Promotion of leaf sheath growth by gibberellic acid in a dwarf mutant of rice

Author(s): Chiaki Matsukura, Shin-ichi Itoh, Keisuke Nemoto, Eiichi Tanimoto and Junji
Yamaguchi
Source: Planta, Vol. 205, No. 2 (June 1998), pp. 145-152
Published by: Springer
Stable URL: https://www.jstor.org/stable/23385302
Accessed: 24-02-2020 21:02 UTC

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Planta (1998) 205: 145-152
Planta
© Springer-Verlag 1998

Promotion of leaf sheath growth by gibberellic acid

in a dwarf mutant of rice

Chiaki Matsukura1, Shin-ichi Itoh1, Keisuke Nemoto2, Eiichi Tanimoto3, Junji Yamaguchi1
'Bioscience Center and School of Agricultural Sciences, Nagoya University, Chikusa-ku, Nagoya 464-01, Japan
2Faculty of Agriculture, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113, Japan
'institute of Natural Sciences, Nagoya City University, Mizuho-ku, Nagoya 467, Japan

Received: 28 August 1997 / Accepted: 16 October 1997

Abstract. The mechanism of gibberellin (GA)-induced endogenous level of IAA (Reid and Davis 1992; Law
leaf sheath growth was examined using a dwarf mutant and Hamilton 1989). However, recent studies have
of rice (Oryza sativa L. cv. Tan-ginbozu) treated in shown that GAs strikingly promote cell division in the
advance with an inhibitor of GA biosynthesis. Gibber intercalary meristem of submerged deepwater rice, and
ellic acid (GA3) enhanced the growth of the second leaf that this is due to the activation of histone HI kinase
sheath, but auxins did not. Measurement of the mitotic and cyclin genes at the initial stage (Sauter et al. 1995).
index and cell size revealed that cell elongation rather It is also known that GA controls the direction of cell
than cell division is promoted by GA3. Gibberellic acid growth by controlling the orientation of cellulose micro
increased the extensibility of cell walls in the elongation fibrils, which seems to be determined by the underlying
zone of the leaf sheath. It also increased the total arrangement of cortical microtubules, and that the
amount of osmotic solutes including sugars in the orientation
leaf of the microtubules is influenced by auxin
and of
sheath, but did not increase the osmotic concentration GA (Shibaoka 1994). Both GA- and auxin-induced
the cell sap, due to an accompanying increasechanges in cell in the mechanical properties of cell walls of
volume by water absorption. In the later stage of stems,
GA3coleoptiles and roots have also been reported
(Olson et al. 1965; Cleland 1967; Masuda 1968; Yama
induced growth, starch granules completely disappeared
moto et al. 1970; Kutschera and Kende 1988; Tanimoto
from leaf sheath cells, whereas dense granules remained
in control plants. These findings indicate that
1994),GA
suggesting that an increase in cell wall extensibility
results in loosening and relaxation of cell walls, thereby
enhances cell elongation by increasing wall extensibility,
osmotic concentration being kept unchanged by allowing
starch turgor-driven elongation to occur.
degradation. In several plant species, including maize (Phinney
1984), pea (Ingram et al. 1984), rice (Suge and Mu
Key words: Gibberellin - Leaf sheath elongationrakami - Cell 1968), and Arabidopsis (Koornneef and van der
extension - Cell wall extensibility - Oryza (dwarf Veen 1980), GA-deficient dwarf mutants have been
mutant) - Starch degradation isolated and used to study GA-induced physiological
processes, since these mutants can be restored to the
normal phenotype by the application of exogenous GAs.
In the GA-deficient dwarf mutant of barley, Ml 17,
Introduction
however, gibberellic acid (GA3) increases the length and
the number of cells in the first leaf only slightly (Zwar
and Chandler 1995).
Gibberellins (GAs) are known to promote cell elonga
Physiological and genetic control of elongation has
tion, and to induce hydrolytic enzymes in the aleurone
not yet been fully elucidated and further analyses are
layer of cereal seeds. Auxin also promotes cell elonga
needed. In the present study, we investigated the effects
tion, and GA has been suggested to increase the
of GA3 on cell elongation, cell division, osmotic values
and cell wall extensibility in the second leaf sheath of a
GA-deficient dwarf mutant of rice, Tan-ginbozu.
Abbreviations: ABA = cw-abscisic acid; 2,4-D = 2,4-dichloro
phenoxyacetic acid; GAn = gibberellin An; GA3 = gibberellic
acid; PAS = Periodic acid-Schiff reaction; PCIB = p-chlorophe
noxy-wobutyric acid; TIBA = 2,4,5-triiodobenzoic acid Materials and methods
Correspondence to: J. Yamaguchi;
E-mail: b42295a@nucc.cc.nagoya-u.ac.jp; Plant materials. Seeds of rice (Oryza sativa L. cv. Tan-ginbozu), a
Fax: 81 (52) 789 5226; Tel: 81 (52) 789 5219 dwarf mutant derived from the cultivar Ginbozu, were a gift from

