 25, 123–130

Flower developmental stage and organ sensitivity of bell

pepper (Capsicum annuum L.) to elevated temperature
A. N. ERICKSON & A. H. MARKHART

Department of Horticulture Science, University of Minnesota, St. Paul, Minnesota, USA

ABSTRACT face temperature most likely has increased by 0·6 ± 0·2 °C

High temperatures adversely affect crop productivity of
several plant species including bell pepper (Capsicum
annuum L. var. annuum). The objectives of this study were:
(1) to determine whether flower ontogeny is adversely
affected by high temperature during different phases of
development, including pre- and post-pollination events;
(2) to determine the duration of high temperature exposure
necessary to cause reduction in fruit set; and (3) to deter-
mine whether injury to the pistil or stamen during develop-
ment is responsible for reduced fruit set during high
temperature. We determined that flower buds at <2·5 mm in
length, corresponding to microspore mother cell meiosis to
tetrad dissolution, and flowers that reached anthesis during
the high temperature exposure had reduced fruit set when
exposed to 33 °C for 48 or 120 h. Flower buds at <2·5 mm in
length also had reduced pollen viability when exposed to
33 °C for 120 h. Morphological examination demonstrated
that meiocytes initiated tetrad formation, but after dissolu-
tion the microspores remained small and clumped without
a thick exine. High temperature exposure at a late-
development, pre-anthesis stage did not affect pistil or
stamen viability, but high post-pollination temperatures
inhibited fruit set, suggesting that fertilization is sensitive
to high temperature stress.
Key-words: high temperature; plant reproduction; mega-
sporogenesis; microsporogenesis; microspore mother cell
meiosis; ontogeny; pistil; pollen viability; pollination;
stamen.
INTRODUCTION
High temperatures can cause significant losses in crop pro-
ductivity of many species due to reduced seed yield and
increased flower abscission (Levy, Rabinowitch & Kedar
1978; Rylski & Spigelman 1982; Konsens, Ofir & Kigel
1991; Wheeler et al. 2000). The adverse effects of high tem-
peratures may increase in frequency, if the occurrences of
extremely hot days increase as predicted with the general
warming of the global climate. As of 2001, global mean sur-
Correspondence: Dr Ami N. Erickson, Front Range Community
College, Westminster Campus, Department of Science and Tech-
nology, West Service Center, 3645 West 112th Ave, Westminster, CO
80031-2199, USA. E-mail: anerickson@hotmail.com
over the last 140 and 100 years, and the recent warming has
been greatest over the mid-latitude continents in winter
and spring. (Houghton & Yihui 2001).
Pepper is one of many crops that are primarily grown in
the mid-latitudes and also is sensitive to high temperature.
Field and controlled environment observations of pepper
production indicate substantial abortion of floral buds
occurs when day temperatures are ≥ 34 °C and/or night tem-
peratures are ≥ 21 °C for extended periods of time (Cochran
1936; Rylski & Spigelman 1982). Controlled experiments
exposing peppers to constant 33 °C with increased vapour
pressure deficit (VPD) or no increase in VPD demon-
strated that reduced fruit set was a result of the increased
temperature and not the increased VPD. At 33 °C, peppers
continued to produce flowers, and flowers did not abscise
until after opening, resulting in significantly reduced fruit
set (Erickson & Markhart 2001). The stages and structures
of pepper flower development and reproduction affected
by high temperature remains undetermined.
Sensitivity of flower development, particularly
microsporogenesis, to high temperatures has been demon-
strated in many other crops. In bean (Phaseolus vulgaris L.)
(Monterroso & Wien 1990) and cowpea (Vigna unguiculata
(L.) Walp.) (Warrag & Hall 1984) high temperature expo-
sure appears to adversely affect microspore development
after tetrad dissolution and prior to anthesis, resulting in
reduced pollen viability. Pollen viability and pollen shed is
reduced by high temperature in corn (Zea mays L.)
(Schoper et al. 1987) and tomato (Lycopersicon esculentum
L. Mill.) (Sato, Peet & Thomas 2000), suggesting that
anther dehiscence may also be inhibited by high tempera-
ture exposure. Identifying whether high temperature has a
similar effect on pepper pollen development and whether
other developmental stages and flower structures are sen-
sitive will contribute to defining cultural solutions and
breeding strategies for improved pepper crop production
during periods of elevated temperature.
The objectives of this study were: (1) to determine
whether flower ontogeny in bell pepper is adversely
affected by high temperature during different phases of
development; (2) to determine the duration of high tem-
perature exposure necessary to cause reduction in fruit set;
(3) to determine whether injury to the pistil or stamen dur-
ing development is responsible for reduced fruit set during
high temperature; and (4) to determine whether pre- and/
or post-pollination events are sensitive to high temperature
exposure.

