Annals of Botany 89: 683±687, 2002
doi:10.1093/aob/mcf112, available online at www.aob.oupjournals.org

Rice (Oryza sativa L.) Cultivars Tolerant to High Temperature at Flowering:
Anther Characteristics
T S U T O M U MA TS U I 1 , * and K EN JI O MA S A 2
1Experimental Farm, Kyoto University, 12±1 Hatchonawate, Takatsuki, Osaka 569±0096, Japan and 2Graduate Schoolof Agricultural and Life Sciences, The University of Tokyo, Tokyo 113±8657, Japan
Received: 12 October 2001 Returned for revision: 12 December 2001 Accepted: 20 February 2002

We examined the relationship between morphological characteristics of anthers and fertility in japonica rice cultivars subjected to high temperature (37´5/26 °C day/night) at ¯owering. Percentage fertility was negatively correlated with the number of cell layers that separated the anther locule from the lacuna that formed between the
septum and the stomium. The cell layers consisted of the remaining septum and degraded tapetum, and serve tokeep the adjacent two locules closed. Anther dehiscence therefore requires the rupture of the cell layers. We
conclude that the tight closure of the locules by the cell layers delayed locule opening, and decreased fertility athigh temperatures.
ã 2002 Annals of Botany Company
Key words: Anther dehiscence, anther size, high temperature tolerance, lacuna between septum and stomium, Oryza
sativa L., rice, septum, tapetum.

INTRODUCTION
Rice is grown mainly in tropical and subtropical zones, anda high temperature at ¯owering can induce ¯oret sterility
and can limit grain yield (Osada et al., 1973; Satake andYoshida, 1978; Matsushima et al., 1982). Since the 1980s,
an increase in the concentration of greenhouse gases, suchas carbon dioxide, in the atmosphere is thought to have been
responsible for increasing the air temperature (Hansen et al.,1984). Amongst other things, global warming is expected toresult in the occurrence of high temperature-induced ¯oretsterility in rice.
Crop scientists have attempted to assess the effects ofincreasing temperature and high carbon dioxide in theatmosphere on the growth and yield of rice using simulation
models (Boote et al., 1994; Horie et al., 1996, 1997;
Matthews et al., 1997). Horie et al. (1996) suggested that the
anticipated high temperature would induce ¯oret sterility
and increase the instability of the rice yield even intemperate regions. These authors also showed that adoptionof high temperature-tolerant cultivars is one of the mosteffective countermeasures to maintain high productivity andstability of rice under the anticipated climate in temperateregions (Horie et al., 1996).
The main cause of ¯oret sterility induced by hightemperatures at ¯owering is anther indehiscence (Satakeand Yoshida, 1978; Mackill et al., 1982; Matsui et al.,1997a, b, 2001). High temperatures at ¯owering inhibit
swelling of the pollen grains (Matsui et al., 2000b), which isthe driving force behind anther dehiscence in rice (Matsui
et al., 1999a, b). Anthers of high temperature-tolerant
cultivars dehisce more easily than those of susceptible
cultivars and contribute to pollination under high-tempera* For correspondence. Fax +81 726 83 1532, e-mail matsuit@kais.kyotou.ac.jp

