Plant male reproductive development is highly organized
and sensitive to various environmental stressors, including
high temperature. We have established an experimental
procedure to evaluate high temperature injury in japonica
rice plants. High temperature treatment (39 ° C/30 ° C)
starting at the microspore stage repeatedly reduced spikelet
fertility in our system. Morphological observations revealed
that pollen viability in plants exposed to high temperatures
was lower than that in control plants. Most pollen grains in
high temperature-treated plants displayed a normal round
shape and stained reddish purple with Alexander’s reagent;
however, the pollen grains were very poorly attached and
displayed limited germination on the stigma. To investigate
gene regulatory mechanisms in the anther in high
temperature environments, DNA microarray analysis was
performed by comparing non-treated samples with samples
treated with 2–4 d of high heat. Genes responsive to high
temperatures were identifi ed from clustering of microarray
data. Among these, at least 13 were designated as high
temperature-repressed genes in the anther. Expression
analyses revealed that these genes were expressed specifi cally
in the immature anther mainly in the tapetum at the
microspore stage and down-regulated after 1 d of high
temperature. The expression levels of Osc6 , OsRAFTIN and
TDR , which are tapetum-specifi c genes, were unaffected
by high temperatures. These results suggest that not all
tapetal genes are inhibited by increased temperatures and
the tapetum itself is not degraded in such an environment.
However, high temperatures may disrupt some of the
tapetum functions required for pollen adhesion and
germination on the stigma.
Keywords: High temperature stress • Male sterility •
Microarray • Rice • Tapetum .
Abbreviations : AD , auricle distance ; GO , gene ontology ;
RT–PCR , reverse transcription–PCR.
Introduction
Rice ( Oryza sativa L.) is one of the world’s most important
cereals; however, grain yields often fl uctuate due to various
environmental stressors. Temperature during reproductive
development is an important factor determining grain yields
in rice ( Satake and Yoshida 1978 , Nishiyama 1984 , Prasad
et al. 2006 ). To date, many studies have focused on low tem-
perature-induced pollen sterility in rice ( Nishiyama 1984 ,
Wen et al. 2002 , Oliver et al. 2005 ) because, compared with
other cereal crops, such as wheat ( Triticum aestivum ) and
barley ( Hordeum vulgare ), rice is much more sensitive to
low temperature due to its tropical origin. However, global
climate change is likely to exacerbate current crop vulnera-
bility to environmental stress. In particular, the sexual repro-
ductive phase is predicted to be vulnerable to the effects of
global warming ( Hedhly et al. 2009 ). Global warming may
increase the instability of rice yields even in temperate
regions, mainly due to the increased probability of male ste-
rility induced by high temperatures ( Horie et al. 1996 ).
Male reproductive development in higher plants is known
to be very sensitive to abiotic stress. In particular, high or low
temperature stress results in a lower seed set due to male
sterility in most crops, including tomatoes ( Peet et al. 1998 ,
Sato et al. 2002 ), cowpeas ( Ahmed et al. 1992 ), wheat ( Saini
et al. 1984 ), barley ( Sakata et al. 2000 , Koike et al. 2003 ,
High Temperatures Cause Male Sterility in Rice Plants with
Transcriptional Alterations During Pollen Development
Makoto Endo 1 , 6 , Tohru Tsuchiya 2 , Kazuki Hamada 3 , Shingo Kawamura 3 , Kentaro Yano 3 ,
Masahiro Ohshima 1 , Atsushi Higashitani 4 , 5 , Masao Watanabe 4 , 5 and Makiko Kawagishi-Kobayashi 1 , ∗
1 Rice Biotechnology Research Team, National Institute of Crop Science, NARO, Tsukuba, Ibaraki, 305-8518 Japan
2 Life Science Research Center, Mie University, Tsu, 514-8507 Japan
3 Faculty of Agriculture, Meiji University, Kawasaki, 214-8571 Japan
4 Graduate School of Life Sciences, Tohoku University, Katahira, Sendai, 980-8577 Japan
5 Faculty of Science, Tohoku University, Aramaki-Aoba, Sendai, 980-8578 Japan
Plant Cell Physiol. 50(11): 1911–1922 (2009) doi:10.1093/pcp/pcp135, available online at www.pcp.oxfordjournals.org
© The Author 2009. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org
6 Present address: Takii Plant Breeding Experiment Station, Konan, Shiga, 520-3231 Japan.
∗ Corresponding author: E-mail, makikokk@affrc.go.jp ; Fax, + 81-29-838-8484 .
1911Plant Cell Physiol. 50(11): 1911–1922 (2009) doi:10.1093/pcp/pcp135 © The Author 2009.