© 2002 Blackwell Science Ltd 123

124 A. N. Erickson & A. H. Markhart
MATERIALS AND METHODS
Plant material
Bell pepper (Capsicum annuum L.) seeds of ‘Ace’ and
‘Bell Boy’ were sown in 20 cm3 plug trays in a soil-less
media (Strong-Lite Germination Mix; Seneca, IL, USA)
and germinated under mist at 25 ± 4 °C. After germina-
tion, plants were transplanted into 0·5-litre azalea pots in
soil-less media (Strong-Lite soil-less Universal Mix;
Sepeca) and were grown in the greenhouse in day/night
temperatures of 25/21 °C, under ambient light and supple-
mental, high intensity discharge lighting for 14 h of 400 W
high-pressure sodium lamps (Lucolux LU400; General
Electric, Cleveland, OH, USA). Plants were watered as
needed and fertilized weekly with 800 mL of water soluble
20 mM · N−4·4 mM · P−16·6 mM · K (Peter’s Lite, Fogels-
ville, PA, USA). After temperature treatments com-
menced, plants at 33 °C were watered daily, and all plants
were fertilized bi-weekly. When the first flower buds
reached anthesis (approximately 3 weeks after initial
transplant) plants were repotted into 10 L azalea pots in
soil-less media and fertilized twice weekly with 1600 mL
Peter’s Lite. Open flowers and fruitlets were pinched until
commencement of experiments.
Pepper plants of each cultivar that had developed at
least seven reproductive nodes (approximately 3 months
after sowing) were placed into four growth chambers at
day/night temperatures of 25/21 °C with a 12 h photoperiod
of 450 ± 50 µmol m−2 s−1 PPFD (photosynthetic photon flux
density) as measured at the top of the plant canopy with a
LI-COR quantum flux sensor (LI-COR Environmental
Division; Lincoln, NB, USA). Irradiation sources consisted
of a combination of 60% of total wattage from cool white
fluorescent lamps and 40% of total wattage from 60 W
incandescent lamps. Relative humidity within chambers
was 70 ± 10%. To allow flower buds to develop in con-
trolled conditions, all visible flower buds were pinched
upon moving to growth chambers, and peppers were
allowed to grow at day/night temperatures of 25/21 °C until
two new flowers on consecutive reproductive nodes
reached anthesis.
Table 1. Developmental stages of bell pepper flowers associated with microspore development, bud size, and days to anthesis at 25/21 °C
as determined with anther squashes and aniline blue stain
Bud stage Characteristics
I Calyx lobes closed, petal not visible,
green buds
II Calyx lobe beginning to separate
III Further separation of calyx, petals
becoming visible
IV Protruding flower petals
V Protruding flower petals almost
twice as long as calyx
Stages of microspore development
Anthers were excised from buds of ‘Ace’ and ‘Bell Boy’
pepper plants grown in growth chambers at day/night tem-
peratures of 25/21 °C to ascertain the representative devel-
opmental stages corresponding with flower bud length.
Anther squashes and aniline blue stain (1% aniline blue in
80% propionic acid) were used to determine pollen devel-
opmental stages. At least 10 buds of each cultivar for each
length category from 1 to 10 mm at intervals of 2 mm were
examined. Bud length was determined by measuring the
distance from the base of the pedicel to the top of the sepal
or corolla depending on which was longer. The relationship
between flower bud length, approximate days to anthesis,
and stage of pollen development is presented in Table 1.
Flower developmental stages sensitive to
high temperature
Four floral developmental stages were related to the length
of the floral bud: stage I, buds with length < 2·5 mm (pre-
meiotic to microspore mother cell (MMC) meiosis); stage
II, buds 2·5–4·5 mm (tetrad dissolution); stage III, buds 4·6–
6·5 mm (young microspores); stage IV, buds 6·6–11 mm
(maturing microspores). A minimum of nine buds from five
plants for each temperature treatment was tagged at each
developmental stage before transferring at mid-photope-
riod to a growth chamber with constant temperature of 33
°C. Five plants of each cultivar remained in chambers with
optimum day/night temperatures of 25/21 °C. After 6, 48, or
120 h of 33 °C treatment, five plants of each cultivar were
returned to 25/21 °C. After the high-temperature treat-
ments were completed, all chambers were set to day/night
temperatures of 25/21 °C, and plants were randomized
between the chambers. Plants were re-randomized weekly
within chambers to reduce chamber effects of variation in
temperature and irradiation.
Plants were examined every 3 d to determine days to
anthesis and fruit set or abscission. Fruits were counted
when 3 cm or larger in length and were removed to reduce
assimilate competition.
Length of Days to
buds (mm) anthesis Stage of microsporogenesis
<2·5 14–17 Pre-meiotic and different
phases of meiosis
3·0–4·0 9–13 Tetrad formation and dissolution
4·5–6·5 6–8 Young, free microspore
7·0–8·0 3–5 Maturing microspores. Exine
becoming thick and decorated
8·5–11·0 1–2 Pollen grains undergoing mitosis;
thick, dark exine
© 2002 Blackwell Science Ltd, Plant, Cell and Environment, 25, 125–130

