Germination induction of dormant Avena fatua caryopses

Full text

Journal
of
Plant
Physiology
176
(2015)
169–179
Contents
lists
available
at
ScienceDirect
Journal
of
Plant
Physiology
journal
h
om
epage:
www.elsevier.com/locate/jplph
Physiology
Germination
induction
of
dormant
Avena
fatua
caryopses
by
KAR1and
GA3involving
the
control
of
reactive
oxygen
species
(H2O2and
O2•−)
and
enzymatic
antioxidants
(superoxide
dismutase
and
catalase)
both
in
the
embryo
and
the
aleurone
layers
Danuta
Cembrowska-Lecha,
Marek
Koprowskib,
Jan
K˛
epczy ´
nskia,∗
aDepartment
of
Plant
Physiology
and
Genetic
Engineering,
Faculty
of
Biology,
University
of
Szczecin,
W˛
aska
13,
71-415
Szczecin,
Poland
bDepartment
of
Heteroorganic
Chemistry,
Centre
of
Molecular
and
Macromolecular
Studies,
Polish
Academy
of
Sciences,
Sienkiewicza
112,
90-363
Łód´
z,
Poland
a
r
t
i
c
l
e
i
n
f
o
Article
history:
Received
21
November
2014
Accepted
25
November
2014
Available
online
22
December
2014
Keywords:
Abscisic
acid
Avena
fatua
Caryopses
germination
Karrikinolide
Reactive
oxygen
species
a
b
s
t
r
a
c
t
Avena
fatua
L.
caryopses
did
not
germinate
at
20 ◦C
in
darkness
because
they
were
dormant.
However,
they
were
able
to
germinate
in
the
presence
of
karrikinolide
(KAR1),
a
key
bioactive
compound
present
in
smoke,
and
also
in
the
presence
of
gibberellin
A3(GA3),
a
commonly
known
stimulator
of
seed
ger-
mination.
The
aim
of
this
study
was
to
collect
information
on
a
possible
relationship
between
the
above
regulators
and
abscisic
acid
(ABA),
reactive
oxygen
species
(ROS)
and
ROS
scavenging
antioxidants
in
the
regulation
of
dormant
caryopses
germination.
KAR1and
GA3caused
complete
germination
of
dormant
A.
fatua
caryopses.
Hydrogen
peroxide
(H2O2),
compounds
generating
the
superoxide
(O2•−),
i.e.
menadione
(MN),
methylviologen
(MV)
and
an
inhibitor
of
catalase
activity,
aminotriazole
(AT),
induced
germination
of
dormant
caryopses.
KAR1,
GA3,
H2O2and
AT
decreased
ABA
content
in
embryos.
Furthermore,
KAR1,
GA3,
H2O2,
MN,
MV
and
AT
increased
␣-amylase
activity
in
caryopses.
The
effect
of
KAR1and
GA3on
ROS
(H2O2,
O2•−)
and
activities
of
the
superoxide
dismutase
(SOD)
and
catalase
(CAT)
were
determined
in
caryopses,
embryos
and
aleurone
layers.
SOD
was
represented
by
four
isoforms
and
catalase
by
one.
In
situ
localization
of
ROS
showed
that
the
effect
of
KAR1and
GA3was
associated
with
the
localization
of
hydrogen
peroxide
mainly
on
the
coleorhiza.
However,
the
superoxide
was
mainly
localized
on
the
surface
of
the
scutellum.
Superoxide
was
also
detected
in
the
protruding
radicle.
Germination
induction
of
dormant
caryopses
by
KAR1and
GA3was
related
to
an
increasing
content
of
H2O2,
O2•−and
activities
of
SOD
and
CAT
in
embryos,
thus
ROS
homeostasis
was
probably
required
for
the
germination
of
dor-
mant
caryopses.
The
above
regulators
increased
the
content
of
ROS
in
aleurone
layers
and
decreased
the
activities
of
SOD
and
CAT,
probably
leading
to
the
programmed
cell
death.
The
presented
data
provide
new
insights
into
the
germination
induction
of
A.
fatua
dormant
caryopses
by
KAR1and
also
by
GA3.
In
A.
fatua,
KAR1or
GA3is
included
in
the
induction
germination
of
dormant
caryopses
through
regulation
level
of
ABA
in
embryos
and
ROS-antioxidant
status
both
in
embryos
and
aleurone
layers.
©
2014
Elsevier
GmbH.
All
rights
reserved.
Abbreviations:
ABA,
abscisic
acid;
AT,
aminotriazole;
DAB,
3,3-
diaminobenzidine;
DPI,
diphenyleneiodonium;
EDTA,
ethylenediaminetetraacetic
acid;
GA3,
gibberellin
A3;
HRMS,
toward
high-resolution
mechanical
spectroscopy;
KAR1,
karrikinolide;
MN,
menadione;
MV,
methylviologen;
NBT,
nitro
blue
tetra-
zolium;
NMR,
nuclear
magnetic
resonance;
ROS,
reactive
oxygen
species;
SOD,
superoxide
dismutase;
TCA,
trichloroacetic
acid.
∗Corresponding
author.
Tel.:
+48
91
444
15
44.
E-mail
address:
jankepcz@wp.pl
(J.
K˛
epczy ´
nski).
Introduction
Seed
dormancy
can
be
defined
as
an
inability
of
viable
imbibed
seeds
to
germinate
under
conditions
that
are
favorable
for
the
ger-
mination
process
(Bewley,
1997).
It
is
commonly
accepted
that
the
balance
between
abscisic
acid
(ABA)
and
gibberellins
(GAs)
and
sen-
sitivity
to
these
hormones
are
responsible
for
the
regulation
of
the
dormancy
state
and
germination
of
dormant
seeds
(Cadman
et
al.,
2006;
Finch-Savage
and
Leubner-Metzger,
2006).
ABA
is
the
most
important
hormone
responsible
for
the
establishment
and
mainte-
nance
of
dormancy
in
seeds.
A
decrease
in
the
ABA
content
in
seeds
http://dx.doi.org/10.1016/j.jplph.2014.11.010
0176-1617/©
2014
Elsevier
GmbH.
All
rights
reserved.

170
D.
Cembrowska-Lech
et
al.
/
Journal
of
Plant
Physiology
176
(2015)
169–179
by
physical
factors,
chemicals
or
genetic
manipulation
reduces
dor-
mancy
(Finch-Savage
and
Leubner-Metzger,
2006).
GAs
have
been
considered
as
promotors
of
progression
from
dormancy
release
through
germination
(Finkelstein
et
al.,
2008).
Dormancy
release
may
involve
a
decline
in
the
ABA
content
and
an
increase
of
GAs
level
(Hilhorst,
2007).
According
to
available
data,
reactive
oxygen
species
(ROS),
such
as
superoxide
anion
(O2•−),
hydrogen
perox-
ide
(H2O2)
and
hydroxyl
radical
(•OH),
are
also
involved
in
the
regulation
of
dormancy
in
seeds
(El-Maarouf-Bouteau
and
Bailly,
2008;
Whitaker
et
al.,
2010;
Diaz-Vivancos
et
al.,
2013).
Applica-
tion
of
exogenous
ROS
or
compounds
generating
ROS
can
break
dormancy
in
seeds
of
several
plant
species.
When
a
certain
level
of
ROS
is
reached,
it
has
a
signaling
function
and
consequently
results
in
completing
seed
germination
(Bailly
et
al.,
2008).
For
example,
dormancy
release
associated
with
accumulation
of
ROS
has
been
noted
in
sunflower
seeds
(Oracz
et
al.,
2007).
An
ade-
quate
level
of
ROS
depends
on
its
production
and
scavenging
by
the
enzymatic
system:
superoxide
dismutase
(SOD),
catalase
(CAT),
peroxidases,
glutathione
reductase
(GR),
monodehydroascorbate
reductase
(MDHAR)
and
non-enzymatic
compounds
such
as
the
reduced
glutathione
and
ascorbate
(El-Maarouf-Bouteau
and
Bailly,
2008).
Cross-talk
between
ROS
and
ABA
or
gibberellin
metabolism
and
signaling
has
been
discussed
in
the
context
of
controlling
barley
caryopses
dormancy
(Bahin
et
al.,
2011).
It
was
suggested
that
in
barley,
releasing
embryo
dormancy
by
hydrogen
peroxide
took
place
through
the
activation
of
gibberellin
A3(GA3)
signaling
and
synthesis
rather
than
through
the
repression
of
ABA
signaling
(Bahin
et
al.,
2011).
Germination
and
postgermination
processes
of
grasses
i.e.
wheat,
barley,
wild
oat,
depend
on
events
in
aleu-
rone
cells
synthesizing
and
secreting
hydrolytic
enzymes,
mainly
␣-amylase
to
starchy
endosperm,
in
order
to
mobilize
storage
materials.
Both
synthesis
and
secretion
of
hydrolytic
enzymes
are
induced
by
gibberellins
synthesized
in
embryos
and
diffused
to
the
starchy
endosperm
(Appleford
and
Lenton,
1997).
The
aleu-
rone
cells,
after
the
completion
of
their
secretory
role,
undergo
programmed
cell
death
(PCD)
(Finnie
et
al.,
2011).
PCD
is
stimu-
lated
by
gibberellins
and
inhibited
by
ABA
(Bethke
et
al.,
1999).
ROS,
especially
H2O2,
are
considered
as
key
inducers
of
PCD.
Appli-
cation
of
H2O2induces
cell
death
in
cells
treated
with
GA,
but
not
in
those
treated
with
ABA
(El-Maarouf-Bouteau
and
Bailly,
2008).
GA3
down-regulates
the
activity
of
antioxidant
enzymes:
CAT,
ascorbate
peroxidase
and
SOD,
to
ensure
a
sufficient
accumulation
of
hydro-
gen
peroxide
prior
to
the
onset
of
cell
death
in
barley
(Fath
et
al.,
2001a;
De
Pinto
et
al.,
2012).
