Plant
Physiol.
(1984)
74,
112-116
0032-0889/84/74/0112/05/$01
.00/0
Terpenoid
Metabolism
in
Plastids'
SITES
OF
PHYTOENE
SYNTHETASE
ACTIVITY
AND
SYNTHESIS
IN
PLANT
CELLS
Received
for
publication
June
13,
1983
and
in
revised
form
September
6,
1983
BILAL
CAMARA*
Laboratoire
de
Regulations
Metaboliques
et
Diffterenciation
des
Plastes,
Tour
53,
2eme
etage,
Universite
Pierre
et
Marie
Curie
(Paris
6),
4
Place
Jussieu,
75230
Paris
Cedex
05
ABSTRACT
The
biosynthesis
of
phytoene
from
prephytoene
pyrophosphate
has
been
localized
exclusively
in
the
plastid
compartment
of
ruptured
proto-
plasts
derived
from
Triticum
leaves
and
Capsicum
fruits.
The
phytoene
synthetase
activity
in
Triticum
leaves
deficient
in
plastid
ribosomes
was
comparable
to
those
obtained
in
normal
leaves.
In
addition,
the
stimulation
of
phytoene
synthetase
activity
observed
in
green
Capsi-
cum
fruit
after
2-(4-chlorophenylthio)triethylamine
hydrochloride
treat-
ment
was
not
abolished
by
chlororamphenicol
and
lincomycin,
in
contrast
to
the
inhibition
observed
after
cycloheximide
treatment.
These
data
conclusively
show
that
phytoene
synthetase
is
localized
exclusively
in
the
plastid
compartment
in
higher
plants
and
that
its
synthesis
is
not
performed
on
70S
ribosomes.
Carotenoids
accumulate
predominantly
in
the
plastid
com-
partment
of
higher
plant
cells.
Increasing
evidence
has
estab-
lished
the
autonomy
of
plastids
in
the
formation
of
carotenoids
from
various
precursors.
The
site(s)
of
transcription
and
trans-
lation
of
the
enzymes
involved
in
the
synthesis
of
these
pigments
is
unresolved.
Earlier
studies
have
been
concerned
with
in
vivo
translation
inhibitor
treatments
or
with
mutations
which
alter
plastid
morphogenesis
and
carotenoid
accumulation
(29,
31).
The
enzyme
which
catalyzes
the
formation
of
the
first
carote-
noid
in
the
pathway,
i.e.
phytoene,
is
known
as
phytoene
syn-
thetase
(25).
The
intraplastidial
location
of
this
activity
has
been
demonstrated
recently
in
chromoplasts
(8,
22).
Here
we
present
evidence
that
this
enzyme
is
exclusively
localized
in
plastids
and
that
its
synthesis,
as
judged
by
the
activity
present
in
ribosome-
deficient
plastids
and
by
translation
inhibitor
treatments,
is
not
performed
on
70S
ribosomes.
MATERIALS
AND
METHODS
Plant
Material.
Wheat
leaves
(Triticum
sativum
L.
var
Flor-
ence
aurore)
and
Pepper
fruits
(Capsicum
annuum
L.
var
Yolo
wonder)
were
used.
Triticum
seeds
were
surface
sterilized
(12)
and
germinated
in
darkness
at
25°C
or
34°C
for
12
d.
The
first
leaf
was
used
for
experimentation.
For
protoplast
preparation,
Triticum
plants
were
grown
at
25°C
for
9
d
under
artificial
light
(5000
lux;
11
h
photoperiod).
Capsicum
fruits,
generously
pro-
vided
by
the
Senegal
Agriculture
Service,
were
harvested
at
mature
green
and
orange
stages.
Subcellular
Fractionation.
Protoplasts
were
isolated
(starting
'Supported
by
grants
from
the
'Direction
de
la
Recherche
Universi-
taire'
and
from
the
'Centre
National
de
la
Recherche
Scientifique.'
from
5
to
10
g
fresh
weight)
and
purified
from
Triticum
leaves,
essentially
by
the
method
of
Lilley
et
al.