After completion of the first replication (R1), a need to
separate stage IV into two stages of development became
apparent due to the variation in time to anthesis within
this stage. In the second replication (R2) stage IV was
divided into two stages: bud length 7–8·5 mm (stage IV)
and bud length 8·6–11 mm (stage V).
The experimental design was a completely randomized
split-plot with treatment duration and cultivar as the main
plot and bud developmental stage as the subplot. Replica-
tion and plant were considered random effects. Each tem-
perature treatment consisted of five plants of each cultivar
with four buds (R1) or three buds (R2, to accommodate the
increase in stage division) for each bud category per plant.
R2 had an unbalanced design with three bud size categories
per plant, resulting in nine tagged buds per plant and a total
of nine buds per category in each treatment. The trials were
analysed using the split-plot, random effects procedure in
JMP version 3 (SAS Institute Inc., Cary, NC, USA). One-
way analysis of variance was conducted on treatment dura-
tion within each flower bud stage to compare developmen-
tal stages IV and V. Statistical analysis of percentage fruit
set was performed after arcsin transformation. Pairwise
comparisons between means were completed using Tukey–
Kramer HSD (P < 0·05).
Pistil and stamen sensitivity during
early development
‘Ace’ and ‘Bell Boy’ pepper plants were randomly sepa-
rated between four growth chambers. After a period of
adjustment (approximately 2 weeks) flowers of < 2·5 mm in
length (Stage I) were tagged in the morning prior to the
temperature treatment. This bud length correlates with
MMC meiosis (Table 1). At mid-photoperiod, the temper-
ature in two of the chambers was changed to a constant
33 °C. Temperature remained at 33 °C for 120 h and was
returned to 25/21 °C at mid-photoperiod on the fifth
day. Plants were re-randomized weekly within growth
chambers to reduce effects of variation in temperature
and irradiation.
Fifteen days after flower buds were tagged, flower buds
that were approximately 11 mm in length with ‘balloon-
ing’ petals, were marked for pollen donation or emascu-
lated in the evening and covered with 3 cm wide wax
paper bags to prevent exposure to pollen and stigma dry-
ing. The following day, emasculated flowers were polli-
nated with pollen donated from open and dehiscing
flowers. Pollen was always taken from a different plant
than that used for the female. Pollen was donated ran-
domly from untreated or 33 °C-treated flowers. Emascula-
tion and pollination were repeated for 3 d until all tagged
flowers were pollinated. The crossing pattern of the tem-
perature treatments (°C) was as follows: (1) female at
control temperature × male heat-treated (25 × 33); (2)
female heat-treated × male at control temperatures (33 ×
25); (3) female at control temperature × male at control
temperature (25 × 25); (4) female heat-treated × male
heat-treated (33 × 33).
© 2002 Blackwell Science Ltd, Plant, Cell and Environment, 25, 123–130
Reactions of bell pepper to elevated temperature 125
Fruit developed until the mature green stage (approxi-
mately 30 d after pollination). Fruit were harvested, fresh
weight and fruit lengths measured, and seed number
determined.
The experiment was arranged as a randomized split-plot
design with cultivar and flower temperature as the main
plot factors and pollen temperature treatment as the sub-
plot factor. Two replications were used, and there were four
(R1) or eight (R2) plants in each plot with 20 (R1) or eight
(R2) flower buds in each subplot. Chamber was considered
a random effect and provided the error term for main plot
factors. Fruit set, fruit fresh weight, fruit length, and seed
number were analysed with a split-plot analysis of variance
using the random effects procedure of JMP version 3 (SAS
Institute Inc.). Percentage fruit set data was arcsin trans-
formed prior to analysis.
Pollen viability
Pollen viability was estimated by staining in 0·4% Trypan
blue (Sigma, St Louis, MO, USA). Flower buds were
treated at 33 °C during early development (Stage 1) for
120 h as in the above experiment. At anthesis, flowers of
each cultivar from each treatment were collected from
eight randomly chosen plants. Flowers were tapped over
glass microslides and approximately 70 µL of 0·4% Trypan
blue were added. Using a light microscope and the 160 ×
microscope field, five microscope fields with >20 pollen,
when possible, were counted and percentage pollen viabil-
ity calculated. Clear, normal-appearing pollen in Trypan
blue stains were considered viable.
Pollen viability was analysed with a split-plot analysis of
variance with chamber and temperature as the main plot
and pepper cultivar as the subplot. Statistical analysis of
percentage pollen viability was performed after arcsin
transformation.
Flower bud morphology
Flowers of ‘Ace’ and ‘Bell Boy’ plants grown in growth
chambers at the temperature regimes indicated above
were used to study morphology of the developing flower
bud, particularly the developing microspores. Flower buds
were harvested after 4, 8 and 17 d from first day of temper-
ature treatment, corresponding with buds of 3 mm in
length (tetrad formation), 4–5 mm in length (tetrad disso-
lution) and 8 mm in length (maturing microspores nearing
dehiscence) (Table 1). Buds were fixed for 3 h in FAA
(5 mL formalin, 5 mL glacial acetic acid, 90 mL 50% ethyl
alcohol) or Histochoice (Amesco, Solon, OH, USA), and
dehydrated and infiltrated in an ethanol and tert-butanol
series. Buds were embedded in wax (Paraplast ® tissue
embedding medium; Oxford Labware, St.Louis, MO,
USA). Wax was sectioned with an AO ‘820’ microtome
(American Optical Co., Buffalo, NY, USA) into 10 µm sec-
tions, stained with safranin red and fast green, and exam-
ined with a light microscope.
126 A. N. Erickson & A. H. Markhart

