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Binding Properties In Vitro of Phytochrome to a Membrane Fraction

Authors:

Abstract

Brief irradiation of a buffer extract of dark-grown zucchini squash seedlings with red light results in the binding of the far-red-absorbing form of phytochrome to a particulate fraction. A low concentration of magnesium (0.1 mM) permits partial far-red reversal of the binding. A higher concentration (10 mM) yields actually enhanced binding after the far-red treatment. Both magnesium and calcium have a strong effect on the vesicularization of the phytochrome-binding particles and on their aggregation into readily sedimentable complexes. At concentrations above 10 mM, binding of the far-red-absorbing form of phytochrome is inhibited. These effects were not observed with sodium or potassium. Increasing the H(+) concentration led to increased binding of the far-red-absorbing form. This form of phytochrome bound at pH 6.5 and 10 mM magnesium is released if either the pH is raised to 8.0 or the magnesium concentration is raised to 50 mM. These properties suggest a new method for phytochrome purification.
Proc.
Nat.
Acad.
Sci.
USA
Vol.
70,
No.
12,
Part
II,
pp.
3861-3865,
December
1973
Binding
Properties
In
Vitro
of
Phytochrome
to
a
Membrane
Fraction
(stereospecific
binding/cation
effects/pH
effects/phytochrome
purification)
D.
MARMP*,
J.
BOISARDt,
AND
W.
R.
BRIGGSt
The
Biological
Laboratories,
Harvard
University,
Cambridge,
Massachusetts
02138
Communicated
by
Sterling
B.
Hendricks,
August
30,
1973
ABSTRACT
Brief
irradiation
of
a
buffer
extract
of
dark-grown
zucchini
squash
seedlings
with
red
light
results
in
the
binding
of
the
far-red-absorbing
form
of
phytochrome
to
a
particulate
fraction.
A
low
concentra-
tion
of
magnesium
(0.1
mM)
permits
partial
far-red
re-
versal
of
the
binding.
A
higher
concentration
(10
mM)
yields
actually
enhanced
binding
after
the
far-red
treat-
ment.
Both
magnesium
and
calcium
have
a
strong
effect
on
the
vesicularization
of
the
phytochrome-binding
particles
and
on
their
aggregation
into
readily
sediment-
able
complexes.
At
concentrations
above
10
mM,
binding
of
the
far-red-absorbing
form
of
phytochrome
is
inhibited.
These
effects
were
not
observed
with
sodium
or
potassium.
Increasing
the
H+
concentration
led
to
increased
binding
of
the
far-red-absorbing
form.
This
form
of
phytochrome
bound
at
pH
6.5
and
10
mM
magnesium
is
released
if
either
the
pH
is
raised
to
8.0
or
the
magnesium
concentra-
tion
is
raised
to
50
mM.
These
properties
suggest
a
new
method
for
phytochrome
purification.
In
the
past
few
years,
strong
evidence
has
been
accumulating
that
an
early
consequence
of
the
phototransformation
of
the
red-light-absorbing
and
biologically
inactive
form
of
phyto-
chrome
(Pr)
to
the
far-red-absorbing
and
biologically
active
form
(Pfr)
might
be
a
change
in
the
functional
properties
of
membranes
(1).
Rubinstein
et
al.
(2)
first
reported
that
a
small
fraction
of
the
phytochrome
extracted
from
dark-
grown
oat
seedlings
was
pelletable.
Marm6
et
al.
(3)
reported
a
correlation
between
phytochrome
binding
and
the
binding
of
naphthylphthalamic
acid.
Naphthylphthalamic
acid
bind-
ing
has
been
proposed
as
a
marker
for
the
plasmalemma
(4).
Irradiation
of
etiolated
squash
seedlings
with
3
min
of
red
light
increased
the
amount
of
pelletable
phytochrome
by
a
factor
of
10
(5,
6).
With
10
mM
magnesium
present
in
the
extraction
medium,
over
40%
of
the
total
extractable
phyto-
chrome
was
readily
sedimented.
The
phytochrome-containing
structures
were
characterized
by
sucrose
density
gradient
centrifugation
and
electron
microscopy
(6).
The
size
of
the
phytochrome-containing
structures
depended
strongly
upon
the
concentration
of
magnesium
present
in
the
extraction
medium
(6).
A
low
concentration
(0.1-1
mM)
yielded
particles
Abbreviations:
0.5
KS,
500
X
g
supernatant;
17
KS,
17,000
X
g
supernatant;
17
KP,
17,000
X
g
pellet;
50
KS,
50,000
X
g
supernatant;
50
KP,
50,000
X
g
pellet;
Pr,
red-absorbing
form
of
phytochrome;
Pfr,
far-red-absorbing
form
of
phytochrome.
*Present
address:
Institut
fur
Biologie
III,
D7800,
Freiburg,
Germany,
Schanzlestrasse
9-11.
tPresent
address:
Laboratoire
de
Photobiologie,
C.