(23).
For
Capsicuim
protoplasts,
a
method
modified
from
Fujiwake
et
al.
(18)
and
Lilley
et
al.
(23)
was
adopted.
Thin
pericarp
slices
were
digested
in
the
presence
of
0.5%
macerozyme,
2%
cellulase,
500
mm
sorbitol,
0.05%
PVP,
0.1
mm
CaCl2,
and
5
mm
Mes
(final
pH
5.5).
The
released
protoplasts
were
monitored
by
light
micros-
copy
and
filtered
through
Blutex
(100
,m
apertures).
The
crude
Capsicum
protoplast
suspension,
after
centrifugation
at
lOOg
for
5
min,
was
suspended
in
the
medium
of
500
mm
sucrose
and
50
mM
Mes,
pH
6,
and
centrifuged
at
lOOg
for
2
min.
The
protoplast
suspension
was
diluted
(1
ml
suspension
+
0.5
ml
50
mm
Mes,
pH
6),
and
protoplasts
were
ruptured
by
four
passages
through
a
10-ml
syringe
fitted
with Blutex
(20
gm
apertures).
The
resulting
suspension
(3
ml)
was
layered
on
the
top
of
a
sucrose
gradient
(30-60%,
w/w)
containing
50
mM
Tris-
HCI
(pH
7.6)
and
centrifuged
at
100,000g
in
a
Beckman
L50
ultracentrifuge
equipped
with
the
SW27
rotor
for
1
h.
Fractions
(1.5
ml)
were
collected
and
enzyme
markers
(see
below)
were
used
to
locate
the
different
cellular
fractions.
Enzyme
Assays.
The
different
assays
were
performed
using
a
cell-free
system
prepared
as
follows:
excised
plant
material
(1
g
fresh
weight/2
ml
medium
containing:
5
mM
MgC92,
5
mm
DTT,
0.25
M
sucrose,
50
mM
Tris-HCI,
pH
7.8)
was
homogenized
in
a
Waring
Blendor
at
full
speed
for
3
x
4
s
and
the
supernatant
obtained
after
centrifugation
at
1SOg
for
5
min
was
used
for
enzyme
assays.
RuBP2
carboxylase
(EC
4.1.1.39)
was
assayed
according
to
Bravdo
et
al.
(6).
NADP-glyceraldehyde
phosphate
dehydroge-
nase
(EC
1.2.1.13)
was
assayed
as
described
by
Bradbeer
et
al.
(5).
Catalase
(EC
1.1
1.1.6)
was
determined
as
described
by
Luck
(24).
The
method
described
by
Hackett
et
al.
(20)
was
used
for
Cyt
c
oxidase
(EC
1.9.3.1).
Phytoene
synthetase
was
assayed
as
described
by
Camara
et
al.
(9).
Fractions
(0.25
ml)
derived
from
the
sucrose
gradients
or
cell-free
extracts
(5
mg
protein)
were
incubated
in
the
presence
of
(2
ml
final
volume)
[3H]prephytoene
pyrophosphate
(
12
Ci/mol,
0.5
,uCi)
(synthesized
by
the
addition
of
trans-diazogeranylgeranial
into
a
chilled
ether
solution
con-
taining
zinc
iodide
and
trans-geranylgeraniol
followed
by
phos-
phorylation
of
the
resulting
alcohol
[9]),
1
mM
KF,
10
mM
MgC92,
5
mM
MnCl2,
10
mm
DTT,
and
buffered
with
50
mM
Tris-HCl,
pH
7.6.
The
reaction
was
incubated
at
25°C
for
4
h
in
the
case
of
fractions
derived
from
the
sucrose
gradients
and
for
1
h
in
the
other
cases.
The
reactions
were
terminated
by
the
addition
of
acetone:
ethanol
(2:1,
v/v).
Phytoene
was
extracted
after
the
addition
of
authentic
standard
(1
mg)
and
purified
by
TLC
as
described
by
Camara
et
al.
(8).
