Download 165 Regeneration of protochlorophyll in dark

Regeneration
dark
grown
of
protochlorophyll
seedlings
illumination
with
in
following
red
and
far
red
light
C.J.P.
Spruit
Laboratorium
and
C.W.
Raven
Plantenfysiologisch Onderzoek, Landbouwhogeschool, Wageningen.*
voor
SUMMARY
The rates
of
regeneration of protochlorophyll (Pchl)
pea and
maize
far red.
In neither
rates
of pigment
final
level
of
studied, following
were
material
reached
sequent
to
a
effect
of
continuous
latent
in
decreases
than after far red.
ded that the
found
we
evidence
red
formative
leaves,
less
for
an
rapidly during the
red
effect
far red
-
was
not due
to
a
of
red
-
far red
control
during
of the
of Pchl
red
light.
the
rate
earlier
part
of
a
of
terminal
It is concluin sub-
greeningprocess
regeneration
by
initial
upon the
only
plant
bean,
followed
regeneration after
of the
the
red
but
that in this
stages
the kinetics
becoming manifest
process,
last
or
control
observed
of
seedlings
grown
alone,
pigment photobleaching in
pre-illumination upon
light, is
of dark
prolonged darkness, indicating
There is considerable
a
in leaves
dose of either red
saturating
In older maize
regeneration.
Pchl,
pigment production
red
have
a
as
but
such,
subsequent
prolonged light period.
1.
INTRODUCTION
Chlorophyll
In
and
is
be
phyllide
the
as
dark grown
under
supposed
and
seedlings
remains
in the
biosynthesis
to
be
to
far
required
below
and
subsequent
the
has
plant evidently
of this compound,
transfer
protochlorophyllide.
rate
the
of Chi
a
formation
(Price
&
Klein
1964). Usually,
*
283rd
Communication
Acta Bot. Neerl.
a
the
19(2), April
a
other steps
no
a,
relatively
a
repressing
since
1961;
low
pigment
an
in
reached
proto-
early step
other intermediates have been
A brief illuminationof dark
treatment
Mitrakos
Plant
to
protochloro-
level of
effect upon
to
grown
chlorophyllide
formation of additional
by
the
strongly dependent
initial
upon
of the dark grown material.
1961;
Henshall & Goodwin
the dark grown plants
for
1960).
appear
ultimately
protochlorophyllide
light
Laboratory
1970
All
second illumination is
experiments
of
a.
level of Chi
of
plants.
the first
Virgin
1961;
in these
delta-amino-
reports in the literature indicate that
during
light quality, given during
via
Bogorad
(e.g.
for the transformation of the
darkness is followed
to
Several
formed
The concentration of this
Build-up
accumulate in normal
be
reared in the dark accumulate
material results in transformation of the
a
to
precursors
chlorophyllide
to
continuous illumination.
reported
supposed
tetrapyrrole
temporary end product.
plants
chlorophyllide
in the
of
protochlorophyllide
light independent
is
plants
series
a
Angiosperms, light
precursor
in
(Chi a)
a
acid
levulinic
Physiological
were
pretreated
either
Research
165
C.
with red
alone,
of Chi
formation in continuous
a
far red
during
of the
1958;
depends
(Virgin
of this
phase by
1961).
These observations
of Pchl.
During
etiolated pea
by
preliminary
far red.
in
small
vitro Pchl
at
size
of the
pigment
have included in
we
considerably
are
this
In
the
article,
forms
present
phyllide
these
ditions of
Therefore,
that
we
have
represent
phytylated
MATERIALS AND
2.1.