The
activity
of
these
enzymes
has
been
maintained
in
ABA
treated
cells
(Fath
et
al.,
2001a).
Germination
of
dormant
and
non-dormant
seeds
can
be
also
induced
by
ecological
factors
such
as
smoke
released
from
fire.
It
has
been
proved
that
smoke
can
enhance
the
seed
germina-
tion
of
1200
species
representing
more
than
80
genera
worldwide
(Dixon
et
al.,
2009).
A
primary
germination
stimulant
has
been
discovered
in
plant-derived
smoke
(Van
Staden
et
al.,
2004)
and
burned
cellulose
(Flematti
et
al.,
2004).
It
was
named
butenolide,
3-methyl-2H-furo[2,3-c]pyran-2-one,
and
now
is
known
as
kar-
rikinolide
(KAR1)
(Dixon
et
al.,
2009).
KAR1,
similarly
to
smoke
can
stimulate
seed
germination
of
fire-prone
and
non-fire-prone
plant
species.
Both
smoke
and
KAR1can
stimulate
germination
or
seedling
growth
of
weeds
and
crops
(Light
et
al.,
2009).
Recently,
five
karrikins,
namely
KAR2–KAR6have
been
found
in
smoke
(Flematti
et
al.,
2009).
Avena
fatua
(wild
oat)
is
a
persistent
weed
in
cereal
production
systems
in
many
regions
of
the
world,
including
Poland.
After
the
harvest,
dormancy
of
caryopses
of
this
grass
can
be
caused
by
the
tissue
surrounding
the
embryo,
the
embryo
itself,
or
both
(Adkins
and
Peters,
2001).
Germination
of
dormant
florets
and/or
caryopses
can
be
induced
by
various
factors,
e.g.
dry
storage
(Foley,
1994;
K˛
epczy ´
nski
et
al.,
2013),
gibberellin
(Adkins
et
al.,
1986;
K˛
epczy ´
nski
et
al.,
2006,
2013),
smoke
(Adkins
and
Peters,
2001;
K˛
epczy ´
nski
et
al.,
2006,
2010)
and
KAR1(Daws
et
al.,
2007;
Stevens
et
al.,
2007;
K˛
epczy ´
nski
et
al.,
2010,
2013).
Earlier
studies
have
shown
that
the
response
of
dormant
A.
fatua
caryopses
to
KAR1requires
ethylene
action
(K˛
epczy ´
nski
and
Van
Staden,
2012)
and
gibberellin
biosyn-
thesis
(K˛
epczy ´
nski
et
al.,
2013).
A
stimulatory
effect
of
KAR1and
GA3on
germination
of
dormant
A.
fatua
caryopses
is
associated
with
increasing
dehydrogenases
and
␣,
␤-amylases
before
radicle
protrusion
(K˛
epczy ´
nski
et
al.,
2013).
Little
information
can
be
found
in
the
literature
on
the
inter-
action
between
KAR1,
which
is
considered
as
a
representative
of
a
novel
class
of
plant
growth
regulators
(Nelson
et
al.,
2009),
and
plant
hormones
in
seeds.
There
is
no
data
on
the
participation
of
ROS
in
the
germination
of
dormant
A.
fatua
caryopses.
Likewise,
no
information
is
available
on
the
interaction
between
KAR1and
ROS
with
respect
to
dormancy
and
germination
of
seeds.
Similarly,
it
is
unknown
whether
KAR1and
GA3can
control
ROS
levels
in
aleurone
layers
of
dormant
A.
fatua
caryopses.
So
far,
the
effect
of
the
above
regulators
on
ROS
content
and
antioxidants
in
embryo
and
aleurone
layers
from
the
same
A.
fatua
caryopsis
has
not
been
determined.
Until
now
the
effect
of
GAs
on
ROS
level
and
antioxidants
was
not
analyzed
in
cereal
embryo
and
aleurone
layers
in
the
same
exper-
iment.
Likewise,
no
one
has
compared
the
effect
of
KAR1,
GA3and
ROS
on
content
of
ABA
in
dormant
embryos
of
grasses.
In
addition,
localization
of
hydrogen
peroxide
and
superoxide
in
embryos
from
caryopses
incubated
in
the
presence
of
KAR1or
GA3is
unknown.
Moreover,
the
effect
of
KAR1and
GA3on
isoforms
activities
of
SOD
and
CAT
was
not
analyzed
in
caryopses.
Therefore
the
aim
of
the
present
study
was
to
examine
whether
germination
induction
of
dormant
A.
fatua
caryopses
by
KAR1and
GA3is
associated
with
the
control
of
ROS
level,
H2O2and
O2•−,
and
the
enzymatic
antioxidants
that
scavenge
them,
CAT
and
SOD,
in
two
components,
embryo
and
aleurone
layers.
In
addition,
the
effect
of
KAR1and
GA3on
in
situ
localization
of
H2O2and
O2•− in
embryo
and
isoenzymes
activity
of
CAT
and
SOD
were
tested.
More-
over,
a
relationship
between
KAR1,
GA3,
H2O2or
ROS
generating
compounds
and
␣-amylase
activity
and
ABA
was
also
analyzed.
Materials
and
methods
Plant
material
Avena
fatua
L.
(wilde
oat)
spikelets
were
collected
in
Poland
near
Szczecin
at
the
time
of
natural
dispersal
in
July
21,
2010.
Spikelets
contained
2–3
florets
covered
with
glumes.
The
floret
was
a
single
caryopsis
(fruit)
covered
by
the
lemma
and
palea
(Simpson,
2007).
After
collection,
florets
were
dried
at
room
temperature
for
7
days
to
a
constant
moisture
content
(ca.
11%)
and
then
stored
at
−20 ◦C
until
they
were
needed.
The
lemma
and
palea
were
removed
for
the
experiments
and
only
the
caryopses
were
used.
Synthesis
of
the
karrikinolide
(KAR1)
KAR1,
3-methyl-2H-furo[2,3-c]pyran-2-one,
was
synthesized
according
to
Nagase
et
al.
(2008).
The
desired
compound
was
syn-
thesized
using
direct
and
region
selective
Ti-cross
aldol
addition
involving
dihydro-2H-pyran-3-(4H)-one
and
methyl
pyruvate
as
the
keep
step,
followed
by
furanone
formation.
The
crude
prod-
uct
was
purified
with
silica
gel
column
chromatography
to
obtain
a
crystal
compound.
Their
structure
was
confirmed
by
1H
nuclear
magnetic
resonance
(NMR)
and
toward
high-resolution
mechani-
cal
spectroscopy
(HRMS)
analysis.
NMR
spectra
were
recorded
on
a
Bruker
AC
200
spectrometer
operating
at
200.13
MHz
(using
CD13
as
a
solvent).
MS
spectra
including
HRMS
were
recorded
on
Finni-
gan
MAT
95
spectrometer.

D.
Cembrowska-Lech
et
al.
/
Journal
of
Plant
Physiology
176
(2015)
169–179
171
Germination
assays
Caryopses,
25
in
each
of
three
replicates,
were
incubated
at
20 ◦C
in
darkness,
in
6-cm
Petri
dishes
on
one
layer
of
filter
paper
(Whatman
No.
1)
moistened
with
1.5
mL
deionized
water
or
var-
ious
solutions.
Caryopses
were
incubated
in
the
presence
of
KAR1
(3
×
10−9M)
or
GA3(10−5M),
either
alone
or
in
combination
with,
diphenyleneiodonium
(DPI)
(10−4M).
Hydrogen
peroxide
(H2O2)
(10−3,
5
×
10−3,
10−2M)
solutions
were
changed
every
24
h.
Amino-
triazole
(AT)
(10−5,
10−4,
10−3M)
solutions
were
present
during
the
whole
period
of
incubation.
Caryopses
were
also
pretreated
with
menadione
(MN)
(10−3M)
for
1,
3,
6
or
12
h,
and
H2O2(1
M)
or
methylviologen
(MV)
(10−5M)
for
6,
12
or
24
h.
After
the
pretreat-
ments
caryopses
were
rinsed
with
100
mL
deionized
water
before
the
germination
test
at
20 ◦C.
The
numbers
of
germinated
caryopses
were
scored
5
days
after
the
start
of
incubation.
Radicle
protrusion
through
the
coleorhiza
was
the
criterion
for
germination.
Abscisic
acid
(ABA)
determination
To
quantify
the
endogenous
level
of
ABA
in
A.
fatua
L.
caryopses,
in
each
of
5
replicates
25
caryopses
were
incubated
at
20 ◦C
in
dark-
ness,
in
6-cm
Petri
dishes
on
one
layer
of
filter
paper
(Whatman
No.
1)
moistened
with
deionized
water
or
with
various
solutions:
KAR1(3
×
10−9M),
GA3(10−5M),
H2O2(1
M)
or
AT
(10−3M).
After
16
and
24
h,
embryos
were
dissected
and
frozen
immediately
in
liquid
nitrogen
and
stored
at
−80 ◦C
prior
to
ABA
analysis.
Twenty-
five
embryos
were
ground
to
a
fine
powder
in
liquid
nitrogen
using
a
Retsch
MM200
laboratory
mill
ball
and
ABA
was
extracted
in
7.5
mL
of
80%
(v/v)
methanol
with
[2H4]-ABA
(1
ng/␮L)
as
an
internal
standard.
After
the
extraction
at
15 ◦C
for
32
h,
methanol
was
removed
by
evaporation
in
a
speed
vacuum
centrifuge.
Sam-
ples
were
then
purified
with
hexane
and
ethyl
acetate.
Next,
the
samples
were
centrifuged
for
20
min
and
the
upper
phase
was
collected
in
a
glass
vial
and
evaporated
to
dryness
in
a
speed
vacuum
centrifuge.
Dry
samples
were
resuspended
in
1
mL
100%
(v/v)
methanol,
methylated
and
evaporated
to
dryness.