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 146 C. Matsukura et al.: Gibberellin-mediated leaf sheath growth in rice

Dr. T. Tashiro (Nagoya University Farm) in 1995 and Dr. M.

Koshioka (National Research Institute of Vegetables,Load=20
Ornamental
g/mm2 0 g/mm2
Plants and Tea). \< >f< >

Measurement of shoot elongation. The effect

C of GA3 on shoot
elongation was quantitatively determined by a omodification of the Elastic
(J) extension
"microdrop method" of Murakami (1968). Twelve
c seeds of Tan Total
ginbozu were surface-sterilized for 30 min
<D
with a 3% NaCIO -*—1 Extension
X
solution, washed three times with sterile distilled
LU water, soaked in
6.9 nM uniconazole for 24 h and then in sterile distilled water for
an additional 24 h after washing out the uniconazole. These seeds Plastic
extension
were placed on a 1 % agar plate and grown under fluorescent lamps
at 30 °C. Gibberellic acid and/or other plant hormones,
0 10 were 15
Time when
applied to the coleoptile of each seedling at the first-leaf stage, (min)
the tip of the blade of the second leaf had just emerged. The length
of the second leaf sheath was measured at
Fig.1-4 d after
1. Scheme to measure GA3
cell wall extensibility. Details are described
application. in the Materials and methods