N.
R.
S.
(L.
A.
No.
203),
Facult6
des
Sciences
de
Rouen,
76130,
Mont-
Saint-Aignan,
France.
$
Present
address:
Department
of
Plant
Biology,
Carnegie
Institution
of
Washington,
Stanford,
Calif.
94305.
3861
too
small
to
reveal
structural
detail
by
electron
microscopy
of
negatively
stained
preparations
(about
100
A).
Higher
con-
centrations
(3-10
mM)
caused
some
vesicularization
of
parti-
cles
(to
structures
400-600
A
in
diameter)
and
aggregation
of
the
vesicles
formed.
Purified
preparations
of
the
vesicles
con-
tain
phospholipids,
steroids,
glycolipids,
and
proteins,
indica-
tive of
their
membrane
nature
(J.
M.
Mackenzie,
Jr.
and
J.
Beck,
personal
communication).
Evidence
that
the
structures
formed
with
higher
magnesium
concentration
are
vesicles
comes
from
comparing
their
appearance
with
that
of
authen-
tic
negatively
stained
liposomes
of
a
similar
size
(refs.
6
and
7;
J.
M.
Mackenzie,
Jr.,
personal
communication).
The
aim
of
the
present
paper
is
to
demonstrate
that
Pfr
can
be
bound
in
vitro
to
a
particulate
fraction
obtained
from
dark-
grown
squash
seedlings.
The
influence
of
divalent
and
monova-
lent
cations
and
of
pH
on
the
binding
of
Pfr
will
also
be
con-
sidered.
MATERIALS
AND
METHODS
Seeds
of
zucchini
squash
(Cucurbita
pepo,
L.,
cv.
Black
Beauty)
were
germinated
on
moist
absorbant
paper
and
grown
in
darkness
for
4
days
at
250.
The
seedlings
were
then
chilled
to
40;
hypocotyl
hooks
about
1
cm
in
length
were
harvested
and
placed
immediately
in
Syracuse
dishes
on
ice.
The
extraction
buffer
contained:
25
mM
N-morpholino-3-
propanesulfonic
acid
(MOPS),
3
mM
disodium
salt
of
ethylene-
diamine
tetraacetic
acid
(EDTA),
14
mM
2-mercaptoethanol,
10
or
0.1
mM
MgC12,
and
250
mM
sucrose,
at
pH
7.35.
A
ratio
of
4
ml
of
extraction
buffer
to
1
g
fresh
weight
of
tissue
yielded
a
final
pH
in
the
homogenate
of
7.0.
Except
where
noted
below,
the
pH
was
always
maintained
thereafter
at
7.0.
Ten
grams
of
tissue
was
chopped
in
the
extraction
buffer
with
a
razor
blade
for
10
min
and
then
ground
gently
in
a
chilled
mortar
for
4
min.
The
resulting
brei
was
filtered
through
four
layers
of
cheesecloth
and
one
layer
of
Miracloth
to
remove
larger
cellular
debris.
Subsequent
treatment
is
shown
in
Fig.
1.
An
additional
10-min
centrifugation
at
500
X
g
removed
the
remaining
cell
fragments.
Centrifugation
of
the
500
X
g
supernatant
(0.5
KS)
at
17,000
X
g
for
30
min
then
removed
the
majority
of
the
organelle
fractions.
Phytochrome
in
a
supernatant
or
a
pellet
was
estimated
in
a
dual-beam
dif-
ference
spectrophotometer
(Ratiospect
R-2).
The
measuring
wavelengths
were
660
and
730
nm.
To
allow
analysis
of
very
small
aliquots
of
samples,
CaCO3
powder
was
used
to
amplify
sample
absorbence
(8).
The
maximum
variation
between
replicate
samples
never
exceeded
10%.
The
use
of
CaCOa
also
essentially
eliminated
any
inherent
light-scattering
dif-
ferences
between
supernatants
and
resuspended
pellets.
Each
experiment
contained
at
least
two
replicates
for
each
Proc.
Nat.
Acad.
Sci.
USA
70
(1973)
40
30
's
m.
201
I:
FIG.
1.
Extraction
procedure
for
phytochrome
and
phyto-
chrome-binding
particles
from
squash
seedlings,
used
to
study
the
influence
of
various
factors
on
phytochrome
binding.
Order
of
treatments
not
necessarily
that
shown.
Incubations
at
250
were
for
15
min.
0.5
KP
and
0.5
KS,
500
X
g
pellet
and
super-
natant,
respectively;
17
KP
and
17
KS,
17,000
X
g
pellet
and
supernatant,
respectively;
50
KP
and
50
KS,
50,000
X
g
pellet
and
supernatant,
respectively.
measurement,
and
all
experiments
were
repeated
at
least
once.
The
manipulations
were
all
carried
out
at
under
dim
green
light
except
as
specified
below.