Table 2. Experimental set up for the pre-anthesis to post-
pollination experiment. Flowers were exposed to 33 °C or
maintained at day/night temperatures 25/21 °C (25 °C) for 3 d prior
to anthesis, reciprocally pollinated, then moved to post-pollination
temperatures of 25/21 °C or 33 °C for 2 d. After 2 d, all plants were
returned to 25/21 °C.
plants for each treatment. Pollen viability was analysed in a
2 × 2 factorial analysis of variance with cultivar and tem-
perature as the main factors with two replications. Statisti-
cal analysis of percentage pollen viability was performed
after arcsin transformation.

Post-pollination temperature
RESULTS
25 °C 25 °C 33 °C 33 °C
Flower developmental stages sensitive to
Pre-anthesis temperature Pre-anthesis temperature
high temperature
Pistil × Stamen Stamen Pistil × Stamen Stamen
To determine which stages of pepper flower development
25 °C 25 °C 25 °C 25 °C
25 °C 33 °C 25 °C 33 °C are sensitive to high temperatures, flower buds at four
33 °C 25 °C 33 °C 25 °C developmental stages (Table 1: I to IV) were maintained at
33 °C 33 °C 33 °C 33 °C 25/21 °C or exposed from 6 to 120 h to 33 °C. Duration of
high temperature exposure affected the different stages of
flower development variably (F9,64 = 4·4, P < 0·01). Treat-
ment at 33 °C for 48 and 120 h of flower developmental
stages I and IV reduced fruit set (Fig. 1). Flower bud stages
Pistil and stamen sensitivity shortly before
II and III were not significantly affected by any duration of
anthesis and after pollination
high temperature exposure. Both cultivars responded sim-
Twenty-four plants of each cultivar were randomly sepa- ilarly (F1,64 = 1·5, P = 0·18).
rated into four chambers. After 2 weeks at control day/night Flower buds at stage I, which were 1·0–2·5 mm in length
temperatures (25 °C/21 °C), two (R1) or three (R2) flowers and beginning to undergo MMC meiosis, experienced a
of 8 mm in length per plant were tagged for pollination at decrease in fruit set from an average of 57% with 6 h of high
anthesis, and temperatures in two of the growth chambers temperature exposure to 14% with 120 h of exposure (Fig.
changed at mid-photoperiod to 33 °C. After 2 d, the tagged 1a). Most flower buds abscised after flowers opened.
flowers were emasculated in the evening, and flowers at the Flower buds at stage IV (6·6–11 mm in length with matur-
same stage as tagged flowers were marked for pollen dona-
tion. The following day, emasculated flowers were polli-
nated with pollen donated from open and dehiscing flowers
(b) Stage II
from either 25/21 or 33 °C-exposed plants. Pollen was 100
(a) Stage I

always taken from a different plant than that used for the
a
female. After pollination, plants were randomly placed at a a
control temperatures (25/21 °C) or at 33 °C for post-polli- 50
ab a
a
nation treatment. After 2 d all treatments at 33 °C were ab
b
Percent of fruit set

returned to control temperatures. The crossing pattern of

the pre-pollination treatments and post-pollination treat- 0
0 6 48 120

ments is defined in Table 2. (c) Stage III (d) Stage IV

100
The experimental design was a randomized split-plot a
a a
design with cultivar and post-pollination temperature as the
a a a
main-plot factors and flower cross as the subplot factor.
50 50 b
There were three plants in each subplot with two replica-
tions in time. Analysis of variance was completed using the c
split-plot, random effects procedure in JMP version 3 (SAS
0 0
Institute Inc.) for fruit set. Statistical analysis of percentage 0 6 48 120 0 6 48 120
fruit set was performed after arcsin transformation. Time (h)