Histochemical analysis. Rice leaf sheaths were fixed with 4%

paraformaldehyde and 0.25% glutaraldehyde in 50 mM sodium Measurement of osmotic concentration. Osmotic concentrations of
phosphate buffer (pH 7.4), dehydrated through a tert-butyl alcohol the leaf sheaths were measured by a vapor-pressure method
series, embedded in Paraplast Plus tissue-embedding medium (Miyamoto and Kamisaka 1988). Groups of 30 leaf sheaths were
(Sherwood Medical, St. Louis, Mo., USA), sectioned longitudinally excised from the seedlings after treatment with or without GA3j as
at 10-12 um with a rotary microtome, and mounted on silane described in histochemical analysis, put in air-tight vials and frozen
coated slides. The sections were stained by Feulgen's reaction with
at -20 °C. Frozen segments were transferred to a funnel with a
a counterstain of Fast Green to visualize nuclei and cell wall as
nylon mesh (106 um pore) fixed to the bottom. The funnel was
shown in Fig. 4, or by the Periodic acid-Schiff (PAS) reaction centrifuged in a vial at 4 °C for 10 min at 1000 g. Aliquots (10 (il)
(Jensen 1962) to visualize starch granules and cell wall as shown
ofinthe filtrate were subjected to analysis in a vapor-pressure
Fig. 9. Cell size was measured with an ocular micrometer. osmometer (model 5520; Wescor, Logan, Utah, USA). The
The percentage of dividing cells was determined for everymeasurement
0.5 was repeated three times for each time and means
mm section from the base to tip of the second leaf sheath in weretwo calculated. Free-space liquid was collected directly from fresh
parenchyma cell files, which were the second and third cell files
leaf sheaths by the same centrifugation procedure. The volume of
inwards from the adaxial epidermis. The cell length was calculated
free-space liquid in the seedling was less than 5% of the total liquid
by dividing the length of each 0.25-mm section from the base toobtained
tip from the frozen and thawed leaf sheaths. Therefore, the
by the number of cells in it; the cell files counted were the same as
osmotic concentration of the total liquid obtained from the frozen
those described above. At least five seedlings were measured and for thawed leaf sheath was regarded as the osmotic concentration
each sample collected at the designated time. Three-dimensional
in the rice leaf sheath.
histograms shown in Fig. 5A and B were constructed using graphic
software (DeltaGraph Pro ver. 3.5; DeltaPoint Inc. Monterey,
Sugar analysis. Samples (0.1-0.3 g fresh weight) were ground in
Calif., USA). 5.5% perchloric acid, extracted as described by Tobias et al. (1992)
and assayed by coupled enzymatic assay methods measuring the
Measurement of cell wall extensibility. Gibberellic acid (300 pmol/ increase in A340 as described by Guglielminetti et al. (1995).
plant) was applied to the seedlings and the shoots were excised from
the seedlings 12, 24 and 34 h after GA3 application. The shoots
from the control plants were also excised at each time. The excised Results
shoots were immediately immersed in methanol at 65 °C for 10 min
and transferred to fresh methanol at 4 °C. Methanol-killed shoots
were re-hydrated with 10 mM Mes buffer (pH 6.0) for 15 min at Effect of plant hormones on growth of the second leaf
sheath
0 °C. After re-hydration, the coleoptiles were removed, and the of the dwarf mutant. Application of exogenous
diameter of the second leaf sheath was measured under a GA3 to the seedlings of Tan-ginbozu, a GA-deficient
microscope at a position 3 mm from the basal end. The dwarf
basal part
mutant of rice, restored the normal phenotype as
of the second leaf sheath was subjected to creep-extension analysis
previously reported (Murakami 1968; Mitsunaga and
using a Rheoner RE-33005 creep tester (Yamaden Co., Tokyo,
Yamaguchi 1993). However, to establish the most
Japan), which recorded the extension of the leaf sheath under a
effective
constant load at 0.5-s intervals. The load was regulated by a conditions for promotion of leaf sheath growth
computer system which controlled a stepping motor and by GA,
a load we
cell. applied various amounts of GA3 and
Leaf sheaths were secured between two clamps, one at examined
a positiontheir effects on growth (Fig. 2A). Gibberellic
2 mm from the basal end and the other at 5 or 7 mm from theapplied
acid base, at as low as 10~13 mol/plant promoted
leaving 3 mm (12- and 24-h-treated seedlings) or 5 mm (34-h of the second leaf sheath, and the effect was
elongation
treated seedlings) in length for extension. The amount of load
saturated at l(T10 mol/plant (Fig. 2B). The elongation
(20 g/mm2) applied to each leaf sheath was determined by the
cross-sectional area calculated from the leaf sheath diameter. The started after a 1-d lag period and ceased 2 d after
maximum speed of extension was 0.5 mm/s. The extension application
of of GA3. The final length of the second leaf
the leaf sheath was recorded for 10 min under the constant load sheath after GA3 treatment was five- to sevenfold longer
(20 g/mm2) and the final extension length (|im) was designated as that without GA3 treatment. We also examined the
than
total extension (Fig. 1). After 10 min of extension, the load was
effects of various types of GA on sheath growth. Active
immediately reduced to 0 g and the shrinkage of the leaf sheath was
GAs such as GA|, GA3, GA4 and GA7 were effective,
recorded for 5 min. The final length after a 5-min shrinkage period
was recorded. The residual extension after the shrinkage was but an inactive type of GA, GA13, showed only a minor
effect (data not shown), confirming the results reported
designated as plastic extension and the difference between total
previously (Reeve and Crozier 1974).
extension and plastic extension was designated as elastic extension.