85%
was
and
be
a
this
steady
pretreatment.
regeneration
illumination of
rates
of Pchl
either red
material
these
in
two
with
contrast
or
the
light
the
red
some
rates
treat-
findings,
Since pea, because of the
darkness,
well
as
Pchl.
as
the rather
suitable
particularly
a
at
made
Maize
terms
material,
which
seedlings,
was
for the
chosen
a
as
intend
and
a
pigment
separate paper. It
a
an
fraction of
1957),
pigment
may
in
absorption
a’’ denotes
the
fate of
under the
forms,
non-
protochloro-
discuss
to
and
be relevant
to
pigment(s) having
We
nm.
found
form of the
phytylated
appear
Likewise, “chlorophyll
non-phytylated
in
not
protochlorophyll
acetone.
665
between
this did
by light (Shibata
be stated
con-
here,
protochlorophyll
our
and that this fraction appears
(Sironval
et
al.
1965).
METHODS
of Pisum sativum
Zea mays
rel.
cv.
the
cv.
“Caldera”
hum. Seeds
added in
experiments
166
the
Plant material
Seedlings
and
80%
invariably
to
2.
in
present experiments,
be non-transformable
not
rich
since
promiscuously
to
a
will
the
mixture, absorbing
our
of
leaves of dark grown bean
pigments,
phytylated
subsequent
nm
state
distant taxonomic group.
distinction
nm
is
in
study.
grown in
especially
about 660
at
evidence and the results of
extensive
weight,
the
peak
different for
rate
of which
length
reactions after
form
to
seems
more
seedlings,
and
more
about 628
various
however,
no
a
in the duration
became interested in the
we
al.
el
elimination of the
saturating irradiation with
spectroscopic
this
study
of the
context.
at
pigment
the
a
dark
significantly
unit
per
our
have been used
maximum
of
larger
from
representative
phytylated
sight
leaves
a
for-
a
(Withrow
is involved in
phytochrome
1967),
not
undertook
content
that
rate
cases,
of Chi
rate
the
the
a
the
estimations indicated that
were
first
we
for
independent
be
phytochrome
Both direct
regeneration
quoted above,
low
of
seedlings (Spruit
Since
ments.
might suggest
study
the
period,
mainly
phase,
spectrum
lag phase,
to
appears
in this materialafter
regeneration
followed
a
induction
of the
W. RAVEN
in continuous illumination: the
a
an
AND C.
number of
a
hours after the first
short illumination has
completion
formation
dark
a
This manifests itself
action
The
After
measured. In
was
some
shows
preceding
a
red.
SPRUIT
slow down the initial
to
above).
pigment
At the
pigment
of
of Pchl
also
see
upon pretreatment.
induction
rate
reported
was
far
by
light
in the formation of Chi
lag phase
production
followed
illumination, given
an
Virgin
1956;
of
after red
given
mation
with red
or
J. P.
were
dark
consisted
“Krombek”,
were
sown
during
of the
in
the
Phaseolus
vulgaris
reared in total darkness
pasteurized
soil.
growth period.
“plumules”
of the
As
far
at
as
cv.
“Widusa”
20 °C and about
necessary,
water
The leaf material used in the
pea
(third
Acta Hot.
and
Neerl.
fourth
unex-
19(2), April
1970
REGENERATION
PROTOCHLOROPHYLL
internodes with
panded
the
OF
the
bean and
vesting
the
done in absolute
was
2.2.
Irradiation
The
phototransformation
the
of
light
filters
and 2.9
2.3.
Leitz
a
4
10
x
10
x
of Pchl
erg/cm
erg/cm
2
was
able.
We
used
a
fluorescent tube
moid”
nr.
62
moid”
nr.
46.
at
sec
slide
descent
lamp,
und
735
529
2.4.
2.2
nm,
Alternatively,
the
by
intensity
4
10
X
of interference
means
The
erg/cm
were
layers
stat
25 °C for the
the
at
2
at
sec
samples
650
nm
for 5 minutes.
given
gram
in
placed
petri
leaves
appropriate
treatment,
of calcium carbonate. The
filter with
80%
milliliters of the
minutes
with
suction.
12
ml
up
The
residue
acetone-water.
same
at
solvent.
