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3 FiguresProperties of Barley Seed Chitinases and Release of Embryo-Associated Isoforms during Early Stages of Imbibition
Abstract
Barley (Hordeum vulgare L.) seeds contain at least five proteins with chitinase (CH) activity. Two of these (CH1 and CH2) are found primarily in the aleurone and endosperm tissues, and the other three (CH3, CH4, and CH5) are enriched in the embryo. From the bran fraction, three of these CHs (CH1, CH2, and CH3) were purified to apparent homogeneity. These three CHs have apparent molecular masses of 27, 34, and 35 kilodaltons and isoelectric points of 9.3, 9.2, and 8.7, respectively. CH2 and CH3 have amino terminal sequences resembling a portion of the chitin-binding domain of lectins and other plant defense proteins. CH1 lacks this domain. All three CHs exhibit antifungal activity and inhibit the mycelial growth of some species of trichoderma and Fusarium in vitro. During the early period of imbibition by seeds, two of the embryo-associated CHs are selectively released into the surrounding aqueous medium.
Figures
Plant
Physiol.
(1992)
99,
1009-1014
0032-0889/92/99/1
009/06/$01
.00/0
Received
for
publication
October
25,
1991
Accepted
February
3,
1992
Properties
of
Barley
Seed
Chitinases
and
Release
of
Embryo-
Associated
Isoforms
during
Early
Stages
of
Imbibition'
Mark
Swegle,
Karl
1.
Kramer,
and
Subbaratnam
Muthukrishnan*
Department
of
Biochemistry,
Willard
Hall,
Kansas
State
University,
Manhattan,
Kansas
66506
(M.S.,
S.M.);
and
United
States
Grain
Marketing
Research
Laboratory,
Agricultural
Research
Service,
United
States
Department
of
Agriculture,
Manhattan,
Kansas
66502
(K.J.K.)
ABSTRACT
Barley
(Hordeum
vulgare
L.)
seeds
contain
at
least
five
proteins
with
chitinase
(CH)
activity.
Two
of
these
(CH1
and
CH2)
are
found
primarily
in
the
aleurone
and
endosperm
tissues,
and
the
other
three
(CH3,
CH4,
and
CH5)
are
enriched
in
the
embryo.
From
the
bran
fraction,
three
of
these
CHs
(CH1,
CH2,
and
CH3)
were
purified
to
apparent
homogeneity.
These
three
CHs
have
apparent
molecular
masses
of
27,
34,
and
35
kilodaltons
and
isoelectric
points
of
9.3,
9.2,
and
8.7,
respectively.
CH2
and
CH3
have
amino
terminal
sequences
resembling
a
portion
of
the
chitin-binding
domain
of
lectins
and
other
plant
defense
proteins.
CH
1
lacks
this
domain.
All
three
CHs
exhibit
antifungal
activity
and
inhibit
the
mycelial
growth
of
some
species
of
Trichoderma
and
Fusarium
in
vitro.
During
the
early
period
of
imbibition
by
seeds,
two
of
the
embryo-associated
CHs
are
selectively
released
into
the
surround-
ing
aqueous
medium.
CHs2
(EC
3.2.1.14)
catalyze
the
hydrolysis
of
1(1,4)
link-
ages
between
N-acetylglucosamine
(2-acetamido-2-deoxy-
glucopyranoside)
residues
in
the
linear
homopolymer,
chitin.
They
are
widely
distributed
enzymes
and
are
found
in
micro-
organisms,
plants,
and
animals.
A
role
for
the
enzymes
in
plant
defense
against
fungal
attack
is
suggested
by
the
ab-
sence
of
chitin
in
higher
plants
(1),
its
presence
in
fungal
cell
walls
(2),
and
the
finding
that
plant
CHs
inhibit
spore
ger-
mination
and
mycelial
growth
of
certain
fungi
in
vitro
(5,
21,
24).
When
a
CH
was
used
in
combination
with
a
((1,3)-
glucanase
(15)
or
a
ribosome-inactivating
protein
(13),
the
mycelial
growth
of
a
wider
range
of
fungal
genera
was
inhibited.
CHs
have
been
isolated
from
tissues
of
many
higher
plants
(5)
including
wheat
and
barley
(Hordeum
vulgare
L.)
seeds
(8,
12,
18)
and
from
barley
leaves
infected
with
fungi
(10).
They
are
constitutively
expressed
at
low
levels
in
leaves
and
at
high
levels
in
roots
(25)
and
seeds
(13).
Increased
levels
of
gene
expression
or
enzymic
activity
have
been
observed
in
leaves
after
inoculation
with
fungi
(22),
bacteria
(6),
or
viruses
1
This
is
contribution
92-180-J
of
the
Kansas
Agricultural
Experi-
ment
Station,
in
cooperation
with
the
Agricultural
Research
Service,
U.S.
Department
of
Agriculture.
2
Abbreviations:
CH,
chitinase;
SPB,
sodium
phosphate
buffer;
DPA,
days
postanthesis;
ATCC,
American
Type
Culture
Collection;
SSC,
standard
saline
citrate;
IEF,
isoelectric
focusing.
