Expression of Expansin Genes Is Correlated with Growth in Deepwater Rice

Abstract

Expansins are a family of proteins that catalyze long-term extension of isolated cell walls. Previously, two expansin proteins have been isolated from internodes of deepwater rice, and three rice expansin genes, Os-EXP1, Os-EXP2, and Os-EXP3, have been identified. We report here on the identification of a fourth rice expansin gene, Os-EXP4, and on the expression pattern of the rice expansin gene family in deepwater rice. Rice expansin genes show organ-specific differential expression in the coleoptile, root, leaf, and internode. In these organs, there is increased expression of Os-EXP1, Os-EXP3, and Os-EXP4 in developmental regions where elongation occurs. This pattern of gene expression is also correlated with acid-induced in vitro cell wall extensibility. Submergence and treatment with gibberellin, both of which promote rapid internodal elongation, induced accumulation of Os-EXP4 mRNA before the rate of growth started to increase. Our results indicate that the expression of expansin genes in deepwater rice is differentially regulated by developmental, hormonal, and environmental signals and is correlated with cell elongation.

Full text

lhe Plant Cell,
Vol.
9, 1661-1671, September 1997
O
1997 American Society of Plant Physiologists
Expression
of
Expansin Genes
1s
Correlated
with Growth
in
Deepwater Rice
Hyung-TaegChoandHansKende'
Michigan State University-Department of Energy Plant Research Laboratory, Michigan State University, East Lansing,
Michigan 48824-1 31 2
Expansins are a family of proteins that catalyze long-term extension of isolated cell walls. Previously,
two
expansin pro-
teins have been isolated from internodes of deepwater rice, and three rice expansin genes,
Os-EXPI, Os-EXP2,
and
os-
EXP3,
have been identified. We report here on the identification of a fourth rice expansin gene,
Os-EXP4,
and on the ex-
pression pattern of the rice expansin gene family in deepwater rice. Rice expansin genes show organ-specific
differential expression in the coleoptile, root, leaf, and internode. In these organs, there is increased expression of
Os-
EXP7, Os-EXP3,
and
Os-EXP4
in developmental regions where elongation occurs. This pattern of gene expression is
also correlated with acid-induced in vitro cell wall extensibility. Submergence and treatment with gibberellin, both of
which promote rapid internodal elongation, induced accumulation of
Os-EXP4
mRNA before the rate of growth started
to increase Our results indicate that the expression of expansin genesdn deepwater rice is differentially regulated by
developmental, hormonal, and environmental signals and is correlhted with-cell elongation.
INTRODUCTION
Because plants are sessile organisms, it is an adaptive ad-
vantage that their growth and development are subject to
control by environmental conditions. Regulatory effects of
the environment are often mediated by hormonal signals
and can therefore be mimicked by application of the appro-
priate plant growth regulator(s). Greatly accelerated internodal
elongation of deepwater rice in response to submergence
exemplifies how an environmental stimulus induces growth
via a series of hormonal interactions (Raskin and Kende,
1984a, 1984b; Hoffmann-Benning and Kende, 1992). Deep-
water rice is a Subsistence crop in large areas of Southeast
Asia that are inundated by flood waters during the monsoon
season. The livelihood of
>100
million people depends on
the successful cultivation of this rice (Mannan, 1988). The
remarkably high growth rates of deepwater rice-up to
25
cm a day (Vergara et al., 1976)-permit this rice to emerge
from;the rising flood waters and taavoid drowning. The yield
of deepwater rice is far below that of improved riBe cultivars,
which lack elongation capacity (Mannan, 1988). Wnderstand-
ing the mdecular basis of'the growth response of'deepwater
rice is necessary for the eventual introduction of.ihcreased
elongation cagacityinto high-yielding modern rice cultivara.
The primary signal for enhanced elongation is the reducsed.'
partia1 pressure of
O2
in the submerged tissue. Under low
O2
tension, ethylene synthesis is enhanced; ethylene renders
the internode more responsive to gibberellin (GA) by lower-
To
whom
correspondence
should
be
addressed. E-mail hkende
@msu.edu;
fax
51
7-353-91
68.
ing the leve1 of endogenous abscisic acid (Raskin and Kende,
1984a, 1984b; Hoffmann-Benning and Kende, 1992). GA is
the immediate growth-promoting hormone and acts by en-
hancing cell elongation and cell division activity in the inter-
calary meristem (Sauter and Kende, 1992; Sauter et al., 1993).
Hormonal and environmental stimuli that promote growth
of plant cells act by inducing stress relaxation
of
the primary
cell wall, which is thought to be principally a network of cel-
lulose microfibrils interconnected by hemicelluloses (Carpita
and Gibeaut, 1993). On the basis
of
this cell wall model, two
wall-loosening processes can be envisionedi breakage of
covalent bonds within cross-linking polymers or disruption
of
noncovalent bonds, such as hydrogen or iollic bonds, be-
tween wall polymers. It has been proposed that cleavage of
covalent bonds is catalyzed by wall hydrolases or endo-
transglycosylases
(Fry,
1989;
Fry
et al., 1992; Nishitani and
Tominaga, 1992). Current evidence does not' support the
notion that these enzymes are able to cause long-term ex-
tensimof isolated cell walls (McQueen-Mason et al., 1993;
Cosgrove and Durachko, 1994). Recently, a new group
of
wall proteins, the expansins, has been identified as wall-
loosening factors that can promote long-term extension
of isolated cell walls (reviewed in McQueen-Mason, 1995;
Cosgrove, 1996). Evidence indicates that expansins cause
cell wall loosening by disrupting hydrogen bonds between
cellulose microfibrils and matrix polymers (McQueen-Mason
and Cosgrove, 1994).
Two expansin proteins have been isolated from deepwater
rice internodes (Cho and Kende, 1997a), and three expansin

1662
The Plant Cell
A
os-ExP1
Os-EXP2
Os-EXP3
OS
-Em4
Os
-EWl
Os-EXPZ
Os-EXP3
Os-ED4
SPZO
SPZ9
Os-EXPl
Os-EW2
Os-EXP3
OS-EXP4
OS-EXPl
Os-EXP2
OS-EXP3
OS-EXP4
os-ExP1
Os-EXP2
Os-EXP3
OS-EXP4
OS-EXP1
OS-EXPZ
OS-EW3
OS-EXP4
os-ExP1
OS-EXP2
Os-EXP3
Os-EXP4
genes have been identified from rice (Shcherban
et
al.,
7995;
Cho and Kende, 1997a). Expansin gene families have also
been found in cucumber and Arabidopsis (Shcherban et al.,
1995). The redundancy
of
expansin genes indicates that ex-
pression of individual expansin genes may be differentially
regulated at various developmental stages and by diverse
Siqnal DeDtide
masrssallllfsafcfl
dsgmekqpamllvlvtlcafa
magssaatscarflallatcllPmeaas
maiagvlfllflarqasa
.#.
FTASG-NKAF
AGYGG-Q*AH
environmental stimuli. This would permit the plant to fine-
tune its response to developmental signals and changes in
the environment. We report here on the identification of a
fourth rice expansin gene, Os-EXP4, whose expression is
highly correlated with growth, and show tissue-specific ex-
pression of expansin genes and their developmental, hor-
monal, and environmental regulation. Our results supporl the
hypothesis that expansins may at least in part determine the
growth rate of plant organs. Therefore, increasing the expres-
sion of the proper expansin genes may raise the growth po-
tential of high-yielding rice cultivars.
RESULTS
Cloning of
Os-EXP4
and Sequence Analysis
of
the
Rice
B
os
-
EXP2
-
os-EXP4
I
Pt-EXP1
cs-EXP1
-
At-EXP2
1
Os-EXP3
Figure
1.
Comparison
of
the Deduced Amino
Acid
Sequences
of
Rice
Expansin Proteins.
(A)
Alignment
of
the
rice
expansin amino acid sequences deduced
from
cDNA
sequences. SP20 and SP29
are
the N-terminal amino
acid
sequences
of
two
expansins isolated from air-grown rice inter-
nodes
(Cho
and
Kende, 1997a). The amino
acid
at
position
6
could
not
be
determined,
and
position
8
of
SP29
(.)
may
be
either
a
T
or
an
S
residue.
Gaps
are
shown
as
dots;
letters
on black and gray back-
ground indicate identical amino acids.
Signal
peptide
sequences
were
predicted using the
PSORT
program (Nakai and Kanehisa,
1992).
(B)
Phylogenetic
tree
of
expansins. Mature protein regions with-
out signal peptides
were
compared using the clustal method
of
DNASTAR Megalign (DNASTAR Inc., Madison,
WI).
GenBank
acces-
sion numbers
are
U30382
(Cs-EXP7),
U30460 (Cs-fXP2),
U30476
(At-EXPI), U30477
(OS-EXP~),
U30478 (At-EXPS), U30479 (OS-EXP3),
and SP29, from rice internodes and have sequenced their
N-terminal amino acids (Cho and Kende, 1997a). The N-ter-
mina1 amino acid sequence
of
SP20 is identical to that en-
coded by Os-EXPI. Because síx
of
eight N-terminal amino
acids of SP29 match an amino acid sequence deduced from
the nucleotide sequence
of
Os-EXP2, we concluded tenta-
tively that SP29 is encoded by a rice expansin gene that is
different from yet very similar to Os-EXP2. To isolate the cDNA
coding for SP29, we screened a cDNA library prepared from
deepwater rice internodes with a probe from the conserved
coding region
of
Os-EXP2. Positive clones were further
screened with gene-specific probes of Os-EXP1 and Os-EXP2.
One cDNA clone that did not hybridize with the gene-specific
probes was sequenced and shown to represent the new
rice expansin gene Os-fXP4 (GenBank accession number
U85246).
Os-EXP4 consists of 1229 bp, with an open reading frame
of 738
bp,
54
bp
of the 5' flanking sequence, and 437 bp of
the 3' flanking sequence. The open reading frame encodes
a protein of
246
amino acids, with a signal peptide predicted
by the PSORT program (Nakai and Kanehisa, 1992) (Figure
1A). The mature protein of 228 amino acids has
a
calculated
molecular mass
of
24
kD,
which agrees well with the molec-
ular mass of SP29 as determined by SDS-PAGE (Cho and
U30480
(At-EXPG), U30481 (At-€XP2),
U64892
(R-€XP7),
U85246
(OS-EXP~),
X85187
(Ps-EXPI), and
Y07782
(Os-EXP7).

Expansin Gene Expression
in
Rice
1663
Os-EXPI
OS-EXP2 Os-EXPS
OS-EXP2
OS-EXP3
OS-EXP4
24.5
26.2
25.925.5
26.421.4
B
Os-EXPI
OS-EXP2
«p
OS-EXP3
•
OS-EXP4
•
Figure
2.
Gene-Specific
Rice
Expansin cDNA Probes.
(A)
The
percentage
of
nucleotide
identity
of the
gene-specific rice
expansin
cDNA
probes.
(B)
DMA gel
blot analysis showing
the
specificity
of the
gene-spe-
cific probes. Plasmid
DMAs
given above
the
blots
were
digested
with restriction enzymes, subjected
to
electrophoresis,
blotted,
and
hybridized under stringent conditions with the gene-specific probes
indicated
at
left.
Kende,
1997a). The deduced amino acid sequence of
Os-EXP4
indicates that
it
indeed encodes SP29 (Figure 1A).
Eight
N-terminal
amino
acids
of
SP29 match
those
encoded
by
Os-EXP4.
At
position
8,
amino acid sequencing yielded
both
T and S
residues;
the
amino acid deduced from
Os-EXP4
is
serine.
The
cleavage sites
of the
signal peptides
of
Os-EXPI
and
Os-EXP4,
predicted
by the
PSORT pro-
gram, are consistent with the N-terminal amino acid se-
quences
of
SP20
and
SP29.
The
deduced amino acid
sequences
of the
rice expansin family show that
Os-EXP2
and
Os-EXP4
are
most closely related
to
each other, with
an
amino acid identity
of
85.8%;
Os-EXP3
is
least related
to the
other rice expansin genes
and
also
to all
other known plant
expansin genes (Figure
1 B).
To
investigate
the
differential expression
of
each rice
ex-
pansin gene
in
various rice tissues, gene-specific probes
were
prepared consisting mainly of the 3' untranslated re-
gions
of the
respective cDNAs. These regions have 21.4
to
26.4% nucleotide identity
to
each other (Figure 2A).
DMA gel
blot analyses performed under the same conditions as used
for
RNA gel
blotting (65°C washing temperature) showed
that
all
four probes were indeed gene specific (Figure 2B).
Genomic Analysis
DNA
gel
blot
analysis
was
used
to
estimate
the
complexity
of
the
expansin gene family
in the
rice genome (Figure
3).
Each
gene-specific probe detected
a
single band, indicating
that
the
four known rice expansin genes exist
as
single cop-
ies
(Figures
3A to
3D). Because some probes contained
short stretches from
the
coding region
and
because rela-
tively low-stringency conditions were used (55°C washing
temperature), some blots showed additional faint bands
whose
sizes corresponded
to
known expansin
bands.
Os-
EXPS
has a
BamHI site
in the
middle
of the
coding region.
Therefore,
a
band
(~1 kb)
shown
in
lane
B + H in
Figure
3C
corresponds
to the 3'
half
of the
gene.
A
probe prepared
from the 5' flanking and coding region of
Os-EXP3
detected
the 5'
half (~1.3
kb) of the
gene (data
not
shown).
The DNA
gel
blot analysis performed with
a
probe from
the
coding
re-
gion
of
Os-EXP1
showed
the
four
expansin
genes
and
sev-
eral
unidentified bands (Figure
3E,
lane
E). The
bands
of
~10.3
and 6.3 kb
(Figure
3E,
lane
E)
could
be
detected
in
other
gel
blot analyses that were performed with probes
from
the
coding
regions
of
other
expansin
cDNAs
(results
not
shown). These additional bands
may
represent other
ex-
pansin genes
or
related genes, such
as
those encoding pollen
B+H
E B+H E B+H E B+H E
B+H E
12.3-
7.2-
2.3-
Figure
3. DNA Gel
Blot Analysis
of the
Expansin Gene Family
in the
Rice
Genome.
Five
micrograms
of
rice genomic
DNA was
digested either with
EcoRI
(E) or
with
BamHI
and
Hindlll (B+H)
and
subjected
to gel
blot
hybridization. Gene-specific probes were used.
(A)Os-EXP).
(B)
OS-EXP2.
(C)
Os-EXPS.
(D)
OS-EXP4.
(E)
Probe from
the
coding region
of
Os-EXP1.
Each
identified
ex-
pansin
band
is
labeled with
its
respective number.
The
length
of the
DNA
markers
is
indicated
at
left
in
kilobases.

1664
The
Plant Cell
Os-EXPI
OS-EXP2
Os-EXP3
OS-EXP4
E37
17S
rRNA
Figure
4.
Tissue-Specific Expression Pattern
of
Rice Expansin
Genes.
Each
lane contains
20
^.g
of
total
RNA
isolated from
the
basal
1-cm
region of uppermost internodes, expanding leaves of mature plants,
and the
apical region
of
coleoptiles
and
roots.
The
transcript levels
of £37
encoding
a
plastid inner envelop protein
and 17S
rRNA
served
as
internal loading controls.
allergens
and
their homologs, which
are
considered
to be a
subfamily
of the
expansins (Cosgrove
et
al., 1997).
also
of
similar magnitude
in
root
and
coleoptile apices.
In
leaves,
which contain more plastids than do other organs,
the
abundance
of £37
mRNA
was
increased. Therefore,
17S
rRNA
was used as an
additional
internal loading control.
Expression
of
Expansin Genes
in
Different
Developmental Regions
of
Coleoptiles,
Roots,
and
Internodes
Os-EXP1
and
Os-EXP4
were expressed
at
higher levels
in
the
apical 1-cm region
of the
coleoptile, where growth
oc-
curs, than
in the
adjacent basal region;
the
transcript level
of
Os-EXP2
was
similar
in
both regions (Figures
5B and
5C).
In
the
root, expression
of
Os-EXP1, Os-EXP3,
and
Os-EXP4
Apical
(1cm)
Basal
(1cm)
Mesocotyl
Os-EXPI
OS-EXP2
Os-EXP4
E37
Organ-Specific Expression
of
Expansin Genes
The
transcript levels
of the
four expansin genes were exam-
ined
in the
expanding region
of
young leaves from
11- to 13-
week-old air-grown plants; in the basal 1-cm region of the
uppermost internode, which contains
the
intercalary mer-
istem
and
elongation zone from
the
same adult plants;
and
in
the
apical region
of
coleoptiles
and
roots from 3-day-old
seedlings (Figure
4).
Air-grown mature plants grow slowly,
whereas
the
coleoptile
and
root
of
seedlings elongate
at a
fast
rate.
In the
coleoptile
and
internode,
Os-EXP1, Os-EXP2,
and
Os-EXP4
were expressed,
but no
detectable
Os-EXP3
signal
was
found
in
either
tissue.
In the
root,
all
expansin
genes were expressed. This
was the
only tissue
in
which
Os-EXP3
mRNA
was
found. Only
OS-EXP2
transcript could
be
detected
at low
abundance
in the
growing zone
of
leaves.
Expansin mRNAs accumulated
to
higher levels
in the
apical regions
of
coleoptiles
and
roots than
in the
more
slowly expanding tissues of internodes and leaves. The tran-
script
level
of
£37, which encodes
a
major 37-kD protein
of
the
inner plastid envelope (Teyssier
et
al., 1996), served
as
control
for the
quantity
of
total
RNA
loaded because
its ex-
pression remains constant in rice internodes induced to
grow
rapidly
by GA
(Van
der
Knaap
and
Kende,
1995);
it is
Apical
D
Basal
$ 100
> 50
Os-EXPI
OS-EXP2
OS-EXP4
Figure
5.
Differential Expression
of
Rice
Expansin Genes
in the
Api-
cal
and
Basal Regions
of the
Coleoptile.
(A)
The
apical
and
basal regions
of the
coleoptile
from dark-grown
3-day-old
rice
seedlings.
(B)
RNA gel
blot analysis. Each lane contains
20
(xg
of
total
RNA
iso-
lated from each region.
£37 was
used
as the
internal loading
control.
(C)
Quantification
of
mRNA levels
of
each expansin gene. Blots
shown
in (B)
were quantified with
a
Phosphorlmager,
and the
rela-
tive
mRNA
levels were
calculated
by
setting
the
value
for the
apical
region
to
100.

Expansin
Gene Expression
in
Rice
1665
B
Os-EXPI
OS-EXP2
Root
hair
(RH)
zone
u (1 cm)
——
Root
hair (RH)
zone
I
(1
cm)
.
Growing
zone
(0.5
cm)
L=,
Os-EXP3
=•
OS-EXP4
=.
E37
17S
rRNA
Os-EXPI
OS-EXP2
OS-EXP3
OS-EXP4
Figure
6.
Differential Expression
of
Rice Expansin Genes
in
Differ-
ent
Developmental Regions of the Root.
(A)
Different developmental regions of the primary root from dark-
grown
3-day-old rice seedlings.
(B) RNA gel
blot analysis. Each lane contains
20
(ig
of
total
RNA
iso-
lated from each region.
E37 and 17S
rRNA were used
as
internal
loading controls.
(C)
Quantification
of
mRNA levels
of
each expansin gene. Blots
shown
in (B)
were quantified with
a
Phosphorlmager,
and the
rela-
tive
mRNA levels were calculated
by
setting
the
value
for the
grow-
ing
region
to
100.
transcripts
was
confined
to the
apical
5-mm region, which
corresponds to the growing zone (Figures 6B and 6C). Os-
EXP2
mRNA
also
accumulated
mainly
in the
apical
region
but was
expressed
in the
root hair zone
as
well.
The
level
of
E37
mRNA
was
lower
in the
root hair zone than
in the
apical
region,
but the
abundance
of 17S
rRNA
remained constant
along
the
root,
confirming
equal
loading
of
total
RNA.
In
air-
grown internodes,
Os-EXP1
and
Os-EXP4
were expressed
at
the
highest levels
in the
basal growing zone, with tran-
script
levels declining
in the
nongrowing
upper
regions (Fig-
ures
7B and
7C).
The
mRNA abundance
of
Os-EXP2
showed
the
opposite tendency;
it was
much lower
in the
basal grow-
ing
zone than
in the
upper nongrowing regions.
Differential
Cell
Wall Extensibility along
the
Developmental Regions
of the
Coleoptile,
Root,
and
Internode
To
correlate expansin action
and
expression
of
expansin
genes,
we
examined
acid-induced
extensibility
of
isolated
cell walls from
the
same developmental regions that were
B
0-1 1-2 2-3
DZ
EZ&
IM
node
-3
Os-EXPI
» ,
-2
OS-EXP2
OS-EXP4
E37
Os-EXPI
OS-EXP2 OS-EXP4
Figure
7. Differential Expression of Rice Expansin Genes along the
Different
Developmental Regions
of the
Uppermost Internode.
(A)
Different developmental regions of the uppermost internode from
11- to
13-week-old rice plants.
DZ,
differentiation zone;
EZ,
elonga-
tion zone;
IM,
intercalary meristem.
(B) RNA gel
blot analysis. Each lane
contains
20
M-9
of
total
RNA
iso-
lated from each region.
E37 was
used
as the
internal
loading
control.
The
numerals
0-1, 1-2,
and 2-3 indicate distance from the node in
centimeters.
(C)
Quantification
of the
message levels
of
each expansin gene.
Blots
shown
in (B)
were
quantified
with
a
Phosphorlmager,
and the
relative
mRNA levels were calculated
by
setting
the
highest value
for
each
blotto
100.

1666
The Plant Cell
A
B
10 min
Coleoptile
-
Apiml
pH4.5
Basal
Root
Growing
pH4.5
,
RHI
RH
I1
C
lnternode
I
I
100
I
i
50
r
80
Apical Basal Growing
RHI
RHII
T
0-1 1-2 2-3
Developmental region Developmental region Distance from node (cm)
Figure
8.
Acid-lnduced Extension
of
the Cell Walls from Different Developmental Regions
of
Coleoptiles, Roots, and Internodes.
(A)
Acid
extension
of
the coleoptile
cell
walls. Extension
of
the middle 5-mm regions
from
the 1-cm-long apical and basal segments
(see
Figure
5A)
was
measured.
(6)
Acid extension
of
the root cell walls. Extension
of
the middle 4-mm regions from each root segment
(see
Figure
6A)
was measured. RH, root
hair.
(C)
Acid extension of the internodal
cell
walls. Extension
of
the entire 1-cm segment of each region
(see
Figure
7A)
was measured.
After
the
cell
wall
preparations
were
incubated in pH
6.8
buffer
for
20
min, the solution was changed (arrows) to pH
4.5
buffer. Coleoptile and in-
ternodal
cell
walls
were
subjected to constant tension
by
using
a
weight
of
10 g.
with
5
g
being
used
for
roots.
The
chart
recorder
tracings are
from
a
single representative experiment. Data are mean values
?SE
(n
=
5
for
the coleoptile;
n
=
4
for the
root
and internode).
used in the gene expression studies. Extensibility
of
the cell
wall from the apical region of the coleoptile was approxi-
mately twofold higher than that from the basal region (Figure
8A). In the root, wall extensibility was entirely confined to
the apical 5-mm region (Figure 88). In internodes, cell wall
extensibility also declined sharply from the growing to the
nongrowing region (Figure 8C).
Accumulation of Expansin Transcripts
in
Response to
GA
and Submergence
We compared the levels of the three internodal expansin
transcripts in air-grown (control), submerged, and GA-treated
internodes from
11-
to 13-week-old plants (Figures 9A and
96).
Os-EXf
7
mRNA levels decreased at the beginning of
incubation, probably as a result
of
excising the stem sec-
tions that contain the growing internode. Between
24
and
48
hr of incubation, the transcript level of
Os-EXf
7
returned to
its initial value in GA-treated internodes; in submerged inter-
nodes,
it
returned to approximately half of its original value.
Os-EXf2
mRNA accumulated gradually during 48 hr of treat-
ment with GA but rapidly between
3
and
6
hr of submer-
gence. In contrast, the abundance of
Os-EXP4
transcript
increased rapidly within
6
hr of incubation with GA and
within 3 hr of submergence.
Because the level of
Os-EXf4
transcript increased earlier
than that of
Os-EXP2
in response to both treatment with GA
and submergence, we determined the time course
of
Os-EXf4
mRNA accumulation during the first
6
hr of submergence
and incubation in GA (Figure
10).
Under both experimental
conditions, the transcript level
of
Os-EXf4
increased within
30 min and reached a maximum after 3 hr.
DlSCUSSlON
Conservation of Expansins during Evolution
The rice expansin gene family does not represent a separate
monocotyledonous branch in the phylogenetic tree of known

Expansin
Gene
Expression
in
Rice
1667
expansins (Figure 1B).
The
closely related rice expansins
Os-EXP2
and
Os-EXP4
form
a
subfamily with
a
Pinus
taeda
expansin
(Pt-EXPI),
a
cucumber expansin
(Cs-EXPI),
and
an
Arabidopsis expansin
(M-EXP2).
The
high similarity
of ex-
pansins from
a
gymnosperm
and
from monocotyledonous
and
dicotyledonous
angiosperms
is
evidence
for the
con-
servation
of
this family
of
proteins during
the
evolution
of
seed
plants.
Os-EXPl
and
another cucumber expansin,
Cs-
EXP2,
are
closely related, whereas
Os-EXP3
showed least
similarity
to
other expansins.
Acid-induced cell elongation occurs
in
algae, mosses, ferns,
gymnosperms,
and
angiosperms
(Taiz,
1984; Cosgrove, 1996).
Because expansins mediate extension
of
isolated
plant
cell
walls under
acid
conditions,
it is
conceivable that they play
a
role
in
cell enlargement
of
both lower
and
higher plants.
Even
though phylogenetically separate organisms have cell
walls
of
different composition, expansins
may act by a
com-
mon
mechanism suggested for higher plant cell walls, namely,
by
disrupting
hydrogen
bonds
between
load-bearing
wall
polymers (McQueen-Mason
and
Cosgrove, 1994).
Differential Expression Pattern
of
Expansin Genes
Expansin
genes
are
differentially expressed
in the
major
vegetative parts of rice plants, and their mRNAs are most
GA
Air-grown
Submerged
Time (hr)
Os-EXPl
OS-EXP2
OS-EXP4
0 6 12 24 48 6 12 24 48 3 6 12 24 48
E37
B
0)
JO
20
0)
OL
Os-EXPl
250
150
100
50
OS-EXP2
400
200
100
48
24
36
48
OS-EXP4
36
Time (hr) Time (hr) Time (hr)
Figure
9.
Accumulation
of
Rice
Expansin
Transcripts
in
GA-Treated
and
Submerged
Internodes.
(A)
RNA gel
blot
analysis.
Each
lane
contains
20
p.g
of
total
RNA
isolated
from
the
basal
2-cm
region
of the
uppermost
internodes
that
had
been
treated
with
50 \M
GA3,
submerged,
or
kept
in air for 0 to 48 hr. £37
served
as
internal
control.
(B)
Quantification
of the
mRNA
levels
of
each
gene.
Blots
shown
in (A)
were
quantified
with
a
Phosphorlmager.

1668
The
Plant
Cell
Time (hr)
OS-EXP4
E37
____GA____
Submerged
0
0.5 1 2 3 6 0.5 1 2 3 6
B
200
§
.» 150
100
«
50
GA
Submerged
0123456
Time (hr)
Figure
10.
Accumulation
of the
Os-EXP4
Transcript
in
GA-Treated
and
Submerged Internodes.
(A)
RNA gel
blot analysis.
Each
lane contains
20
^g
of
total
RNA
iso-
lated
from
the
basal 2-cm region
of the
uppermost
internodes
that
had
been
treated with
50
^M
GA3
or
submerged
for 0 to 6 hr.
(B)
Quantification
of
Os-EXP4
mRNA
levels. Blots shown
in (A)
were
quantified with
a
Phosphorlmager.
abundant
in
actively
growing
organs,
such
as the
coleoptile
and the
primary root (Figure
4). In
expanding leaves, only
low
expression
of
Os-EXP2
has
been found.
It is
conceiv-
able that there
are
leaf-specific expansins
that
have
not yet
been
identified
and
whose transcripts
are not
represented
in
our
cDNA library prepared from deepwater rice internodes.
DNA
gel
blot analysis indicates that there
may
indeed
be
more than four expansin genes
in
rice (Figure
3), in
addition
to the allergen-type p-expanins identified by Cosgrove et al.
(1997).
Expression
of
Os-EXP3
is
confined
to the
very short
apical region
of the
root.
Differential
expression
of
expansin genes also occurs along
the developmental regions of the coleoptile, root, and intern-
ode
(Figures
5 to 7).
Whereas mRNA abundance
of
Os-EXP1,
Os-EXP3,
and
Os-EXP4
is
correlated with cell elongation,
expression
of
Os-EXP2
appears
to be
less linked
to
primary
growth.
In
contrast
to the
expression
of
other expansin
genes,
Os-EXP2
continues
to be
expressed
in the
root hair
zones
where growth
of the
primary root
has
ceased (Figures
6B and
6C).
In the
uppermost internode,
the
expression
level
of
Os-EXP2
is
higher
in the
nongrowing differentiation
zone than
in the
intercalary meristem
and the
elongation
zone
(Figures
7B and
7C).
The
difference
in
expression
of
Os-EXP2
and the other expansin genes
leads
us to suggest
a
distinct
role
for the
Os-EXP2
protein.
The
nongrowing
re-
gions
of the
internode
and the
root
are
both
differentiating
tissues. Therefore,
Os-EXP2
may
play
a
role
in the
differenti-
ation
of the
vascular system
and
perhaps
in the
differentia-
tion
and
growth
of
root hairs.
In
maize roots, cell wall
proteins extracted from
the
basal region showed higher
ex-
tension activity than
did
cell wall proteins from
the
apical
re-
gion
(Wu et
al.,
1996).
It is
possible
that
an
expansin
with
a
function(s) similar
to
that
of
Os-EXP2
is
expressed
at an
ele-
vated level
in the
basal region
of the
primary root
of
maize
as
well.
We
have previously reported
on the
highly localized
oc-
currence
of
expansin protein along
the
inner epidermal layer
and
around immature vascular bundles
of
rice internodes
(Cho
and
Kende, 1997b).
We do not
know
the
role
of ex-
pansins
in
those tissues,
but the
high concentration
of ex-
pansins
in
specific cells further supports
the
notion that
certain expansins,
for
example, those associated with
the
developing vasculature,
may
have physiological functions
other than promoting cell elongation. McQueen-Mason
and
Cosgrove
(1995) showed that
the two
cucumber expansins
have
slightly different
pH
dependence
and
cell wall rheolog-
ical
effects, which
may
indicate differences
in
functions,
and
Rose
et al.
(1997) have identified
a
family
of
expansins that
may
play
a
role
in
cell wall disassembly
of
ripening fruits.
Thus,
there
is
mounting evidence that besides mediating
cell
wall
extension, expansins
may
also function
in
other pro-
cesses
involving cell wall modifications. However,
the
bio-
chemical mechanism
of
expansin action, namely, disruption
of
hydrogen bonds between cell wall polymers,
may be
common
to all
expansin-mediated reactions.
Expansin
Gene Expression
Is
Correlated with Rapid
Internodal Growth
of
Deepwater Rice Induced
by
Submergence
and GA
The
response
of
deepwater rice internodes
to
submergence
illustrates
how a
physiological process,
in
this case rapid
in-
ternodal elongation,
is
controlled
by the
interaction between
plant hormones
(Raskin
and
Kende, 1984b; Hoffmann-
Benning
and
Kende, 1992). Both
the
initial environmental
signal,
submergence, and the immediate growth-promoting
hormone,
GA,
greatly increased
the
mRNA
abundance
of
Os-EXP2
and
Os-EXP4
(Figure
9).
Submergence
and GA
promoted expression
of
Os-EXP4
within
30 min
(Figure 10),
which
is
within
the
observed
lag
time
of 40 min for GA-
induced internodal growth (Sauter
and
Kende, 1992)
and 3
hr
and 20 min for
submergence-induced elongation
(Rose-
John
and
Kende, 1985).
The
accumulation
of
Os-EXP2
mRNA
was
considerably slower than that
of
Os-EXP4
in
both
GA-
treated
and
submerged internodes. These results support
the
hypothesis that Os-EXP4 plays
a
role
in
submergence-
and
GA-enhanced growth
of
deepwater rice.

Expansin Gene Expression in Rice 1669
Expansin Gene Expression and Cell Wall Extensibility
Acid-induced extensibility of native cell walls
is
indicative
for
the expansin content of the
wall
and
for
the
susceptibility of
the cell
wall
to
expansin
action
(Cosgrove, 1996).
These
two
parameters may at least in part also determine
the
growth
rate of the tissue. In oat coleoptiles (Cosgrove and
Li,
1993)
and cucumber hypocotyls (McQueen-Mason, 19954, sus-
ceptibility to expansin rather than expansin content may
be
a determining factor in establishing
the
rate of
growth.
In
contrast, elongation
of
maize roots under
water
stress
(Wu
et al., 1996) and growth of deepwater rice internodes
as
a
function of the developmental stage or as
a
result of sub-
mergence are correlated with both expansin content
and
susceptibility
of
the cell wall to expansin (Cho and Kende,
1997b). The pattern of acid-induced wall extensibility of dif-
ferent developmental regions of the coleoptile, root,
and
in-
ternode (Figure
8)
is
similar
to
the expression pattern of
the
Os-fXP7, Os-EXP3,
and
Os-EXP4
genes (Figures
5
to
7).
This
is
further evidence that growth of these three vegetative
organs
is
determined
at
least in part by the level of expansin.
Conclusions
In
this
study, we
have
shown
that
(1) four expansin
genes
are
differentially expressed
in
the
coleoptile,
root,
and
inter-
node
of
deepwater rice;
(2)
developmental, hormonal, and
environmental signals affect expression of expansin genes
differentially;
and
(3) the expression pattern
of
some of the
expansin genes
is
highly correlated with wall extensibility
and
cell elongation. We propose that individual expansins
play different roles in cell expansion and differentiation and
that
expansin action contributes
to
the growth of the major
vegetative organs of rice and ultimately to survival
of
deep-
water rice under submergence.
METHODS
Plant Material
Rice (Olyza sativa cv Pin Gaew 56) seeds were germinated on two
sheets of moist Whatman No.
1
filter paper in a Petri dish in darkness
at 30°C for 3 days. Seedlings with coleoptiles
2-
to 2.5-cm long and
roots 4- to 6-cm long were selected for RNA isolation and cell wall
extension measurements. For experiments with internodes, rice
plants were grown as described previously (Stünzi and Kende, 1989).
Twenty-centimeter-long stem sections containing the uppermost in-
ternode were excised from
1
1
-
to 13-week-old plants, according to
the method of Raskin and Kende (1984a). For treatment in air and
gibberellic acid (GA,),
15
to
20
sections were incubated in distilled
water or in
50
JLM GA, such that the water level remained below the
basal node. For submergence, the sections, with a weight attached,
were completely immersed in 2.5-liter, 60-cm-deep plexiglass cylin-
ders containing distilled water (Kutschera and Kende, 1988). They
were incubated for up to 48 hr at 27°C under continuous light (cool-
white fluorescent tubes, 53 pmol m-2 sec-’; General Electric, Cleve-
land, OH).
cDNA Cloning and DNA Sequencing
The rice expansin
Os-EXP4
cDNA was cloned from a rice internode
cDNA libraty. A 750-bp probe containing the coding region was gen-
erated from
Os-EXPZ
by digestion using Sal1 and Eagl. Plaque-form-
ing units
(100,000)
of the cDNA library were screened with this probe.
Single positive plaques were screened with
Os-EXP
7-
and
Os-EXf2-
specific probes in the fifth screening. Hybridization of the plaque
blots on nitrocellulose (Protran; Schleicher
&
Schuell) was performed
in
6
x
SSC
(1
x
SSC
is
0.15
M NaCI,
0.015
M sodium citrate),
0.5%
SDS, and
5
X
Denhardt’s solution
(1
X
Denhardt’s solution is
0.01
%
Ficoll,
0.01
%
PVP,
0.01
%
BSA) at 62°C for 16 hr. The nitrocellulose
membranes were washed with 0.2
x
SSC and
0.1
%
SDS at 62°C. A
clone that hybridized with the probe from the
Os-EXf2
coding region
but not with
Os-EXf
7-
and Os-EXPZ-specific 3’ probes was iso-
lated. The insert from the phage DNA was isolated and cloned into
pBluescript SK- phagemid (Stratagene, La.Jolla, CA).
The DNA insert was sequenced using Taq FS DNA polymerase
and fluorescent-dideoxy terminators in a cycle-sequencing method.
The resultant DNA fragments were separated by electrophoresis and
analyzed using an automated Applied Biosystems (Foster City, CA)
373A Stretch DNA sequencer at the W.M. Keck facility of Yale Uni-
versity (New Haven, CT). This new rice expansin cDNA clone was
named
Os-EXP4.
Preparation
of
the Probes
Gene-specific probes were prepared mainly from the 3’ untranslated
regions of the respective transcripts. A 440-bp fragment between the
Hincll and Eagl restriction sites (including 142 bp from the coding re-
gion) was used for the Os-EXP7-specific probe, and a 580-bp frag-
ment between two Eagl restriction sites (including 72 bp from the
coding region) was used for the Os-EXP2-specific probe. Probes
were generated by polymerase chain reaction with oligonucleotide
primers corresponding to 3
‘
gene-specific regions of
Os-EXP3
(5’-GTCGCCCCGTCCAACTGGTTC-3‘
and 5‘-AAlTGGTGGGCA-
AAACAlTCA-3’) and
Os-EXP4
(5’-CCAGTTCTAGCCGCCACCGAC-
ATC-3’ and
5‘-ATTCCGlTGCAAGGCCATCACTCC-3’).
The length
of
the polymerase chain reaction probes was 270 bp for
Os-EXP3
(in-
cluding 35 bp from the coding region) and 457 bp for
Os-EXP4.
A
probe (656 bp) used for genomic DNA gel blot analysis was prepared
from the coding region of
Os-EXP7
by Hincll restriction digestion.
RNA and DNA Gel Blot Analyses
Total RNA was extracted according to the method of Puissant and
Houdeline
(1
990). Twenty micrograms of total RNA was separated on
formaldehyde gels and stained with ethidium bromide to ensure
equal loading of RNA. The RNA was then capillary transferred to ny-
lon membranes (Hybond N; Amersham) with 20
X
SSC for 16 hr. Ra-
diolabeled DNA probes were prepared using a random priming DNA
labeling kit (Boehringer Mannheim). The membranes were prehybrid-
ized at 42°C in
5
x
SSC,
10
X
Denhardt’s solution,
0.1
%
SDS,
0.1
M
potassium phosphate, pH 6.8, and 0.2 mg mL salmon sperm DNA
and hybridized at 42°C in
5
X
SSC, 30% formamide,
10%
dextran

1670 The Plant Cell
sulfate,
10
X
Denhardt's solution,
0.1
M potasium phosphate, pH
6.8, and 0.2 mg mL-I salmon sperm DNA. The membranes were
then washed twice for 40 min with 2
X
SSC and 0.2% SDS and twice
for 40 min with 0.2
x
SSC and
0.1
%
SDS at 65°C. Autoradiography
was performed using Hyperfilm-MP (Amersham) at -80°C with two
amplification screens. The message levels were quantified with a
Phosphorlmager (Molecular Dynamics, Sunnyvale, CA) and analyzed
with the ImageQuant program (Molecular Dynarnics). For the RNA
gel blot analyses with
€37
and 17s rRNA, the membranes were
stripped and probed again with one of the above probes.
For cDNA gel blot analysis, 2 ng of purified plasmid DNA was re-
striction digested, separated by electrophoresis, and transferred to a
nylon membrane. Prehybridization, hybridization, washing, and auto-
radiography of the blots were performed as described for RNA gel
blot analysis.
Genomic DNA was isolated from rice internodes using the method
described in Ausubel et al. (1987). Five micrograms of DNA was di-
gested either with EcoRl or with BamHl and Hindlll, separated in an
0.8%
agarose gel, and transferred to a nylon membrane. Prehybrid-
ization, hybridization, and autoradiography of the blots was per-
formed as described for RNA blot analysis, except that the blots
were washed at 55°C.
Measurement
of
Cell Wall Extension
Cell wall extension (creep) was measured according to the method of
Cosgrove (1989), as modified for rice tissue (Cho and Kende, 1997a).
Coleoptiles, roots, and the uppermost internodes were frozen at
-8O"C, abraded with carborundum (300 mesh; Fisher, Fair Lawn, NJ)
slurry, and thawed. One-centimeter-long sections of the coleoptiles,
5-mm-long sections of the roots, and 1.5-cm-long sections of the in-
ternodes were cut from each region (Figures 5A, 6A, and 7A) and
pressed between filter paper to remove water and cell sap. The sec-
tions were inserted between two clamps spaced
5
mm apart for the
coleoptile, 4 mm apart for the root, and
1
cm apart for the internode,
placed into an extensometer equipped with an angular displacement
transducer (Kutschera and Briggs, 1987), and subjected to constant
tension using a weight of
10
g for the coleoptile and the internode
and 5 g for the root. For acid-induced extension, the segments were
submerged in 50 mM Hepes-Tris buffer, pH 6.8, for 20 min, after which
the buffer was replaced with 50 mM sodium acetate buffer, pH 4.5.
ACKNOWLEDGMENTS
We thank Drs. Yoshiaki Nagamura and Takuji Sasaki (Rice Genome
Research Program, Tsukuba, Japan) for the generous gift
of
rice ex-
pressed sequence tag clones; Dr. Athanasios Theologis (U.S. De-
partment of Agriculture Plant Gene Expression Center, Albany, CA)
for the 17s rRNA probe; and Esther Van der Knaap (Michigan State
University-Department of Energy Plant Research Laboratory) for the
E37cDNA probe and the deepwater rice cDNA library. This work was
supported by Grant No. IBN 9407763 from the National Science
Foundation and Grant No. DE-FG02-91 ER20021 from the U.S. De-
partment of Energy.
Received April29, 1997; accepted July
11,
1997
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DOI 10.1105/tpc.9.9.1661
1997;9;1661-1671Plant Cell
H T Cho and H Kende
Expression of expansin genes is correlated with growth in deepwater rice.
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