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C. Matsukura et al.: Gibberellin-mediated leaf sheath growth in rice 147

On agar 40 T
Uniconazole i GA3 application
(6.9HM) sw T T
Day -2 -1 0 1 2 3 4 5 6
I 1 1 1 1 1 I I I
0 30'
▲ 1 A ▲ 4
0 12 3 4
Days after GA3 application
20'

10"

Second leaf
O)
c
\r ~| Leaf blade 0

control GA3 IAA 2,4-D kinetin ABA

Leaf sheath
B
40

C0 30
0

03
® 20
Applied GA3 (mol/ plant) "O
c
o
o

Fig. 2. A Experimental procedure. The length of the second leaf

w 10
0

sheath of dwarf rice was measured 1, 2, 3 and 4 d after application of

GA3 at various concentrations. For details see text. B Dose response
D)
of GA3 on elongation of the second leaf sheath. Values are
0 0
means ± SD for 12 tested plants GAo GAo GAq GAo
Control GAo

The effects of IAA, 2,4-dichlorophenoxyacetic acidFig. 3A,B. Effects of various plant hormones on leaf sheath
(2,4-D), kinetin and ds-abscisic acid (ABA) were also elongation in dwarf rice. The length of the second leaf sheath w
examined. None of them promoted leaf sheath growth at measured 2 d after the application of plant hormones. The values ar
300 pmol/plant (Fig. 3A). However, ABA (100 or means ± SD for 14 tested plants. A 300 pmol/plant of GA3, IAA
300 pmol) given together with G A (100 pmol) clearly 2,4-D, kinetin or ABA. B 100 pmol/plant GA3 plus IAA, or
100 pmol/plant GA3 plus ABA. IAA1, ABA1: 100 pmol/plant
inhibited GA-induced promotion of leaf sheath growth IAA2, ABA2: 300 pmol/plant
(Fig. 3B, columns GA3 + ABA1,2). Such an antagonistic
effect of ABA on the GA-induced process has been
reported for the GA-induced activation of oc-amylase in longer than the cells in the basal region (Figs. 4, 5), an
cereal seeds (Jones and Jacobsen 1991). The application eight- to tenfold longer than the cells in the leaf sheat
of IAA (Fig. 3B, columns GA3 + IAA1,2) and a-naph without GA3 application (Figs. 4, 5). We also calculated
thalene acetic acid (NAA) (data not shown) to the GA the percentages of cells in mitosis (late prophase t
treated sheath had only a slight inhibitory effect. telophase) for each 0.5-mm-long section of the sheath
Inhibitors of polar auxin transport, 2,4,5-triiodobenzoic (Fig. 5B). Meristematic cells were observed in the basa
acid (TIBA) and /?-chlorophenoxy-wobutyric acid region of the second leaf sheath in both GA3-treated an
(PCIB), and inhibitors of ethylene biosynthesis, (amino untreated seedlings, and the mitotic index in this regio
oxy)acetic acid (AOA) and L-a-(2-amino-ethoxyvinyl) was 3-4% during the first 24 h after GA3 application
glycine (AVG) also had no effects (data not shown). (Fig. 5B,C).
The cells in the meristematic area were nearly the
Anatomical analyses of G A ^-promoted growth of leaf same size at all developmental stages (at 0-1 mm from
sheaths. To investigate the effects of GA on elongation the base in Fig. 5C). At the time of GA3 application, t
and division of cells in dwarf rice seedlings, we whole leaf sheath tissue had meristematic activity
(mitotic index 3-4%), but after GA3 application, the
anatomically examined the second leaf sheath of 4-d
old seedlings for 0^8 h after application of GA3 non-meristematic area increased gradually and the
(Fig. 4). The average length of parenchyma cells in each meristematic activity had disappeared almost completely
0.25-mm-long section was determined along the whole by 48 h after GA3 application (Fig. 5C). Although GA3
second leaf sheath (Fig. 5A). During the first 6 h after application slightly increased the mitotic index (Fig. 5B),
the application of GA3, the parenchyma cells were about it increased the total number of cells in the longitudinal
7.5 |mi in length. Thereafter, the cells rapidly elongated parenchyma cell files of the second leaf sheath by, at
until 48 h (Fig. 5A). The final cell length, except in the most, 1.6-fold (Table 1). We speculate that leaf sheath
basal region, was about 80 |im, which is four- to sixfold growth promoted by GA3 was mainly caused by

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148 C. Matsukura et al.: Gibberellin-mediated leaf sheath growth in rice