The
protein
content
was
2Abbreviations:
RuBP,
ribulose
bisphosphate;
CPTA,
2-(4-chloro-
phenylthio)triethylamine
hydrochloride;
Ethephon,
(2-chloro-
ethyl)phosphonic
acid.
112
PLASTID
TERPENOID
METABOLISM
estimated
after
precipitation
with
10%
TCA
(8),
with
BSA
as
a
standard.
The
Chl
and
carotenoid
contents
were
determined
according
to
Arnon
(1)
and
Davies
(13).
Ribosome
and
Nucleic
Acid
Analysis.
The
fractions
containing
70S
and
80S
ribosomes
were
scanned
at
254
nm
after
centrifu-
gation
in
a
(10-34%)
sucrose
gradient
(14).
The
nucleic
acids
were
extracted
(16),
precipitated
in
the
cold
(-20°C)
by
addition
of
2.5
volumes
of
ethanol,
and
subjected
to
disc
electrophoresis
(16)
using
quartz
tubes.
The
different
fractions
were
monitored
at
260
nm.
Stimulation
of
Phytoene
Synthesis
and
Translation
Inhibitor
Treatments.
Mature
green
fruits
were
cleaned
and
dipped
for
30
min
in
a
solution
containing
30
mg
ml-'
CPTA
or
30
mg
ml-'
Ethephon
in
0.1%
Tween
80.
At
the
completion
of
this
time,
they
were
exposed
to
continuous
light
(5,000
lux
25C)
for
2
to
7
d.
For
experiments
involving
translation
inhibitors,
the
fruits
were
dipped
for
30
min
in
water
or
in
inhibitor
solution
(0.4
mg
ml-'
chloramphenicol,
0.1
mg
ml-'
lincomycin,
0.2
mg
ml-'
cycloheximide).
The
fruits
were
allowed
to
dry
before
CPTA
treatments
conducted
as
described
above.
The
2,000g
fraction
obtained
as
described
before
(8)
was
used
for
phytoene
synthetase
assay.
RESULTS
AND
DISCUSSION
Subcellular
Localization
of
Phytoene
Synthetase
Activity.
The
enzymic
localization
assay
was
performed
after
protoplast
rup-
ture
and
centrifugation
in
a
sucrose
(30-60%,
w/w)
gradient.
The
profile
of
the
different
cellular
fractions
detected
by
marker
enzyme
activities
showed
consistently
three
main
peaks
(Figs.
1-
3).
In
green
plant
material
(
Triticum
leaves
and
Capsicum
fruits),
the
peak
representing
the
plastid
fraction
was
well
identified
by
the
presence
of
CHI
and
RuBP
carboxylase
activity
(Figs.
1
and
2).
The
low
RuBP
carboxylase
activity
present
at
the
top
of
the
gradient
was
due
to
the
stromal
proteins
liberated
from
broken
chloroplasts.
In
ripening
Capsicum
fruits,
containing
chromo-
plasts,
the
plastid
fraction
was
best
located
by
the
position
of
carotenoids
(Fig.
3).
The
two
other
peaks
(Figs.
1-3),
representing
60
00
0~~~~~~
5
1~~0
150
0
0
~ ~
U%
FRACTIONS
FIG.
1.
Distribution
of
phytoene
synthetase
and
marker
enzyme
ac-
tivities
after
mechanical
rupture
of
protoplasts
derived
from
Triticulm
leaves.
One
arbitrary
unit
corresponds
to:
0.1
zimol
min~'
fraction~'
for
Cyt
c
oxidase;
50
Mamol
min~'
fraction~'
for
catalase;
5
nmol
min~'
fraction
'
for
RuBPCase;
and
10
pmol
min
'
fraction
'
for
phytoene
synthetase.
The
sucrose
concentration
of
the
different
fractions
was
determined
by
refractometry.
(n
snI
5
w
5
1S
FRACTIONS
0
0
0
.4
FIG.
2.
Distribution
of
phytoene
synthetase
and
marker
enzyme
ac-
tivities
after
mechanical
rupture
of
protoplasts
derived
from
green
Cap-
siczm
fruits.
