Acta Bot. Need.
October
21(5),
1972,
539-548
p.
Photo-oxidation
leaf
Cucumis
Ph.R.
van
Botanisch
of
discs
leaf
pigments
during
in
chilling
Hasselt
Laboratorium, Universiteit, Groningen
SUMMARY
The
is
effect
of
of the
length
lag phase
The
the
causing
carotene
process.
by
a
fast
at
nature
primary
no
pigments
of
The
one.
has
at
sensitivity
the shortest
and
100% oxygen
of
role
1 °C has
been
lag phase
and
a
,
a
Pigment degradation
slow
degradation rate
photo-oxidation corresponds
to
and is
chlorophyll
are
to
most sensitive
the
to
photo-
b.
disappears after boiling.
chlorophyll degradation
in the
studied.
pigment degradation show
by xanthophyll, chlorophyll
decreases
biphasic
plays
leaf
Kinetics
Carotene
lag phase.
followed
oxidation,
Cucumis
on
followed
(lag phase)
1.
light
photo-oxidative
a
Some
discussed.
possible
It is
factors
that
suggested
protection against photo-oxidation during chilling.
INTRODUCTION
combined with
Light
suboptimal
pigment concentration, especially
val
(1957);
McWilliam & Naylor
High light intensity
Franck & French
causes
(1941);
Sironval & Kandler
found
negligible
a
growth
in
temperatures
causes
Paris
thermophilic plants,
Alberda
(1967);
decrease
a
in
Siron-
(1926);
(1969).
photo-oxidative pigment bleaching (solarization),
Hager
(1957);
(1958), working
effect of temperature
Zurzycki
with
the
on
(1957).
of 100.000 lux,
light intensity
a
of Chlorella
photo-bleaching
between 18 °C and 38 °C. Other authors found that lower temperatures enhance
caused
photo-bleaching
In
studying
(chilling)
more
on
injury
At the
gain
more
of leaf
2.
same
time
insight
of low
we
observed
than
of 7
1957).
chilling
in the
light
caused
in the dark.
took
pigments
of
Hager
place
light during chilling
in the
the
light.
To
degradation
studied.
METHODS
Kleine Groene
mm
14
that
chilling
of leaf
damaging effect
1957;
temperature above freezing point
Scherpe)
were
day temperature of 25 °C and
first leaves of ±
sucrose
was
plants (cv.
Discs
degradation
in the
AND
minimum
20 °C.
effects
cucumber leaf discs
pigments
Cucumis
by high light intensity (Sironval
damaging
thermophilic plants
to
MATERIAL
at a
the
diameter
days
solution in
old
special
were
punched
plants. Samples
P.V.C.
grown in pots in
minimum
a
between the
of discs
dishes with
an
were
a
greenhouse
night temperature of
largest
floated
air-tight
veins
on
perspex
of the
25 ml 1
%
screw-on
lid.
Eight
dishes
were
placed
ethanol-water mixture
a
cooling
thermostat.
kept
in
at
a
circle
in
a
tray.
The
the desired temperature
dishes stood in
by circulating
it
a
30%
through
540
PH.
The dishes
the
were
experiment
them
by bubbling
of oxygen
A
400
filled with
gas mixture
it
(type
the dishes. Gases
bottles with
alkaline
obtained
was
H.P.L.R.;
absorb
to
room
(2°C ±
1
most
in
was
the
traces
dishes under
filled with
of the infrared radiation. The whole
10
distilled
cm
placed
was
a
metal
bottomed
glass
a
During
humidified
were
solution.
by placing
Philips)
HASSELT
freed from
was
pyrogallol
container lined with silver paper. The container
water
N
water.
2
an
VAN
them three times.
by flushing
through
through
of 20.000 lux
lamp
mercury
a
streamed
through washing
by conducting
light intensity
W.
were
gases
R.
in
a
cool
°C).
At different times
of 40 discs
samples
taken
were
out
of the dishes and dried
between filter paper.