The
percent
of
Pfr
bound
was
calculated
as
the
amount
in
the
pellet
divided
by
the
total
of
that
in
the
pellet
plus
that
in
the
supernatant,
times
100.
RESULTS
In
Vivo
and
In
Vitro
Induction
and
Reversal
of
Binding.
If
intact
squash
seedlings
are
irradiated
with
3
min
of
red
light
(saturating
for
Pr
to
Pfr
phototransformation)
just
before
extraction,
about
40%
of
the
phytochrome
is
found
in
the
pelletable
fraction
17,000
X
g
pellet
(17
KP),
provided
that
the
magnesium
concentration
in
the
extraction
medium
is
10
mM
(Table
1).
Further
centrifugation
of
the
17,000
X
g
supernatant
(17
KS)
for
30
min
at
50,000
X
g
brings
down
only
an
additional
4%.
With
0.1
mM
magnesium,
only
20%
is
pelletable,
and
in
the absence
of
any
light
treatment,
only
4%
is
pelletable
(Table
1).
Light
treatment
and
magnesium
concentration
affected
only
the
distribution
of
phytochrome
between
pellet
and
supernatant.
The
total
was
not
altered.
TABLE
1.
Recovery
of
phytochrome
in
pelletable
form
after
red
irradiation
in
vivo,
with
different
magnesium
concentrations
in
extraction
buffer
%Pfr
in
%Pfr
in
Light
treatment
[Mg++],
mM
17
KP
50
KP
3-min
red
10.0
40
4
3-min
red
0.1
20
None
10.0
4
o-a
red
*---
dark
control
10
5
10
20
30
45
60
90
(mn)
120
time
of
pre-centrifugation
FIG.
2.
Effect
of
precentrifugation
of
the
0.5
KS
before
red
irradiation
on
subsequently
pelletable
phytochrome
after
red
treatment
and
a
second
centrifugation
of
30
min
at
50,000
X
g.
In
order
to
determine
whether
phytochrome
binding
could
be
induced
in
vitro,
hooks
from
dark-grown
seedlings
were
extracted
in
the
presence
of
10
mM
magnesium.
The
0.5
KS
was
then
warmed
to
250
and
irradiated
with
saturating
red
light,
before
it
was
rechilled
and
centrifuged
at
17,000
X
g
for
30
min.
As
Table
2
shows,
36%
of
the
phytochrome
ap-
peared
in
the
pellet
(17
KP).
A
second
centrifugation
at
50,000
X
g
for
30
min
brought
down
an
additional
10%.
Dark
controls
contained
negligible
photochrome
in
the
pellets.
Clearly,
binding
could
be
induced
in
vitro.
As
before,
there
was
no
loss
of
total
phytochrome.
The
46%
pelletable
after
red
irradiation
in
vitro
of
the
0.5
KS
compares
favorably
with
the
44%
pelletable
after
red
treatment
in
vivo
(Table
1).
If
red-light
treatment
in
vitro
was
delayed
to
the
17
KS,
the
subsequent
50
KP
contained
34%
of
the
total
phytochrome
(Table
2).
As
before,
the
sample
was
warmed,
irradiated,
and
rechilled
before
centrifugation.
Evidently
the
17,000
X
g
centrifugation
had
removed
about
25%
of
the
structures
to
which
Pfr
could
bind.
If
red-light
treatment
was
further
de-
layed
to
the
50
KS,
a
second
50,000
X
g
centrifugation
pelleted
only
16%
of
the
phytochrome
(Table
2).
Thus,
the
first
50,0C0
X
g
centrifugation
had
removed
almost
half
of
the
phytochrome-binding
structures
remaining
in
the
17
KS.
From
the
results
shown
in
Tables
1
and
2,
an
important
property
of
the
phytochrome-binding
fraction
is
seen.
If
Pfr
becomes
bound
in
vivo,
it
will
sediment
by
centrifugation
at
17,000
X
g
for
30
min
(40%
of
the
phytochrome
in
the
17
KP).
However,
if
phytochrome
is
not
present
as
Pfr
through
this
centrifugation,
the
binding
structures
remain
in
the
17
KS;
if
the
17
KS
is
treated
with
red
light,
there
is
still
enough
binding
material
to
yield
34%
of
the
phytochrome
pelletable
during
a
50,000
X
g
centrifugation
(50
KP).
The
sedimenta-
TABLE
2.
Recovery
of
phytochrome
in
pelletable
form
after
red
irradiation
of
the
supernatants
after
each
of
the
three
centrifugation
steps
Supernatant
%Pfr
in
%Pfr
in
irradiated
[Mg++],
mM
17
KP
50
KP
0.5
KS
10
36
10
17
KS
10
34
50
KS
10
-
16*
*
After
a
second
50,000
X
g,
30-min
centrifugation.
3862
Botany:
Marm6
et
al.
2.
PPhytochrome
Binding
In
Vitro
3863
TABLE
3.
