Hot. Need.
Acta
Protection
against
of
p.
4150
leaf
Cucumis
photo-oxidative
during
Ph.R. van
1976,
25(1), February
pigments
degradation
chilling
Hasselt
Biologisch Centrum, Afdeling Plantenfysiologie, Rijksuniversiteit Groningen,
Haren
SUMMARY
The
possible protection against photo-oxidative degradation
Cucumis
leaf
and
sulphoxide
ineffective.
at
discs
1 °C
glycerol
by
well
as
Photo-oxidation
wide
a
however,
least
at
of chlorophyll
compounds
was
KCN
ferricyanide, ascorbate,
as
was,
of
variety
partly,
and carotene in
investigated.
and
Dimethyl
hydroxylamine
prevented by
were
benzoquinone,
hydroquinone, naphtoquinone, guajacol, benzidine, diaminobenzidine, alizarinesulphonicacid,
and
triphenyltetrazoliumchloride
oxidative
damage
ethylenediamine
It
viologen.
tetraacetic
of Cucumis
protection
of the
protection
1,1-dimethylureea.
trishydroxymethylaminomethane,
acid,
increased
also
was
3-(3,4-dichlorophenyl),
Photo-
promoted by salicylaldoxine, disalicylidenediaminopropane, azide,
was
high pH
at
values.
leaf chloroplasts against
electron
low
It
suggested
and
diquat
that
the
benzyl-
mechanism
of
temperaturephoto-oxidative damage is
from the
transport pathway
is
reducing
to the
oxidizing
side
a
of the
photosystems.
1.
INTRODUCTION
Noack
observed that
(1925)
photo-oxidation
chlorophyll
in Fontinalis leaves is
the leaves in
by soaking
a
protected against
aqueous benzidine solution
0.04%
before irradiation. The leaves became brown because of oxidation of benzidine
while the
from
chlorophyll
protective
effect
Surprisingly
oxidative
damage
protect
Heber
to
of
in
cells
Setchenka
application
et
acceptor
al.
pH
of
was
(1971)
that
1959).
DMSO
of inhibitors of
seem
These
photo-
been
made.
inhibits
compounds
1968, Trunova
isolated
frost
may also
1968).
chloroplast
phosphorylation.
and DMSO
on
the
during chilling
light
induced chloro-
investigated.
was
In
studied.
that the
Hill reaction
spinach chloroplasts
pH
also affects
with
is influenced
6.5 and 8.0 and decreased
than 8.0. In addition,
have
to
(DMSO)
protects
of
The
cells.
the effect
uncoupling
glycerol
showed
in isolated
pH
living
protected chlorophyll
methanolic solution.
sulphoxide
& Bishop
Cucumis leaf discs
maximal between
higher
showed
against freezing-induced
degradation
on
in
freezing injury (Quatrano
against
in
to
as
chemicals
by
dimethyl
(Lovelock
(1967)
addition the effect of
was
investigations
and
well
as
restricted
not
higher plants
glycerol
Ernst
The effect of
electron
is
further
no
animal cells
plant
&
lamellae
phyll
in dried leaves,
evidently
damage
Application
left unaltered. Benzidine also
was
photo-oxidation
at
pH
DCPIP
as
an
by pH. Activity
lower than 6.5 and
photo-oxidation
of
chlorophyll
in
42
PH.
vitro
(Evstigneev
This author found
1972).
in ethanol
chlorophyll-a by benzoquinone
the effect of various inhibitors
in Cucumis leaf discs
on
the
excitation of
quench directly
enhanced
VAN
HASSELT
photo-oxidation
lower than 6.0. In this
pH
photooxidative degradation
of
of
study
pigments
is also described.
during chilling
Two main groups of inhibitors
an
at a
R.
tested:
were
such
chlorophyll
firstly
substances which
benzoquinone
as
substances which affect the electron transport in the
and
chloroplast such
may
secondly
as
DCMU
and SAL.
2.
MATERIAL
2.1
AND
METHODS
Material
Plants
sativus
(Cucumis
greenhouse (minimum
20
(L)
cv.