60.000
a
few
5
nm
cm
nm were
as
well
as
the
of the
at
path length
of
“Cine-
25 W incan-
a
blue
“Plexiglass”
46. Precautions
nr.
samples directly.
light-tight
800
scatter.
and
and the
2°C,
flasks
nm
cells.
the
on
If
in
a
to
was
lined
were
tins in
a
thermo-
extracted
filter
filter
was
were
extracts
kept
the
the
rarely
in
again
and
filtration
extracted
with
washed
then
in the
%
at
with
few
a
optical
the
to
more
as
600
region
model
density readings
wavelengths
amounted
to
For storage
2°C.
throughout
spectrophotometer
other
a
centrifuged
acetone.
dark
a
over
clear supernatant transferred
Beckman
at
by
were
0.01
by grinding
sand,
a
on
nearest
washed
removed
was
possible
as
the
to
little
a
measured
necessary,
This correction
311)
volume with 80
were
readings
quickly
as
were
liquid
Vessels
extracts
subtracted from the
a trace
mm
reach the
model
The combined
g and
hours,
optical density
700
using
to
in
pure acetone,
ml volumetric flask and made up
The
put
samples
amount
periods
of
dishes the lids of which
Corp.
small
25
not
weighed
were
Scale
(Ohaus
together
for 30
did
were
mortar
ml
3
of blue “Cine-
orange-yellow
required periods.
balance
gram. After the
glass
of
through
monophosphor
layer
of “Cinemoid”
sources
small
about 7
a
used, consisting
filtered
layer
found unavoid-
green
in
layer
a
was
regeneration
were
of the
samples
“Centogram”
a
estimations
Pigment
One
and
was
of moist filter paper. These
with 4
2.5.
one
from these
light
Protochlorophyll
at
Corp.)
was
light
of
17), wrapped
safelight
a
plus
0248
nr.
little
a
safelight, consisting
of which
light
Haas)
samples,
TL 40, colour
Strand Electric
The irradiated samples
for
projector
All radiations
nm.
of the
green
(Philips
taken that the
were
to
har-
bands isolated from
spectral
Filtraflex B 40).
2
handling
weak
(The
(Rohm
a
of
pair
of maize. All
Safelights
During subsequent
a
leaf
primary
coleoptiles
effected with
500 W
at
sec
the
material),
whole
darkness.
“Prado”
4
1.4
leaf
the
or
(Balzers, Liechtenstein, type
about
was
attached
leaves
primary
a
DU,
at
800
correction
than 0.01 O.D.
units.
Acta Bot. Neerl.
19(2), April
1970
167
C. J.
2.6.
The
rather
This is
sensitive
a
small
to
Fig.
wavelength
errors,
and
the
by
the
Boardman
(1962).
during
checked
was
each
calculated values
are
spectrometer calibrations
dependent.
1
gives
the concentrations of total Pchl in bean leaves
period following
Pchl
regeneration
confirming
seedlings,
a
red
saturating
proved
or
red
-
was
pigment
than
following
5
rate
of
material,
of Akoyunoglou & Siegelman
(1968).
In young
dependent
appears to
periods
up
jxg;
somewhat different
the
we
of
original
values
scatter
to
the level before
approach
24 hours,
no
rate
significant
or
the
effect
final level
or
5
min.
picture (fig. 2). Regeneration
Pchl level. Because of the small
in bean leaves
red
plus
5
min.
of
of Pchl
rate
varying
far red.
of phototransfortned protochlorophyll. The
days,
amounts
±
23
ug;
of
21
pigment
days,
±
per
23
gram fresh
regeneration
during
age,
Results
a
expressed
100% level
weight:
8
day
never
amounts
within the
Nevertheless,
considerably.
difference in the
no
protochlorophyll
absolute
14
the
observed
min. red
of the amounts
following
a
50% of
formed,
accuracy,
12
The
of the
During regeneration
Regeneration
±
function of the
found.
more
limits of
a
upon the age
strongly
plateau ultimately reached
Pea leaves present
reached
as
far red irradiation.
terminal far red irradiation upon either the initial
of Pchl
Fig. I.
a
the observations
the
illumination.