ture conditions (Satake and Yoshida, 1978; Mackill et al.,
1982; Matsui et al., 2000b, 2001). However, the factors
determining the degree of anther dehiscence are unknown.
Here, we examine the relationship between morphological characteristics of the anther and ¯oret tolerance tohigh temperature at ¯owering in japonica rice cultivars in
relation to sterility. We also investigate the morphologicalcharacteristics of anthers in tolerant cultivars and discuss
why their anthers dehisce so readily.
MATERIALS AND METHODS
Floret sterility due to high temperatures during the ¯owering
periodData concerning ¯oret tolerance to high temperatures at¯owering have been reported previously (Matsui et al.,2001). Nine japonica rice cultivars, including two tolerant
to, and two susceptible to, high temperature were used
(Table 1). Seeds were sown from March to June 1995 so that
panicles would emerge late in August. Seedlings at the5´0±5´5 leaf stage were transplanted in a circular pattern into
4-l pots, with 20 seedlings per pot, and were grown outdoors
under submerged soil conditions (Experimental Farm of
Kyoto University, Osaka, Japan). Each pot was provided
with 0´4 g N, 0´4 g P2O5 and 0´4 g K2O as a top dressing
about 45 d before heading (about 15 d before panicle
initiation). Tillers were removed during the vegetative stage
as they appeared.
Sun-lit phytotrons were used for high-temperature treatments. Plants at the middle heading stage were exposed to37´5 °C for 6 h (1000±1600 h) for six consecutive days.
The night-time temperature (1800±0800 h) was 26´0 °C.
Relative humidity was 60 % in the daytime (1000±1600 h)
and 80 % at night. Air temperature and humidity wereã 2002 Annals of Botany Company

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Matsui and Omasa Ð Anthers of High±temperature-tolerant RiceTA B L E 1. Tolerance of rice cultivars to high temperature at ¯owering based on the studies of Matsui et al. (2001)

Cultivar
1
2
3
4
5
6
7
8
9

Nipponbare
Akitakomachi
Aichinokaori
Yumehikari
KinmazeAkihikari
Aoinokaze
Minamihikari
Hinohikari

Percentage fertility under high temperature

Control
35´0/26´0 °C*

37´5/26´0 °C

40´0/26´0 °C

94´6
93´4
96´1
91´5
95´4
92´6
91´4
92´7
93´4

85´3
80´0
78´2
74´3
65´2
65´9
58´4
45´7
44´1

35´0
50´3
19´8
22´9
22´2
13´2
19´9
19´2
13´7

Tolerance to high temperature
Tolerant
Tolerant
Moderate
Moderate
Moderate
Moderate
Moderate
Susceptible
Susceptible* Day/night temperature

at 1800 h, 6 d later. Three pots of each cultivar were used foreach treatment.
The fertility of all ¯orets on panicles on which ¯orets
started to open on the ®rst day of treatment and ®nishedopening before the last day of treatment was examined atmaturity. More than four panicles (over 150 ¯orets) were
examined per pot. Means of the three pots were used for
regression analysis.
Morphological characteristics of anthers

F I G . 1. Measurement of anther characteristics. A, Exterior of rice anther.
Anther length (L) was taken as the length of the long locule. Antherwidth was calculated as the mean width taken in two directions[(W1+W2)/2]: one parallel to the stomium (W1) and the other (W2) atright angles. B, Schematic transverse section of anther. C, Magni®cationof B around the cell layers separating the locule from the lacuna formed
between the septum and the stomium. The number of cell layers
separating the lacuna from the locule between the septum and thestomium was counted at the point where the number of cell layers was
smallest (the probable breaking point when the septum opens; between
arrowheads). La, Lacuna formed between the septum and the stomium;
En, endothecium; Ep, epidermis; T, tapetum; Lo, locule; St, stomium.

The same nine cultivars were grown in 2000 under theconditions described above. On the day before ¯orets
opened, the third ¯orets from the top of the ®rst branches
were ®xed in FAA after removing the lemmas. Six ¯orets
per cultivar were ®xed.
Three ¯orets (18 anthers) per cultivar were used formeasurement of anther sizes. The length and width ofanthers were measured with a micrometer under a stereomicroscope. Width was taken as the mean of measurements
made parallel and at right angles to the stomium (Fig. 1A).
The other three ¯orets were dehydrated in a methanol±
butanol series before being in®ltrated and embedded inparaf®n. Transverse sections, 8 mm thick, were cut from the¯orets of 18 anthers and stained with toluidine blue-O. The
number of cell layers separating the anther locule from thelacuna that formed between the septum and the stomium
was counted at the point where the number of layers was
smallest (probably the point that will break when the septum
opens; Fig. 1C, cf. Fig. 4). The number of cell layers wasrecorded every 80 mm for all four locules in ®ve sections,from 480 mm to 800 mm from the apex of the anther. Meananther length, width, length 3 width and the number of cell
layers were calculated for each ¯oret. Means of the three¯orets of each cultivar were then used for regression
analysis. Predictor valuables in the multiple regression
analysis were determined by a stepwise regression procedure.
R E S U LT S