Methylated
samples
were
dissolved
in
20
␮L
hexane
and
1
␮L
aliquots
were
analyzed
on
a
gas
chromatograph
(Agilent
6890
N)
equipped
with
a
DB-225
capillary
column
(Abel
Industries)
and
mass
spectromet-
ric
detector
(Agilent
5973
N).
The
mass
spectrometer
was
set
to
monitor
ions
at
m/z
190
and
194.
Evaluation
of
H2O2and
O2•− content
Extracellular
H2O2production
was
determined
according
to
the
method
described
by
Velikova
et
al.
(2000).
Caryopses
were
incu-
bated
either
in
water
or
in
the
solution
of
3
×
10−9M
KAR1or
10−5M
GA3for
up
to
28
h.
After
every
4
h
of
caryopses
imbibition,
the
embryos
and
aleurone
layers
were
isolated
from
the
caryopses.
Twenty-five
caryopses,
embryos
or
aleurone
layers
were
ground
and
homogenized
with
cold
1%
trichloroacetic
acid
(TCA)
(fresh
weight:TCA,
1:10,
w/v).
After
20
min
of
centrifugation
at
14,000
×
g
at
4◦C,
the
resulting
supernatant
was
used
for
spectrophotometric
analysis.
The
reaction
mixture
was
composed
of
0.5
mL
of
10
mM
potassium
phosphate
buffer
(pH
7.0),
1
mL
of
1
M
KI
(in
10
mM
potassium
phosphate
buffer,
pH
7.0)
and
0.5
mL
of
the
collected
supernatant.
The
reaction
started
with
the
addition
of
KI
and
the
sample
was
incubated
for
60
min
at
25 ◦C.
The
absorbance
of
the
end
product
was
measured
at
390
nm.
A
standard
curve
was
pre-
pared
by
using
the
H2O2standard.
The
results
were
expressed
as
␮M
H2O2g−1FW.
Extracellular
O2•− production
was
estimated
using
the
method
developed
by
Elsner
and
Heupel
(1976).
Caryopses
were
incubated
either
in
water
or
in
the
solution
of
3
×
10−9M
KAR1or
10−5M
GA3for
up
to
28
h.
After
every
4
h
of
imbibition
of
the
caryopses,
the
embryos
and
aleurone
layers
were
isolated
from
the
caryopses.
Twenty-five
caryopses,
embryos
or
aleurone
layers,
were
ground
and
homogenized
with
cold
1%
TCA
(fresh
weight:TCA,
1:10,
w/v).
The
homogenate
was
centrifuged
for
20
min
at
14,000
×
g
at
4◦C
and
then
the
supernatant
was
used.
The
supernatant
(1
mL)
was
first
incubated
at
25 ◦C
for
30
min
in
the
presence
of
1
mL
1
mM
hydroxylamine
hydrochloride
(in
50
mM
potassium
phosphate
buffer,
pH
7.8).
The
volume
(0.5
mL)
of
this
reaction
mixture
was
then
incubated
with
0.5
mL
of
17
mM
sulfanilamide
(in
50
mM
potassium
phosphate
buffer,
pH
7.8)
and
0.5
mL
of
7
mM
2-naphtylamine
(in
50
mM
potassium
phosphate
buffer,
pH
7.8)
at
25 ◦C,
for
30
min.
The
absorbance
was
measured
at
540
nm
after
centrifugation
at
14,000
×
g
for
10
min.
A
calibration
curve
was
established
using
sodium
nitrite
(NaNO2).
The
results
were
expressed
as
mM
O2•− g−1FW.
For
hydrogen
peroxide
and
super-
oxide
anion
content
the
data
were
shown
as
means
of
five
biological
replicates
±
SD.
Visualization
of
H2O2and
O2•−
Caryopses
were
incubated
either
in
water
or
in
the
solution
of
3
×
10−9M
KAR1or
10−5M
GA3for
24
or
28
h.
In
situ
local-
ization
of
reactive
oxygen
species
(ROS)
in
embryo
tissue
was
identified
by
incubating
embryos
in
either
3,3-diaminobenzidine
(DAB)
(1
mg/mL
DAB
containing
0.05%
(v/v)
Tween-20
and
10
mM
Na2HPO4)
for
90
min
in
darkness
(Thordal-Christensen
et
al.,
1997)
or
6
mM
nitro
blue
tetrazolium
(NBT
in
10
mM
Tris–HCl,
pH
7.4)
for
10
min
(Beyer
and
Fridovich,
1987).
Dark
yellow
staining
in
the
presence
of
DAB
indicated
polymerization
of
DAB,
requiring
H2O2
and
peroxidase
activity
and
dark
blue
staining
in
the
presence
of
NBT
indicated
O2•− production.
Superoxide
dismutase
(SOD)
and
catalase
(CAT)
extraction
and
activity
Spectrophotometric
analysis
All
enzyme
assays
(SOD
and
CAT)
were
performed
in
the
same
crude
extract.
Caryopses
were
incubated
either
in
water
or
in
the
solution
of
3
×
10−9M
KAR1or
10−5M
GA3for
up
to
28
h.
Twenty-
five
caryopses,
embryos
or
aleurone
layers
were
ground
to
a
fine
powder
in
liquid
nitrogen
using
a
Retsch
MM200
laboratory
mill
ball
and
homogenized
for
10
min
in
0.1
M
potassium
phosphate
buffer
(pH
7.0)
containing
10
mM
ethylenediaminetetraacetic
acid
(EDTA)
and
1%
PVP
(fresh
weight:buffer,
1:10,
w/v).
Homogenates
were
centrifuged
for
20
min
at
15,000
×
g
at
4◦C.
SOD
(EC
1.15.1.1)
activity
was
tested
according
to
Giannopolitis
and
Ries
(1977)
by
the
inhibition
of
NBT
chloride
photoreduction.
The
assay
was
carried
out
using
the
following
reaction
mixture:
0.1
M
potassium
phosphate
buffer
(pH
7.8),
1.3
␮M
riboflavine,
13
mM
methionine,
63
␮M
NBT,
0.1
mM
EDTA
and
100
␮L
of
the
enzymatic
extract.
The
reaction
mixture
was
illuminated
(50
␮mol
m−2s−1)
at
25 ◦C
for
10
min
and
the
absorbance
measured
at
560
nm.
One
unit
of
SOD
activity
was
defined
as
the
amount
of
the
enzyme
required
to
inhibit
the
reduction
of
NBT
by
50%
under
the
specified
conditions.
SOD
activity
of
the
extracts
was
expressed
as
U
mg−1protein.
CAT
(EC
1.11.1.6)
activity
was
measured
according
to
Rao
et
al.
(1996).
The
enzyme
activity
was
monitored
spectrophotomet-
rically
at
240
nm
for
60
s
using
the
following
mixture:
50
mM
potassium
phosphate
buffer
(pH
7.0),
14.3
mM
H2O2and
100
␮L
of
enzymatic
extract.
Purified
CAT
was
used
as
a
calibration
standard.
CAT
activity
was
expressed
as
U
mg−1protein.
Data
for
both
enzyme
activities
were
expressed
as
means
of
five
biological
repli-
cates
±
SD.

172
D.
Cembrowska-Lech
et
al.
/
Journal
of
Plant
Physiology
176
(2015)
169–179
Native-PAGE
and
activity
staining
of
SOD
and
CAT
For
the
analysis
of
SOD
and
CAT
activity
in
native-PAGE,
total
protein
was
extracted
under
non-denaturing
conditions
from
A.
fatua
L.
caryopses,
embryos
or
aleurone
layers
after
0,
12
and
24
h
of
caryopses
incubation
in
the
presence
of
KAR1(3
×
10−9M)
or
GA3(10−5M).
All
samples
were
ground
to
a
fine
powder
in
liquid
nitrogen
using
a
Retsch
MM200
laboratory
mill
ball
and
homoge-
nized
in
an
extraction
buffer
containing
0.1
M
Tricine–Tris
(pH
8.0),
3
mM
MgSO4,
0.1
mM
DTT
and
3
mM
ethylene
glycol
tetraacetic
acid
(EGTA)
for
SOD
or
0.1
M
potassium–phosphate
buffer
(pH
7.8)
containing
1
mM
EDTA,
1%
(w/w)
PVP
and
0.5%
(v/v)
Triton
X-
100
for
CAT
(fresh
weight:buffer,
1:5,
w/v).
Insoluble
material
was
removed
by
centrifugation
at
14,000
×
g
for
10
min
at
4◦C
and
a
supernatant
was
used.
Protein
was
separated
using
native-PAGE
at
4◦C
and
150
V
in
the
Laemmli
(1970)
buffer
system
without
SDS.
For
the
visualization
of
SOD,
native
gel
(12.5%
polyacrylamide
gel)
was
soaked
in
50
mM
potassium–phosphate
buffer
(pH
7.0)
containing
5
mM
H2O2or
3
mM
KCN.
After
rinsing
with
distilled
water,
the
gel
was
stained
with
50
mM
potassium–phosphate
buffer
(pH
7.8)
containing
10
mM
EDTA,
28
mM
TEMED,
30
␮M
riboflavin
and
245
␮M
NBT
for
20
min
in
the
dark
at
25 ◦C,
then
exposed
to
white
light
until
the
SOD
activity
bands
became
visible
(Beauchamp
and
Fridovich,
1971).
For
identification
of
isoforms,
40
␮g
protein
per
well
were
used
and
to
determine
the
effect
of
KAR1or
GA320
␮g
protein
per
well
was
applied.
Izoenzymes
of
CAT
were
visualized
on
a
10%
native-PAGE
gel
by
following
the
method
of
Woodbury
et
al.
(1971).
For
identification,
20
␮g
protein
per
well
was
used.
After
washing
three
times
with
distilled
water,
the
gel
was
soaked
for
10
min
in
50
mM
H2O2,
rinsed
twice
with
distilled
water,
and
then
stained
in
the
solution
of
1%
(w/v)
potassium
ferricyanide
and
1%
(w/v)
ferric
chloride.