Kleine Groene
day temperature
25 °C;
Scherpe)
minimum
were
in
grown
a
night temperature
°C).
Discs
of 7
leaves of
Discs
weeks old
placed
were
incubated
diameter
mm
two
at
in
1 °C in the
were
punched
the
on
2.2
Measurement of the
Chlorophyll (a
80%
acetone
663
at
and
method
+
nm.
used
1974). Carotene
tography and
selt
2.3
b)
extract
645
was
to
solutions in the dark for
test
was
of 40 leaf discs
In
some
of
as
the
from
its concentration
was
in darkness,
light
as
test
hours
solutions and
described
1 °C the leaf discs
at
at room
previously
were
pretreat-
temperature.
concentration
measured
of
a
samples
chlorophyll by
measured
as
in
spectrophotometrically
described before (van
experiments
enabled usage
separated
or
two
pigment
concentration
which
was
veins of the first
largest
25 ml of the
petri-dishes containing
light (20,000 lux)
(van Hasselt 1972). Before exposure
ed
between the
plants.
more
of
Hasselt
a
1972)
refined extraction
10 discs (van
Hasselt
cellulose column chroma-
described earlier (van Has-
1972).
Inhibitors
Chemicals
were
the dark in
The
a
dissolved
following
sulphonic acid,
DCMU,
buffer
(pH 6.0)
and
stored
in
1,2-dehydroxy
agents.
anthrachinon
sulphonic
acid
Az., sodiumazide; Asc., sodiumascorbate; Benz., benzidinedihy-
drochloride; BQ,
benzidine
phosphate
abbrevationsare used for the various chemical
Aliz., alizarin
sodium salt;
in 0.15 M
refrigerator.
1,4-benzoquinone; BV, benzylviologen; DAB, 3,3’-diamino-
tetrahydrochloride; Diquat,
1,1-ethylene-2,2’-bipyridylium (2Br
3-(3,3-dichlorophenyl)-l,l-dimethylurea;
nol-indophenol;
DMSO,
diaminopropane;
dimethyl sulphoxide; DSPD, NN’-disalicylidene-ES-
EDTA,
ethylenediamine
potassiumferricyanide;
Gua.,
ammonium
HQ,
chloride;
);
DCPIP, 2,6-dichloro-phe-
tetraacetic
guajacol-o-methoxyphenol;
1,4-hydroxy
quinone; SAL, salicylaldoxime;
TTC
benzoquinone;
acid;
ferricyanide,
HAM,
NQ,
hydroxyl-
1,4-naphto-
triphenyltetrazoliumchloride; TRIS,
Tris
PROTECTION
OF CUCUMIS
LEAF
PIGMENTS
AGAINST PHOTO-OXIDATIVE
Further abbreva-
(hydroxy-methyl) aminomethane; KCN, potassium cyanide.
tions;PSI
3.
photosystem I;
PS II
43
DEGRADATION
II.
photosystem
RESULTS
3.1
Effect
of
No effect of
trations
of
tions
(fig. I).
than
8
slight
maximal
2.
Since
medium the leaf cells
of
at
were
After 26 hours in the
at
pH
this
pH
3.3
11.
pH
chlorophyll
accelerated.
was
lost
It is
to
of
4
pH
a
Dark
pH
at
discs
6
higher
degradation
as
inhibiting
was
into the
pH
of
chlorophyll
6.0. In the acid
had
region
with the dark value,
compared
at
2. At
pH
the medium.
in
studies
and
at
maximal effect occurred
was
Cucumis
were
increased
especially
leaf discs
carried
stimulating
pronounced
more
degradation
concluded that
degradation
Therefore, further
Effect
at
till
pH
region chlorophyll degradation
at
condi-
light
maximal
lower than 6 and
degradation
minimal
was
the dark. Photo-oxidative
maximal effect
oxidative
higher
towards lower
pH again chlorophyll
light
against
evidently damaged.
light photo-oxidative
was
than in
of the
was
leaked from the
pigments
chlorophyll degradation
In the alcaline
pH
a
occurred.