(33).
The
enzymes
also
can
be
induced
by
wounding
(7)
or
by
exposure
to
ethylene
(4),
fungal
cell
wall
preparations
(7),
or
abiotic
elicitors
such
as
salicylic
acid
and
mercuric
chloride
(19).
Not
only
CHs,
but
also
,B(1,3)-glucanases
and
certain
other
proteins
accumulate
to
high
levels
following
these
treatments
or
pathogen
attacks.
Collectively,
all
of
these
proteins
are
called
b-proteins
or
pathogenesis-related
proteins
(31).
The
rather
large
number
of
factors
that
trigger
the
accumulation
of
these
proteins
and
the
lack
of
a
high
degree
of
pathogen
specificity
in
their
induction
imply
that
they
are
part
of
a
general
response
of
plants
to
stress.
Their
induction
has
been
correlated
with
greater
resistance
to
subsequent
pathogen
attack
(31).
These
proteins
may
be
partially
respon-
sible
for
a
systemically
acquired
resistance
(32).
The
induction
of
CHs
and
other
defense-related
proteins
during
flowering
and
seed
germination
may
protect
plant
tissues
from
micro-
bial
attack
during
abrupt
developmental
changes
(20).
During
a
previous
study
of
genes
expressed
in
the
aleurone
layer
cells
of
germinating
barley
seeds,
we
identified
a
cDNA
clone
encoding
a
CH
(29).
Results
of
western
blot
analysis
indicated
the
presence
of
at
least
two
CHs
in
barley
seeds.
We
have
now
analyzed
the
distribution
of
CHs
in
barley
seed
tissues
in
greater
detail
and
have
purified
and
partially
char-
acterized
three
distinct
enzymes.
One
of
these
purified
CHs
is
located
predominantly
in
the
embryo
and
is
released
from
the
seed
very
early
during
imbibition.
The
other
two
enzymes
are
more
abundant
in
the
aleurone
and
endosperm
tissues.
MATERIALS
AND
METHODS
Plant
Material
Barley
(Hordeum
vulgare
L.
cv
Himalaya)
seeds
were
ob-
tained
from
the
Agronomy
Department,
Washington
State
University.
Barley
plants
were
maintained
in
a
growth
cham-
ber
at
22
to
260C
with
a
15/9-h
light/dark
cycle.
Developing
spikes
were
harvested
after
anthesis
at
intervals
of
5
d
up
to
45
DPA.
Seed
tissues
were
dissected,
immediately
frozen
in
liquid
nitrogen,
and
stored
at
-700C.
For
imbibition
studies,
whole
seeds
were
surface
sterilized
by
mixing
with
70%
(v/v)
ethanol
for
4
min
on
a
magnetic
stirrer.
They
were
rinsed
four
times
with
water,
once
quickly
and
three
times
for
3
min
on
the
magnetic
stirrer.
One
hundred
seeds
were
placed
crease
side
down
in
15-
x
100-
mm
Petri
dishes
containing
5
mL
of
water
and
kept
at
250C.
Samples
that
imbibed
longer
than
12
h
were
supplemented
with
an
additional
5
mL
of
water.
At
each
sampling
time,
all
1009
Plant
Physiol.
Vol.
99,
1992
water
was
removed,
and
volumes
were
recorded.
Samples
for
western
blot
analysis
were
prepared
by
TCA
precipitation
of
2%
of the
volume
of
recovered
material,
which
was
equivalent
to
the
protein
released
from
two
seeds.
Fungal
Material
Cultures
of
Trichoderma
harzianum
(ATCC
52443)
and
Trichoderma
viride
(ATCC
52438)
were
obtained
from
the
ATCC.
All
other
fungi
were
obtained
from
Dr.
John
Leslie,
Kansas
State
University.
Mycelial
growth
inhibition
assays
were
done
on
carrot
juice
agar
plates
at
250C
(21).
An
agar
cube
containing
mycelia
was
placed
at
the
center.
When
colonies
were
about
3
cm
in
diameter,
sterile
paper
discs
were
placed
1
mm
from
the
growing
front.
Test
solutions
were
applied
to
the
discs
in
10-
1sL
volumes,
and
the
plates
were
incubated
until
discs
spotted
with
water
or
buffer
were
overgrown.
CH
Purification
by
Chitin
Affinity
Chromatography
Barley
bran
(250
g)
was
the
starting
material
for
protein
purification.
The
bran
abraded
by
pearling
of
whole
seeds
consisted
of
all
of
the
pericarp
and
embryo,
most
of
the
aleurone,
and
some
of
the
endosperm.
The
bran
was
mixed
with
25
mm
SPB,
pH
7.0,
at
5
mL/g.
After
insoluble
material
was
removed
by
centrifugation
at
13,000g,
the
supernatant
was
fractionated
by
ammonium
sulfate
precipitation.