Colorless
bands
of
CAT
isoenzymes
appeared
as
the
gel
was
stained
blue.
The
experiment
was
performed
in
three
replicates.
˛-Amylase
determination
The
analysis
of
␣-amylase
activity
was
according
to
the
method
described
by
K˛
epczy ´
nski
et
al.
(2006).
After
incubation
in
the
presence
of
KAR1(3
×
10−9M)
or
GA3(10−5M),
H2O2(1
M),
MN
(10−3M),
MV
(10−5M)
or
AT
(10−3M)
for
24
h
caryopses
were
ground
using
a
pre-chilled
mortar
and
pestle
in
ice-cold
extraction
buffer
(fresh
weight:extraction
buffer,
1:10,
w/v).
All
extraction
steps
were
performed
at
4◦C.
The
extraction
buffer
contained
20
mM
Tris–maleate,
pH
6.2,
with
1.0
mM
CaCl2.
The
homogenate
was
centrifuged
at
12,000
×
g
for
5
min
at
4◦C.
The
clear
super-
natant
was
used
for
enzyme
activity
and
the
protein
content
assay.
The
reaction
volume
contained
1.2
mL
enzyme
extract
and
1.2
mL
buffer
was
incubated
for
2
min
at
37 ◦C
in
a
water
bath.
The
reaction
was
initiated
by
adding
0.6
mL
suspension
of
Phadebas
blue
starch
(Magle
Life
Sciences,
Sweden)
(25
mg
mL−1),
vortexed
and
incu-
bated
with
shaking
for
30
min
at
37 ◦C
in
a
water
bath.
The
reaction
was
stopped
by
adding
0.6
mL
of
0.5
M
NaOH.
The
reaction
mix-
ture
was
centrifuged,
and
the
absorbance
of
resultant
supernatant
was
read
at
620
nm
against
a
blank
sample
(Phadebas
incubated
in
the
buffer
alone).
Spectrophotometric
analyses
were
conducted
at
room
temperature
on
an
UV–vis
spectrophotometer
(Thermo
Fisher
Scientific,
Madison,
USA).
Barley
malt
␣-amylase
was
used
as
a
calibration
curve.
Results
are
expressed
as
U
mg−1protein.
One
unit
(U)
was
equivalent
to
the
amount
of
enzyme
liberating
1
mg
maltose
from
starch
at
37 ◦C
and
pH
6.2.
Data
were
expressed
as
means
of
five
biological
replicates
±
SD.
Protein
determination
The
protein
content
in
the
enzymatic
extracts
was
assayed
by
Bradford’s
method
(1976),
using
bovine
serum
albumin
(BSA)
as
a
standard.
Fig.
1.
The
effect
of
hydrogen
peroxide
(H2O2)
or
catalase
inhibitor,
aminotriazole
(AT)
on
the
germination
of
Avena
fatua
L.
caryopses
at
20 ◦C
after
5
days.
H2O2was
applied
for
6,
12,
24
h
or
was
changed
every
24
h
up
to
5
days.
Vertical
bars
indi-
cate
±
SD.
One-way
ANOVA
with
the
Duncan
post
hoc
test
was
used
to
determine
significant
differences.
Mean
values
with
different
letters
(a–d)
are
significantly
different
(P
<
0.05).
Statistical
Analysis
Data
were
analyzed
for
significance
using
one-way
or
two-way
analysis
of
variance,
ANOVA
(Statistica
for
Windows
ver.
9.0,
Stat-
Soft
Inc.,
Tulsa,
OK,
USA).
Duncan’s
multiple
range
test
was
used
for
determination
of
significant
differences
between
values
of
ger-
mination
and
enzymatic
activities
in
A.
fatua
caryopses
(P
≤
0.05).
Similar
results
were
obtained
in
two
independent
experiments.
Results
The
effect
of
H2O2and
compounds
generating,
menadione
(MN)
and
methylviologen
(MV)
or
increasing,
aminotriazole
(AT),
ROS
A
common
representative
of
ROS,
i.e.
H2O2was
applied
either
continuously
or
prior
to
incubation
(Fig.
1).
A.
fatua
L.
caryopses
incubated
in
water
almost
did
not
germinate.
The
continuous
pres-
ence
of
H2O2caused
germination
of
50%
of
caryopses
only
at
5
×
10−3M;
a
lower
concentration
was
insufficient
and
a
higher
was
too
high
for
germination
stimulation.
H2O2was
also
effective
as
an
inductor
when
it
was
applied
for
6,
12
and
24
h
only
at
1
M.
The
highest
stimulatory
effect
appeared
when
the
caryopses
were
treated
for
24
h,
resulting
in
60%
of
the
caryopses
germination.
Likewise,
AT,
the
inhibitor
of
CAT
(Amory
et
al.,
1992),
stimu-
lated
caryopses
germination
at
all
concentrations
used
(Fig.
1).
The
strongest
effect
was
observed
in
the
presence
of
this
compound
at
the
highest
concentration:
when
10−3M
was
used,
55%
germina-
tion
was
noted.
Two
ROS-generating
compounds,
MN
and
MV,
were
also
applied
during
various
periods
of
preincubation
(Fig.
2).
Most
caryopses
preincubated
in
water,
produced
low
(5%)
germination
after
the
transfer
to
water
(control).
Preincubation
in
the
solution
of
MN
at
10−3M
for
6
h
was
the
most
effective
and
increased
germination
after
the
transfer
to
water
by
up
to
35%.
MV,
applied
at
10−5M
for
12
h
was
very
active
and
caused
germination
of
over
60%
of
the
caryopses.
The
effect
of
diphenyleneiodonium
(DPI)
in
the
presence
of
KAR1
or
GA3
The
following
experiment
was
conducted
to
obtain
an
answer
whether
ROS
were
involved
in
the
response
of
caryopses
to
KAR1
and
GA3.
Both
KAR1and
GA3strongly
stimulated
germination
caus-
ing
almost
complete
germination
of
the
dormant
caryopses:
ca.

D.
Cembrowska-Lech
et
al.
/
Journal
of
Plant
Physiology
176
(2015)
169–179
173
Fig.
2.
The
effect
of
menadione
(MN)
or
methylviologene
(MV),
applied
for
various
period,
on
the
germination
of
A.
fatua
L.
caryopses
at
20 ◦C
after
5
days.
Vertical
bars
indicate
±
SD.
One-way
ANOVA
with
the
Duncan
post
hoc
test
was
used
to
determine
significant
differences.
Mean
values
with
different
letters
(a–e)
are
significantly
different
(P
<
0.05).
100%
in
comparison
to
ca.
9%
in
the
case
of
the
untreated
caryopses
(Fig.
3).
DPI,
the
inhibitor
of
NADPH
oxidase
markedly
inhibited
germination
in
the
presence
of
KAR1or
GA3;
about
40–55%
of
the
caryopses
germinated.
The
effect
of
KAR1,
GA3,
H2O2and
AT
on
the
content
of
ABA
Twenty
five
embryos
isolated
from
dry
caryopses
contained
22
ng
of
ABA
(Fig.
4).
Incubation
of
caryopses
in
water
for
16
h
decreased
ca.
2
times
the
content
of
ABA.
Prolongation
of
the
incu-
bation
period
up
to
24
h
did
not
result
in
a
change
of
the
ABA
level.
KAR1and
GA3applied
during
the
incubation
lowered
the
level
of
ABA
in
embryos
for
about
30–40%
(Fig.
4A).
Likewise,
hydrogen
per-
oxide
applied
at
1
M
decreased
the
ABA
content
to
a
similar
value
as
the
above
regulators
did
(Fig.
4B).
AT
caused
a
similar
effect
on
the
content
of
ABA
as
exogenous
H2O2.
The
inhibitory
effect
of
H2O2at
1
M
was
also
evident
after
24
h
of
incubation.
The
effect
of
KAR1and
GA3on
H2O2and
O2•− localization
In
order
to
find
out
whether
KAR1and
GA3could
affect
the
local-
ization
of
ROS,
H2O2and
O2•−,
in
dormant
embryos,
H2O2and
O2•− were
visualized
in
embryos
isolated
from
caryopses,
incu-
bated
either
in
water
or
in
solutions
of
the
above
regulators
(Fig.
5).
H2O2was
localized
by
using
3,3-diaminobenzidine
(DAB)
which
produced
yellow
spots
in
the
presence
of
H2O2(Fig.
5A).
Embryos
Fig.
3.
The
effect
of
KAR1or
GA3in
the
absence
or
presence
of
NADPH
oxidase
inhibitor
(DPI)
on
the
germination
of
A.
fatua
L.
caryopses
at
20 ◦C
after
5
days.
Vertical
bars
indicate
±
SD.
One-way
ANOVA
with
the
Duncan
post
hoc
test
was
used
to
determine
significant
differences.
Mean
values
with
different
letters
(a–d)
are
significantly
different
(P
<
0.05).
isolated
from
dry
caryopses
and
from
caryopses
incubated
in
water
for
24
and
28
h
did
not
or
only
slightly
accumulate
H2O2.
KAR1
caused
homogenous
accumulation
of
H2O2on
the
surface
of
the
coleorhiza
only
after
28
h
of
incubation
(stage
I).
The
highest
inten-
sity
of
color
was
observed
after
28
h
when
the
coleorhiza
was
elongated
(stage
II).
Incubation
for
28
h
in
the
presence
of
GA3
increased
the
intensity
of
coleorhiza
staining
(stage
II).
Spots
were
concentrated
on
the
surface
of
the
coleorhiza
surrounding
the
radi-
cle
(stage
II).
The
radicle
which
protruded
through
the
coleorhiza
was
also
colored
(stage
III).
Nitro
blue
tetrazolium
(NBT)
was
used
to
analyze
O2•− accumulation
(Fig.
5B).
The
area
exhibited
dark
blue
formazan
spots
indicating
that
the
O2•− was
produced.