Regular Paper
at China Academ
y of Agricultural Sciences on M
arch 20, 2010
http://pcp.oxfordjournals.org
D
ow
nloaded from
Oshino et al. 2007 ) and rice ( Satake and Yoshida 1978 ,
Nishiyama 1984 , Prasad et al. 2006 ). In fl owering plants, male
gametogenesis from archesporal cells to mature pollen
involves a series of complicated events ( Goldberg et al. 1993 ).
During male reproductive development, the young
microspore stage is known to be highly sensitive to environ-
mental stress, and cold stress in rice is particularly well
known. The young microspore stage during or just after
microspore release from the tetrad is the most vulnerable to
low temperatures ( Nishiyama 1984 , Oliver et al. 2005 ).
At this stage, the tapetum, the innermost cell layer of the
anther locule, is thought to be fully developed and actively
providing nutrients, components and enzymes for microspore
development. It is known that the tapetum is severely
damaged in rice plants exposed to low temperatures at the
young microspore stage ( Nishiyama 1984 ).
Satake and Yoshida (1978) reported that exposure to
high temperatures induced sterility, and rice plants were
most sensitive to excessive heat during two periods, namely
the fl owering stage and the young microspore stage. In high
temperature injury during fl owering, spikelet sterility is
mainly due to poor anther dehiscence ( Satake and Yoshida
1978 , Matsui et al. 1997 ). Furthermore, it has been reported
that high temperatures on the day of fl owering decrease
the ability of pollen grains to swell, thus resulting in poor
anther dehiscence ( Matsui et al. 2000 ). However, little is
known about how high temperatures at the microspore
stage induce sterility in rice. In order to gain insight into the
mechanisms of sterility induced by high temperatures at
the microspore stage in rice, we observed pollen structure
and germination after high temperature treatment and
analyzed the relative gene expression levels during treat-
ment each day.
Results
High temperature treatment at the microspore
stage reduced spikelet fertility
To investigate the effects of high temperatures on spikelet
fertility, rice plants were exposed to temperatures of 39 ° C in
the daytime and 30 ° C at night for 7 d at three different
developmental stages. The stages were early panicle devel-
opment, glumous fl ower primordium differentiation and
the early microspore stage following meiosis. The results
clearly indicated that the microspore stage was the most
sensitive to heat stress, and spikelet fertility was completely
lost at this point ( Table 1 ). This fi nding was consistent with
a previous study reporting that reduction of spikelet fertility
in rice was caused by high temperatures during the booting
stage, 9 d before heading ( Satake and Yoshida 1978 ). Since
microsporogenesis is known to be highly sensitive to various
stresses, we performed a detailed analysis focused on the
effects of high temperatures at the microspore stage. The
developmental stages were estimated by auricle distance
(AD), which is the distance between the auricle of the fl ag
leaf and that of the penultimate leaf. High temperature
treatments were started at AD = 0 cm, corresponding to the
early microspore stage. Because 5 d of treatment resulted in
complete sterility, we examined the effects of a shorter expo-
sure to high temperatures. Rice plants were exposed to high
temperatures (39 ° C/30 ° C) for 2–4 d, and were subsequently
transferred back to a normal environment (28 ° C/22 ° C) until
seed maturity ( Fig. 1 ). Spikelet fertility drastically decreased
when the plants were treated with high temperatures for
2–4 d ( Table 2 ). Longer exposure showed more severe effects
on spikelet fertility, and the 1 d treatment resulted in only
a limited effect. These results suggest that heat stress at the
microspore stage for 2 d was suffi cient to induce spikelet
sterility and irreversibly damage some aspects of panicle
development.
Pollen grains exposed to high temperatures at the
microspore stage displayed limited germination
on the stigma
To gain insight into how high temperatures reduce spikelet
fertility, the anther, pollen and pistil were carefully observed
through a microscope. At heading, the anther and pistil of
the high temperature-treated plants were indistinguishable
from those of the untreated plants in shape and size (data
not shown). Pollen viability of untreated and plants and
plants treated for 2 or 4 d was tested using Alexander’s
reagent ( Alexander 1969 ). As shown in Fig. 2A , high tem-
perature treatments increased the number of spikelets con-
taining inviable pollen, while all 36 spikelets showed ≥ 75 %
pollen viability in the untreated plants. However, 28 and 33
spikelets out of 36 showed ≥ 50 % pollen viability in the plants
treated for 2 or 4 d, respectively ( Fig. 2A ). The spikelets
in high temperature-treated plants had a signifi cant amount
of pollen grains which stained reddish purple ( Fig. 2B ).
