Annals of Botany 86: 47±51, 2000
doi:10.1006/anbo.2000.1158, available online at http://www.idealibrary.com on

Mechanism of Septum Opening in Anthers of Two-rowed Barley (Hordeum vulgare L.)
T. M AT S U I * {, K . O M A SA { and T. H O R IE }{Experimental Farm, Kyoto University, 12-1 Hatchonawate, Takatsuki, Osaka 569-0096, Japan, {Graduate School of
Agricultural and Life Science, The University of Tokyo, Tokyo 113-8657, Japan and }Graduate School of Agriculture,
Kyoto University, Kyoto 606-8502, Japan
Received: 15 December 1999

Returned for revision: 3 January 2000 Accepted: 15 March 2000

To test the hypothesis that the rapid swelling of pollen grains driven by potassium movement opens the septum inanthers of poaceous plants, we studied (1) the behaviour of pollen grains during unfolding of the locule and (2) thedistribution of potassium in the locule in two-rowed barley. In the ®rst experiment, the unfolding of decapitatedanthers was observed. The pollen grains paved the inner wall of the locule during the unfolding process, suggestingthat the pollen grains bend the locule wall outward when they adhere to the wall. In the second experiment, thedistribution of potassium in transverse sections of locules in dehisced and indehisced anthers was observed. Inindehisced anthers, potassium was detected outside the pollen grains. In contrast, in dehisced anthers, potassium wasdetected inside pollen grains. This suggested potassium ions moved from the inter-pollen space (locular ¯uid) into the# 2000 Annals of Botany Company
pollen grains in the locule at the time of pollen-grain swelling.
Key words: Hordeum vulgare L., locule unfolding, pollen grain swelling, potassium ion, two-rowed barley.

I N T RO D U C T I O NThis article describes how pollen grains swell and the locule
unfolds during the dehiscence of anthers in two-rowedbarley. Historically, anther dehiscence was believed to be asimple desiccatory process of the tissue in general, probably
because of the synchrony between dehiscence and ¯oralopening. However, some scientists recognized the importance of pollen growth for anther dehiscence at thebeginning of the 20th century (cited in Keijzer, 1987a).
Keijzer (1987a) examined the anther dehiscence mechanism
in liliaceous plants microscopically, and clari®ed that thegrowth of pollen mass ruptures the deteriorated tapeta thatclose the two adjacent locules after enzymatic rupture ofseptal cells. Thus, fusion of the locules is complete justbefore anthers dehisce. A similar process was described by
Bonner and Dickinson (1989) in tomato (Lycopersiconesculentum Mill.).
In poaceous plants, Cheng et al. (1979, 1986) related theseptum opening of maize (Zea mays L.) to a tangential
tensile force generated by shrinking of the outer tangential
wall of the epidermis. However, Keijzer et al. (1996) also
showed enzymatic cell-wall lysis in the maize septum andconcluded that this lysis results in the opening of theseptum, and that the increase in pollen grain size in the 48 hprior to anthesis causes the septum to widen.
On the other hand, Matsui et al. (1999a, b) found that thepollen grains of rice (Oryza sativa L.) swell rapidly (within afew minutes) in response to ¯oret opening. They concluded
that the swelling pressure of the pollen forces the locules tounfold, rupturing the enzymatically-weakened septum andjoining the two adjacent cylindrical locules into a single
cylindrical cavity. They considered pressure to be the* For correspondence. Fax 81-726-83-1532.

0305-7364/00/070047+05 $35.00/00

driving force opening both the septum and stomium(Matsui et al., 1999b). Matsui et al. (2000) described asimilar mechanism in cleistogamous two-rowed barley(Hordeum vulgare L.), in which the rapid swelling of pollen
in response to the removal of the lemma opens the septumand stomium. Moreover, they detected potassium in thepollen grains sampled from dehisced antlers and proposedthat this accumulation of potassium ions in the pollen
grains causes the rapid swelling (Matsui et al., 2000). This
theory can explain the synchrony between anther dehiscence and ¯oret opening which occurs in some poaceous
plants, e.g. rice, without considering the desiccation of theanther. However, some important questions still remainunanswered: (1) There is disparity between the rate ofvolume increase in the pollen grains and the theca during
fusion of the two locules. Given the pollen grain diameterof barley measured by Matsui et al. (2000), the volume ofpollen grains in dehisced anthers was 1.7-fold greater thanthat in indehisced anthers, but the theca volume increased
two-fold during fusion of the two locules. How can pollen
swelling force the joining of the locules and swelling of thetheca? (2) What is the source of the potassium found in thepollen grains sampled from the dehisced anthers of barley?
Does the potassium really migrate from outside the pollen
grain into the pollen grain at the time of swelling?
In this paper we answer these questions, and con®rm thetheory that rapid swelling of pollen grains causes unfoldingof the anther locules. First, we observed a decapitatedanther during arti®cially-induced locule unfolding to clarify
the behaviour of the pollen grains. Second, we observed thedistribution of potassium in transverse sections of dehisced
and indehisced anthers to examine the movement ofpotassium in the locule.
# 2000 Annals of Botany Company