168
regeneration
Protochlorophyll
dark
of
RAVEN
RESULTS
3.1.
of
since
precaution,
necessary
somewhat temperature
proved
coefficients used
absorption
calibration of the spectrophotometer
wavelength
AND C. W.
concentrations
pigment
the molar
adopted
measurement.
3.
of
Calculation
We have
P. SPRUIT
dark
as
period
cent
per
represents
old
the
seedlings,
|xg.
Acta Bot.
Neerl.
19(2), April
1970
OF
REGENERATION
Fig.
2.
PROTOCHLOROPHYLL
Regeneration
to
1. The
fig.
of protochlorophyll in
100% level represents
leaves
a
of 7
day
old
pea
pigment regeneration
See also
seedlings.
of ±
3.5 (xg per
legend
gram fresh
we'ght.
Fig.
3.
Regeneration
various
ages.
along the
The
9
or
±
3.5
axis.
the
See
|xg;
14
red followed
days,
by
±
far
In maize of various ages
the
increase in
rapid
Acta Bot.
Neerl.
legend
19(2), April
maize
curves
to
fig.
of the
8 (xg;
17
days,
±
at
8 :xg;
a
Pchl,
no
In
21
±
days,
seedlings
was
significant
dark
in
ages
leaves
are
of
shifted
gram fresh
weight:
7 (xg.
between 5 and
14
low level.
value in 9
original
and
1.
red in pea also.
Upon longer
1970
coleoptiles
for the successive
following pigment regenerations per
{fig. 3), regeneration
80%
red-far red could be found.
old
day
the
uniformly stopped
final level of about
of
9
reading,
further
represents the
days old, regeneration
period
in
protochlorophyll
vertical
100% level
days,
after red
of
To facilitate
fairly rapid,
day
old
leading
differences between red
incubation,
to
a
coleoptiles. During
a
and
difference between
169
C.
Fig
4.
Absorption spectra
About
a.
b.
1
the
light
two
of about
cally
g bean
17
3.2.
In
amounts
red
following
in
Chlorophyll
confirmed the
grown in
complete
a
in
of the
the
days,
dark
1
cell
1
in
80%
final
level,
about
red
most
RAVEN
acetone,
under
the
optimum
final level of
be
not
in
leaves
way, is statisticonditions.
Pchl reached
demonstrated,
whereas in
of the effect decreased.
grown leaves
contain
a
(Spruit
substance
that leaves
1966)
spectroscopically
4. The concentration amounts
but
W.
C.
marked
reached in this
25%
decreases
magnitude
significant
Pchl present,
AND
cm.
apparent which is
to
SPRUIT
cm.
earlier observation
darkness,
and bean
pea
this effect could
coleoptiles,
We have
Chi
becomes
and
17
grown
P.
cm.
cell
The difference in
older than
leaves,
cell 5
Absorption
treatments
that far
in darkness.
of dark
seedlings. Absorption
pea
Absorption
leaves.
days.
significant,
It shows
of extracts
whole
g
leaves.
g pea
4.2
c.
100
J.
there
are
to
of pea,
similar
roughly
25
to
%
considerable variations between individual
batches.
In
experiments,
most
this
sowing.
During
diffuse
daylight.
tained the
To
under
sown
pigment
therefore does
as
whether
green
as
in
water
treatment
control batches
safelight.
same
The
proportion
of the seeds
responsible
for
a
few
during sowing, they
this
seedlings,
our
Exposure
appear
soaked
well
were
for
to
the
imbibed in absolute
likewise
con-
in the material treated in
light during
to
for the
before
exposed
responsible
was
leaf material
as
hours
were
the imbibition
“chlorophyll”
period
found in the dark
seedlings.
