P oc.
Nadl.
Acad.
Sci.
USA
Vol.
88,
pp.
2712-2715,
Ap il
1991
Bo any
P o ein
u no e
as
a
componen
in
he
ligh /da k
egula ion
o
phosphoenolpy u a e
ca boxylase
p o ein-se ine
kinase
ac i i y
in
C4
plan s
( egula o y
phospho yla ion/C4
pho osyn hesis/ligh
ac i a ion/p o ein
kinase/cycloheximide)
JIN-AN
JIAO*,
CRISTINA
ECHEVARR A* ,
JEAN
VIDAL ,
AND
RAYMOND
CHOLLET*§
*Depa men
o
Biochemis y,
Uni e si y
o
Neb aska-Lincoln,
Eas
Campus,
Lincoln,
NE
68583-0718;
Labo a o io
de
Fisiologia
Vege al,
Facul ad
de
Biolog a,
Uni e sidad
de
Se illa,
A enida
de
la
Reina
Me cedes
s/n,
41012,
Se illa,
Spain;
and
Labo a oi e
de
Physiologie
V6gd ale
Mol6culai e,
Uni 6
de
Reche che
Associde
Cen e
Na ional
de
la
Reche che
Scien i ique
1128,
Uni e si
de
Pa is-Sud,
Cen e
d'O say,
Bi imen
430,
O say-Cedex,
F ance
Communica ed
by
William
L.
Og en,
Janua y
3,
1991
( ecei ed o
e iew
No embe
13,
1990)
ABSTRACT
Maize
lea
phosphoenolpy u a e
ca boxylase
[PEPC;
o hophospha e:oxaloace a e
ca boxy-lyase
(phospho-
yla ing),
EC
4.1.1.311
p o ein-se ine
kinase
(PEPC-PK)
phos-
pho yla es
se ine-15
o
i s
a ge
enzyme,
hus
leading
o
an
inc ease
in
ca aly ic
ac i i y
and
a
concomi an
dec ease
in
mala e
sensi i i y
o
his
cy oplasmic
C4
pho osyn hesis
enzyme
in
he
ligh .
We
ha e
ecen ly
demons a ed
ha
he
PEPC-PK
ac i i y
in
maize
lea es
is
slowly,
bu
s ikingly,
inc eased
in
he
ligh
and
dec eased
in
da kness.
In
his
epo ,
we
p o ide
e idence
ha
cycloheximide,
an
inhibi o
o
cy oplasmic
p o-
ein
syn hesis,
when
ed
o
de ached
lea es
o
C4
monoco s
(maize,
so ghum)
and
dico s
(Po ulaca
ole acea)
in
he
da k
o
ligh ,
comple ely
p e en s
he
in
i o
ligh
ac i a ion
o
PEPC-PK
ac i i y
ega dless
o
whe he
he
p o ein
kinase
ac i i y
is
assessed
in
i o
o
in
i o.
In
con as ,
chlo am-
phenicol,
an
inhibi o
o
p o ein
syn hesis
in
chlo oplas s,
has
no
e ec
on
he
ligh
ac i a ion
o
maize
PEPC-PK.
Simila ly,
ea men
wi h
cycloheximide
did
no
in luence
he
ligh
ac i-
a ion o
o he
pho osyn hesis- ela ed
enzymes
in
maize,
in-
cluding
cy oplasmic
suc ose-phospha e
syn hase
and
chlo o-
plas
s omal
NADPH-mala e
dehyd ogenase
and
py u a e,P;
dikinase.
These
and
ela ed
esul s,
in
which
de ached
maize
lea es
we e
ea ed
simul aneously
wi h
cycloheximide
and
mic ocys in-LR,
a
po en
in
i o
and
in
i o
inhibi o
o
he
PEPC
ype
2A
p o ein
phospha ase,
indica e
ha
sho - e m
p o ein
u no e
o
he
PEPC-PK
i sel
o
some
o he
essen ial
componen (s)
(e.g.,
a
pu a i e
p o ein
ha
modi ies
his
kinase
ac i i y)
is
one
o
he
p ima y
le els
in
he
complex
and
unique
egula o y
cascade
e ec ing
he
e e sible
ligh
ac i a-
ion/se yl
phospho yla ion
o
PEPC
in
he
mesophyll
cy o-
plasm
o
C4
plan s.
Ligh
e e sibly
ac i a es
a
numbe
o
pho osyn hesis-
ela ed
enzymes
in
plan s
ia
se e al
di e en
mechanisms
(1-4).
Among
hese
is
he
ligh
ac i a ion
o
lea
cy oplasmic
phosphoenolpy u a e
ca boxylase
[PEPC;
o hophos-
pha e:oxaloace a e
ca boxy-lyase
(phospho yla ing),
EC
4.1.1.31]
in
C4
plan s
by
e e sible
p o ein
phospho yla ion
(5,
6).