chlorophyll
degradation
again increasing
concentration
7 and 8. At
occurred. As in darkness
this
concen-
compounds
chilling.
chlorophyll
different
degradation
pH
at
wide range of
concentration irrespective
chlorophyll
In the dark the
only slightly
was
a
of these
protection
pH
influences the
and
observed within
was
that the
differs from that against
Effect
PH
dimethyl sulphoxide
DMSO
or
(table 1), indicating
freezing
3.2
and
glycerol
glycerol
out at
at
in the
gradually
high pH
during chilling
pH
to
a
photowas
6.0.
chemicals
The effect of various substances which affect electron transport in the chloro-
plast
on
leaf
pigment degradation
Photo-oxidative
discs
Table
during chilling
I.
The
chlorophyll
are
effect
in
expressed
Concentration
was
and
of the
a
and
carotene
by application of Diquat,
dimethyl sulphoxide
leaf discs after
percentage
(M)
accelerated
of glycerol
Cucumis
as
degradation
is shown in tables 2 and 3.
of chlorophyll
on
at
20.000
concentration.
Glycerol
Dimethyl sulphoxide
0
54
54
0.05
58
54
0.1.
53
52
0.5
57
56
1
56
52
2
53
-
Cucumis leaf
photo-oxidative degradation
treatment of 40 hours
initial pigment
in
SAL and DSPD.
lux
at
1
of
C. The data
44
PH.
1.
Fig.
hours
6
Effect of
10 discs
samples of
Table
2.
The
effect
concentration
1
pH
on
chlorophyll
light (20,000 lux)
in
in
or
in
togetherwith
of several
Cucumis
The chemicals
are
of the
percentage
each
1C.
Each
leaf
discs after
point represents
HASSELT
incubation
the
for
26
mean value
of
standard deviation.
photosynthetic
leaf discs
dissolved in
initial
after
a
inhibitors
treatment of 40
the
on
and
chlorophyll
hours at
20,000
lux
or
carotene
darkness
0.15
M
phosphate buffer
at
6,0. The
pH
pigment concentration, obtained
from
data
samples
are
of 40
treatment.
Treatment
control
DSPD.
SAL,
2
10
2
10
Diquat,
10
DCMU,
DAB.
10
TTC,
10
10
2
2
M
M
4
M
5
M
M
red.
Light
Dark
102
17
106
39
94
11
106
1
95
0
9
96
3
76
104
42
103
41
82
blue.
Dark
57
77
M
discs and solution
solution
Carotene
Chlorophyll
Light
1
their
at
VAN
at
C.
as
2
content of Cucumis
darkness
R.
1
2
98
56
96
99
100
1
2
107
110
expressed
discs
for
OF
PROTECTION
Table
3.
The
CUCUMIS
effect
LEAF
AGAINST PHOTO-OXIDATIVE
PIGMENTS
of various
reagentia
on
after a treatment of 30 hours at
20,000
0.15 M
6.0. Data are
phosphate buffer
concentration
and
pH
at
represent
the
lux
mean
or
of 3
chlorophyll degradation
darkness
at 1 °C.
expressed
as
The
in
were
of the initial
together
leaf
Cucumis
reagentia
percentage
of 10 discs
samples
45
DEGRADATION
with
discs
dissolved
in
chlorophyll
their
standard
No
pigment
deviation.
Treatment
Light
control
77
±
1
97
±
16
±
1
94
±
1
57
±
3
104
±
2
28
±
2
96
±
1
78
±
2
98
±
2
72
±
1
101
±
2
79
±
2
99
±
2
24 ±
2
99
±
1
0.5 M
TRIS.
0.01
EDTA,
Az„
0.01
M
M
0.01
HAM.
M
0.01
Ferricyanid*.
KCN,
BV,
0.01
0.01
M
M
M
Dark
DCMU, TTC and DAB inhibited pigment
degradation by
any of the above chemicals
Table 3 shows
of
degradation
that TRIS, EDTA, Az.,
chlorophyll.
effect. The effect of
oxidative
substances tested in
Gua.
were
Again
no
and
4.