The
30
to
60%
fraction
was
dialyzed
against
SPB
and
mixed
in
a
beaker
with
SPB-equilibrated
chitin.
Colloidal
crab
shell
chi-
tin
used
for
this
step
was
prepared
as
described
previously
(21).
After
4
h
of
mixing,
the
chitin
was
washed
three
times
with
30
mL
of
SPB,
followed
by
three
more
washes
with
30
mL
of
25
mm
sodium
citrate,
pH
4.0,
in
a
beaker.
The
chitin
was
then
placed
in
a
column
(1
x
20
cm),
and
bound
material
was
eluted
with
20
mm
acetic
acid,
pH
3.2.
Two
CHs
(CH2
and
CH3)
were
eluted
in
a
single
peak
and
were
subsequently
resolved
in
a
Bio-Rad
Rotofor
(ampholytes
8-10;
Serva)
according
to
the
manufacturer's
directions.
Pooled
fractions
were
dialyzed
against
SPB.
To
isolate
CH1,
which
did
not
bind
to
chitin
under
these
conditions,
the
first
two
batch
supernatants
from
the
proce-
dure
described
above
were
precipitated
with
ammonium
sulfate
(60%)
and
dialyzed
against
50
mm
sodium
citrate,
pH
4.5.
One-
to
2-milliliter
aliquots
(60
mg
protein/mL)
were
applied
to
the
chitin
column
equilibrated
with
the
same
buffer.
Elution
with
1
M
NaCl
in
10
mm
Tris,
pH
7.4,
resulted
in
substantial
purification
of
CH1.
Pooled
fractions
contain-
ing
CH
were
dialyzed
against
50
mm
Tris,
pH
7.5,
and
loaded
onto
a
carboxymethyl-cellulose
(Sigma)
column
(1.5
x
30
cm)
equilibrated
with
the
same
buffer.
A
step
gradient
from
0
to
80
mm
NaCl
at
10
mm
intervals
was
applied.
The
putative
CH,
CH4,
was
recovered
in
the
flow-through
fractions
from
the
carboxymethyl-cellulose
column.
CH1
was
eluted
with
60
mm
NaCl.
Pooled
fractions
were
dialyzed
against
SPB.
Protein
concentration
was
determined
with
the
Bio-Rad
pro-
tein
assay
reagent
with
immunoglobulin
G
as
the
standard.
Amino
Acid
Sequencing
Thirty
micrograms
of
CH1,
CH2,
and
CH3
were
sequenced
with
an
Applied
Biosystems
model
477A
sequencer
by
Dr.
William
Morgan,
University
of
Missouri-Kansas
City.
Amino
acid
derivatives
were
identified
by
HPLC.
PAGE
SDS-PAGE
was
performed
as
described
previously
(27)
using
10%
polyacrylamide
minigels
(Hoefer
model
SE250)
for
immunoblotting
and
12%
gels
(BRL
model
V16)
for
mol
wt
estimation.
Proteins
were
stained
with
Coomassie
brilliant
blue
R-250.
Mol
wt
markers
were
from
Sigma.
Discontinuous
native
PAGE
at
low
pH
was
performed
according
to
the
method
of
Blackshear
(3).
Activity
Gels
Activity
gels
containing
glycol
chitin
were
prepared
and
processed
as
described
by
Trudel
and
Asselin
(30).
Immunoblotting
A
Polyblot
apparatus
(American
Bionetics)
was
used
ac-
cording
to
the
manufacturer's
directions
to
transfer
proteins
to
nitrocellulose
(Schleicher
and
Schuell).
Blots
were
incu-
bated
with
antibodies
raised
in
rabbits
against
CH1
or
a
bean
leaf
CH.
The
bean
leaf
CH
antiserum
was
a
gift
from
Dr.
Richard
Broglie,
Du
Pont
Co.
The
antigen-antibody
complex
was
detected
with
a
goat
anti-rabbit
immunoglobulin
G
conjugated
with
horseradish
peroxidase
(Fisher)
using
the
horseradish
peroxidase
color
development
reagent
(Bio-Rad).
Enzyme
Assays
Tritiated
chitin
(670,000
cpm/mg)
was
prepared
as
de-
scribed
by
Molano
et
al.
(17).
Each
100
,uL
of
standard
assay
mixture
contained
0.5
mg
of
chitin,
5
usmol
of
indicated
buffer,
and
enzyme
solution.
Reactions
were
stopped
after
1
h
at
250C
by
the
addition
of
300
,uL
of
10%
(w/v)
TCA.
After
centrifugation
for
5
min
at
6,000g,
the
tritiated
chitin
oligo-
saccharides
contained
in
200
,uL
of
supernatant
were
meas-
ured.
Specific
activities
were
determined
from
initial
veloci-
ties
at
300C
according
to
the
method
of
Boller
et
al.
(4)
and
expressed
as
nanokatals
per
milligram
of
protein.
One
na-
nokatal
equals
1
nmol
of
2-acetamido-2-deoxy-D-glucopyr-
anoside
equivalents
released
per
second.