Leaf
pigments
mined
different
extract was
and 5 ml
xanthophylls)
obtained
by washing
from the
pigments
to
was
tration
at
in the dark
3.
in
or
a
Zeiss
in weak
was
1
a
extract
cm
of
traces
out as
The
(1955).
measured
glass-pearl
b
were
Bruinsma
petroleum-ether
acetone
water
water
was
deter-
(1963).
made between
was
A
of
layer
extract
using
layer
an
was
se-
adsorbed into
a
sodium
anhydrous
water.
described
450
at
spectrophotometer.
green
follows.
as
and the
with
12)
absorb
Carotene concentration
nm
extract
carried
(1957).
440
distinction
(no
Goodwin
by
C. F.
above the cellulose
Elution of the
by
and
mixture of 10 ml of the 100 %
a
light petroleum
(Whatman
a
60-80 °C) with 500 ml of distilled
described
as
described
as
Mickle
a
chlorophyll
determined
were
in
acetone
and calculated
light petroleum (bp.
cellulose column
sulphate
100%
concentrations
xanthophyll
automatic method
parated
with
5 minutes. Concentrations of
spectrophotometrically
Carotene and
the
extracted
were
for
homogenizer
All
Lederer & Lederer
by
xanthophyll
nm,
operations
were
concen-
carried
out
light.
RESULTS
3.1.
Fig.
The effect
1
shows
of
that
light
light during chilling
carotene
in Cucumis leaf discs. No
the first
16 hours
and
the
after this
only
decrease
lag
phase
in
chlorophyll
the
degradation
causes
degradation
maximum
of
concentration is
degradation
and
chlorophyll
is observed in the dark.
During
relatively
slow
is
(fast phase)
rate
attained.
The
lag phase
as
well
the fast
as
sudden transition from the
appears
3.2.
to
proceed
without
The effect of
an
N
2
phase
lag phase
pigment
was
degradation
some
required
was
in
degradation
the
to
fast
proceed linearly.
Carotene
phase.
There is
a
degradation
atmosphere
compared.
an
N
2
degradation, especially
for
appear
lag phase.
The decrease of the concentration of
nitrogen atmosphere
to
of leaf
chlorophyll
During
atmosphere.
of
carotene
pigments,
at
the
and
carotene
first 24
After 24
hours,
{fig. 2). So,
least
during
in air and in
hours
there
was
however,
oxygen and
a
no
there
light
are
the first 24 hours.
PHOTO-OXIDATION
Fig.
1. Relation
carotene and
OF
LEAF PIGMENTS
between
the
Fig.
2.
of carotene and
Relation
between
duration
in the dark
chlorophyll
tion
IN
the
We may conclude that
discs, during chilling,
is
in
of the
and in
of the
duration
chlorophyll
chilling
the
due
to
the
3 shows that
degradation
little
difference
However,
a
of
chlorophyll
between
oxygen
still
the
concentration
of
pure
light
aerobic
and the
concentra-
nitrogen.
in
Cucumis
photo-bleaching
leaf
process.
concentrations
carotene.
degradation
enhances
the
pigment degradation
higher oxygen concentration
and of
and
in the
atmosphere of
an
3.3. The effect of different oxygen
Fig.
treatment
541
CHILLING
light.
an
induced
light
DURING
chilling treatment
air and in
mainly
LEAF DISCS
CUCUMIS
pigment
rate
causes
During
at
21
degradation
a
shorter lag phase
the fast
%
and
during
phase
100%
the
in
there is
oxygen.
fast
phase:
542
PH.
Fig.
3.
tion
of carotene and
after
Relation
18
between
hours
is inhibited in
phase
of
while
at
carotene
Table
the
duration
chlorophyll
in
of the
in
photo-oxidation
an
atmosphere
21
%
the
oxygen
fast
effect
of
an
N
2
starts
phase
concentration of 50-60
1. The
air
0%,
bleaching
%
21
of
of
light
HASSELT
concentra-
oxygen.
and
chlorophyll
100%
VAN
and the
100%
and
At
1).
carotene
oxygen,
the fast
concentration of 80-90
at a carotene
chlorophyll
degradation
starts
at
%
a
%.
atmosphere during
and carotene at 1°C and 20000
of
of pure N2 {table
chlorophyll degradation
in the
chilling treatment
atmosphere
an
R.
lux. The
the
fast
of
degradation phase
pigment concentration
is
in
expressed
%
chlorophyll
of the initial
concentration.