Effectsof
the
rel
ativesequenceof
magnsium
addition
and
red
and
far-red
treatments
on
amount
of
phytochrome
pelletable
from
the
17
KS
[Mg++],
mM
in
Sequence
of
%Phytochrome*
extraction
buffer
treatments
in
50
KP
10.0
Red
34
10.0
Red,
far-red
48
0.1
Mg++,
red
54
0.1
Mg++
red,
far-red
59
0.1
mg+
+
far-red
7
0.1
Red,
Mg++
48
0.1
Red,
far-red,
Mg++
15
0.1
Far-red,
Mg++
2
*
If
final
light
treatment
was
red,
phytochrome
in
50
KP
was
Pfr.
If
it
was
far
red,
phytochrome
in
50
KP
was
Pr.
First
two
lines,
Mg++
at
10
mM
from
start
of
extraction.
tion
properties
of
the
binding
fraction
are
evidently
altered
by
complexing
with
phytochrome.
The
influence
of
precentrifugation
of
the
0.5
KS
at
50,000
X
g
for
periods
of
from
5
to
120
min
before
red
irradiation
on
subsequently
pelletable
phytochrome
was
investigated
in
more
detail.
The
results
are
shown
in
Fig.
2.
In
each
case,
the
supernatant
obtained
after
precentrifugation
was
irradiated
as
before
and
then
centrifuged
again
at
50,000
X
g
for
30
min
to
assay
for
bound
Pfr.
One
hour
of
such
precentrifugation
is
sufficient
to
remove
almost
all
of
the
binding
structures,
reducing
the
pelletable
phytochrome
to
the
level
of
the
dark
control.
In
vivo
studies
(5)
have
shown
that
the
binding
of
Pfr
to
membranes
could
be
partially
reversed
by
subsequent
far-
red
irradiation
in
vivo,
just
before
extraction.
After
red
plus
far-red
treatment,
about
12%
of
the
phytochrome
(as
Pr)
was
pelletable
(10
mM
magnesium
in
the
extraction
medium)
compared
to
40%
after
red
treatment
only.
The
question
therefore
arose
as
to
whether
one
could
reverse
Pir
binding
in
vitro
with
far-red
light.
The
results
are
shown
in
Table
3.
When
10
mM
magnesium
was
present
from
the
start
of
extraction,
far-red
treatment
of
the
17
KS
after
red
treat-
ment
actually
increased
the
percentage
of
bound
phytochrome
from
34
to
48%.
If
phytochrome
and
the
binding
fraction
were
extracted
in
the
presence
of
1/100
the
magnesium
con-
centration
(0.1
mM),
and
magnesium
was
increased
to
10
mM
in
the
17
KS
just
before
red
or
red
plus
far-red
treatment,
far
red
also
yielded
a
slight
increase
in
binding
(from
54
to
59%).
Far
red
alone
yielded
7%
bound.
However,
when
the
red
plus
far-red
treatment
preceded
the
increase
in
magnesium,
only
15%
of
the
phytochrome
was
bound.
Thus
with
low
mag-
nesium
concentrations,
the
binding
of
Pfr
was
partially
reversible
by
far-red
light.
Far-red
light
alone,
before
mag-
nesium
addition,
gave
only
2%
binding.
When
Pfr
is
bound
in
vitro
as
described
above,
the
complex
is
remarkably
stable.
There
was
no
measurable
destruction
of
phytochrome,
nor
was
there
any
release
to
the
soluble
fraction
during
a
4-hr
incubation
in
the
dark
at
25°.
The
Effects
of
Monovalent
and
Divalent
Cations
on
Pelletable
Pfr.
The
influence
of
K+,
Na+,
Mg++,
and
Ca++,
all
applied
as
chlorides,
on
the
pelletability
of
Pfr
was
investigated
following
the
procedure
described
in
Fig.
1.
All
experiments
were
done
starting
with
the
same
17
KS.
The
ions
were
added,
lon
Concentration
(mM)
FIG.
3.
Influence
of
monovalent
and
divalent
cations
on
the
pelletability
of
Pfr
from
the
17
KS
by
centrifugation
at
50,000
X
g.
Bound
Pfr
was
measured
in
50
KP.
the
pH
was
readjusted
to
7.0,
the
sample
was
warmed
to
25°,
and
red-light
treatment
given,
before
the
second
centrifuga-
tion.
It
was
shown
elsewhere
(6)
that
magnesium
had
a
specific
effect
on
phytochrome
pelletability;
it
altered
the
sedimentation
properties
of
the
Pfr-binding
membrane
fraction.
Increasing
its
concentration
from
0.1
to
3
mM
caused
formation
of
vesicles;
increase
from
3
to
10
mM
caused
the
membrane
vesicles
to
form
large
aggregates.
This
effect
is
also
shown
by
results
obtained
after
in
vitro
induc-
tion
of
binding
at
various
magnesium
concentrations
(Fig.