Lysozyme
activity
was
measured
as
described
by
Mauch
et
al.
(14).
,B-N-acetylglucosaminidase
activity
was
measured
at
pH
4.5
as
described
by
Roberts
and
Selitrennikoff
(21).
Chi-
tosanase
activity
was
tested
by
substituting
glycol
chitosan
(Sigma)
for
glycol
chitin
in
substrate
overlay
gels.
pH
Optimum
Determination
The
effect
of
pH
on
enzymatic
activity
was
determined
with
the
following
buffers
at
50
mm
concentrations:
HCl-KCl
at
pH
2;
sodium
citrate
at
pH
3
to
6;
sodium
phosphate
at
pH
7
to
8;
glycine
at
pH
9
to
10;
and
trisodium
phosphate
at
pH
11.
1010
SWEGLE
ET
AL.
BARLEY
SEED
CHITINASE
RELEASE
DURING
IMBIBITION
Isoelectric
Point
Determination
Samples
were
subjected
to
electrophoresis
in
IEF
grade
agarose
(FMC)
slab
gels
according
to
the
manufacturer's
instructions.
IEF
standards
were
from
Sigma.
RNA
Analysis
Total
RNA
was
recovered
from
tissues
ground
to
a
powder
in
liquid
nitrogen
and
extracted
with
4
mL
of
100
mm
sodium
glycinate
(pH
8.0),
10
mm
EDTA,
100
mm
NaCl,
1%
(w/v)
SDS,
and
0.2%
(w/v)
proteinase
K.
After
the
mixture
was
centrifuged
for
10
min
at
6000g,
the
supernatant
was
brought
to
8
mL
by
the
addition
of
70%
guanidine
isothiocyanate,
mixed
with
2
g
of
CsCl, placed
over
a
2-mL
cushion
of
5.7
M
CsCl
and
100
mm
EDTA,
and
centrifuged
for
20
h
at
174,000g.
Pellets
were
dissolved
in
10
mm
Tris,
pH
8.0,
containing
1
mM
EDTA.
Aliquots
of
10
,g
were
subjected
to
electrophoresis
in
a
1.2%
agarose-formaldehyde
gel
and
blot-
ted
onto
nitrocellulose
with
20X
SSC.
(lX
SSC
is
0.15
M
NaCl,
0.015
M
sodium
citrate.)
The
blot
was
hybridized
for
16
h
with
insert
DNA
from
a
barley
CH
cDNA
clone
(29)
that
was
labeled
using
a
random
primer,
[32P]dCTP,
and
the
Klenow
fragment
of
DNA
polymerase
I.
The
blot
was
washed
twice
for
40
min
at
500C
in
2X
SSC
and
0.1%
SDS,
once
for
40
min
at
650C
in
0.1X
SSC
and
0.1%
SDS,
and
then
exposed
to
X-ray
film
for
58
h.
RNA
size
markers
were
from
BRL.
CH
4-
1
2
3
X6i~,
I"~e
W
.
-,
3-
4
5
6
7
8
9
66
.
.
2-
5-
45
...36
2.
24
20
14
Figure
2.
Native
PAGE
(lanes
1-5)
and
SDS-PAGE
(lanes
6-9)
of
barley
seed
CHs.
CHs
are
identified
in
the
left
margin.
Lane
1,
CH
activity
gel
overlay
of
material
in
lane
2;
lanes
2-9,
stained
with
Coomassie
blue;
lane
2,
30
to
60%
fraction
of
bran
extract
(450
Mug);
lane
3,
CH1
(4
Mug);
lane
4,
CH3
(4
Mg);
lane
5,
CH2
(4
Ag);
lane
6,
CH1
(10
Mg);
lane
7,
CH3
(10
Mug);
lane
8,
CH2
(10
Mg);
and
lane
9,
standard
proteins
with
mol
wts
x
10-3
listed
in
right
margin.
RESULTS
Tissue
Distribution
The
distribution
of
CHs
within
barley
seeds
was
examined
with
substrate
overlay
gels
after
electrophoresis
of
extracts
from
pericarp,
aleurone,
endosperm,
and
embryo
(including
the
scutellum)
tissues
dissected
from
imbibed,
mature
seeds.
Crude
extracts
were
subjected
to
discontinuous
native
PAGE
at
low
pH,
and
the
resulting
gel
was
assayed
for
CH
activity
with
a
glycol
chitin-containing
overlay
gel
(Fig.
1).
Two
CHs,
CH1
and
CH2,
accounted
for
most
of
the
activity
in
the
aleurone
and
endosperm
tissues
(lanes
3
and
4).
A
third
relatively
abundant
CH,
CH3,
was
located
primarily
in
the
embryo
(lane
5).
Two
additional
proteins
that
appeared
to
be
CHs
(CH4
and
CH5)
also
were
found
primarily
in
the
em-
bryo.
Little
CH
activity
was
detected
in
the
pericarp
extract
(data
not
shown).
2
3
4
5
6
CHll
4
--
2
S
1
Figure
1.