One
arbitrary
unit
corresponds
to:
0.
I
,mol
min-'
fraction-'
for
Cyt
c
oxidase;
45
gmol
min-'
fraction-'
for
catalase;
1
nmol
min-'
fraction-'
for
RuBPCase;
and
12
pmol
min-'
fraction-'
for
phytoene
synthetase.
4
4
UJ
;
00
o7
c
z
x
.ii
)
oU
X
u
X
Da
>
lu
2
W
zQ
I
i
0
1O
N
5
10
15
FRACTIONS
FIG.
3.
Distribution
of
phytoene
synthetase
and
marker
enzyme
ac-
tivities
after
mechanical
rupture
of
protoplasts
derived
from
orange
Capsicutm
fruits.
One
arbitrary
unit
corresponds
to:
0.1
Imol
min-'
fraction-'
for
Cyt
c
oxidase;
60
Mmol
min-'
fraction-'
for
catalase;
and
20
pmol
min-'
fraction-'
for
phytoene
synthetase.
the
mitochondrial
and
peroxisomal
fractions,
respectively,
were
identified
by
the
distribution
of
Cyt
c
oxidase
and
catalase
activities.
The
sucrose
density
gradient
centrifugation
pattern
showed
a
strict
association
between
the
plastid
fraction
and
the
phytoene
synthetase
activity
(Figs.
1-3).
Neither
the
mitochon-
drial
fraction
nor
the
peroxisomal
fraction
contained
a
significant
level
of
phytoene
synthetase
activity.
Therefore,
it
appears
that
phytoene
synthetase
in
higher
plant
cells
(leaf
and
fruit)
is
located
in
the
plastids.
Site
of
Synthesis
of
Phytoene
Synthetase.
To
determine
113
Plant
Physiol.
Vol.
74,
1984
whether
or
not
phytoene
synthetase
is
coded
in
the
plastome,
we
used
Triticum
seedlings
deficient
in
plastid
(70S)
ribosomes.
For
this,
the
procedure
developed
and
extensively
used
by
Feierabend
(16)
was
adopted.
Triticum
seedlings
were
grown
in
the
dark
at
25°C
and
34°C
for
12
d.
At
the
completion
of
this
period,
the
etiolated
seedlings
were
illuminated
(5000
lux)
for
12
h
at
the
temperatures
used
for
the
growth
period.
The
first
leaf
of
seed-
lings
maintained
at
34°C
failed
to
green
in
contrast
to
that
of
seedlings
kept
at
25°C.
The
accumulation
of
carotenoids
was
drastically
inhibited
at
34°C.
Compared
to
seedlings
grown
and
illuminated
at
25°C,
only
3%
#3-carotene,
20%
lutein,
15%
vio-
laxanthin,
and
10%
neoxanthin
were
detected
at
34°C.
These
data
complement
those
obtained
by
Rademacher
and
Feierabend
(27)
on
Secale
seedlings.
We
observed
similar
results
during
the
greening
of
Nicotiana
cells
in
culture
at
27°C
and
32°C
(Camara
et
al.,
unpublished
results).
The
pattern
of
the
ribosomal
fractions
for
the
illuminated
plant
material
grown
at
25°C
showed,
after
centrifugation
in
a
linear
sucrose
gradient,
two
prominent
peaks
(Fig.
4).
Their
sedimentation
characteristics
are
comparable
to
those
observed
previously
in
Pisum
apices
(15)
and
in
Secale
seedlings
(16)
for
0.8
E
C
0.2
I
0.8
0
49
0.2
5
20
fractions
35
FIG.
4.
Sedimentation
pattern
of
the
ribosomal
fractions
prepared
from
the
first
leaf
of
Triticuim
seedlings
grown
at
25°C
or
34'C
in
darkness
and
illuminated
(5000
lux)
at
the
same
temperatures
for
12
h.