3).
From
10
to
50
mM,
the
amount
of
bound
phytochrome
de-
creased
remarkably,
although
the
pellets
remained
the
same
size.
Calcium
showed
a
similar
behavior
through
the
whole
concentration
range.
Thus,
the
binding
of
Pfr
seems
to
be
inhibited
by
concentrations
of
calcium
or
magnesium
higher
than
10
mM.
Sodium
and
potassium
did
not
cause
formation
and
aggregation
of
Pfr-binding
vesicles
(Fig.
3).
Pellets
were
scarcely
visible
even
with
concentrations
of
100
mM.
The
effect
of
these
ions
on
binding
itself
needs
more
investigation.
The
Effect
of
pH
on
Pfr
Binding
In
Vitro.
The
effect
of
alteration
of
pH
in
the
presence
of
various
concentrations
of
magnesium
in
the
17
KS
was
investigated
following
the
protocol
shown
in
Fig.
1.
The
results
are
shown
in
Fig.
4.
For
all
five
magnesium
concentrations,
decreasing
the
pH
increased
the
percentage
of
bound
phytochrome
in
the
50
KP.
The
optimum
concentration
of
magnesium
at
pH
6.5
yielded
more
than
80%
of
the
phytochrome
bound.
In
the
absence
of
binding
structures,
the
phytochrome
itself
did
not
precipitate
under
these
conditions.
Pr
binding
(no
light
treat-
ment)
was
measured
at
two
different
magnesium
concentra-
tions.
In
both
cases
(1
and
10
mM),
Pr
binding
was
more
than
an
order
of
magnitude
lower
than
comparable
Pfr
binding.
Magnesium
Reversal
of
the
pH
Effect,
and
pH
Reversal
of
the
Magnesium
Effect.
A
large
volume
of
17
KS
was
initially
adjusted
to
pH
6.5
and
10
mM
magnesium
to
yield
maximum
binding
(Fig.
4).
Binding
was
induced
as
usual
by
red-light
treatment.
Six
aliquots
were
taken.
One
remained
as
the
control;
three
others
were
readjusted
to
pH
7.0,
7.5,
and
8.0;
and
two
others
were
kept
at
pH
6.5,
but
the
magnesium
con-
centration
was
increased
to
30
and
50
mM.
After
subsequent
centrifugation,
the
50
KP
and
50
KS
were
assayed
for
phyto-
Proc.
Nat.
Acad.
Sci.
USA
70
(1973)
Proc.
Nat.
Acad.
Sci.
USA
70
(1978)
402
20-
Oi
3
5
10
30
50
Mg++
Concentration
(mM)
FIG.
4.
Influence
of
pH
at
several
different
magnesium
con-
centrations
on
the
pelletability
of
Pfr
from
the
17
KS.
Ion
and
pH
adjustments
were
made
before
incubation
and
irradiation.
Bound
Pfr
was
measured
in
50
KP.
chrome.
The
percent
pelletable
was
clearly
reduced
either
by
increasing
the
pH
or
by
increasing
the
magnesium
concen-
tration
(Fig.
5).
The
open
circles
show
the
amount
bound
when
the
given
conditions
of
pH
and
magnesium
concentra-
tion
were
achieved
before
red
light
treatment,
and
are
taken
from
Fig.
4.
It
is
conceivable
that
a
longer
incubation
period
might
be
needed
to
release
the
maximal
bound
phytochrome
after
an
increase
either
in
pH
or
magnesium
concentration.
DISCUSSION
After.brief
irradiation
of
etiolated
zucchini
squash
seedlings,
about
40%
of
the
total
extractable
phytochrome
can
be
pelleted.
It
was
shown
elsewhere
(6)
that
this
fraction
of
Pfr
was
bound
to
a
membrane
fraction
that
could
be
isolated
and
purified.
The
recognition
of
Pfr
by
the
receptor
sites
at
the
membrane
surface
must
be
highly
stereospecific
(5).
The
sites
distinguish
between
Pr
and
Pfr
despite
the
apparently
small
conformational
difference
between
the
two
forms
of
phyto-
chrome
(9).
The
present
work
demonstrates
that
even
in
an
in
vitro
system,
the
membrane
fraction
is
still
able
to
recog-
nize
Pfr
stereospecifically.
The
in
vitro
binding
of
Pfr
to
this
membrane
fraction
and
its
sedimentation
properties
are
strongly
affected
by
mag-
nesium
and
calcium.
Potassium
and
sodium
have
no
obvious
effect
on
the
pelletability
of
the
phytochrome-binding
struc-
tures.
As
was
shown
in
studies
on
the
induction
of
binding
in
vivo
(6),
magnesium
seems
to
have
at
least
three
effects:
vesicularization,
aggregation
of
the
vesicles,
and
at
higher
concentrations,
inhibition
of
Pfr
binding.