18
treatment
hrs
air
42 hrs
air
18
hrs
air
+
chlorophyll
71%
10%
57%
carotene
32%
0%
20%
3.4.
The
The
sensitivity
and
4b.
then
chlorophyll
The
to
sensitivity
of different
of different leaf
Carotene
different
a
is
the
and
most
sensitivity
chlorophyll
b
has the
of the
Xanthophyll
responding
oxidation.
and
forms
of
chlorophyll
carotene
and
photo-oxidation
photo-oxidation
pigment,
pigments
lag phase.
rate
to
2
is shown in fig. 4a
followed
by
xanthophyll,
b.
to
photo-oxidation
Carotene has the shortest
longest lag phase.
concentration is left, the
to
sensitive
chlorophyll
the different length of the
pigments
pigments
24 hrs N
When 20-30
%
is
mainly
lag phase
of the initial
due
while
pigment
degradation diminishes.
b,
which
contain
chlorophyll
a,
more
are
oxygen than the
less
sensitive
to
cor-
photo-
PHOTO-OXIDATION
Fig.
4a.
tration
Fig.
4b.
tration
Relation
of
OF
between
carotene,
Relation
LEAF
the duration
the duration
chlorophyll
3.5. The influence of
When the
light intensity
degradation
rate
Therefore it
can
the
lag phase
CUCUMIS
of the
xanthophyll, chlorophyll
between
of carotene,
PIGMENTS IN
during
a
and
LEAF
DISCS
chilling
a
of the
and
treatment in
chlorophyll
chilling
chlorophyll
DURING
543
CHILLING
the
light
and the concen-
light
and the
b.
treatment in the
concen-
b.
light intensity
is reduced
both the
be concluded that
and the fast
phase
to one
half (by
lag phase and
light intensity
of
means
the fast
is
a
of
a
phase
limiting
pigment degradation.
cheese
cloth)
decreases
factor
the
{fig. 5).
during
both
544
Fig.
PH. R. VAN HASSELT
5.
Relation
carotene and
between
chlorophyll
the
in
duration
the
dark
of
(D)
the
and at
chilling
a
treatment
light intensity
3.6. The effect of low temperature pretreatment
A
better
understanding
oxidation would be
in the
We
slow,
Fig.
6.
tration
lag phase
first
of
gained
were
if
between
and
more
protecting
insight concerning
possibility
cold-induced alteration
of carotene
factors
the
on
concentration
and 20000
the
cells
plant
of
lux.
lag phase
from
photo-
the mechanisms involved
available.
considered the
Relation
the
and
of 10000 lux
the
during
duration
chlorophyll
of the
with
of
the
an
induction of the
lag phase. Fig.
chilling
and without
treatment
a
fast
phase by
6 shows that
in the
pretreatment
light
of
a
and
chilling
a
pretreat-
the
concen-
in the
dark.
PHOTO-OXIDATION
of the
ment
The
LEAF
discs
at
for the
The effect of
Fig.
7. Relation
tration
4.
CUCUMIS
LEAF
in the dark has
DISCS DURING
eifect
no
of the
is therefore
se
not
re-
lag phase.
boiling
fig.
living
between
of carotene and
545
CHILLING
photo-oxidation.
on
Low temperature per
present.
7 the
is
lag phase
completely
therefore be concluded that the
can
of the
properties
2°C
occurrence
As demonstrated in
10 minutes. It
PIGMENTS IN
is still
lag phase
sponsible
3.7.
OF
absent in discs boiled for
lag phase
is bound
to
the
cell.
the
duration
chlorophyll in
of the
chilling
boiled
and unboiled
treatment in
leaf
the
and
light
the
concen-
discs.