+GA3
Region -GA3

MM
■

Basal

Fig. 4. Histochemical an
of leaf sheaths of GA3-
dwarf rice. Longitudin
Oh 24 h 48 h
tions were taken throug
leaf sheath parenchym
plants that were either
or not treated with 300
plant of GA3. Basal, par
view of the region 0.25
from the base of the second leaf
Apical sheath. Apical, part of the view
of the region 2.5 mm below the
top of the second leaf sheath.
Sections were stained by
Feulgen's reaction with a
counterstain of Fast Green to
visualized nuclei and cell walls.
Oh 24 h 48 h 12 h 24 h 48 h Bar = 0.02 mm

disappeared
extension of those cells just above the meristematic areawithin 48 h after GA application although
dense
that had already lost meristematic activity but werestarch
stillgranules remained in the non-treated
immature. sheath. (Fig. 9). These results indicate that GA increases
the total amounts of osmotic solutes including sugars
Effect of GA on cell wall extensibility. Figure 6 shows the(especially glucose and fructose) in the leaf sheaths,
time-course change in cell wall extensibility of the secondprobably due to enhanced starch degradation.
leaf sheath after GA3 treatment. Total extensibility,
elastic extensibility and plastic extensibility of the leaf
sheath cell wall were increased by GA3 treatment Discussion
(Fig. 6A-C). Figure 6D summarizes the time-course of
the effect of GA3 on the three parameters of cell wallGibberellin is a major effective hormone for growth of rice
extensibility. The effect of GA3 was apparent 12 h afterleaf sheaths. The GA-deficient type of dwarf mutant of
GA3 application and reached a maximum after 24 h, rice, Tan-ginbozu, is known to be a dx mutant in which
following striking growth of the second leaf sheaththe GA biosynthesis pathway from ent-kaurene to GA53
(Fig. 6E). The increase in plastic extensibility was moreblocked (Moore et al. 1988). The elongation of the
second leaf sheath of this mutant is stimulated by
distinct than that in elastic extensibility at 12 h, but the
promotive effects of GA3 on both extensibilities wereexogenous GA applications, and this mutant has been
similar after 24 h (Fig. 6D). These results indicate a used for a microdrop bioassay of GAs (Murakami
positive relationship between GA-promoted growth and 1968). The experimental system used in this report is a
increase in cell wall extensibility during the early stage of modification of this bioassay system, that is, the
leaf sheath growth. seedlings were pretreated with uniconazole to reduce
the endogenous GA level and to enhance the sensitivity
Changes in osmotic solutes and sugar contents, and starch to exogenous GAs, which may reflect the elongation in a
degradation during leaf sheath growth. The osmotic normal cultivar of rice. Our results showed that the
concentration of the cell sap of the leaf sheath did not growth of the leaf sheath is strongly stimulated by
change significantly after application of GA3 (Fig. 7A). biologically active GA, GA3 (Fig. 2), and that ABA
However, the total amount of osmotic solutes in a whole treatment has an antagonistic effect (Fig. 3B). Other
leaf sheath increased during elongation growth, and phytohormones such as auxin, cytokinin and ethylene
GA3 strikingly stimulated the increase (Fig. 7B). Sugar had no significant effect on GA3-stimulated leaf sheath
analyses showed that the total amount of soluble sugars growth, although IAA reportedly promotes the elonga
(glucose, fructose, and sucrose) in the leaf sheath was tion of pea stem segments in cooperation with GA
strikingly increased by GA3 trgatment (Fig. 8). It is (Tanimoto et al. 1967). Auxin stimulates shoot elonga
noteworthy that starch granules in the leaf sheath cells tion in pea epicotyls and ethylene enhances the

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C. Matsukura et al.: Gibberellin-mediated leaf sheath growth in rice 149

Table 1. Effect of GA3 on total number of c

sheath of rice seedlings

Time (h) Control GA3 application

0 80.7 ± 4.5 80.7 ± 4.5

6 n.d. 77.2 ± 8.4
12 n.d. 264.8 ± 17.4
24 232.1 ± 20.7 (100) 288.8 ± 6.4 (124)
48 284.1 ± 76.5 (100) 453.9 ± 22.5 (159)

Each value is the mean ± SD of six measurements from three leaf

sheaths

GA-associated internode elongation of floating rice

(Ockerse 1970; Yang et al. 1996; Raskin and Kende
1984a). The auxin effects on tissue elongation have been
widely studied in excised mature tissue rather than the
intact and/or immature tissue. Leaf sheath growth in the
dwarf mutant of rice was not, however, affected either by
exogenous auxin or by the inhibitors of polar auxin
transport, TIBA and PCIB (data not shown). The action
of auxin may not be involved in the GA-induced
promotion of leaf sheath growth.