Figure 1. Fruit set response of flowers exposed to different

Pollen viability
Pollen viability was estimated to determine whether matur-
ing microspores were damaged by high temperature just
before dehiscence. Dehiscing flowers of ‘Ace’ were col-
lected 2 d after commencement of the temperature treat-
ment from 25 and 33 °C-exposed plants. Pollen viability was
estimated as described above, except that 1% aniline blue
in 80% propionic acid was used for staining. Four flowers of
each cultivar were collected from four randomly chosen
durations of 33 °C at four different developmental stages. Flower
buds were tagged at bud lengths of (a) < 2·5 mm(Stage I, MMC
meiosis); (b) 2·5–4·5 mm (Stage II, tetrad dissolution); (c) 4·6–
6·5 mm (Stage III, Young microspore); and (d) 6·6–11·0 mm (Stage
IV, Maturing microspore). Flowers were exposed to 0, 6, 48, or 120
h at 33 °C. Values are means ± standard error from untransformed
percentage fruit set from two replications. No differences were
found between ‘Ace’ and ‘Bell Boy’, and the results presented
correspond to the pooled data. Bars with different letters within
each graph are significantly different [Tukey–Kramer HSD (P <
0·05)].

© 2002 Blackwell Science Ltd, Plant, Cell and Environment, 25, 125–130

(a) Pistil at 25°C
100
75
50
Percent of fruit set
25
0 pollinated with pollen from untreated flowers, to 49·3 ±
(b) Pistil at 33°C
25
100
75
50
25
0
25
Pollen temperature (°C)
Figure 2. Percentage fruit set of pepper pistils exposed to 25 °C
(a) or 33 °C (b) for 5 d during early flower development and hand
pollinated with pollen from either 25 or 33 °C-treated flowers.
Values are means from two replications and two cultivars with
standard error bars. Flowers, pollinated with pollen from 33 °C-
treated flowers, had significantly reduced fruit set.
ing microspores) experienced a decrease in fruit set from an
average of 79% with 6 h of high temperature exposure to
39% fruit after 48 h and only 8% fruit after 120 h (Fig. 1d).
Due to the significant increase in flower abortion by
stage IV flowers exposed to 120 h at 33 °C, in comparison
with those exposed to 48 h (Fig. 1d), we separated stage IV
into two developmental stages in replication 2. Flower buds
8·6–11 mm in length with ballooning petals (protruding
flower petals almost twice as long as calyx) were considered
as a fifth developmental stage (Table 1: stage V). A two-way
analysis of variance of duration and developmental stage
(IV versus V) again demonstrated a significant interaction
(F = 5·5, P <0·01). Flower buds of 6·6–8·5 mm in length
(stage IV) experienced a reduction of fruit set from 83·3%
fruit set after 48 h of exposure to 17% after 120 h of expo-
sure. Flower buds of 8·6–11 mm in length (stage V) experi-
enced a reduction of fruit set from an average of 94% after
6 h of temperature exposure to 22% after 48 h and 0% fruit
set after 120 h. Most flower buds at 8·6–11 mm in length
(stage IV) reached anthesis during the 48 h temperature
exposure. Flower buds of 6·6–8·5 mm in length (stage IV)
reached anthesis after the 48 h exposure and during the
120 h high temperature exposure.
Pistil and stamen sensitivity during early
development
To determine whether early pistil or stamen development is
sensitive to high temperature, stage I flower buds (Table 1)
were exposed to 120 h at 33 °C. At anthesis, these flowers
were reciprocally crossed with untreated flowers to deter-
mine fruit set. Fruit set was reduced when pollen was
donated from flowers exposed to 33 °C during stage I (Pol-
len: F1,8 = 53·1, P < 0·01) (Fig. 2). However, high tempera-
© 2002 Blackwell Science Ltd, Plant, Cell and Environment, 25, 123–130
Reactions of bell pepper to elevated temperature 127
ture exposure of the pistil for 5 d during stage I
development did not significantly affect fruit set (F1,8 = 1·2,
P = 0·31). Both cultivars responded similarly to the treat-
ments (F1,8 = 1·2, P = 0·28), and their mean percentage fruit
set was pooled. Mean fruit set percentage ± SE was reduced
from 87·1 ± 4·2%, when pistils from both treatments were
4·2% when pollinated with 33 °C-treated flowers.
33
Fruit fresh weight (F1,8 = 203, P < 0·01) (Fig. 3), fruit
length (F1,8 = 164, P < 0·01), and seed set (F1,8 = 160, P <
0·001) were significantly reduced when flowers were polli-
nated with pollen donated from flowers exposed to 33 °C
during early flower development. The pattern is similar for
33 fruit fresh weight, fruit length and seed set, thus only fruit
fresh weight is presented in Fig. 3. Although cultivar differ-
ences in fruit length interacted with pollen treatment (F1,8 =
7·1, P < 0·01), fruit from both cultivars had reduced length
when meiocytes were exposed to high temperatures. Fruit
length of ‘Ace’ peppers was reduced from a mean (cm) ± SE
of 8·0 ± 0·1% when pollinated with control pollen to 5·5 ±
0·2% when pollinated with 33 °C-treated pollen, and ‘Bell
Boy’ fruit length was reduced from 7·0 ± 0·1 to 5·4 ± 0·2%.
The number of seeds per fruit was affected by 33 °C treat-
ment of both the pistil (F1,9 = 13·0, P < 0·01) and stamen.
Seed number from pistils exposed to high temperature was
reduced by 15% for ‘Ace’, but seed number was further
reduced by 85–90% for ‘Ace’ when either untreated or