CH
activity
gel
analysis
of
crude
extracts
of
barley
tissues.
Lane
1,
Flour
(20
Mg);
lane
2,
bran
(60
Mg);
lane
3,
aleurone
(30
Ag);
lane
4,
endosperm
(30
Mg);
lane
5,
embryo
(90
Mg);
lane
6,
protein
released
from
one
seed
after
18
h
of
imbibition.
To
determine
the
best
milling
fraction
for
use
as
a
starting
material
for
the
purification
of
the
CHs,
mature
barley
seeds
were
pearled
to
remove
the
bran,
which
contained
all
of
the
embryo
and
pericarp,
most
of
the
aleurone
layer,
and
some
of
the
endosperm.
The
remaining
polished
seeds
were
milled
into
flour.
Enzyme
activity
gel
analysis
of
crude
extracts
of
the
flour
and
bran
fractions
showed
that
the
former
contained
primarily
CH1
and
CH2
and
that
the
latter
had
all
five
of
the
enzymes
(Fig.
1,
lanes
1
and
2).
Therefore,
the
bran
fraction
instead
of
the
whole
seed
was
extracted
as
an
initial
enrichment
step
for
purification
of
these
enzymes.
CH
Purification
A
combination
of
purification
methods,
including
chitin
affinity
chromatography
and
preparative
scale
IEF
or
cation
exchange
chromatography,
was
used
to
isolate
CH1,
CH2,
and
CH3
from
bran
extracts.
The
apparent
homogeneity
of
these
preparations
was
demonstrated
by
native
PAGE
and
SDS-PAGE
(Fig.
2).
Yields
of
CH1,
CH2,
and
CH3
were
90,
80,
and
80
,ug,
respectively,
per
g
of
bran
(equivalent
to
20
mg
of
total
extractable
protein).
A
fourth
putative
CH,
CH4,
was
partially
purified
but
was
not
characterized
further.
CH5
was
not
purified.
The
apparent
molecular
masses
of
CH1,
CH2,
and
CH3
were
27, 34,
and
35
kD,
respectively.
Physical
and
Enzymic
Properties
Some
of
the
properties
of
the
three
purified
CHs
are
listed
in
Table
I.
Although
CH2
and
CH3
were
similar
in
size,
they
differed
in
isoelectric
point
and
in
relative
mobility
under
native
PAGE
conditions
(Fig.
2,
lanes
4
and
5).
All
of
the
enzymes
were
basic
proteins
with
acidic
pH
optima
and
were
1
011
1-
Plant
Physiol.
Vol.
99,
1992
Table
I.
Physical
and
Enzymic
Properties
of
Barley
Seed
CHs
CH1
CH2
CH3
Apparent
molecular
mass
(kD)
27
34
35
Isoelectric
point
9.3
9.2
8.7
Chitin-binding
domain
None
Present
Present
pH
optimum
4.5
4
3.5
and
6
pH
range
for
50%
activity
3.5-7
3-7
3-9
Specific
activity'
94
±
9
133
±
12
70
±
6
'
Specific
activities
are
expressed
as
nanokatals
per
milligram
of
protein.
One
nanokatal
equals
1
nmol
of
2-acetamido-2-deoxy-D-
glucopyranoside
equivalents
released
per
second.
Values
shown
are
the
means
(±SD)
of
three
assays.
CH
mRNA
Accumulation
during
Seed
Development
A
barley
aleurone
CH
cDNA
probe
(29)
was
used
for
northern
blot
analysis
of
total
RNA
isolated
from
developing
seed
tissues
(Fig.
3).
Hybridizable
mRNA
was
detected
by
15
DPA
in
both
the
endosperm
and
aleurone
and
was
present
in
both
tissues
through
40
DPA.
The
amount
in
whole
seeds
at
45
DPA
was
less
than
that
observed
at
40
DPA
in
either
tissue
alone,
suggesting
that
a
net
loss
of
these
messages
occurred
at
the
very
end
of
seed
development.
CH
mRNA
was
generally
maintained
at
higher
levels
in
the
aleurone
tissue
than
in
the
endosperm,
except
at
30
DPA,
the
time
at
which
the
mRNA
level
peaked
in
the
latter
tissue.
Hybridiz-
able
mRNA
was
not
detected
in
the
developing
embryo
(data
not
shown).
recognized
by
polyclonal
antibodies
raised
against
either
CH1
or
a
bean
leaf
CH
(data
not
shown).
CHi,
CH2,
and
CH3
were
enzymically
active
when
glycol
chitin
or
tritiated
chitin
was
used
as
a
substrate,
but
they
were
inactive
toward
glycol
chitosan
(data
not
shown).
A
separate
basic
protein
active
toward
glycol
chitosan
was
detected
in
crude
extracts
of
aleurone
and
endosperm
tissues,
but
this
chitosanase
activity
was
not
associated
with
any
of
the
CHs
and
was
not
studied
further.