altered gradually between 0800 and 1000 h, and between
1600 and 1800 h. The treatments started at 1800 h and ended

Percentage fertility under a day/night temperature regime of
37´5/26 °C was signi®cantly correlated with the number ofMatsui and Omasa Ð Anthers of High±temperature-tolerant RiceF I G . 2. Relationship between the number of cell layers separating thelocule from the lacuna formed between the septum and the stomium andpercentage fertility. Numbers represent cultivars (see Table 1). Verticalbars indicate 6 s.e. of three pots. Horizontal bars indicate 6 s.e. of three¯orets.

cell layers separating the locule from the lacuna that formed
between the septum and the stomium (Fig. 2). There was nocorrelation between anther length, width or length 3 widthand percentage fertility, but anther length 3 width wassigni®cantly correlated with the difference between measured percentage fertility and percentage fertility estimatedfrom the number of cell layers separating the locule from thelacuna (r = 0´750, P < 0.05). Under high temperature, 85 %of the variance in the percentage fertility among the nine
cultivars was explained by multiple regression with thenumber of cell layers and the product of anther length andwidth (Fig. 3).
When the locule is separated from the lacuna between the
septum and the stomium by three or more layers, then theselayers consist of parenchyma cells and endothecium cells inthe septum, and tapetal cells (Fig. 4). If it is separated by one
or two layers only, then these consist of endothecium cells
and/or degrading tapetal cells.
D IS C U S SIO N
The difference in the sterility-inducing daytime temperature
between the most tolerant cultivar, Akitakomachi, and themost susceptible cultivar, Hinohikari, used in the present
study has been reported to be about 3 °C (Matsui et al.,2001). Using a crop simulation model, Horie et al. (1996)
showed that even a difference of 1´6 °C in the sterilityinducing temperature could have a serious effect on rice
yield in a temperate region under anticipated global
warming. The present results suggest that the 3 °C difference in tolerance was mainly related to the number of cell
layers separating the locule from the lacuna that formed

685

F I G . 3. Comparison between observed and estimated fertility in rice
under a daytime temperature of 37´5 °C. Numbers represent cultivars(see Table 1). The estimation was made by: y = ± 57´6x1** + 160´5x2* +12´03, where x1 and x2 represent the number of cell layers separating thelocule from the lacuna between the septum and the stomium, and the
product of length and width of anther, respectively. * and ** indicate
signi®cance at 5 and 1 % levels, respectively.

between the septum and the stomium, and secondarily
related to anther size.
It has been reported that anther length is positively
correlated with tolerance to ¯oret sterility induced by a lowtemperature at the booting stage (e.g. Hashimoto, 1961;
Suzuki, 1981, 1982; Tanno et al., 1999), which is the mostserious cool-weather damage in rice. Using cultivars with alarge variation in anther width, Tanno et al. (1999) showedthat the product of anther length and width was correlated
more closely with tolerance to low temperature at thebooting stage than was length alone. The number of pollen
grains in the anther is positively correlated with antherlength in rice cultivars (Suzuki, 1981), and the inner wall of
the anther locule in Poaceae is lined with pollen grains
before the septum opens (Keijzer et al., 1996; Matsui et al.,2000a). Thus, anther width would also be positively
correlated with the number of pollen grains in the anther.
Since the direct cause of ¯oret sterility induced by low
temperature at the booting stage is the decreased number ofgerminated pollen grains caused by anther indehiscence(Shimazaki et al., 1964), it has been assumed that anthersize would be correlated with tolerance to low temperature
because the reduction in the number of dehisced anthers
would be compensated by a large number of pollen grains
per anther (Suzuki, 1981; Tanno et al., 1999). Anther size
might be correlated with tolerance to high temperatureinduced sterility in a similar way, because the direct cause of
spikelet sterility induced by high temperature at ¯owering is
also a decrease in the number of germinated pollen grainscaused by poor anther dehiscence (Satake and Yoshida,1978; Mackill et al., 1982; Matsui et al., 1997a, b, 2001).