DISCUSSION
is needed for
Oxygen
in the
considered,
serves
ning
as
in
process
to
in
N
2
not
-atmosphere
on
H 20
the effect of
a
photo-oxidative
light-induced
rather
substances,
as
an
could be
electron
hydroxylamine,
Kandler
a,
of the
bleaching
degradation
(1958)
but almost
Chlorella. These authors,
ment
The
explanation
serves
be
degradation
carotene
regarded
as
than oxygen,
acceptor.
be
can
in which
02
degradation begin-
carotene
that oxygen
an
process,
set
a
photo-oxidative
the
are
free
by
Preliminary
electron
the
photo-
experiments,
inhibitor of 0 -yielding
2
reactions,
support this last explanation.
chlorophyll
the end
to
and enhances
light-induced pigment degradation
after 24 hours may be
other cell
Another
Sironval &
than
large extent,
a
which
oxidation of
however,
Therefore
electron acceptor.
an
an
acceptors.
do
chlorophyll bleaching
light during chilling.
as
concluded that
sensitive
however,
period. Fig.
as
xanthophyll is
chlorophyll
determined
b
to
less
sensitive
photo-bleaching
pigment degradation
only
in
at
4a shows that the general kinetics of pig-
should be taken into
account.
546
PH.
The observed differences in the
the
to
the first
pigments during
leaf
different abilities
Carotene, the
of
sensitive
most
energy of
of
the
pigments
In addition the carotenoids could
accept the
to
of
At
1
°C,
low
triplet
which is able
mechanism
a
(Donohue
rapid enzymatic
inhibited. As
are
electron
deflected towards carotene,
reduction of the
enzymatic
the other
On
variable
of
the
the
a
reduction
consequence the
flow of the
photosynthetic
which is oxidized
to
carotene
however is inhibited
epoxides
concentration
carotene
that
oxidation
The
rapidly
the
by
decreases
in
Hager
into
(1957)
a
may
to
of
b and
same
carotene
is
observed
it is
Therefore,
inhibitionof
and
xanthophyll
pigments
photo-oxidative
a
photo-
less
are
chlorophyll
a
a
slow
found
Nijhuis
a
a
preliminary
(1968)
at
a
50.000 lux
(room
decrease of
experiments
caro-
we
did
yellow pigments.
caused
enzymatic activity
of the
a
of
to
of
the
part
is better
yellow pigments
the
chloroplast
of the
by
the
low
lag phase
a
hours
energy
might
transfer,
membrane system.
pigment
is
An-
localized inside the
protected against photo-oxidation.
pigment
describe
after 24
pigment-pigment
degradation precedes
is in accordance with results
similar
into
carotene
experiments.
that
phase
days
two
concentration and
time. In
low
damage
could be
transformation
transformation of
a
the concentrations of the
efficiency
consequently
that
to
degradation
during photo-oxidation
Thomas &
the
our
by photo-oxidative
(1958). They
of
out.
be due
lower
a
explanation
membrane and
of
possibility
correlation between
rate
attributed
The fact
b
corresponding
xanthophyll
at
occur
temperature (1 °C) used in
caused
rate
pigments.
stated that in Avena shoots after
discrepancy
The lower
phase of bleaching of
role in the
chlorophyll
the
chlorophyll
increase of
an
find such
other
than
be ruled
not
concentration
This
pigments
The
(1958).
a
can
temperature)
primary
a
photo-
agreement with the
chlorophyll degradation.
of the other
lag phase
in
not
{fig. 3).
degradation
of
plays
is
of the fast
beginning
the
in the inhibition of
carotene
This is in agreement with the results of Hager (1957) and Siron-
Kandler
xanthophyll
tene
such
as
of
lag phase
concentrations
phase
photo-oxidation
to
chlorophyll
not
the
during
the
at
of the fast
carotene
carotene.