Gibberellin-induced leaf sheath growth is mainly caused by

cell elongation. In this study, GA3 promoted cell

Time (h)

Fig. 5. A Effect of GA3 on cell elongation in the second leaf sheath.

Average cell lengths are derived from two parenchyma cell files in each
0.25-mm-section. B Effect of GA3 on cell division Time in(h) the second leaf
sheaths. Average percentages of dividing cells are derived from two
parenchyma cell files in each 0.5-mm-section. C Average
Fig. 6A-E. Time-course of changescell
in celllength
wall extensibility after GA3
(closed squares) and average percentage of The
treatment. dividing cells
second leaf sheath (cross
of rice seedlings was either treated
hatched columns) versus distance from (•)
theor notbase of
treated (O) the
with second
GA3 (300 pmol/plant) leaf
for the designated
sheath (at 0-2.5 mm from the base). Attime.
least five
For details seedlings
see text were
and Fig. 1. A Total extension; B elastic
counted for each sample collected at the designated timeD effect of GA on the elastic and plastic
extension; C plastic extension;
extension; E growth of the second leaf sheath. Data are derived from
Fig. 5A. Values are means ± SD for 12 tested plants

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150 C. Matsukura et al.: Gibberellin-mediated leaf sheath growth in rice

A B

Time (h) Time (h)

Fig. 7A,B. Effect of GA3 on the osmotic concentration in the leaf
sheaths (A) and the total amount of osmotic solutes in a whole leaf
sheath (B). The leaf sheath of dwarf rice seedling was either treated
Fig. 9A,B.
(O) or not treated (□) with 300 pmol/plant of Starch
GA3granules
for the in parenchyma
designated cells of the second leaf
time. Values are means ± SD for four sheaths.
tested The leaf sheath of dwarf rice seedlings was treated (B) or not
plants
treated (A) with 300 pmol/plant of GA3 for 48 h. Longitudinal
sections were stained by the Periodic acid-Schiff (PAS) reaction to
visualize starch granules and cell walls. Bar = 0.02 mm (A, B)

activity in this area was already near the final phase at

the time of GA3 application. It is of interest that the rate
of cell division was not promoted by GA3. Similar
results have been reported for the first leaves of the GA
responsive dwarf mutant of barley, M117 (Zwar and
Chandler 1995). Growth of the leaf sheath characterized
in this study would be typical of that for monocot leaves.
On the other hand, rapid elongation of internodes in
submerged deepwater rice, which is induced by GA, is
based on increases in both the cell production rate in the
intercalary meristem and the cell extension rate in the
elongation zone just above it (Sauter and Kende 1992).
Time after GA3 application (h) that GA promotes cell elongation in most
It is likely
immature cells, but cell division only in particular cells/
Fig. 8. Effect of GA3 on the sugar content in the leaf sheaths. The leaf
tissues.
sheath of dwarf rice seedling was either treated (O) or not treated (□)
with 300 pmol/plant of GA3 for designated time. Glc, glucose; Fru,
Gibberellin-induced
fructose; Sue, sucrose. Values are means ± SD for three cell tested
elongationplants
is due to cell wall
loosening and accompanied by an increase in sugar
contents as a result of starch degradation. A positive
elongation but promoted cell division relationship between
only GA-induced growth
slightly (Figs.promotion
4, 5). The promotive effect of andexogenous
the increase in cell wall extensibility
GA3 on cell was demon
division remained at the level of less than a 1 % increase strated in the leaf sheath (Fig. 6). Similar results have
in the mitotic index (Fig. 5B), and the number of cellsbeen in reported in several plants; GA-increased plastic
a longitudinal cell file in the leaf sheath was increased extensibility in wheat leaves (Keyes et al. 1990) and in
only 1.6-fold by GA application (Table 1), although the cucumber hypocotyls (Taylor and Cosgrove 1989), and
length of leaf sheath was increased 5- to 6-fold. Cellsreduced
of minimum relaxation time (t0) as measured by
the whole leaf sheath showed relatively high meristema stress-relaxation analysis in lettuce hypocotyls (Kamis
tic activity (3-4%, Fig. 5C) at the time of GA applicaaka et al. 1972), pea epicotyl hooks (Nakamura et al.
tion, and nearly the same activity was observed in the 1975) and pea roots (Tanimoto 1994). Kutschera and
basal area during the subsequent 24 h, but the activity Kende (1988) have reported a GA-induced increase in
was lost almost completely in the seedlings after 48 both h, plastic (irreversible) and elastic (reversible) exten
both with and without GA3 application. These results sibilities in the internodes of deepwater rice. In cucum
show that cell division in the leaf sheath is restricted to ber hypocotyls (Taylor and Cosgrove 1989), however,
the basal few millimeters, and that the meristematic the plastic extensibility was more significantly enhanced

