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Light limitation of photosynthesis and activation of Ribulose bisphosphate carboxylase in wheat seedlings

Authors:

Abstract

In limiting light the activation of ribulose-1,5-bisphosphate (RuP(2)) carboxylase [3-phospho-D-glycerate carboxylyase (dimerizing), EC 4.1.1.39] in leaf extracts of 7- to 8-day-old wheat seedlings changed proportionally with the photosynthetic rate of the intact plants. Higher rates of photosynthesis, induced by increasing irradiances, were accompanied by an increase in activation of the leaf RuP(2) carboxylase, while RuP(2) levels remained unchanged. The degree of activation varied from 20% to 60% of full activation at irradiances of 225-1650 muE/m(2).s (photosynthetically active radiation; E = einstein, 1 mol of photons). Between 225 muE/m(2).s and darkness, activation approached 50% while RuP(2) levels dropped more than 90%. During steady-state photosynthesis, levels of the substrate RuP(2) were 250-300 nmol/mg of chlorophyll in the leaves and were similar at all irradiances above 225 muE/m(2).s (25% of light saturation). When velocities of the carboxylase in leaf extracts were corrected for CO(2) levels estimated to exist within the leaf, they compared favorably with the photosynthetic rates of the intact seedlings. Comparison of CO(2) exchange rate, RuP(2) level, and activation of the carboxylase indicates that light limitation of photosynthesis can be due to two factors: the availability of RuP(2) in dark to dim light and activation of the RuP(2) carboxylase in dim light and higher irradiances.
Proc.
Nati.
Acad.
Sci.
USA
Vol.
78,
No.
5,
pp.
2985-2989,
May
1981
Botany
Light
limitation
of
photosynthesis
and
activation
of
ribulose
bisphosphate
carboxylase
in
wheat
seedlings
(CO2
exchange/carbon
reduction
cycle)
JOHN
T.
PERCHOROWICZ*,
DEBORAH
A.
RAYNES*,
AND
RICHARD
G.
JENSEN*tt
Departments
of
tBiochemistry
and
*Plant
Sciences,
University
of
Arizona,
Tucson,
Arizona
85721
Communicated
by
H.
E.
Carter,
February
17,
1981
ABSTRACT
In
limiting
light
the
activation
of
ribulose-1,5-bis-
phosphate
(RuP2)
carboxylase
[3-phospho-D-glycerate
carboxy-
lyase
(dimerizing),
EC
4.1.1.39]
in
leaf
extracts
of
7-
to
8-day-old
wheat
seedlings
changed
proportionally
with
the
photosynthetic
rate
of
the
intact
plants.
Higher
rates
of
photosynthesis,
induced
by
increasing
irradiances,
were
accompanied
by
an
increase
in
activation
of
the
leaf
RuP2
carboxylase,
while
RuP2
levels
re-
mained
unchanged.
The
degree
of
activation
varied
from
20%
to
60%
of
full
activation
at
irradiances
of
225-1650
izE/m2.s
(pho-
tosynthetically
active
radiation;
E
=
einstein,
1
mol
of
photons).
Between
225
,uE/m2.s
and
darkness,
activation
approached
50%
while
RuP2
levels
dropped
more
than
90%.
During
steady-state
photosynthesis,
levels
of
the
substrate
RuP2
were
250-300
nmol/
mg
of
chlorophyll
in
the
leaves
and
were
similar
at
all
irradiances
above
225
,uE/m2.s
(25%
of
light
saturation).
When
velocities
of
the
carboxylase
in
leaf
extracts
were
corrected
for
CO2
levels
es-
timated
to
exist
within
the
leaf,
they
compared
favorably
with
the
photosynthetic
rates
of
the
intact
seedlings.
Comparison
of
CO2
exchange
rate,
RuP2
level,
and
activation
of
the
carboxylase
in-
dicates
that
light
limitation
of
photosynthesis
can
be
due
to
two
factors:
the
availability
of
RuP2
in
dark
to
dim
light
and
activation
of
the
RuP2
carboxylase
in
dim
light
and
higher
irradiances.
Photosynthesis
is
controlled
by
a
variety
of
factors.
Principal
among
these
is
the
biochemical
regulation
of
CO2
fixation
by
ribulose-1,5-bisphosphate
(RuP2)
carboxylase
[3-phospho-D-
glycerate
carboxy-lyase
(dimerizing),
EC
4.1.1.39].
The
kinetic
properties
of
RuP2
carboxylase
suggest
that
the
amount
of
en-
zyme
as
well
as
the
degree
to
which
the
enzyme
is
activated
should
be
considered
as
major
focal
points
for
regulation
of
pho-
tosynthetic
CO2
assimilation
(1).
Investigations
with
isolated
chloroplasts
and
intact
leaf
tissue
have
indicated
that,
under
many
conditions,
control
resides
at
the
carboxylation
step,
be-
cause
the
levels
of
RuP2
can
be
high
and
saturating
(2).
The
expressed
activity
of
the
RuP2
carboxylase
in
vivo
involves
ac-
tivation
ofthe
enzyme
protein
and
catalysis
of
carboxylation
and
oxygenation
(1).
Studies
with
the
purified
RuP2
carboxylase
show
that
it
assumes
a
degree
of
activation
dictated
by
the
pH,
and
Mg2'
and
CO2
concentrations
as
well
as
the
presence
of
effectors
in
the
solution
(3-6).
In
the
chloroplast,
metabolites
might
be
involved
in
the
regulation
of
enzyme
activation
and
consequently
the
rate
of
photosynthesis.
Because
of
the
central
role
of
the
RuP2
carboxylase
in
photosynthetic
CO2
fixation,
we
have
undertaken
the
present
study
to
measure
activation
of
the
RuP2
carboxylase
and
to
determine
its
involvement
during
photosynthesis.
Control
of
the
RuP2
carboxylase
in
intact
leaf
tissue
has
not
been
investigated
beyond
measuring
activities
of
the
enzyme.
Several
components
of
the
carboxylase
system
must
be
exam-
ined
in
order
to
consider
regulation
of
CO2
assimilation
in
intact
plants.
Properties
of
the
enzyme
itself,
including
activation,
and
levels
of
the
substrates,
RuP2
and
CO2,
should
be
measured
and
compared
with
the
observed
CO2
exchange
rates.
It
is
well
known
that
air
levels
of
CO2
limit
the
rate
of
photosynthesis
(7).
However,
increasing
CO2
in
the
atmosphere
is
not
a
prac-
tical
solution
for
increased
photosynthesis,
except
in
enclosed
environments.
Certainly,
at
limiting
irradiances,
where
pho-
tosynthesis
is
reduced,
changes
in
CO2
concentration
are
not
the
cause
of
the
lowered
rate
of
CO2
exchange.
The
synthesis
of
RuP2
or
the
carboxylation
activity
of the
RuP2
carboxylase
as
expressed
in
the
leaf
must
be
the
limiting
factors.
Using
wheat
seedlings,
we
report
changes
in
the
potential
activity
of
the
RuP2
carboxylase
and
its
response
to
irradiance.
These
changes
are
compared
to
the
rate
of
CO2
uptake
and
the
levels
of
RuP2.
With
seedlings
we
are
able
to
achieve
a
steady-state
rate
of
CO2
ex-
change
at
air
levels
of
CO2
over
a
much
longer
time
than
is
pos-
sible
with
chloroplasts
or
protoplasts.
By
using
small
pots
con-
taining
30
to
50
seedlings,
variations
in
the
results
are
reduced
as
compared
to
sampling
individual
leaves.
We
find that
al-
though
RuP2
levels
can
be
limiting
for
photosynthesis
they
are
saturating
under
most
conditions.
The
activation
of
the
RuP2
carboxylase
responds
to
limiting
irradiances
and
appears
as
a
regulating
factor
for
the
rate
of
photosynthetic
CO2
assimilation.
MATERIALS
AND
METHODS
Plant
Material.
Durum
wheat
var.
Mexicali
was
grown
from
seed
in
1:1
(vol/vol)
potting
soil/vermiculite
in
6-ounce
(170-ml)
Styrofoam
pots.
Pots
were
placed
in
a
growth
chamber
under
the
following
conditions:
16-hr
photoperiod
with
30°C
day,
20°C
night,
irradiance
(supplied
by
a
mixture
of
incandescent
and
fluorescent
lamps)
of
225
;iE/m2.s
(E
=
einstein,
1
mol
of
pho-
tons).
Plants
were
used
at
7-8
days.
Gas
Exchange
Procedure.
The
rate
of
CO2
exchange
was
determined
by
using
an
open
infrared
gas
analysis
system.
A
thermocouple
was
inserted
through
the
side
of
the
pot
and
placed
in
contact
with
a
leaf
blade
to
monitor
leaf
temperature.
Light
was
supplied
from
above
the
pot
by
eight
500-W
flood
lamps.
The
bulbs
were
submerged
in
a
water
bath
containing
a
sheet
of
infrared-absorbing
glass
to
reduce
heat
transfer
to
the
leaves.
The
leaf
cuvette
was
a
glass
cylinder
closed
at
one
end
and
tapered
at
the
other
such
that
it
rested
tightly
inside
the
rim
of
a
pot.
Holes
were
punched
around
the
base
of
the
pot
to
allow
some
gas
to
flush
past
the
roots,
thereby
preventing
CO2
produced
by
the
roots
from
interfering
with
the
measure-
ments
of
CO2
exchange.
CO2
concentrations
of
350-360
,uV
liter
of
air
were
prepared
by
mixing
C02-free
air
and
1-2%
CO2
in
air,
using
flow
meters.
CO2
concentrations
were
compared
Abbreviations:
RuP2,
ribulose
1,5-bisphosphate;
E,
einstein;
Chl,
chlorophyll.
f
To
whom
reprint
requests
should
be
addressed.
2985
The
publication
costs
of
this
article
were
defrayed
in
part
by
page
charge
payment.
This
article
must
therefore
be
hereby
marked
"advertise-
ment"
in
accordance
with
18
U.
S.
C.
§1734
solely
to
indicate
this
fact.
2986
Botany:
Perchorowicz
et
al.
to
a
known
standard.
Before
experimentation
was
begun,
plants
were
allowed
to
photosynthesize
for
15
min
after
reaching
a
steady
rate
of
CO2
exchange.
Stomatal
resistance
to
CO2
diffu-
sion
was
determined
with
a
dew
point
hygrometer.
Upon
term-
ination
of
the
experiment,
several
leaves
were
frozen
in
liquid
nitrogen
for
RuP2
determinations.
All
remaining
leaves
were
quickly
cut
off
at
soil
level
and
plunged
into
a
tared
beaker
of
ice
water.
The
beaker
was
reweighed
and
some
ofthe
tissue
was
used
for
assay
of
RuP2
carboxylase
activity.
Assay
of
RuP2
Carboxylase.
Leaf
tissue
(0.5-1
g)
was
ground
with
1
ml
of
buffer
(100
mM
Hepes
at
pH
7.7,
20
mM
KCl)
in
a
glass
homogenizer
maintained
at
2VC.
The
homogenate
was
filtered
through
two
layers
of
Miracloth.
The
assay
mixture
con-
tained
100
mM
Hepes
at
pH
7.7,
20
mM
KCl,
30
mM
MgCl2,
1
mM
dithioerythritol,
and
12
mM
NaH14CO3
(specific
activity
1
Ci/mol;
1
Ci
=
3.7
X
1010
becquerels).
Assays
were
carried
out
at
250C
in
plastic
vials
capped
with
serum
stoppers.
Total
activity
was
measured
by
adding
20
,ul
of
crude
homogenate
to
460
,ul
of
assay
mixture.
After
10
min
(to
attain
full
activation),
20
,u
of
15
mM
RuP2
was
added
to
start
the
reaction.
The
re-
action
was
stopped
after
30
s
by
addition
of
an
equal
volume
of
1
M
HCL.
Initial
activity
was
measured
by
adding
20
,ul
of
crude
homogenate
to
480
1,u
of
assay
mixture
as
for
the
total
activity
determination
except
that
RuP2
was
present.
The
reaction
was
terminated
after
30
s
by
addition
of
acid.
Percent
activation
was
expressed
as
initial
divided
by
total
activity
times
100.
Assay
of
RuP2.
Amounts
of
RuP2
in
tissue
were
determined
by
the
method
of
Latzko
and
Gibbs
(8)
as
modified
by
Sicher
et
al.
(9).
Immunological
Quantitation
of
RuP2
Carboxylase.
Wheat
RuP2
carboxylase
was
isolated
by
using
a
modification
of
the
procedure
of
Hall
and
Tolbert
(10).
Purity
was
confirmed
by
the
presence
of
one
peak
on
an
isokinetic
sucrose
gradient
(17
hr,
26,000
rpm
in
a
Beckman
SW
27
rotor)
(11)
and
by
the
presence
of
two
bands
corresponding
to
the
large
and
small
subunits
on
sodium
dodecyl
sulfate/polyacrylamide
slab
gels
(3.5%
stacking
gel,
10%
running
gel)
(12).
Two
New
Zealand
White
rabbits
were
sensitized
with
three
semimonthly
subcutaneous
or
in-
tramuscular
injections
of
1
ml,
in
four
or
five
sites,
of
Freund's
complete
adjuvant
containing
5
mg
of
purified
tobacco
carbox-
ylase
(13).
Serum
was
collected
semimonthly
after
a
2-week
rest
period
after
sensitizing.
Only
serum
from
sensitized
rabbits
re-
acted
with
crude
wheat
leaf
homogenates
or
purified
wheat
RuP2
carboxylase.
The
RuP2
carboxylase
in
each
leaf
homogenate
was
quanti-
tated
by
radial
immunodiffusion
(14).
Gels
of
1
mm
thickness
contained
1%
agarose,
0.77%
NaCl,
6.6
mM
potassium
phos-
phate
at
pH
7.0,
0.01%
sodium
azide,
and
sufficient
antiserum
to
allow
detection
of
1-4
ug
of
carboxylase.
Wells
3
mm
in
di-
ameter
were
punched
and
5
,l
of
a
known
or
unknown
sample
was
placed
in
each
well.
Gels
were
incubated
at
room
temper-
ature
in
a
humid
atmosphere
for
3-7
days,
after
which
the
area
encompassed
by
each
precipitate
circle
was
determined.
A
stan-
dard
curve
was
plotted
and
unknown
values
were
determined
from
the
regression
line.
RESULTS
Effect
of
Assay
and
Storage
Conditions
on
RuP2
Carboxylase
Activity.
Activity
of
RuP2
carboxylase
in
the
crude
homogenate
had
a
broad
pH
optimum
(reaching
a
maximum
at
7.7-7.8)
when
assayed
in
the
fully
activated
state.
Both
initial
and
total
activ-
ities
were
assayed
at
pH
7.7.
Enzyme
activity
from
tissue
sub-
jected
to
similar
treatments
on
the
same
day
varied
only
a
few
percent.
Until
homogenization,
leaves
were
kept
on
ice,
which
reduced
changes
in
properties
and
preserved
the
physiological
activation
of
the
enzyme.
Storage
at
25°C
or
in
the
presence
of
added
Mg2'
or
NaHCO3
altered
the
enzyme
activation
from
that
found
immediately
after
homogenization
of
the
leaves.
In
contrast,
enzyme
assayed
from
leaves
that
were
kept
in
ice
for
up
to
90
min
prior
to
homogenization
showed
no
change
in
ac-
tivation
(15).
Leaves
were
homogenized
just
prior
to
assay
of
initial
and
total
activities,
Effect
of
Irradiance
on
Photosynthesis,
Levels
of
Ru02
and
Activation
of
RuP2
Carboxylase.
Photosynthesis
was
measured
at
irradiances
from
the
light
compensation
point
(50
gE/m2.s)
to
near
light
saturation
(1650
puE/m2.s).
Fig.
1
compares
the
levels
of
RuP2
and
activation
of
the
RuP2
carboxylase.
At
low
irradiances,
RuP2
was
low
and
might
have
limited
photosyn-
thesis.
However
at
225
/iE/m2.s
and
higher,
RuP2
levels
were
over
300
nmol/mg
of
chlorophyll
(Chl)
(about
12
mM
in
the
chloroplast)
suggesting
that
RuP2
was
saturating
for
CO2
fixa-
tion.
An
estimation
of
the
time
for
utilization
of
this
RuP2
can
be
made.
Although
rates
of
photorespiration
were
not
measured
in
these
experiments,
assuming
that
the
ratio
of
photorespira-
tion
to
apparent
photosynthesis
is
0.5
(16)
and
that
two
RuP2
are
utilized
per
CO2
involved
during
photorespiration,
while
one
RuP2
is
utilized
per
CO2
fixed,
it
can
be
calculated
that
the
time
for
utilization
of
the
RuP2
pool
varies
from
22.2
s
at
225
AE/m2
s
to
5.0
s
at
1600
,uE/m'
s.
Although
CO2
fixation
does
not
appear
to
be
regulated
at
high
irradiances
by
the
levels
of
RuP2,
there
is
a
direct
correlation
between
activation
of
the
RuP2
carboxylase
and
the
rate
of
pho-
tosynthetic
CO2
exchange
at
irradiances
of
225
,uE/m2.
s
and
above
(Fig.
1
A
and
C).
Indeed,
activation
of
the
carboxylase
remains
quite
constant
from
sample
to
sample,
as
indicated
by
the
bars
depicting
one
standard
deviation.
The
maximal
or
total
activity
of
the
RuP2
carboxylase
in
leaf
extracts
from
40
pots
used
in
this
experiment
was
12.47
±
1.53
(mean
±
SD)
,umol
of
CO2
fixed
per
mg
of
Chl
per
min.
Cal-
2
I.
61.4
:
0
-4
to
300
11
200
"
100
._
0
4a
Cd
a-
60
40
20
60
40
20
300
600
900
1200
Irradiance,
gE/m2-s
1500
12
8
4
FIG.
1.
Effect
of
irradiance
on
the
steady-state
net
rate
of
C02
ex-
change
(A),
level
of
RuP2
(B),
and
activation
of
the
RuP2
carboxylase
(C).
Plants
were
brought
to
a
steady-state
net
rate
of
C02
exchange
for
at
least
15
min
before
tissue
samples
were
taken.
Concentrations
of
RuP2
(mM)
were
calculated
by
assuming
a
chloroplast
volume
of
25
,ul/
mg
of
Chl
(2).
Each
point
represents
the
mean
of
at
least
five
deter-
minations;
1
SD
about
the
mean
is
shown.
AB
I
t
C
Proc.
Natl.
Acad.
Sci
-
USA
78
(1981)
Proc.
Natl.
Acad.
Sci.
USA
78
(1981)
2987
culated
on
the
basis
of
the
RuP2
carboxylase
protein
as
deter-
mined
by
radial
immunodiffusion,
the
specific
activity
was
0.
82
±
0.14
,umol
of
CO2
fixed
per
mg
of
RuP2
carboxylase
per
min.
This
represents
about
15.2
mg
of
RuP2
carboxylase
per
mg
of
Chl
in
these
young
wheat
leaves.
Transient
Effects
Occurring
after
Changes
in
Irradiance.
When
irradiance
was
increased
from
225
,uE/m2.
s
to
650
,AE/
m2
s,
RuP2
levels
in
creased
rapidly
and
oscillations
were
ob-
served
(Fig.
2B).
The
oscillation
increased
in
wavelength
and
leveled
off
within
25-35
min
(data
not
shown).
Increased
irra-
diance
also
caused
the
rate
of
CO2
exchange
to
increase
(Fig.
2A).
Activation
of
the
RuP2
carboxylase
increased
about
as
fast
as
the
CO2
exchange
rate
(Fig.
2C).
The
initial
spike
in
RuP2
concentration
occurred
immediately
prior
to
the
time
necessary
for
enzyme
activation
to
increase.
After
a
drop
in
irradiance
from
650
to
225
/iE/m2
s
both
the
CO2
exchange
rate
and
the
level
of
RuP2
dropped
rapidly,
while
activation
of
the
enzyme
responded
rather
slowly.
It
appears
that
upon
a
drop
in
irradiance
RuP2
may
have
become
limiting
for
CO2
fixation.
With
a
chloroplast
volume
of
25
Al/mg
of
Chl,
RuP2
in
the
chloroplast
would
have
been
3-4
mM.
In
these
experiments
from
130
pots,
there
were
7.3
mg
of
carboxylase
protein
per
1
mg
of
Chl
or
4.2
mM
potential
RuP2
binding
sites.
The
concentration
of
RuP2
was
less
than
that
of
the
binding
sites
during
the
first
7-10
min
after
the
change
in
irradiance.
Notice
that
the
subsequent
slow
rise
in
RuP2
followed
the
slow
drop
in
activation
(between
17
and
45
min).
The
specific
activity
of
the
total
activated
carboxylase
remained
independent
of
illu-
mination
at
1.38
±
0.54
Aumol/mg
of
RuP2
carboxylase
per
min.
Although
the
leaf
temperature
in
these
experiments
varied
be-
tween
23°C
and
26.5°C,
RuP2
level,
the
rate
of
CO2
exchange,
and
the
degree
of
activation
of
the
enzyme
were
little
affected
by
temperature
in
this
range.
225
650
*4'.
S..f
0
d
bD
x
Q
a)
o
o
:S
Irradiance,
tLE/m2.s
225
II
Time,
min
FIG.
2.
Influence
of
two
irradiances
on
the
net
rate
of
C02
ex-
change
(A),
level
of
RuP2
(B),
and
activation
of
the
RuP2
carboxylase
(C).
Plants
were
allowed
to
achieve
a
steady-state
net
rate
of
CO2
ex-
change
at
225
/E/m2.s
before
the
experiment
began.
Each
point
rep-
resents
the
average
of
at
least
five
determinations.
Leaf
resistance
also
responded
to
changes
in
irradiance.
The
resistance
at
225
,uE/m2.s
was
4.0-4.3
s/cm2,
while
at
650
it
was
1.1-1.2
s/cm2.
The
change
in
leaf
resistance
that
followed
the
change
in
irradiance
required
5-7
min
to
complete
and
was
much
slower
than
the
change
in
CO2
exchange
rate,
indicating
the
stomatal
aperture
changes
were
not
controlling
photosynthesis.
Light-to-Dark
Effects
on
Enzyme
Activation
and
RuP2
Lev-
els.
When
wheat
seedlings
were
allowed
to
achieve
steady-state
photosynthesis
at
2000
,ttE/m2
s
and
the
light
was
turned
off,
photosynthesis
ceased
and
evolution
of
CO2,
typical
of
respi-
ratory
processes,
was
observed
(Fig.
3A).
The
level
of
RuP2
dropped
rapidly
in
1
min
to
10%
of the
original
value
(Fig.
3B).
When
light
was
again
introduced,
photosynthesis
resumed
and
the
RuP2
level
increased
rapidly.
Note,
however,
that
activation
of
the
enzyme
changed
little
during
this
sequence.
The
level
of
activation
observed
in
the
dark
was
similar
to
that
found
after
10
hr
of
darkness
in
the
growth
chamber.
However,
when
these
seedlings
were
again
illuminated
at
225
AE/m2
s
the
level
of
activation
dropped
to
values
similar
to
those
given
in
Fig.
2.
Photosynthetic
Rate
Compared
to
the
Rate
of
the
RuP2
Car-
boxylase.
The
rate
of
RuP2
utilization,
measured
as
'4Co2
fix-
ation
with
the
enzyme,
was
compared
to
the
rate
of
RuP2
uti-
lization
of
intact
plants
as
estimated
by
CO2
exchange
measurements
at
low
02
levels.
At
air
levels
of
CO2
and
02,
RuP2
is
utilized
by
both
the
carboxylase
and
oxygenase.
At
low
02
levels,
the
CO2
fixation
rate
approaches
the
rate
of
RuP2
utilization
as
the
oxygenase
activity
and
photorespiration
ap-
proach
zero.
Photosynthesis,
as
CO2
exchange,
was
measured
in
0.3%
02
in
N2
with
350
ppm
CO2
at
various
irradiances.
Levels
of
RuP2
in
the
leaves
were
over
200
nmol/mg
of
Chl.
Plants
were
re-
moved
and
chilled
to
ice
temperature,
and
extracts
were
pre-
pared.
The
Km
for
CO2
and
the
Vm.
of
the
RuP2
carboxylase
Light
Dark
Light,
2000
,uE/m2.s
0
E
10
0
UU
0
200
18
6~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~I
~100
Time,
min
FIG.
3.
Effect
of
light-to-dark
transitions
on
the
net
rate
of
C02
exchange
(A),
level
of
RuP2
(B),
and
activation
of
the
RuP2
carboxylase
(C).
Plants
were
brought
to
steady-state
photosynthesis
at
2000
uE/
m2.s
before
the
experiment
began.
Each
point
represents
the
mean
of
at
least
five
determinations.
Botany:
Perchorowicz
et
al
Proc.
Natl.
Acad.
Sci.
USA
78
(1981)
Table
1.
Comparison
of
the
rate
of
CO2
exchange
with
intact
wheat
seedlings
and
the
activity
of
RuP2
carboxylase
at
air
levels
of
CO2
Photo-
RuP2
carboxylase
Calculated
Vmax
of
RuP2
Irradiance,
synthesis,*
velocity,t
internal
CO2,
carboxylase,
gE/m2.s
gmol/mg
Chl-hr
,tmol/mg
Chl-hr
uM
,umol/mg
Chl-hr
225
23.9
±
3.1
26.6
±
2.8
10.0
±
0.1
103
±
11
650
57.9
±
5.7
63.0
±
11.1
9.1
±
0.4
272
±
53
1150
77.3
±
11.0
81.1
±
10.8
8.2
±
0.4
380
±
58
Each
value
is
the
mean
±
SD
from
five
pots
of
wheat.
*
Measured
at
350
ppm
CO2
in
0.3%
02
in
N2
to
eliminate
photorespiration.
t
Calculated
at
the
internal
CO2
concentration
derived
from
diffusive
resistance
measurements
from
the
leaves.
The
observed
Vma,
and
the
Km
for
CO2
of
30
gM
(average
of
the
extracts)
were
used
to
obtain
the
velocity.
was
determined
for
each
extract
and
the
rate
of
CO2
fixation
was
calculated
at
the
internal
CO2
levels
estimated
in
the
plant.
The
internal
CO2
was
estimated
from
leaf
resistance
measurements
(17).
As
indicated
in
Table
1,
the
two
rates,
total
photosynthesis
and
the
RuP2
carboxylase
activity,
are
quite
comparable.
The
activity
of
the
RuP2
carboxylase
appears
to
be
similar
to
the
rate
of
photosynthesis.
The
mean
of
the
various
internal
CO2
levels
and
the
maximal
velocity
of
the
carboxylase
are
also
given
in
Table
1.
DISCUSSION
Previous
work
in
our
laboratory
(2)
with
spinach
chloroplasts
indicated
that
even
though
the
rate
of
CO2
fixation
varied
with
light
intensity,
there
was
often
no
correlation
between
RuP2
levels
and
photosynthesis.
The
results
obtained
with
whole
leaves
in
the
current
study
(Fig.
1)
clearly
show
that
the
limi-
tation
of
steady-state
photosynthesis
by
irradiance
does
not
have
to
be
caused
by
reduced
RuP2
levels
but
can
occur
by
limitation
of
the
carboxylase
activity
in
the
chloroplast.
Below
225
AE/
m2
s
the
levels
of
RuP2
do
appear
to
limit
photosynthesis..
The
observation
that
steady-state
RuP2
levels
are
essentially
the
same
at
irradiances
of
225
juE/m2
s
and
higher
strongly
indi-
cates
that
RuP2
is
not
limiting
the
rate
of
photosynthesis
in
this
range.
As
long
as
RuP2
levels
are
saturating,
RuP2
synthesis
cannot
be
the
limiting
factor
for
the
rate
of
CO2
fixation.
The
processes
necessary
for
the
production
of
RuP2
include
photo-
phosphorylation,
NADPH
production,-
and
the
activity
of
other
enzymes
involved
in
the
Calvin
cycle.
In
air,
CO2
levels
do
limit
photosynthesis.
Calculations
based
on
measurements
of
leaf
resistance
indicate
only
small
changes
in
internal
CO2
as
irradiance
varied
(Table
1).
Thus,
when
RuP2
levels
are
saturating
the
activity
of
the
RuP2
carboxylase
appears
to
be
regulating
the
rate
of
photosynthesis
in
response
to
irradiance.
When
irradiance
is
increased,
wheat
seedlings
are
able
to
increase
RuP2
levels
above
those
at
steady
state
(Fig.
2B).
The
rise
in
the
RuP2
pool
reflects
a
temporary
imbalance
between
CO2
assimilation
and
RuP2
production
by
the
Calvin
cycle.
With
time
the
pool
returns
to
its
steady-state
value,
indicating
a
sep-
arate
controlling
mechanism,
other
than
utilization
by
the
car-
boxylase,
for
determining
maximum
steady-state
RuP2
levels.
During
steady-state
photosynthesis,
RuP2
levels
exceed
the
potential
binding
sites
on
the
enzyme
by
approximately
2-fold.
Only
when
irradiance
is
suddenly
lowered
from
650
to
225
,E/
m2
s
does
RuP2
concentration
in
the
chloroplast
become
less
than
the
number
of
potential
binding
sites
(Fig.
2B).
At
this
time
RuP2
may
well
be
limiting
for
CO2
fixation.
Activation
of
the
RuP2
carboxylase
decreases
as
steady-state
photosynthesis
is
limited
by
irradiance
(Fig.
1).
Although
ac-
tivation
rises
quickly
to
a
new
steady-state
value
when
the
ir-
radiance
is
increased
(Fig.
2C),
it
falls
slowly
after
a
decrease
in
irradiance
from
650
to
225
,uE/m2.
s.
Apparently,
during
this
latter
transition
the
regulation
of
photosynthesis
shifts
from
the
carboxylase
to
the
levels
of
RuP2.
The
enzyme
adjusts
more
slowly
to
the
drop
in
irradiance
than
does
the
Calvin
cycle's
ability
to
produce
RuP2,
resulting
in
limiting
RuP2
while
high
catalytic
activity
is
maintained
by
the
enzyme.
Only
as
enzyme
activation
is
readjusted
does
RuP2
return
to
its
steady-state
value.
Deactivation
of
RuP2
carboxylase
does
not
occur
when
plants
are
darkened.
The
absence
of
light
stops
synthesis
of
RuP2
and
its
levels
drop
rapidly,
bringing
about
the
cessation
of
CO2
fix-
ation.
When
the
light
is
on,
the
increase
in
CO2
exchange
fol-
lows
the
increase
in
RuP2.
Activation
of
the
RuP2
carboxylase
takes
several
minutes
to
stabilize
at
the
steady-state
values
shown
in
Fig.
3C
or
Fig.
1C,
depending
on
irradiance.
This
may
explain
why
only
small
activation
changes
were
measured
with
wheat
protoplasts
by
Robinson
et
al.
(18).
When
darkened
plants
are
illuminated
at
near-saturating
irradiance,
the
change
in
activation
can
be
small.
Control
of
CO2
fixation
shifts
from
low
RuP2
levels
in
dark
to
activation
of
the
RuP2
carboxylase
in
light.
The
dependency
of
photosynthesis
on
activation
of
the
RuP2
carboxylase
may
not
be
seen
if
RuP2
levels
are
limiting.
Low
RuP2
levels
can
occur
when
CO2
is
considerably
higher
than
air
levels,
a
condition
usually
prevalent
when
photosyn-
thesis
is
measured
in
solution
with
isolated
protoplasts
or
chloroplasts.
We
conclude
that
photosynthesis
by
higher
plants,
as
ex-
emplified
by
wheat
seedlings,
can
be
regulated
either
by
the
level
of
RuP2
as;-generated
by
the
Calvin
cycle
and
electron
transport
or
by
activation
of
the
RuP2
carboxylase.
The
molec-
ular
mechanisms
by
which
irradiance
effects
activation
of
the
RuP2
carboxylase
through
the
light-trapping
apparatus
in
the
chloroplast
are
not
fully
apparent,
but
are
thought
to
involve
pH
and
Mg2+
changes.
Specifics
on
the
control
await
further
research.
We
are
grateful
to
David
Zaitlin
for
determination
of
RuP2
carbox-
ylase
purity
and
to
Dave
Parsons
for
supplying
the
wheat
seed.
This
research
was
supported
partially
by
the
Science
and
Education
Admin-
istration
of
the
U.
S.
Department
of
Agriculture
under
Grant
5901-0410-
8-0114-0
from
the
Competitive
Research
Grants
Office,
by
the
Mon-
santo
Agricultural
Products
Co.,
and
by
Grant
PCM77-26284
from
the
National
Science
Foundation.
This
is
University
of
Arizona
Agricultural
Experiment
Station
Paper
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... (Evans et al., 1983;Mächler et al., 1988;Kumar et al., 2002) montre que le taux de la photosynthèse diminue chez les plantes déficientes d'azote et qu'il dépend aussi de la capacité de capturer la lumière. Un faible rayonnement lumineux réduisait la photosynthèse chez les plantes du blé dur (Perchorowicz et al., 1981). En outre, Sage (1990) a suggéré que les limitations de la photosynthèse sont compensées par des régulations au sein des processus impliqués dans la photosynthèse. ...
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The aim of this study was to evaluate the efficiency of three nitrogenous fertilizers applied using wheat and barley grain yield achieved under biotic stress due to crown rot of cereals induced by Fusarium culmorum. The experiment took place in three greenhouses representing different environments, and using a single variety of durum wheat, bread wheat and barley. The effects of inoculation, forms and doses of nitrogen on disease severity, yield and its components were used to reach this aim. Results pointed out that yield of each variety varied according to environments, rates and forms of nitrogen used. Under conditions favorable to disease development, a 24 g L-1 of fertilizers and especially that of urea significantly reduced the yield of durum wheat by 76%. But generally, ammonium nitrate at a rate of 1.5 g L-1 reduced disease severity and allowed a gain in grain yield, within all environments, to reach 222%, 307% and 667% for of durum wheat, bread wheat and barley, respectively. In conclusion, the efficiency of nitrogen fertilization depended on the form of nitrogen, its rate and the environments where they were used.
... Radioactive content of acid-stable 14 C products was determined using a Liquid Scintillation Analyzer (Packard Tri-Carb, PerkinElmer). Rubisco activation state (S) is the ratio of initial to total Rubisco activity [32][33][34] . ...