The
Each
percentage
was
experiment
quinones
and
Cucumis leaf discs
figures presents
initial
phosphate
buffer at
Treatment
(10
2
and
pH
the
is
carotene
pigment
M)
were
compounds
presented
photo-oxidation.
degree by NQ, BQ,
related compounds
after
a
treatment
concentration.
on
the
of 40 hours
concentration
Chlorophyll
19
NQ
51
HQ
88 1
BQ
97'
GUA
in
of
a
20,000
sample
The chemicals
of 40
were
Dark
chlorophyll
HQ.
lux
of
chlorophyll
or
darkness at
discs, expressed
dissolved
3
98
101
17
91
98
45 1
91
102
46 1
89
Benz.
IV
99
Aliz.
58
discs and solution
brown.
discs and
blue.
Dark
101
97
1
Light
98
and
less effective.
degradation
at
photo-
table 4. All
BQ, HQ,
were
and
without
on
Carotene
86
solution
photo-oxidative
KCN
6.0.
control
1
and
and related
inhibited
same
pigment
Light
2
BV increased
HAM
occurred in the dark. Carotene and
inhibited to the
effect of
of the
and
quinones
chlorophyll
pigment degradation
carotene in
1 °C.
this
of
(table 2).
observed in darkness.
effective, followed by Benz.; Aliz. and NQ
most
degradation
Table
of
degradation
degradation
was
Ferricyanide,
application
3
-
—
-
—
-
-
in
0.15
as
M
46
Table
a
R.
PH.
5.
The combined
treatment of 26 hours
of 6
mean
the
mean
samples
of 3
samples
T reatment
2
(10
of 10 discs
concentration.
phyll
effect of
in the
together
5
10
2
2
10
BQ.
M
M
M
Combined
inhibited
BV
±2
35
3
28 ±
±2
33
i
I
22
±2
95 ± 3
62
i
2
83
±3
78
98 ± 3
95
1
104
±3
91
t
i
with
inhibit
4.
1
31
percentage
represent
values
at
the
represent
of the initial
pH
chloro6.0.
Dark
without
were
photo-oxidative
99
±2
95
101
±
1
102
± 3
102
±
1
102
3
99
4
105
1
±
BQ)
effect
in
chlorophyll
=
99
±2
99
4
97
±2
92
3
4
94
± 3
± 2
102
±2
100 ±
1
t
obtain
to
3
dark.
In
degradation.
101
the
photo-oxidative
information
more
in
table 5.
DCMU
light
This
± 2
photo-oxidative
shown
are
SAL
4
accelerated
The results
the
BV
which inhibited
studied
were
i
which
compounds
All
alone
inhibitor, however,
BV, and SAL. TTC and BQ alone did
However,
degradation.
not
2
±
compounds
and
the presence of Az.,
chlorophyll
Az.
32 ±
mechanisms of their action.
BV. and SAL did
the dark
phosphate buffer
M
SAL
91
ineffective in
as
values
Light
Con-
80
treatments
combinations
C.
trol
(DCMU, TTC, and
about the
was
in 0.15
HASSELT
chlorophyll degradation after
deviation,
expressed
dissolved
Light
damage (SAL, BV.and Az.)
damage
standard
are
on
darkness at 1
trol
Control
10
data
were
Az.
TTC,
or
with their
of 10 discs. The
The chemicals
Con-
M)
DCMU,
inhibitors
photosynthetic
light (20.000 lux)
VAN
the
accelarating compounds
alleviate the inhibitionby TTC and BQ
to a
large
Az.,
extent.
DISCUSSION
Photo-oxidation of Cucumis leaf
various
substances
in
three
pigments
ways.
It
is
DCMU; it is unaffected by compounds
accelerated
discussed
4.1
by
SAL
which
Reagentia
(singlet state)
Livingstone
a
Diquat.
The
1 °C is affected
by
like
eg.
ferricyanine
effects
of
these
by application
benzoquinone
and
KCN
and
three groups
of
and
it is
will
be
briefly.