&
carotene
alteration in
no
the
during
oxygen-containing
sensitive
val
of
role
primary
a
different oxygen
beginning
unlikely
and
at
addition,
In
hand
chlorophyll
amount
chlorophyll
be
of the
quenching
buffering system,
a
suggest
The
stop.
a
temperature. Therefore
oxidation
of
energy.
other sub-
light during chilling.
the
at
to
comes
mechanism will be
The
as
reactions
however, enzymatic
assimilation
2
epoxide.
light
and
1959).
photosynthetic
These authors
1968).
the various
carotenoids.
photo-oxidized
C0
serve
electrons from the
excess
al. 1967; Krinsky
et
very efficient
& Nakayama
chlorophyll (Claes
chlorophyll
VAN HASSELT
be attributed
can
harmful
quench
to
may protect
its
by
between
lag phase
photo-oxidation ( fig. 4)
pigment,
against photo-oxidation
stances
of the
length
period
R.
in the
case
similar
a
fast
phase
of Sironval & Kandler
of Chlorella
lag phase
photo-bleaching.
in the
case
of aerobic
OF
PHOTO-OXIDATION
DURING
LEAF PIGMENTS IN CUCUMIS LEAF DISCS
of
photo-bleaching
chlorophyll-protein
in
complexes
547
CHILLING
isolated
Aspidistra
chloroplasts.
When
consider that
we
lag
is
phase
This
due
inhibition
against
from the
As
as
A
serve
it is
before,
inhibitor of
remains
intact
is
electron transport
flect
higher
a
the
leaf
harmful
processes.
pigments
light
energy
buffering system draining electrons
during
the
lag
otherwise
such
as
plays
a
role
primary
lag phase during chilling.
of
occurrence
of leaf
disturbed. An
which
electrons,
tecting
a
protecting
quench
carotene
in the
for the
plausible explanation
more
system
as
could
as
photo-oxidative
substances
that
unlikely
photo-oxidation
phase during photo-oxidation
fast
pigments
phase,
a
lag phase preceding
while
the
during
intact electron
transport
would induce
a
is that the electron
transport
system
of
bleaching
fast
the
phase
might
pigments,
to
de-
pro-
substances.
The different
the
could also
They
damaging
by
well
(the origin of)
pigments.
discussed
an
produced
as
conclude that
can
These substances
photo-oxidation.
and trap oxygen.
we
inhibition of
be
might
concentration
oxygen
lag phase,
the
to
higher
a
reduce the
light intensity
of the
length
of the
lag phase
pigments might correspond
destruction of different factors
photo-oxidative
needed for electron
to
trans-
port.
It
proteins
or
membrane
bound
Further
experiments
photo-oxidation
are
to
these
to
of the
site of
primary
(Krinsky
localized in
are
the
grana lamellae which
& Benson
50% lipids (Weier
complete disappearance
in addition that the
cell
factors
and
50% proteins
well be lamellar
causes
that these
likely
seems
consist of
since
proteins,
1966). They might
boiling
of leaf discs
Several authors
lag phase (fig. 7).
photo-oxidative damage
assume
is localized in the
1968).
elucidate the
origin
of the
lag phase during chlorophyll
in progress.
ACKNOWLEDGEMENTS
The author wishes
the
manuscript,
for
correcting the
to thank
Mrs.
R.
Prof.
Luytink
English
Dr.
M. H.
van
for technical
Raalte
for discussion and critical
assistance,
and
Mrs. I.
Ridge
and
reading
of
Mrs. S. Ides
text.
REFERENCES
Alberda,
Th.
(1969):
concentration
and
The
effect
of low
photosynthesis
temperature
of maize
plants
on
on
dry
matter
production, chlorophyll
different ages. Acta
Bol. Neerl.
18:
39-50.
Bruinsma,
J.
(1963):
Photochem.
Claes,
in
H. & T.
vitro
in
H.
O.
M. Nakayama
14b:
Press London
In
-
of
chlorophylls
a
and
b
in
plant
extracts.
von
(1959):
Das
Carotinen
mit
photooxydative
verschiedenen
Ausbleichen
von
Chlorophyll
Chromophoren Gruppen.
Z.
746-747.
T.
V.,
xanthophylls.
quantitative analysis
2: 241-249.
Gegenwart
Naturforsch.
Donohue,
The
Photobiol.
O.
T.
New
M.
Nakayama
W. Goodwin
York.
&
C.
O.
Chichester
(1967): Oxygen
(ed.), Biochemistry of Chloroplasts II:
reactions
431-441.
in
Acad.
548
PH.
J.
Paris,
A.
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