When
a
preparation
of
Micrococcus
lysodeikticus
cell
walls
was
used
as
a
substrate,
all
three
of
the
CHs
were
found
to
exhibit
a
weak
lysozyme-
like
activity.
CH3
had
the
greatest
lysozyme
activity
(5%)
and
CH1
the
least
(1%)
on
a
per
microgram
basis,
when
compared
to
chicken
egg
white
lysozyme
(data
not
shown).
These
results
indicated
that
the
purified
barley
proteins
are
endochitinases.
The
barley
CHs
also
were
tested
for
fl-N-acetylglucosamin-
idase
activity
with
the
substrate
analog
p-nitrophenyl-fl-D-
N,N'-diacetylchitobiose
(Sigma).
Ten
micrograms
of
each
CH
failed
to
release
p-nitrophenol
during
24-h
incubations
at
370C.
Molting
fluid
from
Manduca
sexta
(tobacco
hornworm),
which
contains
a
f3-N-acetylglucosaminidase
(9),
was
used
as
a
positive
control.
Molting
fluid
samples
containing
1.0
and
0.1
,ug
of
total
protein
released
p-nitrophenol
within
1
and
24
h,
respectively,
under
the
same
assay
conditions.
All
of
the
results
obtained
with
polysaccharide
and
oligosaccharide
substrates
indicated
that
the
barley
enzymes
have
a
different
mode
of
action
from
that
of
an
exocleaving
3-N-acetylglu-
cosaminidase
(21)
and
that
they
act
primarily
as
endocleav-
ing
CHs
with
a
greater
specificity
for
a
,B(1-4)
polymer
of
N-acetyl-D-glucosamine
than
for
a
(3(1-4)
copolymer
of
N-
acetylglucosamine
and
N-acetylmuramic
acid.
Antifungal
Properties
Fungal
growth
assays
on
agar
plates
revealed
that,
at
1
Isg/
disc,
all
three
CHs
inhibited
mycelial
growth
of
T.
harzianum
(data
not
shown),
a
fungal
saprophyte
whose
cell
wall
con-
tains
chitin
(2).
Greater
inhibitions
were
seen
with
10
,ug.
Nearly
identical
results
were
also
obtained
with
T.
viride
and
the
pathogen,
Rhizoctonia
solani.
Weak
inhibition
of
the
growth
of
Fusarium
culmorum
and
Fusarium
graminearum
was
also
observed.
None
of
these
CHs
slowed
mycelial
growth
of
Pythium
myriotylum,
an
oomycete
that
lacks
cell
wall
chitin
(2).
CH
Release
during
Imbibition
During
imbibition
by
surface-sterilized
barley
seeds
in
deionized
water,
CH3
was
released
into
the
medium.
CH3
was
detected
by
immunoblotting
as
early
as
2
h
after
the
seeds
were
wetted
(Fig.
4).
In
one
experiment,
70
ug
of
CH3
was
isolated
by
chitin
affinity
chromatography
from
the
material
released
after
10
h
of
imbibition
by
1000
seeds
in
0
24
10
20
5'
C.-
A
AO
DPA
Figure
3.
CH
mRNA
accumulation
during
seed
development.
Northern
analysis
of
10-Ag
aliquots
of
total
RNA
from
developing
aleurone
(top)
and
endosperm
(bottom)
tissues.
Developmental
stages
identified
as
DPA.
Five-
and
45-DPA
samples
were
extracted
from
whole
seeds
(indicated
by
letter
S).
Mobilities
of
RNA
markers
and
their
sizes
in
kilobases
are
indicated
in
right
margin.
1012
SWEGLE
ET
AL.
BARLEY
SEED
CHITINASE
RELEASE
DURING
IMBIBITION
water.
CH1
appeared
in
the
water
by
24
h,
by
which
time
a
large
number
of
other
proteins
in
addition
to
CH
also
had
been
released.
Enzyme
activity
gel
analysis
of
the
leached
proteins
after
18
h
of
imbibition
revealed
the
presence
of
CH3
as
well
as
one
of
the
other
putative
CHs
(CH4)
found
primarily
in
the
embryo
(Fig.
1,
lane
6).
CH4
may
be
the
additional
immunoreactive
band
observed
as
early
as
4
h
after
seed
imbibition
(Fig.
4).
CH
1
S
V
S
S
I
V
S
R
A
Q
Chitinase
C
S
V
S S
I
V
S
R
A
Q
CH
2
E
Q
X
G
S
CH
3
E
Q
X
G
S
Q
A
G
G
A
Tobacco
Ch
E
Q
C G
S
Q
A
G
G
A
R
C
A
S
G L
C
C
S
K
Chitinase
T
X
*
Q
*
*
* * * *
*
T
*
P
N
X
*
*
*
*
X
Amino
Acid
Sequence
Comparisons
Amino
terminal
sequences
of
CHi,
CH2,
and
CH3
are
compared
with
those
of
other
plant
CHs
and
lectins
in
Figure
5.
CH1
and
another
barley
CH,
CHC
(8),
are
identical
for
the
first
10
residues
at
their
amino
termini.