The
supernatant
obtained
after
centrifugation
at
20,000g
for
30
min
was
layered
on
a
10
to
34%
sucrose
gradient
and
centrifuged
72,000g
for
5
h
(Beckman
SW
27
rotor).
Fractions
(0.8
ml)
were
collected.
E
c
0
0
0.12
FIG.
5.
Electrophoretic
analysis
in
2.5%
acrylamide
of
nucleic
acid
extracted
from
the
first
leaf
of
Triticuin
seedlings
grown
at
25°C
and
34°C
in
darkness
and
illuminated
(5000
lux)
at
the
same
temperatures
for
12
h.
70S
ribosomes
and
80S
ribosomes.
Triticum
seedlings
grown
and
illuminated
at
34C
lacked
the
fraction
corresponding
to
70S
ribosomes.
The
nucleic
acids
extract
obtained
from
Triticum
seedlings
grown
and
illuminated
at
25°C
and
34C
was
subjected
to
disc
electrophoresis
(Fig.
5).
The
electrophoretic
mobility
obtained
after
scanning
the
quartz
tubes
at
260
nm
allowed
the
identifi-
cation
of
different
RNA
fractions.
The
peaks
corresponding
to
16S
and
23S
RNA
were
not
detected
in
seedlings
maintained
at
34C
(Fig.
5),
in
good
agreement
with
results
obtained
in
light-
grown
Triticum,
Hordeum
and
Pisum
seedlings
(16),
and
in
Secale
(8).
These
data
indicate
a
deficiency
of
70S
ribosomes
in
the
plastids
of
seedlings
grown
and
illuminated
at
340C.
The
mechanism
responsible
for
this
deficiency
is
unknown.
Bunger
and
Feierabend
(7)
have
shown
that
it
is
not
caused
by
a
defective
RNA
polymerase.
It
has
been
suggested
that
photodestruction
may
induce
the
formation
of
ribosome-deficient
plastids
(33).
This
possibility
can
be
ruled
out
since
the
deficiency
could
be
noted
in
either
a
light
or
a
dark
regime
(16).
Whatever
the
cause
of
this
deficiency,
the
plastids
derived
from
these
plant
materials
are
unable
to
carry
out
the
synthesis
of
plastid-coded
polypeptides
(3,
17).
With
this
in
mind,
we
tested
the
activity
of
plastidial
NADP-glyceraldehyde
phosphate
dehy-
drogenase
that
is
exclusively
synthesized
on
cytoplasmic
ribo-
somes
(5),
plastidial
RuBP
carboxylase
that
is
partly
synthesized
on
cytoplasmic
and
plastidial
ribosomes
(14),
and
mitochondrial
Cyt
c
oxidase
that
is
partly
synthesized
on
cytoplasmic
and
mitochondrial
ribosomes
(30).
The
RuBP
carboxylase
in
seed-
lings
grown
at
34°C
was
drastically
depressed
(Table
I).
The
NADP-glyceraldehyde
phosphate
dehydrogenase
activity
in
the
extract
made
from
seedlings
grown
at
34C
was
lower
than
that
at
25°C
(Table
I).
On
the
other
hand,
Cyt
c
oxidase
activity
exhibited
a
comparable
level
in
extracts
made
from
seedlings
grown
at
25
and
340C
(Table
I).
The
inhibition
of
RuBP
carbox-
ylase
is
readily
explained
by
the
fact
that
the
synthesis
of
the
large
subunit
coded
by
the
plastid
genome
is
prevented
under
the
experimental
conditions
used,
whereas
that
affecting
NADP-
glyceraldehyde
phosphate
dehydrogenase
could
be
tentatively
correlated
to
the
control
exercised
by
the
plastid
on
cytoplasmi-
cally
synthetized
polypeptides
(5),
as
noted
also
in
Secale
(16,
19).
The
results
obtained
for
phytoene
synthetase
are
also
presented
in
Table
I.
The
activity
in
extracts
made
from
seedlings
grown
at
25C
and
34°C
showed
comparable
levels
of
activity.
This
parallels
the
trend
observed
for
Cyt
c
oxidase.