686

Matsui and Omasa Ð Anthers of High±temperature-tolerant RiceF I G . 4. Transverse section of the septum in which one or two (A, B), or three (C) cell layers separate the locule from the lacuna between the septum
and the stomium. A, Degrading tapetal cells separate the lacuna from the locule (`Nipponbare', high-temperature tolerant); B, degrading endotheciumand degraded tapetal cells separate the lacuna from the locule (`Akitakomachi', high-temperature tolerant); C, degrading tapetal cells, endocecium
cells and parenchyma cells separate the lacuna from the locule (`Minamihikari', high-temperature susceptible). L, Anther locule; P, pollen grain;arrowhead, stomium; *, lacuna formed between the septum and the stomium. Bars = 25 mm.

The number of cell layers separating the locule from thelacuna between the septum and the stomium was moreclosely related to fertility under the high-temperature
treatment than was anther size. Keijzer et al. (1996)
reported that the stomium dissociates from the septum 2 d
before anthesis in maize, and this dissociation ruptures thetapetum membranes. Thus, adjacent maize locules become
one cavity before anthesis (Keijzer et al., 1996). In rice, thestomium dissociates from the septum before anthesis, but
the locules are kept closed until anthesis by parenchyma andendothecium cells in the septum, often supported by thedegrading tapetum (Matsui et al., 1999b). The lacuna that issurrounded by the septum and stomium cells is formed inthis way. Since the locule remains closed until anthesis,
opening of the septum (i.e. locule opening) at ¯owering is anindispensable process for anther dehiscence (Matsui et al.,1999b). The number of cell layers separating the locule fromthe lacuna between the septum and the stomium is thenumber of cell layers responsible for keeping the locules
closed. These cell layers might be responsible for the hightemperature susceptibility of rice through the strength of the
septum and thus the degree of anther dehiscence.
The fundamental cause of poor anther dehiscenceinduced by high temperatures is disturbance of pollen
swelling (Matsui et al., 2000b), which is the driving force
for anther dehiscence (Matsui et al., 1999a, b). Although it
is not known whether the disturbance is caused by water
loss, water loss in the locule would impede pollen swelling
because swelling occurs as a result of water migration intopollen grains. Indeed, low atmospheric humidity disturbs
anther dehiscence in rice (Matsui et al., 1999b). Thus,
delayed anther dehiscence at ¯oret opening might inhibit theswelling of pollen grains through loss of water in the locule
under high temperatures, and might have a feedback effectdelaying dehiscence further. Therefore, the strength of the

septum might be more important for high-temperature
tolerance than the size of the anther which is the dominantfactor in tolerance to sterility induced by low temperature at
the booting stage. However, it must be noted that the rangeof anther sizes used in the present experiment (1´82±
2´24 mm in length and 0´405±0´455 mm in width) was not aswide as that used in studying tolerance to low temperature at
the booting stage, when tolerance was attributed to anthersize (e.g. anther lengths from 1´59 to 2´54 mm, and widths
from 0´395 to 0´515 mm; Tanno et al., 1999). Thus, thenarrow range of anther sizes may have overemphasized theimportance of the number of cell layers in high-temperature
tolerance.
A C K N O W L ED G E ME N T
We thank H. Kagata for technical assistance.
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