Trace
amounts
controls of
170
not
were
in about the
the usual way ,fig. 4.
grown
check
found in the
“chlorophyll”
darkness and
peas
treatment
our
of
a
chlorophyll-like
bean and maize
substance could be observed in the dark
experiments,
but the
quantities
Acta Bot.
Neerl.
were
very small
19(2), April 1970
REGENERATION
and
OF PROTOCHLOROPHYLL
of doubtfull
this respect.
significance.
It appears
plant originates
in the
green
the
early stages
of
3.3.
Yield of
Obviously,
have
We
the
estimated both the
Fig.
5.
loss
Yield
at 0°
due
to
some
was
of
in
Pchl
observed
measure
of
a
this
light
difference
of
loss
spectrum
a
min. red.
It
must
bleaching
of
25°.
a
embryo during
be
likely
in
green. At 0°
primary
Smith
in
optical
stated
here
that the
form of Chi
a
at
at
2
0°.
the
pigments
pigment
absorbing
loss
at
vivo.
caused
pigment
old bean
day
leaves
6
an
(1961)
also
attempted
gives
were
by
for the
yields
an
to
example
preirradiated
one
min. red.
at
The
additional dose of
changes
can
be
very
forms may be involved.
have observed is due
about 685
is lost
relatively higher
have
Fig.
by
pigment
we
of 8
We
a.
absorption
or
a
lower than
Goedheer
The leaves
density,
is
a.
Pchl
separate sample,
25°. Similar
min. followed
that
a
of
of irradiation.
(1948).
in
mole of Chi
temperature effect is
leaves
duration
of the
by spectrophotometry
for
this
likely,
formed Chi
for bean leaves
one
formed and
a
fraction of the
a
with
Most
function
initially
and it appears that several
At any rate, it is
in
position
leaves of this
quantities
two
the illumination
temperature with green light
complex
4.
at
so
reported by
were
photobleaching
pigment
as
during
of Pchl
spectrum gives the changes
9
less
light,
observed than
regeneration
this
the
to
is derived from
figure
protochlorophyllphotoconversion
photoconversion
in the
give
of Chi
scatter. It is evident that
green and red
should
by light
quantity
in this
points
illumination with red
pigment
transported
the ratio of these
irradiation,
5. As each of the
show
different
a
pigment
photoconversion
mole of Pchl converted
always
points
during
and is
cotyledons
protochlorophyll
one
occupies
pea
that the
possible,
development.
converted. After red
one,fig.
Evidently,
least
at
to
photo-
nm.
DISCUSSION
Our results
upon
Pchl
have failed
observations
with
oat
Acta Bot.
to
regeneration
(Spruit
show
during
1967).
significant
the first
effects of the
hours of
A similar result
was
phytochrome
darkness,
obtained
confirming
system
earlier
by Jacques (1968)
seedlings.
Neerl. 19(2), April
1970
171
C. J. P. SPRUIT
Fig.
Difference
6.
luminated
sent the
In young
first hour
of
bean,
This led
and the
initial
It is
final
by
be
an
nature
far
red
illumination, fig.
for
Chi
of the
have
a
pea
and
in due
If
far red
3.
a
nm.
All
preilrepretreat-
period
longer
that is
the
also
possibly
few hours.
a
dark
periods.
depressed by
conclude
must
in the
during
constant
is
photoperiod
that
not
a
the
itself
a
we want
fit in with
to
those, demonstrating
subsequent
a
continuous illu-
falls somewhat outside the scope
make
a
few remarks that may aid in
problem.
reported
that
the
light by
given immediately
a
after red
1968). According
to
maize leaves.
red.
With
We
to
can
of the
shortening
lag
pre-exposure
(Withrow
Virgin
al.
(1961),
findings
1956;
in wheat,
in
the
can
be
Mitra-
in this
far red is
antagonism
red-far red
bean, far red reversal of the
our
red,
Henshall & Goodwin
1963;
confirm the
el
period
to
red
matter
effect
was
will appear
course.