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Matsui et al.ÐMechanism of Locule Unfolding in Two-rowed Barley
M AT E R I A L S A N D M E T H O D S

Plant material
Cleistogamous two-rowed barley (Hordeum vulgare L.,`Saikaikawa-24') was grown under ®eld conditions (Experimental Farm of Kyoto University, Osaka, Japan) from
November to May. Heading was from the end of March tomid-April.
Observation of the unfolding process of decapitated antherlocules and the interior of thecae before and after unfolding
Florets with anthers that could dehisce in response to theremoval of the lemma (hereafter referred to as mature
anthers) were sampled prior to pollination. Such ¯orets

were selected by observing the sequential pollination of¯orets on a panicle. In the same panicle, pollination occurs
from the middle towards both apical and basal ends
(Hoshikawa, 1980). This sequence of pollination corresponds to the order of the anther maturity: anthers in¯orets that pollinate earlier have a greater ability to dehiscethan those in ¯orets that pollinate later (Matsui et al.,2000). Anther maturity was determined as follows. A ¯oretin which the anthers had not dehisced was detached fromthe panicle, the lemma of the ¯oret was removed and theanthers that dehisced within 5 min were regarded as mature
anthers. Immediately after sampling, the lemma wasremoved and the anthers were decapitated. The unfolding
process of the locules was observed under a stereomicroscope at 178C and 90 % relative humidity (RH).

F I G . 1. Unfolding process of the decapitated anther. A, Anthers prior to rupture of the septum. B, Anthers at the beginning of septum rupture.
The septum of the lower theca has been ruptured. C, Anther just after septum rupture. The pollen grains adhere to the locule wall in an orderlymanner. D, Anther a few minutes after rupture of the septum. The pollen grains are released from the locule wall.

Matsui et al.ÐMechanism of Locule Unfolding in Two-rowed Barley49

Mature anthers were also sampled to record the moisture
condition of the thecae. In one group, thecae were arti®cially opened immediately after sampling, while in theother group, thecae were opened after the spontaneous
rupture of the septum. In both groups, moisture condition
was recorded immediately after arti®cial opening of thethecae.

were wet and the pollen grains adhered closely to the loculewall (Fig. 2A). In contrast, in anthers whose septa hadruptured spontaneously after removal of the lemma, theinsides of the locules were dry and the pollen grains werereleased from the locule walls (Fig. 2B).

Detection of potassium ions in the antherIn transverse sections of the locule in indehisced anthers,
potassium salt crystals were formed on the surface of thepollen grains and the remnants of the tapetum (Fig. 3A). In
contrast, the crystals were formed inside the pollen grains in
the fused locule in dehisced anthers (Fig. 3B).

Florets were detached from the panicle several days afteremergence. Subsequently, the lemmas of the ¯orets wereremoved and the anthers were decapitated at 208C and
70 % RH. After 5 min, both dehisced (mature) andindehisced ( pre-mature) anthers were stained with
Macallum's modi®ed solution (Toriyama, 1955) at 08C
for 48 h. The materials were thoroughly washed with cold
water and ®xed in 70 % ethanol before being dehydrated ina methanol±butanol series, and embedded in paran. Tenmicron-thick transverse sections were prepared andobserved, without staining, under a microscope.
R E S U LT S
Behaviour of pollen grains and moisture condition in antherlocules during the unfolding process
After decapitation of the anthers, the septa ruptured
normally. The pollen grains remained stuck to the tapetum
remnants in an orderly manner during the rupture(Fig. 1A±C). After the emergence of the stomium, thepollen grains were released from the tapetum and dispersed
into the thecae ( joined locules) (Fig. 1C, D). Figure 2 shows
the interior of a theca opened arti®cially. Before the septum
ruptured, the insides of the locules of the mature antherDistribution of potassium in transverse sections of the anther