high-temperature-treated pistils, respectively, were polli-
nated with high-temperature-treated pollen. ‘Bell Boy’ and
‘Ace’ responded similarly to both treatments (F1,8 = 0·2, P =
0·68). In addition, all fruit from flowers pollinated with
33 °C-treated pollen were deformed, and many were par-
thenocarpic.
Ace Bell boy
Pistil at 25°C Pistil at 25°C
120
80
40
Weight (g)
0
Pistil at 33°C Pistil at 33°C
25
33
120 120
80 80
40 40
0 0
25 33 25 33
Pollen temperature (°C)
Figure 3. Fresh weight (g) of fruits produced by pistils exposed
to 25 °C or 33 °C for 5 d during early flower development,
corresponding with MMC meiosis, and hand pollinated with pollen
from either the 25 or 33 °C-treated flowers. Values are means from
two replications with standard error bars.
128 A. N. Erickson & A. H. Markhart
(a) (b)
(c) (d)
(e) (f)
Figure 4. Micrographs of anther sections (10 µm thick)
embedded in wax showing different stages of pepper pollen
development from ‘Bell Boy’ flowers maintained at day/night
temperatures of 25/21 °C or exposed to 33 °C for 5 d when buds
reached 1·0–2·0 mm in length. (A) Tetrads from a flower bud 3 mm
in length grown at 25/21 °C. (B) Tetrads from a flower bud 3 mm in
length, day 4 of 33 °C treatment. (C) Young microspores from a
flower bud 4 mm in length grown at 25/21 °C. (D) Young
microspores from a flower bud 4 mm in length, day 2 after 33 °C
treatment. (E) Maturing pollen grains from a flower bud 8 mm in
length grown at 25/21 °C. (F) Aborted pollen grains with a few fully
developed pollen grains from a flower bud 8 mm in length day 10
after the 33 °C treatment. Bar = 10 µm for all figures.
Pollen viability
Pollen from flowers exposed for 120 h during early devel-
opment (Stage I), corresponding with MMC meiosis, were
collected at anthesis to determine pollen viability. Percent-
age viability, estimated by staining with 0·4% Trypan Blue,
was greatly reduced when meiocytes were exposed to high
temperature during meiosis (F1,81 = 179·1, P < 0·01). The
mean ± SE of percentage viability was 91 ± 1% (25 °C) and
11 ± 2% (33 °C) for ‘Ace’ and 81 ± 1% (25 °C) and 21 ± 2%
(33 °C) for ‘Bell Boy’. The two cultivars did have a signifi-
cant interaction with treatment (F1,81 = 13·9, P < 0·01), with
‘Bell Boy’ exposed to 33 °C having a slightly higher pollen
viability. Most non-viable, high-temperature-treated pollen
appeared to be deformed, empty, and clumped, whereas
untreated, normal pollen were triangular and elliptical with
a thick exine layer.
© 2002 Blackwell Science Ltd, Plant, Cell and Environment, 25, 125–130
Pollen morphology
For further morphological examination of pollen develop-
ment, thick wax sections were viewed under a light micro-
scope. On the fourth day of treatment, collected flower
buds were 2·5–3 mm in length. The microspore develop-
mental stages observed at this bud length included early
MMC meiosis to recently released microspore. The major-
ity of meiocytes observed were in the dyad or tetrad stage.
Both untreated and high-temperature-treated anthers suc-
cessfully produced tetrads (Fig. 4A & B). After 8 d of tem-
perature treatment, flower buds were 4–5 mm in length and
all anthers from non-heat-treated flowers contained young
microspores (Fig. 4C). Anthers from high-temperature-
treated flowers also contained free microspores, but these
were misshapen, clumped, and appeared to be recently
released from the tetrads (Fig. 4D). Untreated flowers, col-
lected just prior to anthesis, contained mature pollen grains
(Fig. 4E). High-temperature-treated flowers also contained
a few mature pollen grains, but these were slightly swollen
and deformed. Most microspores from the high-tempera-
ture-treated flowers were shrunken and empty without a
noticeable exine layer (Fig. 4F).
Pistil and stamen sensitivity shortly before
anthesis and after pollination
The second stage of flower sensitivity to high temperature
(Stage IV) was further analysed by exposing flowers to
33 °C for 3 d before anthesis, reciprocally crossing with
untreated flowers at anthesis, and exposing pollinated
flowers to 33 °C for 2 d or maintaining flowers at control
temperatures (Table 2). Post-pollination temperatures sig-
nificantly affected fruit set (F1,16 = 48·8, P < 0·01) whereas
pre-pollination temperature exposure of the pepper flowers
was not a significant factor (F3,16 = 1·4, P = 0·19). The post-
pollination temperature of 33 °C significantly reduced fruit
set (Fig. 5) from an average of 79% at 25/21 °C to an aver-
age of 27% at 33 °C. Both cultivars responded similarly
(F1,16 = 1·6, P = 0·21).
Pollen viability
Pollen viability, as estimated by staining in aniline blue, was
not significantly affected by exposure to high temperatures
during the 3 d prior to anthesis (F1,17 = 0·85, P = 0·37). Per-
centage pollen viability remained high for both untreated
and high-temperature-treated flowers of both cultivars.
DISCUSSION
Two stages of bell pepper flower development were sensi-
tive to high temperature exposure of 33 °C for 48 or 120 h.
Bell pepper fruit set decreased when flowers were exposed
during early flower development, corresponding with
microspore mother cell meiosis (Stage I, Table 1), and dur-
ing late flower development, corresponding with
microspore maturation, anthesis, and pollination (Stage IV