Benzoquinone
that
and
at
prevented
and
Also Amesz & Fork
compound.
(1967)
Quinones
interact with the
apparently
chlorophyll
do
not
well
stimulate
molecules of PS II
by
as
et
al.
(Fujimori
(1969)
&
show
fluorescence in Swiss
Ulva lobata and
as
fluorescence
solution
and Amesz
quinones strongly quench chlorophyll
occurred in the presence of DCMU
low temperature
at
chlorophyll
energy in benzene
chard chloroplasts and in the intact algae
Quenching
both
quenches
chlorophyll triplet
1957).
number of
prevent photo-oxidation
effectively
very
Porphyra perforata.
in the absence of this
electron
transport
but
formation of traps for the
excitation energy.
A
similar mechanism
compounds
(table 4)
in
leaf
Cucumis
may
prevent
discs.
explain
why benzoquinone
photo-oxidative
Interaction
between
degradation
benzoquinone
(BQ)
and
of leaf
and
related
pigments
the
excited
PROTECTION
OF
chlorophyll
inhibit the
CUCUMIS
may
LEAF PIGMENTS
formation of
prevent
of
production
AGAINST PHOTO-OXIDATIVE
triplet chlorophyll
damaging singlet
A similar mechanism is
triplet chlorophyll).
may
quenches
oxygen
for the
suggested
therefore,
and
(because
oxygen
47
DEGRADATION
protective
action
DAB and benzidine.
by
Another
oxidation
Noack
while
is
possibility
by acting
as
that these
preferential
a
the
time
same
DCMU
(Nir
oxidation is
not
in
to
1970).
electron
It
protected
seems
transport
induced
both
against
oxygen.
in
be insensitive
to
therefore that this type of
but
directly
occurs
vitro
photo-oxidation.
shown
was
singlet
vivo and
in
photo-
prevent
quinones
light
lamellae
chloroplast
& Seligman
coupled
is
chlorophyll
Photo-oxidation of DAB
to
and
that benzidine is oxidized
(1925) observed
at
substances
substrate for
photo-
in the presence
of chlorophyll, light, and oxygen.
TTC and related
TTC
compounds
act
by quinone
and
is
suggestion
DAB since
TTC
efficiently quenched by
supported
DCMU inhibits the
5).
by
the
1°C
at
substrate
PS I
Q
(Duysens
DCMU
and
the
at
& Sweers
1963).
to
the reduced side of the
pathway operates
cytochrome
Reagentia
chlorophyll
Burr
as a
PS II
which
of 10
2
do
not
did
synthesis
affecting
the
activity
carboxy
10
explains
1973,
2
DCMU
1974).
PS
during chilling
back
II
and
prevented.
because it
flow of electrons
by
DCMU
more
from the
suggested
of electrons
that such
via
a
cyclic
high potential
1971).
photo-oxidation
influence the
in
2
M
KCN
our
electrons may
excess
presence of DCMU
rate
agreement
KCN
of
inhibits
(Kandler
experiments,
the ineffectiveness of KCN
of
photo-oxidation
with the results of Myers
does however
primarily
dismutase
and under the conditions used in
function. This
a
It has been
affect
completely.
of
damage
is blocked
not
Chlorella.
with
of Chlorella
Hasselt
(tables
DCMU in Cucumis
consequence of the relative
during chilling (table 3),
(1940)
benzoquinone
photosystem.
in the
M KCN
is
this
side of PS II between the first reduc-
by enabling
photosystem.
around
and
ofQ is
consequence reoxidation
b 559 (Cramer & Bohme
Application
&
a
beyond Q
the oxidized side
to
TTC
transport intermediates between
the oxidized side of the
When electron transport
4.2
reducing
As
II
in benzene
degradation of leaf pigments
possibly prevents photo-oxidative
flow back
at
the
for the
1957). Moreover,
photo-oxidative damage by
electron
may prevent oxidation of PS
reduced
both
by
carotene
proposed
photo-oxidative damage (table 5).
described earlier (van
was
inhibits electron transport
ed
that
Hill reaction
and
fluorescence
& Livingstone
finding
photo-oxidative
A similar inhibitionof
discs
in the
chlorophyll
chlorophyll
(Fujimori
inhibit the effect of accelerators of
leaf
of
may be related with the mechanism
(fig. 2), however,
inhibition
and
electron acceptors
as
1953). Inhibition of photo-oxidation
(Dyar
1
to
&
°C,
inhibit
photo-
photosynthesis
Liesenkotter
this
enzyme
by
1963)
will
not
prevent photo-oxidative
damage.