After
24
h
of
incubation
in
water,
the
accumulation
of
the
formazan
on
the
scutellum
was
rather
homogenous
and
not
intense;
it
was
not
similar
as
in
the
case
of
embryos
from
dry
caryopses.
Later,
a
slight
increase
of
color
intensity
was
noted
after
28
h
of
incubation.
KAR1
or
GA3applied
during
24
h
of
the
incubation
markedly
increased
the
accumulation
of
the
formazan
on
the
scutellum.
Staining
was
more
intense,
especially
when
more
advanced
stages
were
consid-
ered
(stage
II).
After
prolongation
of
the
incubation
for
up
to
28
h,
the
intensity
of
scutellum
color
was
similar
when
similar
stages
were
considered
(stages
I
and
II)
and
coleorhiza
was
also
slightly
colored
(stage
II).
In
addition
more
formazan
on
colorhiza
covering
top
of
radical
was
observed.
More
promoted
stages
of
embryo
ger-
mination
(stage
III)
showed
an
additional
surface
with
formazan
Fig.
4.
The
effect
of
KAR1(3
×
10−9M),
GA3(10−5M)
(A),
hydrogen
peroxide
(H2O2)
(1
M)
or
aminotriazole
(AT)
(10−3M)
(B)
on
abscisic
acid
(ABA)
content
in
embryos
isolated
from
A.
fatua
caryopses
incubated
for
16
or
24
h
at
20 ◦C.
Vertical
bars
indicate
±
SD.
Two-way
ANOVA
with
the
Duncan
post
hoc
test
was
used
to
determine
significant
differences.
Mean
values
with
different
letters
(a–c)
are
significantly
different
(P
<
0.05).

174
D.
Cembrowska-Lech
et
al.
/
Journal
of
Plant
Physiology
176
(2015)
169–179
Fig.
5.
In
situ
localization
of
hydrogen
peroxide
(H2O2)
(A)
and
superoxide
anion
(O2•−)
(B)
in
the
embryo
of
A.
fatua
L.
after
incubation
of
caryopses
in
the
absence
or
presence
of
KAR1(3
×
10−9M)
or
GA3(10−5M)
for
24
or
28
h
at
20 ◦C.
Dark
yellow
staining
in
the
presence
of
3,3-diaminobenzidine
(DAB)
indicates
of
H2O2.
Dark
blue
staining
in
the
presence
of
nitro
blue
tetrazolium
(NBT)
indicates
O2•− production.
deposits
on
the
coleorhiza
and
a
very
high
intensity
on
the
surface
of
embryo
radicles.
The
effect
of
KAR1and
GA3on
H2O2and
O2•− production
The
next
experiment
was
conducted
to
determine
the
effect
of
KAR1and
GA3on
the
content
of
H2O2and
O2•− after
various
periods
of
incubation
–
up
to
28
h
(Tables
1
and
2).
The
content
of
H2O2
in
caryopses
and
embryos
isolated
from
caryopses
was
increas-
ing
until
the
end
of
the
incubation
period
of
dormant
caryopses
in
water
(Table
1).
However,
the
content
of
H2O2in
aleurone
layers
in
the
caryopses
incubated
in
water
was
constant
during
the
whole
period
of
imbibition.
KAR1and
GA3increased
the
content
of
H2O2in
the
caryopses
after
16
h
of
incubation.
Both
compounds
increased
the
level
of
H2O2in
embryos
after
most
incubation
periods.
The
effect
of
KAR1and
GA3on
H2O2level
appeared
after
8
and
4
h
respectively.
The
effect
of
these
regulators
on
the
content
of
H2O2
was
higher
in
aleurone
layers
than
in
embryos.
The
content
of
hydrogen
peroxide
in
aleurone
layers
was
increased
by
KAR1and
GA3beginning
from
8
h
respectively.
KAR1and
GA3increased
the
content
of
H2O22–3
times
after
28
h.
The
effect
of
both
compounds
applied
during
the
incubation
of
caryopses
was
mainly
associated
with
the
control
of
H2O2production
in
aleurone
layers;
GA3was
more
effective
than
KAR1especially
in
the
case
of
aleurone
layers.
The
content
of
another
ROS,
namely
O2•−,
increased
continu-
ously
in
caryopses,
embryos
and
aleurone
layers
until
the
end
of
the
incubation
(Table
2).
Both
KAR1and
GA3increased
the
content
of
the
O2•− in
caryopses
and
embryos
beginning
from
4
h
and
in
aleurone
layers
beginning
from
12
h
of
imbibition.
Thus,
the
effect
of
both
compounds
occurred
earlier
in
embryos
than
in
aleurone
layers;
GA3had
a
similar
effect
as
KAR1on
the
content
of
O2•−
in
caryopses
in
most
periods,
but
was
more
effective
than
KAR1in
increasing
the
O2•− content
both
in
embryos
and
in
aleurone
layers.
The
effect
of
KAR1and
GA3on
SOD
and
CAT
activity
To
collect
more
information
on
the
regulation
of
ROS
by
KAR1
and
GA3the
activities
of
antioxidant
enzymes
SOD
and
CAT
were
measured
in
caryopses
or
embryos
and
aleurone
layers
isolated
from
caryopses
after
various
periods
of
incubation
in
water
and
in
KAR1or
GA3solutions.
The
activity
of
SOD
was
similar
in
embryos
during
the
whole
period
of
incubation
in
water
(Table
3).
The
activ-
ity
of
this
enzyme
was
similar
in
aleurone
layers
over
the
time
of
imbibition
of
the
caryopses
in
water.
KAR1and
GA3increased
the
activity
of
this
enzyme
in
caryopses
beginning
from
4
h.
In
embryos
the
stimulatory
effect
of
KAR1occurred
beginning
from
20
h
and
the
effect
of
GA3occurred
beginning
from
4
h.
KAR1and
GA3inhibited
the
SOD
activity
in
aleurone
layers
in
almost
all
periods
of
imbi-
bition
(Table
3).
CAT
in
caryopses,
embryos
and
aleurone
layers
almost
did
not
change
from
8
h
until
the
end
of
the
imbibition
period
in
water
(Table
4).
KAR1increased
the
activity
of
this
enzyme
in
caryopses
beginning
from
16
h
and
GA3did
so
beginning
from
8
h.
In
embryos,
the
stimulatory
effect
of
KAR1and
GA3began
to
occur
from
4
h.
KAR1and
GA3both
began
to
inhibit
the
activity
in
aleurone
layers
from
16
h
of
imbibition.
The
Effect
of
KAR1and
GA3on
electrophoretic
patterns
of
SOD
and
CAT
It
was
found
that
total
SOD
activity
was
represented
by
4
iso-
forms:
SOD-1
and
SOD-2
were
represented
by
Mn-SOD
whereas
SOD-3
and
SOD-4
were
represented
by
Cu/Zn
SOD
(Table
5).
All
isoforms
were
detected
in
dry
caryopses,
embryos
from
dry
cary-
opses,
caryopses
and
embryos
isolated
from
caryopses
incubated
in
water
and
in
KAR1or
GA3solutions
for
12
or
24
h
(not
shown).
The
most
intense
bands
were
related
to
SOD-2.
Intensity
of
other

D.
Cembrowska-Lech
et
al.
/
Journal
of
Plant
Physiology
176
(2015)
169–179
175
Table
1
The
effect
of
KAR1or
GA3on
the
hydrogen
peroxide
(H2O2)
level
in
A.
fatua
L.
caryopses,
embryos
and
aleurone
layers
after
caryopses
incubation
for
different
time
at
20 ◦C.
Vertical
bars
indicate
±
SD.
One-way
ANOVA
with
the
Duncan
post
hoc
test
was
used
to
determine
significant
differences.
Mean
values
with
different
letters
(a–c)
are
significantly
different
(P
<
0.05).
After
30
and
28
h
ca.
10
±
5%
of
the
caryopses
with
radicle
protruded
through
the
coleorhiza
were
noted
as
result
of
KAR1or
GA3treatment.
Treatment
H2O2(␮M
g−1FW)
Time
(h)
0
4
8
12
16
20
24
28
Caryopsis
H2O 84.0
±
3.4
86.0
±
2.8a
92.4
±
5.0a
98.1
±
2.7b
95.4
±
4.3a
98.5
±
3.0a
102.6
±
1.8a
101.4
±
4.0a
KAR1,
3
×
10−9M
–
87.2
±
3.2a
91.4
±
3.4a
90.5
±
2.6a
100.7
±
1.6b
101.1
±
2.4a
103.6
±
3.4a
122.9
±
3.2b
GA3,
10−5M
–
87.6
±
3.1a
93.0
±
3.8a
99.0
±
4.1b
108.0
±
2.3c
111.9
±
4.0b
117.3
±
2.6b
152.4
±
7.3c
Embryo
H2O
36.2
±
1.5
39.3
±
2.4a
41.9
±
3.5a
43.8
±
5.1a
45.8
±
7.3a
43.8
±
3.7a
42.5
±
4.4a
45.8
±
2.7a
KAR1,
3
×
10−9M–
41.3
±
1.2a 49.8
±
2.5b 51.7
±
3.4b 54.2
±
3.6b 55.7
±
3.4b 56.8
±
3.9b 60.2
±
2.6b
GA3,
10−5M–
46.1
±
2.1b 52.3
±
1.2b 55.7
±
1.3b 57.6
±
3.7b
58.7
±
3.2b
60.7
±
2.3b
67.0
±
3.1c
Aleurone
layer
H2O
33.3
±
2.2
33.3
±
1.1ab
32.8
±
1.8a
33.9
±
2.5a
36.5
±
2.0a
34.6
±
1.1a
33.1
±
3.4a
36.0
±
1.2a
KAR1,
3
×
10−9M
–
35.2
±
2.2b
38.0
±
2.6b
39.7
±
3.1b
46.2
±
2.3b
55.2
±
2.0b
64.4
±
2.7b
74.6
±
3.7b
GA3,
10−5M
–
36.9
±
1.9b
57.2
±
1.7c
60.1
±
4.0c
66.7
±
3.4c
69.7
±
1.5c
73.5
±
2.8c
94.7
±
1.2c
Table
2
The
effect
of
KAR1or
GA3on
the
superoxide
anion
(O2•−)
level
in
A.
fatua
L.
caryopses,
embryos
and
aleurone
layers
after
caryopses
incubation
for
different
time
at
20 ◦C.