When
the
extract
prepared
from
plants
given
at
25°C
was
added
to that
derived
from
plants
maintained
at
34C
and
incubated
with
prephytoene
pyrophosphate,
the
activity
was
not
depressed
(Table
I):
this
Table
I.
Enz:'me
Activities
of
the
Cell-Free
Extract
Prepared
fiom
the
First
Leaf
of
Triticutm
Seedlings
Seedlings
were
grown
at
25°C
or
34°C
and
illuminated
for
12
h
at
the
same
temperatures.
A
cell-free
extract
derived
from
Triticimmn
leaf
as
described
in
"Materials
and
Methods"
was
used
for
enzyme
assays.
The
activities
are
expressed
as
nmol
mg-'
protein
min-',
except
for
phytoene
synthetase
expressed
in
pmol
mg-'
protein
min-'.
Enzymes
250C
34°C
34/25
RuBP
carboxylase
68
9
0.13
Cyt
c
oxidase
95
100
1.05
NADP
glyceraldehyde
dehydrogenase
132
72
0.54
Phytoene
synthetase
15
17
1.13
Phytoene
synthetasea
17.5
aIn
order
to
test
the
presence
of
a
possible
inhibitor,
an
aliquot
equivalent
to
0.1
mg
protein
was
taken
from
the
extract
derived
from
leaves
grown
at
25°C
and
added
to
the
incubation
medium
used
for
leaf
material
maintained
at
34C.
Go
25C
I
-
340C
a
I
I
I
a I a
250C
340C
1400oo0
C'h,
en
en
,
cn
,
CM
N-
T-
N
9
eloctrophrsc
mobNity
---.
114
CAMARA
PLASTID
TERPENOID
METABOLISM
9
c-
76
-
.
25
E
z1
25
0
0
12
24
48
h
FIG.
6.
Influence
of
CPTA
treatment
on
phytoene
synthetase
activity
of
cell-free
extracts
(2000g
fraction,
i.e.
plastidial
fraction,
equivalent
to
5
mg
protein)
prepared
from
green
Capsicum
fruits.
The
extraction
and
incubation
with
[3H]prephytoene
pyrophosphate
are
described
in
"Ma-
terials
and
Methods."
Table
II.
Influence
of
Translation
Inhibitors
on
the
CPTA-Stimulated
Phytoene
Synthetase
Activity
of
Green
Capsicum
Fruit
Plastids
Capsicum
plastids
(5
mg
protein)
isolated
after
CPTA
and
inhibitor
treatments
(see
"Materials
and
Methods")
were
incubated
for
1
h
with
[3HJprephytoene
pyrophosphate
as
described
in
"Materials
and
Meth-
ods."
Phytoene
Treatment/
Treatments
Synthesis
Controla
pmol
mg-'
protein
min'
Control
(water
only)
32
Control
(water
+
Tween
80)
34
CPTA
72
2.25
Chloramphenicol
+
CPTA
68
2.12
Lincomycin
+
CPTA
70
2.18
Cycloheximide
+
CPTA
29
0.90
The
value
32
pmol
mg-'
protein
min-'
was
used
(water
only).
experiment
excludes
the
possible
presence
of
an
inhibitor
in
the
extract
made
from
plants
grown
at
normal
temperature
(25C).
Furthermore,
the
withdrawing
effect
of
Chl
synthetase
observed
when
prenyl
precursors
up
to
C20
chain
are
used
as
substrates
does
not
operate
with
prephytoene
pyrophosphate
(Camara
et
al.,
9)
since
prephytoene
pyrophosphate
is
an
intermediate
be-
yond
any
step
available
to
the
Chl
pathway.
This
precursor
is
channeled
towards
carotenoid
biosynthesis,
eliminating
side
re-
actions.
In
these
conditions,
the
activities
reported
represent
the
real
phytoene
synthetase
activities
present
in
the
extracts.