The
inhibition
might
be
explained
by
a
change
in
the
affinity
between
the
ligand
and
the
receptor
on
the
vesicles
caused
by
intra-
or
interchain
crosslinking
through
protein
carboxyl
or
lipid
phosphate
groups
(10)
or
simply
by
ionic
strength.
At
constant
magnesium
concentration
Pfr
binding
depends
strongly
on
the
pH
(Fig.
4).
At
lower
pH
values
the
binding
increases.
Q'
6.5
7.0
7.5
8.0
pH
FIG.
5.
Influence
of
increasing
magnesium
concentration
or
pH
in
releasing
phytochrome
bound
as
Pfr
at
pH
6.5,
magnesium
10
mM.
(Left)
pH
constant,
magnesium
concentration
increased;
(right)
magnesium
concentration
constant,
pH
increased.
Open
circles
represent
the
percent
binding
obtained
when
pH
and
magnesium
concentration
were
adjusted
before
irradiation
(from
Fig.
3).
This
effect
could
be
explained
by
charge
neutralization,
and
thus
compensation
of
repulsive
forces
(10).
There
is
no
obvious
effect
of
pH
on
the
pelletability
of
the
binding
structures.
Although
one
cannot
presently
say
for
certain
that
the
phyto-
chrome
is
bound
to
the
vesicles
seen
in
the
electron
micro-
scope,
the
parallelism
between
vesicle
formation
and
phyto-
chrome
sedimentability
is
striking.
For
the
reconstitution
of
membranes
from
Mycoplasma
laidlawii
(11)
and
for
the
recombination
of
erythrocyte
mem-
branes
and
ATPase
(12),
increasing
the
magnesium
concen-
tration
had
the
same
effect
as
increasing
the
H+
concentra-
tion.
In
the
present
case,
the
effect
is
reversed:
increasing
magnesium
concentration
has
the
same
effect
as
decreasing
the
H+
concentration
(Fig.
5).
It
was
shown
elsewhere
(5)
that
far-red
irradiation
in
vivo
subsequent
to
red
irradiation
would
partially
reverse
the
red
effect;
reducing
bound
phytochrome
from
40
to
12%.
Partial
far-red
reversibility
of
Pfr
binding
can
also
be
demonstrated
in
vitro
(Table
3)
at
low
magnesium
concentration
(0.1
mM).
With
higher
magnesium
concentration,
however
(10
mM),
far
red
subsequent
to
red
actually
increases
the
amount
of
phytochrome
bound
(Table
3).
This
phenomenon
requires
further
investigation.
The
various
effects
described
in
this
paper
suggest
a
simple
procedure
for
purification
of
phytochrome,
directly
based
on
Fig.
1.
Dark-grown
plant
material
is
homogenized
in
buffer
at
low
magnesium
concentration
(0.1
mM).
The
pH
of
the
17
KS
is
adjusted
to
6.5
and
the
magnesium
concentration
to
10
mM
before
saturating
red
plus
far-red
irradiation,
to
yield
maximum
binding.
The
50
KP
is
then
washed
in
buffer
with
a
low
magnesium
concentration
(0.1
mM)
at
pH
7.5
to
disaggregate
the
vesicles
and
release
the
phytochrome.
Mag-
nesium
is
then
added
to
50
mM
to
reaggregate
the
vesicles
under
conditions
in
which
phytochrome
does
not
bind.
Subsequent
centrifugation
removes
the
membrane
fraction
while
the
phytochrome
remains
in
the
supernatant.
The
advantages
of
this
procedure
would
be
potentially
high
yield
and
rapid
preparation
of
highly
purified
phytochrome.
This
procedure
has
been
used
successf.ully
both
at
Rouen
and
at
0OmM
Mg++
60-
40_
0
20-
0
0
I-
-
9-
3864
Botany:
Mann6
et
al.
Proc.
Nat.
Acad.
Sci.
USA
70
(1973)
Harvard.
In
the
former
case,
yields
between
40
and
50%
were
obtained,
and
the
phytochrome
had
a
ratio
A2w/A6a
between
13
and
17.
Precipitation
with
33%
saturated
ammonium
sulfate,
followed
by
redissolving
in
0.1
M
sodium
phosphate
buffer
and
Sephadex
G-200
gel
filtration,
has
given
prepara-
tions
with
a
ratio
near
2.5
(present
authors
with
J.
M.
Mackenzie,
Jr.
and
David
W.
Hopkins,
personal
communica-
tion).
For
comparison,
purified
rye
phytochrome
has
a
ratio
near
1.3
(13).
We
thank
Dr.
David
Hopkins
and
Mr.
John
M.
Mackenzie,
Jr.
for
their
interest
in
this
work
and
for
valuable
discussions.