Vertical
bars
indicate
±
SD.
One-way
ANOVA
with
the
Duncan
post
hoc
test
was
used
to
determine
significant
differences.
Mean
values
with
different
letters
(a–c)
are
significantly
different
(P
<
0.05).
Treatment
O2•− (mM
g−1FW)
Time
(h)
0
4
8
12
16
20
24
28
Caryopsis
H2O
3.5
±
0.5
4.1
±
1.3a
5.1
±
0.4a
7.0
±
0.8a
8.0
±
0.8a
7.5
±
0.5a
8.1
±
1.5a
9.0
±
1.4a
KAR1,
3
×
10−9M
–
6.5
±
1.0b
9.9
±
1.1b
12.1
±
1.7b
16.2
±
1.4b
16.6
±
0.7b
18.0
±
1.5b
18.9
±
1.2b
GA3,
10−5M–
7.7
±
0.7b 13.3
±
1.1c 15.1
±
1.2c 17.3
±
1.1b
17.6
±
0.9b
18.1
±
2.1b
19.0
±
1.7b
Embryo
H2O
1.5
±
0.3
1.3
±
0.2a
1.4
±
0.2a
3.1
±
0.4a
4.7
±
0.4a
4.9
±
0.3a
5.2
±
0.3a
6.1
±
0.4a
KAR1,
3
×
10−9M
–
2.9
±
0.2bc
4.8
±
0.3bc
6.8
±
0.5bc
7.4
±
0.7b
7.6
±
0.5b
7.9
±
0.3b
9.1
±
0.3b
GA3,
10−5M
–
3.4
±
0.4c
5.3
±
0.5c
7.8
±
0.3c
9.3
±
0.6c
10.5
±
0.7c
12.8
±
0.3c
15.0
±
0.7c
Aleurone
layer
H2O
2.1
±
0.3
4.4
±
0.3ab
6.7
±
0.5bc
5.1
±
0.3a
5.7
±
0.2a
6.0
±
0.2a
6.5
±
0.3a
7.1
±
0.5a
KAR1,
3
×
10−9M–
4.8
±
0.3b 5.6
±
0.5a
8.7
±
0.6b
7.9
±
0.4b
8.9
±
0.4b
9.9
±
0.3b
12.0
±
0.8b
GA3,
10−5M
–
5.0
±
0.2b
7.5
±
0.3c
8.4
±
0.4b
11.1
±
0.6c
11.2
±
0.5c
12.0
±
0.8c
17.0
±
0.2c
bands
was
similar
and
independent
of
the
treatment
and
incuba-
tion
time.
KAR1increased
the
intensity
of
the
band
representing
SOD-2
in
caryopses
and
embryos
after
24
h
of
incubation
and
GA3
increased
the
intensity
after
12
and
24
h
also
in
the
case
of
cary-
opses
and
embryos
(Table
5).
Intensity
of
the
SOD-2
band
was
similar
when
the
aleurone
from
dry
caryopses
was
used
or
after
incubation
in
water
for
12
or
24
h.
SOD-2
isoenzyme
intensity
dis-
appeared
after
24
or
12
h
due
to
the
treatment
with
KAR1or
GA3,
respectively.
However,
when
40
␮g
protein
instead
of
20
␮g
was
applied
for
the
electrophoresis
some
intensity
of
SOD-2
after
24
h
incubation
in
the
presence
of
KAR1or
GA3was
observed
but
much
lower
in
comparison
to
the
control
(not
shown).
Only
one
band
of
CAT
activity
was
noted
in
all
samples
of
caryopses,
embryos
and
aleurone
layers
(Table
5).
The
bands
repre-
senting
dry
caryopses
and
embryos
and
the
aleurone
layers
isolated
from
caryopses
incubated
for
12
and
24
h
had
a
similar
intensity.
Table
3
The
effect
of
KAR1or
GA3on
the
superoxide
dismutase
(SOD)
activity
in
A.
fatua
L.
caryopses,
embryos
and
aleurone
layers
after
caryopses
incubation
for
different
time
at
20 ◦C.
Vertical
bars
indicate
±
SD.
One-way
ANOVA
with
the
Duncan
post
hoc
test
was
used
to
determine
significant
differences.
Mean
values
with
different
letters
(a–c)
are
significantly
different
(P
<
0.05).
Treatment
SOD
(U
mg−1protein)
Time
(h)
0
4
8
12
16
20
24
28
Caryopsis
H2O
26.4
±
1.7
24.4
±
1.4a
26.4
±
2.5ab
25.4
±
2.5a
25.4
±
2.7a
25.4
±
2.6a
26.1
±
1.9a
27.2
±
2.0a
KAR1,
3
×
10−9M
–
26.8
±
1.3b
32.8
±
1.5b
33.2
±
4.1b
38.8
±
0.8b
40.3
±
1.7b
43.8
±
2.2c
47.7
±
1.1c
GA3,
10−5M
–
29.7
±
1.7c
36.7
±
1.2c
39.3
±
1.6c
41.5
±
1.2c
41.7
±
0.6b
40.2
±
0.6b
39.7
±
1.4b
Embryo
H2O
24.6
±
0.4
24.0
±
1.0ab
22.2
±
4.1a
23.8
±
1.2ab
23.7
±
3.3ab
24.2
±
3.0a
22.7
±
3.1a
25.1
±
1.2a
KAR1,
3
×
10−9M
–
25.1
±
0.5bc
24.4
±
1.3ab
25.5
±
1.0b
27.0
±
1.0b
32.4
±
2.2b
32.6
±
2.1b
41.2
±
1.2b
GA3,
10−5M
–
26.2
±
0.6c
26.0
±
1.3b
35.3
±
1.1c
47.9
±
1.8c
45.5
±
2.6c
45.3
±
0.7c
44.3
±
0.7c
Aleurone
layer
H2O
19.9
±
1.5
22.0
±
0.9b
21.3
±
2.0c
23.9
±
1.3c
20.3
±
1.3c
19.3
±
1.2b
19.7
±
1.9c
21.6
±
2.1b
KAR1,
3
×
10−9M
–
17.8
±
1.0a
19.2
±
1.0bc
17.5
±
1.2b
14.4
±
0.6b
11.5
±
0.5a
11.7
±
0.4b
8.8
±
0.7a
GA3,
10−5M
–
18.0
±
1.1a
16.0
±
0.3a
15.9
±
0.6a
11.5
±
0.6a
10.8
±
0.5a
9.6
±
0.6a
9.0
±
0.9a

176
D.
Cembrowska-Lech
et
al.
/
Journal
of
Plant
Physiology
176
(2015)
169–179
Table
4
The
effect
of
KAR1or
GA3on
the
catalase
(CAT)
activity
in
A.
fatua
L.
caryopses,
embryos
and
aleurone
layers
after
caryopses
incubation
for
different
time
at
20 ◦C.
Vertical
bars
indicate
±
SD.
One-way
ANOVA
with
the
Duncan
post
hoc
test
was
used
to
determine
significant
differences.
Mean
values
with
different
letters
(a–c)
are
significantly
different
(P
<
0.05).
Treatment
CAT
(U
mg−1protein
min−1)
Time
(h)
0
4
8
12
16
20
24
28
Caryopsis
H2O 7.4
±
1.3
10.0
±
0.9ab
11.4
±
1.9ab
12.9
±
2.7ab
11.6
±
0.5a
12.6
±
0.3a
12.4
±
0.5a
11.8
±
0.8a
KAR1,
3
×
10−9M
–
13.6
±
0.8c
13.4
±
0.5b
15.4
±
0.7b
17.1
±
0.6b
17.5
±
0.7b
18.0
±
0.9b
27.1
±
1.0b
GA3,
10−5M
–
11.9
±
1.4bc
25.6
±
1.1c
26.5
±
0.7c
33.4
±
0.7c
34.3
±
0.7c
35.0
±
0.7c
38.6
±
0.3c
Embryo
H2O
5.2
±
1.1
7.7
±
1.9a
14.2
±
1.2a
13.6
±
0.4a
15.8
±
0.9a
15.2
±
0.9a
15.2
±
0.6a
15.8
±
0.6a
KAR1,
3
×
10−9M–
15.1
±
2.1b 14.5
±
0.4a 16.2
±
0.5b 15.8
±
0.6a 22.6
±
0.7b 24.1
±
1.0b 27.9
±
0.5b
GA3,
10−5M–
16.9
±
2.1b 20.4
±
0.4b 36.4
±
2.3c 39.2
±
2.3b
39.4
±
1.1c
38.5
±
1.3c
48.0
±
5.0c
Aleurone
layer
H2O
7.1
±
2.2
11.9
±
3.8b
13.2
±
0.2b
12.1
±
0.8b
11.2
±
0.3b
11.1
±
0.4b
11.5
±
0.3b
10.7
±
0.6b
KAR1,
3
×
10−9M
–
9.7
±
2.1ab
12.9
±
0.3b
10.7
±
0.5ab
7.0
±
0.2a
7.0
±
0.4a
7.0
±
0.3a
4.8
±
0.5a
GA3,
10−5M
–
8.2
±
0.8a
10.4
±
1.3a
11.8
±
0.9ab
6.1
±
1.1a
6.9
±
0.3a
6.9
±
0.6a
4.9
±
0.4a
KAR1increased
the
intensity
of
the
CAT
band
after
24
h
in
the
case
of
caryopses
and
embryos.
GA3increased
the
intensity
of
the
CAT
band
after
12
and
24
h
incubation
when
caryopses
or
embryos
were
used.