We observed a notable difference in the numbers of pollen
grains on the stigma between the high temperature-treated
and untreated plants. In the untreated plants, we observed
Table 1 Spikelet fertiltiy of rice plants after exposure to high
temperature at different developmental stages
Treatment Spikelet fertillity ( % )
High temperature I 60.9
II 59.8
III 0.0
Untreated 78.6
Rice plants were exposed to high temperature at developmental stages estimated
as I, early panicle development; II, glumous fl ower primordium differentiation;
III, early microspore following meiosis.
1912
M. Endo et al.
Plant Cell Physiol. 50(11): 1911–1922 (2009) doi:10.1093/pcp/pcp135 © The Author 2009.
at China Academ
y of Agricultural Sciences on M
arch 20, 2010
http://pcp.oxfordjournals.org
D
ow
nloaded from
vigorous pollen germination and pollen tube elongation on
the stigma after pollination ( Fig. 3A ). Conversely, the number
of pollen grains on the stigma in the high temperature-
treated plants was drastically reduced and the pollen on the
stigma had barely germinated ( Fig. 3B ). No difference was
detected between the high temperature-treated and
untreated plants in regard to the opening of the spikelets or
anther dehiscence at fl owering (data not shown). It is possi-
ble that most of the pollen grains did not adhere to the
stigma or were washed off during the staining process due to
poor germination. We subsequently performed crosses using
high temperature-treated plants as female parents to inves-
tigate the effects of heat stress on the pistil. Pollen from
untreated plants was shed onto the high temperature-
treated pistil, and thereafter the pollen germination was
observed after staining with aniline blue. The normal extent of
pollen germination and pollen tube elongation was observed
in 20 out of 24 crosses pollinating high temperature-treated
pistils with untreated pollen, and then the progeny from
these crosses grew normally. Representative results are
shown in Fig. 3C . The number of pollen grains on the high
temperature-treated stigma was similar to that observed on
the untreated stigma ( Fig. 3A, C ). These results suggest that
high temperatures at the microspore stage did not damage
the ability of the pistil to receive pollen by the stigma, but
impaired the ability of pollen to attach and/or germinate on
the stigma.
Identifi cation of high temperature-responsive genes
in the anther using DNA microarray
To gain insight into the molecular mechanisms of heat-
induced male sterility, we analyzed transcriptional altera-
tions in the anther under high temperature conditions using
a 22K rice oligo DNA microarray (Agilent Technologies,
Palo Alto, CA, USA). Total RNA was extracted from the
anthers exposed to high temperatures for 2, 3 and 4 d (H2,
H3 and H4) and from untreated anthers with the corre-
sponding timing (C2, C3 amd C4). RNA was also prepared
from seedlings exposed to high temperatures for 4 d (HS)
and from seedlings without treatment (CS) to distinguish
AD
-1~1 cm
Co
nt
ro
l
H
ig
h
te
m
pe
ra
tu
re
tre
at
m
en
t
2 days
3 days
4 days
C2 C3 C4
H2 H3 H4
Heading
CM
H2M
H4M
Fig. 1 A schematic illustration of the high temperature treatment.
Rice plants were grown in a greenhouse at 28 ° C in the daytime and at
22 ° C at night (28 ° C/22 ° C) as a normal condition (thin arrows). The
high temperature treatment started at auricle distance (AD) = −1 to
+ 1 cm, corresponding to the early microspore stage. During the high
temperature treatment, plants were cultivated in a growth chamber
at 39 ° C/30 ° C (thick arrows). RNA was extracted from the anthers
after high temperature treatment from day 2 to day 4 (H2, H3 and H4)
and from the corresponding untreated anthers (C2, C3 and C4). Some
of the high temperature-treated and untreated plants were grown at
28 ° C/22 ° C until maturity (H2M, H4M and CM) in order to analyze
pollen viability and seed fertility.
0
5
10
15
20
25
30
35
40
75–100 50–75 25–50 0–25
Pollen viability of each spikelet (%)
N
um
be
r o
f s
pi
ke
le
ts
CM
H2M
H4M
A
B
CM H2M H4M
Fig. 2 Viability of the high temperature-treated pollen. Anthers were
harvested just prior to anthesis and subjected to Alexander’s staining.
(A) Pollen viability was expressed as a percentage of the stained pollen.
The viability of pollen from 36 spikelets was scored for each plant.
(B) Typical anthers stained with Alexander’s reagent. CM, untreated;
H2M and H4M, high temperature treated for 2 or 4 d, respectively.
Bars = 0.1 mm.
Table 2 Spikelet fertility of high temperature-treated plants
Experiment High temperature-treated Untreated
1 d 2 d 3 d 4 d
1 – a 5.3 0.6 3.9 71.5
2 – 29.8 8.5 8.3 61.0
3 – 17.8 20.4 9.7 81.2
4 63.9 24.5 – 5.5 85.1
a Data not available.