Pchl,
172
We
brief
during
problem
however. A detailed report of
we assume
rate
rate
was
spectrum
induction
an
darkness,
findings
formation
in continuous
weakly antagonistic
doubtful,
of
following
clear how these
of
Akoyunoglou
1964;
material
min. at 653
9
essentially
1961; Price & Klein 1961; Sisler & Klein
only
the
formation is
regeneration
accumulation of Chi
by
evidence for
or no
This aspect of the
Several authors
kos
for
The
difference
induction in Pchl regeneration lasting
of the present article. However,
eliminated
The
RAVEN
red-far red mechanism.
control
the
illumination
level after 24 hours
pigment
the
mination period.
defining
nm.
red-far red effect could be observed after
immediately
red-far red
bean.
grown
653
the irradiation. In older leaves of maize and
of Pchl
rate
not
a
terminal
controlled
of dark
min. at
additional
of Pchl
rate
seems to
only,
to a
an
1
plus
there is little
material,
there
preceding
during
nm
C. W.
0°.
following
In maize
leaves
primary
min. at 529
changes
ments at
regeneration
of
spectrum
for 2
AND
of Chi
that the
a
rate
which condition
of Chi
a
of Pchl formation as such is
accumulation should equal the initial
clearly is
accumulation during
not
not
rate
influenced
of dark
by light,
regeneration
satisfied. In many experiments the initial
illuminationis much lower than the initial
Acta
Bot.
Neerl.
19(2), April
1970
REGENERATION
of
rate
after
OF PROTOCHLOROPHYLL
Pchl
pigment
to
converted
Chi
to
dark grown
Pchl,
light and,
troyed by
light,
the
of the
capacity
with the first stages of
accumulation is
mechanism may
the
account
pigment
red effect.
lag
short
and
irradiation,
provision
may,
in
the
that
is,
are
they
manifest
therefore,
overall
rate
Pchl
saturate
photoproducts
the
Pchl
des-
are
of
rate
Chi
a
concomitant
increases,
a net
of Chi
rate
a
of Pchl formation. This
rate
observed
accumulation,
of the influence of light
We have started work
are
points
during
during
only
suppose
the
re-
biosynthetic
some
for
a
by
way
red
the
prolonged
subsequent
that these effects represent either
from
pigments
and
of Pchl
rate
the
of attack
in
prepared
Chi
pigment forming
intensity
of
development
photodestruction
of
or
a,
these
to
wavelength
a
formative
the
during
system
experimental approach
An
influence the
to
possible
accumulation of
of the
capacity
prolonged irradiation.
study
original
pre-illumination
photodestruction
We
phase
the
regulating
the
to
of Pchl formation. In continuous
rate
mechanism, protecting
photoreversible
the
a
and become
irradiationperiod.
ring
initial
intensity,
for the kinetics of Chi
further
a
The
pre-illumination
a
surpassing
a
formed
follows the
a
sufficient
biosynthetic system forming
reached,
apparatus during
-far
upon
of Chi
production
of the
of Chi
newly
already present, is rapidly
light intensity
less than the
of
rate
initial
continuous illumination.
by
Pchl
chloroplast development. Ultimately,
the failure of
generation,
the
light
kinetics
photobleaching
biosynthesis
time, part
same
accumulation
a
severalfold the
attribute the
to
initial
further
a
Chi
and afterwards.
lag phase
Given
at
depending
is, therefore,
accumulation
inclined
an
the
rule surpasses
a
material,
on,
least
at
At the
photoconversion.
a
From then
a.
of
production
are
as
stimulation of Pchl
light
with
whereas
dark,
combination of
a
Upon irradiation of
of
We
regeneration.
accumulation
the
of illumination
hours
of Pchl
rate
in
regeneration
several
subsequent
may be the
problems
upon Chi
du-
effect,
a accumulation.
this line.
along
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