The
that
inability of ferricyanide
during chilling
no
to
influence
photo-oxidative
electron flow between the
two
damage suggests
photosystems
occurs,
in
48
R.
PH.
agreement with the observation that DCMU
of BV which functions
4.3
Reagentia
BV
accelerated
and
BV
DCMU
Such
did
may
also
0
2
and
2
cyclic
to
electron
effect of DSPD
for
rise
gives
reduction
electron
The
photo-oxidation
for
Diquat
BV
since
(table 5).
in
uptake
1972).
two
by produc-
reasons,
protecting
electron
trans-
the oxidized side of PS E
also
may
explain
oxidizing
effect
accelerating
EDTA and
high pH
these substances
I
the effect of DSPD
of PS
which appears
1969). However,
(Raven
side
at
on
II,
to
photo-oxidative
during chilling
the
oxidizing
Cheniae 1970). In addition
to
may be
similar
an
to
inhibitive
the effect
to
side of PS II
suggested
at
to
(Katoh
of SAL,
HAM
appears
to
site different from the
SAL, Az.,
inhibitive effect of
al.
1972, Trebst 1970,
the electron
chilling temperature,
back flow of electrons from the
inhibit electron
TRIS,
the
et
on
effect
oxidizingsideofPSII.thusexplainingtheaccelerating
damage.
of
damage
related
their inhibitive effect
which will be very low
PS II,
may also inhibit the
flow from
these inhibitors
reducing
to
the
photo-oxidative
on
transport between H z O and PS
inhibition site of other inhibitors (Katoh
1972). The ineffectiveness of HAM in photo-oxidation
HAM
by
of
pool,
insensitive oxygen
1971, Egneus
This
hydrogen peroxide
to
the
Diquat.
as
free radical which
be excluded.
cannot
at a
a
photo-oxidation
DCMU
al.
in
resulting
endogenous
some
the
et
flow from PS
the
on
similar way
a
needed for
stimulation of
account
inhibit the action
to
(table 5).
the concomittant inhibitionof
by
The last mechanism
H 20
from
chloroplasts (Setchenia
port from the reduced
inhibit
Electrons
originate
inhibit the
not
in
side of PS I
oxygen. The last process
1969).
HASSELT
photo-oxidation
damage
reducing
and BV may accelerate
Diquat
tion of H
by
possibly
pool
a
irradiated
the
at
(Baldwin
will
accelerate
unable
was
end of PS I
reducing
photo-oxidative
reoxidized
turn
production
the
which
herbicide is reduced
is in
at
VAN
only inhibits electron flow from H 2 0
PS II
to
and
may
not
be
II
al.
et
explained
the back
if
flow of
electrons.
The existence
cyclic
pathway
against
of
a
cyclic
around PS
photo-oxidative
addition this
protective
pathway
I
operating
would offer
damage
an
of leaves
side of the
under low
mechanism could function
conditions when the flow of energy absorbed
photosynthesis
around PS
by
the
II
for
explanation
resembling
the
stress.
temperature
also
under
pigments
other
into the
reactions is inhibited and electrons accumulate
at
are
reducing
photosystems.
due to Prof. Dr. Ir. P. J. C,
Kuiper
for critical
reading
of the
In
stress
enzymatic
the
ACKNOWLEDGEMENT
Thanks
the
protection
manuscript
PROTECTION
OF
CUCUMIS
LEAF
PIGMENTS
AGAINST PHOTO-OXIDATIVE
49
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