CH2
and
CH3,
on
the
other
hand,
are
not
similar
to
CH1
or
CHC
at
their
amino
termini.
Instead,
they
are
identical
with
or
similar
to
the
amino
termini
of
tobacco,
barley,
and
bean
CH,
as
well
as
wheat
germ
agglutinin,
nettle
lectin,
and
hevein
(26,
28).
A
property
common
to
these
proteins
is
the
ability
to
bind
chitin.
DISCUSSION
Two
barley
seed
CHs,
C
and
T,
were
described
previously
(8,
13).
Both
are
basic
proteins
that
are
present
in
the
aleurone
and
endosperm
tissues
and
that
act
as
endocleaving
enzymes.
A
third
CH,
K,
secreted
by
embryogenic
cell
suspensions
of
barley
was
described
recently
(11).
In
this
report,
we
describe
the
isolation
and
partial
characterization
of
three
barley
seed
CHs,
one
of
which
(CH3)
is
located
primarily
in
the
embryo.
Two
other
proteins
that
also
have
CH
activity
have
been
detected
in
the
embryo,
and
one
of
these
has
been
partially
purified.
All
five
of
these
proteins
are
present
in
the
mature
seed
(Fig.
1).
CH1
and
CH2
are
localized
primarily
in
aleurone
and
endosperm
tissues.
Those
CHs
appear
to
be
similar
if
not
identical
in
tissue
localization,
amino
acid
sequence,
and
physical
properties
to
CHs
C
and
T,
respectively
(11,
12).
CH3
is
a
CH
relatively
abundant
in
barley
seeds
that
probably
has
not
been
reported
previously.
Although
CH3
is
like
CH2
in
size
and
amino
terminal
sequence,
other
prop-
erties
distinguish
it
from
CH2.
CH3
is
active
over
a
broader
pH
range
and
has
a
lower
isoelectric
point
of
pH
8.7.
CH3
2
4 8
12
18
24
24
A
B
C
Figure
4.
Immunoblot
of
proteins
released
during
imbibition.
Two
seed
equivalents
per
sample
were
treated
with
antibodies
raised
against
CH1.
Hours
of
imbibition
are
indicated
at
top.
Lane
A,
CH1
(100
ng);
lane
B,
CH3
(100
ng);
lane
C,
sample
containing
par-
tially
purified
CH4.
The
two
24-h
samples
are
from
two
different
experiments.
Bean
Ch
WGA
*
*
*
*
R
*
*
* *
*
L
*
P
G
*
N
*
*
*
Q
K
*
*
*
*
S
*
*
K
L
*
P
N
N
*
*
*
Q
Figure
5.
Amino
terminal
sequences
of
plant
CHs
and
lectins.
Barley
CH
C
sequence
is
from
Leah
et
al.
(12).
Barley
CHs
T
and
K
sequences
are
from
Kragh
et
al.
(10,
1
1).
Other
sequences
are
from
Stanford
et
al.
(28).
Stars
indicate
identity
with
tobacco
CH
se-
quence.
WGA,
Wheat
germ
agglutinin.
has
a
lower
specific
activity
than
CH1
and
CH2.
Most
im-
portant,
CH3
is
located
predominantly
in
the
embryo,
whereas
CH2
is
found
in
the
aleurone
and
endosperm.
The
properties
of
CH3
closely
resemble
those
of
the
recently
reported
CH
K
from
the
medium
of
barley
cell
suspensions
in
culture
(11).
The
barley
embryo
also
contains
two
other
putative
CHs
(CH4
and
CH5)
that
have
not
been
previously
described.
CH1,
CH2,
and
CH3
digest
tritiated
chitin
and
glycol
chitin,
but
they
do
not
hydrolyze
f-N-acetylglucosaminidase
substrates.
Like
many
plant
CHs
(5,
21,
24,
26),
all
three
purified
CHs
inhibit
the
mycelial
growth
of
several
chitin-
containing
fungi
in
vitro
and
have
basic
isoelectric
points.
CH2
and
CH3
have
N-terminal
lectin-like
chitin-binding
domain
sequences
and
appear
to
represent
class
I
CHs.
CH1
and
CHC
lack
this
domain
and
are
probably
class
II
CHs
(26).
Southern
blot
analyses
indicate
the
presence
of
multiple
CH
genes
in
barley.
Two
reported
barley
CH
cDNA
clones,
clone
10
(ref.
29)
and
cCHI26
(ref.
13),
are
derived
from
distinct
genes,
because
the
nucleotide
sequences
differ
by
10%
and
their
primary
translation
products
differ
in
size.
The
translation
product
corresponding
to
clone
10
may
be
a
precursor
form
for
mature
CH2
or
CH3.
Because
the
tissue
distribution
of
transcripts
detected
by
clone
10
(Fig.
3)
is
identical
with
that
of
CH2
in
the
mature
seed
(Fig.
1),
it
is
likely
that
clone
10
encodes
CH2
rather
than
CH3.