DISCUSSION
Previously, Matsui et al. (1999a, b, 2000) reported that,in rice and two-rowed barley, pollen grains swell rapidly
causing the locules to unfold at the start of antherdehiscence, and that this unfolding causes the two adjacentcylindrical locules to join into a single pollen cavity. At thetime the septum widens, the volume of the theca approximately doubles (Matsui et al., 1999b, 2000). In contrast,
the dierence in pollen grain diameter between indehiscedand dehisced anthers was less than 20 % (less than 75 % involume) (Matsui et al., 1999a, 2000). Given this dierence
between the rate of volume increase of the theca andthe pollen grains, the reports that pollen swelling is thedriving force behind unfolding of the locules remainedinconclusive.
The present study has shown how the pollen grains
unfold the locules. At the time when the two locules fuse,
the pollen grains still adhere to the tapetum in an orderly
manner. This suggests that the pollen grains paving theF I G . 2. Interior of the thecae before (A) and after (B) rupture of the septum.

50Matsui et al.ÐMechanism of Locule Unfolding in Two-rowed BarleyAB

F I G . 3. Distribution of potassium in transverse sections of indehisced (A) and dehisced (B) thecae. Arrowheads indicate the potassium salt
crystals formed outside (A) and inside (B) the pollen grains. Bars  50 mm.

inner wall of the locule bend the locule wall outward by thecombination of the ®xed circumference of the locule andtangential swelling of pollen grains attached to the loculewall. In this way, a 14 % increase in the diameter of pollen
grains can unite the two locules into a cylindrical cavity
when the pollen grain diameter is 25 % of the locule
diameter. Moreover, Keijzer et al. (1996) reported that thepollen grains of maize are tightly packed against theremnants of the tapetum and that the mutual pressure
forces the pollen grains to lose their spherical shape 2 dbefore anthesis. A similar change in the shape of pollen
grains in the locule as a result of mutual pressure has also
been reported in rice (Takeoka et al., 1993). The change inshape would also help unfold the locule with less increase indiameter.
Matsui et al. (2000) detected potassium in pollen grainsfrom dehisced anthers, but not in those from indehisced
anthers, suggesting the participation of potassium ions inthe swelling of pollen grains. The present observation of
the distribution of potassium salt crystals stained with

Matsui et al.ÐMechanism of Locule Unfolding in Two-rowed Barley
Macallum's modi®ed solution revealed that almost all thepotassium in indehisced anthers was found outside thepollen grains and that much of the potassium in dehisced
anthers was present inside the pollen grains. This strongly
supports the theory that potassium ions move from theinter-pollen space into the pollen grains, resulting in pollen
swelling.
Moreover, before the rupture of the septum, the inside ofthe locule is wet and the pollen grains adhere to the locule
wall in an orderly manner. However, after rupture of theseptum, the inside of the locule is dry and the pollen grains
are detached from the walls. This suggests that water moves
from the inter-pollen space into the pollen grains at the timeof locule unfolding; this is also in agreement with the theory
that the movement of potassium ions from the inter-pollen
space into the pollen grains causes pollen swelling. Since thesurface of the pollen grains and inner surface of the locule
wall are hydrophobic (Heslop-Harrison, 1968; Keijzer,1987b) and poaceous pollen grains are stuck to the tapetumwith tiny amounts of pollenkitt (Keijzer et al., 1996),
adhesion between them would be lost rapidly upon
dehydration of the locule and a decrease in contact area
between the tapetum and the grain as a result of a change inpollen grain shape. At the same time, expansion of thelocule after the rupture of the septum would release thetightly-packed pollen grains, which would then detach fromthe tapetum.
There is solid evidence that the plasma membrane ofpollen grains has potassium ion channels (Obermeyer andKolb, 1993) and a proton pump (Weisenseel and Jae, 1976;
Obermeyer et al., 1992). Moreover, Obermeyer and Blatt(1995) showed that K  in¯ux and H  eux are present inquiescent, ungerminated pollen grains in Lilium longi¯orum.
Although such a K  in¯ux is regarded as one of theprocesses of pollen grain germination (Weisenseel et al.,
1975; Obermeyer and Blatt, 1995), it may also regulate theosmotic potential for the swelling of pollen grains inpoaceous plants.

AC K N OW L E D GE M E N T S
We thank H. Kagata and R. Wada for their technicalassistance in the ®eld.

51

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