In
addition,
experiments
using
translation
inhibitors
were
per-
formed
owing
to
the
fact
that
plastids
isolated
from
green
Cap-
sicum
fruits
treated
with
CPTA
for
48
h
showed
a
stimulation
of
phytoene
synthesis
(Fig.
6;
Camara,
unpublished
data),
with-
out
visible
modification
of
plastid
structure
(Camara
and
Bran-
geon,
unpublished
data).
The
ultrastructural
modifications
started
at
the
4th
d,
as
indicated
by
the
concomitant
appearance
of
the
yellow
color
of
the
pericarp,
due
to
lycopene
accumulation.
This
system
was
amenable
for
experiments
with
70S
(chloram-
phenicol
and
lincomycin)
and
80S
(cycloheximide)
translation
inhibitors.
The
results
obtained
(Table
II)
show
that
chloramphenicol
and
lincomycin
did
not
abolish
the
stimulation
of
phytoene
synthesis
from
prephytoene
pyrophosphate,
while
cycloheximide
did.
Sim-
ilar
results
were
obtained
after
72
h
using
Ethephon
(Camara
and
Dogbo,
unpublished
data).
Whether
or
not
this
stimulation
of
phytoene
synthetase
activity
by
CPTA
is
the
result
of
an
increased
protein
synthesis
is
debatable.
Earlier
studies
based
on
RuBP
carboxylase
have
displayed
conflicting
evidence
on
this
point
-(see
Bottomley
for
discussion,
4).
However,
protein
syn-
thesis
cannot
be
excluded,
since
CPTA
induces
lycopene
accu-
mulation
like
that
observed
in
ripening
tomato
fruits;
also,
in
the
latter
case,
changes
in
the
amount
of
mRNA
and
protein
synthesis
occurred
as
shown
by
in
vitro
translation
in
a
wheat
germ
system
(28).
This
trend
is
also
observed
during
ripening
of
avocado
fruits
(10).
The
above
results
are
clearly
taken
as
an
evidence
for
the
absence
of
synthesis
of
phytoene
synthetase
on
plastid
(70S)
ribosomes.
Thus,
phytoene
synthetase
is
coded
exclusively
by
the
nuclear
genome.
However,
although
prominent
nucleo-cy-
toplasmic
participation
during
the
synthesis
of
phytoene
synthe-
tase
is
demonstrated
in
this
work,
one
cannot
exclude
a
possible
plastid
regulation
of
its
synthesis,
as
suggested
for
NADP-glyc-
eraldehyde
phosphate
dehydrogenase
(5).
Presently,
concerning
enzymes
involved
in
the
biogenesis
of
plastid
terpenoids,
the
results
obtained
for
phytoene
synthetase
are
to
be
compared
with
those
obtained
for
NADPH:Pchlide
oxidoreductase
(2,
19),
which
is
synthesized
as
a high
mol
wt
precursor
in
the
cytoplasm.
Obviously,
phytoene
synthetase
be-
longs
to
the
family
of
plastidic
stroma
and
membrane
proteins
made
on
80S
ribosomes
and
ultimately
processed
by
posttrans-
lational
mechanisms
(1
1,
14,
26).
Further
work
on
phytoene
synthetase
is
focused
on
the
nature
of
its
cytoplasmic
precursor
and
its
functional
integration
by
plastids.
Acknowledgments-I
thank
Professor
R.
Moneger
for
his
constant
interest
and
valuable
discussion
during
the
work.
Also,
I
thank
Dr.
H.
Yokoyama
of
the
USDA
Fruit
and
Vegetable
Chemistry
Laboratory,
Pasadena,
CA,
for
the
generous
gift
of
CPTA;
Dr.
J.
E.
Grady
of
the
Upjohn
Company
for
the
generous
gift
of
lincomycin;
and
J.
Pecheur,
CFPI,
France,
for
the
generous
gift
of
Ethephon.
I
am
grateful
to
P.
B.
Auliac
for
providing
electrophoresis
equipment.
I
appreciate
the
technical
assistance
given
by
C.
Agnes,
F.
Bardat,
and
A.
Nargeot-Garcia.
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CAMARA