CAT
activity
in
aleurone
layer
ceased
after
24
h
or
12
h
incu-
bation
due
to
the
treatment
with
KAR1or
GA3respectively.
When
40
␮g
protein
was
applied
for
electrophoresis,
some
intensity
after
incubation
for
12
and
24
h
in
the
presence
of
GA3was
noted,
but
lower
in
comparison
to
the
control
(not
shown).
The
effect
of
KAR1,
GA3,
H2O2,
AT,
MN
and
MV
on
˛-amylase
activity
After
24
h
of
incubation
both
KAR1and
GA3increased
the
activ-
ity
of
␣-amylase
(Fig.
6).
The
activity
of
this
enzyme
increased
2.5
or
5
times
due
to
the
KAR1or
GA3treatment.
Likewise,
H2O2markedly
increased
the
enzyme’s
activity
although
to
a
little
lower
level
than
KAR1.
The
application
of
ROS
generating
MN
or
MV
also
increased
Table
5
The
effect
of
KAR1or
GA3on
the
superoxide
dismutase
(SOD)
and
catalase
(CAT)
isoenzymes
activities
in
A.
fatua
L.
caryopses,
embryos
and
aleurone
layers
after
caryopses
incubation
for
12
or
24
h
at
20 ◦C.

D.
Cembrowska-Lech
et
al.
/
Journal
of
Plant
Physiology
176
(2015)
169–179
177
Fig.
6.
The
effect
of
KAR1(3
×
10−9M),
GA3(10−5M),
hydrogen
peroxide
(H2O2)
(1
M),
menadione
(MN)
(10−3M),
methylviologen
(MV)
(10−5M)
or
aminotriazole
(AT)
(10−3M)
on
the
␣-amylase
activity
in
A.
fatua
L.
caryopses
after
incubation
for
24
h
at
20 ◦C.
Vertical
bars
indicate
±
SD.
Two-way
ANOVA
with
the
Duncan
post
hoc
test
was
used
to
determine
significant
differences.
Mean
values
with
different
letters
(a–e)
are
significantly
different
(P
<
0.05).
the
activity
of
this
enzyme.
Inhibition
of
CAT
activity
by
AT
also
stimulated
the
activity
of
the
␣-amylase.
Discussion
KAR1and
GA3induced
germination
of
dormant
caryopses
associated
with
the
control
of
ABA
level
and
ROS-antioxidant
status
in
embryos
In
order
to
determine
the
role
of
ROS
in
germination
of
A.
fatua
L.
dormant
caryopses,
exogenous
H2O2and
compounds
modulat-
ing
the
content
of
endogenous
H2O2and
O2•− were
applied.
It
was
shown
that
exogenous
H2O2,
a
compound
that
breaks
dor-
mancy
in
sunflower
seeds
(Oracz
et
al.,
2009)
and
barley
caryopses
(Fontaine
et
al.,
1994;
Bahin
et
al.,
2011)
also
induced
germination
of
dormant
A.
fatua
caryopses
(Hsiao
and
Quick,
1984)
(Fig.
1).
The
release
of
dormancy
by
H2O2could
be
associated
with
oxidizing
phenolic
compounds
present
in
the
caryopses
envelopes
(Fontaine
et
al.,
1994).
Alternatively
it
might
mimic
endogenous
H2O2which
could
participate
in
regulating
dormancy
via
control
expression
of
genes.
It
was
found
that
H2O2,
which
partially
removed
dor-
mancy
in
barley,
inhibited
the
expression
of
the
HvGA2ox3
gene
involved
in
the
catabolism
of
GA
and
enhanced
the
expression
of
the
HvGA20ox1
gene
implicated
in
GA
synthesis
(Bahin
et
al.,
2011).
However,
Leymarie
et
al.
(2012)
suggested
that
the
release
of
dor-
mancy
in
Arabidopsis
seeds
by
ROS
did
not
involve
the
metabolism
of
gibberellins,
but
germination
could
be
triggered
through
gib-
berellin
signaling
activation.
Exogenous
H2O2was
not
able
to
remove
dormancy
in
A.
fatua
caryopses
completely
probably
due
to
its
degradation
by
the
CAT
and
peroxidases
as
has
been
suggested
in
the
case
of
barley
embryos
(Bahin
et
al.,
2011).
Germination
stim-
ulation
by
AT
(Fig.
1),
an
inhibitor
of
the
CAT
(Amory
et
al.,
1992)
known
to
increase
the
content
of
H2O2,
might
indicate
the
impor-
tant
role
of
endogenous
H2O2in
releasing
dormancy
in
A.
fatua
caryopses.
In
contrast,
AT
did
not
have
an
effect
on
germination
of
dormant
barley
caryopses
(Bahin
et
al.,
2011).
The
stimulatory
effect
on
caryopses
germination
exercised
by
MN
and
MV
(Fig.
2),
compounds
generating
O2•− (Wise
and
Naylor,
1987;
Hung
and
Kao,
2004),
indicated
that
this
ROS
also
participated
in
dormancy
releasing
as
has
been
found
with
Arabidopsis
seeds
(Leymarie
et
al.,
2012).
However,
in
contrast
to
the
results
obtained
with
Arabidopsis
(Leymarie
et
al.,
2012)
in
the
experiment
with
A.
fatua
the
MV
was
more
effective
than
the
MN.
In
the
case
of
barley,
MV
did
not
have
any
effect
but
MN
stimulated
the
germination
of
dormant
caryopses
(Bahin
et
al.,
2011).
In
order
to
establish
the
relationship
between
KAR1or
GA3and
ROS
the
above
regulators
were
applied
in
combination
with
free
radical
scavengers.
Sodium
benzoate
(SB),
a
general
free
radical
scavenger
(Hung
and
Kao,
2004),
and
Tiron,
O2•− scavenger
(Wise
and
Naylor,
1987),
lowered
the
effect
of
KAR1and
GA3only
on
the
rate
of
A.
fatua
L.
caryopses
germination
(not
shown).
However,
DPI,
the
inhibitor
of
NADPH
oxidase
catalyzing
the
production
of
appoplastic
O2•− from
oxygen
(Sagi
and
Fluhr,
2006),
decreased
markedly
the
percentage
of
germination
in
the
presence
of
KAR1
or
GA3,
suggesting
an
important
role
of
the
O2•− in
germination
induction
by
the
above
regulators
(Fig.
3).
The
key
function
of
NADPH
oxidase
in
germination
of
switchgrass
(Sarath
et
al.,
2007),
sunflower
(Oracz
et
al.,
2009),
barley
(Ishibashi
et
al.,
2010)
and
Arabidopsis
(Leymarie
et
al.,
2012)
has
been
noted
as
well.
A
decrease
in
the
content
of
endogenous
ABA
in
imbibed
embryos
of
A.
fatua
caused
by
KAR1(Fig.
4A)
might
suggest
that
germination
induction
by
this
compound
involves
the
control
level
of
that
inhibitor.
In
contrast,
KAR1did
not
affect
the
ABA
level
dur-
ing
imbibition
of
dormant
Arabidopsis
seeds
although
it
stimulated
germination
(Nelson
et
al.,
2009).
Exogenous
GA3,
similarly
to
KAR1,
lowered
the
ABA
level
in
A.
fatua
embryos
(Fig.
4A).
Gibberellin
also
inhibited
accumulation
of
ABA
during
imbibition
and
released
dor-
mancy
in
Nicotiana
plumbaginifolia
seeds
(Grappin
et
al.,
2000).
The
relationship
between
GA3and
ABA
in
the
regulation
of
dormancy
state
has
been
known;
ABA
is
considered
as
a
positive
regulator
and
GA3as
a
negative
for
seed
dormancy
maintenance
(Kucera
et
al.,
2005;
Feurtado
and
Kermode,
2007).
Earlier
studies
showed
that
KAR1required
gibberellin
biosynthesis
to
show
its
stimula-
tory
effect
on
the
germination
of
dormant
Arabidopsis
thaliana
seeds
(Nelson
et
al.,
2009)
and
A.
fatua
caryopses
(K˛
epczy ´
nski
et
al.,
2013).
Furthermore,
it
was
found
that
KAR1did
not
influence
the
sen-
sitivity
of
A.
thaliana
seeds
to
gibberellins
(Nelson
et
al.,
2009).
Likewise,
there
was
not
apparent
connection
between
gibberellins
and
the
germination
response
of
Brassica
tournefortii
seeds
to
KAR1
(Long
et
al.,
2010).
H2O2,
at
concentration
inducing
germination
of
caryopses
(Fig.
1),
similarly
as
KAR1decreased
the
ABA
content
in
dormant
A.
fatua
embryos
(Fig.
4B).
The
fact
that
AT,
a
well-
known
inhibitor
of
CAT
activity,
decreased
the
ABA
level
(Fig.
4B)
stimulating
the
germination
of
dormant
caryopses
(Fig.
1)
might
suggest
that
endogenous
H2O2also
controlled
the
content
of
that
inhibitor.
Contradictory
results
were
obtained
in
experiments
with
dormant
barley
caryopses,
H2O2decreased
(Wang
et
al.,
1998)
or
increased
(Bahin
et
al.,
2011)
ABA
content
in
embryos.
In
Arabidop-
sis
seeds
releasing
dormancy
by
H2O2did
not
seem
to
involve
the
metabolism
of
ABA
(Leymarie
et
al.,
2012).
The
application
of
DAB
and
NBT
revealed
a
difference
in
the
presence
of
H2O2and
O2•− in
embryos
isolated
from
dormant
cary-
opses
untreated
and
treated
with
KAR1or
GA3(Fig.
5).
KAR1and
GA3caused
accumulation
of
H2O2(Fig.
5A)
in
the
coleorhiza
and
the
radicle,
while
the
O2•− (Fig.
5B)
accumulated
mainly
in
the
scutellum,
partly
in
the
coleorhiza
and
also
in
the
radicle.