Two
segments
of
nine
and
13
amino
acids
predicted
from
the
clone
10
sequence
match
those
determined
directly
from
internal
fragments
of
CH
T,
which
is
most
closely
related
to
CH2
(8,
11).
Additional
sequence
information
will
be
required
to
unequivocally
identify
the
chitinase
corresponding
to
clone
10.
Leah
et
al.
(13)
reported
that
the
CHI26
gene
is
expressed
only
in
aleurone
cells
of
developing
seeds.
Our
results
showed
that
CH
transcripts
are
present
not
only
in
aleurone
cells
but
also
in
endosperm
tissues
during
seed
development
(Fig.
3).
The
CH
released
from
seeds
during
imbibition,
CH3,
is
localized
primarily
in
the
embryo
from
where
it
is
likely,
1013
Plant
Physiol.
Vol.
99,
1992
released.
The
putative
CH,
CH4,
also
is
released
early
during
imbibition
and
is
found
primarily
in
the
embryo
(Fig.
1).
This
protein
is
detected
in
the
material
that
is
released
as
early
as
4
h
after
imbibition
(Fig.
4,
cf.
lanes
4-24
with
lane
C).
The
chitin-binding
lectin,
wheat
germ
agglutinin,
is
released
from
wheat
embryos
during
the
first
hour
of
imbibition
(16).
CHs
thus
represent
a
second
class
of
proteins
that
are
released
from
embryos
of
grass
seeds
during
the
early
stages
of
imbibition
and
interact
with
chitin.
Seed
CHs
presumably
help
protect
seeds
and
seedlings
from
pathogenic
fungi
before
and
during
germination.
The
significance
of
CH
release
during
imbibition
is
not
known.
In
nature,
these
enzymes
would
be
released
into
the
soil
where
they
could
contact
soil-borne
fungi,
possibly
causing
direct
inhibition
of
mycelial
growth
or
the
release
of
chitin
fragments
that
may
act
as
signals
to
elicit
other plant
defense
responses
(23).
Further
study
is
necessary
to
unequivocally
define
the
actual
physiological
role
of
the
seed
CHs
in
plant
development.
ACKNOWLEDGMENTS
We
thank
Drs.
Peter
Wong,
Claude
Selitrennikoff,
and
Sam
Wang
for
their
critical
readings
of
our
manuscript,
Dr.
Richard
Broglie
for
bean
CH
antiserum,
and
Dr.
Bikram
Gill
and
Duane
Wilson
for
their
help
in
growing
and
analyzing
the
barley
plants
used
in
this
work.
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1014
SWEGLE
ET
AL.
- ... Pathogenesis-related proteins were first classified in 1978; this classification was based on serology and sequencing details in tobacco (Van Strien, 1999, Van Loon et al., 1994). Up to now, 17 groups of PR proteins have been identified in various plants which consist of chitinases (Swegle et al., 1992; Velazhahan et al., 2000), glucanases (Velazhahan and Muthukrishnan, 2003 ), proteinaseinhibitor (Leah et al., 1991), thaumatin-like proteins (Roberts and Selitrennikoff, 1990) and defensins (Terras et al., 1992). Thaumatin-like proteins (TLP) shows high sequence similarity to thaumatin, a sweet-tasting protein from the West African shrub Thaumatococcus danielli. ...
- ... Evaluation of plant chitinases in various seeds including Benincasa hispida (Shih et al. 2001), Secale cereale (Taira et al. 2001), Cucumis melo (Witmer et al. 2003), Glicine max (L.) Merr. (Yeboah et al. 1998), Hordeum vulgare L. (Swegle et al. 1992), and Zea mays L. (Huynh et al. 1992) have already been described. To investigate an expression pattern of PR proteins in rape seeds during germination, two different extract buffers (DW and K-PO 4 ) were used to obtain crude enzymes in this study. ...
- ... Two brief reviews of plant chitinases have appeared recently (Collinge et al. 1993; Flach et al. 1992); the review by Flach et al. (1992) includes information on chitinases from other et al. Leah et al. 1991 Jacobsen et al. 1990 Swegle et al. 1992 Kragh et al. 1991 Leah et al. 1987 Kragh et al. 1991 Jacobsen et al. 1990 Kragh et al. 1991 Kragh et al. 1990 Kragh et al. 1991 Swegle et al. 1992 Swegle et al. 1992 I Kurosaki et al. 1989 Kurosaki et al. 1989 Kurosaki et al. 1989 Kurosaki et al. 1989 Kurosaki et al. 1986 Kurosaki et al. 1986 Kurosaki et al. 1986 Kurosaki et al. 1986 Kurosaki et al. 1987a Kurosaki et al. 1987a MCtraux et al. 1988 Majeau et al. 1990 Van Damme et al. 1993 Martin 1991 Job's tears (Croix lachryma-jobi) I Ary et al. 1989 Maize (Zea mays) leaves I Nasser et al. 1988 Nasser et al. 1988 Nasser et al. 1988 Nasser et al. 1988 ...
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