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C. Matsukura et al.: Gibberellin-mediated leaf sheath growth in rice 151

by GA. Our present results indicatedPerata etthat

al. (1997)
both have plastic
reported that the induction of
oc-amylase by
and elastic extensibilities were enhanced by GA3
GA3 in barley
(Fig.embryos
6) is repressed by
to the same extent. In auxin-inducedcarbohydrates
elongationconvertedof from starch, indicating the
oat
existence
coleoptiles, auxin initially increases the of cross-talkextensi
elastic between the signaling for GA
bility (Masuda 1968) and then theand sugar-regulation
plastic of oc-amylase gene expression.
extensibility
(Cleland 1967; Masuda 1968). In contrast, GA3may
Similar regulation initially
take place for starch degradation
enhanced the plastic and then thein elastic
the leaf sheath.
extensibility
(Fig. 6D). The physical meanings of the observed
In the early plastic the leaf sheath cells
stage of germination,
and elastic extensibilities have not yet
play both been
sink defined
and source functions, i.e., carbohydrate
completely. However, Hohl and unloaded
Schopfer from the (1992)
embryos have
after degradation of reserve
proposed a revision of the view thatstarch
the in the endosperm plastic
observed might be initially stored as
extension represents a true plastic extension.
starch Indeed,
granules and eventually consumed for growth and
they have shown by the creep test, using maize metabolism. The appreciable accumulation of starch
coleoptiles cell walls, that auxin-induced increased granules in leaf sheath cells not treated with GA3 might
plastic extension precedes the elastic extension. Thus be due to reduced starch-degradation activity because of
the plastic extensibility observed in Fig. 6C may not be low levels of endogenous GAs. Further studies will be
the real plasticity in terms of physical definition. Further needed for identification and characterization of the
studies on the plastic and elastic extension processes mechanism(s) for unloading of long-distance-transport
involves GA-induced leaf sheath elongation are in ed sucrose and for starch degradation in the leaf sheath
progress in an effort to define the rheological meaning. tissues.
Although the molecular mechanism of change in cell
wall extensibilities is unknown, there are some sugges We thank Dr. Pierdomenico Perata for suggestions, invaluable
tions that metabolic changes in cell wall polymers discussion and critically reviewing the manuscript. We also thank
participate in GA-induced cell wall extension, e.g. Drs. T. Tashiro and M. Koshioka for providing Tan-ginbozu seeds,
enhancement of ß-glucan hydrolysis (Carpita and Kana Dr. K. Katoh for providing the osmometer and the Sumitomo
bus 1988) and endotransglycosylation (Smith et al. Chemical Co. for the gift of uniconazole. This work was supported
in part by Grants-in-Aid No. 05276102 (to J.Y.) for Special
1996). In addition to GA-regulated cellulose microfibril
Research on Priority Areas from the Ministry of Education,
orientation (Shibaoka 1994), the interaction of hemicel Science and Culture, Japan.
lulosic polymers and/or cellulose microfibrils is consid
ered to determine the cell wall extensibility.
The number of starch granules in the leaf sheath
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