P e ious
in
i o
(7,
8)
and
in
i o
(9)
s udies
wi h
maize
lea
PEPC
demons a ed
ha
he
phospho yla ion
o
a
single,
N- e minal
se yl
esidue
(Se -15)
leads
o
an
inc ease
in
ca aly ic
ac i i y
and
a
dec ease
in
eedback
inhibi ion
o
he
a ge
enzyme
by
L-mala e.
Rela ed
indings
om
a
econ-
s i u ed
phospho yla ion
sys em
indica ed
ha
he
ac i i y
o
he
p o ein-se ine
kinase
ha
ca alyzes
his
egula o y
phos-
pho yla ion
o
PEPC
is
no
a ec ed
by
a
numbe
o
pu a i e,
ligh -modula ed
cy oplasmic
e ec o s
(e.g.,
educed
hio e-
doxin
h,
Ca2+,
PPj,
uc ose
2,6-bisphospha e)
and
au ophos-
pho yla ion
(6,
7).
Howe e ,
mo e
ecen
wo k
has
es ab-
lished
ha
he
phosphoenolpy u a e
ca boxylase
p o ein-
se ine
kinase
(PEPC-PK)
is
ac i a ed
by
ligh
and
inac i a ed
by
da kness
in
i o
(10).
Mo eo e ,
his
s iking
egula o y
p ocess
appea s
independen
o
SH
s a us,
Ca2l
le els,
and
a
pu a i e,
igh -binding
PEPC-PK
e ec o
(10).
One
o
he
dis inguishing
ea u es
o
he
e e sible
ligh
ac i a ion
o
PEPC-PK
and
i s
a ge
enzyme,
PEPC,
in
C4
plan s
is
i s
sluggishness
in
i o;
when
compa ed
o
he
in
i o
ac i a ion
o
pho o egula ed
mesophyll
chlo oplas
s omal
enzymes
such
as
py u a e,Pi
dikinase
(PPDK)
and
NADPH-
mala e
dehyd ogenase
(MDH)
(2,
6),
he
o me
a e
bo h
ela i ely
slow
p ocesses,
aking
up
o
1
h ,
a he
han
minu es,
o
comple ion
(10-12).
To
gain
mo e
insigh
in o
his
di e ence
and
he
speci ic
mechanism(s)
by
which
he
PEPC-PK
ac i i y
in
i o
is
slowly,
bu
s ikingly,
inc eased
in
he
ligh
and
dec eased
in
da kness
(10),
de ached
maize
lea es
we e
ed
wo
widely
used
inhibi o s
o
p o ein
syn-
hesis.
PEPC-PK
ac i i y
was
subsequen ly
assessed
ei he
in
i o
[mala e
IC50
alues
o
inhibi ion
o
he
a ge
enzyme
(11,
12)]
o
in
i o
[32p
phospho yla ion
o
pu i ied
da k- o m
PEPC
(7, 10)].
Whe eas
chlo amphenicol
(CAP),
a
70S
ibo-
some-speci ic
inhibi o
o
chlo oplas ic
p o ein
syn hesis,
had
no
e ec
on
he
ligh
ac i a ion
o
PEPC-PK,
cyclohex-
imide
(CHX),
an
inhibi o
o
cy oplasmic
p o ein
syn hesis,
comple ely
blocked
he
ligh
ac i a ion
o
his
p o ein-se ine
kinase.
In
con as ,
he
in
i o
ac i a ion
o
se e al
o he
pho o egula ed
cy oplasmic
[suc ose-phospha e
syn hase
(SPS)]
and
chlo oplas ic
(PPDK,
MDH)
pho osyn hesis-
ela ed
enzymes
was
no
in luenced
by
CHX
ea men .
These
esul s
indica e
ha
he
syn hesis
and
deg ada ion
o
PEPC-PK
pe
se
o
some
o he
essen ial
componen (s)
a e
in ol ed
a
one
o
he
p ima y
le els
in
he
egula o y
cascade
e ec ing
he
e e sible
ligh
ac i a ion/se yl
phos-
pho yla ion
o
PEPC
in
he
mesophyll
cy oplasm
o
C4
plan s.
MATERIALS
AND
METHODS
Ma e ials.
Maize
(Zea
mays
L.,
c .
Golden
C oss
Ban am)
plan s
we e
g own
as
desc ibed
(7,
10).
[y-32P]ATP
[speci ic
ac i i y,
3000
Ci
(111
TBq)/mmol]
was
pu chased
om
Ame sham.
Da k- o m
maize
lea
PEPC
was
pu i ied
by
he
p ocedu e
desc ibed
(7,
8).
All
biochemical
eagen s
we e
ob ained
om
Sigma
excep
o
mic ocys in-LR
(MC)
(Cal-
Abb e ia ions:
PEPC,
phosphoenolpy u a e
ca boxylase;
PEPC-
PK,
phosphoenolpy u a e
ca boxylase
p o ein-se ine
kinase;
SPS,
suc ose-phospha e
syn hase;
PPDK,
py u a e,P1
dikinase;
MDH,
NADPH-mala e
dehyd ogenase;
CHX,
cycloheximide;
CAP,
chlo -
amphenicol;
MC,
mic ocys in-LR.
§To
whom
ep in
eques s
should
be
add essed
a :
Uni e si y
o
Neb aska-Lincoln,
Depa men
o
Biochemis y,
210
BcH,
Eas
Campus,
Lincoln,
NE
68583-0718.
2712
The
publica ion
cos s
o
his
a icle
we e
de ayed
in
pa
by
page
cha ge
paymen .
This
a icle
mus
he e o e
be
he eby
ma ked
"ad e isemen "
in
acco dance
wi h
18
U.S.C.
§1734
solely
o
indica e
his
ac .
P oc.
Na l.
Acad.
Sci.
USA
88
(1991)
2713
biochem).
S ock
solu ions
o
10
mM
CHX
and
310
mM
CAP
we e
p epa ed
in
absolu e
e hanol,
while
0.5
mM
MC
was
dissol ed
in
20%
( ol/ ol)
me hanol.
Feeding
o
P o ein
Syn hesis
and
P o ein
Phospha ase
In-
hibi o s.
P eillumina ed
lea es
(-2
g
esh
w
each)
om
4-
o
6-week-old
maize
plan s
we e
excised
unde wa e ,
in-
se ed
in o
150-ml
beake s
con aining
100
ml
o
dis illed
wa e
(con ol),
5
ILM
CHX,
310
,uM
CAP,
o
10
nM
MC
in
wa e
and
main ained
a
oom
empe a u e.
When
eeding
was
done
in
he
da k,
he
beake s
we e
placed
in
a
da kened
ume
hood
o e nigh .
The
da k
sample
was
hen
p epa ed
om
hese
lea es
and
he
co esponding
ligh
sample
was
collec ed
a e
a
90-min
illumina ion
o
he
issue.
When
eeding
was
done
in
he
ligh ,
de ached
con ol
lea es
ha
had
been
p eillumi-
na ed
o
1.5
h
in
wa e
we e
ei he
main ained
in
wa e
o
ed
inhibi o s
o
4
h
in
con inued
ligh ,
ollowed
by
p ep-
a a ion
o
lea
ex ac s.
Illumina ion
was
p o ided
by
a
o ced-ai
cooled
300-W,
low- empe a u e
lamp
a
an
inciden
ligh
in ensi y
o
600-800
/iE
m
2
s-'
(E,
eins ein)
(400-700
nm).
P epa a ion
o
Lea
Ex ac s.
Samples
(0.3
g
esh
w )
om
he
con ol
o
inhibi o - ea ed
lea
ma e ial
we e
chopped
and
g ound
a
40C
in
a
p echilled
mo a
con aining
washed
sand,
2%
(w / ol)
insoluble
poly inylpy olidone,
and
1.5
ml
o
he
app op ia e
ex ac ion
bu e .
Bu e
A
[0.1
M
T is
HC1,
pH
8.0/20%
( ol/ ol)
glyce ol/10
mM
MgCl2/14
mM
2-me cap oe hanol/1
mM
EDTA]
was
used
o
p epa-
a ion
o
PEPC
and
i s
p o ein-se ine
kinase;
bu e
B
(bu e
A
plus
2
mM
py u a e)
was
used
o
PPDK;
bu e
C
(50
mM
Mops-NaOH,
pH
7.5/15
mM
MgCl2/2.5
mM
di hio h ei ol/1
mM
EDTA/0.1%
T i on
X-100)
was
used
o
SPS;
and
bu e
D
(0.1
M
T is-HCl,
pH
8.0/1
mM
EDTA/14
mM
2-me cap-
oe hanol)
was
used
o
MDH.
The
c ude
lea
homogena es
we e
il e ed
h ough
an
80-, m
nylon
ne
and
cen i uged
o
1.5
min
a
8700
x
g.
The
supe na an
luid
was
ei he
used
immedia ely
(PPDK,
MDH)
o
a e
a
0.2-ml
aliquo
was
apidly
desal ed
a
4°C
on
a
Sephadex
G-25
column
(1
x
5
cm)
equilib a ed
wi h
0.1
M
T is-HCI,
pH
7.5/10
mM
MgCI2/20%
( ol/ ol)
glyce ol
o
PEPC
and
PEPC-PK
o
bu e
C
minus
T i on
X-100
o
SPS.
Ac i i y
Assays.
PEPC
ac i i y
was
de e mined
spec o-
pho ome ically
a
340
nm
and
30°C.
The
assay
mix u e
(12)
con ained,
in
a
o al
ol
o
1
ml,
50
mM
Hepes-KOH
(pH
7.3),
2.5
mM
phosphoenolpy u a e,
5
mM
MgCI2,
1
mM
NaHCO3,
0.2
mM
NADH,
10
uni s
o
mala e
dehyd ogenase,
a ious
concen a ions
o
L-mala e,
and
10
,ul
o
desal ed
ex ac
(added
las ).
Mala e
IC50
alues
we e
aken
as
he
mala e
concen a ion
equi ed o
50%o
inhibi ion
o
PEPC
ac i i y
unde
hese
assay
condi ions.
PEPC-PK
ac i i y
was
mea-
su ed
by
32p
inco po a ion
om
[y32P]ATP
in o
pu i ied
da k- o m
PEPC
(10).
The
phospho yla ion
mix u e
con-
ained
35
,ul
o
desal ed
ex ac ,
10
ug
o
pu i ied
da k- o m
maize
PEPC,
an
adenyla e
kinase
inhibi o
plus
a
c ea ine
kinase/phosphoc ea ine
ADP-sca enging
sys em
(10),
25
,uM
ATP,
and
3
,Ci
o
[y-32P]ATP
in
a
inal
ol
o
60
p1.
A e
45
min
o
incuba ion
a
30°C,
he
eac ion
was
s opped
by
adding
20
,ud
o
SDS
sample
bu e
(0.25
M
T is-HCl,
pH
6.8/8%
SDS/40o
glyce ol/20%
2-me cap oe hanol),
ol-
lowed
by
immedia e
boiling
o
2
min.
Ve ical
SDS/PAGE
was
pe o med
as
desc ibed
(13,
14),
and
au o adiog aphs
we e
p epa ed
om
he
d ied
gels
wi h
Kodak
X-Oma
AR
ilm
and
wo
Ligh ning
Plus
in ensi ying
sc eens
(DuPon )
a
-800C.
SPS,
PPDK,
and
MDH
ac i i ies
we e
measu ed
acco ding
o
e .
15
(a
limi ing
subs a e
concen a ions
plus
10
mM
P1
a
250C),
e .
16
( o wa d
di ec ion
plus
2.5
mM
glucose
6-phospha e
and
2
uni s
o
pu i ied
maize
PEPC
a
300C),
and
e .
17
a
30TC,
espec i ely.
RESULTS
AND
DISCUSSION
E ec s
o
CHX,
CAP,
and
MC
on
he
Ligh -Induced
Changes
in
Mala e
Sensi i i y
o
Maize
Lea
PEPC.
The
IC50
alues
o
PEPC
inhibi ion
by
L-mala e
we e
used
as
an
indi ec
means
o
ollowing
he
e ec
o da k
o
ligh
ansi-
ions
on
he
appa en
in
i o
ac i i y
o
he
PEPC-PK
since
hese
alues
e lec
he
se yl-phospho yla ion
s a us
o
he
a ge
enzyme
bo h
in
i o
(7,
8)
and
in
i o
in
esponse
o
ligh
and
da k
(9-12).
Feeding
5
,uM
CHX
o
de ached
p eillumina ed
maize
lea es
in
he
da k
o e nigh
comple ely
and
ep oducibly
p e en ed
he
subsequen
ligh -induced
inc ease
in
he
mala e
IC50
alue
o
PEPC
wi hou
ha ing
any
signi ican
e ec
on
he
da k- o m
enzyme
(Table
1).
In
con as ,
CAP
ea men
had
no
e ec
on
he
ligh -induced
changes
in
mala e
sensi i i y
o
PEPC
(Table
1).
O e nigh
eeding
o
5
AM
CHX
in
he
da k
o
p eda kened
maize
lea es
had
he
same
inhibi o y
e ec
on
ligh
ac i a ion
o
PEPC.
Resul s
simila
o
hose
p esen ed
in
Table
1
we e
ob ained
when
de ached
lea es
o
so ghum,
ano he
C4
g ass,
and
hal ed
lea es
o
Po ulaca
ole acea,
a
C4
dico ,
we e
ed
CHX
(da a
no
shown).
Gi en
ha
such
inhibi o s
a e
known
no
o
be
absolu ely
speci ic,
hus
possibly
causing
de imen al
side
e ec s
(18),
and
ha
he
3-(3
,4-dichlo ophenyl)-1,
1-dime hylu ea-
sensi i e
ligh
ac i a ion/phospho yla ion
o
PEPC
occu s
in
he
cy oplasm
and
is
somehow
ela ed
o
pho osyn he ic
elec on
anspo
and/o
pho ophospho yla ion
(5,
6,
19),
i
was
impe a i e
o
examine
he
e ec
o
CHX
ea men
on
he
in
i o
ligh
ac i a ion
o
o he
pho osyn hesis- ela ed
enzymes
in
maize.
Cy oplasmic
SPS
and
chlo oplas
s omal
PPDK
a e,
like
PEPC,
ligh -ac i a ed
by
e e sible
phos-
pho yla ion/dephospho yla ion
cycles
(2,
3, 6,
15,
20).
In
con as ,
s omal
MDH
is
ligh
ac i a ed
by
3-(3,4-
dichlo ophenyl)-1,1-dime hylu ea-sensi i e
changes
in
i s
SH
edox
s a us
media ed
by
noncyclic
elec on
low
and
he
chlo oplas ic
e edoxin/ hio edoxin
m
sys em
(1,
2,
17,
21).
No ably,
he
esul s
(Table
2)
indica e
ha
he
ligh
ac i a ion
o
hese
h ee
enzymes
was
no
signi ican ly
a ec ed
by
eeding
5
A&M
CHX
o
de ached
maize
lea es
unde
condi-
ions
iden ical
o
hose
desc ibed
in
Table
1.
Simila ly,
CHX
ea men
o
de ached
lea es
had
no
ob ious
e ec
on
ei he
hei
o al
soluble
p o ein
con en
(mg/g
esh
w )
o
poly-
pep ide
pa e n
(e.g.,
see
Fig.
lA,
lane
2
e sus
3
and
lane
5
e sus
6)
o e
he
du a ion
o
hese
ela i ely
sho - e m
expe imen s.
Thus,
he
inhibi o y
e ec
o
CHX
on
he
appa en
in
i o
ac i i y
o
he
PEPC-PK
(Table
1)
appea s
a he
selec i e
o
he
ligh
ac i a ion
o
his
speci ic
con-
e e
enzyme.
F om
he
esul s
desc ibed
abo e,
i
is
clea
ha
de
no o
syn hesis
o
PEPC-PK
o
some
o he
essen ial
componen (s)
(e.g.,
a
pu a i e
modi ying
p o ein
ha
ac i a es
his
p o ein-
se ine
kinase
in
i o)
is
induced
du ing
a
1.5-h
exposu e
o
ligh .
Thus,
i
was
an icipa ed
ha
i
CHX
we e
ed
o
illumina ed
de ached
lea
issue
a e
a
poin
a
which
su i-
cien
p o ein
(i.e.,
ei he
PEPC-PK
o
he
pu a i e
modi ying
Table
1.
E ec s
o
p o ein
syn hesis
inhibi o s
on
ligh -induced
inc ease
in
he
mala e
IC50
alue
o
maize
lea
PEPC
Mala e
IC50,
mM
Inhibi o
Ligh
(L)
Da k
(D)
L/D
None
(con ol)
0.45
0.18
2.5
CHX
0.20
0.17
1.2
CAP
0.46
0.18
2.6
P eillumina ed
maize
lea es
we e
excised
and
ed
wa e
(con ol),
5
gM
CHX,
o
310
jLM
CAP
in
he
da k
o e nigh .
Da k- o m
PEPC
ex ac s
we e
hen
p epa ed
om
hese
lea es
and
he
co esponding
ligh - o m
ex ac s
we e
made
a e
a
90-min
illumina ion
o
he
issue.
Bo any:
Jiao
e
al.
P oc.
Na l.
Acad.
Sci.
USA
88
(1991)
Table
2.
E ec s
o
CHX
ea men
on
in
i o
ligh
ac i a ion
o
SPS,
PPDK,
and
MDH
in
de ached
maize
lea es
Ligh /da k
ac i i y
a io
Enzyme
Con ol*
+
5
IAM
CHX
SWP
6.5
5.5
PPDK
7.0
6.9
MDH
11.7
12.7
See
Table
1
and
Ma e ials
and
Me hods
o
expe imen al
de ails.
*The
ligh -ac i a ed
ac i i ies
(in
,&mol
pe
min
pe
mg
o
soluble
p o ein)
o
SPS,
PPDK,
and
MDH
we e
0.14,
0.43,
and
0.16,
espec i ely.
Ac i i y
was
de e mined
a
limi ing
subs a e
concen a ions
in
he
p esence
o
he
inhibi o
Pi
(15).
p o ein)
had
al eady
been
syn hesized
in
he
ligh
so
ha
PEPC
was
phospho yla ed
and
in
i s
high
mala e
IC50
o m,
con inued
illumina ion
o
he
inhibi o - ed
issue
would
main-
ain
he
a ge
enzyme
in
i s
high
ac i a ion
(phospho yla ion)
s a e.
This
easoning
assumes
ha
he
ac i i y
o
he
PEPC
ype
2A
p o ein
phospha ase
(22)
is
ela i ely
low
in
he
ligh
and/o
ha
apid
deg ada ion
o
he
newly
syn hesized
PEPC-PK
o
pu a i e
modi ying
p o ein
does
no
ake
place
in
he
ligh .
Howe e ,
when
p eillumina ed
(1.5
h
in
he
ligh )
con ol
lea
issue
was
placed
in
wa e
o
5
,M
CHX
and
main ained
in
he
ligh
o
an
addi ional
4
h ,
he
mala e
IC50
alue
emained
cons an
in
he
absence
o
he
inhibi o
bu
dec eased
o
a
le el
cha ac e is ic
o
da k- o m
PEPC
in
he
p esence
o
CHX
(Table
3).
These
da a
sugges
ha
(i)
he
PEPC
ype
2A
p o ein
phospha ase
is
ac i e
in
he
ligh ,
and
(ii)
he e
is
ne
u no e
o
he
PEPC-PK
o
pu a i e
modi-
ying
p o ein
in
he
ligh
in
he
absence
o
i s
syn hesis,
he eby
leading
o
a
ne
dephospho yla ion
o
PEPC
and
he
esul ing
dec ease
in
i s
mala e
IC50
alue.
Based
on
hese
obse a ions
and
sugges ions,
a
mo e
c i ical
and
e ealing
expe imen
was
designed.
Con ol
de ached
lea es
ha
had
been
p eillumina ed
o
1.5
h
we e
ed
5
AsM
CHX
and
10
nM
MC,
alone
and
in
combina ion,
o
4
h
in
he
ligh .
This
la e
cyclic
hep apep ide
is
a
po en
and
speci ic
inhibi o
o
plan
and
animal
ype
1
and
2A
p o ein
phospha ases
bo h
in
i o
and
in
i o
(23-25).
In
he
absence
o
he
p o ein
syn hesis
inhibi o ,
bo h
he
con ol
and
MC- ea ed
illumina ed
issue
main ained
PEPC
in
i s
high
mala e
IC50
o m
(Table
3).
In
con as ,
in
he
p esence
o
CHX
alone,
he
ac i a ion
(phospho yla ion)
s a e
o
he
a ge
enzyme
collapsed
back
o
a
mala e
IC50
alue
cha ac e is ic
o
he
da k- o m
enzyme
(see
abo e
and
Table
3).
Howe e ,
in
he
p esence
o
bo h
inhibi o s,
he
CHX-induced
dec ease
in
he
mala e
IC50
alue
was
la gely
p e en ed,
clea ly
indica ing
ha
he
p o-
ein
phospha ase
ha
dephospho yla es
ligh - o m
PEPC
in
i o
emains
o ally
ac i e
in
he
ligh
and
is
o
he
ype
1
o
ype
2A
class
(23-25),
as
p e iously
implica ed
by
in
i o
s udies
(22).
Thus,
he
egula o y
phospho yla ion
s a us
o
PEPC
is
mainly
de e mined
by
i s
ligh -ac i a ed
p o ein-
se ine
kinase
(10),
he
la e
o
which
appea s
o
be
ligh /da k
Table
3.
E ec s
o
p o ein
syn hesis
and
p o ein
phospha ase
inhibi o s
on
he
main enance
o
he
high
mala e
IC50
o m
o
PEPC
in
he
ligh
Inhibi o (s)
Mala e
IC50,
mM
None
(con ol)
0.46
CHX
0.17*
MC
0.42
CHX
+
MC
0.35
De ached
con ol
lea es,
p eillumina ed
o
1.5
h
in
wa e ,
we e
A
1
2
3
4
5 6
7
8
9
modula ed
by
he
ela i e
a es
o
i s
syn hesis
and
deg ada-
ion
in
he
cy oplasm
o
bo h
C4
monoco s
(maize,
so ghum)
and
dico s
(P.
ole acea).
E ec s
o
P o ein
Syn hesis
Inhibi o s
on
PEPC-PK
Ac i i y.
One
ques ion
a ising
om
he
mala e-sensi i i y
expe imen s
desc ibed
abo e
(Tables
1
and
3)
is
whe he
CHX
inhibi s
he
appa en
in
i o
PEPC-PK
ac i i y
by
di ec
in e ac ion
wi h
his
con e e
enzyme,
a
pu a i e
modi ying
p o ein,
o
PEPC
pe
se.
To
add ess
his
and
o he
issues,
in
i o
32P
phospho yla ion
assays
o
PEPC-PK
ac i i y
we e
pe o med
wi h
apidly
p epa ed,
desal ed
lea
ex ac s
and
pu i ied
da k- o m
PEPC
as
he
p o ein
subs a e
(10).
Fig.
1
shows
he
e ec
o
CHX
and
CAP
on
ligh
ac i a ion
o
PEPC-PK
ac i i y
when
he
inhibi o s
we e
ed
o
p eillumina ed
de-
ached
maize
lea es
in
da kness
o e nigh .
While
he
ac i i y
o
he
PEPC-PK
is
low
in
da kness
and
high
in
he
ligh
o
he
con ol
(10)
and
CAP- ea ed
issue
(c .
lane
2
e sus
5
and
lane
4
e sus
7,
espec i ely,
in
Fig.
1B),
eeding
5
juM
CHX
comple ely
p e en ed
his
s iking
ligh
ac i a ion
o
he
p o ein-se ine
kinase
(c .
lane
3
e sus
6
in
Fig
1B).
When
ei he
CHX
(5
gM)
o
CAP
(310
LM)
was
added
di ec ly
o
he
phospho yla ion
assay
mix u e
con aining
he
desal ed
ex ac
om
he
illumina ed
con ol
lea
issue,
no
in
i o
inhibi ion
o
PEPC-PK
ac i i y
was
obse ed
(c .
lanes
5, 8,
and
9
in
Fig.
1B).
Simila ly,
when
de ached
con ol
lea es,
p eillumina ed
o
1.5
h
in
wa e ,
we e
ed
CHX
(5
juM)
o
an
addi ional
4
h
in
he
ligh ,
PEPC-PK
ac i i y
was
o ally
inhibi ed
(c .
lanes
2
and
4
e sus
lane
3
in
Fig.
2B).
The
p esence
o
bo h
CHX
and
MC
also
led
o
a
comple e
inhibi ion
o
PEPC-PK
ac i i y
(Fig.
2B,
lane
5),
e en
hough
he
mala e
IC50
alue
o
he
endogenous
PEPC
was
ela i ely
PEPC=_.
_ -
PPDK
B
1
2
3
4
5
6
7
8
9
PEPC-
FIG.
1.
Ligh
ac i a ion
o
PEPC-PK
ac i i y
and
he
e ec s
o
p o ein
syn hesis
inhibi o s
in
i o
and
in
i o.
P eillumina ed
maize
lea es
we e
de ached
and
ed
wa e ,
5
,uM
CHX,
o
310
,uM
CAP
in
he
da k
o e nigh .
The
da k- o m
PEPC-PK
was
ex ac ed
be o e
illumina ion
o
hese
lea es
and
he
co esponding
ligh - o m
p o ein
kinase
was
ex ac ed
a e
a
90-min
illumina ion
(c .
Table
1).
PEPC-PK
ac i i y
in
desal ed
lea
ex ac s
was
de e mined
by
32P
phospho yla ion
o
pu i ied
da k- o m
PEPC
as
desc ibed
in
Ma e-
ials
and
Me hods
and
in
e .
10.
(A)
SDS
gel
s ained
wi h
Coomassie
blue
R-250.
(B)
Co esponding
au o adiog aph
o
A.
Desal ed
ex-
ac s
we e
p epa ed
om
da kened
(lanes
2-4)
and
illumina ed
(lanes
5-9)
lea
issue
ed
wa e
(lanes
2, 5,
8,
and
9),
5
,.M
CHX
(lanes
3
and
6),
o
310
,uM
CAP
(lanes
4
and
7).
Lanes
8
and
9,
expe imen s
in
which
5
,uM
CHX
and
310
,uM
CAP,
espec i ely,
we e
added
di ec ly
o
he
in
i o
assay
mix u e
o
PEPC-PK
ac i i y
ex ac ed
om
illumina ed
con ol
lea
issue.
Lane
1,
pu i ied
da k- o m
PEPC.
The
43-kDa
c ea ine
kinase
monome
[*
(see
e .
10)]
and
he
95-kDa
PPDK
and
110-kDa
PEPC
subuni s
a e
indica ed
in
A.
A ows
in
B
poin
o o he
ligh -enhanced
bu
CHX-insensi i e
phosphop o eins.
subsequen ly
main ained
in
wa e
(con ol)
o
edS
,uM
CHX,
10
nM
MC,
o
bo h
o
an
addi ional
4
h
in
he
ligh
be o e
ex ac ion
and
assay
o
PEPC
ac i i y
wi h
o
wi hou
L-mala e.
*Da k- o m
PEPC
has
a
mala e
IC50
alue
o
-0.2
mM
(see
Table
1).
2714
Bo any:
Jiao
e
al.
P oc.
Na l.
Acad.
Sci.
USA
88
(1991)
2715
A
1
2
3
4
5
PEPC-u*
-*i
a"
PPDK-
B
2
3
4
5
Al
;
I'liL
F
..V
FIG.
2.
E ec s
o
CHX
and
MC
on
PEPC-PK
ac i i y
when
ed
o
.4
h
in
he
ligh
o
de ached
con ol
lea es
ha
had
been
p eillumina ed
o
1.5
h
in
wa e
be o e
ea men
(c .
Table
3).
PEPC-PK
was
assayed
by
32p
phospho yla ion
o
pu i ied
da k- o m
PEPC
as
desc ibed
in
Ma e ials
and
Me hods
and
in
e .
10.
(A)
SDS
gel
s ained
wi h
Coomassie
blue
R-250.
(B)
Co esponding
au o a-
diog aph
o
A.
Desal ed
ex ac s
we e
p epa ed
om
p eillumina ed
con ol
lea
issue
ha
was
main ained
in
wa e
(lanes
2)
o
ed
5
JLM
CHX
(lanes
3),
10
nM
MC
(lanes
4),
o
5
IAM
CHX
plus
10
nM
MC
(lanes
5)
o
an
addi ional
4
h
in
he
ligh .
Lane
1,
pu i ied
da k- o m
PEPC.
The
c ea ine
kinase
(*),
PPDK,
and
PEPC
monome s
a e
indica ed
in
A.
high
unde
hese
condi ions
(Table
3).
These
collec i e
ind-
ings
u he
suppo
he
iew
ha
he
PEPC
ype
2A
p o ein
phospha ase(s)
(22)
emains
ully
ac i e
in
he
ligh ,
e en
in
he
p esence
o
CHX,
and
is
e ec i ely
inhibi ed
by
nano-
mola
concen a ions
o
MC
in
i o.
Excep
in
he
p esence
o
bo h
CHX
and
MC,
whene e
he
in
i o
PEPC-PK
ac i i y
was
low,
so
was
he
mala e
IC50
alue
o
he
endogenous
PEPC
om
he
co esponding
lea
issue
and
ice
e sa
(c .
Table
1
e sus
Fig.
1B
and
Table
3
e sus
Fig.
2B).
I is
no able
ha
while
he
in
i o
ligh
ac i a ion
o
PEPC-PK
ac i i y
is
comple ely
inhibi ed
by
eeding
CHX
o
de ached
lea es
(Fig.
1B),
o he
soluble
lea
p o eins
ha
a e
phospho yla ed
in
i o
by
endogenous
p o ein
kinases
a e
no
a ec ed
by
such
ea men ;
his
includes
e en
hose
polypep ides
whose
in
i o
phospho yla ion
s a us
is
g ea e
in
he
ligh
han
in
he
da k
ex ac s
(see
a ows
in
Fig.
1B).
The e o e,
i
is
e iden
om
he
p esen
s udy
ha
he
inhibi o y
e ec
o
his
cy oplasmic
p o ein
syn hesis
inhib-
i o
is
qui e
speci ic
o
he
PEPC-PK
o
some
o he
essen ial
componen (s)
(e.g.,
i s
pu a i e
modi ying
p o ein),
wi hou
ha ing
signi ican
e ec s
on
o he
p o ein
kinase
and/o
p o ein
phospha ase
ac i i ies
(Fig.
1B)
and
ligh -ac i a ion
sys ems
(Table
2).
CONCLUDING
REMARKS
The
p esen
s udy
demons a es
ha
he
in
i o
ligh /da k
egula ion
o
PEPC-PK
ac i i y
in
C4
lea es
(10)
in ol es
ne
de
no o
cy oplasmic
p o ein
syn hesis
in
he
ligh
and
sub-
sequen
deg ada ion
in
da kness.
A
p esen ,
i
is
no
known
wha
essen ial
componen (s)-e.g.,
PEPC-PK
i sel
o
a
pu a i e
modi ying
p o ein-is
he
a ge
o
his
unique
ligh -modula ion
sys em.
The
inc easingly
complex
PEPC
egula o y
cascade,
in ol ing
a
leas
p o ein
syn hesis/
deg ada ion
and
phospho yla ion/dephospho yla ion
cycles,
is
o ally
di e en
om
o he
well-known
mechanisms
o
e e sible
ligh
ac i a ion
o
pho osyn hesis- ela ed
enzymes
(1-4,
6,
15)
and
may
well
explain
why
he
ligh /da k
modu-
la ion
o
PEPC-PK
(10)
and
i s
a ge
enzyme
PEPC
(11,
12,
19)
is
so
much
slowe
han
ha
o
o he
pho oac i a ed
enzymes
(e.g.,
PPDK,
MDH).
I
is
ob ious
ha
u he
pu i ica ion
o
he
PEPC-PK
(7)
and
he
subsequen
p oduc-
ion
o
monospeci ic
an ibodies
agains
his
p o ein
will
elucida e
whe he ,
indeed,
his
speci ic
enzyme
is
he
a ge
o
his
unique,
p o ein
u no e -based
egula o y
sys em.
In
addi ion,
ou
indings
aise
se e al
in e es ing
ques ions
as
o
how
pho osyn hesis- ela ed
(5,
6,
19)
ligh
and
da k
signals
so
speci ically
in luence
cy oplasmic
p o ein
syn he-
sis
and
deg ada ion,
espec i ely,
and
whe he
sho - e m
p o ein
u no e
is,
like
o he
pos ansla ional
co alen
modi ica ions,
a
gene al
mechanism
o
egula ing
enzyme
ac i i y
in
plan s
in
esponse
o
ex e nal
and
in e nal
s imuli.
Clea ly,
ou
esul s
indica e
ha
his
is
p obably
no
he
case
wi h
espec
o
he
ligh -ac i a ion
sys ems
associa ed
wi h
o he
pho osyn hesis- ela ed
cy oplasmic
and
s omal
en-
zymes.
We
hank
Ms.
Shi ley
Condon
o
he
excellen
echnical
assis -
ance.
This
esea ch
was
suppo ed
in
pa
by
G an s
DMB-8704237
and
DCB-9017726
om
he
Na ional
Science
Founda ion
(R.C.)
and
by
he
Labo a o io
de
Fisiologia
Vege al
a
Se illa
Uni e si y
(C.E.)
and
is
published
as
jou nal
se ies
no.
9418
o
he
Uni e si y
o
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