This
work
was
supported
by
NSF
Grant
GB-30964X
to
W.R.B.,
by
the
Department
of
Plant
Biology,
Carnegie
Institution
of
Wash-
ington,
Stanford,
Calif.,
by
the
Deutsche
Forschungsgemeinschaft
(SFB
46)
and
by
the
D6l6gation
G&n6rale
A
la
Recherche
Scien-
tifique
et
Technique.
During
the
work.
D.M.
held
a
Max
Kade
Foundation
fellowship
and
J.B.
a
NATO
fellowship.
1.
Hendricks,
S.
B.
&
Borthwick,
H.
A.
(1967)
Proc.
Nat.
Acad.
Sci.
USA
58,
2125-2130.
Phytochrome
Binding
In
Vitro
3865
2.
Rubinstein,
B.,
Drury,
K.
S.
&
Park,
R.
B.
(1969)
Plant
Physiol.
44,
105-109.
3.
Marm6,
D.,
Schafer,
E.,
Trillmich,
F.
&
Hertel,
R.
(1971)
Eur.
Ann.
Symp.
Plant
Photomorphogenesis
(Eretria)
p.
36,
abstract.
4.
Hertel,
R.,
Thomson,
K.
St.
&
Russo,
V.
(1972)
Planta
107,
325-340.
5.
Boisard,
J.,
Marm6,
D.
&
Briggs,
W.
R.
(1974)
Plant
Physiol.,
in
press.
6.
Marm6,
D.,
Mackenzie,
J.
M.,
Jr.,
Boisard,
J.
&
Briggs,
W.
R.
(1974)
Plant
Physiol.,
in
press.
7.
Miyamoto,
V.
K.
&
Stoeckenius,
W.
(1971)
J.
Membrane
Biol.
4,
252-269.
8.
Butler,
W.
L.
(1962)
J.
Opt.
Soc.
Amer.
52,
292-299.
9.
Tobin,
E.
M.
&
Briggs,
W.
R.
(1973)
Photochem.
Photobiol.,
in
press.
10.
Razin,
S.
(1972)
Biochim.
Biophys.
Ada
265,
241-296.
11.
Rottem,
S.,
Stein,
0.
&
Razin,
S.
(1969)
Arch.
Biochem.
Biophys.
125,
46-56.
12.
Zwaal,
R.
F.
A.
&
van
Deenan,
L. L.
M.
(1970)
Chem.
Phys.
Lipids
4,
311-322.
13.
Rice,
H.
V.,
Briggs,
W.
R.
&
Jackson-White,
C.
J.
(1973)
Plant
Physiol.
51,
917-926.
... Phytochrome binds to cell membranes (17,19,23) and alters properties of natural (15, 20, 25-27, 29, 30, 33, 34) and model (22) membranes; recent evidence also suggests that changes in membrane properties play a central role in the operation of the biological clock (3,7,12,28,31). Changes in the Pf, level induce phase shifts in endogenous rhythms in Lemna (13) and Phaseolus (1,2) implying that phytochrome interacts with the rhythmic oscillator in these plants. ...
... It is clear that additional experiments are required to test the validity of the theories described above. Hopefully, techniques recently developed for isolating membrane-bound phytochrome from etiolated tissue (17,19) can be adapted to green plants, to permit in vitro tests of phytochrome-rhythmic interactions. ...
Article
Phytochrome, a membrane-localized biliprotein whose conformation is shifted reversibly by brief red or far-red light treatments, interacts with the rhythmic oscillator to regulate leaflet movement and potassium flux in pulvinal motor cells of Samanea. Darkened pinnae exposed briefly to red light (high Pfr level) have less potassium in motor cells in the extensor region, more potassium in motor cells in the flexor region, and smaller angles than those exposed to far-red light (low Pfr level). Increase in temperature from 24° to 37° increases the differential effect of the light treatments during opening (the energetic phase) but not during closure, implying that phytochrome controls an energetic process. It seems likely that phytochrome interacts with rhythmically controlled potassium pumps in flexor and extensor cells. During nyctinastic closure of white-illuminated pinnae, exposure to far-red light before darkening results in larger angles than does exposure to red. As in rhythmic opening, the angles of all pinnae and the differential effect of the light treatments increases with increasing temperature.
... Ethyl alcohol (12), lithium (10), and D 2 0 (11) increase the cycle length and valinomycin (8) induces phase shifts in endogenous rhythms; all these substances probably alter membrane properties. Phytochrome, photoreceptor for changes in the phase (21) and cycle length (6,7) of endogenous rhythms in plants, binds to membranes (19,27,32,35) and regulates ion flux (24,25,33,42,48). Blue light also induces phase shifts (21) and changes in the length of rhythmic cycles (18); recent studies indicate that a blue-absorbing pigment, probably a flavin, also binds to a particulate fraction of tissue extracts (Briggs and Marme, personal communication) and regulates membrane properties in vivo (20). ...