100
(a) Post pollination = 25 ∞C
75
50
Percent of fruit set
25
0
100
(b) Post pollination = 33 ∞C
75
50
25
0
25⫻25 25⫻33 33⫻25 33⫻33
Pre-pollination temperature (∞C)
Figure 5. Percentage fruit set of manually pollinated,
reciprocally crossed, pepper flowers exposed to pre-pollination
temperatures of 25 °C (25/21 °C) or 33 °C for 3 d prior to anthesis
(female × male) and given post-pollination temperatures of (a) 25
or (b) 33 °C for 2 d. Values are pooled means from two cultivars
and two replicates with standard error bars. Only post-pollination
temperatures significantly affected fruit set.
and V, Table 1). Flower development between these stages
was not significantly affected by high temperature. These
results are similar to those of Vara Prasad et al. (2001) who
determined that peanut (Arachis hypogaea L.) is also sen-
sitive to high temperature exposure during microsporogen-
esis and at anthesis.
Early flower development: microspore mother
cell meiosis
When high temperature exposure occurred during early
flower development (Stage I), early pistil development was
minimally affected, resulting in a slight reduction of seed
number, whereas pollen viability was greatly reduced. High
temperature exposure during MMC meiosis reduced fruit
set, fruit size, and seed number when flowers were polli-
nated with high-temperature-treated pollen. Most fruit
from pepper flowers pollinated with high-temperature-
treated pollen were malformed and often parthenocarpic.
High temperature has a more acute affect on stamen devel-
opment than on the development of the pistil in other crops
as well, including cowpea (Warrag & Hall 1984), bean;
(Monterroso & Wien 1990) and tomato (Peet, Willits &
Gardner 1997; Peet, Sato & Gardner 1998).
Exposure to high temperature from MMC meiosis to
tetrad dissolution resulted in greatly reduced pollen viabil-
© 2002 Blackwell Science Ltd, Plant, Cell and Environment, 25, 123–130
Reactions of bell pepper to elevated temperature 129
ity and reduced anther dehiscence. We observed that pep-
per microspores exposed to high temperature completed
meiosis, forming tetrads. Once microspores were released
from the tetrad formation, however, most failed to form a
thick, distinct exine layer and expand to the size of normal
pollen grains. Although a few viable pollen were produced
by high-temperature-exposed flowers, most dehisced pollen
grains were empty, clumped, and smaller than the viable
pollen of non-heat-treated flowers. Ahmed, Hall & DeMa-
son (1992) determined that microspore development in
cowpea was sensitive to high temperature just after tetrad
dissolution from the microspore mother sac, resulting in
small, deformed microspores and a prematurely degener-
ated tapetal layer. Similar morphological differences have
been observed in pepper pollen exposed to chilling temper-
atures during MMC meiosis and tetrad dissolution (Mer-
cado et al. 1997).
Besides providing callase for the digestion of the callose
surrounding the microspore tetrad, the tapetum is believed
to provide nutrition and sporopollenin needed to construct
the pollen exine after tetrad dissolution (Bedinger 1992).
Male sterility due to mutation and environmental stress has
been associated with malfunction of the tapetum in many
plant species (Ahmed et al. 1992; Worral et al. 1992; Jin,
Horner & Palmer 1997). The stage of high-temperature
sensitivity of pepper pollen development appears to occur
when the microspores are most dependent on a functioning
tapetum, and the observed pollen sterility may be due to
inhibition of translocation of nutrients or sporopollenin to
the developing microspores.
Late flower development: pre-anthesis to
post-pollination