These
data
might
indicate
that
KAR1and
GA3controlled
ROS
production
in
embryos
and
suggest
that
the
rate
of
production
of
these
ROS
was
greater
than
the
rate
of
detoxification.
Different
localizations
of
H2O2and
O2•− might
suggest
that
the
function
of
the
above
ROS
were
related
to
different
parts
of
the
embryo.
A
high
accumulation
of
the
O2•− in
the
scutellum
(Fig.
5B),
which
serves
as
a
place
of
gibberellin
synthesis
(Appleford
and
Lenton,
1997)
and
a
transfer
route
for
peptides
and
sugars
(West
et
al.,
1998;
Aoki
et
al.,
2006)
in
cereal
grains,
was
probably
related
to
programmed
cell
death.
Pro-
grammed
cell
death
in
the
scutellum
of
germinated
wheat
grains
has
been
described
by
Domínguez
et
al.
(2012).
It
was
also
shown
that
the
exogenous
gibberellin
did
not
affect
PCD
in
the
scutellum
of
wheat,
but
ABA
had
a
strong
inhibitory
effect
on
germination
and

178
D.
Cembrowska-Lech
et
al.
/
Journal
of
Plant
Physiology
176
(2015)
169–179
PCD.
The
localization
of
the
O2•− on
top
of
the
coleorhiza
might
suggest
special
requirements
for
this
ROS
for
that
part
of
the
cole-
orhiza
which
would
be
broken
by
the
radicle.
Previously
it
was
demonstrated
that
ROS
are
involved
in
lettuce
endosperm
cap
tip
weakening
(Zhang
et
al.,
2014).
Staining
of
the
radicle
with
DAB
and
NBT
demonstrated
that
radicles
were
able
to
produce
both
H2O2
and
O2•−.
The
presence
of
both
H2O2and
O2•− in
the
radicle
may
suggest
its
role
in
radicle
elongation
associated
with
its
protrusion
through
the
coleorhiza.
Previously,
it
has
been
demonstrated
that
ROS
were
involved
in
root
and
radicle
growth
(Liszkay
et
al.,
2004;
Oracz
et
al.,
2009).
It
was
also
suggested
that
ROS
generated
after
the
treatment
of
sunflower
embryos
with
the
dormancy
releasing
compound,
HCN,
played
a
role
in
cell
elongation
related
to
radicle
protrusion
(Oracz
et
al.,
2009).
There
are
data
showing
that
endogenous
ROS
are
responsible
for
the
shift
from
dormant
to
non-dormant
seeds
(Bailly
et
al.,
2008).
KAR1and
GA3increased
the
content
of
H2O2and
O2•− from
an
early
stage
of
imbibition
(Tables
1
and
2).
A
similar
relationship
between
factors
removing
seed
dormancy
and
the
level
of
ROS
was
demonstrated;
for
example,
the
cyanide
causing
dormancy
release
in
sunflower
seeds
triggered
ROS
accumulation
(Oracz
et
al.,
2007).
Dormancy
release
in
those
seeds
by
after-ripening
was
associated
with
increasing
ROS.
On
the
other
hand,
after-ripening
did
not
affect
the
accumulation
of
H2O2in
barley
(Bahin
et
al.,
2011).
The
increase
of
H2O2and
O2•− in
A.
fatua
embryos
caused
by
KAR1and
GA3
supports,
a
conclusion
that
both
ROS
play
important
function
in
ger-
mination
control
of
dormant
caryopses
by
above
regulators.
Since
H2O2was
considered
not
only
as
a
signaling
molecule
but
also
as
a
toxic
cellular
metabolite
(Bailly
et
al.,
2008)
it
could
be
expected
that
the
concentration
of
O2•− and
H2O2in
embryos
should
be
maintained
on
an
adequate
level,
safe
for
the
embryo
cells.
Probably
therefore
KAR1and
GA3increased
the
activity
of
the
SOD
(Table
3),
producing
H2O2from
O2•− and
CAT
(Table
4),
an
enzyme
removing
H2O2.
Among
four
SOD
isoforms,
two
Mn-SOD
and
two
Cu/Zn-SOD,
high
activity
was
noted
only
in
the
case
of
Mn-SOD-2
(Table
5).
The
activity
of
this
isoform
was
increased
by
KAR1and
GA3.
Thus,
stimu-
latory
effect
of
above
regulators
on
SOD
activity
(Table
3)
probably
was
related
to
Mn-SOD-2.
CAT
was
represented
only
by
one
isoform
which
was
controlled
by
the
above
regulators.
KAR1and
GA3induced
germination
of
dormant
caryopses
associated
with
control
of
˛-amylase
in
A.
fatua
caryopses
and
ROS-antioxidant
status
in
aleurone
layers
Germination
processes
of
grasses
are
controlled
by
GA3and
ABA
via
regulation
of
synthesis
and
secretion
of
hydrolytic
enzymes,
mainly
␣-amylase
from
the
aleurone
to
the
starchy
endosperm
(Jones
and
Jacobsen,
1991;
Bethke
et
al.,
1999).
GA3and
KAR1
have
been
found
to
stimulate
␣-amylase
in
A.
fatua
caryopses
(K˛
epczy ´
nski
et
al.,
2013).
A
stimulatory
effect
of
gibberellin
on
␣-
amylase
production
in
aleurone
layers
of
A.
fatua
has
also
been
demonstrated
previously
(Smith
and
Hooley,
2002).
The
activity
of
␣-amylase
was
increased
not
only
by
KAR1and
especially
GA3,
H2O2,
MN,
MV
and
AT
(Fig.
6),
compounds
stimulating
caryopses
germination
(Figs.
1
and
2).
After
finishing
their
important
role,
aleurone
cells
are
useless
and
therefore
die.
Death
of
the
aleu-
rone
cells
in
several
cereal
known
as
programmed
cell
death
(PCD),
is
controlled
by
ROS,
H2O2and
O2•− (Fath
et
al.,
2001a,b,
2002).
The
increase
of
H2O2and
O2•− contents
in
A.
fatua
aleurone
layers
treated
with
GA3or
KAR1(Tables
1
and
2)
is
probably
associated
with
aleurone
layers
preparing
to
die.
An
increased
ROS
concentra-
tion
due
to
the
treatment
with
GA3has
been
previously
reported
by
Fath
et
al.
(2002).
It
has
also
been
found
that
CAT
and
SOD
activity
were
down-regulated
by
GA3in
barley
(Fath
et
al.,
2001a)
to
cause
a
sufficient
accumulation
of
ROS
required
for
cell
death.
Likewise,
KAR1and
GA3decreased
the
activity
of
SOD
and
CAT
in
A.
fatua
aleurone
layers
(Tables
3
and
4).
Data
regarding
gel
staining
activity
of
SOD-2
and
CAT
showed
that
both
KAR1and
GA3caused
marked
decreasing
of
those
enzymes.
Thus,
similarly
to
cereal
aleurone,
in
A.
fatua
aleurone
the
gibberellin
probably
induced
programmed
cell
death
by
increasing
ROS
content
which
was
correlated
to
a
decreasing
activity
of
scavenging
enzymes.
Presented
results
indi-
cate
that
KAR1played
a
similar
role
to
GA3in
the
aleurone
layer
of
A.
fatua
caryopses.
In
summary,
KAR1,
GA3and
ROS
participate
in
the
regulation
of
dormancy
release
and
germination
of
A.
fatua
caryopses.
Ger-
mination
induction
of
dormant
caryopses
involves
an
interaction
between
various
compounds.
The
increase
of
H2O2and
O2•− in
A.
fatua
embryos
caused
by
KAR1and
GA3together
with
data
from
the
experiment
with
the
inhibitor
of
NADPH
oxidase
strongly
indi-
cates
the
involvement
of
ROS
in
germination
control
of
dormant
caryopses
by
the
above
regulators.
Both
KAR1and
GA3,
release
dor-
mancy
by
decreasing
the
content
of
endogenous
ABA
and
maintain
ROS
homeostasis
in
the
embryo.
In
the
embryo
the
ROS
concentra-
tion
was
increased
simultaneously
with
the
enzymatic
scavenging
system
in
order
to
adjust
ROS
to
non-toxic,
but
signaling
levels.
Since
ROS
could
also
decrease
the
content
of
ABA,
multiple
control
of
dormancy
releasing
by
KAR1and
GA3should
be
considered.
Both
KAR1and
GA3probably
induce
programmed
cell
death
in
the
aleurone
and
scutellum
associated
with
an
increase
in
the
concentration
of
ROS,
H2O2and
O2•−,
which
were
related
with
a
decreasing
activity
of
SOD
and
CAT.
A.
fatua
caryopses,
used
in
the
above
experiments,
are
characterized
by
non-deep
physi-
ological
dormancy
which
is
probably
mainly
associated
with
the
presence
of
tissues
surrounding
the
embryo.
Therefore,
the
control
of
␣-amylase
activity
in
caryopses
and
ROS
in
aleurone
layers
can
either
be,
as
usual,
discussed
in
relation
to
germination
or
can
be
considered
as
responsible
for
lowering
the
effect
of
the
aleurone
which
is
probably
partially
responsible
for
imposing
dormancy.
Previously,
it
has
been
postulated
that
the
endosperm/pericarp
tissues
surrounding
the
embryo
are
responsible
for
physiological
dormancy
in
cereal
caryopses
(Finch-Savage
and
Leubner-Metzger,
2006;
Howard
et
al.,
2012).
Concluding,
the
role
of
KAR1and
GA3in
the
induction
germina-
tion
of
A.
fatua
dormant
caryopses
appears
to
be
mediated
by
the
control
of
ABA
level
in
embryos
and
ROS-antioxidant
status
both
in
embryos
and
aleurone
layers.
The
level
of
ABA
could
be
controlled
by
KAR1and
GA3directly
or
via
ROS.
Acknowledgment
The
study
was
supported
by
the
Ministry
of
Science
and
Higher
Education
Grant
NN
310
726340.
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