Article
Samanea leaflets usually open in white light and fold together when darkened, but also open and dose with a circadian rhythm during prolonged darkness. Leaflet movement results from differential changes in the turgor and shape of motor cells on opposite sides of the pulvinus; extensor cells expand during opening and shrink during closure, while flexor cells shrink during opening and expand during closure but change shape more than size. Potassium in both open and closed pulvini is about 0.4 N. Flame photometric and electron microprobe analyses reveal that rhythmic and light-regulated postassium flux is the basis for pulvinar turgor movements. Rhythmic potassium flux during darkness in motor cells in the extensor region involves alternating predominance of inwardly directed ion pumps and leakage outward through diffusion channels, each lasting ca 12 h. White light affects the system by activating outwardly directed K⁺ pumps in motor cells in the flexor region.
Chapter
Attempts to elucidate the molecular mechanism of phytochrome action must reconcile two superficially contradictory sets of data: (a) Physiological studies where rapid, phytochrome-mediated changes in membrane properties have led to the hypothesis that the pigment directly modifies cellular membrane function as its primary action upon photoconversion to pfp.1,2,3,4 and (b) Biochemical and immunocytochemical studies where most of the phytochrome is, respec tively, extractable as a water soluble protein under conventional conditions and apparently uniformly distributed in the cytosol in situ -at least in unirradiated, etiolated tissue.5
Chapter
Knowledge about the influence of light on the form and function of plants developed over the last century. Phytochrome was recognized in 1949 as an essential absorber of light in these photomorphogenic processes. The recognition came entirely from logical deductions based on physiological responses of plants and their propagules. The discovery in 1952 of the photoreversibility of a potential response to light was the key factor leading to the present understanding of phytochrome action.
Chapter
Light is an essential factor in the control of plant development. Red light and blue light are the most effective spectrum regions and it is now a well established fact that they play a dual role in plant responses to the environment: first, they act through photosynthesis to provide a source of energy; secondly, they can modulate physiological responses through the action of two photoreceptors (phytochrome and the blue light pigment). Several reports have shown that phytochrome is responsible for perception of light quality while the blue-light pigment detects light quantity (Gaba and Black 1979,Thomas and Dickinson 1979, Ritter et al. 1981, Smith 1981). However, the mode of action of the pigments has not yet been clearly established. Membranes have been proposed as possible primary sites of action of phytochrome, the best known photoreceptor (Marmé 1977). Several studies have demonstrated that one of the primary events occurring just after light perception by phytochrome involves the transport of ions across membranes and particularly calcium ions (Satter et al. 1970; >Tezuka and Yamamoto 1975; Weisenseel and Ruppert 1977Brownlee and Kendrick 1979a, b; Dreyer and Weisenseel 1979). Studies to elucidate the mechanism of phytochrome action have focused primarily on physicochemical properties of the photoreceptor. It is now well known that the pigment exists in two interconvertible molecular forms. The Pfr form, absorbing in the far-red region of the spectrum, is induced by red light (R) and can be converted to the Pf form, absorbing in the red region, by far-red light (FR).
Article
Phytochrome is a chromoprotein that serves as the photoreceptor for a wide range of photomorphogenic responses in plants. This chromoprotein exists in two photointerconvertible forms at physiological temperatures. One form absorbs maximally near 665 nm (Pr) and is considered inactive, while the other absorbs maximally near 730 nm (Pfr) and is morphogenically active. Since the discovery of this pigment some 25 years ago (Borthwick, 1972), two different, although not necessarily mutually exclusive, approaches to an understanding of its mode of action have been taken. One approach has been to investigate phytochrome-mediated responses in attempts to deduce the nature of the primary events that lead to these responses. A second approach has been to investigate the biophysical and biochemical properties of the pigment so that a direct path to understanding its molecular mechanism of action might become available.
Chapter
Exchange of H+ or OH- between plant cells and their surroundings is associated with a wide range of metabolic processes, and especially with solute transport. One major field of investigation has been the causes and effects of the large balancing fluxes of H+ or OH- which occur during excess absorption of cations (e.g. K+ from K2SO4 solutions) or anions (e.g. Cl- from CaCl2 solutions). A second, biophysical approach, deals with the contribution of H+ and OH- fluxes to the electrical parameters of cell membranes. A third approach considers the separation of H+ and OH- across cell membranes as an energy source for other ion transport processes within the cell. These aspects have been discussed separately in other Chapters of this Volume. The aim of this Chapter is to present the main findings as part of a wider appraisal of the significance of H+ and OH- transport in cellular metabolism. The starting-point is simply that the intracellular distribution of H+ (i.e. the intracellular pH), far from being a haphazard consequence of metabolism, is a closely controlled component of the ion balance within cells. Particular attention is thus paid to the pH-regulating role of H+ and OH- transport. Interactions with other ion transport processes and various morphogenetic phenomena are also considered in relation to the need for pH regulation. Some of the processes discussed in this Chapter (e.g. organic acid synthesis, and NO - 3 reduction) are also discussed in the next Chapter by Osmond.