High temperature exposure during late flower develop-
ment (Stage IV to V), corresponding with microspore mat-
uration, anthesis, and pollination, also resulted in reduced
fruit set. By exposing pepper flowers to 33 °C for 3 d prior
to anthesis or 2 d after pollination, we determined that pol-
len viability prior to pollination was not affected. Instead,
post-pollination events are sensitive to high temperature
and are responsible for decreased fruit set, when stage IV
or V flowers are exposed to elevated temperatures.
These results demonstrate that increasing temperature
from 25 to 33 °C for 3 d prior to anthesis does not affect the
reproductive structures prior to pollination. In contrast,
pollen viability of bean was greatly reduced when exposed
to heat stress just before anthesis (Weaver et al. 1985;
Monterroso & Wien 1990). We examined pepper pollen via-
bility and determined that exposure to high temperature
for 3 d prior to anthesis did not reduce viability.
Exposure to high temperature after pollination, on the
other hand, inhibited one or more post-pollination events,
such as stigma receptivity, pollen tube growth and guidance,
transmission of the gametes, and/or fruit initiation and
development. Cochran (1938) observed that the time from
pollination to fertilization in pepper required a minimum of
42 h, and after fertilization, the zygote required 24–36 h
130 A. N. Erickson & A. H. Markhart
before division could begin. The length of time required for
fertilization to occur corresponds with the 33 °C treatment
length, suggesting the either pollen germination, pollen
tube growth or fertilization is sensitive to high-temperature
exposure. In peanut, pollen tube germination and growth is
retarded by exposure to 39 °C (Vara Prasad et al. 2001). The
inability of mature and germinating pollen of many plant
species to produce heat shock proteins (Mascarenhas &
Crone 1996) may make germinating pollen sensitive to high
temperature. A period of acclimation may moderate the
acute affect of high temperature on fruit set in pepper as
observed in these experiments. The next step in determin-
ing whether the female or male gametophyte or sporophyte
in bell pepper is injured by exposure to high temperatures
during post-pollination events is to determine the timing of
fertilization or fruit development inhibition and to deter-
mine whether pollen germination of pepper can occur at
33 °C.
We conclude that high temperature inhibits the develop-
ment of pollen grains during the period of final tetrad for-
mation to tetrad dissolution, when flower buds are 16–18 d
before anthesis, resulting in pollen sterility. The reduction
of pollen viability effectively reduces fruit size and fruit set.
Injury to the pistil is minimal. In contrast, exposure to high
temperatures just before anthesis does not cause injury to
either the female or male organs. Instead, inhibition of fer-
tilization or early fruit development occurs after pollination
and is responsible for the reduction of fruit set during the
period between anthesis and fruit development. The dura-
tion of high-temperature exposure and the developmental
stages of flowers exposed are important factors in fruit set
success of pepper and should be considered when examin-
ing the effects of heat stress on fruit set and the potential of
global warming to reduce crop productivity.
ACKNOWLEDGMENTS
We thank Vera Krischik, Alan Smith, John Erwin, and
Thomas Soulen for helpful reviews and editorial comments.
University of Minnesota journal series 011210094.
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Received 24 March 2001; received in revised form 30 August 2001;
accepted for publication 30 August 2001