1913
Rice male sterility due to high temperatures
Plant Cell Physiol. 50(11): 1911–1922 (2009) doi:10.1093/pcp/pcp135 © The Author 2009.
at China Academ
y of Agricultural Sciences on M
arch 20, 2010
http://pcp.oxfordjournals.org
D
ow
nloaded from
the specifi c transcriptional alteration along with anther
development from the general heat shock response in the
vegetative organs. These samples analyzed by DNA microarray
are schematically shown in Fig. 1 . To screen high temperature-
responsive genes, we initially chose 1,439 genes that show a
> 2-fold difference of expression in either one of the three
comparisons, H2/C2, H3/C3 or H4/C4. These selected genes
were subsequently classifi ed into 30 clusters according to
their expression pattern ( Fig. 4 ). In clusters 2 and 15, the
genes were down-regulated by high temperature treatments
specifi cally in the anther after just 2 d. Clusters 10 and 29
contained genes whose expression gradually decreased
during the high temperature treatment. Clusters 4, 6 and 30
contained genes that were highly up-regulated after high
temperature treatment for 4 d. The genes belonging to each
cluster are listed in Supplementary Table S1 and the gene
classifi cation based on the gene ontology (GO) in 30 clusters
is shown in Supplementary Fig. S1.
We focused on the genes down-regulated by high tem-
peratures, because it is assumed that the genes important
for pollen development and fertility were impaired under
high temperature conditions. The gene expression in cluster
2 and cluster 15 (containing 15 and 56 genes, respectively)
was severely impaired after only 2 d but it was not greatly
affected after 3 or 4 d of high temperatures. We noted these
genes showing a drastic decrease after 2 d of high tempera-
tures, because the response to 4 d of high temperatures
could have resulted from various indirect effects and 2 d of
heat was suffi cient to induce sterility. According to the GO
classifi cation, some of the genes in clusters 2 and 15 were
presumably involved in the metabolic process and related
to the catalytic activities (Supplementary Figure 1). We
chose all 15 genes in cluster 2 and three genes from cluster
15 (listed in Table 3 ) for the individual gene expression
analysis.
The relative expression levels of these 18 selected genes
were analyzed by real-time reverse transcription–PCR
(RT–PCR) using RNA samples from two independent heat
stress experiments, one of which was the same as used for
a DNA microarray analysis. Among the selected 18 genes,
we analyzed the relative gene expression levels of 16 genes,
because the PCR primers for two genes, AK102387 and
AK073529, did not work. In 15 out of 16 genes tested,
real-time RT–PCR analysis demonstrated the drastic down-
regulation due to high temperatures that had been observed
in microarray analysis. The relative expression levels of these
15 genes are shown in Fig. 5 . A common feature of these
genes (except for AK062288 and AK058903) was that expres-
sion was seen until day 2 without treatment and was drasti-
cally impaired within 1 d of the high temperature treatments.
These 13 genes were therefore designated as high temperature-
repressed genes.
Tissue specifi city of the genes that were immediately
down-regulated in response to the high temperature
treatment
Among the high temperature-repressed genes we identifi ed
YY1 and YY2 as immature anther-specifi c genes which are
mainly expressed in the rice tapetum ( Hihara et al. 1996 ).
The highest expression of YY1 and YY2 (AK107918 and
AK105510, respectively) in non-treated anthers was observed
on days 0 and 1, which correspond to the microspore stage
( Fig. 5 ). This fi nding was consistent with the previous report
by Hihara et al. (1996) . Because the temporal expression
profi le was shared among 13 genes, including YY1 and YY2 ,
we examined whether these genes were expressed in the same
tissue. The expression of genes shown in Fig. 5 was analyzed
by real-time RT-PCR using various tissues. As expected, high
temperature-repressed genes were highly expressed in the
immature anther but were not detected in the root, leaf,
pistil, callus or mature anther ( Table 4 ). Furthermore,
a detailed expression analysis of these genes revealed that
the highest level of expression was in anthers with a fully
developed tapetum at the microspore stage ( Fig. 6 ). To
investigate where these high temperature-repressed genes
A B C
Fig. 3 Pollen germination on stigma. Aniline blue staining of a self-pollinated stigma of an untreated plant (A), a self-pollinated stigma of a plant
exposed to high temperature for 4 d (B) and a cross-pollinated stigma of a plant exposed to high temperature for 4 d with untreated pollen (C).
Bars = 0.1 mm.
1914
M. Endo et al.
Plant Cell Physiol. 50(11): 1911–1922 (2009) doi: