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Plant
Physiol.
(1992)
99,
1226-1231
0032-0889/92/99/1
226/06/$01
.00/0
Received
for
publication
November
1,
1991
Accepted
February
13,
1992
Developmental
Regulation
of
(1-=3,1-.4)-,-Glucanase
Gene
Expression
in
Barley'
Tissue-Specific
Expression
of
Individual
Isoenzymes
Nada
Slakeski
and
Geoffrey
B.
Fincher*
Commonwealth
Centre
for
Protein
and
Enzyme
Technology,
Department
of
Biochemistry,
La
Trobe
University,
Bundoora,
Victoria,
3083
Australia
ABSTRACT
Two
genes
encode
(1-.3,1-.4)-I6-D-glucan
4-glucanohydrolase
(EC
3.2.1.73)
isoenzymes
El
and
Ell
in
barley
(Hordeum
vulgare
L.).
Specific
DNA
probes
have
been
used
in
Northern
analyses
to
examine
the
developmental
regulation
of
individual
(1-.3,1-+4)-
j-glucanase
genes
in
the
aleurone
and
scutellum
of
germinated
grain
and
in
young
leaves
and
young
roots.
In
aleurone
and
scutella
excised
from
germinated
grain,
mRNAs
encoding
both
isoenzymes
are
present
but
developmental
patterns
differ
between
the
two
tissues.
Thus,
levels
of
both
isoenzyme
El
and
Ell
mRNA
increase
significantly
in
the
aleurone
between
1
and
3
days
after
the
initia-
tion
of
germination.
In
the
scutellum,
isoenzyme
El
mRNA
predom-
inates
and
decreases
as
germination
proceeds.
lsoenzyme
El
mRNA
appears
in
young
leaves
approximately
8
days
after
the
initiation
of
germination
and
levels
rise
until
about
20
days.
Enzyme
activity
in
leaf
extracts
parallels
the
development
of
isoenzyme
El
mRNA.
No
isoenzyme
Ell
mRNA
is
detected
in
the
leaves
in
this
period.
Analysis
of
RNA
from
different
leaf
segments
indicates
that
the
isoenzyme
El
mRNA
is
distributed
relatively
evenly
along
the
length
of
the
leaf.
In
young
roots,
mRNA
encoding
(1-_+3,1-+4)-,B-glucan-
ase
isoenzyme
El
is
detected
at
high
levels
3
to
6
days
after
the
initiation
of
germination;
again,
little
or
no
isoenzyme
Ell
mRNA
is
found.
Overall,
transcription
of
the
(1-*3,1-.4)-f-glucanase
isoen-
zyme
Ell
gene
appears
to
be
restricted
to
the
germinating
grain,
whereas
isoenzyme
El
is
expressed
in
a
wider
range
of
tissues
during
seedling
development.
During
the
germination
of
barley
(Hordeum
vulgare
L.)
hydrolytic
enzymes
secreted
from
the
aleurone
layer
and
scutellum
depolymerize
starch
and
reserve
proteins
stored
in
the
cells
of
the
starchy
endosperm.
An
important
preliminary
event
in
this
process
is
the
removal
of
walls
of
the
starchy
endosperm
cells
because
the
wall
acts
as
a
physical
barrier
that
restricts
access
of
a-amylases,
peptidases,
and
other
hydrolases
to
substrates
within
the
cell
(7).
The
walls
in
the
starchy
endosperm
of
barley
are
composed
of
more
than
70%
by
weight
(1--3,1--4)-,
-glucan.
This
polysaccharide
is
de-
polymerized
by
the
action
of
two
(1-*3,1--*4)-f3-glucan
4-
'
This
work
was
supported
by
grants
to
G.B.F.
from
the
Australian
Research
Council
and
the
Grains
Research
and
Development
Corporation.
glucanohydrolases
(EC
3.2.1.73)
that
have
been
purified
and
characterized
and
are
known
to
be
encoded
by
separate
genes
(19,
29,
30).
Both
isolated
aleurone
layers
and
excised
scutella
secrete
(1--3,1-+4)-fl-glucanase
isoenzymes
in
vitro
(26)
and
this
has
been
confirmed
in
vivo
by
hybridization
histochemical
ex-
amination
of
the
location
of
mRNA
encoding
the
enzymes
(21).
Expression
of
(1--3,1--4)-#-glucanase
mRNA
is
de-
tected
first
in
the
scutellar
epithelium.
However,
after
1
d
(1-
3,1-4)-fl-glucanase
mRNA
levels
in
the
scutellum
de-
crease,
but
increase
progressively
in
the
aleurone
from
the
proximal
to
the
distal
end
of
the
grain
(21).
The
cDNA
probe
used
in
these
experiments
(8)
to
monitor
transcription
of
(1-
3,1-
4)-3-glucanase
genes
did
not
distinguish
between
the
two
individual
genes,
which
exhibit
92%
sequence
identity
in
their
protein-coding
regions
(24).
The
genes
encoding
barley
(1--3,1--4)-l3-glucanase
iso-
enzymes
EI
and
EII
have
recently
been
isolated
and
charac-
terized
(18,
24,
28)
together
with
near
full-length
cDNAs
for
both
isoenzyme
EI
and
EII
(24).
Although
the
two
genes
exhibit
a
high
degree
of
sequence
identity
in
their
5'
untrans-
lated
and
protein-coding
regions,
their
sequences
diverge
dramatically
in
the
3'
untranslated
region
(24).
This
permits
the
design
of
oligonucleotide
and
short
DNA
probes
to
spe-
cifically
identify
mRNA
encoding
the
individual
isoenzymes.
In
the
present
work,
we
have
used
isoenzyme-specific
probes
to
examine
the
differential
expression
of
the
two
genes
in
the
aleurone
and
scutellum
of
germinated
grain.
Further,
the
probes
have
been
used
to
investigate
the
regulation
of
(1-
3,1--+4)-fl-glucanase
gene
expression
during
the
devel-
opment
of
young
barley
leaves
and
in
young
roots
and
coleoptiles.
The
results
indicate
that
transcription
of
the
(1-.
3,1-4)-,B-glucanase
isoenzyme
EII
gene
is
'germination-spe-
cific,'
whereas
isoenzyme
El
is
expressed
both
in
the
germi-
nated
grain
and
in
developing
vegetative
tissues.
MATERIALS
AND
METHODS
Plant
Material
Barley
(Hordeum
vulgare
L.,
cv
Clipper)
was
obtained
from
Joe
White
Maltings
(Collingwood,
Victoria,
Australia)
and
from
the
John
Innes
Centre
for
Plant
Science
Research
(Nor-
wich,
UK).
For
germination
experiments,
grains
were
surface-
1226
EXPRESSION
OF
(1--3,1-*4)-fl-GLUCANASES
IN
BARLEY
sterilized
in
0.2%
(w/v)
silver
nitrate
for
20
min,
rinsed
with
0.5
M
NaCl,
washed
exhaustively
with
sterile
distilled
water,
and
soaked
for
16
h
in
sterile
distilled
water.
Grain
was
subsequently
germinated
in
sterile
Petri
dishes
at
220C
in
a
growth
cabinet,
or
on
vermiculite
in
a
glasshouse
under
ambient
conditions
of
light
and
temperature.
At
selected
times
after
the
initiation
of
germination,
tissues
were
har-
vested
for
RNA
isolation
and
for
the
measurement
of
enzyme
activity.
(1--3,1-.4)-#-Glucanase
Activity
Tissues
were
ground
under
liquid
nitrogen
and
extracted
with
50
mm
sodium
acetate
buffer,
pH
5.0
(containing
10
mm
sodium
azide,
10
mm
EDTA,
10
mm
DTT,
and
3
mm
PMSF)
at
40C
for
10
min.
Insoluble
material
was
removed
by
cen-
trifugation
and
the
extract
stored
at
-700C
prior
to
assay.
Protein
concentrations
in
the
extracts
were
measured
with
Coomassie
blue,
using
BSA
as
a
standard.
Enzyme
activity
was
measured
viscometrically,
using
water-soluble
barley
(1--3,1-+4)-f3-glucan
(Biocon
Pty.
Ltd.,
Cork,
Ireland)
as
substrate
(29).
A
unit
of
activity
is
defined
as
the
amount
of
enzyme
causing,
at
400C,
an
increase
of
1.0
in
the
reciprocal
specific
viscosity
per
min
of
a
5
mg/mL
(1-
3,1-
4)-fl-glucan
solution
in
50
mm
sodium
acetate
buffer,
pH
5.0
(containing
10
mm
sodium
azide
and
400
,ug/mL
BSA)
(29).
Specific
activity
is
calculated
as
the
activity
per
milligram
of
protein
in
the
extract.
For
the
estimation
of
cellulose
activity,
carbox-
ymethyl
cellulose
with
a
degree
of
substitution
of
0.54
(Edifas
B50;
ICI
Australia
Pty.
Ltd.)
was
used
as
a
substrate
in
viscometric
assays
(30).
RNA
Isolation
and
Northern
Analysis
Total
RNA
was
extracted
from
vegetative
tissue
ground
to
a
fine
powder
under
liquid
nitrogen
using
hot
phenol/LiCl
(27).
Total
RNA
samples
from
scutella
and
aleurone
layers
excised
from
intact
grain
1
and
3
d
after
the
initiation
of
germination
were
generously
provided
by
Dr.
Harri
Ranki.
The
RNA
samples
(3-20
,g)
were
separated
in
1%
(w/v)
agarose
gels
in
2.2
M
formaldehyde
(23)
and
transferred
to
nitrocellulose
filters
(Hybond-C
Extra,
Amersham).
Control
gels
were
stained
with
ethidium
bromide
for
comparison
of
the
relative
intensities
of
ribosomal
RNA
bands
and
loadings
were
adjusted
where
necessary
to
ensure
that
approximately
equal
amounts
of
RNA
were
applied
to
each
lane
of
the
gel.
Filters
were
prehybridized
and
hybridized
with
specific
cDNA
probes
as
described
by
Slakeski
et
al.
(24).
The
probe
sequences
corresponded
to
the
entire
3'
untranslated
regions
of
the
barley
(1--3,1-+4)-fl-glucanase
isoenzyme
EI
and
EIl
genes
(24),
and
their
specificity
was
routinely
checked
by
spotting
isoenzyme
El
and
EIl
cDNA
preparations
onto
the
nitrocellulose
filters
before
probing.
The
probes
were
pre-
pared
from
the
near
full-length
cDNAs
by
the
PCR2
using
conditions
described
previously
(24)
and
were
approximately
400
and
180
base
pairs
in
length
for
isoenzymes
El
and
ElI,
respectively.
The
fragments
were
recovered
from
agarose
gels
with
GeneClean
(Bio
101
Inc.,
La
Jolla,
CA)
and
labeled
with
2Abbreviation:
PCR,
polymerase
chain
reaction.
a-[32P]dCTP
using
random
sequence
hexanucleotides
as
primers
(6).
The
specific
activity
of
isoenzyme
EI
cDNA
probes
was
usually
5-
to
10-fold
higher
than
the
shorter
isoenzyme
EII
probes.
RESULTS
Expression
in
Germinated
Grain
Isoenzyme
EI
mRNA
of
approximately
1500
nucleotides
in
length
was
found
in
RNA
preparations
from
both
aleurone
layers
and
scutella
that
had
been
dissected
from
intact
barley
grain
1
or
3
d
after
germination
was
initiated
(Fig.
1).
After
1
d,
levels
of
isoenzyme
EI
mRNA
in
the
scutellum
were
relatively
high,
but
decreased
after
3
d.
In
contrast,
isoenzyme
EI
mRNA
in
the
aleurone
RNA
preparations
increased
sig-
nificantly
between
1
and
3
d
(Fig.
1).
Although
comparisons
of
signal
intensities
allow
the
relative
abundance
of
isoen-
zyme
EI
mRNA
to
be
visually
assessed
in
1-
and
3-d
aleurone
preparations,
or
in
1-
and
3-d
scutellar
preparations,
approx-
imately
four
times
more
scutellar
RNA
was
loaded
on
the
LLU
Z
ZD
ZDJ
D
LJ
-
-j
0
Days
<
)
germination:
1
3
1
3
El
Ell
o.
Figure
1.
Northern
analysis
of
RNA
preparations
from
scutella
and
aleurone
excised
from
intact,
germinated
barley
grain
1
and
3
d
after
the
initiation
of
germination.
Approximately
3
,ig
aleurone
and
12
ug
scutellar
RNA
was
loaded
onto
the
gels.
The
nitrocellulose
filter
probed
with
the
(1--3,1--+4)-f3-glucanase
isoenzyme
El
cDNA
fragment
was
exposed
for
18
h
and
the
filter
probed
with
the
isoenzyme
Ell
cDNA
fragment
for
48
h.
1227
SLAKESKI
AND
FINCHER
gel;
therefore,
care
must
be
exercised
in
comparing
the
inten-
sities
of
signals
between
the
aleurone
and
scutellar
prepara-
tions
in
Figure
1.
This
problem
is
compounded
by
the
highly
localized
expression
of
(1--3,1-.-4)-f3-glucanase
genes
in
the
single
layer
of
epithelial
cells
in
the
scutellum
(21),
because
mRNA
from
these
cells
will
be
diluted
with
mRNAs
extracted
simultaneously
from
metabolically
active
parenchymatous
and
vascular
tissues
of
the
scutellum
(7).
It
is
possible,
there-
fore,
that
the
abundance
of
isoenzyme
El
mRNA
in
individual
scutellar
epithelial
cells
may
be
as
high
or
even
exceed
corresponding
levels
in
aleurone
cells.
Careful
examination
of
the
northern
blot
(Fig.
1)
reveals
slight
differences
in
the
sizes
of
isoenzyme
El
mRNAs
from
the
scutellar
and
the
aleurone
extracts.
The
two
transcripts
presumably
represent
the
products
transcribed
from
different
transcription
start
points
on
the
gene
(2,
24)
or
to
differences
caused
by
the
use
of
multiple
polyadenylation
sites
(5).
When
the
specific
isoenzyme
Eli
probe
was
used,
an
in-
crease
in
binding
of
the
probe
to
aleurone
mRNA
was
again
observed
between
1
and
3
d
(Fig.
1).
Isoenzyme
ElI
mRNA
levels
in
the
scutellum
were
relatively
low
and
did
not
change
significantly
between
1
and
3
d.
When
comparing
isoenzyme
El
and
EII
signal
intensities
in
Figure
1,
it
should
be
remem-
bered
that
the
isoenzyme
El
probes
were
labeled
to
a
signif-
icantly
higher
specific
activity
than
the
shorter
isoenzyme
EII
probes.
Development
of
(1-.3,1--*4)-6-Glucanase
in
Young
Leaves
When
barley
seedlings
were
grown
in
the
glasshouse,
(1-
3,1--4)-f3-glucanase
activity
could
not
be
detected
in
aqueous
0.8
0
C.)
._
CL
cn
0.6
0.4
0.2
0
0
10
20
Days
germination
Figure
2.
Development
of
(1--3,1->4)-#-glucanase
enzyme
activity
in
extracts
of
young
leaves.
The
time
scale
represents
the
number
of
days
after
the
initiation
of
germination.
The
first
leaf
emerged
approximately
5
to
6
d
after
the
initiation
of
germination
and
by
15
d,
two
to
three
leaves
were
visible.
]
El!
Figure
3.
Northern
blot
showing
the
development
of
(1--3,1-->4)-
fl-glucanase
mRNA
in
young
leaves
at
increasing
times
after
the
initiation
of
germination.
The
nitrocellulose
filter
probed
with
the
(1-*3,1--*4)-,B-glucanase
isoenzyme
El-specific
DNA
was
exposed
for
18
h
and
the
filter
probed
with
the
isoenzyme
Ell
probe
for
3
d.
Approximately
equal
amounts
of
RNA
(10
Ag)
were
loaded
in
each
lane,
except
for
the
1
7-d
preparation,
where
6
,ug
was
loaded.
extracts
of
young
leaves
until
at
least
8
d
after
the
initiation
of
germination.
However,
activity
increased
rapidly
between
8
and
15
d
and
thereafter
increased
more
slowly
(Fig.
2).
Cellulases
are
also
capable
of
hydrolyzing
the
(1-*3,14)-j3-
glucan
substrate
used
in
this
assay
and
are
commonly
found
in
plant
tissues.
Accordingly,
the
leaf
extracts
were
assayed
for
cellulose
activity
using
carboxymethyl
cellulose
as
a
sub-
strate
(30),
but
no
activity
was
detected.
When
preparations
of
total
RNA
from
leaves
were
sub-
jected
to
northern
analysis,
isoenzyme
El
mRNA
was
first
detected
at
approximately
8
d
after
the
initiation
of
germi-
nation.
Levels
of
the
isoenzyme
El
mRNA
then
increased
quickly
up
to
23
d
and
thereafter
remained
about
the
same
(Fig.
3).
No
mRNA
encoding
(1--3,1-*4)-f3-glucanase
isoen-
zyme
EIl
was
found
in
the
leaf
RNA
preparations
during
the
entire
period
of
the
experiment
(Fig.
3).
Development
of
the
leaves
could
be
accelerated
by
growing
the
seedlings
in
a
growth
chamber
under
constant
light;
in
leaf
extracts
from
these
plants,
isoenzyme
El
mRNA
could
be
detected
6
d
after
the
initiation
of
germination.
It
should
be
emphasized
that
the
'young
leaf'
tissue
from
8-
to
15-d
seedlings
(Figs.
2
and
3)
included
all
leaves
in
the
seedling
and,
therefore,
would
include
leaves
at
different
develop-
mental
stages.
To
define
the
distribution
of
(1--3,1--+4)-,3-
glucanase
expression
within
a
single
leaf,
leaves
of
6-d-old,
growth
chamber-grown
plants
were
carefully
separated
from
the
grain
and
the
coleoptile
and
dissected
into
three
approx-
imately
equal
segments
corresponding
to
the
basal,
medial,
and
apical
portions
of
the
leaves.
Messenger
RNA
encoding
isoenzyme
El
was
detected
in
all
the
extracts
and,
although
it
appeared
to
be
slightly
more
abundant
in
the
apical
regions,
this
could
be
attributed
to
slight
differences
in
the
amount
of
1
228
Plant
Physiol.
Vol.
99,
1992
EXPRESSION
OF
(1--3,1-
,4)-fl-GLUCANASES
IN
BARLEY
RNA
loaded
on
this
gel
(Fig.
4).
Further,
in
similar
segments
from
10-,
13-,
and
16-d-old
leaves,
the
mRNA
was
also
distributed
evenly
along
the
leaf
(not
shown).
Expression
in
Coleoptiles
No
mRNA
corresponding
to
isoenzyme
El
or
Eli
could
be
detected
in
RNA
from
intact,
3-d-old
coleoptiles
(Fig.
5),
or
in
RNA
preparations
of
4-,
5-,
and
6-d-old
etiolated
coleop-
tiles
(results
not
shown).
Similarly,
no
expression
was
de-
tected
in
intact
coleoptiles
or
coleoptile
sections
after
treat-
ment
with
IAA
(25).
Expression
in
Young
Roots
In
northern
analyses
of
total
RNA
from
young
root
tissue,
mRNA
for
isoenzyme
EI
was
detected
at
relatively
high
levels
both
3
and
6
d
after
the
initiation
of
germination
(Fig.
5).
Isoenzyme
EII
expression
was
not
apparent
in
young
roots
at
these
developmental
stages,
even
after
prolonged
autoradi-
ography
(Fig.
5).
Two
possible
promoters
of
the
(1-*3,1-
4)-
13-glucanase
isoenzyme
EI
gene,
designated
the
distal
and
proximal
promoter,
have
been
identified
(24)
and
transcrip-
tion
from
these
promoters
was
tested
by
PCR.
When
the
young
root
RNA
preparations
were
used
as
a
template
for
the
amplification
of
specific
(1--3,1--4)-f3-glucanase
mRNAs
by
PCR
(24),
only
one
transcript
was
detected
and
this
corresponded
to
expression
from
the
distal
promoter
of
the
gene
(data
not
shown).
Thus,
expression
from
the
putative,
proximal
promoter
(24)
remains
to
be
demonstrated.
Coleoptile
Root
3
day
3
day
El
*1
Root
6
day
g
EU
<
Figure
5.
Northern
blots
of
RNA
from
3-d
coleoptiles
and
from
roots
3
and
6
d
after
the
initiation
of
germination.
In
each
case,
10
jig
RNA
was
loaded
onto
the
agarose
gels
and
exposure
times
were
18
h
for
the
filter
probed
with
(1--*3,1--*4)-f3-glucanase
isoenzyme
El
DNA
and
2
d
for
filters
probed
with
the
isoenzyme
ElI
cDNA
fragment.
LEAF
SECTION
A
M
B
El
so
"m
ElI
Figure
4.
Location
of
(1---3,1--*4)-#3-glucanase
isoenzyme
El
mRNA
in
apical
(A),
medial
(M),
and
basal
(B)
segments
of-young
leaves
of
barley
seedlings
using
northern
analysis.
Approximately
20
,Ag
total
RNA
was
loaded
in
each
lane of
the
gel
and
autoradiography
was
for
2
d
(isoenzyme
El
probe)
and
6
d
(isoenzyme
Ell
probe).
DISCUSSION
Transcription
of
genes
encoding
the
two
(13,14)-3-
glucanase
isoenzymes
in
germinated
barley
grain
has
been
demonstrated
by
northern
analysis
using
probes
specific
for
the
individual
gene
products.
In
intact
grain,
isoenzyme
El
mRNA
is
detected
in
the
scutellum,
with
levels
apparently
decreasing
between
1
and
3
d
after
germination
begins
(Fig.
1).
Relatively
low
levels
of
isoenzyme
ElI
gene
transcription
are
detected
in
the
scutellum
(Fig.
1).
These
results
with
intact,
germinated
grain
are
consistent
with
earlier
observa-
tions
that
excised
scutella,
incubated
in
vitro
at
250C,
secrete
(1-+3,1--4)-i3-glucanases
into
the
surrounding
medium
and
that
isoenzyme
El
predominates
(26).
The
identity
of
individ-
ual
isoenzymes
in
the
early
work
was
based
on
relative
electrophoretic
mobility
and
on
the
binding
of
polyclonal
antibodies
on
western
blots
(26).
Thus,
the
secretion
of
indi-
vidual
(1--3,1--*4)-f3-glucanase
isoenzymes
from
isolated
scutella
(26)
appears
to
accurately
reflect
the
transcription
patterns
of
the
corresponding
genes
in
the
scutellum
of
intact,
germinated
grain
(Fig.
1).
It
is
clear,
however,
that
the
tem-
poral
patterns
of
expression
differ.
In
isolated
scutella,
isoen-
zyme
El
is
secreted
for
3
to
4
d
(26),
whereas
in
intact
grain
the
level
of
mRNA
for
isoenzyme
El
in
the
scutellum
is
much
higher
1
d
after
the
initiation
of
germination
than
at
3
d
(Fig.
1).
Similarly,
examination
of
(1--3,1-*4)-,3-glucanase
mRNA
by
hybridization
histochemical
methods
showed
that
tran-
scriptional
activity
of
the
genes
in
the
scutellum
decreased
dramatically
after
1
d
(21).
Although
the
cDNA
probe
used
in
the
in
situ
hybridization
experiments
did
not
discriminate
1229
SLAKESKI
AND
FINCHER
between
isoenzyme
El
and
isoenzyme
EII
mRNA
species
(8,
21),
it
seems
likely
that
the
(1--3,1-
4)-f3-glucanase
mRNA
observed
in
the
scutellar
epithelial
cells
(21)
will
prove
to
encode
isoenzyme
El.
In
the
aleurone
of
intact,
germinating
barley,
both
isoen-
zyme
EI
and
EIl
genes
are
transcribed
and
the
levels
of
mRNA
for
each
isoenzyme
increase
between
1
and
3
d
(Fig.
1
and
ref.
21).
Again,
this
is
consistent
with
the
isoenzymes
secreted
from
isolated
aleurone
layers,
although
in
the
iso-
lated
layers
isoenzyme
EII
predominated
(26,
cf.
Fig.
1).
Primer
extension
analyses
have
also
indicated
that
isoenzyme
EII
mRNA
is
more
abundant
than
isoenzyme
EI
mRNA
in
grain
extracts
that
included
material
from
both
the
scutellum
and
aleurone
(18).
The
participation
of
both
the
aleurone
and
scutellum
in
the
secretion
of
(1--3,1-
4)-fl-glucanases,
to-
gether
with
the
spatial
and
temporal
patterns
of
expression,
are
consistent
with
observed
patterns
of
cell
wall
degradation
in
the
endosperm
(7).
Slakeski
et
al.
(24)
observed
that
the
gene
for
(1--3,1--+4)-
#-glucanase
isoenzyme
EI
is
transcribed
at
relatively
high
levels
in
young
leaves.
This
has
been
confirmed
here
and
it
is
also
shown
that
the
isoenzyme
El
gene
is
transcribed
in
young
roots
(Figs.
3
and
5).
In
these
tissues,
only
the
gene
encoding
(1--3,1-
4)-f3-glucanase
isoenzyme
EI
is
expressed;
no
isoenzyme
EII
mRNA
is
detected
(Figs.
3-5).
The
devel-
opment
of
isoenzyme
EI
mRNA
in
young
leaves
parallels
the
development
of
(1--3,1-.4)-f3-glucanase
enzyme
activity
(Figs.
2
and
3)
and
expression
levels
appear
to
be
essentially
similar
along
the
length
of
the
young
leaf
(Fig.
4).
The
functional
role
of
the
(1--3,1--+4)-#3-glucanases
in
germinated
barley
grain
is
almost
certainly
to
degrade
the
(1--3,1-4)-f3-
glucans
of
the
cell
walls
during
endosperm
mobilization.
However,
the
function
of
the
enzyme
in
developing
leaves
and
roots
(Figs.
2-4)
is
not
yet
clear,
but
is
likely
to
be
related
to
cell
wall
metabolism.
The
walls
of
young
leaves
contain
approximately
16%
(w/w)
(1--3,1-4)-f-glucan
(N.
Sakurai,
personal
communication).
The
high
levels
of
(13,14)-f-
glucanase
mRNA
in
developing
barley
leaves
have
prompted
speculation
that
the
enzyme
participates
in
the
formation
of
intercellular
airspaces
necessary
for
diffusion
of
carbon
diox-
ide,
oxygen,
and
water
vapor
in
parts
of
the
leaf
that
initially
have
no
direct
access
to
the
atmosphere
(24);
airspace
for-
mation
is
prerequisite
for
the
development
of
an
efficient
photosynthetic
system
in
young
leaves.
In
preliminary
exper-
iments,
monoclonal
antibodies
specific
for
(13,14)-,B-
glucanase
isoenzyme
EI
(13)
have
been
used
in
gold-labeling
studies
to
locate
the
enzyme
in
sections
of
developing
barley
leaves
(B.
Wells,
K.
Roberts,
G.B.
Fincher,
unpublished).
Although
airspace
formation
was
clearly
evident,
no
(1-+
3,1-4)4-
glucanase
antigen
could
be
detected
in
the
walls
of
separating
mesophyll
cells,
and
we
conclude
that
the
enzyme
is
not
involved
in
the
formation
of
these
airspaces.
Another
possible
role
for
the
(1--3,1--4)-#3-glucanase
in
developing
leaves
could
be
in
the
'loosening'
of
cell
wall
polysaccharides
during
cell
elongation.
The
process
of
cell
elongation
has
been
examined
in
great
detail
in
elongating
coleoptiles
of
barley
and
maize
(14,
22).
The
(1--3,1-+4)-i3-
glucan
content
of
cell
walls
from
4-d-old
barley
coleoptiles
is
approximately
19%
by
weight
(22)
but
the
level
decreases
progressively
during
coleoptile
growth.
Similarly,
the
(1--
3,1-4)-fl-glucan
content
of
young
barley
leaves
and
oat
coleoptiles
decreases
during
development
(3).
In
maize
co-
leoptile
walls,
levels
of
the
polysaccharide
initially
increase
(4)
but
then
decrease
from
14
to
3%
(w/w)
between
5
and
10
d
(20).
The
removal
of
(1--3,1---4)-3-glucans
from
maize
coleoptile
walls
has
been
attributed
to
an
exo-13-glucanase
that
is
capable
of
degrading
the
polysaccharide
to
glucose
(17),
although
recent
work
in
which
cell
elongation
was
inhibited
by
polyclonal
antibodies
raises
the
possibility
that
endohydrolases
may
also
be
involved
(15).
Presumably,
glu-
cose
released
during
(1-+3,1--4)-,B-glucan
degradation
in
these
tissues,
which
would
require
the
concerted
action
of
(1--3,1-4)-#-glucan
endohydrolases,
exohydrolases,
and
possibly
1-glucosidases
(7,
8),
provides
an
energy
source
to
support
seedling
growth.
In
barley,
no
(1-.3,1-*4)-3-glucanase
transcription
was
detected
in
coleoptiles
(24).
In
the
present
study,
we
have
reexamined
several
RNA
preparations
from
intact
coleoptiles
and
coleoptile
segments
(Fig.
5
and
other
data
not
shown),
together
with
RNA
from
coleoptiles
in
which
cell
elongation
was
significantly
exhanced
by
auxin
treatment
(25),
but
can
find
no
evidence
for
the
transcription
of
(1-
3,1-.4)-#3-glu-
canase
genes,
even
at
low
levels,
for
up
to
8
d
after
the
initiation
of
germination.
This
finding,
coupled
with
the
detection
in
barley
seedling
extracts
of
exo-13-glucanases
(J.
Wang,
P.B.
H0j,
G.B.
Fincher,
unpublished
data),
suggests
that
the
(1--3,1--4)-f-glucan
endohydrolases
may
not
me-
diate
in
auxin-mediated
cell
elongation
in
barley
coleoptiles
and
that
the
removal
of
(1-+3,1--*4)-#i-glucan
from
the
walls
of
elongating
cells
may
be
attributable
to
the
,B-glucan
exo-
hydrolases,
to
cellulases,
or
to
acid-mediated,
nonenzymic
solubilization
(12).
Cellulases
(more
correctly
xyloglucanases)
capable
of
hydrolyzing
wall
xyloglucans
have been
impli-
cated
in
cell
elongation
in
dicotyledons
(11),
and
these
en-
zymes
will
also
hydrolyze
(1--3,1-.4)-,3-glucans
(10).
How-
ever,
no
cellulase
activity
could
be
detected
in
the
extracts
of
young
leaves
or
coleoptiles
examined
here.
Although
we
are
as
yet
unable
to
formally
rule
out
the
possibility
that
the
(1-.+
3,1-34)-f-glucanase
in
leaves
mediates
partial
wall
hydrolysis
during
loosening
and
cell
elongation,
the
absence
of
(1--
3,1-
-4)-3-glucanase
expression
in
elongating
coleoptiles
in-
dicates
that
this
is
unlikely.
Finally,
(1--3,1-+4)-,B-glucanases
could
participate
in
the
differentiation
of
vascular
tissues
in
the
leaves,
based
on
the
changes
that
occur
in
cell
walls
in
these
tissues
and
on
observations
that
any
unlignified
primary
cell
walls
in
the
xylem
disappear
during
sieve
element
development
(9).
Such
a
role
for
(1--3,1-
4)-#-glucanases
in
barley
leaves
remains
to
be
demonstrated,
but
the
possibility
can
now
be
addressed
using
gold-labeling
techniques
and
monoclonal
antibodies
(13)
at
the
electron
microscope
level.
In
summary,
expression
of
the
gene
encoding
barley
(1--
3,1-
4)-f3-glucanase
isoenzyme
EII
has
so
far
been
detected
at
significant
levels
only
in
the
aleurone
of
germinating
grain,
whereas
isoenzyme
El
is
expressed
also
in
the
scutellum,
in
leaves,
and
in
roots.
This
raises
the
question
as
to
why
this
pattern
of
tissue-specific
expression
has
evolved.
Both
iso-
enzymes
exhibit
essentially
the
same
substrate
specificity
(29),
so
there
appears
to
be
no
reason
for
differential
expres-
sion
based
on
enzyme
action
patterns.
It
is
worth
noting
that
1230
Plant
Physiol.
Vol.
99,
1992
EXPRESSION
OF
(1-+3,1-*4)-fl-GLUCANASES
IN
BARLEY
Table
1.
(1-3,1-4)-f3-Glucanase
Expression
in
the
Germinated
Grain
and
Developing
Vegetative
Tissues
mRNA
Transcript
Levels
Tissue
El
Ell
Germinated
grain
Aleurone
1
day
++
trace
Aleurone
3
day
++++ ++++
Scutellum
1
day
++
++
Scutellum
3
day
+
++
Vegetative
tissue
6-d
leaf
trace
6-d
root
++++
3-d
coleoptile
3-d
root
++++
the
aleurone
is
triploid,
whereas
the
other
tissues
examined
here
are
diploid,
but
again
there
is
no
indication
that
this
difference
is
related
to
the
expression
patterns.
It
has
also
been
observed
that
the
promoter
of
the
gene
encoding
isoen-
zyme
EI
contains
a
150-nucleotide
pair
insertion
that
is
absent
in
the
isoenzyme
EII
promoter
and
that
this
segment
could
be
responsible
for
the
differential
expression
patterns
(28).
This
can
now
be
investigated.
We
have
attempted
to
summarize
the
mRNA
levels
in
a
semi-quantitative
fashion
in
Table
I.
Given
the
role
of
GA3
in
the
induction
of
(1--3,1--o4)-j3-glucanase
and
other
pro-
teins
in
tissue
from
germinating
grain
(7,
26),
together
with
the
central
importance
of
auxin
in
plant
vascular
differentia-
tion
(1)
and
in
cell
extension
(16),
the
tissue-specific
regula-
tion
of
individual
barley
(1-+3,1--4)-,B-glucanase
genes
by
phytohormones
has
now
been
examined
and
is
reported
elsewhere
(25).
ACKNOWLEDGMENTS
We
thank
Drs.
Maria
Hrmova
and
Robyn
van
Heeswijck
for
helpful
discussions.
We
are
particularly
grateful
to
Dr.
Harri
Ranki
for
providing
RNA
preparations
from
germinating
barley
grains.
N.S.
acknowledges
receipt
of
a
La
Trobe
University
Post-Graduate
Re-
search
Award.
LITERATURE
CITED
1.
Aloni
R
(1987)
Differentiation
of
vascular
tissues.
Annu
Rev
Plant
Physiol
38:
179-204
2.
Brown
JWS,
Feix
G
(1990)
A
functional
splice
site
in
the
5'
untranslated
region
of
a
zein
gene.
Nucleic
Acids
Res
18:
111-117
3.
Buchala
AJ,
Wilkie
KCB
(1974)
Total
hemicellulose
from
Hor-
deum
vulgare
plants
at
different
stages
of
maturity.
Phyto-
chemistry
13:
1347-1351
4.
Carpita
NC
(1984)
Cell
wall
development
in
maize
coleoptiles.
Plant
Physiol
76:
205-212
5.
Dean
C,
Tamaki
S,
Dunsmuir
P,
Favreau
M,
Katayama
C,
Dooner
H,
Bedbrook
J
(1986)
mRNA
transcripts
of
several
plant
genes
are
polyadenylated
at
multiple
sites
in
vivo.
Nu-
cleic
Acids
Res
14:
2229-2240
6.
Feinberg
AP,
Vogelstein
B
(1983)
A
technique
for
radiolabeling
DNA
restriction
endonuclease
fragments
to
high
specific
activ-
ity.
Anal
Biochem
132:
6-13
7.
Fincher
GB
(1989)
Molecular
and
cellular
biology
associated
with
endosperm
mobilization
in
germinating
cereal
grains.
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